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G Keith

Publications and source records attributed to G Keith.

At least 109 records · Page 6Linked to original sources
PubMed1986

Primary structure of leader RNA and nucleoprotein genes of the rabies genome: segmented homology with VSV.

We have determined the nucleotide sequence of the 3'region of the rabies genome (PV strain). This work is a first step in a project aimed at establishing the complete primary structure. From the 3'nucleotide sequence of the RNA genome, an octadecanucleotide complementary to the 3'extremity was constructed and used to prime cDNA synthesis. Two overlapping recombinant cDNA clones hybridizing with the nucleoprotein mRNA (NmRNA) were isolated and sequenced. The 1500 first nucleotides of the rabies genome cover two transcriptional units: the leader RNA and the NmRNA which was shown to be initiated around residue 59 by S1 nuclease protection experiments. Comparison between rabies PV and CVS strains up to residue 180 suggests a rapid evolution in the leader region. Studies of the sequence relationships between the 3'regions of two Rhabdoviruses, rabies virus and Vesicular Stomatitis Virus (VSV), demonstrate that there is a segmented homology. Stretches of highly conserved amino acids possibly involved in the interaction with the RNA genome were observed in the N protein, despite a wide divergence in the remaining sequence. In addition, the high homology between the transcription start and stop signals reflects the conservation of a similar transcriptional mechanism in these two non segmented negative strand RNA viruses.

Amino Acid Sequence↗
PubMed1986

The primary structure of wheat germ tRNAArg--the substrate for arginyl-tRNAArg:protein transferase.

Besides its major role in protein synthesis, wheat germ arginyl-tRNAArg can serve as an amino acid donor in an enzymatic reaction to bovine serum albumin catalysed by the enzyme arginyl-tRNAArg: protein transferase. The nucleotide sequence of the tRNAArg involved in this reaction was determined to be: pG-A-C-U-C-C-G-U-m1G-m2G-C-C-C-A-A-D-Gm-G-A-X-A-A-G-G-C-m2(2) G-C-U-G-G-U-Cm-U-I-C-G-m2A-A-A-C-C-A-G-A-G-A-D-U-m5C-U-G-G-G-T-psi -C-G-m1 A-U-C-C-C-C-A-G-C-G-G-A-G-U-C-G-C-C-AOH. We suggest that the decapentanucleotide 5'-G-U-Pu-m2G-C-N-C-A-A-D-Gm-G-A-X-A-3', localized in the D-region, interacts specifically with the protein transferase.

Acyltransferases↗
PubMed1986

The primary structure of threonine tRNA (anticodon I-G-U) from bovine liver.

The primary structure of threonine tRNA (anticodon I-G-U) from bovine liver has been determined using 32P postlabeling methods. Its nucleotide sequence is pG-G-C-C-C-U-G-U-m1G-m2G-C-U-A-G-C-D-G-G-D-C-A-A-A-G-C-m2(2)G-C-C- U-G-U-m3 C-U-I-G-U-t6A-A-A-C-A-G-G-A-G-A-D-m5C-C-U-G-G-G-U-psi-C-G-m1 A-A-U-C-C-C-A-G-C-G-G-G-G-C-m5C-U-C-C-A. The anticodon I-G-U, according to the wobble hypothesis, is expected to recognize A-C-U, A-C-C and A-C-A, 3 out of the 4 codons for threonine. However, if the original wobble hypothesis, as it has been shown recently for several yeasts and a fungus, is only restricted to U and C in the ox, it would recognize only 2 codons: A-C-C and A-C-U.

Animals↗
PubMed1986

Walking along the rabies genome: is the large G-L intergenic region a remnant gene?

Rabies cDNA clones, obtained by "walking along the genome" using two successive DNA primers, have allowed the sequence determination of the genes encoding the N, M1, M2, G, and the beginning of the L protein as well as the rabies intergenic regions. Start and stop transcription signals located at the border of each gene encoding a protein have been identified and are similar to the corresponding signals from vesicular stomatitis virus (VSV) and Sendai virus. Except for limited stretches of the nucleoprotein, there is no homology between corresponding structural proteins of these three viruses. Rabies intergenic regions are variable both in length and sequence. Evidence for the existence of a remnant protein gene in the 423 nucleotide long G-L intergenic region is presented. This finding is discussed in terms of the evolution of unsegmented negative-strand RNA viruses.

Amino Acid Sequence↗
PubMed1985

Queuosine modification of the wobble base in tRNAHis influences 'in vivo' decoding properties.

The 'in vivo' decoding properties of four tRNAHis isoacceptors, two from Drosophila melanogaster and two from brewer's yeast, were studied after their microinjection, along with turnip yellow mosaic virus (TYMV) coat protein mRNA, into Xenopus laevis oocytes. The two Drosophila isoacceptors are identical besides containing either a guanosine (G) or the hypermodified nucleoside queuosine (Q) in the wobble position. The brewer's yeast isoacceptors differ by four bases in the anticodon stem, and by one base in the amino acceptor stem. Our results show that, under competing 'in vivo' conditions, the Drosophila tRNAHis with the anticodon GUG clearly prefers the histidine codon CAC to the codon CAU, whereas little preference is observed for the tRNAHis with the anticodon QUG for the codon CAU, and no preference for either codon by the two yeast isoacceptors. Hence, it can be concluded that the presence of the Q-base clearly affects the choice of the codon. This is the first demonstration of an 'in vivo' codon preference by tRNA isoacceptors differing in the modification of the wobble base during the elongation step of protein synthesis. These results imply that one function of the Q-base is at the translational level.

Animals↗
PubMed1985

[Properties of DNA-(cytosine-5-)-methyltransferase in the brain].

Beef brain DNA-(cytosine-5-)-methyltransferase was partially purified by chromatography on Ultrogel AcA34 and Dyematrex Blue A. The purification was of 360 times and the recovery of 75%. The pH optimum of the reaction is 7.6 NaCl inhibits double stranded DNA methylation, but stimulates single stranded DNA methylation up to 50 mM, before inhibiting. EDTA (1 mM) and MgCl2 (4 mM) stimulate DNA methylation. Polyamines inhibit the reaction.

Animals↗
PubMed1984

Sequence of tRNA Ile IAU from brewer's yeast.

The nucleotide sequence of tRNAIle from brewer's yeast Saccharomyces cerevisiae was determined. Its primary structure is pG-G-U-C-U-C-U-U-m1G-m2G- C-C-C-A-G-D-D-G-G-D-D-A-A-G-G-C-A-C-C-G-U-G-C-U-I-A-U-t6 A-A-C-G-C-G-G-G-GA-D-m5 C-A-G-C-G-G-T-psi-C-G-m1 A-U-C-C-C-G-C-U-A-G-A-G-A-C-C-A-C-C-A. Its anticodon is I-A-U. It should therefore recognize the three isoleucine codons and is for this reason probably the only isoacceptor tRNA for isoleucine in brewer's yeast. It presents a large homology with its counterpart from Torulopsis utilis (87%).

Animals↗
PubMed1983

Enzymatic methylation of chemically alkylated DNA and poly(dG-dC) X poly(dG-dC) in B and Z forms.

The enzymatic methylation of chemically alkylated DNA and of poly(dG-dC) X poly(dG-dC) by beef brain DNA(cytosine-5-)-methyltransferase have been tested. The alkylation by dimethylsulfate, which yields mostly 7 methylguanine (m7G) and 3 methyladenine (m3A) do not affect the enzymatic methylation. The dimethylsulfate alkylated poly(dG-dC) X poly(dG-dC) converted into the Z-form in the presence of MgCl2, is just as well methylated as the native or the alkylated polynucleotide in the B-form. The alkylation of DNA or of poly(dG-dC) X poly(dG-dC) by methylnitrosourea yields, in addition to the above base modifications described for dimethylsulfate, methylphosphotriesters and O6-methylguanine. The enzymatic methylation of these substrates modified by methylnitrosourea is decreased. This decrease is proportional to the extent of the chemical alkylation of the substrate.

Alkylation↗
PubMed1983

The nucleotide sequence of methionine elongator tRNA from wheat germ.

Wheat germ methionine elongator tRNA (tRNAmMet) was purified by three column chromatogaphies followed by electrophoresis on polyacrylamide gel. Its sequence is pG-G-G-G-U-G-G-U-m1G-m2G-C-G-C-A-G-D-D-G-G-C-acp3U-A-G-C-G-C-m22G-psi-A-G-G- psi-C-U-Cm-A-U-mt6A-A-psi-C-C-U-G-A-G-m7G-D-m5C-G-A-G-A-T-psi-C-G-m1A-G2-C-C-U- C-U-C-U-C-A-C-C-C-C-A-C-C-A. Two hypermodified nucleosides, methylthreoninocarbonyladenosine (mt6A) and 3(3-amino-3-carboxypropyl)uridine (acp3U), are present in this tRNA.

Base Sequence↗
PubMed1983

Sequence of tRNALeu CmAA from Bacillus stearothermophilus.

The primary structure of Bacillus stearothermophilus tRNALeu was determined and found to be :pGCCGAUGs4UGGCGGAAUDGGCAGm1ACGCGCACGACUCmAAms2i6AA psi CGUGUGGGCUUUGCCCGUGUGGGT psi CGACUCCCACCAUCGGCACCA. The molecule has a large extraloop and contains only 8 minor nucleotides. There is a G at position 21 like in all other sequenced bacterial tRNAsLeu.m1A is in position 22, just before the D stem like in several other procaryotic tRNAs. The anticodon is CmAA and is adjacent to a ms2i6A in the 3'-direction.

Base Sequence↗
PubMed1983

Enzymatic methylation of chicken erythrocyte DNA modified by two carcinogens, 2-(N-acetoxyacetylamino) fluorene and methylnitrosourea.

Both DNA-AAF and MNU-alkylated DNA are methylated less than nonmodified DNA by rat brain nuclei cytosine 5-methyltransferase purified either by chromatography on DEAE cellulose or by Dyematrex. The inhibition of methylation is proportional to the modification of the DNA, and DNA having a given percentage of bases modified with MNU is less methylated than DNA modified to the same extent with AAF. Moreover, DNA-AAF irreversibly inhibits the methylation of native DNA, whereas MNU-alkylated DNA does not inhibit the methylation of native DNA. The AAF-substituted DNA has a higher affinity for the enzyme than native DNA. However, this is probably not due to the AAF-induced local destabilization of the DNA helix, since heat-denatured DNA shows a lower affinity for the enzyme than double-stranded DNA. Addition of DNA-AAF to the enzyme preincubated with native DNA inhibits methylation, but only after a lag period. This agrees with the model in which the methylase walks along the strand to methylate cytosine residues before being detached from the DNA. AAF bound to guanine residues may block the movement of the enzyme along the helix. The in vitro hypomethylation of DNA, caused by carcinogens, could explain the in vivo observations made by several authors and could have significance in gene activity, cellular differentiation, and oncogenesis.

2-Acetylaminofluorene↗
PubMed1983

Optimization of conditions for labeling the 3' OH end of tRNA using T4 RNA ligase.

For several years most primary structure studies of ribonucleic acids have used the [32P] in vitro post-labeling techniques. We adapted our methods from the literature, and simplified them to make them accessible to any laboratory. These procedures are especially useful for preparation and purification of post labeling enzymes: T4 polynucleotide kinase, T4 RNA ligase and of gamma [32P] ATP. We developed a test tube method for 5' [32P] pCp preparation followed by tRNA labeling with T4 RNA ligase. The parameters for optimal labeling were determined. Labeling of 3.10(6) to 5.10(6) Cerenkov CPM per microgram tRNA are currently obtained.

Adenosine Triphosphate↗
PubMed1983

NMR study of slowly exchanging imino protons in yeast tRNAasp.

We have monitored the exchange of imino and amino protons by NMR after quick transfer of yeast tRNAAsp in 2H2O solvent. When the concentration of exchange-catalyzing buffer is not too high, one imino proton exchanges considerably more slowly than any other (e.g., 100 hr versus 4 hr for the second-slowest imino proton at 18 degrees C in 15 mM Mg). This provides excellent conditions for identification, by the nuclear Overhauser effect, of the slowest exchanging proton, which we show to be the imino proton of the U-8 . A-14 reverse Hoogsteen tertiary-structure base pair; other slowly exchanging protons are identified as imino protons from A . U-11 and G . psi-13. In preliminary experiments, we find that the exchange of these protons is catalyzed by cacodylate or Tris buffer. The lifetimes of two other imino protons, ca. 10 min at 28 degrees C, are buffer independent. Slowly exchanging amino protons have also been observed. Correlation with the exchange of the uracil-8 imino proton suggests that they may be from adenine-14.

Base Sequence↗
PubMed1982

Enzymatic replacement in vitro of the first anticodon base of yeast tRNAAsp: application to the study of tRNA maturation in vivo, after microinjection into frog oocytes.

A combination of several enzymes, RNase-T1, nuclease S1, T4-polynucleotide kinase and T4-RNA ligase were used to prepare and modify different fragments of yeast tRNAAsp (normal anticodon G U C). This allowed us to reconstitute, in vitro, a chimeric tRNA that has any of the four bases G, A, U or C, as the first anticodon nucleotide, labelled with (32p) in its 3' position. Such reconstituted (32p) labelled yeast tRNAAsp were microinjected into the cytoplasm or the nucleus of the frog oocyte and checked for their stability as well as for their potential to work as a substrate for the maturation (modifying) enzymes under in vivo conditions. Our results indicate that the chimeric yeast tRNAsAsp were quite stable inside the frog oocyte. Also, the G34 was effectively transformed inside the cytoplasm of frog oocyte into Q34 and mannosyl-Q34; U34 into mcm5s2U and mcm5U. In contrast, C34 and A34 were not transformed at all neither in the cytoplasm nor in the nucleus of the frog oocyte. The above procedure constitutes a new approach in order to detect the presence of a given modifying enzyme inside the frog oocyte; also it provides informations about its cellular location and possibility about its specificity of interaction with foreign tRNA.

Animals↗
PubMed1982

[Incubation of Beef tRNALeu in interferon-treated cells lysate leads to a partial removal of the 3' terminal sequence (author's transl)].

Beef tRNALeu and tRNAArg labeled with [32P]-phosphate at their 3' hydroxyl or 5'-phosphate ends have been incubated in the presence of lysates of interferon-treated Mouse cells. The degradation of the two tRNAs has been followed by polyacrylamide slab gel electrophoresis. In all cases, the incubation leads to a degradation of the tRNAs to a variable extent, but tRNALeu is degraded much faster than tRNAArg. This study, provides evidence that the 3' terminal C - C - A is the primary target of degradation leading to a very fast inactivation of tRNALeu and to the inhibition of translation of exogenous and synthetic mRNAs.

Animals↗
PubMed1981

Partial digestion of tRNA--aminoacyl-tRNA synthetase complexes with cobra venom ribonuclease.

Transfer RNA molecules have been labeled with 32P at the 5' or 3' end and digested with cobra venom ribonuclease, which preferentially cuts double-stranded regions. The products of yeast tRNAPhe and tRNAVal were analyzed by high-resolution gel electrohporesis. In the free state, these tRNAs were cut predominantly in the acceptor and anticodon stems. Minor cuts occurred in the T psi stem in tRNAVal. The topography of zones interacting with their cognate synthetases was studied by determining the tRNA regions shielded by protein. Nearly 100% protection was found in the anticodon and acceptor stem of tRNAVal, while in tRNAPhe only the stem of the anticodon was protected. Noncognate interactions between tRNAPhe and tryptophanyl-tRNA synthetase from beef pancreas were examined. The beef enzyme did not protect tRNAPhe despite the fact that efficient misaminoacylation occurred. The pattern of shielding obtained for each tRNA-synthetase complex was compared with the results of direct ultraviolet cross-linking experiments with these complexes.

Amino Acyl-tRNA Synthetases↗