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Biomedical subjects

T Ohama

Publications and source records attributed to T Ohama.

At least 37 records · Page 2Linked to original sources
PubMed1995

Identification of new selenocysteine tRNA[SER]SEC isoacceptors in human cell lines.

The selenocysteine tRNA population was examined in a human T-cell line and in a human monocytic cell line for the occurrence of additional species of selenocysteine tRNA. At least three additional (and possibly more) selenocysteine isoacceptors were found which occur in minor levels as compared to the two major selenocysteine isoacceptors previously characterized. The possible significance of these newly observed species are discussed.

Cell Line↗
PubMed1994

Utilization of selenocysteyl-tRNA[Ser]Sec and seryl-tRNA[Ser]Sec in protein synthesis.

The UGA selenocysteine (Sec) codon in glutathione peroxidase mRNA and in selenoprotein P and the UGA stop codon in rabbit beta-globin mRNA were employed to study the utilization of Sec-tRNA[Ser]Sec and Ser-tRNA[Ser]Sec in protein synthesis. In vitro Ser-tRNA[Ser]Sec served as a suppressor of the UGA Sec codon as well as the UGA stop codon, while Sec-tRNA[Ser]Sec did not. However, in vivo Sec-tRNA[Ser]Sec did donate Sec to glutathione peroxidase in Xenopus oocytes microinjected with glutathione peroxidase mRNA and Sec-tRNA. A ribosome binding assay was devised to investigate the interaction of aminoacyl-tRNA, rabbit reticulocyte ribosomes, and eukaryotic elongation factor 1 (eEF-1) in response to the appropriate trinucleoside diphosphate template. Ser-tRNA[Ser]Sec bound weakly to ribosomes in the presence of eEF-1 and UGA as compared to Phe-tRNA, Ser-tRNAIGA, and Met-tRNAm which bound more efficiently in the presence of eEF-1 and the appropriate template. No increase in the binding of Sec-tRNA[Ser]Sec was observed under the same conditions as Ser-tRNA[Ser]Sec. The ribosome binding studies substantiated the finding that Ser-tRNA[Ser]Sec serves as a suppressor of UGA codons in protein synthesis, but Sec-tRNA[Ser]Sec does not. In addition, these studies provide strong evidence that a specific elongation factor is required in mammalian cells for insertion of Sec into protein from Sec-tRNA[Ser]Sec.

Acylation↗
PubMed1994

Selenocysteine insertion or termination: factors affecting UGA codon fate and complementary anticodon:codon mutations.

Translation of UGA as selenocysteine instead of termination occurs in numerous proteins, and the process of recording UGA requires specific signals in the corresponding mRNAs. In eukaryotes, stem-loops in the 3' untranslated region of the mRNAs confer this function. Despite the presence of these signals, selenocysteine incorporation is inefficient. To investigate the reason for this, we examined the effects of the amount of deiodinase cDNA on UGA readthrough in transfected cells, quantitating the full-length and UGA terminated products by Western blotting. The gene for the selenocysteine-specific tRNA was also cotransfected to determine if it was limiting. We find that the concentrations of both the selenoprotein DNA and the tRNA affect the ratio of selenocysteine incorporation to termination. Selenium depletion was also found to decrease readthrough. The fact that the truncated peptide is synthesized intracellularly demonstrates unequivocally that UGA can serve as both a stop and a selenocysteine codon in a single mRNA. Mutation of UGA to UAA (stop) or UUA (leucine) in the deiodinase mRNA abolishes deiodinase activity; but activity is partially restored when selenocysteine tRNAs containing complementary mutations are contransfected. Thus, UGA is not essential for selenocysteine incorporation in mammalian cells, provided that codon:anticodon complementarity is maintained.

Anticodon↗
PubMed1994

Selenocysteine tRNA and serine tRNA are aminoacylated by the same synthetase, but may manifest different identities with respect to the long extra arm.

Selenocysteine (Sec) tRNA([Ser])Sec donates Sec to protein, but interestingly, this amino acid is synthesized on tRNA which is first aminoacylated with serine. Thus, the identity elements in tRNA([Ser])Sec for aminoacylation correspond to elements for seryl-tRNA synthetase recognition. As tRNA([Ser])Sec has low homology to the tRNA(Ser) isoacceptors, it would seem then that the identity elements in tRNA([Ser])Sec involve (1) very specific sequences, (2) conformational features, and/or (3) different points or domains for tRNA[Ser]Sec:synthetase and tRNASer:synthetase recognition. Initially, we confirmed that the same synthetase aminoacylates both tRNAs by showing that a mutant tRNA[Ser]Sec which has a blocked 3'-terminus is a competitive inhibitor of tRNASer aminoacylation with a partially purified and a highly purified seryl-tRNA synthetase preparation. The discriminator base (base G73) is essential for aminoacylation of tRNA([Ser])Sec and tRNA(Ser), while the long extra arm plays an important role which seems to be orientation- and length-specific in tRNA(Ser) and, in addition, may manifest sequence specificity in tRNA([Ser])Sec. This difference in the tRNA recognition specificity is discussed. The acceptor stem, DHU stem, and T phi C stem contribute to the recognition process, but to a lesser extent than the discriminator base and the long extra arm.

Animals↗
PubMed1994

Effect of starving and refeeding on lipid metabolism in suncus.

We have previously reported that fatty liver is easily induced in a novel experimental animal, Suncus murinus (suncus) by withholding food, and that apolipoprotein B (apo B) is not actively synthesized in the liver. In the present paper we describe the effect of starving and refeeding on lipid and lipoprotein metabolism in suncus, in order to explore the mechanisms of induction of fatty liver by starving and of its improvement by refeeding. Starvation induced increase in triglyceride content and decrease in glycogen content of the liver. Although the glycogen content returned to the level before starvation at 12 h after refeeding, the triglyceride content decreased gradually but did not reach the prestarvation level even at 24 h after refeeding in suncus. Plasma lipids, glucose, and insulin levels were decreased by starvation and returned to the levels before starvation between 8 and 24 h after refeeding. On the other hand, the plasma levels of free fatty acid and ketone bodies were elevated significantly by starvation and decreased rapidly by refeeding. These responses to starvation and refeeding, except for the change in hepatic triglyceride, are in common with other experimental animals, suggesting that there are no abnormalities in glucose metabolism or in fatty acid metabolism in suncus. In conclusion, the fatty liver induced by starvation in suncus may be caused by impaired triglyceride transport out of the liver, for which apolipoprotein B is mostly responsible, as reported previously.

Animals↗
PubMed1993

Non-universal decoding of the leucine codon CUG in several Candida species.

It has been reported that CUG, a universal leucine codon, is read as serine in an asporogenic yeast, Candida cylindracea. The distribution of this non-universal genetic code in various yeast species was studied using an in vitro translation assay system with a synthetic messenger RNA containing CUG codons in-frame. It was found that CUG is used as a serine codon in six out of the fourteen species examined, while it is used for leucine in the remaining eight. The tRNA species responsible for the translation of codon CUG as serine was detected in all the six species in which CUG is translated as serine. The grouping according to the CUG codon assignments in these yeast species shows a good correlation with physiological classification by the chain lengths of the isoprenoid moiety of ubiquinone and the cell-wall sugar contained in the yeasts. The six Candida species examined in which CUG is used as serine belong to one distinct group in Hemiascomycetes.

Amino Acid Sequence↗
PubMed1993

Unassigned or nonsense codons in Micrococcus luteus.

We previously reported that in Micrococcus luteus, a Gram-positive eubacterium with high genomic G + C content, certain codons ending with A did not appear in coding frames, including termination sites, and tRNAs that translate these codons were not detected. These facts suggest that at least some of them are unassigned (nonsense) codons, i.e. not assigned to any amino acid or to any stop signal. We have investigated whether AGA and AUA, universal Arg and Ile codons, respectively, are really unassigned codons by using a cell-free extract prepared from M. luteus and synthetic messenger RNAs. Translation of synthetic mRNA containing in-frame AGA codons does not result in "read-through" to codons beyond the AGA codons, i.e. translation ceases at codon AGA. Essentially the same result was obtained with mRNA containing AUA in-frame. A sucrose-gradient centrifugation profile of the reaction mixture has shown that practically all of the peptides that have been synthesized are attached to 70 S ribosomes. When in-frame AGA or AUA codons are replaced by UGA codons in mRNA, no read-through occurs beyond UGA, just as in the case of AGA or AUA. However, the synthesized peptide is released from the 70 S ribosomes. These data suggest that AGA and AUA are unassigned codons and differ from UGA in that they are not used for termination.

Adenine Nucleotides↗
PubMed1993

Characterization of serum lipoproteins from Suncus: a candidate animal model for abetalipoproteinemia.

We have previously reported that fatty liver was induced in a novel experimental animal, Suncus murinus (suncus), by 24-h fasting and that apolipoprotein B (apo B) was not actively synthesized in the liver. However, a faint signal of apo B mRNA was detected in the liver, suggesting possible synthesis of apo B. Small amounts of VLDL and LDL have been separated from suncus serum by ultracentrifugation. Electron microscopic study of the lipoproteins revealed the existence of small particles in VLDL. High performance liquid chromatographic analysis of the lipoproteins showed that the peaks of TG and cholesterol were mainly at the HDL fraction. These results indicate the existence of lipoproteins as small as HDL which were rich in TG and floated at the density of VLDL upon ultracentrifugation. Apolipoprotein analysis showed two bands of 500- and 200-kDa proteins in VLDL and LDL. Western blot analysis using antibody against the 500-kDa protein revealed reaction not only with suncus 500- and 200-kDa proteins but also with human apo B-100. In conclusion, a small amount of apo B is transported in the suncus serum as VLDL and LDL, although almost all lipid is packed in HDL-size particles.

Abetalipoproteinemia↗
PubMed1992

Serine tRNA complementary to the nonuniversal serine codon CUG in Candida cylindracea: evolutionary implications.

In the asporogenic yeast Candida cylindracea, the codon CUG is read as serine instead of leucine. This is an unusual instance in which the amino acid assignment of a codon deviates from the universal code. To infer the evolutionary process of this change, the tRNA with the anticodon sequence CAG, which is complementary to and thus responsible for translation of the codon CUG, has been identified. Indeed, this tRNA translates an in-frame CUG codon in a synthetic mRNA as serine in an in vitro translation system. The gene for the tRNA is interrupted by an intron in the anticodon loop. Sequence comparisons of the tRNA and its gene suggest that a single cytidine was inserted into the anticodon loop of the gene for tRNA(Ser)IGA during evolution to produce tRNA(Ser)CAG. The tRNA(Ser)CAG may be produced from its precursor molecule containing the cytidine insertion by splicing.

Amino Acid Sequence↗
PubMed1992

Planarian mitochondria. I. Heterogeneity of cytochrome c oxidase subunit I gene sequences in the freshwater planarian, Dugesia japonica.

We have detected sequence heterogeneity in the cytochrome c oxidase subunit I (COI) gene of freshwater planarian, Dugesia japonica, collected in one locality. A part of the COI gene was amplified via the polymerase chain reaction (PCR) using template DNA prepared from a mixture of 500 individuals or from each of 18 individuals. Analyses of DNA sequences by standard strategies for cloning and sequencing or by direct sequencing clearly show that (1) considerable sequence heterogeneity exists in DNA prepared from the mixed individuals, (2) 11 individuals have almost identical sequences (type A), and (3) 7 individuals have sequences different from one another (Seq-D1 to Seq-D7; collectively called type D). Each of the Seq-D1-D7 sequences except for Seq-D5 shows some heterogeneity even in a single individual (heteroplasmy). A possible cause of the sequence heterogeneities is discussed.

Animals↗
PubMed1992

Planarian mitochondria. II. The unique genetic code as deduced from cytochrome c oxidase subunit I gene sequences.

The cytochrome c oxidase subunit I (COI) gene sequences from planarian (Dugesia japonica) DNA, most probably of mitochondrial origin, are heterogeneous. Taking advantage of the heterogeneity that occurs primarily in silent sites of the COI DNA sequences, amino acid assignments of several codons have been deduced as nonuniversal: UGA = Trp, AAA = Asp, and AGR (R: A or G) = Ser. In addition, UAA, a stop codon in the universal genetic code, is tentatively assumed to be a tyrosine codon, because three of the sequences examined have UAA at the well-conserved tyrosine site of UAY (Y: U or C) in other planarian sequences as well as in the mitochondria of human, Xenopus, sea urchin, Drosophila, Trypanosoma, and Saccharomyces cerevisiae. AUA would most probably be an isoleucine codon in these mitochondria, whereas it is a methionine codon in the majority of nonplant mitochondria.

Amino Acid Sequence↗
PubMed1991

Novel anticodon composition of transfer RNAs in Micrococcus luteus, a bacterium with a high genomic G + C content. Correlation with codon usage.

The number and relative amount of isoacceptor tRNAs for each amino acid in Micrococcus luteus, a Gram-positive bacterium with high genomic G + C content, have been determined by sequencing their anticodon loop and its adjacent regions and by selective labelling of tRNAs. Thirty-one tRNA species with 29 different anticodon sequences have been detected. All the tRNAs have G or C at the anticodon first position except for tRNA(ICGArg) and tRNA(NGASer), in response to the abundant usage of NNC and NNG codons. No tRNA with the anticodon UNN capable of translating codon NNA has been detected, in accordance with a very low or zero usage of NNA codons. The relative amount of isoacceptor tRNAs for an amino acid determined by selective labelling strongly correlates with usage of the corresponding codons. On the basis of these and other observations in this and other eubacterial species, we conclude that the relative amount and anticodon composition of isoacceptor tRNA species are flexible, and their changes are mainly adaptive phenomena that have been primarily affected by codon usage, which in turn is affected by directional mutation pressure.

Anticodon↗