Interspecies Comparison Reveals Evolution of Control Regions in the Nematode Sex-Determining Gene Tra-2

The Caenorhabditis elegans sex-determining gene tra-2 promotes female development and expresses 4.7-, 1.9- and 1.8-kb mRNAs. The 4.7-kb mRNA encodes the major feminizing activity of the locus, a predicted membrane receptor that mediates cell-to-cell communication, named TRA-2A. The tra-2 gene was characterized from a close relative, C. briggsae. The Cb-tra-2 gene expresses only a 4.7-kb mRNA and alternatively spliced variants, which encode TRA-2A homologues. The Cb-TRA-2A and Ce-TRA-2A sequences are highly diverged, sharing only 43% identity, although their hydropathy profiles remain remarkably similar. Three potential regulatory sites of Ce-tra-2 activity were previously identified by analyzing tra-2(eg), tra-2(gf), and tra-2(mx) mutations. Two of these sites, the EG site and MX region, are conserved in Cb-tra-2. By contrast, the two direct repeat elements in the Ce-tra-2 3' untranslated region, which are disrupted in tra-2(gf) mutants, are absent. Injection of Cb-tra-2 antisense RNA into C. briggsae mimics the Ce-tra-2 loss-of-function phenotype. Thus, antisense RNA permits studies of gene activity in nematodes that lack extensive genetics.     

Analysis of the Role of Tra-1 in Germline Sex Determination in the Nematode Caenorhabditis Elegans

In wild-type Caenorhabditis elegans there are two sexes, self-fertilizing hermaphrodites (XX) and males (XO). To investigate the role of tra-1 in controlling sex determination in germline tissue, we have examined germline phenotypes of nine tra-1 loss-of-function (lf) mutations. Previous work has shown that tra-1 is needed for female somatic development as the nongonadal soma of tra-1(lf) XX mutants is masculinized. In contrast, the germline of tra-1(lf) XX and XO animals is often feminized; a brief period of spermatogenesis is followed by oogenesis, rather than the continuous spermatogenesis observed in wild-type males. In addition, abnormal gonadal (germ line and somatic gonad) phenotypes are observed which may reflect defects in development or function of somatic gonad regulatory cells. Analysis of germline feminization and abnormal gonadal phenotypes of the various mutations alone or in trans to a deficiency reveals that they cannot be ordered in an allelic series and they do not converge to a single phenotypic endpoint. These observations lead to the suggestion that tra-1 may produce multiple products and/or is autoregulated. One interpretation of the germline feminization is that tra-1(+) is necessary for continued specification of spermatogenesis in males. We also report the isolation and characterization of tra-1 gain-of-function (gf) mutations with novel phenotypes. These include temperature sensitive, recessive germline feminization, and partial somatic loss-of-function phenotypes.     

马铃薯StAP1基因的分子克隆与表达分析

采用RT-PCR方法从马铃薯品种‘Désirée’中克隆了StAP1cDNA序列（GenBank登录号GU220568）。该基因cDNA开放阅读框长度为735bp,编码一个由244个氨基酸残基组成的蛋白,该蛋白分子量为28.57kDa,理论等电点为8.32。StAP1蛋白含有1个高度保守的MADS结构域,与烟草NAP1具有较高一致性。组织表达分析显示,StAP1在马铃薯植株的顶芽、花和叶中有较高水平的转录表达,在块茎中有微量表达。利用反义StCOL转基因马铃薯植株进一步分析表明,StAP1的表达受StCOL的调控。     

羊草OEE1基因的克隆及盐胁迫下的表达

从羊草(Leymus chinensis )叶片cDNA文库中克隆得到可能编码33 kD的光系统Ⅱ(PSⅡ)外周蛋白(oxygen-evolving enhancer protein1,OEE1)全长cDNA(GenBank登录号为EF583851),命名为LcOEE1.序列分析结果表明,该cDNA全长1 107 bp,5′非编码区为32 bp,3′非编码区为71 bp,编码区长987 bp,编码328个氨基酸.BALSTp比对发现,该基因氨基酸序列与已报道的小麦和水稻中的OEE1序列具有95%和94%的相似性.聚类分析表明,该基因与小麦和水稻的亲缘关系较近,与拟南芥和菠菜OEE1基因的亲缘关系较远.Northern杂交结果表明,在200 mmol/L的NaCl处理7 d的幼叶中,OEE1 mRNA的表达量明显高于未处理的对照,说明羊草中OEEl基因受盐诱导.     

Molecular Cloning and Single Nucleotide Polymorphism Detection of Buffalo DGAT1 Gene

We report molecular cloning and single nucleotide polymorphism detection of the buffalo DGAT1 gene. Diacylglycerol acyltransferase (DGAT1) is considered the key enzyme in controlling the rate of synthesis of triglycerides. The DGAT1 gene was recently identified as a strong functional candidate gene affecting milk yield and composition in cattle. A full-length buffalo DGAT1 genomic DNA was amplified by iterative PCR based on homolog cloning. The buffalo DGAT1 gene comprises 17 exons and spans approximately 8.3 kb. The genomic structures of DGAT1 are highly conserved among mammal species. The deduced protein of buffalo DGAT1 contains 489 amino acids, showing high-sequence similarity with mammal homologs. Through PCR-SSCP analysis and sequencing, seven polymorphic positions were detected in the complete genomic region of buffalo DGAT1, and their frequencies were observed from a collection of 117 buffalo. The SNP (C/T) detected at position 11785 in exon 17 creates a substitution change for the amino acid sequence, resulting in an Ala residue (GCG) transition to a Val residue (GTG) in position 484 of buffalo DGAT1 protein. Information provided in this study will be useful in further studies to determine the role DGAT1 plays in the regulation of milk fat synthesis and quality improvement for milk in buffalo. Jing Yuan and Jun Zhou contributed equally to this work.     

A Novel Dominant Transformer Allele of the Sex-Determining Gene Her-1 of Caenorhabditis Elegans

We have characterized a novel dominant allele of the sex-determining gene her-1 of Caenorhabditis elegans. This allele, called n695, results in the incomplete transformation of XX animals into phenotypic males. Previously characterized recessive her-1 alleles transform XO animals into phenotypic hermaphrodites. We have identified five new recessive her-1 mutations as intragenic suppressors of n695. Three of these suppressors are weak, temperature-sensitive alleles. We show that the recessive her-1 mutations are loss-of-function alleles, and that the her-1(n695) mutation results in a gain-of-function at the her-1 locus. The existence of dominant and recessive alleles that cause opposite phenotypic transformations demonstrates that the her-1 gene acts to control sexual identity in C. elegans.     

大豆胞囊线虫抗性基因定位与克隆研究进展

大豆胞囊线虫（soybean cyst nematode,SCN）是大豆生产上一种危害严重的世界性害虫,能给大豆生产造成极大损失。大豆抗性品种选育是防治其措施中最经济、有效的方法。大豆SCN抗性的分子遗传学研究是开展大豆SCN抗性分子育种的理论基础,本文针对SCN抗性基因定位和克隆两个方面的研究现状进行了综述,并对当前研究中存在的问题及发展前景进行了讨论与展望。     

人类新基因LACE1的克隆和特性初步分析

目的:构建人类新基因LACE1,同时对该基因进行初步的特性和功能研究.方法:通过生物信息学EST拼接技术,RT-PCR等技术,克隆出30个人类未知功能基因.利用RT-PCR技术对该基因的表达谱进行研究,同时结合绿色荧光蛋白与荧光显微镜对该蛋白的定位进行初步分析,并利用MTT初步分析该基因的功能.结果:成功克隆出30个未知功能的人类新基因,其中LACEI(Homo sapiens lactation elevated 1)是一个没有任何功能文献报道的新基因,通过生物信息学分析该基因NCBI:NM一145315.3,其cDNA全长为2 262bp,有13个外显子和12个内含子组成,主要定位于人6号染色体,该基因定位于细胞质中,MTT结果显示该基因能明显抑制细胞增值.结论:人类新基因LACEI是一个抑制细胞增值的相对保守的人类新基因.     

睾丸生精细胞凋亡相关基因TSARG1与Mtsarg1的分子克隆及Mtsarg1基因的表达谱分析

从已获得的在隐睾和正常睾丸对照中表达量有明显差异的EST片段（GenBank登录号：BE644538）出发,利用生物信息学和实验技术,克隆了小鼠睾丸生精细胞凋亡相关新基因Mtsarg1及相应的人类新基因TSARG1,Gen-Bank登录号分别为AF399971和AY032925。小鼠Mtsargl与人类TSARGl基因在氨基酸水平有55％的一致性和61％相似性,与其他已知蛋白质无明显同源性。小鼠10种组织的RT-PCR分析结果表明,Mtsargl基因在睾丸中高表达,在附睾中呈微弱表达,在其他组织不表达,提示Mtsargl和TSARGl基因在生精细胞凋亡或精子发生中具有潜在的重要作用。     

Molecular Cloning and Expression of Aven Gene in Chicken

Aven was originally identified as a protein that regulates apoptosis by binding to apoptotic regulators, Bcl-xL and Apaf-1. Recently was found that Aven protein is a potent activator of ATM, critical for its DNA damage-induced activation. An Aven cDNA clone was isolated from chicken (Gallus gallus) after screening of a cerebellum cDNA library. The full-length cDNA is 1,430 nt in size, encoding for a polypeptide of 352 amino acid residues. The predicted amino acid sequence of the chicken Aven is 69, 46, 45 and 37% identical to those of zebra finch, human, xenopus and zebrafish orthologs, respectively. Expression analysis reveals that the chicken Aven gene is expressed in the adult brain, heart, intestine, kidney, lung, stomach and spleen, as well as in the whole embryos of 4- and 6-days old. Phylogenetic analysis of the Aven ortholog proteins from various organisms clusters the chicken Aven in the same group with other bird Avens.     

HCV E2区基因的分子克隆及序列分析

用RT－PCR KIT从西安血站大样抗HCV阳性血清中筛选出HCV RNA阳性血清,提取HCV的RNA,利用随机引物反转录合成其cDNA并进行半巢式PCR反应。将纯化的PCR产物酶切后与表达载体PET－22b^＋连接,经过双脱氧末端终止法双向测序,得到852bp长的核苷酸序列,通过将该序列与已知不同型的HCV E2序列比较得知,此序列正是HCVⅡ型目的基因。     

Cloning, Sequencing and Mapping of a Manganese Superoxide Dismutase Gene of the Nematode Caenorhabditis elegans

We have cloned, sequenced and mapped a gene (sod-2) encodingmanganese superoxide dismutase [EC 1.15.1.1] from the nematodeCaenorhabditis elegans. The sod-2 was mapped to chromosome Iby hybridization with a YAC polytene filter. The protein-codingregion spans 1129 base pairs including 4 introns and encodesa protein of 221 amino acids (aa) (Mr = 24536) of which thefirst 24 aa are the presumed mitochondorial-targeting signalpeptide. The gene sequence of sod-2 was slightly different froman isoform, sod-3.     

Molecular Cloning and Bacterial Expression of the Catalytic Domain of the SENP1 Gene of Oryzias latipes

In this study, we cloned the catalytic domain of the Oryzias latipes sentrin/SUMO-specific protease 1 (OlSENP1-CD) gene and produced the recombinant OlSENP1-CD protein in Escherichia coli. Experimental procedures designed to reveal the ability of the recombinant protein to show deSUMOylating activity in vitro should be helpful in future studies of other SENPs and the SUMO pathway.     

小鼠Daintain/AIF-1基因启动子的克隆及其荧光素酶报告基因载体的构建

目的：对Daintain/AIF-1（大炎肽/同种异体移植炎症因子-1）基因启动子进行克隆并构建荧光素酶报告基因载体,为进一步研究Daintain/AIF-1的转录调控作用提供了质粒资源。方法：提取单核巨噬细胞系RAW264.7基因组DNA,以其为模板采用PCR方法克隆出Daintain/AIF-1基因5＇端UTR区1.6 kb DNA序列,将该序列同源重组到pGL3-Basic载体上,转化感受态DH5α并酶切鉴定和测序。结果：PCR产物片段与预期结果一致,Daintain/AIF-1基因5＇端UTR区1.6 kb DNA序列连接到pGL3-Basic载体上,构建成pGL3-Basic-Daintain/AIF-1（pGL3-Basic-DT）载体,酶切结果与理论预测值一致,经测序证实无碱基突变。结论：Daintain/AIF-1基因报告基因载体的构建为进一步研究Daintain/AIF-1转录调控作用提供了载体资源。     

羊驼催乳素基因cDNA克隆及序列分析

实验通过克隆分析羊驼催乳素基因的部分序列,对羊驼催乳素基因的结构和功能进行初步探索和揭示。从GeneBank中已报到的脊椎动物催乳素基因保守区设计一对引物,采用Trizol法提取羊驼胎盘总RNA,利用RT-PCR技术扩增出长度约为510 bp的片断。测序后在NCB I工作平台中进行BLASTn同源性比较,得出结论:羊驼催乳素基因与已登录的哺乳动物催乳素基因同源性均超过85%,最高达97%。借助DNAstar分子生物学分析软件绘制了相关动物的遗传进化图,并对羊驼的种属地位进行了进一步验证。。     

HCV E2区基因的分子克隆及序列分析

用RT-PCRKIT从西安血站大样抗HCV阳性血清中筛选出HCVRNA阳性血清,提取HCV的RNA,利用随机引物反转录合成其cDNA并进行半巢式PCR反应。将纯化的PCR产物酶切后与表达载体PET-22b     

烟草病程相关蛋白基因启动子的克隆及序列分析

在植物的防御反应中,会诱导产生一些宿主编码的蛋白,叫做病程相关蛋白。该文通过PCR扩增,从烟草(Nicotiana tobacwn ce.Samsun)中克隆了水扬酸诱导表达的PR—1a基因的两个启动子TP12及TP13。以期用于构建诱导表达基因敲除系统,并用于无性繁殖植物的无标记基因转化。序列分析表明,启动子TP12含1313个核苷酸,与已报道的序列比较,核苷酸的同源性为98．6％;TP13含654个核苷酸,与已报道的序列比较,核苷酸的同源性为99．4％。