人伴肌动蛋白相关锚定蛋白基因cDNA的克隆与功能分析

小鼠伴肌动蛋白相关锚定蛋白(N-RAP)是已知的与肌纤维生成有关的细胞骨架蛋白,但是,与此对应的人类N-RAP基因的cDNA序列及其功能一直是未知的.为了明确N-RAP基因在人类中所起的作用,利用生物信息学分析工具和分子生物学技术,成功地克隆了人N-RAP基因.人N-RAP基因cDNA序列含有一个5 088 bp的开放读码框,与小鼠N-RAP基因有86%的相似性.染色体定位分析表明,人N-RAP基因定位于10q25～q26之间,编码区由41个外显子组成,与HABP2和CASP7紧密连锁.电子表达谱分析表明,肌肉、心脏、脑、脊髓和前列腺有人N-RAP基因不同长度的cDNA序列.人N-RAP与小鼠N-RAP蛋白质序列有88%的相似性,与人Nebulin有约63%的相似性,与小鼠Nebulin有约59%的相似性.结构域分析发现,N端为LIM结构域,从第170位氨基酸开始,连续出现40个Nebulin相关重复序列.RT-PCR实验表明,人N-RAP基因在成人脑、骨骼肌和心脏组织中都有表达,在骨髓中不表达.亚细胞定位研究显示,人N-RAP基因表达于胞浆.基于序列相似性、结构域分析、表达谱分析和亚细胞定位研究,可以推测人N-RAP与小鼠N-RAP同属Nebulin家族,具有类似的功能.     

鼠mPC-1基因的克隆与特性分析

为深入研究人前列腺癌相关基因PC-1的生物功能和进化保守状况,从小鼠肾脏中克隆了全长cDNA序列,命名为mPC-1(GenBank Acc No.AY048852).mPC-1基因cDNA全长为2 193 bp,主要定位于小鼠染色体3A1-A2区域.mPC-1基因最大开放阅读框编码的蛋白质由224个氨基酸组成,与人PC-1蛋白编码区存在82%的序列一致性,含有coiled-coil结构域和PEST结构域.生物信息学分析表明,由6个外显子组成的mPC-1基因与mD52高度同源,其中,第一外显子代表该基因的特异性序列,实验证据显示mPC-1基因具有自己的启动子,推测mPC-l与小鼠mD52可能是重叠基因.对小鼠20种组织器官和不同发育阶段的胚胎组织cDNA的RT-PCR检测证实,该基因主要在前列腺、肾和眼组织中表达,在胃和平滑肌中有少量表达,在其他组织中表达很弱或不表达.而mD52基因则几乎广泛存在于小鼠的各个组织器官中,因此,两个基因虽然序列上高度重叠却是独立调控的.综上所述,mPC-1基因可能是一个与人PC-1基因结构功能类似的新基因.     

类固醇5-α还原酶样基因Srd5α2l2的克隆及表达分析

采用表达序列标签（EST）介导的基因克隆和表达谱分析,从小鼠心脏克隆了一个cDNA为2 348 bp,主要在心脏表达的新基因Srd5α2l2（GenBank Acc No.AF548365）.该基因由12个外显子组成,3′非翻译区富含ATTTA序列,最大开放阅读框编码一个由361个氨基酸组成的假定蛋白,该蛋白质C端含有类固醇5-α还原酶的保守结构域（3-oxo-5-alpha-steroid 4-dehydrogenase,STEROID_DH）.生物信息学分析表明,人cDNA 克隆DKFZp313D0829（AL833108）是Srd5α2l2的人同源基因.经同源性检索,支持Srd5α2l2的全部41条EST中25条来自小鼠心脏组织.RT-PCR检测初步证实,该基因主要在心脏中表达,而在其他组织中不表达或弱表达.综合考虑Srd5α2l2的序列特征和表达谱,Srd5α2l2可能在心脏组织中发挥重要作用.     

人类PKNOX1基因一种新剪接型全长cDNA的克隆与表达分析

为了寻找新的Down’s综合征相关基因,利用生物信息学分析与实验技术相结合的方法,从定位于Down’s综合征关键区内(21q22．3)的EST(GenBank登录号H77399)出发,从人类睾丸组织cDNA文库内克隆到含同源盒结构域转录因子PKNOX1的一种新剪接型全长cDNA,命名为PKNOX1B,GenBank登陆号AYl42115。PKNOX1B基因跨越58．4kb,全长cDNA约2．8kb,有11个外显子和10个内含子,编码405个氨基酸残基的酸性蛋白质,分子量为44．628kDa,等电点6．28。PKNOX1B与PKNOX1的前9个外显子及9个内含子完全相同,由于PKNOX1B在第10与11外显子之间发生了差异剪接,以致其在3’端cDNA序列被截短约2kb,所编码的蛋白质在C端较PKNOX1短30个氨基酸残基。但PKNOX1B保留了与PKNOX1完全相同的同源盒结构域,因而它可能与其他含同源盒结构域基因家族成员一样参与了发育的遗传调控。RT-PCR结果显示PKNOX1B除骨髓组织外在人体组织广泛表达。在睾丸组织中PKNOX1可见5kb,2．9kb,2kb 3种转录本,而在其他组织中仅发现2个较大的转录本,2kb的转录本在睾丸组织呈现特异性的表达,它有可能参与了精子的发生过程。     

鼠mPC-1基因的克隆与特性分析

为深入研究人前列腺癌相关基因PC-1的生物功能和进化保守状况,从小鼠肾脏中克隆了全长cDNA序列,命名为mPC-1（GenBank Acc No.AY048852）.mPC-1基因cDNA全长为2 193 bp,主要定位于小鼠染色体3A1-A2区域.mPC-1基因最大开放阅读框编码的蛋白质由224个氨基酸组成,与人PC-1蛋白编码区存在82%的序列一致性,含有coiled-coil结构域和PEST结构域.生物信息学分析表明,由6个外显子组成的mPC-1基因与mD52高度同源,其中,第一外显子代表该基因的特异性序列,实验证据显示mPC-1基因具有自己的启动子,推测mPC-1与小鼠mD52可能是重叠基因.对小鼠20种组织器官和不同发育阶段的胚胎组织cDNA的RT-PCR检测证实,该基因主要在前列腺、肾和眼组织中表达,在胃和平滑肌中有少量表达,在其他组织中表达很弱或不表达.而mD52基因则几乎广泛存在于小鼠的各个组织器官中,因此,两个基因虽然序列上高度重叠却是独立调控的.综上所述,mPC-1基因可能是一个与人PC-1基因结构功能类似的新基因.     

人分化相关基因Ndr2的克隆与组织表达谱研究

人Ndr1基因参与细胞终末分化 ,并且对肿瘤细胞增殖和肿瘤转移具有抑制作用 .从人 2 2周孕龄胎肝cDNA文库中获得与人Ndr1基因同源的一段表达性序列标签 ,继而从成人脑cDNA文库分离出其全长cDNA(2 12 1bp) ,并将该基因命名为Ndr2 .其染色体定位为 14q11 1- 11 2 ,开放阅读框编码 371个氨基酸 ,且与NDR1蛋白一样 ,含有一个典型的α β水解酶折叠类结构域 (α βhydrolasefold) .Northern杂交和点杂交分析显示 ,该基因与Ndr1一样 ,在脑中高表达 ,在胚胎组织的表达较低 ,在 8种人肿瘤细胞中的表达极低 .然而 ,Ndr2基因的组织表达谱与Ndr1又有鲜明的差异 :其在成人骨骼肌和脑等神经组织中表达最高 ,在唾液腺、肝、肾、心肌和气管中的表达次之 .结果提示 ,NDR2具有与NDR1相似或相关的重要功能 .     

一个新鼻咽癌抑瘤候选基因的克隆及其功能初步分析

从cDNA 代表差异分析法（cDNA representational difference analysis, cDNA RDA）分离的新cDNA片段入手,进一步采用RT-PCR验证,其中发现AF152605片段在74%鼻咽癌(nasopharyngeal carcinoma,NPC)活检组织中表达下调和缺失,DNA印迹显示其代表一转录本为2.1 kb的基因,结合生物信息学,运用文库筛选克隆该基因,命名为NAG4基因,GenBank登录号AF179285,定位于6q22.1～22.33,至少含有两个外显子,并在第一外显子的上游有TATA盒样序列,编码一个508个氨基酸组成的、分子质量为57.4 ku的蛋白质;功能预测NAG4基因产物与小鼠溴区蛋白BP75有84%同源,是含有多个磷酸化位点和溴区结构域的核内转录因子;突变分析表明NAG4基因在HeLa细胞株中发生整码突变.以上结果说明NAG4基因是鼻咽癌抑瘤基因的良好候选者,其表达的下调参与了鼻咽癌的发生.     

家蚕MLP基因的克隆及其结构分析

利用生物信息学的方法快速获得家蚕MLP (Muscle LIM protein, MLP)基因cDNA电子序列, 经RT-PCR生物验证正确, 登录GenBank (No. DQ311195)。MLP基因cDNA长2 327 bp, ORF全长1 485 bp, 编码产生494个氨基酸。该MLP基因组DNA含有11个外显子, 10个内含子, 所有内含子/外显子边界都符合典型的GT/AG剪切模式。MLP基因编码的蛋白富含Gly (14.4%), 分子量约为53.03 kDa, 等电点(PI)为8.29。通过BLAST分析发现该基因编码的家蚕肌肉LIM蛋白, 含有5个保守的LIM结构域, 家蚕的另一种LIM蛋白(AAR23823)含一个LIM结构域, 两者可能是通过可变剪切产生; 后者可能通过竞争作用调节前者在肌细胞中的功能。MLP的克隆为进一步研究其体内功能奠定了基础。     

青岛文昌鱼LIM类基因Bblim同源框区的cDNA克隆、序列分析及其在胚胎发育中的表达

不同卵裂球发育命运的特化、亦即胚胎细胞的分化是动物胚胎发育的重要特征。多数胚胎细胞尽管形态特征完全一致,却具有不同的发育命运。预示着：在这些细胞中存在有决定发育命运的因素－决定子。本工作克隆了青岛文昌鱼LIM类同源框基因的同源框片段。目的在于揭示决定子的分子本质。青岛近海采集性成熟的成年青岛文昌鱼,收集未受精卵、受精卵以及各处不同时期的胚胎,液氮冻存备用。分别制备总RNA。根据其它动物LIM类同源框基因的序列设计引物（Tab.1),连续进行RT－PCR和PCR两次扩增。其中,原肠胚来源的第二次PCR产物经电泳鉴定（Fig.1)后,酶切、克隆入质粒、测序、将该片段所在的基因命名为Bblim基因,该自然称为Bblim同源框。根据Bblim基因同源框的核苷酸序列推导出其相应的氨基酸序列（Fig.2),与其它LIM类同源框基因进行比较（Fig.3)后,认为：Bblim基因可归入lim3类基因。比较胚胎发育各个不同时期第二次PCR产物的含量－即Bblim基因的转录（Fig.4),提示：该基因可能在受精后和原肠成形前后两个发育阶段起作用。此外,Bblim基因的同源域与海鞘Hrlim的同源域高度同源,表达模式也相似,提示,Bblim基因可能是Hrlim基因在文昌鱼中的类似物。     

Chromosomal Location and Some Structural Features of Human Clathrin Light-Chain Genes (CLTA and CLTB)

Two human clathrin light-chain genes have been defined. The gene (CLTA) encoding the LCa light chain maps to the long arm of chromosome 12 at 12q23-q24 and that encoding the LCb light chain (CLTB) maps to the long arm of chromosome 4 at 4q2-q3. Isolation and characterization of partial genomic clones encoding human LCa and LCb reveal the neuron-specific insertions of the LCa and LCb proteins to he encoded by discrete exons, thus proving that clathrin light chains undergo alternate mRNA splicing to generate tissue-specific protein isoforms. The insertion sequence of LCb is encoded by a single exon and that of LCa by two exons. The first of the two neuron-specific LCa exons is homologous to the corresponding LCb exon. An intronic sequence of the LCb gene with similarity to the second neuron-specific exon of the LCa gene has been identified.     

Cloning of the novel isoform of the estrogen receptor beta cDNA (ERbeta isoform M cDNA) from the human testicular cDNA library

Our recent report has revealed the existence of the progesterone receptor (PR) isoform S, which consists of the novel PR exon S and exons 4-8 of the PR gene in the human testicular cDNA library. More recently, we have cloned the human estrogen receptor alpha (ERalpha) isoform S cDNA from the library. The ERalpha isoform S cDNA also contains the novel ERalpha exon S and exons 4-8 of the ERalpha cDNA. Based on these findings, we assumed that the novel isoform of cDNA like the PR- and ERalpha isoforms might exist in the human ER beta (ERbeta). In order to investigate this possibility, we have screened the human testicular cDNA library using the exons 4-8 corresponding sequence of the human ERbeta cDNA. Consequently, we have cloned a novel isoform of the ERbeta cDNA that consists of a previously unidentified 5'-sequence and the exons 5-8 of the ERbeta gene. We termed this isoform cDNA the "ERbeta isoform M cDNA". The 5'-sequence of the ERbeta isoform M cDNA was confirmed to be derived from a novel exon (termed the "exon M") by analysis of the genomic DNA. Moreover, we have analyzed the molecular size of the ERbeta isoform M encoded by the ERbeta isoform M mRNA by transient expression of the ERbeta isoform M cDNA in the 293T cell. The approximately 28 kDa protein, which was recognized by the anti-rat ERbeta antibody against the carboxyl-terminal region, was synthesized in the cells. Thus, we concluded that the ATG in the exon M could be used as the translation initiation codon. This report revealed for the first time the existence of the ERbeta mRNA isoform that is not caused by the skipping of one or more exons, by the alternative usage of the multiple exon 8s, nor by the alternative utilization of the untranslated 5'-exons located on the upstream region of the exon 1.     

Genomic structure and expression of human guanosine monophosphate reductase

Summary In vitro translation in the rabbit reticulocyte system and transient expression in Cos7 cells were performed to characterize the protein encoded by a chromosome 6-linked human cDNA clone, whose nucleotide sequence is homologous to that of Escherichia coli guanosine monophosphate reductase (GMP reductase) cDNA. The molecular weight of the peptide produced by the cDNA was about 37,000 Dalton, and the protein produced in the Cos7 cells exhibited GMP reductase activity, substantiating that the cDNA is for human GMP reductase. The corresponding genomic clones were obtained from two human genomic libraries. The gene spans about 50 Kb and is composed of 9 exons, which encode 345 amino acid residues. Organization of exons and introns was established by DNA sequencing of each exon and splicing junctions. The gene contains two potential SpI binding sites within exon 1, and a functional atypical polyadenylation signal in exon 9.     

Analysis of the human cysteine-rich protein gene (CSRP), assignment to chromosome 1q24-1q32, and identification of an associated MspI polymorphism.

The human cysteine-rich protein (hCRP) is encoded by a highly conserved and widely expressed serum-inducible immediate early response gene. hCRP contains two copies of the "LIM/double zinc-finger" motif. Using a characterized hCRP cDNA probe, we demonstrate that the human CRP gene (CSRP) is present in a single copy and that both mouse and human genomes contain one or more CRP-related genes detected by hybridization at low stringency. Using a panel of human x rodent somatic cell hybrids, the hCRP locus is assigned to chromosome 1. In situ hybridization of 3H-labeled CRP cDNA to human metaphase chromosomes confirms this assignment and permits regional localization to bands 1q24-1q32. A common MspI polymorphism is identified and mapped to intron 4 of the hCRP gene. The chromosomal localization and restriction site polymorphism should prove useful in future studies of the function of this gene.