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 11 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 EA I 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 baculoviruis vector although yields remained low. We followed RT activity during purification using the poly(rC)center dot p(dG)12-18, which specifically detects only RNA-dependent DNA polymerase activity. We could not detect incorporation of dTTP into poly(rC)center dot 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 EA1-1EP 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. (C) 2007 Elsevier B.V. All rights reserved.
- group II intron
- intron-encoded proteins
- genomic walking
- RNA-dependent DNA polymerases