Balancing Selection on Electrophoretic Variation of Phosphoglucose Isomerase in Two Species of Field Cricket: Gryllus Veletis and G. Pennsylvanicus

Two species of crickets, Gryllus veletis and G. pennsylvanicus, share six electrophoretic mobility classes for the enzyme phosphoglucose isomerase (PGI), despite evidence from other genetic markers that the two species are not closely related within eastern North American field crickets. Moreover, the frequencies of the two most common PGI electrophoretic classes (PGI-100 and PGI-65) covary in sympatric populations of these species in the eastern United States, suggesting that PGI may be subject to trans-specific balancing selection. To determine the molecular basis of the electrophoretic variation, we characterized the DNA sequence of the Pgi gene from 29 crickets (15 G. veletis and 14 G. pennsylvanicus). Amino acid substitutions that distinguish the electrophoretic classes are not the same in the two species, and there is no evidence that specific replacement substitutions represent trans-specific polymorphism. In particular, the amino acids that diagnose the PGI-65 allele relative to the PGI-100 allele differ both between G. veletis and G. pennsylvanicus and within G. pennsylvanicus. The heterogeneity among electrophoretic classes that covary in sympatric populations coupled with analysis of patterns of nucleotide variation suggest that Pgi is not evolving neutrally. Instead, the data are consistent with balancing selection operating on an emergent property of the PGI protein.     

Alu insertion polymorphisms for the study of human genomic diversity

We analyze genetic variation at fused1, a locus that is close to the centromere of the X chromosome-autosome (X/4) fusion in Drosophila americana. In contrast to other X-linked and autosomal genes, for which a lack of population subdivision in D. americana has been observed at the DNA level, we find strong haplotype structure associated with the alternative chromosomal arrangements. There are several derived fixed differences at fused1 (including one amino acid replacement) between two haplotype classes of this locus. From these results, we obtain an estimate of an age of approximately 0.61 million years for the origin of the two haplotypes of the fused1 gene. Haplotypes associated with the X/4 fusion have less DNA sequence variation at fused1 than haplotypes associated with the ancestral chromosome arrangement. The X/4 haplotypes also exhibit clinal variation for the allele frequencies of the three most common amino acid replacement polymorphisms, but not for adjacent silent polymorphisms. These patterns of variation are best explained as a result of selection acting on amino acid substitutions, with geographic variation in selection pressures.     

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.     

Clinal variation for amino acid polymorphisms at the Pgm locus in Drosophila melanogaster

Clinal variation is common for enzymes in the glycolytic pathway for Drosophila melanogaster and is generally accepted as an adaptive response to different climates. Although the enzyme phosphoglucomutase (PGM) possesses several allozyme polymorphisms, it is unique in that it had been reported to show no clinal variation. Our recent DNA sequence investigation of Pgm found extensive cryptic amino acid polymorphism segregating with the allozyme alleles. In this study, we characterize the geographic variation of Pgm amino acid polymorphisms at the nucleotide level along a latitudinal cline in the eastern United States. A survey of 15 SNPs across the Pgm gene finds significant clinal differentiation for the allozyme polymorphisms as well as for many of the cryptic amino acid polymorphisms. A test of independence shows that pervasive linkage disequilibrium across this gene region can explain many of the amino acid clines. A single Pgm haplotype defined by two amino acid polymorphisms shows the strongest correlation with latitude and the steepest change in allele frequency across the cline. We propose that clinal selection at Pgm may in part explain the extensive amino acid polymorphism at this locus and is consistent with a multilocus response to selection in the glycolytic pathway.     

Nucleotide polymorphism and natural selection at the pantophysin (Pan I) locus in the Atlantic cod, Gadus morhua (L.)

Molecular studies of nucleotide sequence variation have rarely attempted to test hypotheses related to geographically varying patterns of natural selection. The present study tested the role of spatially varying selection in producing significant linkage disequilibrium and large differences in the frequencies of two common alleles at the pantophysin (Pan I) locus among five populations of the Atlantic cod, Gadus morhua. Nucleotide sequences of 124 Pan I alleles showed strong evidence for an unusual mix of balancing and directional selection but no evidence of stable geographically varying selection. The alleles were highly divergent at both the nucleotide level (differing on average by 19 mutations) and at amino acid level (each having experienced three amino acid substitutions since diverging from a common ancestral allele). All six amino acid substitutions occurred in a 56-residue intravesicular loop (IV1 domain) of the vesicle protein and each involved a radical change. An analysis of molecular variation revealed significant heterogeneity in the frequencies of recently derived mutations segregating within both allelic classes, suggesting that two selective sweeps may be presently occurring among populations. The dynamic nature of the Pan I polymorphism in G. morhua and clear departure from equilibrium conditions invalidate a simple model of spatially varying selection.     

Detection of genetic variation with radioactive ligands. I. Electrophoretic screening of plasma proteins with a selected panel of compounds.

To detect new genetic variation in human plasma proteins, a panel of 63 radioactive substances were screened as potential radioligands using polyacrylamide gel electrophoresis (PAGE) and autoradiography. Vitamins, hormones, drugs, amino acids, purines, pyrimidines, sugars and lipids labeled with 14C or other radionuclides were among those substances tested. A majority bound to albumin and a smaller fraction to prealbumins and lipoproteins. Several vitamins and hormones bound to specific alpha and beta globulins. (1) Electrophoretic polymorphisms of vitamin D-binding protein (group-specific component), a vitamin B12-binding protein (transcobalamin II), and thyroxine-binding alpha-globulin are described elsewhere. (2) Testosterone-binding beta-globulin (TeBG) showed an electrophoretic polymorphism in Caucasians and a possible deficiency allele. (3) Transcortin showed an electrophoretic doublet in all persons tested but no electrophoretic variation. (4) A protein binding derivative of norepinephrine or epinephrine was identified as transferrin. (5) A nonpolymorphic protein running cathodal to albumin and binding a derivative of riboflavin was tentatively identified as a fraction of albumin with mobility altered as a result of interaction with the ligand.     

Tracing the history of an enzyme polymorphism: the case of alcohol dehydrogenase-2 (Adh-2) of the olive fruit fly Bactrocera oleae

In the olive fruit fly Bactrocera oleae, previous studies have described a one-locus three-allele electrophoretic polymorphism of the enzyme alcohol dehydrogenase and provided evidence that the polymorphism is under the influence of selection. A recent study has shown that this species carries a two-locus duplication for alcohol dehydrogenase. Here, we show that the polymorphism maps at one of the duplicated loci, Adh2, and identify the nucleotide and, therefore, the inferred amino acid differences among the three allozymes. At the amino acid level, the polymorphism is of the simplest possible form: there is no intra-allozyme variation, and interallozyme differences are restricted to one amino acid for two pairs of alleles and to two amino acids for the third pair. Consideration of the amino acid residues at the sites that segregate in B. oleae in four congeneric species and the phylogenetic trees produced from the nucleotide sequences of the Adh2 gene of these species point to the same allozyme as the ancestral form of the polymorphism. Interestingly, this allozyme comprises less than 1% of the gene pool of present-day natural populations of B. oleae, where the other two allozymes appear to form a stable polymorphism. Previous studies have shown that the frequency of the rare allozyme rises rapidly in laboratory colonies maintained on artificial diet and declines again when the artificial diet is replaced with olive fruit, the natural substrate of B. oleae. The geographical distribution of several congeneric species suggests that B. oleae originated in the Indian subcontinent, where the olive tree is practically absent. The poor performance of the ancestral allele on the olive fruit suggests the possibility that the decline of this allele and the concomitant rise of the presently common alleles might be associated with the expansion of the insect's geographical distribution to areas where the olive tree has become its main and perhaps sole host. The estimated age of the polymorphism is compatible with this hypothesis, but firmer support could be difficult to obtain.     

Mhc-DQB repertoire variation in hominoid and Old World primate species.

Comparison of 87 distinct Mhc-DQB sequences, obtained from 13 primate species, demonstrates that five out of eight trans-species Mhc-DQB allele lineages are at least 30 million years old and predate divergence of hominoid and Old World primate species. One lineage may be much older because its members are not only traced back in higher primates, but also are present in a New World primate species. Comparing Mhc-DQB repertoire variation in distinct species, allows one to pinpoint when certain polymorphisms were lost or gained in primate evolution. Heterogeneity observed among members of trans-species Mhc-DQB allele lineages can be explained in major part by point mutations, whereas intraexonic crossing-over is a potent mechanism in generating new allele lineages. The stability of Mhc-DQB polymorphisms is influenced by selective forces because distinct allele lineages appear to have accumulated nucleotide substitutions and amino acid replacements at different rates.     

Isoallele Frequencies in Very Large Populations

The frequencies of electrophoretically distinguishable allelic forms of enzymes may be very different from the corresponding frequencies of structurally distinct forms, because many sequence variants may have identical electrophoretic charge. In large populations such frequencies will be determined largely by the number of amino acid sites that are free to vary. The number of distinguishable electrophoretic variants will remain fairly small. Beyond some limiting size, no further effect of population size on allele frequencies is expected, so isolated large populations will have closely similar allele frequencies if polymorphism is due largely to mutation and drift. The most common electrophoretic alleles are expected to be flanked by the next most common, with the rarer alleles increasingly distal. Neither strong selection nor mutation/drift interpretations of enzyme polymorphism are yet disproven, nor is any point between these extremes.     

Four DNA polymorphisms on the short arm of the X chromosome: allele frequencies in a German and in a Turkish population

Four restriction fragment length polymorphisms, revealed by cloned arbitrary X chromosome segments (L1.28, RC8, pD2, 754) were studied in samples (50 individuals each) of a German and a Turkish population. All previously reported alleles of these polymorphisms were found in both populations, except the infrequent RC8 allele B3 (3.0 kb fragment), which was absent in both groups. The observed minor alleles were found to be rarer in the German series than in the Turkish group, but there was no conclusive evidence of essentially different allele frequencies in either population. However, the frequencies of the RC8 allele B2 (5.3 kb fragment) were differing at the 5% significance level. The allele frequencies of the four polymorphisms are presented and compared with those reported from other European regions.     

Evolution of HLA class II molecules: Allelic and amino acid site variability across populations

Analysis of the highly polymorphic beta1 domains of the HLA class II molecules encoded by the DRB1, DQB1, and DPB1 loci reveals contrasting levels of diversity at the allele and amino acid site levels. Statistics of allele frequency distributions, based on Watterson's homozygosity statistic F, reveal distinct evolutionary patterns for these loci in ethnically diverse samples (26 populations for DQB1 and DRB1 and 14 for DPB1). When examined over all populations, the DQB1 locus allelic variation exhibits striking balanced polymorphism (P < 10(-4)), DRB1 shows some evidence of balancing selection (P < 0.06), and while there is overall very little evidence for selection of DPB1 allele frequencies, there is a trend in the direction of balancing selection (P < 0.08). In contrast, at the amino acid level all three loci show strong evidence of balancing selection at some sites. Averaged over polymorphic amino acid sites, DQB1 and DPB1 show similar deviation from neutrality expectations, and both exhibit more balanced polymorphic amino acid sites than DRB1. Across ethnic groups, polymorphisms at many codons show evidence for balancing selection, yet data consistent with directional selection were observed at other codons. Both antigen-binding pocket- and non-pocket-forming amino acid sites show overall deviation from neutrality for all three loci. Only in the case of DRB1 was there a significant difference between pocket- and non-pocket-forming amino acid sites. Our findings indicate that balancing selection at the MHC occurs at the level of polymorphic amino acid residues, and that in many cases this selection is consistent across populations.     

The silkmoth late chorion locus. I. Variation within two paired multigene families

The 140 X 10(3) base late chorion locus of Bombyx mori contains two 15-member multigene families arranged in tightly linked pairs, which are divergently transcribed (the high-cysteine A (HcA) and the high-cysteine B (HcB) families). Previous DNA hybridization experiments have indicated that all members of these gene families contain a complex pattern of shared sequence variation. The sequence analysis in this paper involving all 15 gene pairs allows a comprehensive examination of the nature of this variation. Average sequence homology between gene pairs is: 95% for the protein-encoding regions; 93% for the common 272 base-pair 5' flanking region; 87% for the introns; and 88% for the 3' untranslated regions. Considering the great degree of sequence homology in the coding regions, an unexpectedly high level of variation is found in the deduced protein sequences. Over 50% of the nucleotide substitutions in the protein-encoding regions lead to amino acid replacements, most of which involve a change in charge or effect the secondary structure of the protein. In addition, significant differences in length between the proteins occur in the carboxyl-terminal arm. In both families, the major portion of this arm is composed of Cys-Gly-Gly and Cys-Gly subrepeats forming a (Cys-Gly-Gly)2-(Cys-Gly)2 major repeat. Differences in the number of complete and partial repeats results in deduced protein sequences that contain arms varying from 32 to 54 amino acid residues for members of the HcA family and 14 to 88 residues for the HcB family. The high level of variation in protein composition indicates a lack of strong selective pressure. We suggest the high level of DNA sequence homology maintained by these genes in the coding as well as in the non-coding regions is the result of sequence exchange between family members.     

Strain typing in Leishmania donovani by using sequence-confirmed amplified region analysis

To differentiate strains of Leishmania donovani, allelic markers at the DNA level were developed by sequence-confirmed amplified region analysis (SCAR). Homologous fragments from different strains of L. donovani were amplified by PCR using random primers and subsequently screened for single-strand conformation polymorphisms. Direct sequencing revealed 55 sequence polymorphisms in eight co-dominant DNA markers; 38 of them were single point mutations. Heterozygosity was evident for 69% and fixed heterozygosity for 25% of all polymorphisms. At most polymorphic sites one of the segregation genotypes was missing. Nineteen unique multilocus genotypes were identified among 29 strains of L. donovani. One genotype was represented by eight Sudanese strains; also two strains from Sudan as well as two strains from Kenya, respectively, shared identical genotypes. All other strains had individual multilocus genotypes. Calculation of genetic distances showed a correlation between multilocus genotypes and the geographical origin of these strains. African strains were found in one well-supported cluster with Kenyan and Sudanese strains clearly separated. SCAR markers seem to represent a random sample of neutral genetic variation present in natural populations. They are co-dominant because they can detect all possible allele combinations in a diploid organism and may, therefore, be very useful for population genetic analysis in Leishmania.     

Allelic and haplotypic diversity at the major histocompatibility class II within domesticated Australian Atlantic salmon (Salmo salar L.)

Variation within the major histocompatibility (MH) class II alpha gene ( Sasa-DAA ) was compared between domesticated Australian Atlantic salmon and their ancestral Canadian population. The level of Sasa-DAA and MH class II beta gene ( Sasa-DAB ) sequence variation was also examined within the Australian population and compared with that published for European Atlantic salmon populations. In contrast to variation previously reported for non-coding microsatellite loci, a high level of MH class II allelic variation has been maintained within the domesticated Australian populations. Furthermore, a high level of Sasa-DAA and Sasa-DAB allele sequence diversity was also observed and exceeded that reported for other cultured Atlantic salmon populations. The number of Sasa-DAB allele sequences (14) surpassed the number of Sasa-DAA allele sequences (9) to produce 14 unique class II haplotypes. We conclude that the Australian Atlantic salmon populations show high MH class II allelic and haplotypic variation compared with both its ancestral Canadian population and other cultured Atlantic salmon populations.     

Phylogenetic Analysis of the Non-structural (NS) Gene of Influenza A Viruses Isolated in Kazakhstan in 2002-2009

Although the important role of the non-structural (NSI and NEP) gene of influenza A in virulence of the virus is well established,our knowledge about the extent of variation in the NS gene pool of influenza A viruses in their natural reservoirs in Kazakhstan is incomplete.17 influenza A viruses of different subtypes were studied in this paper.Seven types of haemagglutinin and five different neuraminidase subtypes in eight combinations were found among the isolated viruses.A comparison of nucleotide sequences of isolated viruses revealed a substantial number of silent mutations,which results in high degree of homology in amino acid sequences.By phylogenetic analysis it was shown that two distinct gene pools,corresponding to both NS allele A with 5 Clades and B,were present at the same time in Kazakhstan.The degree of variation within the alleles was very low.In our study allele A viruses had a maximum of 5％ amino acid divergence in Clade while allele B viruses had only 4％ amino acid divergence.     

Long-term balancing selection at the west nile virus resistance gene, Oas1b, maintains transspecific polymorphisms in the house mouse

Oligoadenylate synthetases (OASs) are interferon-inducible enzymes that participate in the first line of defense against a wide range of viral infection. Recent studies have determined that Oas1b, a member of the OAS gene family in the house mouse (Mus musculus), provides specific protection against flavivirus infection (e.g., West Nile virus, dengue fever virus, and yellow fever virus). We characterized the nucleotide sequence variation in coding and noncoding regions of the Oas1b gene for a large number of wild-derived strains of M. musculus and related species. Our sequence analyses determined that this gene is one of the most polymorphic genes ever described in any mammal. The level of variation in noncoding regions of Oas1b is an order of magnitude higher than the level reported for other regions of the mouse genome and is significantly different from the level of intraspecific variation expected under neutrality. Furthermore, a phylogenetic analysis of intronic sequences demonstrated that Oas1b alleles are ancient and that their divergence predates several speciation events, resulting in transspecific polymorphisms. The amino acid sequence of Oas1b is also extremely variable, with 1 out of 7 amino acid positions being polymorphic within M. musculus. Oas1b alleles are comparatively more divergent at synonymous positions than most autosomal genes and the ratio of nonsynonymous to synonymous substitution is remarkably high, suggesting that positive selection has been acting on Oas1b. The ancestry of Oas1b polymorphisms and the high level of amino acid polymorphisms strongly suggest that the allelic variation at Oas1b has been maintained in mouse populations by long-term balancing selection.     

Nucleotide polymorphism at the xanthine dehydrogenase locus in Drosophila pseudoobscura.

Sequential polyacrylamide electrophoresis has revealed 20 allozymes of xanthine dehydrogenase (XDH) in Drosophila pseudoobscura. DNA sequence determination of seven isolates of the Xdh locus that represent six allozyme classes are presented here. Of the 5,456 sites examined, 180 are polymorphic, with 27 polymorphisms occurring at nonsynonymous, or replacement, sites. An average of nine amino acids differ between XDH allozyme classes, with 85% of the polymorphic amino acids singly represented. The level and pattern of variation observed at Xdh argue that the effective population size of the species is quite large--i.e., on the order of 2 x 10(6)--and that the populations sampled are quite ancient. In addition, as judged by two statistical tests, the levels of nucleotide polymorphism observed at Xdh are compatible with predictions from the neutral theory of molecular evolution.     

A double monoclonal IgG1 kappa and IgG2 kappa in a single myeloma patient. Variation in clonal products and therapeutic responses

Two electrophoretically homogeneous immunoglobulins were detected in the serum of a patient with multiple myeloma. The heavy chains were of different classes (gamma 1 and gamma 2). The light chains of both were kappa, but had different electrophoretic mobilities on polyacrylamide gels. Amino acid sequence analysis, carbohydrate determinations, and biosynthetic experiments indicated that the difference seen in their electrophoretic mobility was due to the glycosylation of one but not the other kappa-chain. The primary structure of both chains demonstrated that they both used a V kappa 1 and a J kappa 2 gene segment and the same C kappa allele, Km(1,2), and that both contained the same junctional three amino acid deletion. However, they varied by 19 amino acids in the first 94 amino terminal residues encoded for by the V kappa gene, with some of the substitutions requiring two base changes in the appropriate codons. Moreover, the malignant "clones" producing the two proteins differed in their responses to chemotherapy. These data indicate that, although the two clones producing the serum proteins were different at the time of study, they may have arisen from the same precursor clone.