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Biomedical subjects

Y Taya

Publications and source records attributed to Y Taya.

At least 109 records · Page 6Linked to original sources

A rat tRNA gene cluster containing the genes for tRNAPro and tRNALys. Analysis of nucleotide sequences of the genes and the surrounding regions.

A lambda clone carrying a rat DNA fragment of 11.9 kb was isolated from a rat gene library with total rat tRNA as a probe. Nucleotide sequence analysis revealed that the DNA fragment contained six tRNA genes, three for tRNAPro and three for tRNALys. Of the six genes all but one tRNAPro gene have the same polarity. Each tRNA gene is separated by a DNA region of 0.1 to 3.6 kb. The 5'-flanking regions of the six rat genes in the cluster do not have any significant sequence homology, but in the 3'-flanking region, each gene has a short T cluster, which is supposed to be a transcription termination signal.

Animals↗

The human fibroblast and human immune interferon genes and their expression in homologous and heterologous cells.

The genetic information coding for human fibroblast interferon (IFN-beta) has been cloned both as a DNA copy (cDNA) and as a genomic clone. Human IFN-beta is made as a precursor and consists of a signal sequence 21 amino acid residues long followed by the mature protein 166 amino acids long. A single site for glycosylation is present. The human IFN-beta gene does not contain introns. Transfection of monkey cells with a chimeric SV40 derivative containing the human IFN-beta cDNA clone under control of the late SV40 promoter leads to secretion of high levels of IFN-beta. When a genomic clone is used in the same vector, IFN-beta synthesis can be further enhanced up to 30-fold by treatment with poly(rI) . poly(rC); this shows that a cis-active control element is present in the clone. An efficient expression system in Escherichia coli was worked out based on a plasmid containing the promoter PL of bacteriophage lambda, which is regulated by a temperature-sensitive repressor. This promoter is followed by a segment derived from bacteriophage MS2 that contains the ribosome-binding site of the replicase gene. The latter, however, is replaced by the human IFN-beta gene. Upon induction, high levels (about 5 x 10(9) IU 1(-1)) of IFN-beta are synthesized by the bacteria; this corresponds to about 2% of the total bacterial protein. The human immune (type II) interferon (IFN-gamma) gene has similarly been cloned. Partly purified mRNA derived from human spleen cells that had been induced with staphylococcal enterotoxin A was used as starting material. A full-length cDNA clone was sequenced. The total cDNA sequence is about 1150 nucleotides long; it contains a single open reading frame coding for 166 amino acids, the first 20 of which constitute the transmembrane signal. There are two sites for glycosylation. The amino acid sequence is quite different from that of IFN-alpha or IFN-beta, although a few similarities can be noted. The untranslated 3'-terminal region is about 550 nucleotides long. The IFN-gamma gene was expressed in monkey cells, again by using the SV40-derived vector, and the secreted product was characterized as true human IFN-gamma. A genomic clone in the form of a bacteriophage lambda derivative was also obtained. The IFN-gamma gene extends over at least 5 kilobases and contains at least two introns.

Amino Acid Sequence↗

Molecular cloning of human immune interferon cDNA and its expression in eukaryotic cells.

Starting with mRNA derived from Staphylococcal enterotoxin A induced human splenocytes, dsDNA was synthesized and inserted into unique BamHI site of the eukaryotic expression vector pSV529 (1). A recombinant plasmid containing human immune interferon (IFN-gamma) cDNA was identified by hybridization of plasmid inserted DNA bound onto nitrocellulose filters with mRNA derived from SEA-induced splenocytes, translation of the eluted RNA in Xenopus laevis oocytes and assaying for IFN activity. Plasmids containing the entire human IFN-gamma cDNA sequence were identified by colony hybridization and were sequenced. A unique coding region was identified which predicted a protein of 166 amino acids, the 20 N-terminal amino acids of which presumably represent a signal peptide. After transfection of monkey cells with plasmid DNA isolated from one of the recombinant clones (pHIIF-SV-gamma 1), IFN was excreted into the culture medium. This IFN was not distinguishable from human IFN-gamma by serological criteria or by cell target species specificity.

Amino Acid Sequence↗

Cloning and structure of the human immune interferon-gamma chromosomal gene.

Two clones containing the human immune interferon-gamma (IFN-gamma) chromosomal gene were isolated from a human DNA library present in lambda Charon4A phage. DNA from these clones specified biologically active interferon upon injection into the nuclei of Xenopus laevis oocytes. Analysis of the clones revealed that they were derived from the same chromosomal segment. Restriction fragments that hybridized with 32P-labeled cDNA probes were subcloned into plasmids and the complete sequence of the IFN-gamma gene was determined. Unlike IFNs-alpha and -beta, IFN-gamma does contain introns. Their presence was also revealed by electron microscopy. It is intriguing that the smallest of the three introns is located just in the middle of the Glu-Glu sequence which is conserved among all three forms of interferon at approximately the same position. The promoter region was found to contain a prototype TATA box, many palindromic structures and several repeating sequences and two symmetrical structures. Particularly interesting was the existence of two sequences homologous to those present in the chicken albumin and the human IFN-beta gene promoter region. A sequence GTGTTG common to several other genes was found in the region approximately 10 nucleotides downstream from the polyadenylation site.

Animals↗

Isolation and characterization of IFN-gamma mRNA derived from mitogen-induced human splenocytes.

Human splenocytes from large cultures (5 1) were stimulated with Staphylococcus enterotoxin A (SEA). After 2.5 d of conditioning, poly A+RNA was isolated from these cultures and its in-vitro translational capacity was evaluated in a wheat germ extract. Injection of the RNA into Xenopus laevis oocytes yielded a low but reproducible amount of IFN activity, which was neutralizable only by an antiserum to gamma interferon (IFN-gamma) and not by antisera to alpha interferon (IFN-alpha) and/or beta interferon (IFN-beta). Formamide sucrose gradient centrifugation analysis indicated that the mRNA which codes for IFN-gamma sediments at around 15 S. The profile, however, suggested a size heterogeneity of IFN-specific mRNA. Electrophoresis of cDNA synthesized on these mRNA fractions indicated that the mRNA coding for gamma IFN has a length of between 900 and 1400 nucleotides.

Animals↗

Antibody responses of horses to equine influenza viruses during a postepizootic period in Japan.

The antibody responses to equine influenza viruses were investigated during a postepizootic period of the disease. Serum samples were collected from a total of 128 horses on three occasions during the years 1967-77. No significant increase of hemagglutination-inhibition antibody titers to subtypes 1 and 2 of equine influenza virus were detected in any of the sera tested. The maternal hemagglutination-inhibition antibody titers of foals decreased over a four month interval. A marked increase of the titers was recognized in only the equine influenza virus vaccinated horses. These findings suggest that equine influenza virus was not prevalent in the horse populations during the observation period. In such conditions, the dissemination of equine influenza viruses in the horses is discussed in relation to introduction of the disease from abroad. We also examined whether the doctrine of original antigenic sin, an immunological phenomenon recognized in human influenza, was applicable for equine influenza. However, no marked increase of hemagglutination-inhibition antibody titer to the primary infecting subtype in the 44 horses was observed after administration of the heterologous subtype vaccine.

Animals↗

Correlation between acrasins and spore germination inhibitors in cellular slime molds.

Discadenine,3-(3-amino-3-carboxypropyl)-N6-delta 2-isopentenyladenine, which inhibits spore germination, was previously found in Dictyostelium discoideum. Studies on the distribution of discadenine in different species of cellular slime molds by high-pressure liquid chromatography showed that discadenine is present in D. discoideum, Dictyostelium purpureum, and Dictyostelium mucoroides, but not in Dictyostelium minutum, Polysphondylium violaceum, or Polysphondylium pallidum. Discadenine synthetase, which is involved in biosynthesis of discadenine with N6-delta 2-isopentenyladenine as substrate, was only detected in cells of the former three species. In addition, discadenine inhibited spore germination only in these three species. These results clearly demonstrate that discadenine is produced as an inhibitor of spore germination in the species of cellular slime molds in which the acrasin is cyclic adenosine 5'-monophosphate (AMP). This means that there is a structural and biochemical correlation between the spore germination inhibitor and the acrasin, since 5'-AMP, a direct precursor in discadenine biosynthesis, can be derived from cyclic AMP by hydrolysis with cyclic AMP phosphodiesterase.

Adenine↗

Occurrence of 1-methyladenosine and absence of ribothymidine in transfer ribonucleic acid of Mycobacterium smegmatis.

The minor base composition of Mycobacterium smegmatis tRNA has been studied. Thin-layer chromatographic patterns of a ribonuclease T2 digest of mycobacterial tRNA indicated the presence of appreciable amounts of 1-methyladenosine (which is commonly present only in eucaryotic tRNA), dihydrouridine, and 7-methylguanosine. Ribothymidine was absent. The S-adenosylmethionine-dependent tRNA methylases of M. smegmatis catalyzed the formation of 1-methyladenosine when Escherichia coli tRNA was used as acceptor. Similarly, E. coli extracts methylated the tRNA of M. smegmatis, forming ribothymidine.

Adenosine↗

Cisplatinum and taxol induce different patterns of p53 phosphorylation.

Posttranslational modifications of p53 induced by two widely used anticancer agents, cisplatinum (DDP) and taxol were investigated in two human cancer cell lines. Although both drugs were able to induce phosphorylation at serine 20 (Ser20), only DDP treatment induced p53 phosphorylation at serine 15 (Ser15). Moreover, both drug treatments were able to increase p53 levels and consequently the transcription of waf1 and mdm-2 genes, although DDP treatment resulted in a stronger inducer of both genes. Using two ataxia telangiectasia mutated (ATM) cell lines, the role of ATM in drug-induced p53 phosphorylations was investigated. No differences in drug-induced p53 phosphorylation could be observed, indicating that ATM is not the kinase involved in these phosphorylation events. In addition, inhibition of DNA-dependent protein kinase activity by wortmannin did not abolish p53 phosphorylation at Ser15 and Ser20, again indicating that DNA-PK is unlikely to be the kinase involved. After both taxol and DDP treatments, an activation of hCHK2 was found and this is likely to be responsible for phosphorylation at Ser20. In contrast, only DDP was able to activate ATR, which is the candidate kinase for phosphorylation of Ser15 by this drug. This data clearly suggests that differential mechanisms are involved in phosphorylation and activation of p53 depending on the drug type.

Androstadienes↗