GENETIC AN-4LYSIS OF ASPARTATE AMINOTRANSFERASE ISOZYMES FROM HYBRIDS BETWEEN DROSOPHILA MELANOGASTER AND DROSOPHILA SIMULANS AND MUTAGEN-INDUCED ISOZYME VARIANTS E. H. GRELL Biology Division, Oak Ridge National Laboratory, Oak Ridge, Tennessee 37830 Manuscript received April 25, 1975 ABSTRACT
The aspartate aminotransferases (designated GOT1 and GOT2) are two enzymes of Drosophila melanogaster for which naturally occurring electrophoretic variants were not found. There is an electrophoretic difference between D. melanogaster and D . simulans. Since the F, hybrid offspring of these species are sterile, a genetic analysis of the ordinary type cannot be done on differences between the two species. A method was devised to make “partial hybrids” in which one chromosome arm is homozygous for melanogaster genes in an otherwise hybrid background. By using this method, Gotl was localized to 2R and Got2 to 2L. Once a gene can be assigned to a chromosome, it may be followed in crossing schemes and mutations from mutagen treatments may be looked for. At the locus of Gotl a mutation with low activity was recovered and designated Gotlzo. It was located a t a genetic map position of 75 on 2R. A Got2 mutant with a greater migration to the anode was recovered and designated Got2J.I t was located at a genetic map position of 3.0, and in the salivary chromosome was between 22B1 and 22B4 inclusive.
HE extensive electrophoretic polymorphism of enzymes in Drosophila usuTally provides the basic material for genetic analysis and experimentation with these enzymes. If true polymorphism is not found, one may search for the rare mutant enzyme in natural populations and laboratory stocks. A mutant of any enzyme probably can be found if a search includes a large enough number of flies, but in practice it is not always possible to find a variant. Without a genetic variant no genetic study is possible. The only alternative to finding a natural variant is the induction of a variant by the action of a mutagen. One Drosophila melanogaster isozyme system in which there appears to be no easily detectable electrophoretic varability is the aspartate aminotransferase system. Like most other animals, Drosophila has two isozymes of aspartate aminoet al. transferase, the soluble and the mitochondrial forms. In humans (DAVIDSON 1970; CHEN and GIBLETT1971) and in mice (CHAPMAN and RUDDLE1972; DELORENZO and RUDDLE 1970), alleles which specify electrophoretically different isozymes of both forms have been found at two separate loci. I n these previous genetic investigations the enzymes were given the symbol GOT, for glutamate Research sponsored by the U S Atomrc Energy Commission nned contract wlth the Union Carbide Corporation. Genetics 83: 753-7G4 August, 1976
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oxaloacetate transaminase; this convention will be continued here. The gene symbol Got will be used. Drosophila simulans is more closely related to D.melanogaster than any other species, but many genetic differences between them exist. The recovery of hybrids between the species is restricted in several ways (STURTEVANT 1921) . D. melarrogaster females and D.simulans males mate quite readily, but the reciprocal combination mates much less frequently. If normal free-X melanogaster females are mated with simulans males, the sons with a melanogaster X do not survive beyond a larval stage. If attached-X melanogaster females are mated to simulans males, only the sons survive. It is necessary for viability that a hybrid zygote have a simulans X chromosome. All of the hybrids are entirely sterile and have rudimentary gonads. I n order to achieve at least a limited capability for genetic analysis of differences between melanogaster and simulans, MULLERand PONTECORVO (1940) devised the method of making “partial hybrids.” These are the offspring of triploid melanogaster females and X-irradiated simulans males. The triploid mother carries a recessive marker on each chromosome and the father has wild-type alleles. The triploids produce some egg nuclei which contain a haploid set of chromosome plus one or more extra chromosomes. If such a hyperploid egg is fertilized by a sperm in which the X-ray treatment has caused a loss of the chromosome or chromosomes that are overrepresented in the egg, a diploid zygote is formed which can develop into an adult Drosophila. Partial hybrids with several chromosome contents were studied by PONTECORVO ( 1942, 1943). Partial hybrids can be used to assign a structural locus of an enzyme to a chromosome if there is an electrophoretic difference between melanogaster and simulans. A fly homozygous for one melanogaster chromosome and heterozygous for other chromosomes will contain only the melanogaster enzyme if the structural locus is on the homozygous melanogaster chromosome. In this report we discuss a refinement which has been devised to allow localization to a chromosome arm rather than to a whole chromosome. Melanogaster and simulans stocks have been synthesized which do not require the use of X-irradiated simulans males and produce many of the desired types per female. After the chromosomal arm which carries the locus of the enzyme is known, a mutation of that locus can he induced in D. melanogaster. When a mutant is obtained more precise location and further manipulation are possible. MATERIALS A N D METHODS
Drosophila stocks: Descriptions and genetic locations of marker mutations can be found in LINDSLEY and GRELL(1968). The combinations of free autosomal arms and compounds arms were synthesized. The compound autosome stocks as originally described by RASMUSSEN(1960) and LEWIS (1967) contained two identical autosomal arms attached to one centromere. Flies containing both a compound 2 L [ C ( 2 L ) ] and compound 2R [ C / Z R ) ] or both ( C 3 L ) and ( C 3 R ) are the usual way in which the compounds are kept in stock. The original free 2R ( F ( S R ) I ] derived as a crossover between In(2LR)Zt”S and a normal chromosome. The original free 3R [ F ( 3 R ) 1 ] was obtained from a 3;4 translocation. The F(2L)I and F ( 3 L ) l were derived from compounds by X irradiation.
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To separate the two arms of the large autosomes of D. simulans, male simulans with the marker Ubxnt were irradiated with 4000 R of X rays and mated to stw females. F, males with the Ubxm marker were backcrossed to stw females. The offspring of individual F, males were examined, and those showing linkage between the Y chromosome and an autosomal marker were saved. Each simulans Y-2 translocation was crossed to melanogaster C ( 2 L ) ; F ( 2 R ) / F ( 2 R ) and F ( 2 L ) / F ( Z L ) ; C ( 2 R ) stocks, and each simulans Y-3 translocation was crossed to melanogaster C ( 3 L ) ; F ( 3 R ) / F ( 3 R ) and F ( 3 L ) / F ( 3 L ) ; C ( 3 R ) stocks. The crosses that produced offspring were examined for marker genes on the compounds and electrophoresed to score for the aspartate aminotransferase phenotype. TOinduce mutations of isozyme loci, compound autosome stocks were used. This allowed two treated genes (one on each of the two identical arms of the compound) to be scored at the same time, increasing the efficiency of the search by a factor of 2. The increased efficiency is significant in a n electrophoretic search for mutants and overrides the inconvenience of the method. Preliminary genetic locations were made using the stocks with the dominant markers, S Sp Tft nwD PinYt. The enzyme mutation was made heterozygous with a marked chromosome, and crossovers between markers were selected and tested for the enzyme allele. After rough locations were determined, closer markers were used to obtain a more precise location. Cytological location used chromosomal deficiencies and a series of duplications. The duplicaand tions were derived from T(Y;2)'s broken at different places along chromosome 2 (LINDSLEY et al. 1972). The rationale was that the whole chronlosomes of the fly had one allele SANDLER and the fragments were derived from chromosomes with another allele. The flies with both allelic forms of the enzyme had the locus of the enzyme on the chromosome fragment and thus the position of the locus was distal to the chromosome break. Electrophoresis: Polyacrylamide gels 11% in. long were cast from 5% cyanogum 41. Most of the work was done with a buffer system of 0.04 M Tris adjusted to p H 8.5 with H,PO,; for observations with a lower pH the buffer was 0.016 M Tris adjusted to pH 6.5 with H,PO,. Buffer in the electrode vessels was the same as that in the gels. Samples were applied in slits made i n the gel. A potential of 700V applied across the gel gave a current of around 20mA, which was applied for 3 hours with cooling by circulating ice water. et al. (1970) with The staining method for aspartate aminotransferases was that of DAVIDSON slight modification. The mixture contained 225 mg L-aspartic acid, 125 mg a: ketoglutaric acid, 200 mg disodium dihydrogen EDTA, 0.5 mg pyridoxal phosphate and 100 mg fast blue BB i n 100 ml of 0.05 M Tris with a final p H of 7.5. Incubation of gels in the staining mixture of 0" with shaking for 3 to 4 hours gave the best results, although staining at r m m temperature (-22") for 1 hour gave satisfsctory staining. Regions of aspartate aminotransferase activity stained blue, and the gels acquired a brownish-pink background stain. RESULTS
T h e GOT isozymes of Drosophila: Drosophila. like other animals, have two isozymes of aspartate aminotransferase (GOT). The GOT1 isozyme migrates to the anode more rapidly than GOT2 during acrylamide gel electrophoresis (Figure 1) . Electrophoresis of samples of flies from 150 stocks of D. melanogaster did not reveal a single natural variant of either isozyme, therefore it was concluded that these enzymes do not often exhibit electrophoretic polymorphism in melanogaster. This does not imply that there are no polymorphic populations or that there are no naturally cccurring mutants in laboratory stocks or natural populations. Electrophoresis of D. simulans revealed that GOT1 and GOT2 of this species are electrophoretically distinguishable from the corresponding isozymes of D. melanogaster (Figure 2 ) . Both enzymes of simulans migrate more slowly to the
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I
GOTl
GOT2
D. melunoguster FIGUREl . - G e l after electrophoresis of Drosophila extract stained to make areas of GOT activity visible. The GOTl isozymes are nearer the anode and the GOT2 isozymes are nearer the cathode. anode than the melanogaster enzymes. In addition, GOTl of simulans smears badly during electrophoresis at pH 8.5, although better isozyme patterns are obtained at pH 6.5 (Figure 3). Hybrids of melanogaster and simulans have the isozymes of both parents, but the simulans forms are present in smaller amounts than the melanogaster forms. Hybrids also contain hybrid enzymes of intermediate migration on gels. At this point one cannot differentiate between a oneor a two-gene difference between the species. One gene may specify a polypeptide in both GOTl and GOT2 and thereby account for the similar but shifted patterns of melanogaster and simulans. On the other hand the two species may differ by two genes, in which case the parallel relationship could be fortuitous. Assignment of Got loci to specific chromosome arms: The two chromosomes which may be readily tested for a Got locus with preexisting stocks are the X chromosome and the fourth chromosome. The two large autosomes require a new approach. To test the X chromosome, melariogaster-simulans hybrid females were electrophoretically compared with males from attached-X melanogaster mothers and simulans fathers. The one X chromosome of the males comes from their simulans father. The GOTl and GOT2 patterns of these two types were both hybrids. If a locus had been on the X chromosome, the hybrid males would have had at least one GOT pattern which did not contain melanogaster isozyme; therefore there was no Got locus on the X chromosome.
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75 7
>GOT1
>
GOTZ
me/ mefiim
sim
pH 8.5 FIGURE 2.-Isozymes of D.melmogaster, D.simulans and F, hybrids. Electrophoresis was at pH 8.5. GOT2 isozymes are clearly different in the two species, and the hybrid contains a hybrid enzymes.
The presence oE a locus on chromosome 4 was assessed by use of a stock constructed by MULLER and PONTECORVO as a product of their synthesis of partial hybrids. It has a simulans 4 (sim-4) in a melafiogasterbackground. The sim-4 is balanced with the dominant, ciD,and is kept balanced because sim-4/sim-4 homozygotes are rather weak and males are sterile. The GOT1 and GOT2 isozyme patterns of sim-4/sim-4 and sim-4/ciDin the melanogaster background are identical to wholly melanogaster flies. Females of the genotype sim-4/ciDwere mated to simulans males. All offspring had hybrid patterns of both GOT1 and GOT2. Therefore the conclusion was that the fourth chromosome contained no Got locus. For the major autosomes (chromosomes 2 and 3 ) , the presence of a Got locus was tested one chromosome arm at a time. To test the left arm of chromosome 2, melanogaster C(2L), dp; F(2R)f, bw/F(2)f,bw females were crossed to T(Y;2)3/stw simulans males. To test the right arm of chromosome 2, the melanogaster mothers were F(2L), dp/F(2R), cn and the simulans fathers were T(Y;2)2/stm.To test 3L the mothers were C(3L), h'; F(3R)f, e ro ca/F(3R)f, e ro ca and the fathers were simulans T(Y;3)f,Ubxm/ca*.To test 3R the mothers were F(3L), hP/F(3L),h'; C(3R), sr and the simulans males were T(Y;3)3,
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FIGURE3.-GOT isozymes of D.melanogasierj D.simulans and an F, hybrid. Electrophoresis was at pH 6.5. GOT1 isozymes as well as GOT2 isozymes are different in the t w o species.
2
L dP
5 7
2
D. melanogoster ?
-I +
bW
R
x
21
+
-
+
2R
+
D.sim/uns T ( Y ; 2 ) t
b w (me1 2R)
+ +
(sim
2R)
2L homozygous D. melonogoster; other chromosomes heterozygous
FIGURE 4.-Scheme used to localize the loci of the enzymes from partial hybrids between D. melamguster and D. simulans. In this example, 2L (left arm of the second chromosome) is homozygous in the partial hybrids and all other chromosome arms are heterozygous. The melanoguster mothers contain a compound 2L marked with d p (dumpy) and free 2 R s marked with bw (brown). The simulans fathers are heterozygous for a translocation between the Y chromosome and the second chromosome.
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TABLE 1
GOT patterns present in hybrid Drosophila with different homozygous chromosome arms GOT1
GOT2
Chromosome arm homozygous in hybrid background
pattern
pattern.
simulans X melanogaster 2L melanogaster 2R simulans 3L melanogaster 3L melanogaster 3R simulans 4
hybrid hybrid me1 hybrid hybrid hybrid hybrid
hybrid me1 hybrid hybrid hybrid hybrid hybrid
Ubxm/caz.The objective in these crosses was to obtain flies homozygous for one melanogaster arm in an otherwise hybrid background (Figure 4). The compound chromosomes were the homozygous arms. They were marked with recessive markers to ensure that the offspring might be unambiguously classified. The translocation in the simulans fathers uncoupled the two arms of the chromosome EMS
I
II
m Got2 FIGURE 5.-Crossing scheme to pick up mutants of Got2. Males in Generation I are injected with EMS. Males carrying a treated chromosome are backcrossed to females like their mothers. These males of generation I1 are placed with mates in vials with one male per vial. One fly from each vial is tested by electrophoresis. Several males from a positive vial are individually mated, and they and their progeny are tested electrophoretically to make certain that the mutation is recovered.
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E. € GRELL I .
and allowed formation of sperm without a chromosomearm. In the case of crosses between C(3L), he; F(3R)I, e ro ca/F(3R)I, e ro ca, two kinds of flies were produced. One class, hP; Ubx"', was homozygous for the melanogaster 3L and hybrid for 3R. The other class was wild type for the markers, homozygous for the simulans 3L and hybrid for 3 R. Table 1 is a summary of the results of the crosses; it shows that flies with a homozygous melanogaster 2R have only the melanogaster GOTl isozyme. Flies homozygous for melanogaster 2 L have only the melanogaster GOT2 isozyme. All other types have hybrid patterns of GOTl and GOT2. These results indicate that the Gotl locus is on 2R and the Got2 locus is on 2L. The alleles of melanogaster are designated Gotldfand Got2", while the alleles of simulans are designated GotZs and Got2*. Mutation of GotlMand (;OW: The scheme for mutagenesis is shown in (Figure 5 ) . For mutagenesis of Got2' in 2L, C(2L), nubL; C(2R), cn were mated to EMS-treated C(2L), cl; C(2R), sp* males. F, males of the genotype C(2L), cl; C(2R), cn were selected and backcrossed to C(2L), nub*;C(2R), cn females. One C(2L), cl; C(2R), cn son from each backcross mating was homogenized and electrophoresed. A total of 320 mutagenized compound 2L chromosomes were tested for mutations of Got2. One mutation was found which caused GOT2 to migrate to the anode more rapidly than the standard G O n M(Figure 6). The new allele has been designated G 0 t 2 ~Got2H/GorPJ . heterozygotes have a threebanded pattern. For mutagenesis of Gotl in 2R, (2L), cl; C(2R), vg females were mated to EMS-treated C(2L), dp; C(2R), cn males. F, males of the genotype C(2L), cl; C(2R), cn were backcrossed to C(2L), cl; C(2R), v g females. One son from each
FIGURE 6.-Gel of the standard melanogaster allele G o W , a heterozygote of Got"/GofZJ and the newly induced mutant allele Got2J.
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backcross was electrophoresed. No mutant with altered electrophoretic migration was discovered among 450 tested males; however, one culture yielded flies that had a GOTl which was somewhat lighter and more smeared than that ordinarily specified by Gotlu. These flies proved to be heterozygous for a mutant allele designed Got2 l o . Homozygotes of Got2 lowere very low in GOTl activity (Figure 7). The homozygotes appeared to be normal in appearance, viability and fertility in spite of having very little GOTl activity. Genetic locations of Gotl and Got2: Since the locations of Gotl and Got2 are known to be on 2R and 2L, respectively, one may ignore other possibilities at the outset. To locate Got1 on 2R, females of the genotype Gotl'"/TftGot2" nwDPinYt were mated to bwD males. Crossovers between the visible markers were selected and crossed to Got1 l o . Then flies with crossover chromosomes heterozygous with Got2'" were electrophoresed and scored for the presence of Got2" or Goti'" in the crossover chromosome. This experiment indicated that Got2 was closely linked to nwDand to the left of it. For a more precise location, L GotZU/Got1'"nwD PinYt females were constructed and mated to bwDmales. Males crossovers between L and nwDwere selected and crossed to Got21" females. Of 31 crossovers, 15 were crossovers between L and Got2 and 16 were crossovers between Got2 and nwD. The distance between L and nu?' was 6 map units; the distance between L and Got2 was 3 map units; and the genetic location of Got2 was near 75 on the map of the second chromosome (Figure 8). The genetic location of Got2 was roughly determined by making females heterozygous for S Got2* Sp Tft and GozJ2 and mating to G 0 t 2 ~ males. Crossovers were selected and electrophoresed, and Got2 was found to be closely linked to S . For a precise location, S G 0 t 2 shv/+ ~ G0t2~ -I- females were mated to -I-GotPMshv males. Of 28 crossovers between S and shv, 19 were between S and Got2 and 9 were between Got2 and shv. The interval between S and shv was 2.5
GOT 1 GOT2
Go/1
Go/1 ' O
FIGURE7.-Gel of the EMS-induced mutant, Goil'O, with GoilY, the allele from which it was derived.
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E. H.GRELL
1
I
-
I
72.0
54.8
75
79,b
107#2
FIGURE8.-Map
of a segment of 2R showing position of Goti on genetic map with some of the markers used in its location.
Got2 shv ho 0#1,
1#3
3.0
3,a 4.0
FIGURE9.-Map of a segment of 2L showing the position of Got2 on the genetic map and some of the markers used to locate it. map units; Got2 was 0.68 of that interval, or 1.7 map units from S . The standard map position of S is 1.3and the position of Got2 is 3.0 (Figure 9). Cytological location: Since Got2 was near the tip of 2L, and near S, there were chromosome aberrations which aided in a cytological location. The T(Y;2)'s of LINDSLEY and SANDLERet d.(1972)were a source of fragments which included the tip of 2L up to various points near the site of Got2 (Figure 10). To test for the presence of Got2 on the fragment, C(Z)RM, y ; T(Y;2), y+ Bs/SMI females were mated to C(2L),GotP,cl; C(2fi),cn males. Female offspring with C(I)RM, the desired part of the translocation, and C(2L), GotJ, cl; C(2R), cn were electrophoresed and scored for the presence of Got2" on the fragment. Two fragments with breaks in 21D and 22A did not carry Got2", while three fragments with breaks in 22B, 22C and 22D did carry G o P . The break in 22B
FIGURE 10.-Chromosome aberrations used to locate Got2 on the salivary chromosome map. The drawing of the salivary chromosome is taken from LEWIS(1945). The line at the bottom represents L m s ' Df(2L)S2. The gap in the line corresponds to the limits of the deficiency. The lines above the chromosome drawing represent the fragments derived from Y-2translocations described in LINDSLEY et al. (1972).The numbers indicate the salivary chromosome section in which the second chromosome is broken. Translocation D70 is broken in 21D,J69 in 22A, H56 in 22B, B112 in 22C and R136 in 22D.
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appears to be just after band 22B4, so Got2 must be in 22B4 or to the left of it. The left limits are assessed by Df (2L)S2. The deficiency lacks a segment from 21D1 io 22A6 (LEWIS 1945), but still carries Got2"; therefore Got2 must be to the right of 22A6, which would place it in 22B1 or to the right. The present information indicates that Got2 is in the interval from 22B1 to 22B4 inclusive. The Got1 locus in 2R has not been located on the salivary chromosome map. It is in the middle of the chromosome arm, and there is less material in stock that can be used to help locate it. DISCUSSION
The objective of this work was to begin a genetic analysis of aspartate aminotransferase (GOT) isozymes in Drosophila metanogaster. The task was made difficult because there is no detectable electrophoretic ploymorphism of either of the two isozymes of aspartate aminotransferase. These is a difference in the electrophoretic migration of homologous GOT isozymes of D.melanogaster and D.simulans, and the ability to locate this difference to a choromsome arm is helpful in mutagenesis. Without any knowledge of location, all chromosomes would have to be marked and followed. With chromosome arms identified, however, there is the added advantage that two treated alleles are scored for mutation in every fly that is electrophoresed if compound autosomes are used. In obtaining mutants from EMS treatment it is necessary to test a fly from the F, generation, because when F, flies are tested, mosaic flies are found which have a mutation in their somatic cells but which does not extend into germ cells and is not transmitted. The method for the synthesis of partial hybrids of melanogaster and simulans i s very easy once the necessary stocks are constructed. LITERATURE CITED
CHEN, S . H. and E. R. GIBLETT,1971 Genetic variation of soluble glutamic-oxaloacetic transaminase in man. Am. J. Hum. Genet. 23: 419-424.
V. M. and F. H. RUDDLE, 1972 Glutamate oxaloacetate transaminase (GOT) genetics CHAPMAN, in the mouse: Polymorphism of GOT-1. Genetics 70: 299-305. M. C. RATTAZZI, F. H. RUDDLEand H. A. LUBS,1970 Genetic DAVIDSON, R. G., J. A. CORTNER, polymorphism of human mitochondrial glutamic oxaloacetic transaminase. Science 169 : 391-392. DELORENZO, R. J. and F. H. RUDDLE, 1970 Glutamate oxalacetate transaminase (GOT) genetics in Mus musculus: Linkage, polymorphism and phenotypes of the GOT-I and GOT-2 loci. Biochem. Genet. 4: 259-274.
LEWIS,E. B., 1945 The relation of repeats to position effects in Drosophila melanogaster. Genetics 30: 137-166. -, 1967 Genes and gene complexes. In: Heritage from Mendel. Edited by R. H. BRINK.The University of Wisconsin Press, Madison, Wisconsin. LXNDSLEY, D. L. and E. H. GRELL,1968 Genetic variations of Drosophila melanogaster. Publ. 627. Carnegie Institute of Washington, Washington, D. C. LINDSLEY, D. L. and L. SANDLER et al., 1978 Segmental aneuploidy and the genetic gross structure of the Drosophila genome. Genetics 71: 157-184.
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MULLER, H. J. and G. PONTECORVO, 1940 Recombinants between Drosophila species the F, of which are sterile. Nature 146: 199-200. PONTECORVO, G., 1942 Hybrid sterility i n artificially produced recombinants between D . mel1943 anogaster and D . simulans. Proc. Roy. Soc. London, Ser. B 61: 385-397. -, Viability interactions between chromosomes of D.melanogaster and D . simulans. J. Genet. 45: 51-66. RASMUSSON, I. E., 1960 Report, Drosophila Info. Serv., Vol. 34,p. 53. STURTEVANT, A. H., 1921 Genetic studies on Drosophila simulrms. I. Introduction. Hybrids with Drosophila melanogzster. Genetics 5 : 488-500. Corresponding editor: B. H. JUDD
The aspartate aminotransferases (designated GOT1 and GOT2) are two enzymes of Drosophila melanogaster for which naturally occurring electrophoretic variants were not found. There is an electrophoretic difference between D. melanogaster and D . simulans. Since the F, hybrid offspring of these species are sterile, a genetic analysis of the ordinary type cannot be done on differences between the two species. A method was devised to make “partial hybrids” in which one chromosome arm is homozygous for melanogaster genes in an otherwise hybrid background. By using this method, Gotl was localized to 2R and Got2 to 2L. Once a gene can be assigned to a chromosome, it may be followed in crossing schemes and mutations from mutagen treatments may be looked for. At the locus of Gotl a mutation with low activity was recovered and designated Gotlzo. It was located a t a genetic map position of 75 on 2R. A Got2 mutant with a greater migration to the anode was recovered and designated Got2J.I t was located at a genetic map position of 3.0, and in the salivary chromosome was between 22B1 and 22B4 inclusive.
HE extensive electrophoretic polymorphism of enzymes in Drosophila usuTally provides the basic material for genetic analysis and experimentation with these enzymes. If true polymorphism is not found, one may search for the rare mutant enzyme in natural populations and laboratory stocks. A mutant of any enzyme probably can be found if a search includes a large enough number of flies, but in practice it is not always possible to find a variant. Without a genetic variant no genetic study is possible. The only alternative to finding a natural variant is the induction of a variant by the action of a mutagen. One Drosophila melanogaster isozyme system in which there appears to be no easily detectable electrophoretic varability is the aspartate aminotransferase system. Like most other animals, Drosophila has two isozymes of aspartate aminoet al. transferase, the soluble and the mitochondrial forms. In humans (DAVIDSON 1970; CHEN and GIBLETT1971) and in mice (CHAPMAN and RUDDLE1972; DELORENZO and RUDDLE 1970), alleles which specify electrophoretically different isozymes of both forms have been found at two separate loci. I n these previous genetic investigations the enzymes were given the symbol GOT, for glutamate Research sponsored by the U S Atomrc Energy Commission nned contract wlth the Union Carbide Corporation. Genetics 83: 753-7G4 August, 1976
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E. H. GRELL
oxaloacetate transaminase; this convention will be continued here. The gene symbol Got will be used. Drosophila simulans is more closely related to D.melanogaster than any other species, but many genetic differences between them exist. The recovery of hybrids between the species is restricted in several ways (STURTEVANT 1921) . D. melarrogaster females and D.simulans males mate quite readily, but the reciprocal combination mates much less frequently. If normal free-X melanogaster females are mated with simulans males, the sons with a melanogaster X do not survive beyond a larval stage. If attached-X melanogaster females are mated to simulans males, only the sons survive. It is necessary for viability that a hybrid zygote have a simulans X chromosome. All of the hybrids are entirely sterile and have rudimentary gonads. I n order to achieve at least a limited capability for genetic analysis of differences between melanogaster and simulans, MULLERand PONTECORVO (1940) devised the method of making “partial hybrids.” These are the offspring of triploid melanogaster females and X-irradiated simulans males. The triploid mother carries a recessive marker on each chromosome and the father has wild-type alleles. The triploids produce some egg nuclei which contain a haploid set of chromosome plus one or more extra chromosomes. If such a hyperploid egg is fertilized by a sperm in which the X-ray treatment has caused a loss of the chromosome or chromosomes that are overrepresented in the egg, a diploid zygote is formed which can develop into an adult Drosophila. Partial hybrids with several chromosome contents were studied by PONTECORVO ( 1942, 1943). Partial hybrids can be used to assign a structural locus of an enzyme to a chromosome if there is an electrophoretic difference between melanogaster and simulans. A fly homozygous for one melanogaster chromosome and heterozygous for other chromosomes will contain only the melanogaster enzyme if the structural locus is on the homozygous melanogaster chromosome. In this report we discuss a refinement which has been devised to allow localization to a chromosome arm rather than to a whole chromosome. Melanogaster and simulans stocks have been synthesized which do not require the use of X-irradiated simulans males and produce many of the desired types per female. After the chromosomal arm which carries the locus of the enzyme is known, a mutation of that locus can he induced in D. melanogaster. When a mutant is obtained more precise location and further manipulation are possible. MATERIALS A N D METHODS
Drosophila stocks: Descriptions and genetic locations of marker mutations can be found in LINDSLEY and GRELL(1968). The combinations of free autosomal arms and compounds arms were synthesized. The compound autosome stocks as originally described by RASMUSSEN(1960) and LEWIS (1967) contained two identical autosomal arms attached to one centromere. Flies containing both a compound 2 L [ C ( 2 L ) ] and compound 2R [ C / Z R ) ] or both ( C 3 L ) and ( C 3 R ) are the usual way in which the compounds are kept in stock. The original free 2R ( F ( S R ) I ] derived as a crossover between In(2LR)Zt”S and a normal chromosome. The original free 3R [ F ( 3 R ) 1 ] was obtained from a 3;4 translocation. The F(2L)I and F ( 3 L ) l were derived from compounds by X irradiation.
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To separate the two arms of the large autosomes of D. simulans, male simulans with the marker Ubxnt were irradiated with 4000 R of X rays and mated to stw females. F, males with the Ubxm marker were backcrossed to stw females. The offspring of individual F, males were examined, and those showing linkage between the Y chromosome and an autosomal marker were saved. Each simulans Y-2 translocation was crossed to melanogaster C ( 2 L ) ; F ( 2 R ) / F ( 2 R ) and F ( 2 L ) / F ( Z L ) ; C ( 2 R ) stocks, and each simulans Y-3 translocation was crossed to melanogaster C ( 3 L ) ; F ( 3 R ) / F ( 3 R ) and F ( 3 L ) / F ( 3 L ) ; C ( 3 R ) stocks. The crosses that produced offspring were examined for marker genes on the compounds and electrophoresed to score for the aspartate aminotransferase phenotype. TOinduce mutations of isozyme loci, compound autosome stocks were used. This allowed two treated genes (one on each of the two identical arms of the compound) to be scored at the same time, increasing the efficiency of the search by a factor of 2. The increased efficiency is significant in a n electrophoretic search for mutants and overrides the inconvenience of the method. Preliminary genetic locations were made using the stocks with the dominant markers, S Sp Tft nwD PinYt. The enzyme mutation was made heterozygous with a marked chromosome, and crossovers between markers were selected and tested for the enzyme allele. After rough locations were determined, closer markers were used to obtain a more precise location. Cytological location used chromosomal deficiencies and a series of duplications. The duplicaand tions were derived from T(Y;2)'s broken at different places along chromosome 2 (LINDSLEY et al. 1972). The rationale was that the whole chronlosomes of the fly had one allele SANDLER and the fragments were derived from chromosomes with another allele. The flies with both allelic forms of the enzyme had the locus of the enzyme on the chromosome fragment and thus the position of the locus was distal to the chromosome break. Electrophoresis: Polyacrylamide gels 11% in. long were cast from 5% cyanogum 41. Most of the work was done with a buffer system of 0.04 M Tris adjusted to p H 8.5 with H,PO,; for observations with a lower pH the buffer was 0.016 M Tris adjusted to pH 6.5 with H,PO,. Buffer in the electrode vessels was the same as that in the gels. Samples were applied in slits made i n the gel. A potential of 700V applied across the gel gave a current of around 20mA, which was applied for 3 hours with cooling by circulating ice water. et al. (1970) with The staining method for aspartate aminotransferases was that of DAVIDSON slight modification. The mixture contained 225 mg L-aspartic acid, 125 mg a: ketoglutaric acid, 200 mg disodium dihydrogen EDTA, 0.5 mg pyridoxal phosphate and 100 mg fast blue BB i n 100 ml of 0.05 M Tris with a final p H of 7.5. Incubation of gels in the staining mixture of 0" with shaking for 3 to 4 hours gave the best results, although staining at r m m temperature (-22") for 1 hour gave satisfsctory staining. Regions of aspartate aminotransferase activity stained blue, and the gels acquired a brownish-pink background stain. RESULTS
T h e GOT isozymes of Drosophila: Drosophila. like other animals, have two isozymes of aspartate aminotransferase (GOT). The GOT1 isozyme migrates to the anode more rapidly than GOT2 during acrylamide gel electrophoresis (Figure 1) . Electrophoresis of samples of flies from 150 stocks of D. melanogaster did not reveal a single natural variant of either isozyme, therefore it was concluded that these enzymes do not often exhibit electrophoretic polymorphism in melanogaster. This does not imply that there are no polymorphic populations or that there are no naturally cccurring mutants in laboratory stocks or natural populations. Electrophoresis of D. simulans revealed that GOT1 and GOT2 of this species are electrophoretically distinguishable from the corresponding isozymes of D. melanogaster (Figure 2 ) . Both enzymes of simulans migrate more slowly to the
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I
GOTl
GOT2
D. melunoguster FIGUREl . - G e l after electrophoresis of Drosophila extract stained to make areas of GOT activity visible. The GOTl isozymes are nearer the anode and the GOT2 isozymes are nearer the cathode. anode than the melanogaster enzymes. In addition, GOTl of simulans smears badly during electrophoresis at pH 8.5, although better isozyme patterns are obtained at pH 6.5 (Figure 3). Hybrids of melanogaster and simulans have the isozymes of both parents, but the simulans forms are present in smaller amounts than the melanogaster forms. Hybrids also contain hybrid enzymes of intermediate migration on gels. At this point one cannot differentiate between a oneor a two-gene difference between the species. One gene may specify a polypeptide in both GOTl and GOT2 and thereby account for the similar but shifted patterns of melanogaster and simulans. On the other hand the two species may differ by two genes, in which case the parallel relationship could be fortuitous. Assignment of Got loci to specific chromosome arms: The two chromosomes which may be readily tested for a Got locus with preexisting stocks are the X chromosome and the fourth chromosome. The two large autosomes require a new approach. To test the X chromosome, melariogaster-simulans hybrid females were electrophoretically compared with males from attached-X melanogaster mothers and simulans fathers. The one X chromosome of the males comes from their simulans father. The GOTl and GOT2 patterns of these two types were both hybrids. If a locus had been on the X chromosome, the hybrid males would have had at least one GOT pattern which did not contain melanogaster isozyme; therefore there was no Got locus on the X chromosome.
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75 7
>GOT1
>
GOTZ
me/ mefiim
sim
pH 8.5 FIGURE 2.-Isozymes of D.melmogaster, D.simulans and F, hybrids. Electrophoresis was at pH 8.5. GOT2 isozymes are clearly different in the two species, and the hybrid contains a hybrid enzymes.
The presence oE a locus on chromosome 4 was assessed by use of a stock constructed by MULLER and PONTECORVO as a product of their synthesis of partial hybrids. It has a simulans 4 (sim-4) in a melafiogasterbackground. The sim-4 is balanced with the dominant, ciD,and is kept balanced because sim-4/sim-4 homozygotes are rather weak and males are sterile. The GOT1 and GOT2 isozyme patterns of sim-4/sim-4 and sim-4/ciDin the melanogaster background are identical to wholly melanogaster flies. Females of the genotype sim-4/ciDwere mated to simulans males. All offspring had hybrid patterns of both GOT1 and GOT2. Therefore the conclusion was that the fourth chromosome contained no Got locus. For the major autosomes (chromosomes 2 and 3 ) , the presence of a Got locus was tested one chromosome arm at a time. To test the left arm of chromosome 2, melanogaster C(2L), dp; F(2R)f, bw/F(2)f,bw females were crossed to T(Y;2)3/stw simulans males. To test the right arm of chromosome 2, the melanogaster mothers were F(2L), dp/F(2R), cn and the simulans fathers were T(Y;2)2/stm.To test 3L the mothers were C(3L), h'; F(3R)f, e ro ca/F(3R)f, e ro ca and the fathers were simulans T(Y;3)f,Ubxm/ca*.To test 3R the mothers were F(3L), hP/F(3L),h'; C(3R), sr and the simulans males were T(Y;3)3,
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FIGURE3.-GOT isozymes of D.melanogasierj D.simulans and an F, hybrid. Electrophoresis was at pH 6.5. GOT1 isozymes as well as GOT2 isozymes are different in the t w o species.
2
L dP
5 7
2
D. melanogoster ?
-I +
bW
R
x
21
+
-
+
2R
+
D.sim/uns T ( Y ; 2 ) t
b w (me1 2R)
+ +
(sim
2R)
2L homozygous D. melonogoster; other chromosomes heterozygous
FIGURE 4.-Scheme used to localize the loci of the enzymes from partial hybrids between D. melamguster and D. simulans. In this example, 2L (left arm of the second chromosome) is homozygous in the partial hybrids and all other chromosome arms are heterozygous. The melanoguster mothers contain a compound 2L marked with d p (dumpy) and free 2 R s marked with bw (brown). The simulans fathers are heterozygous for a translocation between the Y chromosome and the second chromosome.
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TABLE 1
GOT patterns present in hybrid Drosophila with different homozygous chromosome arms GOT1
GOT2
Chromosome arm homozygous in hybrid background
pattern
pattern.
simulans X melanogaster 2L melanogaster 2R simulans 3L melanogaster 3L melanogaster 3R simulans 4
hybrid hybrid me1 hybrid hybrid hybrid hybrid
hybrid me1 hybrid hybrid hybrid hybrid hybrid
Ubxm/caz.The objective in these crosses was to obtain flies homozygous for one melanogaster arm in an otherwise hybrid background (Figure 4). The compound chromosomes were the homozygous arms. They were marked with recessive markers to ensure that the offspring might be unambiguously classified. The translocation in the simulans fathers uncoupled the two arms of the chromosome EMS
I
II
m Got2 FIGURE 5.-Crossing scheme to pick up mutants of Got2. Males in Generation I are injected with EMS. Males carrying a treated chromosome are backcrossed to females like their mothers. These males of generation I1 are placed with mates in vials with one male per vial. One fly from each vial is tested by electrophoresis. Several males from a positive vial are individually mated, and they and their progeny are tested electrophoretically to make certain that the mutation is recovered.
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and allowed formation of sperm without a chromosomearm. In the case of crosses between C(3L), he; F(3R)I, e ro ca/F(3R)I, e ro ca, two kinds of flies were produced. One class, hP; Ubx"', was homozygous for the melanogaster 3L and hybrid for 3R. The other class was wild type for the markers, homozygous for the simulans 3L and hybrid for 3 R. Table 1 is a summary of the results of the crosses; it shows that flies with a homozygous melanogaster 2R have only the melanogaster GOTl isozyme. Flies homozygous for melanogaster 2 L have only the melanogaster GOT2 isozyme. All other types have hybrid patterns of GOTl and GOT2. These results indicate that the Gotl locus is on 2R and the Got2 locus is on 2L. The alleles of melanogaster are designated Gotldfand Got2", while the alleles of simulans are designated GotZs and Got2*. Mutation of GotlMand (;OW: The scheme for mutagenesis is shown in (Figure 5 ) . For mutagenesis of Got2' in 2L, C(2L), nubL; C(2R), cn were mated to EMS-treated C(2L), cl; C(2R), sp* males. F, males of the genotype C(2L), cl; C(2R), cn were selected and backcrossed to C(2L), nub*;C(2R), cn females. One C(2L), cl; C(2R), cn son from each backcross mating was homogenized and electrophoresed. A total of 320 mutagenized compound 2L chromosomes were tested for mutations of Got2. One mutation was found which caused GOT2 to migrate to the anode more rapidly than the standard G O n M(Figure 6). The new allele has been designated G 0 t 2 ~Got2H/GorPJ . heterozygotes have a threebanded pattern. For mutagenesis of Gotl in 2R, (2L), cl; C(2R), vg females were mated to EMS-treated C(2L), dp; C(2R), cn males. F, males of the genotype C(2L), cl; C(2R), cn were backcrossed to C(2L), cl; C(2R), v g females. One son from each
FIGURE 6.-Gel of the standard melanogaster allele G o W , a heterozygote of Got"/GofZJ and the newly induced mutant allele Got2J.
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backcross was electrophoresed. No mutant with altered electrophoretic migration was discovered among 450 tested males; however, one culture yielded flies that had a GOTl which was somewhat lighter and more smeared than that ordinarily specified by Gotlu. These flies proved to be heterozygous for a mutant allele designed Got2 l o . Homozygotes of Got2 lowere very low in GOTl activity (Figure 7). The homozygotes appeared to be normal in appearance, viability and fertility in spite of having very little GOTl activity. Genetic locations of Gotl and Got2: Since the locations of Gotl and Got2 are known to be on 2R and 2L, respectively, one may ignore other possibilities at the outset. To locate Got1 on 2R, females of the genotype Gotl'"/TftGot2" nwDPinYt were mated to bwD males. Crossovers between the visible markers were selected and crossed to Got1 l o . Then flies with crossover chromosomes heterozygous with Got2'" were electrophoresed and scored for the presence of Got2" or Goti'" in the crossover chromosome. This experiment indicated that Got2 was closely linked to nwDand to the left of it. For a more precise location, L GotZU/Got1'"nwD PinYt females were constructed and mated to bwDmales. Males crossovers between L and nwDwere selected and crossed to Got21" females. Of 31 crossovers, 15 were crossovers between L and Got2 and 16 were crossovers between Got2 and nwD. The distance between L and nu?' was 6 map units; the distance between L and Got2 was 3 map units; and the genetic location of Got2 was near 75 on the map of the second chromosome (Figure 8). The genetic location of Got2 was roughly determined by making females heterozygous for S Got2* Sp Tft and GozJ2 and mating to G 0 t 2 ~ males. Crossovers were selected and electrophoresed, and Got2 was found to be closely linked to S . For a precise location, S G 0 t 2 shv/+ ~ G0t2~ -I- females were mated to -I-GotPMshv males. Of 28 crossovers between S and shv, 19 were between S and Got2 and 9 were between Got2 and shv. The interval between S and shv was 2.5
GOT 1 GOT2
Go/1
Go/1 ' O
FIGURE7.-Gel of the EMS-induced mutant, Goil'O, with GoilY, the allele from which it was derived.
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E. H.GRELL
1
I
-
I
72.0
54.8
75
79,b
107#2
FIGURE8.-Map
of a segment of 2R showing position of Goti on genetic map with some of the markers used in its location.
Got2 shv ho 0#1,
1#3
3.0
3,a 4.0
FIGURE9.-Map of a segment of 2L showing the position of Got2 on the genetic map and some of the markers used to locate it. map units; Got2 was 0.68 of that interval, or 1.7 map units from S . The standard map position of S is 1.3and the position of Got2 is 3.0 (Figure 9). Cytological location: Since Got2 was near the tip of 2L, and near S, there were chromosome aberrations which aided in a cytological location. The T(Y;2)'s of LINDSLEY and SANDLERet d.(1972)were a source of fragments which included the tip of 2L up to various points near the site of Got2 (Figure 10). To test for the presence of Got2 on the fragment, C(Z)RM, y ; T(Y;2), y+ Bs/SMI females were mated to C(2L),GotP,cl; C(2fi),cn males. Female offspring with C(I)RM, the desired part of the translocation, and C(2L), GotJ, cl; C(2R), cn were electrophoresed and scored for the presence of Got2" on the fragment. Two fragments with breaks in 21D and 22A did not carry Got2", while three fragments with breaks in 22B, 22C and 22D did carry G o P . The break in 22B
FIGURE 10.-Chromosome aberrations used to locate Got2 on the salivary chromosome map. The drawing of the salivary chromosome is taken from LEWIS(1945). The line at the bottom represents L m s ' Df(2L)S2. The gap in the line corresponds to the limits of the deficiency. The lines above the chromosome drawing represent the fragments derived from Y-2translocations described in LINDSLEY et al. (1972).The numbers indicate the salivary chromosome section in which the second chromosome is broken. Translocation D70 is broken in 21D,J69 in 22A, H56 in 22B, B112 in 22C and R136 in 22D.
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appears to be just after band 22B4, so Got2 must be in 22B4 or to the left of it. The left limits are assessed by Df (2L)S2. The deficiency lacks a segment from 21D1 io 22A6 (LEWIS 1945), but still carries Got2"; therefore Got2 must be to the right of 22A6, which would place it in 22B1 or to the right. The present information indicates that Got2 is in the interval from 22B1 to 22B4 inclusive. The Got1 locus in 2R has not been located on the salivary chromosome map. It is in the middle of the chromosome arm, and there is less material in stock that can be used to help locate it. DISCUSSION
The objective of this work was to begin a genetic analysis of aspartate aminotransferase (GOT) isozymes in Drosophila metanogaster. The task was made difficult because there is no detectable electrophoretic ploymorphism of either of the two isozymes of aspartate aminotransferase. These is a difference in the electrophoretic migration of homologous GOT isozymes of D.melanogaster and D.simulans, and the ability to locate this difference to a choromsome arm is helpful in mutagenesis. Without any knowledge of location, all chromosomes would have to be marked and followed. With chromosome arms identified, however, there is the added advantage that two treated alleles are scored for mutation in every fly that is electrophoresed if compound autosomes are used. In obtaining mutants from EMS treatment it is necessary to test a fly from the F, generation, because when F, flies are tested, mosaic flies are found which have a mutation in their somatic cells but which does not extend into germ cells and is not transmitted. The method for the synthesis of partial hybrids of melanogaster and simulans i s very easy once the necessary stocks are constructed. LITERATURE CITED
CHEN, S . H. and E. R. GIBLETT,1971 Genetic variation of soluble glutamic-oxaloacetic transaminase in man. Am. J. Hum. Genet. 23: 419-424.
V. M. and F. H. RUDDLE, 1972 Glutamate oxaloacetate transaminase (GOT) genetics CHAPMAN, in the mouse: Polymorphism of GOT-1. Genetics 70: 299-305. M. C. RATTAZZI, F. H. RUDDLEand H. A. LUBS,1970 Genetic DAVIDSON, R. G., J. A. CORTNER, polymorphism of human mitochondrial glutamic oxaloacetic transaminase. Science 169 : 391-392. DELORENZO, R. J. and F. H. RUDDLE, 1970 Glutamate oxalacetate transaminase (GOT) genetics in Mus musculus: Linkage, polymorphism and phenotypes of the GOT-I and GOT-2 loci. Biochem. Genet. 4: 259-274.
LEWIS,E. B., 1945 The relation of repeats to position effects in Drosophila melanogaster. Genetics 30: 137-166. -, 1967 Genes and gene complexes. In: Heritage from Mendel. Edited by R. H. BRINK.The University of Wisconsin Press, Madison, Wisconsin. LXNDSLEY, D. L. and E. H. GRELL,1968 Genetic variations of Drosophila melanogaster. Publ. 627. Carnegie Institute of Washington, Washington, D. C. LINDSLEY, D. L. and L. SANDLER et al., 1978 Segmental aneuploidy and the genetic gross structure of the Drosophila genome. Genetics 71: 157-184.
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MULLER, H. J. and G. PONTECORVO, 1940 Recombinants between Drosophila species the F, of which are sterile. Nature 146: 199-200. PONTECORVO, G., 1942 Hybrid sterility i n artificially produced recombinants between D . mel1943 anogaster and D . simulans. Proc. Roy. Soc. London, Ser. B 61: 385-397. -, Viability interactions between chromosomes of D.melanogaster and D . simulans. J. Genet. 45: 51-66. RASMUSSON, I. E., 1960 Report, Drosophila Info. Serv., Vol. 34,p. 53. STURTEVANT, A. H., 1921 Genetic studies on Drosophila simulrms. I. Introduction. Hybrids with Drosophila melanogzster. Genetics 5 : 488-500. Corresponding editor: B. H. JUDD
