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I Winicov

Publications and source records attributed to I Winicov.

At least 37 records · Page 2Linked to original sources
PubMed1987

Histone variants and acetylated species from the alfalfa plant Medicago sativa.

The histones from the alfalfa plant Medicago sativa have been characterized in terms of type variants and levels of acetylation. Histones were isolated directly from total plant tissue (callus), eliminating the need to develop methods for nuclear isolation. An acid-urea-polyacrylamide gel with a transverse Triton X-100 gradient resolved and identified in a single gel at least one type of histone H4, two variant forms of histone H2B, two variant forms of histone H3, and four variant forms of histone H2A from a crude histone preparation. Histone H4 was present 25% in an unmodified state and 75% as monomodified, presumably as monoacetylated histone. Both histone H3 variants displayed five bands, consistent with up to four internal sites of acetylation. The two H3 variants differed in their steady-state level of acetylation, suggesting that they may reside in different chromatin environments. Several histone H1 species were identified by solubility and cross-reactivity with antiserum raised against the globular part of bovine H1(0), indicating conservation of epitopes between histone H1 of mammals and higher plants.

Acetylation↗
PubMed1986

Preferential nucleosome placement on pBR322 restriction fragments.

Two restriction fragments of DNA containing the regulatory feature GTG/CAC were experimentally associated with core histones. The reconstituted DNA-histone complexes consisted of different forms of mononucleosomes. Lambda exonuclease and Fnu4HI were used to probe the structure of each distinct nucleoprotein complex. For each of the DNA fragments, one form of particle was produced that showed preferred placement of the core octamer on the DNA. The GTG/CAC base triplets may play some role in determining the final histone core positions in these reconstitutes.

Base Sequence↗
PubMed1986

Accurate in vitro initiation of beta-globin gene transcription in induced Friend-cell nuclei.

The initiation of transcription by RNA polymerase II in isolated murine erythroleukemia cell nuclei was investigated by isolating newly synthesized gamma-thio (gamma-S-)-triphosphate-labeled transcripts by Hg-agarose chromatography. The 5' terminus of transcripts initiated in vitro with [gamma-35S]ATP or [gamma-35S]GTP was identified as the thiotetraphosphate in alkaline hydrolysis products from Hg-agarose-selected RNA. Additional control experiments analyzing the nuclear transcription of two well characterized tRNA genes showed that each gene was initiated with the proper triphosphate, either gamma-S-ATP or gamma-S-GTP, indicating little, if any, exchange of the gamma-S-labeled substrate to the other triphosphates. As determined by S1 mapping, newly synthesized beta-globin gene transcripts initiate only with gamma-S-ATP. Their 5'-terminus is located at the cap site, and their synthesis is inhibited by 1 microgram alpha-amanitin/ml. In reactions containing gamma-S-ATP but not gamma-S-GTP, several additional initiation sites are observed that are located in the 5'-flanking region. We conclude that RNA polymerase II can initiate transcription at the cap site in isolated nuclei.

Amanitins↗
PubMed1984

5'-Hydroxyl RNA kinase from mouse L cells.

A 5'-hydroxyl RNA kinase from mouse L cells has been partially purified and characterized. The enzyme transfers the gamma-phosphorus from ATP to 5'-hydroxyl termini of RNA much more efficiently than DNA substrates, and is virtually inactive on 3'-CMP. The molecular mass of the predominant kinase activity is estimated to be 93-96 kDa from denaturing and non-denaturing polyacrylamide gel analyses. A minor band of lower molecular mass has been also observed. The enzyme activity requires Mg2+ and is inhibited by both Mn2+ and Zn2+. Antibodies to small nuclear ribonucleoproteins have no effect on this activity.

Animals↗
PubMed1983

Nuclear beta-globin RNA transcripts initiated in vitro with gamma-thio nucleoside triphosphate.

We have been able to obtain significant levels of apparently in vitro-initiated beta-globin transcripts from isolated nuclei using mouse Friend cells. The level of hybrid selected beta-globin transcripts was increased 3-9-fold in nuclei after the cells were induced with dimethylsulfoxide. The newly initiated transcripts were detected through incorporation of gamma-thio ATP and isolation by Hg-agarose chromatography. The 5'-terminus was identified by recovery of the thio tetraphosphate in hydrolysis products from in vitro initiated RNA using gamma-35S ATP.

Adenosine Triphosphate↗
PubMed1982

Dichlorobenzimidazole-riboside inhibition of nuclear RNA accumulation initiated with gamma-thio analogues of ATP and GTP.

The gamma-thio analogues of ATP and GTP, ATP[S] and GTP[S], have been used as affinity probes to measure RNA synthesis initiated in vitro in L cell nuclei. 5,6-Dichlororibofuranosylbenzimidazole was found to inhibit total RNA synthesis in vitro and the amount bound by mercury-affinity chromatography. Initiation in vitro was also shown by [35S]ATP[S] and [35S]GTP[S] incorporation in RNA molecules with a larger size distribution. Although 66% of RNA molecules labeled with [35S]GTP[S] and 74% of those labeled with [35S]ATP[S] were 3-12 S n size, the remainder of the label was recovered in RNA molecules larger than 12 S and a significant portion in RNA larger tha 18 S. The specificity of the initiation process seems to be indicated by the finding of molecules of the size of pre-tRNA on gel electrophoresis which were labeled with [35S]GTP[S] but not [35S]ATP[S] under our experimental conditions. 5,6-Dichlororibofuranosylbenzimidazole severely inhibited the incorporation of [35S]GTP[S] and [35S]AT[[S] by all size classes, indicating that it can decrease accumulation of RNA initiated in vitro in the L cell nuclear system.

Adenosine Triphosphate↗
PubMed1982

Nuclear ligation of RNA polynucleotide kinase products.

RNA polynucleotide kinase has been shown to transfer [gamma 32P] from ATP to 5-OH termini of endogenous nuclear RNA. The products of this reaction have been isolated in RNA larger than 125 after in vitro incubation of mouse L cell nuclei. About 20%-30% of these 5'-OH kinase products are polyadenylated. A sizeable fraction of the [gamma 32P] label from ATP is also found in internal phosphodiester bonds after 30-minute nuclear incubation in vitro. The possibility of substantial [32P] 1) 2mM nucleoside triphosphates in the incubation medium, 2) limited nearest-neighbor distribution 3' and 5' to the phosphodiester bond compared with that from [alpha 32P] UTP, 3) different nearest-neighbor distribution for RNA molecules greater than 125 and 12-3S, 4) relative insensitivity of the [gamma 32P] incorporation to alpha-amanitin as compared with total RNA synthesis, 5) internal [32P] appearance in RNA greater than 12S in less than five minutes of incubation, and 6) less than 0.03% to 0.6% of the total [32P] in the alpha position of nucleoside triphosphates after 30 minutes of incubation. The [gamma 32P] incorporation was dependent on high ATP concentration and was insensitive to competition by inorganic phosphate. These results are consistent with the levels of 5' RNA polynucleotide kinase activity in L cell nuclei and suggest the presence of an RNA ligase that can utilize the termini generated by the 5'-OH RNA kinase in a ligation reaction.

Amanitins↗
PubMed1982

Nuclear ligation of RNA 5'-OH kinase products in tRNA.

Mouse L-cell nuclei incorporate gamma-32P from ATP in vitro predominantly in 5'-monophosphoryl termini and internal phosphodiester bonds with a nonrandom nearest-neighbor distribution. In the presence of 1 microgram of alpha-amanitin per ml the gamma-32P showed a time-dependent appearance in RNA bands which migrated with mature tRNA species but not with pre-tRNA and 5S RNA. The gamma-32P was found in internal phosphodiester bonds as shown by alkaline phosphatase resistance and was identified in 3'-monophosphates after RNase T2, T1, and A digestion. The specificity of this incorporation was indicated by a limited number of labeled oligonucleotides from a T1 digest and identification of 70 to 80% of the 32P label as Cp on complete digestion of the eluted tRNA band. We also observed transiently [gamma-32P]ATP-labeled RNA bands (in 5'-monophosphate positions) that were 32 to 45 nucleotides long. The results presented suggest splicing of several mouse L-cell tRNA species in isolated nuclei which involve the RNA 5'-OH kinase products as intermediates.

Adenosine Triphosphate↗
PubMed1981

Liver and kidney nuclear RNA synthesis and modifications in dimethylnitrosamine-treated rats.

RNA synthesis was measured in nuclei isolated from rat liver and kidney 22 h post injection of 30 mg dimethylnitrosamine/kg body weight. In nuclear preparations were shown by electron microscopy to consist of clean hepatocytes and the liver nuclei showed no apparent necrosis at that time. In vitro RNA synthesis and methylation were proportional to time and nuclear concentration, as well as dependent on exogenous nucleoside triphosphates and S-adenosylmethionine. 60-70% of the in vitro synthesis was inhibited by 1 microgram/ml alpha-amanitin. Total liver nuclear RNA synthesis was increased after dimethylnitrosamine exposure, but, unlike RNA synthesis in nuclei after partial hepatectomy, both alpha-amanitin-sensitive and -resistant synthesis were increased. Differences were found between dimethylnitrosamine-treated liver and kidney nuclear RNA synthesis which was sensitive to inhibition by 1-10 microgram/ml alpha-amanitin, presumably a product of RNA polymerase III. Nuclear RNA methylation with S-adenosylmethionine, which was dependent on new RNA synthesis, differed between dimethylnitrosamine-treated rat liver and kidney nuclei. The endogenous RNA methyl substituents labeled in vitro showed differences in levels of methylation of bases, the 2'-O position of ribose and caps in comparison between control and dimethylnitrosamine-treated nuclei from both liver and kidney. Significant differences were obtained in both nuclear RNA transcription and methylation in vitro between the two tissues in response to pretreatment of the rat in vito dimethylnitrosamine.

Animals↗
PubMed1979

Nuclear transcription in vitro. Sensitivity to inhibition by ribosyldichlorobenzimidazole and rifamycin AF/013.

L cell nuclear preparations were shown to transcribe RNA for periods up to 1 h at 37 degrees C. Nearly 70% of the transcription products were sensitive to inhibition by 1 microgram/mL of alpha-amanitin, indicating that they were transcribed by RNA polymerase II. Analysis of polyphosphorylated termini of in vitro synthesized RNA showed the presence of a phosphatase activity which prevents quantitative recovery of these termini. The finding of in vitro labeled polyphosphorylated termini in RNA greater than 12 S after short periods of incubation shows initiation in vitro for this size class. The labeling of these polyphosphorylated termini is decreased in the presence of rifamycin AF/013. The use of two apparent inhibitors of initiation, rifamycin AF/013 and 5,6-dichloro-1-beta-D-ribofuranosylbenzimidazole (DRB), has allowed detection of in vitro initiated transcripts of heterogeneous nuclear RNA. Both of these inhibitors act primarily at later times of incubation, in contrast to alpha-amanitin which acts on elongation and inhibits in vitro RNA synthesis immediately. The selective pattern of DRB inhibition on hnRNA is retained in vitro and some accumulation of large-size molecules is observed. It can be estimated that about 30% of the greater than 12S hnRNA sequences transcribed in vitro are sensitive to DRB and 48% of greater than 12S RNA are sensitive to rifamycin AF/013 inhibition.

Cell Nucleus↗
PubMed1977

RNA phosphorylation: a polynucleotide kinase function in mouse L cell nuclei.

Analysis of [gamma-32P]ATP in vitro labeled nuclear RNA shows transfer of gamma-32P from ATP to form the 5'-terminal monophosphate for large RNA molecules. This finding is an actively transcribing nuclear system capable of guanylation and methylation reactions indicates that polynucleotide kinase activity in the eukaryotic nucleus may be functional in kinase reactions involving RNA. It further suggest a participation in the posttranscriptional modification reactions involved in RNA processing. All four nucleosides were found to act as acceptors at the 5' termini of RNA. It is also shown that both ATP and GTP can serve as donors in the nuclear polynucleotide kinase reaction.

Adenosine Triphosphate↗
PubMed1976

Synthesis methylation, and capping of nuclear RNA by a subcellular system.

A subcellular system is described which is capable of in vitro synthesis of large nuclear RNA and the formation of both cap I [m7G(5')pppXmpYp] and capII [m7G(5')-pppXmpYmpZp] structures. This system, which consists of partially purified intact nuclei and residual cytoplasmic tags, carries out both guanosine addition, utilizing GTP, and the appropriate methylation reactions, utilizing S-adenosylmethionine as the methyl donor. The general structure of the caps was verified by analyses of methylated derivatives recovered after RNase T2 hydrolysis and after digestion with P1 nuclease, bacterial alkaline phosphatase,and nucleotide pyrophosphatase. Cap formation in large nuclear RNA species was found to be closely associated with transcription, as indicated by alpha-manitin sensitivity and a requirement for the presence of all four nucleoside triphosphates. Recovery of a class of cap II structures, in which only the methyl group at position Y is labeled, as well as capII structures in which all methylated constituents are labeled, indicates the presence of at least two independent methylation events in the in vitro system.

Amanitins↗
PubMed1975

Stereospecificity of hydrogen transfer by phosphoglycerate dehydrogenase.

Phosphoglycerate dehydrogenase (EC 1.1.1.95) has been shown to be A site specific in its hydrogen transfer capacity unlike other dehydrogenases which use phosphorylated substrates. The experiments have been carried out using a coupled assay system with yeast alcohol dehydrogenase. The specific activity measurements of the reaction products indicate the possible influence of an isotope effect on this system.

Alcohol Oxidoreductases↗
PubMed1975

The role of 5-S RNA in temperature-sensitive ribosomal RNA maturation.

The synthesis of 5-S RNA was found to be unchanged at both the permissive (33.5 degrees C) and non-permissive (38.5 degrees C) temperatures in a temperature-sensitive Baby Hamster Kidney cell line (BHK 21 ts 422 E) as measured relative to synthesis of 18-S rRNA. The 5-S RNA is shown to be associated with nucleolar ribonucleoprotein particles even though rRNA processing does not yield a functional 28-S rRNA at the non-permissive temperature. The amount of 5-S RNA found associated with the 80-S ribonucleoprotein particles was the same at the permissive and non-permissive temperatures, indicating that an aberrant 5-S RNA contribution to rRNA processing is not a primary cause for the temperature-sensitive lesion of rRNA maturation in this mutant cell line. The amount of 5-S RNA in nucleolar 80-S RNA particles indicated that the association of 5-S RNA with the rRNA precursor particle occurs before the cleavage step at which 32-S precursor RNA is produced.

Cell Fractionation↗
PubMed1975

The mechanism of end product inhibition of serine biosynthesis. V. Mechanism of serim inhibition of phosphoglycerate dehydrogenases.

The reduction of enzyme-bound DPN constitutes a half-reaction of phosphoglycerate dehydrogenase and has been investigated fluorometrically. Serine was found to inhibit the half-reaction to the same extent and with the same degree of cooperation as the steady state reaction. This finding identifies the ternary complex conversion as the point in the reaction sequence at which serine inhibition occurs. Delta H determinations for the half-reaction showed no difference whether serine was or was not present and led to the conclusion that the inhibitory effect of serine could only manifest itself through the delta S term in the expression for the formation of the activated transition state complex. DL-3-P[2-2H]glyceric acid showed no primary isotope effect in the half-reaction. This result excludes hydrogen transfer as the rate-limiting step in the half-reaction and confirms that an isomerization step, affected by serine, exists in the ternary complex conversion scheme. The deuterated 3-P-glyceric acid shows an isotope effect of 2 in the steady state reaction.

Alcohol Oxidoreductases↗