Studies of esterase 6 in Drosophila melanogaster. XVIII. Biochemical differences between the slow and fast allozymes

Most natural populations of Drosophila melanogaster are polymorphic for two major electrophoretic variants at the esterase-6 locus. The frequency of the EST 6F allozyme is greatest in populations in warmer latitudes, whereas the EST 6S allozyme is predominant in colder latitudes. Latitudinal clines in electromorph frequencies are found on three continents. Purified preparations of the allozymes have been characterized for their pH optimum, substrate specificity, organophosphate inhibition, alcohol activation, thermal stability, and kinetic parameters. These and previous analyses of the EST 6 allozymes reveal that the two variants have differences in their physical and kinetic properties that may provide a basis for the selective maintenance of the polymorphisms and an explanation of the clinal variation observed in natural populations.      

An Assessment of "Hidden" Heterogeneity within Electromorphs at Three Enzyme Loci in Deer Mice

Allelic heterogeneity within protein electromorphs at three loci was examined in populations of deer mice (Peromyscus maniculatus) collected from five localities across North America. We used a variety of electrophoretic techniques (including several starch and acrylamide conditions, gel-sieving, and isoelectric focusing) plus heat denaturation. Of particular interest was the supernatant glutamate oxalate transaminase system (GOT-1; aspartate aminotransferase-1 of some authors), which under standard electrophoretic conditions had been shown to exhibit basically a two-allele polymorphism throughout the range of maniculatus. The use of all of the above techniques failed to uncover any additional variation for GOT-1 in these populations. Similarly, no new scorable variation was resolved at the essentially monomorphic malate dehydrogenase-1 locus by additional conditions of electrophoresis. In marked contrast to the results for the above two enzymes, the use of multiple conditions of electrophoresis resolved the 8 standard-condition electromorphs of esterase-1 into a total of 23 variants showing strong geographic differentiation in frequency. These 23 electromorphs were further divided into a total of 35 variants by thermal stability studies. However, the allelic nature of all of the thermal stability esterase variants remains to be documented. The results of this study, taken together with the remarkable geographic heterogeneity for this species in ecology, morphology, karyotype and mitochondrial DNA sequence, suggest that some form of balancing selection may be acting to maintain the GOT-1 polymorphism.     

Molecular Basis of Polymorphism at the Esterase-5b Locus in Drosophila Pseudoobscura

Sequence variation was studied in a 2.2-kb region encompassing the esterase-5B locus in Drosophila pseudoobscura from two California populations. In these populations, two common electrophoretic classes and many less frequent variants occur, and it was formerly shown by KEITH (1983) that allele frequencies differed from random distribution under an infinite allele model. Nucleotide polymorphisms were determined in 16 sequences representing 14 electrophoretic classes. There was no significant sequence differentiation between populations, and both synonymous and nonsynonymous polymorphisms are distributed homogeneously along the sequence. The data show that the two major electrophoretic classes are heterogeneous at the amino acid level with no diagnostic amino acid(s) distinguishing them. At the nucleotide level, members of one major class are more similar to members of other electrophoretic classes than they are to each other. It appears that random combinations of the neutral amino acid polymorphisms and other undefined physical properties of the proteins generate the different electrophoretic classes and maintain considerable variation at Est-5B.     

Esterase-7, a common constituent of numerous mouse tissues.

1. Electrophoretic and staining techniques for the relatively specific demonstration of esterase-7 in mouse tissues are described. Esterase-7 was found to be a common constituent of most mouse tissues. 2. The electrophoretic polymorphisms determined by three alleles a, b and c were studied in nine mouse strains. 3. The banding pattern of esterase-7 is being described and the isoelectric points of the allelic bands are being estimated. 4. Recombination studies on a further 129 animals confirmed that the Es-7 locus is tightly linked to Es-2 and Es-11. 5. Esterase-7 precipitates with antiesterase-2 from the rabbit. 6. It differs from esterase-2 by its individual substrate preference and by its susceptibility to inhibition by acylating agents.     

Examination of Allelic Variation at the Hexokinase Loci of DROSOPHILA PSEUDOOBSCURA and D. PERSIMILIS by Different Methods

Recently a number of electrophoretic techniques have been applied to reveal the presence of additional genetic variation among the electrophoretic mobility classes of the highly polymorphic xanthine dehydrogenase (XDH ) and esterase-5 (est-5) loci. We examined the hexokinase loci of Drosophila pseudoobscura and D. persimilis using a variety of techniques to determine whether further allelic variation could be revealed for these much less polymorphic loci and to analyze the nature of the known variation at the hexokinase-1 (hex-1) locus. The following studies were conducted: 135 strains of the two species from six localities were examined with buffer pH ranging from 5.5 to 10.0; 40 strains of D. pseudoobscura and 9 strains of D. persimilis from Mather were studied using starch gel concentrations ranging from 8.5 to 15.5% and were examined for differences in heat stability and reactivity to the thiol reagent pCMSA; strains were also tested for susceptibility to urea denaturation and differences in relative activities. Major findings of the work are: (1) No additional allelic variation could be detected at any of the hexokinase loci by applying these techniques. The finding of abundant hidden genetic variation in XDH and est-5 does not extend to all enzyme loci. (2) Evidence from studies using pCMSA indicates that the hex-1 alleles 0.6, 0.8, 1.0 and 1.2 of the two species form a series of unit charge steps. Since the 0.94 allele of D. persimilis has mobility intermediate between 0.8 and 1.0, it is argued that routine electrophoretic techniques are sensitive to at least some conservative amino acid substitutions. (3) Strong correlations were found at the hex-1 locus between low allelic frequency, reduced relative activity and reduced stability to heat and urea denaturation. Since the three sibling species, D. pseudoobscura, D. persimilis and D. miranda, all appear to share a common high frequency allele (1.0) at that locus, these findings are taken as evidence that the observed allelic frequencies are a result of directional selection and mutation, rather than any form of balancing selection.     

Dynamics of Correlated Genetic Systems. IV. Multilocus Effects of Ethanol Stress Environments

Four replicate populations of Drosophila melanogaster, two reared on medium supplemented with ethanol and two reared on standard medium, were electrophoretically monitored for 28 generations. During the first 12 generations, allelic, genotypic and gametic frequencies were determined for eight polymorphic enzymes: GOT, alpha-GPDH, MDH, ADH, TO, E6, Ec and ODH. Samples from generation 18 and 28 were electrophoretically typed for ADH and alpha-GPDH. In addition, samples from generation 27 were analyzed for the presence of inversion heterozygotes. The experimental results showed rapid gene-frequency divergence between control and treatment populations at the Adh locus in a direction consistent with the activity hierarchy of Adh genotypes. Gene-frequency divergence between control and treatment populations also occurred at the alpha-Gpdh locus, although the agreement among replicates appeared to have broken down by generation 28. No differential gene-frequency change occurred at any of the six remaining marker loci. Furthermore, values of linkage disequilibria among all linked pairs of genes were initially small and remained small throughout the course of the experiment. Taking these facts into account, it is argued that the gene-frequency response observed at ADH is most probably caused by selection at the Adh locus. The gene frequency response at alpha-Gpdh can also be be accounted for in terms of the effect of ethanol on energy metabolism, although other explanations cannot be excluded.     

Sequential gel electrophoretic analysis of esterase-2 in two populations of Drosophila buzzatii

Sequential electrophoresis, using three different buffer systems on cellulose acetate gels, was used to characterize the allelic variation for esterase-2 in two populations of D. buzzatii in Australia that are separated by 550 km. Twenty-five alleles were detected, of which nine were unique to one population, eight unique to the other, and only eight were common to both populations. Allele frequencies within each population were significantly different between the two major chromosome sequences (standard and j inversion), and for each chromosome sequence allele frequencies were significantly different between populations. Observed allelic frequency distributions were not significantly different from those predicted for selective neutrality using the homozygosity test statistic. However, estimates of the effective sizes of the populations derived from their observed differentiation, together with the history of the species in Australia, provide support for some form of balancing selection affecting at least some of the alleles.     

Frequency Distribution of Esterase-5 Alleles in Two Populations of DROSOPHILA PSEUDOOBSCURA

Statistical tests comparing allele frequencies in natural populations with those predicted by various theories of genic variation depend critically on the accurate enumeration of alleles. This study used a series of five sequential electrophoretic conditions to characterize the allele frequency distributions of esterase-5 in two large population samples of Drosophila pseudoobscura from California. In Standard chromosome lines 12 electromorphs were discriminated using a single electrophoretic condition. When four additional criteria were used, the number of electromorphs increased to 41, 33 in one population and 22 in the other. Both populations had the same two alleles in high frequency, with other alleles present in frequencies of 6% or less. Although each population had a number of unique alleles, a χ² contingency test demonstrated no significant genetic divergence between them. A statistical comparison of allele frequencies in both populations with that predicted by neutral models suggests that the individual and combined distributions deviate from neutrality in the direction of purifying selection.—Sex-Ratio chromosomes differed markedly from Standard chromosomes in both allelic content and diversity. In 32 Sex-Ratio chromosomes from one population only three alleles were found, all of which were detected under the initial "standard" electrophoretic conditions. Moreover, none of these alleles was found in the Standard chromosome lines.     

A hybrid zone between Chorthippus parallelus parallelus and Chorthippus parallelus erythropus (Orthoptera: Acrididae): morphological and electrophoretic characters

Two subspecies of the grasshopper Chorthippus parullelus have distributions that abut in the Pyrenees. We have studied morphological and electrophoretic variation in II populations near Mont Louis, Pyrénées-Orientales, France. The two subspecies differ in several morphological characters and at one enzyme locus, esterase-2, and in this area they hybridize forming a cline in morphology less than 5 km wide. Examination of museum specimens suggests that this hybrid zone extends for most of the length of the Pyrenees, possibly breaking down in the extreme west. As the two subspecies are known to differ in mating behaviour this hybrid zone is well suited to the study of reinforcement.     

Geographic heterogeneity in natural selection on an MHC locus in sockeye salmon

Balancing selection maintains high levels of polymorphism and heterozygosity in genes of the MHC (major histocompatibility complex) of vertebrate organisms, and promotes long evolutionary persistence of individual alleles and strongly differentiated allelic lineages. In this study, genetic variation at the MHC class II DAB-beta1 locus was examined in 31 populations of sockeye salmon (Oncorhynchus nerka) inhabiting the Fraser River drainage of British Columbia, Canada. Twenty-five percent of variation at the locus was partitioned among sockeye populations, as compared with 5% at neutral genetic markers. Geographic heterogeneity of balancing selection was detected among four regions in the Fraser River drainage and among lake systems within regions. High levels of beta1 allelic diversity and heterozygosity, as well as distributions of alleles and allelic lineages that were more even than expected for a neutral locus, indicated the presence of balancing selection in populations throughout much of the interior Fraser drainage. However, proximate populations in the upper Fraser region, and four of six populations from the lower Fraser drainage, exhibited much lower levels of genetic diversity and had beta1 allele frequency distributions in conformance with those expected for a neutral locus, or a locus under directional selection. Pair-wise FST values for beta1 averaged 0.19 and tended to exceed the corresponding values estimated for neutral loci at all levels of population structure, although they were lower among populations experiencing balancing selection than among other populations. The apparent heterogeneity in selection resulted in strong genetic differentiation between geographically proximate populations with and without detectable levels of balancing selection, in stark contrast to observations at neutral loci. The strong partitioning and complex structure of beta1 diversity within and among sockeye populations on a small geographic scale illustrates the value of incorporating adaptive variation into conservation planning for the species.     

Inheritance of isozyme variation and heterozygosity in Pinus ponderosa.

Techniques are presented to detect 23 isozyme loci in the long-lived perennial plant, ponderosa pine. Meiotically derived megagametophyte from seeds is used to examine directly the segregation of allelic variants. Approximately seven seeds were initially examined for 12 enzymes from each of 47 trees from ten stands throughout the northern Rocky Mountain region. Additional seeds were also examined from selected families to confirm the inheritance of observed electrophoretic variants at 13 polymorphic loci and to estimate linkage relationship. Significant norandom segregation was consistently detected for three pairs of loci: ADH-1:AAT-2, ADH-1:PGI-1, and LAP-2:6PG-1. Preliminary estimates of population parameters reveal a relatively high average heterozygosity (H = 0.123). This is partitioned into a high amont of genetic variation within local stands, with only approximately 12% of the total heterozygosity resulting from genic difference between stands.     

Genetic Variability at the Esterase-6 Locus in Natural Populations of DROSOPHILA SIMULANS in Relation to Environmental Heterogeneity

The allozymic variation at the esterase-6 locus was examined in fourteen samples of natural populations of Drosophila simulans collected in two different localities. The samples were collected from different species of fruits in the two localities and from banana baits located some meters apart from the fruits. The analysis was done by sequential electrophoresis, using varied gel concentrations and buffers. By this technique, we detected 27 alleles at the esterase-6 locus, where only five alleles were detected by the usual method. The results obtained suggest that habitat choice alone may not be a selective factor, but may be selective in combination with other environmental factors.     

Genetic variation in pea (Pisum) dehydrins: sequence elements responsible for length differences between dehydrin alleles and between dehydrin loci in Pisum sativum L.

 The electrophoretic patterns of dehydrins extracted from mature seeds of a range of pea (Pisum) species revealed extensive variation in dehydrin polypeptide mobility. Variation was also observed among lines of P. sativum. Crosses between lines with different dehydrin electrophoretic patterns produced F₁ seeds with additive patterns, and segregation in the F₂ generation was consistent with a 1 : 2 : 1 ratio, indicating allelic variation at each of two dehydrin loci (Dhn2, Dhn3). Genetic linkage was observed between Dhn2 and Dhn3, and the segregation ratios indicated preferential transmission of one allele at the Dhn3 locus. Dehydrin cDNA clones were characterised that encoded the allelic variants at Dhn2 and Dhn3. Their deduced amino-acid sequences were very similar to each other as well as to the product of the Dhn1 locus reported previously. Comparisons were made between the sequences of allelic variants at a single locus, and between the products of different loci. Differences in the electrophoretic mobilities between allelic variants at Dhn2 and Dhn3 were associated with differences in polypeptide length resulting principally from tandem duplications of 21 (Dhn2) or 24 (Dhn3) amino-acid residues. These duplications accounted for much of the difference in length between dehydrins encoded by the different loci. The conserved core of one of the duplicated regions varied in copy number, and small insertions/deletions of amino acids near this core also contributed to length variation both between allelic forms and between loci. Dehydrins possess characteristic highly conserved amino-acid sequence motifs, yet vary considerably in length. Mechanisms involving sequence duplication appear to be responsible for generating the length differences observed between allelic variants as well as between the products of different loci. Received: 12 June 1997 / Accepted: 29 October 1997     

Distinctions among Allelic Variants Associated with Chromosome 3 Inversions in DROSOPHILA PSEUDOOBSCURA and DROSOPHILA PERSIMILIS

Efforts were made to discriminate new genetic variants among electrophoretic alleles that are associated with chromosome 3 inversions of Drosophila pseudoobscura and D. persimilis. Apparent genetic similarities for electrophoretic alleles between these two species and among the common inversions they carry were reexamined by altering gel concentration and buffer pH. At the amylase locus, the 1.09 electrophoretic allele could be further separated into two allelic classes that differentiated the WT and KL arrangements. Similarly, the 0.84 electrophoretic allele was divided into two allelic classes, one characteristic of the Santa Cruz phylad arrangements, TL and SC, and the other found in strains of the Standard phylad arrangements and CH. Uncommon amylase alleles proved to be different alleles in the two species. No new allelic variants, however, could be found among strains with the amylase 1.00 allele, the commonest allele in the Standard phylad of both species. No major new allelic variation was detected for acid phosphatase-3 and larval protein-10 that revealed any further differentiation among species or inversions. Variation at all three loci in strains of the Bogota population remained genetically similar to variation in strains of mainland D. pseudoobscura.     

Nearly identical allelic distributions of xanthine dehydrogenase in two populations of Drosophila pseudoobscura

In a previous study, Keith (1983) showed by sequential gel electrophoresis of the esterase-5 protein in Drosophila pseudoobscura that a highly polymorphic locus with many alleles can have very similar frequency distributions in populations separated by 500 km. The present work studies another highly polymorphic locus, xanthine dehydrogenase, in the same California population samples, using the same technique to distinguish allelic classes. Twelve electromorphs were found in one population and 15 in the other. Both populations shared a single very frequent (approximately 60%) allele, as well as five other alleles in low but similar frequencies. In addition, each population had an array of unique alleles present only once in one population sample but absent in the other. A statistical test against the stationary distribution for neutral alleles shows that, if the populations are at equilibrium, then purifying selection is operating on xanthine dehydrogenase. The extremely close similarity in frequency distributions of the alleles between populations for both the xanthine dehydrogenase and esterase-5 loci, despite differences in allele frequency distribution between loci, strongly emphasizes the importance of migration in influencing genic diversity in these populations.     

An Experimental Investigation of the Unit Charge Model of Protein Polymorphism and Its Relation to the Esterase-5 Locus of DROSOPHILA PSEUDOOBSCURA, DROSOPHILA PERSIMILIS, and DROSOPHILA MIRANDA

The relationship between charge changes and electrophoretic mobility changes is investigated experimentally. The charge of several proteins is altered by reaction with small molecules of known structure and the change in electrophoretic mobility is measured. The method of Ferguson plots is used to separate charge and shape components of mobility differences. The average effect of an amino acid charge change on the mobility of the esterase-5( 1.00) allele of Drosophila pseudoobscura is estimated to be 0.046. This estimate is then used to apply the step model of Ohta and Kimura (1973) to electrophoretic mobility data for the esterase-5 locus of D. pseudoobscura and D. miranda. The variation in electrophoretic mobility at this locus was found to be in agreement with the predictions of the step model.     

Genetics of esterases in Drosophila. III. Influence of different chromosomes on esterase pattern in Drosophila

The present report presents the results of starch and polyacrylamide gel electrophoretic studies of the influence of the X chromosome on the expression of esterase-6 in D. melanogaster × D. simulans hybrids heterozygous for locus Est-6 as well as studies of the influence of autosomes on esterase expression in Drosophila of the virilis group. A differential expression of esterase-6 has been detected in D. melanogaster × D. simulans hybrid males. A differential decrease in the activity of esterase-6 (both F and S allozymes) derived from D. melanogaster has been noted. In hybrid females, the activity of parental esterases is the same. It is suggested that the X chromosome regulates the expression of esterase-6 in D. melanogaster. Analysis of individuals obtained in different schemes of crosses between different species of Drosophila of the virilis group by use of stocks marked with mutations in various chromosomes indicates that other autosomes (in particular, autosomes 4 and 5) also influence the phenotypic expression of esterases (which are controlled by genes located on the second chromosome).     

Natural selection and the genetic differentiation of coastal and Arctic populations of the Atlantic cod in northern Norway: a test involving nucleotide sequence variation at the pantophysin (PanI) locus

To examine the role of contemporary selection in maintaining significant allele frequency differences at the pantophysin (PanI) locus among populations of the Atlantic cod, Gadus morhua, in northern Norway, we sequenced 127 PanIA alleles sampled from six coastal and two Barents Sea populations. The distributions of variable sites segregating within the PanIA allelic class were then compared among the populations. Significant differences were detected in the overall frequencies of PanIA alleles among populations within coastal and Arctic regions that was similar in magnitude to heterogeneity in the distributions of polymorphic sites segregating within the PanIA allelic class. The differentiation observed at silent sites in the PanIA allelic class contradicts the predicted effects of widescale gene flow and suggests that postsettlement selection acting on cohorts cannot be responsible for the genetic differences described between coastal and Arctic populations. Our results suggest that the marked differences observed between coastal and Arctic populations of G. morhua in northern Norway at the PanI locus reflect the action of recent diversifying selection and that populations throughout the region may be more independent than suggested by previous studies.     

Genetics and polymorphism of a duplicated malate dehydrogenase (MDH) locus in the grasshopperOxya japonica japonica (Orthoptera:Acrididae:Oxyinae)

A biochemical genetic study of the enzyme malate dehydrogenase (MDH) was conducted in the grasshopperOxya j. japonica. Analysis of MDH electrophoretic variation in this species of grasshopper shows that one of the two autosomal loci for MDH in grasshoppers, the Mdh-2 locus, controlling the anodal set of MDH isozymes, is duplicated. Results of breeding studies confirm this and the observed polymorphism at theMdh-2 locus in the two populations ofOxya j. japonica studied can be attributed to three forms of linked alleles at the duplicated locus in equilibrium in both populations. In this respect, all individuals of this species possess heterozygous allelic combinations at the duplicatedMdh-2 locus, which may account for the spread of the duplicated locus in the populations of this species of grasshopper.This research was supported by a grant (Vote F) from the University of Malaya, Kuala Lumpur.     

Genic Heterogeneity within Electrophoretic "Alleles" and the Pattern of Variation among Loci in DROSOPHILA PSEUDOOBSCURA

An investigation, similar to our previously reported xanthine dehydrogenase study, was undertaken to examine the extent of hidden genic variation at nine loci (five larval proteins, three esterases and one aldehyde oxidase) by sequential application of various electrophoretic criteria employing pH, gel concentration and buffer variation. Polymorphic loci appear to fall into two distinct groups: weakly polymorphic, including larval protein 6, 7, 8, 10 and 13 and esterase-1 and -6; and highly polymorphic, including esterase-5, Xdh and possibly Ao. Monomorphic loci may belong to a third group different from all polymorphic loci. Bogota, a geographical isolate that is reproductively isolated from the mainland population, was found to be genetically distinct at four of the ten loci examined in detail so far, including Xdh, whereas previously it was found to be genetically distinct at none. These results are discussed in the light of balancing selection, neutral and mutation-selection hypotheses of genic variation in natural populations.