三角帆蚌金属硫蛋白基因的克隆及序列分析

采用RACE技术,获得了三角帆蚌(Hyriopsis cumingii)金属硫蛋白基因的全长cDNA序列.该序列全长467 bp,由长92 bp的5'UTR(untranslated region),159 bp的3'UTR,和216 bp的开放阅读框(openreading frame,ORF)组成.共编码71个氨基酸,分子量大约为7.1 ku,理论等电点为7.24.该蛋白序列中半胱氨酸含量最丰富(29.6%),其次是甘氨酸(14.1%),存在软体动物金属硫蛋白的特征序列CKCXXXCXCX,且C-末端的氨基酸序列也符合软体动物金属硫蛋白标签序列C-X-C-X(3)-C-T-G-X(3)-C-X-C-X(3)-C-X-C-K.蛋白序列特征分析表明,该序列与其他贝类的金属硫蛋白基因具有很高的相似性,具备金属硫蛋白的典型特征,是金属硫蛋白家族的成员.     

球根白丝膜菌γ-actin基因的克隆及表达分析

根据真菌肌动蛋白(actin)基因保守区序列设计引物,用简并PCR法和RACE技术分离得到球根白丝膜菌(Leucocortinarius bulbiger)γ-肌动蛋白基因(Lb-act)的全长cDNA序列。该序列全长为1 357 bp,包含一个1 137 bp的开放阅读框(ORF),编码378个氨基酸,5'端非翻译区(5'UTR)92 bp,3'UTR长度128 bp。Port Param软件在线分析结果表明,该cDNA所编码的蛋白质理论等电点为5.12,相对分子质量为95.022 kD,具有真菌γ-actin基因3个保守特征序列。Blast同源性检索结果表明,Lb-act氨基酸序列与担子菌肌动蛋白序列有较高的相似性,其与双色蜡蘑的肌动蛋白氨基酸序列的亲缘关系最近。Lb-act基因在不同碳源及磷水平培养条件下表达量基本一致,验证了该基因作为分子内标的可靠性。     

鸡二价金属转运蛋白1 cDNA的克隆与序列分析

鸡二价金属转运蛋白1（divalent metal transporter 1, DMT1）在动物胃肠道锰吸收过程中起重要作用.根据哺乳动物Dmt1同源蛋白氨基酸序列的保守性设计引物,应用3′RACE(rapid amplification of cDNA ends)技术,扩增并克隆获得鸡小肠Dmt1 cDNA 3′端1 289bp和1 092bp的2种片段,发现其3′端翻译区和非翻译区存在差异. 根据鸡Dmt1 cDNA 3′端片段的测序结果设计引物,扩增获得1个与3′端片段部分重叠的鸡Dmt1 cDNA 5′端907 bp片段,并对其进行了克隆测序. 根据鸡小肠Dmt1 3′RACE片段和5′RACE片段序列信息进行拼接,从而获得鸡小肠Dmt1 cDNA全序列信息.结果表明,鸡小肠Dmt1 cDNA有2种形式,1种全长为1 972个核苷酸,其中5′非翻译区为104个核苷酸,编码区1 695个核苷酸,3′非翻译区为173个核苷酸,编码1个含564个氨基酸残基的蛋白质;另1种形式为1 775个核苷酸,其中5′非翻译区为104个核苷酸,编码区1 593个核苷酸,3′非翻译区为78个核苷酸,编码1个含530个氨基酸残基的蛋白质.据鸡Dmt1 cDNA推测出的2种形式蛋白质的氨基酸序列与人、大鼠和小鼠的Dmt1蛋白具有高度同源性,它们的同源性分别为82%、82%、80%,和 84%、84%、83%. 对推测氨基酸序列进行疏水性和跨膜区分析表明,Dmt1蛋白为1种跨膜整合蛋白,具有膜转运蛋白糖基化位点和底物结合位点的保守序列.     

SNF2家族新成员Ercc61的cDNA克隆与表达分析

SNF2家族蛋白在基因组复制、修复与表达中具有重要作用.报道了SNF2家族新成员Ercc61(excision repair crosscomplementing rodent repair deficiency,complementation group 6-like)的cDNA克隆、特性与表达分析.通过表达序列标签(EST)搜索和组装,获得了cDNA全长4002 bp的新基因Ercc6l(GenBank Acc.No AY172688),然后通过RT-PCR在小鼠胚胎心脏成功克隆了该基因.Ercc6l在小鼠基因组中由两个外显子和一个内含子组成,定位于X染色体,最大开放阅读框(ORF)编码一个含l 240个氨基酸的假定蛋白质.该假定蛋白质含有SNF2蛋白的8个保守基序(SNF2结构域).通过与SNF2家族各亚家族的成员进行多重比对,初步确认Ercc6l属于ERCC6亚家族成员.将Ercc6l编码区克隆到pEGFP-C3然后转染HeLa,3T3和B16细胞,融合蛋白主要定位于胞浆.BLAST搜索检索出69条小鼠EST与Erccol同源,这些EST主要来自胚胎和肿瘤组织.对小鼠不同发育时期的多种组织进行RT-PCR,发现Ercc6l在胚胎期强表达,出生产后表达显著下调.这些结果提示Ercc6l在胚胎发育和肿瘤发生中可能具有重要作用.     

编码新型转录因子样蛋白的小鼠及人类同源cDNA的克隆(英文)

 通过检索GenBank的表达序列标签 (EST)数据库并结合cDNA末端快速扩增法 (RACE) ,从小鼠胸腺克隆到一个新的cDNA序列 ,并从人类肝癌组织中克隆出了其同源cDNA .根据读码框架分析 ,这两个cDNA分别编码 541和 555个氨基酸的蛋白质 两个蛋白质之间氨基酸序列一致率为77% ,和已知蛋白无显著同源性 .分子生物学软件和网上分析表明 ,两个蛋白质所含功能序列与STAT家族成员极为相似 ,均含有包括酪氨酸蛋白激酶在内的多种蛋白激酶的磷酸化位点和核定位信号 (NLS) ,可能是一种新型转录因子 .RT PCR分析显示 ,两个基因在正常组织中选择性表达 ,其分布相似 ,而且都具有一定程度的与分化或增殖相关的趋势 .     

SNF2家族新成员Ercc6l的cDNA克隆与表达分析(英)

SNF2家族蛋白在基因组复制、修复与表达中具有重要作用. 报道了SNF2家族新成员Ercc6l (excision repair cross-complementing rodent repair deficiency, complementation group 6-like)的cDNA克隆、特性与表达分析.通过表达序列标签（EST）搜索和组装,获得了cDNA全长4 002 bp的新基因Ercc6l(GenBank Acc.No AY172688),然后通过RT-PCR在小鼠胚胎心脏成功克隆了该基因.Ercc6l在小鼠基因组中由两个外显子和一个内含子组成,定位于X染色体,最大开放阅读框（ORF）编码一个含1 240个氨基酸的假定蛋白质.该假定蛋白质含有SNF2蛋白的8个保守基序（SNF2结构域）.通过与SNF2家族各亚家族的成员进行多重比对,初步确认Ercc6l属于ERCC6亚家族成员.将Ercc6l编码区克隆到pEGFP-C3然后转染HeLa,3T3 和B16细胞,融合蛋白主要定位于胞浆.BLAST搜索检索出69条小鼠EST与Ercc6l同源,这些EST主要来自胚胎和肿瘤组织.对小鼠不同发育时期的多种组织进行RT-PCR,发现Ercc6l在胚胎期强表达,出生产后表达显著下调.这些结果提示Ercc6l在胚胎发育和肿瘤发生中可能具有重要作用.     

苋菜IRT1基因克隆、序列及表达分析

通过对镉超积累苋菜品种天星米铁转运蛋白基因( IRT1)的克隆、序列及表达分析,旨在为植物修复镉污染土壤奠定基础.依据同源克隆原理,通过RACE技术克隆苋菜IRT1基因及生物信息学方法分析基因序列结构和功能,Northern杂交研究基因表达.苋菜IRT1基因cDNA全长1135 bp,包含完整的阅读框,编码322个氨基酸.苋菜IRT1蛋白与已知铁转运蛋白相似性在53.70％-63.04％,具有铁转运蛋白典型的功能结构特征,即N端含有1个信号肽、氨基酸序列上具有完整的ZIP家族功能结构域( Pfam:Zip)和7个跨膜结构域(TMs).苋菜IRT1蛋白还具有1个COG0428超级家族(转运二价金属离子功能)、2个蛋白激酶C磷酸化位点和2个酪蛋白Ⅱ磷酸化位点.低铁胁迫时苋菜根中IRT1基因表达量增加,加镉处理没有改变IRT1基因表达量.因此,推断苋菜IRT1基因是ZIP家族的一员,具有转运二价金属离子功能,将基因在GenBank中注册,序列号为:GU363501,命名为AmIRT1.     

一个新的鼠STE20家族激酶的克隆和鉴定

从鼠肝cDNA文库克隆了一个新的STE20类蛋白激酶,Mess1.其cDNA长1.7 kb,编码了一个497个氨基酸残基的多肽,与人MST2具有95%的氨基酸相同.Mess1蛋白氨基末端激酶催化区的序列与STE20同源,其羧基末端包含了一簇丝氨酸/苏氨酸和谷氨酸丰富的序列,被认为具有介导与SH2功能区结合的作用.MESS1可能通过与含有SH2功能区的蛋白质相互作用参与细胞内信号转导.     

大头金蝇β-actin基因克隆与多克隆抗体制备

为深入研究大头金蝇Chrysomya megacephala(Fabricius)关键功能基因的表达调控,运用RT-PCR和RACE技术,克隆获得大头金蝇β-actin基因cDNA全长序列(GenBank登录号为KC207081),并对其进行生物信息学分析。大头金蝇β-actin基因cDNA全长1 355 bp,其中开放阅读框(ORF)为1 131 bp,编码376个氨基酸,5'UTR长度为87 bp,3'UTR约为110 bp。ORF编码的蛋白质分子量为41.8 D,等电点5.286,氨基酸序列与其他昆虫β-actin一致性高达97%-99%,且含有由6个β-actin蛋白家族特有的保守模式(motif)所构成的指纹。根据对β-actin氨基酸序列的亲水性、抗原性和表面可及性分析预测,设计并合成β-actin抗原多肽(CysGPYARVKRHQKGLKT),免疫新西兰兔获得多克隆抗体,其效价远高于1∶64 000。采用Western Blot技术检测大头金蝇各发育阶段的β-actin蛋白表达,结果表明β-actin表达恒定。本文的研究结果为大头金蝇功能基因的深入研究提供了坚实的基础。     

鳜小肽转运载体PepT1基因分子特征及其表达研究

小肽转运载体（PepT1）是低亲和力、高容量的肽转运载体,在小肽的吸收过程中发挥着重要的作用。研究采用同源克隆和RACE技术克隆了鳜鱼（Siniperca chuatsi） PepT1基因全长cDNA序列,其cDNA序列全长为2480 bp,包含43 bp的5'UTR序列,232 bp的3'UTR序列,以及2205 bp开放阅读框,编码735个氨基酸。 氨基酸序列同源性分析结果显示,鳜鱼与石斑鱼（Epinephelus aeneus）、鲈鱼（Dicentrarchus labrax）PepT1间同源性均为89%,与其他非鱼类物种的同源性则在46%56%。经预测,鳜鱼PepT1编码蛋白的分子量为64.8 kD,等电点为8.97,该蛋白具有与哺乳动物同源蛋白相似的12 个螺旋跨膜结构,并且在跨膜区9和10之间有一个大的外环;跨膜区氨基酸高度保守,并存在有5个膜外N-糖基化位点和3个膜内含蛋白激酶C基序的相同区域。实时荧光定量表达分析表明,鳜鱼PepT1基因在前肠和中肠中表达量显著高于后肠（P0.05）,这说明前、中肠是鳜鱼肠道吸收小肽的主要部位;在胚后不同发育阶段鳜鱼前肠均能检测到PepT1基因的表达,并且在10 g个体中表达量最高,之后随着体重的增加其表达量维持在一个稳定水平。本研究结果首次报道了鳜鱼PepT1基因全序列及其分子表达特征,为鱼类营养及生理学的研究提供有价值的参考资料。       

Molecular cloning and expression of mouse placental lactogen I complementary deoxyribonucleic acid

The mouse midpregnancy lactogen or placental lactogen I (mPL-I) is encoded by a 1.0-kilobase mRNA that appears transiently during gestation, with maximal amounts accumulating in the placenta at day 10 of pregnancy. Several cDNA clones for mPL-I have been isolated from a lambda gt11 expression library constructed from day 10-placental RNA. The cDNA sequence indicates that mPL-I is synthesized as a 224 amino acid precursor, and is secreted as a 194 amino acid glycosylated hormone. The deduced amino acid sequence of mPL-I is highly homologous to the known members of the PRL family in the mouse, and hybridization analysis indicates that the mouse genome contains several mPL-I genes. Introduction of the mPL-I cDNA in an expression vector into cultured mouse cells results in the synthesis and secretion of glycosylated mPL-I protein that is recognized by anti-mPL-I antiserum and is biologically active.     

Mouse glandular kallikrein genes. Nucleotide sequence of cloned cDNA coding for a member of the kallikrein arginyl esteropeptidase group of serine proteases

A library of cloned cDNA to male mouse submaxillary gland poly(A)-containing RNA was constructed in the plasmid pBR322. Inserts containing sequences estimated to be in the 1-5% abundance class were identified by hybridization to radiolabeled cDNA and examined by nucleotide sequence analysis. A sequence coding for a peptide with 57% homology to the only complete kallikrein sequence reported to date (from pig pancreas) was identified by a computer search program. This insert appears to code for the COOH-terminal 149 amino acids of a protein presumed therefore to be a serine protease. Comparison of the predicted amino acid sequence of this protein with analogous sequences in the three characterized members of the mouse submaxillary gland kallikrein arginyl esteropeptidase group of enzymes revealed extensive homology, although not complete identity. Thus, there are at least four members of this enzyme family expressed in the mouse submaxillary gland.     

The gene encoding a major component of the lateral elements of synaptonemal complexes of the rat is related to X-linked lymphocyte-regulated genes.

The lateral elements of synaptonemal complexes (SCs) of the rat contain major components with relative electrophoretic mobilities (M(r)S) of 30,000 and 33,000. After one-dimensional separation of SC proteins on polyacrylamide-sodium dodecyl sulfate gels, these components show up as two broad bands. These bands contain closely related proteins, as judged from their peptide maps and immunological reactivity. Using affinity-purified polyclonal anti-30,000- and anti-33,000-M(r) component antibodies, we isolated a cDNA encoding at least one of the 30,000- or 33,000-M(r) SC components. The protein predicted from the nucleotide sequence of the cDNA, called SCP3 (for synaptonemal complex protein 3), has a molecular mass of 29.7 kDa and a pI value of 9.4. It has a potential nucleotide binding site and contains stretches that are predicted to be capable of forming coiled-coil structures. In the male rat, the gene encoding SCP3 is transcribed exclusively in the testis. SCP3 has significant amino acid similarity to the pM1 protein, which is one of the predicted products of an X-linked lymphocyte-regulated gene family of the mouse: there are 63% amino acid sequence similarity and 35% amino acid identity between the SCP3 and pM1 proteins. However, SCP3 differs from pM1 in several respects, and whether the proteins fulfill related functions is still an open question.     

Prediction of the coding sequences of mouse homologues of KIAA gene: IV. The complete nucleotide sequences of 500 mouse KIAA-homologous cDNAs identified by screening of terminal sequences of cDNA clones randomly sampled from size-fractionated libraries.

We have been conducting a mouse cDNA project to predict protein-coding sequences of mouse homologues of human KIAA and FLJ genes since 2001. As an extension of these projects, we herein present the entire sequences of 500 mKIAA cDNA clones and 4 novel cDNA clones that were incidentally identified during this project. We have isolated cDNA clones from the size-fractionated mouse cDNA libraries derived from 7 tissues and 3 types of cultured cells. The average size of the 504 cDNA sequences reached 4.3 kb and that of the deduced amino acid sequences from these cDNAs was 807 amino acid residues. We assigned the integrity of CDSs from the comparison with the corresponding human KIAA cDNA sequences. The comparison of mouse and human sequences revealed that two different human KIAA cDNAs are derived from single genes. Furthermore, 3 out of 4 proteins encoded in the novel cDNA clones showed moderate sequence similarity with human KIAA proteins, thus we could obtain new members of KIAA protein families through our mouse cDNA projects.     

Cloning of a human collagen-binding protein, and its homology with rat gp46, chick hsp47 and mouse J6 proteins.

Several cDNA clones encoding a collagen-binding protein were isolated from human fibroblasts. The cDNA encoded a 417 amino acid protein, containing two potential N-linked oligosaccharide binding sites and a C-terminal RDEL sequence, which has been shown to act as an endoplasmic retention signal in other systems. The derived amino acid sequence of the protein shows close homology with gp46 from rat skeletal myoblasts, J6 protein from mouse F9 embryonal carcinoma cells and hsp47 from chick embryo fibroblasts. It also shows sequence similarity with members of the serpin family.     

Cloning of a Chinese hamster protein homologous to the mouse t-complex protein TCP-1: structural similarity to the ubiquitous 'chaperonin' family of heat-shock proteins

The complete cDNA sequence of a Chinese hamster ovary cell protein, homologous to a mouse t-complex protein, TCP-1, has been determined. The deduced amino acid sequences of the mouse/Chinese hamster TCP-1 proteins exhibit significant identity to the 60-65 kDa heat shock 'chaperonin' family of proteins present in prokaryotes and in the eukaryotic cell organelles.     

Cloning and expression of a PR5-like protein from Arabidopsis: inhibition of fungal growth by bacterially expressed protein

Pathogenesis-related (PR)-5 proteins are a family of proteins that are induced by different phytopathogens in many plants and share significant sequence similarity with thaumatin. We isolated a complementary DNA (ATLP-3) encoding a PR5-like protein from Arabidopsis which is distinct from two other previously reported PR5 cDNAs from the same plant species. The predicted ATLP-3 protein with its amino-terminal signal sequence is 245 amino acids in length and is acidic with a pI of 4.8. The deduced amino acid sequence of ATLP-3 shows significant sequence similarity with PR5 and thaumatin-like proteins from Arabidopsis and other plants and contains a putative signal sequence at the amino-terminus. The expression of ATLP-3 and a related gene (ATLP-1) that we previously isolated from Arabidopsis was induced by pathogen infection and salicylic acid, a known inducer of pathogenesis-related genes. Southern blot analysis indicates that the ATLP-1 and ATLP-3 are coded by single-copy genes. To study the effect of ATLP-1 and ATLP-3 proteins on fungal growth, the cDNA regions corresponding to putative mature protein were expressed in Escherichia coli and the cDNA encoded proteins were purified. ATLP-1 and ATLP-3 proteins cross-reacted with anti-osmotin and anti-zeamatin antibodies. ATLP-3 protein showed antifungal activity against several fungal pathogens suggesting that ATLP-3 may be involved in plant defense against fungal pathogens.