Thermophilic bacterial DNA polymerases with reverse-transcriptase activity.
Conserved motifs found in known bacterial polI DNA polymerase sequences were identified, and degenerate PCR primers were designed for PCR amplification of an internal portion of polI genes from all bacterial divisions. We describe here a method that has allowed the rapid identification and isolation of 13 polI genes from a diverse selection of thermophilic bacteria and report on the biochemical characteristics of nine of the purified recombinant enzymes. Several enzymes showed significant reverse-transcriptase activity in the presence of Mg2+, particularly the polymerases from Bacillus caldolyticus EA1, Caldibacillus cellovorans CompA.2, and Clostridium stercorarium.
Shandilya
Harini
H
Invitrogen Corporation, 1600 Faraday Avenue, Carlsbad, CA 92008, USA.
Griffiths
Kate
K
Flynn
Elizabeth K
EK
Astatke
Mekbib
M
Shih
Po-Jen
PJ
Lee
Jun E
JE
Gerard
Gary F
GF
Gibbs
Moreland D
MD
Bergquist
Peter L
PL
D001407:Bacillus / Q000201:enzymology / Q000235:genetics; D001419:Bacteria / Q000201:enzymology* / Q000235:genetics; D001483:Base Sequence; D003001:Cloning, Molecular; D003013:Clostridium / Q000201:enzymology / Q000235:genetics; D004256:DNA Polymerase I / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D006358:Hot Temperature; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D008969:Molecular Sequence Data; D012194:RNA-Directed DNA Polymerase / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid
D017931:DNA Primers; D004269:DNA, Bacterial; D011994:Recombinant Proteins; D012194:RNA-Directed DNA Polymerase; D004256:DNA Polymerase I; D008274:Magnesium