REGULATION OF GENE FUNCTION: A COMPARISON OF X-LINKED ENZYME ACTIVITY LEVELS 1N NORMAL AND INTERSEXUAL TRIPLOIDS OF DROSOPHILA MELANOGASTERl

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1 REGULATION OF GENE FUNCTION: A COMPARISON OF X-LINKED ENZYME ACTIVITY LEVELS 1N NORMAL AND INTERSEXUAL TRIPLOIDS OF DROSOPHILA MELANOGASTERl JOHN C. LUCCHESIZ AND JOHN M. RAWLS, JR. Department of Zoology and Curriculum in Genetics, University of North Carolina, Chapel Hill, North Carolina Manuscript received September 18, 1972 Transmitted by Arthur Chovnick ABSTRACT We have measured gene function in normal and male-like intersexual triploids of Drosophila melanogaster by assaying crude extracts of whole flies or thoraces for levels of an X-linked (6-phosphogluconate dehydrogenase) and an autosomal (NADP-dependent isocitrate dehydrogenase) enzyme activity. Our observations lead us to conclude that each dose of the X-linked gene is more active in the cells of these intersexes than it is in normal triploid or diploid female cells. These results indicate that a level of activity intermediate between the normal male and female levels is possible for X-linked genes. IN Drosophila, the similarity in expression of X-linked genes in females, having two X chromosomes, and in males, having one X, is attributed to dosage compensation (MULLER 1950; STERN 1960) and has been documented at the level of transcription (MUKHERJEE and BEERMANN 1965) as well as at the level of enzymatic activity (SEECOF, KAPLAN and FUTCH 1969; TOBLER, BOWMAN and SIMMONS 1971; BAILLIE and CHOVNICK 1971). We have recently measured X-linked gene activity in triploid females (bearing three X chromosomes and three sets of autosomes) and found that each X-linked gene dose in these flies is as active as it is in a diploid female genome (LUCCHESI and RAWLS 1972). This communication presents the results of a study of autosomal and X-linked gene activity in phenotypically male-like triploid intersexes (bearing two X chromosomes and three sets of autosomes). Our observations indicate that enzyme activity levels are equivalent in such intersexes and in triploid females and, therefore, that X-linked gene activity expressed on a per gene basis is greater in these triploid intersexes than it is in triploid or diploid females. MATERIALS AND METHODS The enzymes used in this study were 6-phosphogluconate dehydrogenase (GPGD) (E.C ) and NADP-dependent isocitrate dehydrogenase (IDH) (E.C ). The former is X-linked, with its structural gene locus at 0.9 (YOUNG 1966); the latter is autosomal, with its This investigation was supported by Research Grant GM and Genetics Training Grant 2 T1 GM-685 of the National Institutes of Health. Recipient of a Research Career Development Award (K4-GM-13, 277) from the National Institutes of General Medical Sciences. Genetics 73 : March, 1973.

2 460 J. C. LUCCHESI AND J. M. RAWLS, JR. s?ructural gene locus at 27.2 on chromosome 3 (Fox 1971). In one experiment males and females from a wild-type (Samarkand) stock were used. In the other experiments, triploid females bearing the X-chromosome constitution yz sc w" ec/fmg,y:"cl scb dm B were used; thesefmales have._ an attached-x and a free X chromosome in addition to three sets of autosomes (XX/X;AAA). When mated to wild-type (Samarkazd) males they produce thrq classes of phenotypically distinguishable daughters-triploids (X$/X;AAA) and diploids (XX/Y;AA and X/X;AA)-and two classes of male-like intersexes-xx/y;aaa and X/X;AAA, with the latter type being more mmmon in our cultures. In another cross. T(1+3)w"C'1, U f/y males were used; these males have a small segment of their X chromosome, bearing the structural gene for GPGD (Pgd+) and the wild-type allele of w:' (U+) inserted into chromosome 3. The presence of this insertion in a genome will be designated Dp(X), while theadeleted X chromosome 5 noted Df(X). Among the progeny of this cross. intersexes w$h two (XX/Y;AAA) or thrse (XX/Dp(X)/Y;AAA) Pgdf doses, triploid females yith two (XX/Df (X);AAA) or three (XX/Df (X)/Dp(X);AAA) doses, and diploid females (XX/Y;AA) are all phenotypically distinguishable. It must be noted that the w+ allele present cn Dp(X) is subjected to a position effect leading to a variegated eye phenotype, in the appropriate gmotypes. This position effect does not appear to spread significantly to the locus of Ped+ as evidenced by the dosage response obtained by SEECOF, KAPLAN and FUTCH (1969) in males and females with this duplication. A full description of the genetic symbols mentioned above can be found in LINDSLEY and GHELL (1968). Fly cultures were maintained at 25 i 1 "C on standard cornmeal-molasses-agar medium, seeded with li\e yeast and containing propionic acid and tegosept-m as mold inhibitors. Flies 24 to 48 hours old were collected, counted, and weighed. In some instances, they were transected with a sharp blade arid separated into a fraction containing thoraces with attached heads, legs and wings and a fraction containing abdomens. Crude extracts were prepared and the levels of 6PGD and IDH activities were measured spectrophotometrically as described elsewhere (LUCCIIESI and R~wr.s 1972). Protein concentration was determined after a method of Lownu, e/ al. (1951) using bovine serum albumin as a standard. Enzyme activities are expressed as mpmoles of NADP reduced/min/mg protein. Replicate determinations consisted of enzyme assays performed on separate extracts. Means and their standard errors were computed and compared using Student's t test. Comparisons with a p are considered significantly different. RESULTS The distribution of various enzymes has been shown to differ between adult Drosophila males and females due to the presence of ovarian tissue in the abdomens of the latter (STEELE, YOUNG and CHILDS 1969). Since the triploid interzexes generated by the crosses used in this study were externally male-like. we decided (1) to perform the critical enzyme assays on extracts obtained from the thoraces of transected intersexes and triploid females, and (2) to select an autosomal and an X-linked enzyme whose respective levels of activity would be similar in normal males and females. As can be seen from the data presented in Table 1, the ratio of GPGD activity in male and female thoracic fractions (1.1) is similar to the ratio of IDH activity (1.2). Correlating the level of activity of a structural gene to the level of activity of its enzyme product may be invalid if the latter were subject to certain forms of feedback control. Therefore, to insure that the observed levels of GPGD activity in triploid intersexes reflect the level of function of its X-linked structural gene (Pgd+), intersexes bearing two (X-X/Y;AAA) and three (XX/D (X/Y;AAA) doses, and triploids bearing two (XX/Df (X);AAA) and three ( z X/Df (X)/Dp (X);AAA) doses of Pgdf were obtained and assayed. The results. presented as

3 GENE FUNCTION IN DROSOPHILA TABLE 1 Distributions of enzyme activities* in wild-type mules and females 461 GPGD IDH Sample Abdomens Thoraces) Abdomens Thoracest Males 18.5 f Females 11.8 t *.4 *Expressed as mean mpmoles NADP reduced x 102/min/mg protein t se. (number of determinations per mean equals 3). t Thoraces with attached heads, legs, and wings. the whole fly data of Table 2, show dependence of GPGD levels on structural gene dosage within the intersex and the triploid female genomes. These results, incidentally, show that the Pgd+ locus in Dp(X) is not subjected to a position effect significant enough to mask the additive effect of this duplication on the level of GPGD activity. Table 2 includes GPGD and IDH activity measurements on thoracic fractions of triploid intersexes, and of diploid and triploid females. No significant differences were found among these different genotypes. DISCUSSION In order to make an accurate comparison of GPGD activity in intersexes and in triploid females, specific activity measurements must be correlated to the total number of Pgd+ structural genes present per unit of extracted tissue. Since cell size is very similar in triploid intersexes and in triploid females (DOBZHANSKY 1929), the same number of nuclei per unit of tissue can be expected in these two types of flies. Therefore, the activity per Pgd+ gene dose for intersexes can be obtained by dividing their mean specific activity value by two (since there are two X chromosomes per nucleus in these flies), and for triploid females by dividing their mean specific activity value by three (since these flies have three X chromosomes per nucleus). Using the thoraces data of Table 2, these calculations yield GPGD values of 2.35 for intersexes and 1.4 for triploid females, which leads us to conclude that the mean expression of each X-linked gene dose is higher in intersex than in female triploid cells. This conclusion is quite consistent with results of measurements of X-chromosome activity performed by autoradiographic monitoring of 3H-uridine incorpora tion along larval salivary gland polytene chromosomes (MARONI and PLAUT 1972, 1973). A similar comparison can be made by normalizing GPGD specific activity measurements in relation to IDH, since intersexes and triploids each contain three doses of the IDH structural gene per cell and, incidentally to this type of analysis, exhibit the same level of thoracic IDH specific activity. The 6PGD/IDH specific activity ratios for thoraces (Table 2) are 0.35 for intersexes and 0.35 for triploids. Since intersexes have two and triploid females have three X chromosomes per nucleus, dividing these ratios by two and three, respectively, yields normalized levels of activity per Pgdf dose of in intersexes and of in

4 462 Y. C. LUCCHESI AND J M. RAWLS, JR. I $ B 2 w h z

5 GENE FUNCTION IN DROSOPHILA 463 triploid females. It is satisfying to note that if the same calculations are performed with the specific activity values obtained from whole-fly extracts of triploid female controls and triploid intersex controls (Table 2), the activity values per Pgd+ dose are in intersexes and in triploids. Needless to say, the concordance of these results with those obtained above with data from thoraces indicates that the 6PGD and IDH intrafly distributions exhibited by triploid females are altered to the same extent in intersexes. We have previously presented evidence that each X-linked gene dose is as active in a normal diploid female cell as it is in a triploid female cell (LUCCHESI and RAWLS 1972). In light of this fact, the comparison of gene activity between the male-like intersexes and triploid females reported herein can be held as evidence that each dose of an X-linked gene is more active in the cells of these intersexes than it is in a normal diploid female cell; the three sets of calculations presented above yield an average increase in activity of 58% for Pgd+. Lastly, we wish to point out that regardless of the particular mechanism by which it is mediated, dosage compensation requires that the activity of each X-linked gene dose in a diploid female genome be half that of the single gene dose in a diploid male genome. Our measurements of Pgd+ activity in male-like triploid intersexes indicate that a level of activity intermediate between the normal male and female levels is possible. We are grateful to PROF. D. P. COSTELLO for helpful criticism and suggestions during the preparation of this manuscript. LITERATURE CITED BAILLIE, D. L. and A. CHOVNICK, 1971 Studies on the genetic control of tryptophan pyrrolase in Drosophila melanogaster. Molec. Gen. Genet. 112 : DOBZHANSHY, TH., 1929 The influence of the quantity and quality of the chromosomal material on the size of the cells in Drosophila melanogaster. Arch. Entwicklungsmech. Organ. 115: FOX, D. J., 1971 The soluble citric acid cycle enzymes of Drosophila melanogaster. I. Genetics and ontogeny of NADP-linked isocitrate dehydrogenase. Biochem. Genet. 5: LINDSLEY, D. C. and E. H. GRELL, 1968 Genetic variations of Drosophila mehnogmter. Carnegie Inst. of Washington Pub. No. 627: LOWRY, 0. H., N. J. ROSEBROUGH, A. L. FARR and R. J. RANDALL, 1951 with the Folin phenol reagent. J. Biol. Chem. 193: Protein measurement LUCCHFSI, J. C. and J. M. RAWLS, 1973 Regulation of gene function: A comparison of enzyme activity levels in relation to gene dosage in diploids and triploids of Drosophila melanogaster. Biochem. Genet, 9: MARONI, G., and W. PLAUT, 1972 A cytological analysis of dosage compensation in D. melanogaster triploids. Genetics 71: s37. -, 1973 Dosage ccumpensation in Drosophila mhogaster triploids. I. Autoradiographic study. Chromosoma (In press). MURHWEE, A. S. and W. BEERMANN, 1965 Synthesis of RNA by the X-chromosomes of Drosophila melanogaster and the problem of dosage compensation. Nature 207 : MULLER, H. J., 1950 Evidence of the precision of genetic adaptation. Harvey Lecture Series XLIII, ,l: C. C. Thomas, Springfield.

6 464 J. C. LUCCHESI AND J. M. RAWLS, JR. SEECOF, R. L., W. D. KAPLAN and D. G. FUTCH, 1969 Dosage compensation of enzyme activities in Drosophila melanogaster. Proc. Nat. Acad. Sci. 62: STEELE, M. W., W. J. YOUNG and B. CHILDS, 1969 Genetic regulation of glucose-6-phosphate dehydrogenase activity in Drosophila melanogaster. Biochem. Genet. 3 : STERN, C., 1960 Dosage compensation-development of a concept and new facts. Can. J. Genet. Cytol. 2: TOBLER, J., J. T. BOWMAN and J. R. SIMMONS, 1971 Gene modulation in Drosophila: Dosage compensation and relocated U+ genes. Biochem. Genet. 5: YOUNG, W. J., 1966 X-linked electrophoretic variation in 6-phosphogluconate dehydrogenase in Drosophila rnelanogaster. Hered. 57 :