藏系绵羊ZFX/ZFY基因片段进化分析

目的:获得X染色体、Y染色体在进化上是独立进行的依据.方法:用PCR方法扩增并克隆藏系绵羊ZFX/ZFY基因片段并测序,对ZFX/ZFY基因片段进行了序列比对.结果:扩增的ZFX/ZFY基因片段长度为447bp,该对基因比对结果是总的同源相似率为80.76%,ZFY基因的同源性在96.4～99.3%之间,而ZFY基因的同源性在95.3～98.4%之间;在相应148个氨基酸中,ZFX基因的氨基酸差异为2个,而ZFY基因的氨基酸差异为12个.结论:在进化过程中ZFY基因比ZFX基因更活跃,而ZFX基因较为保守,为研究X染色体、Y染色体在进化上的差异提供参考.     

牦牛与其他物种ZFX/ZFY基因片段间的进化关系

利用PCR扩增、克隆和序列分析法对牦牛ZFX/ZFY基因第11外显子部分片段进行了研究,并同来自于NCBI GenBank中人、猩猩、普通牛等9个物种的ZFX/ZFY基因核苷酸及其氨基酸序列进行了进化分析.结果表明,牦牛ZFX、ZFY基因间核苷酸序列同源性为94.1%,显示同一物种同源基因ZFX/ZFY间存在变异;比较的10个物种间ZFX基因核苷酸序列同源性为87.7%、ZFY基因为81.7%,相应ZFX、ZFY氨基酸同源性分别为96.6%、91.0%,ZFY基因的变异性大于ZFX基因,显示X染色体与Y染色体可能是独立进化.     

1株梅花鹿源狂犬病街毒株全基因组测序与分析

对1株梅花鹿源性狂犬病街毒株(DRV)进行全基因组克隆,对全长cDNA进行测序分析.RT-PCR扩增克隆覆盖全基因组9个重叠基因片段,基因组3'和5'末端采取3,-RACE和5'-RACE方法,9个重叠基因片段序列拼接得到DRV全基因组cDNA序列,共11 863个核苷酸.DRV毒株全基因组构成与其他狂犬病毒基因组构成相似,由5个编码区组成,基因起始位点和终止位点高度保守,在核蛋白和糖蛋白的重要抗原位点有个别氨基酸发生变异,对已完成全基因组测序的几个基因1型毒株分别进行了N、P、M、G、L基因核苷酸及氨基酸的同源性比较.与其他具有代表性的毒株进行N基因序列比较建立的系统进化树表明,DRV毒株属于基因1型,与中国人用疫苗株3aG同源性最高为94%,与分类位置未确定的北高加索毒株(WCBV)的同源性最低为71%.本研究结果可为狂犬病毒各项分子生物学研究提供理论参考.     

东方粘虫颗粒体病毒超氧化物歧化酶基因的克隆与分析

为获得东方粘虫颗粒体病毒(Pseudelatia separata granulovirus,PsGV)基因组序列,采用随机克隆方法,建立PsGV的质粒基因文库,并通过对插入片段进行克隆鉴定和序列分析,获得编码超氧化物歧化酶蛋白的基因(PsGV-sod)。该基因阅读框为462bp,共编码153个氨基酸。核苷酸和氨基酸同源性比较结果表明该基因与其他颗粒体病毒同源性较高,通过保守基序分析,认为其为铜锌超氧化物歧化酶。     

枳壳CBF转录激活因子基因片段的克隆

根据不同植物CBF同源基因的保守区设计合成简并引物,采用PCR技术首次从枳壳基因组中分离出一个DNA片段并克隆到pMD18-T载体中.序列测定和分析表明,该片段长464bp,与拟南芥3个CBF基因的核酸序列及其推导的氨基酸序列分别具有79%和67%～69%的同源性,而且推导的氨基酸序列含有同源性更高的AP2DNA结合域和CBF蛋白的两段特征序列PKK/RPAGRxKFxETRHP和DSAWR.结果 表明,本研究克隆的片段为枳壳CBF基因片段.     

J亚群禽白血病病毒蛋鸡分离株SD07LK1全基因组核苷酸序列的比较分析

[目的]分析我国ALV-J蛋鸡分离株的来源和进一步演变趋势.[方法]以J亚群禽白血病病毒(ALV-J)蛋鸡分离株SD07LK1感染的鸡胚成纤维细胞(CEF)基因组:DNA作为其前病毒基因组模板,根据已发表序列设计合成9对引物,经PCR扩增出9段连续的、相互部分重叠的DNA片段和闭合环形前病毒两末端LTR的连接区段,并分别连入T载体进行克隆、测序.[结果]用:DNAstar软件对测序结果进行剪辑和拼接,首次完成了ALV-J蛋鸡分离株SD07LK1的前病毒全基因组核苷酸序列.[结论]将该序列与另外已完成的全基因组序列的比较表明,ALV-J的整个基因组gag和pol基因相对保守,各毒株间对应基因的同源性分别在95.O%以上,env基因的同源性仅为88.6%～94.0%.     

鸡脂肪酸结合蛋白基因的克隆和测序分析

根据哺乳动物脂肪酸结合蛋白基因序列设计一对引物对鸡基因组进行PCR扩增,将163bp扩增片段进行克隆和测序,并与猪的脂肪酸结合蛋白（fatty acid binding protein,FABP）基因序列进行同源性比较。该基因因片段与猪的心脏脂肪酸结合蛋白（heart fatty acid binding protein,H-FABP）基因有68%的同源性,与猪的脂肪型脂肪酸结合蛋白（adipocyte fatty acid binding protein,A-FABP）基因有75%的同源性,演绎成氨基酸之后与猪的脂肪型脂肪酸结合蛋白相应的氨基酸有75%的同源性。Northern结果表明该基因只在脂肪组织中表达。     

利用锌指基序的保守性从人脑组织分离新的C_2H_2型锌指蛋白基因表达序列

本文利用锌指基序的保守性设计引物,在低严谨条件下扩增人基因组总DNA,获得8个长度呈梯度的PCR扩增产物电泳条带。取其中第2和第3电泳条带,回收DNA片段并将它们克隆,经测序和查新比较,共获得60个含有锌指蛋白基因基序的单一的序列,其中23个为新的锌指蛋白基因DNA片段。以它们为探针,杂交筛选人脑组织cDNA文库,得到初筛cD-NA克隆44个。对其中28个初筛cDNA克隆进行复筛之后,得到20个cDNA单克隆。对这些阳性克隆进行测序,读出18个含有锌指蛋白基因基序的序列,国际联网查新之后,证明其中16个是新的锌指蛋白基因片段。这些新的锌指蛋白基因片段为今后克隆有意义的全长锌指蛋白cDNA提供了重要的实验材料。     

山羊卵泡刺激素α亚基cDNA的分子克隆与序列分析

从新屠宰的雌山羊脑垂体中提取总RNA ,反转录获得cDNA .以此cDNA为模板用PCR法扩增目的片段 ,获得长为 380bp的山羊卵泡刺激素α亚基cDNA片段 .将它克隆至pMD 18 T Verctor.随机挑选 3个阳性重组子进行测序 ,将测序结果与绵羊、牛、猪等多种哺乳动物该基因的核苷酸序列及相应氨基酸序列进行比较 .结果表明 ,山羊卵泡刺激素α亚基基因氨基酸序列与绵羊、水牛的同源性最高 ,达 96 % ,与牛的同源性达 95 % ,与人的同源性较低 ,为 74 % .山羊卵泡刺激素α亚基基因编码区的核苷酸与绵羊的同源性最高 ,达 95 % ,与水牛、牛的同源性达 94 % ,与马和大鼠的同源性较低 ,为 85 % .总体来看 ,在哺乳类动物中FSHα亚基基因同源性还是很高的 .     

嗜热菌Aquifex pyrophilus中mbh 2基因簇的鉴定和部分基因的克隆及测序

基于嗜热菌Aquifex aeolicus的mbhS2基因,设计合成特异性引物,以Aquifex pyrophilus 的染色体DNA为模板,应用PCR技术扩增出目的片段.序列测定结果显示,其推导出的氨基酸序列与A.aeolicus的相应序列具有85%的同源性.以此PCR片段为探针,从A.pyrophilus 的Nco I部分基因组文库(4～6kb)中筛选出含5kb大小插入片段的阳性克隆,进而对其进行了亚克隆及测序.结果表明,该插入子包含A.pyrophilus mbj2基因簇的小亚基基因mbhS2、orf1基因和部分orf2基因.所推导出的氨基酸序列与A.aeolicus的MbhS2和Orf963相比较的同源性分别为81%和60%.     

Zfa is an expressed retroposon derived from an alternative transcript of the Zfx gene.

ZFY, a gene on the Y chromosome encoding a zinc finger protein, has been proposed as a candidate for the human testis determining gene. Sequences related to ZFY, called ZFX, are present on the X chromosome of a wide range of placental mammals. Unlike most mammals the mouse has four genes homologous to ZFY; two on the Y chromosome, Zfy-1 and Zfy-2, an X-linked gene, Zfx, and an autosomal gene, Zfa. We show here that Zfa has arisen recently by retroposition of one of at least three alternatively spliced mRNAs transcribed from the Zfx gene. Zfa is an unusual retroposon in that it has retained an open reading frame and is expressed, although its function may be limited or altered by the presence of a potentially inactivating mutation in the third of its zinc fingers. This mutation must have occurred at the same time or soon after the retroposition event as it is also present in the Zfa gene of Mus spretus. Interestingly the third finger of the M. musculus musculus Zfy-2 gene has also sustained a mutation suggesting that this gene family may be rapidly evolving in mice.     

Cloning and comparative analysis of the bovine, porcine, and equine sex chromosome genes ZFX and ZFY.

A growing body of evidence suggests the involvement of sex chromosome genes in mammalian development. We report the cloning and characterization of the complete coding regions of the bovine Y chromosome ZFY and X chromosome ZFX genes, and partial coding regions of porcine and equine ZFX and ZFY genes. Bovine ZFY and ZFX are highly similar to each other and to ZFX and ZFY from other species. While bovine and human ZFY proteins are both 801 amino acids long, bovine ZFX is 5 amino acids shorter than human ZFX. Like in humans, both bovine ZFY and ZFX contain 13 zinc finger motifs and belong to the Krueppel family of C2H2-type zinc finger proteins. The internal exon-intron organization of the bovine, porcine and equine ZFX and ZFY genes has been determined and compared. Within this region, the exon lengths and the positions of the splice sites are conserved, further suggesting a high evolutionary conservation of the ZFX and ZFY genes. Additionally, new alternatively spliced forms of human ZFX have been identified.     

Differential rates of evolution for the ZFY-related zinc finger genes,Zfy, Zfx,and Zfa in the mouse genus Mus

A comparative study of the last exon of the zinc finger genes Zfx, Zfy, and Zfa from species of mice in the genus Mus was conducted to assess the extent of gene-specific and chromosome-specific effects on the evolutionary patterns among related X-, Y-, and autosomal-linked genes. Phylogenetic analyses of 29 sequences from Zfx, Zfa, and Zfy from 10 taxa were performed to infer relatedness among the zinc finger loci, and codon-based maximum likelihood analyses were conducted to assess evolutionary pattern among genes. Five models of nucleotide sequence evolution were applied and compared using a likelihood ratio test. Estimates of nonsynonymous to synonymous changes (dN/dS) for these genes suggest that amino acid substitutions are occurring at a more rapid rate across the autosomal- and Y-specific lineages compared to the X-specific lineage, with the Y-specific lineage showing the highest rate under certain models. The data suggest the action of gene-specific effects on evolutionary pattern. In particular, Zfa and Zfy genes, both with presumed restricted expression, appear less functionally constrained relative to ubiquitously expressed Zfx. Slightly elevated dN/dS for Zfy genes in comparison to Zfa also suggest Y-specific effects.     

Isolation of a zinc finger motif (ZNF75) mapping on chromosome Xq26.

We report here the partial characterization of a new human zinc finger (ZNF75) gene of the Kruppel type mapping to the long arm of the X chromosome. A cosmid clone was isolated from a library specific to the Xq24-qter region by hybridization to a degenerate oligonucleotide representing the link between two contigous fingers of the C2H2 type. The sequence of the pertinent cosmid fragments demonstrated five consecutive zinc finger motifs, all pertaining to the Kruppel family. A reading frame starting at least 75 amino acids before the first zinc finger and ending 11 amino acids after the last one was identified; comparison with other ZF genes suggests that this genomic fragment represents the carboxy-terminal exon of the gene. Homology of approximately 55% in the zinc finger region was detected with many zinc finger genes including mouse Zfp-35 and human ZFN7 cDNA clones. Mapping using a panel of sematic cell hybrids and chromosomal in situ hybridization localized the gene to Xq26, in a region not previously known to contain zinc finger genes.     

过表达miR-9对黄鳝受精卵中基因转录组水平的影响

为了更深入地研究鱼类天然性逆转的生理学机制, 研究通过克隆黄鳝(Monopterus albus) miR-9的前体序列, 及在黄鳝受精卵中过表达miR-9的生物学研究, 最终筛选得到157个基因与miR-9的过表达相关, 其中松弛素信号通路(ko04926)为显著富集的KEGG条目。tektin 4基因(tekt4)、环腺苷酸环化酶合成酶2a(adcy2a)、Ⅰ型细胞骨架角蛋白13(k1c13)和视黄醇脱氢酶5(rdh5)等基因的表达量在miR-9过表达后出现不同程度的升高; 成纤维细胞生长因子13b(fgf13b)和促甲状腺激素释放激素受体2(trhr2)等基因的表达量在miR-9过表达后出现不同程度的降低。rdh5基因在黄鳝眼睛中具有最高的表达水平(P<0.01), 而在脑、血液、精巢和卵巢中几乎没有表达(P<0.01)。松弛素家族基因rln3a和rln3b在黄鳝成鱼中的组织表达模式类似, 均在脑和精巢组织中具有较高的表达水平。推测miR-9可能与黄鳝视觉功能的发育或维持相关; miR-9可能通过松弛素信号通路调控黄鳝精巢的发育过程。研究有助于探讨miR-9对内分泌系统相关基因的调控通路, 为阐明黄鳝性别分化的分子机制提供理论依据。     

Regional assignments of the zinc finger Y-linked gene (ZFY) and related sequences on human and mouse chromosomes

Recent chromosome walking experiments have identified a candidate gene (ZFY) for the testis-determining factor on the human Y chromosome (Page et al., 1987). We report here the regional assignments of the ZFY gene and related sequences in the human and the mouse. By in situ hybridization, we assigned ZFX and ZFY to human chromosome bands Xp21 and Yp11.3, respectively. Although the mouse harbors two Zfy genes, only one site at band A1 of its Y chromosome was significantly labeled. The mouse Zfx gene and the Zfa gene on chromosome 10 were assigned to bands XD and 10B5, respectively. These assignments of the ZFX gene in human and mouse add another marker to the conserved syntenic group for evaluating the evolutionary relationship of the human and mouse X chromosomes.     

黄鳝P-450芳香化酶基因的克隆及序列分析

P-450芳香化酶(P450arom)是催化雄激素生物合成雌激素的关键酶。本文采用RT-PCR和RACE(Rapid amplifi- cation of cDNA ends)法,首次分离和克隆了雌雄同体鱼黄鳝卵巢中P450 arom基因。该基因cDNA全长1802bp(不包 括poly(A)),5'端非翻译区有49bp,3'端202bp(不包含poly(A)),阅读框(Open reading frame,ORF)1551bp,翻译成517 个氨基酸,计算的蛋白质分子量58．2kDa。同源性分析显示,黄鳝卵巢P450arom的氨基酸序列与其他鱼卵巢 P450arom具有63％-80％同源性,与其他鱼脑P450arom为58％-60％同源,与人胚盘和鸡卵巢P450arom则为 50％-52％同源;但在芳香化酶高保守区(包括1-螺旋区,芳香化酶特异保守区和血红素结合区)的同源性高达 76％-92％。系统发育分析表明芳香化酶基因是单起源,黄鳝卵巢芳香化酶基因与鳉鱼卵巢的关系最近,与鱼类卵 巢P450arom属于同一分支的,与鱼类脑及鸡和人的属于不同分支。