Takashi Uemori

A novel DNA polymerase in the hyperthermophilic archaeon, Pyrococcus furiosus: gene cloning, expression, and characterization

In many respects Archaea are much more like eukaryotes than prokaryotes with respect to the conservation of many of the components involved in transcription, translation and DNA replication. So far, only a few DNA polymerases with structures similar to those of eukaryotic DNA polymerase α have been found in Archaea. The identification and characterization of all the DNA polymerases of one archaeon would add considerably to our knowledge of the basic mechanisms of DNA replication in these organisms.

Amplification of the 16S-23S spacer region in rRNA operons of mycoplasmas by the polymerase chain reaction

Summary Nucleotide sequences of the spacer region between 16S and 23S DNA in the ribosomal RNA operons of mycoplasmas were identified by analysis of products of the polymerase chain reaction (PCR) amplified from the corresponding regions of 12 species of this family. Three common PCR primers, F1, F2, and R1, were designed by analysis of similarity between these sequences. Primers F1 and R1 produced fragments of 340 to 660 bp when the DNA of each species was used as the template. Specific amplification of the spacer region was confirmed by a second round of PCR in which the amplified products were used as the templates and F2 and R1 were used as the primers. No discrete band was observed in electrophoresis when human or mouse DNA served as the template with use of primers F1 and R2, which suggests that many mycoplasmal species that sometimes contaminate a culture of eukaryotic cells can be detected by the PCR.

Detection of Mycoplasma hyopneumoniae DNA by the polymerase chain reaction.

DNA amplification by the polymerase chain reaction (PCR) was examined to detect DNA of Mycoplasma hyopneumoniae, an etiological agent of porcine pneumonia. A pair of synthetic primers was selected that specify the amplification of a 520-basepair DNA fragment in a reiterative sequence of M. hyopneumoniae genome. The PCR product was detected by direct gel electrophoresis or by blot hybridization to a synthetic oligonucleotide probe. The specificity of PCR for M. hyopneumoniae was confirmed by lack of cross-reactivity to DNA from other porcine mycoplasmas.

Characterization of a functional domain of human calpastatin

Expression plasmids were constructed from the cDNA of human calpastatin to examine the contribution to the inhibition of calpain of highly conserved sequences in each of four repetitive domains. A series of deletion derivatives of domain 1 proteins, truncated at either the amino or carboxy terminus, were produced in E. coli. Deletion from the amino terminus past the amino terminal conserved sequence decreased the inhibition. When the middle conserved sequence, the M-sequence, was further deleted, no inhibition was detected, but deletion from the carboxy terminus past the carboxy terminal conserved sequence did not decrease the inhibition until the M-sequence was reached. Nuclear magnetic resonance and circular dichroism spectra showed that domain 1 has an unfolded structure. Peptides that contained the M-sequence and some neighboring sequences were synthesized to measure the minimum size of the inhibitory peptide, which was the M-sequence with the next six residues on the amino terminal side.

Rapid detection and differentiation of the major mycoplasma contaminants in cell cultures using real-time PCR with SYBR Green I and melting curve analysis.

A quantitative real‐time polymerase chain reaction (PCR) procedure followed by melting curve analysis, using the green fluorescence dye SYBR Green I, was developed for rapid detection and differentiation of mycoplasma contaminants in cell cultures. This method showed that the detection of the target sequence was linear over a range from 104 to 10 colony‐forming units (CFU) of the mycoplasma cells. Analysis of the melting temperature of the PCR products allowed differentiation of the major mycoplasma contaminants. These results demonstrate that the protocol described in the present study can decrease the time to obtain reproducible results by simultaneous detection and differentiation of the Mycoplasma species contaminating cell cultures.

Identification of a mismatch-specific endonuclease in hyperthermophilic Archaea.

The common mismatch repair system processed by MutS and MutL and their homologs was identified in Bacteria and Eukarya. However, no evidence of a functional MutS/L homolog has been reported for archaeal organisms, and it is not known whether the mismatch repair system is conserved in Archaea. Here, we describe an endonuclease that cleaves double-stranded DNA containing a mismatched base pair, from the hyperthermophilic archaeon Pyrococcus furiosus. The corresponding gene revealed that the activity originates from PF0012, and we named this enzyme Endonuclease MS (EndoMS) as the mismatch-specific Endonuclease. The sequence similarity suggested that EndoMS is the ortholog of NucS isolated from Pyrococcus abyssi, published previously. Biochemical characterizations of the EndoMS homolog from Thermococcus kodakarensis clearly showed that EndoMS specifically cleaves both strands of double-stranded DNA into 5′-protruding forms, with the mismatched base pair in the central position. EndoMS cleaves G/T, G/G, T/T, T/C and A/G mismatches, with a more preference for G/T, G/G and T/T, but has very little or no effect on C/C, A/C and A/A mismatches. The discovery of this endonuclease suggests the existence of a novel mismatch repair process, initiated by the double-strand break generated by the EndoMS endonuclease, in Archaea and some Bacteria.

The Polymerase Chain Reaction for Mycoplasma pulmonis

in vitro DNA amplification by polymerase chain reaction was examined to detect Mycoplasma pulmonis. A pair of synthetic oligonucleotide primers was constructed, and used to amplify a unique sequence of M. pulmonis DNA. Amplified products were detected by agarose gel electrophoresis and verified by blot hybridization with a synthetic oligonucleotide probe. This system detected cellular DNA of M. pulmonis but not M. arthritidis or M. neurolyticum, and thus appears to be useful for M. pulmonis diagnosis.

Applications of nucleic acid chaperone activity of CspA and its homologues.

In Escherichia coli, the cold shock response is exerted upon temperature change from 37 to 15°C and is characterized by induction of several cold shock proteins including its major cold shock protein, CspA. E. coli CspA family consists of nine members, CspA to CspI. CspA and some of its homologues play a critical role in cold acclimation of cells as RNA chaperones by destabilizing secondary structures in RNAs. Here, we showed that the nucleic acid melting activity of Csp proteins can be used to facilitate reactions, such as RT-PCR or RNA cleavage reactions by endoribonucleases, which are hindered by presence of secondary structures in the DNA/RNA substrate used. The low substrate specificity of Csps together with their compatibility with various enzymes and their stability and activity over a broad temperature range makes them ideal candidates to be used for a variety of processes.