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PubMed1991

Apolipoprotein B mRNA sequences 3' of the editing site are necessary and sufficient for editing and editosome assembly.

Apolipoprotein B (apoB) mRNA is edited in rat liver and intestine through the direct conversion of cytidine to uridine at nucleotide 6666. Recently, we have proposed the 'Mooring Sequence' model, in which editing complexes (editosomes) assemble on specific apoB mRNA flanking sequences to direct this site-specific editing event. To test this model, apoB mRNA deletion and translocation mutants were constructed and analyzed. Specific sequences 3' of the editing site were absolutely required for editing, while specific sequences and bulk RNA 5' of the editing site were required for efficient editing. Translocation of apoB 3' flanking sequences induced editing of an upstream cytidine, demonstrating that 3' sequences are necessary and sufficient to direct editing in vitro. 3' flanking sequences were also shown to be necessary and sufficient for editosome complex assembly. These data provide strong support for a 'Mooring Sequence' model in which 3' apoB flanking sequences direct editosome assembly and subsequent editing in vitro, while 5' flanking sequences enhance these functions.

Animals↗
PubMed1990

RNA editing of apolipoprotein B mRNA. Sequence specificity determined by in vitro coupled transcription editing.

Apolipoprotein (apo) B-48 mRNA is produced by in vivo RNA editing which involves a C----U conversion of the first base of the codon CAA for Gln-2153, changing it to UAA, an in-frame stop codon. We have reproduced the editing reaction in vitro using nuclear extracts. Efficient RNA editing was demonstrated by using apoB mRNA segments as substrate or in a coupled transcription-editing reaction using apoB minigenes as template. ApoB minigenes were constructed by ligating the adenovirus major late promoter to a fragment of apoB-100 DNA containing the editing site and used for the transcription-editing reaction. We defined the sequence specificity of the editing reaction using site-specific single and multiple base mutants constructed by the polymerase chain reaction. Among 22 different mutant apoB-100 minigene constructs containing mutations in the bases immediately flanking the edited C-6666, 20 were edited in the coupled transcription-editing reaction. The results suggest a relatively lax sequence specificity for apoB mRNA editing. Our observation may have important implications for apoB-48 biogenesis as well as for the editing process as a general biologic regulatory mechanism.

Animals↗
PubMed1999

Differential enhancement of Cry2A versus Cry11A yields in Bacillus thuringiensis by use of the cry3A STAB mRNA sequence.

Previously we demonstrated that the yield of Cry3A (70 kDa) can be increased as much as 10-fold when cry3A including its upstream STAB-SD mRNA stabilizing sequence is expressed in Bacillus thuringiensis under the control of cyt1A promoters. To determine whether the cyt1A promoters/STAB-SD combination (cyt1AP/STAB) has broader applicability, we used it to synthesize two other Cry endotoxins in the 70-kDa mass range, Cry2A and Cry11A. Combination of cyt1AP/STAB with orfs 2 and 3 of the cry2A operon yielded about 4. 4-fold the amount of Cry2A obtained with the wild-type cry2A operon. The yield of Cry11A obtained with a construct that contained the cyt1AP/STAB, cry11A and the 20-kDa protein gene was 1.3-fold the amount obtained with a construct similar to the wild-type operon. These results demonstrate that the cyt1AP/STAB combination can enhance synthesis of different Cry proteins significantly, but that the level of enhancement varies with the specific protein synthesized.

Animals↗
PubMed1984

Mechanism of induction of human chorionic gonadotropin in lung tumor cells in culture. Increased levels of alpha-human chorionic gonadotropin-specific mRNA sequences and benzo(a)pyrene-induced hypomethylation.

Human lung cells (ChaGo) derived from a bronchogenic carcinoma produce human chorionic gonadotropin (hCG), predominantly the alpha subunit of the glycoprotein hormone, under culture conditions. Treatment of the cells with the polycyclic aromatic hydrocarbons, benzo(a)pyrene (BaP) or dimethylbenzanthracene, at concentrations which do not affect cell growth or macromolecular synthesis, stimulates the production of hCG in these cells. The levels of alpha hCG-specific mRNA (mRNA alpha hCG) sequences in total poly(A)+ RNA isolated from control and drug-treated ChaGo cells are determined by the dot hybridization technique using 32P-labeled, cloned cDNA alpha hCG probe. A concentration-dependent increase in the levels of mRNA alpha hCG sequences in BaP or dimethylbenzanthracene-treated ChaGo cells has been observed. The increase in the level of mRNA alpha hCG sequences can be detected after treatment of the cells with either of the drugs for 24 h, and this level attains its maximum within 48-72 h following drug treatment. A comparative study of the restriction endonuclease (MspI/HpaII) digestion patterns of the control and BaP-treated cell DNA suggests that the internal "C" residues of the -CCGG- sequences in the alpha hCG gene of untreated cells are highly methylated; whereas the internal C residues of the same MspI/HpaII recognition sequences in the alpha hCG gene are comparatively less methylated in BaP-treated cell DNA.

Benzo(a)pyrene↗
PubMed2001

Programmed +1 frameshifting stimulated by complementarity between a downstream mRNA sequence and an error-correcting region of rRNA.

Like most retroviruses and retrotransposons, the retrotransposon Ty3 expresses its pol gene analog (POL3) as a translational fusion to the upstream gag analog (GAG3). The Gag3-Pol3 fusion occurs by frameshifting during translation of the mRNA that encodes the two separate but overlapping ORFs. We showed previously that the shift occurs by out-of-frame binding of a normal aminoacyl-tRNA in the ribosomal A site caused by an aberrant codonoanticodon interaction in the P site. This event is unlike all previously described programmed translational frameshifts because it does not require tRNA slippage between cognate or near-cognate codons in the mRNA. A sequence of 15 nt distal to the frameshift site stimulates frameshifting 7.5-fold. Here we show that the Ty3 stimulator acts as an unstructured region to stimulate frameshifting. Its function depends on strict spacing from the site of frameshifting. Finally, the stimulator increases frameshifting dependent on sense codon-induced pausing, but has no effect on frameshifting dependent on pauses induced by nonsense codons. Complementarity between the stimulator and a portion of the accuracy center of the ribosome, Helix 18, implies that the stimulator may directly disrupt error correction by the ribosome.

Amino Acid Sequence↗
PubMed1988

Nucleotide sequence determination of point mutations at the mouse HPRT locus using in vitro amplification of HPRT mRNA sequences.

Cloning of genomic and cDNA sequences of mammalian genes has made it possible to analyze at the molecular level mutations induced by radiation and chemical mutagens. The X-linked HPRT gene is very suitable for these investigations because in addition to the availability of cell culture systems, HPRT mutants can also be obtained directly from the lymphocytes of mouse and man. Recently a new technique has been introduced by Saiki and co-workers which allows the cloning and sequencing of small specific DNA segments from total genomic DNA after in vitro amplification of those segments up to 200,000-fold (Saiki et al., 1985). We have adapted this so-called polymerase chain reaction (PCR) procedure in such a way that the entire mouse HPRT-coding region could be amplified, cloned and sequenced. Instead of genomic DNA, we have used RNA as template in the PCR reactions. This allows us to detect point mutations in HPRT exon sequences in a very efficient way, since the DNA sequence of all 9 exons, which are scattered over 34 kb of DNA, can be obtained from only one amplification experiment. We studied the nature of 3 N-ethyl-N-nitrosourea (ENU)-induced HPRT mutants from cultured mouse lymphoma cells. One contains an A:T----G:C transition, the second an A:T----T:A transversion, whereas the third mutant is the result of abnormal splicing events, probably due to a mutation in the 3' splice site of the first intron.

Animals↗
PubMed1983

V kappa gene family in (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies that express CGAT (or pGAT) public idiotypic specificities. Protein and mRNA sequencing of eight monoclonal V kappa chains.

A large proportion of (Glu60 Ala30 Tyr10)n (GAT)-specific antibodies expresses public idiotypic specificities, termed CGAT (or pGAT), that require the presence of both the heavy and the light chains in order to be expressed. We report in this paper the complete sequence of eight V kappa regions pertaining to eight anti-GAT monoclonal antibodies derived from three strains of mice: BALB/c, DBA/2, and C57BL/6. The methodology used a combination of NH2-terminal amino acid and mRNA nucleotide sequencing. All eight sequences analyzed, although highly homologous and all pertaining to the same V kappa 1 subgroup, allowed definition of three germline genes that are likely to be present in all three strains of mice and also in NZB. It seems likely, however, that any given strain may not necessarily use all three genes for making anti-GAT antibodies. The search for structural correlates of idiotypes could not be framed in a simple picture, but our data suggest that similar idiotopes may result from different interacting primary structures, leading to structural homologies that should be visualized at three-dimensional level.

Amino Acid Sequence↗
PubMed1986

Genetic determinants of growth phase-dependent and adenovirus 5-responsive expression of the Chinese hamster thymidine kinase gene are contained within thymidine kinase mRNA sequences.

We have constructed a chimeric thymidine kinase (TK) minigene, pHe delta 6Ha, which combines the complete coding and 3' noncoding regions of a Chinese hamster TK cDNA with the promoter region and 5' untranslated region of the TK gene of herpes simplex virus type 1. We have transformed rat 4 cells to Tk+ with this gene and analyzed the pattern of TK gene expression in these transformants under various conditions of in vitro cell culture. We find that TK gene expression in these Tk+ transformants is growth phase dependent, responsive to adenovirus 5 infection, and indistinguishable in character under a variety of cell culture conditions from the pattern of TK gene expression in rat 4 cells transformed to Tk+ with the genomic Chinese hamster TK gene clone lambda HaTK.5. We are led to the conclusion that the genetic elements which mediate growth phase-dependent TK gene expression are contained entirely within the sequences of the mature cytoplasmic hamster TK mRNA.

Adenoviruses, Human↗
PubMedDate not supplied

Primary structure of human transferrin receptor deduced from the mRNA sequence.

In vertebrates all iron is taken up via the carrier protein transferrin. The carrier first binds its receptor and the receptor-ligand complex is then internalized via coated pits. The transferrin receptor is a transmembrane glycoprotein (apparent molecular weight (MW) 180,000) composed of two disulphide-bonded sub-units (each of apparent MW 90,000) It contains three N-linked glycan units and is post-translationally modified with both phosphate and fatty acyl groups. Here we have determined the nucleotide sequence of the coding region of the human transferrin receptor mRNA and from this deduced the amino acid sequence of the protein. The receptor does not contain an N-terminal signal peptide but there is a membrane-spanning segment 62 amino acids from the N-terminus. It therefore has a somewhat unusual configuration with a small N-terminal cytoplasmic domain and a C-terminal extracellular domain of 672 amino acids.

Amino Acid Sequence↗
PubMed1979

Production of mRNA in Chinese hamster cells: relationship of the rate of synthesis to the cytoplasmic concentration of nine specific mRNA sequences.

We constructed cloned DNA sequences complementary to unselected mRNAs [poly(A)+ cytoplasmic RNA] from Chinese hamster ovary cells and used them in RNA:DNA hybridization experiments. Each cloned DNA hybridized a single mRNA from 1.3-3.5 kb in length. The relative rates of labeling (transcription rates) of nuclear RNA complementary to each individual DNA segment varied approximately 10 fold. The relative cytoplasmic concentration of the same specific RNA sequences in the mRNA after an equilibrium labeling of the cells varied approximately 100 fold. In addition, we estimated the sizes of the nuclear RNA precursor molecules to these cytoplasmic mRNAs. Four main conclusions arise from these studies. First, the primary RNA transcripts, which range in size from 2.4-13.5 kb, are 2-6 times larger than the mRNAs; second, each cloned DNA segment is complementary to only one species of mRNA; third, for the RNA complementary to at least three of the nine cloned DNA segments, the relative cytoplasmic content is considerably different from the relative rate of nuclear RNA synthesis, suggesting the post-transcriptional events are involved in the determination of the cytoplasmic concentrations of some mammalian mRNAs; and fourth, the fraction of total nonribosomal nuclear RNA complementary to the nine cloned DNA segments is in most cases 10 fold less than the fraction of cytoplasmic mRNA complementary to the same cloned DNA segments, suggesting the synthesis of many hnRNA molecules that are qualitatively different from those which eventually contribute mRNA to the cytoplasm.

Animals↗
PubMed1982

Distinct alpha-tropomyosin mRNA sequences in chicken skeletal muscle.

Recombinant plasmids have been isolated which contain sequences complementary to two distinct alpha-tropomyosin mRNA species present in chicken leg muscle. The proteins coded for by these different mRNAs could be distinguished by their electrophoretic behaviour in the presence of 3.5 M urea. The properties of the minor alpha-tropomyosin of chicken leg muscle were similar to those reported for the alpha-tropomyosin of slow twitch chicken skeletal muscle. Sequence analysis of available plasmids showed that the deduced protein sequences of both types of alpha-tropomyosin were very similar and closely related to the known protein sequence of rabbit alpha-tropomyosin. However considerable variation in nucleotide coding sequence of the two alpha-tropomyosin mRNAs was found.

Animals↗
PubMed1992

Cooperation of pre-mRNA sequence elements in splice site selection.

We have recently demonstrated that short internal exons in pre-mRNA transcripts with three exons and two introns are ignored by splicing machinery in vitro and in vivo, resulting in exon skipping. Exon skipping is reversed when the pyrimidine content of the polypyrimidine tract in the upstream intron is increased (Z. Dominski and R. Kole, Mol. Cell. Biol. 11:6075-6083, 1991). Here we show that skipping of the short internal exon can be partially reversed by mutations which modify the upstream branch point sequence of the 5' splice site at the end of the exon to their respective consensus sequences. When the modified elements are combined with one another in the same pre-mRNA, exon skipping is fully reversed. Full reversion of exon skipping is also observed when these elements are combined individually with the upstream polypyrimidine tract strengthened by three purine-to-pyrimidine mutations. The observed patterns of splice site selection are similar in vitro (in nuclear extracts from HeLa cells) and in vivo (in transfected HeLa cells). We also show that the length of the downstream intron plays a role in splice site selection. Our data indicate that the interplay between the sequence elements in pre-mRNA controls the outcome of each splicing event, providing the means for very subtle regulation of alternative splicing.

Base Composition↗
PubMedDate not supplied

Construction of recombinant plasmids containing rat thyroglobulin mRNA sequences.

Two plasmids containing rat thyroglobulin cDNA sequences have been constructed and characterized. A plasmid with a 500-bp insert (pRT6) was isolated and identified as thyroglobulin-specific on the basis of the tissue specificity of the inserted sequence and of its ability to retain thyroglobulin mRNA on a nitrocellulose filter. The cDNA insert in pRT6 was subsequently used to screen a rat thyroid cDNA library constructed with large cDNA. A plasmid was found containing a 1700-bp insert. The polarity and the fidelity of the insert is demonstrated by S1 mapping.

Animals↗
PubMed1983

Cloned mRNA sequences for two types of embryonic myosin heavy chains from chick skeletal muscle. I. DNA and derived amino acid sequence of light meromyosin.

Two myosin heavy chain cDNA clones (251 and 110), constructed from chick embryonic skeletal muscle mRNA, were subjected to extensive DNA sequence analysis. A complete description of the DNA sequence of clone 251 was obtained. This 1.5-kilobase pair cDNA sequence specified the COOH-terminal 439 amino acids of the myosin heavy chain, and included the entire 3' nontranslated region. The translated and 3' nontranslated sequences were purine- (64%) and AT-(71%) rich, respectively. The derived amino acid sequence of clone 251 correlated well with sequences obtained by direct amino acid sequencing of adult rabbit back muscle myosin heavy chain protein (87% homology), as well as with cloned myosin heavy chain sequences from other species. Comparison of clone 251 with a partial DNA sequence of clone 110 revealed significant structural differences both in the translated, and 3' nontranslated regions. This data indicates that these two clones represent two distinct myosin heavy chain genes. The protein sequence specified by clone 251 corresponds to the light meromyosin portion of the myosin heavy chain rod. These sequences, like other myosin heavy chain rod sequences, are alpha-helical and exhibit 7- and 28-residue periodicities in the linear distribution of nonpolar, and basic and acidic amino acids, respectively.

Amino Acid Sequence↗
PubMed2000

Tools for evaluating ubiquitin (UbC) gene expression: characterization of the rat UbC promoter and use of an unique 3' mRNA sequence.

UbC is one of three members of the ubiquitin gene family. We have cloned the rat UbC promoter and used primer extension analysis to map the UbC site of transcription initiation to 63 bp upstream of the putative first intron. We used a rat UbC promoter-luciferase reporter minigene to transfect H9c2 cardiomyocytes, HepG2 hepatocytes, CaCo2 colon cells, NIH3T3 fibroblasts or L6 myocytes and found the rat UbC promoter has constitutive activity. We also showed that dexamethasone stimulated the UbC promoter in L6 myocytes. Finally, we showed that a UbC-specific sequence at the 3' end of the rat UbC mRNA transcript can be used to selectively and quantitatively measure UbC: (1) mRNA using a RNase protection assay, and (2) transcription using a nuclear run-off assay to measure the rate of transcription of the UbC gene. These findings will be useful in studying the regulation of the UbC gene.

3' Untranslated Regions↗
PubMed1988

Enzymatic amplification of myosin heavy-chain mRNA sequences in vitro.

We have developed a procedure that detects the presence of mRNA coding for human beta-myosin heavy chain in small amounts of total, unfractionated RNA isolated from heart or skeletal muscle. The protocol is based on the enzymatic amplification in vitro of a selected 106-bp myosin isotype-specific subregion of this mRNA. The method, which is a modification of the so-called "polymerase chain reaction," requires two synthetic oligonucleotide primers (20-mers), reverse transcriptase, and DNA polymerase I (Klenow fragment). Two principle steps are involved: (i) the selected mRNA subregion is converted into a double-stranded cDNA, and (ii) this cDNA is amplified in 22 synthetic cycles. After gel electrophoresis and blotting the amplification product is identified by hybridization with a third oligonucleotide recognizing the region between the two primer annealing sites, and by restriction mapping. Only mRNA from muscle tissue promoted formation of the amplified 106-bp fragment. We estimate that less than 30,000 beta-myosin heavy-chain mRNA molecules are sufficient to produce a signal. The procedure is fast, specific, and very sensitive. It may be used in muscle gene expression studies with small numbers of cells or even in single muscle fibers.

Animals↗
PubMed1985

Differential regulation of trypsinogen mRNA translation: full-length mRNA sequences encoding two oppositely charged trypsinogen isoenzymes in the dog pancreas.

In the absence of changes in functional mRNA levels, stimulation of the pancreas with caerulein, a peptide analog of cholecystokinin, has been previously shown to increase the synthesis of anionic but not cationic trypsinogen. To look for structure-function correlations, a high-yield, full-length cDNA library has been constructed from canine pancreatic poly(A)+ mRNA. Full-length clones coding for the two major trypsinogen isoenzyme forms have been identified by colony hybridization and verified by in vitro translation of hybrid-selected mRNA in the presence of microsomal membranes and an optimal redox potential. Disulfide-bonded translation products were separated and identified by two-dimensional isoelectric focusing-sodium dodecyl sulfate-gel electrophoresis. Nucleotide sequence analysis allowed us to deduce the amino acid sequences for the anionic and cationic forms of canine trypsinogen, which contain 232 and 231 residues, respectively (77% amino acid identity), and the 15-residue amino terminal signal sequences (53% amino acid identity) associated with the two presecretory forms. Measurements of relative and absolute mRNA levels, when related to relative protein synthesis values, indicated that the translational efficiency of anionic trypsinogen mRNA exceeded that of cationic trypsinogen mRNA by 1.5- to 2.9-fold under basal conditions. Analysis of the 5' noncoding regions of trypsinogen mRNAs revealed a striking conservation of sequence (10 of 12 bases) between dog and rat anionic trypsinogen forms. This contrasted markedly with the divergence of the 5' noncoding regions observed between dog anionic and cationic trypsinogen mRNAs.

Amino Acid Sequence↗
PubMed1980

Molecular cloning of DNA complementary to a mouse alpha-fetoprotein mRNA sequence.

DNA complementary to mouse yolk sac messenger RNA has been inserted at the PstI site of the plasmid pBR322 by annealing of the oligo(dG)-tailed plasmid DNA with the oligo(dC)-tailed mouse DNA. Transformation of Escherichia coli strain RRI with this annealed DNA yielded clones bearing recombinant plasmids. The clones were screened for DNA complementary to mouse alpha-fetoprotein (AFP) messenger RNA sequences by hybridization with a cDNA probe transcribed from an AFP mRNA of over 90% purity. Out of nine plasmids that were isolated and analyzed by restiction mapping, all had homologous insert DNA of various lengths. The plasmid with the longest insert, pAF6, contained 1.65 kb of added DNA, which is about 70% of the AFP mRNA. This clone was positively identified by a hybridization-translation procedure to contain a cDNA sequence for AFP. A restriction map of this clone and the orientation of the message are presented.

Animals↗