Characterization Of Channel Catfish Ictalurus Punctatus T Cell Receptor Genes And Alloantigen Dependent Cytotoxic T Lymphocytes

Author : Evgueni Ivanovich Kountikov
Publisher :
ISBN 13 :
Total Pages : 262 pages
Book Rating : 4.:/5 (785 download)

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Download or read book Channel Catfish (Ictalurus Punctatus) CD45 written by Evgueni Ivanovich Kountikov and published by . This book was released on 2004 with total page 262 pages. Available in PDF, EPUB and Kindle. Book excerpt: Complementary DNA for the channel catfish protein tyrosine phosphatase CD45 molecule has been cloned and characterized. Similar to the CD45 homologs in other species, the catfish cDNA encoded all characteristic features of CD45: leader peptide; an alternatively spliced amino-terminus rich in O-glycosylation sites; a cysteine rich domain; three fibronectin three domains; a transmembrane region and two phosphotyrosine phosphatase domains. However analysis of CD45 cDNAs from various catfish cells demonstrated that catfish CD45 is unique and quite different from previously characterized CD45 molecules in that it contains a large number of alternatively spliced exons. Sequence analyses of cDNAs derived from the catfish clonal B cell line 3B11 indicated that this cell line alone can express up to thirteen alternatively spliced exons. Furthermore sequence similarity among the alternatively spliced exons suggested duplication events. To establish the exact number of alternatively spliced exons and further characterize their organization, the entire gene for catfish CD45 has been sequenced. The analysis demonstrated that the catfish CD45 functional gene spans 32 kb and contains 49 exons. In comparison, the human CD45 gene consists of 34 exons and the pufferfish gene consists of 30 exons. The difference in the catfish, otherwise structurally conserved, gene is mainly due to presence of 18 alternatively spliced exons. Those exons are apparently derived through several duplications. In addition, duplication events were likely involved in generating six CD45 pseudogenes that are found in the catfish genome. These six pseudogenes are all truncated on the 3 ' end. The presence of a CD45 pseudogene in the pufferfish genome, organized very similarly to the catfish pseudogenes, suggests that this gene duplication is an early event (400 million years ago) in fish. To characterize catfish CD45 at the protein level, specific monoclonal antibodies were produced. CD45 protein from a clonal B cell line was purified and used for immunization and screening purposes. Five mAbs were found reactive to the surface expressed CD45 protein and capable of immunoprecipitating phosphotyrosine phosphatase activity from B cell lysates. Fluorescence activated cell sorting showed that these mAbs are specific to at least two distinct CD45 isoforms. The specificity of two of the mAbs, SF8 and HB4, was confirmed by protein sequencing by tandem mass spectrometry of peptides derived from immunoprecipitated protein. The SF8 mAb was also used to demonstrate that catfish T cells express smaller CD45 isoforms then catfish B cells, a situation reminiscent of what occurs in mammals.