Download Gene Expression in Adult Metafemales of Drosophila

Copyright 0 1989 by the Genetics Society of America
Gene Expression in Adult Metafemalesof Drosophila melanogaster
James A. Birchler,'? JohnC. Hiebert' andMark Krietzmant
'The Biological Laboratories, Haroard University, Cambridge, Massachusetts 02138, and tDepartment o f Genetics, University o f
Calijornia, Berkeley, Calijornia 94720
Manuscript received November 3 0 , 1988
Accepted for publication April 27, 1989
ABSTRACT
The expression of selected X-linked and autosomal genes was examined in metafemales ( 3 X 2 A )
compared to diploid sisters. Three enzyme activities (glucose-6-phosphatedehydrogenase, 6-phosphogluconate dehydrogenase, p-hydroxyacid dehydrogenase) encoded by X-linked genes are not significantly different in the two classes of flies. In contrast, three autosomally encoded enzyme activities
(alcohol dehydrogenase, a-glycerophosphate dehydrogenase, isocitrate dehydrogenase) are reduced
in metafemales. Protein and DNA comparisons between metafemales and diploid sistersshow a
lowered level of total protein whereas the total DNA measurements are similar. Thus, the total cell
number in metafemales is basically unchanged but gene expression is reduced. Phenotypic analysis of
three autosomal loci, glass (gl),purple ( p r ) and pink-peach (Pp), show that all three have lowered
expression in metafemales while the X-linked loci, white-apricot (w") and Bar (B),are dosage compensated. Quantitative dot blot analysis of messenger RNA levels of the second chromosomal locus,
alcohol dehydrogenase (Adh),and theX chromosomal locus, rudimentary ( r ) ,show that Adh has reduced
expression and r is partially compensated per total RNA in metafemales. It is proposed that the
increased dosage of the X chromosome inversely affects both the X and autosomal gene expression
but the simultaneous increased dosage of the structuralgenes on theX results in dosage compensation.
The reduced levels of expression of autosomal genes could contribute to the great inviability of
metafemales.
I
N most higher eukaryotic organisms, a change of
individual chromosome number from the normal
diploid results in reduced vigor and, in extreme cases,
inviability (BRIDGES
1916; BLAKESLEE
1934). These
syndromes have generally been attributed t o an imbalance of gene products, due to structuralgene dosage effects that presumably upset metabolicfunctions
(PATTERSON,
BROWNand STONE 1940; MULLER
1950). Exceptions to this rule involvesex chromosomes that exhibit dosage compensation (MULLER
1932). That is, changes in dosage of these chromosomes, that otherwise would be lethal, are equalized
in the level of expression between the two sexes. In
Drosophila, the single X chromosome in the male
produces a nearly equivalent level of product as do
the two chromosomes in the female.
The metafemales, on the other hand,with three X
chromosomes and two sets of autosomes, are highly
inviable.If their X chromosomes exhibit a dosage
response, then their inviability can be explained by
the classical imbalance notion. However, if the three
X chromosomes show dosage compensation, then an
explanation for their inviability must be sought elsewhere.
Beginning with STERN(1960), several investigators
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Genetics 122: 869-879 (August, 1989)
have examined the question of whether X-linked genes
exhibit dosage compensation in metafemales (ANANIEV et al. 1974; LUCCHESI,
RAWLSand MARONI 19'74;
STEWART
and MERRIAM 1975; DEVLIN,HOLMand
GRIGLIATTI
l984,1985a,b; 1988). The formerauthors conclude that the dosage compensation is not
present. However, the latter three sets concur that
for those examples of genes tested, dosage compensation is operative. That is, the total level ofexpression
of the three x's in metafemales is more or less equivalent to the level of expression in the normal diploid.
Without further information, the inviability of metafemales and thecompensation oftheir X chromosomes
presents a paradox.
We have reexamined the question of X and autosomal expression inadult metafemales withparticular
regard to the absolute levels. Previous studies have
standardized enzyme activity measurements to total
fly protein. While such a method is valuable for many
comparisons, it is necessary to determine this parameter in experiments in which the total protein level
mightalso be altered. In view of the finding in a
variety of plant species (BIRCHLER
1979, 1983), Drosophila (DEVLIN,HOLMand GRIGLIATTI
1988)and
mouse (KLOSE and PUTZ 1983) that trisomycauses
reductions in the expression of linked and unlinked
structural genes, total protein might not be the most
appropriate standard since its level could be changed
870
J. A. Birchler, J. C. Hiebert
and
also in metafemales, particularly considering that a
rather substantial fraction (20%) of the genome is
trisomic.
In addition, autoradiographic studies involving incorporation of tritiated uridine into nascent RNA on
polytene chromosomes of metafemales
and normal
sisters have all standardized the values obtained for
the X chromosome to those observed over the autosomes. The reason for such standardization was that
the autosomal values would serve asan internal control for uptake of label, for example, as well as other
parameters. The underlying assumption was that autosomal gene expression would not be altered in metafemales. The present study reexamines this assumption in lightof the fact that trisomy produces an
inverse effect on unlinked genes in the various organisms noted above.
The results show that X-linked expression of two
phenotypic mutants, three enzyme structural genes
are nearly
and rudimentary locusmessengerRNA
equal infemales and metafemales. Autosomally enalcohol dehydrogenase messenger
coded
enzymes,
RNA, three hypomorphic eye color mutants and total
protein, however, exhibit significantly less expression
in metafemales. Two types of test indicate that the
number of cells in metafemalesand normals are quite
similar under the conditions usedin the molecular
analyses. These involve examination of the number
( 1 929)
of winghairs per unit area, which DOBZHANSKY
found to be an indicator of cell sizeand number, and
secondly an estimation of DNA content in the two
types of flies.
Neither test gave a significant difference.
Thus, it appears that on a per cell basis, the expression
of most genes is reduced in metafemalesregardless of
chromosomallocation. The X-linkedgenes exhibit
dosage compensation because the increased number
of structural genes cancels the inverse effect of the
triple-X genotype. The autosomalgenes, however,
show reduced expression since their copy number is
unchanged. It is hypothesized that this condition contributes to the inviability of metafemales.
MATERIALS AND METHODS
Fly culture: Flies were grown on instant medium from
Carolina Biological Supply Co. at 20". An exception was
the flies grown at the initial stage of the project for phenotypicanalysisof purple, which were grown at 25". The
metafemales homozygous for pink-peach were found in a
population also grown at the higher temperature. Trials at
25' and 20" gave greater survival of metafemales at the
latter temperature in accordance with the findings of DOBZHANSKY (1928). For the molecular analyses, metafemales
and control sisters were collected at 1-24 hr posteclosion,
frozen in liquid nitrogen and maintained at -80 ".This age
period was chosen to minimize differences in protein content due to the enlargement of the ovaries in older diploid
females as well as to minimize loss of metafemales due to
mortality.
Geneticcrossesandestimation
of metafemale fre-
M. Krietzman
quency: Metafemales were collected from crosses of FM7,
y2 v B/Y males by attached X , C(I)DX, y w f / Y females. Both
parental lines were isogenic for chromosomes two and three,
which were derived from an Oregon R wild stock. This
cross generates four types of zygotes:(1) normal attached X
females carrying a Y chromosome; (2) normal males with
the balancer X (FM7) with a Y chromosome; (3) triplo-X
metafemales carrying both the attached X chromosome and
the FM7 X chromosome; and (4) nullo-X, duplicated Y zygotes that are lethal. The normal female progeny have white
eyes and the normal males, vermilion Bur. The exceptional
metafemales are wild type in color and only slightly Bar,
thus allowing ready identification in a field ofcollected flies.
A furtherdescription of the mutants and chromosomes used
can be found in LINDSLEY
and GRELL(1968).
To estimate the frequency of metafemales, the female
progeny from 100 vials was counted. From a total of 16,768
females, the frequency of metafemale survival to eclosion
was 1.6 X lo-' (27 metafemales) with these growth conditions and genetic background. In addition, seven other
exceptional females were recovered. Progeny tests indicated
that these were triploid females or females carrying a detached X from the compound chromosome heterozygous
with the FM7 chromosome from the male parent.
Proteindetermination: Total protein was estimated
using Bradford reagent (BRADFORD
1976) withbovine
serum albumin as a standard.
Enzymeactivitymeasurement: The assays for the six
enzymes examined were as described: alcohol dehydrogenase, ADH (WOODRUFF
and ASHBURNER
1979); a-glycerophosphate dehydrogenase, aGPDH (RAWLSand LUCCHESI
1974); isocitrate dehydrogenase, IDH (RAWLSand LUCCHESI 1974; STEWART
and MERRIAM 1974); 6-phosphogluconate dehydrogenase, 6PGDH; glucose-6-phosphate dehydrogenase, G6PDH (RAWLSand LUCCHESI1974; STEWART
and
MERRIAM 1974)
and
8-hydroxyacid
and GRELL1978) with the
dehydrogenase, @HAD(TOBLER
following modifications. Flies were homogenized in groups
of 5 in 250 pl (or 10 in 500 pl) of phosphate buffer (50 mM,
pH 7.5; 5 X lo-' mM 8-mercaptoethanol) in an Eppendorf
centrifuge tube using a custom made pestle. Samples were
centrifuged at 10,000 X g for 5 min and the supernatant
used as enzyme preparation.
All reactions were performedat 30" in 1.0 ml total
volume. Extract volumes for each enzyme and length of
incubation were as follows: ADH, 25 pl, 5 min; IDH, 25 pl,
10 min; aGPDH, 10 pl, 5 min; GGPDH, 25 pl, 20 min;
GPGDH, 20 pl, 20min; PHAD, 25 pl, 20 min. The reactions
were stopped by the addition of 50 pl of 6% sodium dodecyl
sulfate (SDS) with vigorous vortexing. The enzyme activity
was determined by optical density measurements at 340 nm
on a Zeiss spectrophotometer. Background absorbance was
determined for each sample by incubating the appropriate
volume of enzyme extract with 50 pl of SDS and subsequent
addition of this mixture to the standard volume of assay
solution. Under these conditions, the increase in optical
density was linear for all enzymes withtime and concentrations up to at least five times the standard conditions. All
reagents were purchased from Sigma.
DNA estimation: Total DNA was estimated using the
Hoechst fluorescence assay of LABARCA
and PAIGEN
(1980).
Calf thymus DNA (Sigma)was used as a standard. Ten flies
were homogenized in 500 pl of phosphate-buffered saline
solution in replicas of ten for each data point. Background
fluorescence for each extract was determined before the
addition of Hoechst reagent.
In order to evaluate the Hoechst DNA estimation for
meta and diploid females, it was necessary to determine the
MetafemalesGene
in Expression
TABLE 1
DNA estimate for males, females and contributionof the Y
chromosome
Genotype
n
FM7/FM7
FM7/Y
FM7/0
XYlXY
WfY
XY
10
10
10
10
10
10
aglFly
f SE
0.815 f 0.021
0.700 f 0.010
0.641 f 0.009
0.732 f 0.021
0.727 f 0.017
0.608 f 0.017
Each extract consisted of 10 flies/500 pl extraction buffer, n =
number of extracts for each class of fly. Values are expressed as
micrograms per individual fly. Calf thymus DNA was used as a
standard. Flies were generated from crosses of heterozygous FM7/
XY females to FM7IY and XY/Y males, respectively.
relative contribution of the Y chromosome to this assay as
well as the relative DNA measurement by this method in
males and females. For this, virgin female flies heterozygous
I)EN,
for theFM7, y2 v B balancer X chromosome and the In(
y, attached XY chromosome were crossed in separate experiments by two types of males. In the first case, they were
crossed by FM7/Y males. This produces a progeny with
homozygous FM7 females, heterozygous XY/FM7 females,
FM7/Y males and X Y / Y males. In the second type of cross,
the FM7/XY females were mated withZn(I)EN, y, XY/O
males. The four progeny types are homozygous XY females,
FM7/XYfemales, XY/O males and F M 7 / 0 males. From these
two crosses, homozygous FM7 females, FM7/Y males and
F M 7 / 0 males as well as XY/XY females, XY/Y males and XY
males were subjected to theHoechst fluorescent DNA assay.
The results are shown in Table 1. By this method the, Y
chromosome contributes between 8-16% of the male DNA
content and maleshave approximately 83-86% asmuch
DNA as femalesin this genetic background. The estimation
of DNA mass per individual fly (-1 pg per female) by this
method is similar to others (ROBERTSON
1978).
ROBERTSONnoted thatdifferent genetic backgrounds
influence the cell number to a modest degree. The measurements to control for the contribution of the Y chromosome come, by necessity, from related but not segregating
populations. The comparison of normal and metafemales
was performed on flies from the same population but from
a different genetic background than the one used for estimation of the contribution of the Y . While the number of
cells (and hence DNA measure) may differ between the
population used to generate metafemales and to make a Y
chromosome comparison, it is considered that the relative
measure within the latter is informative in interpreting the
relative measure in the former(euploid versus metafemales).
Cell size and number measurements:
Three metafemales
and five normal sisters were collected from the stock used
for molecular analysis (C( I)DX, y w f / Y females and C( I)DX,
y w f / F M 7 metafemales) and grown at 20". Each wing was
mounted on a slide and a countwas made of the number of
hairs within an area of 0.81 mm2 in four defined locations
on each wing. The mean & SE of 24 counts in metafemales
was 7.67 f 0.28. The mean of 40 counts in euploid sisters
was 7.93 f 0.15. A t-test analysis shows that these means
are not significantly different.
Electrophoresis: Extracts of the isogenic stock were subjected tostarch gel electrophoresis [Tris-borate-EDTA (ethylenediamine-tetraacetic acid)] according to YOUNG (1966).
The allozymes of the autosomal loci are Adh-F, CUGPDH-S
and ZDH-F. For the X encoded loci, the alleles are G6PDHA, 6PGDH-A andthestandard
(monomorphic) allele at
87 1
@HADfor both the C(I)DX, y wfand FM7, y2 v B chromosomes.
RNA extraction: RNA was extractedfrom metafemales, diploid sisters, Adh deficiency
(Df(ZL)Adfl3/
Bf((2L)Adh"u379) (WOODRUFF
and ASHBURNER
1979)and
Oregon R stocks by the method of COX (1968) with the
following modifications. Adh deficiency andOregonR
stocks were homogenized in 10 volumes of 8 M guanidineHCI, 0.01 M EDTA (pH 7.0) using a Tissumizer. The
metafemales and diploid sisters were homogenized in a
microfuge tube with a custom made pestle. Thirty metafemales and 50 diploid sisters collected from the same culture
and of the same age were used. After precipitation with onehalfvolumeof absolute ethanol for 1 hr, the
pellet was
resuspended in 4 M guanidine-HCl, 0.01 M EDTA (pH 7.0)
and precipitated with one-half volume ofabsolute ethanol a
total of five times. The pellet remaining after the washes
was extracted with sterile chelexed diethyl pyrocarbonate
(DEPC) treated water (1 ml/g original mass). After 10 min
of centrifugation in a microfuge, the supernatant was retained and the pellet extracted with DEPC-treated water at
56" using 2 ml/g of original mass. Following centrifugation
as before, the pellet was extracted at room temperature
using the 2 ml/g ratio. Aliquots were taken from the three
water extractions for determinations of absorbance at 260
nm to determine fractions with RNA. T o these fractions,
sodium acetate was added to a final concentration of 0.1 M
and followed by twovolumes of absolute ethanol. After
precipitation, centrifugation and resuspension in sterile,
chelexed DEPC treated water, the yield was determined by
absorbance at 260 and 280 nm. The RNA was reprecipitated as before. After centrifugation, the pellet was dissolved
atthe desired concentration by the addition of sterile,
chelexed DEPC-treated water.
RNA isolated from metafemales and euploid females was
diluted to the same target concentration. The final dilution
was used in triplicate for absorbance readings at 260 and
280 nm to determine the relative metafemale vs. female
concentrations. They were 0.331 and 0.313 pg/pl for metafemale euploid females, respectively. RNA preparations
from Adh deficiency and Oregon R flies were repeatedly
diluted until a final concentration of 0.3 13 pg/pl(==euploid
female) was achieved as determined by triplicate absorbance
readings at 260 nm.
For the determination of mRNA levels from therudimentary locus, 15 metafemales and 15 euploid sisters were used
for an RNA extraction as described above. The final concentrations as determined from triplicate readings were
0.166 and0.168 pg/pl for metafemales and euploid females.
Dot blots:Pieces ofnitrocellulose 8 X 20 cm were marked
in 2-cm squares with a lead pencil. The sheets were wetted
in water and then soaked in 20 X SSC (1X = 0.15 M NaCI,
0.01 5 M Na-citrate) for 4 hr priorto RNA application
(THOMAS
1980). The filters were then blotted to dampness
on 3"
Whatman paper. The RNA was applied to the
center of the squares in two microliter aliquots in ten replicas
each for metafemales, euploid females and Adh deficiency.
The filters were placed between two sheets of 3MM Whatman paper and baked under vacuum for 2 hr at 80". For
rudimentary, the aliquots were 5 pl each and no Adh deficiency RNA was used.
Hybridization: Filters were wetted in 5 X SSC ( l x SSC
= 0.15 M NaCI; 0.0 15 M Na-citrate) and prehybridized (150
pl/cm2) in a solution of 50% formamide, deionized with AG
501-X8 (Bio-Rad) resin, 5X SSC, 10 m M polyvinylpyrrolidine, 1% bovine serum albumin, 0.5% SDS and 0.2 mg/ml
calf thymus DNA (Sigma) for 4 hr at 60". Hybridization
was started by addition of "P-labeled probe at 1 X 1O6 cpm/
872
J. A. Birchler, J. C. Hiebert and M. Krietzman
mlof
solution. Hybridization was conducted for 16 hr
followed by four washes consistingof 0.1 X SSC, 5 mM
Na2HP04,0.015% pyrophosphate, 0.2% SDS (pH 7.0) each
for 30 min at 75". Then the filters were washed at room
temperature twice for 30 min in 3 mM Tris-HCI (pH 9.0).
Filters were dried, subjected to autoradiography with Kodak
XRP-1 film overnight at 70" to test for gross background,
then cut into individual squares, dried under a heat lamp
and counted in 5 ml of toluene plus 4% Scintiprep (Fisher)
in a Beckman LS 1801 scintillation counter. Control concentration curves for both Adh and rudimentary conducted
under these conditions using total Oregon R RNA demonstrated that thequantitation is linear up to atleast 5 pg/dot.
Probe preparation: Single-stranded RNA probes were
generated from an SP6 vector (GREEN,MANIATISand MELTON 1983) into which had been subcloned the HindIII to
1980) such that
EcoRI 3' half of the ADH gene (GOLDBERG
the RNA transcribed from the plasmid is antisense to Adh
messenger RNA. The reaction mix consisted of the following: 40 mM Tris-HCI (pH 7.5), 6 mMMgC12, 2 mM spermidine, 0.5 mM ATP, 0.5 mM CTP, 0.5 mM GTP, 0.4 mM
UTP, approximately 150 pCi ["PIUTP (3000 Ci/mmol)
(NewEngland Nuclear), 40 units RNAsin (Promega Biotech), 15 units SP6 polymerase (Bethesda Research Labs),
0.5 pg linearized plasmid in a total volume of 20 PI. The
reaction was incubated at 37" for 1 hr at which point the
volume was adjusted to 50 PI with 0.01 M Tris-C1, 0.001 M
EDTA (pH 8.0). One microliter was removed and diluted
to 50 pi in water. Twenty microliters of the diluted sample
were spotted onto two Whatman #1 2.3 cm discs. One was
dried directly and the second washed twice in 250 ml of 5%
trichloroacetic acid plus 1% sodium pyrophosphate, once in
250 ml of absolute ethanol and finally in 250 ml anhydrous
ether. The washed filter was dried under a heat lamp and
both counted in a Beckman LS 1801 scintillation counter
todeterminethe
percent of incorporation oflabel into
RNA.
The completed reaction mixture was applied to a spin
column of Sephadex G-50. The RNA recovered from the
column was precipitated by addition of 1 pg of Escherichia
coli transfer RNA per 50 pl recovered from the column,
one-tenth volume 2 M sodium acetate and two volumes of
absolute ethanol. After centrifugation and resuspension in
100 pi of sterile DEPC-treated H20, the appropriate volumes were added to the hybridization bags.
Antisense rudimentary probe was prepared by the same
method using a plasmid constructed by B. ZERGES and P.
SCHEDL
containing an SP6 promoter and a cDNA to rudiet al. 1984).
mentary (SEAGRAVES
Ribosomal RNA probe: A HindIII fragment (-1 kb) of
WELLAUER
and LONG
the 28s ribosomal RNAgene (DAWID,
1978) was subcloned into an SP6 vector such thatthe
transcribed R N A is complementary to native 28s rRNA.
The reaction mixture for transcription was identical to that
for Adh with the exception that the addedlabel was approximately 10 pCi of ["PIUTP (3000 Ci/mmol).
Densitometry: RNA from Oregon Rwas applied to nitrocellulose, prepared as described above, via a Bio-dot (BioRad) apparatus following the procedure outlined by the
manufacturer. A concentration curve consisting of 1X the
amount of RNA in the euploid female sample and 0.5 and
0.25 X dilutions were prepared forprobing with anti-rRNA.
Two micrograms ofRNA perdot were used in the 1X
sample. After hybridization, the filters were exposed to
Kodak XRP-1 film without intensifying screens. The developed film was scanned with an LKB 2202 Ultroscan Laser
Densitometer to determine the linearity of the curve for
rRNA.
RNA from metafemale, euploid female and Adh deficiency flieswas applied to nitrocellulose sheets as above and
probed with labeledanti-rRNA. Developed filmwas scanned
as described above. Integration and determination of relative peak areas were performed by the LKB Gelscan program.
RESULTS
Beforeproceeding tothe comparisons of metafemales and diploid females, an analysis of the relative
expression of the various X chromosomes used, was
conducted in order to evaluate thedataonmetafemales. The two chromosomeswereattached
X,
C(I)DX,y w f , and thebalancer FM7, y2 v B. Since the
metafemales have both chromosomes whereas the euploid control females have only C(I)DX, it was necessary to determine the relative expression of the six
enzymes studied from the compound X and from the
FM7 chromosome.For this, two comparisons were
made. In the first, females of C(I)DX, y w f / Y were
assayed with FM7/Y males, both of which were from
the same culture and isogenic for Oregon R autosomes. Secondly, FM7 homozygous females were compared to FM7/Y males that carry the same isogenic
autosomes as the preceding stock. These two comparisons tested whether C(1)DX or FM7 has an unusual
modifying effect on any of the autosomal loci monitored.
The results of these two comparisons are shown in
Table 2. T h e relative male/female expression in both
cases is quite similar. T h e values given in the table are
expressed as activity per individual. Consequently, the
male values are lower than thefemale. However, when
corrected for thefact that males have between 83 and
86% asmuch DNA as females, the expression of
autosomal genes is slightly greater in males, or equal
to females, and the X encoded enzymes, G6PDH and
GPGDH, approach near complete dosage compemation. Beta-hydroxyacid dehydrogenase, on the other
hand, shows ahigher expression in males in both
comparisons and represents a case of overcompensation. T o test whether this difference could be attributedtodifferential
expression in organsfound in
males and absent in females, thoraces of both sexes
from the FM7 stock were examined for thecomparative expression of these six enzymes. T h e results indicatethat thehigher male expression is found in
isolated thoraces as well and suggests that this is a
property of the expression of this enzyme (Table 3).
In a recent paper by DEVLIN, HOLM
and GRIGLIATTI
(1985b), this property of PHAD was also observed in
larvae.
The comparisons show that the degree of dosage
compensation is similar in both cases; hence the compound X and the FM7 chromosome exhibit similar
expression. As noted above, the allozymes in the two
chromosomes are the same, as judged by electropho-
Gene Expression in Metafemales
873
TABLE 2
Male/female comparisonsof C(2)DX and FM7 chromosomes
Whole flies
FM7/FM7
Enzyme
FM7/Y
G6DPH
6PGDH
BHAD
ADH
IDH
aCPDH
Whole flies
Thoraces
C(I)DX/Y
6.158f0.209
5.450f0.177
7.219 f 0.257
5.852 f 0.370
11.604 f 0.434
4.839f0.145
4.210f0.080
4.051 fO.064
7.138 f 0.193
5.032 f 0.354
9.244 f 0.241
3.939f0.129
6.029f0.177
5.370f0.145
5.659 f 0.273
4.051 f 0.241
8.424 f 0.241
5.514f0.305
4.132f0.129
3.971 f 0 . 0 9 6
6.334 f 0.370
2.910 f 0.129
7.267 f 0.145
5.145f0.209
4.084f0.113
3.119f0.113
1.945 f 0.193
1.736 f 0.145
4.678 f 0.193
7.379f0.482
2.605f0.064
2.106f0.048
2.186 f 0.145
1.527 f 0.080
3.955 f 0.080
5.900f0.113
Enzyme activities are expressed as micromoles of NADH or NADPH produced per ml (X102)in the reactions described in
Values represent the mean f SE of ten extracts each from ten flies or thoraces, respectively.
MATERIALS
AND METHODS.
TABLE 3
duce a dosage response just as in males where the
reduction
in the number of whole X chromosomes
Metafemale/female enzyme activity and protein comparisons
results in dosage compensation.
Enzyme
Merafemale
Euploid female
n
Ratio
The three autosomally encoded enzymes, in contrast,
all show reductions in metafemales below the
G6PDH
3.087 f 0.354 3.135 f 0.354 20 0.98
the null
diploid female values. A statisticaltestof
3.360 f 0.129 3.617 f 0.225 20 0.93
6PGDH
BHAD
4.084 f 0.322 5.064 f 0.370 20 0.81
hypothesis that the enzyme levelsare equivalent, with
4.116 f 0.273 5.177 f 0.322 20 0.80
ADH
an alternative that they are lower in metafemales(i.e.,
5.595 f 0.225 7.524 f 0.322 20 0.74
IDH
one-tailed t test) gives a result that each is significantly
aGPDH
4.662 f 0.402 6.125 f 0.354 20 0.76
reduced in metafemales at the 95% or greater confiProtein (mg/ml) 0.475 f 0.017 0.595 f 0.022 20 0.80
dence level.
Enzyme activities are expressed as micromoles of NADH or
Consistent with this observation is the fact that the
NADPH produced per ml (X102) in the reactions described in
MATERIALS AND METHODS. Protein values are milligrams per ml.
total protein values are significantly reduced on a per
Bovine serum albumin served as a standard. Values represent the
fly basis in metafemales relative to diploid sisters. If,
mean f SE of 20 extracts each prepared from fiveflies of the
assuggested by the above data,the expressionof
respective class.
many genes per cell were reduced approximately to
retic criteria. Because of the similarity of expression,
the inverse of the X chromosomal dosage, i e . , twothe evaluation of the metafemale values is simplified,
thirds of normal, then the total protein per fly would
requiring no correction for gross differential allelic
be lower. Ifthe X chromosomal genes, whichaccount
expression.
for approximately 20% of the total, were roughly
The metafemale data are presented in Table 3. For
unchanged and the remaining 80% were reduced in
the three X-linked enzymes, there are no significant
expression, the total protein comparisonwould be
differences in statistical tests betweenthe metafemales
0.73. The observed valueof
0.80 (Table 3) apvalues and those of the normal diploid female. The
proaches this figure. This value measures thousands
largest difference for X-encoded
enzymes
is for
of proteins whose individual levels might be greater
PHAD, which is actually less in
the metafemale. As
or lesser. Moreover, the steady state levels are not
noted above, this enzymeis expressed at higher levels
necessarily indicative ofrates of synthesisand secondin males than in females.It might also bethe case that
ary ramifications may occur. Yet the total protein
thisenzyme is overcompensated in metafemalesas
levels are reduced significantly as wouldbe predicted
well, thereby exhibiting less enzyme in the triplo-X
from the results of individual enzymemeasurements.
flies than in diploids. The probability that this deviaThe determination of the absolute values in metation is a matterof chance is between 5 and 10%. Thus, females and theirdiploid sistersdepends upon a measitfalls outside the limitsofsignificance
and it is
ure of the numberof cells inthe two types. DOBZHANconcluded that the three
enzymes exhibit similar levels
SKY (1929) determined that the number and size of
of expression despite the increased dosage of their
cells in the wings of metafemalescompared to normal
structural lociinmetafemales.Previous
studies, as
wasbasically the same in flies grown at 20". As an
well as our own (data not shown), on each of these
additional test, the total DNA values in the two types
loci have indicated that varying the regions surroundof females were compared using the Hoechst fluoresing the structuralgenes givesa dosage effect (TOBLER cence assay developed by LABARCA
and PAICEN
and GRELL1978; STEWARTand MERRIAM 1974;
(1980). If one uses the value for the contribution of
RAWLSand LUCCHESI
1974). However, in metafethe Y chromosome to the assay ofbetween 8 and16%,
males varyingthe whole X chromosome does not prothen metafemales (C(I)DX/X) would have from 95 to
J. A. Birchler,J. C. Hiebert and M. Krietzman
874
TABLE 4
wa and compared themto diploid wa sisters. The result
DNA estimate in metafemales andeuploid sisters
was that the intensity of pigment in the two types was
similar. This experiment was repeated and the same
response was found. Also, the X-linked Bar eye locus
exhibits a phenotype indicating that it is dosage compensated in metafemales (MARGOLIS, 1934;present
experiments).
It was of interest todeterminewhether
hypomorphic alleles ofautosomal loci would exhibita
phenotypically recognizable reduction in expression
in metafemales. Three autosomal genes affecting eye
pigment level that have known hypomorphic alleles
( p r ) and ( p ) (LINDSLEYand
are glass(gl),purple
GRELL1968). Accordingly, a test was set up using
these todeterminetheir
phenotypic expression in
metafemales. This experiment was intended to be a
parallel to STERN'Sphenotypic test for metafemale
dosagecompensation of the X-linked hypomorphic
white-apricot.
Metafemales were constructed that were homozygous for purple (pr) and cinnabar (cn). T h e purple
mutation is a hypomorphic lesion in the early steps in
the pteridine biosynthetic pathway (YIM, GRELLand
JACOBSON 1977) and the cinnabar mutant blocks the
production of ommachromes. Consequently,the p r cn
double mutant allows detection of modulations of the
purple locus expression without the complicating factor of brown pigments. Crosses were setup using
C(I)RM,y; p r cn females and +/E p r cn males. The
metafemaleprogeny
are exceptional y+ females.
Twelve were recovered in the screen and compared
to sisters of the same age. The metafemales had a
lighter eye color that persisted for the duration of
their life (Figure 1).
With respect to glass (gl), attachedX
females
C(I)DX,y f / Y ; g160J9were mated to males of +/E g16*j9
genotype. The progeny were screened for y + r metafemales. Two wererecovered andboth showed a
lowered intensity of pigment than their diploid sisters
(Fig. 1).
Lastly, the pink-peach (Pp) allele of the pink locus
was examined. A stock of C(I)DX, y f / Y females and
++/Y males homozygous for Pp was constructed. T o
test that pP is in fact a hypomorphic mutation, a stock
of Df(3R)pZ5,red e/TM3, Sb Ser pP e was crossed to the
pinkpeach stock. T h e Pp/Df(3R)pz5
flies have a lighter
eye color than@ / T M 3 , Pp flies. Thus, this allele fulfills
the criterion for a hypomorph.T h e five metafemales
recovered from the attached Xstock had a lightereye
color than their euploid sisters (Figure 1).
It perhaps should be noted that the observed
reduction in phenotypic expression for all three autosomal loci is not due to some unusual higher expression in euploid females. In fact, in all three cases of
g160j9,pr and p P , the male expression has more pigment
than the euploid female. A higher expression of au-
Genotype
n
Metafemale
Euploid female
5
5
cg/Fly
& SE
1.03 ? 0.041
1.12 k 0,092
Each extract consisted of 10 fliesl500 pl extraction buffer, n =
number of extracts for each class of fly. Values are expressed as
micrograms per individual fly. Calf thymus DNA was usedas a
standard. Metafemales and euploid sisters were collected from a
C ( I ) D X ,y w f / Y ; FM7/Y stock grown at 20".
102% as much DNA as sibling euploids (C(I)DX/Y),
assuming no gross difference incell number. The
observed value was 92%. The assays showed no significant difference in total DNA content per fly between metafemales and normals (Table 4).
The cell number counts have been repeated with
the particular stock, that was used for the molecular
studies and thatwas grown under thesame conditions.
The comparison relies on thefact that each cell of the
wing produces a single hair. The mean metafemale
value was 9.47 hairs per square millimeter and 9.79
fornormal diploids. These measurements are not
significantly different in statistical tests. Thus, under
our experimental conditions, a similar result to that
of DOBZHANSKY
was found.
The cell number and size data were collected from
flies grown at 20", which allows maximum survival.
Therefore, these measurements were performed on
metafemales that are morehealthy than those grown
at other temperatures. Since we find alterations of
gene expression under these conditions, it is possible
thatmoresevere
effects would befound at other
intemperatures (e.g., 25"). As thetemperature
creases, the cellsize butnotnumber
decreases in
normal stocks (ROBERTSON
1959). It is not excluded
from our data, thepossibility that cell size or number
could be altered in specific tissues of
metafemales even
at 20". This, however, would necessarily occur in a
minority of tissuesgiven the DNA measurements. The
data of MARGOLIS(1934) on facet number in metafemales, when viewed with an understanding of the
action of the Bar mutation, indicate that the metafemale condition alone does not alter the facet number compared to normal females. Thus, in these two
examples of individual tissues (wings, eyes), there is
not a detectable alteration of cell or facet number in
metafemales. Clearly, there is noreduction incell
number in metafemales to two-thirds of the euploid,
which is the level required to explain the observed
values on the basis of altered cell number.
Phenotypic test of autosomal expression in metafemales: Dosage compensation of X-linkedlociin
metafemales was first observed phenotypically for the
hypomorphic apricot allele of the white locus (w").
STERN(1 960) produced
metafemales homozygous for
Gene Expression in Metafemales
875
TABLE 5
Comparison of ADH messenger RNA levels in normal and
metafemales
Filter No.
488
1
2
3
Mean cpm
metafemale
0.74323
0.70141
0.72
Mean cpm
normal female
435
200
676
Mean
*
SD
Ratio
0.7270.02
Mean cpm values are the average of ten dots from which the
background value determined from ten dots of Adh deficiency on
the same blot were subtracted. The metafemale values have been
corrected relative to the normal female by the ratio of RNA in the
two samplesas determined by triplicate OD260 readings (as described
in MATERIALS AND METHODS).
FIGURE1 .-Phenotypic test of autosomal expression in metafey; pr cn; (right)metafemale,
males. (Top,left) normal female, C(I)RM,
C(I ) R M , y/+; pr cn. Middle, left, normal female, C( I)DX, g16'j9;
right, metafemale, C(I)DX,y f/++;g16'J9. (Bottom, left) normal feyf/E P p ; (right) metafemale, C(I)DX,yf/++; p'.
male, C(I)DX,
yf/c
tosomal hypomorphic mutants in males is often observed and the degreeto which it occurs is influenced
by modifiers (BIRCHLER1984).
The examination of five mutants all with effects in
the eye (w", B , p r , i6p, gl) provides an analysis within a
single tissue that examines the effects of both X and
autosomalgenes. The molecularanalyses by their
nature are most easily performed on whole fly preparations. The phenotypic analysis, however, addresses
the question in a single tissue and an analogous result
was found.
RNA analysis: If the expression of autosomal genes
were reduced on a per cell basis and the number of
cells per metafemale is comparable to euploid sisters,
then the amount of specific messenger RNAs from
autosomal genes would bereduced in total RNA
preparations. Since total RNA is predominantly comprised of ribosomal RNA, the above observations
would predict that if each cell has similar levels of
ribosomalRNA in metafemales and normals but a
reduction of many autosomally encoded messenger
RNAs, then measurements of specific messenger RNA
quantities encoded by an autosomal gene would be
reduced per total RNA in metafemales.
T o test this, total RNA was prepared from meta
and normal females, dotted onto nitrocellulose and
probed with "P-labeled RNA, antisense to Adh, transcribed from an SP6 promoter. After hybridization
and washing, the filter was subjected to autoradiography to screen for gross problems with background
and then the squares of nitrocellulose were cut, dried
and subjected to liquidscintillation spectrometry.
Three filters were individually hybridized. Each contained ten replica dots of metafemales,euploid female
and Adh deficiency RNA which was used as a background control. The results are shownin Table 5.
The average metafemale/euploid ratio from the three
filters is 0.72. The 99% confidence interval (0.710.73) indicated that this value is highly significantly
different from 1.OO.
To check that comparable amounts of ribosomal
RNAwere present in the preparations, total RNA
from euploid, metafemale and Adh deficiency flieswas
applied to nitrocellulose with a dot blot apparatus,
hybridized withanti-28s ribosomal RNA asdescribed
in MATERIALS AND METHODS. After autoradiography,
the film was scanned with a laser densitometer. When
probed with anti-28s ribosomal RNA, the three samples have a similar level of hybridization. A control
l X , 0.5X, and
concentration series, consistingof
0.25X the RNA amounts in the euploid sample, demonstrated that differences of these magnitudes are
readily discernible (Figure 2).
T o test whether Adh mRNAlevels in thisstock
differed between malesand females, RNAwas probed
from FM7/Y and C ( l ) D X , y w f / Y on three separate
blots each containing ten dots for each sex. The mean
male/female ratio for the threeblots was 1.54 +: 0.22
(sE). Thus, as with the phenotypic mutants, there is a
higher level of expression in males.
J. A. Birchler,J. C. Hiebert
and
876
f; i n
f
m
Bi
ix
fX
1x
FIGURE2.-Densitometric scan of quantitative dot blots for ribosomalRNAin metafemale, euploid female and Adh nulltotal
RNApreparations. a, Scan of autoradiograph of Adh null (n),
euploid female ( f ) and metafemale (m) RNA preparations probed
with anti-28s ribosomal RNA. The arbitrary integrated areas of
the respective peaks are 1475, 1674 and 1647. b, Scan of autoradiograph of dilution series. The 1X preparation of total Oregon R
RNA is equivalent to the euploid female sample ina. The integrated
area differs since the two samples (a, b) were probed and autoradiographed independently. The % X and %X samples represent dilutions of 1X. The integrated areas in ascending order are 299, 884
and 1517.
As a further test of the relative expression of RNAs
in metafemales, anindependentRNApreparation
from metafemales and diploid sisters from the same
stock was probed with antisense RNA synthesized on
a vector carrying a
cDNA clone from the X-linked
rudimentary locus (SEAGRAVES
et al. 1984). This gene
provided a test of whether the X, autosomal and total
RNA values are of the same relative proportions as
expected from the othermeasures of gene expression
and cell number. T o test the degree of compensation
in males and females, RNA was probed from FM7/Y
males and C(l)DX, y , w f / Y females on four separate
blots. T h e mean ratio of male/female values was 0.75
f 0.08 (sE). Thus, there is partial dosage compensation of rudimentary RNA levels in this background.
A filter containing nine dots
of both metafemale
and normalRNA gave a ratio of 1.24. The metafemale mean cpm above background was 86 f 10 (SE)
and the female mean cpm 69 f 1 1 (sE). T h e ratio is
intermediatebetweentheexpectation
of complete
dosage compensation (1.OO) and dosage effect (1.50),
as is the case between males and females. Clearly, the
expression of rudimentary mRNA is not reduced relative to total RNA as is Adh messenger RNA. Thus,
the same basic relative expression of X and autosomal
genes is found at the RNA level.
DISCUSSION
An analysisof gene expression in adult metafemales
was conducted and it was found that three X-linked
enzyme activities and phenotypic mutants are dosage
compensated between triplo-X and diploid females,
M.Krietzman
whereas autosomal enzyme activities, phenotypic
expression and messenger RNA levels are reduced.
These observationscoupled with the totalprotein
estimate suggest that the expression of many genes is
reduced in metafemales since both cell counts (DOBZHANSKY 1929 and above) and DNA estimates (above)
indicate a similar number and size of cells in the two
types of females grown at 20 O . It is important to note,
however, that both DOBZHANSKY'S
cell counts and the
DNA measurements were performed on flies grown
under conditions most favorable for metafemale survival. It ispossible that metafemales grown under
other conditions might be moreseverely affected and
thus these parameters would differ from normal females.
Metafemales grown at 25" have classically been
observed to bereduced insize relative to diploid
sisters. At 20", there is no obvious size difference.
The wing margin excisions and disarrangedeye facets
that are often found
in metafemales grown at 25 " are
absent at20". Sterility is a characteristicfound at both
temperatures. Metafemales grown at 25 " probably
differ to some degree in cell number or size but this
was not examined in this study because the bulk of
the observations are from flies grown at 20".
Normal and metafemales were analyzed in the molecular studies shortly after eclosion to minimize any
potential differences between the two that might occur due to thedevelopment of the ovaries in normal
females as they agecompared to the sterilemetafemales. It is unlikely that any differences due tothese
considerations significantly alter the magnitude
of the
effects observed.Forexample,
if there weremore
ribosomal RNA present in euploid females due toany
greater mass of the ovaries but no corresponding
Adh
messenger RNA increase(since ADH is not expressed
at high levels in ovaries and eggs), thenthe Adh
mRNA per total RNA values would be lower in normal females than in metafemales. The opposite result
was found. Also in this regard, it should be notedthat
metafemales grown at 20" are considerably more
1928;
vigorous than those grownat 25 " (DOBZHANSKY
present experiments). This has allowed a more reasonable comparison betweenthe two types of females.
T h e analysis of alcohol dehydrogenase messenger
RNA levels per total RNA showed a highly significant
reduction in metafemales compared to normals. In
contrast, mRNA levels from the X-linked rudimentary
locus were measured andfoundto
give ametafema1e:female ratio of 1.24. While this value falls
between the extremes expected for compensation and
dosage effect,it is clear that rudimentary mRNA is not
equal to Adh levels as would be expectedif all mRNAs
were reduced in metafemales relative to ribosomal
RNA and that autosomal levels were equivalent to
those originating from the X. These measurements
Gene Expression in Metafemales
TABLE 6
Summary of the phenotypicexpression of mutants in
metafemales
X-linked mutants
Dosage
compensation
white-apricot
X
Bar
X
Dosage
effect
Reference
STERN(1960); present experiments
MARCULIS(1 934);
present experiments
Inverse
Autosomal effect
mutants
purple
glass-60j9
pink-peach
Stubble
Unaffected
X
X
X
X
Reference
Present experiments
Present experiments
Present experiments
BEATONet al.
( 1988)
are independent of per fly or DNA measurements
and therefore do nothave an absolute point of reference. However, their relativelevels support the observations on thephenotypic, enzymeactivity and protein
levels that a major fractionof autosomal gene expression is lowered in metafemales and a major fraction
of X chromosomal expression is dosage compensated.
Because previousstudies of enzyme activities in
metafemales have been standardized to total protein
or by producing X/autosomal ratios, it is difficult to
compare results. Indeed, these former studies do show
indications of autosomal reductions or elevations of
all specific enzyme activities, which could represent
modulations of various activities or total protein. T h e
standardization to protein obscured the fact that autosomal expression is reduced. T o o u r knowledge
there had been no previous phenotypic tests of the
expression of autosomal hypomorphic alleles in metafemales. However,recently BEATONet al. (1988)
observed that the third chromosomal mutant, Stubble,
Sb, was reduced in expression in metafemales. A summary of the phenotypic expression of mutants examined in metafemales is given in Table 6.
One example in the literature allows an absolute
evaluation of X and autosomalgene expression in
normal and metafemales. The autoradiographic data
for overall transcription rates in salivary gland polytene chromosomes of LUCCHESI,
RAWLSand MARONI
(1 974)show that the numberof silver grains over the
x's in normal and metafemales are within 2% of each
other. However, the number of grains over the autosomes is reduced by 15%. In this case, the chromosomes serveasabsolutepoints
of reference.Such
measurementsaverage
the transcription of many
genes that may or may not be affected. Nevertheless,
if the results are considered in this way, they are
consistent with the measurements reported here.
877
The present study was conducted in adults. Using
this stage as opposed to larvae permits the identification of exceptional flies carrying the genetic markers
indicative of the metafemale progeny, but that are,in
fact, diploid or triploid females. Diploid females carrying an X chromosome from their fathers result
when
the compound X detaches and oneof the products is
joined at fertilization with an X bearing sperm. In the
stock used in these experiments, the percentage
of
third instar females carrying genetic markers indicative of metafemales is about 1. In the absence of
rigorous distinguishing characters between metafemales and euploids (resulting from detachments), metafemale analyses in larvae may not accurately reflect
the relative levels of X and autosomal geneexpression
due to contamination of the presumptive metafemale
class with euploids. This would tend to equalize any
differences between the two. An analysis conducted
in adults circumvents this problem.
In a recent article, DEVLIN, HOLM and
GRIGLIATTI
(1 988)
analyzed the expression of eight autosomalloci
in larvae of metafemales as well as trisomics for the
left arm of chromosomes two and three. The specific
activities of two autosomalgenes were reduced in
metafemales. In contrast, theautosomal trisomics producedmore obvious inverse effects uponunlinked
genes. The interpretation afforded these data
was that
the X and autosomes do not exercise an equivalent
regulatory effect upon autosomal geneexpression and
that the basis of X chromosomaldosage compensation
in metafemales and autosomal dosage compensation
in autosomal trisomics is brought about by distinct
mechanisms.
However, in light of our results in adult females, it
is potentially the case that the inverse effect of the
trisomic X condition is, in fact, effective upon more
autosomal genes thantrisomics 2L and 3L. This would
produce a lower total protein level which would tend
to cancel the inverse effects when the measurements
are expressed as specific activity, which they were.
The X chromosomal genes believed to be compensated in their analysis gave a slight dosage effect and
the presumed
unaffected
autosomal
genes
were
slightly reduced below the diploid level. Because the
values were normalized to total protein, it is possible
that if the values were expressed in absolute rather
than relative terms, the data would show that X chromosomal genes would bestronglydosagecompensated and theautosomal genes reduced
in metafemales
compared to normal. As noted above, the percentage
of metafemales at the thirdinstar is approximately 1,
whereas trisomics for 2L and 3L die primarily in the
pupal stage (FITZ-EARLE
and HOLM 1979).Since metafemales, as a general rule, die must earlier in development than 2L and 3L trisomics, it seems likely that
metafemales would be more severely affected. Alter-
878
J. A. Birchler,J. C. Hiebert and M. Krietzman
natively, some developmental difference might exist
between larval and adult metafemales with regard to
the effects of X chromosomal trisomy on autosomal
gene expression. A progressive severity as development proceeds is consistent with the general rule that
aneuploidy is more detrimental in later stages.
One interpretation of the present study is that gene
expression on all chromosomal regionsis reduced and
the observed compensation on the X results from the
increased dosage of the structural genes on this chromosome. In this sense, X chromosome compensation
in metafemales appears to be analogous to cases of
dosage compensation in maize (BIRCHLER
1979, 1981;
BIRCHLER
and NEWTON1981) as well as to autosomal
and GRIGLIATTI
dosage compensation (DEVLIN, HOLM
1982, 1985a,b, 1988;DEVLIN, GRIGLIATTI
and HOLM
1984; BIRCHLER
1983b). In thesecases, the structural
gene dosage response is cancelled by an inverse effect
simultaneously produced by the varied chromosomal
region.
It has been suggested that the inverse response is a
reflection of some aspect of the trans-acting regulatory
systems that operate in higher eukaryotes (BIRCHLER
1985). It was proposed that geneexpression is brought
about by a complex of factors that produce modulations when the stoichiometry of the individual components is varied. The chromosomal segments that
produce the inverse response would contain a structural gene, which exhibits a dosage effect, for one or
more of these factors thus producingthe altered ratio
of components. In the case described above, the inverse regulatory genes on the extra X chromosome,
which themselves must not be compensated, reduce
the expression of many structural genes on the
X,
which would result in the phenomenon recognizedas
dosage compensation. These regulatory genes would
also cause reductions in the expression of autosomal
genes.
We suggest that the greatinviability of metafemales
is due in part to the reduction in expression of many
autosomal structural genes. The lower levels ofnearly
80% of the cellular productsprovide insufficient
quantities for normal metabolism. An alternative explanation would be that there are gene
dosage effects
for X-linked genes in metafemales, which might account for their inviability. In this case, the surviving
flies are exceptional since they exhibit compensation.
This, however, would require a uniquemechanism to
operate in only a small fraction of the metafemale flies
and does not explain the observed autosomal reductions.
Research supported by grants from the National Science Foundation (J.A.B.) and March of Dimes (J.A.B.) and in part
by a grant
fromtheNationalInstitutesofHealthtoKENNETHPAIGEN,
in
whose laboratory portions of this work
was performed. Discussions
E ATHLEEN NEWTON,DAVID SCHOTT,
with LEONARDRABINOW,
T JLLE HAZELRIGG,
BOBLEVISand FORDANIELLE THIERRY-MIEG,
SPENCER
were helpful. T h e assistance of CARLA INOUYE,
TED
LEE, SAHAR BAZZAZ, RON COHEN,
CEDRICH o a n d H O N G H u is
gratefully acknowledged.The authors are grateful
D. GOLDBERG,
to
B. ZERGES and P. SCHEDLfor providing Adh and CRUDplasmids.
in preparation
The help of
E. VALMINUTO
and K. MCCREE-DIAZ the
of the manuscriptis greatly appreciated.
REST
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Communicating editor: V. G. FINNERTY