Biochemical Genetics, %l. 15, Nos. 9/10, 1977
Isolation and Genetic Characterization of Alcohol Dehydrogenase Thermostability Variants Occurring in Natural Populations of
Drosophila melanogaster Bonnie SampselP
Received 12 Dec. 1976--Final21 Mar. 1977
D r o s o p h i l a m e l a n o g a s t e r collected from natural populations were examinedf o
thermostability variants within electrophoretic mobility classes of two enzymes. In alcohol dehydrogenase, two discrete forms of the "slow" allozyme and three discrete forms of the "fast" allozyme were revealed by postelectrophoretic treatments ranging from 15 sec at 40 C to 40 sec at 43 C. All variants have been mapped to within O.7 unit of the A d h locus. Results of a geographic survey indicate that two alleles giving rise to fast-moderate and slow-moderate allozymes are common everywhere; other variants have a collective frequency ranging from 0% to 7%. In a test of the possibility that the rare A d h alleles could be generated by intragenic recombination between the two common alleles, electrophoresis and heat treatment of progeny recombinant for flanking markers o f A d h revealed no new allozymes. Among 27 stocks containing slow ~-glycerophosphate dehydrogenase allozymes and 109 fast stocks, heat treatments revealed no additional variation. KEY WORDS: allozymes; thermostability; alcohol dehydrogenase; Drosophila melano-
gaster; natural populations. INTRODUCTION T h e use o f h e a t t r e a t m e n t s to d i s c r i m i n a t e between allozymes with similar e l e c t r o p h o r e t i c mobilities has a d d e d new r e s o l u t i o n to the study o f genetic This work was supported in part by NSF Grant No. DEB-76-01903 to Roger Milkman. The author is a trainee on NIH Training Grant No. T32-GM07091. Department of Zoology, The University of Iowa, Iowa City, Iowa.
971 This journal is copyrighted by Plenum. Each article is available for $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011.
972
Sampsell
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ADH Thermostability Variants in D. melanogaster Populations
973
variation. Investigation of thermostability differences has produced evidence of additional alleles at a number of loci in several Drosophila species (see Table I). For some loci, the alleles uncovered by heat treatments may occur in natural populations at fairly high frequencies. Bernstein et al. (1973) and Cochrane (1976) observed the same thermostability variants of xanthine dehydrogenase (XDH) and esterase-6 in relatively small samples from different locations. For other enzymes, the temperature-sensitive variants may be rare or occur only sporadically. Natural populations of D. rnelanogaster are typically polymorphic for two electrophoretically distinguishable alleles of A d h - - s l o w and fast (O'Brien and MacIntyre, 1969; Berger, 1970; Vigue and Johnson, 1973). It now appears that both of these mobility classes are heterogeneous and that as many as four alleles can be distinguished using a combination of electrophoresis and heat treatments (Th6rig et al., 1975; Milkman, 1976). Vigue and Johnson (1973) observed a clinal change along the eastern coast of the United States, with the frequency o f A d h s increasing from 0.50 to 0.90 as they moved southward. They also found that the fast and slow allozymes differ in specific activity, substrate specificity, and heat stability, and they hypothesized that these functional differences might provide the basis for differential selection among alternative Adh genotypes in varying environments. Evidence of increased levels of genetic variation has led to theories about the processes maintaining the polymorphism and the mechanisms for generating new alleles. For example, Watt (1972) postulated that new alleles may arise at an already polymorphic locus, not only by mutation but also by intragenic recombination between two alMic forms that have alternative nucleotide sequences at two sites. Such new alleles could be most easily recognized if their products had properties (such as mobility or thermostability) different from those of the parents. Gelbart et al. (1976) have observed several instances of ry ÷ alleles formed by intragenic recombination or gene conversion in which the XDH allozyme had a different electrophoretic mobility than that resulting from either of the alleles present in the heterezygous mother. The Adh locus has two common alleles (designated in this article as "slow-moderate" and "fast-moderate," where "moderate" describes the heat resistance of the enzyme) which occur in most populations at frequencies of 10~ or more. Although the exact amino acid sequence specified by the two alleles is not known, the existence of at least three rare forms in addition to the common two suggests variation at more than one site. The purpose of this investigation was to examine flies from natural populations for possible thermostability variants at two loci, to determine the genetic basis of any variants observed, and to carry out the first large-scale geographic survey on the frequencies of thermally classified allozymes. In
974
Sampsell
addition, an experiment was designed to test whether one or more of the rare ADH variants could be formed by intragenic recombination in females heterozygous for the slow-moderate and fast-moderate forms. MATERIALS AND METHODS Isolation of Variants
In the initial stages of the investigation, the magnitude of the thermostability differences which might occur was not known, and there was a question whether it would be possible to recognize two allozymes with different heat resistances but identical mobilities if they occurred in a heterozygous condition. It was decided that all flies would be tested as homozygotes or, if necessary, as heterozygotes with alleles with appropriate known properties. Male D. melanogaster collected during the summer of 1975 in Cedar Rapids, Iowa, were crossed with females of a balanced marker stock carrying Cy and Adh Fm (the common fast allele) on one second chromosome and bw vl and Adh sm (the common slow allele) on the other second chromosome. The conventional crossing scheme was employed to obtain stocks homozygous for a single wild second chromosome. After the wild males had mated, they were electrophoresed to determine their genotypes for Adh and c~Gpdh. When the wild fly was heterozygous for either enzyme, it was generally possible to isolate both of his second chromosomes. Once strains homozygous for individual wild second chromosomes were derived, four flies per strain were tested. All electrophoresis was performed using 1- by 6-inch cellulose acetate strips run for 45 min at 210 V in a Gelman deluxe chamber atpH 8.8 (Gelman high-resolution buffer). Staining was carried out for 8 min at room temperature in a staining mixture prepared from stock solutions to have the following final concentration per milliliter of stain: 26 mg tris, 0.8 mg EDTA, 0.6 mg NAD, 0.12 mg phenazine methosulfate, 0.6 mg nitroblue-tetrazolium, and either 1.6 mg of c~-glycerophosphate or 48 mg of see-butanol. The final pH of the stain was 9.0. Single flies were homogenized in 0.02 ml of half-strength Gelman H.R. buffer in wells of an Adamkewicz multiple sample homogenizer (Adamkewicz and Milkman, 1970). Use of the Adamkewicz applicator made it possible to determine the mobility genotype of individual flies for the two enzyme loci under investigation and to make replicate strips from each set of homogenates which could be subjected to a range of heat treatments. Control strips were stained immediately after e!ectrophoresis, while strips to be heat treated were placed in a Saran envelope, immersed in a water bath maintained to within 0.1 C for the designated period, and then stained.
ADH ThermostabilityVariants in D. melanogaster Populations
975
Empirically it was found that a treatment of 15 sec at 40 C would permit us to distinguish between the sensitive and moderate slow forms of A D H which had previously been identified in this laboratory (Milkman, 1976). Treatments of 20 sec and 40 sec at 43 C resulted in considerable inactivation of most A D H allozymes and could thus reveal the presence of any relatively resistant forms. Therefore, these three treatments were used to survey all strains, c~-Glycerophosphate dehydrogenase was more heat resistant than ADH, and treatments of 20 and 40 sec at 53 C were chosen for their ability to produce considerable inactivation in most strains. In the numerous cases in which isolated wild chromosomes could not be made homozygous presumably because of the presence of a lethal allele somewhere on the second chromosome, it was still possible to investigate the wild Adh allele. The A D H molecule in Drosophila is a dimer, and flies heterozygous for fast and slow alleles show three bands (SS, FS, and FF) that reflect the possible combinations of the two subunits. Thus by choosing flies with the appropriate Cy or bw vl markers, mobility heterozygotes could be tested and the resistance of the homodimer band formed by the wild allele determined. After the strips had been stained for one or the other enzyme, the spots were visually scored on a 0-4 scale (0, no spot; 4, very dark spot). The values for the four flies from each strain were averaged on the control and treated strips, and the percentage loss in staining intensity was calculated for each treatment. Strains which showed unusually high or low losses were retested using up to 32 flies per strain. In addition to the strains derived from the Cedar Rapids flies, a set of stocks which had been established from flies collected near Raleigh, North Carolina, was also tested for A D H variants. These stocks, which were kindly supplied by Dr. Rollin Richmond, either were homozygous for the second chromosome or were balanced against the chromosome carrying Cy and Adh Fro. In order to identify the alleles which produce the various A D H allozymes, the following symbols have been used. The common alleles in each mobility class which show intermediate thermostability compared to that of the rare variants have been designated Adh Fm and Adh sin, respectively. In addition, three new variants have been designated Adh Fr for fast-resistant, Adh FS for fast-sensitive, and Adh ss for slow-sensitive. Geographic Survey Once stocks were established for each of the five Adh alleles, crosses were made to generate heterozygotes of known composition, and these (except Adhr'/Adh rS) were tested with various heat treatments. It was determined that
976
Sampsell
all combinations gave unique responses to the pair of screening treatments of 15 sec at 40 C and 20 sec at 43 C (AdhVr/Adh F~ heter0zygotes should be extremely rare in population samples). Therefore, a more direct procedure was employed in the study of flies collected in a variety of geographic locations. After being crossed to Cy/bw vl females in the case of males, or being allowed to found isofemale lines in the case of females, wild flies were electrophoresed, and replicate strips were tested with the two survey treatments. In most cases, a clear determination of the genotype of the wild fly was achieved, but if there was any doubt F1 flies were available for retesting. This modified procedure did not permit the immediate identification of additional allozymes of a more resistant or sensitive nature than those known, but it did allow a large number of flies to be surveyed rapidly for the five categories already described. Further testing of the variant stocks will, moreover, allow the resolution of more extreme forms. Also, the direct testing of wild flies had an additional advantage over that of making lines homozygous for individual second chromosomes. Direct testing prevented the loss of lines, due to sterility factors of lethals, that accompanies the derivation of homozygous lines.
Mapping and Recombination Experiments To analyze the genetic basis of the thermostability variants, several laboratory stocks homozygous for visible markers on the second chromosome and for different Adh alleles were employed. [Markers included black (b)--48.5; elbow (el)--50.0; reduced scraggly (rdS)--51.2; purple (pr)--54.5; and cinnabar (en)--57.5.] Adh vr was localized by crossing flies with the fast-resistant allozyme to b Adh vm cn flies. Heterozygous females of the form + A d h V r + / b Adh F" cn were backcrossed to b Adh Fm cn males, and recombinants for b and cn and parental-type offspring were tested for the presence of a resistant A D H allozyme using a treatment of 20 sec at 43 C. Adh ss was mapped using the b Adh F'n cn stock in the same manner as described for Adh Ft. Recombinants between b and cn as well as offspring with parental phenotypes were tested using a treatment of 15 sec at 40 C to verify the presence of the sensitive-slow allele. Adh F~ was mapped by first crossing flies with the fast-sensitive enzyme to a b Adh Fm pr stock. The F1 females, heterozygous for the two forms of fast (AdhVm/AdhF~), were next crossed to b Adh sm pr males. Recombinants between b and pr as well as parental types were tested for 15 sec at 40 C to determine the resistance of their F F homodimer band. This treatment totally inactivated an F~F~ band but did not affect an FmF m band. To test the hypothesis that AdhVm/Adh sm heterozygotes could give rise to one of the rare variants by intragenic recombination, a marker stock with
ADH Thermostability Variants in D. melanogaster Populations
977
Table IL Reduction in ~GPDH Staining after 40 sec at 53 C
Number c~Gpdh of genotype strains SS FF
27 109
Percent reduction 0-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-89 90-100 0~ 1
0 0
1 1
5 4
3 14
4 12
5 26
4 18
l 21
4 12
"Indicates number of strains which showed an average loss in each range. flanking markers e l and r d s which was homozygous for A d h rm was used. Wild-type females homozygous for A d h sm were mated to e l A d h Fm r d ~ males, and F~ females were backcrossed to the same marker stock. Progeny which were recombinant for the region between el and r d s were electrophoresed and heat treated to see if any new nonparental allozymes were present. RESULTS Isolation of Thermostability Variants
The response observed for strains homozygous for slow and fast forms of c~GPDH to the treatment of 40 sec at 53 C is given in Table II. The slow and fast allozymes appeared to be very similar in their average thermostability. No clear-cut v a r i a n t s - - o f either more or less heat resistance--were observed among the 136 lines tested. The strains did show a range of losses in staining intensity, but retesting of lines which initially displayed a very high or very low average loss gave results much closer to the mean for all strains. Since the differences in thermostability did not persist in retesting, the variation was either not genetically based or was not controlled by genes on the second chromosome. The responses of flies electrophoretically homozygous for A d h are shown for two different treatments in Table IlL Two features are clearly evident in these results. First, on the average, the slow allozyme was more heat resistant than the fast form. This finding is in agreement with reports by a number of other investigators who tested crude extracts from strains of known electrophoretic genotypes (Gibson, 1970; Vigue and Johnson, 1973; D a y e t al., 1974). Second, two of the fast strains exhibited extreme heat resistance and appeared to represent a discretely different category of thermostability. Although these two were electrophoretically indistinguishable from the other fast allozymes, their staining intensity showed essentially no reduction after 40 sec at 43 C. Moreover, a third stock which is not listed in Table III, since it could be tested only as a balanced heterozygote,, also appeared to have a fast-resistant form of A D H .
978
Sampsell
r&
Isolation and Genetic Characterization of Alcohol Dehydrogenase Thermostability Variants Occurring in Natural Populations of
Drosophila melanogaster Bonnie SampselP
Received 12 Dec. 1976--Final21 Mar. 1977
D r o s o p h i l a m e l a n o g a s t e r collected from natural populations were examinedf o
thermostability variants within electrophoretic mobility classes of two enzymes. In alcohol dehydrogenase, two discrete forms of the "slow" allozyme and three discrete forms of the "fast" allozyme were revealed by postelectrophoretic treatments ranging from 15 sec at 40 C to 40 sec at 43 C. All variants have been mapped to within O.7 unit of the A d h locus. Results of a geographic survey indicate that two alleles giving rise to fast-moderate and slow-moderate allozymes are common everywhere; other variants have a collective frequency ranging from 0% to 7%. In a test of the possibility that the rare A d h alleles could be generated by intragenic recombination between the two common alleles, electrophoresis and heat treatment of progeny recombinant for flanking markers o f A d h revealed no new allozymes. Among 27 stocks containing slow ~-glycerophosphate dehydrogenase allozymes and 109 fast stocks, heat treatments revealed no additional variation. KEY WORDS: allozymes; thermostability; alcohol dehydrogenase; Drosophila melano-
gaster; natural populations. INTRODUCTION T h e use o f h e a t t r e a t m e n t s to d i s c r i m i n a t e between allozymes with similar e l e c t r o p h o r e t i c mobilities has a d d e d new r e s o l u t i o n to the study o f genetic This work was supported in part by NSF Grant No. DEB-76-01903 to Roger Milkman. The author is a trainee on NIH Training Grant No. T32-GM07091. Department of Zoology, The University of Iowa, Iowa City, Iowa.
971 This journal is copyrighted by Plenum. Each article is available for $7.50 from Plenum Publishing Corporation, 227 West 17th Street, New York, N.Y. 10011.
972
Sampsell
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ADH Thermostability Variants in D. melanogaster Populations
973
variation. Investigation of thermostability differences has produced evidence of additional alleles at a number of loci in several Drosophila species (see Table I). For some loci, the alleles uncovered by heat treatments may occur in natural populations at fairly high frequencies. Bernstein et al. (1973) and Cochrane (1976) observed the same thermostability variants of xanthine dehydrogenase (XDH) and esterase-6 in relatively small samples from different locations. For other enzymes, the temperature-sensitive variants may be rare or occur only sporadically. Natural populations of D. rnelanogaster are typically polymorphic for two electrophoretically distinguishable alleles of A d h - - s l o w and fast (O'Brien and MacIntyre, 1969; Berger, 1970; Vigue and Johnson, 1973). It now appears that both of these mobility classes are heterogeneous and that as many as four alleles can be distinguished using a combination of electrophoresis and heat treatments (Th6rig et al., 1975; Milkman, 1976). Vigue and Johnson (1973) observed a clinal change along the eastern coast of the United States, with the frequency o f A d h s increasing from 0.50 to 0.90 as they moved southward. They also found that the fast and slow allozymes differ in specific activity, substrate specificity, and heat stability, and they hypothesized that these functional differences might provide the basis for differential selection among alternative Adh genotypes in varying environments. Evidence of increased levels of genetic variation has led to theories about the processes maintaining the polymorphism and the mechanisms for generating new alleles. For example, Watt (1972) postulated that new alleles may arise at an already polymorphic locus, not only by mutation but also by intragenic recombination between two alMic forms that have alternative nucleotide sequences at two sites. Such new alleles could be most easily recognized if their products had properties (such as mobility or thermostability) different from those of the parents. Gelbart et al. (1976) have observed several instances of ry ÷ alleles formed by intragenic recombination or gene conversion in which the XDH allozyme had a different electrophoretic mobility than that resulting from either of the alleles present in the heterezygous mother. The Adh locus has two common alleles (designated in this article as "slow-moderate" and "fast-moderate," where "moderate" describes the heat resistance of the enzyme) which occur in most populations at frequencies of 10~ or more. Although the exact amino acid sequence specified by the two alleles is not known, the existence of at least three rare forms in addition to the common two suggests variation at more than one site. The purpose of this investigation was to examine flies from natural populations for possible thermostability variants at two loci, to determine the genetic basis of any variants observed, and to carry out the first large-scale geographic survey on the frequencies of thermally classified allozymes. In
974
Sampsell
addition, an experiment was designed to test whether one or more of the rare ADH variants could be formed by intragenic recombination in females heterozygous for the slow-moderate and fast-moderate forms. MATERIALS AND METHODS Isolation of Variants
In the initial stages of the investigation, the magnitude of the thermostability differences which might occur was not known, and there was a question whether it would be possible to recognize two allozymes with different heat resistances but identical mobilities if they occurred in a heterozygous condition. It was decided that all flies would be tested as homozygotes or, if necessary, as heterozygotes with alleles with appropriate known properties. Male D. melanogaster collected during the summer of 1975 in Cedar Rapids, Iowa, were crossed with females of a balanced marker stock carrying Cy and Adh Fm (the common fast allele) on one second chromosome and bw vl and Adh sm (the common slow allele) on the other second chromosome. The conventional crossing scheme was employed to obtain stocks homozygous for a single wild second chromosome. After the wild males had mated, they were electrophoresed to determine their genotypes for Adh and c~Gpdh. When the wild fly was heterozygous for either enzyme, it was generally possible to isolate both of his second chromosomes. Once strains homozygous for individual wild second chromosomes were derived, four flies per strain were tested. All electrophoresis was performed using 1- by 6-inch cellulose acetate strips run for 45 min at 210 V in a Gelman deluxe chamber atpH 8.8 (Gelman high-resolution buffer). Staining was carried out for 8 min at room temperature in a staining mixture prepared from stock solutions to have the following final concentration per milliliter of stain: 26 mg tris, 0.8 mg EDTA, 0.6 mg NAD, 0.12 mg phenazine methosulfate, 0.6 mg nitroblue-tetrazolium, and either 1.6 mg of c~-glycerophosphate or 48 mg of see-butanol. The final pH of the stain was 9.0. Single flies were homogenized in 0.02 ml of half-strength Gelman H.R. buffer in wells of an Adamkewicz multiple sample homogenizer (Adamkewicz and Milkman, 1970). Use of the Adamkewicz applicator made it possible to determine the mobility genotype of individual flies for the two enzyme loci under investigation and to make replicate strips from each set of homogenates which could be subjected to a range of heat treatments. Control strips were stained immediately after e!ectrophoresis, while strips to be heat treated were placed in a Saran envelope, immersed in a water bath maintained to within 0.1 C for the designated period, and then stained.
ADH ThermostabilityVariants in D. melanogaster Populations
975
Empirically it was found that a treatment of 15 sec at 40 C would permit us to distinguish between the sensitive and moderate slow forms of A D H which had previously been identified in this laboratory (Milkman, 1976). Treatments of 20 sec and 40 sec at 43 C resulted in considerable inactivation of most A D H allozymes and could thus reveal the presence of any relatively resistant forms. Therefore, these three treatments were used to survey all strains, c~-Glycerophosphate dehydrogenase was more heat resistant than ADH, and treatments of 20 and 40 sec at 53 C were chosen for their ability to produce considerable inactivation in most strains. In the numerous cases in which isolated wild chromosomes could not be made homozygous presumably because of the presence of a lethal allele somewhere on the second chromosome, it was still possible to investigate the wild Adh allele. The A D H molecule in Drosophila is a dimer, and flies heterozygous for fast and slow alleles show three bands (SS, FS, and FF) that reflect the possible combinations of the two subunits. Thus by choosing flies with the appropriate Cy or bw vl markers, mobility heterozygotes could be tested and the resistance of the homodimer band formed by the wild allele determined. After the strips had been stained for one or the other enzyme, the spots were visually scored on a 0-4 scale (0, no spot; 4, very dark spot). The values for the four flies from each strain were averaged on the control and treated strips, and the percentage loss in staining intensity was calculated for each treatment. Strains which showed unusually high or low losses were retested using up to 32 flies per strain. In addition to the strains derived from the Cedar Rapids flies, a set of stocks which had been established from flies collected near Raleigh, North Carolina, was also tested for A D H variants. These stocks, which were kindly supplied by Dr. Rollin Richmond, either were homozygous for the second chromosome or were balanced against the chromosome carrying Cy and Adh Fro. In order to identify the alleles which produce the various A D H allozymes, the following symbols have been used. The common alleles in each mobility class which show intermediate thermostability compared to that of the rare variants have been designated Adh Fm and Adh sin, respectively. In addition, three new variants have been designated Adh Fr for fast-resistant, Adh FS for fast-sensitive, and Adh ss for slow-sensitive. Geographic Survey Once stocks were established for each of the five Adh alleles, crosses were made to generate heterozygotes of known composition, and these (except Adhr'/Adh rS) were tested with various heat treatments. It was determined that
976
Sampsell
all combinations gave unique responses to the pair of screening treatments of 15 sec at 40 C and 20 sec at 43 C (AdhVr/Adh F~ heter0zygotes should be extremely rare in population samples). Therefore, a more direct procedure was employed in the study of flies collected in a variety of geographic locations. After being crossed to Cy/bw vl females in the case of males, or being allowed to found isofemale lines in the case of females, wild flies were electrophoresed, and replicate strips were tested with the two survey treatments. In most cases, a clear determination of the genotype of the wild fly was achieved, but if there was any doubt F1 flies were available for retesting. This modified procedure did not permit the immediate identification of additional allozymes of a more resistant or sensitive nature than those known, but it did allow a large number of flies to be surveyed rapidly for the five categories already described. Further testing of the variant stocks will, moreover, allow the resolution of more extreme forms. Also, the direct testing of wild flies had an additional advantage over that of making lines homozygous for individual second chromosomes. Direct testing prevented the loss of lines, due to sterility factors of lethals, that accompanies the derivation of homozygous lines.
Mapping and Recombination Experiments To analyze the genetic basis of the thermostability variants, several laboratory stocks homozygous for visible markers on the second chromosome and for different Adh alleles were employed. [Markers included black (b)--48.5; elbow (el)--50.0; reduced scraggly (rdS)--51.2; purple (pr)--54.5; and cinnabar (en)--57.5.] Adh vr was localized by crossing flies with the fast-resistant allozyme to b Adh vm cn flies. Heterozygous females of the form + A d h V r + / b Adh F" cn were backcrossed to b Adh Fm cn males, and recombinants for b and cn and parental-type offspring were tested for the presence of a resistant A D H allozyme using a treatment of 20 sec at 43 C. Adh ss was mapped using the b Adh F'n cn stock in the same manner as described for Adh Ft. Recombinants between b and cn as well as offspring with parental phenotypes were tested using a treatment of 15 sec at 40 C to verify the presence of the sensitive-slow allele. Adh F~ was mapped by first crossing flies with the fast-sensitive enzyme to a b Adh Fm pr stock. The F1 females, heterozygous for the two forms of fast (AdhVm/AdhF~), were next crossed to b Adh sm pr males. Recombinants between b and pr as well as parental types were tested for 15 sec at 40 C to determine the resistance of their F F homodimer band. This treatment totally inactivated an F~F~ band but did not affect an FmF m band. To test the hypothesis that AdhVm/Adh sm heterozygotes could give rise to one of the rare variants by intragenic recombination, a marker stock with
ADH Thermostability Variants in D. melanogaster Populations
977
Table IL Reduction in ~GPDH Staining after 40 sec at 53 C
Number c~Gpdh of genotype strains SS FF
27 109
Percent reduction 0-9 10-19 20-29 30-39 40-49 50-59 60-69 70-79 80-89 90-100 0~ 1
0 0
1 1
5 4
3 14
4 12
5 26
4 18
l 21
4 12
"Indicates number of strains which showed an average loss in each range. flanking markers e l and r d s which was homozygous for A d h rm was used. Wild-type females homozygous for A d h sm were mated to e l A d h Fm r d ~ males, and F~ females were backcrossed to the same marker stock. Progeny which were recombinant for the region between el and r d s were electrophoresed and heat treated to see if any new nonparental allozymes were present. RESULTS Isolation of Thermostability Variants
The response observed for strains homozygous for slow and fast forms of c~GPDH to the treatment of 40 sec at 53 C is given in Table II. The slow and fast allozymes appeared to be very similar in their average thermostability. No clear-cut v a r i a n t s - - o f either more or less heat resistance--were observed among the 136 lines tested. The strains did show a range of losses in staining intensity, but retesting of lines which initially displayed a very high or very low average loss gave results much closer to the mean for all strains. Since the differences in thermostability did not persist in retesting, the variation was either not genetically based or was not controlled by genes on the second chromosome. The responses of flies electrophoretically homozygous for A d h are shown for two different treatments in Table IlL Two features are clearly evident in these results. First, on the average, the slow allozyme was more heat resistant than the fast form. This finding is in agreement with reports by a number of other investigators who tested crude extracts from strains of known electrophoretic genotypes (Gibson, 1970; Vigue and Johnson, 1973; D a y e t al., 1974). Second, two of the fast strains exhibited extreme heat resistance and appeared to represent a discretely different category of thermostability. Although these two were electrophoretically indistinguishable from the other fast allozymes, their staining intensity showed essentially no reduction after 40 sec at 43 C. Moreover, a third stock which is not listed in Table III, since it could be tested only as a balanced heterozygote,, also appeared to have a fast-resistant form of A D H .
978
Sampsell
r&
