Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.7.7 (DNA polymerase)
 17,007 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

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.
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PMID:Thermophilic bacterial DNA polymerases with reverse-transcriptase activity. 1519 5

A number of thermophilic bacteria have been surveyed for possessing reverse transcriptase genes using a degenerate primer approach derived from an alignment of known group II intron encoded reverse transcriptases (RT) from mesophilic prokaryotes and eukaryotes. Six out of 34 thermophilic isolates gave a PCR product that was indicative of an RT internal fragment on sequencing. A putative RT from Bacillus caldolyticus strain EA1 was isolated by genomic walking and cloned into an Escherichia coli expression vector. The recombinant protein proved to be insoluble and was unable to be recovered from the insoluble fraction of lysates of E. coli. The RT was successfully expressed in a baculovirus vector although yields remained low. We followed RT activity during purification using the poly(rC)*p(dG)(12-18), which specifically detects only RNA-dependent DNA polymerase activity. We could not detect incorporation of dTTP into poly(rC) )*p(dG)(12-18) when using uninfected Sf21 lysates and conclude that the substrate is not a template for DNA-dependent DNA polymerase. Although a high level of RT activity was detected in the total cell protein, when compared to the activity detected in the soluble fraction, only about 10% of the activity was soluble. Sequence comparisons showed significant differences between the EA1-IEP and a Geobacillus RT expressed by others. We conclude that it may be necessary to isolate the IEP RT as a ribonucleoprotein to obtain sufficient material for further analysis.
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PMID:Reverse transcriptases: intron-encoded proteins found in thermophilic bacteria. 1736 95