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J Sekiguchi

Publications and source records attributed to J Sekiguchi.

At least 91 records · Page 5Linked to original sources
PubMed1994

Requirements for noncovalent binding of vaccinia topoisomerase I to duplex DNA.

Vaccinia DNA topoisomerase binds duplex DNA and forms a covalent adduct at sites containing a conserved sequence element 5'(C/T)CCTT decreases in the scissile strand. Distinctive aspects of noncovalent versus covalent interaction emerge from analysis of the binding properties of Topo(Phe-274), a mutated protein which is unable to cleave DNA, but which binds DNA noncovalently. Whereas DNA cleavage by wild type enzyme is most efficient with 'suicide' substrates containing fewer than 10 base pairs distal to the scissile bond, optimal noncovalent binding by Topo(Phe-274) requires at least 10-bp of DNA 3' of the cleavage site. Thus, the region of DNA flanking the pentamer motif serves to stabilize the noncovalent topoisomerase-DNA complex. This result is consistent with the downstream dimensions of the DNA binding site deduced from nuclease footprinting. Topo(Phe-274) binds to duplex DNA lacking the consensus pentamer with 7-10-fold lower affinity than to CCCTT-containing DNA.

Base Sequence↗
PubMed1994

Stimulation of vaccinia topoisomerase I by nucleoside triphosphates.

The rate of relaxation of supercoiled DNA by purified vaccinia topoisomerase I is stimulated 20-fold by 5 mM ATP. A similar effect is elicited by GTP, CTP, UTP, dATP, and adenosine 5'-(beta, gamma-imido)triphosphate. ATP-mediated rate enhancement requires salt as a coactivator. ADP and inorganic pyrophosphate also stimulate relaxation 10-20-fold, whereas AMP and inorganic phosphate have little effect. A model for allosteric activation of topoisomerase by nucleotides is suggested.

Binding Sites↗
PubMed1994

Effect of degS-degU mutations on the expression of sigD, encoding an alternative sigma factor, and autolysin operon of Bacillus subtilis.

Primer extension analysis of transcripts of the Bacillus subtilis autolysin (cwlB) operon indicated that SigD-dependent transcripts from the Pd promoter are missing in the degU32(Hy) and degS200 (Hy) mutants. The degU32(Hy) mutation caused a 99% reduction in the expression of a sigD-lacZ translational fusion gene constructed in the B. subtilis chromosome. The phosphorylated form of the DegU protein seems to be a regulator for expression of the sigD gene.

Bacillus subtilis↗
PubMed1993

Bacillus subtilis mutant deficient in the major autolytic amidase and glucosaminidase is impaired in motility.

The purified autolytic endo-beta-N-acetylglucosaminidase of Bacillus subtilis AC327 was cleaved with cyanogen bromide, and the N-terminal amino acid sequence of one of the peptide fragments was determined. Then, a DNA fragment containing a part of the glucosaminidase gene was cloned into Escherichia coli JM109 using synthetic oligonucleotides as probes whose sequences had been deduced from the N-terminal amino acid sequence. Zymographic analysis showed that the resultant glucosaminidase-deficient strain lacked a 35-kDa lytic band in addition to a 90-kDa lytic one corresponding to the glucosaminidase. A double mutant strain deficient in the major two autolysins (amidase and glucosaminidase) exhibited greatly impaired motility on a swarm plate whereas the single mutant strains were motile.

Acetylglucosaminidase↗
PubMed1993

Nucleotide sequence of the large mitochondrial rRNA gene of Penicillium chrysogenum.

The nucleotide sequence of a large rRNA (1-rRNA) gene and its flanking regions in the cloned fragments of mitochondrial (mt) DNA from Penicillium chrysogenum NRRL1951 (Sekiguchi J., Ohsaki, T., Yamamoto, H., Koichi, K. and Shida, T. (1990) J. Gen. Microbiol. 136, 535-543) was determined and compared with those in Aspergillus nidulans and Neurospora crassa mitochondrial DNAs. The P. chrysogenum mt 1-rRNA gene has a 1678 bp intron which intervenes between a 2835 bp 5' exon and a 581 bp 3' exon, and extensive homology exists between overall sequences of mt 1-rRNA genes of P. chrysogenum and A. nidulans. The P. chrysogenum intron contains a large open reading frame which encodes a polypeptide comprising of 399 amino acids. The intron sequence also suggests that the intron belongs to a self-splicing group IA.

Base Sequence↗
PubMed1993

Molecular cloning, sequence analysis, and characterization of a new cell wall hydrolase, CwlL, of Bacillus licheniformis.

We have cloned a DNA fragment containing the gene for a cell wall hydrolase from Bacillus licheniformis FD0120 into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame (ORF; designated as cwlL), which is different from the B. licheniformis cell wall hydrolase gene cwlM, and encodes a polypeptide of 360 amino acids with a molecular mass of 38,994. The enzyme purified from the E. coli clone is an N-acetylmuramoyl-L-alanine amidase, which has a M(r) value of 41 kDa as determined by SDS-polyacrylamide gel electrophoresis, and is able to digest B. licheniformis, B. subtilis and Micrococcus luteus cell walls. The nucleotide and deduced amino acid sequences of cwlL are very similar to those of ORF3 in the putative operon xpaL1-xpaL2-ORF3 in B. licheniformis MC14. Moreover, the amino acid sequence homology of CwlL with the B. subtilis amidase CwlA indicates two evolutionarily distinguishable regions in CwlL. The sequence homology of CwlL with other cell wall hydrolases and the regulation of cwlL are discussed.

Amino Acid Sequence↗
PubMed1993

Use of the osteocutaneous free scapular flap on the lower extremities.

Reports on the use of osteocutaneous free scapular flap transfers have focused primarily on mandible reconstruction. Yet the scapula is a nearly straight bone, and it is also useful in the reconstruction of long bones. We would like to report on 23 patients undergoing osteocutaneous free scapular flap transfers to the lower extremities in whom we obtained extremely satisfactory results. These 23 surgical patients included 10 with pseudoarthrosis with osteomyelitis, 3 with pseudoarthrosis due to trauma, and 5 with osteomyelitis of the lower extremities, as well as 5 with other miscellaneous problems. Surgery was successful in all patients. There was a recurrence of osteomyelitis in 2 patients. In 1 patient, the problem was minor and was treated on an outpatient basis. In the other patient, an infection arose in surgical dead space, and a second operation was required. The level of weight bearing achieved with the osteocutaneous free scapular flap is the same as obtained with a vascularized free fibular graft used for the treatment of pseudoarthrosis. We therefore believe that the osteocutaneous free scapular flap is extremely effective in the treatment of chronic and severe lower leg problems.

Adolescent↗
PubMed1993

Molecular cloning of a sporulation-specific cell wall hydrolase gene of Bacillus subtilis.

Southern hybridization analysis of Bacillus subtilis 168S chromosomal DNA with a Bacillus licheniformis cell wall hydrolase gene, cwlM, as a probe indicated the presence of a cwlM homolog in B. subtilis. DNA sequencing of the cwlM homologous region showed that a gene encoding a polypeptide of 255 amino acids with a molecular mass of 27,146 Da is located 625 bp upstream and in the opposite direction of spoVJ. The deduced amino acid sequence of this gene (tentatively designated as cwlC) showed an overall identity of 73% with that of cwlM and of 40% with the C-terminal half of the B. subtilis vegetative autolysin, CwlB. The construction of an in-frame cwlC-lacZ fusion gene in the B. subtilis chromosome indicated that cwlC is induced at 6 to 7 h after sporulation (t6 to t7). The spoIIIC (sigma K) mutation and earlier sporulation mutations greatly reduced the expression of the cwlC-lacZ fusion gene. Northern hybridization analysis using oligonucleotide probes of the cwlC region indicated that a unique cwlC transcript appeared at t7.5 and t9. Transcriptional start points determined by primer extension analysis suggested that the -10 region is very similar to the consensus sequence for the sigma K-dependent promoter. Insertional inactivation of the cwlC gene in the B. subtilis chromosome caused the disappearance of a 31-kDa protein lytic for Micrococcus cell walls, which is mainly located within the cytoplasmic and membrane fractions of cells at t9. The CwlC protein hydrolyzed both B. subtilis vegetative cell walls and spore peptidoglycan.

Amino Acid Sequence↗
PubMed1993

High-level transcription of the major Bacillus subtilis autolysin operon depends on expression of the sigma D gene and is affected by a sin (flaD) mutation.

Transcription of the major Bacillus subtilis autolysin gene (cwlB) was investigated. Deletion of the region upstream of the gene cluster lppX-cwbA-cwlB led to a loss of promoter activity. Primer extension analysis suggested that the cwlB operon is transcribed by E sigma D and E sigma A, the former transcripts being predominants at the exponential growth phase. Expression of the lppX-lacZ fusion gene was reduced by about 90% in a sigD-null mutant. A sin (flaD1) mutation caused a severe defect in transcription of the lppX-cwbA-cwlB operon. The sin (flaD1) mutation also reduced expression of a sigD-lacZ fusion gene constructed in the B. subtilis chromosome. Since the sigD-null mutant exhibits motility and autolysin deficiencies and filamentation, similar phenotypes in the sin (flaD1) mutant may be caused by reduction in expression of the sigma D protein.

Amino Acid Sequence↗
PubMed1993

Chemical synthesis and properties of an oligodeoxyribonucleotide containing a 2-deoxyribosylformamide residue.

To elucidate the conformational properties of DNA with a 2-deoxyribosylformamide residue (dF), an oligodeoxyribonucleotide containing this abasic residue in a specific position of the nucleotide sequence was synthesized by the standard solid-phase phosphotriester method. Deprotection of the synthesized oligonucleotide was performed under routine alkaline and acidic conditions. The presence of a dF residue in the oligomer was confirmed by ion-spray mass spectrometry. A dF residue was found to affect considerably the stability of the DNA duplex, as determined from the melting behavior of the dF-containing duplex.

Base Sequence↗
PubMed1992

Characterization of the Bacillus subtilis CwbA protein which stimulates cell wall lytic amidases.

The Bacillus subtilis cell wall binding protein, CwbA, stimulated the cell wall lytic activities of the B. subtilis and B. licheniformis autolysins (CwlA and CwlM, respectively) in addition to that of the major B. subtilis autolysin (CwlB). Even though the substrate for the enzyme reaction was changed from B. subtilis cell wall containing a teichoic acid to Micrococcus luteus cell wall containing a teichuronic acid, the stimulatory effect of CwbA on CwlA activity was observed.

Bacillus subtilis↗
PubMed1992

Genetic structure, isolation and characterization of a Bacillus licheniformis cell wall hydrolase.

A DNA fragment containing the gene for a cell wall hydrolase of Bacillus licheniformis was cloned into Escherichia coli. Sequencing of the fragment showed the presence of an open reading frame which encodes a polypeptide of 253 amino acids with a molecular mass of 27,513. The gene was designated as cwlM, for cell wall lysis. The deduced amino acid sequence indicated that there is a repeated sequence consisting of 33 amino acid residues in the C-terminal region. Deletion of the C-terminal region did not lead to any loss of cell wall lytic activity. The gene product purified from E. coli cells harboring a cwlM-bearing plasmid exhibited a M(r) value of 29 kDa on SDS-polyacrylamide gels, and characterization of the specific substrate bond cleaved by CWLM indicated that the enzyme is an N-acetylmuramoyl-L-alanine amidase (EC 3.5.1.28). The enzyme hydrolyzed the cell wall of Micrococcus luteus more efficiently than those of B. licheniformis and B. subtilis, but the truncated CWLM (lacking the C-terminal region) had lost this preference. CWLM prepared from B. subtilis cells harboring a plasmid containing cwlM had a similar M(r) value to that from E. coli. Amino acid sequence homologies between CWLM and other amidases, and their protein structures are discussed.

Amino Acid Sequence↗
PubMed1992

Restoration of the mandible by full-thickness calvarial bone flap.

Two patients with reconstruction of a massive mandibular defect with vascularized full-thickness calvarial bone flaps are reported. In Patient 1, the mandibular body developed osteomyelitis and once was replaced with a metallic prosthesis. The prosthesis later perforated the skin and was removed. Full-thickness calvarial bone flaps were elevated bilaterally to reconstruct the mandibular body. In Patient 2, the mandible was totally destroyed by invasion of squamous cell carcinoma. The lower one-half of the face was resected and replaced with a large island scalp flap with full-thickness calvarial bone.

Adult↗
PubMed1992

Molecular cloning and sequencing of the upstream region of the major Bacillus subtilis autolysin gene: a modifier protein exhibiting sequence homology to the major autolysin and the spoIID product.

The upstream region of the N-acetylmuramoyl-L-alanine amidase gene (cwlB; a major Bacillus subtilis autolysin) was cloned into Escherichia coli by chromosome walking. Sequencing of the region showed the presence of two open reading frames, one (designated as cwbA) which starts at a UUG codon and encodes a polypeptide of 705 amino acids with an M(r) of 76,725, and the other (designated as lppX), upstream of cwbA, comprising 102 amino acids and having a signal sequence characteristic of a lipoprotein. Purification of the CwbA protein and determination of its N-terminal amino acid sequence revealed that it contains a presumed signal peptide which is processed after Ala at position 25 from the N-terminal, and that the M(r) of the mature form is 75,000. The amino acid sequences of the N-terminal and C-terminal regions of CwbA were found to be highly homologous with those of the cell wall binding domain of CwlB and the spoIID gene product, respectively. CwbA stimulated the major autolysin activity approximately threefold in vitro. These data indicate that CwbA is the modifier protein of the major autolysin reported by Herbold, D. R. & Glaser, L. (1975; Journal of Biological Chemistry 250, 1676-1682). In-frame fusion between the lppX and lacZ genes demonstrated that lppX is translated in vivo and expressed during the exponential growth phase.

Amino Acid Sequence↗
PubMed1992

Characterization of four-stranded DNA and RNA fragments.

We have investigated the structures formed by deoxyribo- and ribooligonucleotides containing guanine-rich sequences. Inter- and intrastrand guanine tetrads are not only more stable in the presence of K+ than they are in the presence of Na+, but also more compact.

DNA↗
PubMed1991

Purification and characterization of a cell wall hydrolase encoded by the cwlA gene of Bacillus subtilis.

A cell wall hydrolase of Bacillus subtilis was prepared from Escherichia coli cells harboring a plasmid containing the B. subtilis cwlA gene and purified by hydroxyapatite column chromatography and HPLC through TSK-gel G3000SWXL. In contrast to the molecular mass of 29,919 Da deduced from its nucleotide sequence, the purified CWLA is a 23 kDa protein. Characterization of the specific substrate bond cleaved by CWLA indicated the enzyme is an N-acetylmuramyl-L-alanine amidase. A 32-kDa precursor protein was detected on zymography of a crude cell homogenate. Some of the enzymatic properties of CWLA are also described.

Bacillus subtilis↗
PubMed1991

Restoration of the anterior neck surface in the burned patient by free groin flap.

In treating extensive burn contractures of the anterior neck, we obtained good results using free flaps. Ninety-nine patients with neck contractures were treated with free flaps. The ages of the patients range from 2 to 64 years. We used 84 free groin flaps, 10 free latissimus dorsi musculocutaneous flaps, 2 free deltopectoral flaps, 2 free scapular flaps, and 1 free anterolateral thigh flap. In all patients, contractures were sufficiently released and no recurrences followed after a mean follow-up of 8.7 years. After defatting procedures, natural profiles and good appearances were restored, especially in patients in whom groin flaps were used.

Adolescent↗