Susumu Nishimura

Dramatic events in ciliate evolution: alteration of UAA and UAG termination codons to glutamine codons due to anticodon mutations in two Tetrahymena tRNAsGln

The three major glutamine tRNAs of Tetrahymena thermophila were isolated and their nucleotide sequences determined by post‐labeling techniques. Two of these tRNAsGln show unusual codon recognition: a previously isolated tRNAGlnUmUA and a second species with CUA in the anticodon (tRNAGlnCUA). These two tRNAs recognize two of the three termination codons on natural mRNAs in a reticulocyte system. tRNAGlnUmUA reads the UAA codon of α‐globin mRNA and the UAG codon of tobacco mosaic virus (TMV) RNA, whereas tRNAGlnCUA recognizes only UAG. This indicates that Tetrahymena uses UAA and UAG as glutamine codons and that UGA may be the only functional termination codon. A notable feature of these two tRNAsGln is their unusually strong readthrough efficiency, e.g. purified tRNAGlnCUA achieves complete readthrough over the UAG stop codon of TMV RNA. The third major tRNAGln of Tetrahymena has a UmUG anticodon and presumably reads the two normal glutamine codons CAA and CAG. The sequence homology between tRNAGlnUmUG and tRNAGlnUmUA is 81%, whereas that between tRNAGlnCUA and tRNAGlnUmUA is 95%, indicating that the two unusual tRNAsGln evolved from the normal tRNAGln early in ciliate evolution. Possible events leading to an altered genetic code in ciliates are discussed.

Fractionation of a mutagenic principle from broiled fish by high-pressure liquid chromatography

A mutagenic principle present in the basic fraction of broiled sardines cooked in the ordinary way was fractionated by high-pressure liquid chromatography using a reversed-phase column. Results indicated that the major mutagenic principle is not identical with any of the mutagens previously isolated from amino acid pyrolysis products, and that a new, unidentified mutagen exists in broiled sardines.

Selective inhibition of formation of suppressor glutamine tRNA in moloney murine leukemia virus-infected NIH-3T3 cells by Avarol

Avarol is a sesquiterpenoid hydroquinone, which displays no inhibitory potencies on mammalian DNA polymerases alpha, beta, and gamma, on mammalian RNA polymerases I, II, and III, or on reverse transcriptases from Moloney murine leukemia virus (Mo-MuLV) and from HIV. For a further elucidation of the antiviral effect of Avarol, we used NIH-3T3 cells infected with Mo-MuLV as a model system. The results show that in uninfected NIH-3T3 cells Avarol (i) causes a 50% reduction of the growth rate only at the high concentration of 29.6 microM and (ii) is accumulated in the cytoplasm close to the nucleus. At the much lower concentrations of 1-3 microM, Avarol causes an almost complete inhibition of viral progeny release. Moreover, it is shown that at 3 microM Avarol, the increase of the Mo-MuLV-induced UAG suppressor glutamine tRNA (tRNA(UmUGGln) was reduced to the normal level. Dot blot hybridization studies revealed that Avarol displays no inhibitory activity on cellular and viral mRNA synthesis. Taking the processing pathway of viral polyprotein Pr180gag,pol to p80 (reverse transcriptase) as an example, our Western blotting experiments showed that the final maturation process, conversion of p110 to p80, is inhibited in Avarol-treated cells. From these data we conclude that Avarol prevents the suppression of the UAG termination codon at the gag-pol junction of the retroviral genome. The functional consequence of this event is very likely an inhibition of the readthrough of the retroviral protease gene which overlaps the pol and gag genes, resulting in the reduction of the protease synthesis which is necessary for the viral proliferation.

The change in optical activity of amino acid-specific Escherichia coli transfer RNA containing 4-thiouridylate by chemical modifications

Abstract Circular dichroism (CD) spectra of five species of 4-thiouridine containing amino acid-specific tRNA from Escherichia coli were measured in the near ultraviolet region. In the 300–400 nm region, near 335 nm, which correlated with a minor absorption maximum, tRNA II Tyr showed a particular profile of CD having three bands, whereas all other tRNA (tRNA F Met , tRNA I Val , tRNA Phe , and tRNA Arg showed essentially similar spectra having two bands. This suggests that two adjacent 4-thiouridylate residues in tRNA II Tyr have quite different optical activity from the others due to the excitation splitting of the CD band by the nearest neighbor interaction. Thus, the CD profiles in the near ultraviolet region show dissymmetry near the thionucleotide in tRNA molecule. Oxidation of tRNA II Tyr with iodine showed a gross change of CD in the near ultraviolet region, the sign of which converted from positive to negative; tRNA F Met and tRNA I Val showed their characteristic bands in the near ultraviolet region after iodine oxidation. Treatment of tRNA with cyanogen bromide gave essentially similar profiles of CD as for the intact molecules. Treatment with S -benzylthioisothiourea resulted in an unique CD spectrum for tRNA II Tyr , just a reversed profile of its iodine oxidation. No significant change in CD in the ultraviolet region was detected even after subjecting these tRNA to the three previously mentioned chemical modifications.

Formation of 8-Hydroxydeoxyguanosine in DNA and Carcinogenesis

Oxygen radicals are produced by ionizing radiation and many other environmental carcinogens. They are also produced endogenously in cells by oxygen metabolism. It seems important therefore to study DNA damage by oxygen radicals and its relation to carcinogenesis.

ADP-Ribosylation of Human c-Ha- ras Protein by Hen Liver ADP-Ribosyl Transferase

A portion of the amino acid sequence of human c-Ha-ras protooncogene product p21 is highly homologous with the corresponding region of a family of guanine binding membrane proteins, G-proteins, that are involved in signal transduction (1). Therefore, an analogous function is suggested for ras proteins and G-proteins. Although G-proteins are ADP- ribosylated by cholera toxin (2) or pertussis toxin (3), there has been no data of ADP-ribosylation of c-Ha-ras product by bacterial toxins. On the other hand, both Tsai et al. (4) and our group (5) found ADP-ribosylation of Escherichia coli synthesized c-Ha-ras protein by eukaryotic ADP-ribosyl transferases. Here we identify the amino acid residue which is ADP- ribosylated by a purified hen liver enzyme.