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.     

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.     

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.     

Analysis of the active center of hydrogenase from Desulfovibrio vulgaris Miyazaki by magnetic measurements

Hydrogenase [hydrogen: ferricytochrome c3 oxidoreductase, EC 1.12.2.1] solubilized and purified from the particulate fraction of Desulfovibrio vulgaris Miyazaki F (IAM 12604) contains 8 iron and 8 labile sulfide ions in one molecule which is composed of two unequal subunits (Mr: 60,000 + 29,000). It does not contain nickel atoms. The EPR (electron paramagnetic resonance) spectrum has an isotropic signal at g = 2.017 which is independent of the temperature. The peak-to-peak width of the signal is about 20 G. The signal intensity is nearly equivalent to 1 unpaired electron per molecule. No other signals can be detected in the field range between 2,240 and 4,240 G (which corresponds to g-values between 2.91 and 1.54). Ferricyanide has only a little effect on the shape and intensity of the EPR signal. The hydrogenase reduced under H2 is EPR silent. The M?ssbauer spectrum has no hyperfine splitting at 4K. The isomer shift and quadrupole splitting at 77K are 0.38 and 0.87 mm/s, respectively. Based on these magnetic measurements, the structure of the active center of hydrogenase was suggested to be [4Fe-4S]3+ + [4Fe-4S]2+.     

Glucagon-related peptides in the rat hypothalamus

Summary Immunohistochemically, nerve fibers and terminals reacting with anti-N-terminal-specific but not with anti-C-terminal-specific glucagon antiserum were observed in the following rat hypothalamic regions: paraventricular nucleus, supraoptic nucleus, anterior hypothalamus, arcuate nucleus, ventromedial hypothalamic nucleus and median eminence. Few fibers and terminals were demonstrated in the lateral hypothalamic area and dorsomedial hypothalamic nucleus. Radioimmunoassay data indicated that the concentration of gut glucagon-like immunoreactivity was higher in the ventromedial nucleus than in the lateral hypothalamic area. In food-deprived conditions, this concentration increased in both these parts. This was also verified in immunostained preparations in which a marked enhancement of gut glucagon-like immunoreactivity-containing fibers and terminals was observed in many hypothalamic regions. Several immunoreactive cell bodies were found in the ventromedial and arcuate nuclei of starved rats. Both biochemical and morphological data suggest that glucagon-related peptides may act as neurotransmitters or neuromodulators in the hypothalamus and may be involved in the central regulatory mechanism related to feeding behavior and energy metabolism.     

Intact antigen receptor-mediated generation of inositol phosphates and increased intracellular calcium in CD4 CD8 T lymphocytes from MRL lpr mice

The predominant T lymphocytes that accumulate in the peripheral lymphoid tissues of mice homozygous for the lpr gene bear the phenotype CD3+CD4-CD8-. By certain functional criteria these cells would appear to have impaired CD3-mediated signal transduction, in that they do not respond to alloantigen and produce little if any detectable IL-2 or other lymphokines. However, the signal pathway appears adequate for achieving other T cell functions, including induction of high affinity IL-2R, and thymic deletion. To clarify the basis of this seeming discrepancy, we examined transmembrane signal transduction in T cell subsets of lpr/lpr (lpr) and +/+ mice, as defined by increased [Ca2+]i and the generation of inositol phosphates (InsPs). Stimulation of lpr CD4-CD8- cells with anti-CD3 antibody produced prompt and sustained increases in the concentration of [C2+]i and in InsPs. Similar responses occurred in mature T cells from lpr and +/+ mice, except for the somewhat slower kinetics of their increased [Ca2+]i. In marked distinction to the anti-CD2-mediated response, Con A, even in high doses, could not stimulate any increase of [Ca2+]i in lpr CD4-CD8- cells, and only modest increases in InsPs. Mature T cells, whether of lpr or +/+ origin, yielded normal increased [Ca2+]i with Con A. The reason for the differences in signal transduction between anti-CD3 and Con A stimulation of lpr CD4-CD8- cells may relate to the absence of surface structures on these immature T cells that are required for activation by Con A but not by anti-CD3. The data demonstrate that the CD3 complex in lpr CD4-CD8- T cells can couple to phospholipase C to hydrolyze phosphoinositides. These activation properties of lpr CD4-CD8- T cells have interesting functional parallels to thymocytes at the time of thymic selection, as well as tolerance induction of mature T lymphocytes.     

Partial inhibition of protein synthesis accelerates the synthesis of porphyrin in heme-deficient mutants of Escherichia coli

Mutants of Escherichia coli defective in the HemA protein grow extremely poorly as the result of heme deficiency. A novel hemA mutant was identified whose rate of growth was dramatically enhanced by addition to the medium of low concentrations of translational inhibitors, such as chloramphenicol and tetracycline. This mutant (H110) carries mutation at position 314 in the hemA gene, which resulted in diminished activity of the encoded protein. Restoration of growth of H110 upon addition of the drugs mentioned above was due to activation of the synthesis of porphyrin. However, this activation was not characteristic exclusively of cells with this mutant hemA gene since it was also observed in a heme-deficient strain bearing the wild-type hemA gene. The activation did not depend on the promoter activity of the hemA gene, as indicated by studies with fusion genes. It appears that partial inhibition of protein synthesis via inhibition of peptidyltransferase can promote the synthesis of porphyrin by providing an increased supply of Guamyl-tRNA for porphyrin synthesis. Glutamyl-tRNA is the common substrate for peptidyltransferase and HemA.