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Biomedical subjects

A Nakazawa

Publications and source records attributed to A Nakazawa.

At least 145 records · Page 8Linked to original sources
PubMed1986

Isolation and characterization of cDNA for chicken muscle adenylate kinase.

A cDNA clone for muscle adenylate kinase was isolated from a cDNA library of chick skeletal muscle poly(A)+ RNA, and the DNA sequence was determined. The cDNA insert had 854 nucleotides, which consisted of the 5'-untranslated sequence of 57 nucleotides, the sequence of 582 nucleotides coding for 194 amino acids, and the 3'-untranslated sequence of 163 nucleotides and the poly(A) tail of 52 nucleotides. The amino acid sequence predicted from the nucleotide sequence was highly homologous with the reported sequences of human, calf, porcine, and rabbit muscle adenylate kinases. RNA blot analysis of poly(A)+ RNA from various chicken tissues revealed a single species of mRNA of approximately 850 nucleotides and its tissue-specific distribution. The induction of muscle adenylate kinase mRNA synthesis during the chick embryogenesis was also demonstrated by the blot analysis. Southern blot analysis indicated a single gene for muscle adenylate kinase in the chicken genome.

Adenylate Kinase↗
PubMed1986

Effect of base substitutions in the colicin E1 gene on colicin E1 export and bacteriocin activity.

Base substitutions have been introduced into the segment of the colicin E1 gene corresponding to the polypeptide region between the 404th and the 502nd residues which was considered to participate in colicin E1 export and bacteriocin activity. The methods used were in vitro localized mutagenesis with sodium bisulphite and in vivo mutagenesis using either nitrosoguanidine or ethyl methane sulphonate. Cells carrying mutagenized plasmids were screened by their inability to form a clear zone on a lawn of colicin E1 sensitive cells. Mutation sites were determined from the nucleotide sequence analysis and the altered amino acid residues were reduced. The mutant proteins were analysed for their ability to be exported to the periplasmic space and for their bacteriocin activity. Out of eight mutants obtained, three had a single amino acid replacement. Mutant proteins that had Ser and Glu in place of Pro-462 and Gly-502, respectively, showed a decrease in both the export and the bacteriocin activity. A mutant protein having Arg in place of Gly-439 showed a decrease only in the bacteriocin activity. These results suggest that the target region of colicin E1 contributes to the export as well as the bacteriocin activity but the two functions are supported in part by different amino acid residues of the protein.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of the regulatory gene xylS on the Pseudomonas putida TOL plasmid and identification of the protein product.

The xylS gene is a regulatory gene which positively controls expression of the genes on the TOL plasmid for degradation enzymes of benzoate or m-toluate in Pseudomonas putida. Cloning of the gene in Escherichia coli and determination of the nucleotide sequence revealed an open reading frame of 963 bp which corresponds to a protein with an Mr of 36,502. The xylS gene was recloned onto a tac-promoter vector, and the product was identified by the maxicell procedure as a protein with an approximate Mr of 37,000. The predicted amino acid sequence of XylS protein showed a basic character and contained a region similar to those in other DNA-binding proteins.

Amino Acid Sequence↗
PubMed1986

Nucleotide sequence of a DNA segment promoting transcription in Pseudomonas putida.

A DNA segment that promotes gene expression in Pseudomonas putida was identified in pTN8, a mutant plasmid of an RP4-TOL recombinant. A promoter on the segment was cloned with a promoter-probe vector containing the xylE gene of the TOL plasmid. The xylE gene was expressed under the control of the promoter, and the gene product catechol 2,3-dioxygenase was constitutively synthesized. As analyzed by an S1 nuclease protection assay, the amount of mRNA produced in P. putida was more than that in Escherichia coli. Fine S1 nuclease mapping and reverse transcriptase mapping revealed three tandem transcription start sites in both P. putida and E. coli. The nucleotide sequence preceding the transcription start sites was determined; a part of this sequence contained a sequence homologous to E. coli promoter sequences. A tentative consensus sequence for P. putida constitutive promoters is proposed.

Base Sequence↗
PubMed1985

Positive regulation of the colicin E1 gene by cyclic AMP and cyclic AMP receptor protein.

In previous experiments, we showed that the in vivo transcription of the colicin E1 gene was dependent on cyclic AMP in adenylate cyclase-defective mutant cells of Escherichia coli (Ebina, Y. and Nakazawa, A (1983) J. Biol. Chem. 258, 7072-7078). We now show that cyclic AMP and cyclic AMP receptor protein stimulated the in vitro transcription of the gene in the presence of spermidine. As determined in DNase I protection experiments, two binding sites for the complex of cyclic AMP and the receptor protein were identified about 60 base pairs (CRP-1) and 110 base pairs (CRP-2) upstream from the transcription initiation site of the colicin E1 gene. CRP-1 had a higher affinity for the complex than that of CRP-2. Substituting an unrelated DNA sequence for CRP-2 reduced the efficiency of in vitro stimulation of the gene by cyclic AMP and the receptor protein. These potential binding sites for the cyclic AMP-cyclic AMP receptor protein complex probably participate in the stimulation of the colicin E1 gene transcription.

Base Sequence↗
PubMed1985

Development of myokinase mRNA during embryogenesis of the chick.

During chick embryogenesis, the mRNA for myokinase, as determined by in vitro translation, appears on the 16th day. The mRNA levels do not change drastically in succeeding stages and increase immediately after hatching. In contrast, myokinase activity and its protein gradually increase from the 16th day. Thus, myokinase mRNA that is translatable in vitro seems to accumulate transiently in the late stage of embryogenesis. In sucrose density gradient centrifugation, the mRNA sedimented as one peak with a coefficient of 10S.

Adenylate Kinase↗
PubMed1985

Determination of the transcription initiation site and identification of the protein product of the regulatory gene xylR for xyl operons on the TOL plasmid.

The xylR gene is a regulatory gene on the TOL plasmid, which acts in a positive manner on xyl operons for degradation of toluene and xylenes in Pseudomonas putida. A DNA fragment containing the xylR promoter region was cloned on promoter-probing vectors, and its nucleotide sequence was determined. The transcription initiation site of the xylR gene was determined in cells of P. putida and Escherichia coli by S1 nuclease and reverse transcriptase mapping. Two initiation sites were detected which were identical in both P. putida and E. coli. The amounts of mRNA synthesized in both bacterial cells were almost the same and independent of the inducers for xyl operons. The consensus sequences for E. coli promoters were found in the region preceding the respective transcription initiation sites. The product of the xylR gene was identified by the maxicell system as a protein with an approximate molecular weight of 67,000.

Base Sequence↗
PubMed1984

Factors necessary for the export process of colicin E1 across cytoplasmic membrane of Escherichia coli.

Factors necessary for the export process of colicin E1 across the cytoplasmic membrane of Escherichia coli were investigated. beta-Galactosidase activities from gene fusions between the colicin E1 and lacZ genes were recovered in the inner membrane fraction of E. coli when the region containing the internal signal-like sequence of colicin E1 [M. Yamada et al. (1982) Proc. Natl Acad. Sci. USA 79, 2827-2831] was present, but were found in the soluble fraction when the region was eliminated. The colicin E1 export was reduced upon insertion mutation in a gene that is located downstream from the colicin E1 gene in the same operon and responsible for mitomycin-C-induced killing of the host cell. A frame shift mutation of the colicin E1 plasmid was constructed to direct the protein which had lost the COOH-terminal 13 residues of original colicin E1 and was altered in 6 residues of the new COOH-terminal portion. The aberrant colicin E1 that was inducibly synthesized remained inside the cells. These results indicate that colicin E1 is exported with the aid of a product of the downstream gene and that the COOH-terminal portion is necessary for the export. The binding of colicin E1 to the cytoplasmic membrane through the internal signal-like sequence may be a step in the protein export process.

Autoradiography↗
PubMed1984

Transcription analysis of the lexA gene of Escherichia coli: attenuation and cotranscription with the neighboring region.

The lexA gene of Escherichia coli encodes a repressor of the genes whose expression is induced by the agents that result in DNA damage. In vivo transcripts of the lexA gene consisted of two species; mRNA-1 of 673 bases and mRNA-2 of approximately 3,000 bases. The transcription in vivo started at a site which was two-base pairs downstream from the in vitro initiation site reported previously. The majority of the transcription stopped at a series of T residues preceeded by a dyad symmetry located immediately after the lexA gene. A small fraction of the transcription passed through the termination site to form the mRNA of downstream gene(s) which would be related to the "SOS functions".

Bacterial Proteins↗
PubMed1984

Nucleotide sequence of the promoter region of the xylDEGF operon on TOL plasmid of Pseudomonas putida.

The transcription initiation site of the xylDEGF operon on the TOL plasmid of Pseudomonas putida mt-2 was determined in P. putida and in Escherichia coli by S1 nuclease and reverse transcriptase mapping. The induced synthesis of mRNA started at the same start point in both P. putida and E. coli, although the amount of mRNA in E. coli cells was less than that in P. putida. The nucleotide sequence of the region surrounding the start point was also determined. The ribosome-binding site (RBS) complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E. coli preceded the predicted start codon for the xylD gene. The consensus nucleotide sequence for E. coli promoters was not found in the region preceding the transcription start point. On the other hand, the sequences of the "-10" and the "-35" regions of the xylDEGF operon revealed some homology with the respective, previously determined sequences of the xylABC operon of the TOL plasmid.

Base Sequence↗
PubMed1984

Nucleotide sequence surrounding transcription initiation site of xylABC operon on TOL plasmid of Pseudomonas putida.

The xylABC operon on the TOL plasmid directs the synthesis of enzymes for conversion of toluene to benzoate and is positively controlled by the regulatory gene xylR. In the study here the nucleotide sequence was determined for the regulatory region of this operon. The in vivo transcription initiation site of the operon was determined by S1 nuclease and reverse transcriptase mapping. RNA was prepared from m-methylbenzyl alcohol-induced cells of Pseudomonas putida and Escherichia coli carrying pTN2, a derivative of the TOL plasmid containing the structural and regulatory genes of the entire toluene-degrading pathway. The amount of E. coli mRNA was estimated to be only 10% of that of P. putida mRNA. Consensus sequences of the -10 region (Pribnow box) and the -35 region (RNA polymerase recognition site) in E. coli genes were not found in the region preceding the transcription initiation site, whereas a sequence complementary to the 3' end of the 16S rRNA of Pseudomonas aeruginosa and E. coli existed in front of the predicted start codon of the xylA gene. These results explain the inefficient expression of TOL genes in E. coli.

Base Sequence↗
PubMed1983

LexA protein is a repressor of the colicin E1 gene.

LexA protein is a repressor of several chromosomal genes involved in the SOS response in Escherichia coli. In previous experiments, we found that LexA protein may also be a repressor of the colicin E1 gene. We now present evidence that the purified LexA protein strongly repressed the in vitro transcription of the colicin E1 gene. As determined in DNase I protection experiments, LexA protein bound with a high affinity to the approximately 40-base pair long sequence between the Pribnow box and the start codon of the colicin E1 gene. The sequence of the binding site was composed of two overlapped "SOS boxes" to which the LexA protein bound in a cooperative manner.

Bacterial Proteins↗
PubMed1983

Cyclic AMP-dependent initiation and rho-dependent termination of colicin E1 gene transcription.

We have analyzed the initiation and termination sites of transcription in vivo of the colicin E1 gene in Escherichia coli cells by S1-mapping assay and RNA blot hybridization. According to the S1-mapping assay, the transcription was initiated at about 75 base pairs upstream from the NH2-terminal codon of the gene. The initiation site corresponded with one of the two promoters which were previously determined by in vitro transcription experiments (Ebina, Y., Kishi, F., Miki, T., Kagamiyama, H., Nakazawa, T., and Nakazawa, A. (1981) Gene 15, 119-126). Transcription in vivo of the colicin E1 gene was stimulated by cyclic AMP in the adenylate cyclase-defective mutant cells. Two transcripts of the colicin E1 gene, approximately 1700 and 2200 nucleotides, were detected by the blot hybridization. Since initiation of the transcription started at one site in vivo, these results indicated two termination sites. The location of the termination sites were approximately 60 and 560 base pairs downstream from the COOH-terminal codon of the gene as judged by S1-mapping assay. In vitro transcription experiments with rho-factor strongly suggested that the termination in the proximal terminator was rho-dependent. In the terminator structure, there is the sequence CAAACAAA which is homologous to a common sequence CAATCAA found in other rho-dependent terminators.

Base Sequence↗
PubMed1983

Complete nucleotide sequence of the metapyrocatechase gene on the TOI plasmid of Pseudomonas putida mt-2.

Metapyrocatechase which catalyzes the oxygenative ring cleavage of catechol to form alpha-hydroxymuconic epsilon-semialdehyde is encoded by the xylE gene on the TOL plasmid of Pseudomonas putida mt-2. We have cloned the xylE region in Escherichia coli and determined the nucleotide sequence of the DNA fragment of 985 base pairs around the gene. The fragment included only one open translational frame of sufficient length to accommodate the enzyme. The predicted amino acid sequence consisted of 307 residues, and its NH2- and COOH-terminal sequences were in perfect agreement with those of the enzyme recently determined (Nakai, C., Hori, K., Kagamiyama, H., Nakazawa, T., and Nozaki, M. (1983) J. Biol. Chem. 258, 2916-2922). A mutant plasmid was isolated which did not direct the synthesis of the active enzyme. This plasmid had a DNA region corresponding to the NH2-terminal two-thirds of the polypeptide. From the deduced amino acid sequence, the secondary structure was predicted. Around 10 base pairs upstream from the initiator codon for metapyrocatechase, there was a base sequence which was complementary to the 3'-end of 16 S rRNAs from both E.coli and Pseudomonas aeruginosa. A preferential usage of C- and G-terminated codons was found in the coding region xylE, which contributed to the relatively high G + C content (57%) of this gene.

Amino Acid Sequence↗