基于ND4基因部分片段探讨中国4个家驴品种的母系构成

为了分析中国家驴的遗传多样性,对保种和开发利用这一固有遗传资源提供有益帮助,并对其母性起源提供一些基础资料,作者对我国4个家驴品种(德州、凉州、云南、蒙古)209个个体的mtDNAND4基因编码区409bp片段进行了扩增、单链构象多态(SSCP)检测与测序分析,共检测到5种单倍型8个多态位点,其单倍型多样度为0.4699,核苷酸多样度为0.00308,表明我国家驴的遗传多态性比较丰富。通过对各单倍型序列按照脊椎动物线粒体编码规则翻译成氨基酸序列进行比对发现,部分核苷酸变异引起了氨基酸的变异。构建的简化加权中值网络聚类图显示,4个家驴品种的样品来自两个母系源头,并且,4个中国家驴品种都是由这两个母性世系混杂而成的,即未发现由同一种世系构成的品种。这与以前基于D-loop区序列对于中国家驴的研究结果相似,即地理位置、世系构成以及母性起源之间没有明显的相互关系。     

Compensated deleterious mutations in insect genomes

Relatively little is known about the importance of amino acid interactions in protein and phenotypic evolution. Here we examine whether mutations that are pathogenic in Drosophila melanogaster become fixed via epistasis in other Dipteran genomes. Overall divergence at pathogenic amino acid sites is reduced. However, approximately 10% of the substitutions at these sites carry the exact same pathogenic amino acid found in D. melanogaster mutants. Hence compensatory mutation(s) must have evolved. Surprisingly, the fraction 10% is not affected by phylogenetic distance. These results support a selection-driven process that allows compensated amino acid substitutions to become rapidly fixed in taxa with large populations.     

Transitions to asexuality result in excess amino acid substitutions

Theory predicts that linkage between genetic loci reduces the efficiency of purifying selection. Because of the permanent linkage of all heritable genetic material, asexual lineages may be exceptionally prone to deleterious-mutation accumulation in both nuclear and organelle genes. Here, we show that the ratio of the rate of amino acid to silent substitution (Ka/Ks) in mitochondrial protein-coding genes is higher in obligately asexual lineages than in sexual lineages of the microcrustacean Daphnia pulex. Using a phylogeny-based approach to quantify the frequency of mutational-effect classes, we estimate that mitochondrial protein-coding genes in asexual lineages accumulate deleterious amino acid substitutions at four times the rate in sexual lineages. These results support the hypothesis that sexual reproduction plays a prominent role in reducing the mutational burden in populations.     

Replacements of Pro86 in phage T4 lysozyme extend an alpha-helix but do not alter protein stability

To investigate the relation between protein stability and the predicted stabilities of individual secondary structural elements, residue Pro86 in an alpha-helix in phage T4 lysozyme was replaced by ten different amino acids. The x-ray crystal structures of seven of the mutant lysozymes were determined at high resolution. In each case, replacement of the proline resulted in the formation of an extended alpha-helix. This involves a large conformational change in residues 81 to 83 and smaller shifts that extend 20 angstroms across the protein surface. Unexpectedly, all ten amino acid substitutions marginally reduce protein thermostability. This insensitivity of stability to the amino acid at position 86 is not simply explained by statistical and thermodynamic criteria for helical propensity. The observed conformational changes illustrate a general mechanism by which proteins can tolerate mutations.     

Amino acid composition of proteins as a product of molecular evolution

The average amino acid composition of proteins is determined by the genetic code and by random base changes in evolution. Small but significant deviations from expected composition can be explained by selective constraint on amino acid substitutions. In particular, the deficiency of arginine in proteins has been caused by constraint, during evolution, on fixation of mutations substituting arginine for other amino acids.     

植物病原真菌对几类重要单位点杀菌剂的抗药性分子机制

单位点杀菌剂是植物病害管理的重要组成部分,随着单位点杀菌剂的大量、广泛使用,抗性问题也随之产生。目前为止,有植物病原菌对各大类单位点杀菌剂均具抗性的报道。本文作者主要阐述了生产中常用的5类单位点杀菌剂,包括苯并咪唑类杀菌剂（MBCs）、二甲酰亚胺类杀菌剂（DCFs）、14α-脱甲基酶抑制剂（DMIs）、QoIs和琥珀酸脱氢酶抑制剂（SDHIs）的作用机理及抗性分子机制的研究进展,并进一步论述了抗药性产生的机理及抗性治理原则。MBCs作用于β-微管蛋白,抗性主要与靶标蛋白基因的点突变有关,突变造成的氨基酸变化多集中于第50、167、198、200和240等5个位置,主要突变位点为第198位,同一菌株通常只发生一个氨基酸变异,不同位点的点突变甚至同一位点的不同氨基酸替代均会引起抗性水平的差异;DCFs的作用靶标尚不清楚,病原真菌对其抗性可能与双元组氨酸激酶OS基因的点突变有关;DMIs通过抑制14α-脱甲基酶最终影响麦角甾醇的合成,抗性主要与Cyp51的点突变或过量表达或运输体的过量表达相关,Cyp51点突变是抗DMI的主要机制,同一突变对不同的三唑类杀菌剂敏感性表现不尽相同,不同位置的点突变在同一病原菌中对不同三唑类杀菌剂的敏感性影响也不同。点突变数量在不同的真菌中表现不同,有单个发生,也有多个同时发生,且对抗药性具有积累效应;QoIs作用于电子传递链的复合物III,抗性主要与Cytb的点突变有关,与抗性相关的点突变主要发生在Cytb的120-155和255-280两个编码区,其中G143A和F129L为最主要的点突变;SDHIs作用于电子传递链的复合物II,抗性主要与SdhB、SdhC或SdhD的点突变有关,大部分病原真菌对SDHIs的抗性与SdhB点突变有关,SdhB点突变发生位置比较单一,在多种病原菌中突变均发生在相同的组氨酸上即H272, 而SdhC和SdhD突变位点比较多。     

养殖场分离的耐氟喹诺酮类药物的大肠杆菌基因突变研究

【目的】探讨从养殖场动物、环境和饲养员分离的大肠杆菌的gyrA 和parC 基因突变特征。【方法】用琼脂稀释法测定环丙沙星和恩诺沙星对菌株的最小抑菌浓度。PCR扩增gyrA 和parC 基因的喹诺酮耐药决定区，扩增的片段长度分别为525 bp和487 bp，PCR产物直接测序。【结果】在63株突变株中，在GyrA 亚基发生的氨基酸替代有Ser83→Leu（62株）和Asp87→Asn（52株）、Asp87→Tyr（2株）、Asp87→His（2株）；ParC 亚基的氨基酸替代有Ser80→Ile（47株）、Ser80→Arg（2株）和Glu84→Val（3株）、Glu84→Lys（4株）、Glu84→Gly（5株）、Glu84→Ala（1株）。环丙沙星对菌株的MIC小于0.125μg·ml-1时，GyrA和ParC亚基均没有任何变异；环丙沙星的MIC为0.125～0.25 μg·ml-1时，GyrA亚基出现单一氨基酸替代；环丙沙星的MIC为0.5～32μg·ml-1时，出现GyrA 83位和87位双替代或者GyrA83和ParC80位双替代；环丙沙星的MIC为4～128μg·ml-1，发生GyrA 双替代和ParC单替代；环丙沙星的MIC在16～128μg·ml-1，发生GyrA双替代和ParC 双替代。【结论】不同来源的耐氟喹诺酮类药物的大肠杆菌GyrA和ParC具有多种氨基酸替代类型，而且GyrA和ParC突变位点的数量与菌株对氟喹诺酮类耐药水平呈正相关。     

Comment on "Transitions to asexuality result in excess amino acid substitutions"

Paland and Lynch (Reports, 17 February 2006, p. 990) showed that in Daphnia pulex, the ratio of amino acid replacement to silent substitution in mitochondrial genes is higher in asexual lineages than in sexual lineages. If this base-composition bias is maintained by selection, it too should alter following transitions in reproductive mode. Analysis reveals no such change in the genomes of D. pulex.     

Classic selective sweeps were rare in recent human evolution

Efforts to identify the genetic basis of human adaptations from polymorphism data have sought footprints of "classic selective sweeps" (in which a beneficial mutation arises and rapidly fixes in the population).Yet it remains unknown whether this form of natural selection was common in our evolution. We examined the evidence for classic sweeps in resequencing data from 179 human genomes. As expected under a recurrent-sweep model, we found that diversity levels decrease near exons and conserved noncoding regions. In contrast to expectation, however, the trough in diversity around human-specific amino acid substitutions is no more pronounced than around synonymous substitutions. Moreover, relative to the genome background, amino acid and putative regulatory sites are not significantly enriched in alleles that are highly differentiated between populations. These findings indicate that classic sweeps were not a dominant mode of human adaptation over the past ~250,000 years.     

Target-selected inactivation of the zebrafish rag1 gene

The zebrafish has become a favorite organism for genetic analysis of vertebrate development, but methods for generating mutants by reverse genetic approaches have been lacking. We report a method to obtain stable mutants of a gene based on knowledge of the gene sequence only. Parental fish were mutagenized with N-ethyl-N-nitrosourea; in 2679 F1 fish, the rag1 gene was analyzed for heterozygous mutations by resequencing. In total, we found 15 mutations: 9 resulted in amino acid substitutions and 1 resulted in a premature stop codon. This truncation mutant was found to be homozygous viable and defective in V(D)J joining. Although presumably immune deficient, these homozygous rag1 mutant fish are able to reach adulthood and are fertile. As sperm samples from all 2679 F1 fish were collected and cryopreserved, we have in principle generated a mutant library from which mutants of most zebrafish genes can be isolated.     

马麝朊蛋白(PrP)基因的克隆和序列分析

从马麝的全血中提取基因组总DNA,用所设计引物以聚合酶链式反应扩增出细胞型朊蛋白(PrPC)基因,并克隆到pMD18-T载体。序列分析表明所克隆的马麝PrP基因片段大约为771bp,包含了朊蛋白基因的完整编码区序列,即包含在单一外显子内的完整开放阅读框,与国外报道的同科动物PrP基因序列基本相同。马麝PrP基因含5个短而富含G-C的元件,可编码5个八肽(九肽)重复Pro-His-Gly-Gly-Gly-Trp-Gly-Gln或Pro-Gln/His-Gly-Ala/Gly-Gly–Gly-Trp-Gly-Gln,其氨基酸序列含有24个氨基酸的N-端信号肽和23个氨基酸的C-端信号肽。与白尾鹿(Odocoileusvirginianus)和麋鹿(Cervuselaphus)的PrP基因相比,其核苷酸序列和推导氨基酸序列同源性分别为97.4%、97.9%和98.1%、97.7%。共发生15个碱基替换,其中10个为同义码替换,5个为异义突变,即G57A、S100N、N173S、T177N和M208I。     

Fitting discrete probability distributions to evolutionary events

The assumptions underlying the use of the Poisson distribution are essentially that the probability of an event is small but nearly identical for all occurrences and that the occurrence of an event does not alter the probability of recurrence of such events. These assumptions do not seem to be met for evolutionary events since (i) the probability of fixing nucleotide codon substitutions is not equal for all substitutions at a codon, and probably varies for the same substitution in different lineages; (ii) the probability of fixing codon substitutions varies among positions of a cistron; and (iii) the fixation of a nucleotide codon substitution at one position in a cistron modifies, and may even promote, the fixation of a codon substitution elsewhere along the cistron. Natural selection presumably is the causative factor that acts to modify the probability of a nucleotide codon substitution's being fixed in a population. The use of the negative binomial distribution is consistent with the evidence that selective pressure on amino acid or nucleotide codon positions varies both among codon positions of a cistron and at a particular position during evolutionary time. If the number of fixations of nucleotide codon substitutions per position of cistrons encoding cytochromes c are phyletically inferred (phylogeny based on a paleontological record) rather than phenetically inferred (based on paired comparisons of extant species' differences in the absence of a phylogeny) the distribution of these fixation data cannot be described adequately by a single Poisson distribution. The fit of these same data to a negative binomial distribution is very satisfactory. It has been argued that the fit of phenetically inferred fixation data, which do not take account of parallel or reverse fixations, to the Poisson distribution was supportive evidence for the hypothesis that protein evolution results from the fixation of selectively neutral codon substitutions. This argument now appears to be undercut by the evidence that data on nucleotide codon fixation are more probably distributed according to the negative binomial distribution. The fact that fixation data can be described by a particular discrete probability distribution does not of itself provide insight into the mechanisms of the evolutionary process. However, the facts-(i) that the assumptions underlying the use of the negative binomial distribution adequately deal with the varying probability of fixing amino acid or nucleotide codon substitutions at and among the positions of a cistron and (ii) that the negative binomial distribution provides an excellent fit for the phyletically inferred fixation data-suggest that the negative binomial is a very appropriate discrete probability distribution for describing evolutionary events. Amino acids or their nucleotide codon substitutions may be fixed at a position of a cistron as though selectively neutral relative to the codon being replaced, even though the codon position will not be selectively neutral, since many amino acids cannot function there. The negative binomial distribution treats this situation well whereas a single Poisson distribution could only be satisfactory if all codon positions that could vary were selectively neutral.     

H1N1亚型猪流感病毒HA蛋白225位氨基酸对病毒致病性的影响

[目的]流感病毒的致病性是由多个基因共同决定的,笔者之前的研究结果发现当两株具有相似基因特征的H1N1亚型猪流感病毒进行HA基因替换以后,病毒对小鼠的致病性发生了改变.通过确定影响病毒致病性的关键氨基酸位点,为进一步揭示流感病毒致病力差异奠定了基础.[方法]对ZD71和SY130的HA蛋白氨基酸序列进行比对,确认氨基酸...     

ADD1基因PCR-SSCPs标记与猪肌内脂肪含量及背膘厚的关系

采用PCR SSCPs方法在苏太猪脂肪细胞定向分化因子 1(ADD1)基因第 347和 374位点发现单核苷酸多态性(SNP) ,分别为ADD1第 90和 99位氨基酸密码子的第 3位碱基 ,但这两个位点碱基的替换均没有引起氨基酸序列的变化。通过对 10 0头苏太猪肥育猪背最长肌中肌内脂肪含量的相关分析 ,发现杂合子AB肌内脂肪含量最高 ,极显著地高于AA纯合子 (P <0 0 1) ,BB纯合子肌内脂肪含量介于AB和AA之间 ,并有高于AA纯合子的趋势 (P =0 11)。各基因型间背膘厚没有显著差异。提示ADD1基因该位点上的PCR SSCPs可能作为选择肌内脂肪含量 ,而不影响背膘厚的一个辅助选择标记     

Genetic diversity and differentiation of four goat lineages based on analysis of complete mtDNA d-loop

The complete sequences of mtDNA D-loops from 362 individuals were analyzed in order to investigate the genetic diversity and differentiation of their lineages. The results indicated that all of the analyzed sequences were differentiated into four clear lineages (A, B, C, and D). Lineages C and D might originate from Lineages B and A, respectively. The genetic diversity of complete mtDNA D-loop of four lineages was very abundant. The 76 bp insertion and the 17 bp deletion were detected in the longest and the shortest sequences, respectively. The 76 bp insertion was a repeat like motif found in many other animals. Lineages C and D were differentiated into two subclades (C1 and C2) and (D1 and D2), respectively. Lineage C might originate from Asia, and Lineage D might originate from Fertile Crescent.     

Spectral tuning of pigments underlying red-green color vision

Variations in the absorption spectra of cone photopigments over the spectral range of about 530 to 562 nanometers are a principal cause of individual differences in human color vision and of differences in color vision within and across other primates. To study the molecular basis of these variations, nucleotide sequences were determined for eight primate photopigment genes. The spectral peaks of the pigments specified by these genes spanned the range from 530 to 562 nanometers. Comparisons of the deduced amino acid sequences of these eight pigments suggest that three amino acid substitutions produce the approximately 30-nanometer difference in spectral peaks of the pigments underlying human red-green color vision, and red shifts of specific magnitudes are produced by replacement of nonpolar with hydroxyl-bearing amino acids at each of the three critical positions.     

人工诱导猪链球菌氟喹诺酮耐药株的靶位突变分析

以4株临床分离的对环丙沙星和恩诺沙星敏感的猪链球菌2型菌株为研究对象,采用体外递增药物浓度的方法分别诱导了其对环丙沙星和恩诺沙星耐药的菌株,按CLSI推荐方法测定了环丙沙星和恩诺沙星对亲本敏感株和诱导耐药株的MIC,测定了亲本株和诱导耐药株的生长曲线,并采用PCR和基因测序的方法分析了诱导耐药株的DNA回旋酶(GyrA和GyrB)和拓扑异构酶Ⅳ(ParC和ParE)耐药决定区(QRDR)的基因突变和氨基酸序列变化.结果表明:浓度递增法成功诱导了猪链球菌对环丙沙星和恩诺沙星耐药性,其MIC分别由05 mg·L-1上升至128 mg·L-1;与敏感株比较,恩诺沙星与环丙沙星诱导的耐药菌在gyrA和gyrB,或parC和parE耐药决定区的氨基酸序列有突变,除了已报道的与氟喹诺酮耐药相关的ParC的Ser79Phe,GyrA的Ser81Arg,GyrB的Asp315Asn、Ser285Leu和Glu354Lys及ParE的Pro278Ser点突变外,在诱导菌中还出现了一些不曾报道的突变位点和氨基酸缺失,如GyrA的Gln118His和ParE的Asn297Tyr突变,GyrB的288～291位和ParC的62位氨基酸缺失.结果提示:逐步增加药物浓度可以诱导猪链球菌对氟喹诺酮类抗菌药耐药性,并导致主要靶位发生突变.     

Predicting the evolution of human influenza A

Eighteen codons in the HA1 domain of the hemagglutinin genes of human influenza A subtype H3 appear to be under positive selection to change the amino acid they encode. Retrospective tests show that viral lineages undergoing the greatest number of mutations in the positively selected codons were the progenitors of future H3 lineages in 9 of 11 recent influenza seasons. Codons under positive selection were associated with antibody combining site A or B or the sialic acid receptor binding site. However, not all codons in these sites had predictive value. Monitoring new H3 isolates for additional changes in positively selected codons might help identify the most fit extant viral strains that arise during antigenic drift.     

Identification of p53 gene mutations in bladder cancers and urine samples

Although bladder cancers are very common, little is known about their molecular pathogenesis. In this study, invasive bladder cancers were evaluated for the presence of gene mutations in the p53 suppressor gene. Of 18 tumors evaluated, 11 (61 percent) were found to have genetic alterations of p53. The alterations included ten point mutations resulting in single amino acid substitutions, and one 24-base pair deletion. In all but one case, the mutations were associated with chromosome 17p allelic deletions, leaving the cells with only mutant forms of the p53 gene products. Through the use of the polymerase chain reaction and oligomer-specific hybridization, p53 mutations were identified in 1 to 7 percent of the cells within the urine sediment of each of three patients tested. The p53 mutations are the first genetic alterations demonstrated to occur in a high proportion of primary invasive bladder cancers. Detection of such mutations ex vivo has clinical implications for monitoring individuals whose tumor cells are shed extracorporeally.     

ROS-generating mitochondrial DNA mutations can regulate tumor cell metastasis

Mutations in mitochondrial DNA (mtDNA) occur at high frequency in human tumors, but whether these mutations alter tumor cell behavior has been unclear. We used cytoplasmic hybrid (cybrid) technology to replace the endogenous mtDNA in a mouse tumor cell line that was poorly metastatic with mtDNA from a cell line that was highly metastatic, and vice versa. Using assays of metastasis in mice, we found that the recipient tumor cells acquired the metastatic potential of the transferred mtDNA. The mtDNA conferring high metastatic potential contained G13997A and 13885insC mutations in the gene encoding NADH (reduced form of nicotinamide adenine dinucleotide) dehydrogenase subunit 6 (ND6). These mutations produced a deficiency in respiratory complex I activity and were associated with overproduction of reactive oxygen species (ROS). Pretreatment of the highly metastatic tumor cells with ROS scavengers suppressed their metastatic potential in mice. These results indicate that mtDNA mutations can contribute to tumor progression by enhancing the metastatic potential of tumor cells.