SUBTRACTION PCR CLONING OF NOVEL LYMPHOCYTE-SPECIFIC PROTEIN TYROSINE PHOSPHATASE GENES

新型淋巴细胞特异性蛋白酪氨酸磷酸酶基因的减法PCR克隆

• 批准号: 06807033
• 负责人: KISHIHARA Kenji
• 金额: $ 1.02万
• 依托单位: KYUSHU UNIVERSITY
• 依托单位国家: 日本
• 项目类别: Grant-in-Aid for General Scientific Research (C)
• 财政年份: 1994
• 资助国家: 日本
• 起止时间: 1994 至 1995
• 项目状态: 已结题
• 来源: https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-06807033/
• 关键词: LYMPHOCYTE; PROTEIN TYROSINE PHOSPHATASE; PCR; CLONING

We've tried to cloned novel protein tyrosine phosphatase (PTP) genes whose products are involved in a signal transduction mechanism in lymphocytes for two years. A subtraction PCR method was developed and improved to clone the new genes. Finally, we've obtained two PCR products (B9 and J15) which are parts of new gene candidates estimated by homology analysis of gene banks. B9 and J15 are obtained from a murine thymoma (BW5147) and human T-lymphoma (Jurkat), respectively. Unfortunately, human homologue of complete B9 PTP gene has been reported in the way of analyzing the gene. Therefore, we concentrated to analyze the J15 genes. At first, we tried to clone a full length of the J15 gene in a cDNA library derived from human peripheral mononuclear lymphocytes (PBL). As the result of the screening, ca. 4 kb cDNA containing the J15 sequence was obtained. The size of the cDNA was consistent with the result of Northern blot analysis. The results of structural analysis of the J15 cDNA clone we … More re as follows. (1) The clone contains a hydrophobic amino acid-rich sequence typical of a transmembrane region. (2) The cytoplasmic domain contains a tandem repeat of two PTP domains. (3) The J15 clone derived from Jurkat has a deletion of amino acid residues in one of the PTP domains in comparison to the sequence of the J15 clone from PBL.The properties of the J15 PTP described above strongly suggest that the J15 PTP is a novel transmembrane PTP such as CD45 and LAR.Moreover, an additional band of ca. 5 kb was detected by the Northern blot analysis, implying that The J15 PTP may have an isoform. Furthermore, the J15 gene expression was also detected in non-lymphoid cells and organs. The finding that the J15 clone derived from Jurkat has a deletion of amino acid residues may be interesting if the deletion inactivates the PTP activity because a mechanism of transformation of Jurkat cells is still unknown. The results of this project will be presented at major annual meetings for immunology, biochemistry and molecular biology in this year, 1996. Our manuscript of this project is in submission. Less

两年来，我们一直试图克隆新的蛋白酪氨酸磷酸酶(PTP)基因，其产物参与淋巴细胞的信号转导机制。建立并改进了一种用于克隆新基因的消减PCR方法。最后，通过基因库的同源性分析，我们得到了两个新的候选基因的部分聚合酶链式反应产物(B9和J15)。B9和J15分别取自小鼠胸腺瘤(BW5147)和人T淋巴瘤(Jurkat)。不幸的是，在分析B9 PTP基因的方法中，已经报道了人类完整的B9 PTP基因的同源性。因此，我们集中分析了J15基因。首先，我们试图从人外周血单个核细胞(PBL)的cDNA文库中克隆出J15基因的全长。作为筛选的结果，获得了含有J15序列的约4kb的cDNA。该基因的大小与Northern印迹分析结果一致。J15cDNA克隆WE…的结构分析结果更多内容如下。(1)该克隆含有典型的跨膜区富含氨基酸的疏水序列。(2)胞质结构域含有两个PTP结构域的串联重复序列。(3)来自Jurkat的J15克隆与来自PBL的J15克隆相比，在PTP结构域的一个氨基酸残基缺失。上述J15 PTP的性质强烈地表明J15 PTP是一种新的跨膜PTP，如CD45和LAR。此外，Northern印迹分析还检测到一条约5kb的额外条带，表明J15 PTP可能具有异构体。此外，J15基因在非淋巴样细胞和器官中也有表达。从Jurkat衍生的J15克隆具有氨基酸残基缺失的发现可能是有趣的，如果该缺失使PTP活性失活，因为Jurkat细胞的转化机制尚不清楚。该项目的成果将于1996年在免疫学、生物化学和分子生物学的主要年度会议上公布。我们关于这个项目的手稿正在提交中。较少