Smith, Martin Alexander; (1995) Purification and cDNA sequencing of C4l: An actin-associated protein. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The actin network is crucial for many cellular events such as cell movement, phagocytosis, cell division and movement of cell surface receptors. Control of the actin network lies with a large number of actin-associated proteins that regulate the polymerisation status, interaction and geometry of actin. Protein C4 is an actin-associated polypeptide doublet described by Shapland et al (1988; 1993). C41 is the only protein C4 isoform present in motile cells such as lymphocytes and transformed mesenchymal cells, and the objectives of this work were to see if C41 is associated with actin filament bundles in transformed cells and to characterise the molecule at the level of the gene. My initial studies have shown that C41 is associated with vestigial actin filament bundles found in transformed mesenchymal cells. To further investigate C41 I have purified the molecule to more than 90[percent] homogeneity from human T cell lymphoma (HTCL), and then used degenerate oligonucleotides to obtain internal C41 sequence from reverse transcribed HTCL mRNA (RT-PCR). C41 gene-specific oligonucleotides were used to obtain both the 5'- end (anchor-PCR) and 3'- end (3'- rapid amplification of cDNA ends) from reversed transcribed HTCL mRNA. Cloning and sequencing of these overlapping PCR products gives the full length coding sequence for C41 from HTCL. The translated product of C41 open reading frame is 199 amino acids in length, with a calculated molecular weight of 22,381 Da and an estimated pi of 8.09. Northern blotting reveals that C41 is expressed as a single message of 1.44 Kb which is apparently up- regulated in oncogenically transformed lymphocytes. Database searches indicate that C41 is a previously undescribed molecule. Regions of homology, including a potential actin-binding domain and phosphorylation sites, which are shared between C41 and other proteins such as rat transgelin (Prinjha et al, 1994), chicken SM22[alpha] (Pearlstone et al, 1987), rat NP25 (unpublished), chicken calponins alpha and beta (Takahashi and Nadal-Ginard, 1991), and Drosophila mp20 (Ayme-Southgate et al, 1989) suggest that all of these proteins may be classified as members of a new transgelin-like multigene family.

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