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Biomedical subjects

H Nashimoto

Publications and source records attributed to H Nashimoto.

At least 19 recordsLinked to original sources
PubMed1992

Diversity of immunodominant 56-kDa type-specific antigen (TSA) of Rickettsia tsutsugamushi. Sequence and comparative analyses of the genes encoding TSA homologues from four antigenic variants.

There are several antigenic variants in Rickettsia tsutsugamushi, and a type-specific antigen (TSA) of 56-kilodaltons located on the rickettsial surface is responsible for the variation. The primary structures of the protein in two variants, Gilliam and Karp, have been reported independently by us and Stover et al. by cloning and sequencing the corresponding genes (Ohashi, N., Nashimoto, H., Ikeda, H., and Tamura, A. (1990) Gene (Amst.) 91, 119-122; Stover, C. K., Marana, D. P., Carter, J. M., Roe, B. A., Mardis, E., and Oaks, E. V. (1990) Infect. Immun. 58, 2076-2084). In the present study, genes encoding the TSA homologues of the other four variants, Kato, Kawasaki, Kuroki, and Shimokoshi, which are all distinguishable serologically, were cloned and sequenced, and consequently, it became possible to compare the primary structures of the six antigenic variants. The sequence analyses revealed a complete open reading frame encoding 55,308-56,745-dalton proteins with 521-532 amino acids, in which a putative signal peptide consisting of 22 amino acids was recognized at the NH2-terminal end. Transcription of the gene is regulated by several tandem promoters. All TSA molecules have the characteristics of transmembrane proteins with alternating hydrophobic and hydrophilic regions, and contain four variable domains with spans of 16-40 amino acids which are located in the hydrophilic regions in the molecule and show different amino acid sequences among the strains. Phylogenetic classification among the R. tsutsugamushi strains based on TSA homologues supports the antigenic relationships known in the closely and distantly related strains.

Amino Acid Sequence↗
PubMed1992

Cloning and characterization of a repetitive sequence from Pneumocystis carinii.

Four repetitive sequence clones measuring 10.9-23.4 kb in length were isolated from the genomic library of Pneumocystis carinii. Restriction enzymes mapping and cross-hybridization studies revealed that these clones are interrelated and that they derive from the common repeat unit, which is specific for P. carinii. Dot-blot analysis suggested that the copy number of the repeat sequence is about 100, assuming that the genome size is 1.5 x 10(7) bp. Interestingly, the repetition unit extended over at least 23.4 kb and included long, 5.2-kb inverted repeats, for example, A-B-A'-C, in which A' is the inversion of A.

Blotting, Southern↗
PubMed1990

Cloning and sequencing of the gene (tsg56) encoding a type-specific antigen from Rickettsia tsutsugamushi.

A type-specific antigen (TSA) in Rickettsia tsutsugamushi is a 56-kDa protein located on the rickettsial surface. The gene (tsg56) encoding the TSA was isolated from R. tsutsugamushi Gilliam strain. Sequencing revealed an open reading frame of 1572 bp encoding a 524-amino acid (aa) protein with an Mr of 56,053. The deduced aa sequence consists of a 22-aa signal sequence and the mature TSA sequence (502 aa; Mr 53,803), whose N-terminal portion has the aa sequence determined directly in our previous study [Ohashi et al., Infect. Immun. 57 (1989) 1427-1431]. Alternate localization of hydrophobic and hydrophilic regions was recognized in the TSA molecule, suggesting that the TSA is a transmembrane protein. The codon usage in the A + T-rich (59%) tsg56 gene reflects a high A + T content of R. tsutsugamushi. The 5'-flanking region contains sequences similar to those of ribosome-binding sites and promoters of Escherichia coli.

Amino Acid Sequence↗
PubMed1989

Common sites for recombination and cleavage mediated by bacteriophage T4 DNA topoisomerase in vitro.

We have previously shown that purified T4 DNA topoisomerase promotes illegitimate recombination between two lambda DNA molecules, or between lambda and plasmid DNA in vitro (Ikeda, H. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 922-926). Since the recombinant DNA contains a duplication or deletion, it is inferred that the cross-overs take place between nonhomologous sequences of lambda DNA. In this paper, we have examined the sequences of the recombination junctions produced by the recombination between two lambda DNA molecules mediated by T4 DNA topoisomerase. We have shown that there is either no homology or there are 1-5-base pair homologies between the parental DNAs in seven combinations of lambda recombination sites, indicating that homology is not essential for the recombination. Next, we have shown an association of the recombination sites with the topoisomerase cleavage sites, indicating that a capacity of the topoisomerase to make a transient double-stranded break in DNA plays a role in the illegitimate recombination. A consensus sequence for T4 topoisomerase cleavage sites, RNAY decreases NNNNRTNY, was deduced. The cleavage experiment showed that T4 topoisomerase-mediated cleavage takes place in a 4-base pair staggered fashion and produces 5'-protruding ends.

Base Sequence↗
PubMed1985

Suppressors of temperature-sensitive mutations in a ribosomal protein gene, rpsL (S12), of Escherichia coli K12.

Temperature-sensitive (ts) mutations were isolated within a ribosomal protein gene (rpsL) of Escherichia coli K12. Mutations were mapped by complementation using various transducing phages and plasmids carrying the rpsL gene, having either a normal or a defective promoter for the rpsL operon. One of these mutations, ts118, resulted in a mutant S12 protein which behaved differently from the wild-type S12 on CM-cellulose column chromatography. Suppressors of these ts mutations were isolated and characterized; one was found to be a mutation of a nonribosomal protein gene which was closely linked to the RNAase III gene on the E. coli chromosome. This suppressor, which was recessive to its wild-type allele, was cloned into a transducing phage and mapped finely. A series of cold-sensitive mutations, affecting the assembly of ribosomes at 20 degrees C, was isolated within the purL to nadB region of the E. coli chromosome and one group, named rbaA, mapped at the same locus as the suppressor mutation, showing close linkage to the RNAase III gene.

Bacteriophage lambda↗
PubMed1985

DNA sequencing of the Escherichia coli ribonuclease III gene and its mutations.

A 0.7 kb DNA fragment of the Escherichia coli K12 chromosome was shown to contain the structural gene for RNAse III (rnc). The DNA sequence of the gene was determined and its alteration in an RNAse III defective mutant, AB301-105, was identified. DNA sequence analysis also showed that a secondary-site suppressor of a temperature-sensitive mutation in the E. coli ribosomal protein gene, rpsL, occurred within the rnc gene, providing genetic evidence for the interaction of ribosomal proteins with RNAse III, which in turn acts on the nascent ribosomal RNA during assembly of ribosomes in E. coli.

Amino Acid Sequence↗
PubMed1984

Characterization of an amber mutation in the structural gene for ribosomal protein L15, which impairs the expression of the protein export gene, secY, in Escherichia coli.

We have previously described a temperature-sensitive mutant, ts215, which is defective in protein secretion. Complementation studies indicated that the mutation was located at the distal part of the spc ribosomal protein operon and the gene secY is required for efficient protein secretion. We now report a more complete genetic and biochemical analysis of the ts215 mutant. These studies revealed that the ts215 mutant has an amber mutation in the gene rp10 for ribosomal protein L15, which is located upstream and adjacent to secY. The amber mutation exerts a polar effect on secY causing a defect in protein secretion. These conclusions were supported by the following observations. The mutant strain carries a phi 80 prophage containing a temperature-sensitive suppressor, supFts6. The strain contains decreased amounts of L15 and is suppressible by a temperature-independent nonsense suppressor. In addition, L15 contains an extra tyrosine residue when suppressed by supF. DNA sequence analysis revealed the presence of a single base change in rp10 resulting in an amber codon at the 38th codon of L15. The mutant phenotype is complemented by a plasmid carrying only the secY gene under lac promoter control. The mutant cells complemented by secY can grow and synthesize proteins at normal rates and abundances at 42 degrees C, despite the fact that their ribosomes contain barely detectable levels of L15. These results indicate that ribosomal protein L15 is dispensable for protein synthesis and cell growth. In contrast, the decreased level of expression of the secY gene leads to defective protein secretion and defective cell growth.

Electrophoresis, Polyacrylamide Gel↗
PubMed1983

A temperature-sensitive mutant of E. coli exhibiting slow processing of exported proteins.

A temperature-sensitive E. coli mutant with a mutation in the spc ribosomal protein operon was found to have a conditional defect in the processing of precursor proteins destined for the periplasmic space or the outer membrane. At high temperatures, significant amounts of precursor proteins having unprocessed signal sequences are detected in the mutant cell by pulse-labeling. The precursors are processed at very slow rates during a subsequent chase. Genetic analysis indicates that the mutation impairs the function of a gene, termed secY, located at the promoter-distal part of the spc operon. The secY gene is distinct from those genes previously known to specify ribosomal proteins, yet it is within the spc operon. It is suggested that the product of the secY gene is a component of the cellular apparatus that is essential for protein secretion across the cytoplasmic membrane. The gene secY is probably identical with prlA, previously identified as a suppressor of signal sequence mutations.

Bacterial Proteins↗
PubMed1979

Cloning of an EcoRI fragment carrying E. coli tufA gene.

EcoRI fragments of the transducing phage lambda fus3 DNA have been linked to the ColEl derivative plasmid RSF2124 (ColEl-Apr) DNA using bacteriophage T4 ligase. Among the plasmids formed, one designated pTUAl was found to contain the E. coli tufA gene. The proof for the presence of tufA gene in pTUAl is based on the following observations: (1) ability of pTUAl DNA and is EcoRI fragments to direct synthesis of EF-Tu in a cell-free protein synthesizing system; and (2) RNA . DNA hybridization of RNA transcribed from phage lambda rifd18 carrying tufB with DNA from pTUAl.

Coliphages↗
PubMed1970

Structure and function of bacterial ribosomes. XI. Dependence of 50S ribosomal assembly on simultaneous assembly of 30S subunits.

Some spectinomycin-resistant mutants of Escherichia coli are cold-sensitive. They are unable to assemble both 30S and 50S ribosomal subunits at low temperatures. They accumulate two kinds of incomplete particles, related to 30S and 50S subunits respectively. A single mutation, causing an alteration in a 30S ribosomal component, is responsible for these phenotypes. These results show that assembly of 50S subunits in vivo is dependent on simultaneous assembly of 30S subunits. On the other hand, the assembly of 30S subunits appears to be independent of 50S assembly.

Bacterial Proteins↗