Use of degenerate primers and touchdown PCR to amplify a halogenase gene fragment from Streptomyces venezuelae ISP5230.
Consensus amino acid sequences of FADH(2)-dependent bacterial halogenases were used to design PCR primers amplifying a halogenase gene fragment from the chloramphenicol producer Streptomyces venezuelae ISP5230. The sequence-specific degenerate primers (MPF1 and MPR2) were used with a touchdown PCR procedure in the first PCR-assisted cloning of a halogenase gene fragment. In the region of the 290-bp PCR product containing the reverse primer, the deduced amino acid sequence exhibited characteristics of a beta-alpha-beta fold present in FAD-binding sites of certain monooxygenases. When used to probe Southern blots of restriction-enzyme-digested DNA, the [alpha-(32)P]dCTP-labeled PCR product hybridized specifically with DNA fragments from genomic DNA of S. venezuelae ISP5230. Primers MPF1 and MPR2 also allowed amplification by PCR of approximately 290-bp DNA fragments from several other streptomycetes. The fragments from Streptomyces aureofaciens NRRL2209 and Streptomyces coelicolor A3(2) showed sequence identity with halogenase genes from these species. Thus, the PCR primers are of potential value for amplification and subsequent isolation of actinomycete halogenase genes.
Piraee
M
M
Biology Department, Dalhousie University, Halifax, Nova Scotia, Canada B3H 4J1.
Vining
Leo C
LC
D000595:Amino Acid Sequence; D015139:Blotting, Southern; D017931:DNA Primers / Q000235:genetics*; D005798:Genes, Bacterial / Q000235:genetics*; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics*; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000145:classification / Q000201:enzymology / Q000235:genetics*
D017931:DNA Primers; D010088:Oxidoreductases