Masanori Oka

Cloning, nucleotide sequence, and expression in Escherichia coli of DNA polymerase gene (polA) from Thermus thermophilus HB8

Abstract A gene coding for a thermostable DNA polymerase I (Tth Pol I) was cloned from Thermus thermophilus HB8, and its nucleotide sequence was identified. The Tth Pol I gene ( poIA ) has an open reading frame of 2505 base pairs available to encode a peptide of 834 amino acids. Another incomplete open reading frame was also found upstream from the poA gene. In the deduced amino acid sequence of Tth Pol I, which shows 87% similarity to that of Thermus aquaticus DNA polymerase, there is the leucine zipper structure from Leu458 to Leu486. The Tth Pol I gene was subcloned in a high-expression vector. Escherichia coli cells harboring the hybrid plasmid produced about 100,000 units of thermostable DNA polymerase in a 200-ml culture.

Construction and characterization of N-terminally truncated DNA polymerase from Thermus thermophilus

Abstract Various plasmids harboring the truncated DNA polymerase gene ( polA ) from Thermus thermophilus HB8 ( Tth polymerase) were constructed. The most thermostable Tth Δ NF 2 polymerase [the gene product of polA Δ NF 2, which lacked a 751-bp region (region flanked by initiation codon and Fsp I site in the polA gene)] was selected, and purified from the recombinant Escherichia coli . SDS polyacrylamide gel electrophoresis revealed that the molecular weight of the Tth Δ NF 2 polymerase is 58–61 kDa, which is approximately 30 kDa smaller than that of the wild-type enzyme. The specific activity of the 5′-to-3′ polymerization of the Tth Δ NF 2 polymerase was 63% of that of the Tth polymerase. However, no 5′-to-3′ exonuclease activity was detected in this mutant enzyme (less than 1% of the specific activity of wild-type enzyme). The activities of the wild-type and mutant enzymes were maximal at 75°C. Approximately 50% of the enzyme activity was retained even after heat treatment of the Tth Δ NF 2 polymerase at 70°C for 2 h, but the thermostability of the mutant enzyme was slightly lower than that of the wild-type enzyme. Both the Tth Δ NF 2 and Tth polymerases were capable of non-templated addition of deoxyribonucleotide to a 3′-hydroxyl group of blunt-ended DNA.

Host-dependent activation of IS1203v excision in shiga toxin-producing Escherichia coli

IS1203v is an insertion sequence (IS) which is identical to the most abundant IS elements in the genome of Escherichia coli O157:H7. However, there is no sequence homologous to IS1203v in the genome of E. coli K-12. We constructed a system to analyze the excision frequency of IS1203v, and demonstrated that the frequency in E. coli O157:H7 was approximately 10(5) times higher than that in E. coli K-12. We also investigated the excision frequencies of IS1203v in various E. coli isolates, and showed that the excision frequencies of IS1203v-possessing strains were approximately 10(3) times higher than those of IS1203v-nonpossessing strains. The results suggest that the IS1203v-possessing strains use a common system to enhance IS1203v excision.

Functional similarities of a thermostable protein-disulfide oxidoreductase identified in the archaeon Pyrococcus horikoshii to bacterial DsbA enzymes

We have isolated and characterized a gene for a putative protein-disulfide oxidoreductase (phdsb) in the archaeon Pyrococcus horikoshii. The open reading frame of phdsb encodes a protein of 170 amino acids with an NH2-terminal extension similar to the bacterial signal peptides. The putative mature region of PhDsb includes a sequence motif, Cys-Pro-His-Cys (CPHC), that is conserved in members of the bacterial DsbA family, but otherwise the archaeal and bacterial sequences do not show substantial similarity. A recombinant protein corresponding to the predicted mature form of PhDsb behaved as a monomer and manifested oxidoreductase activities in vitro similar to those of DsbA of Escherichia coli. The catalytic activity of PhDsb was thermostable and was shown by mutation analysis to depend on the NH2-terminal cysteine residue of the CPHC motif. Thus, in spite of their low overall sequence similarities, DsbA-like proteins of archaea and bacteria appear to be highly similar in terms of function.

Double evaluations of chemicals using a cocktail of fused recombinant receptors

Some chemicals have multipotential as endocrine-disrupting chemicals (EDCs). For example, some chemicals act as both estrogens and antiandrogens. Numbers of such chemicals should be evaluated from many aspects; however, labor and expenses are generally limited. We have developed two expression systems for the wild type of human estrogen receptor alpha and the wild type of human androgen receptor fused with a maltose binding protein. They are soluble and have binding activities. They showed dose-responses to natural hormones and well-known potential EDCs. After we established each assay condition for a competitive binding assay using each receptor, we found that two assay systems can be carried out simultaneously under limited and harmonized conditions. Under harmonized conditions using a cocktail of two types of receptors, we could estimate natural hormones and potential EDCs. Interference between two assay systems was not observed under these conditions. We believe that some competitive binding assays can be carried out using a cocktail of receptors at the same time if interference among different assay systems can be avoided by choosing ideal conditions.