Download Mosaic: A Position-Effect Variegation Eye

Mosaic: A Position-Effect Variegation
Eye-Color Mutant in the Mosquito
Anopheles gambiae
M. Q. Benedict, L. M. McNitt, A. J. Cornel, and F. H. Collins
The Mosaic (Mos) mutation, isolated in the F1 of 60Co-irradiated mosquitoes, confers
variegated eye color to third and fourth instar larvae, pupae, and adults of the
mosquito Anopheles gambiae. Mos is recessive in wild pink eye (p1) individuals,
but is dominant and confers areas of wild-type pigment in mutant pink eye backgrounds. Mos is located 14.4 cM from pink eye on the X chromosome and is associated with a duplication of division 2B euchromatin that has been inserted into
division 6 heterochromatin. Various combinations of Mos, pink eye alleles, and the
autosomal mutation red eye were produced. In all cases, the darker pigmented
regions of the eye in Mos individuals show the phenotypic interactions expected
if the phenotype of those regions is due to expression of a p1 allele. Expression
of Mos is suppressed by rearing larvae at 328C relative to 228C. All of these characteristics are consistent with Mos being a duplicated wild copy of the pink eye
gene undergoing position-effect variegation.
From the Centers for Disease Control and Prevention,
4770 Buford Hwy., Division of Parasitic Diseases, MS F22, Atlanta, GA 30341. L. M. McNitt is currently at the
University of California, San Diego, School of Medicine,
La Jolla, California. A. J. Cornel is currently at the University of California, Department of Entomology, Mosquito Control Research Laboratory, Parlier, California.
F. H. Collins is currently at the University of Notre
Dame, Department of Biological Sciences, Notre Dame,
Indiana. We thank Dr. Nora Besansky of the University
of Notre Dame for providing the Mos mutant. We also
gratefully acknowledge the generous support of the
John D. and Catherine T. MacArthur Foundation and
the UNDP/World Bank/WHO Special Programme for Research and Training in Tropical Diseases. James Gathany of CDC photographed the Mos adults. Address correspondence to M. Q. Benedict at the address above
or e-mail: [email protected].
The American Genetic Association 91:128–133
128
Normal gene expression within an individual animal varies according to tissue- and
organ-specific developmental patterns due
in part to differing gene regulation. However, expression within an individual, tissue, or organ may deviate from normal
patterns due to cell lineage-specific changes in genotype or aberrant expression resulting in phenotypic variegation. One
manifestation of the latter is position-effect variegation (PEV).
Position-effect variegation refers to ‘‘the
mosaic expression of a gene lying near a
breakpoint in a chromosome rearrangement . . . most easily demonstrable for
cell-autonomous phenotypes’’ (Spofford
1976). The subject has been reviewed
( Henikoff 1990; Weiler and Wakimoto
1995) and commented upon ( Henikoff
1994; Spradling and Karpen 1990) extensively. In addition to PEV being most evident for cell-autonomous phenotypes in
which expression is spatially restricted, it
is also more easily observed in external
visible phenotypes that have a two-dimensional nature, such as cuticle or eye pigmentation. The cause of such variegated,
or mosaic, expression is generally an induced rearrangement that brings a wildtype allele of a gene located in euchromatin
into close proximity of heterochromatin.
Relocation of such genes into the proximity of the heterochromatin suppresses
gene expression in a variegated fashion re-
sulting in clusters of mutant phenotype
cells derived from a common lineage. The
basis of differential expression most commonly suggested is varying condensation
of chromatin in the vicinity of the rearrangement, however, underreplication of
DNA in polytene chromosomes has been
seen ( Karpen and Spradling 1990). In general, no genotypic differences are believed
to exist between variegating genes in cell
clusters in which the gene is expressed
and those in which it is not. Rather the
molecular basis of suppression of gene activity is due to spreading condensation or
‘‘heterochromatinization’’ of the adjacent
euchromatin in the rearrangement. The effect is therefore usually cis-acting, since
the homologue in which the rearrangement has occurred also carries the variegating allele. Furthermore, the lineage-specific modifications of gene expression are
unique to each individual and are not inherited.
Two other bases of phenotypic mosaicism are known: genetic mosaicism, and
excision/insertion of transposable elements (Ashburner 1989). Unlike PEV, both
of these mechanisms result in genotypic
changes in the cells expressing altered
phenotypes and occasionally in germ-line
changes. Genetic mosaicism due to somatic nondisjunction or mitotic recombination produces variegated phenotypes,
usually affecting a low frequency of cell
Table 1. Numbers of progeny of various phenotypic classes resulting from Mosaic crosses
Mos1
Mos
No.
of
families
Cross
A
B
C
D
E
F
G
H
(p Mos / 3 p Mos ?) F1 inbred
(p1 Mos1 / 3 p Mos ?) F1 inbred
(p Mos / 3 p1 Mos1 ?) F1 inbred
pw Mos1 /3 (pw Mos /3 p1 Mos1 ?) F1 ?
(p Mos / 3 p1 Mos1 ?) /3 pw Mos1 ?
(pw Mos / 3 p1 Mos1 ?) F1 /3 pw Mos1 ?
(p1 Mos1 / 3 p Mos ?) F1 / 3 pw Mos1 ?
pw Mos / p5 Mos1 /3 pw Mos1 ?
w
1
7
8
15
8
6
8
7
10
p (p5)
/
?
238
0
288
117
108
261
94
94
(33)
p1
pw
/
?
284
0
127
158
221
239
104
89
(36)
p (p5)
x2 deviation
pw
/
?
/
?
/
?
Total
Sex
Mos
p
17
36
56
119
14.575*
0.468
284
10.239*
31
46
43
49
0.458
8.287*
7.172*
0.007
0.019
2.273
1.008
2.153
0.153
0
491
558
1232
566
465
776
480
1115
1.9609
169
347
0
0
0
0
245
281
1.138
3.458
2.700
0.109
132
209
137
112
205
121
8
15
20
19
(236) (258)
27.05*
2.914
* P , .05.
lineages. Likewise, insertion of transposable elements into wild alleles often results in mutations, and somatic excision of
such elements may restore the wild phenotype in a mosaic fashion as exemplified
by the Mos1 Mariner element ( Bryan et al.
1987).
The malaria vector Anopheles gambiae
is a model organism for genetic and molecular biology studies, the goal of which
is interfering with its ability to transmit
malaria parasites (Collins and Besansky
1994). However, published morphological
mutants available for genetic study of A.
gambiae at this time number only five: red
eye ( Beard et al. 1994), pink eye ( Benedict
et al. 1996; Mason 1967), white ( Benedict
et al. 1996; Besansky et al. 1995; Mason
1967), collarless (Mason 1967), and dieldrin resistance ( Davidson and Hamon
1962). Of these, only white has been
cloned, mainly for use as a genetic transformation marker ( Besansky et al. 1995).
During a genetic screen to induce X
chromosome eye-color mutations, a variegation mutant named Mosaic (Mos) was
induced by gamma-irradiation ( Besansky
et al. 1995). We report the inheritance, karyotype, temperature effects on expression, and probable basis of this mutant
phenotype.
Materials and Methods
Mosquito Strains, Culture, and
Mutagenesis
Sex determination in A. gambiae is similar
to that of Drosophila melanogaster; XX individuals are female, and XY are male.
Two X-linked eye-color genes, white ( Besansky et al. 1995) and pink eye (Mason
1967), have been described. Three pink
eye alleles—p, pw, and p5—produce pink,
white, or scarlet eye color ( Benedict et al.
1996), respectively, and the various pink
eye alleles are codominant.
Eight mosquito strains were used for all
experiments: (1) G3, a wild-eye strain isolated in Gambia, was the source of p1 Mos1
in crosses B–G ( Table 1). (2) WE is pure
breeding for the pink eye allele pw and was
obtained from the London School of Medicine and Tropical Hygiene ( Beard et al.
1994; Benedict et al. 1996). This was the
source of pw Mos1 in crosses A, D, E, F, G,
and H. (3) PE breeds true for p and was
obtained from the same source as WE and
the mutant has been described elsewhere
(Mason 1967). (4) p5; c; r is homozygous
for a pink eye allele that confers a bright
red eye color and darkens to near wildtype in adults ( Benedict et al. 1996). It is
also homozygous for the autosomal recessive alleles c of collarless on chromosome
2 (Mason 1967) and r of red eye on chromosome 3 ( Beard et al. 1994). (5 and 6)
Strains m2 and m5 carry the w1 and w2 mutations of the white gene, respectively
( Benedict et al. 1996). (7) rrcc is homozygous for red eye and collarless and is p1.
(8) The origin of Mos has been described
briefly ( Besansky et al. 1995); a female
with variegated eye color was found in the
F1 of 60Co-irradiated p1 males that were
crossed to p / p females. Other combinations of alleles for eye-color interaction
studies were derived from these strains by
standard crossing schemes.
Mosquito larvae were reared at 278C and
fed 2:1 TetraMin Baby-E Fish Foody:brewer’s yeast mixed with water as a 2% w/v
slurry ( Benedict 1997). Adults were held
at 278C, approximately 80% RH, and fed on
human blood and 10% Karoy syrup in water. Matings to establish mutant lines were
performed in 1 pint paper cups with
screen-covered tops, or for subsequent inheritance crosses, in 1 gallon cages. Phenotypes were determined in the pupal
stage.
Temperature effects on Mos expression
were analyzed by rearing similarly to
above, except at either 228C or 328C
(618C) from approximately 24 h before
hatching until the pupal stage. Eyes of pupae less than 24 h old were examined using a stereomicroscope. The proportion of
the eye that was pigmented was estimated
by examination of either eye chosen at
random and classification into three
groups: ,33% ( low), 33–66% (medium),
and $67% ( high) pigmented (e.g., 0%
would be totally mutant, and 100% would
be wild type). Three replicate experiments
were performed, and in each temperature
group 134–319 individuals were scored.
The proportion of individuals in the high
pigment class was compared with the other classes combined by ANOVA with two
independent variables: replicate and temperature. Significance was defined as P ,
.05.
Crosses and Cytogenetic Analysis
Linkage and inheritance crosses were performed between newly emerged females
and males, and a blood meal was offered
3–6 days after emergence. Sexes were separated either by visual examination of terminalia in the pupal stage, or after CO2 anesthesia of adults approximately 16 h old.
All crosses were analyzed by standard chisquare analysis and significance levels
were defined as P , .05. Heterozygous females for cytology were obtained from the
F1 of Mos pw males crossed to G3 females.
Ovarian nurse-cell polytene chromosomes
were prepared according to Green and
Hunt (1980), and in situ hybridizations of
biotin-labeled cDNAs were performed by
the method of Kumar and Collins (1994).
Results
Origin and Phenotype of Mos
Among the F1 progeny of a cross between
pink eye females and irradiated G3 males,
Besansky et al. (1995) found an exception-
Benedict et al • A PEV Mutant in Anopheles gambiae 129
Figure 1. (A) Wild-type and (B,C) Mosaic adults. Note the well-defined regions of variegation in the Mosaic adults,
variations from symmetry, and the clustering of pigmented ommatidia. The pink color of the lighter portion of the
eyes of these individuals reflects their p phenotype.
al female with variegated eye color in
which patches of wild-type ommatidia (a
green iridescent surface over reddish
black) were seen amid a pale pink background. This female, whose mutation was
named Mosaic, was crossed to p1 males
and a polymorphic stock consisting of
wild, Mos, and pale pink eyed individuals
was established. Subsequently isofemale
lines that contained only pink eye color or
the variegated progeny were combined to
establish pure-breeding stocks. From the
variegated stock, 21 families were subcultured, and all 889 female and 920 male
progeny were also variegated. The phenotype of the pink eye-color strain was
confirmed to be due to an allele of p (data
not shown) and appears to be the p allele
expected from the original mutant isolation.
The Mos phenotype is apparent beginning at the third instar, when the adult
imaginal eye becomes visible, but is more
easily observed in the pupal and adult
stages ( Figure 1). Well-defined regions of
pigmentation vary in extent among individuals from being so extensive as to be
Figure 2. Effect of temperature on the proportion of
the eye that is pigmented. Three replicate experiments
were performed (diamond-1, circle-2, triangle-3). Rearing temperature and the proportion-pigmented classes
are shown on the X axis. Numbers of individuals
scored at either 228C or 328C in the low, medium, and
high classes for replicates 1, 2, and 3, respectively,
were 228C: low, 50, 118, 125; medium, 67, 116, 117; high,
28, 85, 36; and 328C: low, 17, 69, 51; medium, 49, 87, 94;
high, 68, 137, 84.
130 The Journal of Heredity 2000:91(2)
nearly wild type to so few and small that
the eye is almost totally mutant. The vast
majority of individual ommatidia are completely wild or mutant, but partial wild expression is sometimes observed. The
patches of pigmentation are generally
large, well-defined, and relatively few in
number, but rarely pigmentation is peppered across the eye in addition to the
large patches.
Since elevated culture temperature is
known to suppress PEV, Mos expression
was determined when larvae were reared
at temperatures at the low and high end
of the reasonable laboratory rearing temperature range; either 228C or 328C. Culturing pure-breeding Mos pw at 328C beginning 24 h before hatching increased the
proportion of individuals with a high proportion of pigmented eye relative to 228C
(p . F 5 0.0168; Figure 2). On the other
hand, replicate was not a significant independent variable. Therefore rearing at an
elevated temperature suppressed Mos variegation.
In order to observe the phenotypic interactions of Mos, red eye, and pink eye
alleles, various combinations were produced by standard crossing methods. In
the absence of Mos, the red eye and pink
eye phenotypes interact to produce pale
peach or pumpkin color (p r and p5 r, respectively; Figure 3). When Mos was introduced, in all cases the phenotype observed in the regions of the eye that were
more darkly pigmented due to Mos was
that expected if Mos was a p1 allele, but in
the paler areas the phenotype was that expected for the various alleles present at
the pink eye locus only. In both the darker
and the paler regions, the interactions between pink eye and red eye were also that
expected for p1 or only of the alleles present at the pink eye locus, respectively.
Genetic Analysis of Mosaic
Numerous genetic crosses were performed to determine the mode of inheri-
Figure 3. Interactions between Mosaic, pink eye, and
red eye. Representations of the phenotypic interactions
and genotypes of hemizygous male X chromosomes
(for Mos and pink eye) and the autosomal gene red eye
(r1 is fully dominant over r) . (A) Typical wild-type eye
color is nearly black with a green iridescent surface, a
color achieved in (B) red eye individuals, but only after
the adults are approximately 48 h old ( Beard et al.
1994). Until this time, the color is brick red. (C) Similarly p5 individuals have bright red eyes until the adults
are approximately 48 h old ( Benedict et al. 1996). (D)
The pink eye allele p5 interacts with r to produce orange color ( Benedict et al. 1996). (E,F) Regardless of
the red eye genotype, pw individuals have white eyes
due to pw epistasis over red eye ( Beard et al. 1994). (G)
Mos p5 individuals have p1 pigmented eyes in the more
darkly pigmented regions, but p5 color elsewhere. (H)
However, in rr individuals, the darkly pigmented regions have the same phenotype as in ( B), that is, p1 r.
(I) The epistasis of pw over r is observed in the lighter
color eye regions, which are white, contrasting with
the darker regions, which are phenotypically p1 r.
tance and patterns of expression of Mos.
No significant heterogeneity chi-square
values were observed in any crosses described in the text here or in Table 1. Occasional chi-square deviations for sex and
Mos are noted in the crosses listed in Table 1, although we find that the rate of development and general vigor of Mos individuals is similar to wild type.
Several crosses clearly demonstrate
that Mos is sex linked. For example, when
pw Mos1 females were crossed to p Mos
males, all F1 females were p Mos and males
Figure 4. Ovarian polytene X female chromosomes of (A) a Mos homozygote showing the duplication ( D), heterochromatic regions of division 6 ( H), and the limits of divisions 2B and 6 marked. (B) A drawing that represents
our interpretation of the chromosome shown in (A). (C) A normal Mos1 homozygote chromosome probed with
c51 showing the site of hybridization and the uninterrupted heterochromatic puff in division 6. (D) A Mos homozygote probed with c51 showing the normal site of hybridization (2B) and the location that hybridizes in the
duplication (*) flanked by diffuse heterochromatin.
were pw Mos1 (6 families, 125 females, 120
males). Similarly, when p Mos females
were crossed to p1 Mos1 males or pw Mos
females were crossed to p1 Mos1 males, all
F1 males and females ‘‘switched’’ to the
maternal and paternal phenotypes, respectively (18 families, 638 females, 655
males). Crosses A and B also confirm sex
linkage. From the above, and all crosses in
Table 1 except D and H, it is also clear that
Mos is recessive to wild type in p1 phenotype individuals. In no case were p1 Mos
individuals identified (with the exception
of a weak interaction in r / r individuals
discussed below). Crosses to the various
pink eye mutant alleles (p, pw, and p5) demonstrate that Mos is a dominant allele in
all mutant phenotype backgrounds: in pw
/ p (above) and pw / p5 (cross H) female
heterozygotes.
Sex linkage, dominance in a mutant pink
eye background, and the recombination
rate observed (discussed below) made it
possible to exchange pink eye alleles independently of Mos. This was done by a
scheme to obtain progeny from recombinant females in which the Mos phenotype
could not be determined phenotypically,
as follows: Mos p / Mos p females were
crossed to 11 males, and their F1 female
progeny testcrossed to Mos1 pw males.
Wild-eye progeny females were again testcrossed to Mos1 pw males. The large majority of these females were predicted to
carry either one nonrecombinant X chromosome, or less commonly a recombinant
Mos p1 chromosome. Families of progeny
reflecting these predictions were obtained
(data not shown), that is, most families
contained no Mos progeny, but in one family, a few Mos progeny appeared. These
were crossed to Mos1 pw females ( TC3) to
propagate a strain from which Mos pw was
purified. The same strategy was utilized to
create a Mos p5 strain.
Several crosses suggested that recombination occurs between Mos and pink
eye. For example, one half of the expected
recombinants can be observed in crosses
A, B, C, E, F, and G. However, our best estimate of the distance between pink eye
and Mos was 14.4 cM in an appropriate
testcross (cross H, linkage x2 5 563.99).
This is similar to the distance of 16.2 cM
inferred in cross G, wherein half of the recombinants could be identified, but higher
than the distances of 9.0 cM and 9.8 cM
inferred in the similar crosses C and E, respectively.
We conducted crosses to determine if
Mos would be expressed in a white mutant
background. The genetic scheme to determine this was based on the expected recombination between white and Mos as follows: since the distance between pink eye
and Mos is 14.4 cM and the distance between white and pink eye has been estimated at distances as great as 3.5 cM
( Benedict et al. 1996), the distance between Mos and white was expected to be
between 10.9 and17.9 cM, depending on
the order of the three loci. Therefore when
heterozygous p5 w1 Mos / p1 w Mos1 females were testcrossed to p1 w Mos1
males (where w is either w1 or w2), 35–47
recombinant w / Mos individuals would be
expected among 570 testcross progeny if
Mos were expressed in a w background.
However, no Mos w were detected, rather
all Mos were p5. Therefore w is epistatic
over Mos, as previously shown for pink
eye ( Benedict et al. 1996).
Even though Mos generally appeared recessive, in certain genetic crosses we observed occasional cases of weak semidominance. Therefore crosses were performed
to determine whether Mos in the heterozygous state could be observed in various
pink and red eye genetic backgrounds. Although the effect was weak, Mos was
slightly evident in the heterozygous state
in Mos p5 / Mos1 p1; r / r pupae and adults
compared to Mos1 p1 / Mos1 p1 ; r / r and
Mos1 p5 / Mos1 p1 ; r / r. However, the following were phenotypically indistinguishable (all r1 / r1): Mos pw / Mos1 p1 ; Mos1
pw / Mos1 p1, and Mos1 p1 / Mos1 p1. We
caution, however, that the slight degree of
semidominant expression in an r / r background would make it difficult to consistently distinguish individuals of this genotype from wild type.
Cytogenetic Analysis of Mos
Examination of the ovarian nurse-cell
polytene chromosomes of Mos / Mos1 and
Mos / Mos females revealed an insertion of
euchromatin into the heterochromatic
portion of division 6 on the X chromosome, however, no other lesion was visible ( Figure 4A,B). The insertion was clearly flanked by two heterochromatic puffs
that are typical of division 6. This insertion appeared to contain two to four
bands, depending on the chromosome
preparation. Heterozygous females invariably had one homologue containing a normal division 6 and one with the euchromatic insertion. Based on the genetic
behavior of Mos, we suspected that a pink
eye duplication might be involved so we
compared the appearance of the region of
the chromosome in which pink eye is located, division 2B ( Zheng et al. 1993), to
the insertion. The appearance of the insertion was indeed similar to 2B, however,
this determination was very subjective
and required confirmation. Fortunately
two cDNAs, c51 and c81, have been
mapped to unique sites in division 2B by
in situ hybridization (Cornel A and Collins
F, unpublished data), and we suspected on
the basis of mapping that they might flank
the pink eye gene (see Discussion). Both
Benedict et al • A PEV Mutant in Anopheles gambiae 131
cDNAs were hybridized independently to
ovarian polytene chromosomes of Mos homozygotes, and c51 consistently hybridized to both the insertion and division 2B
( Figure 4D). The c81 probe was observed
to hybridize consistently only to division
2B, although once, hybridization was observed near the junction of the insertion
at the opposite end from the site of hybridization of c51 (data not shown).
Discussion
Mos has classical characteristics of PEV,
most obviously the variegating eye-color
phenotype due to a chromosomal rearrangement that juxtaposes a euchromatic
gene located in division 2B into division 6
heterochromatin. Incubation of individuals at elevated temperatures during critical developmental periods typically suppresses PEV, and Mos shares this effect.
Finally, Mos is recessive relative to wild
type.
Five lines of evidence suggest that the
Mos phenotype is due to a pink eye duplication into division 6 on the X chromosome: (1) The aberration we observe in
division 6 has been observed in all Mosaic
females examined, but never in any wildtype individuals. (2) Mos is expressed in
mutant pink eye backgrounds, but not in
wild or white backgrounds. (3) Mos is expressed in both spontaneous and induced
pink eye mutants. This reduces the probability that Mos is due to a trans-acting regulatory mutation affecting pink eye expression. (4) In all combinations of pink eye
alleles with Mos and red eye, the color of
the more darkly pigmented regions of the
eye is that expected if Mos is a p1 allele.
(5) cDNA c51 that hybridizes to a unique
location in the vicinity of pink eye in wildtype mosquitoes also hybridizes to the
Mos-associated euchromatic insertion.
We arrived at the conclusion that cDNAs
c51 and c81 were located near pink eye as
follows: the white gene and microsatellite
AGHX99 had been mapped by both microsatellite analysis and in situ hybridization
to 2A and 2C, respectively, and pink eye to
the interval between ( Besansky et al.
1995; Zheng et al. 1993). This interval containing division 2B was also the location
in which both c51 and c81 had been located by in situ hybridization (Cornel A
and Collins F, unpublished data).
The distance between Mosaic and pink
eye is consistent with available microsatellite mapping data ( Zheng et al. 1993).
The microsatellite linkage map of the X
chromosome is 49 cM long and all of the
132 The Journal of Heredity 2000:91(2)
microsatellite markers are located on the
euchromatic right arm ( Zheng et al.1993,
1996). This means that the distance from
pink eye to the Mos aberration must be
greater than the distance from pink eye to
the most distant marker in the direction
of Mos, AGXH678. The distance from pink
eye to AGXH678 is 8.5 cM, and this microsatellite has been located in division 6.
Therefore, since Mos is located 14.4 cM
from pink eye, Mos is located distal to the
last marker, a deduction consistent with
the cytology.
The simplest explanation for the origin
of Mos is that it was induced by transposition roughly of division 2B containing a
p1 allele into division 6 of the X chromosome. This event would have deleted the
wild pink eye allele so that complementation with the pink eye gene on the other
homologue would fail in the screen from
which the mutant was isolated. We suspect this original deletion was lost via recombination during establishment of the
Mos stocks over several generations.
Variegation mutants have been used
successfully in D. melanogaster to study
eye differentiation (reviewed by Becker
1966). Due to the clonal propagation of
eye cells accumulating pigment, visualizing and compiling the patterns of ommatidia that are pigmented reveals the underlying pattern of eye development.
Anopheline morphology lends itself particularly well to such studies since the pupal eye undergoes extensive development
and is easily visible nondestructively
through the transparent puparial cuticle.
Furthermore, the very distinct demarcations of pigmentation in Mos individuals
would make this mutant an ideal candidate for such studies.
The extremely well-delineated pigmented regions in Mos mutants reflect the expression mechanisms of the gene(s) involved. Whereas cell-autonomous eye-color
mutants must be expressed in the cell
whose phenotype is affected, nonautonomous mutants may affect the phenotype
of cells in which the allele is not expressed
via a transferred product. The Mos phenotype suggests that pink eye is cell autonomous, but no experiments have been
performed to verify this.
On the basis of comparisons with Drosophila eye-color genes, it is possible to
narrow the candidates for the pink eye
gene. Since ommochromes are the only
visible eye pigments of anophelines
( Beard et al. 1994), the number of genes
affecting eye color is considerably reduced relative to D. melanogaster, which
also have pteridine pigments. It is reasonable to believe that the suite of genes affecting ommochrome eye pigments in
anophelines is similar to that of D. melanogaster, and cell-autonomous genes that
could have phenotypic effects like pink
eye / Mos might be identified on the basis
of similar characteristics. A search of the
Drosophila database, FlyBase, produced
only one other gene of D. melanogaster
that has these characteristics: scarlet.
Scarlet is part of a heterodimeric transmembrane ABC transporter complex with
white ( Ewart et al. 1994). Pink eye, like A.
gambiae white, is epistatic over red eye,
consistent with the phenotype expected
of ABC transporter-complex mutations. Finally, like scarlet, pink eye of A. gambiae
does not eliminate male accessory gland
and testis sheath pigmentation ( Benedict
et al. 1996).
The easily detected phenotypic effects
of many position-effect variegation mutants provide a powerful tool with which
to study underlying mechanisms of gene
regulation, differentiation, and chromatin
structure. In these experiments, the striking phenotype of the Mos mutation lends
itself particularly well to these studies.
Furthermore, the cytological aberration
associated with Mos may provide a useful
means for positional cloning of the pink
eye gene.
References
Ashburner MA, 1989. Drosophila: a laboratory handbook. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.
Beard CB, Benedict MQ, Primus JP, Finnerty V, and Collins FH, 1994. Eye pigments in wild-type and eye-color
mutant strains of the African malaria vector Anopheles
gambiae. J Hered 86:375–380.
Becker HJ, 1966. Genetic and variegation mosaics in the
eye of Drosophila. Curr Top Dev Biol 1:155–171.
Benedict MQ 1997. Care and maintenance of anopheline mosquito colonies. In: The molecular biology of
insect disease vectors (Crampton JM, Beard CB, and
Louis C, eds). New York, Chapman & Hall; 3–12.
Benedict MQ, Besansky NJ, Chang H, Mukabayire O,
and Collins FH, 1996. Mutations in the Anopheles gambiae pink-eye and white genes define distinct, tightly
linked eye-color loci. J Hered 87:48–53.
Besansky NJ, Bedell JA, Benedict MQ, Mukabayire O,
Hilfiker D, and Collins FH, 1995. Cloning and characterization of the white gene from Anopheles gambiae. Insect Mol Biol 4:217–231.
Bryan GJ, Jacobson JW, and Hartl DL, 1987. Heritable
somatic excision of a Drosophila transposon. Science
235:1636–1638.
Collins FH and Besansky NJ, 1994. Vector biology and
the control of malaria in Africa. Science 264:1874–1875.
Davidson G and Hamon J, 1962. A case of dominant
dieldrin resistance in Anopheles gambiae Giles. Nature
196:1012.
Ewart GD, Cannell D, Cox GB, and Howells AJ, 1994.
Mutational analysis of the traffic ATPase (ABC) transporters involved in uptake of eye pigment precursors
in Drosophila melanogaster: Implications for structurefunction relationships. J Biol Chem 269:10370–10377.
Green CA and Hunt RH, 1980. Interpretations of variation in ovarian polytene chromosomes of Anopheles funestus Giles, An. parensis Gillies, and An. aruni. Genetica 51:187–195.
Henikoff S, 1990. Position-effect variegation after 60
years. Trends Genet 6:422–426.
Henikoff S, 1994. A reconsideration of the mechanism
of position effect. Genetics 138:1–5.
Karpen GH and Spradling AC, 1990. Reduced DNA polytenization of a minichromosome region undergoing
position-effect variegation in Drosophila. Cell 63:97–
107.
Kumar V and Collins FH, 1994. A technique for nucleic
acid in situ hybridization to polytene chromosomes of
mosquitoes in the Anopheles gambiae complex. Insect
Mol Biol 3:41–47.
Mason GF, 1967. Genetic studies on mutations in species A and B of the Anopheles gambiae complex. Genet
Res Camb 10:205–217.
Spofford JB, 1976. Position-effect variegation in Drosophila, 1976. In: The genetics and biology of Drosophila (Ashburner M and Novitski E, eds). New York:
Academic Press; 955–1018.
Spradling AC and Karpen GH, 1990. Sixty years of mystery. Genetics 126:779–784.
Weiler KS and Wakimoto BT, 1995. Heterochromatin
and gene expression in Drosophila. Ann Rev Genet 29:
577–605.
Zheng L, Benedict MQ, Cornel AJ, Collins FH, and Kafatos FC, 1996. An integrated genetic map of the African
human malaria vector mosquito Anopheles gambiae.
Genetics 143:941–952.
Zheng L, Collins FH, Kumar V, and Kafatos FC, 1993. A
detailed genetic map for the X chromosome of the malaria vector, Anopheles gambiae. Science 261:605–608.
Received April 14, 1998
Accepted August 25, 1999
Corresponding Editor: Ross MacIntyre
Benedict et al • A PEV Mutant in Anopheles gambiae 133