Purification and characterization of phosphoglucose isomerase allozymes from Daphnia magna.

Phosphoglucose isomerase (PGI, EC 5.3.1.9) is polymorphic in many populations. Frequently, it has been shown that naturally occurring allozymes exhibit strong deviations form Hardy-Weinberg expectations, suggesting fitness relevant mutations. To investigate the nature of this allozymic variation, PGI was purified from Daphnia magna to high purity yielding a specific activity of 135.2 U/mg. The kinetic parameters of the allozymes were characterized depending upon ionic strength, pH and viscosity. The half-saturation constants of the allozymes were all equal, while the specific activity of the PGI from heterozygotes was consistently higher than the PGI of the homozygotes, independent of pH, ionic strength and viscosity of the solution.     

Studies of esterase 6 in Drosophila melanogaster. I. The genetics of a posttranslational modification

A locus has been found, an allele of which causes a modification of some allozymes of the enzyme esterase 6 in Drosophila melanogaster. There are two alleles of this locus, one of which is dominant to the other and results in increased electrophoretic mobility of affected allozymes. The locus responsible has been mapped to 3-56.7 on the standard genetic map (Est-6 is at 3-36.8). Of 13 other enzyme systems analyzed, only leucine aminopeptidase is affected by the modifier locus. Neuraminidase incubations of homogenates altered the electrophoretic mobility of esterase 6 allozymes, but the mobility differences found are not large enough to conclude that esterase 6 is sialylated.This work was supported by NIH Grant No. GM23706 and PHS Grant SO7RR7031 to Rollin C. Richmond and by NIH Genetics Training Grant No. 82 to Indiana University.     

The genetic structure of endangered populations in the Cranberry Fritillary,Boloria aquilonaris (Lepidoptera,Nymphalidae): RAPDs vs allozymes

The genetic population structure of the Cranberry Fritillary Boloria aquilonaris was studied using both RAPDs (random amplified polymorphic DNA) and allozymes. In Belgium, B. aquilonaris has a naturally fragmented distribution that has been accentuated due to human activity during the last century. The genetic population structure of this butterfly was analysed at the regional (several Ardenne uplands) and at the landscape level (several populations within an Ardenne upland). Both population genetic markers confirmed results from a previous CMR study at the landscape scale. At the regional scale however, important incongruences were observed between RAPDs and allozymes. The average gene diversity for the RAPD data was twice that of the allozyme data. The degree of population subdivision was also much greater for RAPDs than for allozymes. The UPGMA clusters produced by each of these markers differed significantly. We believe that, given the higher rate of mutation of RAPDs and the greater number of loci assayed by this method, RAPDs reveal a more accurate and recent population genetic structure than allozymes.     

Genetic structure of avian populations--allozymes revisited

Selection on allozymes has sometimes been advanced as one explanation for the low levels of population differentiation detected in avian populations by the use of enzymatic markers. Comparisons of the amount of population subdivision (estimated by FST values or analogous indices) measured by enzymatic and mitochondrial DNA (mtDNA) markers in birds were seen as evidence for this because mtDNA typically produces a more structured picture of population subdivisions. In fact, when taking into account the smaller effective population size of mtDNA, nuclear and mitochondrial markers give concordant results. Some discrepancies still exist, but I suggest that some might originate from different amounts of nuclear vs. mitochondrial gene flow due to partial reproductive isolation. Variable number of tandem repeat (VNTR) loci do not provide a dramatically different picture of population structures in birds compared to allozymes. Although more tests are needed, such as comparing the amount of genetic structure detected in the same populations with allozymes and microsatellites, the low levels of population subdivision measured with allozymes in birds seem to reflect historical and demographic processes and would not appear to result from any peculiarities of bird enzymatic loci.     

Isolation and genetic characterization of alcohol dehydrogenase thermostability variants occurring in natural populations of Drosophila melanogaster

Drosophila melanogaster collected from natural populations were examined fo thermostability variants within electrophoretic mobility classes of two enzymes. In alcohol dehydrogenases, 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 0.7 unit of the Adh 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 Adh alleles could be generated by intragenic recombination between the two common alleles, electrophoresis and heat treatment of progeny recombinant for flanking markers of Adh revealed no new allozymes. Among 27 stocks containing slow alpha-glycerophosphate dehydrogenase allozymes and 109 fast stocks, heat treatments revealed no additional variation.     

A rapid fluorescence technique for electrophoretic identification of hypoxanthine phosphoribosyltransferase allozymes

A method for the rapid identification of hypoxanthine phosphoribosyltransferase allozymes is described. It involves electrophoresis on cellulose acetate gel, followed by detection of the phosphoribosyltransferase activity through a fluorescent end product formed in a specific coupled reaction with inosine 5′-monophosphate dehydrogenase. This method has been used successfully to distinguish from each other the allozymes of man, mouse, Chinese hamster, Syrian hamster, and chicken, and should be useful for the biochemical characterization of cellular mutants and somatic cell hybrids utilizing hypoxanthine phosphoribosyltransferase as a selective marker.     

Inhibition of phosphoglucose isomerase allozymes from the wing polymorphic waterstrider,Limnoporus canaliculatus,by pentose shunt metabolites

Inhibition of phosphoglucose isomerase (PGI) allozymes from the wing-polymorphic waterstrider, Limnoporus canaliculatus, by three pentose-shunt metabolites was studied at several different temperatures. This was done to determine if the allozymes exhibited a differential ability to participate in lipid biosynthesis via differential partitioning of carbon flux through the pentose shunt versus glycolysis. 6-Phosphogluconate and erythrose-4-phosphate proved to be strong competitive inhibitors of PGI, while sedoheptulose-7-phosphate was a very weak inhibitor. The PGI allozymes from L. canalicualtus were differentially inhibited by 6-phosphogluconate at two of the three temperatures studied. However, this property does not appear to be an adaptive difference between the allozymes but, rather, a correlated effect resulting from variation in substrate binding. Estimates of reaction rates for the allozymes indicate that the differences in inhibition result in no detectable differences in reaction velocities. Thus, no evidence in support of the hypothesis that PGI allozymes from Limnoporus canaliculatus were adapted to function in different metabolic capacities via differential inhibition was obtained in this study. However, the importance of this characteristic in allozymic adaptation in natural populations remains an open question.Supported by NSF Grant DEB 7908802 and UPHS Grant GM 21133 to R. K. Koehn and an NSF dissertation improvement grant to A. J. Zera.     

Taxonomic relationships between four species of the Mullidae family revealed by three genetic methods: allozymes, random amplified polymorpic DNA and mitochondrial DNA

Genetic distances were lower between Upeneus moluccensis and Pseudopeneus prayensis than between those species and Mullus barbatus and Mullus surmuletus . However, for allozymes, the two Mullus species were found genetically more distinct from U. moluccensis than from P. prayensis , but RAPD and mtDNA analysis showed the opposite. RAPDs revealed less interspecific divergence compared with allozymes and the results they produced were more consistent with mtDNA analysis. Although RAPDs did not add any supplementary taxonomic information, they proved valuable tools for quick and reliable species discrimination compared with allozymes and mtDNA.     

Differential expression of allelic carboxylesterase-2 genes in oocytes of loach (Misgurnus fossilis L.) and heterogeneity of loach oocytes and eggs for the expression of allelic carboxylesterase-2 genes

Tissue and cell differences have been found in the expression of allelic carboxylesterase-2 (E-2) (carboxylic ester hydrolase, E.C. 3.1. 1.1) genes of the loach. The relative activity of two allozymes in the brain and muscles of heterozygotes is similar, whereas in oocytes and eggs of the same fish the activity of one of the allozymes is considerably greater than that of the other. Oocytes and eggs of some heterozygotes were shown to be heterogeneous for the expression of E-2 alleles. The phenomenon implies that roe produced by a heterozygous female can contain two or more egg types: in some the alleles are equally expressed, while in others one of the alleles is predominantly expressed.     

Mitochondrial DNA sequences support allozyme evidence for cryptic radiation of New Zealand Peripatoides (Onychophora)

A combination of single-strand conformation polymorphism analysis (SSCP) and sequencing were used to survey cytochrome oxidase I (COI) mitochondrial DNA (mtDNA) diversity among New Zealand ovoviviparous Onychophora. Most of the sites and individuals had previously been analysed using allozyme electrophoresis. A total of 157 peripatus collected at 54 sites throughout New Zealand were screened yielding 62 different haplotypes. Comparison of 540-bp COI sequences from Peripatoides revealed mean among-clade genetic distances of up to 11. 4% using Kimura 2-parameter (K2P) analysis or 17.5% using general time-reversible (GTR + I + Gamma) analysis. Phylogenetic analysis revealed eight well-supported clades that were consistent with the allozyme analysis. Five of the six cryptic peripatus species distinguished by allozymes were confirmed by mtDNA analysis. The sixth taxon appeared to be paraphyletic, but genetic and geographical evidence suggested recent speciation. Two additional taxa were evident from the mtDNA data but neither occurred within the areas surveyed using allozymes. Among the peripatus surveyed with both mtDNA and allozymes, only one clear instance of recent introgression was evident, even though several taxa occurred in sympatry. This suggests well-developed mate recognition despite minimal morphological variation and low overall genetic diversity.     

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.      

Genetic mapping and characterization of aldehyde oxidase of Anopheles albimanus (Diptera: Culicidae)

Aldehyde oxidase (Ao) of Anopheles albimanus Wiedemann was mapped on chromosome 3. The sequence is hexokinase-1--19.2 +/- 1.8--stripe--28.3 +/- 2.2--beta-hydroxy acid dehydrogenase--3.6 +/- 0.3--aldehyde oxidase--2.6 +/- 0.4--esterase-8--6.1 +/- 1.9--esterase-4--?--esterase-6 (phosphoglucomutase). Aldehyde oxidase is 26.1 +/- 2.5 from phosphoglucomutase and 27.2 +/- 1.6 from esterase-6. The one-band electromorph of Ao in homozygotes and the three-band type in heterozygotes suggest that the enzyme is a dimer. The isoelectric points of slow and fast allozymes are 5.5 and 4.8, respectively. A variety of electrophoretic techniques was used to determine if the allozymes of Ao can be differentiated on a basis other than mobility. The slow, fast, and hybrid genotypes were analyzed for differences in thermostability, reactivity to thiol reagent, susceptibility to urea denaturation, substrate specificities, and response to chelating agents. The relative effect of p]H on allozymes was tested by varying the pH of the staining buffer over a range of 4-12. No significant differences were detected among allozymes and no additional allelic variations were observed.     

Concordance of allozyme and microsatellite differentiation in a marine fish, but evidence of selection at a microsatellite locus

Previous studies have reported higher levels of divergence for microsatellites than for allozymes in several species, suggested to reflect stabilizing selection on the allozymes. We compared the differentiation patterns of 11 allozyme and nine microsatellite loci using 679 spawning Atlantic herring (Clupea harengus) collected in the Baltic and North Seas to test for differential natural selection on these markers. Observed distributions of F statistics for the two types of markers are conspicuously dissimilar, but we show that these differences can largely be explained by sampling phenomena caused by different allele frequency distributions and degrees of variability. The results show consistently low levels of differentiation for both marker types, with the exception of one outlier microsatellite locus with a notably high F(ST). The aberrant pattern at this locus is primarily due to two alleles occurring at markedly high frequencies in the Baltic, suggesting selection at this locus, or a closely linked one. When excluding this locus, the two marker types show similar, weak differentiation patterns with F(ST) values between the Baltic and the North Seas of 0.001 and 0.002 for allozymes and microsatellites, respectively. This small heterogeneity, and weak isolation by distance, is easier to distinguish statistically with microsatellites than with allozymes that have fewer alleles and skewed frequency distributions. The allozymes, however, also detect surprisingly low levels of divergence. Our results support suggestions that previously described differences between marker types are primarily caused by a small number of outlier loci.     

The structure of morphometric and allozyme variation in relict populations of Gypsophila fastigiata (Caryophyllaceae) in Sweden

Patterns of variation and the structuring of diversity in seed phenotype and allozymes were investigated in Sweden Gypsophila fastigiata, a perennial herb with a disjunct ‘Late Glacial relict’ distribution in eastern and northern Europe. The overall patterns of variation in allozymes and seed morphology are congruent and are significantly correlated with geographic distance. However, most of the congruence between the distance matrices based on allozymes and seed morphology is attributable to regional differentiation between the isolated Öland and Dalarna metapopulations. On a local scale, within the two metapopulations, seed shape variation is partially correlated with geography whereas allozyme variation is unrelated to the geographic disposition of the sampling sites. Despite the fact that regional metapopulations can be discriminated on the basis of seed shape and allozyme frequencies, relatively little of the total diversity in seed morphology and allozymes is due to differentiation between regions. Partitioning of diversity into its within- and among-regional, site and individual components, showed that the majority of diversity (52% for seed shape and 91% for allozymes) is stored within sites. Seventy-three percent of the variation in allozymes is due to variation within individuals (i.e. heterozygosity) whereas phenotypic variation in seed shape within individuals is low (0.4% of the total diversity). The events that led to the large scale disjunction of the Late Glacial distribution of G. fastigiata may have shaped the pattern of differentiation observed among the regional isolates of the species. However, the species' history of local population disjunction during post-glacial and historic times has not had a substantial impact on the spatial organization of allozyme and morphometric diversity within regions. Extensive local gene flow may have allowed the regional metapopulations to have functioned as extended effective populations–on a time scale of tens or hundreds of years–and may have hindered the loss of within-site diversity.     

Electrophoretic evaluation of the taxonomic status of two species of sea anemone

Puerto Rican populations of two species of sea anemones (Bunodosoma cavernata and B. granulifera) which had previously been considered one were assayed electrophoretically for enzymes encoded by 12 loci. The two species shared no common allozymes at 6 of the 12 loci. Genetic distance and identity values based on these allozymes were computed for the Puerto Rican populations and for B. cavernata from Florida and B. granulifera from Panama. The Puerto Rican populations of both species had much higher genetic identities for their geographically distant conspecifics than for each other. These results indicate that the two species are reproductively isolated and should be considered as separate valid species. Average heterozygosities are presented which are the first published for coelenterate species.     

Genetic anomalies associated with Cerion hybrid zones: the origin and maintenance of new electromorphic variants called hybrizymes

Natural hybrid zones involving the West Indian pulmonate land snail Cerion are characterized by the occurrence, in low to moderate frequencies, of allozymes that are unique to interspecific hybrid zones. As such electrophoretically detected genetic anomalies have also been reported in hybrid zones involving mammals, birds, reptiles, amphibians, and insects this appears to be a general phenomenon. These unexpected allozymes are inappropriately called 'rare alleles' and the term hybrizyme is introduced. The origins of hybrizymes are discussed in terms of suppressor-mutation systems, transposon-induced hybrid dysgenesis and intragenic recombination, but available evidence will not resolve this issue. Similarly, it is not clear whether the relatively high frequencies of particular hybrizymes are due to selection or genetic drift or some combination of these agents. Finally, the evolutionary significance of hybrizymes and the possible role of hybridization in introducing new genetic variation into populations are discussed.     

Comparison of purified acid phosphatase allozymes inDrosophila virilis: Differences in carbohydrate content and composition of the allozymes

Three acid phosphatase (EC 3.1.3.2) allozymes (ACPH¹, ACPH², and ACPH⁴) ofDrosophila virilis show different activities as measured by electrophoretic techniques. Recently, it was suggested that these differences are attributable to the variable ability of the allozymes to be incorporated into lysosomes (Narise, S.,Genet. Res. Cambr., 45:143, 1985). Immunoelectrophoresis demonstrated that the activity differences between these electrophoretic variants coincided with differences in the amount of the enzyme protein in soluble fractions but not in whole cell-free extracts. These results support the idea that acid phosphatase allozymes inD. virilis are cell-localization variants. We examined the problem by structural analysis of both the protein and the carbohydrate moieties of these allozyme glycoproteins, since lysosomal enzymes are known to become localized in lysosomes through their carbohydrate moieties. The three ACPH allozymes were purified to homogeneity from their respective homozygotes and compared with respect to amino acid composition and carbohydrate content and composition. Amino acid compositions were similar, while content and compositions of neutral sugars were significantly different. The neutral sugar content of ACPH¹ was 9.2%; that of ACPH², 21.0%; and that of ACPH⁴, 7.3%. A trace of hexosamines, but noN-acetylneuraminic acid, was found in the ACPH allozymes. Isoelectric points varied corresponding to their electrophoretic mobilities, which were not changed by treatment with alkaline phosphatase and neuraminidase.     

A Generic Estimation of Population Subdivision Using Distances between Alleles with Special Reference for Microsatellite Loci

Several estimators of population differentiation have been proposed in the recent past to deal with various types of genetic markers (i.e., allozymes, nucleotide sequences, restriction fragment length polymorphisms, or microsatellites). We discuss the relationships among these estimators and show how a single analysis of variance framework can accomodate these qualitatively different data types.     

Comparative Assessment of Genetic Variability in the Populations of Endemic and Endangered Yellow Catfish, Horabagrus brachysoma (Teleostei: Horabagridae), Based on Allozyme, RAPD, and Microsatellite Markers

The comparative assessment of genetic diversity using allozymes, random amplified polymorphic DNA (RAPD), and microsatellite markers was conducted in endemic and endangered yellow catfish (Horabagrus brachysoma) sampled from three locations in Western Ghats river systems of India. Among the three markers, microsatellites show more polymorphism, having 100% polymorphic loci, whereas allozymes show the least (56%). In RAPD, 60.5% of fragments were polymorphic. Observed heterozygosity and F(ST) values were very high in microsatellites, compared with the other markers. Microsatellite and RAPD markers reported a higher degree of genetic differentiation than allozymes among the populations depicted by pairwise F(ST)/G(ST), AMOVA, Nei's genetic distance, and UPGMA dendrogram. The three classes of markers demonstrated striking genetic differentiation between pairs of H. brachysoma populations. The data emphasize the need for fishery management, conservation, and rehabilitation of this species.     

Temperature-dependent kinetic variation among phosphoglucose isomerase allozymes from the wing-polymorphic water strider, Limnoporus canaliculatus

Phosphoglucose isomerase (PGI) allozymes were isolated from the wing- polymorphic water strider, Limnoporus canaliculatus, and were characterized biochemically with respect to temperature-dependent kinetic and thermostability properties. At higher temperatures, the allozymes exhibited significant differences in Michaelis constant (Km) values for substrates of both the forward and reverse reaction directions. Results were consistent with expectations of adaptive kinetic differentiation based on the latitudinal variation of PGI allele frequencies. PGI genotypes also differed with regard to maximal velocity (Vmax)/Km ratios at higher temperatures. These differences were due primarily, if not exclusively, to allozyme-dependent variation in Km values. The allozymes also exhibited dramatic differences in thermostability. However, no thermostability differences were observed when the substrate analogue 6-phosphogluconate was present in the incubation medium. The data from this study, together with data from Mytilus edulis and Metridium senile on temperature-dependent kinetic variation among PGI allozymes, form a consistent picture of natural selection influencing the clinal variation of alleles at this locus in these three phylogenetically distant organisms. More definitive support of this hypothesis, however, must await additional studies on the physiological effects of the allozymic variation as well as direct measurements of fitness differences among the enzyme genotypes.