The Alkali-Stable Dinucleotide Sequences in 18 s + 28 s Ribonucleates From Wheat Germ

Singh, Harwant; Lane, B. G.

doi:10.1139/o64-112

Cited by 86 publications

(44 citation statements)

References 20 publications

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“…The knowledge of the chemical behaviour, structural and biological significance of 2'-O-methylnucleoside residues is still fragmentary. 2'-0-Methylated oligonucleotides are more stable under alkaline conditions than other oligoribonucleotides [3]. The [5], p-chlorophenoxyacetyl for the 5'-hydroxyl group [6], methoxymethylidene for the 2',3'-cis-diol system [7], and benzoyl for the exo-amino group of cytidine.…”

Section: Introductionmentioning

confidence: 99%

The modified nucleosides of tRNAs. II. Synthesis of 2^′-O-methylcytidylyl (3^′-5^′) cytidine

Markiewicz

Wiewiórowski

1975

Nucl Acids Res

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Section: Introductionmentioning

confidence: 99%

The modified nucleosides of tRNAs. II. Synthesis of 2^′-O-methylcytidylyl (3^′-5^′) cytidine

Markiewicz

Wiewiórowski

1975

Nucl Acids Res

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“…Oligonucleotides containing guanylyl residues only at the 3'-termini were generated by exhaustive enzymatic digestion of highmolecular-weight wheat germ RNA prepared by a modification of the procedure of Singh and Lane (17). RNA as necessary to maintain pH between 7 and 8.…”

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confidence: 99%

Differential activity staining: its use in characterization of guanylyl-specific ribonuclease in the genus Ustilago.

Blank¹,

Dekker²

1975

Proc. Natl. Acad. Sci. U.S.A.

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Guanylyl-specific ribonuclease can be identified by a novel technique employing electrophoresis in polyacrylamide slabs followed by differential activity staining. The technique requires as little as 7 ng of enzyme which may be grossly admixed with contaminants, including other ribonucleases.Upon electrophoresis and activity staining, a variety of ribonucleases can be visualized as light or clear bands in a colored background formed by toluidine blue complexed with oligonucleotide substrate. Guanylyl-specific ribonuclease, which is detectable when using an oligonucleotide substrate of random base sequence, does not yield a band when using oligonucleotides bearing guanylyl residues at the 3'-termini only and containing, therefore, no susceptible internucleotide bonds; in contrast, a ribonuclease with a different base specificity or no base specificity yields a band with either substrate. This differential activity staining method for establishing guanylyl specificity permits estimation of the extent of nonspecific cleavage of internucleotide linkages by a putatively guanylyl-specific enzyme and is at least as sensitive as conventional procedures for determination of base specificity.With this new technique guanyloribonuclease has been identified in the unfractionated culture medium of 10 organisms belonging to the phytopathogenic fungal genus Ustilago. It is suggested that guanylyl-specific ribonuclease is widely distributed among Ustilago species; its electrophoretic properties may be revealing of phylogenetic relationships among these plant parasites and among their hosts.The general technique of differential activity staining, developed for determination of the base specificity of ribonucleases, may be widely applicable to analysis of enzymes catalyzing depolymerization reactions.Since discovery of the classic RNase T1, guanylyl-specific ribonucleases have been found in bacteria, in the three classes of fungi, and in a slime mold (1-5). Most of these enzymes are extracellular proteins of molecular weight about 11,000; the amino-acid composition of six (1-4) and the amino-acid sequence of two of them (1, 6) have been reported. Although they are most widely known as an analytical tool for determination of nucleotide sequence in RNA (1, 7), the guanylyl-specific ribonucleases are highly interesting in their own right. On-going studies of these enzymes include analysis of their mechanism of action (1) and comparison, at a basic level, with that of ribonuclease A (8); determination of structure-function relationships among the various G-specific RNases and the related, purine-preferring RNase U2 (9); and investigation of their distribution, biological function, and regulation (10, 11).Among known guanylyl-specific ribonucleases are two excreted by members of the phytopathogenic fungal genus Ustilago-RNase Ui from U. sphaerogena and an enzyme from the corn smut U. maydis (1, 10). We report here that extracellular guanylyl-specific ribonuclease is produced by Abbreviation: G-specific, guanylyl-specific. eight...

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“…Moreover, we have found that the addition of wheat germ RNA to the reaction mixture greatly stimulates the transfer of methyl groups from S-adenosyl methionine to the 2'-oxygen of RNA nucleosides. Wheat-germ ribosomal RNA and alkali-stable dinucleotides were prepared by the method of Singh and Lane (8). Rat liver nuclei were prepared as described (9).…”

mentioning

confidence: 99%

“…Rat liver nuclei were prepared as described (9). DNA was determined with diphenylamine (10 (8). The dinucleotides were then eluted as a second fraction (Fig.…”

mentioning

confidence: 99%

“…1 was desalted by readsorption to DEAE-cellulose, followed by elution with the volatile buffer, 1 M pyridinium formate, pH 4.5 (11). The pyridinium formate was removed in a flash evaporator, and the alkali-stable dinucleotides were completely degraded to sugar-methylated ribonucleosides, basemethylated ribonucleosides, and nonmethylated ribonucleosides by the combined action of Crotalus adamanteus phosphodiesterase and E. coli alkaline phosphatase (7,8,12). The reaction mixture (1 ml) contained the following: 0.1 M (NH4)2CO3, pH 9.2; 5 mM MgCl2; 700 usg phosphodiesterase; 50 ,ug alkaline phosphatase; and 5-15 A260 units of alkalistable dinucleotides.…”

mentioning

confidence: 99%

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2′- O -Methylation of Adenosine, Guanosine, Uridine, and Cytidine in RNA of Isolated Rat Liver Nuclei

Al-Arif

Sporn

1972

Proc. Natl. Acad. Sci. U.S.A.

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Nuclei isolated from rat liver, when incubated with methyl-labeled S-adenosylmethionine, incorporated label into 2'-O-methyladenosine, 2'-0-methylguanosine, 2'-O-methyluridine, and 2'-0-methylcytidine of endogenous nuclear RNA. Addition of ribosomal RNA rrom wheat germ to the reaction markedly stimulated 2'-O-methylation of total RNA. The relative incorporation of label into the four different 2'-O-methyl ribonucleosides was greatly chahged by the addition of wheat germ RNA. There was much more 2'-O-methylation of the purine ribonucleosides, relative to the pyrimidine ribonucleosides, in the reaction stimulated by wheat germ RNA.Newly synthesized RNA is enzymatically modified in mam-:malian cell nuclei before it is able to function in its cytoplasmic role in protein synthesis (1-3). One way in which newly synthesized RNA is "processed" is the insertion of methyl groups on the 2'-oxygen atom of ribose (sugarmethylation) of all four-of the RNA nucleosides, to yield nucleoside 2'--methyl ethers (4). Although isolated mammalian nucleolar preparations have been reported to contain such sugar-methylating activity (5, 6), the products of sugarmhethylation of RNA; namely Am, Gm, Um, and Cm, have not been conclusively identified. We have recently developed a simple paper chromatographic method for the separation of the four commonly occurring sugar-methylated ribonucleosides from each other, as well as from all of the commonly occurring base-methylated ribonucleosides (7). Using this method, we now report, for the first time, that an enzyme system found in isolated rat liver nuclei will transfer a methyl group from S-adenosyl methionine to the 2'-oxygen of A, G, U, and C in endogenous nuclear RNA. Moreover, we have found that the addition of wheat germ RNA to the reaction mixture greatly stimulates the transfer of methyl groups from S-adenosyl methionine to the 2'-oxygen of RNA (Fig. 1) with 100 ml of 0.085 M Tris-formate, pH 7.8-7 M urea (8). The dinucleotides were then eluted as a second fraction (Fig. 1) t. The molarity of all Tris-formate buffers is expressed in terms of the Tris, rather than formate, concentration.

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The Alkali-Stable Dinucleotide Sequences in 18 s + 28 s Ribonucleates From Wheat Germ

Cited by 86 publications

References 20 publications

The modified nucleosides of tRNAs. II. Synthesis of 2^′-O-methylcytidylyl (3^′-5^′) cytidine

The modified nucleosides of tRNAs. II. Synthesis of 2^′-O-methylcytidylyl (3^′-5^′) cytidine

Differential activity staining: its use in characterization of guanylyl-specific ribonuclease in the genus Ustilago.

2′- O -Methylation of Adenosine, Guanosine, Uridine, and Cytidine in RNA of Isolated Rat Liver Nuclei

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The Alkali-Stable Dinucleotide Sequences in 18 s + 28 s Ribonucleates From Wheat Germ

Cited by 86 publications

References 20 publications

The modified nucleosides of tRNAs. II. Synthesis of 2′-O-methylcytidylyl (3′-5′) cytidine

The modified nucleosides of tRNAs. II. Synthesis of 2′-O-methylcytidylyl (3′-5′) cytidine

Differential activity staining: its use in characterization of guanylyl-specific ribonuclease in the genus Ustilago.

2′- O -Methylation of Adenosine, Guanosine, Uridine, and Cytidine in RNA of Isolated Rat Liver Nuclei

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The modified nucleosides of tRNAs. II. Synthesis of 2^′-O-methylcytidylyl (3^′-5^′) cytidine

The modified nucleosides of tRNAs. II. Synthesis of 2^′-O-methylcytidylyl (3^′-5^′) cytidine