Cloning and analysis of the DNA polymerase-encoding gene from Thermus caldophilus GK24.
The gene encoding Thermus caldophilus GK24 (Tca) DNA polymerase was cloned into Escherichia coli using the structural gene coding for Thermus aquaticus YT-1 (Taq) DNA polymerase as a hybridization probe. The nucleotide sequence of the cloned DNA was determined. The primary structure of the Tca DNA polymerase was deduced from the nucleotide sequence. The Tca DNA polymerase comprised 834 amino acid residues and its molecular mass was determined to be 93,810. On alignment of the whole amino acid sequence, Tca DNA polymerase showed a high sequence homology with the E. coli DNA polymerase I-like DNA polymerases, and 86% identity with Taq DNA polymerase, 38% with E. coli and Streptococcus pneumoniae (Spn) DNA polymerase I. An extremely high sequence identity was observed in the region containing the polymerase activity. The codon usage in the Tca DNA polymerase gene was in fact similar to the characteristic usages in the genes for proteins from bacteria of genus Thermus: the G+C content in the third position of the codons was as high as 93%. The Tca DNA polymerase gene was expressed under the control of tac promoter on a high copy plasmid, pTCA in E. coli.
Kwon
S T
ST
Department of Genetic Engineering, Sung Kyun Kwan University, Suwon, Korea.
Kim
J S
JS
Park
J H
JH
Kim
H K
HK
Lee
D S
DS
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D003001:Cloning, Molecular; D003062:Codon / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics*; D004259:DNA-Directed DNA Polymerase / Q000737:chemistry / Q000235:genetics*; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015870:Gene Expression; D005798:Genes, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D010957:Plasmids; D016133:Polymerase Chain Reaction; D015183:Restriction Mapping; D017386:Sequence Homology, Amino Acid; D013824:Thermus / Q000201:enzymology* / Q000235:genetics*
D000596:Amino Acids; D003062:Codon; D017931:DNA Primers; D004269:DNA, Bacterial; D004259:DNA-Directed DNA Polymerase