Molecular cloning and nucleotide sequence of the DNA polymerase gene from Thermus flavus.

The thermostable properties of the DNA polymerase activity from Thermus aquaticus (Taq) have contributed greatly to the yield, specificity, automation and utility of the polymerase chain reaction method for amplifying DNA (1). We report here the cloning and nucleotide sequence of a DNA polymerase gene from another thermophilic bacterium — Thermus flavus (Tfl). The cloning of the DNA polymerase gene was perfomed by the rapid filter assay as described previously (2). The nucleotide sequence was determined by dideoxy chain termination method of Sanger (3) using Taq DNA polymerase (Taq Poll) or modified T7 DNA polymerase and substituting deaza-7-dGTP for dGTP. The resulting recombinant E.coli plasmid pTfl4 involved an open reading frame of 831 codons. In addition to the ORF of the DNA polymerase gene we have cloned the upstream (551 b.p.) and downstream (359 b.p.) regions. In the upstream region there is a near-consensus ribosome binding site at position (—20 to -15) and no sequences are homologous to known E. coli transcription regulatory sequences. Therefore, we assume, that the observed expression of the Tfl polymerase (Tfl Poll) gene can be due to transcription from the vector borne /ac-promoter and translation reinitiation provided by the Shine—Dalgarno sequence of the cloned gene. Comparison of the Tfl Poll and Taq Poll amino acid sequences revealed a significant amino acid sequence similarity. Comparing the Tfl poll amino acid sequence to the E.coli Poll sequence shows, that amino acid residues essential for E.coli Poll activity are conserved in Tfl Poll.