Nucleotide Variation at the Gpdh Locus in the Genus Drosophila

The Gpdh locus was sequenced in a broad range of Drosophila species. In contrast to the extreme evolutionary constraint seen at the amino acid level, the synonymous sites evolve at rates comparable to those of other genes. Gpdh nucleotide sequences were used to infer a phylogenetic tree, and the relationships among the species of the obscura group were examined in detail. A survey of nucleotide polymorphism within D. pseudoobscura revealed no amino acid variation in this species. Applying a modified McDonald-Kreitman test, the amino acid divergence between species in the obscura group does not appear to be excessive, implying that drift is adequate to explain the patterns of amino acid change at this locus. In addition, the level of polymorphism at the Gpdh locus in D. pseudoobscura is comparable to that found at other loci, as determined by a Hudson-Kreitman-Aguade test. Thus, the pattern of nucleotide variation within and between species at the Gpdh locus is consistent with a neutral model.     

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 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.     

Extensive amino acid polymorphism at the pgm locus is consistent with adaptive protein evolution in Drosophila melanogaster

PGM plays a central role in the glycolytic pathway at the branch point leading to glycogen metabolism and is highly polymorphic in allozyme studies of many species. We have characterized the nucleotide diversity across the Pgm gene in Drosophila melanogaster and D. simulans to investigate the role that protein polymorphism plays at this crucial metabolic branch point shared with several other enzymes. Although D. melanogaster and D. simulans share common allozyme mobility alleles, we find these allozymes are the result of many different amino acid changes at the nucleotide level. In addition, specific allozyme classes within species contain several amino acid changes, which may explain the absence of latitudinal clines for PGM allozyme alleles, the lack of association of PGM allozymes with the cosmopolitan In(3L)P inversion, and the failure to detect differences between PGM allozymes in functional studies. We find a significant excess of amino acid polymorphisms within D. melanogaster when compared to the complete absence of fixed replacements with D. simulans. There is also strong linkage disequilibrium across the 2354 bp of the Pgm locus, which may be explained by a specific amino acid haplotype that is high in frequency yet contains an excess of singleton polymorphisms. Like G6pd, Pgm shows strong evidence for a branch point enzyme that exhibits adaptive protein evolution.     

中国人白细胞介素-12 cDNA基因的克隆及序列分析与比较

为研究中国人ＩＬ－１２的基因特征,采用逆转录巢式聚合酶链反应（ＲＴ－ｎＰＣＲ）从中国人脐带血单核细胞中分别克隆了Ｐ３５、Ｐ４０两亚基ｃＤＮＡ基因,包括完整的前体蛋白编码序列,其中Ｐ３５ ｃＤＮＡ编码２１９个氨基酸的多肽,Ｐ４０ ｃＤＮＡ编码３２８个氨基酸的多肽,与国外序列（ＮＫＳＦ、ＣＬＭＦ）比较结果发现：所克隆序列Ｐ３５同ＮＫＳＦ相比,第４４ａａ密友子由ＧＴＣ（Ｖａｌ）→ＧＴＧ（Ｖａｌ）,但未改     

Identification of the base-pair substitution responsible for a human acid alpha glucosidase allele with lower "affinity" for glycogen (GAA 2) and transient gene expression in deficient cells

The lysosomal enzyme termed acid alpha glucosidase (GAA), or acid maltase, is genetically polymorphic, with three alleles segregating in the normal population. The rarer GAA 2 allozyme has a lower affinity for glycogen and starch but not for lower-molecular-weight substrates. The GAA 2 allozyme can be detected by "affinity" electrophoresis in starch gel, since the lower affinity for the starch matrix results in a more rapid migration to the anode. Previously, we have isolated and sequenced the cDNA for GAA and transiently expressed the cDNA in deficient fibroblasts. In order to determine the molecular basis for the GAA 2 allozyme, we constructed a cDNA and a genomic DNA library from a GAA 2 cell line and determined the nucleotide sequence of the coding region. Only a single base-pair substitution of an A for a G at base-pair 271 was found, resulting in substitution of asparagine for aspartic acid at codon 91. This amino acid substitution is consistent with the more basic pI of the GAA 2 enzyme. The base-pair substitution also abolishes a Taq-I site, predicting the generation of a larger DNA fragment. This larger Taq-I fragment was also seen in two other individuals expressing the GAA 2 allozyme. A 5' fragment containing the base-pair substitution was ligated to the remaining 3' cDNA from a GAA 1 allele and cloned into an expression vector, and the hybrid cDNA was transiently expressed in SV40-transformed GAA-deficient fibroblasts. The enzyme activity exhibited the altered mobility of the GAA 2 allozyme, as demonstrated by electrophoresis in starch gel.(ABSTRACT TRUNCATED AT 250 WORDS)     

In vitro characterization of a human calcitonin receptor gene polymorphism

Calcitonin is a 32 amino acid peptide hormone that inhibits bone resorption by stimulating calcitonin receptors (CTR) located on the surfaces of osteoclasts. A polymorphism at nucleotide 1340 of the human calcitonin receptor gene (CALCR) lies within the coding region and has the potential to change the amino acid at codon 447 from leucine to proline. In the present study, we scanned the coding region, portions of the 5'-flanking and 3'-flanking sequences, and the intron-exon boundaries of the human CALCR gene for additional polymorphisms, and determined the frequency of the codon 447 polymorphism in several ethnic groups. Because a leucine to proline change has the potential for significant structural alteration, receptor genes encoding either leucine or proline at residue 447 were transiently expressed in COS-7 cells to determine the binding and functional consequences of this polymorphism. Our complete polymorphism scan of the CALCR gene identified 11 polymorphic sites in the gene and confirmed the presence of the previously identified nucleotide T1340C (codon 447) polymorphism. Ten of the 11 polymorphisms were single nucleotide polymorphisms (SNPs). For the codon 447 polymorphism, the prevalence of the TT genotype (leucine) was 59% in Caucasians, 27% in African-Americans, 0% in Asians, and 20% in Hispanics. The presence of this SNP appears to have no statistically significant difference with the receptor's ability to bind calcitonin or signal when activated with the hormone.     

Molecular typing of Toxoplasma gondii strains by GRA6 gene sequence analysis

The utility of sequence polymorphisms in the dense granule antigen GRA6 gene as typing markers for Toxoplasma gondii was investigated. The coding region of GRA6 was amplified, sequenced and compared for 30 Toxoplasma strains from eight different zymodemes (Z1-Z8). Sequence alignment identified nucleotide polymorphisms at 24 positions out of 690 bp, which correlated with murine-virulence. Types I, II, and III could be distinguished from each other on the basis of three, 10, and six variable positions, respectively. Two deletions of 15 bp and 3 bp existed in the avirulent (type II) strains. With one exception, all polymorphic positions resulted in amino acid substitutions, and the two gaps of 15 bp and 3 bp caused the deletion of six amino acids in type II strains. Intra-specific polymorphisms were also found in the virulent group. A high degree of sequence polymorphism correlating with the phenotypes of T. gondii strains points to the GRA6 gene being a good marker for strain characterisation and typing of the isolates of this apicomplexan. The large variety of amino acid changes supports the view that the GRA6 protein plays an important role in the antigenicity and pathogenicity of T. gondii. The existence of polymorphic restriction sites for endonuclease MseI was used to develop a PCR-RFLP method which could simply differentiate the three different groups (types I, II, III) of T. gondii.     

Genetic Variability of Flight Metabolism in DROSOPHILA MELANOGASTER . III. Effects of Gpdh Allozymes and Environmental Temperature on Power Output

The effect of allozyme variation at the sn-glycerol-3-phosphate dehydrogenase (Gpdh) locus on variation in the mechanical power output of the flight muscles of Drosophila melanogaster was investigated. The influence of different rearing and flight temperatures and of their interactions with the Gpdh allozymic genotypes (allotypes) on flight ability also were analyzed. Populations from three continents were used, and Gpdh allotypes were generated from crosses between randomly paired isofemale lines made autozygous for each of the two alleles by inbreeding. Measurements made during tethered flight, together with wing morphology, were used to estimate power output using both Weis-Fogh's and Ellington's formulas. Analyses of variance (ANOVA) indicated significant main effects for both environmental components (rearing and flight temperatures) but for only one of the three genetic components (genetic backgrounds within continent); Gpdh allotypes and populations (continent of origin) were not significant. The interaction between rearing and flight temperature was highly significant, indicating some physiological adaptation. The effect of Gpdh allozymes depended on both rearing and flight temperature and was either significant or marginally so, depending on which set of formulas was used. In either case, the S/S allotype showed a 2-4% greater power output than the F/F allotype at low temperature for both interactions. In addition, the S/S allotype showed significantly greater power output than the F/F allotype among flies raised at 15 degrees and flown at 15 degrees, whereas the reverse was true for flies raised at 30 degrees and flown at 30 degrees. Significant differences among the three allotypes for GPDH activity level were found in general, with S/S having the highest, F/S intermediate and F/F the lowest activity, and an inverse relationship existed between rearing temperature and activity. The temperature effects on power output are consistent with the geographical and seasonal variation observed at the Gpdh locus in nature. In general, the results show that Gpdh can be considered a minor polygene affecting quantitative variation in the power output during flight and that genotype-by-environment interaction is an important component of that effect.     

Triglyceride pools, flight and activity variation at the Gpdh locus in Drosophila melanogaster

We have created a set of P-element excision-derived Gpdh alleles that generate a range of GPDH activity phenotypes ranging from zero to full activity. By placing these synthetic alleles in isogenic backgrounds, we characterize the effects of minor and major activity variation on two different aspects of Gpdh function: the standing triglyceride pool and glycerol-3-phosphate shuttle-assisted flight. We observe small but statistically significant reductions in triglyceride content for adult Gpdh genotypes possessing 33-80% reductions from normal activity. These small differences scale to a notable proportion of the observed genetic variation in triglyceride content in natural populations. Using a tethered fly assay to assess flight metabolism, we observed that genotypes with 100 and 66% activity exhibited no significant difference in wing-beat frequency (WBF), while activity reductions from 60 to 10% showed statistically significant reductions of approximately 7% in WBF. These studies show that the molecular polymorphism associated with GPDH activity could be maintained in natural populations by selection in the triglyceride pool.     

Diversifying selection underlies the origin of allozyme polymorphism at the phosphoglucose isomerase locus in Tigriopus californicus

The marine copepod Tigriopus californicus lives in intertidal rock pools along the Pacific coast, where it exhibits strong, temporally stable population genetic structure. Previous allozyme surveys have found high frequency private alleles among neighboring subpopulations, indicating that there is limited genetic exchange between populations. Here we evaluate the factors responsible for the diversification and maintenance of alleles at the phosphoglucose isomerase (Pgi) locus by evaluating patterns of nucleotide variation underlying previously identified allozyme polymorphism. Copepods were sampled from eleven sites throughout California and Baja California, revealing deep genetic structure among populations as well as genetic variability within populations. Evidence of recombination is limited to the sample from Pescadero and there is no support for linkage disequilibrium across the Pgi locus. Neutrality tests and codon-based models of substitution suggest the action of natural selection due to elevated non-synonymous substitutions at a small number of sites in Pgi. Two sites are identified as the charge-changing residues underlying allozyme polymorphisms in T. californicus. A reanalysis of allozyme variation at several focal populations, spanning a period of 26 years and over 200 generations, shows that Pgi alleles are maintained without notable frequency changes. Our data suggest that diversifying selection accounted for the origin of Pgi allozymes, while McDonald-Kreitman tests and the temporal stability of private allozyme alleles suggests that balancing selection may be involved in the maintenance of amino acid polymorphisms within populations.     

Mining single nucleotide polymorphisms from EST data of silkworm, Bombyx mori, inbred strain Dazao

We made use of 81,635 expressed sequence tags (ESTs) derived from 12 different cDNA libraries of the silkworm, Bombyx mori, inbred strain Dazao (P50), to identify high-quality candidate single nucleotide polymorphisms (SNPs). By PHRAP assembling, 12,980 contigs containing 11,537 contigs assembled by more than one read were obtained, and 101 candidate SNPs and 27 single base insertions/deletions were identified from 117 contigs assembled from 1576 high-quality reads base-called with PHRED and screened on the basis of the neighborhood quality standard (NQS). Simultaneously, we also predicted 40 SNPs in coding regions (cSNPs), of which 26 were predicted to lead to amino acid non-synonymous variations and 14 synonymous substitutions. Also, the 1.66:1 ratio of transition/transversion is different from that of other insects. As the first SNP analysis of a Lepidoptera, B. mori, the single nucleotide polymorphic density is estimated to be 1.3 x 10(-3) by sequence diversity. This analysis shows that expressed sequences from multiple libraries may provide an abundant source of comparative reads to mine for cSNPs from the silkworm genome.     

中国部分地方鸡种B-LⅡβ(β_1外显子)基因分子遗传多态性研究

应用ＰＣＲ－ＲＦＬＰｓ方法对１０个鸡种的Ｂ－ＬⅡβ（β１外显子）基因进行分子遗传多态性研 究,综合４种限制性内切酶的酶切情况,共检测到３７种基因组合型,并且在个体间以及品种间 的基因型频率、基因组合型频率都存在较大的差异。同时各酶切位点的Ｈａｒｄｙ－Ｗｅｉｎｂｅｒｇ平衡状态在品种上也表现不一致。克隆测序结果表明：β１外显子分子遗传多态性更多地体现在氨 基酸水平,作为抗原结合区,其丰富的多态性与抗原多样性相一致。     

Spontaneous mutations affecting glycerol-3-phosphate dehydrogenase enzyme activity in Drosophila melanogaster.

Significant genetic variance in glycerol-3-phosphate dehydrogenase (GPDH) activity was observed between chromosome lines of Drosophila melanogaster that had each accumulated spontaneous mutations for approximately 300 generations. No restriction map variation was found in a 26-kb region surrounding the entire Gpdh gene. The restriction analysis used is capable of detecting insertions/deletions larger than 0.05 kb. The survey would also detect chromosomal recombinations that include the entire Gpdh coding region. Therefore, if the spontaneous mutations that affected the enzyme activity are located inside the Gpdh gene region, then they are base pair substitutions or structural changes that are smaller than the limit in resolution described above.     

苎麻细胞质雄性不育"三系"ISSR特异片段克隆和序列分析

利用ISSR分子标记技术对苎麻细胞质雄性不育"三系"mtDNA进行多态性分析;在选用的38个IS-SR引物中,有6个引物的扩增产物在不育系、保持系和恢复系之间存在差异。对这些特异性片段进行克隆和序列测定,结果表明:片段21-MS全长956bp,包含一个525bp的完整编码区,共编码174个氨基酸。片段31-M/R全长778bp,包含一个404bp的不完整编码区,共编码134个氨基酸;其核苷酸和氨基酸序列与已报道的多种植物中的番茄红素β-环化酶基因分别存在71～76和73～77的同源性。     

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.