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

1995 Fiscal Year Final Research Report Summary

• Project/Area Number: 06807033
• Research Category: Grant-in-Aid for General Scientific Research (C)
• Allocation Type: Single-year Grants
• Research Field: Immunology
• Research Institution: KYUSHU UNIVERSITY
• Principal Investigator: KISHIHARA Kenji MEDICAL INSTITUTE OF BIOREGULATION, KYUSHU UNIVERSITY, ASSISTANT PROFESSOR, 生体防御医学研究所, 助手 (80214774)
• Co-Investigator(Kenkyū-buntansha): YOSHIDA Hiroki MEDICAL INSTITUTE OF BIOREGULATION, KYUSHU UNIVERSITY, ASSISTANT PROFESSOR, 生体防御医学研究所, 助手 (40260715)
• Project Period (FY): 1994 – 1995
• Keywords: LYMPHOCYTE / PROTEIN TYROSINE PHOSPHATASE / PCR / CLONING

Research Abstract

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 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.