PMID- 17363195
OWN - NLM
STAT- MEDLINE
DCOM- 20070613
LR  - 20070416
IS  - 0378-1119 (Print)
IS  - 0378-1119 (Linking)
VI  - 393
IP  - 1-2
DP  - 2007 May 15
TI  - Reverse transcriptases: intron-encoded proteins found in thermophilic bacteria.
PG  - 137-44
AB  - 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.
FAU - Ng, Bernie
AU  - Ng B
AD  - Department of Chemistry and Biomolecular Sciences, Macquarie University, North 
      Ryde, NSW 2109, Australia.
FAU - Nayak, Sunil
AU  - Nayak S
FAU - Gibbs, Moreland D
AU  - Gibbs MD
FAU - Lee, Jun
AU  - Lee J
FAU - Bergquist, Peter L
AU  - Bergquist PL
LA  - eng
SI  - GENBANK/EF165030
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20070220
PL  - Netherlands
TA  - Gene
JT  - Gene
JID - 7706761
RN  - 0 (Bacterial Proteins)
RN  - EC 2.7.7.49 (RNA-Directed DNA Polymerase)
SB  - IM
MH  - Amino Acid Sequence
MH  - Bacillus/*enzymology
MH  - Bacterial Proteins/chemistry/isolation & purification/*metabolism
MH  - Baculoviridae
MH  - Cloning, Molecular
MH  - Escherichia coli
MH  - Introns/*genetics
MH  - Molecular Sequence Data
MH  - RNA-Directed DNA Polymerase/chemistry/isolation & purification/*metabolism
EDAT- 2007/03/17 09:00
MHDA- 2007/06/15 09:00
CRDT- 2007/03/17 09:00
PHST- 2006/12/19 00:00 [received]
PHST- 2007/02/05 00:00 [revised]
PHST- 2007/02/05 00:00 [accepted]
PHST- 2007/03/17 09:00 [pubmed]
PHST- 2007/06/15 09:00 [medline]
PHST- 2007/03/17 09:00 [entrez]
AID - S0378-1119(07)00082-0 [pii]
AID - 10.1016/j.gene.2007.02.003 [doi]
PST - ppublish
SO  - Gene. 2007 May 15;393(1-2):137-44. doi: 10.1016/j.gene.2007.02.003. Epub 2007 Feb 
      20.