种新的蛋白酪氨酸磷酸酶基因的克隆、定位和组织表达谱研究

从人胎肝cDNA文库分离出一长度为5248bp的cDNA克隆,该基因包含26个外显子和25个内含子,染色体定位于在某些肿瘤细胞中易缺失的3p21.1-21.33.其可读框编码1636个氨基酸,该蛋白属于蛋白酪氨酸磷酸酶(PTP)家族,其C端有一个典型的PTP结构域,N端含有约800氨基酸残基的BRO1样结构域及随后2个可能的SH3结构域结合位点,在这两个结构域之间及C末端还各有一个脯氨酸富集区.Northern杂交和点杂交分析显示,该基因以大约5.4kb的单一转录物广泛表达于人体各种组织,而且在人部分肿瘤细胞中高表达.结果提示,人源PTP-TD14是一个新的蛋白酪氨酸磷酸酶。 Abstract:A human cDNA of 5248bp encoding a novel protein tyrosine phosphatase PTP-TD14(1636aa) has been isolated from fetal liver.The gene is located at chromosome 3p21.3,an area frequently deleted in many types of cancer,and composed of at least 26 exons and 25 introns.The phosphatase has unique features in its domain structure:a tyrosine phosphatase domain,a C-terminal PEST motif,two SH3-binding motifs,two proline-rich region and an N-terminal domain similar to yeast BRO1 (a yeast protein that is involved in the mitogen-activated protein kinase signaling pathway).Northern blot and dot blot hybridizations indicate that it is expressed ubiquitously in human 50 tissues and 7 cancer cell lines.Thus,it is a novel protein tyrosine phosphatase gene located on 3p21.3.     

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 .     

水稻花药绒毡层特异表达基因RA39的克隆与表达特性分析

利用cDNA减法杂交、差异杂交筛选和RACE等技术,从水稻(Oryza sativa L. ssp. japonica)中克隆了一个新的绒毡层特异性cDNA,其编码基因被命名为RA39.该cDNA长1 013 bp, 编码由298个氨基酸残基组成的多肽.RA39是一个单拷贝基因,在绒毡层细胞中特异性表达,在小孢子母细胞减数分裂期的绒毡层细胞中有较高的表达活性.用PSORT和PPSEARCH软件进行的结构分析揭示出RA39蛋白的N端是一个由17个氨基酸残基组成的信号肽,该蛋白包含一个跨膜区和一个胞质尾区两个主要结构域以及多个蛋白激酶的磷酸化位点.     

水稻花药绒毡层特异表达基因RA39的克隆与表达特性分析

利用cDNA减法杂交,差异杂交筛选和RACE等技术。从水稻（Orza sativaL.ssp.japonica)中克隆了一个新的绒毡层特异性cDNA,其编码基因被命名为RA39。该cDNA长1013bp。编码由298个氨基酸残基组成的多肽RA39是一个单拷贝基因。在绒毡层细胞中特异性表达。在小孢子母细胞减数分裂期的绒毡层细胞中有较高的表达活性。用PSORT和PPSEARCH软件进行的结构分析揭示出RA39蛋白的N端是一个由17个氨基酸残基组成的信号肽,该蛋白包含一个跨膜区和一个胞质尾区两个主要结构域以及多个蛋白激酶的磷酸化位点。     

一个大鼠心肌缺血-再灌诱导表达上调的新基因Mip1的克隆和特性分析

采用生物信息学方法和5′cDNA末端快速扩增法(RACE)技术相结合,克隆了一个大鼠心肌缺血-再灌诱导表达上调的新基因Mip1,并经RT-PCR测序及多组织膜RNA印迹证实.生物信息学分析显示,MIP-1定位于大鼠染色体1q12区,含5个外显子和4个内含子,开放阅读框(ORF)为1 827 bp,编码608个氨基酸,其编码蛋白N端含KRAB结构域,C端含14个连续的C2H2型锌指蛋白结构域,氨基酸第277位至293位为双向的核定位信号,多组织膜RNA印迹显示该基因在脑组织表达最高,其次是心脏,在其他组织表达较低或无表达.进一步深入研究该基因的功能具有重要生物学意义.     

人类锌指结构新基因ZNF18的克隆和表达谱分析

在很多转录因子中发现的锌指结构,被认为在人类心脏的发育和相关疾病的发生过程中发挥重要的作用。本文报道了克隆和表达分析人类新的锌指蛋白基因ZNF18。该基因cDNA长2 767 bp,编码一个有549个氨基酸的蛋白,这一蛋白含有一个SCAN结构域,一个KRAB结构域和5个连续的C2H2型锌指结构域。ZNF18蛋白与小鼠Zfp535有77％的同源性。ZNF18基因定位于人染色体17p12~p13,包含9个外显子和8个内含子。以ZNF18全长编码区为探针进行Northern杂交,结果显示ZNF18在成体小鼠各组织中广泛表达,但在心脏中低丰度表达。整体原位杂交结果显示,ZNF18基因在小鼠胚胎的表达有很高的动态性。ZNF18主要在E7.5小鼠胚胎的胚外组织表达,E8.5出现了胚胎躯干前端表达。ZNF18从E9.0开始在胚胎的心脏和尾部表达,尤其在E10.5胚胎的心脏高丰度表达。这提示ZNF18基因与心脏发育过程可能有密切的关系。     

斜纹夜蛾核型多角体病毒BamHI—J片段序列分析

报道了斜纹夜蛾核型多角体病毒（SpltMNPV)BamHI-J片段的序列结构。该片段定位于SpltMNPV基因组25.8-29.9图单位（msp unit）,包括4个完整的开放读码框,几丁质酶基因（chiA）的3′端部分序列和一个同源区（hr）的部分序列。4个完整的读码框包括lef-8基因,杆状病毒J结构域蛋白基因（baculovirus J domain protein gene,bjdp),ORF570和ORF165。序列分离表明：ORF570与毒蛾核型多角体病毒（Lymantria dispar MNPV）的解旋酶－2基因有31％的氨基酸同源性。ORF165为SpltMNPV特有。J结构域蛋白在其他杆状病毒基因组中尚未见报道,其氨基酸序列N端存在J结构域,推断该蛋白质具有与DnaJ蛋白类似特征。lef-8基因编码的氨基酸与已报道的杆状病毒基因组中的lef-8基因编码的氨基酸具有高的同源性,且其C端具有与其他杆状病毒LEF－8类似的保守序列CIKICGIHGQKG。     

小鼠睾丸和卵巢特异表达基因Zfp474的克隆与特征分析

运用NCBI中的数据库消减杂交(Digital Differential Display,DDD)分析方法,从小鼠睾丸组织中分离了一个含有C2HC／C3H结构的新型锌指蛋白基因——ZIM74(GenBank登录号：AY350709)。通过推导和进一步的RT-PCR实验证实：该基因含4个外显子,gDNA在染色体上跨度29869bp,定位于小鼠染色体18D1。cDNA编码一个含347个氨基酸的新蛋白,带有C2HC／C3H结构域。Northern杂交结果显示：该基因含有2．37kb大小的唯一转录本,主要在睾丸中强表达,卵巢中有表达,而在其他组织中该基因无表达。结果提示：Zfp474基因对精于发生和卵母细胞的发育可能起重要作用。     

新基因人MOB cDNA的克隆与功能分析

人MOB基因被认为是在脑组织中高表达的5次跨膜而功能未知的膜蛋白.从胎儿和成人肾脏消减杂交cDNA文库中获得了一条新的表达序列标签（expressed sequence tag,EST）序列,该序列对应于功能未知的MOB基因.利用生物信息学分析工具和分子生物学技术,从胎儿肝脏中成功地克隆了人MOB基因cDNA序列,同时也获得了含有完整开放读码框的大鼠和鸡MOB的电子全长序列.人MOB基因定位于10q11.1～11.2之间,含有一个1 242 bp的开放阅读框,第415位开始的ATG可能是翻译起始位点.核酸序列相似性搜索发现,其他种属中存在与之高度相似的EST序列,如爪蟾（79%）、羊（87%）、猪（94%）、牛（93%）.蛋白质序列相似性搜索发现,人MOB与大鼠、小鼠和鸡MOB有97%、97%、91％的相似性,与其他一些不同种属来源的假想蛋白有45%～73%的相似性.不同物种之间MOB基因核酸和蛋白质序列的高度相似说明,MOB是进化上高度保守的蛋白质.保守结构域分析发现,人、小鼠、大鼠和鸡MOB结构域组成完全一致,N端均与不育α基序（sterile alpha motif,SAM）结构域显著匹配,随后出现匹配显著性稍差的几个结构域.核酸和蛋白质序列的保守性提示,除N端约70个氨基酸组成SAM结构域外,其余的保守氨基酸可能形成一个新的保守的MOB结构域.表达谱分析表明,人MOB基因在所有被检组织和细胞中都表达.亚细胞定位研究显示,人MOB广泛表达于细胞内,主要在细胞核.DNA含量测定结果表明,人MOB基因过表达并不影响HeLa细胞的细胞周期和凋亡.总之,人MOB蛋白是一个在多种组织和细胞中广泛表达、细胞内广泛分布、进化上十分保守的蛋白质,可能通过特定蛋白质之间的相互作用,参与多种发育过程的调节.其过表达不影响HeLa细胞的周期和凋亡.     

菊芋类金属硫蛋白基因htMT2的克隆及其表达特征分析

从菊芋 (HelianthustuberosusL .)块茎cDNA文库中得到了一个新的植物类金属硫蛋白基因htMT2的cDNA序列 ,全长 5 0 9bp ,包括 2 4 0bp的开放阅读框、6 2bp的 5′端非翻译区、2 0 7bp的 3′端非翻译区。通过PCR获得了 2个htMT2编码区的部分基因组片段htMTG_1及htMTG_2 ,长度分别为 986bp和 982bp。分析表明两个基因组片段均包含 3个外显子及 2个内含子 ,编码一个由 79个氨基酸残基组成的多肽 ,与从htMT2推测的多肽完全一致 ,该多肽具有植物类金属硫蛋白的典型结构特征 ,N端及C端结构域富含Cys ,分别具有 8个和 7个Cys残基 ,上述两个结构域被一个无Cys的中间区分开。Southern杂交结果表明 ,htMT2在菊芋基因组中以小基因家族的形式存在。Northern杂交结果表明htMT2在叶片、叶柄、茎及块茎中均有表达 ,在茎中有较高水平的表达 ,但在根中未检测到杂交信号。经Cu2 处理后 ,htMT2在茎中的表达量显著降低。与其他 2型金属硫蛋白的序列同源性比较及htMT2对金属离子处理的反应均表明 ,htMT2是一种新的植物类金属硫蛋白基因。     

HD-PTP: A novel protein tyrosine phosphatase gene on human chromosome 3p21.3

A human cDNA encoding a novel protein tyrosine phosphatase has been isolated. The phosphatase has unique features in its domain structure: a "Zn-hand" domain containing several SH3-binding motifs, a tyrosine phosphatase domain, a C-terminal PEST motif, and an N-terminal domain similar to yeast BRO1, an apoptosis-related mammalian AIP1 and to a RHO-binding protein, Rhophilin. The gene is located at chromosome 3p21.3, an area frequently deleted in many types of cancer, especially within the functionally defined narrow region. The gene may be a human homolog of the rat PTP-TD14 gene reported by others, which can suppress H-ras-mediated transformation. We identified a hemizygous missense mutation in a lung cancer cell line. Thus, the phosphatase gene may be a candidate for one of the tumor suppressor genes located on 3p21.3.     

Interaction of Axl receptor tyrosine kinase with C1-TEN,a novel C1 domain-containing protein with homology to tensin

Axl receptor tyrosine kinase is implicated in several malignancies and is the receptor for the vitamin K-dependent growth factor Gas6. From a yeast two-hybrid screen of protein-protein interactions with the Axl cytoplasmic domain, we detected a previously uncharacterised SH2 domain-containing protein. We cloned two novel splice variants of this protein that give rise to 1409- and 1419-amino acid proteins, differing only in their N-terminal residues and yielding a 150-kDa protein product by in vitro translation. The Axl-interacting C-terminus contains a tandem SH2 and PTB domain combination homologous to the focal adhesion protein tensin. We detected interaction of Axl with both domains in mammalian cells by co-immunoprecipitation and two-hybrid analyses. In addition, the protein possesses an N-terminal putative phorbol ester-binding C1 domain as well as a central tyrosine phosphatase motif. Thus, we have named the protein C1 domain-containing phosphatase and TENsin homologue (C1-TEN). Northern blot analysis of C1-TEN in human tissues revealed highest expression in heart, kidney, and liver. In summary, we have identified a novel multi-domain intracellular protein that interacts with Axl and which may furthermore be involved in other signal transduction pathways.     

Protein tyrosine phosphatase containing SH2 domains: characterization, preferential expression in hematopoietic cells, and localization to human chromosome 12p12-p13.

Protein tyrosine phosphorylation has been implicated in the growth and functional responses of hematopoietic cells. Recently, approaches have been developed to characterize the protein tyrosine phosphatases that may contribute to regulation of protein tyrosine phosphorylation. One novel protein tyrosine phosphatase was expressed predominantly in hematopoietic cells. Hematopoietic cell phosphatase encodes a 68-kDa protein that contains a single phosphatase conserved domain. Unlike other known protein tyrosine phosphatases, hematopoietic cell phosphatase contains two src homology 2 domains. We also cloned the human homolog, which has 95% amino acid sequence identity. Both the murine and human gene products have tyrosine-specific phosphatase activity, and both are expressed predominantly in hematopoietic cells. Importantly, the human gene maps to chromosome 12 region p12-p13. This region is associated with rearrangements in approximately 10% of cases of acute lymphocytic leukemia in children.     

Analysis of the human TrkB gene genomic organization reveals novel TrkB isoforms, unusual gene length, and splicing mechanism.

We determined the gene structure of the human TrkB gene. The gene is unusually large and spans at least 590 kbp. It contains 24 exons. Using alternative promoters, splicing, and polyadenylation sites, the gene can create at least 100 isoforms, that can encode 10 proteins. RT-PCR and Northern blot analysis reveals that only three major protein isoforms are generated by the gene: the full length receptor, an isoform lacking the tyrosine kinase domain, and a novel isoform lacking the tyrosine kinase domain but containing a Shc binding site. This novel isoform, TrkB-T-Shc is generated by the use of a new alternative exon 19. It is expressed only in brain. TrkB-T-Shc protein is located in the plasma membrane. Coimmunoprecipitation experiments show that TrkB-T-Shc is not phosphorylated by the full length receptor, indicating that it could be a negative regulator of TrkB signaling in the brain.     

Two substrate-targeting sites in the Yersinia protein tyrosine phosphatase co-operate to promote bacterial virulence

YopH is a protein tyrosine phosphatase and an essential virulence determinant of the pathogenic bacterium Yersinia. Yersinia delivers YopH into infected host cells using a type III secretion mechanism. YopH dephosphorylates several focal adhesion proteins including p130Cas in human epithelial cells, resulting in disruption of focal adhesions and cell detachment from the extracellular matrix. How the C-terminal protein tyrosine phosphatase domain of YopH targets specific substrates such as p130Cas in the complex milieu of the host cell has not been fully elucidated. An N-terminal non-catalytic domain of YopH binds p130Cas in a phosphotyrosine-dependent manner and functions as a novel substrate-targeting site. The structure of the YopH protein tyrosine phosphatase domain bound to a model phosphopeptide substrate was solved and the resulting structure revealed a second substrate-targeting site ('site 2') within the catalytic domain. Site 2 binds to p130Cas in a phosphotyrosine-dependent manner, and co-operates with the N-terminal domain ('site 1') to promote efficient recognition of p130Cas by YopH in epithelial cells. The identification of two substrate-targeting sites in YopH that co-operate to promote epithelial cell detachment and bacterial virulence reinforces the importance of protein-protein interactions for determining protein tyrosine phosphatase specificity in vivo, and highlights the sophisticated nature of microbial pathogenicity factors.     

Solution structure of the ubiquitin-binding domain in Swa2p from Saccharomyces cerevisiae

The SWA2/AUX1 gene has been proposed to encode the Saccharomyces cerevisiae ortholog of mammalian auxilin. Swa2p is required for clathrin assembly/dissassembly in vivo, thereby implicating it in intracellular protein and lipid trafficking. While investigating the 287-residue N-terminal region of Swa2p, we found a single stably folded domain between residues 140 and 180. Using binding assays and structural analysis, we established this to be a ubiquitin-associated (UBA) domain, unidentified by bioinformatics of the yeast genome. We determined the solution structure of this Swa2p domain and found a characteristic three-helix UBA fold. Comparisons of structures of known UBA folds reveal that the position of the third helix is quite variable. This helix in Swa2p UBA contains a bulkier tyrosine in place of smaller residues found in other UBAs and cannot pack as close to the second helix. The molecular surface of Swa2p UBA has a mostly negative potential, with a single hydrophobic surface patch found also in the UBA domains of human protein, HHR23A. The presence of a UBA domain implicates Swa2p in novel roles involving ubiquitin and ubiquitinated substrates. We propose that Swa2p is a multifunctional protein capable of recognizing several proteins through its protein-protein recognition domains.     

A novel higher plant protein tyrosine phosphatase interacts with SNF1-related protein kinases via a KIS (kinase interaction sequence) domain

A novel protein phosphatase in Arabidopsis thaliana was identified by database searching. This protein, designated AtPTPKIS1, contains a protein tyrosine phosphatase (PTP) catalytic domain and a kinase interaction sequence (KIS) domain. It is predicted to interact with plant SNF1-related kinases (SnRKs), representing central regulators of metabolic and stress responses. AtPTPKIS1 has close homologues in other plant species, both dicots and monocots, but is not found in other kingdoms. The tomato homologue of AtPTPKIS1 was expressed as a recombinant protein and shown to hydrolyse a generic phosphatase substrate, and phosphotyrosine residues in synthetic peptides. The KIS domain of AtPTPKIS1 was shown to interact with the plant SnRK AKIN11 both in vivo in the yeast two-hybrid system, and in vitro in a GST-fusion 'pull down' assay. The genomes of Arabidopsis and other plants contain further predicted proteins related to AtPTPKIS1, which could also interact with SnRKs and act in novel regulatory and signalling pathways.