PROKARYOTIC METALLOTHIONEIN GENE CHARACTERIZATION AND EXPRESSION - CHROMOSOME CRAWLING BY LIGATION-MEDIATED PCR

A strategy is described for the characterization of a novel gene employing the polymerase chain reaction, inosine-containing oligonucleotide primers, and ligation mediated, or anchored polymerase chain reaction (APCR). The same primers, designed from the known protein sequence, are used to amplify the coding region of the gene and subsequently to 'chromosome crawl' by APCR. This strategy was applied to the characterization of a prokaryotic metallothionein gene, designated smtA, from the cyanobacterium Synechococcus PCC 6301 ( = Anacystis nidulans). The abundance of smtA transcripts was examined in extracts from cells exposed to heat shock and elevated concentrations of cadmium, zinc and copper ions. There was no detectable change in smtA transcript abundance following exposure to heat shock, while exposure to all three metal ions led to an increase in abundance. A smtA homologue was also identified in Synechococcus PCC 7942 ( = Anacystis nidulans R2).