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F M Rottman

Publications and source records attributed to F M Rottman.

At least 55 records · Page 3Linked to original sources
PubMed1984

Effect of undermethylation on mRNA cytoplasmic appearance and half-life.

S-Tubercidinylhomocysteine (STH) is a structural analog of S-adenosylhomocysteine and a potent inhibitor of S-adenosylmethionine-dependent methyltransferase reactions. We investigated the effects of STH on HeLa cell mRNA metabolism. Dual labeling studies reveal that STH dramatically inhibits the methylation of HeLa mRNA in a dose-dependent manner. Analysis of the modified nucleosides and 5'-terminal cap structures in radiolabeled mRNA by high-pressure liquid chromatography indicated that internal N6-methylation of adenosine was reduced by 65% at 50 microM STH and by 83% at 500 microM STH. The N6-methylation of adenosine contained in cap structures was similarly reduced at both concentrations of STH. Substantial amounts of cap structures lacking 2'-O-methylated nucleosides (m7GpppN, cap zero) were detected at the higher level of STH. To test the possibility that methylation affects mRNA stability, cytoplasmic mRNA half-life was measured in a pulse-chase experiment. The half-life of undermethylated mRNA, produced as a consequence of STH treatment, was unchanged compared with the control. To determine whether mRNA methylation is coupled to nuclear processing or transport, the time of cytoplasmic appearance of polyadenylated RNA in STH-treated HeLa cells was compared with untreated cells. STH caused a significant lag in the time of appearance of the polyadenylated RNA, suggesting that mRNA methylation may be required for efficient processing or transport.

Chromatography, High Pressure Liquid↗
PubMed1983

Characterization and nucleotide sequence of the gene for the common alpha subunit of the bovine pituitary glycoprotein hormones.

The gene coding for the common alpha subunit of the bovine pituitary glycoprotein hormones was isolated from a bovine genomic library. The gene spans roughly 16.5 kbp, contains three intervening sequences, and codes for a message of approximately 730 nucleotides. The complete coding region of the gene was sequenced as well as 315 nucleotides of 5' flanking sequence and the entire intron C. Only a single base difference was found when the sequence of the gene was compared with that of the cDNA. Genomic blotting experiments suggest the presence of a single alpha subunit gene. Comparison of the bovine and human alpha subunit genes indicated that the high level of homology observed in the coding regions has been maintained throughout the 5' and 3' untranslated regions, and at least 90 nucleotides of the 5'flanking regions. Additionally, there is an 18 base pair sequence present in both the 5' flanking and 5' untranslated regions of the gene that is homologous to a region of the chick ovalbumin gene. This ovalbumin sequence has been suggested as a binding site for the progesterone receptor-complex.

Amino Acid Sequence↗
PubMed1983

Developmental expression of growth hormone and prolactin genes in the bovine pituitary.

Cell-free translation was used to initially characterize the major mRNA species present in the bovine anterior pituitary as a function of development. The only detectable change in translation products, which occurred during the transition from fetus to adult, was a reversal in the relative ratio of pituitary growth hormone and prolactin. Subsequent hybridization analysis with cloned growth hormone and prolactin cDNA probes indicated that growth hormone mRNA comprised over 40% of the total fetal mRNA and was 50- to 100-fold higher than prolactin mRNA. The steady state levels of growth hormone mRNA remained relatively constant throughout gestation. In contrast, prolactin mRNA levels, which were low early in gestation, increased during development to become the principal mRNA in the adult pituitary. Since growth hormone and prolactin are synthesized and secreted by specialized cells (somatotrophs and mammotrophs, respectively) immunochemical staining was used to determine whether the changes in the mRNA levels for these two hormones were a reflection of specific cell proliferation. For growth hormone, there was a close correlation between the number of somatotrophs and the relative levels of growth hormone mRNA. In contrast, the increase in prolactin mRNA exceeded the increase in the number of mammotrophs. Thus, the cellular concentration of growth hormone mRNA remains relatively constant during development, while the cellular concentration of prolactin mRNA increases by more than an order of magnitude.

Aging↗
PubMed1982

Cloning and nucleotide sequencing of the bovine growth hormone gene.

A gene coding for bovine growth hormone was isolated from a bovine genomic library. The nucleotide sequence of the coding regions of the gene was found to be identical with that of a nearly full-length growth hormone cDNA clone. The gene sequence is approximately 1800 bp in length and contains four intervening sequences. The second intervening sequence of 227 nucleotides does not contain a repetitive element similar to that observed in the rat growth hormone gene. A comparison of the 5' and 3' flanking and untranslated regions of the bovine, human and rat growth hormone genes revealed many areas of highly conserved sequence. Especially noteworthy was the observation that all three genes had a 38 nucleotide homologous sequence within their 5' flanking regions located about 100 bp upstream from their transcription initiation sites.

Amino Acid Sequence↗
PubMed1982

Nucleotide sequence of bovine prolactin messenger RNA. Evidence for sequence polymorphism.

Hybrid molecules containing DNA sequences complementary to bovine pituitary mRNA were constructed in the Pst I site of pBR322 by the dC . dG tailing technique. Recombinant plasmids containing bovine prolactin (bPRL) sequences were amplified in bacteria and identified by hybridization to purified [32P]bPRL cDNA sequences. Nucleotide sequence analysis was performed on the inserts from two of the positive clones. One clone, pBPRL72, contained a 982-base pair insert that included 67 nucleotides of the 5'-untranslated region, the complete coding region of the preprolactin protein (690 nucleotides), and the entire 3'-untranslated region (150 nucleotides) of bPRL mRNA. The nucleotide sequence analysis of clone pBPRL72 predicted the sequence of a 30-amino acid signal peptide and confirmed the published amino acid sequence of the protein with one exception. A comparison of the pBPRL72 cDNA sequence with a second bPRL clone, pBPRL4, revealed four silent nucleotide differences. Three of the base changes occurred in the third position of amino acid codons, and one occurred in the 3'-noncoding region. The sequence polymorphism suggests the existence of alleles or multiple loci for bPRL that do not alter the protein structure.

Amino Acid Sequence↗
PubMed1982

Variation in the polyadenylylation site of bovine prolactin mRNA.

The poly(A) site of bovine prolactin (bPRL) mRNA was examined by phased priming of cDNA synthesis with oligodeoxynucleotides of the general sequence d(pT8-N-N'). The existence of multiple poly(A)-adjacent sequences in bPRL mRNA was indicated by the production of specific chain-termination fragments with at least three d(pT8-N-N') sequences. Comparison of the sequence bands produced by initiation of cDNA synthesis on the bPRL mRNA template with d(pT8-A-G), d(pT8-G-A), and d(pT8-C-G) revealed a shifted pattern of identical fragments. The shift in position of related sequence bands on the gel suggested that the difference in length of the three major bPRL mRNA species occurred within a span of 12 nucleotides. Sequence analysis conducted with the three d(pT8-N-N') primers gave identical nucleotide sequences for the 3' noncoding region of the bPRL mRNA species and suggested that the mRNA molecules were heterogeneous in length. The existence of multiple poly(A) sites was confirmed by determination of the nucleotide sequence of bPRL cDNA clones containing two of the major poly(A)-adjacent sequences predicted by the oligodeoxynucleotide primers. The mRNA molecules containing these multiple poly(A)-addition sites were shown to be present in the bPRL mRNA obtained from a single pituitary gland. The variation in the poly(A) junction of bPRL mRNA may be a reflection of the processing events at the 3' terminus of mRNAs.

Animals↗
PubMed1980

Ontogeny of pituitary hormone mRNAs in the bovine fetus. Quantitation of pre-prolactin mRNA as a function of gestation.

The mRNA coding for pre-prolactin (pPRL) of the adult bovine anterior pituitary was purified to 85% homogeneity and used as a template for cDNA synthesis with reverse transcriptase from avian myeloblastosis virus. The cDNA sequences complementary to pPRL mRNA were further purified by employing a limited back-hybridization step and treatment with S1 nuclease. The pPRL cDNA preparation was judged to be homogeneous by comparing its hybridization kinetics to those of ovalbumin and globin mRNA standards and by thermal melt analysis of the pPRL mRNA:cDNA hybrids. Total cellular RNA was extracted from individual bovine fetal pituitaries of either sex, ranging from 90 to 200 days of gestation, and examined for its pPRL mRNA concentration by hybridization with an excess of pPRL cDNA. The hybridization assay was capable of detecting picogram amounts of pPRL mRNA, e.g. amounts less than 0.002% of input total cellular RNA. These results indicated that from 90 to 200 days of gestation, the levels of pPRL mRNA relative to total cellular RNA in bovine fetal pituitaries increase exponentially. This increase in pPRL mRNA occurs to the same extent in either sex, with the most dramatic shift (over 10-fold) occurring between 120 and 145 days of gestation.

Animals↗
PubMed1980

Construction and characterization of a cDNA clone containing a portion of the bovine prolactin sequence.

Poly(A)-containing RNA from the bovine anterior pituitary has been used as a template for the enzymatic synthesis of double-stranded cDNA. The resulting double-stranded cDNA was inserted into the Pst I site of pBR322 with the oligo(dG)-oligo(dC) tailing technique and subsequently cloned in E. coli chi 1776. Clones containing sequences complementary to prolactin mRNA were identified by colony hybridization with partially purified prolactin cDNA. A 250 base pair sequence from one prolactin positive clone was extensively characterized and shown to contain the coding information for amino acids 119-192 of authentic bovine prolactin. The recombinant DNA from this clone was covalently attached to diazotized aminocellulose and used to purify prolactin mRNA from a mixture of mRNAs.

Animals↗
PubMed1979

Specific inhibition of capped mRNA translation in vitro by m7G5'pppp5'G and m7G5'pppp5'm7G.

A unique set of diguanosine cap analogues containing a 5'-5' tetraphosphate linkage instead of the normal triphosphate was synthesized by chemical methylation of G5'pppp5'G. Both 7-methylguanosine products, m7G5'pppp5'G and m7G5'pppp5'm7G, acted as potent inhibitors of capped brome mosaic virus (BMV) RNA translation in the homologous wheat germ protein synthesis system. Inhibition of in vitro protein synthesis required the presence of the 7-methyl group on guanosine and was specific for capped mRNA. In comparison with the partial cap analogue, m7GTP, the methylated diguanosine tetraphosphate structures were 25-50 fold more potent inhibitors of in vitro protein synthesis. Analysis of the in vitro translation products of the four species of BMV RNA showed a differential sensitivity to inhibition by m7G5'pppp5'm7G.

Methylation↗
PubMed1979

Purification of pre-prolactin mRNA from bovine anterior pituitary glands.

Total cytoplasmic RNA, isolated from bovine anterior pituitary glands, directed the synthesis of primarily two proteins in a wheat germ cell-free system. Analysis of wheat germ cell-free products by sodium dodecyl sulfate-slab gel electrophoresis, direct immunoprecipitation with prolactin aatibody, and prolactin- and growth hormone-specific radioimmunoassay indicated that the two proteins were pre-prolactin and pre-growth hormone. Agarose-urea-gel electrophoresis of poly(A)-containing RNA revealed the presence of a large symmetrical RNA peak with an estimated size of approximately 15 S. The RNA contained in this peak was eluted from the gel and translated in the wheat germ cell-free system. Analysis of the translation products by electrophoresis, direct immunoprecipitation, and radioimmunoassay resulted in the detection of only pre-prolactin. The extent of pre-prolactin mRNA purification was assessed by synthesizing DNA complementary to gel-purified pre-prolactin mRNA and then following the hybridization of this cDNA to gel purified pre-prolactin mRNA. The result of this hybridization is consistent with the cell-free translation studies, namely that pre-prolactin mRNA is the predominant mRNA component of pituitary poly(A)-containing RNA.

Animals↗
PubMed1977

RNA transport in isolated myeloma nuclei. Transport from membrane-denuded nuclei.

Nuclei prepared from MOPC-21 cells were treated with the nonionic detergents Triton X-100 or Nonidet P-40. Chemical analysis revealed that nearly 90% of the nuclear phospholipid was removed by detergent treatment. The membrane-denuded nuclei remained intact with preservation of nuclear pore complexes as demonstrated by electron microscopy. Ribonucleic acid transport from detergent-treated nuclei proceeded at the same rate and to the same extent as in control nuclei. Normal nuclear restriction of nucleic acids was unaltered by removal of the nuclear membranes. The effect of temperature on transport of RNA from freshly isolated myeloma nuclei with intact nuclear envelopes was studied. No temperature transition was associated with the transport process. These data indicate that the transport of macromolecules from isolated myeloma nuclei is independent of the nuclear membrane.

Adenosine Triphosphate↗
PubMed1976

The sensitivity of RNA polymerases I and II from Novikoff hepatoma (N1S1) cells to 3'-deoxyadenosine 5'-triphosphate.

The synthesis of ribosomal precursor RNA in Novikoff hepatoma (N1S1) cells is very sensitive to cordycepin (3'-dA). The synthesis of hnRNA, however, is resistant to inhibition concentrations of 3'-dA that completely block the synthesis of 45S ribosomal RNA precursor. We have examined the RNA polymerases present in these cultured cells with regard to their sensitivity to cordycepin 5'-triphosphate (3'-dATP) in an effort to explain the differential inhibition of RNA synthesis observed in vivo. RNA polymerases I and II were characterized on the basis of their chromatographic behavior on DEAE-Sephadex, as well as the response of their enzymatic activities to ionic strength, the divalent metal ions Mn2+ and Mg2+, and the toxin alpha-amanitin. For both enzymes the inhibition of in vitro RNA synthesis by 3'-dATP was competitive for ATP. The km values for ATP and the K1 values for 3'-dATP for the two enzymes were quite similar. RNA polymerase II, the enzyme presumed responsible for hnRNA synthesis, was actually slightly more sensitive to 3'-dATP than RNA polymerase I, the enzyme presumed responsible for ribosomal precursor RNA synthesis. Similar data were obtained when the RNA polymerases were assayed in isolated nuclei. These results indicate that the differential inhibition of RNA synthesis caused by 3'-dA in vivo cannot be simply explained by differential sensitivity of RNA polymerases I and II to 3'-dATP.

Adenine Nucleotides↗
PubMed1976

Methylation of high-molecular-weight subunit RNA of feline leukemia virus.

The high-molecular-weight subunit RNA of feline leukemia virus (Rickard strain) (FeLV-R) was analyzed for the presence of methyl groups. After purification of native 50-60S FeLV-R RNA on nondenaturing aqueous sucrose density gradients. FeLV-R 28S subunit RNA, doubly labeled with [14C]uridine and [methyl-3H]methionine, was isolated by centrifugation through denaturing sucrose density gradients in dimethyl sulfoxide. As calculated from their respective 3H/14C ratios. FeLV-R 28S RNA was methylated to the same degree as host cell poly(A)+ mRNA. When the 28S FeLV-R RNA was hydrolyzed to completion with RNase T2 or alkali, all of the methyl-3H chromatographed with mononucleotides on Pellionex-WAX, a weak anion exchanger. The methyl-labeled material co-chromatographed with 6-methyladenosine if the mononucleotide fraction obtained by Pellionex-WAX chromatography was hydrolyzed to nucleosides by bacterial alkaline phosphatase or with 6-methyladenine if purine bases were released from the mononucleotides by acid hydrolysis. In another experiment in which FeLV-R 28S RNA uniformly labeled with 32P was hydrolyzed and then analyzed by Pellionex-WAX chromatography, all of the 32P label again co-chromatographed with mononucleotides. Thus FeLV-R 28S RNA does not appear to contain a 5' structure, either methylated or nonmethylated similar to those recently reported for cellular and some animal virus mRNA's.

Adenine↗
PubMed1975

Characterization of Novikoff hepatoma mRNA methylation and heterogeneity in the methylated 5' terminus.

KOH digestion of methyl-labeled poly(A)+ mRNA purified by (dT)-cellulose chromatography produced mononucleotide and multiple peaks of a large oligonucleotide (-6 to -8 charge) when separated on the basis of charge by Pellionex-WAX high-speed liquid chromatography in 7 M urea. Heat denaturation of the RNA before application to (dT)-cellulose was required to release contaminants (mostly 18S rRNA) that persisted even after repeated binding to (dT)-cellulose at room temperature. Analysis of the purified poly(A)+ mRNA by enzyme digestion, acid hydrolysis, and a variety of chromatographic techniques has shown that the monucleotide (53%) is due entirely to N6-methyladenosine. The large oligonucleotides (47%) were found to contain 7-methylguanosine and the 2'-0-methyl derivatives of all four nucleosides. No radioactivity was found associated with the poly(A) segment. Periodate oxidation of the mRNA followed by beta elimination released only labeled 7-methylguanine consistent with a blocked 5' terminus containing an unusual 5'-5' bond. Alkaline phosphatase treatment of intact mRNA had no effect on the migration of the KOH produced oligonucleotides on Pellionex-WAX. When RNA from which 7-methylguanine was removed by beta elimination was used for the phosphatase treatment, distinct dinucleotides (NmpNp) and trinucleotides (NmpNmpNp) occurred after KOH hydrolysis and Pellionex-WAX chromatography. Thus Novikoff hepatoma poly(A)+ mRNA molecules can contain either one or two 2'-0-methylnucleotides linked by a 5'-5' bond to a terminal 7-methylguanosine and the 2'-0-methylation can occur with any of the four nucleotides. The 5' terminus may be represented by m7G5'ppp5' (Nmp)lor2Np, a general structure proposed earlier as a possible 5' terminus for all eucaryotic mRNA molecules (Rottman, F., Shatkin, A., and Perry, R. (1974), Cell 3, 197). The composition analyses indicate that there are 3.0 N6-methyladenosine residues, 1.0 7-methylguanosine residue, and 1.7 2'-0-methylnucleoside residues per average mRNA molecule.

Animals↗