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Characterization of 32b1, a partial cDNA encoding novel sequence having high similarity with the immediate early gene, Cyr61

University of British Columbia

The 32b1 cDNA clone was isolated from a λgtll library made from a Jurkat T lymphocyte cell line during a search for a transactivating factor for the HIV-LTR that was responsible for Ras responsiveness. The clone was 1.3kb in length and the fulllength clone, postulated from Northern blot experiments done in human tissues, was estimated to be approximately 2.6 kb. This led to a comprehensive search of different libraries of human blood leukocytes, lung and brain cell lines to find the missing 5'end. Although the original segment of cDNA clone was isolated many times, novel sequence was never found experimentally. A search in the NCBI database revealed new 5' end sequence as well as evidence linking 32b1 with wound repair. Further examination of the gene induction kinetics were carried out via Northern Hybridizaiton analysis. Unexpectedly, the gene was not found in peripheral blood leukocytes and none of the cell lines that the cDNA clone was isolated from was seen to express it. The only cell line that did express it was a Rat-2 fibroblast cell line where a message having high sequence similarity with 32b1 was seen to be induced by serum, EGF but not by the phorbol ester, PMA. The stability of the rat mRNA transcript was also studied from induction patterns after actinomycin D treatment with and without cycloheximide. It was found to have an transient half life of less than 30 minutes. The induction of the 32b1 mRNA message was prolonged after treatment with cycloheximide which implicated that translation had an effect on its mRNA stability. These results allowed certain conclusions to be made about 32b1 that can be a basis in further research into its function in cell proliferation. The focus of this research was to complete the cDNA sequence, find the missing 5' end of the cloned cDNA and to characterize the expression of the gene.

Thesis/Dissertation

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2009-03-11

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http://hdl.handle.net/2429/5929

Biochemistry and Molecular Biology

University of British Columbia

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