PubMed Health⌕ Search

Biomedical subjects

G Keith

Publications and source records attributed to G Keith.

At least 19 recordsLinked to original sources
PubMed1992

Presence and coding properties of 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um) in the wobble position of the anticodon of tRNA(Leu) (U*AA) from brewer's yeast.

The unknown modified nucleoside U* has been isolated by enzymatic and HPLC protocols from tRNA(Leu) (U*AA) recently discovered in brewer's yeast. The pure U* nucleoside has been characterized by electron impact mass spectroscopy, and comparison of its chromatographic and UV-absorption properties with those of appropriate synthetic compounds. The structure of U* was established as 2'-O-methyl-5-carbamoylmethyluridine (ncm5Um). The yeast tRNA(Leu) (U*AA) is the only tRNA so far sequenced which has been shown to contain ncm5Um. The location of such a modified uridine at the first position of the anticodon restricts the decoding property to A of the leucine UUA codon.

Anticodon↗
PubMed1992

The protein of M(r) 21,000 constituting the prosome-like particle of duck erythroblasts is homologous to apoferritin.

In duck erythroblasts, two major populations of untranslated messenger (m) RNP can be separated by sucrose gradient centrifugation in low ionic strength. One of these contains globin mRNA associated to protein factors, among them the prosomes. The other, sedimenting in the 35S zone, contains non-globin mRNA. From this '35S' mRNP, a new RNP particle called the prosome-like particle was isolated and characterized [Akhayat, O., Infante, A. A., Infante, D., Martins de Sa, C., Grossi de Sa, M.-F. & Scherrer, K. (1987) Eur. J. Biochem. 170, 23-33]. The PLP is a multimer of a protein of M(r) 21,000, and contains small RNA species. The particle is tightly associated with repressed mRNA and inhibits in vitro protein synthesis. We show here that the protein of M(r) 21,000, constituting the prosome-like particle, is apoferritin. Different approaches confirm the RNP character of this particle and provide evidence that some of its RNA species are tRNA. The hypothesis is discussed as to whether (apo-)ferritin might serve other functions in addition to iron storage.

Amino Acid Sequence↗
PubMed1992

The major RNA in prosomes of HeLa cells and duck erythroblasts is tRNA(Lys,3).

Two-dimensional gel electrophoresis of HeLa cell prosomal RNAs, 3'-end labeled by RNA ligase, revealed one prominent spot. Determination of a partial sequence at the 3'-end indicated full homology to the 18 nucleotides at the 3'-end of tRNA(Lys,3) from rabbit, the bovine and the human species. An oligonucleotide complementary to the 3'-end of tRNA(Lys,3) hybridized on Northern blots with prosomal RNA from both HeLa cells and duck erythroblasts. In two-dimensional PAGE, the major pRNA of HeLa cells co-migrated with bovine tRNA(Lys,3). Reconstitution of the CCA 3'-end of RNA from both human and duck prosomes, by tRNA-nucleotidyl-transferase, confirmed the tRNA character of this type of RNA. Furthermore, it revealed at least one additional tRNA band about 85 nt long among the prosomal RNA from both species. Finally, confirming an original property of prosomal RNA, we show that in vitro synthesized tRNA(Lys,3) hybridizes stably to duck globin mRNA, and to poly(A)(+)- and poly(A)(-)-RNA from HeLa cells.

Animals↗
PubMed1992

Structural specificity of Rn nuclease I as probed on yeast tRNA(Phe) and tRNA(Asp).

A single-strand-specific nuclease from rye germ (Rn nuclease I) was characterized as a tool for secondary and tertiary structure investigation of RNAs. To test the procedure, yeast tRNA(Phe) and tRNA(Asp) for which the tertiary structures are known, as well as the 3'-half of tRNA(Asp) were used as substrates. In tRNA(Phe) the nuclease introduced main primary cuts at positions U33 and A35 of the anticodon loop and G18 and G19 of the D loop. No primary cuts were observed within the double stranded stems. In tRNA(Asp) the main cuts occurred at positions U33, G34, U35, C36 of the anticodon loop and G18 and C20:1 positions in the D loop. No cuts were observed in the T loop in intact tRNA(Asp) but strong primary cleavages occurred at positions psi 55, C56, A57 within that loop in the absence of the tertiary interactions between T and D loops (use of 3'-half tRNA(Asp)). These results show that Rn nuclease I is specific for exposed single-stranded regions.

Base Composition↗
PubMed1992

Pleiotropic effect of a point mutation in the yeast SUP4-o tRNA gene: in vivo pre-tRNA processing in S. cerevisiae.

The expression of mutant tyrosine-inserting ochre suppressor SUP4-o tRNA genes in vivo in S. cerevisiae was examined as a basis for further studies of tRNA transcription and processing. In vivo yeast precursor tRNAs have been identified by filter hybridization and primer extension analysis. We have previously shown that a mutant SUP4-o tRNA gene with a C52----A52 transversion at positive 52 (C52----A52(+IVS) allele) was transcribed but that the primary transcript was not processed correctly. We show here that 5' and 3' end processing as well as splicing are defective for this mutant but that the 5' end processing is restored when the intron is removed from the gene by oligonucleotide directed mutagenesis (C52----A52(-IVS) allele). Our results imply that the C52----A52 transversion by itself cannot account for the lack of susceptibility to RNase P cleavage but that the overall tertiary structure of the mutant tRNA precursor is destabilized by the intron/anticodon stem. A second consequence of the C52----A52 transversion is to prevent complete maturation of the tRNA precursor at its 3' end since intermediates containing incompletely processed 3' trailers accumulate in the yeast cells transformed with the C52----A52(-IVS) allele. A correct structure of the T stem might therefore define a structural feature required for the recognition of the 3' processing activity.

Base Sequence↗
PubMed1992

Use of a dot blot hybridization method for identification of pure tRNA species on different membranes.

The characterization of a tRNA in purification procedures usually involves aminoacylation assays but recently, the hybridization by dot blot with specific oligonucleotides as probes has been used for the tRNA identification. We present here an optimization of a dot blot hybridization method for the tRNA detection by comparing the efficiency of eight different nylon membranes. Neutral 0.22 microns porosity membranes (Nytran, Biodine A) give the best detection efficiency when small quantities of material (less than 40 ng of tRNA) are dotted on filter; by contrast, neutral 0.45 microns porosity membranes (such as Hybond N) are the most efficient when larger quantities of tRNA are dotted on the filter. The described technique allows to detect less than 20 pg of a pure tRNA species. Its use in the identification of Saccharomyces cerevisiae initiator tRNA(Met) in counter-current distribution fractions is shown.

Autoradiography↗
PubMed1992

Inhibition of murine leukemia viruses by nuclease-resistant alpha-oligonucleotides.

We studied the antiviral activity of nuclease-resistant alpha-anomeric oligonucleotides. An alpha-oligonucleotide (20-mer) targeted to the primer binding site (PBS) of murine retroviruses inhibited viral spreading. The inhibition only occurred when the cells had been electropermeabilized in the presence of the oligonucleotide. The PBS sequence is involved in reverse transcription and in translation. The data suggest that the oligonucleotide could perturb reverse transcription activity. Thus, either the oligonucleotide induced a decrease in initiation or it inhibited the extension of the minus or plus strands DNA during reverse transcription. These results show that reverse transcription may be an interesting target for antisense oligonucleotides.

3T3 Cells↗
PubMed1991

O-ribosyl-phosphate purine as a constant modified nucleotide located at position 64 in cytoplasmic initiator tRNAs(Met) of yeasts.

The unknown modified nucleotide G*, isolated from both Schizosaccharomyces pombe and Torulopsis utilis initiator tRNAs(Met), has been identified as an O-ribosyl-(1"----2')-guanosine-5"-phosphate, called Gr(p), by means of HPLC, UV-absorption, mass spectrometry and periodate oxidation procedures. By comparison with the previously published structure of Ar(p) isolated from Saccharomyces cerevisiae initiator tRNA(Met), the (1"----2')-glycosidic bond in Gr(p) has been postulated to have a beta-spatial conformation. The modified nucleotide Gr(p) is located at position 64 in the tRNA(Met) molecules, i.e. at the same position as Ar(p). Since we have also characterized Gr(p) in Candida albicans initiator tRNA(Met), the phosphoribosylation of purine 64 can be considered as a constant nucleotide modification in the cytoplasmic initiator tRNAs(Met) of all yeast species so far sequenced. Precise evidence for the presence of Gr(p) in initiator tRNAs(Met) of several plants is also reported.

Candida↗
PubMed1991

Effect of cobalamin derivatives on in vitro enzymatic DNA methylation: methylcobalamin can act as a methyl donor.

5-Methylcytosine synthesis in DNA involves the transfer of methyl groups from S-adenosyl-methionine to the 5'-position of cytosine through the action of DNA (cytosine-5)-methyltransferase. The rate of this reaction has been found to be enhanced by cobalt ions. We therefore analyzed the influence of vitamin B12 and related compounds containing cobalt on DNA methylation. Vitamin B12, methylcobalamin, and coenzyme B12 were found to enhance significantly the de novo DNA methylation in the presence of S-adenosylmethionine for concentrations up to 1 microM, but at higher concentrations these compounds were found to inhibit DNA methylation. Methylcobalamin behaves as a competitive inhibitor of the enzymatic methylation reaction (Ki = 15 microM), the Km for S-adenosylmethionine being 8 microM. In addition, the use of radioactive methylcobalamin shows that it can be used as a methyl donor in the de novo and maintenance DNA methylation reactions. Thus, two DNA methylation pathways could exist: one involving methylation from S-adenosylmethionine and a second one involving methylation from methylcobalamin.

Animals↗
PubMed1991

Site-specific hypomethylation of c-myc protooncogene in liver nodules and inhibition of DNA methylation by N-nitrosomorpholine.

The protooncogene c-myc was investigated in N-nitrosomorpholine-induced rat liver nodules to elucidate the role of altered DNA methylation in chemical carcinogenesis. Furthermore, Micrococcus luteus DNA and chicken erythrocyte DNA were modified in vitro by reactive metabolites of N-nitrosomorpholine, generated by P450-dependent monooxygenases. The modified DNAs were less methylated in vitro than control DNAs by DNA-(cytosine-5)-methyltransferase (DNA methylase). The DNA methylase assay and 32P-postlabeling analysis revealed lowered levels of DNA methylation in nodular DNA. In nodular tissue, c-myc messenger RNA levels were found to be increased compared to normal liver. DNA methylation analysis using the restriction endonucleases HpaII/MspI indicated hypomethylation in the first intron of c-myc DNA in liver nodules. The results suggest that genotoxic lesions may cause stably inherited, aberrant DNA methylation patterns which may be responsible for site-specific hypomethylation of the c-myc protooncogene in liver nodules.

Animals↗
PubMed1991

Sequence of a new tRNA(Leu)(U*AA) from brewer's yeast.

The nucleotide sequence of a new tRNA(Leu)(anticodon U*AA) from Saccharomyces cerevisiae which could recognize exclusively the UUA codon has been determined. Its primary structure is: pGGAGGGUUGm2GCac4CGAGDGmGDCDAAGGCm2(2)GGCAGACmUU*AAm1GA++ + psi CUGUUGGACGGUUGUCCGm5CGCGAGT psi CGm1A(orA)ACCUCGCAUCCUUCACCA. This tRNA has a large extraloop and contains 15 modified nucleotides. So far it is the third isoacceptor tRNA for leucine in yeast. It has 61% homology with tRNA(Leu)(anticodon m5CAA) and 63% homology with tRNA(Leu)(anticodon UAG), the two other known yeast tRNAs(Leu).

Base Sequence↗
PubMed1991

Purification and characterization of recombinant equine infectious anemia virus reverse transcriptase.

A 1.67-kb segment of the equine infectious anemia virus pol gene, encoding a 66-kDa reverse transcriptase (RT), was cloned and expressed in Escherichia coli. Recombinant RT, purified by a combination of metal chelate affinity chromatography and ion-exchange chromatography, displays both RNA-dependent DNA polymerase and RNase H activity. The affinity of purified RT for its replication primer, tRNA(3Lys) was equivalent to that observed for human immunodeficiency virus RT. Our data suggest that an additional domain between RT-RNase H and integrase on the equine infectious anemia virus pol open reading frame is not an integral component of the RT polypeptide.

Animals↗
PubMed1990

Expression and site-directed mutagenesis of the catalytic domain of human poly(ADP-ribose)polymerase in Escherichia coli. Lysine 893 is critical for activity.

Bacterially expressed fusion proteins containing the COOH-terminal domain of the human poly(ADP-ribose)polymerase were analyzed by means of a novel assay, the "activity blot," which allows the detection of transferred polypeptides involved in poly(ADP-ribose) synthesis. Deletion analysis demonstrated that the 40-kDa COOH-terminal region of the enzyme is an autonomous catalytic domain exhibiting both the polymerizing and branching activities in the absence of DNA. Site-directed mutagenesis demonstrated that lysine 893 is essential for these catalytic processes. In addition, sequence similarities obtained with the NAD(P)+ amino acid dehydrogenases suggest that (i) lysine 893 may interact with the substrates of poly(ADP-ribose)polymerase and (ii) the COOH-terminal part of the 40-kDa fragment may also contain a Rossman fold structure.

Amino Acid Sequence↗
PubMed1990

Characterization of a chemically synthesized RNA having the sequence of the yeast initiator tRNA(Met).

A 75-unit long oligoribonucleotide corresponding to the sequence of the Saccharomyces cerevisiae initiator tRNA was synthesized chemically. The crude RNA was purified, and the sequence was verified by RNA sequencing techniques. A particularly useful purification step involved hydrophobic chromatography on BND-cellulose. The purified RNA could be aminoacylated to 28% of a bona fide initiator tRNA(Met) sample and threonylated to 76% of the level observed with native tRNA(fMet) from E. coli.

Base Sequence↗
PubMed1990

Heterogeneities in vertebrate tRNAs(Trp) avian retroviruses package only as a primer the tRNA(Trp) lacking modified m2G in position 7.

Bovine, rabbit and chicken tRNA(Trp) species and tRNA(Trp) packaged in avian myeloblastosis virus were separated and purified using two-dimensional gel electrophoresis and their primary structures were determined. Two major tRNA(Trp) species (1 and 2) were identified in beef and rabbit, two minor ones (3 and 4) in beef and only one minor in rabbit. Their structures differ by 4 nucleotide substitutions located in the D, S and T loops (positions 16, 47, 57 and 59). Species 3 and 4 differ from one another by only one nucleotide at position 2. Differences between tRNA(Trp) species were also observed in the extent of methylation of some nucleotides. Chicken tRNA(Trp) presents only one species similar to the mammalian type 1 tRNA(Trp). In the case of the three studies animals this tRNA could be separated into two subspecies, which differ by a post-transcriptional modification of nucleotide 7 in the acceptor stem: G or m2G. However only the nonmethylated species is used as the primer of DNA-RNA directed retrotranscription since it is only that form which was found in avian retroviruses. The methylation of G to m2G at position 7 could thus prevent the recognition of tRNA(Trp) by retroviral protein(s) responsible for the selective packaging of the primer tRNA(Trp).

Animals↗
PubMed1990

Use of two-dimensional thin-layer chromatography for the components study of poly(adenosine diphosphate ribose).

Two-dimensional thin-layer chromatography on cellulose plates has been used for separating and quantifying the three adenosine derivatives: AMP, phosphoribosyl AMP (PRAMP), and (PR)2AMP obtained by venom phosphodiesterase digestion of poly(ADP-ribose). In vitro synthesized polymer, up to 300 derivatives in length were studied. Some parameters of the complexity of poly(ADP-ribose) could be deduced from our results: (i) The first branching point appears in fragments of approximately 21 derivatives in length. (ii) The branching points are located at regular distances of approximately 41 derivatives from each other.

Adenosine Monophosphate↗
PubMed1978

Methylation of yeast tRNAPhe by enzymes from cytoplasm, chloroplasts and mitochondria of Phaseolus vulgaris.

Pure yeast tRNAPhe was used as a substrate to compare the tRNA methylating activities in Phaseolus vulgaris cytoplasm, chloroplasts and mitochondria, in the presence of S-adenosyl[Me-3H]methionine. The resulting [Me-3H]-tRNAPhe was then analyzed, using the techniques of nucleotide sequence determination. Cytoplasmic and mitochondrial enzymes catalyze the methylation (into m5C) of C48 present in the extra-loop, while chloroplast enzyme preparations catalyze the modification (into m1A) of A14 present in the dihydrouridine loop of tRNAPhe.

Base Sequence↗