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At least 235 records · Page 13Linked to original sources
PubMed1985

Respiratory syncytial virus fusion glycoprotein: nucleotide sequence of mRNA, identification of cleavage activation site and amino acid sequence of N-terminus of F1 subunit.

The amino acid sequence of respiratory syncytial virus fusion protein (Fo) was deduced from the sequence of a partial cDNA clone of mRNA and from the 5' mRNA sequence obtained by primer extension and dideoxysequencing. The encoded protein of 574 amino acids is extremely hydrophobic and has a molecular weight of 63371 daltons. The site of proteolytic cleavage within this protein was accurately mapped by determining a partial amino acid sequence of the N-terminus of the larger subunit (F1) purified by radioimmunoprecipitation using monoclonal antibodies. Alignment of the N-terminus of the F1 subunit within the deduced amino acid sequence of Fo permitted us to identify a sequence of lys-lys-arg-lys-arg-arg at the C-terminus of the smaller N-terminal F2 subunit that appears to represent the cleavage/activation domain. Five potential sites of glycosylation, four within the F2 subunit, were also identified. Three extremely hydrophobic domains are present in the protein; a) the N-terminal signal sequence, b) the N-terminus of the F1 subunit that is analogous to the N-terminus of the paramyxovirus F1 subunit and the HA2 subunit of influenza virus hemagglutinin, and c) the putative membrane anchorage domain near the C-terminus of F1.

Amino Acid Sequence↗
PubMed1982

Rat pancreatic kallikrein mRNA: nucleotide sequence and amino acid sequence of the encoded preproenzyme.

We have cloned via recombinant DNA technology the mRNA sequence for rat pancreatic preprokallikrein. Four cloned overlapping double-stranded cDNAs gave a continuous mRNA sequence of 867 nucleotides beginning within the 5'-noncoding region and extending to the poly(A) tail. The mRNA sequence reveals that pancreatic kallikrein is synthesized as a prezymogen of 265 amino acids, including a proposed secretory prepeptide of 17 amino acids and a proposed activation peptide of 11 amino acids. The activation peptide, although similar in length, is distinct from those of the other classes of pancreatic serine proteases. The amino acid sequence of the predicted active form of the enzyme is closely related to the partial sequences obtained for other kallikrein-like serine proteases including rat submaxillary gland kallikrein, pig pancreatic and submaxillary gland kallikreins, the gamma subunit of mouse nerve growth factor, and rat tonin. Key amino acid residues thought to be involved in the substrate-cleavage specificity of kallikreins are retained. Hybridization analysis showed relatively high levels of kallikrein mRNA in the rat pancreas, submaxillary and parotid glands, spleen, and kidney, indicating the active synthesis of kallikrein in these tissues.

Amino Acid Sequence↗
PubMed2003

Antagonistic signals within the COX2 mRNA coding sequence control its translation in Saccharomyces cerevisiae mitochondria.

Translation of the mitochondrially coded COX2 mRNA within the organelle in yeast produces the precursor of Cox2p (pre-Cox2p), which is processed and assembled into cytochrome c oxidase. The mRNA sequence of the first 14 COX2 codons, specifying the pre-Cox2p leader peptide, was previously shown to contain a positively acting element required for translation of a mitochondrial reporter gene, ARG8(m), fused to the 91st codon of COX2. Here we show that three relatively short sequences within the COX2 mRNA coding sequence, or structures they form in vivo, inhibit translation of the reporter in the absence of the positive element. One negative element was localized within codons 15 to 25 and shown to function at the level of the mRNA sequence, whereas two others are within predicted stem-loop structures formed by codons 22-44 and by codons 46-74. All three of these inhibitory elements are antagonized in a sequence-specific manner by reintroduction of the upstream positive-acting sequence. These interactions appear to be independent of 5'- and 3'-untranslated leader sequences, as they are also observed when the same reporter constructs are expressed from the COX3 locus. Overexpression of MRS2, which encodes a mitochondrial magnesium carrier, partially suppresses translational inhibition by each isolated negatively acting element, but does not suppress them in combination. We hypothesize that interplay among these signals during translation in vivo may ensure proper timing of pre-Cox2p synthesis and assembly into cytochrome c oxidase.

Amino Acid Sequence↗
PubMed1982

Primary structure of two distinct rat pancreatic preproelastases determined by sequence analysis of the complete cloned messenger ribonucleic acid sequences.

The mRNA sequences for two rat pancreatic elastolytic enzymes have been cloned by recombinant DNA technology and their nucleotide sequences determined. Rat elastase I mRNA is 1113 nucleotides in length, plus a poly(A) tail, and encodes a preproelastase of 266 amino acids. The amino acid sequence of the predicted active form of rat elastase I is 84% homologous to porcine elastase 1. Key amino acid residues involved in determining substrate specificity of porcine elastase 1 are retained in the rat enzyme. The activation peptide of the zymogen does not appear related to that of other mammalian pancreatic serine proteases. The mRNA for elastase I is localized in the rough endoplasmic reticulum of acinar cells, as expected for the site of synthesis of an exocrine secretory enzyme. Rat elastase II mRNA is 910 nucleotides in length, plus a poly(A) tail, and encodes a preproenzyme of 271 amino acids. The amino acid sequence is more closely related to porcine elastase 1 (58% sequence identity) than to the other pancreatic serine proteases (33-39% sequence identity). Predictions of substrate preference based upon key amino acid residues that define the substrate binding cleft are consistent with the broad specificity observed for mammalian pancreatic elastase 2. The activation peptide is similar to that of the chymotrypsinogens and retains an N-terminal cysteine available to form a disulfide link to an internal conserved cysteine residue.

Amino Acid Sequence↗
PubMed1996

Both 5' and 3' sequences of maize adh1 mRNA are required for enhanced translation under low-oxygen conditions.

Alcohol dehydrogenase-1 (ADH1) synthesis in O2-deprived roots of maize (Zea mays L.) results from induced transcription and selective translation of ADH1 mRNA. The effect of ADH1 mRNA sequences on message stability and translation was studied in protoplasts of the maize cell line P3377.5' capped and 3' polyadenylated mRNA constructs containing the firefly gene (luc) for luciferase (LUC) or the Escherichia coli gene (uidA) for beta-glucuronidase (GUS) coding region were synthesized in vitro and electroporated into protoplasts that were cultured at 40 or 5% O2. A LUC mRNA with a 17-nucleotide polylinker 5' untranslated region (UTR) was expressed 10-fold higher under aerobic conditions than under hypoxic conditions. Expression of five chimeric ADH1-GUS mRNAs was measured relative to this LUC mRNA. An mRNA containing the 5'-UTR and the first 18 codons of adh1 in a translational fusion with the GUS coding region and followed by the 3'-UTR of adh1 was expressed 57-fold higher at 5% O2. Progressive deletion of adh1 5'-UTR and coding sequences reduced expression of the GUS-mRNA at 5% O2, but had little impact on expression of 40% O2. Enhancement of expression in hypoxic protoplasts conferred by the adh1 5'-UTR and the first 26 codons decreased more than 3-fold when the adh1 3'-UTR was removed. In addition, the adh1 3'-UTR slightly inhibited expression in aerobic protoplasts. The physical half-lives of the GUS and LUC mRNAs were similar under both anaerobic and hypoxic conditions, indicating that expression levels were largely independent of mRNA stability. Thus, both adh1 5' and 3' mRNA sequences are required for enhanced translation in protoplasts under O2 deprivation.

Alcohol Dehydrogenase↗
PubMed1999

mRNAs have greater negative folding free energies than shuffled or codon choice randomized sequences.

An examination of 51 mRNA sequences in GenBank has revealed that calculated mRNA folding is more stable than expected by chance. Free energy minimization calculations of native mRNA sequences are more negative than randomized mRNA sequences with the same base composition and length. Randomization of the coding region of genes yields folding free energies of less negative magnitude than the original native mRNA sequence. Randomization of codon choice, while still preserving original base composition, also results in less stable mRNAs. This suggests that a bias in the selection of codons favors the potential formation of mRNA structures which contribute to folding stability.

Codon↗
PubMed2004

Hyphantria cunea ferritin heavy chain homologue: cDNA sequence and mRNA expression.

We have sequenced a cDNA clone encoding a 26-kDa ferritin subunit, which was heavy chain homologue (HCH), in fall webworm, Hyphantria cunea. The HCH cDNA was obtained from the screening of a cDNA library using a PCR product. H. cunea ferritin is composed of 221 amino acid residues and their calculated mass is 26,160 Da. The protein contains the conserved motifs for the ferroxidase center typical for heavy chains of vertebrate ferritin. The iron-responsive element sequence with a predicted stem-loop structure is present in the 5'-untranslated region of ferritin HCH mRNA. The sequence alignment of ferritin HCH shows 68.9 and 68.7% identity with Galleria mellonella HCH (26 kDa ferritin) and Manduca sexta HCH, respectively. While G type insect ferritin vertebrate light chain homologue (LCH) is distantly related to H. cunea ferritin HCH (17.2-20.8%), the Northern blot analysis revealed that H. cunea ferritin HCH was ubiquitously expressed in various tissues and all developmental stages. The ferritin expression of midgut is more responsive to iron-fed, compared to fat body in H. cunea.

Amino Acid Sequence↗
PubMed1982

Identification of a sequence responsible for periodic synthesis of yeast histone 2A mRNA.

Sequences required for the regulated expression of a yeast histone 2A (H2A) gene have been investigated by using fusions between this gene and the Escherichia coli lacZ gene. Fusions containing the entire spacer region in which divergent transcription of the H2A and H2B genes is initiated result in low-level constitutive synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in yeast. Regulated expression (which is characterized by periodic synthesis during the S phase of the cell cycle) is restored when a 1.3-kilobase HindIII fragment containing a small region of the 3' end of the H2B gene is present in either orientation. The regulatory activity in this region appears to be coincident with a sequence that supports autonomous replication in yeast.

Base Sequence↗
PubMed1994

Expression of (cac)n/(gtg)n simple repetitive sequences in mRNA of human lymphocytes.

Di- and trinucleotide tandem repeat sequences are ubiquitously interspersed and are often polymorphic in the human genome. We have analyzed the transcription of simple (cac)n/(gtg)n repeats in the cDNA of RNA from human lymphocytes. When using such motifs as probes in RNA hybridization experiments, distinct signals are scarcely demonstrable. In order to investigate mRNA sequences that contain such simple repeats, 1 million phage clones from cDNA libraries were screened with the probe (CAC)5. From 50 hybridizing phages, 38 clones were successfully isolated and characterized. The lengths of the transcripts ranged from 120 bp to 3.5 kb. More than 15 different additional simple repeat motifs were found immediately next to or distant to the (cac)n/(gtg)n repeat. In 12 clones, significant homologies were identified with a wide variety of unrelated genes, such as a processed pseudogene of human ubiquitin, serin protease inhibitor genes, a gene candidate from yeast, and sequence-tagged sites of man and mouse. Of the clones, 18% represented mRNA of MHC class I promotor binding protein; 79% displayed novel single copy sequences or partial similarity to many different organelle and nuclear genomes of animal, fungal, bacterial, and viral sequences. These data indicate that short (cac)n/(gtg)n stretches (n < or = 6) are sometimes contained in open reading frames, but more often in the 3' and 5' untranslated portions of mature mRNAs. Longer stretches of perfect simple (cac)n/(gtg)n repeats can rarely be recovered, even from the hnRNA of human lymphocytes.

Amino Acid Sequence↗
PubMed1978

Cloning of a double-stranded cDNA that codes for a portion of chicken preproalbumin. A general method for isolating a specific DNA sequence from partially purified mRNA.

A scheme is presented for cloning a double-stranded cDNA molecule that codes for a portion of chicken preproalbumin. This method, which does not require pure mRNA or cDNA, has widespread applicability. Chicken preproalbumin was identified as a Mr = 72,000 polypeptide by immunoprecipitation of proteins synehesized in a wheat germ cell-free translation system from total, guanidine.HCl-extracted, rooster liver RNA. After removal of the bulk of the ribosomal RNA by poly(U)-Sephadex G-10 chromatography, albumin mRNA was enriched approximately 2-fold by centrifugation through low salt, isokinetic sucrose gradients, until it represented about 30% of the mRNA sequences present. Double-stranded cDNA prepared from this mRNA was then inserted into the Pst 1 site of the plasmid PBR322 by the "G-C tailing" technique and the recombinant DNA was used to transform Echerichia coli stran X1776. Transformants containing putative albumin DNA sequences were identified by colony hybridization with a cDNA probe that was highly enriched for albumin cDNA sequences. This probe was isolated by hybridizing the partially purified RNA preparation to its cDNA, under conditions of RNA excess, to a R0t value such that only the most abundant cDNA sequences had hybridized. Unhybridized, less abundant, sequences were destroyed by subsequent S1 nuclease digestion. The identity of clones that hybridized to this abundant class cDNA was established by DNA-mRNA hybrid-arrested cell-free translation. Hybridization of nick-translated, albumin-containing, plasmid DNA to total liver poly(A)+ RNA, that had been separated on methyl mercury agarose gels and transferred to diazobenzyloxymethyl paper, established that avian albumin mRNA has a molecular weight of 850,000. This molecular weight corresponds to approximately 2,600 nucleotides, or 600 nucleotides longer than the size required to code for the preproalbumin polypeptide.

Animals↗
PubMed1998

The KH domain of the branchpoint sequence binding protein determines specificity for the pre-mRNA branchpoint sequence.

The yeast and mammalian branchpoint sequence binding proteins (BBP and mBBP/SF1) contain both KH domain and Zn knuckle RNA-binding motifs. The single KH domain of these proteins is sufficient for specific recognition of the pre-mRNA branchpoint sequence (BPS). However, an interaction is only apparent if one or more accessory modules are present to increase binding affinity. The Zn knuckles of BBP/mBBP can be replaced by an RNA-binding peptide derived from the HIV-1 nucleocapsid protein or by an arginine-serine (RS)7 peptide, without loss of specificity. Only the seven-nucleotide branchpoint sequence and two nucleotides to either side are necessary for RNA binding to the chimeric proteins. Therefore, we propose that all three of these accessory RNA-binding modules bind the phosphate backbone, whereas the KH domain interacts specifically with the bases of the BPS. Proteins and protein complexes with multiple RNA-binding motifs are frequent, suggesting that an intimate collaboration between two or more motifs will be a general theme in RNA-protein interactions.

Amino Acid Sequence↗
PubMed1990

Quantification analysis of 5'-splice signal sequences in mRNA precursors. Mutations in 5'-splice signal sequence of human beta-globin gene and beta-thalassemia.

Concerning the signals which direct excision of introns from mRNA precursors in higher eukaryotic genes, consensus 9-nucleotide sequence, (CA)AG/GT(AG)AGT, has been proposed with the 5'-splice site, but actual 5'-splice site sequences differ from it in a greater or lesser degree. We analyzed 5'-splice site sequence of human beta-globin gene by quantification method (categorical discriminant analysis) proposed previously. Analysis of 13-nucleotide sequences and deleted sequences showed that 9-nucleotide sequences in the consensus region are almost sufficient to define 5'-splice signal. To confirm this view, we examined a number of beta-globin mutant genes, where nucleotide changes occur at the authentic 5'-splice site of the first intron and cause beta-thalassemia phenotype. Our method could explain why such mutations abolish the 5'-splice site and cryptic 5'-splice sites are activated.

Base Sequence↗
PubMed1998

AML1-like transcription factor induces serine elastase activity in ovine pulmonary artery smooth muscle cells.

In previous studies, we showed that induction of pulmonary artery (PA) smooth muscle cell (SMC) elastase activity by serum-treated elastin (STE) requires DNA transcription. We therefore used differential mRNA display to identify transcripts expressed coincident with elastase induction. Twenty-four individual transcripts were differentially expressed from a screen of approximately 2000 mRNA sequences. An mRNA with sequence homology to the human transcription factor AML1 was identified and subsequently cloned from ovine PA SMCs. Since AML1 binds to a consensus sequence in the promoter of neutrophil elastase, we pursued the possibility that AML1 is a candidate transcription factor for SMC elastase. We documented by immunohistochemistry that serum stimulation induces increased expression of AML1 in the nucleus of PA SMCs. We also showed that STE induction of elastase activity is associated with early expression of AML1 mRNA and protein and that AML1 consensus sequence DNA binding activity is increased in nuclear extracts of STE-treated cells. In addition, AML1 antisense oligonucleotides reduced serum induction of elastase activity. Our study thus provides the first functional evidence of AML1 transcriptional activity related to elastase genes and offers novel insights into the broader biological significance of AML1 in nonmyeloid cells.

Animals↗
PubMed1982

Modulation of tubulin mRNA levels by interferon in human lymphoblastoid cells.

Blot hybridization with labeled tubulin cDNA showed that treatment of Ramos cells, a human cell line of lymphoblastoid origin, with either alpha or beta interferon (IFN) induced a marked increase in the amount of tubulin mRNA sequences. The level of tubulin mRNA sequences increased rapidly after exposure of cells to IFN-alpha and reached a maximum after 1 h of treatment, which was four times the control level. Treatment with IFN-beta induced a maximal increase after 4 h; the amount of tubulin mRNA sequences was seven times higher than the control level. The mRNA extracted from IFN-treated and nontreated cells was translated in vitro in a reticulocyte lysate cell-free system containing [35S]methionine. Electrophoretic analysis of the labeled cell-free products showed an increase in the amount of translatable tubulin mRNA that parallels the time course of induction of tubulin mRNA sequences. Two-dimensional gel electrophoresis of the labeled protein products directed by mRNA indicates that IFN caused a more pronounced increase in the level of alpha-tubulin than beta-tubulin mRNA. Treatment with colchicine, which disrupts the cell microtubules, caused a marked decrease in the tubulin mRNA content. Concomitant treatment of the cells with colchicine and IFN abolished the interferon-dependent induction of tubulin mRNA.

Actins↗
PubMed1982

[Mapping of 2 cloned DNA fragments containing repetitive mouse genome regions, actively transcribing into pre-mRNA].

Sequence organization of two cloned mouse genomic DNA fragments which are efficiently transcribed into pre-mRNA was studied. In both cases, the arrangement of sequences hybridizing to polysomal mRNA (or cDNA), double-stranded RNA (dsRNA-B) isolated from pre-mRNA and oligo (dT), was established. In one fragment, the regions transcribed are represented by three different repetitive mRNA (or cDNA) hybridizing sequences interspersed into the AT-rich tract, the type B-2 sequence and the unique DNA sequence which also transcribed into mRNA. Nuclear pre-mRNA of 28S is hybridized to three repetitive mRNA hybridizing regions. The significance of such a complex array of repeated sequences inside eukaryotic transcription units and their possible role in mRNA processing and splicing is discussed.

Animals↗
PubMed1981

Identification and mapping of two polypeptides encoded within the herpes simplex virus type 1 thymidine kinase gene sequences.

mRNA's homologous to the herpes simplex virus type 1 DNA restriction endonuclease fragment BamHI p, which contains the thymidine kinase gene, have been identified and mapped by hybrid-arrested translation and mRNA selection. Such mRNA's, when translated in vitro, directed the synthesis of polypeptides of apparent molecular weights 43,000 (VI43) and 39,000 (VI39). mRNA for enzymatically active thymidine kinase was enriched by more than 20-fold after selection. Mapping was carried out with restriction endonuclease fragments of BamHI p, and locations of the 5' and 3' termini of VI43 mRNA were deduced. Analysis of nucleotide sequences around the 5' terminus revealed several consensus sequences commonly found at the start of eucaryotic mRNA's and which are presumably involved in initiation of transcription by RNA polymerase II. Translation of mRNA's for VI43, VI39, and the thymidine kinase enzyme was arrested only by a 1,170-base-pair region of BamHI p. Since this region is insufficient for adjacent genes, coding sequences for VI43 and VI39 must overlap; the possible relationship of these two polypeptides is discussed. A virus-induced product equivalent to VI39 was detected in infected cells.

Base Sequence↗
PubMed1985

Three intergenic regions of coronavirus mouse hepatitis virus strain A59 genome RNA contain a common nucleotide sequence that is homologous to the 3' end of the viral mRNA leader sequence.

cDNA clones that represent various portions of the coronavirus mouse hepatitis virus strain A59 genome RNA have been constructed. cDNAs were synthesized by transcription of genome RNA by using either oligo(dT) or random oligomers of calf thymus DNA as primers. These cDNAs were converted into double-stranded DNA and cloned into pBR322 by standard techniques. The resulting cloned viral DNA fragments were mapped to viral genes by hybridization with Northern blots of intracellular RNA from mouse hepatitis virus strain A59-infected cells. These cDNA clones map in six of the seven viral genes. Clone g344, 1.8 kilobases, is the largest and encompasses gene 5 (which encodes a nonstructural protein) and gene 6 (which encodes the E1 viral glycoprotein) as well as the intergenic regions preceding genes 5, 6, and 7. Sequencing of parts of this cloned DNA show that these three intergenic regions contain a common 11-nucleotide sequence. This sequence shares homology with the 3' end of the viral mRNA leader sequence. Thus, this common intergenic sequence may contain a binding site for a leader RNA that hybridizes to negative-strand viral RNA at the beginning of each gene to prime mRNA synthesis. The different degrees of homology between the leader and its putative binding site may influence the differential rates of transcription of the various viral mRNAs.

Animals↗
PubMed1988

Assay of a ribonuclease that preferentially hydrolyses mRNAs containing cytokine-derived UA-rich instability sequences.

mRNA molecules encoding a number of inflammatory cytokines, as well as certain proto-oncogenes, contain a conserved UA-exclusive sequence in the 3' untranslated region that confers message instability in vivo. This sequence may comprise a critical regulatory element, governing the level of these mRNA molecules, and determining the efficiency with which they are translated. Through the use of a double-label RNAse assay, we have determined that lysates prepared from mouse macrophages selectively degrade mRNA molecules containing the 3' untranslated UA sequence found in the mRNA encoding human cachectin/TNF. The degree of instability is dependent upon the number of copies of inserted UA sequence present in the target mRNA molecule (a Xenopus beta-globin mRNA). mRNAs containing randomly generated UA sequences are more labile than unmodified globin mRNA, but less susceptible to degradation than mRNAs containing the authentic cachectin-derived sequence. mRNA molecules containing synthetic UG-exclusive sequences are normally stable or protected in vitro. The destruction of UA-containing mRNA is inhibited by random adenylate/uridilate copolymers, but not by guanylate/uridilate copolymers. Boiling or proteinase K treatment destroys the selective nucleolytic activity of macrophage lysates. We propose that the nuclease measured here may serve to regulate cellular levels of mRNA molecules encoding cachectin, other inflammatory cytokines, and certain proto-oncogene products.

Adenine↗