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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 The publication costs of this article were partly defrayed by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. 81734 solely to indicate this fact. 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. 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