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At least 937 records · Page 52Linked to original sources

PubMed1992

Proteolytic processing of a Murray Valley encephalitis virus non-structural polyprotein segment containing the viral proteinase: accumulation of a NS3-4A precursor which requires mature NS3 for efficient processing.

The proteolytic processing of a non-structural polyprotein segment from the cytoplasmic domain of NS2A to the C terminus of NS5 of Murray Valley encephalitis (MVE) virus was examined, when expressed from cDNA via a vaccinia virus recombinant, in transiently transfected COS cells, or synthesized by cell-free translation. Cleavages mediated by the virus-encoded proteinase domain in NS3 at the junctions of NS2A-2B, NS2B-3 and NS4B-5 were catalysed efficiently. However, the cleavage at the NS3-4A junction, also mediated by the NS3 proteinase, was greatly delayed. Little or no NS3 was found, but an 85K precursor molecule accumulated; this was identified as NS3-4A. Termination codons were introduced by site-directed mutagenesis at the junctions of the NS3-4A, NS4A-4B and NS4B-5 genes to generate C-terminal truncations of the MVE virus polyprotein segment. In expression studies of these constructs the predicted NS3-mediated proteolytic cleavages were catalysed, except for that at the NS3-4A junction. In co-infections and co-transfections with constructs encoding the MVE virus nonstructural polyprotein region truncated at the C termini of NS3 or NS4A, efficient processing at the NS3-4A site was induced. Thus it appears that the MVE virus polyprotein is cleaved inefficiently in cis at the NS3-4A junction, whereas the site is processed efficiently in trans by mature NS3. The NS3-4A precursor is also seen in flavivirus-infected cells. Its function remains to be determined, but it could play a role in the replication of flavivirus, in view of the importance of polyprotein processing in the regulation of gene expression of positive-stranded RNA viruses, the modulation of processing at the NS3-4A site by NS3 or NS3-containing precursors described in the present study and the importance of NS3 as an integral part of the viral polymerase complex.

Animals↗

PubMed1984

Molecular cloning of a cDNA for human pyruvate carboxylase. Structural relationship to other biotin-containing carboxylases and regulation of mRNA content in differentiating preadipocytes.

An oligonucleotide probe specific for the amino acid sequence at the biotin site in pyruvate carboxylase was used to screen a human liver cDNA library. Nine cDNA clones were isolated and three proved to be pyruvate carboxylase clones based on nucleotide sequencing and Northern blotting. The biotin site amino acid sequence of human pyruvate carboxylase agreed perfectly with that of the sheep enzyme in 14 consecutive positions. The highly conserved amino acid sequence, Ala-Met-Lys-Met, found at the biotin site in most biotin-containing carboxylases was also present in human pyruvate carboxylase. The termination codon was located 35 residues 3' to the lysine residue at which the biotin is attached. Therefore, the biotin cofactor is covalently linked near the carboxyl-terminal end of the carboxylase protein. These data are consistent with that observed for other biotin-containing carboxylases and strongly suggests that the genes encoding the biotin-containing carboxylases may have evolved from a common ancestral gene. Northern blotting of mRNA isolated from human, baboon, and rat liver demonstrated that the pyruvate carboxylase mRNA was 4.2 kilobase pairs in length in all species examined. Southern blot analysis of genomic DNA isolated from human-Chinese hamster somatic cell hybrids localized the pyruvate carboxylase gene on the long arm of human chromosome 11. The human cDNA was also used to quantitate pyruvate carboxylase mRNA levels in a differentiating mouse preadipocyte cell line. These data demonstrated that pyruvate carboxylase mRNA content increased 23-fold in 7 days after the onset of differentiation.

Adipose Tissue↗

PubMed1998

Molecular cloning and characterization of the bovine and human tuftelin genes.

The bovine tuftelin gene was cloned and its structure determined by DNA sequence analysis and comparison to bovine tuftelin cDNA. The analyses demonstrated that the cDNA contains a 1014 bp open reading frame encoding a protein of 338 residues with a calculated molecular weight of 38,630 kDa and an isoelectric point of 5.85. Although similar, these results differ from those previously published [Deutsch et al. (1991) J. Biol. Chem. 266, 16021-16028] which contained a different conceptual amino acid sequence for the carboxy terminal region and identification of a different termination codon because of the absence of a single guanine residue in the published sequence. The protein does not appear to share homology or domain motifs with any other known protein. The bovine gene consists of 13 exons ranging in size from 66 to 1531 bp, the latter containing the encoded carboxy terminal and 3' untranslated regions. These exons are embedded in greater than 28 kbp of genomic DNA and codons are generally not divided at exon/intron borders. Sequence analysis of the cDNA and products produced by reverse transcriptase/polymerase chain reaction demonstrated that exons 2, 5 and 6 are alternatively spliced. The 3' portion of the human gene was also isolated and characterized by DNA sequencing, which demonstrated agreement between the bovine and human sequences in the segment in question. The difference between the presently reported sequence and that of the previously published one suggests the possibility of an unusual type of polymorphism which would result in markedly different amino acid sequences at the carboxy terminal region of the protein. The human tuftelin gene was localized to chromosome 1q21 by in situ hybridization.

Alternative Splicing↗

PubMed1992

Gene family for an elicitor-induced sesquiterpene cyclase in tobacco.

The initial step in the conversion of the isoprenoid intermediate farnesyl diphosphate to the sesquiterpenoid phytoalexin capsidiol in elicitor-treated tobacco tissues is catalyzed by an inducible sesquiterpene cyclase [5-epi-aristolochene synthase (EAS)]. Two independent cDNA clones (cEAS1 and cEAS2) encoding EAS were isolated from an elicitor-induced tobacco cDNA library by differential hybridization and subsequently were characterized by hybrid selection--in vitro translation. Insertion of cEAS1, a partial cDNA clone encoding 175 C-terminal amino acids, into an Escherichia coli expression vector resulted in accumulation of a fusion protein immunodetectable with EAS-specific polyclonal antibodies. The cDNA clones were used to isolate two full-length EAS genes that mapped 5 kilobases (kb) apart on one 15-kb genomic clone. The nucleotide sequences of the structural gene components were identical from 388 base pairs (bp) upstream of the transcription initiation site to 40 bp downstream of the translation termination codon, suggesting a relatively recent duplication event. The genes consist of 1479-bp open reading frames, each containing five introns and specifying 56,828-Da proteins. The N-terminal amino acid sequence deduced from the genomic clones was identical to the first 16 amino acids of the EAS protein identifiable by Edman degradation. RNA blot hybridization with cEAS1 demonstrated a mRNA induction time course consistent with the induction of the EAS mRNA translational activity with maximum levels 4-6 h after elicitation. EAS mRNA was not detected in control cells. DNA blot-hybridization analysis of genomic DNA revealed a copy number of approximately 12-15 for EAS-like genes in the tetraploid tobacco genome. The conservation of a putative allelic prenyl diphosphate binding motif is also discussed.

Alkyl and Aryl Transferases↗

PubMed1997

Novel HEXA mutation in a Bedouin Tay-Sachs patient associated with exon skipping and reduced transcript level.

An Israeli-Bedouin infant from a consanguineous family was diagnosed with Tay-Sachs disease (TSD). The patient was found to carry the novel +3tIVS4 mutation in homozygosity. Direct sequencing of the cDNA showed that the +3tIVS4 mutation caused complete skipping of exon 4 resulting in a stop codon 17 bp downstream. We postulate that the insertion disrupts base pairing between the consensus sequence and U1 snRNA causing exon skipping. The resultant transcript contains a premature termination codon and is severely reduced in amount. An extended nucleotide portion of the 5' end of IVS4 was sequenced.

Arabs↗

PubMed1999

The cleavable carboxyl-terminus of the small coat protein of cowpea mosaic virus is involved in RNA encapsidation.

The site of cleavage of the small coat protein of cowpea mosaic virus has been precisely mapped and the proteolysis has been shown to result in the loss of 24 amino acids from the carboxyl-terminus of the protein. A series of premature termination and deletion mutants was constructed to investigate the role or roles of these carboxyl-terminal amino acids in the viral replication cycle. Mutants containing premature termination codons at or downstream of the cleavage site were viable but reverted to wild-type after a single passage through cowpea plants, indicating that the carboxyl-terminal amino acids are important. Mutants with the equivalent deletions were genetically stable and shown to be debilitated with respect to virus accumulation. The specific infectivity of preparations of a deletion mutant (DM4) lacking all 24 amino acids was 6-fold less than that of a wild-type preparation. This was shown to be a result of DM4 preparations containing a much increased percentage (73%) of empty (RNA-free) particles, a finding that implicates the cleavable carboxyl-terminal residues in the packaging of the virion RNAs.

Binding Sites↗

PubMed2003

Identification of two novel mutations in the human growth hormone receptor gene.

Deletions and mutations in the growth hormone receptor gene are the underlying etiology of Laron syndrome (LS). Most of the patients are distributed in and originating from the Mediterranean and Middle-Eastern countries. Thirty-nine mutations have been described so far. We hereby report 2 novel nonsense mutations, one in exon 2 found in three Jewish-Iraqi patients from Israel; and another in exon 6 found in an Italian girl. DNA sequencing of exon 2 revealed a G to A transition at nucleotide 83 in the fourth codon of the signal peptide (W-15X). In exon 6, a T to A transversion was found in amino acid 141 (L141X). Both mutations introduced a premature termination codon that led to a truncated non-functioning receptor. In addition we found in the Jewish-Iraqi patients, a mutation in exon 7 (R211H, previously described) and in the Italian family the polymorphism Gly168, in exon 6.

Body Height↗

PubMed2006

A novel therapeutic approach for genetic diseases by introduction of suppressor tRNA.

The appearance of the premature translation termination codons (PTCs) in the transcript is the major cause of human genetic diseases. PTC-containing transcripts are rapidly degraded through nonsense-mediated decay (NMD) pathway. If such mRNA transcripts were translated in frame like normal transcripts, it would afford not only restoration of the level of full-length protein but also prevention of mRNA degradation by the NMD pathway. Here we describe a novel approach to read through PTC-containing mRNAs using suppressor tRNA that is introduced to cells by transfection. Luciferase reporter gene assay showed that nonsense and four-base codons were suppressed by the corresponding suppressor tRNAs derived from human tRNA(Ser). We also demonstrated that transfection of the suppressor tRNA to Ullrich disease fibroblasts, possessing a frameshift mutation in the collagen VI alpha2 gene, induced the upregulation of the collagen VI alpha2 mRNA and accumulation of the collagen VI protein. PTC suppression potentially provides a novel therapeutic means to rescue various PTC-related diseases.

Anticodon↗

PubMed1992

A putative two-component regulatory system involved in secondary metabolism in Streptomyces spp.

A DNA fragment stimulating actinorhodin, undecylprodigiosin, and A-factor production in Streptomyces lividans 66 was cloned from Streptomyces coelicolor A3(2). Nucleotide sequencing revealed the presence of an open reading frame of 225 codons, named afsQ1, that showed great similarity in amino acid sequence to the response regulators of typical prokaryotic two-component regulatory systems responsible for adaptive responses. The termination codon, TGA, of afsQ1 overlapped the initiation codon, GTG, of a second open reading frame, afsQ2, of 535 codons. The afsQ2 gene product showed homology with the sensory histidine protein kinases of two-component systems. In agreement with the assumption that the AfsQ1 and AfsQ2 proteins comprise an aspartate-histidine phosphotransfer system, an amino acid replacement from Asp to Glu at residue 52 of AfsQ1, generated by site-directed mutagenesis, resulted in loss of the protein's ability to stimulate antibiotic production in S. lividans. Primer extension experiments indicated that transcription of the afsQ1 and afsQ2 genes initiates at the translational start codon (GTG) of the afsQ1 gene. The afsQ1 and afsQ2 genes were physically mapped at a chromosomal position near the actinorhodin biosynthetic gene cluster (act) by hybridization to Southern blots of restriction fragments separated by pulsed-field gel electrophoresis. Disruption of either afsQ1 or afsQ2 on the S. coelicolor chromosome by use of phage phi C31KC515 led to no detectable change in secondary metabolite formation or morphogenesis. The afsQ1 gene on pIJ922 suppressed the S. coelicolor absA mutation and caused actinorhodin production but did not suppress the absB mutation. Southern blot hybridization showed that sequences homologous to afsQ1 and afsQ2 are present in almost all of the actinomycetes examined.

4-Butyrolactone↗

PubMed1991

Characterization of the genome of rice tungro bacilliform virus: comparison with Commelina yellow mottle virus and caulimoviruses.

Rice tungro disease is caused by an infection of two different viruses, rice tungro spherical virus (a (+) sense RNA virus) and rice tungro bacilliform virus (RTBV) with a genome of circular double-stranded DNA. The genome of an RTBV isolate from the Philippines was cloned, sequenced, and found to be 8000 bp in length. It contains four open reading frames (ORFs) on a single strand, with ORF 1 having an internal termination codon (TAA). The 5' and 3' ends of a polyadenylated viral RNA transcript, of genome length, were mapped by primer extension and cDNA sequence analysis, respectively. The transcript is terminally redundant by 265-268 nucleotides. Purified virus particles contain two major proteins with molecular masses of 37 and 33 kDa, although only the 37-kDa protein was detected in the infected rice tissues. The N-terminal amino acid sequence of the 33-kDa protein was determined and its coding region was identified on the RTBV genome. The identity of the coat protein gene was further confirmed by expressing a region of the genome in Escherichia coli, the products of which reacted with anti-RTBV antibody. The unusually long ORF 3 of RTBV is predicted to encode a polyprotein of 194.1 kDa that includes: the coat protein(s), viral proteinase, reverse transcriptase, and ribonuclease H. The sections of the polyprotein show varying degrees of similarity to the counterparts of Commelina yellow mottle virus (a member of the proposed badnavirus group) and caulimoviruses. The functions of the other three ORFs are unknown.

Amino Acid Sequence↗

PubMed2006

An mRNA sequence derived from a programmed frameshifting signal decreases codon discrimination during translation initiation.

Sequences and structures in the mRNA can alter the accuracy of translation. In some cases, mRNA secondary structures like hairpin loops or pseudoknots can cause frequent errors of translational reading frame (programmed frameshifting) or misreading of termination codons as sense (nonsense readthrough). In other cases, the primary mRNA sequence stimulates the error probably by interacting with an element of the ribosome to interfere with error correction. One such primary mRNA sequence, the Ty3 stimulator, increases programmed +1 frameshifting 7.5 times in the yeast Saccharomyces cerevisiae. Here we show that this stimulator also increases the usage of non-AUG initiation codons in the bacterium Escherichia coli but not in S. cerevisiae. These data suggest that in E. coli, though not in yeast, an element of the ribosome's elongation accuracy mechanism ensures initiation accuracy.

Base Sequence↗

PubMed1992

Molecular characterization of beta-thalassemia in Czechoslovakia.

We have identified different beta-thalassemia mutations in 93 members of 34 families of Czech or Slovakian descent using gene amplification, hybridization with specific 32P-labeled oligonucleotide probes, sequencing of amplified DNA, and gene mapping. The G----A mutation at IVS-I-1 was found in 18 families; other Mediterranean mutations were IVS-II-1 (G----A), IVS-II-745 (C----G), IVS-I-110 (G----A), and codon 39 (C----T); these were present in 9 additional families. The G----T mutation at codon 121, known to cause Heinz-body beta-thalassemia, was present in 3 families, and the frameshift at codons 82/83 (-G), first described in the Azerbaijanian population, in 2 families. A newly discovered allele was a frameshift at codons 38/39 (-C). One beta-thalassemia allele was incompletely characterized. We observed in 2 families a T----C mutation at position +96 UTR (untranslated region) relative to the termination codon; this mutation likely is a rare polymorphism. alpha-Thalassemia was rare; only one person carried the -alpha 3.7 heterozygosity, and one other had a yet to be identified alpha-thalassemia-1, while seven had the alpha alpha alpha anti 3.7 triplication.

Base Sequence↗

PubMed1992

Precise mapping and in vitro translation of a trifunctional subgenomic RNA of barley yellow dwarf virus.

The barley yellow dwarf virus genome consists of a single 5.7-kb RNA encoding six open reading frames (ORFs). The four ORFs in the 3' half of the genome were proposed to be expressed via subgenomic mRNAs. Here, we show that the PAV serotype of barley yellow dwarf virus generates two subgenomic RNAs of 2.9 and 0.8 kb in infected plants and protoplasts. The 5' end of the larger subgenomic RNA (sgRNA1) was precisely mapped to base 2769 by ribonuclease protection and primer extension methods. Synthetic sgRNA1, containing the exact 5' and 3' ends, was generated by in vitro transcription, translated in vitro, and shown to serve as a message for three ORFs. Translation initiated at the first two AUG codons in sgRNA1, yielding the 22-kDa viral coat protein (CP) and a 17-kDa protein encoded by an overlapping, out-of-frame ORF. In addition, readthrough of the amber stop codon of the CP ORF gave rise to a 72-kDa fusion product of the CP ORF and an in-frame 50K ORF immediately 3' of the CP termination codon. The 0.8-kb sgRNA2 was present in greater abundance than sgRNA1 and likely serves as a message for a 6.7K ORF at the 3' end of the genome. Sequences that may control subgenomic RNA synthesis and the translational events are compared with those of other plant viruses.

Base Sequence↗

PubMed1993

Detection of a molecular defect in 40 of 44 patients with haemophilia B by PCR and denaturing gradient gel electrophoresis.

Oligonucleotides were computer designed to amplify by the polymerase chain reaction (PCR) the coding region, splice junctions, 112 bp of the 5' flanking region and 279 bp surrounding the polyadenylation site of the factor IX gene for analysis by denaturing gradient gel electrophoresis (DGGE). Forty-four unselected haemophilia B patients were studied of whom 24 had severe haemophilia and 20 had a mild to moderate form of the disease. Potential mutations were identified in 40 (91%) of the 44 cases. A defect could not be detected in three severe and one mild haemophiliac by DGGE analysis and direct sequencing of all the PCR fragments from these patients revealed no nucleotide alteration supporting the DGGE results. A total of 37 point mutations, two complete gene deletions and a duplication of 26 bp were found. The 37 point mutations included 35 single nucleotide substitutions, a deletion and an insertion of one nucleotide. The 35 single nucleotide substitutions included 26 missense mutations, seven nonsense mutations, a G (-6) to A transition in the promoter region and a G (30154) to A transition within the donor splice site of the last intron. Fifteen of these nucleotide substitutions involved CpG dinucleotides. Fifteen point mutations were found at codons where nucleotide substitutions had not been detected before. An insertion of a single nucleotide T at position 6370 and deletion of a G at nucleotide 30845 resulted in frameshift mutations creating stop codons at amino acid positions -2 and 250, respectively. A duplication of 26 bp (17747-17772) in exon V was found in a severe haemophilia patient resulting in a termination codon in exon VI. The detection of the mutation by the combined use of PCR, DGGE and direct sequencing was important for carrier diagnosis of 20 families with no prior history of haemophilia B.

Amino Acids↗

PubMed1985

Analyses of the mRNA transcription processes of snowshoe hare bunyavirus S and M RNA species.

The time course of synthesis of snowshoe hare bunyavirus small (S)- and medium (M)-sized viral RNA (vRNA), viral cRNA (vcRNA), and mRNA species was analyzed by using single-stranded DNA probes representing the S- and M-coded gene products. In the presence of puromycin, an inhibitor of protein synthesis, the subgenomic S mRNA species were detected, but not full-length S vcRNA or S vRNA species. No M-related RNA species were identified in puromycin-treated cells. In the absence of puromycin, full-length M and S vRNA, S vcRNA, and subgenomic S mRNA species were observed, as well as apparently full-length M vcRNA species, presumably including the approximately similar-sized M mRNA species. The 5' ends of the S and M mRNA species have been shown to be heterogeneous and some 12 to 17 bases longer than the ends of their corresponding presumptive replicative vcRNA species, in agreement with an earlier report that they represent nonviral primer sequences (D. H. L. Bishop, M. E. Gay, and Y. Matsuoko, Nucleic Acids Res. 11:6409-6418, 1983). The 3' ends of the M and S mRNA species were found to be shorter by some 60 and 100 nucleotides, respectively, than those of their corresponding full-length vcRNA species. Comparison of the 3' noncoding regions of the S and M vcRNA species revealed that there are conserved sequences following the translation termination codons of the two RNA species. One of these conserved sequences is a pyrimidine-rich template sequence that is approximately 20 nucleotides beyond the deduced S mRNA transcription termination site.

Base Sequence↗

PubMed1991

Regulatory elements of the erythropoietin gene.

Because the human hepatoma cell line Hep3B produces erythropoietin (Epo) in a regulated fashion, it can be used to investigate the cis-acting regulatory elements of the Epo gene. Comparison of primate and mouse sequences shows strong homology not only in the coding sequence but also within the 5' flanking region, the first intron, and the 3' flanking region. These portions of the Epo gene were inserted 5' and 3' to a reporter gene, human growth hormone (GH). 5A is a 1,192-base pair (bp) HindIII-Xbal fragment that extends from 378 bp 5' to the cap site through the first intron. To obviate the problem of false initiation of translation from the Epo ATG start codon, this site was changed to TAG by site-directed mutagenesis. 3A is a 255-bp Accl-BglII fragment that extends 67 bp upstream from the Epo termination codon and covers most of the 3' noncoding region of homology. The plasmid DNAs were transfected by electroporation into Hep3B cells with RSVCAT as an internal standard to correct for transfection efficiency. One aliquot of cells was exposed to 50 mumol/L CoCl2 or to 1% O2. At the end of the incubations, GH and Epo were measured in the cell media and the cell pellet was assayed for CAT. Production of GH was stimulated 1.7-fold by cobalt or hypoxia. Furthermore, addition of 3A to the GH gene, irrespective of orientation, stimulated GH production 2.6-fold with CoCl2 and 2.3-fold with hypoxia. Stable cell lines were produced by cotransfection of the above constructions, along with the selectable marker pSV-Neo. In two clones, exposure to hypoxia resulted in much more marked (16-fold) induction of GH. Stimulus of both GH and Epo production by hypoxia was partially abrogated by carbon monoxide. These results demonstrate the presence of promoter and enhancer elements within the human Epo gene that are appropriately responsive to hypoxia and cobalt.

Animals↗

PubMed1994

A conserved AUG triplet in the 5' nontranslated region of poliovirus can function as an initiation codon in vitro and in vivo.

Poliovirus translation is initiated at AUG743, 154 nt downstream of a conserved heptanucleotide CUUAUGG at the 3' border of the internal ribosome entry site. AUG586 is part of this motif and is normally not an initiation codon, but was activated following alteration of its context from CUUAUGG to ACCAUGG. Initiation at AUG586 was efficient and yielded a 7.2-kDa polypeptide translated in an open reading frame that overlapped AUG743 by 38 nt, but the presence of this activated codon reduced initiation at AUG743 by only 50%. Growth of a mutant poliovirus W1-5NC-1 containing the CUU-->ACC substitutions was impaired and was not alleviated by a termination codon placed four triplets downstream of AUG586 in the virus W1-5NC-2. The virus W1-5NC-6 contained the substitution U584A and had a similar sp phenotype; the phenotype of W1-5NC-1 is thus probably due to substitution within the conserved CUUAUGG motif per se rather than to activation of AUG586. A sp mutant virus W1-5NC-3 was derived from W1-5NC-1 by deletion of nt 588-745, indicating that AUG586 could initiate translation in vivo. These observations indicate that although AUG586 can be activated by upstream substitutions, it is nevertheless readily bypassed by ribosomes in mRNAs containing wt downstream elements, resulting in initiation at AUG743.

Base Sequence↗

PubMed1999

Mutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pigmentosa.

The autosomal dominant retinitis pigmentosa (RP) locus, designated RP1, has been mapped through linkage studies to a 4-cM interval at 8q11-13. Here we describe a new photoreceptor-specific gene that maps in this interval and whose expression is modulated by retinal oxygen levels in vivo. This gene consists of at least 4 exons that encode a predicted protein of 2,156 amino acids. A nonsense mutation at codon 677 of this gene is present in approximately 3% of cases of dominant RP in North America. We also detected two deletion mutations that cause frameshifts and introduce premature termination codons in three other families with dominant RP. Our data suggest that mutations in this gene cause dominant RP, and that the encoded protein has an important but unknown role in photoreceptor biology.

Amino Acid Sequence↗