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PubMed1981

Calcium specifically stimulates prolactin synthesis and messenger RNA sequences in GH3 cells.

Addition of CaCl2 (0.4 mM) to GH3 cells incubated in a serum-free, Ca2+-free medium increased relative prolactin synthesis (prolactin synthesis/total protein synthesis) 7- to 150-fold by 3-4 days, but had no effect on relative growth hormone synthesis. Half-maximal and maximal stimulation of relative prolactin synthesis occurred at about 0.07 and 0.2 mM CaCl2, respectively. Neither MgCl2 (0.4 mM) nor KCl 0.4 mM) affected relative prolactin synthesis. 32P-labeled rat prolactin and growth hormone cDNA plasmids were employed to measure by RNA gel blot hybridization changes in cytoplasmic levels of the corresponding mRNA sequences. Both prolactin mRNA sequence levels and translatable prolactin mRNA increased in parallel with the CaCl2-induced increase in relative prolactin synthesis. CaCl2 increased prolactin mRNA sequences by 7- to 200-fold, without affecting growth hormone mRNA sequence levels. Dexamethasone (100 nM) decreased prolactin mRNA sequences in cells incubated with CaCl2, antagonized the CaCl2-induced increases in prolactin mRNA sequences independently of the CaCl2 concentration.

Animals↗

PubMed1997

Latent sequence periodicity of some oncogenes and DNA-binding protein genes.

A method of latent periodicity search is developed. We use mutual information to reveal the latent periodicity of mRNA sequences. The latent periodicity of an mRNA sequence is a periodicity with a low level of similarity between any two periods inside the mRNA sequence. The mutual information between an artificial numerical sequence and an mRNA sequence is calculated. The length of the artificial sequence period is varied from 2 to 150. The high level of the mutual information between artificial and mRNA sequences allows us to find any type of latent periodicity of mRNA sequence. The latent periodicity of many mRNA coding regions has been found. For example, the retinoblastoma gene of HSRBS clone contains a region with a latent period equal to 45 bases. The A-RAF oncogene of HSARAFIR clone contains a region with a latent period equal to 84 bases. Integrated sequences for the regions with latent periodicity are determined. The potential significance of latent periodicity is discussed.

Animals↗

PubMed1997

Enhancement of recombinant glucoamylase expression by introducing yeast GAL7 mRNA termination sequence.

Glucoamylase gene (STA1) of Saccharomyces diastaticus was expressed in recombinant Saccharomyces cerevisiae systems. The yeast, GAL7 mRNA termination sequence, was introduced in the 3' noncoding region of the STA1 structural gene which was under the control of the SUC2 promoter and STA1 secretion signal sequence. This plasmid was named YEpSSG7 and was introduced into yeast S. cerevisiae MMY2 to construct recombinant S. cerevisiae MMY2SSG7. The GAL7 mRNA termination sequence enhanced the glucoamylase expression level by 3-5 times depending on the culture conditions compared to the result from the strain S. cerevisiae MMY2SUCSTA which did not contain the GAL7 mRNA termination sequence. Such an enhancement was not due to plasmid stability or plasmid copy number effects. Such an enhancement was primarily due to the fact that GAL7 mRNA termination sequence stabilized the STA1 mRNA 3' end.

Deoxyribonuclease EcoRI↗

PubMed1987

Porcine D-amino acid oxidase: determination of the mRNA nucleotide sequence by the characterization of genomic and cDNA clones.

Oligodeoxynucleotide probes derived from the published amino acid (aa) sequence for D-amino acid oxidase (DAO) [Ronchi et al. J. Biol. Chem. 259 (1982) 8824-8834] were used to screen cDNA libraries made from porcine kidney cortex and liver. Whereas no clones were obtained from kidney mRNAs, 20 independent ones were isolated from the liver library. Surprisingly, all of them carried only partial cDNAs for DAO starting around aa 100 in the coding sequence and extending for up to 250 bp in the 3'-noncoding sequence. One of these clones, pULB9103, was used to screen a porcine genomic library and allowed the isolation of DAO gene clone phULB001. Four exons encoding aa 1-151 were identified and sequenced, as well as the relevant exon-intron junctions. The mRNA sequence coding for DAO has been reconstituted from the genomic and cDNA sequences; its analysis by computer did not reveal any significant secondary structure, or particular feature, which could explain the failure to obtain full-length cDNAs.

Amino Acid Sequence↗

PubMed1987

Syntactic pattern analysis of 5'-splice site sequences of mRNA precursors in higher eukaryote genes.

The signals which direct the excision of introns from eukaryotic pre-mRNA are not yet well understood. In order to define the signals for 5'-splice sites of mRNA splicing, nucleotide sequences including 5'-splice junctions of mammalian pre-mRNAs are analysed by means of syntactic pattern analysis. Taking this approach, we infer the grammatical rules which specify 5'-splice sites and construct a finite automaton which is the recognizer of the nucleotide sequences at 5'-splice sites. By scanning the automaton along nucleotide sequences, we can identify the positions of 5'-splice junctions with a degree of discrimination of up to 94-97% in the known genes, while the degree of prediction is in the range 50-55% in new genes.

Algorithms↗

PubMed1993

Rapid identification of beta-globin structural mutations by sequencing the mRNA from peripheral blood reticulocytes.

A simple and rapid method for the molecular detection of beta-globin structural mutations is described using a reverse transcription-polymerase chain reaction of reticulocyte mRNA and direct sequencing of the product. The amplified segment (employing a sense primer 5'-ATTTGCTTCTGACACAACTGT-3', located at position + 1 with respect to the Cap site and an antisense primer 5'-TCCAGATGCTCAAGGCCCTTC-3', located at position + 1772 with respect to the Cap site) encompasses the cDNA sequence including the three globin exons. Employing this method we were able to characterize two hemoglobin structural variants: Hb S (beta 6 (A3) Glu-Val: GAG-GTG) and Hb Porto Alegre (beta 9 (A6) Ser-Cys: TCT-TGT). The approach described in this paper should be very useful to detect hemoglobin structural variants because the RNA extraction is simple, rapid and does not require cesium chloride, guanidinium and proteinase K. In addition, the direct sequencing of the RT-PCR product permits the screening of the entire globin genes with only two reactions.

Base Sequence↗

PubMed1976

Role of nonhistone chromosomal proteins in the regulation of histone gene expression.

Histone gene expression was studied during the cell cycle of continuously dividing HeLa S3-cells and following stimulation of confluent monolayers of WI-38 human diploid fibroblasts to proliferate. The presence of histone messenger RNA (mRNA) sequences was assayed by hybridization to a 3H-labeled single-stranded DNA complementary to histone mRNA's. In HeLa S3-cells, histone mRNA sequences were found in the nucleus and associated with polyribosomes during S phase but not during G1. Transcripts of S-phase chromatin contained histone mRNA sequences but those of G1 chromatin did not. Similarly, in WI-38 cells association of histone mRNA sequences with polyribosomes and transcription of histone mRNA sequences from chromatin parallel DNA replication. Together these results suggest that the regulation of histone gene expression resides, at least in part, at the transcriptional level. Chromatin reconstitution studies provide evidence that nonhistone chromosomal proteins play a key role in activation of histone gene transcription during the period of the cell cycle when DNA is replicated. Phosphate groups associated with the S-phase nonhistone chromosomal proteins appear to be functionally involved in the control of histone gene readout. Although WI-38 human diploid fibroblasts transformed by SV40 exhibit morphological and biochemical modifications characteristic of neoplastic cells, transcription of histone mRNA sequences remains unaltered.

Base Sequence↗

PubMed1979

Evidence that the RNA polymerase usually does not make a complete transcript of the late strand of simian virus 40 DNA.

An important model for the transcription of the late (L) strand of simian virus 40 DNA is that, late after infection of permissive monkey cells, the RNA polymerase makes a complete transcript of the L DNA strand before terminating transcription. The purpose of the current work was to test a prediction of this model, namely, that the rate of synthesis of all RNA sequences from the L DNA strand should be equal. About one-half of the L DNA strand is transcribed into late mRNA sequences and the other half into late dRNA sequences, which do not leave the nucleus. Using glucosamine to reduce the size of the intracellular UTP pool before and after a pulse-label with radioactive uridine, a pulse-chase experiment was performed to determine the half-lives of these sequences. The half-life of the late dRNA sequences was determined to be 4 min. The late mRNA sequences were degraded more slowly, on the average, than the late dRNA sequences. In a parallel experiment with similarly treated cells, it was shown that after a 2-min label with radioactive uridine there was only 0.2 times as much radioactivity in the late dRNA sequences as in the late mRNA sequences in the total cellular RNA population. The results could be combined to calculate that the rate of synthesis of the late dRNA sequences was at most 0.3 times that of the late mRNA sequences. Consequently they provide strong evidence that when the RNA polymerase transcribes the late mRNA sequences, it usually terminates transcription before all the late dRNA sequences are transcribed.

Base Sequence↗

PubMed1985

Complete nucleotide sequence of mRNA for caerulein precursor from Xenopus skin: the mRNA contains an unusual repetitive structure.

The complete nucleotide sequence of mRNA for caerulein precursor in the skin of Xenopus laevis was determined. The sequence was composed of 705 bp of coding region, accounting for 234 amino acids, 58 bp of 5'-untranslated region and 158 bp of 3'-untranslated region containing two putative poly(A) signals. It coded for four caerulein peptides interspersed with three 147 bp segments (intercaerulein segment; ICS). Analyses of several caerulein encoding cDNAs revealed some interesting features of caerulein mRNA species, which were highly heterogeneous and consisted of a repetition of two fundamental RNA sequences, a 45-nucleotide caerulein fragment and a 147-nucleotide ICS. The result of Northern blotting indicated that caerulein mRNA was only present in frog skin, not in stomach, upper intestine or liver. It appears that caerulein has different physiological function(s) from mammalian gastrin and cholecystokinin-pancreozymin (CCK). The relationship of caerulein to mammalian gastrointestinal hormones is discussed.

Amino Acid Sequence↗

PubMed1991

The protein-coding region of c-myc mRNA contains a sequence that specifies rapid mRNA turnover and induction by protein synthesis inhibitors.

The stability of certain mRNAs is known to be affected by translation. Some mRNAs appear to be protected from rapid degradation by translation, whereas degradation is coupled to translation for other mRNAs. The molecular determinants of this selective effect of translation are unknown. One example of this effect is the induction of early-response gene mRNAs in the presence of translation inhibitors. To define the molecular basis of induction of early-response gene mRNA expression by inhibitors of protein synthesis, we have performed a mutational analysis of one member of the early response gene family, the c-myc gene. We find that induction by cycloheximide is due to stabilization of c-myc transcripts. The requirements for increased expression of c-myc mRNA by cycloheximide are the presence of the sequence encoding c-myc amino acids 335-439 on a mRNA that can be translated; all other portions of the c-myc gene are dispensable, and this sequence can confer induction of mRNA expression by protein synthesis inhibitors on a heterologous gene. By direct measurement of mRNA turnover in the absence of transcription-blocking drugs, we show that this sequence can function as a selective mRNA destabilizing element, that turnover mediated by this element is translation dependent, and turnover mediated by this element is inhibited by actinomycin D. Our results support the hypothesis that degradation of c-myc mRNA is coupled to translation, that the sequences specifying this form of degradation are contained in the protein-coding sequence, and that translation inhibitors induce expression of c-myc mRNA by blocking turnover mediated by this element.

Cell Line↗

PubMed1986

Opposing effects of glucagon and triiodothyronine on the hepatic levels of messenger ribonucleic acid S14 and the dependence of such effects on circadian factors.

We have studied the effect of glucagon on the expression of a triiodothyronine (T3) and carbohydrate-inducible mRNA sequence (mRNA-S14) in rat liver that undergoes a threefold diurnal variation (peak, 2200 h; nadir, 0800 h). Glucagon injection into euthyroid rats (25 micrograms/100 g body wt i.p., three doses at 15-min intervals) during the nocturnal plateau of mRNA-S14 caused a monoexponential disappearance of this sequence (t1/2, 90 min) accompanied by a 90% reduction in the transcriptional rate in a nuclear run-off assay, indicative of a near total reduction of synthesis. This effect was markedly attenuated in rats treated with T3 (200 micrograms/100 g body wt i.p.) 24 h before glucagon injection. When T3 was given 15 min after glucagon, the glucagon-initiated decline in mRNA-S14 was reversed within 90 min, suggesting a rapid interaction between the two hormones in the evening. Curiously, administration of T3 alone at this hour did not affect a significant increase in mRNA-S14. At 0800 h, however, T3 caused the expected brisk induction of this sequence, whereas glucagon was without effect. In essence, glucagon affected mRNA-S14 synthesis only in the evening, while T3 increased levels of this sequence above the baseline only in the morning. T3, however, reversed the effect of prior glucagon injection at night. The observed alterations in hormonal responsivity could underly the diurnal variation of mRNA-S14 expression. Moreover, the data suggest the hypothesis that T3 may act on S14 gene expression by antagonizing factors that inhibit its transcription.

Animals↗

PubMed1982

Cell-type-specific actin mRNA populations in dictyostelium discoideum.

We have analyzed actin mRNA sequences present in the terminal stages of the development of Dictyostelium discoideum. Four different actin mRNA sequences were detected in migrating pseudoplasmodia. Nucleotide sequence analysis of primer-extension products derived from the four mRNA sequences showed that they each encoded an actin protein with the same eight N-terminal amino acids and that they did not derive from transcription of any previously characterized actin gene. Preculmination pseudoplasmodia of Dictyostelium contain two distinct populations of committed cells, termed prespore and prestalk cells. We show that prestalk cells contain all four of the actin mRNA sequences found in pseudoplasmodia, while prespore cells contain only three of the sequences, and mature spores contain only two. Thus there is a differential loss of actin mRNA sequences during spore-cell differentiation in Dictyostelium.

Actins↗

PubMed1991

Multiple determinants of functional mRNA stability: sequence alterations at either end of the lacZ gene affect the rate of mRNA inactivation.

The Escherichia coli lacZ gene was used as a model system to identify specific sequence elements affecting mRNA stability. Various insertions and substitutions at the ribosome-binding site increased or decreased the rate of mRNA inactivation by up to fourfold. Deletion of a dyad symmetry, which may give rise to a very stable secondary structure in the mRNA immediately downstream of the gene, decreased the functional stability of the lacZ message. The magnitude of the latter effect was strongly dependent on the sequences at the ribosome-binding site, ranging from practically no effect for the most labile transcripts to a threefold decrease in stability for the most stable one. The results suggest that the wild-type lacZ message is inactivated predominantly by attacks near the ribosome-binding site, presumably in part because the putative secondary structure downstream of the gene protects against 3'-exonucleolytic attack. Taken together, the data for all of the modified variants of lacZ were shown to be quantitatively compatible with a general model of mRNA inactivation involving multiple independent target sites.

Base Sequence↗

PubMed1993

Compilation of E. coli mRNA promoter sequences.

An updated compilation of 300 E. coli mRNA promoter sequences is presented. For each sequence the most recent relevant paper was checked, to verify the location of the transcriptional start position as identified experimentally. We comment on the reliability of the sequence databanks and analyze the conservation of known promoter features in the current compilation. This database is available by E-mail.

Base Sequence↗

PubMed1979

Isolation and characterization of the chicken ovomucoid gene.

The chicken ovomucoid gene has been isolated by screening a chicken DNA library with a plasmid containing ovomucoid mRNA sequences. Twelve recombinant phages carrying ovomucoid mRNA sequences were isolated. Two of them, extending farthest into the 5' and 3' direction respectively, were characterized by restriction mapping and Southern hybridization as well as by electron microscopic analysis of hybrids between the cloned DNA and ovomucoid mRNA. Seven intervening sequences interrupt the ovomucoid mRNA sequence in chromosomal DNA. From these data a minimal size of 5.6 kb can be estimated for the length of the ovomucoid gene.

Animals↗

PubMed1987

Gene expression of the chondroitin sulfate proteoglycan core protein PG19.

We have examined genomic sequences and mRNA species hybridizing to a cDNA clone of a yolk sac carcinoma chondroitin sulfate proteoglycan designated PG19. Genomic blot hybridizations with cDNAs covering the majority of the PG19 mRNA sequence revealed 15 to 17 gene fragments. Similar analysis with probes representing either the propeptide or the combined core protein COOH-terminal domain and 3' untranslated sequences revealed single genomic fragments indicating that a single gene codes for the PG19 proteoglycan. Genomic blot analysis with cDNA sequences coding for the serine-glycine repeat of the core protein identified the same gene fragments observed with the entire PG19 cDNA, indicating that this coding region is homologous with sequences present in multiple genes. The same probes were also used to examine mRNA expression. In addition to the PG19 mRNA, several PG19-related mRNAs could be seen. These PG19-related mRNAs had homology with the serine-glycine coding sequence of the PG19 cDNA. These mRNAs may be coding for proteoglycans. The mRNA coding for PG19 appeared to be uniquely expressed in parietal yolk sac and mast cell lineages. The PG19 mRNA existed in different forms in parietal yolk sac and mast cell lines due to cell-type-specific differences in the length of the 5' untranslated sequences. These results indicate that expression of the PG19 proteoglycan gene is regulated both in terms of cell-type-specific transcription and selection of a transcriptional start site.

Aggrecans↗

PubMed1977

Non-histone chromosomal proteins: their role in the regulation of histone-gene expression.

Histone-gene expression was studied during the cell cycle of continuously dividing HeLa S3 cells and after stimulation of confluent monolayers of WI-38 human diploid fibroblasts to proliferate. The presence of histone-mRNA sequences was assayed by hybridization to a 3H-labelled single-stranded DNA complementary to histone-mRNA molecules. In HeLa S3 cells histone mRNA sequences were found in the nucleus and associated with polyribosomes during S-phase, but not during G1-phase. Transcripts of S-phase chromatin contained histone-mRNA sequences, but those of G1-phase chromatin did not. Similarly, in WI-38 cells association of histone-mRNA sequences with polyribosomes and transcription of histone-mRNA sequences from chromatin parallel DNA replication. Taken together these results suggest that the regulation of histone-gene expression resides, at least in part, at the transcriptional level. Chromatin-reconstitution studies provide evidence that non-histone chromosomal proteins play a key role in activation of histone-gene transcription during the period of the cell cycle when DNA is replicated. Phosphate groups associated with the S-phase non-histone chromosomal proteins appear to be functionally involved in the control of histone-gene readout.

Animals↗

PubMed2006

Transterm--extended search facilities and improved integration with other databases.

Transterm has now been publicly available for >10 years. Major changes have been made since its last description in this database issue in 2002. The current database provides data for key regions of mRNA sequences, a curated database of mRNA motifs and tools to allow users to investigate their own motifs or mRNA sequences. The key mRNA regions database is derived computationally from Genbank. It contains 3' and 5' flanking regions, the initiation and termination signal context and coding sequence for annotated CDS features from Genbank and RefSeq. The database is non-redundant, enabling summary files and statistics to be prepared for each species. Advances include providing extended search facilities, the database may now be searched by BLAST in addition to regular expressions (patterns) allowing users to search for motifs such as known miRNA sequences, and the inclusion of RefSeq data. The database contains >40 motifs or structural patterns important for translational control. In this release, patterns from UTRsite and Rfam are also incorporated with cross-referencing. Users may search their sequence data with Transterm or user-defined patterns. The system is accessible at http://uther.otago.ac.nz/Transterm.html.

Databases, Nucleic Acid↗