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PubMed1988

Translation initiation potential of the 5' proximal AUGs of the polycistronic P/C mRNA of Sendai virus. A multipurpose vector for site-specific mutagenesis.

To determine the translation initiation potential of the 5' proximal four AUGs of the polycistronic P/C mRNA of Sendai virus, we have developed an efficient system for oligonucleotide-directed site-specific mutagenesis utilizing a plasmid vector which contains the replication origin of the single-stranded DNA phage f1 and the phage SP6 promoter. Utilizing uridine containing single-stranded DNA templates of the plasmid vector carrying the P/C gene we introduced point mutations in all four of the putative initiator codons (AUGs) of the P/C gene. Based on the analysis of in vitro translation products from the SP6 polymerase-directed transcripts of the mutants, the first and second AUG codons were assigned to the P and C proteins, respectively. Proteins detected in vitro, Y1 and Y2, initiated from the third and fourth AUG codons, respectively, and originated within the C reading frame. Since the C' protein is encoded in the same reading frame as the C protein but did not initiate from any of the 5' proximal AUGs, the synthesis of the C' protein may start from a non-AUG initiator codon in the P/C gene.

Base Sequence↗

PubMed1984

Structure and expression of a pea nuclear gene encoding a chlorophyll a/b-binding polypeptide.

A nuclear gene AB80 has been isolated from a phage lambda Charon 4 library of pea DNA. The sequence of the gene has been determined and it has been shown to contain an uninterrupted reading frame of 269 amino acids, corresponding to a precursor to a constituent polypeptide of the light-harvesting chlorophyll a/b-protein complex. Primer extension and S1 nuclease studies defined a cap site for AB80. The first methionine codon 3' from this site is 69 nucleotides away and is the initiating codon of the open reading frame. A "TATA" sequence occurs 31 nucleotides 5' from the cap site. A second TATA sequence is found 7 nucleotides on the 5' side of the initiating methionine codon and the sequences surrounding this TATA sequence are strikingly similar to those surrounding the first TATA sequence. The mature polypeptide encoded by AB80 differs by 5 amino acids from the polypeptide corresponding to a previously characterized cDNA sequence pAB96. This result is indicative of heterogeneity within the constituent polypeptides of the light-harvesting chlorophyll a/b-protein complex. The sequence Arg-Lys-Ser-Ala-Thr-Thr-Lys-Lys occurs at, or near, the NH(2)-terminus of the mature polypeptide encoded by AB80. This basic peptide is of interest because of its apparent involvement in changes in excitation-energy distribution in chloroplast membranes. Some general similarities, but no extensive sequence homology, is found on comparing the transit sequence for the precursor to the chlorophyll a/b-binding polypeptide with the transit sequences previously determined for the precursors to the small subunit of ribulose-1,5-bisphosphate carboxylase.

Journal Article↗

PubMed1999

Translation initiation at the CUU codon is mediated by the internal ribosome entry site of an insect picorna-like virus in vitro.

AUG-unrelated translation initiation was found in an insect picorna-like virus, Plautia stali intestine virus (PSIV). The positive-strand RNA genome of the virus contains two nonoverlapping open reading frames (ORFs). The capsid protein gene is located in the 3'-proximal ORF and lacks an AUG initiation codon. We examined the translation mechanism and the initiation codon of the capsid protein gene by using various dicistronic and monocistronic RNAs in vitro. The capsid protein gene was translated cap independently in the presence of the upstream cistron, indicating that the gene is translated by internal ribosome entry. Deletion analysis showed that the internal ribosome entry site (IRES) consisted of approximately 250 bases and that its 3' boundary extended slightly into the capsid-coding region. The initiation codon for the IRES-mediated translation was identified as the CUU codon, which is located just upstream of the 5' terminus of the capsid-coding region by site-directed mutagenesis. In vitro translation assays of monocistronic RNAs lacking the 5' part of the IRES showed that this CUU codon was not recognized by scanning ribosomes. This suggests that the PSIV IRES can effectively direct translation initiation without stable codon-anticodon pairing between the initiation codon and the initiator methionyl-tRNA.

Amino Acid Sequence↗

PubMed2002

Identification of an essential pseudoknot in the putative downstream internal ribosome entry site in giardiavirus transcript.

Enhanced translation of giardiavirus-luciferase chimeric mRNA in Giardia lamblia requires the initial 264-nt viral capsid coding region as a putative internal ribosomal entry site (IRES). Essential structural elements in this site include (1) a downstream box (DB) complementary to the anti-DB at the 3' end of 16S-like rRNA, (2) stem-loops I, II, III, and IVA, and (3) a pentanucleotide 5'-UCUCC-3' immediately downstream from stem loop IVA. A search for the structural role of the pentanucleotide suggested that it may form a pseudoknot with another pentanucleotide 5'-GGAGA-3' in loop II. Alteration of the two pentanucleotides by site-directed mutagenesis resulted in a drastic reduction in translation of the transcript. But the loss was recovered by compensatory changes in the two sequences, suggesting Watson-Crick base pairings between them. Results from in vitro enzymatic and chemical structural probing supported the presence of such a pseudoknot 143 nt downstream from the initiation codon. Minor repositioning of this codon led invariably to a complete loss of translation, suggesting that the initiation site is confined within a rigid position defined by all the structural elements in the IRES including the pseudoknot. This is the first pseudoknot of its kind shown to play an important role in a downstream IRES of a viral transcript. The finding is particularly interesting because it could reflect a unique feature of translation initiation in Giardia, which is known to have exceedingly short (1-6 nt) 5' untranslated regions in its mRNAs.

Animals↗

PubMed1993

Superinfection exclusion (sieB) genes of bacteriophages P22 and lambda.

The superinfection exclusion gene (sieB) of Salmonella phage P22 was mapped with phage deletion mutants. The DNA sequence in the region was reexamined in order to find an open reading frame consistent with the deletion mapping. Several discrepancies with the previously published sequence were discovered. The revised sequence revealed a single open reading frame of 242 codons with six likely translation initiation codons. On the basis of deletion and amber mutant phenotypes, the second of these six sites was inferred to be the translation initiation site of the sieB gene. The sieB gene encodes a polypeptide with 192 amino acid residues with a calculated molecular weight of 22,442, which is in reasonable agreement with that estimated from polyacrylamide gels. The transcription start site of sieB was identified by the use of an RNase protection assay. The sieB promoter thus identified was inactivated by a 2-base substitution in its -10 hexamer. The sieB gene of coliphage lambda was also identified. The promoter for lambda sieB was identified by homology to that of P22 sieB.

Amino Acid Sequence↗

PubMed2005

Physical and biochemical properties of mammalian DNase X proteins: non-AUG translation initiation of porcine and bovine mRNAs for DNase X.

DNase X is the first human DNase protein identified as being homologous with DNase I. In the present study we describe the isolation of several mammalian DNase X cDNAs and the molecular characterization of their coding proteins. A sequence comparison reveals some conserved characteristics: all the mammalian DNase X proteins have an N-terminal signal peptide, a potential N-linked glycosylation site and a C-terminal hydrophobic domain. Human DNase X, ectopically expressed in HeLa S3 cells, is located in the ER (endoplasmic reticulum) and is modified by an N-linked glycosylation at Asn-243. Gene expression analyses show that the high expression level in muscular tissues, a known feature of human DNASE X, is also observed in mouse DNase X. Interestingly, the translation of porcine and bovine DNase X proteins occurs in the absence of an in-frame AUG initiation codon. We show that their mRNAs utilize a conserved CUG triplet for translation initiation.

Amino Acid Sequence↗

PubMed1985

Cloning and nucleotide sequence of the trxA gene of Escherichia coli K-12.

The Escherichia coli K-12 trxA gene, the gene encoding thioredoxin, has been cloned and sequenced. The DNA sequence includes 280 base pairs upstream and 46 base pairs downstream of the coding region. The downstream sequence contains the -35 region of the promoter of the rho gene. Northern analysis of the trxA mRNA and S1 nuclease mapping indicate the presence of two promoters for the trxA gene. Initiation from either promoter results in an mRNA containing two potential translation initiation codons, one of which could initiate synthesis of a protein 18 amino acids longer than the mature trxA gene product. The 3' end of the gene, including the last eight codons, contains a stable stem-loop structure (delta G = -12.9 kcal) typical of a rho-independent transcription termination signal.

Amino Acid Sequence↗

PubMed1984

Peptide maps and N-terminal sequences of polypeptides from early region 1A of human adenovirus 5.

Experiments exploring the reasons for a multiplicity of products from early region 1A of adenovirus 5 are described. Labeled early region 1A products from wild-type virus were synthesized in infected cells and in a cell-free system programmed with mRNA from infected cells, immunoprecipitated specifically with an antipeptide serum, E1A-C1, directed against the C-terminal sequence of E1A products, and separated by gel electrophoresis. Two-dimensional maps of [35S]methionine-labeled peptides were consistent with antigens of 52,000 daltons (52K) and 48.5K being from the 13S mRNA and antigens of 50K, 45K, and 35K from the 12S mRNA. Partial N-terminal sequences of 52K, 50K, 48.5K, and 45K synthesized in vitro showed that each of these antigens was initiated at the predicted ATG at nucleotide 560 in the DNA sequence. These results eliminate multiple initiation sites and proteolytic cleavage at the N-terminal end as sources of antigen diversity. Peptide maps and N-terminal sequences were obtained in a similar way for E1A products from the Ad5 deletion mutant dl1504, which lacks the normal initiator codon. As predicted, these polypeptides are initiated at the next ATG, 15 codons downstream in the wild-type sequence. These results are discussed in relation to Kozak's ribosomal scanning model.

Adenoviruses, Human↗

PubMed1990

Ribosomal frameshifting in the yeast retrotransposon Ty: tRNAs induce slippage on a 7 nucleotide minimal site.

Ribosomal frameshifting regulates expression of the TYB gene of yeast Ty retrotransposons. We previously demonstrated that a 14 nucleotide sequence conserved between two families of Ty elements was necessary and sufficient to support ribosomal frameshifting. This work demonstrates that only 7 of these 14 nucleotides are needed for normal levels of frameshifting. Any change to the sequence CUU-AGG-C drastically reduces frameshifting; this suggests that two specific tRNAs, tRNA(UAGLeu) and tRNA(CCUArg), are involved in the event. Our tRNA overproduction data suggest that a leucyl-tRNA, probably tRNA(UAGLeu), an unusual leucine isoacceptor that recognizes all six leucine codons, slips from CUU-Leu onto UUA-Leu (in the +1 reading frame) during a translational pause at the AGG-Arg codon induced by the low availability of tRNA(CCUArg), encoded by a single-copy essential gene. Frameshifting is also directional and reading frame specific. Interestingly, frameshifting is inhibited when the "slip" CUU codon is located three codons downstream, but not four or more codons downstream, of the translational initiation codon.

Amino Acid Sequence↗

PubMed1997

A whole genome shotgun gene fusion method for isolation of translation initiation sites in Escherichia coli: identification of Haemophilus influenzae translation initiation sites in E. coli.

We have developed a new method for isolating translation initiation sites based on the expression of Haemophilus influenzae Rd gene fusions with the Escherichia coli galactokinase (galK) gene. We cloned random DNA fragments of H. influenzae Rd DNA into a plasmid vector containing the galK coding sequence from which the translation initiation site (the ribosome binding site and translation initiation codon) had been removed. A subset of the cloned DNA fragments contained translation initiation sites that, when fused to the galK gene, produced active galactokinase and complemented the host galK mutation. Molecules expressing galactokinase activity were isolated and characterized by DNA sequence analysis, and the sequences were aligned with the recently completed whole genomic sequence of H. influenzae Rd. Translation initiation sites for known, hypothetical, and new genes were identified. Translation initiation sites internal to the coding sequences of a number of genes were identified, suggesting that internal translation initiation sites are common, especially in large genes. This shotgun method provides functional information on translation initiation sites and helps to define gene coding sequences.

Amino Acid Sequence↗

PubMed1991

AUG codons in the RNA leader sequences of the yeast PET genes CBS1 and SCO1 have no influence on translation efficiency.

We report that the major transcription start sites of the yeast PET gene SCO1 are located at positions -149 and -125 relative to the AUG initiation codon of the SCO1 reading frame. The leader sequences of the resulting mRNAs possess a single AUG codon at position -49, which initiates a short open reading frame of three amino acids. The recent finding of a similar situation in the case of the PET gene CBS1 prompted us to address the question as to whether these AUG codons might play some role in the expression of these PET genes. After removal of the upstream AUG codons by site-directed mutagenesis, expression was monitored by use of lacZ fusions and compared to the respective wild-type constructs. Our data show that under all growth conditions tested the leader-contained AUG initiation codons have no significant influence on the expression of both PET genes.

Amino Acid Sequence↗

PubMed1979

Nucleotide sequence coding for the N-terminal region of the matrix protein influenza virus.

After polyadenylation in vitro of the influenza virus RNA segment which contains the coding information for the matrix protein, a cDNA copy can be made using the primer p(dT)8-dA and reverse transcriptase. The sequence of 166 nucleotides of the cDNA was determined by a modification [Brownlee, G. G. & Cartwright, E. M. (1977) J. Mol. Biol, 114, 93--117] of the plus/minus method [Sanger, F. & Coulson, A. R. (1975) J. Mol. Biol. 94, 441--481] and adaptation of the "dideoxy" method [Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. U.S.A. 74, 5463--5467] for sequencing DNA. The cDNA sequences is of the same sense as the mRNA for matrix protein and contains a potential initiating codon, d(ATG), at position 26--28. When matrix protein purified from virus particles was digested with chymotrypsin or trypsin and the amino acid compositions of separated peptides determined, one peptide containing nine amino acids found which had a composition corresponding to that predicted by the cDNA sequence following the first methionine codon, confirming that protein synthesis initiates at this position. The compositions of four other peptides matches those predicted from the nucleotide sequence. There is no processing of the N terminus of the protein before incorporation into the virus particle except for removal of the N-terminal methionine and addition of a "blocking" group on the resulting N-terminal serine residue.

Amino Acid Sequence↗

PubMed2006

Variation in retinitis pigmentosa-11 (PRPF31 or RP11) gene expression between symptomatic and asymptomatic patients with dominant RP11 mutations.

Dominant mutations in the mRNA splicing factor gene PRPF31 (RP11) cause retinitis pigmentosa with reduced penetrance. We studied the expression of RP11 in lymphoblast cell lines from 10 patients, including three who were clinically asymptomatic, with six distinct RP11 mutations. Five of the six mutations were characterized and all five created premature nonsense codons or eliminated the normal initiation codon. Semiquantitative RT-PCR indicated that an average of only 17% of the RP11 mRNA was derived from the mutant allele, likely because the mutant mRNA transcripts were degraded by nonsense-mediated decay. Gene expression levels were measured by Affymetrix and CodeLink microarrays and, for RP11 transcripts, also by real-time PCR. Combined wild-type-plus-mutant RP11 mRNA expression from symptomatic patients was 52 to 77% of that in controls (p < or = 0.0005). Clinically asymptomatic carriers had levels of RP11 mRNA similar to controls and 29-42% higher than in clinically affected patients (0.0001<p<0.05, varying according to measurement technique). Expression levels of seven housekeeping genes (4-15 exons each) and 11 single-exon histone genes showed no substantial differences between affected patients with RP11 mutations and controls. Our results indicate that penetrance of dominant RP11 mutations correlates with the expression level of the remaining wild-type RP11 allele. Because RP11 mutations are apparently null alleles and because nonpenetrance correlates with high wild-type allele expression, the phenotypic effect of RP11 mutations is likely due to haploinsufficiency. The similar mRNA expression levels from genes with and without introns suggest that there is no generalized RNA splicing abnormality in RP11 patients.

Alleles↗

PubMed1995

Site-specific factor involved in the editing of the psbL mRNA in tobacco plastids.

In tobacco plastids, functional psbL mRNA is created by editing an ACG codon to an AUG translation initiation codon. To determine if editing may occur in a chimeric mRNA, the N-terminal part of psbL containing the editing site was translationally fused with the aadA and kan bacterial genes. The chimeric constructs were introduced into the tobacco plastid genome by targeted gene insertion. Editing of the chimeric mRNAs indicated that the 98 nt fragment spanning the psbL editing site contains all cis information required for editing. Expression of the chimeric gene transcripts led to a significant decrease in the editing efficiency of the endogenous psbL mRNA. However, the efficiency of editing in the transplastomic lines was unchanged for four sites in the rpoB and ndhB mRNAs. Reduced efficiency of psbL editing, but not of the other four sites, in the transplastomic lines indicates depletion of psbL-specific editing factor(s). This finding implicates the involvement of site-specific factors in editing of plastid mRNAs in higher plants.

Amino Acid Sequence↗

PubMed1990

A precore-defective mutant of hepatitis B virus associated with e antigen-negative chronic liver disease.

The pathogenesis of chronic liver disease (CLD) due to persistent hepatitis B virus (HBV) infection has not been defined, but the disease activity is believed to correlate with the presence of hepatitis B e-antigen (HBeAg) antigenemia and high viremia. The molecular characterization of an HBV mutant isolated from an HBeAg-negative patient with severe CLD required amplification of the circulating HBV DNA (2 pg/ml) by the polymerase chain reaction (PCR). Direct sequencing of the nucleotides from five independent amplifications of the conserved precore region consistently revealed a G to A mutation in each of the two terminal codons of the precore region. Codon 28 was mutated from tryptophan-encoding TGG to a translational stop codon, TAG; codon 29 preceding the core initiation codon was changed from GGC to GAC. For biologic evaluation of these mutations on HBV replication and expression of HBeAg in vitro, HepG2 cells were transfected with cloned, recircularized mutant HBV DNA. The transfected cells contained subviral core particles in the cytoplasm and secreted mature HBV, without HBeAg, into the medium. The findings present the first evidence that complete HBV genomes can be amplified by PCR and are replication-competent in vitro. The data also indicate that HBeAg is not necessary for replication of HBV and furthermore suggest that HBeAg is not required for the progression of HBV-induced CLD.

Adult↗

PubMed1994

Molecular cloning of the Arabidopsis thaliana sedoheptulose-1,7-biphosphatase gene and expression studies in wheat and Arabidopsis thaliana.

We report here the isolation and nucleotide sequence of genomic clones encoding the chloroplast enzyme sedoheptulose-1,7-bisphosphatase (SBPase) from Arabidopsis thaliana. The coding region of this gene contains eight exons (72-76 bp) and seven introns (75-91 bp) and encodes a polypeptide of 393 amino acids. Unusually, the 5' non-coding region contains two additional AUG codons upstream of the translation initiation codon. A comparison of the deduced Arabidopsis and wheat SBPase polypeptide sequences reveals 78.6%, identity. Expression studies showed that the level of SBPase mRNA in Arabidopsis and wheat is regulated in a light-dependent manner and is also influenced by the developmental stage of the leaf. Although the Arabidopsis SBPase gene is present in a single copy, two hybridizing transcripts were detected in some tissues, suggesting the presence of alternate transcription start sites in the upstream region.

Amino Acid Sequence↗

PubMed1985

The gene for yeast ribosomal protein S31 contains an intron in the leader sequence.

Analysis of the primary structure of the gene for yeast ribosomal protein S31 revealed two unusual features. First, an intron of 312 nucleotides is located within the 5'-untranslated region. Second, the coding sequence for the known amino-terminal peptide of the protein starts 13 codons downstream of the ATG initiation codon, suggesting that S31 is synthesized as a precursor which undergoes post-translational processing to the mature protein. Primer extension analysis showed that transcription of the S31 gene starts at multiple sites. The 5'-flanking region of the gene contains several, previously described, conserved sequence elements that may play a role in the coordinate expression of yeast ribosomal protein genes.

DNA Restriction Enzymes↗

PubMed1996

Expression of the thioredoxin gene (trxA) in Rhodobacter sphaeroides Y is regulated by oxygen.

The structural gene (trxA) coding for thioredoxin in the photosynthetic bacterium Rhodobacter sphaeroides has been cloned and sequenced previously. In the present study, the role of oxygen in trxA expression in R. sphaeroides Y was investigated using mRNA analyses and plasmid-borne trxA'-lacZ+ translational and transcriptional fusions. Northern analysis revealed a trxA-specific transcript of approximately 420-460 nucleotides, indicating that trxA is transcribed as a single gene. By studying the beta-galactosidase activity in strains harboring various phi(trxA'-lacZ+) fusion constructs, the promoter region of the trxA gene was localized within a 64-bp region located 97 nucleotides upstream of the trxA initiator codon. A single trxA transcription initiation site was mapped by primer extension, 27 bp upstream of the trxA gene. Based on these results and the DNA sequence analysis, we propose that a sigma70 consensus sequence serves as a trxA promoter. Results from oxygen shift experiments, as deduced from both mRNA analysis and fusions of the trxA promoter region to lacZ indicate that transcription of the R. sphaeroides trxA gene is regulated by high oxygen tension. DNA sequences involved in this oxygen regulation were also localized in the 64-bp region containing the trxA promoter. Based on our findings the hypothetical biological function of thioredoxin from R. sphaeroides is discussed.

Base Sequence↗