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Biomedical subjects

J Sekiguchi

Publications and source records attributed to J Sekiguchi.

At least 55 records · Page 3Linked to original sources
PubMed1997

Characterization of Bacillus subtilis mutants resistant to cold shock-induced autolysis.

Bacillus subtilis vegetative cells undergo autolysis when exposed to cold shock treatment. A mutant (CA1) resistant to cold shock was isolated, and its DNA was used for the transformation of B. subtilis 168AR. The transformant (TR1) and CA1 had almost completely lost major vegetative autolysins (Cw1B and Cw1G) and motility, and showed a filamentous cell morphology during the exponential phase. Expression of the sigD-lacZ fusion was reduced in TR1. But the introduction of a SigD overproducing plasmid, pHYSigD, into TR1 led to a considerable increase in the amount of autolysin, a normal cell morphology (short rod), and the cold shock-sensitive phenotype. However, motility was not restored in the transformant. The roles of pleiotropic genes in cold shock-induced autolysis are discussed.

Bacillus subtilis↗
PubMed1997

Mutational analysis of 39 residues of vaccinia DNA topoisomerase identifies Lys-220, Arg-223, and Asn-228 as important for covalent catalysis.

Vaccinia DNA topoisomerase, a 314-amino acid type I enzyme, catalyzes the cleavage and rejoining of DNA strands through a DNA-(3'-phosphotyrosyl)-enzyme intermediate. To identify amino acids that participate in the transesterification reaction, we introduced alanine substitutions at 39 positions within a conserved 57amino acid segment upstream of the active-site tyrosine. Purified wild type and mutant proteins were compared with respect to their activities in relaxing supercoiled DNA. The majority of mutant proteins displayed wild type topoisomerase activity. Mutant enzymes that relaxed DNA at reduced rates were subjected to kinetic analysis of the strand cleavage and religation steps under single-turnover and equilibrium conditions. For the wild type topoisomerase, the observed single-turnover cleavage rate constant (kcl) was 0.29 s-1 and the cleavage-religation equilibrium constant (Kcl) was 0.22. The most dramatic mutational effects were seen with R223A; removal of the basic side chain reduced the rates of cleavage and religation by factors of 10(-4.3) and 10(-5.0), respectively, and shifted the cleavage-religation equilibrium in favor of the covalently bound state (Kcl = 1). Introduction of lysine at position 223 restored the rate of cleavage to 1/10 that of the wild type enzyme. We conclude that a basic residue is essential for covalent catalysis and suggest that Arg-223 is a constituent of the active site. Modest mutational effects were observed at two other positions (Lys-220 and Asn-228), at which alanine substitutions slowed the rates of strand cleavage by 1 order of magnitude and shifted the equilibrium toward the noncovalently bound state. Arg-223 and Lys-220 are conserved in all members of the eukaryotic type I topoisomerase family; Asn-228 is conserved among the poxvirus enzymes.

Amino Acid Sequence↗
PubMed1997

Domain structure of vaccinia DNA ligase.

The 552 amino acid vaccinia virus DNA ligase consists of three structural domains defined by partial proteolysis: (i) an amino-terminal 175 amino acid segment that is susceptible to digestion with chymotrypsin and trypsin; (ii) a protease-resistant central domain that contains the active site of nucleotidyl transfer (Lys-231); (iii) a protease-resistant carboxyl domain. The two protease-resistant domains are separated by a protease-sensitive interdomain bridge from positions 296 to 307. Adenylyltransferase and DNA ligation activities are preserved when the N-terminal 200 amino acids are deleted. However, the truncated form of vaccinia ligase has a reduced catalytic rate in strand joining and a lower affinity for DNA than does the full-sized enzyme. The 350 amino acid catalytic core of the vaccinia ligase is similar in size and protease-sensitivity to the full-length bacteriophage T7 DNA ligase.

Adenosine Triphosphate↗
PubMed1997

Site-specific ribonuclease activity of eukaryotic DNA topoisomerase I.

Type I topoisomerases alter DNA topology by cleaving and rejoining one strand of duplex DNA through a covalent protein-DNA intermediate. Here we show that vaccinia topoisomerase, a eukaryotic type IB enzyme, catalyzes site-specific endoribonucleolytic cleavage of an RNA-containing strand. The RNase reaction occurs via transesterification at the scissile ribonucleotide to form a covalent RNA-3'-phosphoryl-enzyme intermediate, which is then attacked by the vicinal 2' OH of the ribose sugar to yield a free 2', 3' cyclic phosphate product. Introduction of a single ribonucleoside at the scissile phosphate of an otherwise all-DNA substrate suffices to convert the topoisomerase into an endonuclease. Human topoisomerase I also has endoribonuclease activity. These findings suggest potential roles for topoisomerases in RNA processing.

Base Sequence↗
PubMed1997

Nick sensing by vaccinia virus DNA ligase requires a 5' phosphate at the nick and occupancy of the adenylate binding site on the enzyme.

Vaccinia virus DNA ligase has an intrinsic nick-sensing function. The enzyme discriminates at the substrate binding step between a DNA containing a 5' phosphate and a DNA containing a 5' hydroxyl at the nick. Further insights into nick recognition and catalysis emerge from studies of the active-site mutant K231A, which is unable to form the covalent ligase-adenylate intermediate and hence cannot activate a nicked DNA substrate via formation of the DNA-adenylate intermediate. Nonetheless, K231A does catalyze phosphodiester bond formation at a preadenylated nick. Hence, the active-site lysine of DNA ligase is not required for the strand closure step of the ligation reaction. The K231A mutant binds tightly to nicked DNA-adenylate but has low affinity for a standard DNA nick. The wild-type vaccinia virus ligase, which is predominantly ligase-adenylate, binds tightly to a DNA nick. This result suggests that occupancy of the AMP binding pocket of DNA ligase is essential for stable binding to DNA. Sequestration of an extrahelical nucleotide by DNA-bound ligase is reminiscent of the base-flipping mechanism of target-site recognition and catalysis used by other DNA modification and repair enzymes.

Adenosine Monophosphate↗
PubMed1997

Viability of transgenic rat embryos after freezing and thawing.

In-vivo viability of frozen-thawed embryos derived from transgenic rats, as well as the transmission and the expression of transgenes in the resultant newborn rats, was investigated. Three strains of transgenic rats, carrying human growth hormone gene connected downstream to the promoter region of the bovine alpha-lactalbumin gene (alpha LA/hGH), bovine beta-casein gene (beta CN/hGH) or bovine alpha-S1 casein gene (alpha S1CN/hGH), were used. Two-cell stage embryos (non-transgenic Wistar female x heterozygous transgenic male) were placed in 10% (v/v) dimethylsulfoxide (DMSO) solution and cooled from -7 to -30 degrees C at -0.5 degree C/min before being plunged into liquid nitrogen. After 2 to 4 years storage, the embryos were thawed by rapid warming. The intact embryos were transferred into the oviducts of Day 1 pseudopregnant recipients. The postthaw survival rate of frozen embryos was high in all 3 transgenic strains (88 to 92%), which was similar to that of control (non-transgenic) frozen embryos (95%). Development to newborn rats following transfer of embryos derived from the 3 strains (64 to 68%) was also similar to that of control embryos (60%). These transgenes (alpha LA/hGH, beta CN/hGH and alpha S1CN/hGH) were detected in the DNA extracts from tail tissue of the newborn rats, but the transmission rates (41, 23 and 32%, respectively) were lower than 50% which is expected in the Mendelian fashion. In a transgenic line carrying alpha S1CN/hGH, hGH levels of secretion into the milk of transgenic newborn rats derived from frozen-thawed embryos and her transgenic offspring were the same mg/ml-level as that of their founder rat. Two-step freezing of embryos derived from transgenic rats was therefore an effective method for the long-term cryopreservation of transgene.

Animals↗
PubMed1997

Quadruplex formation of oligonucleotides containing G clusters and their nuclease resistance.

Oligoribonucleotides containing four G clusters, (UGnU)4 (n = 3-5), were synthesized and characterized by electrophoretic analysis and resistance to nucleases. Electrophoretic analyses indicate that the oligomers except for (UG3U)4 can form antiparallel-stranded monomeric quadruplexes in the presence of potassium cation. The oligomers used in this study are protected from endonuclease P1 and venom phosphodiesterase degradation. In addition, the oligomers indicate resistance to nucleases in human serum and E. coli S-100 fraction.

Base Composition↗
PubMed1996

The Bacillus subtilis chromosome region near 78 degrees contains the genes encoding a new two-component system, three ABC transporters and a lipase.

The nucleotide sequence of a 9444-bp segment around the 78 degrees region of the Bacillus subtilis (Bs) chromosome has been determined. Nine putative orfs were identified. The deduced amino acid sequences of the products of two of them (yfiJ and yfiK) exhibit high similarity to those of a sensor protein, DegS, and a transcriptional regulatory protein, DegU, of Bs, respectively. Three of them (yfiL, yfiM and yfiN) seem to be ABC transporter genes. One orf (designated as lipB), the closest to the sspE among the nine orfs, is the second lipase gene in Bs.

ATP-Binding Cassette Transporters↗
PubMed1996

Cleavage of single- and double-stranded DNAs containing an abasic residue by Escherichia coli exonuclease III (AP endonuclease VI).

The Escherichia coli exonuclease III (AP endonuclease VI) is a DNA-repair enzyme that hydrolyzes the phosphodiester bond 5' to an abasic site in DNA. To study how the enzyme recognizes the abasic site, we used oligonucleotides containing a synthetic abasic site at any desired position in the sequence. We prepared oligonucleotides containing an abasic residue such as 2'-deoxyribosylformamide, 2'-deoxyribose, 1',2'-dideoxy ribofuranose or propanediol. Duplex oligonucleotides containing an abasic residue used in this study were cleaved on the 5' side of the abasic site by exonuclease III in spite of the varieties of the bases opposite and adjacent to the abasic site. In addition, we observed that the enzyme cleaved single-stranded oligonucleotides containing an abasic site on the 5' side of the abasic site. These findings suggest that the enzyme may principally recognize the DNA-pocket formed at an abasic site. The indole ring of the tryptophan 212 residue of the exonuclease III is probably intercalated to the abasic site. The tryptophan in the vicinity of the catalytic site is conserved in the type II AP endonuclease from various organisms.

DNA, Bacterial↗
PubMed1996

Cloning and nucleotide sequence of the mono- and diacylglycerol lipase gene (mdlB) of Aspergillus oryzae.

Aspergillus oryzae IFO4202 produces at least two extracellular lipolytic enzymes L1 and L2 (cutinase, and mono- and diacylglycerol lipase, respectively). Southern hybridization of restriction enzyme-digested genomic DNA fragments with 23-mer oligonucleotides synthesized according to the amino acid sequence of the L2 as probe suggested the presence of the L2 gene (tentatively designated as mdlB) and an additional weakly hybridizing region. A fragment containing the genomic mdlB gene was cloned in Escherichia coli. Nucleotide sequencing of the fragment revealed an open reading frame, comprising 1021 nucleotides, which contains two introns (51 and 52 nucleotides). Putative polyadenylation signals were found 182 and 287 bp downstream of the stop codon. The deduced amino acid sequence of the mdlB gene corresponds to 306 amino acid residues including a leader sequence of 28 amino acids and is highly similar to that of the mdlA gene of Penicillium camembertii. Three residues presumed to form the catalytic triad (serine, aspartic acid and histidine) of lipases were also conserved.

Amino Acid Sequence↗
PubMed1996

Cloning and sequencing of a 27.8-kb nucleotide sequence of the 79 degrees-81 degrees region of the Bacillus subtilis genome containing the sspE locus.

The nucleotide sequence of a 27830-bp DNA segment in the 79 degrees-81 degrees region of the Bacillus subtilis genome has been determined. This region contains 29 complete ORFs including the sspE gene, which encodes a small acid-soluble spore protein gamma and locates on the one side terminal of our assigned region. A homology search for the products deduced from the 29 ORFs revealed that nine of them exhibit significant similarity to known proteins, e.g. proteins involved in an iron uptake system, a multidrug resistance protein, a chloramphenicol resistance protein, epoxide hydrolase, adenine glycosylase, and a glucose-1-dehydrogenase homolog.

Amino Acid Sequence↗
PubMed1996

Covalent DNA binding by vaccinia topoisomerase results in unpairing of the thymine base 5' of the scissile bond.

We have used potassium permanganate to probe contacts between vaccinia DNA topoisomerase and thymine residues in its 5'-CCCTT downward arrow DNA target site. Two major conclusions emerge from the experiments presented: (i) permanganate oxidation of the +2T base of the scissile strand interferes with topoisomerase binding to DNA, and (ii) the +1T base of the scissile strand becomes unpaired upon formation of the covalent topoisomerase-DNA intermediate. Disruption of T:A base pairing is confined to the +1-position. Covalently bound DNAs that have experienced this structural distortion (such DNAs being marked by oxidation at +1T) are fully capable of being religated. We suggest that a protein-induced DNA conformational change is a component of the strand passage step of the topoisomerase reaction.

Base Sequence↗
PubMed1996

Identification of contacts between topoisomerase I and its target DNA by site-specific photocrosslinking.

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, binds and cleaves duplex DNA at sites containing the sequence 5'-(T/C)CCTT. We report the identification of Tyr70 as the site of contact between the enzyme and the +4C base of its target site. This was accomplished by UV-crosslinking topoisomerase to bromocytosine-substituted DNA, followed by isolation and sequencing of peptide-DNA photoadducts. A model for the topoisomerase-DNA interface is proposed, based on the crystal structure of a 9 kDa N-terminal tryptic fragment. The protein domain fits into the DNA major groove such that Tyr70 is positioned close to the +4C base and Tyr72 is situated near the +3C base. Mutational analysis indicates that Tyr70 and Tyr72 contribute to site recognition during covalent catalysis. We propose, based on this and other studies of the vaccinia protein, that DNA backbone recognition and reaction chemistry are performed by a relatively well-conserved 20 kDa C-terminal portion of the vaccinia enzyme, whereas discrimination of the DNA sequence at the cleavage site is accomplished by a separate N-terminal domain, which is less conserved between viral and cellular proteins. Division of function among distinct structural modules may explain the different site specificities of the eukaryotic type I topoisomerases.

Amino Acid Sequence↗
PubMed1996

Cloning and sequencing of a new holin-encoding gene of Bacillus licheniformis.

A Bacillus licheniformis DNA fragment which exhibits homology with the upstream region of the cell-wall hydrolase-encoding gene, cwlL, was cloned into Escherichia coli (Ec). Nucleotide sequencing indicated that there are two open reading frames (tentatively designated as xpaG1 and xpaG2) which encode polypeptides of 89 and 88 amino acids (aa) (10044 and 9764 Da, respectively). Ec cells harboring two compatible plasmids (pMWB1 and pHSGKH) containing the Bacillus subtilis cell-wall hydrolase-encoding gene, cwlA, and xpaG1-G2, respectively, exhibited higher extra-cellular cell-wall hydrolase activity than did cells harboring pMWB1 and a control plasmid, pHSG398. The aa sequence homology of XpaG2 with other polypeptides indicated that xpaG2 is a holin-encoding gene. Moreover, Ec C600 harboring a plasmid containing xpaG1-xpaG2 led to leakage of beta-galactosidase into the extracellular fraction.

Amidohydrolases↗
PubMed1996

Mechanism of inhibition of vaccinia DNA topoisomerase by novobiocin and coumermycin.

Vaccinia DNA topoisomerase, a eukaryotic type I enzyme, has unique pharmacological properties, including sensitivity to the coumarin drugs novobiocin and coumermycin, which are classical inhibitors of DNA gyrase, a type II enzyme. Whereas coumarins inhibit gyrase by binding the GyrB subunit and thereby blocking the ATP-binding site, they inhibit vaccinia topoisomerase by binding to the protein and blocking the interaction of enzyme with DNA. Noncovalent DNA binding and single-turnover DNA cleavage by topoisomerase are inhibited with K1 values of 10-25 microM for coumermycin and 350 microM for novobiocin. Spectroscopic and fluorescence measurements of drug binding t enzyme indicate a single binding site on vaccinia topoisomerase for coumermycin (KD = 27 +/- 5 microM) and two classes of binding sites for novobiocin, one tight site (KD1 = 20 +/- 5 microM) and several weak sites (KD2 = 513 +/- 125 microM; n = 4.9 +/- 0.7). Addition of a stoichiometric amount of DNA to a performed coumermycin-topoisomerase complex quantitatively displaces the drug, indicating that coumermycin binding and DNA binding to topoisomerase are mutually exclusive. A simple interpretation is that the site of drug binding coincides or overlaps with the DNA-binding site on the topoisomerase. Both novobiocin and coumermycin alter the susceptibility of vaccinia topoisomerase to proteolysis with either chymotrypsin or trypsin; similar effects occur when topoisomerase binds to duplex DNA.

Aminocoumarins↗
PubMed1996

Resolution of Holliday junctions by eukaryotic DNA topoisomerase I.

The Holliday junction, a key intermediate in both homologous and site-specific recombination, is generated by the reciprocal exchange of single strands between two DNA duplexes. Resolution of the junctions can occur in two directions with respect to flanking markers, either restoring the parental DNA configuration or generating a genetic crossover. Recombination can be regulated, in principle, by factors that influence the directionality of the resolution step. We demonstrate that the vaccinia virus DNA topoisomerase, a eukaryotic type I enzyme, catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT decreases, that are opposed within a partially mobile four-way junction. Cruciforms are resolved unidirectionally and with high efficiency into two linear duplexes. These findings suggest a model whereby type I topoisomerases may either promote or suppress genetic recombination in vivo.

Base Sequence↗
PubMed1996

Finger replantation in a 12-month-old child: a long-term follow-up.

A 16-year follow-up of a digital replantation in a 12-month-old child is described. Postoperative growth and functional recovery were excellent and the patient had no difficulties in activities of daily living. Some factors such as epiphyseal restoration or compensatory bone growth may be important for the good result obtained in this case.

Amputation, Traumatic↗