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Biomedical subjects

A Hirashima

Publications and source records attributed to A Hirashima.

At least 37 records · Page 2Linked to original sources
PubMed1994

Molecular cloning and characterization of prolyl endopeptidase from human T cells.

The prolyl endopeptidase (PEP) gene of human T cells was amplified by the PCR method and cloned in Escherichia coli. The complete gene consisted of 2,130 nucleotides corresponding to 710 amino acid residues with a calculated molecular mass of 80,750. The nucleotide sequence of this clone revealed that T cell PEP DNA is 48, 50, and 91% homologous to those of Flavobacterium meningosepticum, Aeromonas hydrophila, and porcine brain PEP, respectively. This gene was fused to the lacZ sequence from E. coli and expressed as a fused protein in E. coli. This fused protein exhibited PEP activity, which was inhibited by Z-Pro-prolinal, a specific inhibitor of PEP. The fused protein was purified on a beta-galactosidase specific affinity column. A polyclonal antibody was raised against the purified protein. Immunological characterization suggested that this protein is different from cytosol-soluble PEP.

Amino Acid Sequence↗
PubMed1994

A study on the function of the glycine residue in the YGDD motif of the RNA-dependent RNA polymerase beta-subunit from RNA coliphage Q beta 1.

Q beta replicases in which the Gly residue of the beta-subunit in the motif sequence, YGDD, was replaced with Ala, Ser, Pro, Met, or Val lost their replicase activity in vivo. In an in vitro Mg(2+)-dependent RNA-synthesizing system using poly(rC) or MDV-poly(+) RNA (a derivative of the naturally occurring small RNA that accumulates in the cells during Q beta phage infection) as templates, the lysates from the cells expressing such defective replicases exhibited only 2-6% of the enzyme activity of the lysate from those expressing wild-type replicase. However, the defective replicases, especially A357, with Ala substituted for the Gly, recovered enzyme activity when Mn2+ was added to the reaction mixture. Furthermore, the characteristics of the MDV-poly(+) RNA-dependent RNA synthesis by A357 replicase were similar to those by wild-type replicase in the presence of Mn2+. Gel retardation assay showed that all of the defective replicases could bind MDV-poly(+) RNA. These results suggest that the Gly residue in this motif of Q beta replicase is involved in Mg(2+)-catalyzed polymerization. In the Mn(2+)-catalyzed polymerization, A357 and S357 replicases can act as well as the wild-type replicase.

Allolevivirus↗
PubMed1990

Interference with viral infection by RNA replicase deleted at the carboxy-terminal region.

Escherichia coli cells harboring an altered Q beta RNA replicase which has amino acid substitutions of the glycine residue at position 357 in the conserved sequence Tyr356-Gly357-Asp358-Asp359 of the beta-subunit protein lost the replicase activity but interfered with proliferation of Q beta phage [Inokuchi and Hirashima (1987) J. Virol. 61, 3946-3949]. To examine the mechanism of the interference, we further analyzed various mutants lacking the carboxy-terminal region of the beta-subunit protein. The cells expressing the beta-subunit gene with up to 17% deletion from the carboxy-terminus of the protein prevented the proliferation of Q beta phage. However, in the case that the deletion extended beyond 25% from the carboxy-terminus, the cells showed no interference. In addition, when the interference took place, the phage coat protein synthesis was inhibited. These results indicate that the region between amino acids 440 and 487 of the beta-subunit protein is involved in the interference and suggest that the defective replicase inhibits the phage coat protein synthesis by competing with the ribosomes at the initiation site of the coat gene.

Alanine↗
PubMed1990

Nucleotide 2',3'-cyclic monophosphokinase from actinomycetes.

Streptomyces nucleotide 3'-pyrophosphokinase does not only transfer the 5'-beta, gamma-pyrophosphoryl group of ATP, ATP 3'-pyrophosphate or dATP to a variety of nucleotides at the 3'-OH site, but also adds 2',3'-cyclic terminal monophosphate to some suitable nucleotides with the use of diadenosine 5',5'-polyphosphates (n = 3-5). Examples are pA greater than p, ppA greater than p, pG greater than p, CpG greater than p, etc.

Adenosine Triphosphate↗
PubMed1989

Nucleotide sequence from the ssRNA bacteriophage JP34 resolves the discrepancy between serological and biophysical classification.

The nucleotide sequence of the coat and lysis genes of the single-stranded RNA bacteriophage JP34 is presented. Serological inactivation studies classified this phage as an intermediate between groups I and II. We show that the nucleotide similarity with group I is less than 45% but more than 95% for group II, classifying JP34 as a member of group II. The altered serotype of JP34 is most likely due to the change of three critical amino acids of the coat protein to residues present in group I phage MS2 at the homologous positions. Serological characterization of RNA bacteriophages is thus not unambiguous. Phylogenetic sequence comparison between JP34, GA, and MS2 confirms the existence of a conserved helix in the coat gene of group I and group II phages. We also show that the JP34 coat and lysis genes can be expressed in cDNA clones and that the translation of the lysis gene is coupled to coat gene translation analogous to the regulation found in the group I phages.

Amino Acid Sequence↗
PubMed1989

Artificial immune system against viral infection involving antisense RNA targeted to the 5'-terminal noncoding region of coliphage SP RNA.

We previously reported the utilization of antisense RNA in the development of a novel immune system against RNA coliphage SP proliferation (Hirashima et al. [1986] Proc. Natl. Acad. Sci. U.S. 83, 7726-7730). We attempted to determine the most effective (i.e., those eliciting antiviral activity) sequences for targeting micRNAs within the 5'-terminal noncoding region of 54 nucleotides (nt). It was found that a 30-nt micRNA against the sequence from base 32 to 61 exhibited nearly complete inhibition of phage production. Upon further dissection of this sequence, it was concluded that the most effective micRNA against phage SP production should contain the sequences complementary to the Shine-Dalgarno (SD) sequence of the first gene and its 13-nt upstream sequence. The addition of downstream sequences had little effect. These results suggest that the micRNA functions by preventing the binding of ribosomes to the SD sequence of the first gene. The addition of further upstream sequences had a significant negative effect on the micRNA function, indicating that the removal of such impeditive sequences from a micRNA is an important strategy for the development of a potent micRNA immune system.

Base Sequence↗
PubMed1989

Molecular characterization of a cell wall-associated proteinase gene from Streptococcus lactis NCDO763.

Streptococcus lactis NCDO763 harbours a plasmid designated pLP763. The cells harbouring pLP763 are able to grow to a higher density in milk because of their proteinase-positive phenotype (Prt+). The 6.2 kb HindIII-PstI fragment from pLP763 was found to be responsible for the Prt+ phenotype. The DNA fragment contains an incomplete large open reading frame (ORF). Further sequence analysis downstream from the PstI site revealed that the ORF consists of 5706 bases. It was found that the deduced amino acid sequence consisting of 1902 amino acid residues was extremely similar to that of the Wg2 proteinase, a serine protease from Streptococcus cremoris, suggesting that both genes were derived from a common ancestral gene.

Amino Acid Sequence↗
PubMed1988

Analysis of the complete nucleotide sequence of the group IV RNA coliphage SP.

We report the nucleotide sequence of the Group IV RNA bacteriophage SP. The entire sequence is 4276 nucleotides long. Four cistrons have been identified by comparison with the related Group III phage Q beta. The maturation protein contains 449 amino acids, the coat protein contains 131 amino acids, the read-through protein contains 330 amino acids and the replicase beta-subunit contains 575 amino acids. SP is 59 nucleotides longer than Q beta. We have analyzed both sequence and structural conservation between SP and Q beta and shown that the sequences for the coat and central region of the replicase are strongly conserved between the two genomes. We also show that the S and M replicase binding sites of Q beta are strongly conserved in SP. Interestingly, the base composition of SP and Q beta differ significantly from one another, and most of the differences can be accounted for by a strong preponderance of U in the third position of each codon of Q beta relative to SP. We also compare conserved hairpins associated with potential coat protein and replicase binding sites.

Base Sequence↗
PubMed1987

Interference with viral infection by defective RNA replicase.

RNA-dependent RNA and DNA polymerases have a conserved segment, Tyr-X-Asp-Asp (G. Karmer and P. Argos, Nucleic Acids Res. 12:7269-7282, 1984). To investigate the function of this segment, we changed the Gly residue at position 357 in the conserved sequence Tyr-356-Gly-357-Asp-358-Asp-359 of the replicase of RNA coliphage Q beta to Ala, Ser, Pro, Met, or Val and examined the replicase activity in vivo. Cells carrying the variant plasmids lost the replicase activity and severely inhibited the proliferation of phage Q beta (group III) and related phage SP (group IV) by suppressing phage RNA synthesis. In contrast, substitution of the Gly residue at 390 showed only a slight inhibitory effect, although replicase activity was also lost. These results suggest that the cells harboring an altered replicase at the conserved segment can interfere specifically with the wild-type phage and different but related phage infections.

Coliphages↗
PubMed1986

Engineering of the mRNA-interfering complementary RNA immune system against viral infection.

Creation of an artificial mRNA-interfering complementary RNA (micRNA) immune system, utilizing anti-sense RNAs to inhibit viral gene expression, has been shown to be an effective way to prevent viral infection. In the RNA coliphage SP, the gene for the maturation protein was found to be the best target for this type of immune system; mRNA-interfering complementary RNAs specific to the genes for coat protein and replicase were less effective in preventing infection. The greatest inhibitory effect was observed with a 240-base sequence encompassing the 24-base noncoding region of the maturation gene plus the 216-base coding sequence. Significantly, even a 19-base sequence covering only the Shine-Dalgarno sequence (ribosome-binding region) without the coding region exerted a strong inhibitory effect on phage proliferation. In contrast to the highly specific action against phage SP exhibited by the longer mRNA-interfering complementary RNA, the specificity with the shorter mRNA-interfering complementary RNA was broadened to phages Q beta and GA as well as SP, all of which are classified in the different groups of RNA coliphages. Therefore, this type of anti-viral reagent may be designed to have a particular breadth of specificity, thus increasing its value in various research and possibly clinical applications.

Base Sequence↗
PubMed1986

The complete nucleotide sequence of the group II RNA coliphage GA.

The complete nucleotide sequence of the RNA coliphage GA, a group II phage, is presented. The entire genome comprises 3466 bases. Three large open reading frames were identified, which correspond to the maturation protein gene (390 amino acids), the coat protein gene (129 amino acids) and the replicase beta-subunit protein gene (531 amino acids). In addition, untranslated regions occur at the 5' (135 bases) and 3' (122 bases) ends of the molecule. Two intercistronic untranslated regions occur between the cistrons for the maturation and coat proteins, and between the coat and beta-subunit proteins. We have compared the nucleotide sequence of GA RNA with the published sequence of MS2 RNA, and show that they are related. The comparative structures of two important regulatory regions are presented; the coat protein binding site which is involved in translational repression of the replicase beta-subunit protein gene, and a hairpin in a region proximal to the lysis protein gene.

Base Sequence↗
PubMed1986

Characterization of an intergroup serological mutant from group II RNA phage GA.

Starting from the group II RNA phage GA which has an amber mutation in the maturation protein cistron, a spontaneous mutant of group II phage GA, whose serological and electrophoretic properties became similar to those of group I phage MS2, was isolated and analyzed. The mutant has now become sensitive to anti-MS2 serum and resistant to anti-GA serum. Analysis of the nucleotide sequence of the coat protein gene revealed that G----A transition was the main change. The deduced amino acid sequence showed that five amino acids were substituted in the mutant, and three of the five became identical to MS2, resulting in increased molecular weight of the coat protein. However, it did not complement MS2. These results suggested that the serological change from group II phage GA type to group I phage MS2 type is induced spontaneously at high frequency by minor nucleotide changes in coat protein gene, and confirmed the previous results at the RNA level that MS2 and GA were related although the closeness between them seems somewhat remoter than that of groups III and IV (18, Inokuchi et al, unpublished data for the nucleotide sequence of group IV phage SP).

Amino Acid Sequence↗
PubMed1983

Biochemical characterization of RNA coliphage MX1.

In order to clarify the taxonomic status of an RNA coliphage, MX1 (a serological intermediate between groups III and IV), we examined (i) read-through protein synthesis in a cell-free protein-synthesizing system, (ii) the peptide map of the coat protein and (iii) the RNA sequence in the 3'-terminal region of MX1 RNA. We found that the characteristics of MX1 were closer to those of group III phages than to those of group IV. For example, the gel elecrophoretic pattern of the protein coded for by MX1 RNA was the same as that of group III phage proteins. Peptide fingerprints of the coat protein of MX1 showed that seven tryptic peptides overlapped with corresponding peptides of Q beta (group III phage), whereas only two peptides overlapped with those of SP (group IV phage). Furthermore, in the base sequence of the first 200 nucleotides from the 3'-end of MX1 RNA, about 70% of the nucleotides were homologous to those of the Q beta RNA, whereas the homology to SP RNA was only 53%. These results suggest that MX1 is more closely related to group III phages than to group IV phages and we propose that it should be assigned to the former group.

Base Sequence↗