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

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。建立并改进了减法PCR克隆新基因的方法。最后，我们获得了两个PCR产物（B9和J15），它们是通过基因库同源性分析估计的新候选基因的一部分。B9和J15分别从小鼠胸腺瘤（BW5147）和人t -淋巴瘤（Jurkat）中获得。不幸的是，在分析该基因的方法中，已经报道了完整的b9ptp基因的人类同源物。因此，我们集中对J15基因进行分析。首先，我们尝试从人外周血单核淋巴细胞（PBL） cDNA文库中克隆J15基因的全长。筛选得到含有J15序列的cDNA约4 kb。cDNA的大小与Northern blot分析结果一致。J15 cDNA克隆的结构分析结果如下。(1)该克隆含有一个典型的跨膜区富含氨基酸的疏水序列。(2)细胞质结构域包含两个PTP结构域的串联重复序列。(3)与来自PBL的J15克隆相比，来自Jurkat的J15克隆在PTP结构域的一个氨基酸残基缺失。上述J15 PTP的性质有力地表明J15 PTP是一种新型的跨膜PTP，如CD45和LAR。此外，Northern blot分析还检测到约5 kb的额外条带，这意味着J15 PTP可能具有同工型。此外，在非淋巴样细胞和器官中也检测到J15基因的表达。来自Jurkat的J15克隆存在氨基酸残基缺失，如果缺失使PTP活性失活，这一发现可能是有趣的，因为Jurkat细胞的转化机制尚不清楚。该项目的结果将在1996年免疫学、生物化学和分子生物学的主要年度会议上提出。我们这个项目的手稿已经提交了。少

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