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Biomedical subjects

M Itaya

Publications and source records attributed to M Itaya.

At least 37 records · Page 2Linked to original sources
PubMed1997

[Compartment analysis of 123I-iomazenil brain on early and delayed SPECT].

We investigated the characteristics of 123I-Iomazenil (IMZ) SPECT images in 12 adults (six males and six females, with a mean age of 56.1 years). The washout rate of 123I-IMZ from the brain was estimated from two SPECTs done 15 min and 3 hr after injection. Although the washout was relatively slow, the rates differed in each intracerebral region, suggesting that the distribution of 123I-IMZ was gradually changing. Furthermore, assuming 123I-IMZ kinetics in the brain for the three-compartment, two-parameter model, the transition rate constant (K1) from the blood to the brain and the binding potentials (BP) of benzodiazepine to the receptor were calculated. The BP and K1 values were compared with 123I-IMZ SPECT counts and CBF values by 123I-IMP. The BP values correlated more closely with the counts on the delayed SPECT than those on the early SPECT. It was confirmed that delayed SPECT images reflect better the distribution of the benzodiazepine receptor than early images do. On the other hand, the K1 values correlated highly with CBF obtained by 123I-IMP, and this finding suggested that super-early SPECT images might be remarkably influenced by the distribution of CBF.

Adult↗
PubMed1996

A method to invert DNA segments of the Bacillus subtilis 168 genome by recombination between two homologous sequences.

We developed a method that allows rapid isolation of mutant Bacillus subtilis 168 carrying an inversion of a specific DNA segment of the genome. Two incomplete neomycin resistance gene cassettes were integrated at both ends of the 1652-kb segment to be inverted. Reciprocal recombination within the 590-bp homologous region of these two cassettes created an intact neomycin resistance gene with concomitant inversion of the 1652-kb segment flanked by the two cassettes. Structure of the mutant genome was verified by analyzing the physical map for rare cutting endonucleases, SfiI, NotI, I-CeuI, and I-SceI. The inversion rate was estimated to be 6.9 +/- 1.4 x 10(-8)/cell/cell division at 37 degrees C. The method should be in principle applicable not only to other regions of the B, subtilis genome but also to other bacterial genomes.

Anti-Bacterial Agents↗
PubMed1995

Toward a bacterial genome technology: integration of the Escherichia coli prophage lambda genome into the Bacillus subtilis 168 chromosome.

A novel approach to the cloning large DNAs in the Bacillus subtilis chromosome was examined. An Escherichia coli prophage lambda DNA (48.5 kb) was assembled in the chromosome of B. subtilis. The lambda DNA was first subcloned in four segments, having partially overlapping regions. Assembly of the complete prophage was achieved by successive transformation using three discrete DNA integration modes: overlap-elongation, Campbell-type integration, and gap-filling. In the B. subtilis chromosome, DNA was elongated, using contiguous DNA segments, via overlap-elongation. Jumping from one end of a contiguous DNA stretch to another segment was achieved by Campbell-type integration. The remaining gap was sealed by gap-filling. The incorporated lambda DNA thus assembled was stably replicated as part of the 4188 kb B. subtilis chromosome under non-selective conditions. The present method can be used to accommodate larger DNAs in the B. subtilis chromosome and possible applications of this technique are discussed.

Bacillus subtilis↗
PubMed1995

An estimation of minimal genome size required for life.

The number of indispensable chromosomal loci for a bacterium, Bacillus subtilis was estimated. Seventy-nine randomly selected chromosomal loci were investigated by mutagenesis. Mutation at only six loci rendered B. subtilis unable to form colonies. In contrast, mutants for the rest of the 73 loci retained the ability to form colonies. Mutant B. subtilis with multiple-fold mutations of those dispensable loci (7-, 12- or 33-fold) were not impaired in their ability to form colonies on nutritionally adequate medium, indicating that up to 33 dispensable loci were simultaneously abolished. Given the statistical analyses for the frequency of indispensable loci (6 out of 79), total indispensable genetic material would be included within about 562 kbp. The hypothetical minimum genome size lies in the range of those currently determined smallest genomes for bacteria.

Bacillus subtilis↗
PubMed1995

How to alter the bacterial genome structure.

Bacterial chromosomes, mostly of circular form, have an unique primary structure that are stably maintained. We initiated a systematic study to induce changes of the structure of the Bacillus subtilis chromosome. There are two main goals: (i) to obtain general concepts for possible plasticity of the bacterial genome and (ii) to apply the proposed genome technology to bacteria.

Bacillus subtilis↗
PubMed1994

A novel strategy for stabilization of Escherichia coli ribonuclease HI involving a screen for an intragenic suppressor of carboxyl-terminal deletions.

A strategy to genetically select Escherichia coli ribonuclease HI mutants with enhanced thermostability is described. E. coli strain MIC3001, which shows an RNase H-dependent, temperature-sensitive growth phenotype, was used for this purpose. Introduction of the rnhA gene permits the growth of this temperature-sensitive strain, whereas the gene for the truncated protein, 142-RNase HI, which lacks the carboxyl-terminal 13 residues, cannot. Analyses of the production levels and the stability of a series of mutant proteins with COOH-terminal truncations suggested that 142-RNase HI is nonfunctional in vivo because of a dramatic decrease in the protein stability. Polymerase chain reaction-mediated random mutagenesis of the rnhA142 gene, encoding 142-RNase HI, followed by selection of revertants, allowed us to isolate 11 single amino acid substitutions that render 142-RNase HI functional in vivo. Of them, eight substitutions were shown to enhance the thermal stability of the wild-type RNase HI protein, and of these, six were novel. The genetic selection strategy employed in this experiment was thus shown to be effective for identifying amino acid substitutions that enhance the thermal stability of E. coli RNase HI. Such a strategy would be versatile if a protein of interest could be destabilized by a deletion or a truncation and a conditional-lethal strain were available.

Amino Acid Sequence↗
PubMed1994

Investigating the role of conserved residue Asp134 in Escherichia coli ribonuclease HI by site-directed random mutagenesis.

The role of the conserved Asp134 residue in Escherichia coli ribonuclease HI, which is located at the center of the alpha V helix and lies close to the active site, was analyzed by means of site-directed random mutagenesis. Mutant rnhA genes encoding proteins with ribonuclease H activities were screened by their ability to suppress the ribonuclease-H-dependent, temperature-sensitive growth phenotype of E. coli strain MIC3001. Based on the DNA sequences, nine mutant proteins were predicted to have ribonuclease H activity in vivo. All of these mutant proteins were purified to homogeneity and examined for enzymic activity and protein stability. Among them, only the mutant proteins [D134H]RNase H and [D134N]RNase H were shown to have considerable ribonuclease H activities. Determination of the kinetic parameters revealed that replacement of Asp134 by amino acid residues other than asparagine and histidine dramatically decreased the enzymic activity without seriously affecting the substrate binding. Determination of the CD spectra indicated that none of the mutations seriously affected secondary and tertiary structure. The protein stability was determined from the thermal denaturation curves. All mutant proteins were more stable than the wild-type protein. Such stabilization effects would be a result of a reduction in the negative charge repulsion between Asp134 and the active-site residues, and/or an enhancement of the stability of the alpha V helix. These results strongly suggest that Asp134 does not contribute to the maintenance of the molecular architecture but the carboxyl oxygen at its delta 1 position impacts catalysis.

Amino Acid Sequence↗
PubMed1994

Multiple copies of the proB gene enhance degS-dependent extracellular protease production in Bacillus subtilis.

Bacillus subtilis secretes extracellular proteases whose production is positively regulated by a two-component regulatory system, DegS-DegU, and other regulatory factors including DegR. To identify an additional regulatory gene(s) for exoprotease production, we performed a shotgun cloning in the cell carrying multiple copies of degR and found a transformant producing large amounts of the exoproteases. The plasmid in this transformant, pLC1, showed a synergistic effect with multiple copies of degR on the production of the extracellular proteases, and it required degS for its enhancing effect. The DNA region responsible for the enhancement contained the proB gene, as shown by restriction analyses and sequence determination. The proB gene encoding gamma-glutamyl kinase was followed by the proA gene encoding glutamyl-gamma-semialdehyde dehydrogenase at an interval of 39 nucleotides, suggesting that the genes constitute an operon. pLC1 contained the complete proB gene and a part of proA lacking the proA C-terminal region. It was also found that proB on the chromosome showed a synergistic effect with multiple copies of degR. We consider on the basis of these results that the metabolic intermediate, gamma-glutamyl phosphate, would transmit a signal to DegS, resulting in a higher level of phosphorylated DegU. Possible involvement of DegR in this process is discussed.

1-Pyrroline-5-Carboxylate Dehydrogenase↗
PubMed1994

First evidence for homologous recombination-mediated large DNA inversion on the Bacillus subtilis 168 chromosome.

A Bacillus subtilis 168 strain carrying an inversion of about 1600 kb-long chromosomal DNA was isolated. Physical and genetic analyses demonstrated that the inversion was generated as a result of homologous recombination between two homologous sequences integrated at the met and leuB loci. This is the first clear evidence of a large stable chromosomal inversion induced by homologous recombination in B. subtilis.

Bacillus subtilis↗
PubMed1993

Integration of repeated sequences (pBR322) in the Bacillus subtilis 168 chromosome without affecting the genome structure.

The Escherichia coli plasmid pBR322 sequence (4363 bp) was integrated at the met, pro, or leuB locus of the Bacillus subtilis chromosome without duplication of the flanking chromosomal regions. The integrated pBR322 was stably maintained as part of the chromosome regardless of its orientation or location. It was found that a DNA segment as large as 17 kb cloned in pBR322 can be readily transferred to the B. subtilis chromosome by transformation. It was demonstrated that a second pBR322 sequence could be effectively introduced at different regions of the chromosome by sequential transformation using chromosomal DNA isolated from a strain that had already acquired a pBR322 sequence at a different locus. Similarly, a third pBR322 sequence could be introduced. By this method, two or three pBR322 sequences can be incorporated at unlinked loci without affecting the overall structure of the B. subtilis genome.

Bacillus subtilis↗
PubMed1993

Stability and asymmetric replication of the Bacillus subtilis 168 chromosome structure.

Chromosomal DNAs from a number of strains derived from Bacillus subtilis 168 were digested with restriction endonucleases NotI or SfiI, and the locations of chromosomal alterations were compared with the recently constructed standard NotI-SfiI restriction map (M. Itaya and T. Tanaka, J. Mol. Biol. 220:631-648, 1991). In general, the chromosome structure of B. subtilis 168 was found to be stable, as expected from the genetic stability of this species. DNA alterations, typically deletions, are formed in three limited loci on the chromosome. One of these alterations was characterized as a spontaneous deletion formed between rrn operons, and another occurred as a result of prophage SP beta excision. I found that oriC and terC are not located on precisely opposite sides of the chromosome. Replication in the counter clockwise direction was 196 kb longer than replication in the clockwise direction. The characteristic of length difference is not changed by deletion formation.

Bacillus Phages↗
PubMed1993

Physical mapping of multiple homologous genes in the Bacillus subtilis 168 chromosome: identification of ten ribosomal RNA operon loci.

Ten ribosomal RNA operons (rrn) of the Bacillus subtilis 168 strain were precisely located in the complete physical map, i.e., the NotI and SfiI restriction map [M. Itaya and Tanaka, J. Mol. Biol., 220, 631-648 (1991)]. Mapping was done by identification of the NotI and the SfiI fragments to which the rrnO operon hybridized. This protocol proved that homologous multiple genes dispersed on the chromosome can be physically mapped by using a single membrane filter. A previously unidentified deletion formation was shown to be that caused by homologous recombination between rrnJ-W.

Bacillus subtilis↗
PubMed1993

Construction of the Bacillus subtilis chromosome physical map and the strategy for mapping newly isolated genes in one membrane filter for hybridization.

A complete physical map of the Bacillus subtilis 168 chromosome was constructed. The merging of this physical map is expected not only to provide important insights into the organization and rearrangement of genes of this species but also to be a powerful means for the genome analysis. One of the most practical aspects is rapid and accurate mapping of newly isolated genes using a single membrane filter for hybridization. This protocol proved that not only unique genes but also multiple homologous genes dispersed on the chromosome can be physically mapped.

Bacillus subtilis↗
PubMed1992

Expression, purification, and characterization of a recombinant ribonuclease H from Thermus thermophilus HB8.

Thermus thermophilus ribonuclease H was overexpressed and purified from Escherichia coli. The determination of the complete amino acid sequence allowed modification of that predicted from the DNA sequence, and the enzyme was shown to be composed of 166 amino acid residues with a molecular weight of 18,279. The isoelectric point of the enzyme was 10.5, and the specific absorption coefficient A0.1%(280) was 1.69. The enzymatic and physicochemical properties as well as the thermal and conformational stabilities of the enzyme were compared with those of E. coli RNase HI, which shows 52% amino acid sequence identity. Comparison of the far and near UV circular dichroism spectra suggests that the two enzymes are similar in the main chain folding but different in the spatial environments of tyrosine and tryptophan residues. The enzymatic activities of T. thermophilus RNase H at 37 and 70 degrees C for the hydrolysis of either an M13 DNA/RNA hybrid or a nonanucleotide duplex were approximately 5-fold lower and 3-fold higher, respectively, as compared with E. coli RNase HI at 37 degrees C. The melting temperature, Tm, of T. thermophilus RNase H was 82.1 degrees C in the presence of 1.2 M guanidine hydrochloride, which was 33.9 degrees C higher than that observed for E. coli RNase HI. The free energy changes of unfolding in the absence of denaturant, delta G[H2O], of T. thermophilus RNase H increased by 11.79 kcal/mol at 25 degrees C and 14.07 kcal/mol at 50 degrees C, as compared with E. coli RNase HI.

Amino Acid Sequence↗
PubMed1992

Physical distance between the site of type II DNA binding to the membrane and oriC on the Bacillus subtilis 168 chromosome.

The precise physical locations of the oriC region and the region for type II DNA binding to the membrane on the Bacillus subtilis 168 chromosome were determined. The DNA regions were physically mapped by creating new restriction sites (NotI and SfiI) within these regions. The physical distance between oriC and the type II DNA-binding region was verified with the creation of a novel sequence cleaved by endonuclease I-SceI in each of the above regions. Complete removal of the defined type II membrane-binding region produced no noticeable phenotype.

Bacillus subtilis↗
PubMed1992

Isolation and characterization of the groES and groEL genes of Bacillus subtilis Marburg.

The complete set of groES and groEL gene homologues from Bacillus subtilis Marburg 168 was identified, cloned, and characterized. The nucleotide sequence indicated the presence of two open reading frames corresponding to the groES and groEL genes. The presumptive GroES and GroEL proteins were calculated to be polypeptides of 10,175 and 57,175 Da, respectively, and showed extensive sequence similarities with the known GroES and GroEL proteins of Escherichia coli and Mycobacterium tuberculosis. A heat-inducible transcript initiated upstream of the groES coding region was identified by primer-extension analysis of in vivo transcripts, indicating that the two genes consist of an operon. At least six heat-shock inducible proteins were identified in the cell extract of heat treated B. subtilis. Two proteins of 10 and 60 kDa overproduced in B. subtilis cells carrying a multi-copy groES and groEL plasmid were demonstrated to correspond to two out of the six heat-shock inducible proteins. The groES and groEL genes of B. subtilis were physically mapped on the 60 degrees region of a 360 degrees map and genetically mapped at the position of 40% linkage with the purB locus using PBS1 transduction of the groEL genes tagged with a chloramphenicol resistance (chlr) marker.

Amino Acid Sequence↗