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Biomedical subjects

B Groner

Publications and source records attributed to B Groner.

193 records · Page 11Linked to original sources
PubMed1979

Identification of mouse mammary tumor virus-specific mRNA.

Complementary DNA corresponding to the RNA genome of mouse mammary tumor virus was used to identify viral RNA contained in polysomes of a virus-producing mammary tumor cell line. Separation of polysomal mRNA by agarose gel electrophoresis, transfer of the RNA to diazobenzyloxymethyl paper, and hybridization with 32P-labeled mouse mammary tumor virus complementary DNA revealed three viral RNA size classes of 10, 8.8, and 4.4 kilobases in length, respectively.

Animals↗
PubMed1978

Preferential transcription of the ovalbumin gene in isolated hen oviduct nuclei by RNA polymerase B.

The synthesis of ovalbumin mRNA sequences was studied in isolated nuclei from hen oviduct. Two different methods of analysis were used to distinguish in vitro synthesized from preexisting mRNA sequences: (i) Mercurated ribonucleotides were used for in vitro RNA synthesis, and the newly synthesized RNA was purified by chromatography on sulfhydryl-agarose and hybridized to radioactive ovalbumin cDNA. (ii) [3H]UTP was used to label the in vitro synthesized RNA. Hybridization to unlabeled mercurated cDNA, RNase A digestion, and subsequent purification of the hybrids on SH-agarose allowed the quantitation of newly synthesized ovalbumin mRNA sequences. Approximately 0.1% of the newly synthesized RNA was identified as ovalbumin RNA by both methods. The synthesis of ovalbumin RNA progressed during the incubation of nuclei and was sensitive to actinomycin D and low concentrations of alpha-amanitin. The preferential in vitro transcription of the ovalbumin gene (1000-fold over random transcription of the chicken genome) by RNA polymerase B (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6) suggests that the specificity of in vitro RNA synthesis is retained in isolated nuclei.

Amanitins↗
PubMed1977

Immunoadsorption of specific chicken oviduct polysomes. Isolation of ovalbumin, ovomucoid, and lysozyme messenger RNA.

The messenger RNA coding for the egg white proteins ovalbumin, ovomucoid, and lysozyme were isolated by immunoadsorption of polysomes synthesizing these proteins. Monospecific antibodies against ovalbumin, ovomucoid, and lysozyme, raised in rabbits, were reacted with chicken oviduct polysomes. The antibody-polysome complexes were isolated by immunoadsorption onto sheep anti-rabbit antibodies coupled to an insoluble matrix. The specifically bound polysomes were eluted and the mRNA was obtained by poly(U)-Sepharose chromatography. The three specific RNAs were further purified by preparative gel electrophoresis. The purity of the mRNA preparations was demonstrated by analytical gel electrophoresis, the capability to direct the synthesis of specific protein products in a wheat germ cell-free system, and by hybridization to cDNA transcribed from mRNAoa and mRNAomu. Purified mRNAoa was shown to contain less than 0.1% mRNAomu and purified mRNAomu was about 99% pure with respect to mRNAoa. Purified mRNAly was contaminated with mRNAoa to 0.34% and with mRNAomu to 2.9%.

Animals↗
PubMed1977

Frequency distribution of messenger sequences within polysomal mRNA and nuclear RNA from rat liver.

DNA complementary to polysomal poly(A)-containing mRNA (cDNA) of male rat liver was used to study the diversity of messenger sequences in the nucleus and in polysomes. 1. Hybridization of cDNA against an excess of its own polysomal mRNA template revealed that about 10,000 different mRNA species are expressed in the liver tissue. They are distributed in a wide frequency range derived from approximately 0.5% of the total genome. 2. Hybridization of the cDNA against total nuclear RNA shows that messenger sequences comprise less than 1% of the mass of total nuclear RNA. Messenger sequences have a different frequency distribution in nucleus and cytoplasm. 3. In hybridizations using cDNA, which had been fractionated into sequences representing abundant and scarce polysomal mRNA molecules, it was found that although abundant cytoplasmic messenger sequences are also abundant in the nucleus, they exist in a significantly lower frequency range in the nuclear compartment.

Animals↗
PubMed1977

Size distribution of rat liver nuclear RNA containing mRNA sequences.

Total rat liver poly(A)-containing polysomal mRNA was size-fractionated on polyacrylamide gels in 98% formamide. Complementary DNA (cDNA) was prepared from the 8--14-S mRNA fraction and separated into sequences representing abundant and non-abundant mRNAs. The cDNA complementary to the abundant small mRNA of the rat liver cell (approximately 20 species) was hybridized to nuclear RNA of different lengths to determine the size distribution of nuclear RNA molecules which contain these messenger sequences. It was found that: 1. All abundant 8--14-S poly(A)-containing mRNAs have larger nuclear precursor molecules; 20% of the different messenger sequences are found in nuclear RNA of several times their cytoplasmic length. 2. 70% of the mass of the examined nuclear messenger sequences is in RNA molecules of a size similar to their polysomal mRNA; 30% are in larger than 18-S RNA and 2% are between 37 S and 44 S. 3. The majority of small messenger-containing RNA molecules in the RNA prepared from isolated nuclei are of true nuclear origin, since their frequency distribution differs significantly from that of the polysomal 8--14-S mRNA.

Animals↗
PubMed1977

Isolation of specific messenger RNA by adsorption of polysomes to matrix-bound antibody.

A procedure is presented for the purification of specific mRNAs, which exploits the ability of antibodies prepared against a native protein to bind to the nascent polypeptide on the polysome. Rather than precipitating these soluble antibody-polysome complexes with anti-antibody, which can lead to nonspecific trapping of polysomes, we have linked the anti-antibody to an insoluble matrix. Thus, the antibody-polysome complex binds to the anti-antibody support and nonspecific polysomes can easily be removed by several washes. We have found para-aminobenzyl cellulose (PAB cellulose), to be a suitable matrix for this purpose. This support can bind large quantities of anti-antibody and it displayed no detectable nonspecific affinity for polysomes or RNA. Using this procedure, we have obtained an apparently homogeneous preparation of ovalbumin mRNA.

Animals↗
PubMed1977

A RNA-dependent RNA polymerase activity: implications for chromatin transcription experiments.

Mercurated nucleoside triphosphates have been used for transcription of chicken oviduct chromatin with E. coli RNA polymerase. The newly synthesized RNA was purified from preexisting RNA by SH-agarose chromatography and analyzed for the content of specific mRNA sequences. The apparent preferential production of ovalbumin mRNA sequences was not inhibited by actinomycin D, although total RNA synthesis was reduced by more than 90%. Furthermore, when globin mRNA alone, or added to oviduct chromatin, was incubated in the transcription assay, a significant fraction of this mRNA was retained on SH-agarose. The copurification of chromatin associated RNA with in vitro synthesized mercurated RNA was mainly due to a RNA-dependent synthesis of complementary sequences by the bacterial enzyme. Although denaturation of the transcripts prior to SH-agarose chromatography leads to a reduced contamination with endogenous ovalbumin specific RNA, we are unable to show that the messenger-specific RNA sequences purified with the newly mercurated RNA results from a DNA-dependent reaction.

Animals↗
PubMed1975

Polyadenylate metabolism in the nuclei and cytoplasm of Saccharomyces cerevisiae.

A procedure has been designed for the simultaneous isolation, in a single step, of the nuclei and cytoplasm of Saccharomyces cerevisiae alphas288c spheroplasts. We have examined the polyadenylate poly(A)-containing RNA in these fractions and their kinetics of synthesis. Nuclear RNA saturates with [3H] adenine within 10 min. Labeled RNA appears very quickly in the cytoplasm, exceeding the amount of labeled nuclear RNA within 2 min after the addition of [3H] adenine. Nuclear poly(A)-containing RNA is approximately 10% of the total cellular poly(A)-containing RNA obtained from spheroplasts labeled for 15 min. Nuclear poly(A)-containing RNA is not as large as the giant heterogeneous nuclear RNA of animal cells. The distribution of molecular size in nuclear and cytoplasmic populations of poly(A)-containing RNA is very broad with the average size of the nuclear species being moderately larger than the cytoplasmic species. Three distinct size classes of poly(A), with different apparent kinetic properties, are obtained from yeast poly(A)-containing RNA. Their electrophoretic mobility suggests molecular lengths of approximately 20, 40, and 60 nucleotides (Groner, G., Hynes, N., and Phillips, S. (1975) Biochemistry, 13, 5378-5383). Each of these poly(A) classes are present in the mRNA from large and small polyribosomes.

Adenine↗
PubMedDate not supplied

Interactions in the transcriptional regulation exerted by Stat5 and by members of the steroid hormone receptor family.

The pathways which connect extracellular signals with the regulation of the activity of transcription factors are being investigated in molecular detail. Extensive progress has been made in the description of the mode of action of steroid hormones and of cytokines. Steroid hormones associate intracellularly with latent receptor molecules, cause the dissociation of masking proteins, the dimerization of receptors, and their binding to specific hormone response elements in the promoters of target genes. Cytokines also activate latent transcription factors (Stats--signal transducers and activators of transcription), but act through an enzymatic mechanism. Tyrosine kinases associated with the transmembrane cytokine receptors phosphorylate Stat molecules. The phosphorylated monomers dimerize and assume specific DNA binding ability. Both classes of transcription factors bind to different response elements and regulate different target genes and both signals, cytokines and steroid hormones, can affect growth differentiation and homeostasis of different cell types. Here, we describe that Stat5, a molecule activated by several essential cytokines, functionally interacts with members of the steroid receptor family. We find that glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor synergize with Stat5 in the induction of the transcription from the beta-casein gene promoter. The estrogen receptor diminishes Stat5 mediated induction and the androgen receptor has no effect. Conversely, Stat5 negatively interferes with glucocorticoid receptor, mineralocorticoid receptor and progesterone receptor induced transcription from the MMTV LTR and the estrogen receptor induced transcription from an ERE-containing promoter.

Animals↗