Hidden Electrophoretic Variation at the Xanthine Dehydrogenase Locus in a Natural Population of DROSOPHILA MELANOGASTER

Sixty-two isochromosomal lines of D. melanogaster were screened for cryptic electrophoretic variation at the xanthine dehydrogenase (XDH) locus. Sequential polyacrylamide vertical slab gel electrophoresis was performed using four electrophoretic criteria. A total of 15 classes of electromorphs were revealed. D. melanogaster appears to exhibit as much polymorphism at this locus as other extensively studied Drosophila species.--No evidence for loci on the X or second chromosomes which modified XDH mobility was found. Six of the electromorphs were mapped to the Xdh (ry) structural locus. Eight of the remaining nine classes exhibited mobility variation consistent with structural variation at the Xdh locus. The final class exhibited aberrant patterns and is under further study.     

Exon variability of gene encoding glycerol-3-phosphate dehydrogenase of Ixodes ricinus ticks

We have previously found apparent differences in Gpdh allele frequences between borrelia infected and uninfected Ixodes ricinus as revealed by native gel electrophoresis of allozyme polymorphisms. The present study deals with the genetic basis of the observed allozyme polymorphism. Multiple sequence alignment of 36 Gpdh open reading frames identified a total of 40 polymorphic nucleotide sites. Of the 40 polymorphic nucleotide sites, 34 were silent (did not result in amino acid residue change), while six were active causing a change in the amino acid chain. All polymorphic amino acid sites were situated within the N-terminal NAD-binding domain, whereas the C-terminal substrate-binding domain was highly conserved. Analysis of the obtained Gpdh sequences and GPDH allozyme polymorphisms for individual ticks pointed to amino acid changes at positions 61 (glycine-to-glutamic acid), 64 (serine-to-cysteine) and 102 (glycine-to-arginine) as a key for differential mobility of GPDH allozymes in an electric field. Our findings are discussed in the context of the molecular basis of I. ricinus host finding behavior.     

Gametic associations between inversion and allozyme polymorphisms in Drosophila buzzatii.

Gametic disequilibria between second chromosome polymorphic arrangements and seven linked allozyme loci were estimated in seven populations of Drosophila buzzatii from Argentina. Significant and consistent associations across populations were detected for Est-1, Est-2, Aldox, and XDH: Phenograms based on Nei's genetic distance showed that chromosomes carrying the 2ST arrangement were more similar to each other, irrespective of the population from which they were extracted, than to chromosomes carrying the derived 2J and 2JZ3.Restriction of recombination in heterokaryotypes seems to be the best explanation for the significant linkage disequilibria between inversions and the loci located inside the rearranged segments, for example, Est-1 and Aldox, or close to the break points, for example, Est-2. However, epistatic interactions between Xdh, which is outside the inversions and not near the break points, and loci tightly linked to the inversions, is the most likely explanation for the association between Xdh and chromosomal arrangements. Some of the associations detected in endemic Argentinean populations are coincident with data obtained in colonizing populations of the Old World and Australia. Thus historical processes that took place in the original area of the species' distribution can account for these linkage disequilibria in colonized populations of D. buzzatii.     

Comparative molecular population genetics of the Xdh locus in the cactophilic sibling species Drosophila buzzatii and D. koepferae

The Xdh (rosy) gene is one of the best studied in the Drosophila genus from an evolutionary viewpoint. Here we analyze nucleotide variation in a 1875-bp fragment of the second exon of Xdh in Argentinian populations of the cactophilic D. buzzatii and its sibling D. koepferae. The major electrophoretic alleles of D. buzzatii not only lack diagnostic amino acids in the region studied but also differ on average from each other by four to 13 amino acid changes. Our data also suggest that D. buzzatii populations belonging to different phytogeographic regions are not genetically differentiated, whereas D. koepferae exhibits a significant pattern of population structure. The Xdh region studied is twice as polymorphic in D. buzzatii as in D. koepferae. Differences in historical population size or in recombinational environment between species could account for the differences in the level of nucleotide variation. In both species, the Xdh region exhibits a great number of singletons, which significantly departs from the frequency spectrum expected under neutrality for nonsynonymous sites and also for synonymous sites in D. buzzatii. These excesses of singletons could be the signature of a recent population expansion in D. buzzatii, whereas they may be simply explained as the result of negative selection in D. koepferae.     

Spatial and demographic population genetic structure in Catasetum viridiflavum across a human-disturbed habitat

Spatial and temporal genetic structures were examined across sites on islands and mainland (continuous forest) populations of an epiphytic orchid, Catasetum viridiflavum, using 17 polymorphic allozyme loci. I tested whether patches on islands or at mainland sites comprised small local populations or a large population. Low among population differentiation was observed across the landscape suggesting that the species-specific pollinator and tiny wind-dispersed seeds maintain interconnections among distant patches. Temporal genetic structure among stage classes, and among breeding individuals are important components of the maintenance of genetic variation in this orchid. The natural history of this species including small breeding populations, probable high frequency of mating among relatives, and the high rates of seed movement among sites contribute to the high FIS. These data show that physically isolated patches in this epiphytic orchid comprise a single larger genetic population, which is independent of the physical distances among sites. Although quite different in ecological and life history characteristics, the genetic structure of this orchid demonstrates a pattern similar to temperate and tropical trees in fragmented landscapes.     

Evidence for Linkage Disequilibrium Maintained by Selection in Two Natural Populations of DROSOPHILA SUBOBSCURA

One island and one mainland population of Drosophila subobscura were found polymorphic at the XDH (xanthine dehydrogenase) and the AO (aldehyde oxidase) loci. It was observed that one allele at the XDH locus, which has a low frequency in both populations, is nonrandomly associated with the alleles at the AO locus. Two lines of evidence support the thesis that this linkage disequilibrium is due to epistasis rather than random drift: (1) D or r, measures of the disequilibrium, have the same sign and magnitude in both populations. (2) The linkage disequilibrium is not due to inversions. Inversions segregating on the chromosome carrying XDH and AO have been separated into two classes, between which exchange of alleles at the two loci is suppressed. Linkage disequilibrium for XDH and AO was observed within each class. In the absence of any exchange of alleles, these disequilibria must have arisen and been maintained independently. The suggestion is made that the epistatic disequilibrium results from the close structural and physiological relationship which exists between the two enzymes.     

Nucleotide sequence of the Xdh region in Drosophila pseudoobscura and an analysis of the evolution of synonymous codons

The nucleotide sequence of the Xdh region of Drosophila pseudoobscura is presented. The Xdh gene structure and organization are compared with the homologous region in D. melanogaster. This locus is shown to have similar organization in the two species, although an additional intron and three insertion/deletion events are described for the D. pseudoobscura coding region. The encoded proteins are predicted to have very similar charges and hydrophobic/hydrophilic domains even though 11% of the amino acids are different. A gene 5' to Xdh, putative l(3)s12, is suggested from sequence similarity between the species. Synonymous differences at the Xdh locus between the two species are analyzed using a new method described in the preceding paper by Lewontin. This analysis shows that synonymous positions within the Xdh locus are evolving at very different rates, being dependent on level of codon redundancy. A comparison of synonymous divergence between D. melanogaster and D. pseudoobscura in five additional genes reveals variation in the level of synonymous substitution.      

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.     

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ISSR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956 bp , 包含一个525 bp 的完整编码区, 共编码174 个氨基酸。片段31-M􊄯R 全长778 bp , 包含一个404 bp 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。     

Context-Dependent Survival Differences among Electrophoretic Genotypes in Natural Populations of the Marine Bivalve Spisula Ovalis

We investigated the relationships between allozyme genotypes at nine polymorphic loci and survival in a natural population of the bivalve Spisula ovalis sampled on three occasions (1993, 1994, and 1995) in three different sites (2855 individuals analyzed). This species displays annual growth lines allowing identification of annual cohorts. Therefore we could avoid cohort mixing, a frequent bias in such studies, and evaluate the consistency of the observed effects across cohorts and sites. Significant viability differences were observed both among alleles and between heterozygotes and homozygotes at some loci. Multiple-locus heterozygosity was positively correlated with viability in the 1993-1994 period, but not in the 1994-1995 interval. The observed selective effects were significantly dependent on the cohort and the site considered. A bibliographic survey suggests that such variability is a common feature of studies analyzing heterozygosity-survival relationships. Two explanations are consistent with our results. First, allozyme genotypes may have direct effects on viability that interact with subtle environmental variation in a complex and unpredictable way. Second, allozyme genotypes may be transiently associated with other viability genes responsible for heterotic effects. In any case, the results militate against allozyme loci being themselves consistently overdominant for viability in natural populations.     

Gluconobacter oxydans木糖醇脱氢酶基因的克隆表达及木糖醇的转化分析

从氧化葡萄糖酸杆菌(Gluconobacter oxydans)的基因组DNA上扩增出木糖醇脱氢酶基因xdh,构建了诱导型表达载体pSE-xdh,导入E.coli JM109后获得了高效表达木糖醇脱氢酶基因的重组菌JM109/pSE-xdh。通过HisTrap HP亲和层析和SephacrylS 300分子筛两步纯化从细胞中得到纯酶,并对酶学性质进行研究。XDH最适还原反应的pH值为5.0,最适还原反应的温度为35℃;最适氧化反应的pH值为11.0,最适氧化反应的温度为30℃。重组菌中的XDH依赖NADH,对NADH的米氏常数Km=57.8 mmol/L,最大反应速率Vmax=1209.1 mmol/(ml·min)。重组菌的XDH酶活力为13.9 U/mg。利用重组菌和原始菌混合静止细胞转化D 木酮糖,16 h 28.0 g/L D木酮糖生成16.7 g/L木糖醇,而原始菌单独转化只生成8.3 g/L木糖醇。     

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。     

Cloning and Tissue Expression of 6-Pyruvoyl Tetrahydropterin Synthase and Xanthine Dehydrogenase from <Emphasis Type="Italic">Poecilia reticulata</Emphasis>

Guppy is a popular ornamental fish owing to its diverse body and fin coloration. More than 40 established color varieties have been selectively bred. The complementary DNAs for 2 enzymes that are involved in the de novo synthesis of pteridines and purines, which are important for the production of color pigments, were cloned from the caudal fin. Two cDNA isoforms for 6-pyruvoyl tetrahydropterin synthase (PTPS), with an open reading frame of 130 and 147 amino acids, respectively, were cloned from the Red Tail variety. The deduced amino acid sequence of the longer isoform shows an overall identity of about 65% to the mammalian PTPS sequences. The cDNA for xanthine dehydrogenase (XDH) was cloned from the Yellow Tail variety, and consists of an open reading frame of 1331 amino acids. Although it shows a higher overall identity to bovine aldehyde oxidase (AO; 54%) than to chicken XDH (51%), it has a NAD-binding domain that is specific to XDHs. Northern blot analysis indicated that both PTPS and XDH messenger RNAs were highly expressed in the liver, but absent in the muscle. In the caudal fins, guppy varieties with a higher proportion of xanthophores and erythrophores showed higher expression of PTPS, while XDH mRNA levels were too low to indicate obvious differential expression among the color guppy varieties. The results implied that high expression of PTPS is correlated with the biosynthesis of pteridines in the erythrophores and xanthophores, while the association between the putative guppy XDH with specific chromatophores is less clear.     

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.     

Cloning and partial characterization of the xanthine dehydrogenase gene of Calliphora vicina, a distant relative of Drosophila melanogaster

In vitro enzymatic assays have shown that an enzyme with typical xanthine dehydrogenase (XDH) activities and electrophoretic mobility slightly different from that of Drosophila XDH is present in Calliphora tissues. A Calliphora genomic sequence has been isolated by low-stringency hybridization to the Drosophila rosy gene (XDH), and partially sequenced. This sequence has been shown to be unique, polymorphic, and it maps on chromosome I. Sequence comparisons provide compelling evidence that it belongs to the XDH gene of Calliphora. Interspecies transformation experiments, aimed at investigating functional as well as structural divergence of the XDH genes of Calliphora and Drosophila, are now possible.     

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.     

GEOGRAPHIC DISTRIBUTION OF MOLECULAR VARIANCE WITHIN THE BLUE MARLIN (MAKAIRA NIGRICANS): A HIERARCHICAL ANALYSIS OF ALLOZYME,SINGLE-COPY NUCLEAR DNA,AND MITOCHONDRIAL DNA MARKERS

This study presents a comparative hierarchical analysis of variance applied to three classes of molecular markers within the blue marlin (Makaira nigricans). Results are reported from analyses of four polymorphic allozyme loci, four polymorphic anonymously chosen single-copy nuclear DNA (scnDNA) loci, and previously reported restriction fragment length polymorphisms (RFLPs) of mitochondrial DNA (mtDNA). Samples were collected within and among the Atlantic and Pacific Oceans over a period of several years. Although moderate levels of genetic variation were detected at both polymorphic allozyme (H = 0.30) and scnDNA loci (H = 0.37), mtDNA markers were much more diverse (h = 0.85). Allele frequencies were significantly different between Atlantic and Pacific Ocean samples at three of four allozyme loci and three of four scnDNA loci. Estimates of allozyme genetic differentiation (θ_O) ranged from 0.00 to 0.15, with a mean of 0.08. The θ_O values for scnDNA loci were similar to those of allozymes, ranging from 0.00 to 0.12 with a mean of 0.09. MtDNA RFLP divergence between oceans (θ_O = 0.39) was significantly greater than divergence detected at nuclear loci (95% nuclear confidence interval = 0.04–0.11). The fourfold smaller effective population size of mtDNA and male-mediated gene flow may account for the difference observed between nuclear and mitochondrial divergence estimates.     

Regulation of nitrogen catabolic enzymes in chick liver: effects of insulin.

Previous studies have shown that a group of nitrogen catabolic enzymes including xanthine dehydrogenase, purine nucleoside phosphorylase, and tyrosine aminotransferase are all increased in chick liver by dietary protein as well as single amino acids (e.g. methionine) and certain antimetabolites (e.g. hydrazine). A similar enzyme response pattern can be obtained with insulin. This hormone causes an enhanced rate of XDH synthesis and gives nonadditive results with protein, hydrazine and methionine. Furthermore, a vitamin B6 dependency was observed in responses to both high protein diets and insulin, all suggesting a common regulatory mechanism. In this system dietary protein and insulin may act similarly by increasing the availability of amino acids to the liver -- in one case by supplying amino acids through the diet and in the other by increasing amino acid uptake.     

Extensive clonality in the endangered shrub Haloragodendron lucasii (Haloragaceae) revealed by allozymes and RAPDs

The occurrence of clonality in threatened plants can have important implications for their conservation. In this study, allozymes and RAPDs were used to determine the extent of clonality in the endangered shrub Haloragodendron lucasii (Haloragaceae), which is known from only four sites within an 8 km range. Allozyme markers identified only six multilocus genotypes among the 53 ramets sampled across the four sites, although a total of 54 different genotypes were possible with the three polymorphic allozyme loci detected. The polymorphic bands detected in the RAPD analysis were capable of producing 2⁴⁶ genotypes, but again only six multilocus genotypes were delineated. The allozyme and RAPD data were congruent at three of the four sites. At the fourth site two genotypes were detected by each marker; however, once combined, three multilocus genotypes were observed. The probabilities that the observed number of replicates of each combined allozyme and RAPD genotype could be generated by sexual reproduction were less than 10^–18, leaving little doubt that clonality is the explanation for the observed patterns of genotypes. The genetic conclusions are supported by root excavations which show potential for vegetative reproduction and the observation of no sexual reproduction in the species. The recognition of extensive clonality in H. lucasii has had immediate implications for the conservation management of the species and resulted in changes to the management priorities for the species. Thus it is clear that appropriate genetic studies can play an important role in the management of threatened species.