Download GENETIC AND MOLECULAR ANALYSIS OF THE garnet EYE

Survey
yes no Was this document useful for you?
   Thank you for your participation!

* Your assessment is very important for improving the work of artificial intelligence, which forms the content of this project

Document related concepts

Minimal genome wikipedia, lookup

Epigenetics of neurodegenerative diseases wikipedia, lookup

Ridge (biology) wikipedia, lookup

Biology and consumer behaviour wikipedia, lookup

Genetic drift wikipedia, lookup

Skewed X-inactivation wikipedia, lookup

Quantitative trait locus wikipedia, lookup

NEDD9 wikipedia, lookup

Oncogenomics wikipedia, lookup

Genetic engineering wikipedia, lookup

Neuronal ceroid lipofuscinosis wikipedia, lookup

Gene therapy wikipedia, lookup

Public health genomics wikipedia, lookup

Saethre–Chotzen syndrome wikipedia, lookup

Mutation wikipedia, lookup

Vectors in gene therapy wikipedia, lookup

Gene nomenclature wikipedia, lookup

Polycomb Group Proteins and Cancer wikipedia, lookup

History of genetic engineering wikipedia, lookup

Epigenetics of diabetes Type 2 wikipedia, lookup

Population genetics wikipedia, lookup

Genome evolution wikipedia, lookup

Gene desert wikipedia, lookup

Gene therapy of the human retina wikipedia, lookup

X-inactivation wikipedia, lookup

Therapeutic gene modulation wikipedia, lookup

RNA-Seq wikipedia, lookup

Nutriepigenomics wikipedia, lookup

Point mutation wikipedia, lookup

Gene wikipedia, lookup

Site-specific recombinase technology wikipedia, lookup

Epistasis wikipedia, lookup

Dominance (genetics) wikipedia, lookup

Gene expression profiling wikipedia, lookup

Gene expression programming wikipedia, lookup

Epigenetics of human development wikipedia, lookup

Genome (book) wikipedia, lookup

Artificial gene synthesis wikipedia, lookup

Designer baby wikipedia, lookup

Genomic imprinting wikipedia, lookup

Microevolution wikipedia, lookup

Transcript
GENETIC AND MOLECULAR ANALYSIS OF THE garnet EYE COLOUR GENE
OF Drosophila melanogaster
by
VETT LLOYD
BSc., The University of British Columbia
MSc., The University of Geneva
A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DEGREE OF
DOCTOR OF PHILOSOPHY
in
THE FACULTY OF GRADUATE STUDIES
Genetics Programme
We accept this thesis as conforming
to the required standard
THE UNIVERSITY OF BRITISH COLUMBIA
July 1995
© Vett Lloyd, 1995
In presenting this thesis in partial fulfilment of the requirements for an advanced
degree at the University of British Columbia, I agree that the Library shall make it
freely available for reference and study. I further agree that permission for extensive
copying of this thesis for scholarly purposes may be granted by the head of my
department or by his or her representatives. it is understood that copying or
publication of this thesis for financial gain shall not be allowed without my written
permission.
(Signature)
Department of
cO6 Y
The University of British Columbia
Vancouver, Canada
Date
DE-6 (2/88)
4.
/2
Abstract.
The garnet eye colour gene of Drosophila melanogaster is one the group of
genes called the transport group of eye colour genes. The garnet gene
resembles other members of the transport group of eye colour genes in its
phenotype and shows extensive genetic interactions with them. The most
significant interaction is between garnet and a cryptic allele of the white gene,
first identified as a mutation called enhancer of garnet (we(g)). The phenotype
of garnet mutations and the extreme sensitivity to decreased levels of the white+
gene product suggest that garnet, as well as other members of the transport
group of eye colour genes, act as positive regulators of the white gene. This
interaction may occur at the protein level. A simple model for the physical
interactions between the gene products of garnet, white and other members of
the transport group is proposed.
A critical test of this model requires molecular cloning and analysis of the
individual members of the transport group of eye colour genes. Preliminary
molecular analysis of the garnet gene is reported in chapter two. The garnet
gene is expressed in many different tissues at different stages in development.
Two messages are produced from the garnet gene in wild type embryos.
Conceptual translation of a 4kb c-DNA reveals a novel protein.
In the final chapter I describe the use of the garnet gene to study an example of
epigenetic gene regulation. I have examined a mini-chromosome which
variegates for the garnet gene. The variegation of this mini-chromosome is
extremely unusual in that it depends on the sex of the fly transmitting the mini
chromosome. In this way it conforms to the conventional definition of parental
genomic imprinting. I examined a number of possible mechanisms which might
be responsible for the parental imprinting of the mini-chromosome The results
suggest that heterochromatin formation is responsible for the somatic
expression of the genomic imprint, but a different system may operate to
establish the imprint.
III
TABLE OF CONTENTS
Abstract
ii
Table of contents
iv
List of Tables
Vi
List of Figures
x
Acknowledgments
xv
Dedication
xvi
General introduction
1
Materials and Methods
39
Chapter One
Interactions between garnet and
other eye colour genes.
Introduction
45
Results
47
Discussion
102
Chapter Two
Analysis of the garnet gene
Introduction
116
Results
118
iv
Discussion
Chapter Three
187
Imprinting of a mini-chromosome in
Drosophila melanogaster.
Introduction
205
Results
220
Discussion
269
Bibliography
287
Appendix 1
Determination of eye pigment levels
306
Appendix 2
Cloning of the garnet gene
312
V
Index of Tables:
page
Table 1. A list of genes which affect eye colour in Drosophila
melanogaster.
3
Table 2. Genes proposed as members of the transport group of
eye colour genes.
32
Table 3. Eye colour genes with known or proposed functions.
36
Table 4. The Effect of 3C deficiencies on white and enhancer
of garnet.
54
Table 5. Complementation between e(g) and different white
alleles.
60
Table 6. Rescue of the enhancer of garnet effect by white+
transgenes.
64
Table 7. Sensitivity of the enhancer of garnet effect to
garnet dosage.
68
Table 8. Effect of we(g) dosage on the enhancer of garnet
71
effect.
Table 9. The effect of the enhancer of garnet mutation on
other eye colour mutants.
78
vi
Table 10. Interactions between garnet and other eye colour
genes.
82
Table 11. Summary of interactions between other eye colour
genes and enhancer of garnet and garnet.
84
Table 12. Phenotypes of garnet double mutants with different
white alleles.
89
Table 13. Epistatic interactions between the wa3 and the
alleles.
2
g
91
Table 14. Effect of zeste on garnet.
96
Table 15. Effect of zeste modifiers on the zeste-garnet
genotype.
100
Table 16. Pigment levels of various garnet alleles.
123
Table 17. Quantitative assessment of pteridine pigments
after chromatographic separation of pigments.
128
Table 18. Effect of various garnet alleles on colour of
131
malpighian tubules.
Table 19. Effect of various garnet alleles on testes sheath
134
colour.
vii
Table 20. The phenotype of various garnet alleles in
combination with a deficiency.
137
Table 21. Summary of lesions in different garnet alleles.
158
Table 22. List of genes with sequence similarity to the
garnet gene.
179
Table 23. List of published garent alleles.
189
Table 24. List of other names given to imprinting phenomenon.
208
Table 25. Imprinting and human medical conditions.
212
Table 26. Parental effects in Drosophila melanogaster.
215
Table 27. The effect of different garnet alleles on the imprint.
242
Table 28. Maternal effect of the garnet gene.
245
Table 29. Variegation of the Dp(1;f)LJ9 mini-chromosome:
The effect of developmental temperature.
255
Table 30. Variegation of the Dp(1;f)LJ9 mini-chromosome:
257
The effect of sodium butryrate.
viii
Table 31. Variegation of the Dp(1;f)LJ9 mini-chromosome:
The effect of Y chromosome dosage:
259
Table 32. Imprinting of the Dp(1;f)LJ9 mini-chromosome:
The effect of developmental temperature.
262
Table 33. Imprinting of the Dp(1;f)LJ9 mini-chromosome:
The effect of sodium butryrate.
264
Table 34. Imprinting of the Dp(1;f)LJ9 mini-chromosome:
The effect of Y chromosome dosage.
267
Table 35. The effect of attatched versus free sex chromosomes
on imprinting of Dp(1;f)LJ9.
284
ix
Index of Figures.
page
Figure 1. Diagram of the eye, structure and organization of
different cell types in the ommatidia and organization of the primary
and secondary pigment cells of Drosophila melanogaster.
11
Figure 2. Diagram of the biosynthetic pathway of xanthommatin
production in Drosophila melanogaster.
16
Figure 3. A possible pathway of pteridine pigment biosynthesis
in Drosophila melanogaster.
18
Figure 4. Three different models for pteridine biosynthesis in
Drosophila melanogaster.
20
Figure 5. Phenotypes of three severe garnet alleles in
conjunction with the enhancer of garnet mutation.
49
Figure 6. Cytological localization of the enhancer of garnet
52
mutation.
Figure 7. Diagram of the structure of the white gene of
57
Drosophila melanogaster.
Figure 8. Comparison between the severity of different white
alleles and their effect on garnet.
73
x
Figure 9. Analysis of garnet transcription by in situ hybridization
to rosy null tissues.
86
Figure 10. The effect of zeste mutant alleles on garnet
phenotype.
94
Figure 11. The phenotype of zeste-garnet combinations
in females and males.
98
Figure 12. Nolte’s model for the interaction between eye
colour genes.
110
Figure 13. Model of the physical interactions between the
products of white, brown, scarlet and the transport group of
eye colour genes, including garnet.
113
Figure 14. Spectrum of eye colour phenotypes of different
alleles of the garnet gene.
120
Figure 15. Chromatographic analysis of pteridine pigments
of garnet alleles.
126
Figure 16. Life span of wild type and various garnet mutants.
140
Figure 17. Diagram of the mutation rate to garnet upon the
144
S6-1 strain.
xi
Figure 18. Diagram of the mutation rate and phenotypes of derivative
garnet mutations derived from the original gP mutation.
146
Figure 19. Southern transfer and hybridization analysis of the
gP allele and gP derivative mutations.
150
Figure 20. Southern transfer and hybridization analysis of the
wild type garnet region and the g
1 allele.
153
Figure 21. Southern transfer and hybridization analysis of
garnet mutants.
155
Figure 22. Northern transfer and hybridization analysis of
wild type embryos and garnet mutant adults.
160
Figure 23. Restriction fragment length analysis of lambda
phage clones containing garnet and flanking sequences.
163
Figure 24. Restriction fragment map of garnet and
surrounding region.
165
Figure 25. Strategy used to sequence the imaginal c-DNA
168
clone of the garnet gene.
Figure 26. Sequence and conceptual translation of the
imaginal disc c-DNA clone of the garnet gene.
XII
170
Figure 27. Analysis of garnet transcription by in situ
hybridization to various tissues.
183
Figure 28. Southern analysis of regions of sequence similarity
to the garnet gene in Drosophila melanogaster.
186
Figure 29. Map of the garnet gene generated by intragenic
recombination.
193
Figure 30. Diagram of the structure and origin of the
Dp(1;f)LJ9 mini-chromosome.
222
Figure 31. Meiotic stability of the Dp(1;f)LJ9 mini-chromosome.
224
Figure 32. The garnet phenotype in flies with maternally
and paternally derived Dp(1;f)LJ9 mini-chromosome.
228
Figure 33. The garnet phenotype in malpighian tubules of flies with
maternally and paternally derived Dp(1;f)LJ9 mini-chromosome.
231
Figure 34. Phenotype of narrow abdomen and tiny in flies bearing
maternally and paternally derived Dp(1;f)LJ9 mini-chromosomes. 234
Figure 35. Variegation of narrow abdomen and tiny in genotypically
identical flies bearing either maternally or paternally derived
236
Dp(1;f)LJ9 mini-chromosome.
XIII
Figure 36. The Y chromosome does not cause the imprint.
240
Figure 37. Test of the physiological compensation model.
249
Figure 38. Under-representation of the garnet gene in the
Dp(1;f)LJ9 mini-chromosome.
252
Figure 39. Model of parent-dependent spread of heterochromatin
responsible for the imprint of the Dp(1;f)LJ9 mini-chromosome.
xiv
273
Acknowledgments:
I would like to thank those who have helped me.
It is not possible to list here all those who have given me support and
inspiration, emotional, intellectual and financial, over the many years that this
work was in progress. Nevertheless, those who have helped me know who they
are. They have my gratitude, and I hope, will allow me to opportunity to
reciprocate.
Although any work of science depends on the prior work of others, the specific
contributions of some must be mentioned.
The garnet gene was cloned by Dr. D. Sinclair, working in the laboratory of Dr.
G. Tener, biochemistry U.B.C. Dr. Sinclair initiated the molecular analysis of the
garnet gene, brought the parental effect of Dp(1;f)LJ9 to my attention and
provided me with advice and encouragement at every step. The gP allele,
which facilitated cloning of the garnet gene was isolated by R. Wennberg, in the
laboratory of Dr. T. Grigliatti. The program used to analyze the pigment data was
written by Dr. J. Berger. As with any genetic work, I am indebted to the fly stock
centres, at Bloomington, Indiana, Bowling Green and Umea, Sweden.
A number of undergraduate students also contributed to this work in the course
of doing a directed studies project. D. Dyment and K Swanson contributed data
to the pigment assay controls in appendix one. M. Maharaj made some of the
stocks used to test the effect of the e(g) mutation on other eye colour genes. G.
Mahon mobilized the CaSpeR element construct and characterized the new
insertion strains that were used to test the rescue of the enhancer of garnet
effect. A. Rivers assisted in the sequencing of garnet genomic clones. B. Lee
contributed to the test of the enhancer of garnet effect of different white alleles.
L. Harvey tested the effect of some garnet alleles on longevity. The details of the
experiments are given in the appropriate section.
In addition to these students, I also had the opportunity to supervise a number of
other undergraduate students, from whom I learned at least as much as they
from me. Although their work is unpublished they are: Melanie Klenk, Keyvan
Hyunda, Cara Warrington, Carol Lee, Gurdip Lalli, Lynn Ma, Chaucer Wong,
and Mahnaz Kermati.
Finally, I would like to thank my supervisor Dr. T. Grigliatti for allowing me the
rare privilege of complete freedom of research.
xv
Quotation:
There is no answer. There has never been an answer, there will never be an
answer. That’s the answer.
-Gertrude Stein.
xvi
General Introduction
The garnet gene.
1
General introduction.
The problem:
The eye colour genes of Drosophila melanogaster have attracted the attention
of biologists for as long as the organism has been used as an experimental
system for genetics. Aside from their intrinsic beauty, eye colour genes have
played a role in the genesis of many important genetic concepts: sex linkage,
position effects, pleiotropy, the chromosome theory of inheritance, autonomous
and non-autonomous gene action, intra allelic complementation, taxonomy, the
action of genes through hormones and developmental regulatory cascades.
One of the features of Drosophila eye colour mutants which first appealed to
biologists was the number of mutations which have an effect on eye colour.
While an invaluable genetic resource, the remarkable number of eye colour
genes also posed practical and theoretical problems. The overriding problem
posed by eye colour genes is the sheer number of them. Breme and Demerec
(1942) list 45 eye colour genes. In 1950, 38 eye colour mutants were extant
(Nolte 1950). By 1976, 51 eye colour genes were known (Phillips and Forrest
1976). The list shown in Table 1 is derived from the list of all mutants
described in Drosophila melanogaster up to 1992 (Lindsley and Zimm 1992). It
lists 110 genes; even if pattern and secondary effects are excluded, there are
still 85 genes whose primary effect is on eye colour. Nor are these 110 genes
likely to be all the genes which affect eye colour. Amongst eight eye colour
mutations isolated from a cellar and a vineyard in Italy, in a study of naturally
occurring eye colour variation, three defined novel eye colour genes
(Calatayud, Jacobson and Ferré, 1989). A system allotted considerable genetic
resources would be expected to be essential or at least important for the
2
Table 1. Genes which affect eye colour in Drosophila melanogaster.
The first two columns list the name and gene designation. The eye colour of the
mutant is indicated in the third column. Where there is an allelic series the
colour of the first allele is given. This is only an approximate indication of the
eye colour which varies with allele, age and often sex. The final column gives
the pigment group which is affected. Frequently the pigment or pigment group
affected is not known and is only inferred from the eye colour. This instance is
indicated by a question mark. In some cases the effect on eye pigment is a
secondary effect of patterning defects which change cell fate specification as a
result of which pigment cells fail to differential. These instances are indicated by
“pattern”. In other instances alteration in eye colour or pigmentation is a
secondary result of other alterations such as decreased body size or increased
melanization. These instances are indicated as “secondary”. This list was
derived from Lindsley and Zimm (1992).
3
Table 1. Genes which affect eye colour in Drosophila melanoqaster
mutant
amy
bis
bo
bos
bre
bri
buo
bur
bw
ca
car
cast
cd
cho
cm
ci
cm
cmd
cml
cn
cop
cr-3
dcm
diI--3
dk
dke
Dke-2
dn
dor
Dr
drb
dyb
E(z)
g
gi
Hn
je
kar
kpn/awd
lix
It
ltd
Ixd
amethyst
bistre
bordeaux
bordosteril
bright-eye
bright
burnt-orange
burgundy
brown
claret
carnation
cast
cardinal
chocolate
cinnamon
clot
carmine
carminoid
caramel
cinnabar
copper
cream-3
dark carmine
dilute-3
dark
dark-eye
darkened eye
doughnut
deep orange
Drop
dark red brown
dusty body
enhancer of zeste
garnet
glass
Henna
jelly
karmoisin
killer of prune
little isoxanthopterin
light
Iightoid
low xanthopterin dehydrogenase
4
colour
pigment
affected
purple
brown
purple
brown
orange
orange
orange
brown
brown
both
brown
brown
bright
brown
brown
dark
orange
orangish
brown
orange
brown
pale
brown
paler
darker
brown
darker
pattern
pale orange
darker
darker
brown
pale
paler
darker
dark brown
pink
orange
wild type
wild type
pale
pale
wild type
pteridines?
pteridines?
both?
pteridines?
ommochromes?
ommochromes?
ommochromes?
ommochromes
pteridines
both
both?
pteridines?
both
pteridines?
ommochromes
pteridines
both
both?
pteridines?
ommochromes
pteridines?
both?
pteridines?
both?
both
pattern
pattern?
pattern
both
pattern
both?
secondary?
both
both
pattern?
pattern?
both?
pteridines
pteridines
pteridines
both
both
pteridines
ma
mah
ma!
man
Me
me!
mk
mot-28
mot-K
mot-321
mot-36c
msd(gl)
mtb
mud!
mur
nrs
ocr
or
osh
p
pd
Pdr
Pec
pers
pn
po
port
port-b
pr
Pu
pur
pw
pw-c
pwn
pym/ade2
ral
ras
rb
rdb
red
rl
rm
rs
rud
rv
rwi
ly
Sa
sb
maroon
mahogany
maroon-like
mandarin
Moire
melanized
murky
mottled 28
mottled of K
mottled 321
mottled 36
modifier of sexual dimorphism of gi
matt brown
mudlike
murrey
narrow scoop
ochracea
orange
outshifted
pink
purpleold
purpleolder
Pupilla ecentrica
persimmon
prune
pale occelli
port
port-b
purple
Punch
purplish
pink wing
pink wing c
pawn
polymorph
raisin
raspberry
ruby
reddish-brown
red malpighian tubules
rolled
rimy
rose
ruddle
raven
red wine
rosy
Salmon
soft brown
5
brown
brown
brown
orange
both
darker
darker
mottled
mottled
mottled
mottled
darker
brown
brown
purple
darker
orange
orange
brown
pink
dark pink
rosy-like
pattern
orange
brown
brighter
pale
browny
browny
pale
ruby
pink
lighter
brown
browny
brown
browny
paler
brown
wild type
darker
brown
pink
browny
dark
browny
browny
browny
browny
pteridines
pteridines?
pteridines
ommochromes?
pattern
secondary
secondary
pattern
pattern
pattern
pattern
secondary
secondary?
both?
both?
secondary
ommochromes
both
secondary
both
both
both?
pattern
ommoch romes?
pteridines
pattern?
both?
pteridines?
pteridines
pteridines
pteridines
secondary?
secondary?
secondary
pteridines
pteridines?
pteridines
both?
both?
both?
pattern
pteridines?
pteridines?
pteridines?
secondary
pteridines?
both?
both?
pteridines?
se
st
sf-3
she
som
St
swy
syn
te
trl
It
U
ups
v
yin
w
We
z
very dark
brown
brown
brown
dull brown
orange
darker
brown
dark
purple
lighter
mottled
dull, rough
orange
browny
white
paler
lighter
sepia
safranin
safranin-3
sherry
sombre
scarlet
swarthy
syndrome
tenerchaetae
translucent
tilt
Upturned
upright
vermilion
yin
white
Washed eye
zeste
6
pteridines
pteridines?
pteridines?
pteridines?
secondary
ommochromes
secondary
secondary
secondary
pattern
secondary
pattern
pattern
ommochromes
pteridines?
both
pattern?
both
viability of the organism. Yet few eye colour genes affect the viability of the fly.
The clearest example is that of the white gene. Flies with no pigments due to
null mutations in this gene are completely viable and fertile. In fact, the first
mutation isolated in Drosophila melanogaster was a such a complete loss of
function mutation for the white gene (Morgan in 1910 cited in Lindsley and
Zimm 1992). That it was isolated from a wild population suggests no great loss
of biological vitality.
The sheer number of mutations which alter eye colour was seen as a problem
by early authors (e.g. Nolte 1 952b) and has not yet been adequately resolved.
Even a cursory inspection of the two pigment biosynthetic pathways (Figure 2
and Figure 3) reveals that there are approximately 15 enzymatic steps
required to produce the pigments found in the wild type eye. Thus, even
allowing for co-factors, there remains a considerable excess of genes involved
in the production of the pigments of the wild type eye. In addition to enzymes
and co-factors, it is reasonable to suppose that some of this apparent “excess”
of eye colour genes are concerned with transport, sequestration and control of
deposition of the eye colour pigments. The study of any eye colour gene must
ultimately address the question of pleiotropy and redundancy of these genes.
Both the number of genes and the dispensability of many of them suggest a
diverse range of functions, many of which may be shared by other genes.
Despite their historical importance, the disposition and biogenesis of the
pigments in the eye, the chemical structure of the pigments, the biosynthetic
pathways responsible for the pigments, the genes involved and the complex
physiological, developmental and tissue interactions required to produce wild
type eye colour remain obscure and still subject to debate. In addressing these
7
questions, the physical context of pigments, the pigment cells of the eye, and
the complex developmental regulation of pigment biosynthesis, as revealed by
the intersection of genetic and biochemical studies on the pigments, must be
briefly summarized.
The eye.
The eye is the most thoroughly studied of the four pigmented structures in
Drosophila melanogaster (excluding melanized structures). The structural and
developmental complexity of the eye is underscored by studies which show that
approximately two thirds of randomly selected lethals have defects in the eye (of
those two thirds, one third is responsible for general cell viability functions, the
other third is eye specific, Thaker and Kankel 1992). The development and
structure of the compound eye have been the focus of extensive investigation. I
will briefly summarize this work as it relates to pigment deposition. Both the
signals leading to specification of cell fate in the development of the eye, and
the nervous connections between the eye and the brain are subjects of intense
research. These subjects have been thoroughly and often reviewed
(Meyerowitz and Kanker 1978, Renfranz and Benzer 1989, Pak and Grabowski
1980, Zipursky et al 1984, Tomlinson 1985, Venkatesh, Zipursky and Benzer
1985, Ready 1989, Zipursky 1989, Campus-Ortega 1988, Ranganathang,
Harris and Zuker 1991) and will not be treated thoroughly here.
Development of the eye. The compound eye of Drosophila melanogaster, like
that of many insects, is composed of hundreds of reiterated units, the ommatidia
(Figure 1). The eye analage arises from approximately 20 cells which
invaginate from the embryonic ectoderm and which eventually form the eye
imaginal disc (reviewed by Venkatesh, Zipursky and Benzer 1985, Zipursky
8
1989, Ranganathang, Harris and Zuker 1991). During embryogenesis, first and
second instar stages, the eye imaginal disc cells proliferate but remain
undifferentiated. During the proliferation stage, the eye disc is attached to the
brain by the optic stalk. Later elaboration of the nervous system results in
precise spatial correspondence between individual ommatidia and their
connections in the brain. During the third instar a dramatic wave of
morphogenetic activity sweeps over the disc. This morphogenic furrow is
associated with differentiation of the various cell types of the eye. The pigment
and cone cells are among the last to differentiate and are recruited from the
undifferentiated epithelium by underlying photoreceptor cells. As with other cell
types in the eye, cell fate is not clonally determined but is determined by
position-dependent induction. Finally, during pupation the eye imaginal disc
evaginates to form the adult compound eye.
Structure of the eye. The compound eye of the adult Drosophila is composed of
700-800 ommatidia. The structure of three adjacent ommatidia is shown in
Figure lB. The distal end of each ommatidium consists of the corneal lens and
pseudo cone which functions to gather and focus light. Proximal to the dioptic
apparatus are the 8 photoreceptor cells with central rhabdomeres which
transduce light to nervous impulses. Each rhabdomere has different wavelength
specificity and connects to the brain via a complex network of neural
connections. Surrounding each ommatidium is a sleeve of pigment cells
(arranged as shown in Figure 1C). The pigment cells act to regulate light
exposure and to optically isolate the ommatidia. The pigment granules within
the pigment cells are not static. In bright light they move towards the
rhabdomere thus reducing the amount of light reaching the rhabdomeres
9
Figure 1. Diagram of the eye, structure and organization of different cell types
in the ommatidia and organization of the primary and secondary pigment cells
of Drosophila melanogaster.
A. Schematic diagram of the head of Drosophila melanogaster.
B. Diagram of a longitudinal section through the eye of Drosophila
melanogaster showing three adjacent ommatidia. The various cell types which
compose the ommatidia are shown. Redrawn from Nolte 1950, figure 12.
C. Schematic diagram of the organization of primary and secondary pigment
cells and the ommatidial lens. Redrawn from Nolte 1950, figure 11.
10
The eye of Drosophila melanogaster
A
C
outline of ommatidia lens
primary pigment cell
—
B
secondary pigment cell
e
cell
o
cell nucleus
• pigment granule
membrane
o
p
/0 4—post retinal cells
—layer of monopolar cells
.
external optic glomerulus
external chiasma
11
whereas in low light they migrate to the periphery of the ommadium increasing
the light exposure but decreasing visual acuity.
Pigment cells There are two types of pigment cells, the primary and secondary
pigment cells. The primary pigment cells lie more distally in the eye directly
surrounding the pseudo cone, while the secondary pigment cells lie more
proximally, principally surrounding the photoreceptor cells. These two types of
pigment cells cooperate to completely encase each individual ommatidium
(although each pigment cell is shared by adjacent ommatidia). The pigment
cells have an abundance of pigment granules in their cytoplasm, however, they
are not the only cells with pigment granules. The photoreceptor cells also have
pigment granules in their lateral cytoplasm, although fewer than the pigment
cells. The post retinal and basal cells may also have some pigment granules
(Mainz 1938, Nolte 1950, Reaume, Knecht and Chovnick 1991), although this
has been the source of some dispute.
Pigment granules. Early light and electron microscopy work defined differences
in pigment granule morphology (Shultz 1935, Nolte 1950, Shoup 1966). There
are two types of (normal) pigment granules, named type one and type two. Both
types of granules are ribosome-sized, multi-subunit (Hearl, Dorsett and
Jacobson 1983, Hearl and Jacobson 1984) membrane-bound organelles which
originate in close proximity to the golgi apparatus and are likely derived from it
(Shoup 1966), although alternative origins have been proposed (Reaume,
Knecht and Chovnick 1991). Type one pigment granules first appear
approximately two days before eclosion, which corresponds to the first time that
the ommochrome pigment can be detected (Schultz 1935, Nolte 1950, 1954a).
They are electron dense, grow in size after eclosion and contain only
12
ommochromes. This type is found in primary pigment cells and most probably in
the photoreceptor cells. Type two granules are complex membrane bound,
fenestrated structures found only in the secondary pigment cells. They are the
site of pteridine pigment deposition. Development of these granules and the first
detectable appearance of the pteridine pigments coincide at approximately one
day before eclosion. These granules do not change size in development but do
become denser after eclosion. Change in both the size and morphology of type
one and two pigment granules continues for a few days after eclosion of the
pharate adult, concomitant with increase in the amount of ommochrome and
pteridine pigments. Eye colour mutants often show a variety of complex
changes in the colour and morphology of these pigment granules. For example,
histological analysis of the g
3 allele, the only garnet allele for which
histological information has been recorded (Nolte 1950), shows normal
numbers, distribution, development and morphology of pigment granules. The
only change from wild type was alteration in the intensity of colour of both the
type one and type two granules. This change presumably corresponds to
deficiency for both the pteridine and ommochrome pigments.
The discovery that pigments were associated with proteins (Schultz 1935) led to
the realization that the pigment granules were largely proteinatious. Coordinate
appearance of the pigment granules and pigments in development (Schultz
1935, Nolte 1950, 1954a), in conjunction with the proteinatious nature of the
pigment granules led to speculation that pigment granules are not just passive
sites of pigment deposition but are complexes composed of the biosynthetic
pigment enzymes as well as the pigments themselves (Phillips, Forrest and
Kulkarni, 1973). This hypothesis has been contested (Sullivan, Grillo and Kitos
1974) based on the finding that the enzymes in the xanthommatin pathway are
13
found in different subcellular compartments whereas others are free in the
cytosol. In addition, there is evidence that suggests that the pleiotropic effect of
different eye colour mutants on the various enzymes, on which the enzyme
complex model was based, is a result of failure to differentiate enzymatic and
non-enzymatic conversion of pigment intermediates (Wiley and Forrest 1981).
Nevertheless, more recent work suggests that pigment granules contain at least
some of the enzymes involved in pigment biosynthesis (Dorsett, Yim and
Jacobson 1978, Hearl, Dorsett and Jacobson 1983, Hearl and Jacobson 1984).
Biosynthesis of the eye colour pigments: The eye colour of the wild-type
Drosophila eye is derived from the deposition of two biochemically distinct types
of pigment, the ommochromes and the pteridines. Both types of compounds are
widely present in nature in both the plant and animal kingdom. The
ommochromes are fairly simple compounds derived from the amino acid
tryptophane. The pteridine compounds, however, are remarkably complex. The
pteridines were first isolated by Hopkins (1889) from the wings of an English
brimstone butterfly but chemical identification of the red pigment in Drosophila
as a pteridine was not made until 1940 (Lehderer 1940). The structure of some
of the pteridines is still subject to dispute.
While the fundamental steps of the biosynthetic pathway for their production
appears conserved from the prokaryote, Escheria coil through Drosophila to
humans, the details are still under intense investigation (Phillips and Forrest
1976, Pfleiderer 1993). Figures 2 and 3 show the biosynthetic pathways for
the production of these two compounds. While the biosynthetic pathway for the
ommochrome pigment, xanthommatin, has been determined, based in large
part on Drosophila genetics, the pathway for the pteridine pigments remains
14
Figure 2. Biosynthetic pathway of xanthommatin production in Drosophila
melanogaster.
The structure of the various intermediates in xanthommatin biosynthesis and the
enzymes responsible for their production are shown. Adapted from Phillips and
Forrest 1976.
15
BIOSYNTHESIS OF XANTHOMMATIN
NH2
TRYPTOPHAN
•CH2/\COOH
tryptophan pyrrolase
vermilion
+
N-FORMYL KYNURENINE
NH2
formamidase
KYNURENINE
cinnabar ÷
3-HYDROXYKYNURENINE
COOH
HCI4H2
phenoxazinone synthetase
XANTHOMMATIN
DIHYDROXANTHOMMATIN
16
Figure 3. A possible pathway for pteridine pigment biosynthesis in Drosophila
melanogaster.
The structure of the various intermediates involved in pteridine biosynthesis and
some of the enzymes and genes responsible for their production are shown.
Adapted from Brown et al. 1978, Brown 1989, Calatayud, Jacobson and Ferré
1990 and Pfleiderer 1993.
17
A possible pathway for pteridine pigment biosynthesis i n
Drosophila melanogaster
0
N
NJXj>
2
H
rj,-LP
guanosine triphosphate
+
OHOHHP
HA’d-dLcL-P
GTP cyclohydrolase
Pu + cm, ma-I,
JN)
1
H2NI
Ii
dihydroneopterin-P3
sepiapterin synthase A
pr
sepiapterin synthase B
-N
F-12N
H4 pterin
*
6-acetyl-homopterin
aka-pyrimidodiazepine
0+
H2N
H’
N1X)OH
2
H
H2 pterin
dihydrobiopterin
0
HH H
0*
HH H
HA()H
H2N’ &HI
isoxanthopterin
xanthopterin
H4 biopterin
drosopterin
18
biopterin
Figure 4. Three models proposed for pteridine biosynthesis in Drosophila
melanogaster.
Figure 4 shows three pathways proposed for pteridine biosynthesis. The
structures of the pigments and intermediates are as shown in Figure 3.
A. The pathway proposed by Calatayud, Jacobson and Ferré 1989. This is the
pathway shown in Figure 3.
B. The pathway proposed by Brown 1989.
C. The pathway proposed by Ferré et al. 1983.
19
Three pathways proposed for pterindie biosynthesis I n
Drosophila melanogaster
A. pathway proposed by Calatayud, Jacobson and Ferre, 1989.
GTP
H2 neoperinP3
pyruvoyl -144 pterin
Iactoyl-H4-pterin
H4-ptenn
I
H2-rin
y-H2Lmopterin
pterin
isoxanttpterin
Htbiopterin
‘terins
droso
H4-biopterin
B. Pathway proposed by Brown, 1989
spiapterin
biopterin
GTP
H2 hydroneopterin
H2-Pterin
PyruvoyI-t4Pterin
Lactoyl-H4 Pterin
H4 Biopterin
Isoxanthopterin
osopterin
H2Bitterin
S epiapterin
C. Pathway proposed by Ferre et al., 1983.
GTP
4
4
H2-6-(V, 2’-dioxopropyl)-pterin?
H2-Neopterin
H2-Biotteri n
Pterin
I soxanthopterin
p”
Biopterin
“Drosoprins”
20
H4-Biopterin
somewhat speculative. Figure 4 shows three recent models of the pteridine
pigment biosynthetic pathway.
The first major advance in understanding the formation of pigments was the
realization that the wild type pigmentation was composed of two pigment types;
a red-orange pigment and a yellow-brown pigment. In 1924 Johannsen noted
that there were two types of pigment granules, one red and the other yellow
(Johannsen 1924). This observation was related to two biochemically distinct
pigment groups by Casteel (1929), Schultz (1935) and Mon (1937). Studies of
combinations of eye colour mutants provided complementary evidence of two
independent genetic systems contributing to the wild type eye colour of
Drosophila. In 1931, Wright and his genetics class crossed a variety of eye
colour mutants. They observed that white-eyed flies arose from a cross between
brown and scarlet and between brown and vermilion. (Wright 1931). Based on
these observations of synthetic white mutants he proposed that the wild type
pigment was a compound of two independent genetic pathways. These, he also
related to the two types of pigment granules reported by Johannsen. A similar
observation led Glass to the same conclusion (Glass 1934), as did the
breakdown of a white-eyed mutant into two component eye colours (Nolte 1943
and 1944). Mainz (1938) generated extensive combinations of eye colour
mutants and examined their effects on the histology of the eye. His results
generally supported the idea of two independent pigment pathways. Nolte,
however, could not reproduce these results (Nolte 1950, 1 952a, 1 959a) and
disputed this conclusion as well as some of the morphological observations.
Although the pathways leading to the red and brown pigments may be
biochemically, and genetically distinct, both pigments are associated with the
21
pigment granules of the pigment cells of the eye and thus might be expected to
interact at least at a physiological level. This association may be manifest by
altered pigment granule morphology in mutants with simple enzymatic lesions
which should alter only one of the two pathways (Nolte 1950, 1952, Reaume,
Knecht and Chovnick, 1991). The independence, or lack thereof, of the eye
pigmentation pathways is still a subject of debate (Schwinck, 1975 1978, Ferré
etal. 1983).
Finally, the biosynthesis of eye pigments should be discussed in context of the
complex inter-relation of the different tissues and developmental stages
involved (reviewed by Tearle 1991). For example, production of the one
ommochrome pigment, xanthommatin involves four organs and two
developmental stages. During the larval stages tryptophan is absorbed by the
fat body and malpighian tubules and the excess converted into 3hydroxykynurenine and kynurenine, respectively. During metamorphosis these
organs release these stored compounds which, in addition to tryptophan
derived from protein catabolism during metamorphosis, is taken up by the eye
and occelli. The interplay between the various organs involved in pteridine
biosynthesis and metabolism is likely equally complex, but is unfortunately
largely unknown. Analysis of the biosynthesis of the pigments is further
complicated by the fact that not all of the pigmented organs have all the
necessary biosynthetic enzymes. For example, all four of the enzymes
necessary for production of xanthommatin are found in the eye pigment cells
whereas the occelli has only the last two and nether organ synthesizes xanthine
dyhydrogenase, the product of the rosy locus, which does nevertheless affect
the pigmentation of these organs.
22
Much of the impetus for investigation into these compounds stems from their
role in nucleic-acid metabolism. For example tetrahydrobiopterin is an essential
cofactor in amino-acid metabolism, neu rotransmitter biosynthesis and
molybdoenyzymes in general (Bel and Ferré 1986 and McLean, Boswell and
O’Donnell 1990). Since these compounds are derived from GTP, alterations in
their metabolism is of relatively dire consequence in humans. In this regard
Drosophila has been an immensely useful research tool. Not only have certain
eye colour mutations (Henna and Punch) been proposed as models for
metabolic defects such as phenylketonuria (probably more as a conceptual
than physiologically accurate model) but the pigments provide an abundant,
readily isolated source of an otherwise scarce and highly labile material. These
have proved to be both difficult to isolate from mammals, as would be expected
for a cofactor and metabolic regulator, and to synthesize chemically (Brown and
Fan 1975). The central role of these compounds in cellular metabolism may
provide the biological impetus for sequestering them in the specialize
organelles, the pigment granules. This function also makes the complex tissue
and developmental regulation less surprising.
History of the garnet gene
Eye colour genes of Drosophila hold an prominent place in the history of
genetic analysis. The first mutant isolated in Drosophila was an allele of the
white gene (Morgan in 1910 cited in Lindsley and Zimm 1992). The first mutant
allele of garnet was recorded not long after by Bridges in his pioneering work
on the chromosomal theory of inheritance (Bridges 1916). Since then it has
featured in innumerable genetic studies. As a result of its long history a
literature review of genetic studies involving the garnet gene is essentially
23
tantamount to a survey of the history of genetic analysis in Drosophila
melanogaster. Consequently the following survey of genetic analysis involving
the garnet eye color gene will necessarily be somewhat cursory.
The first mutant allele of garnet was isolated on February 19, 1915 as a
spontaneous mutation in a sable mutant stock (Bridges 1916). This mutation
) was established as an allele of a sex linked gene. As a sex linked marker it
1
(g
was an invaluable tool in a number of studies. It was instrumental in showing
that the behavior of genetic elements paralleled exactly the movements of
chromosomes in both regular and irregular meiosis. Thus garnet played a
pivotal role in the demonstration of the chromosome theory of inheritance. In the
same study garnet was used to demonstrate that recombination occurs at the
four strand stage, that non-homologous chromosomes (the X and Y) will pair
regularly and, by defining the mechanism of non-disjunction, emphasized the
regularities of meioses. Finally, interestingly in context of chapter 3, in this same
study the garnet gene was used to demonstrate the general rule of equivalence
of maternal and paternal genomes.
The eye colour genes next resurfaced in the literature in context of the profound
problem of how the actions of genes can determine the final phenotype of an
organism. Investigators seized upon the eye colour mutations of Drosophila as
an amenable model system in “higher” eukaryotes for the one-gene-one
enzyme theory. It was initially expected that the relationship between the many
genes which affected eye colour could be defined genetically whilst their
products were studied biochemically. These studies were not as immediately
fruitful as was hoped. Genetic studies revealed only that there were two largely
independent pathways (Schultz 1935, Mainz 1938) and biochemical analysis
24
was hindered by the complex, colourless, highly labile and light sensitive nature
of the many intermediate products of these biosynthetic pathways. In addition,
the sheer number of genes affecting eye colour posed tactical as well as
conceptual problems (Nolte 1952b, Lucchesi 1968).
Spurred by Sturtevant’s pioneering work on the cellular autonomy of gene
action in somatic mosaics (Sturtevant 1932), between 1935 and 1937 Ephrussi
and Beadle produced a flurry of reports (Ephrussi and Beadle 1 935a, Ephrussi
and Beadle 1 935b Ephrussi and Beadle 1 935c, Beadle and Ephrussi 1 935a,
Beadle and Ephrussi 1935b, Beadle and Ephrussi 1936, Beadle and Ephrussi
1937, Ephrussi and Beadle 1 937a, Ephrussi and Beadle 1 937b, Beadle 1937)
which focused on the relationship between gene action and development. They
attempted to dissect genetic and biochemical interactions between eye colour
genes using a series of tissue transplants between donors and hosts of different
genotypes. Their results showed that most of the eye colour genes, including
garnet, behaved autonomously upon transplantation. These studies, among
others, were instrumental in nucleating concepts of diffusable versus cell
autonomous substances, the former providing a working model for the action of
hormones, tissue specific interactions (Beadle 1937), fluctuating levels of
substances in development (Hamly and Ephrussi 1937), conservation of
biological functions across species (Howlan, Glancy and Sonnenbilick 1937)
and pleiotropy (Hadorn and Mitchell 1951, Hadorn 1962). Although important in
emphasizing many concepts in development, a contemporary review of these
studies stated “Because of the rather heavy injection of new theoretical
considerations and laboratory symbolism, the uninitiated person is to be
warned that he may find himself bewildered in trying to follow these
investigators to their final conclusions or to comprehend what their conclusions
25
actually are.” (Blakeslee 1938), suggesting that this profusion of studies may not
have been met with unabated enthusiasm.
While these studies were vital to the development of our current understanding
of genetics, the role of the gamet gene was somewhat incidental. It was simply
a convenient marker for the X chromosome. The garnet gene also featured
prominently in a debate between Chovnick and Hexter on the complexity of
genetic loci. In contrast to the previous studies, an intrinsic physical property of
the garnet gene, its relatively large size, was the basis of its role in the
imbroglio.
This debate centered around the question of whether or not garnet was a
complex locus. The debate arose from the concept that genes were, by
definition, indivisible by recombination. Genetic complexity was defined
functionally; if recombination occurred between various alleles, defined as such
by similar position, phenotype and failure to complement, the alleles were
termed pseudo-alleles (a term coined by McClintock 1944, for another purpose
entirely). A gene with pseudo-alleles was then termed a complex gene and was
proposed by Lewis (1951) to consist of a series of duplicated genes. This point
was raised as an issue in assessing the universality of genetic material. It had
been established that alleles of prokaryotic genes could recombine (Benzer
1955). If this was also true of a “typical” eukaryote, Drosophila melanogaster, an
important property of genetic material would be conserved between prokaryotes
and eukaryotes.
The core of the conflict revolved around the inability to recover double mutant
chromosomes from intragenic recombination (Hexter 1956, 1 958a, 1 958b and
26
Chovnick 1956, 1957, 1 958a, 1 958b and 1961, reviewed rather more soberly
by Carlson 1959). The situation was further complicated by gene conversion
events, a phenomenon which at that time, was not documented in Drosophila
melanogaster. Although impossible to determine exactly the source of this
discrepancy, it might be posited that the double mutant chromosome arose from
g5 allele was converted to g+, as suggested
d
a conversion event in which the 3
by Hexter (1958). The four other garnet alleles used in these studies were g
’
1
,g
2
g
3 and g
. Data presented in chapter 2 (Table 21) may explain why it was
4
d
5
3 allele that might have converted. The g
only the g
3 and g
4 alleles are
insertions and thus would be expected to convert at a much lower frequency
than point mutants (Chovnick 1964). The failure to recover recombinants or
convertants between g
1 and g
3 is compatible with molecular data which
indicate that these insertions are highly similar and possibly identical (Figure
20 and 21). In retrospect, both intragenic recombination, as established in
prokaryotes (Benzer 1955) and conversion, shown in Drosophila virills
(Demerec 1928) probably occurred at the garnet locus. Although these issues
were finally resolved by his studies of another eye colour gene, the rosy gene
(Chovnick 1964), the debate concerning the complexity of the garnet gene is
notable for being the smallest pseudo allelic locus studied and for furnishing the
first evidence of gene conversion in Drosophila melanogaster.
The “transport” group of mutants:
The garnet gene alters the levels of both the pteridine and ommochrome groups
of pigments. The garnet gene is not alone in this property. A number of
investigators have suggested that the eye colour mutants which alter both types
of pigments are functionally related. Based primarily on this phenotypic
27
criterion, Nolte (1955) grouped the mutants carmine, carnation, claret, garnet,
light, maroon, pink, prune, purple, purploid, rosy and ruby together as the
arbitrarily named ruby group. On the basis of genetic and histological analysis
of these genes he further subdivided the groups (and their proposed function)
into the sub-groups shown in Table 2. The difficulty with grouping these
mutants based on fine gradations of eye colour is that the groupings become a
rather arbitrary reflection of the allele used. The eye colour of the garnet alleles,
for example, ranges from a pale orange which Nolte would likely have classed
as a member of the light group, to weak alleles that would be classed as
members of the red or dark groups. The hazard of this approach is illustrated by
results reported for interactions between white and carnation. These gave very
different results depending on the alleles used. Inspection of the data presented
in Nolte’s 1955 paper allows a slightly different grouping, which, while also
dependent on phenotype, is based on an effect on one versus both pigment
systems and histology (Table 2). This grouping should be less dependent on
the choice of allele. Encouragingly, I show extensive genetic interactions within
the first group identified by this criteria.
The temptation to categorize eye colour mutations lured others. Schwinck
(1975) again classed eye colour mutations phenotypically. Her criterion was
based on relative concentrations of the drosopterin pigments. Her less
colourfully named grouping, the group two mutations, is shown in the third row
of Table 2. These mutations were unique in that they were the only mutants to
respond to implants of phenylalanine by increasing drosopterin production. This
might indicate a common metabolic defect. Tearle (1991) also proposed a
functional grouping based on phenotype, this one based which organs were
28
Table 2. Genes proposed as members of the transport group of eye colour
genes.
The first and second column shows the name given to the group and the
reference. The third column shows the names of the genes proposed as
members of that particular group.
The ruby group proposed by Nolte (1956) was latter subdivided in to smaller
groups (indicated here by parenthesis) for which he proposed slightly different
functions. The first group contains, rosy, pinkand purploid and is defined by
normal pigment granule morphology and a brown eye colour. The second
group contains claret and maroon, has normal pigment granule morphology
and a light brown eye colour. The third group consists of carnation, carmine,
garnet, ruby and light and is defined by normal pigment granule morphology
and decreased levels of both red and brown pigments. The fourth group
consists solely of purple and is defined by normal pigment granule morphology,
less red pigment and elevated levels of brown pigment. The final group consists
solely of prune and has abnormal pigment granule morphology.
I have regrouped the genes carnation, carmine, claret, garnet, light, pink and
possibly maroon and purploid as one subgroup defined as having normal
pigment granule morphology and less of both classes of pigments. A second
subgroup consists of rosy and possibly maroon and purploid with normal
pigment granule morphology, less red pigments and normal levels of brown
pigments. The last two groups are identical to Nolte’s last subgroups.
29
The group II mutants proposed by Schwink (1975) is defined by their increased
drosopterin production in response to implants of phenylalanine. The transport
group proposed by Sullivan and Sullivan (1975) consists of white, scarlet, claret
and lightoid for which defects in keynurine transport were demonstrated, and
carnation, garnet, light, maroon and pink for which transport defects were
proposed. The group defined by their unusual response to transplantation of
eye discs consists of carnation, carmine, claret, garnet, pink, ruby, maroon-like
and rosy. The grouping proposed by Tearle (1991) is based on the organs
affected by mutations in these genes. Those he listed as group 1A affect
pigmentation of all organs examined.
Finally the group defined as participating in synthetic lethal interactions
rosy ry-SOD,
3
Hennar
,
includes: purple-Purploider, prune-Killer of prune, 6
light-carnation, deep orange-carnation, deep orange-rosy, deep orange
purploid, deep orange-cinnabar-brown, deep orange-fused. Some of these
combinations may reflect special situations. prune is lethal when combined with
Killer of prune (Sturtevant 1956). The latter gene is a dominant allele of the awd
(abnormal wing disc) gene and is a nucleotide diphosphate kinase. The prune
gene product has been identified as a ras GTPase activating protein. The
dominant Kpn allele may cause excessive stimulation of ras-like proteins (Teng,
Engele and Venkatesh 1991). It would not be surprising if such an interaction
3 and iy
6 has also been touted as a synthetic
was lethal. The combination Hnr
6
lethal combination (Taira 1960), however this interaction is specific to the ry
allele; it does not occur with other rosy alleles (Goldberg, Schalet and Chovnick
1962). The gene fused disrupts pteridine metabolism but is not normally
considered an eye colour gene. The synthetic lethality of deep-orange with the
30
cinnabar brown double mutant chromosome is specific to this chromosome and
is not seen with any combination of double mutants.
31
32
Sullivan and
Sullivan (1975)
Beadle and
Eph russi (1936)
Schwink (1975)
Green (1955)
Reedy and
Cavalier (1971)
Bridges (1922,
purple-Purploider
cited in Bridges and Breme 1944)
Lucchesi (1968)
deep orange-carnation, deeporange-rosy, deeporange-fused
deeporange-purploid, deep orange-cinnibar, brown
r
3
Hennar
6
osy
Tiara (1960),
Goldberg, Schalet and Chovnick (1962)
Sturtevant (1956) prune-Killer of prune
Nash (1971)
light-carnation
Nickla, et al (1980)
Hilliker et al. (1992) rosy-SOD
Tearle (1991)
claret, carmine, deep orange, garnet, light, lightoid, orange, pink, ruby,
scarlet white
The transport
group
Aberrant disc
transplantation
epistatic
interactions
Synthetic
lethals
Pigmentation of
all organs
Schwink (1975)
Group II
(carnation, garnet, light, maroon, pink)
, ruby and garnet
3
whitea
bright, brown, clot, lightoid, cardinal, claret, mahogany, rosy, scarlet,sepia
carnation, carmine, claret, garnet, pink, ruby, maroon-like, rosy
(white, scarlet, claret lightoid)
carnation, garnet, maroon-like, orange, pink, rosy, ruby
(carnation, carmine, claret, garnet, light, pink, ruby, maroon? purploid?)
(rosy, maroon?, purploid?) (purple) (prune)
this work
The ruby group
revisited
(prune)
Nolte (1956, 1959) (rosy, pink, purploid)
(claret, maroon)
(carnation, carmine, garnet, ruby, light) (purple)
The ruby group
Membership in the “transport” group of eye colour mutations.
name
reference
members of group
affected by mutations. His group 1A affects pigmentation of all organs
examined.
At approximately the same time, Sullivan and Sullivan (1975) documented
defects in metabolite transport in white and scarlet mutants. They also
discovered similar defects in claret and lightoid mutants. Although not examined
in this study, they proposed that the genes carnation, garnet light, maroon and
pink were, in addition to claret and light, involved in metabolite transport.
Other suggestions of functional similarly between these genes have arisen from
a number of studies. Beadle and Ephrussi (1936) noted that while most mutant
imaginal eye disc transplants were cell autonomous, transplants between
carmine, carnation, claret, garnet, pink and ruby and either vermilion or
cinnabar (the latter non-cell autonomous due to diffusion of the “hormone”- the
metabolic ommochrome intermediate keynurenine) produced exceptional
results. Eye discs from vermilion mutants transplanted into hosts mutant for most
eye colour genes, become wild type. Presumably, the mutant discs are able to
scavenge enough kynurenine to generate wild type levels of pigment. Different
results were seen with members of the “transport” group. Discs from vermilion
donors transplanted into carnation and garnet hosts developed an intermediate
phenotype, whereas when transplanted into carmine, claret, pink and ruby
hosts the vermilion discs develop a mutant phenotype. Minor differences
between mutant and intermediate phenotypes were likely due to the
hypomorphic nature of the mutant used. For example the g
2 allele used in these
experiments is a moderate hypomorphic allele. These results suggest that the
transport group of mutations are physiologically distinct from other eye colour
mutants. The authors implied that this difference could be due to differences in
33
levels of the “hormone”, although this is not consistent with their data. Schwink
(1975) suggest that similar intermediate phenotypes seen in wild type eye discs
implanted into maroon-like or rosy hosts is due to negative feedback of the
biosynthetic pathways by excess products. However, the transport defect
identified by Sullivan and Sullivan (1975) seems a more appealing cause for
these aberrant results. Nevertheless, these results suggest a physiological
equivalence between many of the members of the transport group.
Finally, there have been a few other hints of functional similarity between
various members of the transport group. Green (1955) found that some alleles
of garnet and ruby were unique in showing no additive interactions in
combination with certain white alleles. Certain members of these groups also
show highly specific synthetic dominant (Nolte 1 952a) and synthetic lethal
interaction (Luccessi 1968, Nash 1971, Nickla 1977).
I shall use the term “transport group” genes to refer to members of this group as
they show extensive phenotypic similarity and share a number of physiological
properties, of which a role in transport is the best defined. A direct role in
transport has, however, been shown for only one of these mutants, and this may
not be their primary role. Thus this term is adopted more for its descriptive value
than as a claim of function.
Summary:
The study of eye colour genes is now less fashionable. But, many of the
problems noted by the early investigators still remain and the biological function
34
Table 3. Eye colour genes with known or proposed functions.
The first two columns indicate the designation and name of those genes for
which functions are known or proposed. A
“+“
in the third column indicates that
the gene has been cloned. The function proposed for the gene and the
reference is given in the fourth and fifth columns, respectively. For those genes
which encode an enzymatic function, the name of the enzyme is given in part B
on the next page.
35
36
v
w
z
St
ly
se
rosy
sepia
scarlet
vermillion
white
zeste
brown
claret
carnation
cardinal
cinnamon
clot
cinnabar
Drop
garnet
glass
karmoisin
killer of prune
littleisoxanthopterin
light
lightoid
low xanthine
dehydrog.
mal maroon like
pink
p
pn prune
pr
purple
Pu
Punch
pym polymorph
ras rasberry
ri
rolled
bw
ca
car
cd
cm
cI
Cn
Dr
g
gl
kar
kpn
lix
It
ltd
lxd
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
-
+
-
-
+
+
function
transmembrane channel
transport?/nervous systerm?
transport?/nervous system?
enzyme-ommochrome pathway
molybdenum containing cofactor
enzyme-pteridine pathway
enzyme-ommochrome pathway
cell-cell signaling
transport?/nervous system?
transcription factor
enzyme-ommochrome pathway
regulation of GTP hydroxylase
enzyme-purine synthesis
transport?/nervous system?
transport?/nervous system?
molybdenum containing enzyme
cofactor
molybdenum containing cofactor
transport?/nervous system?
regulation of GTP hydroxylase (Pu)
enyme-ommochrome pathway
enyme-pteridine pathway
enzyme-purine pathway
enzyme-pteridine synthesis
cell-cell signaling-rhabdomere
formation
enzyme
enzyme-pteridine pathway
transmembrane channel
enyme-ommochrome pathway
transmembrane channel
transcription factor
A. Eye colour genes with known functions
gene
cloned
Keith et al 1987
Wiederrecht and Brown, 1984
Tearle et al. 1989
Nissani 1 975/Searles and Voelker 1986
Dressen et al. 1988
Pirrotta 1988
Warner, Watts and Finnerty, 1986
Jones & Rowls 1988
Teng, Engele and Venkatesh, 1991
Searle & Voelker 1986
Mackay and O’Donnell, 1983
Henikoff et al. 1986
Nash-personal communication
Zipursky et al 1993
Dreesen Johnson and Henikoff 1988
Yamamota et al. 1988
Sullivan and Sullivanl975
Sullivan, Grill and Kitos, 1974
Kamdor, Shelton and Finnerty, 1994
Wiederrecht, Paton and Brown 1984
Ghosh and Forrest 1967/Howells in Tearle 1991
Renfranz and Benzer, 1989
this thesis
Moses, Ellis and Rubin, 1989
Sullivan, Grille and Kitos, 1974
Teng, Engele and Venkatesh, 1991
Ordono, Silva and Ferre, 1988
Sullivan and Sullivanl975/Devlin et al. 1989
McCarthy and Nicklal 980
Schott, Baldwin and Finnety, 1986
reference
37
v
B.
gen
e
ci
cn
kar
kpn
lix
pr
Pu
pym
ras
ry
se
dihydroneopterin triphosphate pyrimidodizepine
kynurenine 3-hydroxylase
phenoxazenine synthetase
nucleoside diphosphate synthetase
dihydropterin oxidase
sepiapterin synthase
guanosine triphospate cyclohydrolase
formyiglycine amide ribotide aminotransferase
inosine monophosphate dehydrogenase
xanthine dehydrogenease
6-pyrovoyltetrahydropterin 2-amino 4-oxo-b-acetyl-7,8 dihydro-3H ,9Hpyrim ido
[4,5,6]-[1 ,4] diazepine syntetase
tryptophane pyrrolase
enzymes
of most remain unknown. Those eye colour genes which have been analyzed in
detail have provided insight into a diversity of biological functions (Table 3).
Not only does this diversity suggest a potential resolution of the historical
paradox of the apparently excessive number of eye colour genes, but it also
suggests that the classical system of Drosophila eye pigmentation is a powerful
assay for a diverse array of biological functions. The sensitivity of this system is
such that it has the potential to allow the genetic detection even of those genes
such as the transport group which may have extensive functional redundancy.
The crucial, if still undefined, functions of these genes can only be revealed
though
detailed analysis of the genes involved.
This thesis examines the genetic and molecular properties of one of the eye
colour genes, the garnet gene. Chapter one deals with the complex genetic
interactions between eye colour genes, specifically those of the transport group,
which are likely involved in some aspect of inter or intracellular transport of
metabolites. Membership of garnet gene in this group is indicated by extensive
genetic interactions between the garnet gene and other members of this group.
Molecular analysis of the garnet gene is presented in chapter two. In the final
chapter the garnet gene is used as a genetic and molecular marker to examine
the question of epigenetic gene regulation. Historically, the garnet gene has
been an invaluable tool for those studying a variety of biological and genetic
processes. Further analysis of the garnet gene and other members of the
transport group should lead to insights into the ubiquitous and essential role of
cellular communication in development.
38
MATERIALS AND METHODS
All crosses were performed at 220 C unless otherwise stated. Culture medium
was standard cornmeal/sucrose media supplemented with antibiotics and
0.04% tegosept as a mold inhibitor. Crosses were generally carried out in 8
dram shell vials with groups of 3-5 virgin females crossed to an equal number of
males. For each experiment 3-6 replicates were made. Crosses were
subcultured twice at 4-5 day intervals before the parents were discarded. Each
set of crosses was scored independently. The data from replicate crosses within
a group were subsequently pooled.
Mutant strains and chromosomes: The mutations and rearranged
variegating chromosomes used in this study are described in Lindsey and
Zimm (1992). The g
62 and gS3 alleles were generously provided by Dr. A
Schalet via Dr. D. Sinclair. The Dp(1;f)LJ9 mini-chromosome is carried
balanced against an attached XX and attached xy chromosome. As the minichromosome carries a region which is diplo-lethal in males the attached XY
stock carries the deficiency g-I on the X chromosome. The mini-chromosome
was generously proved by Dr. G. Waring via Dr. D. Sinclair.
Crosses:
Details of the crosses will be described in the appropriate figure and table
legends.
Assays to quantitate eye pigment.
Red pigments, five heads:
39
The amount of pigment deposited in the eye was measured separately for 25
females and 25 males. Flies three to seven days post-eclosion were
decapitated by vigorously banging the frozen flies. Flies were held in the dark at
700C for 4 hours-six months before decapitation. No appreciable loss of
pigment occurred during this time (data not shown). The heads were placed in
wells of a microtitre plate and 30 uL of 0.25M 13-mercaptoethanol in 1%
aqueous NH4OH per well was added. The eye pigment was released by
sonication for three seconds and a five uL aliquot was removed from each well
and applied to a piece of Whattman No. 3 filter paper. The amount of pigment in
the dried spot was determined fluorometrically using a MPS-1 Zeiss
microscope. The software program for processing the data from the
photomultiplier attached to the microscope was written by Dr. J. Berger. Five
groups with five heads per group were measured for each genotype and sex. In
each case, the amount of pigment in each of the five spots was averaged and
then expressed relative to wild type.
Red pigments, single heads:
The amount of pigment in single fly heads was quantified as above except as
follows: only one head was used per microtitre well; 2OuL of solution was
removed after sonication and spotted onto filter paper; a minimum of 10
individuals per genotype were measured.
Red pigments, spectrophotometric assay:
The spectrophotometric assay was adapted from the procedure of Real, Ferré
and Mensua (1985). Five samples of five heads each, per genotype and sex,
were placed in an eppendorf tube with 150 uL of 30% ethanol, acidified to pH 2
and shaken on an orbital shaker for 24 ± 4 hours. The absorbance of the
40
pigment was then read at 480 nm.
Brown pigments, spectrophotometric assay:
This assay was also adapted from the procedure of Real, Ferré and Mensua
(1985) and Euphrussi and Harold 1944. Three samples of twenty heads per
genotype and sex where placed in an eppendorf tube with 450 uL 2M HCI and
0.66% sodium metabisulfite (wlv) and sonicated. 0.9 mL of n-butanol
(equilibrated with H20 and 0.66% sodium metabisulfite) was then added and
the extract was shaken for 30 minutes on an orbital shaker. After centrifugation
(5 mm. at full speed in a microcentrifuge) the aqueous layer was removed and
500 uL of H20 and 0.66% sodium metabisulfite was added. The mixture was
again shaken for 30 minutes, centrifuged and the aqueous layer removed. This
wash was repeated twice. The absorbance of the organic layer was measured
at 492 nm.
Quantification of separated red pigments by thin layer chromatography:
The red pigments were separated and then quantified by a modification of the
method described in Hadorn and Mitchell (1951) and Hadorn (1962). Red
pigments were extracted as described for the five head red pigment assay
above. An aliquot of 5 uL was spotted one inch from the bottom of a cellulose
chromatography plate (Kodak) and allowed to dry. Further aliquots of the same
sample were placed on the existing spot and allowed to dry, for a total of 6 x 5
uL of solution. The pigments were then separated in a solution of n-butanol and
1% ammonium hydroxide (2:1) for 3-4 hours. The pigments on the dried plates
were identified by their distinctive colour under UV light, position and by
comparison to the results of Hadorn and Mitchell (1951) and Hadorn (1962).
The amount of pigment in each spot was quantitated by scanning the
41
chromatography plates with the fluorescent microscope (wavelength
500 nm).
Using this procedure it was possible to identify 9 pigment spots.
Assessment of malpighian tubule colour.
Malpighian tubule colour was assessed essentially as described by Brehme
and Demerec (1942). Malpighian tubules where dissected from healthy
wandering third instar larvae in 0.7% NaCl. The isolated malpighian tubules
were immediately viewed against a black background. Their colour was
assessed in comparison with wild type, carnation, carmine and white mutants.
Assessment of testes sheath colour.
Testes were dissected in 0.7% NaCl from freshly killed males and immediately
examined for colour when placed against a dark blue background. As with
malpighian tubule colour, the colour of the testes sheath fades rapidly in 0.7%
NaCI, tap water or in frozen whole flies.
Molecular analyses:
Genomic DNA was extracted by the method of Jowett (1986) with minor
modification. Following RNAse digestion the DNA was isolated by spooling from
the ethanol-aqueous interface and dissolved in TE. Restriction endonuclease
digestion of the DNA was performed overnight, twice, with intervening
phenol/chloroform extraction and ethanol precipitation.
Isolation of RNA: RNA was isolated from the appropriate stages and genotypes
using either the “hot phenol” method of Jowett (1986) or the guanidine
42
thiocyanate method (Fluka). The latter technique was more effective.
Isolation of lambda clones: lambda clones and DNA were isolated according to
standard procedures (Sambrook, Fritsch and Maniatis 1989).
Isolation of plasmid DNA: Small scale plasmid DNA preparations were isolated
by the alkaline lysis method (Sambrook, Fritsch and Maniatis 1989) or using
“magic mini-preps” (P romega) following manufacturer’s instructions. Large
scale plasmid preparations were prepared by PEG precipitation (Sambrook,
Fritsch and Maniatis 1989) or using “Qiagen” columns following the
manufacturer’s instructions.
Southern and Northern transfers and hybridization: Southern and Northern
transfers and hybridization were performed according to standard procedures
(Sambrook, Fritsch and Maniatis 1989) and following manufacturer’s
(Amersham) instructions.
Labeling of DNA: DNA was radioactively labeled using 32
P and either the nick
translation kit (Amersham)or the random priming kit (Amersham) following the
manufacturer’s instructions. For some experiments DNA was labeled with UTP
flourescein using the ECL (enhancered chemioluminescence) random prime kit
(Amersham) and detected using anti-flourescein antibody conjugated to HRP
which catalyzed a light-emitting reaction, following the manufacturer’s
instructions.
Sequencing: Short overlapping segments of DNA for sequence analysis were
generated using the Exo Ill directed deletion kit following manufacturer’s
43
instructions. In some cases primers to previously determined sequences were
prepared by the UBC oligosynthesis laboratory and used for sequencing.
Sequencing was done using the double stranded dideoxy chain termination
method using 35 labeled DNA. Initially the Sequenase (United States
Biochemicals) kit was used. Subsequently 17 polymerase (Pharmacia) was
substituted for the Sequenase enzyme and all reagents were made following
recipes supplied with the T7 enzyme. The DNA fragments for sequence
determination were separated on a 6% or 8% Acrylamide:bis acrylamide (40:1)
gel or on a 6% “long ranger” (N-methly-acrylamide) (United States
Biochemicals) gel in 0.5% TBE buffer. The genebank accession number for the
garnet c-DNA is U31351.
Sequence similarity search: The search of sequence data bases was carried
out using the blastN and blastX algorithms provided by the national center of
biotechnology information. This search included all eukaryotic sequences
present in the Swiss protein, EMBL and genebank data banks.
In situ hybridization: Hybridization to RNA in whole mount embryos and various
tissues was done essentially as described by Tautz and Pfeifle (1989) with the
exception that hybridization was carried out at 55°C. DIG labeled RNA probes
were made following the manufacturer’s instructions (Boeringer Mannheim) and
detected with alkaline phosphatase-conjugated anti-DIG antibody and NBT/X
phosphate colour reaction.
Photography: Flies and fly tissues were photographed using the Wild
stereomicroscope adapter at 16 or 40 X magnification on a dark blue
background. Kodak Eckatchrome Ti 60 colour slide film was used.
44
Chapter 1
Interactions between garnet and
other eye colour genes
45
Introduction-chapter 1. Interactions between garnet and other eye colour genes:
The large number of genes which affect eye colour suggests that eye
pigmentation is a powerful assay system, able to detect alterations in a wide
variety of biological functions (Table 3). Study of these genes, while having a
long history is still in its early stages and further work on this group is certain to
be rewarding.
Of the eye colour genes, those of the transport group are particularly intriguing.
Membership in this group has been assigned somewhat haphazardly, based on
an arbitrary assessment of phenotype and some suggestive histological and
physiological experiments. The garnet gene has been implicated as a member
of the transport group but detailed analysis of either the gene itself or of
interactions between garnet and other members of this group is lacking. The
choice of garnet as a representative member of the transport group, while in
part arbitrary, was promoted by the extensive previous work on this gene and
the existence of a second site mutant which enhanced the mutant eye colour
phenotype of severe garnet alleles. This interaction provides an attractive
avenue for genetic analysis of the garnet gene.
This chapter presents data on the nature of this second site mutation, the
enhancer of garnet mutation (e(g)), its interaction with garnet and with other
members of the transport group of eye colour mutants, and finally, interactions
between this latter group and garnet. This analysis supports the inclusion of
garnet as a member of the transport group of eye colour genes and suggests a
simple model for the biological role of this class of genes. The molecular
analysis presented in chapter two lays the ground work for testing of this model.
46
Results-Interactions between garnet and other eye colour genes.
The enhancer of garnet mutation: Rather fortuitously, the P-element bearing
strain (S6-1) that generated the garnet allele, gP, which permitted the cloning of
the garnet gene, also contained a second site mutation which made the
phenotypes of severe garnet alleles appear more extreme. This mutation was
called enhancer of garnet (e(g)). The enhancer of garnet mutation has no
independent phenotype. Individuals bearing only the enhancer of garnet
mutation have no alteration in eye colour, viability or fertility (data not shown).
However, in combination with severe alleles of the garnet gene the enhancer of
garnet mutation reduces the amount of eye pigmentation by approximately half
(Figure 5a, b and c and Table 16). This sex linked gene mapped, by
recombination, near the site of a previously isolated enhancer of garnet
mutation (Payne and Denny, 1921) and is presumably allelic to this mutation.
Direct test by complementation is not possible as the original enhancer of
garnet mutation is no longer extant. Other than its phenotype in conjunction with
garnet, little information is available on this mutation.
Cytological position of enhancer of garnet: As a first step in identifying the
nature of the enhancer of garnet lesion, the cytological position of the enhancer
of garnet mutation was determined. The enhancer of garnet mutation had been
mapped by recombination to approximately map position 4 (Wennberg 1988).
This position corresponds roughly to division 3 on the cytological map. Three
deficiencies and one duplication encompassing most of division 3 were
obtained from the stock center (Bowling Green) and tested for complementation
with the enhancer of garnet mutation. None of the heterozygotes between these
deficiencies and the double mutant e(g) g
2 chromosome showed an eye colour
47
Figure 5. Phenotypes of three severe garnet alleles and these garnet alleles
in conjunction with the enhancer of garnet mutation.
d, 5
3
g
O
e and g
The garnet alleles g5
2 are shown on the left of each panel from
top to bottom, respectively. On the right are shown these same alleles in
conjunction with the second site enhancer of garnet mutation.
48
.
e(g) g53d
e(g) g5Oe
e(g) g2
49
phenotype (data not shown), consistent with the absence of any detectable
phenotype for enhancer of garnet homozygote. In contrast, when the g
2 allele
was recombined onto these deficiencies and retested against a e(g) g
2
chromosome, all the deficiencies showed a strong enhancer of garnet
phenotype. The deficiency/enhancer of garnet (Df e(g) g
/e(g) g
2
) phenotype is
2
more severe than the homozygote enhancer of garnet (e(g) g
/e(g) g
2
)
2
phenotype indicating that the original enhancer of garnet mutation is
hypomorphic. The presence of a duplication for this region rescued the
enhancer of garnet mutation on the e(g) g
2 chromosome reducing it to a g
2
phenotype (Figure 6 and Table 4). These results imply that the enhancer of
garnet lesion lies within the area of overlap of these four rearrangements. The
only area held in common by these deficiencies and duplication is region 3C3,
although given the limits of cytological resolution the area of overlap might
extend to bands on either side as 3C2 and 3C3 are often difficult to distinguish.
These data effectively place enhancer of garnet in cytological position 3C2
-
3C4.
The division 3 region has been extensively analyzed at the genetic and
cytological level (Lindsley and Zimm 1992). No mutation corresponding to
enhancer of garnet has been reported for this region but this is not unexpected
as the mutation has only an indirect phenotype. The position of enhancer of
garnet is, however, extremely close to that of the white gene which is located at
position 3C2. Furthermore, there is complete congruence between removal of
the white gene and the occurrence of the enhancer of garnet phenotype or
rescue of these phenotypes (Figure 6B). Initial tests of allelism between
enhancer of garnet and white indicated complementation; the phenotype of w
g+/e(g) 2
g flies is completely wild type. Nevertheless the proximity of the white
50
Figure 6. Cytological localization of the enhancer of garnet mutation.
A. A diagram of the first part of polytene chromosome division 3 is shown
(adapted from Lindsley and Zimm 1992). Above is indicated the position of the
zeste and white genes and the location determined for the enhancer of garnet
lesion. Below is shown the cytological limits of three deficiencies; Df(1)
DF(1)N8, Df(1) wrJl and Df(1) wrJ3, and one duplication; Dp(1;3)N
238 used to
map the enhancer of garnet mutation. The black bars indicate deficiencies, the
striped bar the duplication and the clear bars areas of uncertainty in the
cytological determination of the extent of the deficiencies.
B. The phenotype of the deficiency with respect to white and enhancer or
garnet is summarized. A
gene, a
“-“
“+“
indicates presence of wild type function for that
indicates absence of that gene function. See Table 4 for data and
crosses.
51
postion
Cytological
A.
of
e(g)
white 3C2
zeste
e ) 3C2-3
s1E_
19\T1Th
•
I
I
4
I
I
.
I
—
I
’
1
r
Iii
Iii
I
I
I
I
I
b41
1
‘4 •3D
I
11
I
Df(1)N8
1
I’,
I
I
Df(1) 3C2-3 ‘3E3-4
I
ii
I
I
t
3A
I
I
I
I
-
ni
Df(i)w
Df(1) 3A1-2
-
3C2-3
B. Complementation of white
and enhancer of garnet
w
Df(1)N8
Df(1) rJ1
rJ3
Df(1) w
38
Dp(1,3)
e(g)
-
I
-h
I
I
I
I
I
rJ3
I
Df(i)w
DfcI) 3C3-3C1 2
238
Dp(i;3)N
Dp(1;3) 3B2-3;3D6-7;8ODF
+
+
52
0
Table 4. The Effect of 3C deficiencies on white and enhancer of garnet.
The first column lists the three 3C deficiencies used to cytologically localize the
e(g) lesion. The second and third column list the visual phenotype and red
pigment values (as percent wild type) for the appropriate deficiency over a white
null (w
). The fourlh and fifth columns list the phenotype and red pigment
1
values (as percent wild type) of the appropriate deficiency combined with the g
2
allele heterozygous with the e(g) g
2 chromosome. The last row lists the
equivalent values derived using the e(g) g
2 chromosome for comparison.
CROSSES:
1. To generate the deficiency garnet chromosomes,
P
Df(1)*/In(1)dI49 ®e(g)g
/Yo’
2
Fl
Df(1)*/e(g) g
2
F2
Df(1)*g
/
2
!n(1)d149, g4
selected by garnet phenotype.
pair matings with e(g) g
/YcJ sibs to isolate deficiency bearing
2
chromosome (no e(g) g
cf progeny).
2
0 In(1)d149/Yo’(sibs)
2. To determine phenotypes:
A. With respect to the white gene:
Df(1)*®w/YQ
P
Fl
Df(1)*/w4
phenotype determined.
B. With respect to enhancer of garnet phenotype.
P
Df(1)* g
/In(1)d149
2
Fl
Df(1) g
/e(g) g
2
2
phenotype determined.
1
/YcJ
2
0 e(g) g
*
53
Effect of 3C deficiencies on white and e(g)
Deficiency
Df/white
pigment
Df(1)N8
0
Df(1)WrJ1
phenotype
/e(a) g
2
Lg
2
pigment
phenotype
completely white
11
pale yellow
0
completely white
ND
yellow
Df(1)WrJ3
0
completely white
ND
pale yellow
e(g) g
2
88±9
wild type
54
21
+
+
1
3
orange
gene to the enhancer of garnet mutation led me to test for allelism in a less
direct manner.
enhancer of garnet is a subliminal allele of white. Two types of tests of allelism
between white and enhancer of garnet were made. In the first series of tests,
g
O
e and the 3
g5 mutations were recombined onto
d
the enhanceable g
, 5
2
chromosomes bearing 11 different alleles of the white gene. The white alleles
represent a variety of different types of lesions in the white gene (Figure 7).
These recombinant chromosomes were then tested in trans with the appropriate
e(g) g * mutation to see if the white alleles would complement the enhancer of
garnet allele. Table 5 presents measurements of the levels of red eye
pigments of these genotypes. In general, all the white alleles acted as extreme
enhancers of the three different enhanceable garnet mutations. The one
g
O
e allele. These individuals were
exception was wsat in combination with the 5
g
O
e homozygotes. This
visually, and by pigment assay, indistinguishable from 5
result was repeatable (data not shown). This suggests some allele specificity in
the interactions between white and garnet, which is borne out by the lack of
obvious correlation between the severity of the white allele and its effect on
garnet (Figure 8 and see below). Specifically, although the Sat allele is a
more severe allele of white than the e(g) allele (column 3 of Table 5) it has
virtually no effect on garnet expression. Also interesting, is the result with wa.
The wa allele is a moderate hypomorphic allele of white, yet it has a very strong
enhancer of garnet effect on all three garnet alleles. This effect is more
1 and 1
w
1 18).
pronounced than that of the white null alleles (w
As a second type of test of allelism between white and enhancer of garnet the
white+ transgene was tested for its ability to rescue the enhancer of garnet
55
Figure 7. Diagram of the structure of the white gene of Drosophila
melanogaster.
The heavy line represents the molecular map of the white gene (from Levis and
Bingham 1985). The size, position and direction of transcription of this gene is
shown above the line. The position and identity of inserts in the various white
alleles used to complement the enhancer of garnet mutation are shown above
and below the line (adapted from Lindsley and Zimm 1992). The molecular
lesions associated with sat and w are unknown. The two dashed lines below
the heavy line represent the white DNA included in the P-element constructs
P[(w,ry)]A4-3 (from Hazelrigg, Levis and Ruben 1984) and CaSpeR (from
Pirrota et al 1985).
56
Structure of the white gene
w+ transcript
_A_A __A__
3’ 44
5’
h ch
____
8
will
()
w
RH
-5
R
K
H E
+10
w
P[(w,ry)]A4-3
Sst
___WIJ(
CaSpeR
R=EcoRi
H = Hind III
K = Kpn I
S = Sail
57
Table 5. Complementation between e(g) and different white alleles.
The left half of the table shows the 11 alleles of white tested for
complementation against e(g), their visible phenotype and total red pigment
levels (expressed as a percent of wild type pigment levels). The right side of the
table gives pigment levels for the appropriate combinations of the genotype w
g*/e(g) g*, where
*
indicates the given allele.
The last two rows list the results obtained with the e(g) allele and wild type
allele for white for comparison.
As an incidental note, these results show that when the amount of pigment in
the w g+ / e(g) g
2 strain is assayed the conventionally recessive white alleles
are slightly dominant. In contrast the garnet mutations are truly recessive. These
data agree with previously reported pigment levels of the white and garnet
genes. Nolte (1 959c) in a theoretical treatment of the significance of dominance,
found w/w had slightly less pigment than wild type (w+/wj. In the same study,
Nolte found garnet to be “truly” recessive. This has caused some difficulties in
the interpretation and significance of dominance of alleles but these problems
pose conceptual rather than functional problems.
CROSS:
1. To generate recombinant w g* chromosomes:
g*/g*® w*/YQ
Fl
F2
+
g*/ w’ +
1
® g*/y’f (sibs)
w*g*/Y0
58
(select by phenotype of test cross to g*/g*
and w*/w*
,
balanced to make
stock)
2. To perform the complementation cross:
P
e(g)g*/e(g)g* ® w*g*/Yo
Fl
e(g) g*/w* g*
assayed for pigment levels.
Note: This complementation test was performed three times, once by Barney
Lee as part of his directed studies course. The values of the individual tests
have been averaged as they did not differ greatly.
59
Complementation between e(g) and different white alleles
white allele
2
g
w*/w*
5Oe
w* g*/e(a)
53d
g*
1118
w
<1
completely white
11±1
1
w
<1
completely white
13±1
wa
3± .5 dull orange
5±1
wbf
2±.5 faint yellow
10±1
7±1
3±1
9±1
3±1
3±1
wBXW.7±.1 dull red
6±1
2±1
ND
ND
5±1
2±1
wch
>5 ± .1 pink-orange
12±2
8±1
ND
w’
ND
12±1
8±1
4±1
we
4±.5 pinky
11±1
7±1
ND
1
w
.6±.1 faint pink
9±1
3±1
ND
10±1
22±1
4±1
6±1
8±1
ND
e(g)g*
32±3
14±1
11±1
w÷g*
38±2
26±1
13±2
sat 7± 1
w
dull red
browny orange
.6±.1 faint pink
60
effect on garnet. Two constructs were chosen to provide the white+ transgene.
The first transgene construct contained the full white
+
gene coding region and
approximately 3kb 5’ and 3’ white regulatory sequence (along with rosy+)
inserted within a P-element located at position 1 OOF on 3R (Hazelrigg, Levis
and Ruben 1984). This particular construct shows an unusual pattern of white
+
gene expression. The anterior portion of the eye is normally pigmented (red)
while a crescent at the posterior margin, approximately one quarter of the eye,
remains unpigmented. This construct showed partial rescue of the enhancer of
garnet effect (Table 6). The pattern of rescue was similar to the pattern of
white+ expression. The posterior portion of the eye in which white+ was not
expressed appeared more lightly coloured (enhanced) than the anterior. The
second white+ construct used was the CaSpeR P-element transformation
vector. This construct contains all the coding region of the white gene but has a
deletion of most of the large first intron and all but 300 bp of the 5’ regulatory
region and 630 bp of the 3’ region (Pirrotta, Stellar and Buzzetti 1985). As the
white gene in this construct lacks most of its 5’ regulatory sequences, the
expression of white tends to be weak and highly dependent on the site of
insertion of the construct. Initial tests to determine if this construct would rescue
the enhancer of garnet phenotype were inconclusive (Table 6-top line). The
original insert had marginally more pigment than its e(g) g
; TM3 siblings (26 ±
2
2 vs 23 ± 3) but the difference was not significant. This failure to compensate for
the enhancer of garnet effect might have been due to the weak expression of
the construct. In order to determine if this construct was able to rescue the
enhancer of garnet effect when more strongly expressed, the element was
mobilized and over 30 lines that showed strong expression of the white+ gene
were generated. Results of this experiment, for eight different insert lines, are
shown in Table 6. In all eight lines the white construct was capable of
61
Table 6. Rescue of the enhancer of garnet effect by white+ transgenes.
The first column gives the designation of the individual white transgene inserts.
The first nine are different inserts of the CaSPeR insert, the first of which, 4-1, is
the original insert. These inserts also contain the cdc-2 homolog of Drosophila
melanogaster. The 4-3 transgene contains the complete white gene and most
of its 5’ and 3’ regulatory sequences. This insert was generously provided by
Bob Levis. The last two lines provide control values for e(g) g2 and g2 males.
The e(g) g2 control is an internal control derived from the average values of the
e(g) g2/Y; ÷/TM3 siblings of the experimental crosses.
The second column indicates the strength of the white transgene expression by
pigment assay (as percent wild type) and the last column indicates the ability to
rescue the enhancer of garnet effect in a e(g) g
2 background also determined
by pigment assay.
CROSSES:
1. To generate the different inserts of the CaSpeR 4-1 transgene;
P
67 casper 4-1/casper 4-1
w
;
67
/w
Fl
(transposase source)
/w +/÷
w
;
67 casper/delta 2-3 c? 0 1
w
/Y;
F2
.1.
0 w-/Y delta 2-3/TM3’
‘I,
67 casper/+
w
;
1
/w
scored for strong white+ expression and balanced as homozygotes stocks or
over FM7.
62
To determine the chromosome on which the transgene was inserted;
P
/Y; casper insertJTM3 or w’/Y; casper insert/-i-; -i-/TM3cJ ®
1
w
/wm CyO/Tft
wm
;
4
Segregation of the white insert relative to the dominant markers indicates the
chromosome into which the transgene is inserted.
Of the 27 independent inserts all were located on the third chromosome. No
further effort was made to map them.
2. To assess the strength of the white transgene expression;
P
/Y; insert/TM3cf 0 1
1
w
/w +/÷
w
;
“
/
1
w
w’orY;insert/+
Fl
progeny assayed for pigment levels.
3. To assess the ability of the white+ transgene insert to rescue the enhancer of
garnet effect:
P
e(g) g
/ e(g) g
2
; +/+
2
F1
/Y; insert ‘ and e(g) g
2
/Y; TM3/+
2
e(g) g
progeny assayed for pigment levels.
‘I
/Y; insertJTM3 ‘
1
0 w
a’
Generation of the different casper transgene inserts and determination of the
chromosome into which the transgene had inserted was done by Gwendoyln
Mahon as part of an undergraduate directed studies project.
63
Rescue of enhancer of garnet phenotype by white+ transgenes
w+ insert
effect on white
effect on e(g)
w-/Y:insert/÷
e(g) a2/Y:
insert/+
4-1
21±2
26±2
1-16
32±6
97±5
1-18
68±6
32±4
1-22
91±4
39±5
1-23
96±3
34±3
2-6
49±2
37±2
2-23
82±6
43±5
3-1
22±2
31±5
3-29
23±3
36±3
4-3
27±2
e(g) g2IY; TM3/+
g2/Y
23±3
30±2
64
rescuing the enhancer of garnet effect and restoring the full garnet phenotype.
While all of these new insertions expressed the white
+
gene more strongly than
the original insert, there was again no clear correlation between the strength of
white gene expression and rescue of the enhancer of garnet phenotype
(Figure 8 and see below). The final note of interest arising from this latter set of
experiments is that the interaction between garnet and enhancer of garnet
appears to involve the coding region of the white gene. The white gene in the
CaSpeR construct possesses little regulatory white+ sequences so it clearly
cannot be providing additional white regulatory regions. Yet it is able to rescue
the enhancer of garnet phenotype. This suggests that interaction between
garnet and enhancer of garnet is restricted to the coding region and is possibly
a post-transcriptional process. Examination of white transcription in a garnet
mutant background might resolve this question.
In summary, the enhancer of garnet mutation cytologically maps to the same
position as the white gene. Eleven white alleles, when combined with the
enhanceable garnet alleles, show a severe enhancer of garnet phenotype, in
conjunction with the enhancer of garnet and garnet mutations. The enhancer of
garnet phenotype can be rescued by a white+ transgene containing white+
coding region. Thus the enhancer of garnet mutation appears to be a cryptic
allele of the white gene.
The nature and dose sensitivity of interaction between enhancer of garnet and
garnet: There are a number of systems of genes and specific modifier genes
which have been described in Drosophila. Many of these consist of
65
transposable- or retro-element-induced mutations in one gene, the activity of
which is modified by a mutation in another gene. There are five such modifier
genes; Su(Hw) which modifies mutations produced by insertion of the gypsy
element (Modelell, Bender and Meselson 1983), su(f) which also modifies
gypsy-generated mutations (Parkhurst and Corces 1985), su(wa) which
modifies copia expression (Bingham and Judd 1981), su(pr) and su(s) which
modify mutations produced by the 412 element (Searles and Voelker 1986).
Initially it seemed possible that the interaction between garnet and enhancer of
garnet was of this nature. The first evidence that this was not the case came
g
O
e and g53d are
from the observation that the three enhanceable alleles, g
, 5
2
also the most extreme alleles (Table 16). In addition, none of these garnet
alleles was associated with insertion of any foreign DNA (Figure 21). In order
to determine if sensitivity to the enhancer of garnet mutation was dependent on
some specific property of these three alleles (other than insertion of a
transposable element) or due merely to the dose of functional garnet gene
product, the enhancer of garnet mutation was recombined onto chromosomes
carrying four of the weaker garnet alleles (g
,g
1
, g
3
4 and gP). Dosage of the
garnet gene product was then manipulated by making these chromosomes
heterozygous with a deficiency for the garnet gene. Table 7 shows that
although these alleles are not discernibly sensitive to the enhancer of garnet
mutation as homozygotes, when the dose of the garnet gene is further reduced
by a deficiency for the garnet gene, these alleles become dominantly sensitive
to the enhancer of garnet mutation. Dominant sensitivity to enhancer of garnet
was also observed when garnet gene dosage was reduced with the severe
g53d allele instead of a deficiency (Table 7-legend). Thus the enhancer of
garnet effect is highly sensitive to the dosage of the garnet gene and does not
appear to be allele specific. As the weak garnet alleles used are hypomorphs
66
Table 7. Sensitivity of the enhancer of garnet effect to garnet dosage
The effect of the enhancer of garnet mutation on three strong garnet alleles, g
’
2
Oe and g53d, and two weaker garnet alleles, g
5
g
1 and gP, was determined by
pigment assay. The first column lists the garnet allele. The next two columns
report the eye pigment values for the garnet allele over a deficiency for that
region and the same genotype heterozygous for enhancer of garnet,
respectively. The last two columns list the eye pigment values of the appropriate
garnet and enhancer of garnet and garnet homozygotes for comparison. The
enhancer of garnet effect was also sensitive to garnet dosage when the garnet
gene dosage was reduced using the extreme g53d allele. All values are
expressed as percent wild type pteridine pigments.
d/÷ gP genotype has 24±4 % WT pteridine pigment levels.
3
The e(g) g5
53 gP genotype has 13 ±2 % WT pteridine pigment levels.
g
The e(g) d/e(g)
In comparison with 50 ± 5 for the gP homozygote.
CROSS:
To generate the deficiency genotypes,
P
Df(1)HA97/FM7 ® g/Yor e(g) g/Yo’
Fl
Df(1)HA97/g or e(g) g
‘I,
g*/g*
c( and
progeny assayed
e(g) g*/ e(g) g*
homozygotes are taken from stocks.
67
Sensitivity of garnet alleles to the enhancer of garnet effect and garnet dosage
garnet allele
g/Df(g)
e(g) g/+ Df(g)
e(g) g/e(g) g
1
g
40±3
19±3
30±4
57±2
2
g
40±3
25±3
32±3
37±2
Oe
5
g
28±2
24±2
14±1
26± 1
53
g
d
16±3
9+2
11±1
13+1
qP
38±4
23±2
56±3
50±5
68
(Table 20), the sensitivity is presumably related to the amount of functioning
garnet gene product.
To further examine the sensitivity of the enhancer of garnet and garnet
interaction to the amount of gene product, the expression of the weaker garnet
alleles was tested in genotypes where the dose of the enhancer of garnet
mutation was reduced using a deficiency for the enhancer of garnet region.
Table 8 shows that only a single dose of mutant enhancer of garnet (DI e(g) g /
e(g) g), enhances the phenotype of weak garnet alleles. In contrast to the
previous results, the interaction is not dominant, the deficiency for the enhancer
of garnet region does not enhance the garnet phenotype in the presence of a
wild type allele of enhancer of garnet. Thus interaction between garnet and
enhancer of garnet is very sensitive to the dose of the garnet gene but rather
less sensitive to the dose of enhancer of garnet.
The complementation tests performed between different white alleles and the
enhancer of garnet mutation provide a reasonable set of data to quantify the
dose dependence between garnet and enhancer of garnet. Figure 8 shows a
comparison between the strength of different white alleles and their effect on
garnet. Two points are immediately evident. There is no correlation between the
severity of the white allele and its enhancing effect on garnet. There is however
a good correlation between the strength of the enhancing effect and the severity
of the garnet allele. Although this pattern might appear fortuitous, since only
three garnet alleles were tested as opposed to the eleven white alleles, the
results generally support the findings described above. The interaction between
69
Table 8. Effect of we(g) dosage on the enhancer of garnet effect.
The effect of reducing the dosage of the enhancer of garnet locus with a
deficiency for that region, on different alleles of garnet is shown. The first
column gives the garnet allele examined. The g
2 allele is a strong,
“enhancable” allele. The other three, g
’g
1
3 and gP are weaker alleles not
normally responsive to the enhancer of garnet mutation (as double
homozygotes). The next two columns give pigment values (as percent wild type
pigment) of these garnet alleles (shown as g*) in conjunction with a deficiency,
heterozygous with either a wild type e(g) allele and g* or the e(g) mutation and
g*, respectively. The last two columns show values for garnet and enhancer of
garnet homozygotes for comparison.
CROSSES:
1. To generate Df(1)N8 garnet strains;
P
Df(1)N8, w-, N-/In(1)d149, Hw g4
1.
0 g/YcJ
Fl
Df(1)N8+/+ g
F2
Df(1)N8 g/In(1)d149
recombinants selected by garnet phenotype and notched wing.
1
0 In(1)dI49/Y’ (sibs)
2. To generate e(g) g stocks;
P
/ye(g)cvg
ye(g)cvg
2
‘I,
Fl
®yacscpn wvg
1 f/YQ’
or ecctvg
YcJ
3
ycvvgPf/Y
or
/y cv v g
2
®y e(g) cv g
y e(g) cv g
1
cf
2
g*
recombinants selected by appropriate markers.
y e(g)
3. To generate the Df(1)N8 g/ + g or e(g) g individuals,
P
Df(1)N8 g*/In(1)d149
Fl
Df(1)N8 g*/e(g) g*
or
“
0 g/Yor e(g)
70
g*ty
Effect of e(g) dosage on the enhancer of garnet effect
Dfe(g)g*
+
g*
Dfe(ci)g*
g*
e(g)
28±5
g*
e(cx)g*
e(g)g*
7±1
37±2
32+3
13±2
5±1
57±2
ND
3
g
24±2
14±2
45±1
ND
gP
16±1
8±1
50±5
ND
2
g
71
Figure 8. Comparison between the severity of different white alleles and their
effect on garnet.
A. The effect of different white alleles (combined with three different garnet
g
O
e and g
alleles, g53d 5
(e.g. w*g*/e(g) g’ as heterozygotes with the
2
enhancer of garnet mutation and the same garnet allele (relevant crosses and
genotypes given the legend to Table 5). The percent wild type pigment of the
different white alleles are shown on the X axis. The percent wild type pigment of
the white garnet / e(g) garnet heterozygote are shown for the three garnet
alleles, on the Y axis.
B. The effect of different white+ transgenes in rescuing the enhancer of garnet
effect. The crosses and relevant genotypes given in the legend to Table 6. The
/Y; [white+]/+. The percent wild type pigment of
2
generic genotype is y e(g) cv g
the white+ transgene is shown on the X axis. The pigment level of the “rescued”
genotype is shown on the Y axis.
72
Comparison between the strength of different white alleles and
their complementation of enhancer of garnet.
J-
—
-
*g2
g50e
20-
—
15-
+g53d
—
10-
—
—
r
I
I
5.;
I__
—
—
—
—
—
—
I
—
I
n_
—
0
—
23456789 10
pigment of white allele
Comparison between strength of white transgene and
rescue of enhancer of garnet effect.
‘+z,-
lb
40-—————
C
+
—
I
I
C.)
•1
-C
30-—
25---20
0
-
-
-
-
+
+
102030405060708090100
pigment of white transgene
73
enhancer of garnet and garnet is dramatically sensitive to the dose of the
garnet gene but the effect of the white gene is somewhat allele specific.
Interactions between enhancer of garnet and other eye colour genes: The
genetic interaction between enhancer of garnet and garnet points to some
previously undetected interaction between their gene products. The white gene
product has been proposed, based on both molecular and genetic studies, to
function as transmembrane channels in conjunction with the products of the
brown and scarlet genes (Dressden, Johnson and Henikoff 1988). Thus the
white gene product appears to interact with at least two other eye colour gene
products. As the enhancer of garnet allele of white was an invaluable tool in
examining interactions between white and garnet, I decided to test other eye
colour genes for their sensitivity to the enhancer of garnet allele.
The enhancer of garnet mutation was combined with 26 other eye colour genes.
Since it was practical to test only one allele of each gene, and it is generally not
known which, if any, alleles of the different eye colour genes are amorphic,
there is a possibility that some interactions would not be observed. In order to
increase the sensitivity of this test, the enhancer of garnet mutation was placed
in trans to a deficiency for enhancer of garnet region. Table 9 shows the
results of this survey. Of the 26 eye colour genes examined, the mutant
phenotype of nine, possibly ten, is enhanced by the enhancer of garnet
mutation. These genes share with garnet the property of altering levels of both
brown and red pigments. Significantly, brown and scarlet fail to show an
interaction with the enhancer of garnet mutation. Thus the interaction between
garnet and enhancer of garnet appears to define a different type of interaction
than the structural role proposed for brown and scarlet. Interestingly, raspberry
74
Table 9. The effect of the enhancer of garnet mutation on other eye colour
mutants.
The e(g) mutation was tested in combination with 25 eye colour genes. The first
two columns list the symbol and name of the eye colour gene respectively. The
next two columns list the amount of total pteridine pigments measured for the
given eye colour mutant as a homozygote and for the same homozygous
mutation in combination with the a hemizygous e(g) mutation, respectively. All
values are expressed as percent wild type pteridine pigment. The generic
genotypes are mutant/mutant vs. e(g) mutant! Df e(g) mutant or e(g)/Df e(g);
mutant/mutant respectively. The values for the homozygous mutant genotype
were derived from internal controls for mutations on the second and third
chromosome and external controls (mutant/mutant flies grown concurrently with
the test crosses) for those on the first chromosome. The next column gives the
percentage difference of the two measurements as hemizygouse(g) with mutant
I mutant alone. The last column indicates whether or not this difference
constitutes responsiveness to the enhancer of garnet mutation.
The e(g) mutant phenotypes are heterozygous for the DF(1)N8 deficiency. This
deficiency has two effects on pigment levels unrelated to hemizygosity for the
white locus. Flies bearing the N8 deficiency are generally smaller and have a
transient, slightly brown eye phenotype shortly after eclosion. These effects
have been corrected for using DF(1)N8/ e(g); mutant/Balancer siblings where
possible or an arbitrary cut off point of 75% of the control, mutant alone, value.
In general there was little ambiguity.
75
CROSSES:
1. For X-linked eye colour genes:
A.
P
Df(1)N8/In(1)d149
Fl
Df(1)N8 +/ + mutant
F2
Df(1)N8 mutant?/In(1)d149 0 mutant/Yo’
(test cross individual virgin females to select double mutants)
1
® mutant/Ycy
® In(1)d149/Yo’ (sibs)
‘I
B.
P
/y e(g) cv g
2
y e(g) cv g
2
Fl
ye(g)cvg2÷/÷÷+÷mutant®ye(g)cvg2/Yf(sibs)
F2
y e(g)? cv+ g+ mutant/Ye’ selected.
Stocks were made by crossing to In(1)d149 balancer and the presence of
garnet and the eye colour mutant were confirmed by the appropriate test
cross.
e(g),
0 mutant/Yo’
‘I,
The Df e(g) mutant chromosomes and the first two generations of the e(g)
mutant crosses were made by Mitra Maharaj.
C. The experimental cross:
P
Df(1)N8 mutant/In(1)d149
Fl
Df e(g) mutant’ e(g) mutant
test genotype.
“
0 y e(g) mutant/Yo’
2. For second chromosome mutants:
A.
P
Df(1)N8/In(1)d149; GIa/ + 0 +/Y; mutant/mutant
B.
P
; Tft/CyO
2
y e(g) cv g
/y e(g) cv g
2
a’
0 +/Y, mutant/mutanto’
76
C.
/Y; mutant/CyOcf
2
®y e(g) cv g
Fl
Df(1)N8/÷; Gla/mutant
F2
; mutant/mutant
2
Df(1)N8/y e(g) cv g
; mutant/CyO
2
Df(1)N8/y e(g) cv g
; mutant/mutant
2
+ /y e(g) cv g
; mutant/CyQ
2
+/y e(g) cv g
‘I,
(experimental genotype)
(NB effect control)
(mutant control)
(wild type control)
3. For third chromosomes mutants:
A.
P
B.
P
C.
Fl
® +/Y mutant/mutanto’
Df(1)N8/In(1)d149; H/TM3
; H/TM3
2
/y e(g) cv g
2
y e(g) cv g
Df(1)N8/+; TM3/mutant
0
® ÷/Y mutant/mutantc?
/Y; mutant/TM3cI
2
y e(g) cv g
; mutant/mutant
2
Df(1)N8/y e(g) cv g
; mutantiTM3
2
Df(1)N8/y e(g) cv g
; mutant/mutant
2
+ /y e(g) cv g
; mutant/TM3
2
+/y e(g) cv g
77
(experimental genotype)
(NB effect control)
(mutant control)
(wild type control)
The effect of enhancer of garnet on other eye colour mutants
gene
bordeaux
bo
brown
bw
claret
ca
carnation
car
cardinal
Cd
carmine
cm
cinnabar
cn
dke-c dark eye
dor deep orange
light
It
lightoid
ltd
ma! maroon-like
Moire
Me
mel melanized
Mot-K Mottled of K
mw mottler of w
pink
p
prune
pn
purple
pr
raspberry
ras
ruby
rb
ry
rosy
sepia
se
scarlet
St
vermilion
v
white
wo
occelli
phenotype of
e(g) mutant
mutant
74±5
1 .1±.2
36±4
39±4
97±4
59±10
103±3
74±4
18±2
37± 1
90± 1
50±5
80±3
50±5
100 ±2
1.3± .3
32±2
49±3
37±3
29±2
38±2
60±2
111 ±3
76±5
106 ±4
92 ±7
108±2
1.5± .4
24±4
28±2
113 ±5
10±1
96±2
91 ± 16
5± 1
9± 1
17± 1
105±2
85±6
105±2
79 ±7
1.6 ±.5
14±3
49±2
6±1
41±2
7± 1
38±3
132±2
71±4
103 ±3
99±4
78
difference
effect
144%
104%
66%
72
106
13
93
123
29
25
19
208
106
208
79
127
44
100
16
142
19
63
119
93
97
106
NO
NO
YES
YES?
NO
YES
NO
NO
YES
YES
YES
NO
NO
NO
NO
NO
YES
NO
YES
NO
YES
YES
NO
NO
NO
NO
and maroon-like, lesions in the pteridine pathway, appear suppressed by the
enhancer of garnet mutation. The significance of this suppression is not clear.
Thus, enhancement of mutant eye phenotype by the enhancer of garnet
mutation appears to be a property shared by many eye colour genes in the
“transport” class.
Interactions between garnet and other eye colour genes: As the garnet gene is
not alone in its interaction with the enhancer of garnet allele, and as most of
these belong to the transport group of eye colour mutants, it was of immediate
interest to test the garnet gene for interactions with other eye colour genes.
Nolte (1 952b) found that while alleles of the genes garnet, carnation, carmine
and ruby are all normally recessive, in pairwise combinations they show
synthetic dominance. I extended these analysis to include 14 other eye colour
genes (Table 10). Interactions between pairwise combinations of garnet and
the other eye colour genes were assessed by looking for synthetic dominance
of garnet (garnet/÷, mutant/mutant< +/+; mutant/mutant), synthetic dominance of
the other gene (garnet/garnet or/Y; mutant/-i- <garnet/garnet or garnet/Y; +1+),
synergistic or additive, versus epistatic effects on eye pigmentation of the
double mutants, and effects on viability and fertility of double homozygotes. Of
those mutations which interacted with enhancer of garnet (claret, carmine, deep
orange, light, lightoid, pink, purple, ruby, rosy and probably carnation) all but
purple also interacted with garnet. In no case was an interaction with garnet
observed with a gene that failed to interact with enhancer of garnet. Taken
together with the data of Nolte (1 952b) there is considerable congruence
between those genes which interact with enhancer of garnet and those which
interact with garnet (Table 11). Finally it is interesting to note that, while the
partial sterility seen between garnet and other genes probably reflects a
79
Table 10. Interactions between garnet and other eye colour genes.
Interactions between garnet and other eye colour genes was monitored in three
ways:
1. Epistatic versus additive or synergistic interactions (as monitored both
visually and by pigment assay).
2. Synthetic dominance.
3. Synthetic lethality or sterility of the double homozygotes.
The first two columns indicate the symbol and name of the eye colour gene
tested with in combination with garnet. The next four columns show pteridine
pigment values (expressed as percent wild type levels) for; females and males
homozygous or hemizygous for the other eye colour mutation and female and
male double mutant individuals. To determine if either garnet or the other eye
colour mutant acted in a dominant fashion in a mutant background of the other
gene, the following genotypes were generated; ÷/g; mutant/mutant to determine
if garnet acted in a dominant manner in a mutant background and g/g or g/Y;
mutant/÷ to determine if the mutant became dominant in a garnet background.
The next three columns show pteridine pigment values (expressed as percent
wild type levels) for; individuals homozygous for the other eye colour mutation
and heterozygous for garnet, and individuals homozygous or hemizygous for
garnet and heterozygous for the other eye colour mutant. The next two columns
indicate the fertility of the double homozygotes and notes on their phenotypes.
The last column is a summary of the various models of interaction. A
“+“
indicates interaction, either of epistatic/synergistic, pigment interactions,
synthetic dominance in either direction, synthetic sterility or extensive
80
secondary phenotypes generally indicative of cell death. One
“+“
is given for
each type of interaction observed.
CROSSES:
1. To generate double homozygotes:
For eye colour mutants on the X chromosome;
P
g mutant/In(1)d149 0 g mutant/YJ
2
(double mutant stocks generated in cross 1 B described in legend to
Table 8.
For second and third chromosome mutants;
53 za g53d; E(var) 18. l2Sp/CyO
g
y za d/y
or E(var)303 ru h eg pP K1ffM3
0 In(1)IN(1)BM1/Y;
mutant/mutantcf
53 mutant/CyO or TM3
g
Fl
In(l)!N(1)BM1/y za d,.
y za g53d/y,.
mutant/CyO or
TM3’
P
2. Genotypes used to examine synthetic dominance;
For X-linked eye colour genes,
p
P
g53d/g53d 0 2
g mutant/Yo’
and
mutant/mutant 0 2
g mutant/Yo’
For second and third chromosome eye colour mutants the genotype g53d/g53d
d; mutant/Balancer were
53
or g53diy; mutant/Balancer and In(1)!N(1)BM1/g
generated as siblings of the crosses described above to make double
homozygotes. Cross 2 and 3 described in the legend of Table 9 generate
equivalent phenotypes with the g
2 allele and second and third chromosome
mutations. In all cases the conclusions derived from these crosses were the
g5 allele.
d
same as with the 3
81
82
Interactions between garnet and other eye colour genes
gene
pigment
synthetic
single mutant
double mutant
dominance
female
male
female male ÷7q;m/m j/q;+/m
bw
brown
1 + .2
0
1±.1 0
2±1 5±.5
ca
claret
36±2
43±2 9±1
6±1
35±3 8±1
cd
cardinal
101±4 ND
ND
5±1
101±3
95±3
cn
cinnabar 103 ± 3
ND
108±2 ND
ND
106±1
It
light
42±5 2±1
1±.5
20±1 7±1
37± 1
ltd
lightoid
0
78±10
68±6 0
50±2 8±1
Me
Moire
ND
ND
ND
82±3
80±3
79±5
MotK Mottled 100±2 101±3 ND
ND
ND
95±1
pink
p
ND
ND
34±2
5±1
25±2
45±3
pr
purple
ND
30± 10 40±10 4±1
7±1
37±4
iy
rosy
ND
63±3
6±1
64±10 ND
54±3
se
sepia
ND
ND
111±3
ND
112±5
113±4
St
scarlet
87± 10
81±10 10±2 7±2
69±5 13±2
wo
white
ND
ND
ND
ND
92±7
94±4
occelli
q/Y÷/m
3±.5
6±1
ND
ND
7±1
7±1
ND
ND
ND
ND
16±2
ND
16±2
ND
f&m fertile
male semi-sterile
male fert. f. ND
ND
f&m fertile
male semi-sterile
ND
ND
male semi-sterile
f&m fertile
f. sterile
ND
f&m fertile
ND
fertility
+++
++
++
+
-9
SUMMARY
Table 11. Summary of interactions between other eye colour mutations and
both garnet and enhancer of garnet.
The first two columns give the symbol and the name of the eye colour gene. The
third list the pigment pathway effected or the general nature of the defect (from
Table 1). The final two columns list whether there was an interaction with
either enhancer of garnet or garnet respectively. A gene was considered to
interact with garnet if it showed any one or more of epistatic/synergistic
interactions, synthetic dominance, sterility or unusual phenotypes of double
hete rozygotes.
83
Summary of eye colour genes interacting with e(g) and garnet.
mutant
pigment
interaction interaction
affected
with e(g)
with garnet
claret
both
ca
YES
YES
carnation
both
car
YES
ND
cd
cardinal
both
NO
NO
carmine
both
cm
YES
NO
dor
deep orange
both
YES
ND
garnet
both
YES
NA
g
light
both
It
YES
YES
ltd
lightoid
both
YES
YES
pink
both
YES
YES
p
pd
purpleoid
both
ND
NO?
rb
ruby
both?
YES
ND
rosy
both?
YES
YES
iy
YES*
w
white
both
NA
cn
cinnabar
ommochrome NO
NO
scarlet
ommochrome NO
NO?
St
vermilion
ommochrome NO?
v
ND
bordeaux
ND
bo
pteridines?
NO
NO
bw
brown
pteridines
NO
pn
prune
pteridines
NO?
ND
pr
purple
pteridines
YES
NO
ras
raspberry
pteridines
NO
ND
sepia
pteridines
NO
NO
se
maroon-like
ND
mal
pteridines
NO
NO
ND
dke
dark-eye
pattern
Me
Moire
pattern
NO
NO
Mottled of K
pattern
NO
NO
Mot-K
rolled
NO?
ND
rI
pattern
mel
melanized
secondary
NO
ND
NO
ND
pw-c
pink wing c
secondary?
YES*
z
zeste
regulatory
?
84
Figure 9. Analysis of garnet transcription by in situ hybridization in a rosy null
genetic background.
Panel A shows garnet message in brain (b), salivary gland (sg), eye antennal
discs (e), leg discs (I), wing discs (w), fat body (f) and trachea, of ry
/ry
506
third instar larvae.
Panel B shows garnet message in leg discs (I), a wing disc (w), and possibly a
haltere disc (h?) of ry
/ty third instar larvae at higher magnification.
506
/ry third instar larvae hybridized with
506
Panel C shows two leg discs of ty
sense probe as controls.
85
....•
3
8
I
6s
r
4
q
4
•
t
V
cc
secondary effect of the small, weak, poorly viable males that died a few days
after eclosion, only the rosy gene, when combined with garnet, shows both
complete sterility and female sterility. The interaction between garnet and rosy
was investigated in more detail by examining garnet transcription in imaginal
discs, brain and other tissues derived from rosy null (ry
) third instar larvae.
506
Figure 9 shows these results. Although this is not a quantitative assay, it would
seem that garnet transcription is not noticeably altered in the imaginal discs of
rosy null larvae. Interestingly, however, there appears to be no garnet
transcription in the brain of rosy null third instar larvae, a tissue which shows
garnet expression in wild type larvae (Figure 27).
Interaction between garnet and white: Two of the garnet interactions deserve
additional comments. The interactions between garnet and enhancer of garnet
have already been detailed at some length. As the enhancer of garnet mutation
appears to be an allele of white it is clear that garnet and white show a fairly
complex genetic interaction. It is only by virtue of being a subliminal allele of
white that it was possible to investigate these interactions. The conventional
alleles of white (necessarily) effect eye pigmentation so that the spectrum of
interactions seen with other eye colour mutations is quite limited.
Nolte (1 952b) noted that g
3 interacted additively with the white alleles, wCh,
00 This analysis was extended to include 7 additional
w
, wa, wsat, wbl and .
0
w
1118 completely white), one weak
1 and w
white alleles, two extreme (w
(wBVlfX
1 and
which produces a dull red colour), and four moderate alleles (wbf, we, w
g
O
e and
wt, pinky-yellow eyes) in combination with three garnet alleles, g
,5
2
53 (Table 12). The results are consistent with those reported by Nolte. The
g
d
white and garnet alleles act additively to reduce eye pigmentation. One
87
Table 12. Phenotypes of garnet double mutants with different white alleles.
Oe
5
The phenotype of 11 different white alleles alone and combined with g
’ g
2
and g53d is shown. The first column shows the white allele, the next column
lists its visual phenotype and below, the value obtained from pigment assay for
the white allele alone (as percent wild type pteridine pigment). The last column
lists the phenotype and pigment value of the appropriate white allele and the
d
5
g
3 allele of the genotype w* g53d/y where
*“
indicates the given white
allele. There was in general no great difference between the three garnet
alleles in combination with the white alleles.
88
Additive interactions between white and garnet alleles
white allele phenotype
white and garnet double
mutant phenotype
1 178
w
completely white
<1
completely white
<1
1
w
completely white
<1
completely white
<1
wa
dull orange-red
3±1
completely white
<1
wbf
faint yellow
1±.5
completely white
<1
Bwx
dull red
1±.2
pale pink-orange
ND
wch
faint pink/orange
completely white
<1
00
w
dull red
6±1
faint yellow
2±1
we
pink/orange
3±.5
completely white
<1
1
w
faint pink
completely white
<1
sat
browny-purple
8±1
pale orange
ND
w
faint pink
.6±1
completely white
<1
89
Table 13. Epistatic interaction between g
2 and wa3.
The first row gives the genotype of males with either single or double mutant
combination of g
2 and wa3. The second row gives the values for pteridine eye
pigments (as percent pteridine wild type pigment) for each of these genotypes.
90
Epistatic interaction between g
2 and a3
genotype
/Y
2
g
wa3/Y
a3 g
/Y
2
pigment
38±3
25 ±3
28 ±...2
91
exception has been noted to this rule. Green (1959) noted, while attempting to
functionally distinguish white alleles, that a2 and wa3 in combination with g
2
can not be distinguished from the single mutant. Table 13 shows pigment
determinations for a2, g
2 and the double mutant combination. These results
confirm that, in contrast to the situation with all other combinations, there is no
additive interaction between these alleles. (Green also claims this effect is also
found for another member of the “transport” group of eye colour mutations, ruby
and
.)
3
vva
These results further emphasize the allele specificity of the white-
garnet interaction. Further study of this aberrant epistatic interaction might help
reveal the nature of the physical nature of the white-garnet interaction.
Interaction between garnet and zeste. The zeste gene was originally identified
as a modifier of the white gene. In the presence of a mutant zeste gene, paired
copies of the white gene function less effectively, decreasing pigmentation.
Thus zeste acts as a pairing-dependent positive regulator of white. Molecular
analysis of the zeste gene has shown it to be a protein which acts as a
transcription factor which enhances the expression of white and many other
genes (Pirrota 1988). Mutations in the zeste gene have a dramatic effect on
g
O
e and 3
g5 flies without and with
d
garnet expression. Figure 10 shows g
, 5
2
the z
1 and the za alleles. Table 14 shows the corresponding pigment levels.
Clearly mutants in the zeste gene further reduce the amount of pigmentation of
mutant garnet individuals. The zeste-garnet interaction appears sensitive to
1 allele does reduce garnet expression in
chromosome pairing. While the z
males, with a single X chromosome, in females where the two X chromosomes
may pair, eye pigmentation is essentially abolished (Figure 11). Thus both the
zeste gene and the enhancer of garnet mutation reduce garnet expression.
Both the cytological mapping of enhancer of garnet (Figure 6) and by
92
Figure 10. The effect of zeste mutant alleles on the garnet phenotype.
The effect of the za and z
1 alleles on the phenotype of flies mutant for the three
53 5
g
g
O
e and g
garnet alleles, d,
2 (from top to bottom respectively). Each
photograph shows the eye colour of the garnet allele alone, the same garnet
Qe
5
alleles when combined with the za allele (and also the z
1 allele with the g
garnet allele) and the appropriate e(g)-garnet combination for comparison.
93
e(g) g53d
Oe
5
1 g
z
e(g)
Oe
5
za g
2
e(g) g
2
g
94
Table 14. Effect of zeste on garnet.
The effect of zeste on garnet expression was determined by visual inspection
and by determination of pteridine pigment levels (expressed as percent wild
type levels) for two alleles of zeste (z
1 and za) and the three most severe alleles
0
5
0 and g5
d).
3
of garnet (g
, g
2
Cross: The zeste and garnet mutations were combined as follows:
g*/g* ®yz*/Yc/
Fl
yz’ ÷/÷
+
g*
® g*fyf(sjs)
yz*g/Ycf
progeny selected as yellow, hence probably zeste, and garnet
individuals. These were balanced over the ln(1)d149 balancer to
generate
stocks.
F2
95
Effect of zeste on garnet
garnet allele
zeste genotype
yzg
yzag
g
1
yz
g53d/g53d
3d/y
5
g
16± 1
19±1
10 + 1
12±2
<1 ± 1
10±1
Oe/g
5
g
Q
e
Oe/y
5
g
29±2
33±2
18±3
19±5
<1 ± 1
10±3
/g
g
2
31±2
38±2
14 + 1
17+1
<1 ± 1
21±2
/Y
2
g
96
Figure 11. The phenotype of zeste-garnet combinations in females and males.
Oe and g
5
The phenotypes of three garnet alleles, g53d, g
2 (from top to bottom)
alone and in combination with the z
1 allele are shown. The phenotypes of
homozygous and hem izygous z’- garnet flies differ noticeably. The females
(where pairing between the two X chromosomes is possible) have essentially
white eyes whereas the males show a less dramatically enhanced garnet
mutant phenotype.
97
d/y
g
3
1 5
z
d
g
3
3 5
d/zl
5
zl g
121
e
g
O
e/zl 5
g
O
5
e/Y
g
O
1 5
z
1 g
z
IY
2
z
1
/
2
1 g
z
Table 15. Effect of zeste modifiers on the zeste-garnet genotype.
The effect of two modifiers of zeste, E(z) 1 and Su(z) 302 was determined on
d
5
3 genotypes.
theyza g
1g
2 and yz
Cross:
P
yzi g*/In(1)d149; +1+ oryza g*/Jn( 1)d149; ÷/÷
Su(z)302/TM3 a’
where
*
0 +/Y E(’z,)1 or
indicated either g53d or g
.
2
The g
4 allele occurs in the complex inversion In(1)In(1)d149.
The progeny of this cross were scored visually and by determination of
pteridine pigment levels (values expressed as percent wild type).
99
Effect of modifiers of zeste on garnet
garnet genotype
E(z)1
Su(z)302
yzlg
d/÷;Mod.*/÷
53
d/+; TM3/+
3
1 g5
yz
53 Mod./+
g
1 d/Y;
yz
d/Y;TM3/÷
53
yzlg
19±1
96±2
1 ± .1
11±1
101±2
101 ± 1
15± 1
15±1
/+; Mod./+
2
y za g
/+;TM3/÷
2
yzag
/Y;Mod./+
2
yzag
/Y; TM3/+
2
y za g
97±2
94±2
10±3
9±1
103± 1
100±1
25±2
18± 1
/+; Mod.!-,4
yz g
y z+ g4/÷; TM3/+
/Y;Mod./÷
4
yzg
/Y;TM3/÷
4
yz+g
102± 1
97±2
29±3
14±1
104±2
101 ±3
38±4
35±1
*
Mod.
=
Modifier of zeste, either E(z) 1 or Su(z)302.
100
complementation between zeste and enhancer of garnet (data not shown)
indicate that the enhancer of garnet mutation is not an allele of zeste. This
interaction is sensitive to modifiers of zeste. The enhancer of zeste mutation
d
5
3 in males and is, furthermore, capable of
1 g
further reduces pigment of z
inducing dominant garnet expression in z
1 g53d/÷
+
females (Table 15). The
d
5
3 allele by z
enhancement of the g
1 occurs in males as well as females
indicating that in this case, pairing of X chromosomes is not necessary.
Interestingly the eye phenotype of these flies is a tine grained mosaic of brown
spots on an orange background which becomes more pronounced with age.
Genetically the interaction between zeste and white resembles that between
garnet and white. At least superficially, both appear to be necessary for full
white gene expression. Although the zeste gene has been extensively
investigated and is known to modify the transcription of a number of genes,
there is no mention in the literature of an interaction between zeste and garnet.
This interaction could occur either directly, if zeste acts as a transcriptional
enhancer for the garnet gene, or indirectly, if it occurs due to compromising the
function of the white+ gene. The latter may be more likely as there are no zeste
binding sites at garnet. These possibilities could be distinguished genetically by
providing another wild type copy of the white gene as a transgene which would
be insensitive to pairing-mediated zeste effects.
101
Discussion-Interactions between garnet and other eye colour genes.
The enhancer of garnet mutation:
The first clue to the extensive genetic interactions between garnet and other
genes was the discovery of the enhancer of garnet mutation. The chance of
finding not only a mutant white allele, but a cryptic white allele, in the strain in
which the gP mutation was induced is truly remarkable. This mutation was
pivotal in unraveling the complex network of interactions between white and
many other eye colour mutations. These interactions could not have been
discovered with a conventional allele of white where the phenotype of the white
allele would mask that of the interaction.
The enhancer of garnet mutation is a cryptic allele of white:
Three lines of evidence indicate that the enhancer of garnet mutation is an
allele of the white gene. Firstly, their cytological positions coincide. Secondly 11
different alleles of white acted as strong enhancer of garnet mutations in
combination with the enhancer of garnet mutant and the three alleles of garnet
whose phenotypes are sensitive to the enhancer of garnet mutation. Finally
white+ transgenes were able to rescue the enhancer of garnet effect. Thus the
enhancer of garnet mutation should properly be designated we(g)
Dosage studies with deficiencies for the white gene indicate that the original
enhancer of garnet mutation was hypomorphic. In conventional
complementation tests with different white alleles and white deficiencies, the
enhancer of garnet mutation complements white mutations. Mutations in the
102
white gene, however, fail to complement the enhancer of garnet phenotype.
Thus the enhancer of garnet mutation is a cryptic allele of the white gene. As a
cryptic allele it has no independent phenotype.
The we(g) mutation reveals interactions between white and members of the
transport group.
The we(g) mutation was the key tool in the exploration of the interaction
between garnet and white. Combinations between we(g) and mutations in 26
other eye colour genes were examined in the hope that e(g) would reveal
similar interactions between the white gene and other eye colour mutations. Of
these 26 combinations, nine or possibly ten showed interactions which mimic
the interaction seen between garnet and enhancer of garnet. More importantly,
only those eye colour genes identified as members of the transport group
showed this interaction. Neither brown nor scarlet, likely structural components
of the transmembrane pore complex, nor genes encoding enzymes in the
ommochrome biosynthetic pathway (vermilion and cinnabar) or pteridine
biosynthetic pathway (maroon-like, raspberry and sepia), showed this
interaction.
These results imply that the white gene interacts not only with garnet but also
with claret, carnation, carmine, deep orange, light, lightoid, pink, purple, rosy
and ruby. These genes are largely the same as those described as the ruby
group by Nolte (1955), group two by Schwink (1975) and the transport group by
Sullivan and Sullivan (1975). (Two of the eye colour genes which have been
implicated as members of this group, orange and maroon, were not tested
because mutant alleles were not available).
103
Conceptually, this interaction could arise from a situation where the white gene
product interacted separately with all these genes. Alternatively, the white gene
product, and the products of the transport group of eye colour genes might
interact as members of a large macromolecular complex. The second model
predicts that the transport group of genes would interact with each other as well
as with the white gene. This was found to be the case. Pairwise combinations
between garnet and many of these genes showed a variety of novel
phenotypes, including synthetic sterility, cell death phenotypes, synthetic
dominance and synergistic/additive effects on eye pigmentation. Although these
genes have been previously linked by phenotypic, physiological and
histological analysis, genetic analysis of interactions between these genes has
been haphazard. There is remarkable congruence between the genes which
interact with enhancer of garnet and those which interact with garnet. This
strongly suggests that the white gene interacts in the same fashion with all of
these gene products.
One member of the transport group deserves additional comment; the rosy
gene. The rosy gene is exceptional in this group because it is the only one
which is known to encode a defined enzymatic function. The rosy gene encodes
xanthine dehydrogenase. Unlike other eye colour genes which encode
enzymes, no role for xanthine dehydrogenase has been confirmed in either the
ommochrome or pteridine biosynthetic pathways, (Reaume, Knecht and
Chovnick, 1991). Interactions between rosy and other members of the transport
group might suggest a structural rather than enzymatic role for this gene
product. While structural enzymes are hardly unprecedented, enzymatic activity
appears necessary for both normal eye pigmentation and correct localization of
104
xanthine dyhydrogenase in the pigment granules (Reaume, Knecht and
Chovnick, 1991). It is also possible that rosy has a general function only
indirectly related to eye pigmentation (Hilliker et a!. 1992). It may be important to
note in this context that the interaction between garnet and rosy was unusual in
that it was the only combination that resulted in complete female sterility. Further
investigation may well reveal that the rosy gene plays a slightly different, and
possibly a key role in the function of this group of mutations.
The nature of the interaction between garnet and white:
Null mutations in the white gene show no pigment deposition in any tissue.
Thus the white gene product is necessary for the correct localization of both the
pteridine and ommochrome pigments. Early models advanced the white gene
as a common element which interacted with the product of the brown and
vermilion, cinnabar and scarlet genes to deposit both red and brown pigments
(Nolte 1952b). The role of the white gene in pigment deposition has been
reformulated based on its similarity to a number of transmembrane channel
complex proteins such as bacterial permeases, mammalian multiple drug
resistance genes and the cystic fibrosis gene product (Dressen, Johnson and
Henikoff 1988). These proteins function as transmembrane pores or parts
thereof. One model for its function in eye pigmentation is based on a physical
interaction with the products of the brown and scarlet genes, respectively, to
form the principal component of the transmembrane channel complexes which
allow transport of pteridines and ommochrome pigments, pigment intermediates
and metabolites into the pigment cells. The role of the garnet gene in this
process is unknown.
105
The extensive complementation testing between garnet and different white
alleles allowed me to examine the sensitivity of the enhancer of garnet effect to
the dosage of both the white and the garnet genes. The enhancer of garnet
effect appears to be very sensitive to the dosage of the garnet gene. The
interaction is sufficiently sensitive that if the dosage of the garnet gene is
severely reduced, such as when a mutant garnet allele is heterozygous with a
deficiency for the garnet region (e(g) g / + Df(g)), the enhancer of garnet
mutation acts in a dominant manner. In contrast, the enhancer of garnet effect
appears to be sensitive to the specific allele of white rather than dosage of the
white gene. The allele specificity may reflect the mechanism of the interaction.
In at least one case, that of the wa allele, the lesion at the white gene is
associated with aberrant transcripts. If these altered transcripts are translated,
the strong enhancer of garnet effect might indicate a protein-protein interaction,
conceivably via titration of active garnet gene product. Characterization of the
3 allele, which shows an epistatic interaction
lesion at the white locus in the wa
with garnet might be illuminating. A further suggestion that the interaction
between white and garnet may occur post-translationally can be inferred from
the rescue of the enhancer of garnet effect by white+ transgenes with
essentially only white coding region. Regardless of the nature of the
interaction, genetic interactions between white and garnet clearly indicate a
functional link between these genes.
Severe garnet mutations and weak white mutations are very similar in
phenotype. In this context, it is interesting to note that the chromatography
profile of a garnet allele (g
) is indistinguishable from that of a hypomorph of
2
white (Hadorn and Mitchel 1951). Furthermore, Nolte (1959b) noted that
change in red and brown pigments of the wbl allele produced by temperature
106
change parallels the changes in these pigments between different garnet
alleles. This similarity in phenotype, in conjunction with the proposed structural
role of white in forming the transmembrane channel complex suggests that
garnet is a positive regulator of white function. Specifically if garnet is non
functional, the white gene functions, but much less efficiently. If there is any
further compromise of this system, such as with the we(g) or other white mutant
allele, pigment deposition, which in the presence of a functional garnet gene
product would be unaffected or only slightly diminished, is essentially
eliminated.
Models of gene interaction in the eye:
The most complete model proposed for the role of the many eye colour genes in
the structure and pigmentation of the eye was proposed by Nolte (1952b,1959),
based on data obtained from his decade of histological and genetic work on
these genes. Nolte proposed that the genes which altered levels of both the red
and brown pigments, the “transport group”, were involved in general aspects of
protein metabolism, specifically protein catabolism. The massive rise in protein
catabolism that accompanies metamorphosis and the inability to excrete toxic
by-products could result in the evolution of pathways whereby toxic by-products
were converted into pigments. Genes which regulate or participate in these
functions might alter the amounts of pigment precursors and thus final pigment
levels. Nolte (1 952b) derived a genetic pathway that invoked two partially
independent systems, controlled by brown and scarlet, which interacted with the
white gene. In this scheme garnet, along with ruby, carmine and carnation
operated in separate but parallel pathways to provide appropriate levels of
precursors. Later he included the genes claret, maroon, pink, purple, purploid,
107
prune and rosy (Nolte 1955) and light (Nolte 1 954b) as other members with
similar functions. The genetic interactions, the synthetic dominance and
synergistic interactions, between these genes were the impetus for this model
and they are certainly consistent with the proposal of an interrelated network of
somewhat redundant pathways where mutation in one would lower the
threshold for others. Such a system would also explain why lesions in such a
general and important function were not lethal. Figure 12 shows the functions
derived from genetic interactions as proposed by Nolte (1952b). Nolte’s later
(1959b) variation of the model included available information on tissue
specificity, developmental control of pigment formation and multiple feedback
loops.
Little more can be added conceptually to this model. The analysis of some eye
colour genes over the past thirty years has, however, suggested more specific
roles. This information, in conjunction with the results of this work, allows me to
postulate the model shown in Figure 13. The essential features of this model
are:
1. The white, brown and scarlet genes are structural components of a
transmembrane pore complex.
2. The white gene product interacts separately with the scarlet and brown gene
products.
3. The transport group of eye colour genes also interact with the white gene
product.
4. This interaction is different from that of brown and scarlet, likely fulfilling a
non-structural role.
5. Interaction between the transport group and the white gene is proposed to
occur at the protein level.
108
Figure 12. Nolte’s model for interactions between eye colour genes.
Figure 12 shows a simplified models of interactions between eye colour genes
adapted from models proposed by Nolte (1952b and 1959). The lower circle
indicates biochemical reactions proposed to occur in the body whereas the
upper portion of the figure indicates reactions proposed to occur in the eye.
Roles of a number of eye colour genes are indicated. The genes garnet,
carmine, carnation and ruby are shown at the bottom performing similar,
redundant functions involved with metabolism of eye pigment precursors. The
white gene is shown interacting with the products of both the brown and scarlet
gene to produce the red and brown pigments respectively. The cinnabar and
vermilion genes are indicated as enzymatic lesions.
109
Nolte’s model of eye colour gene interactions
Red
chromoprotein
chromoprotein
• reactions
in
eye
protein
brown
pigment
rec’
pigment(s)
sors
1
precu
precursors
cn +
f
reactions
in body
t
precursors
prersors
v+f
tryptophan
precursors
/
g+cmar4rb
110
+
6. The products of the transport group genes are proposed to form a complex.
7. The rosy gene may have a different and possibly key role in this process.
8. The function of this complex is to enhance the function of the transmembrane
pore complex.
9. These gene products may associate in various combinations to perform
similar functions in other tissues, other locations in the cell, or at other times in
development
These points are discussed in more detail below.
The white, brown and scarlet genes appear to encode transmembrane transport
proteins. The phenotypes of mutations in these genes, the interactions between
these genes, and their physical properties lead to a simple model in which the
white gene product associates with either the brown or the scarlet gene
products to form the transmembrane channel complexes responsible for
transport of the pteridine and ommochrome pigments or pigment intermediates.
The role of the “transport” group of mutants appears somewhat different. The
garnet, light (Devlin et al. 1990) and pink (Jones and Rawls 1988) genes have
been cloned. The garnet gene does not show a strong similarity to
transmembrane proteins, nor has such similarity been published for the light
gene, however, A.J. Howells (cited in Tearle 1991) reports that the pink gene
may have some sequence similarity to the white gene. Genetically, neither
brown nor scarlet interact with the we(g) mutation, suggesting that the transport
group gene products participate in a different type of interaction with the white
gene product from that hypothesized for the brown and scarlet gene products.
Rescue of the enhancer of garnet effect by the coding region of white leads me
to postulate that this interaction involves the protein products of these genes,
although the data are inferential for the garnet gene and there are no equivalent
111
Figure 13. Model of the physical interactions between the products of the
white, brown, scarlet and the transport group of eye colour genes, including
garnet.
The product of the white gene is proposed to interact with the products of the
brown and scarlet genes, separately, to form transmembrane channels in the
cell membrane to allow the ingress of the pteridine and ommochrome pigments
(or intermediates), respectively. The products of the garnet, carnation, carmine,
light, lightoid and pink genes are shown interacting in some ill-defined
macromolecular complex, the function of which is to enhance the activity of the
white gene product. The product of the rosy gene is shown as participating by
some independent mechanism.
112
-
C)
p
0
0
CD
3
0
0
C)
3
9
0
Z
O3.
C,
a
CD
D
CD
CD
0
=
0
C•)
-‘
-h
0
CD
a
0
D
-‘
data concerning other members of the transport group. The effect of e(g)
other members of the transport group of eye colour genes parallels that of we(g)
on garnet. Furthermore, those members of the transport group of eye colour
mutations which are sensitive to we(g) also show genetic interactions with
garnet. This suggests that these “transport” group of genes are functionally
linked. I have interpreted this functional interaction as a macromolecular
complex. Transmembrane permeases are typically large multi-subunit
complexes but other possibilities for the physical nature of the interaction exist.
The rosy gene shows a slightly different spectrum of interactions with garnet,
and is the only member of this group which is known to encode an enzyme. For
this reason, as well as its extensive participation in synthetic lethal interactions, I
have postulated that the rosy gene performs a different, and possibly
controlling, function. This function might conceivably be involved in metabolic
feedback. The phenotypes of null and extreme members of all of the transport
group of genes resemble moderate mutations of the white gene. This
phenotype suggests the function of these genes, or of the complex, is to
enhance the activity of the transmembrane pore complex. I have indicated that
this involves interactions between the gene products. It is, however, possible
that these genes all act as transcriptional enhancers of the white gene. Finally,
the action of this proposed complex may not be restricted to the plasma
membrane of the eye pigment cells. At least one of the enzymes necessary for
ommochrome pigment synthesis is found in the mitochondria (Sullivan, Grub
and Kitos, 1974). Hence the resulting pigment precursor must be transported
across the mitochondrial membrane. The accumulation of pigment in the
mitochondria of rosy mutants also implies the need for transport across this
membrane (Bonse 1967). And, as the pigment granules themselves are also
membrane bound, transport across this membrane would also be necessary for
114
normal pigmentation. The synthetic lethal interactions between certain
members of the transport group, as well as the synthetic sterility or garnet and
rosy, suggests that different members of the complex may associate in other
tissues or at other times in development to perform slightly different functions.
This transport system may in fact have a fairly ubiquitous distribution. The fact
that the sole phenotype of mutants in the system is an alteration in eye
pigmentation may be the results of functional redundancy within the transport
group of eye colour genes and the requirement for massive amounts of pigment
and pigment intermediate transports, within a short time, for normal eye
pigmentation. Thus while the eye cells may place the greatest demand on this
transport system, it does not mean that this system is confined to that tissue.
The expression of the white gene is extensively regulated. Pairing dependent
regulation of the white gene by the zeste gene product is well documented
(Pirrotta 1988). Additional regulators have been described which appear to
regulate not only white but also the brown and scarlet genes, also proposed to
encode structural components of the transmembrane channel complex
(Rabinow et a!. 1991, Birchler et al. 1994). These regulators all appear to act at
the transcriptional level. If the interaction between garnet and white involves
post-transcriptional regulation this will describe a novel form of regulation of the
white gene. Testing of this model will require molecular analysis of the transport
group of eye colour genes. As an initial step in this process, chapter 2 presents
phenotypic characterization of the different garnet mutants and preliminary
molecular characterization of this gene.
115
Chapter 2.
Analysis of the garnet gene
116
Introduction-Chapter 2. Analysis of the garnet gene.
Any attempt to test a model of the biological role of eye colour genes must rest
upon the detailed analysis of individual genes. The garnet gene is a member of
a particularly interesting class, the transport group of eye colour genes. These
genes are defined by alterations in the levels of both the ommochrome and the
pteridine pigments. This phenotype likely stems from lesions in inter- and intra
cellular transport and communication. Chapter 1 presented genetic analysis of
the interactions between the garnet gene and other eye colour genes, which led
to a simple model for the function of these genes. This chapter presents detailed
phenotypic analysis of a number of garnet alleles, evidence for the cloning of
this gene and preliminary molecular analysis. This work lays the necessary
groundwork to determine the function of garnet, to test the model presented in
chapter 1, and leads to further studies on the garnet gene presented in chapter
three.
117
Results: Characterization of the garnet eye colour gene.
Phenotype of garnet mutants:
The garnet gene was originally described as an eye colour mutation and this is
the most obvious phenotype of the garnet mutants. Figure 14 shows the
spectrum of eye colours found in a variety of garnet alleles. Eyes of garnet
mutant flies range from the slightly brown colour of weak alleles through the
browny-orange of the intermediate alleles to the pale orange of the extreme
d allele. This latter allele is unusual that in that it displays a fine grained
3
g5
mottling of red spots on a pale background as the flies age. Although the
various garnet alleles have been used for many years, quantitative information
of the amounts of red and brown pigments exists for only a few garnet alleles
,g
1
(g
,g
2
3 and g
, Nolte 1959). Table 16 gives a quantitative estimate of the
4
levels of pteridine (red) pigments and the ommochrome (brown) pigment
xanthommatin for 18 garnet alleles using four different methods (comparisons
between these methods is discussed in more detail in Appendix 1). Nolte
(1950, 1952b and 1959) gives values for the relative amount of red and brown
pigments in the g
,g
1
,g
2
3 and g
4 alleles as 38, 15, 23 and 57 % (red pigments)
and 56, 32, 47 and 23 % (brown pigment) respectively. He used a sequential
pigment extraction technique to obtain these values. It should be noted that
Ephrussi and Harold (1944), as well as the controls presented in both these
papers show that this technique is problematic. Nevertheless these data agree
reasonably well with those shown in Table 16.
118
Figure 14. Spectrum of eye colour phenotypes of different alleles of the garnet
gene.
The top portion of the figure shows the phenotypes of wild-type (Canton S) and
two garnet alleles, gP and g53d. The two garnet alleles shown represent two
extremes of the spectrum of eye colours due to mutations in the garnet gene.
The g53d allele is the most extreme of the garnet alleles and has a pale orange
eye. The gP allele is a very weak allele. Flies bearing the gP mutation have
slightly browner eyes than wild type upon eclosion which then darken to wild
type within three days.
The lower portion of the figure shows the spectrum of eye colours seen in the
g
O
e, g
garnet alleles, g
, g53d, 5
4
,
6
1 gS3 and gP. A wild type male
, g
1
, g
3
,g
2
(Canton S) is shown in the center of the figure for comparison.
119
oI
Determination of pigment levels in garnet mutants.
Table 16 presents values for total relative absorbance of the pteridine and the
ommochrome pigments, all expressed as percent of the appropriate wild type
pigment levels. As there is only one ommochrome pigment in Drosophila
melanogaster, dihydroxanthommatin, the “brown pigment” column gives an
adequate estimate of the amount of xanthommatin in these mutant strains.
There are however, at least 28 different pteridine pigments and intermediates
(Ferré et al. 1983, 1986). All of the pteridine pigment procedures used to
generate the values given in Table 16 measure principally the content of the
drosopterin pigment (Appendix 1). In order to assess the effect of these
mutants on at least some of the other pteridine pigments, the red pigments were
separated by thin layer chromatography and the relative amount of each
pigment was determined. Adequate discrimination was obtained for only 9
pigments. These results are shown in Table 17 and Figure 15. The garnet
alleles tested generally decrease the amount of all the pigments although the
various alleles have variable effects on the individual pigments. Quantitative
assessment of pigment levels was meaningful only for the drosopterin pigments
(see appendix 1) but visual assessment of pigment levels generally support
this conclusion. The g53d allele appears to be the most severely affected. No
novel pigments or intermediates are detected in any of the garnet mutations.
Ferré et al. (1983, 1986) examined pigments for 52 eye colour genes, the g
1
allele among them. The values they reported for the amount of these nine
121
Table 16. Pigment levels of various garnet alleles.
The first column shows the garnet allele and sex of the mutant assayed. The
next four columns give quantitative determination of the red and brown pigment
levels. The first three values are measurements of total pteridine levels by three
different methods. The last column is a measurement of the brown pigment
xanthommatin. Pterins (5 heads) refers to a measurement of pteridine levels by
the 5-head microflourimeter method. Pterins (1 head) is measurement by the
single head microflourimeter method and pterins (spec.) refers to measurement
by spectoflourimeter. Details of the experimental method are given in the
materials and methods. Comparison between the methods for red pigment
determination is given in appendix 1.
122
Pigment levels of garnet alleles
allele
pterins
(5 heads)
pterins
(1 head)
pterins
(spec.)
ommochromes
(xanthommatin)
1
g
57±2
72±4
4±1
9±1
9±1
15±3
25±3
66±1
g
2
2
g
32±2
37±2
2±1
2±1
3±.3
57±2
ND
15±1
45±1
50±3
17±2
21±2
18±3
56±18
70±8
52±5
g
4
4
g
25±1
38±3
7±1
10±1
10±1
12±3
28±5
37±3
g5Oe
g5Oe
24±2
33±3
1±1
3±1
3±4
35±4
ND
19±4
g53d
g53d
15±2
21±3
12±1
3±1
4±.5
19±4
13±2
7±4
gS3
gS3
43±4
44±3
23±3
26±1
26±3
29±7
41±5
31±3
e(g)g2
cf
2
e(g)g
29±2
32±3
ND
1±1
ND
34±3
15±9
4±3
e(g)g50e 12±1
OecJ 15±1
5
e(g)g
1±1
1±1
2±.3
17±2
13±2
4±2
e(g)g53° 10±1
dcJ 12±1
53
e(g)g
2±1
2±1
3±.3
12±2
6±2
4±2
3
g
cca
123
S6-1
S6-1
96±2
97±6
47±3
75±5
43±5
82±18
75±5
77±6
gP[P]
50±5
3±1
10±3
ND*
gP-Rev1c
93±4
99±4
60±6
101±11
gPReV2
93±4
78±5
86 ±22
ND
gP-Rev3cJ
87±6
ND
88 ±20
78±9
gPReV4
89±5
72±7
72 ±20
98±6
2
gPO
3±1
1±1
2±.5
9±1
gPdcc?
60±3
44±3
54± 10
31 ±2
’
2
gPX
60±2
48±3
49±10
62±4
c?
124
Figure 15. Chromatographic analysis of pteridine pigments of garnet alleles.
Figure 15 shows some of the separated pteridine pigments. The various garnet
mutants from which the pigments were isolated are shown below the
chromatograms. The names of the pigments are shown on the left of the figure.
In addition to the seven pigments identified, there were three unidentified
pigments. Two of these migrate slowly in the solvent and fluoresce blue in ultra
violet light, the third has a greater mobility and fluoresces yellow. These
products might also be degradation products of the pteridine pigments
generated during isolation and chromatography.
125
isoxanthopterin
xanthopterin
residue
drosopterins
unknown blue spot 1
unknown blue spot 2
N)
0
-
unknown yellow spotM
sep iapte ri r
hydroxypteridiflel
2-amino-4-
b lo pte r
CD CD
CI)-
CD
ci
40
________________________________
cJ-1
isosepiapterin.E
jJ
CA)
D
(
—
-+oq
J
—
CD
)C)
c
-
CD
-+0
40
4Q01-+Q(71
CA)
0
CD
-
CD
0 CD
CD
I)
CD
‘—
CD
CD
F’3Q1
Q Q
‘(D
CD
CD
CDCD
—
CD
CD
CD
0-
)
CD
-
I
Q Ci)
I
I
I
-+0
(C)
(
CD
CD
I
CD CD
DDtJ
I
I
I
CD
Ci)
DC))
I
(C)
I... ...•.
____________________________________________________________________
Table 17. Quantitative assessment of pteridine pigments after
chromatographic separation of pigments.
The first column indicates the genotype of various garnet mutants. The
remaining columns show the percent of wild type levels of the indicated
pteridine pigment.
127
128
g5Oe
g5Oe
g53d
g53d
gS3
gS3f
gP,’
S6-1
S6-1
gP-R1
gP-R3
gP-R4
gP-O2
1
gP-dc
gP-X2f
g3
g3cf
g4
g4f
allele
g1
g1f
5
24
16
26
3
13
0
0
2
0
26
18
18
17
106
200
53
112
0
62
71
8
18
46
33
8
8
0
1
3
1
53
52
9
54
108
92
117
97
1
76
77
0
0
0
0
0
0
14
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
167
0
0
0
0
0
0
0
0
0
0
0
0
0
50
0
0
20
0
0
0
43
0
14
0
0
0
0
20
20
40
0
40
0
0
20
0
0
20
0
0
0
129
0
71
0
0
0
0
0
20
60
0
20
0
0
60
0
0
0
0
0
0
267
133
100
33
0
0
0
50
50
50
0
50
0
0
0
Effect of garnet alleles on pteridine pigments
pigment residue droso- mystery mystery isoxanth yellow xantho- sepiapterin spot 1
spot 2
o-pterin spot
pterin pterin
33
0
0
0
0
0
167
0
37
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
0
0
0
0
0
0
0
0
0
0
0
0
15
23
54
15
0
15
86
57
121
58
8
23
0
73
64
27
38
36
36
145
18
2-NH4- biopterin isosepia
pterin
4-OHpteridine
pigments generally agree with the results here, although their method may
provide for greater accuracy.
Malpighian tubule phenotype of garnet alleles.
There are four pigmented structures in the adult fly body, the eye, ommatidium,
the malpighian tubules and the testes sheath. Beadle (1937a, 1937b) has
described the colour of g
2 malpighian tubules. Breme and Demerec (1942)
examined the malpighian tubule colour of 25 different eye colour mutations.
Their survey included four garnet alleles, g’, g
, g
2
3 and g
. Table 18 shows
4
the results of a similar survey extended to include an additional 13 garnet
alleles. The effect of the two garnet alleles surveyed by Breme and Demerec
(1942) on pigmentation agree with my estimate of pigmentation, however, the
results from the control mutations used to compare colour suggest that they
were somewhat more discriminating than I in detecting very low levels of
pigmentation. All of the garnet mutations reduce pigment deposition in
malpighian tubules and, in general, the reduction in colour is more extreme
than observed in the eye. It is not possible to distinguish the colour of the
malpighian tubules of most of the more extreme alleles, as they all appear
essentially colourless even though their phenotypes in the eye are readily
distinguishable. In contrast, the subliminal allele gX, which is associated with an
inversion, shows a weak variegated pigment phenotype in the malpighian
tubules although the eye phenotype is visually indistinguishable from wild type
(data not shown). It is also interesting that the gP revertants (discussed below)
do not appear to be complete revertants based on malpighian tubule
phenotype.
129
Table 18. Effect of various garnet alleles on colour of malpighian tubules.
The first column shows the garnet allele. The second and third give the colour of
the malpighian tubules as determined visually or where reported in the
literature (Breme and Demerec 1942).
130
Survey of malpighian tubule colour of garnet alleles
mutant
colour
reported colour
CS
orange
bright yellow
car
very pale/clear
pale yellow
cm
colourless
very pale yellow
w
colourless
colourless
1
g
colourless
very pale yellow
2
g
very pale/clear
very pale yellow
3
g
colourless
very pale/clear
e
g
O
5
very pale/clear
g53d
colourless
1
g
6
very pale/clear
71
g
2
yellow
gEMS
pale yellow
gS3
colourless
gX
orange/variegated?
2
e(g)g
colourless
e
g
O
e(g) 5
colourless
d
3
e(g) g5
colourless
gP
pale yellow
131
gPReV1
yellow
gPReV2
yellow/orange
gPReV3
yellow
gPReV4
yellow
S6-1
orange
O.R
orange
132
Table 19. Effect of various garnet alleles on testes sheath colour.
The first column shows the garnet alleles. The second indicates the colour of
the testes sheath immediately after dissection.
133
Survey of testes sheath colour of garnet alleles
mutant
colour
CS
bright yellow
1
g
pale yellow
2
g
pale yellow
3
g
yellow
4
g
yellow
Oe
5
g
very pale yellow
g53d
bright yellow
61
g
very pale yellow
271
g
bright yellow
gEMS
pale yellow
gS3
pale yellow
gX
bright yellow
e(g) g
2
pale yellow
Qe
5
e(g) g
very pale yellow
e(g) g53d
yellow
gP
yellow
gPReV1
bright yellow
gPReV2
bright yellow
gPReV3
bright yellow
gPReV4
bright yellow
S6-1
bright yellow
OR
bright yellow
134
Testes sheath phenotype of garnet alleles.
In general, all the mutants decreased the pigmentation of the testes sheath
(Table 19). The effect of the different alleles on testes sheath colour was
generally more severe than the effect on the eye pigmentation but less severe
d
5
3 allele provides the one
than the effect on malpighian tubule colour. The g
exception. Three different strains carrying the g53d mutation all showed
essentially wild-type levels of testes sheath pigmentation. This suggests a
tissue specific pattern of expression in this allele.
The search for a garnet null allele.
g5 allele confers the least pigmentation of all the
d
As observed above, the 3
d
5
3 or any other allele might
garnet alleles. In order to determine whether the g
be a null allele of the garnet gene, I quantitated the effect on eye pigmentation
for different garnet alleles when heterozygous with a deficiency. Table 20
shows the results of red pigment levels of 10 garnet alleles when combined with
a deficiency that includes the garnet locus. The phenotype of the g53d allele is
the most severe. Comparison between the pigment level of a homozygous g53d
strain (Table 16) and the hemizygous g53d condition (Table 20) shows no
pronounced difference. Thus, by the criterion of Muller (1936), the g53d allele
behaves as an amorphic allele with regard to eye pigmentation (this conclusion
3d allele
5
is also supported by Northern analysis of the g
-
Figure 22).
Comparison of the pigment levels of other garnet alleles as homozygotes or
hemizygotes suggest that these alleles are all hypomorphic lesions.
135
Table 20. The phenotype of various garnet alleles in combination with a
deficiency.
The first column shows the garnet allele. The next two columns show the
pteridine pigment values of the appropriate hemizygous garnet allele, as
determined by microflourimetric assay (5 heads) and by spectrophotimetric
assay respectively.
CROSS:
P
Df(1)HA97/FM7 ® g*/jFF
“
Fl
Df(1)HA97/g*
progeny assayed.
where g* indicates the given allele of garnet.
136
Pigment levels of various garnet alleles in combination with a deficiency
allele
percent wild type red pigment
microflourimeter
spectrophotometer
1
g
40±3
8±4
2
g
33±3
4±1
3
g
40±3
9±3
4
g
32±4
6±1
Oe
5
g
28±2
5±2
d
3
g5
16±3
3±1
61
g
34±3
7±3
gP
38±4
8±3
2
gS
34±4
8±1
3
gS
38±3
10±2
137
Aging and garnet.
Shephard et a!. (1989) have reported that a null mutation for the rosy gene has
a decreased adulte life span. Furthermore, Hilliker et al. (1992) have reported
that mutants of the rosy and maroon-like genes show hypersensitivity to oxygen
stress in addition to a decreased life span and Humphreys, Duyf, Hilliker and
Phillips have isolated an allele of the pink gene, another member of the
transport group of eye colour mutations, in a screen for mutations that are
hypersensitive to the free radical generating chemical paraquat (J. Humphreys,
personal communication). Since, longevity should be a sensitive assay for
biochemically uncharacterized lesions that effect cell viability, I tested the
longevity of three of the most severe garnet alleles, with and without the e(g)
mutation, at two temperatures. Figure 16 A and B shows the longevity (%
survival) of wild type and different garnet genotypes at 22° and 29°. Some of
the garnet mutant strains do die earlier than wild type. But reduced vigor is not
particularly surprising in a highly inbred population of flies. More significantly,
the relative longevity of the various garnet mutant alleles differs at 22° and 29°
and the presence of the e(g) mutation, which profoundly reduces the level of
pigmentation, has no appreciable effect on the time of death or shape of the
death curve. Finally flies mutant for the garnet gene lived longer than the wild
d
5
3 outlived
type strains in some instances (at 29° g53d and at 22°, all but g
the wild type flies of the appropriate sex). Thus there is no evidence that garnet
affects longevity.
138
Figure 16. Adult life span of wild type and various garnet mutants.
A. Longevity of wild type (Canton S) and g2, g5Oe and g53d females and males
is shown at 22°
.
Minimum population size is 90 for each genotype.
B. Longevity of wild type (Canton S) and g2, e(g) g2, g5Oe e(g) g5Oe, g53d and
e(g) g53d females and males is shown for at 29°. Minimum population size is
30 for each genotype.
The key for the different genotypes is shown on the graph.
The 29° aging experiment and the first portion of the 22° experiment was done
by Layne Harvey as part of a directed studies project.
139
C
I
0.
a)
C
G)
U)
C
>
1
day
Lifespan of garnet mutants
22 °
--
-H
g53d male
—6-
g2 male
g2female
g5Oe male
g5oe female
g53d female
CS male
CS female
-e
—.-
C)
0
>
29°
day
Lifespan of garnet mutants
CS fern.
g5Oem
g5Oef
g53d m
g53d f.
e(g) g53d f
—.*— e(g)g2m
—+-- e(g)g2f
—+— e(g) g5Oe m
—+-- e(g) g5Oe f
—0— e(g)g53dm
—v—
—- g2m
—K— g2f
—rne-—
—a-—e—
-R- CSrn.
—•—
Cloning of the garnet gene:
Isolation of unstable garnet mutants from dysgenic crosses.
The original gP allele was isolated from crosses involving a natural P-element
bearing strain, S6-1 (Wennberg 1988). This strain has two P-elements in
section 12 of the polytene chromosome, the cytological location of the garnet
gene (Wennberg 1988). Weak garnet mutations arise frequently in this strain
(Figure 17). The relatively high frequency of garnet mutations arising from this
strain is likely due to the proximity of P-elements to the garnet gene (Towers et
a!. 1993). These weak P-element induced mutations, as well as the original gP
allele, remain active and will further mutate to a variety of garnet phenotypes
upon out crossing to non-P bearing strains. Figure 18 shows the frequency
with which new, secondary and tertiary garnet mutations occur. The secondary
garnet mutations are also subject to further mutation upon outcrossing to non-P
bearing strains, although at a much reduced rate. Figure 18B summarizes
extensive lineage and out crossing experiments with these strains. Only the
original gP mutation, four wild-type and four secondary mutations which were
derived from the original gP allele, have been analyzed at the molecular level.
Cloning of the garnet gene:
Cloning of the garnet gene was facilitated by the original gP mutation isolated
by R. Wennberg (Wennberg 1988). The garnet gene was cloned by Dr. D.
Sinclair. Details of the cloning are provided in Appendix 2. Briefly, P-element
containing clones were isolated from a size fractionated library made from the
gP mutation. One such clone hybridized to position 1 2C, the cytological position
142
Figure 17. Diagram of the mutation rate to garnet upon outcrossing the S6-1
strain.
A. The top part of this figure shows the lineage of garnet mutants generated by
outcrossing the P-element containing strain S6-1. The number and phenotypes
of the progeny are shown for five generation of outcrossing. The phenotypes
are given relative to the garnet phenotype, g indicates wild type eye colour, gP
indicates a weak garnet mutation similar to the original gP allele and gM or
gMOd indicates a moderate garnet eye phenotype.
B. The rate of conversion and interconversion to the various garnet phenotypes
is summarized.
143
Generation of garnet mutants in the S6-1 background
24 “+“
18 “+“
sterile
A.
terile
/ terile
_..-31
•‘+
44
A(437
••
31
/
(3.1—aP(
(3.219?)
__43
I / /\.(4a94
1/42
I, /
“‘
/
4
, /
p./
all
Fl
-i-’
V..V24
+“
‘Zç38
“+“
—
“÷.
P
9
\‘1J2S
S...
\ \
‘,
\
\
\
\
all—30g+
(3.2.2+,4l_409+
(gP)
ll.-40 gP
(gP)
afl.-4OgP
all.-4OgP
‘all—4OgP
/,‘all.-40 gP
.p sterile
g+,1 gModl-4OgP
2÷Y3lgP
terile
.afl-4O gP
66 gP
(g.Ste
p
9
112g÷
gMod/8i
Og
allgP
69 gP
‘i_’all_gMod
1 g÷
sterile \\all..gMod
aIl.-gMod
all—gMod
_Øi.-lgMod lg÷/—4OgP
Øll.-40 gP
Z*sterile
0 +/—4OgP
allgP
÷
-i-
-
3 gP
.+
gP
‘
30 ÷
40 +‘•
39 ÷
33+.
37
(437
44 +,
48
6
p
.p24gP
S6-1 C(1)DX [MI
stock
•4(
(4
gP
g÷/—40 gP
g+/.40 gP
+“
26 +
26 ‘+
33
22
c 45+
sterile
÷“
49 .,+
48+
33+
6+
gX
35
cl/
48÷
43+
sterite
4
26 ÷
\c sterile
—0—48 ÷
_p-84 +
42 +‘
50 “+
sterile
c sterile
—‘.sterile
(8.1=gP)
(4
B
Summary of garnet mutation rate in S6-1 background.
4P g+
0.6-2%
S6-1
F5
F4
g”P
gM
144
weak garnet mutation
gP
=
gM
= moderate garnet mutation
“+
garnet revertant
Figure 18. Diagram of the mutation rate and phenotypes of garnet mutations
derived from the original gP allele.
A. The top part of this figure shows the lineage of garnet mutants generated by
outcrossing the gP mutation. The number and phenotypes of the progeny are
shown for six generation of outcrossing. The phenotypes are again
operationally defined relative to the garnet phenotype, g indicates wild type
eye colour, gP indicates a weak garnet mutation similar to the original gP allele,
gM indicates a moderate garnet eye phenotype and gX indicates an extreme or
strong garnet phenotype.
B. The rate of conversion and interconversion to the various garnet phenotypes
is summarized.
145
2gX/ sterile
Generation of new garnet mutants from the original gP allele
F3
Øall 43X
1 gXI io—P
2gXL_L...iE.
A.
F2
I
gP
43
p
9
32 gP
J
sterile
I27
,
I
/
I
som4
Fl
P
9
c(x
0
C(1)DX
[P1
(stock)
F4
M]
/ I
24gP ,1
69
P /
45 gP
49 gP /
54 gP
gP
P\
9
0
most
gP
g+
some gX
(discarded)
/
,
,
I
‘-
—
,/19X lgMod/ 21
3 gf’
.r2gX,—2OgP,--2Og+
2gX/40+gP
all 40-fgX
.all 40+gX
1 gL3
sterile
steril*terilalI 40+gP
40
-all
38gP
40÷g÷
c 2gMod/
34j4.g
sterile
10+9+! 32 gI-’
10+9+! _240+g+
40+aM
37 gP
—
‘4OgP’’ 3g+48g
33gf\\
9
‘
I’
all40+gX
_,all 40+
P
9
2g
36 gP
‘
M/
10+
“ ‘
sterile
,.-
steri e
all 40+gX
sterile
40+9+
all 40+gM
309
,io+gif
10+g+.10+gMI
stenre
1X1g+/40+gP
I 4o+gP
all
lgXI4
—20g+,-.2OgP
..20g+....2OgP
gp
‘:
steriL
,all 40+gM
X140+ciM
M
9
40+
all 4oi-gM
38 yP
all 40+gM
Il 40+gP
19X/40+9M
9ll4O+aP
\
36 gP
all 40+gM
p
9
lr4o+
9.2 (+
3g+/ 39 gIll 40+gP
all 40+gM
9.3 (+)
gX!40+çP ,1gXJ40+gP
59 gP
47
lg+,-.2OgX,-.2OgP
2aXI
35
g
B.
all 40+gP
40+gP
Summary of interconversion rates
33 g1 gX,2gP/40+g+1 g+!40+gP
of garnet mutants
45sterile
.-20g+,-2OgP
all 40+gP
3 gP 1 X3g+/40
1
g+
sterile
a? 4b+gP
20a+ -.2OgP
all 40’-i-gP
0 30/0
all 40+gP
gP = weak garnet allele
1%
\all4O+gP
3 2%
gP
gM
all 40+gP
gM= moderate garent allele
all 40+gP
sterile
gX = extreme garent allele
F2
!
‘
.
9+
gX
146
=
garnet revertant
_______
___
__
_
____
____
__
F3
‘
F5
F4
F6
•_
Jp
ral
I
40+g
X
all 43X
sterl
40+gM
i5ja.
2gX 1
all 40+9X
sterile
all 40+gX
grn3all
2g!,gM
terile
II 40+
P
0
27
igX 1g+/40+OP
X/40+OP
9
10 ,4
II 40+g
43
X
0
4
4o
P
9
sterile
II 40÷
—p- steri
le
2gX -.20 P -20 +
ig
—P
—i-
—
..
—
______
______
____
c
-b 40+q* ..40+(JX
sliall 40+gP
2gX1g40+gX
\38 gP
2gMod/ P
sterile
1
0-i-g
all 50+ gX
lgM/50+gX
all 50+ gX
/50 iX
9
all 50+ gX
all 50+ gX
all 50+ gX
c::: _zE
+
1140+9
40+i+
_
all 50+ gX
O+gX
+/
1O-i-g-i4
P
37 0
39 g
__
2:gP
36 g
a
terile all 40
gX,10g40+g
all 40+0 P
ic X/40+9P
0+g+/
-_..10irJ+ 0
10+
M
/
P
c-*.41 9
38 gP
1140
4AP
36
pP
39 g
P
9
UP
ll40
cSO
2Xl
P
0
40+
II 40-i-gP
X/40+c
0
X
140+9P
40+
M
all 9
X/40+g
M
0
all 40+
all 40-i-gM
1
140÷
X
0
gM2g+/40+gX
40÷ Ox
zi+gP
P
22 9
all 40+gP
X,2gP/40+ +
0
33 OP 1
lg+/40+
P
9
sterile
-.
P
9
i-,—
20
20
all 40+gP
4xall
I
gxg÷/
all
all 40-i-gP
all 40+9 P
all 9
40-iP
all 40+gP
sterile
__
all
all
all
all
50+
50+
50+
50+
Ox
gM
gM
M
9
all 50+ gP
all 50÷ 0P
2g+/50+gP
qX/40+yM
all 50+ çjX
40+rJMMul40
.Ig
I 40+rjM tVi40+gMM
cjP
+pP
2gP,9gX/50+OM
all 50+gX
all 50+gM
P
all 50+ 9
,ll 40-i gX
40+
-
1 gM/50+clX
all.,0÷gX
M/50+9X
0
all 50+ Ox
-
a 1l40+gP
all 50-i-gM
a
63
iOgP/50+g+
147
of the garnet gene. Unique sequence DNA flanking the P element was used to
isolate lambda clones containing inserts of wild type genomic DNA. These
inserts shared a 6.6 Eco RI fragment. This fragment was subcloned and used to
isolate c-DNA clones from an imaginal disc library.
Four lines of evidence indicate that this 6.6 Eco RI fragment identifies at least
part of the garnet gene: The spontaneous gP mutant has an insertion into this
fragment, the size of which is altered in revertants. The P-element in the gP
mutant is inserted into a region corresponding to a 3’ intron of a transcript from
this region. Nine, out of fourteen garnet mutants examined, have alterations,
detectable by Southern analysis, in this fragment. Finally, of the two garnet
mutants examined by Northern analysis both show alteration in the two
transcripts derived from this region. These data are discussed below.
Molecular analysis of the gP allele and its derivatives. The gP mutation is
associated with a P-element insertion into the garnet gene. The P-element that
is inserted in the gP allele is approximately 2Kb in length (Figure 19). Thus it
is not a complete P-factor. The four revertants of the gP allele all show
alterations in the size of the P element inserted as do the spontaneous garnet
P-extreme mutations (Figure 19). Neither the g-revertants nor the gP...
extreme alleles are associated with complete loss of the original P-element
insert. Thus while the revertants are wild type (based on a visual assessment of
eye colour) the P-element insert is not completely removed in any of them. This
incomplete molecular reversion is consistent with incomplete phenotypic
reversion as assessed by pigmentation of the malpighian tubules. Since the
2kb insert in the gP allele causes only a moderate reduction in the function of
148
Figure 19. Southern analysis of the gP allele and gP derivative mutations.
The top portion of this figure shows the results of Southern analysis of the gP
mutation and four phenotypically wild type revertants. DNA from wild type
(Canton S), the gP allele and four revertants was isolated, restricted with Eco RI,
separated on a 0.8% agarose gel, transferred to nylon membrane and probed
with the 6.6 Eco RI putative garnet fragment. The wild type DNA shows a band
at 6.6 kb as expected, as well as a bands at higher molecular weight likely due
to incomplete digestion. The size of the 6.6 kb band is increased to 8.2 kb in the
gP mutation. Similar analysis with DNA restricted with Bam HI and Hind III show
that this change in mobility is not due to polymorphism for an Eco RI site (data
not shown). That this change in mobility is caused by insertion of a P-element is
shown by hybridization of this band to P-element DNA (data not shown). The
size of the 6.6 kb Eco RI band is also altered in each of the gP revertants. In
every case the size of the insert in the gP allele is diminished but in no case is
the insert completely removed.
The two lower figures show the results of Southern analysis of more
spontaneous derivatives of the gP allele. In this case the membrane was
probed with the 4 kb imaginal disc c-DNA clone. The gP’X2, gPX3, gPX5 and
2 alleles are moderate or strong garnet alleles. The gPX2dC allele is a
gPO
subliminal garnet allele (males and homozygous females appear wild type but
females heterozygous with a strong garnet allele show a weak mutant
2 strain. With the
phenotype) which occurred spontaneously in the gPX
possible exception of the gPX2 allele each of these gP derivatives show
gPO alleles might
5 and 2
alterations in the size of the gP insert. The gPX
represent small deletions in the garnet gene due to imprecise excision of the P
element.
149
gP
Cs
gP—Rev2 gP-Rev3 gPRev4
gPRevl
82kb
66k*
Cs
gP
P-X2 gP-X3gP.X5 gP-dC gP-02
9
PX2-dc
8
65kb
—
150
65-
the garnet gene (based on the weak mutant phenotype of this allele) it is
possible that internal deletions of the P-element might relieve whatever
impediment this original insertion produced.
Sequence extending outwards from the termini of the P-element in the cloned
garnet region of the gP mutation identified the position of the insert as
corresponding to the most 3’ intron at position 3234 of the imaginal disc c-DNA
from the garnet region (see below).
Southern analysis of other garnet alleles.
Southern analysis was performed on wild type and fourteen garnet mutants to
determine the presence and nature of lesions in the 6.6 kb Eco RI putative
garnet fragment. Genomic DNA from wild type (C.S) and g
1 mutants was
digested with Eco RI, Barn HI and Hind Ill, size fractionated and probed with the
6.6kb Eco RI fragment (Figure 20) and the 4kb imaginal disc c-DNA (data not
shown). Southern blots of wild type DNA digested with Eco RI and probed with
the 6.6 kb Eco RI putative garnet fragment yield the expected 6.6kb fragment.
Barn HI digestion of wild type DNA gives two fragments one roughly 8kb and the
other approximately 10 kb. Only the smaller of these is detected by the c-DNA
probe (data not shown) indicating that the c-DNA is encoded exclusively by the
sequences to the left of the Barn HI site of the 6.6kb Eco RI fragment. Hind III
digestion of wild type DNA gives two fragments detected by the 6.6kb Eco RI
sequence as a probe, one of 4kb and one of 2.5 kb. Only this 2.5 kb fragment is
detected using the c-DNA as a probe (data not shown). Alterations in the size of
restriction fragments indicates that the g
1 mutation is due to an insertion of
approximately 2kb into this smaller 2.5kb Hind III fragment. Less detailed
151
Figure 20. Southern analysis of the wild type garnet region and the g’ allele
Wild type (Canton S) DNA from the garnet region was restricted with Eco RI,
Barn Hi and Hind lii and probed with a portion of the 6.6 Eco RI fragment.
Similar analysis was performed on the g
1 allele. In the g
1 allele mobility of a
band is diminished in each of the digests indicating the presence of an insertion
in this mutation. Of note, the mobility of the smaller Hind III fragment, which
corresponds to the 3’ end of the transcribed region, is altered in the g’ allele.
A schematic diagram of the restriction map of the garnet gene and segment
used as a probe is shown in below.
152
1
g
Cs
HE
B
H
E
B
.412
48
‘p
.47
.44
.43
1 (&gq
g
B
H
L1
RR\H
HAB
ii\Aii
5’
1 kb
probe
153
RH
I I
B
Figure 21. Southern analysis of garnet mutants.
53
g
The top portion of this figure shows Southern analysis of gl, g2, g3, g4, d,
Oe and 3
5
g
gS The lower portion of the figure shows Southern analysis of ,
.
61
g
gEMS, gim, gS2, gXand T(1;Y)B166. DNA from these flies was isolated,
restricted with Eco RI, separated on a 1% agarose gel, transferred to nylon
membrane and probed with the 6.6 Eco RI garnet fragment. The g
1 and g
3
alleles appear to have identical insertions into this fragment. The g
,,
4
61
g
gEMS and gim also have insertions into the 6.6 Eco RI fragment. The g
53 allele
53 g
g
Oe,
5
appear to have a small deletion in the 6.6 Eco RI fragment. The d,
2 and gX alleles show no change in the mobility of the 6.6 Eco RI fragment.
gS
The g
2 allele shows altered mobility of the Eco RI fragment. That this is due to a
restriction fragment length polymorphism of the 3’ Eco RI site, is shown by
restriction analysis of the g
2 allele with other restriction enzymes, such as Bam
HI, shown at the lower right. Finally, the translocation B 166, shown as a
g
O
e, is broken within the 6.6 Eco RI fragment.
heterozygote with 5
154
4
41
a)
Co
T
V
ci
Co
‘Co
V
14
I
—
C’,
Co
U,
0.
U’
Co
co—i’
p
analysis was pertormed to identify the nature of 12 other garnet alleles (Figure
21).
Oe, g53d, ,
5
Genomic DNA from g
,g
2
,g
3
,g
4
61 gEMS, gim, gS2, gS3 and
g
T(1;y)B 166 mutants was restricted with Eco RI, separated by electrophoresis,
Southern blotted and probed with the 6.6 kb Eco RI fragment. Of the 13 garnet
alleles examined in this way, six show insertions into this fragment, one has a
deletion, one a translocation break and five show no structural alterations
detectable by Southern analysis. Interestingly, Eco RI digest of the g’ and g
3
alleles generated apparently identically sized fragments. This implies that these
alleles possess identically sized insertions into the same region of the garnet
g
O
e and 3
g5 alleles which
d
gene sequence. It is also interesting that the g
,5
2
are the most extreme alleles and also those sensitive to the enhancer of garnet
mutation, showed no structural alterations in garnet. These data are
summarized in Table 21.
Analysis of transcripts arising from the 6.6kb Eco RI putative garnet region.
This 6.6Kb. Eco RI fragment was used as a probe to investigate the size and
abundance of messages transcribed from this region. Figure 22 shows
messages detected by this probe from wild type (Oregon R) embryos and g
3
and g53d newly eclosed adults. Two messages are detected with this probe in
wild type embryos, one approximately 3.5 and the other 4 kb in length. Both of
these messages are absent from adults of the severe allele, g53d, and only
one, possibly slightly larger than the 4kb transcript is present, at reduced levels
in the g
3 adults. The absence of both messages from the g53d allele suggests
that both messages are derived from the garnet gene and that no other m-RNAs
are transcribed from this region in embryos or adults.
156
Table 21. Summary of lesions in the garnet locus in different garnet alleles.
The first column indicates the garnet allele. The second and third columns
show, respectively, the type of lesion and where relevant, the approximate size.
(NA
=
not applicable.)
157
Summary of lesions at the garnet locus in different garnet alleles
allele
type of lesion
size
1
g
insertion
4kb
2
g
point mutation?
NA
3
g
insertion
4kb
insertion
1 kb
Oe
5
g
point mutation?
NA
g53d
point mutation?
NA
61
g
insertion
3kb
gEMS
insertion
2kb
gim
insertion
1 kb
gP
insertion
2kb
gPReV1
insertion
.5kb
gPReV2
insertion
1.5kb
gPReV3
insertion
1 kb
gPReV4
insertion
1.5kb
gS2
point mutation?
NA
gS3
deletion
0.5kb
gX
point mutation?/position effect?
T(1;Y)B 166 translocation breakpoint in garnet
158
Figure 22. Northern analysis of wild type embryos and garnet mutant adults.
g5 individuals and wild type (Oregon R)
d
RNA from adult (0-3 days) g
3 and 3
embryos was isolated, electrophoretically separated, transferred to nylon
membrane and probed with the garnet imaginal disc c-DNA. There are two
messages in wild type embryos. One message, perhaps slightly greater than
the wild type 4 kb message is seen in g
3 adults. No messages are seen in g53d
adults. The lower figure shows the same four lanes probed with the RP 49 gene
as a control for equal loading and RNA integrity.
e=em bryo
a=adult
159
I
A
D
A
0•
cD
(DIP
0.
Co
In summary, the presence of a P-element insert in the 6.6 Eco RI fragment in the
gP mutation, which is altered in garnet-P revertants, the number of garnet
mutants with alterations occurring in this same fragment and the disruption of
the transcripts from this region in garnet mutants all support the contention that
this fragment encodes at least part of the garnet gene.
Molecular analysis of the garnet gene.
Restriction map of garnet and the genomic region encompassing garnet.
The unique sequence DNA flanking the insertion site of the P-element of the gP
mutant was used to isolate lambda phage containing inserts of the homologous
genomic regions of wild type Drosophila melanogaster. Five different lambda
phage were isolated and subjected to restriction analysis using Barn HI, EcoRl,
Hind Ill and Sal I to generate a crude map of the region surrounding the garnet
gene (Figure 23). A summary of these restriction maps is shown in Figure
24. The map includes approximately 30 kb 5’ and 15 kb 3’ to the garnet gene.
The phage all held a 6.6 Kb Eco RI fragment in common. This fragment was
subcloned and subjected to more detailed restriction and sequence analysis.
Detailed restriction map of the garnet region:
A restriction map of the 6.6Kb Eco RI fragment using the enzymes, Eco RI, Barn
HI, Sal I, Hind III and Kpn I is shown in Figure 24. Below this figure is shown a
restriction map of the imaginal disc c-DNA clone isolated using this 6.6Kb Eco
RI fragment as a probe. The restriction analysis of the c-DNA also include the
restriction enzymes Sst I, Pst I, Sph I, and CIa I.
161
Figure 23. Restriction fragment analysis of lambda phage clones containing
the garnet gene and flanking sequences.
Restriction analysis of five lambda phages which were isolated from a genomic
library screened with single copy sequences adjacent to the P-element
responsible for the gP mutation. The five lambda clones, g212, g?13, g?14,
g22O and gA21 were digested with the restriction enzymes, Barn HI, Eco RI,
Hind Ill and Sal I, single and in pairwise combinations. The DNA fragments
were electrophoretically separated, transferred to nylon membrane and probed
with the same g?. DNA. The information derived from these restriction digests as
well as from probing these blots with portions of the garnet gene (data not
shown) was used to generate the restriction map shown in Figure 24.
162
41
9
0
I
r%3
(0
•4
48
I
I
*
•
I.
I
I
o1
*1
•11
.,
•
II
•
.
(0
I
• •fl
*
I
S
—
•
,.J•
I jISf
..
Figure 24. Restriction map of garnet and surrounding region.
A. The top of this figure shows the extent of the five lambda phage clones which
encompasses the garnet region. Below is a crude restriction map of this region.
The 6.6 Eco RI fragment which includes the garnet transcript is indicated by a
heavier bar.
B. The 6.6 Eco RI fragment which identifies the garnet gene is shown in
greater detail. The site of the P-element insertion in the gP mutant, as well as
the location of three introns, the site of the putative hydrophobic domains and of
the polyglutamine repeats is shown. The arrow below indicates the location and
direction of transcription for the 4 kb c-DNA clone isolated from the imaginal
disc library as well as additional restriction enzyme sites. The sequence of the
most 3’ intron into which the P-element is inserted in the gP allele is
TATGCCGCGATNTTTGNANNATCGAAGAGTATG*TTCCAG where the
indicates the insertion point of the P-element.
164
Restriction map of garnet and surrounding region
A.
gM4
g2O
gM3
gx21
—
B
RB
II
—
gx12
—
BRH HBBR
I
III
9-SRRH
liii II
II U
BSHSR Ffl-IS1BB RHBHSRBHRSRHBSR BBR
11111 III 11111 I I liii liii liii I III
5kb
R/
/
/
/
/
/
/
/
/
/
/
/
/
/
/
//
/
/
/
\
\
\
\
\
HB
I I
I
BHO
R
R
Sst SsçxbaH S Sst
ClaCla CIa
I
I
II I I II
III
CIa=Cial
H=HindIIl
Sst=SstI
B=BamHI
Ssp=Sspl
S=SaIl
Xba=XbaI
R
I
•
I
R=EcoRI
\
\
B
ELiBIII
I
=
5’
\
\
HS
Inn
/
/
/
R
3’
H
hydrophobic domains
introns
polyglutamine region
1kb
165
Sequence analysis of the garnet gene. The DNA sequence was determined for
a 1.2 kb DNA fragment isolated from an embryonic library, a 4kb imaginal disc
c-DNA, and part of the 6.6Kb EcoRl genomic fragment which encompasses the
bulk of the garnet gene. The sequencing strategy used to sequence the
imaginal c-DNA and the equivalent section of genomic DNA is presented in
Figure 25.
Sequence analysis of the approximately 1.2 kb DNA fragment derived from the
embryonic c-DNA library showed that, relative to the imaginal disc c-DNA
sequence, the 3’ end of this DNA segment is in the intron at position 3234 and
the 5’ end, marked by a poly C tail, occurs 3’ to the disc c-DNA, presumably in
the genomic region flanking the coding region (data not shown). As this DNA
fragment encompasses both intronic and genomic sequences, lacks a
substantial open reading frame, and is in the opposite orientation to the
imaginal disc-derived c-DNA, it seems likely that this fragment is a genomic
contaminant in the c-DNA library. This explanation is consistent with the
absence of a 1kb message in embryos (Figure 22).
The sequence of the 4kb c-DNA isolated from a 3rd instar imaginal disc library
is shown in Figure 26. The sequence is 3825 bp in length. There is an ATG at
position 297 which could initiate a potential polypeptide of 1054 amino acids.
No poly A tail is present in this c-DNA clone however the size indicates that it
might nevertheless be complete.
Comparison between the sequence of the genomic and imaginal c-DNA
revealed that the c-DNA extends 221 bp 5’ to the 6.6 Eco RI genomic fragment.
Thus the genomic fragment does not contain all of the imaginal c-DNA.
166
Figure 25. Strategy used to sequence the imaginal c-DNA clone of the garnet
gene.
This figure diagrams the segments of the imaginal c-DNA clone that were
sequenced. The thin scale bars represent the size, in base pairs, of the imaginal
c-DNA sequence. The underlying bars represent independent sequence
determination where the location of the bars indicates which section was
sequenced, the arrow indicates the strand sequenced and the thickness of the
bar represents the number of independent sequence determinations of this
segment. The solid bars indicate c-DNA sequence, the stippled bars indicate
sequences from the corresponding genomic region. Introns are shown as
triangles above the scale bar, in the appropriate location.
167
Sequencing stratagy for the
I
0
III
liii
I ‘‘‘I
400
II
‘‘I
I 111111111 I liii
100
200
IJ
III
liii
500
1111111111
1200
I
I
I
900
800
garnet gene
III
1300
III
I
II
I I
600
I
I
-
I
I
I
I
II
IF 1
I
-
I 111111111 I
300
joo
I
700
11111
I
1000
11111
I liii
1400
liii
I
-
11J
[
800
1111111111
1100
1200
4
I
liii
1500
II
I
—
I
I
I
1600
-
*
1IIIIIIIIII 1111111111 IIIIIIIII•I 111111111 I
1700
1800
1900
1600
2000
1111111111
2000
1111111111
2400
r
ri
3200
I till
3600
I
III
II
I 11111
2200
11111
111111111
2300
I
2400
IIøI
E;.*
2800
III
2100
i
III
2500
2900
iii
i
i
II
I till
3700
1111111
600
I
i
liii
3000
I
III
I—I
II
111111111
34n0
np
-
ill
11111
I
‘‘‘
3800
I
I
I
III
—
—
168
‘
I liii
2700
I III
3100
I
I
::spo
I
3900
—
III
I
I
I
11111
III
11111
—
genomic
c-DNA
—
lx
2X
3X
I
2800
I
3200
I
600
Figure 26. Sequence and conceptual translation of the imaginal disc c-DNA
clone of the garnet gene.
The sequence of the imaginal c-DNA clone from the garnet gene is shown. The
sequence is 3825 bp long. Below the sequence is shown the conceptual
translation of the imaginal c-DNA clone. A long open reading frame starts at an
ATG at position 297 which could encode a polypeptide of 1054 amino acids.
The direction of transcription was determined by tissue in situ hybridization with
RNA probes (shown in Figure 27). This putative polypeptide has three
indifferent hydrophobic domains and a stretch of polyglutamine residues. A
schematic summary of the sequence motifs found in this putative polypeptide is
shown in Figure 24. The genebank accession number for this sequence is
U31 351.
169
garnet Sequence
10
20
30
40
50
1234567890 1234567890 1234567890 1234567890 1234567890
AAATTCCGCG ACCCCGTCCT CTCGGGAATC CGTCATAGCT GAGTATTCAC
50
CGGAAAACCC GAAATACGCT CAAGTCACCG GCTTTAAATC AATCATCCGG
100
TTTAATGTAG CTGAACGTTT TGCCTAACGC TAATGACAAA GAATCGCAAG
150
CACCAAGCAC CAAAAAAAAG TTAATGATTG GACTCGTGAT TAATATGAGT
200
GTTCTAGGTA TTCTTCCGGA ATTCGTACTG TAAGGAGATT TATAAAAATA
250
CAATTAGCCT TTTTTAATTC TGCGCCAGCT TTCAGCTCGT TGATTACATG
M
300
TCCAATTCAA TGTCCAGGCT CGATGCGGAT CATTTGGATC GTATTTGCCA
SNSM
SRL
DAD
HLDR
ICH
350
TCCTTGTCCT CGCTGTCCGA CATGGAGACA CTTAGGGCAT GTCCAGCGTG
PCP
RCPT
WRH
LGH
VQRG
400
GTGTTCACAA TGTGCAATGG TTTCGGTTCA CCTTCCGATT CGGAGCGCTG
VHN
VQW
FRFT
FRF
GAL
450
TTGTATGCCA CTTTATTTTT GGACCTTTTT GCTCTTTTTA CCCTTTTTAT
LYAT
L FL
OLE
AL FT
LFM
500
GCTTCTTCTT GCCATCCTTG CTGCCCTGCG CCGCCTGCTG TTCCTGCAGG
L L L
A I L A
ALR
RLL
FLQV
550
TACTTATCCG ACCGTTTGGT GATGCCCACG GCCAGAGCGG CCACACCCTC
LIR
PFG
DAHG
QSG
HTL
600
170
garnet Sequence
10
20
30
40
50
1234567890 1234567890 1234567890 1234567890 1234567890
CATGTCCAGT GGCAGCTCCG TAATGGGTAT GTCATCAATA TTGTCATACT
HVQW
QLR
NGY
VINI
VIL
650
GATCCGCATT CGAGGCACCA CGTCCGTGGG GCGTGGACTT GAGGTAGTGA
IRI
RGTT
SVG
RGL
EVVR
700
GGATTGTTCG ACTGCTCAAT GAGACGAGCC ATCTGGCCGC TCCAGTTGCT
IVR
LLN
ETSH
LAA
PVA
750
CGGGGCGTGA GCTCCCAGAT GCTGCGCAAC CAGCTGTCCG CGTCGACCGA
RGVS
SQM
LRN
QLSA
STD
800
TGCCATGGCC ATGGACACGA CGACGGAGGG CGGCATTCCG GTGGCCATTG
AMA
MDTT
TEG
GIP
VAlE
850
AGATTGTCCA GGAGATGACG CTGCTGTTCA CAGGCGAGCT AATACCGGTG
IVQ
EMT
LLFT
GEL
IPV
900
GCGCCCAAGG AACCGGCATG CCCCTGCCAG ATGGTCTCGA TCTGGA(i IF
APKE
PAC
PCQ
MVSI
WTL
950
GAGTGGATTA ATGCACCGCC ACCGGAAGAT GCGGCAACAG AGTTCATCCT
SGL
MHRH
RKM
RQQ
SSSS
1000
CGGAGCACGA CAAGGACGCA GCTATTCGGT TAGTGCCACC CGAGGCAGGA
EHD
KDA
AIRL
VPP
EAG
1050
ACGGGGAGCT GCATAGCGCG AAATTACCGC CCGTTTGGTG ATGCCCACCC
TGSC
IAR
NYR
PFGD
AHP
1100
GCAGACGGCN CACACCCTCC ATGTCCAGTG GCAGCTCCGT AATGGGTATT
QTA
HTLH
VQW
QLR
NGYF
1150
TCGATCAATA TTGTCTACTT ACTGATCCGC ATTTCGAGGC ACCACGTCGC
DQY
CLL
TDPH
FEA
PRR
1200
171
garnet Sequence
10
20
30
40
50
1234567890 1234567890 12467R9 1234SE7RqO 1234567890
CGTG GGCTGC GTACTTTGAA GGTAGTGAGG ATTGTTCGAC TGCTCAATGA
RGLR
TLK
VVR
IVRL
LNE
1250
GACGAGCCAT CGCCGCTCCA GTTGCTCGGG CGTGAGCTCG AGACTCTGTC
TSH
RRSS
CSG
VSS
RLCR
1300
GCNCTTTTCG CCGCCATCAG CTCCCGTTCC TGGCCTTTCG TGGCACTAAA
XFR
RHQ
LPFL
AFR
GTK
1350
CGAATAGCTC GTCCTTGGTC GTGCTCCGAG GATCAACTGC TTGCCGCATC
RIAR
PWS
CSE
DQLL
AAS
1400
TTCCGGTGGC GGTGCATTAA TCCACTCGTC CAGATCCGAG ACCATCTGGA
SGG
GALl
HSS
RSE
TIWT
1450
CGGGGCACCT TCCGTTGCCT TGGGGCGCCA CCGGTATTAG CTCGCCTGTG
GHL
PLP
WGAT
GIS
SPy
1500
AACAGCAGCG TCATCTCCTG GACAATCTCA ATGCGCACGC GGAATGCCGC
NSSV
ISW
TIS
MRTR
NAA
1550
CCTCCGTCGT CGTGTCCATG GCCATGGCAT CGGTCGACGT GGACAGCTGG
LRR
RVHG
HGI
GRR
GQLV
1600
TTGCGCAGCA TCTCGATCAG CATGCAAGCG GAATTGGCTC GCTCTTGCAC
AQH
LDQ
HASG
IGS
LLH
1650
CTCAATGTCG CTGGAACCAT TGAAGTGCTG AAGCTTGTCC AGCACATGGT
LNVA
GTI
EVL
KLVQ
HMV
1700
CGCAGAGCTA CCAATGATAA TGGTAATACT CAGAATACAA TCATTTGAAA
AEL
PMIM
VIL
RIQ
SFEN
1750
ATACGTATAG AGAGAGTCAC TTACTGTCAC CAAGCCAGGC AGATCCTGAA
TYR
ESH
LLSP
SQA
DPE
1800
172
garnet Sequence
10
20
30
40
50
1234567890 1234567890 1234567890 123457R90 1234S7Rø
GCTCTAAACA CGTGGTGGCC AGGCGAGCGA ACAGCTTCAT CAGCTTCTGC
ALNT
WWP
GER
TASS
ASA
1850
ACATAGACAC CTGGATGTGA CCGGGCCAGT AGCTTCGGAC GAGCAGATGT
HRH
LDVT
GPV
ASD
EQML
1900
TAAGCGTCTC TCGCATCCTC CAGTTCGCCA GCAAACTCGC CAACGATCCA
SVS
RIL
QFAS
KLA
NDP
1950
GGCGGCGGCG TAGAGCACCT CGTACATGGA ATTACTTTGC GCCGAAACGG
GGGV
EHL
VHG
ITLR
RNG
2000
TGAACGTGTC AGCATGATTG GTCATCTCGT TGACGGCAAA TTGCCGGACC
ERV
SMIG
HLV
DGK
LPDH
2050
ACAGGCACTG CAATGGCCNT NGTTNTCGCT ATAGGCGCCG TGCGTGGTNG
RHC
NGX
XXRY
RRR
AWX
2100
AGCTCGACGA CGACAGTTAG ATACCACTCG AAGTTGGTCA CATACAGATA
SSTT
TVR
YHS
KLVT
YRY
2150
CGAACTCTGC GCGCAATATC TCGATCACCT TGTAAAGCCA ATTCGTCCCG
ELC
AQYL
DHL
VKP
IRPD
2200
ATAGGCCGAA CCCTCTGACC CGCTGCCATG TGGCCCAGCA ATCGCTTAAC
RPN
PLT
RCHV
AQQ
SLN
2250
AATCTCCATA AGGTTCTTCT TCGAGACCAT GCCGTAGAGC AGGTCCAGGG
NLHK
VLL
RDH
AVEQ
VQG
2300
CGCGCAGACG TATCGATTCG TCCTTGTCGT CCAGGCAGGC GAGTATGAGA
AQT
YRFV
LVV
QAG
EYEI
2350
TCCTTGTGCG CCTGCACACT CTTCGGGTGC GTCTTCAGGA TTTTCGACAT
LVR
LHT
LRVR
LQD
FRH
2400
173
garnet Sequence
10
20
30
40
50
1234567890 1234567890 1234567890 1234567890 1234567890
GGCCAACAGT CCCAGATATT TCAAGTTCTG GTCCGCGTCC TCGATGAGGA
GQQS
QIF
QVL
VRVL
DED
2450
TGCGCAGCTT CTGCACGCAG AGCTGAATGG AGGCACTGTG GTTGGGCATG
AQL
LHAE
LNG
GTV
VGHA
2500
CCGCTGCTAA TGCTGATAAC GACCGCGATG ACCGTGTTGA TGCACTCATA
AAN
ADN
DRDD
RVD
ALl
2550
CAGAGACTCA TGGCGAGTGC TGTGATAGTT GGGTTTGGTT TTTGGTTCGT
QRLM
ASA
VIV
GFGF
WFV
2600
I I iGi iii GT1 I
ICATTT TTTGTTGGTG TTCGGTTAGT TTTAGTAATT
VFIF
CWC
SVS
FSNF
2650
I I I I TTGTTT [Ti [TI F I TT GTGATTTGTA TTAGTTTGAG AAAAAAAAAA
2700
GTAGGAACAT GTATTAAGAG TAAGCAGCGG GTAAAACGCA CAACATTGCA
VGTC
IKS
KQR
VKRT
TLH
2750
CTACAACAAT AGCAATAGCA AATTAGGCAA TGGCAACAAC AACGGTAGCA
YNN
SNSK
LGN
GNN
NGSI
2800
TATCGATAGC ATACTATACT ATACATATAC TACTAGCTAC TTGCGGTACG
SIA
YYT
IHIL
LAT
CGT
2850
AAGGTAACAA TTAGCGATTA TTGCGATAGA CATTGGGGAA AGAGAACATT
KVTI
SOY
COR
HWGK
RTL
2900
GCAAAGCAAC GCAGCAATGG CAACCAAAAA GAAAAACGTC ACTAAACAAC
QSN
AAMA
TKK
KNV
TKQQ
2950
AGCAACAACA ACAACAACTG CAACGGCTCG TCATTTGTTT TTCTTTCGCT
QQQ QQL QRLV IC.F SFA
3000
FCF
FCF
FEE
VICI
174
SLR
KKK
garnet Sequence
50
30
40
20
10
1234567890 1234567890 1234567890 1234567890 1234567890
TCATCCGATT TGACTAGTTT AGAACTTTGG ATCTCAATGA GTGTGCTCGA
VLD
ELW
ISMS
TSL
SSDL
3050
TAAGCAAAAA ATCGATAGGC AAACGATGAA TTATAGAAAC AAAGACAAAC
YRN
KDKL
TMN
IDRQ
KQK
3100
TTAAGCAGTA TGGCGACAGT CATAAGTTGA GCGAGTGGGA GAGAGAAAGA
RER
EWE
HKLS
GDS
KQY
3150
GATAGACAGA GAGAGAGAGA GAGAGAGAGT ACGCTAGAGC TAGAGAATTG
TLEL
ENC
RES
ERE
DRQR
3200
TACAGTAAAT GATATAACGA ATATATCCAG TCACACGACA ATCATCGAGC
HTT
IIEQ
ISS
TVN
DITN
3250
AGCTTCAATT ATCGATCATT GATATCGACC TTTTAATCGG TCACTTTCGA
HFR
LIG
DIDL
LQL
511
3300
TTTGATTTTT CGAATTTTTT CTTTGCTTTC GCCTTGCTTT GTTGCAATCG
CNR
ALLC
FAF
NFF
FDFS
3350
TTTTCCACAC ATTCTTGGGA AATCGTATCG TATTTTACAT TTTCAGTTCA
ILH
FQFS
SYR
FPH
ILGK
3400
GTTCAGTTGA TTTGTATTTG TATTTTTGTT TTGTTTTGTT TTGTTTGTTT
CLF
VLF
YFCF
LYL
SVD
3450
GTTTTGCAAT GATTTTAAGA CTTGCCTATG AATTAGATTT GTGAGTGGTT
VLQ.
3500
CTATTAATTT CTTTCCTAGC CGGGGTTCTA AGGGGGTTAA AGCGCCAAAC
3550
TGCAAATGCA AAGAGAAAAA GAAACAAGCA AGAAATTATA AATTACATAC
3600
175
garnet Sequence
10
20
30
40
50
1234567890 1234567890 1234567890 1234567890 1234567890
AATCGAAATC ATCAACGTCC TTATCCACAA CTAAAACTAG AACTAAAACT
3650
AAAGCTAAAA CCGAAAACGA AACTAGAAAA TGAAGTGTTC AAGAAAATGG
3700
TAAACTGGAA CTGGAATACT CTAAGAAGTA ATTTAACTTT CTCTTAAACT
3750
GGTCCTGGTC CTTTTCCATT CGGATCGAAT CTCTTGATGA TATGTCTATA
3800
TATTTTTGTG TATCTCAGGG TGGAA
3825
176
Comparison between the c-DNA sequence and genomic sequence reveals at
least four introns, the most 3’ of which is the site of the P-element insert in the
gP mutation.
Conceptual translation of the garnet c-DNA. The direction of transcription was
determined by in situ hybridization of digoxigenin labeled RNA probes to
imaginal discs of third instar larvae. The garnet c-DNA from the Hind Ill site at
1720 and the Sst I site at 2143 was subcloned into pBS KS. Sense and
antisense RNA was produced by transcription of this garnet fragment by T3 and
T7 polymerase respectively. The T7 transcribed probe detected message
whereas the T3 transcribed probe did not. Thus the direction of transcription is
as shown in Figure 26.
There is a reasonable open reading frame in this orientation, starting at position
297 encoding a potential polypeptide of 1054 amino acids. Conceptual
translation of the long open reading frame yields the protein shown in Figure
26. This protein has a number of motifs. There is a poly glutamine stretch (8
repeats) at position 3145. There are three reasonable hydrophobic domains.
Comparison between this sequence and the EMBL and Swiss protein data
banks reveals no informative similarities (Table 22). The first two sequences
show essentially exact correspondence with the garnet gene sequence. The
slight discrepancies in sequence can be ascribed to sequencing errors. As they
are sequence tagged sites from the European genome mapping project this
data will assist in aligning the genetic and molecular maps for the X
chromosome but is not otherwise informative. The other sequences show short
177
regions of similarity restricted to repeated sequence motifs. Thus it would
appear that if garnet -homologous sequences exist in other organisms, (garnet
Table 22. Genes with sequence similarity to garnet.
The first column gives the name and accession code of the gene. The second
and third columns give the region of sequence similarity, relative to the garnet
imaginal c-DNA sequence and the other gene, respectively. The next columns
give the percent similarity within this region and an indication if the similarity is
restricted to a repeated sequence motif.
178
179
7.
Rat Na+,K+ ATPase
alpha2 subunit gene
and 5’ flank seq.
5833-5925
2222-2319
3429-3531
1728-1 808
3745-3775
51988-51933
584-653&45125-46260
33732-33785
20477-20502
910-977
2834-292 1
2839-2911
3092-3179
2575-2670
3094-3190
2572-2654
3395-3425
3059-3186
2558-2669
3383-3435
3770-3798
3094-3161
2600-2687
2565-2637
79%
65%
67%
70%
70%
70%
79%
75%
65%
63%
75%
65%
78%
85%
76%
74%
1188-1253
3717-3765
3721-3768
4262-4300
3096-3175
3395-3446
2600-2670
3534-3572
95%
% similarity
97%
1-221
region of similarity
in other gene
363-428&428-468 2-68&1 30-171
2352-2514
region of similarity
in garnet
4.
C. grisous dhfr
origin of replication
emb X520341 CGDHFRORI
5.
Rat MHC Class I Ag gene
RT1-u haplotype
gb M64795 RATMHRT 1
6.
Mouse beta-globin complex
bho,bhl,bl,b2,bh3 and bh3
emb X14061 MMBGCXD
1.
Drosophila
melanogaster STS
emb Z31953 DM189B8T
2.
Drosophila
melanogaster STS
emb Z32301 DM7C5T
3.
Rat 5.5kb DNA fragment
containing repetitive DNA
emb Xl 3424 RN55REP
gene
Genes with sequence similarity to garnet.
GAGA repeat
T repeat
GUT repeat
GAGA repeat
T repeats
GUT repeat
AT repeat
GAGA repeat
T repeats
T repeats
GAGA repeat
T repeats
GAGA repeat
T repeats
T repeats
A repeats
non-repeat
non-repeat
motif
180
10.
Rat hepatic steroid hydroxylase 3091-3216
hAl gene
2566-2641
gb M33312 RATCYP2A1
3111-3196
687-75 1
3376-3435&2587-2646
180-258
9.
Mouse myoglobin exon 1
and flanking regions
emb X04405 MMMYOGG1
17713-17834
4891-4966
577-663&6399-6518
9845-9918
3092-31 88
2583-26667
8.
Mouse Hox 3.1, 3.2
genes and intergenic region
gb M35603 musHOXMAA
8043-8067
492-516
emb D90049 RNATPA25
70%
67%
79%
65%
73%
70%
84%
GAGA repeat
GTTT repeat
GAGA repeat
GUT repeat
GAGA repeat
T repeats
GTT repeat
is present in sibling species of Drosophila melanogaster (Sturtevant et al. 1925,
SturLevant and Novitski 1941)) these have not been entered into the data bank.
Tissue distribution of garnet transcripts. The tissue specificity of garnet
transcription was investigated by in situ hybridization to embryos and various
organs present in third instar larvae and adults. Figure 27 presents these
results. The garnet gene is clearly transcribed in the eye-antennal imaginal
disc, the anlage of the adult eye. Interestingly it is also present, in other imaginal
discs as well as other tissues such as the larval brain and ovarioles. It is also
abundantly expressed in embryos. Thus garnet is expressed in a variety of
tissues during at least three stages of development.
Do sequences similar to garnet exist in Drosophila melanogaster?
Genetic evidence suggests that the garnet gene is one of a group of genes that
are functionally redundant. Functionally redundant loci might have sequence
similarity. To test this possibility, genomic DNA from wild-type flies was probed
with the 6.6 Eco RI fragment under conditions of reduced stringency. No extra
bands were seen under conditions where sequences sharing approximately
66% identity should be detected (Figure 28). Thus, there do not seem to be
any sequences in the Drosophila melanogaster genome which are highly
similar to the garnet gene.
181
Figure 27. Analysis of garnet transcription by in situ hybridization to various
tissues.
A. The first three images (viewed top to bottom, left to right) show garnet
message in blastula, gastrula and neurula stages of Drosophila melanogaster
embryos.
B. The next two pictures show garnet message in the leg and eye-antennal
imaginal discs.
C. Below is shown garnet transcription in an ovariole.
D. The three pictures on the lower right show the same tissues hybridized with
the sense probe (T3 probe) after 90 minutes of staining.
E. garnet transcription in the brain and the eye-antennal imaginal disc (for
comparison) of wild type third instar larvae.
The embryos and the larval brain were stained for ten minutes before the
staining reaction was stopped. The imaginal discs and ovarioles were stained
for 90 minutes.
182
-
a
a
V
3
Figure 28. Southern analysis of regions of sequence similarity of the garnet
gene in Drosophila melanogaster.
DNA from wild type flies was isolated, restricted with Kpn I and Xho I (which do
not cut within the 6.6 Eco RI fragment which contains the garnet gene),
separated on a 0.8% agarose gel, transferred to nylon membrane and probed
with the 6.6 Eco RI garnet fragment with reduced stringency. Under the
conditions used sequences with approximately two thirds similarity should have
been detected. With the exception of a high molecular smear evident in the Xho
I lane, likely due to incomplete digestion, no additional bands were seen. The
gel was overloaded with DNA and then the filter overexposed to allow detection
of any weakly hybridizing bands.
185
981
IL
‘I
Iudi
19
Ioq
Discussion-garnet
Phenotype of the garnet alleles.
The garnet alleles affect both pteridine and ommochrome pigments. This is not
a phenotype that can easily be explained as a simple enzymatic deficiency. As
the two pigment biosynthetic pathways are biochemically distinct, an enzymatic
lesion should alter only one group of pigments. In addition there is no evidence
for novel pigments, or pigment intermediates, that might be expected to
accumulate in a blocked pigment biosynthetic pathway.
The garnet mutant alleles alter pigmentation of all of the three major pigmented
structures in Drosophila melanogaster, the eye, the malpighian tubules and the
testes sheath. Pigmentation of the malpighian tubules is generally more
severally altered than that of the testes sheath, which in turn is more severely
compromised than that of the eye. This difference could reflect the timing of
differentiation, the type or amount of pigment in these organs.
53 allele presents an important exception to this pattern of decreased
g
The d
pigment levels. Although this allele is the most severe mutant allele, and
genetically behaves as an amorph, based on dosage tests in the eye, it exhibits
essentially wild-type levels of pigmentation in the testes sheath. Interestingly,
Tearle (1991) reports that this allele does not have a pronounced effect on
pigmentation of the occelli either. The obvious conclusion is that this particular
allele is not a typical amorph or hypomorph but has an alteration in tissue
specific regulation of garnet expression. Many alleles of the garnet gene have
been isolated and described (Table 23). Mutant alleles have been isolated as
spontaneous mutations, and induced by treatment with X-rays, gamma rays,
187
Table 23. Published alleles of garnet.
The first column gives the allele designation. The second column gives the
inducing agent, when known. The third and fourth columns gives the name of
the investigator responsible for isolating the allele and the reference. This list
was adapted from Lindsley and Zimm (1992).
188
Published garnet alleles
allele
origin
discoverer
g
1
2
g
”
2
g
3
g
4
g
B
7
gl
spontaneous
spontaneous
unknown
spontaneous
X-rays
X-rays
X-rays
SMS
SMS
SMS
SMS
spontaneous
spontaneous
spontaneous
spontaneous
spontaneous
spontaneous
spontaneous
spontaneous
spontaneous
spontaneous
X-rays
unknown
unknown
spontaneous
spontaneous
X-rays
EMS
spontaneous
spontaneous
X-rays
X-rays
X-rays
X-rays
spontaneous
EMS
spontaneous
Bridges
Bridges 1916
Bridges
Lindsley and Zimm 1992
unknown
Lindsley and Zimm 1992
Bridges
Lindsley and Zimm 1992
Glass
Lindsley and Zimm 1992
Valencia
Valencia 1966
Sobels
Lindsley and Zimm 1992
Sobels
Lindsley and Zimm 1992
Sobels
Lindsley and Zimm 1992
Sobels
Lindsley and Zimm 1992
Sobels
Lindsley and Zimm 1992
Wallace
Lindsley and Zimm 1992
Bridges
Lindsley and Zimm 1992
Emerson
Lindsley and Zimm 1992
Bridges
Lindsley and Zimm 1992
Ives
Lindsley and Zimm 1992
Duncan
Lindsley and Zimm 1992
Mossige
Lindsley and Zimm 1992
Ecken
Lindsley and Zimm 1992
Mather
Lindsley and Zimm 1992
Bridges
Lindsley and Zimm 1992
Green
Lindsley and Zimm 1992
King
King 1950
unknown
Lindsley and Zimm 1992
Hexter
Hexter 1958
Lindsley and Zimm 1992
Williams
Lindsley and Zimm 1992
Ives
Maddorn
Hayman, Madden 1967
Schwinck
Schwinck, Schwinckl 972
Najera 1985
Najera
Demerec
Lindsley and Zimm 1992
Demerec
Lindsley and Zimm 1992
Hoover
Lindsley and Zimm 1992
Hoover
Lindsley and Zimm 1992
Gottschewski Lindsley and Zimm 1992
unknown
Lindsley and Zimm 1992
Waddle
Lindsley and Zimm 1992
°
2
g
61
2615
g
2641
g
2810
g
284
g
°
g29h
Od
3
g
d
2
g3
33
g
J
33
g
’
34
g
e
g37f
k
7
g3
g38b
42
g
a
9h
4
g
Oe
5
g
g53d
g55k
b
4
g6
g68d
Ok
7
g
’
7
g
9
2712
g
2716
g
2719
g
2711
g
°
ge
gEMS
gF
189
reference
gim
g’
gSl
2
gS
gS3
gtUh4
gtUh2
gW
gX
unknown
unknown
spontaneous
spontaneous
spontaneous
spontaneous
spontaneous
spontaneous
X-rays
unknown
unknown
Schalet
Schalet
Schalet
Kuhn
Kuhn
Muller
Muller
190
Lindsley and Zimm
Lindsley and Zimm
Chovnick 1961
Schalet 1986
Schalet 1986
Kuhn 1972
Kuhn 1972
Lindsley and Zimm
Lindsley and Zimm
1992
1992
1992
1992
chemical mutagenesis (Table 23) and P-elements (Wennberg, 1988 and this
work). It is interesting that of the 56 published alleles and the many P-element
derived alleles described in this work, none appear to be true null mutations, Of
the over 300 mutants described for the white gene, which has a similarly sized
transcript, approximately a third 98/344) are phenotypic nulls. This might
suggest that a null mutation of the garnet gene has a phenotype other than
reduced eye pigmentation, although the paucity of null garnet mutants may not
be beyond the bounds of bad luck.
Genetic and molecular limits of the garnet gene:
Perhaps the most useful issue of the Chovnick/Hexter debate on the complexity
of the garnet locus was a recombination fine structure genetic map of the garnet
gene (Figure 29). This map was generated by selecting rare wild type intra
allelic recombinants between different garnet alleles. Each of these studies
involved visually scoring more than a hundred thousand (Hexter 1958
-583,416, Chovnick 1958-762,429 and Chovnick 1961
-
176,526) flies for rare
wild type recombinants in a background of brown-red eye mutants, the
phenotypes of some of which approach wild type with age. The value of working
on a gene where such extensive work has been performed, by others, cannot
be overstated.
The intra-allelic genetic map shown in Figure 29 should, in principle, provide
a basis for correlating the genetic and molecular limits of the garnet gene.
Unfortunately, the gSl allele which defines the right-most limit of the gene is no
longer extant. The molecular lesions responsible for the three left-most alleles
are not known. They are not associated with deletions or insertions. The g
1 and
3 alleles are however associated with seemingly identical insertions which
g
191
Figure 29. Map of the garnet gene generated by intragenic recombination.
The top line indicates the genetic limits of the garnet gene. Below is shown the
order of six garnet alleles, derived from Chovnick 1958, Hexter 1958 and
Chovnick 1961. If the g
3 lesions are in the 3’ region of garnet as
1 and g
suggested by the results of Figure 21 then the direction of transcription can be
oriented relative to the genetic map as shown.
192
RECOMBINATIONAL MAP OF THEgarnet LOCUS
d
5
g
3
I
I
2
g
Oe
5
g
I
1
g
5’
I
gSl
3•
TELOMERE
CENTROMERE
193
have occurred at apparently the same point in the garnet gene. These alleles
are genetically inseparable. This restriction fragment is near the 3’ end of the
gene and is consistent with their recombination position in the rightmost third of
the map. It is also interesting that the 3 leftmost, or possibly 5’, alleles are the
most extreme. The g53d allele might be a lesion in the 5’ regulatory region. If
this were so it would indicate that the orientation of the garnet gene relative to
the centromere is with transcription away from the telomere.
Evidence that the cloned region corresponds to the garnet gene:
The weak P-element induced gP allele was used to clone the garnet gene.
There are three lines of evidence that indicate that the cloned region,
specifically the 6.6 kb Eco RI fragment does in fact contain the garnet gene.
First, a partial P-element interrupts this fragment in the gP allele. This P-element
is altered in both revertants and extreme secondary derivatives of the gP
mutation. Sequence analysis of wild type garnet DNA, the equivalent region of
the gP mutation and a large c-DNA arising from this region places the P
element in a 3’ intron. Secondly, garnet mutations have a high frequency of
alterations in this fragment. Of the 14 garnet alleles examined by Southern
analysis, 9 had either insertions or deletions in this 6.6 Eco RI fragment. Finally,
analysis of transcripts from this region identify two transcripts from wild type
flies, both of which are absent in the extreme g53d mutant and one of which is
absent and the other possibly altered in the hypomorphic g
3 allele. Thus it
seems reasonable to propose that the garnet gene is located within the 6.6 Eco
RI fragment and that the c-DNA which comprises approximately two thirds of this
region, and corresponds to the only major open reading frame within this DNA
segment identifies the garnet gene product. Final proof will, however, require
rescue of the garnet mutant phenotype by P-element mediated transformation.
194
Expression pattern of the garnet gene:
Tissue in situ analysis indicates that the garnet gene is transcribed at many
stages in development including embryos, third instar larvae and adults. It is not
highly transcribed at any developmental stage. Pigment deposition in the eye
and testes sheath starts two to three days before eclosion, about midway
through the pupal stage, and darkening continues up to a week after eclosion
(Schultz 1935). Pigmentation of the malpighian tubules can be detected from
the first instar larvae onwards (Breme and Demerec 1942). The presence of
garnet m-RNA in third instar larvae and young adults is consistent with a role in
eye pigmentation. However, the ubiquitous, low levels of garnet transcription in
embryos is less obviously related to pigmentation. Interestingly, this pattern of
expression is also seen for the light gene (Devlin, Bingham and Wakimoto
1990), another member of the transport group. The tissue distribution of the
garnet transcript is also somewhat surprising. As expected the garnet gene can
be detected in the eye-antennal disc of third instar larvae. It is, however, also
present, albeit at somewhat reduced levels, in the leg and wing imaginal discs,
larval brain as well as in ovarioles and embryos. In addition, garnet mutations
cause diminished pigmentation of the adult eye, malpighian tubules, fat body,
occelli, and testes sheath, suggesting that garnet is also expressed in these
tissues.
Sequence analysis of the garnet gene.
Sequence analysis of the garnet gene has not been particularly revealing.
Conceptual translation of the 4kb imaginal disc c-DNA yields a putative protein
of 1054 amino acid residues. This putative protein has three indifferent
195
hydrophobic domains and a polyglutamine stretch. Polyglutamine repeats have
been found in a number of neurogenic genes but are not necessarily
diagnostic. They have also been implicated in parental imprinting (Green 1993).
In these instances the polyglutamine repeats tend to be long and are not
necessarily either translated or transcribed. Polyglutamine repeats have also
been implicated in protein-protein interactions. This function might be related to
the proposed interactions between the garnet gene product and those of the
other eye colour genes.
Function of the garnet gene.
The considerable genetic resources devoted to the production, developmental
and tissue specific regulation of eye pigments raises the question of their
function. The function of the garnet gene product has not been explicitly
addressed by any author. Nevertheless, there is extensive genetic evidence,
detailed in chapter 1, that the product of the garnet gene interacts with not only
the white gene, but with other members of the transport group of eye colour
genes. Various investigators have proposed functions for this group of genes.
The function of pigments in optically isolating the ommatidia and providing for
light adaptation seems incontestable, Interestingly, a similar function in both
short and long term light adaptation has been proposed for pteridines found in
the mammalian retina (Cremer-Bartels 1975), although the evidence for this is
not compelling. Nevertheless, many authors have sought additional roles for the
eye colour genes.
196
garnet and cell metabolism: The first conceptual approach to studying the
function of eye colour genes derived, appropriately, from attempts to resolve the
paradox of the apparently excessive number of eye colour genes. The first, and
obvious, attempt to deal with this problem was to group the mutations by similar
phenotypes. Nolte (1 954a) proposed that there were in fact only 6 groups of
eye colour mutations, the vermilion group, the light group, the dark group, the
red group, the variegating group and the ruby group, of which garnet is a
member. Detailed studies of the red and brown pigments of these groups (Nolte
1 954b, 1955, 1959) led to the realization that while the vermilion group might
be united in generally disrupting ommochrome synthesis (Nolte 1954a), the rest
of these groups did not represent biochemically or functionally related genes.
Nolte (1 954b, 1 959b) then proposed that the genes of the ruby group represent
lesions in general aspects of cell physiology. Specifically, he proposed that they
are involved with the protein catabolism that generates precursors for pigment
production. This hypothesis is mirrored in various forms in most of the ensuing
proposals for the function of the transport group of genes.
garnet as a transport gene. Sullivan, Grillo and Kitos (1974) and Sullivan and
Sullivan (1975) provided data for a specific variation of Nolte’s hypothesis. They
proposed that this group of genes encoded products responsible for metabolite
transport. Based on the results of a series of experiments where isolated
organs, eye discs and malpighian tubules, were cultured in vitro in labeled
kynurenine, an intermediate in the ommochrome pathway, they proposed that
many of the eye colour mutants were defective in transport of metabolites and
pigment intermediates.
197
The data presented by Sullivan and Sullivan on possible transport defects in
eye colour mutations did not include the garnet gene. They did however,
propose a list of criteria that would identify a gene primarily concerned with
pigment transport. These criteria are: cellular autonomy, effects on both pigment
pathways, effects on all of the pigmented organs, and diminished pigment
levels in conjunction with normal biosynthetic enzyme activity. Cell autonomy
would be expected for a membrane based gene product such as a
transmembrane channel protein. Alterations in both ommochrome and pteridine
pigments suggests that the transport apparatus handles more than one
metabolite or compound. This is not unprecedented (Christensen 1973). One
example, possibly quite relevant to the transport of pigments in Drosophila, is
provided by the mouse pallid locus. Defects in this gene are associated with
diminished transport of tryptophane, L-dopamine and Manganese ions
(discussed by Wiley and Forrest 1981). Alterations in the pigmentation of
different organs would suggest reasonably ubiquitous use of the transport
mechanism. The genes white, brown, scarlet, lightoid, claret, carnation, light,
maroon and pink fulfill all of these criteria. The garnet gene fulfills the first three
of these criteria; the last has not been fully tested (although Glassmann (1956)
reported that the g
2 allele has normal levels of the enzyme kynurenine
formanidase). Nevertheless, an alteration in the transport of kynurenine or any
other compound remains to be shown for garnet.
In retrospect, in light of the complex tissue and developmental interactions, the
known excretory function of the malpighian tubules and fat body, the cell
autonomous nature of many mutations and importance of transport in cell
function, a role in metabolite transport for some of the eye colour genes may
seem obvious. Nonetheless, these authors were the first to furnish data for the
198
role of transport mechanism in the final production of wild type eye
pigmentation. Finally, it should be noted that transport probably involves the
mitochondrial membrane, the pigment granule membrane and possibly the
golgi body membrane, as well as the plasma membrane. Defects in this
transport may have a variety of consequences, such as alterations in
intracellular storage or movement of precursors and excretion of waste
compounds, as well as diminished accumulation of pigments.
garnet in the brain. More recently, McCarthy and Nickla (1980) have proposed
that the genes carnation and light, both members of the transport group, are
involved in a variety of (unspecified) functions and have an essential role in the
development and function of the nervous system. Their studies were based on
extensive genetic and histological examination of double mutant light-carnation
individuals. Flies homozygous for either one of these mutations survive,
whereas, flies homozygous for both die. The lethal phase of the double mutant
is protracted and depends on dosage and activity of light (Nickla, 1977). The
synthetic lethal focus maps to the ventral blastoderm, site of the presumptive
ventral nervous system (Nickla, Lilly and McCarthy, 1980), and double mutant
individuals display abnormal brain morphology (McCarthy and Nickla, 1980).
These authors propose that in addition to a role in pigmentation, carnation and
light perform an essential function in neural development. The garnet
transcription seen in the larval brain, as well as the apparent absence of this
transcription in rosy null larvae, might suggest a similar function for the garnet
gene in this tissue, however further genetic and histological analysis is
required. It should be noted that unlike the carnation-light double mutant, the
rosy-garnet double homozygotes are viable, and other than female sterility,
display no obvious behavioral or physical defects.
199
The suggestion that groups of eye colour genes represent an essential and
redundant function is supported by findings that certain pairwise combinations
of these genes behaved as synthetic lethals. Synthetic lethal combinations
have been known in Drosophila for some time but are not common and offer a
powerful tool to identify functional identity between redundant genes. Although
synthetic lethal combinations not involving eye colour genes exist, the
representation of not only eye colour mutations but specifically of mutants of the
transport group of eye colour genes (Table 2) is intriguing. Notwithstanding, a
systematic search for synthetic lethal combinations amongst pairwise
combinations of eye colour genes has never been done and is not a task to be
undertaken casually; the number of eye colour mutations, even discounting the
numerous alleles of most, and the need to examine multiple allele combinations
would make this an onerous task. A search for interactions amongst the smaller
set of the transport group of eye colour genes might, however, prove revealing.
The garnet gene does not display lethal interactions with at least prune, light,
rosy or deep orange (other members of the transport group have not been
tested). It does however display a full spectrum of other interactions with these
genes, including female sterility, synthetic dominance and a variable spectrum
of cell death phenotypes. These other types of interactions may suggest
specialized roles for the garnet gene.
garnet and intracellular transport Very recently the g
2 allele has been
identified as an enhancer of the quartet mutation (C. Cheney- personal
communication). The quartet mutation is a female sterile mutation which seems
to be involved in localization of the nanos posterior determinant in eggs. The
abnormal phenotype of the quartet mutation is proposed to result from a defect
200
in intracellular transport. The action of the g
2 allele as an enhancer of this
defect might implicate the garnet gene as being involved in intra- as well as
intercellular transport. Preliminary results indicate that nanos transcription may
g5 homozygote (S. Gorski,
d
be severely reduced in the female sterile e(g) 3
personal communication). While the females sterile phenotype could be due to
a general and non-specific physiological effect, examination of nanos
localization in the female sterile garnet and rosy-garnet double mutants may
prove to be informative and is being pursued by the Cheney laboratory
The metabolic role of some of the pigments and intermediates is an area of
intense research. In the earliest work on the chemical nature of these
compounds, Schultz (1935) speculated that as they were highly susceptible to
oxidation-reduction reactions, their function related somehow to this property.
More recently Hilliker et a!. (1992) have observed that mutants for the rosy gene
have increased sensitivity to oxygen stress and a reduced life span. This
suggests an evolutionary impetus for the development of pigments might stem
from an alternative use of metabolic waste products. Metamorphosis is a
metabolically active portion of the insect life cycle. In holometabolic insects,
during this period, excretion of all but gaseous waste products is restricted.
Conversion of toxic by-products of amino acid and nucleic acid catabolism into
stable, non-toxic molecules which could be deposited in high concentrations in
different organs such as the eye, would be useful. If these products served or
enhance some other functions in these organs, there should be considerable
evolutionary impetus to develop such a system. While this may be true of a
subset of eye colour genes this function is unlikely to be restricted to this class
and there is no evidence that mutations in the garnet gene alter longevity.
201
While the suggestion of intercellular transport, cell communication and neural
function are certainly compatible, these studies have yet to do more than imply
an important, but undefined, redundant biological role for this group of eye
colour mutants. Resolution of the biological role of garnet gene will require
further study.
Testing the model of the function of the garnet gene: In summary, the biological
function of the “transport” group of eye colour genes, including garnet remains
speculative. Their proposed functions in general cellular metabolism,
intercellular transport, intracellular transport and a role in neural formation and
function are certainly not mutually exclusive. Genetic analysis suggests that this
group of genes possess a ubiquitous essential and redundant, if unknown
function. Based on the similarity between the phenotypes of mutants in the
transport group of genes and white hypomorphic mutants, similar effects on
pigment accumulation and hypersensitivity to the cryptic we(g) allele described
in chapter 1, I suggest that all of these gene products associate with the product
of the white gene. The unusual epistatic interaction found between the a2 and
the g
2 allele, as well as the similar interaction reported for wa3 and ruby might
provide an avenue to investigate the physical nature of this interaction. The
functional redundancy of the transport group of genes, implied by the pleiotropic
genetic interactions compared with their rather weak phenotypes as single loci
mutations, suggests that they may coordinately perform some essential
function(s). In contrast, the phenotype of other combinations, such as light and
carnation, suggest that they may perform other more specialized functions. The
absence of synthetic lethal combinations involving the garnet gene suggests
that this gene may operate only with many other gene products. However, the
female sterile phenotype of the double mutant rosy-garnet combination
202
intimates that this pair of genes might have a more unique role in the female
germ line. One may envision a complex involved generally in aspects of
transmembrane transport, various members of this complex associating in
different cell types, in different subcellular compartments, and at different stages
of development to perform variations of this function. These proposed functions
remain completely speculative and await further investigation. The cloning of
the garnet gene should lead to further definition of the biological role of not only
the garnet gene but possibly also that of the “transport” group of eye colour
mutations. Direct physical proof of the existence of a macromolecular complex
which regulates a transmembrane pore as proposed above, must await direct
analysis of the garnet gene product. Antibodies to the garnet gene product
could be used to examine the subcellular location of the garnet gene product.
Co-localization and co-immunoprecipitation with anti-garnet and anti- white
antibodies or use of the yeast dihybrid selection system would offer a direct
means of ascertaining if the genetic interaction between these genes was
mirrored by a structural association. In the interim, the genetic and molecular
analysis of the garnet gene makes this gene a useful tool to investigate other
phenomenon.
In the next chapter, I describe a system where the garnet gene is used to study
genomic imprinting in Drosophila melanogaster. Genomic imprinting has
recently attracted attention by the association between imprinting and some
human genetic syndromes but it has been described in insects and is a well
defined phenomenon the sciarids and coccids. In the final chapter I describe a
mini-chromosome which is imprinted. The imprinting is manifest as parent
dependent expression of the garnet gene. The ease of examining eye colour to
monitor imprinting as well as the sophisticated genetics of Drosophila
203
melanogaster have allowed tests of a number of possible mechanisms of
imprinting. The garnet gene has a long history of being used as a tool to
examine other interesting biological phenomenon. The next chapter continues
in this tradition.
204
Chapter 3.
Imprinting of a mini-chromosome
in Drosophila melanogaster
205
Introduction-imprinting of a mini-chromosome in Drosophila melanogaster
1 encompasses a number of processes
The phenomenon of genomic imprinting
whereby a gene or a region of a chromosome is reversibly modified so that it retains a
“memory” of its genetic history. The term imprinting was originally coined by Crouse
(1960) to refer to the complex behavior of the X-chromosome in the dipteran insect
Sciara. She defined imprinting as the “differential behavior of the members of a pair of
homologous chromosome which is predetermined several to many cell generations
before the stage in development at which resulting behavioral differences become
obvious.” This definition is fairly broad. As a result, the term imprinting has been
applied to a vast number of exceptions to normal Mendelian segregation of traits.
Conversely, for historical and traditional reasons, phenomena, that would be defined
as imprinting by contemporary criteria, have been given a variety of other names.
Table 24 provides a list of these terms, the organism with which they have been used
and the probable type of imprinting. A working definition of imprinting has been
proposed by Reik (1992) as a process whereby “epigenetic information is introduced
into chromosomes and is stabley replicated together with the chromosomes as cells
divide”. But even this definition is sufficiently broad that it encompasses a number of
biological oddities which are undoubtedly mechanistically distinct. This problem was
first addressed by Monk (1990) who proposed subgroups to encompass four general
classes of phenomenon which have been collectively called genomic imprinting.
These include species-specific imprinting, differentiation, epimutation and parental
imprinting.
1 The term “imprinting” properly refers to a phenomenon in behavioral psychology
whereby an immature animal learns appropriate behavior from adults. For the
purposes of this thesis it will be understood that the term imprinting refers to the
phenomenon of genomic imprinting.
206
Table 24. Terms used for genomic imprinting.
The first column gives the term used to describe the phenomenon of genomic
imprinting. The second column gives the general group of organisms with which this
term has been used and the third gives the type of imprinting as defined by Monk
(1990). With the exception of the phrase “Non-Mendelian ratios” (Hall 1990) and
parental effects (Baker 1963) all these terms are explained in greater detail in Heslop
Harrison (1990).
207
Other terms used for genomic imprinting
Name of phenomenon
organism group
Parental effects
Insects (Drosophila)
type of imprinting
parental
Block transference of characters plants
species
Genetic affinity
plants
species
Suppression
plants
species
Selectivity of expression
plants
species
Cryptic structural differentiation
plants
species
Skewed back cross ratios
plants
species
Homeosis
plants
species
Character pseudo-linkage
plants
species
Non-Mendelian ratios
humans
parental
208
Strain or species specific imprinting
The term imprinting has been used to refer to the variable phenotypes of the hybrids
resulting from crosses between different subspecies or strains. This type of imprinting
has been called species or strain-specific imprinting (Monk 1990) as the hybrid
phenotype appears influenced by a “memory” of the parental species. The difference
between a mule (horse mother, donkey father) and a hinney (donkey mother, horse
father) is the classical example of this type of imprinting. This type of imprinting likely
reflects the preferential action of maternally deposited activators (Castro-Sierra and
Ohno 1968) or repressors (Schmidtke, KuhI and Engel 1976) on the subtly different
regulatory regions of the genes of the two subspecies or strains. As such, speciesspecific imprinting is dependent of different information encoded in the DNA of the two
species and is not an epigenetic process.
Somatic imprinting or differentiation.
The term imprinting has also been used to refer to the processes of determination and
differentiation whereby the developmental potential of a mitotic clone is restricted
(Paro 1990, KIar 1987, 1990). From both a mechanistic and theoretical point of view
the processes involved in somatic versus germ line “memory” are expected to differ.
Mechanistically, the packaging of DNA is grossly different between the germ line and
soma. More importantly, meiotic products must remain totipotent in order to produce
the complete spectrum of cell types present in the next generation. Somatic cells do
not face such demands so that loss of totipotence implied by parental imprinting
posses no conceptual difficulties. While determination remains a central question in
developmental biology, renaming this process “imprinting” adds nothing to our
understanding of the processes involved.
209
Permanent imprinting.
The term imprinting has also been used to describe parent-specific, permanent
changes in gene activity. This phenomenon is manifest as a permanent alteration in
gene activity after passage though one parent or genetic background (Hadchouel et al
1987, Reuter 1985, Dorn et al. 1993) and has been called epimutation by Holliday
(1987) and allele-specific imprinting by Monk (1990). The mechanism where by a
permanent alteration is produced in one parent and then maintained remains
somewhat obscure. In the situation described by Hadchouel et al (1987) the gene
inactivation was associated with methylation of multiple CpG islands of the hepatitis B
surface antigen transgene, but only after passage through the female germ line. These
investigators proposed that if methylation was reversed at only a low frequency in
males, possibly due to chromatin remodeling in spermatogenesis, then the extensive
methylation in females would constitute a virtually permanent alteration. The
mechanism responsible for this phenomenon in Drosophila remains unknown
although it has been implicitly associated with changes in chromatin structure.
Parental imprinting
In parental imprinting the activity of the imprinted gene is determined by the sex of the
parent transmitting that gene. This type of imprinting has also been termed gamete
specific imprinting (Monk 1990). Parental imprinting is distinguished by its transient
nature, specifically, the imprint is completely reversed in one generation by passage
through the other sex. The result of parental imprinting is the functional
nonequivalence of the maternal and paternal genome.
In mammals the consequence of this non-equivalence is drastic. Embryos (either
parthenogenic, gynogenic or androgenic) or tissues (such as ovarian tumours and
210
Table 25. Human diseases in which imprinting has been implicated.
The first column gives the name of the disease or condition in which imprinting has
been implicated. The second column gives the reference.
211
Human diseases and conditions in which imprinting has been implicated
Condition or syndrome
Reference
Angelman/Prader Willis syndrome
Nicholls et al 1989/Hall 1990
Carmillia De Lange syndrome
Clarke 1990/Hall 1990
Duchenne muscular dystrophy
Hall 1990
Familial glomus tumours
Clarke 1990
Floating harbour syndrome
Clarke 1990
Fragile X mental retardation
Laird 1987
Huntington’s chorea
Laird 1990/Sapienza 1990
Hydatidiform moles
Kajii and Ohama 1977
Myotonic dystrophy
Clarke 1990/Hall 1990
Narcolepsy
Hall 1990
Neurofibromatosis
Clarke 1990
Osteogenic sarcoma
Toguchida et al. 1989
Ovarian tumours
Linder et al 1975
Philadelphia chromosome
Haas, Argyriou and Lion 1992
Retinoblastoma
Clarke 1990
Rhabdomyosa rcoma
Dryja et al 1989
Rubinstein-Taybi syndrome
Clarke 1990/Hall 1990
Russel-Silver dwarfism
Hall 1990
Sotos syndrome
Clarke 1990
Weaver syndrome
Clarke 1990
Wiedemann-Beckman syndrome
Hall 1990
Wilm’s tumour
Clarke 1990
Wolf-Hirschor syndrome
Clarke 1990
212
hydratidiform moles) that are derived from two complete maternal or paternal genomes
are not viable even though they have the full complement of genetic information. This
lethality is thought to be due to the cumulative effect of many imprinted genes, at least
some of which are growth regulators acting early in development. Parental imprinting
has been most thoroughly described in mammals and is currently being intensely
investigated. Much of the impetus for these investigations comes from the implication
of imprinting as the underlying cause of a number of human syndromes. Table 25
lists diseases and medical conditions which are associated with either aberrant
imprinting or the aberrant transmission of imprinted regions. The evidence for
involvement of imprinting in these conditions is well defined in some cases (e.g. fragile
X mental retardation) and considerably more inferential in others.
Parent-dependent gene expression or parental imprinting is, however, found in many
other eukaryotes, including plants (Kermicle and Alleman 1990), C. elegans (Gilchrist
and Moerman 1992), and a variety of insects including the Homopteran Coccids,
mealy bugs and other amoured scale insects (Chandra and Brown 1975), the
Hymenopteran wasp, Nasonia vitripennis (Nur et aL 1988), the Coleopteran coffee
berry borer beetle, Hypothenemus hampei (Brun et a!. 1995), and the Dipterans, the
fungus gnat Sciara
(Crouse 1960) and the genetically well characterized fruit fly
Drosophila melanogaster (see below). The consequence of parental imprinting is far
less drastic in these groups of organisms. For example, gynogenic Drosophila
melanogaster are completely viable and fertile (Fuyama 1984) as are, apparently
androgenic flies (Muller 1958).
Parental imprinting in Drosophila:
Imprinting phenomena have been recognized and studied in Drosophila, albeit under
the name of parental effects, for more than 50 years. All of the reported parental
213
Table 26. Imprinting (parental effects) in Drosophila.
Six examples of parental effects published for Drosophila are shown. The first column
give the direction of the imprint. Following the terminology of Reik (1992) the parent
transmitting the inactivated allele or chromosome is indicated. The second and third
columns give the name of the rearrangement which shows the parental effect and the
reference. The last example occurs in D. hyde!, all the others are found in D.
melanogaster.
214
Imprinting (parental effects) in Drosophila
Direction of imprint
Chromosome
Reference
Paternal
7
T(1;2)dorvar
Demakova and Belyaeva
1988
Paternal
Dp(1;4)wm254.58aBaker and Spofford 1959
Spofford 1959
Hesser 1961
Spofford 1961
Baker 1963
Maternal
Dp(1;3)wVCQ
Maternal
Khesin and Bashkirov
1978
Khesin and Bashkirov
1978
Paternal
In(1)sc and
8
Dp(1;f)1187
Prokofyeva-Belgovskaya
1947
Karpen and Spradling
1990
Paternal
Dp(1;f)LJ9
this work
Maternal
In(1)wm2
Hess 1970
215
(imprinting) effects (Table 26) involve chromosome rearrangements that exhibit
position effect variegation. Position effect variegation is a process whereby a fully
functional gene becomes inactivated due to its relocation adjacent to a broken
segment of constitutive (Spofford 1976) or facultative (Cattanach 1970)
heterochromatin. As the gene inactivation is correlated with the adoption of
heterochromatic morphology in the appropriate section of the salivary gland
chromosomes (Hartmann-Goldstein 1967), it is thought that the genetic inactivation is
due to the spread of heterochromatin which packages and condenses the normally
euchromatic region in such a way that necessary transcription factors cannot access
the gene. The result is variable genetic inactivity.
Imprinting as seen in Drosophila shows many intriguing similarities to the imprinting
described in mammals. The key features of genomic imprinting have been defined by
Metz (1938), based on his work with the Dipteran insect Sciara, but are true of
imprinting phenomenon in all organisms. The primary criterion of imprinting is that the
process affects genetically identical DNA, and is thus a strictly epigenetic
phenomenon. The second criterion is that the imprint persists for only one generation.
Thus the imprint is reversed by passage through meiosis. Genetically this is evidenced
by parental effects but no grandparental effects. This latter feature may be a
conceptual definition rather than a genuine mechanistic distinction. Long term effects
such as seen in epimutation (Darn et al 1993), or medium term effects with diminishing
grandparental effect (such as seen with wmVC0; Khesin and Bashkirov 1978) may be
merely mechanistic variations on a theme. The third criterion for parental imprinting is
that the imprint is mitotically stable. This stability generates functionally distinct clonal
regions. These clones are readily apparent in Figure 32 and have been noted as
intrinsic features of imprinting in mice (Allen et al. 1988), maize (Kermicle and Aliman
1990) and insects (Nur 1990). The clonal nature of the gene expression results in cell
216
to cell variability within a tissue. This variability suggests a stoichiometric process
operates in imprinting and may provide a clue to the mechanism responsible for
imprinting. A fourth criterion is the physical continuity and extent of the imprint. The
imprinted region frequently affects entire regions of a chromosome and may
encompasses more than one gene. Such an effect is self-evident in coccids where the
imprint encompasses an entire chromosome (Nur 1970) but is also seen in mammals.
For example, the closely linked genes thought to be involved in the pathology of
Prader-Willis syndrome, SNRPN, SNF127, PAR-i and PAR-5, are coordinately
transcribed, replicated and maternally imprinted (Gunaratne et al. 1995). The
neighboring genes H19 and !GF2 (Reik 1992) are also imprinted, although in this
case in opposite directions. Cohen (1962) found that the closely linked genes white,
split, notchiod, facet and roughest were all maternally imprinted in the wm254.58a
rearrangement. Imprinting of contiguous genes is also seen with the mini-chromosome
examined here, the garnet gene and the nearby genes narrow abdomen and tiny
(Figure 35) are paternally imprinted. It should be noted, however, that most examples
of parental imprinting in mammals have been reported for isolated genes. The relative
rarity of mammalian imprints which encompass more than one gene may reflect the
lower resolution of genetic studies of the imprint in mammals or possibly the greater
genome size of mammals as opposed to Drosophila melanogaster which might act to
limit a domain to one gene. Finally, the formation of aberrant chromatin structures
appears to be the most universal aspect of imprinting. Heterochromatin formation is
likely responsible for the altered gene expression seen in position effect variegation
which is involved in all of the reported cases of imprinted effects in Drosophila.
Likewise imprinting seen in Coccids and Sciara (Chandra and Brown 1975) clearly
involve large segments of cytologically visible heterochromatin. Cytological studies of
the imprinted heterochromatic chromosomes of coccids have shown that the imprint
can spread and can be somewhat variable (Nur 1970). Heterochromatin is also clearly
217
involved in mammalian X-chromosome inactivation (Lyon 1993) which is imprinted in
both marsupials and some tissues of eutherian mammals. Paramutation (an
epigenetic phenomenon probably closely related to imprinting) seems to be
associated with altered chromatin structure in maize (Patterson, Thorpe and Chandler
1993). Aberrant chromatin structure has been proposed as the causal feature leading
to mis-expression of the fmrl gene responsible for fragile X mental retardation (Laird
1987) and a number of other recent studies have implicated chromatin binding
proteins in the establishment of the imprint in mice (Sasaki et al 1992, Bartolomei et al
1993, Brandeis et al 1993, Ferguson-Smith et al 1993, Stoger et al 1993, Chaillet et al.
1995) and humans (Monk 1988, Barlow 1994, Varmusa and Mann 1994).
This chapter describes a mini-chromosome which shows parent specific imprinting.
The imprint is manifest as clonally repressed expression of the wild type garnet gene
when the mini-chromosome is inherited from the father. The expression of at least two
other genes on this mini-chromosome is also imprinted. The immediate cause of the
repressed garnet expression is position effect variegation. This variegation is
unconventional in that it is strictly dependent on the sex of the transmitting parent.
Thus it would appear that chromatin packaging in this mini-chromosome is imprinted.
Using the cloned garnet gene sequence to analyze this mini-chromosome I have
tested and eliminated a number of factors which might cause the imprint. To assess
the role of heterochromatin in the imprinting process, I have tested the stability of the
imprint in this region using chemical, environmental and genetic modifiers of position
effect variegation which are thought to alter heterochromatic formation. These
modifiers of heterochromatin formation and integrity alter the expression of the imprint
but not the initial decision of whether or not to imprint. This implies that while
heterochromatin is involved in the maintenance and somatic expression of the imprint,
218
it probably does not establish the imprint. Thus the imprinting decision may be under
independent genetic control.
219
RESULTS-imprinting
The Dp(1;f)LJ9 mini-chromosome.
Origin:
The Dp(1;f)LJ9 mini-chromosome is derived from the In(1)sc
29 chromosome. The
29 chromosome is an inversion between the tip of the X chromosome (1 B) and
In(1)sc
the region adjacent to the garnet gene (13A2-5) which places this region near the tip
of the X chromosome. The Dp(1;f)LJ9 mini-chromosome was induced by X-ray
treatment and is a deletion of most the X chromosome euchromatin from In(1)sc
29
(Hardy et al 1984). Only the euchromatic bands from 12A10 to 13A2 and the distal tip,
1A1 to 1 B remain. This region is appended to the centric heterochromatin of the X
chromosome. The general structure and origin of the Dp(1;f)LJ9 mini-chromosome is
diagrammed in Figure 30. Although not mentioned by Hardy eta!, (1984) it may be
assumed that the heterochromatin was broken by the X-ray treatment as the most
proximal euchromatic gene (su(f)) is missing from the mini-chromosome and
variegation for a number of genes adjacent to the heterochromatin is observed (see
below).
Mitotic and meiotic stability.
As the centric heterochromatin (or a region within) is involved in the normal
segregation of chromosomes, I tested the stability of the mini-chromosome in both
meiotic and mitotic cell divisions (Figure 31). The mini-chromosome shows a very
low rate of non-disjunction from males (0.1-0.3%, n=1 546). This rate is comparable to
that observed for a standard X chromosome (Bridges 1916). The rate of non
220
Figure 30. Diagram of the structure and origin of the Dp(1,i)LJ9 mini-chromosome.
The top figure diagrams the wild type chromosome. The middle figure shows the
structure of the In(1)sc
29 chromosome and the lowest figure shows the structure of the
Dp(1;f)LJ9 mini-chromosome. The relative positions of the narrow abdomen, tiny and
garnet genes are shown.
221
Structure and origin of the Dp(1:f)LJ9 mini-chromosome
WILD TYPE X CHROMOSOME
\F/
1B
In(1)sc
1 B/i 3A2 tAO
I
2b
ic/i 3B
29
X RAYS
n&tyg
1B113A2
Ti
12A10
THE MINI-CHROMSOSOME
Dp(1 ;f)LJ9
222
Figure 31. Meiotic stability of the Dp(1;f)LJ9 mini-chromosome.
The top portion of the figure diagrams the production of regular and non-disjunction
gametes from the attached-X and duplication bearing females (X”X/Dp(1;f)LJ9). The
frequency with which the various genotypes arise is shown in the Punnit square. The
total number of flies scored for the maternal cross was 745.
Cross: X’X/Dp(1;f)LJ9
0
d
3
g5
/y y za d/Dp(1,.f)LJ9
53
g
0 y za &
a’
The lower portion of the figure diagrams the production of regular and non-disjunction
gametes from the attached XY duplication bearing male (XY/Dp(1;f)LJ9). The
frequency with which the various genotypes arise is shown in the Punnit square. The
frequency of the y za g53d/Q genotype arising from the paternal cross is given as a
range because all of the five individuals of this genotype arose from one vial
containing only three females. Thus this genotype may represent a pre-meiotic loss of
the mini-chromosome. The total number of flies scored for the paternal cross was
1546.
Cross: X’Y/Dp(1;f)LJ9
a’ ®
d/y
5
3 za g53d
y za g
223
53
g
y za d/Dp(1,’f)LJ9c/
DISJUNCTION IN FEMALES
regular
gametes
53d
rn
Q37
y
Dp
0.47
RIP
CROSS: XIDp(1;f)LJ9
non-disjunction
gametes
0
Yô(+Op
tRIP
0.10
006
RIP
y
4 53di y
DISJUNCTION IN MALES
)t’
53d
0.56
regular
gametes
Dp
0.44
CROSS: )&/Dp(1 ;f)LJ9 x
224
non-disjunction
gametes
fv+Dp
0
0
0.0010.003
diyz
yz 53
disjunction from females is 16% (n=745) which presumably reflects the inability of the
mini-chromosome to pair and recombine with the full length attached X-chromosomes
in the female. Mitotic non-disjunction was monitored by looking for mosaic patches of
mutant yellow tissue in a fly bearing a mutant yellow gene on its normal X
53 za g
g
d/Dp(1;f)L.j9,
5
3 y or y za d/y
d/Dp(1;f)LJ9,
5
3 y) as the
chromosome (y za g
mini-chromosome carries the wild-type allele of yellow. Only 2 instances of mosaicism
were seen in over 10,000 flies suggesting that mitotic non-disjunction does not
happen at an appreciable rate (interesting but probably not relevant is the fact that
these two mosaics were perfect bilateral gynandromorphs and came from the same
parent). Thus it would appear that the mini-chromosome is transmitted faithfully
through both meiosis and mitosis suggesting that the bulk of the centric
heterochromatin, telomere and any other functions necessary for normal disjunction
are not compromised.
Imprinted variegation of the mini-chromosome.
The variegation is dependent on the sex of the transmitting parent:
When females carrying the mini-chromosome (XX/Dp(1;f)LJ9) are crossed to yza
d/y males, the male progeny (y za d/Dp(1;f)LJ9)
3
g5
53 appeared wild type. This is the
g
expected phenotype as the mini-chromosome carries the wild type genes for yellow
and garnet (the za allele has no independent phenotype and serves only to lighten the
garnet mutant phenotype in the background). In contrast, when the males carrying the
mini-chromosome were crossed to females of the same y za g53d strain, the wild-type
garnet gene on the mini-chromosome showed variegated expression in the
d/Dp(1,i)LJ9)
5
3 sons (Figure 32). In most cases the
genotypically identical y za g
variegation was expressed as no, one, two or three large wild type spots (garnet+) on
225
Figure 32. The garnet phenotype in flies with paternally or maternally inherited Dp
(1;f)LJ9 mini-chromosomes.
53 The
g
A. The top figure shows two male flies of identical genotype; yza d/Dp(1;f)LJ9.
fly on the left, with variegated eyes, has a paternally transmitted mini-chromosome
whereas the fly on the right bears a maternally transmitted mini-chromosome.
g53d/y y za d/Dp(1;f)LJ9
1
53
g
® y za 0
Maternal cross: X”X/Dp(1;f)LJ9
Paternal cross: X’Y/Dp(1;f)LJ9
‘
53 za g53d >
g
® y za d/y
53
g
y za d/Dp(1;f)LJ9f
d/y
5
3 za
B. The lower figure shows two female flies of identical genotype; y za g
53 The fly on the left, with variegated eyes, has a paternally transmitted
g
d/Dp(1;f)LJ9.
mini-chromosome whereas the fly on the right bears a maternally transmitted minichromosome.
53 za d/Dp(1;f)LJ9
g
53
g
Maternal cross: y za d/y
a
53 za g5
g
dj
3
y za d/y
‘I,
d/y
5
3 za d/Dp(1,.f)LJ9
53
g
y za g
53 za g5
g
d
3
Paternal cross: y za d/y
53
g
® y za d/y/Dp(1;f)LJ9
1
d/y
5
3 za d/Dp(1;f)LJ9
53
g
y za g
In all cases the genotypes being compared differ only in the parental origin of the mini
chromosome. They are otherwise genotypically identical and isogenic. The yellow
mutation was used to monitor the presence of the mini-chromosome without bias as to
eye phenotype. The zestea allele was used to lighten the background garnet eye
226
d allele. In the absence of wild type garnet expression from the mini3
colour of the g5
d
3
chromosome the background eye colour is a pale orange due to the za and g5
mutations. In cells in which the garnet+ gene on the mini-chromosome is expressed
the eye colour is wild type. In most of the following experiments males of yza
53 were used to monitor the imprint as they were the genotype that arose
g
d/Dp(1;f)LJ9
from both standard maternal and paternal crosses. These phenotypes persist for one
generation only. For example, both the females shown in part B will transmit non
variegating mini-chromosomes regardless of whether they themselves show
variegation for the garnet gene.
227
N
the pale orange background colour. These spots appeared to correspond to clonally
determined regions of the eye (Jannings 1970). This mosaic expression was probably
not due to mitotic loss of the mini-chromosome in the eye since the mini-chromosome
is mitotically stable in the rest of the fly. This imprinted expression is seen in both male
and female progeny (Figure 32) and persists for only one generation (Table 35).
To summarize, genotypically identical progeny, produced by reciprocal crosses and
thus differing only in the parental origin of the sex chromosomes and the minichromosome, show very different phenotypes. When the mini-chromosome is derived
from the male parent the garnet gene variegates extensively. In contrast, genetically
identical offspring resulting from the reciprocal cross in which the mini-chromosome is
derived from the female parent, showed no or very weak variegation (Figure 32).
Thus the parental origin determines the extent of variegation observed in the
genotypically identical progeny. Therefore the expression of the wild type garnet gene
is dependent on the sex of the parent transmitting the mini-chromosome. This situation
constitutes a classical example of genomic imprinting. The many parallels between
this example of imprinting and that seen in coccids and mammals will be discussed
below.
Tissue specificity of the imprint:
This parent-dependent expression is not limited to the eye. Examination of malpighian
tubules in individuals bearing maternally versus paternally derived mini-chromosomes
demonstrate that the garnet gene expression in this tissue also variegated extensively
when the mini-chromosome is transmitted by the father (Figure 33). Malpighian
tubules from individuals in which the mini-chromosome is maternally derived show
only occasional unpigmented spots. Testes sheaths from individuals of these same
genotypes showed a uniform wild type pigmentation as expected since the g53d allele
229
Figure 33. garnet phenotype in malpighian tubules bearing maternally and
paternally derived Dp(1;f)LJ9 mini-chromosome.
The top figure shows a typical malpighian tubule from a male of the genotype yza
3 where the mini-chromosome was inherited from the father. The
d/Dp(1;f)LJgf.
5
g
dark areas are individual pigmented cells. The clear areas are regions in which no
pigment is produced.
The lower figure shows a typical malpighian tubule from a male of the identical
3 In this instance the mini-chromosome was inherited
d/Dp(1;f)LJ9.
5
genotype y za g
from the mother. Only one unpigmented region is seen. Frequently there are none
although up to three unpigmented regions have been found within one tubule from
individuals of this genotype.
Wild type malpighian tubules are always uniformly pigmented, unless damaged in
removal (data not shown).
Crosses:
g53d/Y
&
...). y za d/Dp(1;f)LJ9f
53
g
® y za 0
Maternal X’X/Dp(1;f)LJ9
Paternal: XY/Dp(1;f)LJ9
‘
d/y
5
3 za g53d —y za d/Dp(1,.f)LJ9f
53
g
® y za g
230
I—.
N)
()
4-
•1
4
I
does not affect testes sheath pigmentation.
Imprinted expression of narrow abdomen and tiny
To determine if the parental effect is peculiar to the garnet gene or is due to some
general feature of the mini-chromosome, I tested two other, closely linked, cell
autonomous genes narrow abdomen (na) and tiny (ty) to determine if the expression
of these genes is also dependent on parental origin. When the mini-chromosome was
inherited from the father, the wild type gene on the mini-chromosome showed variable
hypomorphic expression (Figure 34 and 35) in males mutant for narrow abdomen or
tiny on the standard X chromosome (ty/Dp(1;f)LJ9 or na/Dp(1;f)LJ9). In contrast, when
the mini-chromosome was maternally inherited, the genotypically identical progeny
were generally wild type. Thus the imprinting effect seems to encompass at least three
genes in the small euchromatic portion of the mini-chromosome. The “imprinting”
effect for all these genes was paternal. Interestingly, the severity of the imprint, as
assessed by the difference between gene expression when either maternally or
paternally inherited, decreased with distance from the centromere and the centric
heterochromatin.
Imprinting and position effect variegation:
The clonal phenotype of the garnet variegated expression is distinctly reminiscent of
position effect variegation. The variable hypomorphic expression of the genes tiny and
narrow abdomen is also indicative of position-effect variegation. As it is probable that
the centric heterochromatin was broken when the mini-chromosome was generated I
tested several different types of modifiers of position effect variegation to determine if
they modified the mosaic expression of the garnet gene. They did, (see below)
suggesting that the mosaic expression of the garnet, narrow abdomen and tiny genes
232
Figure 34. Phenotype of narrow abdomen and tiny in flies bearing maternally or
paternally derived Dp(1;f)LJ9 mini-chromosomes.
The top portion of the figure shows two genetically identical flies bearing a mutation for
the narrow abdomen (na) gene on the standard X-chromosome and the wild type
allele of narrow abdomen on the mini-chromosome (na/Dp(1;f)LJ9). The individual on
the left bears a maternally derived mini-chromosome whereas the genotypically
identical fly on the right has a paternally derived mini-chromosome. Arrows point to the
abdomen which is considerably more extended (more mutant) in the flies bearing a
paternally derived mini-chromosome.
Crosses:
Maternal X’X/Dp(1;f)LJ9
® na/Ye’—> na/Dp(1;f)LJ9cI
Paternal: XY/Dp(1;f)LJ9 010 na/In(1)d149 —na/Dp(1;f)LJ9o’
The lower figure shows thoracic bristles from flies with a mutation for the tiny (ty) gene
on the regular X-chromosome and the wild type tiny gene on the mini-chromosome
(ty/Dp(1;f)LJ9). The individual on the left bears a maternally derived mini-chromosome.
The genotypically identical fly on the right bears a paternally derived mini
chromosome. Arrows point to the thoracic bristles which are smaller and finer (more
mutant) in the flies bearing a paternally derived mini-chromosome.
Crosses:
Maternal X’X/Dp(1;f)LJ9
0 g
2 ty/Yo’—> na/Dp(1;f,)LJ9o’
Paternal: X’Y/Dp(1;f)LJ9 010 g
2 ty/In(1)d149 .-÷na/Dp(1;f)LJ9o’
233
nalDp(1;f)LJ9
PAT
MAT
tyiDp(1,f) LJ9
MAT
PAT
234
Figure 35. Expression of garnet, narrow abdomen and tiny in genotypically identical
flies bearing either paternally or maternally derived Dp(1;f)LJ9 mini-chromosome.
This figure shows the level of variegation for three closely linked wild type genes,
garnet (position 44.4), narrow abdomen (position 44.5) and tiny (position 45.2), on the
Dp(1;f)LJ9 mini-chromosome.
The level of expression of these genes was assessed visually as follows: The level of
expression of each of these genes was visually estimated. Each fly, or eye in the case
of garnet expression, was assigned a score of 0, 1/2 or 1 for extreme mutant,
hypomorphic and wild type expression respectively. These values were averaged and
this value is presented with the calculated standard error of the mean.
A fly was scored as tiny if its bristles appeared severely minute. In practice this meant
approximately half the length of regular bristles on the sibs. A fly was scored as narrow
abdomen if the abdomen appeared exceptionally long and thin. A fly was scored as a
mutant for garnet if its eyes were completely or extensively variegated. As the scoring
is somewhat subjective the crosses were scored by using flies grown on the same set
of media at the same time and the crosses were scored blind.
235
Parent-dependent expression ofnarrow abdomen. tiny jjsgarnet
EXPRESSION OF GENES ON THE
MINI-CHROMOSOME
mini-chromosome
derived from
mother
mini-chromosome
derived from
father
narrow abdomen
(na)
tiny
garnet
(ty)
(g)
0.67±0.02
0.52±0.02
0.86±0.02
0.51 ±0.02
0.24±0.02
0.06 ±0.02
236
transmitting parent, the immediate cause of the imprinting of the garnet, narrow
abdomen and tiny genes is imprinted position effect variegation. The possible causes
of this imprinted variegation are explored below.
Possible causes of the imprinting effect.
There are a number of possible causes of imprinting. Obviously the mechanism of
imprinting must be established on the haploid genome and thus rely on some feature
of meiosis or gametogenesis that differs between females and males or on the
subsequent fates of the gametes until fusion of the pronuclei. I directly tested the
involvement of a number of factors in the establishment and maintenance of the
imprint. The following possible causes of the imprinting effect were tested:
1. The Y chromosome
2. Allele specific interactions
3. Maternal effect of the imprinted gene
4. Maternally transmitted modifiers of variegation
5. Unusual euchromatic sequence or DNA structure near the garnet gene
6. Special heterochromatic features
Details of experiments designed to test these possibilities are provided below.
1. The Y chromosome.
Imprinting is clearly associated with the sex of the parent from which the imprinted
chromosome is derived. Thus, imprinting must be a consequence of either
physiological or genotypic differences between the parents. The only geneotypic
difference between the parents is the presence of a Y chromosome in males in place
237
of one of the X chromosomes. The Y chromosome is a large heterochromatic body,
known to affect many processes such as chromosome pairing, expression of
heterochromatic genes and position effect variegation. Therefore the parental effect
might be due to a direct interaction between the Y chromosome and the minichromosome. If this were the case, the mini-chromosome should be imprinted when
transmitted by XXY females. This hypothesis was tested by comparing variegation of
genotypically identical XIDp progeny from XXY and standard XX females. The
phenotype of the progeny of these crosses was identical (Figure 36). Thus the minichromosome responds to the sex of the parent rather than the presence or absence of
a Y chromosome.
2. Allele specific interactions.
Imprinting of the mini-chromosome could be a consequence of an interaction between
g5 allele on the
d
the wild type allele of garnet on the mini-chromosome and the 3
regular X chromosome. If this were the case, a parental imprinting effect would not be
seen with other garnet alleles. To determine if the imprinting effect was allele specific,
the variegation of the mini-chromosome was examined in strains heterozygous for the
wild type garnet gene on the mini-chromosome and eight different garnet alleles on
the regular X chromosome. In every case variegation was evident when the minichromosome was introduced paternally but not when introduced maternally (Table
27). This suggests that the imprinting effect is not due to some combination of the
garnet alleles. This result and the imprinting of the nearby narrow abdomen and tiny
genes also argue against specific allelic interaction as the cause of the imprint.
3. Maternal effect of the garnet gene.
It is possible that the differential expression of the garnet gene (and the other two
genes) results from maternal rescue of the mutant phenotype rather than paternal
238
Figure 36. The Y chromosome does not cause the imprint.
The possibility that the imprint was caused directly by the Y chromosome, as opposed
to the sex of the parent, was tested by examining the variegation of the standard test
53 when the mini-chromosome was derived from
g
genotype, yza d/Dp(1;f)LJ9,
females with or without a Y chromosome. The first experimental column shows the
results of transmission of the mini-chromosome from males (which necessarily
possess a Y chromosome). The next two experimental columns show the level of
53 progeny in which
g
garnet expression in genotypically identical yza d/Dp(1;f)LJ9
the mini-chromosome was derived from females with and without a Y chromosome,
respectively. The values shown in the middle of the figure give an indication of the
level of expression of the garnet gene as an indicator of the imprint. The expression of
the garnet gene was monitored both visually and quantitatively by microflourimetric
assay. The eye phenotype is indicated schematically at the bottom of the figure.
Values are taken from Table 34.
Crosses:
Paternal: X’Y/Dp(1;f)LJ9
‘
d/y
5
3 za 3
g5 —>y za d/Dp(1;f)LJ9cJ
d
53
g
® y za g
Maternal : WITH Y CHROMOSOME
X’X/Dp(1;f)LJ9
53
g
0 any male y za d/yf
‘I,
X’X/Y/Dp(1;f)LJ9
53
g
®y za d/y
‘I
53
g
y za d/Dp(1;f)LJ9f
Maternal: WITHOUT Y CHROMOSOME
X’X/Dp(1;f)LJ9
d/Y
5
g
&
3 y za d/Dp(1;f)LJ9
53
g
0 y za 0
239
The imprint is not caused by the Y chromosome
F’
)CkY
xY
Dp(1 ;f)U9
Dp(1 ;f)LJ9
0
I
1
y zag 53d,Dpu9
visual
estimate
pigment
assay
zag53d,Dpg
0. 10 ± 0.02
Dp(1 ;f)LJ9
zag53d,p9
1.0±0
0.92 ± 0.01
94±4
100± 3
VARIEGATED
WILD TYPE
WILD TYPE
(IMPRINTED)
(NOT IMPRINTED)
41 ±2
S
240
(NOT IMPRINTED)
Table 27. The effect of different garnet alleles on the imprint.
The first column lists the garnet allele. The second and third columns indicates
variegation when the Dp(1;f)LJ9 mini-chromosome is transmitted maternally or
paternally, respectively.
Cross:
Maternal cross:
X’X/Dp(1;f)LJ9
Paternal cross:
X’Y/Dp(1;f)LJ9cJ ®
®
g*/yl
>
g*/Dp(1;f)LJgf
g*/g* > g*/Dp(’1;f)LJ9c/
where g* is the given allele of garnet.
241
The effect of different garnet alleles on the imprint
garnet allele on the
regular X
chromosome
phenotype of
g/Dp
Maternal
phenotype of
g/Dp
Paternal
1
g
wild type
variegated
2
g
wild type
variegated
3
g
ND
variegated
4
g
ND
variegated
Oe
5
g
wild type
variegated
d
3
g5
wild type
variegated
61
g
wild type
variegated
gEMS
wild type
ND
gP
wild type
ND
gS3
wild type
variegated
242
regardless of parental origin, but if the mini-chromosome were the only source of wild
type garnet+ product, and if the mother were able to deposit wild type garnet product
in the egg, a conventional maternal effect could be misinterpreted as paternal
imprinting. This explanation is unlikely. Firstly, more than fifty different garnet alleles
have been examined and none show a maternal effect (Lindsley and Zimm 1992).
Secondly, the tissue profile of the garnet gene failed to reveal high levels of garnet
mRNA in ovaries and eggs (Figure 27) as would be expected for a maternally
deposited product. Finally this argument would have to be extended to the other
imprinted genes, narrow abdomen and tiny. Nevertheless, to completely exclude this
argument I genetically tested the garnet gene for a maternal effect on eye
pigmentation. No maternal effect was detected (Table 28). I also directly tested the
mini-chromosome for maternal effect rescue by a wild-type copy of the garnet gene. If
the imprinting effect was due to maternal rescue of the garnet mutant phenotype,
providing a wild type garnet allele (elsewhere than on the mini-chromosome) should
eliminate the imprinting effect. Specifically, female parents with a wild type garnet
allele should deposit enough wild-type product in the egg cytoplasm to rescue the
variegation of the garnet allele on the Dp(1;f)L9 mini-chromosome derived from the
paternal parent. This was not the case (Table 28). Thus a maternal effect for the
garnet gene can be excluded as a cause of the imprint.
4. Physiological compensation.
The physiological compensation model is a variant of the maternal effect hypothesis.
This model posits that the female parent produces, and transmits to the egg, factors
capable of suppressing variegation, but only in response to the variegating Dp(1;f)LJ9
mini-chromosome in her cells. This scenario is formally equivalent to that suggested to
explain the imprinting effect in the imprinted diseases Huntington’s Chorea (Bird, Caro
and Pillins 1974) and fragile X syndrome (Van Dyke and Weiss 1986). Adapted to this
243
Table 28. Maternal effect of the garnet gene.
The potential maternal effect of the garnet gene was examined in two ways. The first
53 males (where the Dp
g
test involved generating genotypically identical za d/Dp
chromosome was paternally derived) produced from females heterozygous (column 2)
or homozygous (column 3) for the garnet gene. The amount of variegation for garnet
was assayed visually (first row of data) as described in the legend to Figure 34 and
by microflourometric measurement of pteridine pigments (second data row, the values
are expressed as percent wild type pigmentation).
Cross: X’Y/Dp(1;f)LJ9cf 0 ÷÷+/y za g53d
d/y
5
3 za g5
d
3
or y za g
153
g
y za d/Dp(1;f)LJ9cf
The lower portion of the table shows a direct test for a maternal effect of the garnet
gene. The amount of pigment was quantitated in homozygous daughters (first row of
data) and hemizygous sons (second row of data) derived from females heterozygous
(column 2) or homozygous for garnet (column 3). All values are again given as
percent wild type pigment levels.
d/y
5
3 za g53d or +/y za g53d
g53d/y ® y za g
f
Cross: y za 1
1
53
g
y za d/Dp(1;f)LJ9
244
Maternal effect of the garnet gene
1. Effect on imprint
53
g
y za d/Dp(1;f)LJ9
visual estimate
pigment assay
2. Effect on garnet
pigment
53 za g53d
g
y za d/y
d/Y
53
yzag
heterozygous
mother
53
g
+ /y za d
0.14 ± 0.03
73±8
homozygous
mother
d
53
y zag53d/yzag
0.10 ± 0.02
41 ±2
heterozygous
mother
d
53
+/yzag
4±1
6±1
homozygous
mother
d
53
yzag53d/yzag
10±1
12±2
245
example of imprinting, this model posits that flies bearing the variegating minichromosome experience a physiological stress. They compensate for this stress by
producing substances that reduce the level or frequency of variegation. (These
hypothetical substances would have to be germ line specific as the female soma
shows variegation when bearing a paternally transmitted mini-chromosome, Figure
37b.) It these substances were additionally transmitted cytoplasmically then females
that possessed a mini-chromosome would produce and transmit substances capable
of rescuing the variegation, and thus their progeny would appear wild type. Males
bearing a mini-chromosome would obviously be unable to transmit such substances to
their progeny.
Initially, this is seemed a plausible explanation for the imprinting effect. Variegation
can be deleterious (unpublished observations) and many modifiers of position effect
variegation are early acting and are maternally deposited (Sinclair et al 1992, Dorn et
al. 1993). To examine this model, crosses were performed in such a way that
genotypically identical progeny were generated, all of which receive the minichromosome from their paternal parent. The crosses differ in whether or not the
maternal parent bore a mini-chromosome and thus might possess and transmit the
53 progeny from normal mothers
g
hypothetical compensatory molecules. y za d/Dp
with a paternally derived mini-chromosome had an arbitrary visual score of variegation
of 0.05 ± 0.01 corresponding to pigment levels of 48±2% (see figure legend).
Genotypically identical progeny from mothers possessing a variegator had a score of
0.07 ± 0.04, corresponding to pigment levels of 46 ± 9% (Figure 37). These values
are not satirically different. Thus there is no evidence that the imprint is due to maternal
transmission of substances capable of suppressing variegation.
246
Figure 37. Test of the physiological compensation model.
A. The top portion of this figure diagrams the physiological compensation model which
has been invoked to the explain reduced severity of Huntington’s chorea when the
disease gene is maternally inherited. The model essentially postulates that maternal
transmission compensatory substances (shown as smiling faces in the leftmost figure)
produced in response to the abnormal condition, mitigates the impact of the disease in
the offspring of these “conditioned” females. In genetic terms this is essentially modifier
gene products with a maternal effect. This models has been proposed implicitly by
Bird, Caro and Pillings (1975) and Van Dyke and Weiss (1980) and explicitly by David
Baillie (personal communication).
B. The lower figure shows a test of the Drosophila equivalent of this model. Essentially
the model postulates that mini-chromosome-bearing mothers will transmit cytoplasmic
factors to the embryo which are capable of mitigating the extent of variegation. The first
two rows show the maternal and paternal genotypes. The third row indicates the
parent contributing the mini-chromosome to the embryo. The fourth row shows the
potential for contribution of maternal modifiers which might modify the imprint. The last
rows show the phenotype and amount of variegation of the diagnostic y za
3 progeny as a measure of the imprint, assessed both by
d/Dp(1;f,)LJ9
5
g
microflourimeter and visual assay. Pigment values for the microflourimeter assay are
expressed as percent wild type levels.
247
Crosses.
Experimental 1. X’X/Dp(1;f)LJ9
0
d
3
g5
....>
/y y za d/Dp(1,.f)LJ9f
53
g
® y za &
53 za d/Dp(1;f)LJ9
g
53
g
Experimental 2*. y za d/y
® XY/Dp(1;f)LJ9cf
53
g
—>y za d/Dp(1;f,)LJ9cf
Experimental 3. X’Y/Dp(1;f)LJ9
53 za g5
g
d
3
cf ® y za d/y
53
g
>y za d/Dp(1;f)LJ9
*
Two mini-chromosomes are lethal due to presence of the male diplo-lethal region. As
53 must have a paternally derived mini-chromosome as
g
a result the y za d/Dp(1;f,)LJ9
the y za g53d homolog must come from the mother.
248
The physiological compensation model
A.
MOTHER
+1+
LESS
(later onset)
B.
MATERNAL
PATERNAL GENOTYPE
y zag5sq,Y
MATERNAL GENOTYPE
SOURCE OF
MINI-CHROMOSOME
MATERNAL
CONTRIBUTION
percent
wild type
pigment
visual
estimate
PHENOTYPE
MORE
PATERNAL
MINI-CHROMOSOME
MATERNAL CYTOPLASM
53
z
y d,yIDp
XXIDpLJ9
XX/Dp
mother
(earlier onset)
PATERNAL
XYIDp
a53d 53d
yzg iyzg
father
father
potential
modifiers
potential
modifiers
none
98±1
46±9
48±2
0.82 ± 0.02
0.07 ± 0.04
0.05 ± 0.01
WILD TYPE
VARIEGATED
249
VARIEGATED
5. Unusual structure of the euchromatic region encompassing the garnet gene.
It is possible that the garnet gene possesses some unusual feature which would
permit the ingress of heterochromatin when the mini-chromosome was received from
the male. The DNA sequence of the garnet gene itself is generally unremarkable
(Figure 26). Nevertheless, it is possible that there is some gross structural
abnormality in the region surrounding the garnet gene which could allow the
aggressive invasion of heterochromatin, although in a parental specific manner.
Localized changes in somatic copy number of genes showing position effect
variegation have been observed in polytene tissue (Karpen and Spradling 1990). If
altered copy number were the cause of the differential expression of the garnet gene,
one would predict that individuals with maternally, versus paternally, derived minichromosomes would differ in DNA content in the vicinity of the garnet gene. To test
this, DNA was extracted from flies which had either a paternally or a maternally
derived mini-chromosome and an X chromosome marked with the g
2 allele, which has
a restriction polymorphism 2 kb from the 3’ end of the garnet gene. These individuals
were tested for alteration in copy number of the garnet gene by Southern blot analysis.
Figure 38 shows that while there is considerable under-representation of DNA at the
garnet locus, the degree of under-representation does not correlate with the amount of
variegation at the phenotypic level, nor with the parental origin of the minichromosome. Thus the under-representation seems unrelated to the imprinting and
may simply reflect the limited amount of euchromatin present in the mini-chromosome.
The control of under representation at the garnet locus of this mini-chromosome is
complex. The results shown in Figure 38 are derived from males bearing an X
chromosome marked with y e(g) cv g
2 and the mini-chromosome. Very few males of
this genotype are obtained because the mini-chromosome appears to variegate for a
diplo-male-lethal locus identified in this region (Stewart and Merriam 1973, Belote and
250
Figure 38. Under-representation of the garnet gene in the Dp(1;f)LJ9 minichromosome.
This figure shows Southern analysis of whole, newly eclosed, y e(g) cv g
/ Dp(1;f)LJ9
2
males where the mini-chromosome was inherited maternally (first lane
paternally (second lane
-
-
MAT.) or
PAT.). DNA was extracted from (0-1 day) males of the
appropriate genotype, restricted with Eco RI, separated on a 1% agarose gel,
transferred to nylon membrane and probed with the garnet c-DNA derived from the
imaginal disc library. Whole flies were used as a source of DNA as the garnet gene
seems to be expressed in a variety of tissues (Figure 27) and males of this genotype
were sufficiently rare to exclude analysis of isolated tissues. The g
2 allele which marks
the normal X chromosome is associated with a Eco RI restriction polymorphism 2 kb 3’
to the garnet gene and thus serves as an internal control. This generates the lower
mobility, 8.5 kb band. The wild type garnet gene on the mini-chromosome produces
the higher mobility, 6.6 kb, band. The lowest band is a 0.2 kb Eco RI band generated
by both the normal X chromosome and the mini-chromosome.
251
I
0,
Lucchesi 1980). The lethality seems to occur principally at the pupal stage, structurally
normal pharate adults form but do not eclose. This diplo-lethality appears sensitive to
the zeste gene as it is alleviated by za and z
1 alleles. When this experiment was
repeated using a za g
/Dp progeny for molecular
2
2 strain, to increase the number of g
analysis, the under representation was not observed (data not shown). Thus the
under-representation appears to be zeste sensitive. Whether there is a causal
relationship between zeste mediated pairing, transcriptional repression, the male
diplo-lethality and sequence under-representation is unclear. The question of underrepresentation is further complicated by recent results which suggest that the observed
“under-representation” is an artifact of DNA modification which inhibits transfer to solid
support during Southern blotting (Glasser and Spradling 1994). Whether this is also
true for the under-representation around the garnet gene in the Dp(7;f)LJ9 minichromosome is currently being investigated by the Glasser lab.
6. The role of heterochromatin.
There are a number of features shared by imprinting and position effect variegation
which might suggest a mechanistic link between the two phenomena. As
heterochromatin formation has been posited to cause position effect variegation and
as heterochromatin seems widely involved in imprinting, I decided to test factors
known to modify position-effect variegation for both their effect on the variegation,
which is the mode of the expression of the imprint, and also on the transmission of the
variegation, that is, the imprint itself.
There are three general groups of factors that affect position effect variegation. They
are: environmental factors such as temperature, chemical factors, such as sodium
butyrate and genetic factors such as the presence of extra heterochromatin in the cell,
usually in the form of an additional Y chromosome. I tested all these factors for their
253
Table 29. Variegation of the Dp(1,i)LJ9 mini-chromosome: The effect of
developmental temperature.
Cultures generating g/Dp(1;f)LJ9 males, where the mini-chromosome was transmitted
either maternally or paternally, were raised at 18, 22, 25 and 290. The amount of
pigment in the eyes of these progeny was determined by microflourimeter assay. Non
specific effects of temperature on pigment (such as effects on fly size or number) were
controlled by assaying siblings. No such effects were noted (data not shown). The top
row indicates the temperature of the culture. MAT and PAT indicate the parental origin
of the mini-chromosome. Values derived from the paternal cross are shown in italics
50 or g
g
for contrast. The garnet allele of the non-Dp bearing parent, either g53d, 0
, is
2
indicated in the first column. ND
=
not done. All values are given as percent wild type
pteridine pigments.
Crosses:
Maternal cross: X”X/Dp(1;f)LJ9
d/y
5
3 or y za 5
g
O
e/yf or
® yz g
/Yo’
2
y e(g) cv g
yza g”/Dp(1,’f)LJ9o’
d/y
5
3 za g53d
Paternal cross: X”Y/Dp(1;f)LJ9o’ ® y za g
Oe
5
Oe/y za g
5
y za g
or
or
/y e(g) cv g
2
2
y e(g) cv g
‘I
yza
254
The effect of culture temperature on variegation of Dp(1;f)LJ9.
g5
d
3
MAT
PAT
Oe
5
g
MAT
PAT
2
g
MAT
PAT
culture temperature
22°
18°
25°
29°
72±8
44± 14
73+8
37± 13
64±6
36±7
39±5
28±4
70±8
47±17
80±9
50±6
71±8
52±9
44±7
35±5
ND
83±9
93±10
89±9
105±8
84±15
90±11
84±7
255
Table 30. Variegation of the Dp(1,i)LJ9 mini-chromosome: The effect of sodium
butyrate.
3 males, where the mini-chromosome was
d/Dp(1;f)LJ9
5
Cultures which generated g
transmitted either maternally or paternally, were supplemented with no, 100, 150, 200,
250 or 300 mM sodium butyrate (top row). The amount of pigment in the eyes of these
progeny was determined by microflourimeter assay (row five and six, all values
expressed as percent wild type pteridine levels). Non specific effects of temperature on
pigment (such as effects on fly size or number) were controlled by assaying
phenotypically wild type XX//Y siblings. These results are shown in the second row.
Flies variegating for In(1)wm4 were grown alongside the Dp(1;f)LJ9 crosses on
identical sets of supplemented media as a control for the effectiveness of butyrate
treatment (row three and four). ND
not done.
Crosses:
Maternal cross: X’X/Dp(1;f)LJ9
f
1
® y za g53d/y
13
d/Dp(1;f)LJ9cf
5
y za g
53 za g53d
g
Paternal cross: X’Y/Dp(1;f)LJ9o’ ® y za d/y
‘I
3
d/Dp(1;f)LJ9
5
y za g
m4 cross: 4
/wm
wm
/Ycf> m4
4
® wm
256
and
ci’.
The effect of butyrate on variegation of Dp(1;f)LJ9.
X’XhY
wm4
’
0
wm4
d/DpLJ9
53
yzag
(maternal)
d/DpLJ9
53
yzag
(paternal)
0 mM
98±4
14±3
1±1
butyrate concentration
100 mM 150 mM 200 mM 250 mM 300 mM
ND
ND
98±4
91±3
87±3
18±4
23±3
40±11 65±10
34±4
19±6
18±6
9±1
18±13 13±10
87±6
81 ±4
71 ±3
64±6
ND
ND
40±2
44±5
38±3
31±2
34±2
34±3
257
Table 31. Variegation of the Dp(1;f)LJ9 mini-chromosome: The effect of Y
chromosome dosage:
Effect of Y chromosome dosage on garnet variegation of Dp(1;f)LJ9 is shown for
individuals in which the mini-chromosome was derived either maternally or paternally.
The first and second columns show the amount of variegation assayed both visually
(row 1) and by microflourimeter assay (row 2, all values expressed as percent wild
type pteridine levels) for genotypes which are X/Dp and X/Dp + Ywhere the minichromosome is paternally derived. The third and fourth columns show the equivalent
genotypes with a maternally derived mini-chromosome.
53 That of the X/Dp
g
Genotype of the X/Dp flies is yza d/Dp(1;f)LJ9.
+
Yflies is
53
g
y za d/Dp(1;f)LJ9/y.
Crosses:
53
g
® y za d/y
Maternal cross: X’XJY/Dp
‘I
53 and y za d/Dp(1;f)LJ9/ycJ
g
53
g
y za d/Dp(1,.f)LJ9
These progeny were separated by progeny testing.
53
g
Paternal cross: y za d/y/Dp(1;f)LJ9
‘®
d/y
5
3 za g53d
y za g
‘I,
yza d/Dp(1;f)LJ9cJ
53 and yza d/Dp(1;f)LJ9/ycf
g
53
g
These progeny were separated by progeny testing.
258
The effect of Y chromosome dosage on variegation of Dp(1;f)LJ9.
Paternal
visual estimate
pigment assay
Maternal
X/Dp
X/Dp
0.07 ± 0.03
37 ± 9
0.97 ± 0.03
99 ±4
+
Y
259
X!Dp
X/Dp
1.0 ± 0
79±5
1.0±0
94±4
+
Y
ability to affect both the extent and the transmission of variegation; that is the
maintenance and establishment of the imprint. All these factors which modify classical
position effect variegation, also modified the variegation associated with this instance
of imprinting. Tables 29, 30, and 31 show the effect of temperature, sodium
butyrate, and the presence of an extra Y chromosome on variegation, respectively.
The effect of developmental temperature and butyrate was the reverse of the canonical
response. While a Hreverseu response to temperature is hardly unprecedented
(Spofford 1976) the biological significance is unclear. Growth on butyrate
supplemented media also enhanced the variegation of the garnet gene on the minichromosome, again, an unconventional response. The effect of these genetic
modifiers of position effect variegation was, with the exception of the Y chromosome,
not particularly dramatic. However, in general, the modifiers of position-effect
variegation did modify the variegation of the garnet gene on the mini-chromosome.
I next tested these same modifiers to determine if they could affect the imprint, that is
the meiotic transmission of the variegation. Table 32, 33, and 34 show the results of
these tests. In no case did any of these conditions, which alter the variegation, alter the
parental imprint. This conclusion is naturally subject to the criticism that
heterochromatin is a complex, and largely uncharacterized structure. Those factors
involved in the establishment of the imprint may in fact be components of
heterochromatin but simply different from those tested here.
The effect of the Y chromosome is particularly striking. Although the strongest
modifiers of variegation, capable of completely obliterating variegation of a paternally
derived mini-chromosome, it had no effect on the transmission of the variegation the
-
imprint. The effect of the Y chromosome on imprinting was examined in four ways: A
260
Table 32. Imprinting of the Dp(1;f)LJ9 mini-chromosome: The effect of developmental
temperature.
Cultures generating g*/Dp(1;f)LJ9 males, where the mini-chromosome was transmitted
both maternally and paternally, were raised at 18, 22, 25 and 29°. In addition the
parents were raised throughout their lives (from egg onwards) at 18, 22, 25 and 29°
The amount of pigment in the eyes of these progeny was determined by
microflourimeter assay, all values are expressed as percent wild type pteridine levels.
Non specific effects of temperature on pigment (such as effects on fly size or number)
were controlled by assaying siblings. No such effects were noted (data not shown).
Data from experiments with three different garnet allele in the non-Dp bearing parent,
d, g
3
Oe or g
5
either g5
2 is shown separately in each table. The temperature at which
the parents were raised is shown on the left. The culture temperature of the cross
which generated the diagnostic g/Dp(1;f)LJ9 progeny is shown at the top. Values
derived from the paternal cross are shown in italics for contrast. ND
=
not done. There
is limited information for the 290 series of paternal crosses as the XY/Dp males were
generally sterile when raised at this temperature.
Crosses:
Maternal cross: X’X/Dp(1;f)LJ9
g5
d
/yf or
® y za 3
1 or y e(g) cv g
0
/YQ’
2
y za g53d/y
d/y
5
3 za g5
d
3
Paternal cross: X’Y/Dp(1;f)LJ9o’ ® y za g
Oe
5
Oe y za g
5
y za g
261
or
or y e(g) cv g
/y e(g) cv g
2
2
Imprinting of the Dp(1;f)LJ9 mini-chromosome: The effect of developmental
temperature.
d
5
g
3
temperature
22°
89±3
37±2
87±3
36±3
96±4
34±4
92±30
16±4
25°
63±5
50±3
61±2
54±3
58±3
27±2
85±4
ND
29°
33± 10
39±2
49±2
30±3
54±1
41±2
59±4
ND
MAT
PAT
MAT
PAT
MAT
PAT
MAT
PAT
g
O
5
e
culture temperature
18
22
96±1
87±2
37±2
69±4
89±4
89±3
81±5
56±3
88±4
95±3
58±7
73±3
108±8
89±3
ND
ND
25°
69±4
58±2
75±4
53±3
60±3
52±2
83±3
ND
29°
61±2
41±3
60±2
33±4
58±2
52±1
59±5
45±6
18° MAT
parental
PAT
22 MAT
temperature
PAT
25 MAT
PAT
29° MAT
PAT
culture temperature
18°
22°
112±6
98±3
67± 10
96±3
102±3
103±6
80±5
79±4
112± 10 107±5
ND
ND
ND
105±5
ND
ND
25°
99±9
ND
108±3
87±4
100±3
114± 30
113±6
ND
29°
96±4
99±5
99±3
92±3
104±2
100± 3
133±30
ND
180 MAT
PAT
parental
22° MAT
PAT
temperature
25° MAT
PAT
29° MAT
PAT
parental
temperature
18°
22
25°
29°
culture
18
94±3
42±4
85±4
27±3
80±4
30±2
91±5
ND
262
Table 33. Imprinting of the Dp(1,’f)LJ9 mini-chromosome: The effect of sodium
butryrate.
Cultures which generated g/Dp(1;f)LJ9 males, where the mini-chromosome was
transmitted either maternally or paternally, were raised on media supplemented with 0
or 200 mM sodium butyrate. In addition the parents were also raised on either 0 or 200
mM sodium butyrate. The amount of pigment in the eyes of these progeny was
determined by microflourimeter assay. All values are expressed as percent wild type
pteridine levels. The first set of numbers reflects the amount of pigment in yza
53 progeny with a maternally inherited mini-chromosome. The second
g
d/Dp(1;f)LJ9
set shows pigment levels of this genotype when the mini-chromosome is paternally
inherited. ND
=
not done.
Crosses:
Maternal cross: X’X/Dp(1;f)LJ9
53
g
f
1
® y za d/y
—*
53
g
y za d/Dp(1;f)LJ9cJ
d/y
5
3 za g53d
Paternal cross: X’Y/Dp(1;f)LJ9o’ ® y za g
—
263
yza d/Dp(1;f)LJ9cJ
53
g
Imprinting of the Dp(1;f)LJ9 mini-chromosome: The effect of sodium butyrate.
parent concentration
progeny concentration
0 mM
0 mM
0 mM
200 mM
200 mM
200 mM
200 mM
0 mM
Maternal minichromosome
Paternal minichromosome
87±6
64±6
67±3
77±3
40±2
31 ±2
ND
71 ±5
264
Table 34. Imprinting of the Dp(1;f)LJ9 mini-chromosome: The effect of Y chromosome
dosage.
Four tests of the effect of the Y chromosome on imprinting of Dp(1;f) LJ9 were
performed.
1. The direct paternal test was intended to determine if an additional Y chromosome in
53
g
the Dp bearing father affected imprinting. Genotypically identical y za d/Dp(1,’f)LJ9
progeny, both with a paternally transmitted mini-chromosome, where generated from
an XY
+
Dp and an XYY
+
Dp fathers (first and second data columns). The results were
assessed by visual inspection and by microflourimeter pigment assay (first and second
data row, values are expressed as percent wild type pteridine levels). y za
53 progeny were distinguished from their y za g
g
d/Dp(1,.f)LJ9
d/Dp(7;f)LJ9/y
5
3 siblings
by progeny testing.
Cross: XX/Dp ® )Q’Y (any male)
X’)QY/Dp
0 X’Y/Dpo’
‘I,
53 za g5
g
d
3
XY/Dp or X”Y/Y/Dpo’ ®y za d/y
53
g
y za d/Dp(1;f)LJ9f
2. A direct maternal effect was tested by examining progeny derived from XX vs. XXY
d/Dp(1;f)LJ9
5
3 progeny,
duplication bearing mothers. Genotypically identical y za g
both with a maternally transmitted mini-chromosome, where generated from an XX
Dp and an XXY
+
+
Dp mothers (first and second data columns). The results were
assessed by visual inspection and by microflourimeter pigment assay, the latter values
d/Dp(1;f)LJ9
5
3 progeny
are expressed as percent wild type pteridine levels. y za g
d/Dp(7,.f)LJ9/y
5
3 siblings by progeny testing.
were distinguished from their yza g
265
Cross: XX/Dp ® any male
X”X/Dp or X”XJY/Dp
d/yçj&
5
3 y za d/Dp(1;f)LJ9cf
53
g
®y za g
3. The third test was of a paternal effect on the mini-chromosome variegation
53
g
unrelated to the mini-chromosome. Genotypically identical y za d/Dp(1;f)LJ9
progeny, both with a maternally transmitted mini-chromosome, where generated from
an XY and an XYY (no mini-chromosome) fathers (first and second data columns). The
results were assessed by visual inspection and by microflourimeter pigment assay, the
53
g
latter values are expressed as percent wild type pteridine levels. y za d/Dp(1;f)LJ9
53 siblings by progeny
g
progeny were distinguished from their yza d/Dp(1,.f)LJ9/y
testing.
Cross: X’Y/O
XY/Y
‘I
d/y
5
3 za d/Dp
53
g
X’Y/O vs. X’Y/Yd ® y za g
_>
53
g
y za d/Dp(1;f)LJ9c
4. The final test was of a maternal effect on the mini-chromosome variegation
53
g
unrelated to the mini-chromosome. Genotypically identical y za d/Dp(7;f)LJ9
progeny, both with a paternally transmitted mini-chromosome, where generated from
an XX and an XXY (no mini-chromosome) mothers (first and second data columns).
The results were assessed by visual inspection and by microflourimeter pigment
assay, the latter values are expressed as percent wild type pteridine levels. y za
53 siblings
g
d/Dp(1;f)LJg
5
g
3 progeny were distinguished from their y za d/Dp(7;f)LJ9/y
by progeny testing.
d/y
5
3 za g53d
cross: X’Y/Dp ® y za g
1
y za g53d/y za g53d or y za g53d/xy
53
g
® X’Y/Dpo’— y za d/Dp(7;f)LJ9
266
Imprinting of the Dp(1;f)LJ9 mini-chromosome: The effect of Y chromosome dosage.
1. Direct paternal effect
XJY/Dp vs. X/Y/Y/Dp
visual
estimate
pigment
assay
XY+Dp
father
XY+Y+Dp
father
0.10±0.02
0.12±0.02
41 ±2
48±3
3. Paternal Y effect
XYvs. XYYx Dp females
visual
estimate
pigment
assay
2. Direct maternal effect
X’X’/Dp vs. X’X//X’X/
XX+Dp
mother
XX+Y+Dp
mother
visual
0.92±0.01
estimate
pigment 100 ±3
assay
1.0±0
94±4
4. Maternal V effect
XXvs. XXY x Dp males
XY ® Dp
mother
XYY 0 Dp
mother
0.73 ± 0.03
1.0±0
75 ± 10
81 ± 10
XX 0 Dp XXY 0 Dp
father
father
visual
0.05 ± 0.02
estimate
pigment 25 ±5
assay
267
0.03 ± 0.03
34 ± 11
direct effect in the male, and female (both the Y chromosome and mini-chromosome
present in the same individual); and as an indirect maternal, and paternal effect (Y
chromosome and mini-chromosome not present in the same parent). The presence of
an extra Y chromosome did not alter the imprint regardless of whether it was present
with or without the mini-chromosome, or in males or females. There was no evidence
of a maternal or paternal Y chromosome effect on variegation as reported by Khesin
and Bashirov (1978). If we accept for the moment, that these modifiers of position effect
variegation act via heterochromatin, then it seems that heterochromatin is involved in
the somatic propagation of the imprint. Clearly an additional Y chromosome can
eliminate the garnet variegation, which is the signature of the imprint of the Dp(1;f)LJ9
mini-chromosome.
268
DISCUSSION-imprinting
In this chapter I have described a mini-chromosome in Drosophila melanogaster
which exhibits genomic, parent-specific or gamete-specific imprinting. The imprint is
independent of the sex of the progeny and is completely reversible. This imprint is
manifest as parent-dependent expression of the garnet eye colour gene, and of at
least two other genes on the mini-chromosome, tiny and narrow abdomen. Imprinting
of the mini-chromosome was first noted as the parent-specific expression of the garnet
gene. While other genes on the mini-chromosome also appear to be imprinted, the
imprint at the garnet gene is the most dramatic expression of the imprint because of its
striking mosaic phenotype in the eye. This mosaic phenotype arises because the
garnet gene is cell autonomous and the inactivation of the paternally derived garnet
allele is not complete. Imprinting of a non-cell autonomous gene would be manifest as
a quantitative difference in the level of gene expression which would be less readily
apparent.
The Dp(1;f)LJ9 mini-chromosome shows extensive variegation (inactivation) for the
garnet+ gene when it is transmitted by a male. When this mini-chromosome is
transmitted by a female the garnel gene is fully expressed. Thus according to the
terminology of Reik (1992) the mini-chromosome is paternally imprinted. It should be
noted that this terminology is arbitrary. There is no data to indicate whether imprinting
acts to inactivate otherwise active genes or visa versa or whether the ground state of
the garnet+ gene on the mini-chromosome is variegated or fully expressed.
The immediate cause of disruption of garnet expression is position effect variegation.
That the mosaic imprinted phenotype is due to conventional position effect variegation,
is shown by the response of this variegator to the standard factors which modify
269
position effect variegation. The variegation is unusual in that is dependent of the sex of
the transmitting parent thus the variegation itself is imprinted. As this example of
genomic imprinting involves position effect variegation, the mechanism whereby
position effect variegation causes gene inactivation is of considerable importance.
While number of models of the mechanism of position effect variegation have been
proposed (Fran kham 1988, Karpen 1994), there is experimental evidence to support
three general classes of models; the somatic elimination model, the nuclear
compartmentalization model and chromatin or heterochromatin formation models. I
have shown that neither the expression or the imprint of the garnet gene correlates
with under-representation of this gene (Figure 38), arguing against a role for somatic
elimination. Of the two other models, experimental evidence slightly favours the
chromatin formation model although a role for functional nuclear localization is
intriguing and can not be ruled out. These two models are discussed in more detail
below, in context of the mechanism of imprinting.
If we accept, for the moment, that position effect variegation is due to the illicit spread
of heterochromatin than this striking phenotype could arise from either, a parent
dependent ability to induce variegation, or from a parent-dependent difference in the
distance of spread or aggressiveness of the invading heterochromatin. The latter is
more likely for the following reasons. There is unlikely to be a parent specific
difference in the ability of the mini-chromosome to form centric heterochromatin. If this
were the case, increased non-disjunction of paternally derived mini-chromosome
would be expected. Frequent non-disjunction has been observed with another mini
chromosome which was broken near the centromere and may have variegated for
centromere function (Wines and Henikoff 1992). However, such mitotic non-disjunction
was not observed with the Dp(1;f)LJ9 mini-chromosome used in this study. Nor is the
imprint likely to result from parent specific recognition of heterochromatic boundary
270
sequences (if in fact such sequences exist) as the same sequence would be present
or absent on the mini-chromosomes whether it was derived from males or females.
Finally, the effect of temperature on the imprint suggests that the maternally derived
mini-chromosome can variegate under some circumstances, If individuals with a
maternally derived mini-chromosome are raised at high temperature a few individuals
show limited inactivation of the garnet gene (Table 29). Thus I propose that
variegation occurs on the mini-chromosome regardless of its parental origin, but the
distance over which heterochromatin spreads, and thus the likelihood of inactivating
the garnet reporter gene is dependent on the parental origin. This situation is
diagrammed in Figure 39. Implicit in this model is that the imprint is evident only
within a limited range along the mini-chromosome. Also implicit in this model is the
proposal that the imprint is nucleated with in the centric heterochromatin and spreads
distally. The resulting parent-dependent differences in heterochromatin formation
become less pronounced with distance from the centromere. This is in accordance
with the extent of the imprinting of the three neighboring genes, garnet, narrow
abdomen and tiny (Figure 35).
I tested a number of factors to determine the cause of the imprinting. The imprinting is
not allele specific. Nor is it a trivial artifact of maternal action of the marker gene,
garnet. More interestingly, the imprint does not seem to be due to maternallycontributed compensatory substances, induced by physiological stress, as has been
proposed for the imprinting effects associated with Huntington’s Chorea and fragile X
mental retardation (Bird, Caro and Pilling 1974, VanDyke and Weiss 1986,
respectively). Investigation of the role of heterochromatin in imprinting initially seemed
promising given the widely observed involvement of heterochromatin in imprinting and
position effect variegation. Tests of the stability and generation of the imprint in the
271
Figure 39. Model of the parent-dependent spread of heterochromatin responsible for
imprinting of the Dp(1;f)LJ9 mini-chromosome.
This figure diagrams the percent of cells showing phenotypic inactivation due to the
spread of heterochromatin as a function of distance from the heterochromatic
boundary. The effect of culture temperature is shown as altering the distance of this
spread rather than the occurrence of variegation.
272
Model of the mechanism of parent-dependent variegation of Dp(1;f)LJ9
100%
variegation of
maternally derived
mini-chromosome
variegation of
paternally derived
mini-chromosome
percent
inactive
cells
0%
Distance from heterchromatic boundary
garnet tiny narrow
abdomen
273
presence of environmental, chemical and genetic factors which modify position effect
variegation, and are proposed to modify heterochromatin formation and integrity,
suggest that at least in this instance of imprinting, the role of heterochromatin is
restricted to the somatic expression of the imprint but is not involved in the
establishment of the imprint.
While these data might challenge the role of chromatin structure in initiation the
imprint, heterochromatin formation is clearly necessary for the somatic manifestation or
maintenance of the imprint. This was shown by the response of the variegation of the
mini-chromosome to conventional modifiers of position effect variegation. Three
factors which generally modify position effect variegation (temperature, butyrate, and Y
chromosome dosage) altered the variegation of this mini-chromosome. The effects of
these modifiers deserve additional comment. The effect of temperature is the reverse
of the conventional effect. Usually high temperature suppresses position effect
variegation. A “reverse” response to high temperature is not unprecedented by any
means (Spofford 1967), however, the other examples of reverse temperature
sensitivity have been attributed to temperature sensitive hypomorphic alleles on the
non-variegating homolog. This cannot be the case for the garnet variegation on the
mini-chromosome as the temperature effect is seen with several alleles, none of which
are temperature sensitive. While the mechanism of position effect variegation is
unknown, the canonical response to temperature suggests that the process of
heterochromatin formation is limiting. The simplest interpretation of the reverse
response of the Dp(1;f)LJ9 variegation to temperature might suggest that it is
euchromatin formation which is limited in this chromosome. Why or how this might
occur is not clear. The unconventional response to butyrate supplemented media is
also interesting. Sodium butyrate has been proposed to suppress position effect
variegation by altering the de-acetylation of histones and so disrupting chromatin
274
condensation. If this is in fact occurring, it is not clear why this would enhance
heterochromatin formation on the mini-chromosome. Finally, the moderation of
variegation by temperature and sodium butyrate was quite modest. This difference in
effectiveness between modifiers of position effect variegation is in contrast to their
effect on conventional (non-parental) variegators (Clegg et al. 1992).
In contrast, the effect of an extra Y chromosome (X/Y/Dp versus X/Dp) was much more
dramatic, essentially eliminating variegation even for paternally derived minichromosomes. It may be that the Y chromosome effects variegation by mechanisms
distinct from that of the other modifiers. In this regard, Talbert and Henikoff (1994) have
argued that the suppressing effect of the Y chromosome on position-effect variegation
is due to occlusion of the heterochromatin-forming compartment of the nucleus rather
than directly on heterochromatin formation. The Y chromosome might also alter the
somatic pairing of the mini-chromosome. In contrast to these models, Zuckerkandel
(1974) proposed that the Y chromosome suppresses position effect variegation by
acting as a binding site for heterochromatic-specific proteins. It is possible that the Y
chromosome is a particularly potent suppresser of Dp(1;f)LJ9 variegation because it is
a powerful competitor for the specific heterochromatic proteins which bind to the minichromosome. One possible candidate might be the proteins which bind to the Stellate
gene cluster or proteins which bind to repetitive sequences near to the garnet gene
(see below). In the absence of data on the mechanism of position effect variegation,
any number of possibilities can be envisioned.
Why do only some variegators show parental effects?
All published parental effects in Drosophila involve position effect variegation (Table
26). This may indicate an obligatory requirement for the large scale chromosome
275
rearrangements associated with position effect variegation. This scenario is hard to
justify conceptually. Absence of published reports of parental imprinting of transgenes
in Drosophila, as is seen in mice, may reflect nothing more than investigators’ aversion
to non-Mendelian expression which defeats the purpose for which transgenes are
usually generated. There have been sporadic unpublished reports of parent
dependent transgene expression as seen in mice (C. Bazinet, personal
communication, C. Berg, personal communication).
Although all published parental effects in Drosophila (excluding conventional
maternal effects) involve variegating rearrangements, by no means is every example
of position effect variegation associated with parental effects (Spofford 1976). Another
oddity evident from Table 26 is that all the examples of parental effects in Drosophila
involve the X chromosome. They do not however share any other common
chromosomal element nor is the heterochromatin of the X chromosome always
involved. Thus this may be simply a spurious coincidence.
As imprinting amongst variegating rearrangements is decidedly a rarity, this raises the
question of what features distinguish those variegating rearrangements which do
show parent-specific variegation. The existence of cloned and characterized DNA
sequences from the vicinity of the garnet gene made this an obvious candidate to
investigate any sequence or larger scale peculiarities which might explain the
imprinting.
Specific sequence motifs within the garnet gene are unlikely to be the cause of the
parental imprinting. Although the 3’ region does contain a polyglutamine repeat
sequence which has been associated with some imprinted genes in humans (Green
1993) the repeat seems too short and inconsequential to explain the imprinted spread
276
of heterochromatin through at least three genes. The trinucleotide repeats found in the
fragile X syndrome, spinal and bulbomuscular atrophy, Huntington’s disease,
spinocereballar ataxia type I and myotonic dystrophy are present as between 5-50
copies in the normal alleles and 40-4000 copies in the imprinted disease causing
alleles. Large scale structural features of the mini-chromosome might also be
associated with the imprint. The Steilate genes are highly repetitive gene clusters
present on the X and Y chromosomes and degenerate members are present in the
proximal heterochromatin of the X chromosome (Shevelyov 1992, E. V.
Benevolenskaya, personal communication). The Stellate genes are highly transcribed
in spermatogenesis but are of unknown function (Livak 1990, Palumbo et al. 1994).
The proximity of Stellate sequences to the garnet gene, as well as possible
degenerate Steilate sequences in the X heterochromatin, may meant that the garnet
gene is flanked by multiple Stellate repeats. Given the propensity of heterochromatin
to engage in non-homologous pairing (Eberl, Duyf and Hilliker 1993, Talbert, LeCiel
and Henikoff 1994), the ability of repeated sequence to nucleate heterochromatin
formation (Dorer and Henikoff, 1994), the fact that many imprinted transgenes in
mammals are present as multiple repeats (Surani, Reik and Allen 1988) and the
presence of repeat sequence within a region of an imprinted transgene defined as the
cis-acting imprinting signal (Chaillet at al. 1995), the Stellate sequences might induce
aberrant heterochromatin structure or nuclear localization of the mini-chromosome
which could lead, somehow, to imprinting. Intriguingly, the euchromatic and
heterochromatic Stellate sequences can be seen to form ectopic fibres, which indicate
illicit pairing, in polyene chromosome preparations (Palumbo et al. 1994). The many
Stellate repeats on the Y chromosome might explain the remarkable effectiveness of
the Y chromosome in suppressing variegation of the mini-chromosome it these
sequences compete for binding proteins, Of course the presence, number and function
of the degenerate heterochromatic Stellate sequences in the mini-chromosome
277
remains to be confirmed. A similar argument could be made for any other middle or
highly repetitive sequences present near garnet and on the Y chromosome. In this
regard it is interesting that the 12DE region adjacent to garnet is peppered with
repetitive sequences (Leung et al. 1987).
Although the consequences of imprinting are dramatically manifest in development,
neither the evolutionary forces which lead to imprinting or the mechanisms are known.
The former issue has occupied the attention of many biologists, but in the absence of
data on the mechanism, all evolutionary scenarios remain completely speculative. The
mechanism of imprinting is of considerable interest in itself, and is also of medical
importance given the number of human diseases in which imprinting has been
implicated.
Mechanism of imprinting:
While it is evident that differences in packaging genetic material in eggs and sperm
may facilitate differential gene expression in early embryogenesis, a simple response
to the different physiology and morphology of the germ cell formation and structure
may not suffice as a mechanism for imprinting. In any case, this explanation fails to
address the question of why and how some genes show parent specific expression
whereas others do not. Based on observations that pairing, recombination and gene
conversion events coincide with condensation at pachytene stage of meiosis, Monk
and Grant (1990) and Hall (1990) have proposed that imprinting is a side effect of
these events. Details of how or why this might occur are unclear and as such this
model is difficult to test empirically. Functional differences in nuclear localization, in
various guises, has been proposed as a potential basis for imprinting.
278
Modulation of gene expression by nuclear compartmentalization has been inferred
from both cytological and genetic evidence. The existence of nuclear compartments is
indisputable but ascribing specific functions to them remains problematic. For
example, the nucleolus is an obvious example of a specialized nuclear compartment.
It has been suggested that other structures found in the nucleus, such as the coiled
bodies, perichromatin fibrils and interchromatin granules, correspond to other
functional compartments associated with splicing. Recent evidence, however,
suggests that this is not the case (Mattaj, 1994). The first intimation that nuclear
compartments might have specific functions in gene regulation stemmed from
observation of the specific orientation of chromosomes in the nucleus, with telomeres
and centromeres apposed to the nuclear membrane (RabI 1885). A similar
chromosome organization in yeast is correlated with variable gene inactivation
resembling position effect variegation (Laurenson and Rhine 1992), however, a causal
relationship between either telomere-nuclear membrane apposition in S. cerevisiae or
centromere-nuclear membrane apposition in S. pombe remains to be shown. Heslop
Harrison (1990) has proposed that imprinting results from differential location of the
two parental or species haploid sets of chromosomes in the zygotic nuclei. There are
many well documented and striking examples of differential location of the two
chromosome sets in plant inter-specific hybrids (summarized by Heslop-Harrison
1990) however, these studies did not correlate the location of the two genome sets
with gene expression. Several investigators have argued for a similar effect of nuclear
localization on gene expression in position effect variegation (Hessler 1957, Wakimoto
and Hearn 1990, Talbert, LeCiel and Henikoff 1994) however cytological correlations
are generally lacking. If a causal relationship between nuclear localization and gene
expression could be shown in position effect variegation, it might serve to support
hypotheses suggesting a connection between nuclear location and gene expression
in imprinting.
279
In mammals and plants, DNA methylation has long been known to be linked to gene
inactivation. There are correlations between methylation and the gene inactivation
resulting from imprinting in mammals. Unfortunately the convenience of determining
the methylation status of a gene has led to methylation being treated synonomously
with imprinting without other evidence of genetic activity. But, the correlation between
methylation and gene activity is by no means absolute. Such a correlation is seen in
only one of six examples of imprinted transgenes (Surani, Reik and Allen 1988) and it
has been shown in a number of cases that the decision to inactivate a gene occurs
before methylation is evident. The Hprt gene on the imprinted X chromosome is
inactivated several days before differential methylation is established (Lock, Takagi
and Martin 1987). Likewise, differential methylation of the imprinted genes H19, Igf2
and Igf2r does not generally persist through meiosis, thus can not constitute the initial
imprinting signal. For example, the parent-specific methylation associated with
imprinted expression of H19 occurs late in embryogenesis and is not propagated
through the male meiosis (Ferguson-Smith et al 1993). Most of the parent-specific
methylation pattern in the promoter and coding region of Igf2r/Mpr is established late
in embryogeneis and is erased in meiosis. There is, however, in this case, one site in
an intron which maintains its methylation status though the female meiosis (Stoger et
al 1993). In contrast, the imprinted gene Igf2 seems devoid of parent specific
methylation in the immediate vicinity of the gene, and the gene is identically
methylated in eggs and sperm (Sasaki et al 1992, Brandeis et al 1993). Thus
methylation seems not to constitute the initial decision or even an early event in
imprinting. The role of methylation in imprinting may be relegated to maintenance of
the decision. Finally, methylation is not causally involved paramutation in maize
(Patterson, Thorpe and Chandler 1993), a phenomenon that resembles imprinting, nor
does it correlate with either imprinting or heterochromatin formation in coccids
280
(Scarbrough, Hattman and Nur 1984) nor is it found in Dipteran insects where there
are striking examples of imprinting. Thus methylation is unlikely to play a role in this
case, or others, of imprinting in Drosophila.
The role of chromatin structure in imprinting is less experimentally tractable than that of
methylation, but, its near universal involvement in imprinting and the involvement of
position effect variegation in imprinting in Drosophila, makes it a logical candidate for
investigation. The role of heterochromatin formation in position effect variegation has
been extensively investigated. Evidence for a chromatin-based model of position effect
variegation stems from cytological, genetic and molecular work. A correlation between
gene inactivation and the cytological appearance of heterochromatin in the
corresponding region of the chromosome (Hartmann-Goldstein 1967, Spofford 1976)
has been observed for many genes. A role for chromatin formation in position effect
variegation is further suggested by the sensitively of position effect variegation in
Drosophila (and the seemingly related telomere mediated position effect in S.
cerevisiae) to alterations in histone levels. A number of other genetic modifiers of
position effect variegation have also proven to encode chromatin associated proteins
(reviewed by Orlando and Paro, 1995). Finally, alterations in nucleosome positioning,
phasing and chromatin accessibility have been found associated with position effect
variegation (Wallrath and Elgin, 1995). Thus there is reasonable evidence to support
the assertion that gene inactivation associated with position effect variegation is
related to changes in chromatin structure. Tartof and Bremer (1990) have extended
these observations to imprinting and have made the rather stringent prediction that
imprinted regions will correspond to regions of intercalary heterochromatin. No ectopic
pairing sites, a sign of intercalary heterochromatin have been reported around the
garnet gene. Nevertheless, the role of chromatin structure in imprinting can not be
disregarded. The results presented in chapter 3 of this thesis imply that
281
heterochromatin in Drosophila may perform a maintenance function, similar to that
played by methylation in mammals. There is evidence that the maintenance stage in
mammals can be resolved into early and late stages. Intriguingly, preliminary evidence
suggests that the early maintenance stage may be mediated by chromatin binding
proteins (Monk 1988, Barlow 1994, Ohlsson, Barlow and Surani, 1994). This may
suggest a parallel between the maintenance mechanisms of the imprint between
mammals and Drosophila. Different mechanisms of late somatic memory might simply
reflect differences in the life cycles of mammals versus Drosophila. Most cells of a
mammal continue to undergo cell division throughout the adult portion of life. The
continuation of cell division requires a high fidelity memory mechanism. Covalent
modification of DNA based by methylation, clearly, would fulfill this function. In
contrast, adult flies are mitotically quiescent, thus there might be less rigorous
requirement for the memory mechanism and a “chaotic” system such a chromatin
partitioning might suffice.
A major impediment to resolution of the precise role of heterochromatin in imprinting
is that the structure of chromatin and molecular architecture of heterochromatin
remains undefined, leaving any models about the role of heterochromatin necessarily
vague. One approach to resolving the mechanism of imprinting is to isolate genetic
modifiers of the imprinting process by screening for mutations which enhance,
eliminate or switch the parental specificity of imprinted genes. Genes which modify
imprinting would be expected to encode products which are components of the
machinery which recognizes the sexual context of the imprinted region and thus might
act in one sex only (which depends on whether gene activity or inactivity is the default
state). Some of these gene products would also have to recognize and isolate the
region to be imprinted, make the initial decision to imprint and then, at least initially,
propagate and enforce the imprinted state. Some progress has been made in
282
Table 35. Effect of attached versus free sex chromosomes on imprinting of
Dp(1;f)LJ9.
53 progeny, with either a paternally
g
Genotypically identical y za d/Dp(1;f)LJ9
transmitted mini-chromosome (columns 2 and 3) or a maternally transmitted minichromosome (columns 4 and 5), were generated from stocks with either attached sex
chromosomes (column 2 and 4) or freely segregating sex chromosomes (column 3
and 5). The results were assessed by visual inspection (first data row) and by
microflourimeter pigment assay (second data row, values are expressed as percent
53 progeny were distinguished from their
g
wild type pteridine levels). yza d/Dp(1;f)LJ9
d/Dp(1,.f)LJ9/y
5
3 siblings by progeny testing.
y za g
Crosses:
d/y
5
3 za g53d
53 ®y za g
g
paternal: X’Y/Dp or y za d/y/Dpo’
53 d/Dp
g
53
g
maternal: X’X/Dp or yza d/yza
yza d/yor
53 X’Y/OO’
g
283
Effect of attached versus free sex chromosomes on imprinting of Dp(1;f)LJ9.
Paternal cross
attached
chromosomes
visual
0.10 ± 0.02
estimate
pigment 41±2
assay
free
chromosomes
Maternal cross
attached
chromosomes
free
chromosomes
0.071 ± 0.03
0.92 ± 0.01
0.73 ± 0.03
37±9
100±3
85±3
284
identifying the location of imprinting genes (imprinting genes are those genes which
control the imprinting process as opposed to imprinted genes) in mice, (Allen, Norris
and Surani 1990, Babinet et al 1990, Cattanach and Beechey 1990, DeLoia and
Softer 1990, Reik, Howlett and Surani 1990, Surani et al 1990, Engler et al 1991,
Foreijt and Gregorova 1992 and Sapeinza et al 1992, Chaillet et al. 1995) and
humans (Sapienza -personal communication). However these genes have not been
otherwise characterized. As imprinting in mammals is early acting and an important
developmental process, the genes which effect it are likely to be both pleiotropic and
lethal when mutant. Such genes cannot easily be detected by crossing different mice
strains each homozygous for potential modifier genes. Human genetics poses similar
but more extreme problems. For the purpose of identifying, cloning and characterizing
such genes, the sophisticated genetic and molecular tools available in Drosophila
would make this organism an excellent system for examining and dissecting imprinting
phenomenon. The existence of genetic modifiers of imprinting in Drosophila is
suggested by the data shown in Table 35. This table shows that the extent of the
imprint differs slightly, but reproducibly, when the mini-chromosome is transmitted by
females with free versus attached X chromosomes. This difference in the extent of
variegation may represent segregation of sex linked imprinting genes with minor
effects in the genetic background of these two strains.
In summary, the garnet gene has been used as a tool to examine various biological
processes. This chapter describes a mini-chromosome which is subject to parent
specific imprinting. This imprint is manifest as a parent-dependent variegation of the
garnet gene. The result is imprinted expression of the garnet gene. The genetic and
molecular information on the garnet gene allowed me to test several factors which
have been postulated to cause imprinting. The primary finding is that heterochromatin,
which is implicitly involved in imprinting in a wide variety of organisms, may act as a
285
memory mechanism, not a primary determinant of the imprint. If heterochromatin is
relegated to a “memory” mechanism, as is methylation in mammals, then there must
be an independent system which is involved in establishing the imprint. This process
is undoubtedly complex but isolation of modifiers of imprinting should help to
illuminate the mechanics and possibly the evolutionary and developmental rational of
the phenomenon of genomic imprinting.
286
Bibliography
Allen, N. D., D. G. Cran, S. C. Barton, S Hettle, W. Reik and M. A. Surani. 1988.
Transgenes as probes for active chromosomal domains in mouse development.
Nature 333: 852-855.
Allen, N. D., M. L. Norris and M. A. Surani. 1990. Epigenetic control of transgene
expression and imprinting by genotype-specific modifiers. Cell 61: 853-861.
Altenburg, L. S. and E. Alternberg. 1959. The mutagenicity of 2,5-bis-ethylene
hydroquinone in Drosophila. Genetics 44: 498.
Babinet, C., V. Richoux, J.-L. Guénet and J.-P. Renard. 1990. The DDk inbred
strain as a model for the study of interactions between parental genomes and
egg cytoplasm in mouse pre implantation development. p. 81-87 In: Genomic
imprinting (development 1990 supplement). Ed: M. Monk and A. Surani.
Company of Biologists Ltd., 1990, Cambridge.
Baker, W. K.. 1963. Genetic control of pigment differentiation in somatic cells.
Am. Zool. 3: 57-69.
Baker, W. K. and J. B. Spofford. 1959. Heterochromatic control of position-effect
variegation in Drosophila. Biological Contributions. Univ. Texas PuIb.
5914:135-154.
Barlow, D. P. 1994. Imprinting: a gamete’s point of view. Trends Genet. 10: 194199.
Bartolomei, M. S., A. C. Webber, M. E. Brunkow and S. M. Tilghman. 1993.
Epigenetic mechanisms underlying the imprinting of the mouse H19 gene.
Genes and Devel. 7: 1663-1673.
Beadle, G. W. 1937a. Development of eye colors in Drosophila: Fat bodies and
malpighian tubes in relation to substances. Genetics 22:587-611.
Beadle, G. W. 1 937b. The inheritance of the color of malpighian tubes in
Drosophila melanogaster. Amer. Nat. 71: 277-279.
Beadle, G. W. and B. Ephrussi. 1935a. Différenciation de Ia couleur de l’oeil
cinnabar chez a Drosophile (Drosophila melanogaste,). Comptes Rendus
Acad. Sci. Paris 201: 642-646.
Beadle, G. W. and B. Ephrussi. 1935b. Transplantation in Drosophila. Proc. Nat.
Acad. Sci. (USA) 21:642-646.
Beadle, G. W. and B. Ephrussi. 1936. The differentiation of eye pigments in
Drosophila as studied by transplantation. Genetics 21: 225-247.
287
Beadle, G. W. and B. Ephrussi. 1937. Development of eye colors in Drosophila:
Diffusible substances and their interrelations. Genetics 22: 76-86.
Bel, Y. and J. Ferré. 1986. Biosynthesis of pteridines and metabolism of
aromatic amino acids in Drosophila melanogaster. p. 335-338 In: Chemistry
and biology of pteridines. Walter de Greuyer and Co. Berlin, N.Y.
Belote, J. M. and J. C. Lucchesi. 1980. Male-specific lethal mutations of
Drosophila melanogaster. Genetics 96: 165-186.
Benzer, S. 1955. Fine structure of a genetic region in bacteriophage. Proc. NatI.
Acad. Sci. USA. 41 :344-354.
Bingham, P. M., and B. H. Judd. 1981. A copy of the copia transposable element
is very tightly linked to the wa allele of the white locus of D. melanogaster. Cell
25: 705-711.
Birchier, J. A., U. Bhadra, L. Rabinow, R. Linsk and A. T. Nguyen-Huynh. 1994.
Weakener of white (Wow), a gene that modifies the expression of the white eye
color locus and that suppresses position effect variegation in Drosophila
melanogaster. Genetics 137: 1057-1070.
Bird, E. D., A. J. Caro and J. B. Pilling. 1974. A sex related factor in the
inheritance of Huntington’s chorea. Ann. Human Genet. 37: 255-260.
Blakeslee, R. W. 1938. Report of the Director. Carnagie Institute of Washington
Yearbook. 42: p156-i 61.
Bonse, A.. 1967. Untersuchungen uber die chemische Natatur und die Bildung
der Harnkong lomerate in die Malpighischen Gefassen der Mutante rosy von
Drosophila melanogaster. Z. Naturforsh. 22: 1027-1029.
Brandeis, M., T. Kafri, M. Ariel, J. R. Chaillet, J. McCarrey, A. Razin and H.
Cedar. 1993. The ontogeny of allele-specific methylation associated with
imprinted genes in the mouse. EMBO J. 12: 3669-3677.
Brehme, K. S. and M. Demerec. 1942. A survey of malpighian tube color in the
eye color mutants of Drosophila melanogaster. Growth 6: 351-355.
Bridges, C. B. 1916. Non-disjunction as proof of the chromosome theory of
heredity. Genetics 1:1-52 and 107-163.
Bridges, C. B. and K. A. Breme. 1944. The mutants of Drosophila melanogaster.
Carnagie Inst. Wash. PubI. 552.
Brown, G. M. 1989. Biosynthesis of H4Biopterin and related compounds. In
Biology and Chemistry of Pteridines. Ed: H.-Ch. Curtius, S. Ghisla, N. Blau.
Walter de Gruyter and Co. Berlin, N. York. p. 199-21 2.
288
Brown, G. M. and C. L. Fan. 1975. The synthesis of pterins catalyzed by
enzymes from Drosophila melanogaster. In: Chemistry and Biology of
Pteridines. Ed: W. Pfleiderer. Walter de Gruyter, Berlin, N. York.
Brown, G. M., G. G. Krivi, C. L. Fan, and T. R. Unnasch. 1978. The biosynthesis
of pteridines in Drosophila melanogaster. In Biology and Chemistry of
Pteridines. Ed: Kisliuk and Brown. Elsevier North Holland, Inc. p. 81-86.
Brun, L. 0., P. Borsa, V. Gaudichon, J. J. Stuart, K. Aronstein, C. Coustau and R.
H. ffrench Constant. 1995. Functional haplodiploidy. Nature 374:506.
Calatayud, M. T., D. A. Jacobson and J. Ferré. 1989. New eye colour mutants
affecting the biosynthesis of pteridine and ommochromes in Drosophila
melanogaster. In Chemistry and Biology of Pteridines. Ed: H.-Ch. Curtius, S.
Ghisla and N. Blau. Walter de Gruyter and Co. N. York, Berlin. p. 591 -594.
Campos-Ortega, J. A. 1988. Cellular interactions during early neurogenesis of
Drosophila melanogaster. Trends Neurosci. 11: 400-405.
Carlson, E. A. 1959. Comparative genetics of complex loci. Quart. Rev. Biol.
34:33-67.
Casteel, D. B. 1929. Histology of the eyes of X-rayed Drosophila. J. Exp. Zool.
53: 373-385.
Castro-Sierra, E. and S. Ohno. 1968. Allelic inhibition at the autosomally
inherited gene locus for liver alcohol dehydrogenase in chicken-quail hybrids.
Biochem. Genet. 1: 323-335.
Cattanach, B. M. and C. V. Beechey. 1990. Autosomal and X-chromosome
imprinting. p. 53-72. In: Genomic imprinting (development 1990 supplement).
Ed: M. Monk and A. Surani. Company of Biologists Ltd., 1990, Cambridge.
Cattanach, B. M. and J. N. Perez. 1970. Parental influence on X-autosome
translocation-induced variegation in the mouse. Genet. Res. Camb. 15:43-53.
Chaillet, J. R., D. S. Badar and P. Leder. 1995. Regulation of genomic imprinting
by gametic and embryonic processes. Genes Dev. 9: 1177-1187.
Chandra, H. S. and S. W. Brown. 1975. Chromosome imprinting and the
mammalian X chromosome. Nature 253: 165-168.
Chandra, H. S. and V. Nanjundiah. 1990. The evolution of genomic imprinting.
p. 47-53. In: Genomic imprinting (development 1990 supplement). Ed: M. Monk
and A. Surani. Company of Biologists Ltd., 1990, Cambridge.
Chovnick, A. 1957. Pseudoallelism at the garnet locus in Drosophila
melanogaster. Genetics 42:365.
289
Chovnick, A. 1958. Aberrant segregation and pseudoalleleism at the garnet
locus in Drosophila melanogaster. Proc. Nati. Acad. Sd. USA. 44:333-337.
Chovnick, A. 1958. Structural and functional aspects of pseudoallelism in
Drosophila melanogaster. Proc. 10th mt. Cong. Genetics. 2:49-50.
Chovnick, A. 1961. The garnet locus in Drosophila melanogaster. 1.
Pseudoallelism. Genetics 46:493-507.
Chovnick, A. 1989. Intragenic recombination in Drosophila: The rosy locus.
Genetics 123:621-624.
Chovnick, A., A. Schalet, R. P. Kemaghan and M. Krauss. 1964. The rosy cistron
in Drosophila meIanogaster genetic fine structure analysis. Genetics 50:12451259.
Chovnick, A., R. J. Lefkowitz and D. R. McQuinn. 1956. Complexity at the garnet
locus in Drosophila melanogaster. Genetics 41:637.
Christensen, H. N. 1973. On the development of amino acid transport systems.
Fed. Proc. 32: 19-37.
Clarke, A. 1990. Genetic imprinting in clinical genetics. p. 131-139. In: Genomic
imprinting (development 1990 supplement). Ed: M. Monk and A. Surani.
Company of Biologists Ltd., 1990, Cambridge.
Cohen, J.. 1962. Position-effect variegation at several closely linked loci in
Drosophila melanogaster. Genetics 47:647-659.
Cremer-Bartels, G. 1975. Pteridines in the mammalian retina and light effects. p.
861-870. In: The chemistry and Biology of Pteridines. Ed: W. Pfleiderer. Walter
de Gruyter, Berlin, N. York.
Crouse, H. V.. 1960. The controlling element in sex chromosome behavior in
sciara. Genetics 45: 1429-1443.
DeLoia, J. A. and D. Solter. 1990. A transgene insertional mutation at an
imprinted locus in the mouse genome. p. 73-79. In: Genomic imprinting
(development 1990 supplement). Ed: M. Monk and A. Surani. Company of
Biologists Ltd., 1990, Cambridge.
Demakova, 0. V. and E. S. Belyaeva. 1988. Effect of mating direction on the
7 in Drosophila melanogaster. Dros.
position effect variegation of T(1;2)dorvar
Info. Service. 67:19-20.
Dobzhansky, T. 1946. Genetics of natural populations. XIII. Recombination and
variability in populations of Drosophila pseudoobscura. Genetics 31: 269-290.
290
Dorer, D. R. and S. Henikoff. 1994. Expansions of transgene repeats cause
heterochromatin formation and gene silencing in Drosophila. Cell 77:993-1002.
Dorn, R., J. Szidonya, G. Korgo, M. Schnert, H. Taubert, E. Archoukieh, B.
Tschiersch, H. Morwietz, G. Wustmann, G. Hoffman and G. Reuter. 1993. P
transposon-induced dominant enhancer mutations of position-effect variegation
in Drosophila melanogaster. Genetics 133: 279-290.
Dorn, R., V. Krauss, G. Reuter and H. Saumweber. 1993. The enhancer of
position-effect variegation of Drosophila, E(var)3-93D, codes for a chromatin
protein containing a conserved domain common to several transcriptional
regulators. Proc. Nat. Acad. Sci. USA 90:11376-11380.
Dorsett, D. L., J. J. Yim and K. B. Jacobson. 1978. Biosynthesis of drosopterins
in the head of Drosophila melanogaster. In: The chemistry and Biology of
Pteridines. Ed: R. L. Kisliuk and G. M. Grown. Elsevier, North Holland. p. 99-104.
Dressen, T. D., D. H. Johnson and S. Henikoff. 1988. The brown protein of
Drosophila melanogaster is similar to the white protein and to components of
active transport complexes. Mol. Cell Biol. 8:5206-5215.
Dryja, T. P., S. Mukai, R. Petersen, J. M. Rapaport, D. Walton and D. W. Yandell.
1989. Parental origin of mutations of the retinoblasoma gene. Nature 339: 556558.
Eberl, D. F., B. J. Duyf and A. J. Hilliker. 1993. The role of heterochromatin in the
expression of a heterochromatic gene, the rolled locus of Drosophila
melanogaster. Genetics 134: 277-292.
Engler, P., D. Haasch, C. A. Pinkert, L. Doglio, M. Glymour, R. Brinster and U.
Storb. 1991. A strain-specific modifier on mouse chromosome 4 controls the
methylation of independent transgene loci. Cell 65: 939-947.
Ephrussi, B. and G. W. Beadle. 1935a. La transplantation des disques
imaginaus chez Ia Drosophile. Comptes Rendus Acad. Sci. Paris. 201: 98-99.
Ephrussi, B. and G. W. Beadle. 1935b. La transplantation des ovaires chez Ia
Drosophile. Bull. Biol. 69: 492-502.
Ephrussi, B. and G. W. Beadle. 1935c. Sur les conditions de
l’autodifferenciation des carateres mendeliens. Comptes Rendus Acad. Sci.
Paris. 201: 1148-1150.
Ephrussi, B. and G. W. Beadle. 1937a. Development of eye colors in
Drosophila: Production and release of cn+ substance by the eyes of different
eye color mutants. Genetics 22: 479-483.
291
Ephrussi, B. and G. W. Beadle. 1 937b. Development of eye colors in
Drosophila: Transplantation experiments on the interaction of vermilion with
other eye colors. Genetics 22: 65-75.
Ephrussi, B. and J. L. Herold. 1944. Studies of eye pigments of Drosophila. I.
Methods of extraction and quantitative estimation of the pigment components.
Genetics 29: 148-175.
Ferguson-Smith, A. C., H. Sasaki, B. M. Cattanach and M. A. Surani. 1993.
Parental-origin-specific epigenetic modification of the mouse H19 gene. Nature
362: 751-755.
Ferré, J., F. Silva, M. D. Real and J. L. Mensua. 1983. Comparative study of the
eye colour mutants of Drosophila meIanogaster Quantitation of the eyepigments and related metabolites. In Chemistry and Biology of Pteridines. Ed: J.
A. Blair. Walter de Gruyter and Co. N. York, Berlin. p. 669-673.
Ferré, J., F. Silva, M. D. Real and J. L. Mensua. 1986. Pigment patterns in
mutants affecting the biosynthesis of pteridines and xanthomatin in Drosophila
melanogaster. Biochem. Genet. 24: 545-569.
Flavell, R. B. and M. O’Dell. 1990. Variation in inheritance cytosine methylation
patterns in wheat at the high molecular weight glutinin and ribosomal RNA gene
loci. p. 15-20. In: Genomic imprinting (development 1990 supplement). Ed: M.
Monk and A. Surani. Company of Biologists Ltd., 1990, Cambridge.
Forejt, J. and S. Gregurova. 1992. Genetic analysis of genomic imprinting: An
imprintor- 1 gene controls inactivation of the paternal copy of the mouse Tme
locus. Cell 70: 443-450.
Frankham, R. 1988. Molecular hypotheses for position-effect variegation: antisense transcription and promotor occlusion. J. Theor. Biol. 135: 85-1 07.
Fuyama, Y. 1984. Gynogenesis in Drosophila melanogaster. Jpn. J. Genetics
59: 91-96.
Gilchrist E. J. and D. G. Moerman. 1992. Mutations in the sup-38 gene of
Caenorhabditis elegans suppress muscle-attachment defects in unc-52
mutants. Genetics 132: 431-442.
Glaser, R. L, and A. C. Spradling. 1994. Unusual properties of genomic DNA
molecules spanning the euchromatic heterochromatic junction of a Drosophila
minichromosome. Nuc. Acids. Res. 22: 5068-5075.
-
Glass, H. B. 1934. A study of dominant mosaic eye-color mutants on Drosophila.
I. Phenotypes and loci involved. Amer. Nat. 68: 107-114.
Glassman, E. 1956. Kynurenine formamidase in mutants of Drosophila.
Genetics 41: 5666-574.
292
Goldberg, A., A. Schalet and A. Chovnick. 1962. On the lethality of double
3 and various ry mutant alleles. Dros. Info. Serv. 36: 67-68.
mutants of Hnr
Green, H. 1993. Human genetic diseases due to codon reiteration: Relationship
to an evolutionary mechanism. Cell 74: 955-956.
Green, M. M. 1959. Spatial and functional properties of pseudoalleles at the
white locus in Drosophila melanogaster. Heredity 13:303-315.
Gumaratne, P. H., A. Mansukhani, S. E. Lipari, H. C. Loiu, P. W. Matindale and
M. L. Goldberg. 1986. Molecular cloning, germ-line transformation and
transcriptional analysis of the zeste locus of Drosophila melanogaster. Proc.
Nat. Acad. Sd. USA 83: 701-705.
Gunaratne, P. H., M. Nakao, D. H. Ledbetter, J. S. Sutcliffe and A. C Chinault.
1995. Tissue-specific and allele-specific replication timing controls in the
imprinted human Prader-Willi syndrome region. Genes Dev. 9: 808-820.
Haas, 0. A., A. Argyriou-Tirita and T. Lion. 1991. Parental origin of the
chromosomes involved in the translocation t(9;22). Nature 359: 414-41 6.
Hadchouel, M., H. Farza, D. Simon, P. Tiollais and C. Pourcel. 1987. Maternal
inhibition of hepatitis B surface antigen gene expression in transgenic mice
correlates with de novo methylation. Nature 329: 454-456
Hadorn E. 1962. Fractionating the fruitfly. Sci. Am (April): 101-110.
Hadorn, E. and H. K. Mitchell. 1951. Properties of mutants of Drosophila
melanogaster and changes during development as revealed by paper
chromatography. Proc. Nat. Acad. Sci. 37: 650-665.
Hall, J. G. 1990. How imprinting is relevant to human disease. p. 141-148. In:
Genomic imprinting (development 1990 supplement). Ed: M. Monk and A.
Surani. Company of Biologists Ltd., 1990, Cambridge.
Hardy, R. W., D. L. Lindsley, K. J. Livak, B. Lewis, A. L. Siversten, G. L. Joslyn, J.
Edwards and S. Bonaccorsi. 1984. Cytogenetic analysis of a segment of the Y
chromosome of Drosophila melanogaster. Genetics 107: 591-610.
Harnly, M. H. and B. Ephrussi. 1937. Development of eye colors in Drosophila:
Time of action of body fluid on cinnabar. Genetics 22:393-401.
Hartmann-Goldstein, I. J.. 1967. On the relationship between
heterochromatinization and variegation in Drosophila, with special reference to
temperature sensitive periods. Genet. Res. 10: 143-1 59.
Hayman, D. L. and R. H. Maddern. 1969. Report-D. melanogaster-new mutants.
Dros. Info. Service 44:69.
293
Hearl, W. G., D. Dorsett and K. B. Jacobson. 1983. The common precursor of
sepiapterin and drosopterin in Drosophila: enzymatic and chemical synthesis.
In Chemistry and Biology of Pteridines. Ed: J. A. Blair. Walter de Gruyter and Co.
N. York, Berlin. p. 397-401.
Hearl, W. G., K. B. Jacobson. 1984. Eye pigment granules of Drosophila
melanogaster. Isolation and characterization for synthesis of sepiapterin and
precursors of drosopterin. Insect Biochem. 14:329-335.
Henikoff, S., D. Nash, R. Hards, J. Bleskan, J. F. Wodford, F. Naguib and D.
Patterson. 1986. Two Drosophila melanogaster mutations block successive
steps of de novo purine synthesis. Proc. Nat. Acad. Sci. USA 83: 3919-3923.
Heslop-Harrison. 1990. Gene Expression and parental dominance in hybrid
plants. p. 21-28. In: Genomic imprinting (development 1990 supplement). Ed: M.
Monk and A. Surani. Company of Biologists Ltd., 1990, Cambridge.
Hess 0. 1970. Independence between modification of genetic position effects
and formation of Iampbrush loops by the Y chromosome of Drosophila hydel.
Mol. Gen. Genet. 107: 224-242.
Hessler, A. 1958. V-type position effects at the light locus in Drosophila
melanogaster. Genetics 43: 395-403.
Hessler, A. Y.. 1961. A study of parental modification of variegated position
effects. Genetics 46: 463-484.
Hexter, W. H. 1956. Pseudoallelism at the g locus. Dros. Info. Serv. 30:121.
Hexter, W. M. 1958a Probable gene conversion in Drosophila. Proc. 10th Int.
Cong. Genetics. 2:120.
Hexter, W. M. 1 958b. On the nature of the garnet locus in Drosophila
melanogaster. Proc. NatI. Acad. Sci. USA. 44:768-771.
Hilliker, A. J., B. Duyf, D. Evans and J. P. Phillips. 1992. Urate-null rosy mutants
of Drosophila melanogaster are hypersensitive to oxygen stress. Proc. Nat.
Acad. Sci. 89: 4343-4347
Holliday R. 1990. Genomic imprinting and allelic exclusion. p. 125-129. In:
Genomic imprinting (development 1990 supplement). Ed: M. Monk and A.
Surani. Company of Biologists Ltd., 1990, Cambridge.
Holliday, R. 1987. The inheritance of epigenetic defects. Science 238: 163-170.
Hopkins, F. G. 1889. Note on yellow pigments in butterflies. Nature 40: 335.
294
Howland, R., E. A. Glancy and B. P. Sonnenblick. 1937. Transplantation of wild
type and eye disks among four species of Drosophila. Genetics 22:434-442.
Janning, W. 1970. Bestimmung des heterochromatisierungsstodiums beim
white-positionseffekt mittles röntgen induzierter mitotischer rekombination in der
augenanlage von Drosophila melanogaster. Mol. Gen. Genet. 107: 128-149.
Johannsen, A. 0. 1924. Eye structure in normal and eye-mutant Drosophila. J.
Morph. 39: 337-350.
Jones, W.K. and J. M. Rawls. 1988. Genetic and molecular mapping of
chromosome region 85A in Drosophila melanogaster. Genetics 120: 733-742.
Jowett, T. 1986 Preparation of nucleic acids p.275-286. In: Drosophila a
practical approach. Ed. D. E. Roberts. IRL Press, Oxford, Washington.
-
Kajii, T. and K. Ohama. 1977. Androgenic origin of hydatidiform mole. Nature
268: 633-634.
Kamdor, K. P., M. E. Shelton and V. Finnerty. 1994. The Drosophila
molybdenum cofactor gene cinnamon is homologous to three EscherichIa coli
cofactor proteins and to the rat protein gephyrin. Genetics 137: 791 -801.
Karpen, G. H.. 1994. Position-effect variegation and the new biology of
heterochromatin. Curr. Op. Genet. and Dev. 4: 281-291.
Karpen, G. H. and A. C. Spradling. 1990. Reduced DNA polytenization of a
minichromosome region undergoing position-effect variegation in Drosophila.
Cell 63: 97-107.
Keith, T.P., M. A, Riley, M. Kreitman, R. C. Lewantin, D. Curtis and G. Chambers.
1987. Sequence of the structural gene for xanthine dehydrogenase (rosy locus)
in Drosophila melanogaster. Genetics 116: 67-73.
Kermicle, J. L. and M. Alleman. 1990. Gametic imprinting in maize in relation to
the angiosperm life cycle. p. 9-14. In: Genomic imprinting (development 1990
supplement). Ed: M. Monk and A. Surani. Company of Biologists Ltd., 1990,
Cambridge.
Khesin, R. B., and V. N. Bashkirov. 1978. Maternal influence upon the V-type
gene position effect in Drosophila melanogaster. Molec. Gen. Genet. 163: 327334.
KIar, A. J. 1987. Differentiatied parental DNA strands confer developmental
asymmetry on daughter cells in fission yeast. Nature 326: 466-470.
KIar, A. J. 1990. Regulation of fission yeast mating-type interconversion by
chromosome imprinting. p3-8. In: Genomic imprinting (development 1990
295
supplement). Ed: M. Monk and A. Surani. Company of Biologists Ltd., 1990,
Cambridge.
Kuhn, D. T. 1972. Report-D. melanogaster-new mutants. Dros. Info. Service
49:38.
Laird, C. D. 1987. Proposed mechanism of inheritance and expression of the
human fragile-X syndrome of mental retardation. Genetics. 117: 587-599.
Laird, C. D. 1990. Huntington’s disease: proposed mechanism of mutation,
inheritance and expression. Trends Genet. 6: 242-247.
Laurenson, P. and J. Rine. 1992. Silencers, silencing and heritable
transcriptional states. Microbiol. Rev. 56: 543-560.
Lederer, E. 1940. Les pigments des invertebres. Biol. Rev. Camb. Phil. Soc. 15:
273-306.
Lewis, E. B. 1951. Pseudoallelism and gene evolution Cold Spring Harbor
Symp. Quant. Biol. 16:159-174.
Linder, D., B. McCaw, X. Kaiser and F. Hecht. 1975. Parthenogenetic origin of
benign ovarian teratoma. New EngI. J. Med. 292: 63-66.
Lindsley, D. L. and G. G. Zimm. 1992. The genome of Drosophila melanogaster.
Academic Press. N.Y.
Livak, K. J.. 1990. Detailed structure of the Drosophila melanogaster Stellate
genes and their transcripts. Genetics 124: 303-31 6.
Lock, L. F., N. Takugi and G. R. Martin. 1987. Methylation of the Hprt gene in the
inactive X occurs after chromosome inactivation. Cell 48: 39-46.
Lucchesi, J. C. 1968. Synthetic lethality and semi-lethality among functionally
related mutants of Drosophila melanogaster. Genetics 59: 37-44.
Lyon, M. F. 1993. Epigenetic inheritance in mammals. Trends Genet. 9: 123128.
Mackay, W. J. and J. M. O’Donnell. 1983. A genetic analysis of the pteridine
biosynthitic enzyme, guanosine triphosphate cyclohydrolase, in Drosophila
melanogaster. Genetics 105: 35-53.
Mainx, F. 1938. Analyse der genwirkung durch faktoren kombination. Versuche
mit den augenfarbenfaktoren von Drosophila melanogaster. Z. lnductiv.
Abstammungs-Vererbungslehre (Mol. Gen. Genet.) 75: 256-276.
Mattaj, I, W.. 1994. Splicing in space. Nature 372:727-728.
296
Matzke, M. A. and A. J. M. Matzke. 1995. Homology-dependent gene silencing
in transgenic plants: what does it really tell us? Trends Genet. 11: 1-3.
McCarthy, A. and H. Nickla. 1980. Morphology of the carnation-light synthetic
lethal focus in Drosophila melanogaster. Experimentia 36:1361-1362.
McClintock, B. 1944. The relation of homozygous deficiencies to mutations and
allelic series in maize. Genetics 29: 478-502.
McLean, J. R., R. Boswell and J. O’Donnell. 1990. Cloning and molecular
characterization of a metabolic gene with developmental functions in
Drosophila. Genetics 126: 1007-1019.
Metz, C. W.. 1938. Chromosome behavior, inheritance and sex determination in
Sciara. Am. Naturalist 72:485-520.
Meyerowitz, E. M. and D. R. Kankel. 1978. A genetic analysis of visual system
development in Drosophila melanogaster. Dev. Biol. 62: 112-142
Modelell, J., W. Bender and M. S. Messelson. 1983. Drosophila melanogaster
mutations suppressible by the suppressor-of-Hairy-wing are insertions of a 7.3
kb mobile element. Proc. Nat. Acad. Sci. USA 80: 1678-1 682.
Monk, M. 1988. Genomic imprinting. Genes and Dev. 2: 921-925.
Monk, M. and M. Grant. 1990. Preferential X-chromosome inactivation, DNA
methylation and imprinting. In: Genomic imprinting (development 1990
supplement). Ed: M. Monk and A. Surani. Company of Biologists Ltd., 1990,
Cambridge.
Moore, T., and D. Haig. 1991. Genomic imprinting in mammalian development:
A parental tug-of-war. Trends Genet. 7: 45-49.
Mori, K. 1937. A study on the development of pigments in various eye color
mutants of Drosophila. Japan. J. Genet. 13: 81-99.
Moses, K., M. C. Ellis and G. M. Rubin. 1989. The glass gene encodes a zincfinger protein required by Drosophila photoreceptor cells. Nature 340: 531-536.
Mottus, R., R. Reeves and T. A. Grigliatti. 1980. Butyrate suppression of position
effect variegation in Drosophila melanogaster. Mol. Gen. Genet. 178: 465-469.
Muller, H. J. 1958. An androgenic homozygous male. Dros. Info. Serv. 32: 140.
Nájera, C. 1985. Report-D. melanogaster-new mutants. Dros. Info. Service
61 :21 5.
Narayanan, Y. and J. A. Weir. 1964. Paper chromatography of pteridines of
prune and clot stocks of Drosophila melanogaster. Genetics 50: 387-392.
297
Nash, W. G. 1971. deep orange and carnation: Another lethal gene combination
in D. m. Dros. Info. Serv. 47:73.
Nicholls, R. B., J. H. M. Knoll, M. G. Butler, S. Karam and M. Lalande. 1989.
Genetic imprinting suggested by maternal heterodisomy in non-deletion Prader
Willi syndrome. Nature 342: 281 -285.
Nickla, H. 1977. Maternal effects determine effect lethal phase of carnation-light
synthetic lethal in Drosophila melanogaster. Nature 268:638-639.
Nickla, H., T. Lilly and A. McCarthy. 1980. Gene activity in the carnation-light
synthetic lethal in Drosophila melanogaster. Experimentia 36:402-403.
Nolte, D. J. 1943. Appearance of unexpected eye colors. Dros. Info. Serv. 17:63.
Nolte, D. J. 1944. White of y wan interaction-product. Dros. Info. Serv. 18: 54.
Nolte, D. J. 1950. The eye pigmentary system of Drosophila: The pigment cells.
J. Genetics 50:79-99.
Nolte, D. J. 1 952a. The eye pigmentary system of Drosophila: II. Phenotypic
effects of gene combinations. J. Genetics 51:130-141.
Nolte, D. J. 1952b. The eye pigmentary system of Drosophila: III. The action of
eye-colour genes. J. Genetics 51:142-186.
Nolte, D. J. 1 954a. The eye pigmentary system of Drosophila: IV. The pigments
of the vermilion group of mutants. J. Genetics 52:111-126.
Nolte, D. J. 1954b. The eye pigmentary system of Drosophila: V. The pigments
of the light and dark groups of mutants. J. Genetics 52:127-135.
Nolte, D. J. 1955. The eye pigmentary system of Drosophila: VI. The pigments of
the ruby and red groups of mutants. J. Genetics 53:1-10.
Nolte, D. J. 1959a. The eye pigmentary system of Drosophila: VII. The white
locus. Heredity 13:219-231.
Nolte, D. J. 1959b. The eye pigmentary system of Drosophila: VIII. Series of
multiple alleles. Heredity 13:233-241.
Nolte, D. J. 1 959c. The eye pigmentary system of Drosophila: IX. Heterozygous
effects of eye-colour genes. Heredity 13:219-231.
Nur, U. 1970. Translocations between eu- and heterochromatic chromosomes,
and spermatocytes lacking a heterochromatic set in male mealy bugs.
Chromosoma 29: 42-61.
298
Nur, U. 1990. Heterochromatinization and euchromatinization of whole
genomes in scale insects (Coccoidea: Homoptera). P. 29-34. In: Genomic
imprinting (development 1990 supplement). Ed: M. Monk and A. Surani.
Company of Biologists Ltd., 1990, Cambridge.
Nur, U., J. H. Werren, D. G. Eickbush, W. D. Burke and T. H. Eichbush. 1988. A
“selfish” B chromosome that enhances its transmission by eliminating the
paternal genome. Science 240: 512-514.
Ohlsson, R., D. Barlow and A. Surani. 1994. Impressions of imprints. Trends
Genet. 10: 41 5-41 7.
Orlando, V. and R. Paro. 1995. Chromatin multiprotein complexes involved in
the maintenance of transcription patterns. Curr. Op. Genet. and Dev. 5: 174-179.
Pak, W. L., Grabowski, S. R. 1980. Physiology of the visual and flight systems.
In: Genetics and Biology of Drosophila, Ed. M. Ashburner and E. Noviski.
Academic Press, London, New York, Sidney, Toronto and San Francisco.
Palumbo, G., S. Bonaccorsi, L. G. Robbins and S. Pimpinelli. 1994. Genetic
analysis of Stellate elements of Drosophila melanogaster. Genetics 138: 11811197.
Parkhurst, S. M. and V. G. Corces. 1985. forked, gypsys and suppressors in
Drosophila. Cell 41: 429-437.
Paro, R. 1990. Imprinting a determined state into the chromatin of Drosophila.
Trends in Genetics 6: 116-121.
Patterson, G. I., C. J. Thorpe and V. L. Chandler. 1993. Paramutation, an allelic
interaction, is associated with a stable and heritable reduction of transcription of
the maize b regulatory gene. Genetics 135: 881 -894.
Payne, F. and M. Denny. 1921. The heredity of orange eye color in Drosophila
melanogaster. Am. Nat. 55: 377-381.
Pfleiderer, W. 1993. Natural pteridines A chemical hobby. In: Chemistry and
biology of pteridines and folates. p. 1-16. Eds: J. E. Ayling, M. G. Nair and C. M.
Baugh. Plenum Press, N. York.
-
Phillips, J. P and H. S. Forrest. 1976. Ommochromes and Pteridines. p. 541617. In: Genetics and Biology of Drosophila. Ed. Ashburner and Noviski,
Academic Press, N.Y.
Phillips, J. P, H. S. Forrest and Kulkarni. 1973. Terminal synthesis of
xanthommatin in Drosophila melanogaster. Ill. Mutational pleiotropy and
pigment granule association of phenoxazinone synthesis. Genetics 73: 45-56.
299
Pirrota, V., H. Stellar and M. P. Bozzetti. 1985. Multiple upstream regulatory
elements control the expression of the Drosophila white gene. EMBO J. 4:
3501-3508.
Prokofyeva-Belgovskaya, A. A.. 1947. Heterochromatinization as a change of
chromosome cycle. J. of Genetics 48:80-98.
Rabinow, L., A. T. Nguyen-Huyah and J. A. Birchler. 1991. A transacting
regulatory gene that inversely affects the expression of the white, brown and
scarlet loci in Drosophila Genetics 129: 463-480.
Rabi, C. 1885. Uber Zelltheilung. Morphologisches Jahrbuch 10: 214-330.
Rangunathang, R., W. A. Harris and C. S. Zuker. 1991. The molecular genetics
of invertebrate phototransduction. Trends Neurosci. 14: 486-493.
Ready, D. P. 1989. A multifaceted approach to neural development. Trends
Neurosci. 12: 102-110.
Real, M. D., J. Ferré and J. L. Mensua. 1985. Methods for the quantitative
estimation of the red and brown pigments of Drosophila melanogaster. Dros.
Info. Serv. 61:198-1 99.
Reame, A. G., D. A. Knecht and A. Chovnick. 1991. The rosy locus in Drosophila
melanogaster Xanthine dehydrogenase and eye pigments. Genetics 129:
1099-1109.
Reedy, J. J. and F. P. Cavalier. 1971. Epistasis in eye colors of Drosophila
melanogaster. J. Heredity 62:131-134.
Reik, W. 1992. Genomic imprinting in mammals. p. 203-229. In: Results and
problems in cell differentiation 18. Ed. W. Hennig. Springer-Verlag. Berlin,
Heidelberg.
Reik, W., S. K. Howlett and M. A. Surani. 1990. Imprinting by DNA methylation:
From transgenes to endogenous gene sequences. p. 99-106. In: Genomic
imprinting (development 1990 supplement). Ed: M. Monk and A. Surani.
Company of Biologists Ltd., 1990, Cambridge.
Renfranz, D. J. and S. Benzer. 1989. Monoclonal antibody probes discriminate
early and late mutant defects in development of the Drosophila retina. Dev.
Biol.. 136: 411-429.
Reuter, G., I. Wolff and B. Friede. 1985. Functional properties of the
heterochromatic sequences inducing m4 position-effect variegation in
Drosophila melanogaster. Chromosoma 93: 132-139.
Sambrook, J., E. F. Fritsch, and T. Maniatis. 1989. Molecular cloning: A
laboratory manual. Ed: C. Nolan, Cold Spring Harbor Laboratory Press, N.Y.
300
Sapienza, C. 1990. Sex-linked dosage-sensitive modifiers as imprinting genes.
p. 107-113. In: Genomic imprinting (development 1990 supplement). Ed: M.
Monk and A. Surani. Company of Biologists Ltd., 1990, Cambridge.
Sapienza, C., J. Paquette, P. Pannunzio, S. Albrechtson and K. Morgan. 1992.
The polar-lethal Ovum Mutant gene maps to the distal portion of the mouse
chromosome 11. Genetics 132: 241-246.
Sasaki, H., B. A. Jones, R. J. Chaillet, A. C. Ferguron-Smith, S. C. Barton, W.
Reik and M. A. Surani. 1992. Paternal imprinting: potentially active chromatin of
the repressed maternal allele of the mouse insulin-like growth factor II (IGF2)
gene. Genes Dev. 6: 1843-1 856.
Scarbrough, K., S. Hattman and U. Nur. 1984. Relationship of DNA methylation
level to the presence of heterochromatin in mealy bugs. Mol. Cell Biol. 4: 599603.
Schalet, A. 1957. Spontaneous mutations at specific X chromosome loci in
Drosophila melanogaster. Genetics 42:393.
Schalet, A. P. 1986. The distribution of, and complementation relationships
between spontaneous X-linked recessive lethal mutations recovered from
crossing long-term laboratory stocks of Drosophila melanogaster. Mutation Res.
163: 115-144.
Schmidtke, J. P. KuhI and W. Engel. 1976. Transitory hemizygosity of paternally
derived alleles in hybrid trout embryos. Nature 260: 31 9-320.
Schott, D. R., M. C. Baldwin and V. Finnerty. 1986. Molybdenum hydroxylases in
Drosophila. Ill. Further characterization of the low xanthine dehydrogenase
gene. Biochem. Genet.. 24:509-527.
Schultz, J. 1935. Aspects of the relation between genes and development in
Drosophila. Am. Nat. 69: 30-54.
Schwinck, I and L. Schwinck. 1972. Report-D. melanogaster-new mutants.
Dros. Info. Service 49:38.
Schwinck, I. 1975. Aurodrospterins in eye colour mutants of Drosophila
melanogaster. In: The chemistry and Biology of Pteridines. Ed: W. Pfleiderer.
Walter de Gruyter, Berlin, N. York. p. 919-929.
Schwink, I. 1978. Drosopterins and the in vivo modulation of their synthesis by
implantation of metabolites in eye colour mutants of Drosophila melanogaster.
In: The chemistry and Biology of Pteridines. Ed: R. L. Kisliuk and G. M. Grown.
Elsevier, North Holland. p. 141-146.
301
Searles, L. L. and R. A. Voelker. 1986. Molecular characterization of the
Drosophila vermillion locus and its suppressible alleles. Proc. Nat. Acad. Sci.
USA 83: 404-408.
Shepherd, J. C. W., U. Walldorf, P. Hug and W. J. Gehring. 1989. Fruit flies with
additional expression of elongation factor EF-1 oc live longer. Proc. Nat. Acad.
Sci. USA 86: 7520-7521.
Shevelyov, Y. Y.. 1992. Copies of a Stellate gene variant are located in the X
heterochromatin of Drosophila melanogaster and are probably expressed.
Genetics 132: 1033-1037.
Shoup, J. R. 1966. The development of pigment granules in the eye of wildtype
and mutant Drosophila melanogaster. J. Cell Biol. 29: 223-249.
Sinclair, D. A., A. A. Ruddell, J. K. Brock, N. J. Clegg, V. K. Lloyd and T. A.
Grigliatti. 1992. A cytogenetic and genetic characterization of a group of closely
linked second chromosome mutations that suppress position-effect variegation
in Drosophila melanogaster. Genetics 130: 333-344.
Spofford, J. B. 1967. Single-locus modification of position-effect variegation in
Drosophila melanogaster. I. white variegation. Genetics 57: 751-766.
Spofford, J. B.. 1959. Parental control of position-effect variegation: I. Parental
heterochromatin and expression of the white locus in compound-X Drosophila
melanogaster. Proc. Nat. Acad. Sci. 45: 1003-1007.
Spofford, J. B.. 1961. Parental control of position-effect variegation. II. Effect of
sex of parent contributing white-mottled rearrangement in Drosophila
melanogaster. Genetics 46:1151-1167.
Spofford, J. B.. 1976. Position-effect variegation in Drosophila. p. 955-1018. In
Genetics and Biology of Drosophila. Ed. Ashburner and Novitski. Academic
Press. N.Y.
Stewart, B. and J. R. Merriam. 1973. Segmental aneuploidy of the X
chromosome. Dros. Info. Service 50: 167-170.
Stoger, R., P. Kubicka, C.-G. Lui, T. Katri, A. Razin, H. Cedar and D. P. Barlow.
1993. Maternal-specific methylation of the imprinted mouse lgf2r locus identifies
the expressed locus as carrying the imprinting signal. Cell 73:61 -71.
Sturtevant, A. H. 1932. The use of mosaics in the study of the developmental
effects of genes. Proc. 6th Int. Cong. Genet., Vol. 1: 304-307.
Sturtevant, A. H. 1956. A highly specific complementary lethal system in
Drosophila melanogaster. Genetics 41: 118-123.
302
Sturtevant, A. H. and E. Novitski. 1941. The homologies of the chromosome
elements in the genus Drosophila. Genetics 26: 517-541.
Sturtevant, A. H., C. B. Bridges, T. H. Morgan, L. V. Morgan and J. U. Chili. 1929.
Garnet. p. 24 In: Contributions to the genetics of Drosophila simulans and
Drosophila melanogaster. Carnagie Inst. Wash. PubI. 399. Carnagie Inst.
Washington.
Sullivan, D. T. and M. C. Sullivan. 1975. Transport defects as the physiological
basis for eye color mutants of Drosophila melanogaster. Biochem. Genet. 13:
603-613.
Sullivan, D. T., R. J. Kitos and M. C. Sullivan. 1973. Developmental and
genetics studies on kynurenine hydroxylase from Drosophila melanogaster.
Genetics 75: 651-661.
Sullivan, D. T., S. L. Grillo, and R. J. Kitos. 1974. Subcellular localization of the
first three enzymes of the ommochrome synthetic pathway in Drosophila
melanogaster. J. Exp. Zool. 188: 225-234.
Surani, M. A., R. Kothary, N. D. Allen, P. B. Singh, R. Fundel, A. C. Reguson
Smith and S. C. Barton. 1990. Genomic imprinting and development in the
mouse. p. 89-98. In: Genomic imprinting (development 1990 supplement). Ed:
M. Monk and A. Surani. Company of Biologists Ltd., 1990, Cambridge
Surani, M. A., W. Reik and N. D. Allen. 1988. Transgenes as molecular probes
for genomic imprinting. Trends. Genet. 4: 59-62.
Taira, T. 1960. Is the double recessive Hflr3 ry homozygote a synthetic lethal?
Dros. Info. Service 34: 107.
Talbert, P. B., C. D. S. LeCiel and S. Henikoff. 1994. Modification of the
Drosophila heterochromatic mutation brownDomlflaflt by linkage alterations.
Genetics 136: 559-571.
Tartof, K. D. and M. Bremer. 1990. Mechanisms for the construction and
developmental control of heterochromatin formation and imprinted chromosome
domains. p35-45. In: Genomic imprinting (development 1990 supplement). Ed:
M. Monk and A. Surani. Company of Biologists Ltd., 1990, Cambridge.
Tautz, D. and C. Pfeifle. 1989. A non-radioactive in situ hybridization method for
the localization of specific RNAs in Drosophila embryos reveals transitional
control of the segmentation gene hunchback. Chromosoma 98:81-85.
Tearle, R. G., J. M. Belote, M. Mckewan, B. S. Baker and A. J. Howells. 1989.
Cloning and characterization of the scarlet gene of Drosophila melanogaster.
Genetics 122: 595-606.
303
Tearle, R.. 1991. Tissue specific effects of ommochrome pathway mutations in
Drosophila melanogaster. Genet. Res. Camb. 57:257-266.
Teng, D. H. F., C. M. Engele and T. ft Venkatesh. 1991. A product of the prune
locus of the Drosophila is similar to mammalian GTPase-activating proteins.
Nature 353: 437-440.
Thaker, H. M., Kankel, D. R. 1992. Mosaic analysis gives an estimate of the
extent of genomic involvement in the development of the visual system in
Drosophila melanogaster. Genetics 131:883-894.
Toguchida, J., K. Ishizaki, M. S. Sasaki, Y. Nakumara, M. Ikenaga, M. Kato, M.
Sugimot, Y. Kotoura and T. Yamamuro. 1989. Preferential mutation of the
paternally derived RB gene as the initial event in sporadic osteosarcoma.
Nature 338: 156-158.
Tomlinson, A. 1985. Cellular interactions in the developing Drosophila eye.
Development 104: 183-193.
Tower, J., G. H. Karpen, N. Craig and A. C. Spradling. 1993. Preferential
transposition of Drosophila P-elements to nearby chromsomal sites. Genetics
133: 347-359.
Valencia, R. M. 1966. Report-D. melanogaster-new mutants. Dros. Info. Service.
41:58.
Van Dyke, D. L., and L. Weiss. 1986. Maternal effect on intelligence in fragile X
males and females. Am. J. Med. Genet. 23: 723-737.
Varmuza, S. and M. Mann. 1994. Genomic imprinting
time bomb. Trends. Genet. 10: 118-123.
-
defusing the ovarian
Venkatesh, T. R., Zipursky, S. L., Benzer, S., 1985. Molecular analysis of the
development of the compound eye in Drosophila. Trends in nuerosci. 8:251257.
Wakimoto, B. and M. Hearn. 1990. The effects of chromosome rearrangements
on the expression of heterochromatic genes in chromosome 2L of Drosophila
melanogaster. Genetics 125: 141-151.
Walirath, L. L. and S. C. R. Elgin. 1995. Position effect variegation in Drosophila
is associated with an altered chromatin structure. Genes and Dev. 9:1263-1277.
Warner, C. K., D. T. Watts and V. Finnerty. 1980. Molybdenum hydroxylases in
Drosophila. I. Preliminary studies of pyridoxal oxidase. Mol. Gen. Genet. 180:
449-453.
Wennberg, R. A. 1988. A novel requirement for the X-chromosome in P-M
hybrid dysgenesis and the interaction of the garnet and enhancer of garnet loci
304
in Drosophila melanogaster. MSc. Thesis. University of British Columbia.
Canada.
Wiederrecht, G. J. and G. M. Brown. 1984. Purification and properties of the
enzymes from Drosophila melanogaster that catalyze the conversion of
dihydroneopterin triphosphate to the pyrimidodiazepine precursor of the
drosopterins. J. Biol. Chem. 259: 14121-14127.
Wiederrecht, G. J., D. R. Paton and G. M. Brown. 1984. Enzymatic conversion of
dihydroneopterin triphosphate to the pyrimido diazepine intermediate involved
in the biosynthesis of the drosopterins of Drosophila melanogaster. J. Biol.
Chem. 259:2 1 95-2200.
Wiley, K. and H. S. Forrest. 1981. Terminal Synthesis of xanthommatin in
Drosophila melanogaster. IV. Enzymatic and non enzymatic catalysis. Biochem.
Genet. 19:1211-1221.
Wines, D. R. and S. Henikoff. 1992. Somatic instablity of a Drosophila
chromosome. Genetics 131: 683-691.
Wright, 5. 1932. Complementary factors for eye color in Drosophila. Am. Nat.
66: 282-283.
Yamamoto, A. H., D. J. Komma, C. D. Shaffer, V. Pirrotta and S. A. Endow. 1989.
the claret locus in Drosophila encodes products required for eye color and for
meiotic chromosome segregation. EMBO J. 8: 3543-3552.
Zipursky, S. L. 1989. Molecular and genetic analysis of Drosophila eye
development: sevenless, bride of sevenless and rough. Trends in Neurosci.
12:183-1 89.
Zipursky, S. L., T. R. Vankatesh, D. B. Teplow and S. Benzer. 1984. Neuronal
development in the Drosophila retina: monoclonal antibodies as molecular
probes. Cell 36: 15-26.
Zuckerkandl, E. 1974. Recherches sur les proprietes et l’activité biologique de
Ia chromatine. Biochimie 56: 937-954.
305
Appendix 1.
Determination of eye pigment levels.
As the principal phenotype of the garnet and other eye colour genes described
in this thesis is alterations in eye pigments, it was necessary to accurately and
precisely quantify the amount of eye pigments.
A. Efficiency and Selectivity of pigment extraction.
The first step in quantitation of eye pigments is to extract these pigments
efficiently and specifically from the eye. The different biochemical properties of
the pteridine (red) and ommochrome (brown) pigments have been exploited to
differentially extract those pigments from fly eyes. A number of such procedures
have been published, that of Real, Ferré and Mensua (1985) was adapted for
this work. It was necessary to show that these procedures efficiently extracted
the intended pigments, either the pteridines or xanthommatin, without
contamination by the other class of pigment. Figure A shows the pigments
extracted from wild type (Oregon R), brown and vermilion mutants as a test of
the efficiency and selectivity of the extraction method. The brown mutant should
have no pteridine pigments whereas the vermilion should not have the
ommochrome pigment. The top series of data show that the procedure for
pteridine pigment extraction is both efficient and highly selective. Essentially no
ommochrome pigment is extracted. The second data series show that the
procedure for ommochrome pigment extraction is less selective, some pteridine
pigments are extracted. It also appears less efficient as the levels of the
306
ommochrome pigment, xanthommatin, extracted from the brown mutant are
consistently lower than from wild type.
The extraction method for pteridine pigments was not only more specific and
reproducible but considerably less time consuming than the method for the
ommochrome pigment. Consequently, only the pteridine pigments were
assayed in most experiments.
A.
Efficiency of red pigment extraction
Q
%O.R
100
<1%
98±5
Efficiency of brown pigment extraction
O.R.
average
reading
106±8
73±3
5±2
%O.R.
100±8
69±8
5±2
B. Contribution of the different pteridine pigments to total pteridine pigment
level.
Unlike the single ommochrome pigment, there are numerous (28+) pteridine
pigments, each of which may contribute to the reading obtained for the total
pteridine pigments. To determine the degree to which each of these pigments
contributed to the total fluorescence recorded for the pteridines,
307
chromatography plates on which the pteridine pigments had been partially
separated was scanned under UV illumination using the same conditions used
to quatitate total pteridine pigment levels. The percent of total fluorescence
attributed to each pigment is shown in Figure B. The major contribution is from
the drosopterin pigments (70%) and the second largest is from the unseparated
residue (15%). Based on the distinctive orange colour, florescence of the
residue is probably due largely to drosopterins. Thus approximately 85% of total
fluorescence is due to drosopterin pigments. While the system could probably
be optimized to preferentially detect other pigments by changing the extraction
technique and the UV illumination wavelength, the drosopterins are stable
molecules and a convenient indicator of pteridine pigment levels.
B.
Fluorescence of separated pteridine pigments.
percent total pigment
pigment
residue
drosopterin
mystery spot 1
mystery spot 2
isoxanthopterin
mystery spot 3
xanthopterin
sepiapterin
2-amino-4-hydroxypteridine
biopterin
isosepiapterin
15
70
2
1
1
2
2
1
1
1
5
C. Comparison between different methods of pteridine pigment quantification.
Most published methods of pigment quantification rely on a change in UV
absorbance as measured by spectrophotometer. I found this method to be too
cumbersome and slow to be suited for large numbers of pigment level
308
determinations. A method involving quantification of fluorescence by a
microflourimeter allowed rapid scanning of multiple spots of extracted pigments.
The top portion of Figure C shows a comparison between pigment levels
determined by spectrophotometric assay (Y axis) and by microflourometric
assay (X axis). Each reading represents the results of pigment quantification
from one group of frozen fly heads. The graph incorporates data from two
separate experiments. One set of data was produced by Kevin Swanson, and
undergraduate research assistant.
The relationship between the two methods seems to be slightly sinusoidal. This
indicates that at high pigment levels the spectrophotometric method provides
more discrimination. However, the lower portion of the graph shows that for low
pigment levels (<20% by the microflourometric method) that the
microflourometric method is the more accurate. These results are not
unexpected as the two methods exploit different properties of the pigments.
Nevertheless, the relationship between the two methods is roughly linear. The
greater speed afforded by the microflourometric assay and the lower variability
made the microflourometric assay the method of choice for pigment
quantitation.
Although the microflourometric assay was more sensitive than the
spectrophotometric assay, it still involved combining five heads to extract
sufficient pigment. Combining heads posses no problem for genotypes where
all individuals should have the same amount of pigment. For mosaic
phenotypes, such as the flies with variegating genotypes studied in chapter 3,
each individual has different amounts of pigmentation. As combining heads
would obscure these differences, I adapted the microflourometer assay to
309
measure pigment from individual heads. The lower figure shows a comparison
of microflourometer readings using one head (Y axis) and 5 heads (X axis).
Each assay was again performed on the same set of frozen heads and
incorporate two independent sets of experiments. One set of data was provided
by David Dyment, an undergraduate student working on a directed studies
project. The results of the two assays are roughly comparable. There is
however, discrepancy between the two assays systems at higher pigment
levels. The deviation appears as a random scatter about the mean suggesting
that it represents genuine differences between the pigment of individual heads
and the average reading of five heads. Data derived from uniformly pigmented
heads shows far less variation (data not shown).
310
Comparison between microflou rimeter and spectrophotometer
assays for pteridine pigment levels
10090
U,
c
V
(1$
a,
I
L.
a,
a,
E
a,
0
€0
0.
(I)
—
— —
n
0’)—
4
—
70
—
n_
-
—
I—
— —
.
‘—
—
—
—
.
—
—
—
—
50— — — — —
—
).4
40—
30--—-.‘.1
——
—I-
_I
2”
— —
I
10—
-
0—
—
0
—
—
10 20 30 40 50 60 70 80 90 100
microflourimeter readings (5 head assay)
+
Comparison between five and one head + +
microflourimeter assay
100
+
I ::41
90
÷
,
-
——
*4
*
—
80
70
60
50
0
E
a)
-c
1
40
30
20
10
0
:
-
-
-
--k--
+
+
+
+
—-÷-
L±
—
I’ll
—
—
—
—
—
II
0102030405060708090100
5 head microflourimeter assay
311.
+
Appendix 2.
Cloning of the garnet gene.
The cloning of the garnet gene forms the basis of the molecular analysis
recorded in chapter 2 of this thesis. This appendix is included to record the
cloning of the gene. The garnet gene was cloned by Donald A. Sinclair using a
P-element induced garnet gene isolated by Richard Wennberg (Wennberg
1988).
A P-element induced allele of garnet, and four revertants thereof, was isolated
from a naturally occurring P-element strain, S6-1 (see Figure 15). After
replacement of the autosomes and most of the X-chromosome by
recombination, two P-elements remained, one at cytological position 1 2B, the
location of the garnet gene, and one at 1 2E. Genomic DNA isolated from the gP
mutation, restricted with Eco RI and probed with the P-element containing Hind
Ill fragment of the p1125.1 plasmid revealed an approximately 8.5 kb fragment.
A library made from size fractionated DNA from the gP strain yielded eight P
element containing clones when probed with the same P-element containing
probe. Hybridization of these clones to polytene chromosmes showed that
seven of these clones represented the 1 2E P-element and the last one, at
cytological position 12B, potentially identified the garnet gene. Using the non P
element containing DNA from this clone, a series of lambda phage clones
where isolated from a wild type genomic (EMBL 3) library (Figure 17 and 30).
A 6.6 Eco RI fragment common to all the phage clones was subcloned into
puclg. This fragment was used to probe two c-DNA libraries (Figure 27) and a
number of spontaneous garnet mutations (Figure 19 and 22).
312