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Biomedical subjects

J Kohli

Publications and source records attributed to J Kohli.

At least 73 records · Page 4Linked to original sources
PubMed1984

An antisuppressor mutation of Schizosaccharomyces pombe affects the post-transcriptional modification of the "wobble" base in the anticodon of tRNAs.

The screening of antisuppressor mutants of the yeast Schizosaccharomyces pombe has been successfully accomplished with high resolution liquid chromatographic methods for the analysis of tRNA nucleosides. Antisuppressor mutations reduce or abolish the function of nonsense suppressor-tRNAs or other informational suppressors. Nonradioactive or 35S-labeled unfractionated tRNA from various strains was digested to nucleosides and analyzed by high performance liquid chromatography. The mutant sin3 has lost the nucleoside 5-(methoxycarbonylmethyl)-2-thiouridine from its tRNA in comparison to parental strains. In eukaryotes this nucleoside is found at the first position of the anticodon (wobble position) in several isoacceptor tRNAs that preferentially recognize codons ending with adenosine. The sin3 mutation reduces the efficiency of UGA and UAA suppressor tRNASer and suppressor tRNALeu. The genetic cosegregation of modification loss, antisuppressor phenotype, and a change in cell size is demonstrated. This indicates that a single mutation in the structural gene for a tRNA modification enzyme causes the three different phenotypes.

Anticodon↗
PubMed1984

The Schizosaccharomyces pombe sup3-i suppressor recognizes ochre, but not amber codons in vitro and in vivo.

The inefficient suppressor sup3-i of the fission yeast Schizosaccharomyces pombe is an ochre suppressor. Sup3-i was derived from the efficient serine inserting UGA suppressor sup3-e. The cloning and sequencing of the sup3-i gene indicate that the suppressor is different from the parent sup3-e by a C----T substitution in the sequence coding for the middle position of the anticodon. In vitro translation assays supplemented with purified sup3-i tRNA and programmed with Xenopus globin mRNAs lead to the accumulation of a readthrough product in response to UAA termination signals, but not in response to UGA termination codons. Transformation of Saccharomyces cerevisiae nonsense mutant strains with plasmid DNA carrying the S. pombe sup3-i gene, led to ochre, but not amber or UGA suppression in vivo.

Alleles↗
PubMed1981

Usage of the three termination codons in a single eukaryotic cell, the Xenopus laevis oocyte.

Oocytes from Xenopus laevis were injected with purified amber (UAG), ochre (UAA), and opal (UGA) suppressor tRNAs from yeasts. The radioactively labeled proteins translated from the endogenous mRNAs were then separated on two-dimensional gels. All three termination codons are used in a single cell, the Xenopus laevis oocyte. But a surprisingly low number of readthrough polypeptides were observed from the 600 mRNAs studied in comparison to uninjected oocytes. The experimental data are compared with the conclusions obtained from the compilation of all available termination sequences on eukaryotic and prokaryotic mRNAs. This comparison indicates that the apparent resistance of natural termination codons against readthrough, as observed by the microinjection experiments, cannot be explained by tandem or very close second stop codons. Instead it suggests that specific context sequences around the termination codons may play a role in the efficiency of translation termination.

Animals↗
PubMed1981

Usage of the three termination codons: compilation and analysis of the known eukaryotic and prokaryotic translation termination sequences.

The published translation termination sequences have been compiled and analysed to aid the interpretation of experiments on termination codon usage in the Xenopus oocyte (Bienz et al. 1981). There are significant differences between prokaryotes and eukaryotes concerning the usage of the three termination codons and of tandem stops. In addition viruses show termination strategies that differ from those of their hosts. Preferred context sequences flanking termination codons are described. Contexts vary within the last codon according to the nature of the termination codon, but are uniform within the first triplet following the terminators.

Animals↗
PubMed1980

Nonsense suppression in eukaryotes: the use of the Xenopus oocyte as an in vivo assay system.

Amber, ochre, and opal nonsense suppressor tRNAs isolated from yeast were injected into Xenopus laevis oocytes together with purified mRNAs (globin mRNA from rabbit, tobacco mosaic virus-RNA). Yeast opal suppressor tRNA is able to read the UGA stop codon of the rabbit beta-globin mRNA, thus producing a readthrough protein. A large readthrough product is also obtained upon coinjection of yeast amber or ochre suppressor tRNA with TMV-RNA. The amount of readthrough product is dependent on the amount of injected suppressor tRNA. The suppression of the terminator codon of TMV-RNA is not susceptible to Mg++ concentration or polyamine addition. Therefore, the Xenopus laevis oocyte provides a simple, sensitive, and well buffered in vivo screening system for all three types of eukaryotic nonsense suppressor tRNAs.

Animals↗
PubMed1980

In vitro suppression of UGA codons in a mitochondrial mRNA.

Although both prokaryotic and eukaryotic messenger RNAs can be easily translated in heterologous protein-synthesizing systems, attempts to achieve correct synthesis of mitochondrial proteins by translation of mitochondrial mRNAs in such systems have failed. In general, the products of synthesis are of low molecular weight and presumably represent fragments of mitochondrial proteins. These fragments display a strong tendency to aggregate. Explanations have included the use by mitochondria of codons requiring a specialized tRNA population and the fortuitous occurrence within genes of purine-rich sequences resembling bacterial ribosome binding sites. In addition, the long 5'-leader sequences present in many mitochondrial (mt) RNAs may also contribute to difficulties in mRNA recognition by heterologous ribosomes. Recent sequence analysis of human mtDNA suggests that the genetic code used by mammalian mitochondria deviates in a number of respects from the 'universal' code, the most striking of these being the use of the UGA termination codon to specify tryptophan. That this may also apply in yeast mitochondria has been shown by Fox and Macino et al., thus providing an obvious and easily testable explanation for the inability of heterologous systems to synthesize full-length mitochondrial proteins. We confirm this explanation and describe here the in vitro synthesis of a full-length subunit II of yeast cytochrome c oxidase in a wheat-germ extract supplemented with a partially purified mitochondrial mRNA for this protein and a UGA-suppressor tRNA from Schizosaccharomyces pombe.

Codon↗
PubMed1979

The nucleotide sequence of a UGA suppressor serine tRNA from Schizosaccharomyces pombe.

The UGA suppressor tRNA produced by Schizosaccharomyces pombe strain sup3-e was purified to homogeneity. It can be aminoacylated with a serine by a crude aminoacyl-tRNA synthetase preparation from S. pombe cells. By combining post-labeling fingerprinting and gel sequencing methods the nucleotide sequence of this tRNA was determined to be: pG-U-C-A-C-U-A-U-G-U-C-ac4C-G-A-G-D-G-G-D-D-A-A-G-G-A-m2G2-psi-U-A-G-A-N-U-U-C-A-i6A-A-psi-C-U-A-A-U-G-G-G-C-U-U-U-G-C-C-C-G-m5C-G-G-C-A-G-G-T-psi-C-A-m1A-A-U-C-C-U-G-C-U-G-G-U-G-A-C-G-C-C-A OH. The anticodon sequence u ca is complementary to the UGA codon.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Identification and nucleotide sequence of the sup8-e UGA-suppressor leucine tRNA from Schizosaccharomyces pombe.

Using the translation of rabbit globin mRNA in wheat germ extracts as an assay for ochre and opal suppression, a UGA suppressor tRNA from Schizosaccharomyces pombre strain sup8-e was purified by column chromatography and two-dimensional gel electrophoresis. The purified tRNA can be aminoacylated with leucine by a crude aminoacyl-tRNA synthetase preparation from a wild type S. pombe strain, and has high activity in the suppressor assay. By a combination of post-labeling fingerprinting and rapid gel sequencing methods the nucleotide sequence of this suppressor tRNA was determined to be: pG-C-G-G-C-U-A-U-G-C-C-ac4C-G-A-G-D-G-G-D-G-D-A-A-G-G-G-m22G-G-C-A-G-A-psi-U-U*-C-A-m1G-C-C-C-U-G-C-U-G-U-U-G-U-A-A-A-A-C-G-m5C-G-A-G-A-G-T-psi-C-G-m1A-A-C-C-U-C-U-C-U-G-G-C-C-G-C-A-C-C-AOH. The anticodon sequence U*CA is complementary to the UGA codon. An interesting feature of the suppressor tRNA is an expanded anticodon loop of nine nucleotides owing to an A-C nonpair at the first anticodon stem position.

Ascomycota↗
PubMed1979

Characterization of a UGA-suppressing serine tRNA from Schizosaccharomyces pombe with the help of a new in vitro assay system for eukaryotic suppressor tRNAs.

Two different allele-specific suppressor mutants of the fission yeast Schizosaccharomyces pombe produce opal (UGA) suppressor tRNAs. This was shown by the use of a new in vitro assay for eukaryotic nonsense suppression: a wheat germ extract is programmed with rabbit globin mRNAs and the readthrough products are studied. alpha-Globin is elongated upon addition of ochre (UAA) suppressor tRNAs, whereas beta-globin yields a readthrough product with opal suppressor tRNAs. This simple and very sensitive assay allowed the purification of the opal suppressor tRNA from S. pombe strain sup3-e. The pure tRNA can be aminoacylated with serine; thus, we conclude that this suppressor tRNA inserts serine in response to the UGA termination codon of pure rabbit beta-globin mRNA.

Alleles↗
PubMed1979

The nucleotide sequence of the major glutamate transfer RNA from Schizosaccharomyces pombe.

The nucleotide sequence of glutamate tRNA1 from Schizosaccharomyces pombe was determined to be pU-C-C-G-U-U-G-U-m1G-G-U-C-C-A-A-C-G-G-C-D-A-G-G-A-U-U-C-G-U-C-G-C-U-U-U*-C-A-C-C-G-A-C-G-G-G-A-G-m5C-G-G-G-G-T-psi-C-G-A-C-U-C-C-C-C-G-C-A-A-C-G-G-A-G-C-C-AOH. The sequence differs markedly from that of S. cerevisiae tRNAGlu. S. pombe glutamate tRNA1 can be aminoacylated by the homologous glutaminyl-tRNA synthetase as well as by the corresponding enzyme from S. cerevisiae.

Amino Acyl-tRNA Synthetases↗
PubMed1979

Avian metabolism of halogenated biphenyls.

1. The metabolism of several halogenated biphenyls has been studied using poultry as a model system. The substrates 4-chlorobiphenyl and 4-bromobiphenyl each yielded two metabolites (4'-chloro-4-biphenylol, 4'-chloro-3,4-biphenyldiol; 4'-bromo-4-biphenylol, 4'-bromo-3,4-biphenyldiol), while 4,4'-dichlorobiphenyl yielded only one metabolite (4,4-dichloro-3-biphenylol). 2. Metabolites were not found in eggs collected for 7 days following the administration of the substrate.

Animals↗
PubMed1978

Nucleotide sequence of phenylalanine transfer RNA from Schizosaccharomyces pombe: implications for transfer RNA recognition by yeast phenylalanyl-tRNA synthetase.

The nucleotide sequence of Schizosaccharomyces pombe tRNAPhe was determined to be pG-U-C-G-C-A-A-U-G**-G*-U-G-psi-A-G-D-D-G-G-G-A-G-C-A-psi-G*-A-C-A-G-A-Cm-U-Gm-A-A-Y-A-psi-m5C-U-G-U-U-G-m7G-U*-C-A-U-C-G-G-T-psi-C-G-A-U-C-C-C-G-G-U-U-U-G-U-G-A-C-A-C-C-AOH. This sequence differs from that of S. cerevisiae tRNAPhe in 27 nucleotides. Saccharomyces cerevisiae phenylalanyl-tRNA synthetase aminoacylates both the homologous tRNAPhe and S. pombe t-NAPhe; the reactions have similar Km and Vmax values. However, the nucleotide sequence in the D stem is different in the two tRNAs. This region was proposed by Roe, B., et al. [(1973) Biochemistry 12, 4146--4154] to be the major recognition site for yeast phenylalanyl-tRNA synthetase, but the present results cast doubt on the validity of this hypothesis.

Amino Acyl-tRNA Synthetases↗