IRENE ABRAHAM3 AND JOHN C. LUCCHESI4. Department of ... and a âdotâ autosome (STURTEVANT and NOVITSKI 1941; PATTERSON .... CO + (E~t-5~)l.
DOSAGE COMPENSATION OF GENES ON THE LEFT AND RIGHT ARMS OF THE X CHROMOSOME OF DROSOPHILA PSEUDOOBSCURA AND DROSOPHILA WILLISTONI’I~ IRENE ABRAHAM3
AND
JOHN C. LUCCHESI4
Department of Zoology and Curriculum in Genetics, University of North Carolina, Chapel Hill, North Carolina 27514 Manuscript received May 10, 1974 Revised copy received July 22, 1974 ABSTRACT
W e have investigated the occurrence of dosage compensation in D. willistoni and D. pseudoobscura, two species whose X chromosome is metacentric with one arm homologous to the X and the other homologous to the left arm of chromosome 3 of D. melanogaster. Crude extracts were assayed for isocitrate dehydrogenase (XR), glucose-6-phosphate dehydrogenase (XL?), 6-phosphogluconate dehydrogenase (XL?), and a-glycerophosphate dehydrogenase (chromosome 2) in D. willistoni, and for esterase-5 (XR), glucose-6-phosphate dehydrogenase (XL?) , 6-phosphogluconate dehydrogenase (XL?) and amylase (chromosome 3 ) in D. pseudoobscura. Our results indicate that a mechanism for dosage compensation is operative in both arms of the X chromosome of these two species.
I N Drosophila, evidence has been accumulating for many years that X-linked gene expression is generally equal in males and females (for reviews, see MULLER 1950; STERN1960; LUCCHESI 1973). This phenomenon of “dosage compensation” has been studied most extensively in Drosophila melanogaster, the species in which it was discovered. MULLER (1950) posed the question of whether dosage compensation would be present in other members of the genus, especially in those species which have a different X chromosome constitution. He suggested that an investigation of such species might provide not only information regarding the pervasiveness of the phenomenon but also some insight into its evolution and the nature of the underlying regulatory mechanism. The ancestral karyotype of the genus Drosophila is thought to consist of six separate chromosomal elements: an acrocentric X , four acrocentric autosomes and a “dot” autosome (STURTEVANT and NOVITSKI 1941; PATTERSON and STONE 1952). The rod X chromosome is considered to be the most primitive form of the X element since it is considerably more common in the genus than the metacentric X derived from fusion of the rod X and a rod autosome. Species in the These stuhes represent a porhon of a t h m s submitted by the senior author to the Department of Zoology, Umversity of North Carolina, in partlal fulfillment of the requirements for the degree of Doctor of Philosophy This investigation was supported by Research Grant GM-15691 of the Nahonal Inshtutes of Health. Present address Department of Biology, Yale Univeruty, New Haven, Connecticut Recipient of a Research Career Development Award (K4 GM 13,277) from the Nahonal Institutes of General Medical Sciences Genetics 78: 1119-11.26 December, 1974
1120
I. A B R A H A M A N D J. C. LUCCHESI
obscura group offer additional evidence on this point. The most primitive member of this group, Drosophila subobscura (BUZZATI-TRAVERSO and SCOSSIROLI 1955; LAKOVAARA, SAURA and FALK1972), has the ancestral rod X chromosome while other members of the group, including Drosophila pseudoobscura, have metacentric X chromosomes. We have investigated the occurrence of a regulatory system for dosage compensation in species that have undergone chromosomal fusions during evolution between the X chromosome and an autosome. In the species studied, the autosomally derived element is present in haploid condition in the male as one arm of the new X chromosome. Drosophila willistoni and D.pseudoobscura exemplify two such species; both have metacentric X chromosomes with the left a n n (designated XL) homologous to the rod X and the right arm ( X R ) homologous to an and TAN1937). Since autosomal arm (3L) of D.melanogaster (STURTEVANT enzyme mapping in these two species has not been extensive we have assumed, in some instances, chromosomal locations of enzymes based on the general evidence of chromosomal homologies deduced by comparing the location of visible and TAN1937; STURTEVANT and NOVITSKI phenotypic mutations ( STURTEVANT 1941). These studies have shown f o r D. melanogaster, D. willistoni, and D. pseudoobscura, as well as f o r other species, that the chromosomal arms have largely retained their identity although fusions between arms and numerous inversions within arms have taken place. While the possibility of small translocations exists, there is no unequivocal evidence that such rearrangements have in fact occurred. Furthermore, recent mapping of enzyme loci in D. pseudoobscura and D. willistoni (HUBBY and LEWONTIN 1966; PRAKASH and LEWONTIN 1968; PRAKASH, LEWONTIN and HUBBY 1969; LAKOVAARA and SAURA 1972) generally confirm homology assignments and the relative conservativeness of the gene array on chromosomal arms of the species in question. In D. willistoni, the structural gene f o r NADP-dependent isocitrate dehydrogenase (IDH) is located at 1-60 on XR (LAKOVAARA and SAURA1972). While glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate dehydrogenase (6PGD) are not mapped, their X-linked location in D.melanogaster suggests that they may be associated with XL in D.willistoni. In D. pseudoobscura, esterase-5 (Est-5) and and LEWONTIN 1966; GGPD have been localized in the X chromosome (HUBBY PRAKASH, LEWONTIN and HUBBY 1969). We have mapped the former to X R and assumed, for reasons of homology. that the latter as well as 6PGD have remained associated with X L . We have compared the activity of all of the above enzymes in males and females, at two different developmental stages. Autosomal enzymes measured for control purposes were a-glycerophosphate dehydrogenase (a-GPDH), which is located at 2-59 in D. willistoni (LAKOVAARA and SAURA 1972), and amylase located on chromosome 3 in D.pseudoobscura (PRAKASH and LEWONTIN 1968). Our evidence supports the contention that X-linked loci in both arms of the X chromosomes of these two species are dosage compensated. MATERIALS A N D METHODS
Genetic stocks and culture conditions: Two D.pseudoobscura mutant stocks, y sn U CO sh, and bd se 11 s p tt, were obtained f r o m North Carolina State University. A y sn U CO se sh line was
S CHROMOSOME DOSAGE C O M P E N S A T I O N
1121
synthesized from the above stocks. Mutants and their linkage relationships are discussed in STURTEVANT and TAN(1937) and STURTEVANT and NOVITSKI(1941). Mutant abbreviations are usually the same as those used for D.melanogaster mutants with similar phenotypes (LINDSEY and GRELL1968). Wild-type stocks were obtained from the University of Texas at Austin. Electrophoretic variants for Est-5 were kindly supplied by DR. J. L. HUBBY.D.willistoni wild-type stocks were received from the University of Texas and from DR.F. J. AYALA. Both species were raised in half pint bottles or in vials on a medium consisting of: 75 m l water, 8 ml light Karo syrup, 5.6 g malt, 5.3 g cornmeal, 1.25 g Brewer's yeast, .7 g soy flour, .78 g agar, .35 ml ethanol, .35 ml propionic acid, and .I5 g tegosept (DR. S. PRAKASH, personal communication) ; the medium was seeded with live baker's yeast. D. willistoni cultures were maintained at 25i.1" and D. pseudoobscura cultures at 2 0 k 1 " . Individuals used for enzyme assays were collected as third instar larvae and as 1-24 hour post-emergence adults.
Esterase-5 mapping in D. pseudoobscura: Flies from a wild-type stock exhibiting a slow E s t 8 (Est-5s) variant were crossed with flies from the marker stock y sn U CO sh having a fast Est-5 (Est8F) variant. Female progeny from this cross, heterozygous for the markers and Est-5 vanants, were backcrossed to the marker stock. Crossover progeny extracts were separated by electrophoresis and stained for Est-5 as described below. A second experiment was performed in order to obtain recombination information with a marker more closely linked to CO than those i n the above cross. Females from a stock y sn U CO se sh Esi-5" were crossed to wild-type males with the slow variant and progeny females were backcrossed to marker stock males. Crossover progeny were tested electrophoretically for Est-5 mobility as follows Single adult flies were homogenized 1966). Polyacrylamide disc electrophorwith a Lucite plastic single fly homogenizer (JOHNSON esis was performed according to the method of ORNSTEIN(1964) and DAVIS(1964). The gels were stained according to HUBBY and LEWONTIN(1966), using a-naphthylacetate as substrate and Fast Red TR as coupling dye. Spectrophotometric determinations of enzyme aciiuity: Adult flies were homogenized at a concentration of 20 mg/ml in Dounce homogenizers with loose pestles or Dual conical homogenizers with ground glass pestles. Homogenization media were 0.05 M tris(hydroxymethy1) aminomethane, tris-HCl, (pH 7.2) for the dehydrogenases, or twice-distilled water for the amylase assay. Flies were homogenized at a concentration of 65 mg/ml of 0.1 M tris-borate (PH 8.9) buffer, containing 1 m M disodium ethylene dinitrolotetraacetate (EDTA) and 5% sucrose, for the esterase-5 assay. Larvae were homogenized under the same conditions as adult flies except that all homogenization media were 1 mM phenylthiourea in order to inhibit tyrosinase activity. Thirty minutes after homogenization extracts were centrifuged at 12,000 x g for 40 minutes and the clear supernatant, excluding the lipoid layer, was used as the enzyme extract. All the steps described above were carried out at 0-4". In order to measure Est-5 levels exclusively, crude extract was separated by electrophoresis, the gels were stained for esterase activity with a-naphthylacetate and Fast Red TR, the Est-5 band was cut out and the dye*elutedfrom it and measured spectrophotometrically. A full descrip1974). One unit of esterase activity is tion of this method has appeared elsewhere (ABRAHAM defined as that amount of enzyme which liberates 1 pmole of a-naphthol/minute a t 28". The amylase activity assay was based on the saccharogenic method of BERNFELD (1955) and modifications by DOANE(1969). One unit of amylase activity is defined as that amount of enzyme that causes the release of the equivalent of 1 pmole of maltose per minute. All dehydrogenase assays were based on the formularies of LUCCHESIand ~ W L (1973) S and measured the generation of NADH or NADPH. One unit of dehydrogenase activity is defined as the w w i n t of enzyme reducing 1 nmole of NAD+ or NADP+ per minute a t 29". Protein determinations were made by the method of LOWRY et al. (1951) using bovine serum albumin as the standard. Statistical procedures: Means of specific activities and their standard deviations for males were compared with those of females using Student's t test a t the 5% level of significance. Confidence limits (95%) for the ratios of the means were computed using Student's t test.
1122
I. ABRAHAM AND J. C. L U C C H E S I
TABLE 1 Genetic LGcalization of the Est-5 locus in Drosophila pseudoobscura ~
~~
3laternal genotypes
Recombinants
Between CO and sh
Between U and U (Est-SF)
+
+
CO
+ (Est-ST)I + sh (Est-5s)j134
CO
(Est-5s)I l4
f sh (Est-Sr)] CO + ( 1 E ~ t1- 5 ~3) Between CO and -~ - se
Between U and CO
+ (Est-5")
U
+ U
CO
se (Est-5F) (Est-5s)
+++
+
CO
(Est-5")i9 I
CO
(Est-5"')
CO
f
+
+ (E~t-5S)l"~
U
CO
+ (E~t-5~)l se
( E S ~ - ~ S ) ( ~ ~ ~
se
(Est-5r)
+ (Est-5S)j3
TABLE 2 Enzyme activities in males and females of Drosophila willistoni
Sex
Specific activity* mean f S.E.
N
IDH
M
201 t 10
10
222 & 5
a-GPDH
F M
178% 9
9 10
220 -c 8
9
GPGD
F M
67 C 3
10
F M
70k 2 422 3
9 10
F
41t 2
9
B. 1-24 post-emergence adults IDH M 2 2 2 C 10
6
EnZyI,,e
Significance between XI & F means
95% confidence interval of ratio of AI/F means
NS
32-1.02
A. Third instar larvae
G6PD
(U-GPDH
F M
223C7 686 i. 32
3
F
4 8
.70- .93
NS
.85-1.07
NS
35-1.20
NS
28-1.12
NS
.89-1.17
NS
.91-1.23
NS
.78-1.11
8
GPGD
M
669 i- 22 65C 4
G6PD
F M
612 2 48C 3
9 5
F
50k 3
5
* Expressed as mean units per mg protein.
5
N = number of determinations; M = male; F = female; S and NS = significance and non-
significance, respectively, at the 5% level.
1123
X CHROMOSOME DOSAGE COMPENSATION RESULTS
Results of the two genetic mapping experiments f o r the structural gene of Est-5 in D.pseudoobscura are presented in Table 1 . These data indicate that the Est-5 locus is closely linked to CO, within 2.4 map units. The gene CO maps in the right portion of the X chromosome (STURTEVANT and TAN1937) and exhibits a striking similarity to the mutant gs located on 3L of D.melanogaster; gs and CO are probably homologous genes. I n D. melanogaster, Est-6, mapped at 3-36.8 (WRIGHT 1963), is located 1.7 map units to the right of gs. Therefore Est-5 of D.pseudoobscura and Est-6 of D.melanogaster are probably also homologous genes, strengthening the existing evidence for the homology of X R in D.pseudoobscura to 3L in D.melanogaster. Specific activity levels of GGPD, IDH and (Y-GPDH in D.willistoni third instar larvae or 1-24 h old emerged adults of both sexes are reported in Table 2. With TABLE 3 Enzyme activities in males and females of Drosophila pseudoobscura
Enzyme
Sex
A. Third instar larvae Est-5t M
Specjfic activity* mean k S.E.
N
1.97 f .W
5
F M
1.92 t .03
4
2.10 f .09
5
GPGD
F M
2.06 f .I2 52.1 f 2.2
5 12
46.2 f 2.7
G6PD
F M
63.5 f 2.8
12 I2
F
71.4 f 3.1
12
B. 1-24 post-emergence adults Est-5+ M 4.15 f .I4
4
F M
A
3.91 iz ,043
5
4.48
3
* .39 4.33 * .I6
GPGD
F M
66.9 k 2.3
G6PD
F M
65.1 1.6 98.3 f 4.6
12 6
101.4 f 3.4
Amylase
F M
,3011 f 018
6 6
.282 f .019
6
F
*
M & F means
Significance between
9 5 % confidence interval of ratio of M/F means
NS
.9%1.15
NS
94-1.14
NS
.97-1.32
NS
.77-1.02
NS
.97-1 .I5
NS
.79-1.30
NS
.94-1.12
NS
.85-1.10
NS
.87-1.32
4 11
* Expressed as mean units per mg protein. +Two separate runs of males and females were performed at each developmental stage for Est-5. These runs are listed separately. Abbreviations as in Table 2.
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I. ABRAHAM A N D J. C. LUCCHESI
the exception of a-GPDH in larvae, none of the enzyme activities measured differed significantly between males and females. In D. pseudoobscura, specific activities of GGPD, GPGD, and Est-5 were determined in male and female larvae or emerged adults. In addition, amylase was measured in adults only. The results, presented in Table 3, reveal indistinguishable levels of activity in males and females for all of the enzymes measured. DISCUSSION
MULLER(1950) pointed out that an understanding of the evolution of mechanisms for genetic adaptation, including that for dosage compensation, may be sought among species whose X chromosome consists of two arms, one derived from the original rod-shaped X (of species such as D.melanogaster), the other corresponding to an original autosomal arm. He suggested that the left arm of the X in D.pseudoobscura would exhibit dosage compensation, but that the right arm might not. The right a n n is generally believed to be more recently evolved as a sex chromosome than the left arm and MULLERbelieved that it might not have been subjected to the forces of natural selection for a sufficient time to have evolved and perfected such a mechanism. Preliminary observations of chromosomal RNA synthesis on the X chromosome of D.pseudoobscura measured by monitoring 3H-uridine incorporation along polytene chromosomes have yielded somewhat conflicting results on this point (CHATTERJEE 1971;DUTTAGUPTA et al. 1973). The results presented here indicate that the two XR-linked enzymes studied are, in fact, dosage compensated. Est-5 in D. pseudoobscura and IDH in D. willistoni have statistically indistinguishable activities in males and females, at either the third instar larval stage o r in 1-24 h old adults. I n the genus Drosophila the basic configuration of six chromosomal arms is virtually inviolable (PATTERSON and STONE1952). The karyotypes appear to have changed by rearrangements of the chromosomal arms via inversions and fusions. Of the 54 different fusions established during the evolution of the genus, 12 have been between the X and an autosome, according to the tabulation of PATTERSON and STONE.Following an X-autosome fusion, the transformation of the autosomal arm into a bona fide sex chromosome is achieved only after the autosomally derived arm is present in a single dose in the male karyotype and is present in two doses in the female karyotype. This apparently entails the dosage compensation of the genes on the translocated autosomal element and the degeneration and eventual loss of the homologous non-translocated, free autosomal element. Two factors combine to bring about the disappearance of the free autosome. The first is the absence or low level of crossing over in Drosophila males. As only the male would receive the free autosome, this element would degenerate over the generations due to the collective weight of the second factor, deleterious mutations. In the absence of the ‘‘corrective” mechanism of recombination, such mutations would accumulate on the sex-linked autosomal element. This is the process thought to have induced the degeneracy of the Y chromosome, in the sense of its lack of genetic content, in Drosophila and other
X CHROMOSOME DOSAGE C O M P E N S A T I O N
1125
species. As HUXLEY (1928) pointed out, the general reduction of crossing over in heterogametic species has led to accentuation of the differentiation of sex chromosomes which were once probably homologous chromosomes. Pari passu with the degeneration of the non-translocated autosomal element just discussed, selective pressures would favor the dosage compensation of loci on the autosomal arm translocated to the X, in order to maintain proper genic balance in the male. It is not evident a t this time that the same mechanism is responsible for the regulation of gene activity of the XR and XL of D.willistoni and D.pseudoobscura and the X of D.melanogaster. Nevertheless, since the same phenotypic end, i.e. the equalization of X-linked gene products in males and females, appears to be achieved in all of these species, they can all be considered to exhibit dosage compensation. We are grateful to DRS.WINIFRED W. DOANEand GUSTAVO MARONIfor helpful discussions and suggestions during the preparation of this manuscript. LITERATURE CITED
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L., 1964 Disc electrophoresis-I. Background and theory. Ann. N.Y. Acad. Sci. 121: ORNSTEIN. 321-349. PATTERSON, J. T. and W. S. STONE,1952 Evolufion in the genus Drosophila. Macmillan Company, New York. PRAKASH, S. and R. C. LEWONTIN, 1968 A molecular approach to the study of genic heterozygosity in natural populations. 111. Direct evidence of coadaptation in gene arrangements of Drosophila. Proc. Natl. Acad. Sci. U.S. 59: 398-405. S., R. C. LEWONTIN and J. L. HUBBY,1969 A molecular approach to the study of PRAKASH, genic heterozygosity in natural populations. IV. Patterns of genic variation in central, marginal and isolated populations o'f Drosophila pseudoobscura. Genetics 61 : 841-855. STERN,C., 1960 Dosage compensation-development of a concept and new facts. Can. J. Genet. Cytol. 2: 105-118.
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