PubMed Health⌕ Search

Biomedical subjects

F Rottman

Publications and source records attributed to F Rottman.

At least 19 recordsLinked to original sources
PubMed1996

Alternative splicing of exon 3 of the human growth hormone receptor is the result of an unusual genetic polymorphism.

Two isoforms of the human growth hormone receptor (hGHR), which differ in the presence (hGHRwt) or absence (hGHRd3) of exon 3, are expressed in the placenta. Specifically, three expression patterns are observed: only hGHRwt, only hGHRd3, or an approximately 1:1 combination of both isoforms. We investigated several potential regulatory mechanisms which might account for the expression of the hGHR isoforms. The frequency of hGHRd3 expression did not change when placentas from differing stages of gestation were examined, suggesting splicing was not developmentally regulated. However, when hGHR isoform expression patterns were examined in each component of a given placenta, it was evident that alternative splicing of exon 3 is individual-specific. Surprisingly, the individual-specific regulation of hGHR isoforms appears to be the result of a polymorphism in the hGHR gene. We analyzed hGHRwt and hGHRd3 expression in Hutterite pedigrees, and our results are consistent with a simple Mendelian inheritance of two differing alleles in which exon 3 is spliced in an "all-or-none" fashion. We conclude the alternative splicing of exon 3 in hGHR transcripts is the result of an unusual polymorphism which significantly alters splicing of the hGHR transcript and that the relatively high frequency (approximately 10%) of homozygous hGHRd3 expression suggests the possibility it may play a role in polygenic determined events.

Alleles↗
PubMed1995

Accurate and efficient N-6-adenosine methylation in spliceosomal U6 small nuclear RNA by HeLa cell extract in vitro.

Human U6 small nuclear RNA (U6 snRNA), an abundant snRNA required for splicing of pre-mRNAs, contains several post-transcriptional modifications including a single m6A (N-6-methyladenosine) at position 43. This A-43 residue is critical for the function of U6 snRNA in splicing of pre-mRNAs. Yeast and plant U6 snRNAs also contain m6A in the corresponding position showing that this modification is evolutionarily conserved. In this study, we show that upon incubation of an unmodified U6 RNA with HeLa cell extract, A-43 residue in human U6 snRNA was rapidly converted to m6A-43. This conversion was detectable as early as 3 min after incubation and was nearly complete in 60 min; no other A residue in U6 snRNA was converted to m6A. Deletion studies showed that the stem-loop structure near the 5' end of U6 snRNA is dispensable for m6A formation; however, the integrity of the 3' stem-loop was necessary for efficient m6A formation. These data show that a short stretch of primary sequence flanking the methylation site is not sufficient for U6 m6A methyltransferase recognition and the enzyme probably recognizes secondary and/or tertiary structural features in U6 snRNA. The enzyme that catalyzes m6A formation in U6 snRNA appears to be distinct from the prolactin mRNA methyltransferase which is also present in HeLa nuclear extracts.

Adenosine↗
PubMed1994

Characterization and partial purification of mRNA N6-adenosine methyltransferase from HeLa cell nuclei. Internal mRNA methylation requires a multisubunit complex.

N6-Methyladenosine is found at internal positions of mRNA in higher eukaryotes. This post-transcriptional modification occurs at a frequency of one to three methylation/average mRNA molecule in mammalian cell lines and is sequence-specific. A highly conserved consensus recognition site for the methyltransferase has been determined from both viral and cellular messages, consisting of the sequence Pu(G/A)AC(U/A) (with A being methylated). Despite the ubiquity and the specificity of this modification, little is known about the mechanism of formation of N6-methyladenosine. Utilizing an in vitro methylation system from HeLa cell nuclear extracts, and a substrate RNA derived from the mRNA coding for bovine prolactin, the mRNA N6-adenosine methyltransferase has been characterized and partially purified. Unique among other characterized nucleic acid methyltransferases, the enzyme is composed of three components which are separable under non-denaturing conditions. The molecular masses of the components are 30, 200, and 875 kDa as determined by gel filtration and glycerol gradient sedimentation. The 200-kDa component appears to contain the S-adenosylmethionine-binding site on a 70-kDa subunit. The 875-kDa component has affinity for single-stranded DNA-agarose, suggesting that it may contain the mRNA-binding site. N6-Adenosine methyltransferase is not sensitive to treatment with micrococcal nuclease, nor to immunodepletion using an anti-trimethylguanosine antibody, suggesting that it does not contain an essential RNA component.

Animals↗
PubMed1986

Inhibition of vaccinia RNA guanine 7-methyltransferase by compounds designed as multisubstrate adducts.

Several potential inhibitors of mRNA guanine 7-methyltransferase, which were designed from mechanism-based considerations, were evaluated against the vaccinia virus capping enzyme complex. Of the compounds tested, 5'-deoxy-5'[6-(2-aminopyrrolo[2,3-d]-pyrimidine-4-one) methylthio]adenosine (9) had good selective inhibitory activity against vaccinia mRNA guanine 7-methyltransferase, exhibiting an IC50 of 9.2 X 10(-5) M. Structure-activity considerations suggest that specific inhibition of RNA methyltransferases by low molecular weight multisubstrate adduct inhibitors may be achievable.

Chemical Phenomena↗
PubMed1986

Isolation of cDNA clones for differentially expressed genes of the human parasite Schistosoma mansoni.

Little is known about the mechanisms that control transformations during the life cycle of Schistosoma mansoni. To enable isolation of DNA sequences encoding developmentally regulated antigens a cDNA expression library in the vector lambda gt11 amp3 was constructed from adult mRNA and immunologically screened with sera from infected individuals. We report here on the properties of three recombinant clones that derive from developmentally regulated genes. Clone 10-3 encoded a beta-galactosidase fusion protein present in high abundance in infected Escherichia coli. Clones 7-2 and 8-2 also produced immunologically recognized proteins; however, the peptides did not appear to be beta-galactosidase fusion proteins. The expression of mRNAs hybridizing to these cDNAs was examined in the different stages of the parasite life cycle. Messenger RNA corresponding to clone 10-3, approximately equal to 1000 bases in length, was present in higher abundance in male worms than in females but was not detected in schistosome eggs. A 900-base mRNA hybridizing to clone 7-2 was observed in adult worms and eggs. Both clone 10-3 and clone 7-2 hybridized to smaller mRNAs in cercariae and freshly transformed schistosomula than in adult worms. Clone 8-2 contained tandem cDNA inserts. One cDNA hybridized to a 1700-base mRNA present in all stages, while the second hybridized to an 800-base mRNA specific to adult female worms.

Animals↗
PubMed1986

Stage and species specificity of antigens encoded by two geographic strains of Schistosoma mansoni mRNA.

Proteins translated in vitro from Schistosoma mansoni adult worm mRNA were assessed for their antigenic specificities compared to different stages, strains and species of the parasite. RNA was extracted from both Puerto Rican and Brazilian parasites and directed the synthesis of high molecular weight proteins. Preabsorption of immune human serum with schistosomula was used to determine whether the in vitro translated proteins contained antigens shared between the adult and this immature stage. Three antigens (Mr 36,000, 29,000, 18,000) were observed to be present in both stages. When adult worm mRNA from 2 different geographic strains of S. mansoni (Puerto Rican and Brazilian) were compared, certain antigenic differences were found in their in vitro translation products (proteins at Mr 78,000, 26,000, 24,000, 22,000, 15,500), suggesting that different antigenic pools may exist in nature. The species specificity of the in vitro proteins was assessed using individual sera from humans whose species of schistosome infection and egg counts were known. Immunoprecipitation with these sera demonstrated that a large number of immunologically cross-reactive proteins were shared between S. mansoni and Schistosoma haematobium but not Schistosoma japonicum. Antigens or antigen complexes at Mr 47,000 and 37,000 were detected only in the immunoprecipitations using anti-S. mansoni sera, whereas an antigen of Mr 39,000 was precipitated only by anti-S. haematobium sera. The recognition of any 1 antigen or group of antigens, however, did not distinguish between intensities of infection.

Animals↗
PubMed1980

Use of oligodeoxynucleotide primers to determine poly(adenylic acid) adjacent sequences in messenger ribonucleic acid. 3'-Terminal noncoding sequence of bovine growth hormone messenger ribonucleic acid.

Twelve synthetic oligodeoxynucleotide primers of the general sequence d(pT8-N-N') were tested in a reverse transcriptase reaction for specific initiation of complementary deoxyribonucleic acid (cDNA) synthesis at the poly(adenylic acid) junction of a messenger ribonucleic acid (mRNA) template. Only the sequence d(pT8-G-C) functioned as a specific primer of cDNA synthesis with an enriched fraction of bovine growth hormone mRNA from the anterior pituitary gland and produced unique fragments in a dideoxy sequencing reaction. The nucleotide sequence obtained by this method extended into the protein coding region of bovine growth hormone mRNA and was confirmed by chemical sequencing of the cDNA initiated with [5'-32P]d(pT8-G-C). The 3'-untranslated region of bovine growth hormone mRNA is 104 nucleotides in length and contains regions of significant homology with both rat and human growth hormone mRNAs, including the region surrounding the common AAUAAA hexanucleotide. The method presented here for selection of the d(pT8-N-N') primer complementary to the poly(A) junction of mRNA is of general applicability for nucleotide sequence analysis of partially purified mRNAs.

Animals↗
PubMed1979

Cytoplasmic location of undermethylated messenger RNA in Novikoff cells.

Novikoff cells in culture were labeled with L-[methyl-3H]methionine and [U-14C]uridine in the presence of (a) TubHcy2, (b) AdoHcy, (c) homocysteine, (d) tubercidin, or (e) without any additions. Only in cultures labeled in the presence of TubHcy were undermethylated cap structures observed to represent a significant portion of [3H]methyl radioactivity. Novikoff cells in culture were then simultaneously labeled with L-[methyl-3H]methionine and [32P]orthophosphate in the presence or absence of TubHcy. Total cytoplasmic, polysomal and monosomal poly(A)-containing RNAs were analyzed. Both monosomal and polysomal mRNA fractions from TubHcy-treated cells contain partially methylated cap structures, suggesting that 2'-O-methylation of the nucleoside adjacent to the pyrophosphate linkage in caps is not required for transport, ribosomal binding or translation. Comparison of nuclear and cytoplasmic cap structures from normal and inhibited cultures indicate that an altered mRNA population is generated in the presence of TubHcy.

Animals↗
PubMed1978

Presence of 5'-terminal cap structures in virus-specific RNA from feline leukemia virus-infected cells.

The F-422 line of feline thymus tumor cells, chronically infected with the Rickard strain of feline leukemia virus (R-FeLV), was labeled with 32P, and the total cytoplasmic RNA was isolated. The RNA was centrifuged through sucrose gradients, and R-FeLV virus-specific RNA (vRNA) was located by hybridization of portions of the gradient fractions to R-FeLV complementary DNA. vRNA classes with average sedimentation coefficients of approximately 36S, 28S, 23S, and 15S were identified. Each class of RNA was recovered by hybridized with mercurated R-FeLV complementary DNA, and the hybrids were chromatographed on columns of sulfhydryl-Sepharose to separate them from unhybridized cellular RNA. Although insufficient amount of 36S and 28S vRNA were obtained for further analysis, the 23S and 15S VRNA classes were analyzed to determine the nature of their 5' termini. Each of these vRNA classes was found to contain stoichiometric amounts of cap structures per unit length of RNA, consistent with the presence of one cap per molecule. The structure of the 23S vRNA cap was found to be m7G5'ppp5'GmpAp, whereas that of the 15S vRNA cap was m7G5'ppp5'GmpGp.

Cell Line↗
PubMed1977

In vivo inhibition of Novikoff cytoplasmic messenger RNA methylation by S-tubercidinylhomocysteine.

The analogue S-tubercidinylhomocysteine (STH) has been used to study the methylation of mRNA in vivo. Partial inhibition of cytoplasmic poly(A)-RNA methylation was observed using a level of inhibitor which still permitted cell growth. Characterization of the partially methylated mRNA indicated the presence of cap structures lacking 2'-O-methylnucleosides, m7GpppN', which are normally not found in mammalian mRNA. Inhibition of additional methylated sites in mRNA at the second 2'-O-methynucleoside, and at internal N6-methyladenosine was also observed Methylation of 7-methylguanosine was not affected under the conditions used in these experiments. The methylnucleoside composition of cap structures differed in STH-inhibited and uninhibited cells. These results indicate that a completely methylated cap is not required for transport of mRNA into the cytoplasm. Furthermore, it may now be possible to assess in vivo the sequential nature of mRNA methylation and its potential role in mRNA processing.

Carcinoma, Hepatocellular↗
PubMed1977

Enzymatic synthesis of oligonucleotides of defined sequence: synthesis of a segment of yeast iso-1-cytochrome c gene.

The deoxyribooligonucleotide, d(pT-T-A-G-C-A-G-A-A-C-C-G-G), constituting a segment of yeast iso-1-cytochrome c gene, has been synthesized by a combination of chemical and primarily enzymatic methods. The starting primer, d(pT-T-A-G1, was chemically synthesized by the phosphodiester method and was extended stepwise, by reactions catalyzed by polynucleotide phosphorylase.

Cytochrome c Group↗
PubMed1976

Kinetics of Novikoff cytoplasmic messenger RNA methylation.

Methylation patterns of Novikoff cytoplasmic mRNA were determined as a function of labeling time with L-[methyl-3H]methionine. The 5'-terminal m7G could be released from whole mRNA by treatment with nucleotide pyrophosphatase. Subsequent alkaline phosphatase treatment of this mRNA, followed by KOH digestion, yielded N'mpNp and N'mpNp from cap 1 (m7GpppN'mpN) and cap 2 (m7GpppN'mpN''mpN), respectively. Our results indicate that the relative amounts of labeled cap structures do change with time and that the amount of internal N6-methyladenosine decreases, relative to 5'-cap structures, as the cytoplasmic mRNAs age and the average size decreases. The formation of cap-2 structures by the addition of second 2'-O-methyl group at position N''m appears to be cytoplasmic event. Thus, after very short labeling times, greater than 80% of the labeled methyl groups in cap 2 are found in this position. These results, along with earlier data obtained on L-cell heterogeneous nuclear RNA methylation, are consistent with a model in which the nucleus is the cellular site of three mRNA methylation events producing 5'-terminal m7G, the first 2'-O-methylnucleoside (N'm) found in cap-1 structures and internal N6-methyladenosine. Subsequently, these nuclear methylations are followed by the cytoplasmic methylation at N''m. Analysis of the methynucleoside composition of cap-1 structures, along with comparable "core" structures (m7GpppN'm) generated from cap-2 by removal of N''m, indicates that at any single labeling time the methylnucleoside composition of a given cap-1 and the cap-2 "core" structure is remarkably similar. On the other hand, comparisons of the methylnucleoside composition of the cap structures at different labeling times indicate an increase in Cm in the first 2'-O-methylnucleoside (N'm) with time.

Carcinoma, Hepatocellular↗
PubMed1975

The methylated constituents of L cell messenger RNA: evidence for an unusual cluster at the 5' terminus.

An analysis of the methylated constituents of L cell mRNA by a combination of chromatographic methods and enzymatic treatments indicates that they comprise both 2'-O-methyl nucleosides and N6-methyl adenine, and/or 1-methyl adenine, and suggests that the 2'-O-methyl nucleotides, Ym, are part of an unusual class of sequences forming the 5' terminus of mRNA. These sequences seem to contain two 2'-O-methyl residues and a terminal residue that is not phosphorylated but, nevertheless, is blocked with respect to polynucleotid kinase reactivity. A strong candidate is a sequence of the type XppY1mpY2mpZp..., where X represents a blocking group which is itself occasionally methylated. The sequences isolated from total poly(A)+ mRNA contain all four species of 2'-O-methylated nucleoside, indicating some variability among different mRNA species. The methylated sequences do not appear to be enriched in the mRNA which hybridizes with repetitive DNA. The average L cell mRNA molecule also contains three residues of N6-methyl adenine. These residues are not part of the poly(A) segment, but appear to be located internal to the poly(A) near the 3' end of the mRNA molecules.

Adenine↗