Cloning of cDNAs for two beta-tubulin isotypes expressed in murine T cell lymphoma, L5178Y and analysis of their translation products.

Abstract

Three beta-tubulin isoforms, M beta Ib, M beta Ia and M beta II, were detected by isoelectric focusing gel electrophoresis (IEF) in extracts of cultured cells of a mouse T cell lymphoma, L5178Y. To investigate the origin(s) of the isoforms, we have isolated cDNA clones encoding beta-tubulin from a cDNA library prepared from poly (A)+ RNA of L5178Y cells. Seventeen cDNA clones carrying the entire coding sequences of beta-tubulin were isolated and classified into two distinct isotypes, represented by two clones designated pMT27 and pMT49, according to the results of restriction mapping. On the basis of the nucleotide sequences of the two cDNAs, pMT27 and pMT49 were identified as mouse beta-tubulin isotypes 3 and 5, respectively. By using in vitro translation products of hybrid-selected mRNAs and of the SP6 in vitro transcripts of the cDNAs, polypeptides encoded by the two cDNA clones were analyzed by IEF. We found that pMT27 and pMT49 encode M beta Ib and M beta Ia, respectively. In addition, M beta II was detected in translation products of mRNA specifically hybridized to pMT49, but not in those of the in vitro transcript of pMT49 DNA. These results suggest that M beta II is the translation product of mRNA whose 3'-untranslated region is highly homologous to that of pMT49.