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At least 325 records · Page 18Linked to original sources
PubMed1981

Identification of ceruloplasmin messenger RNA sequences in heterogeneous nuclear RNA from rat liver.

The distribution of the sequences of ceruloplasmin mRNA in different fractions of heterogeneous nuclear RNA from rat liver was studied using cDNA transcripts of highly purified mRNA as hybridization probe. The content of ceruloplasmin mRNA sequences in poly(A)-containing and poly(A)-free subfractions of heterogeneous nuclear RNA is respectively 1 and 27 molecules per a hepatocyte. Heterogeneous nuclear RNA carrying the sequences of ceruloplasmin mRNA sedimented in sucrose gradients containing formamide, as a broad zone around the 56S peak. Denaturing electrophoresis followed by the transfer of RNA onto diabenzyloxymethyl paper and hybridization with [32P]-cDNA revealed multiple high molecular weight fractions of ceruloplasmin pre = mRNA (9.0, 6.6, 2.2 and 1.6 megadaltons) in the non-adenylated fraction of nuclear RNA and a single 1.1-1.2 megadalton zone in poly(A)-containing nuclear RNA, the latter being equal in size to the mature ceruloplasmin mRNA from liver polysomes.

Animals↗
PubMed1994

Coupling between mRNA synthesis and mRNA stability in Escherichia coli.

Transiently stable products derived from the endonuclease cleavage of transcripts from the secEnusG and rplKAJLrpoBC operons have been identified. Cleavage sites for RNase III occur in the leader of the secEnusG transcript and in the L12-beta intercistronic space of the rplKAJLrpoBC transcript. A single RNase E cleavage site was located in the L1-L10 intergenic space. Inactivation of RNase III and RNase E results respectively in a one- to twofold and a greater than 10-fold stabilization of five mRNA sequences from within the secE, nusG, L11-L1, L10 and beta encoding cistrons. The relative amounts of each of these five mRNA sequences were found to be nearly constant when measured either in the presence or absence of cleavage by RNase III or RNase E. This clearly implies that any increases in the stability of these mRNA sequences resulting from the inactivation of processing by RNase III or RNAase E are counterbalanced by changes in the mRNA synthesis rates. The mechanism that links mRNA synthesis to mRNA decay is not known.

Bacterial Proteins↗
PubMed1975

Human globin gene expression and linkage in bone marrow and fetal liver.

During embryonic development there is a transition from embryonic and fetal to adult beta-type globin chains. The high-molecular-weight RNA found in nuclei from embryonic and adult human erythropoietic tissues, fetal liver, and bone marrow, have been investigated for the presence of gamma(fetal)- and beta(adult)-globin messenger RNA sequences by molecular hybridization. Unlike alpha- and beta-globin mRNA sequences, gamma-globin mRNA sequences are absent from both total and high-molecular-weight nuclear RNA isolated from adult bone marrow. The amount of cytoplasmic gamma-globin mRNA is proportional to the level of gamma-chain synthesis, demonstrating that translational control is not a major control mechanism in the expression of globin genes. Since the gamma-, delta-, and beta-globin genes are known to be closely linked genetically, transcriptional control can discriminate between similar gene sequences that are spatially adjacent to one another.

Bone Marrow↗
PubMed1991

Amplification and sequencing of mRNA encoding acidic fibroblast growth factor (aFGF) from porcine heart.

Progredient stenosis of coronary arteries can induce angiogenic processes, which are probably regulated by polypeptide growth factors like aFGF. Using applications of reverse transcription-polymerase chain reaction, we amplified and sequenced an mRNA encoding aFGF in the porcine myocardium. A DNA fragment of expected size encoding aFGF was amplified with human and bovine aFGF specific oligonucleotide primers in porcine heart. Identity of amplified PCR product to aFGF sequence was confirmed by internal reamplification, Southern hybridization and sequencing of asymmetrically amplified PCR products. The nucleotide sequence analysis of porcine aFGF revealed a homology of 94% to the human and 92% to the bovine cDNA sequences respectively. The amino acid sequence was homologous to the known sequences except for three alterations in the human and thirteen in the bovine aFGF sequences.

Amino Acid Sequence↗
PubMed2004

Translational regulation via 5' mRNA leader sequences revealed by mutational analysis of the Arabidopsis translation initiation factor subunit eIF3h.

Eukaryotic translation initiation factor 3 (eIF3) consists of core subunits that are conserved from yeast to man as well as less conserved, noncore, subunits with potential regulatory roles. Whereas core subunits tend to be indispensable for cell growth, the roles of the noncore subunits remain poorly understood. We addressed the hypothesis that eIF3 noncore subunits have accessory functions that help to regulate translation initiation, by focusing on the Arabidopsis thaliana eIF3h subunit. Indeed, eIF3h was not essential for general protein translation. However, results from transient expression assays and polysome fractionation indicated that the translation efficiency of specific 5' mRNA leader sequences was compromised in an eif3h mutant, including the mRNA for the basic domain leucine zipper (bZip) transcription factor ATB2/AtbZip11, translation of which is regulated by sucrose. Among other pleiotropic developmental defects, the eif3h mutant required exogenous sugar to transit from seedling to vegetative development, but it was hypersensitive to elevated levels of exogenous sugars. The ATB2 mRNA was rendered sensitive to the eIF3h level by a series of upstream open reading frames. Moreover, eIF3h could physically interact with subunits of the COP9 signalosome, a protein complex implicated primarily in the regulation of protein ubiquitination, supporting a direct biochemical connection between translation initiation and protein turnover. Together, these data implicate eIF3 in mRNA-associated translation initiation events, such as scanning, start codon recognition, or reinitiation and suggest that poor translation initiation of specific mRNAs contributes to the pleiotropic spectrum of phenotypic defects in the eif3h mutant.

5' Untranslated Regions↗
PubMed1999

Amino acid sequence of a putative transposase protein of the medaka fish transposable element Tol2 deduced from mRNA nucleotide sequences.

Tol2 is a terminal-inverted repeat transposable element of the medaka fish Oryzias latipes. It is one of a few elements of this class so far demonstrated to be active in vertebrates, thus providing a unique tool for establishing a gene tagging system. For the purpose of identifying its transposase, we analyzed the structures of mRNAs originating from the Tol2 element. The results indicated that transcription of Tol2 is initiated at several sites, the four open reading frames in Tol2 roughly corresponding to exons, and that two main forms of mRNAs, covering exons 1-4 and exons 2-4, are present in medaka fish cells. One or both of these mRNAs are likely to encode a transposase, the amino acid sequence of which was deduced.

Amino Acid Sequence↗
PubMed1977

Activation of histone gene transcription by nonhistone chromosomal proteins in WI-38 human diploid fibroblasts.

The regulation of histone gene expression was examined after confluent, nondividing WI-38 human diploid fibroblasts were stimulated to proliferate. Histone mRNA sequences were assayed by hybrid formation with an 3H-labeled single-stranded DNA complementary to histone mRNAs. Histone mRNA became associated with polyribosomes concomitant with the activation of DNA synthesis. The ability of chromatin from WI-38 cells to serve as a template for in vitro transcription of histone mRNA sequences parallels the onset of DNA replication. A role for nonhistone chromosomal proteins in the control of histone gene readout is suggested because, when chromatin from confluent WI-38 cells was dissociated and then reconstituted in the presence of S phase nonhistone chromosomal proteins, a 500-fold activation of histone mRNA sequence transcription was observed.

Cell Division↗
PubMed1981

Thyroid hormone attenuates and augments hepatic gene expression at a pretranslational level.

We have attempted to ascertain the proportion of the rat hepatic genome that is under the selective influence of thyroid hormones and to describe the response patterns of individual mRNA sequences in the transition between hypothyroidism and euthyroidism and between euthyroidism and hyperthyroidism. Poly(A)+RNA was extracted from livers of thyroidectomized, intact, euthyroid rats and of thyroidectomized rats rendered euthyroid and hyperthyroid with daily doses of triiodothyronine. The extracted RNA was translated in a reticulocyte lysate system in the presence of [35S]methionine, and the products were analyzed by two-dimensional gel electrophoresis. Triiodothyronine attenuates as well as augments the expression of certain genes at a pretranslational level. This could represent either a direct or an indirect action of the hormone. Triiodothyronine influences approximately 8% of the 231 mRNA sequences visualized, stimulating activity in 11 and inhibiting activity in 7 sequences. Translational activity of at least one mRNA sequence decreased in both thyroidectomized and hyperthyroid animals, compared to euthyroid levels. The relationship of mRNA response to receptor occupancy varied with examples of linear and amplified responses and responses that were maximal at less than full nuclear occupancy.

Animals↗
PubMed1987

Expression of the gene for the beta subunit of mouse thyrotropin results in multiple mRNAs differing in their 5'-untranslated regions.

The gene encoding the beta subunit of mouse thyrotropin (TSH beta) has been isolated from a mouse genomic library, and its nucleotide sequence has been determined. Blot hybridization analysis of restriction enzyme digests of mouse DNA indicates that there is a single mouse TSH beta gene. The gene is 4.8 kilobases in length and contains five exons, which are 27, 47, 41, 163, and 328 base pairs long. Exons 1, 2, and 3 encode only 5'-untranslated mRNA sequences and are separated by introns that are 150 and 380 base pairs long. The protein-coding mRNA sequences are found in exons 4 and 5 and are interrupted by a 460-base pair intron. The position of this intron, between the codons for amino acids 34 and 35, has been conserved in all the known glycoprotein hormone beta subunit genes. Exons 3 and 4 are separated by a large 3.2-kilobase intron. When primer extension analysis, using an oligonucleotide primer complementary to exon 4 sequences, was employed to locate the transcription start site, four products were obtained. Nucleotide sequencing of these products showed that they were derived from separate TSH beta mRNAs that differed in the lengths of their 5'-untranslated regions. These 5'-untranslated mRNA sequences are derived from different combinations of exons 1, 2, and 3, each spliced to exons 4 and 5. The longest 5'-untranslated sequence, 116 nucleotides long, includes exons 1, 2, and 3 and the first base of exon 4; the shorter 5'-untranslated regions, 75, 69, and 28 nucleotides long, arise by splicing out the second and/or the third exon sequences. In contrast to the mouse TSH beta gene, transcription of the rat TSH beta gene from the analogous start site has been reported to give only a single mRNA, with a 5'-untranslated region of 28 nucleotides. Divergence of the mouse and rat TSH beta gene sequences at RNA splice sites can account for the absence of exon 2, but not exon 3, sequences in rat TSH beta mRNA. Primer extension and RNase protection analyses also showed that the mouse TSH beta gene contains a second transcription start site, located 43 base pairs upstream of the first start site, in a position corresponding to that in the rat TSH beta gene. Each start site in the mouse gene is flanked by characteristic TATAA box and CAAT box sequences.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed2001

Characterization of the Crithidia fasciculata mRNA cycling sequence binding proteins.

The Crithidia fasciculata cycling sequence binding protein (CSBP) binds with high specificity to sequence elements in several mRNAs that accumulate periodically during the cell cycle. Mutations in these sequence elements abolish both cycling of the mRNA and binding of CSBP. Two genes, CSBPA and CSBPB, encoding putative subunits of CSBP have been cloned and were found to be present in tandem on the same DNA molecule and to be closely related. CSBPA and CSBPB are predicted to encode proteins with sizes of 35.6 and 42.0 kDa, respectively. Both CSBPA and CSBPB proteins have a predicted coiled-coil domain near the N terminus and a novel histidine and cysteine motif near the C terminus. The latter motif is conserved in other trypanosomatid species. Gel sieving chromatography and glycerol gradient sedimentation results indicate that CSBP has a molecular mass in excess of 200 kDa and an extended structure. Recombinant CSBPA and CSBPB also bind specifically to the cycling sequence and together can be reconstituted to give an RNA gel shift similar to that of purified CSBP. Proteins in cell extracts bind to an RNA probe containing six copies of the cycling sequence. The RNA-protein complexes contain both CSBPA and CSBPB, and the binding activity cycles in near synchrony with target mRNA levels. CSBPA and CSBPB mRNA and protein levels show little variation throughout the cell cycle, suggesting that additional factors are involved in the cyclic binding to the cycling sequence elements.

Amino Acid Sequence↗
PubMed1986

[Production of enzymes and enzyme inhibitors in neoplastic tissues].

The mechanisms of ectopic enzyme and enzyme inhibitor-production in neoplastic tissues were investigated. No evidence was obtained to suggest any difference in the structural genes of amylase-producing tumors and normal lymphocytes. mRNA sequence coding for amylase precursor in tumor tissues was identical to that of salivary amylase, suggesting that the amylase in amylase-producing tumors was identical to, or closely resembled the amylase in salivary gland. High incidence of elevation of serum pancreatic secretory trypsin inhibitor (PSTI) was observed in patients with various malignant tumors. PSTI-positive malignant cells were also frequently found in malignant tissues. A comparison of human PSTI mRNA sequence with mouse epidermal growth factor (EGF) mRNA sequence showed that they were 46% homologous. Human PSTI stimulated [3H]thymidine incorporation into DNA in human fibroblasts at concentrations present in human serum.

Amylases↗
PubMed1980

Physicochemical measurement of the base composition of mRNA-related sequences of the human alpha and beta globin genes.

Hybrids formed between human alpha and beta globin cDNA and total human cellular DNA have been studied by thermal denaturation and cesium chloride density gradient centrifugation. From these studies, the weight average G + C content of human alpha globin cDNA has been determined to be 62% +/- 2% and that of human beta globin cDNA 51% +/- 2%. These values correlate well with the results of G + C content of the human alpha and beta globin cDNAs as determined by direct nucleotide sequence analysis of the cDNAs. Thermal denaturation and cesium chloride density gradient centrifugation of DNA-cDNA hybrids can therefore provide accurate information on the base composition of mRNA related sequences of any single copy gene for which a relatively pure cDNA can be obtained, without the necessity for direct nucleotide sequence analysis.

Base Composition↗
PubMed2002

Transcription regulatory sequences and mRNA expression levels in the coronavirus transmissible gastroenteritis virus.

The transcription regulatory sequences (TRSs) of the coronavirus transmissible gastroenteritis virus (TGEV) have been characterized by using a helper virus-dependent expression system based on coronavirus-derived minigenomes to study the synthesis of subgenomic mRNAs. The TRSs are located at the 5' end of TGEV genes and include a highly conserved core sequence (CS), 5'-CUAAAC-3', that is essential for mediating a 100- to 1,000-fold increase in mRNA synthesis when it is located in the appropriate context. The relevant sequences contributing to TRS activity have been studied by extending the CS 5' upstream and 3' downstream. Sequences from virus genes flanking the CS influenced transcription levels from moderate (10- to 20-fold variation) to complete mRNA synthesis silencing, as shown for a canonical CS at nucleotide (nt) 120 from the initiation codon of the S gene that did not lead to the production of the corresponding mRNA. An optimized TRS has been designed comprising 88 nt from the N gene TRS, the CS, and 3 nt 3' to the M gene CS. Further extension of the 5'-flanking nucleotides (i.e., by 176 nt) decreased subgenomic RNA levels. The expression of a reporter gene (beta-glucuronidase) by using the selected TRS led to the production of 2 to 8 microg of protein per 10(6) cells. The presence of an appropriate Kozak context led to a higher level of protein expression. Virus protein levels were shown to be dependent on transcription and translation regulation.

3' Flanking Region↗
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↗
PubMed1999

Tissue-specific differences in the role of RNA 3' of the apolipoprotein B mRNA mooring sequence in editosome assembly.

Site-specific editing of apolipoprotein B (apoB) mRNA by the cytidine deaminase, APOBEC-1 is proposed to require interactions of auxiliary protein(s) with an eleven nucleotide element, the mooring sequence, located 3' of the C --> U editing site. An analysis of the RNA sequence dependence for protein-RNA interactions and editosome assembly in rat liver and the small intestine demonstrated that the mooring sequence was a minimal requirement for these interactions. Sequences 3' of the mooring sequence either interacted with 66 kDa and 44 kDa proteins or enhanced the interactions of these proteins with the mooring sequence. The data also suggested tissue-specific differences in the relative importance of the 3' cis-acting 'enhancer' elements in the efficiency or stability of editosome assembly. We propose that the previously demonstrated differences in apoB mRNA editing efficiency and its regulation in liver and intestine may in part be due to differences in auxiliary protein interactions with apoB mRNA 3' of the mooring sequence.

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↗
PubMed1981

Reduction of prostatic binding protein-messenger ribonucleic acid sequences in rat prostate by castration.

Messenger RNA coding for the three subunits of prostatic binding protein was isolated from polysomal RNA of rat ventral prostate by oligo (dT)-cellulose affinity chromatography and purified by repeated sedimentations through sucrose gradients under denaturing conditions. The purified mRNA migrated as a 9S peak in sucrose gradient centrifugation and hybridized with its cDNA within 2 log Rot units. In a cell-free reticulocyte lysate system, the mRNA directed the synthesis of three polypeptides of 12000, 9000, and 8000 daltons. These translation products were identified as the subunits of prostatic binding protein by immunoreaction with antibodies to this protein. Quantitation of prostatic binding protein-mRNA sequences in normal and castrated rats by hybridization with the cDNA probe showed that 3-day castration reduced the prostatic binding protein-mRNA sequences to less than 2% of the normal level. Similar hybridization was performed by using the cDNA to determine the level of prostatic binding protein coding sequences in polysomal poly(A) RNA following castration. The results showed a first-order rate constant of 3.92 X 10-2 h-1 for reduction of prostatic binding protein-mRNA sequences in polysomes. The period of castration required to reduce the level of these sequences to 50% of the normal level was calculated to be 17.6 h.

Androgen-Binding Protein↗
PubMed2004

Plasma gamma-globin gene expression suggests that fetal hematopoietic cells contribute to the pool of circulating cell-free fetal nucleic acids during pregnancy.

BACKGROUND: Reports of placental mRNA sequences in the plasma of pregnant women suggest that the placenta is the predominant source of cell-free fetal nucleic acids in maternal plasma during pregnancy. We developed an assay for gamma-globin mRNA concentrations to determine whether hematopoietic cells also contribute to the pool of fetal mRNA in maternal plasma. METHODS: Frozen paired plasma samples obtained from 40 women before and within 20 min after elective first-trimester termination of pregnancy (TOP) were analyzed. Fresh plasma samples from eight nonpregnant individuals were included as controls. Plasma gamma-globin mRNA was measured by use of real-time reverse transcription-PCR and analyzed with gestational age. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA was used to confirm the presence of cell-free RNA in each sample. RESULTS: gamma-Globin and GAPDH mRNA sequences were detected in every plasma sample. The concentrations of both messages were significantly increased in pregnancy (P <0.01). The concentrations of gamma-globin mRNA were decreased in most women after TOP, but gamma-globin mRNA was increased in some patients when TOP was performed later than 9 weeks of gestation. CONCLUSIONS: gamma-Globin mRNA sequences can be detected and measured in fresh and frozen plasma samples. Plasma gamma-globin and GAPDH mRNA concentrations are affected by pregnancy. The increased posttermination gamma-globin mRNA concentrations seen in some patients suggest that the source of this message is fetal hematopoietic cells. Further study in pregnant women after 9 weeks of gestation is necessary to evaluate the potential of gamma-globin mRNA as a marker for fetomaternal hemorrhage.

Female↗