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PubMed1993

Conservation of gene structure and arrangement between vaccinia virus and orf virus.

A 3.3-kb BamHI fragment from the center of the orf virus (OV) NZ2 genome has been sequenced, revealing three major open reading frames (ORFs) with homology to vaccinia virus (VAC) genes. These ORFs have been designated F2L, F3R, and F4R and the proteins they encode were found to be homologous to VAC genes H4L (RNA polymerase-associated protein RAP94), H5R (35-kDa virion envelope antigen) and H6R (topoisomerase), respectively. The OV ORFs are arranged on the genome in an almost identical manner to their VAC counterparts revealing the common evolutionary origin of the two viruses despite the extreme difference in their G+C content. Like its VAC counterpart, F3R was shown to be transcribed early and late during infection. S1 and primer extension analysis located the 5' ends of F3R early transcripts to a position 15-16 nt and 5-10 nt, respectively, downstream from an AT-rich sequence resembling a VAC early promoter. The 5' ends of F3R late transcripts were located to an A within the sequence 5'-TAAAG, 41 nt downstream from the early promoter and 17 nt upstream from the initiation codon. S1 analysis of F2L, which is transcribed only late in infection, revealed transcripts initiating from within the sequence 5'-TAAATG. No transcriptional start point could be detected for F4R but the VAC late transcriptional initiation sequence TAAAT was found close to the predicted translational start point. Another late promoter-like sequence, 5'-TAAATG, was found at the 3' end of F2L. This preceded a short ORF tentatively designated as F1L and predicted to be the beginning of a homologue of VAC H3L.

Amino Acid Sequence↗

PubMed1991

The delta subunit of the chloroplast ATPase is plastid-encoded in the diatom Odontella sinensis.

A 5.2 kb PstI restriction fragment containing the atpA gene cluster of the plastic genome of the centric diatom Odontella sinensis was cloned. Sequencing revealed a reading frame of 561 bp separating the genes atpF and atpA, which is preceded by a putative ribosome binding site. The third nucleotide of the codon for the last amino acid of atpF is the first nucleotide of the initiation codon of the 561 bp reading frame. The amino acid sequence deduced from the nucleotide sequence of this gene (atpD) is colinear with delta subunits of different F0F1-ATPases and shows an overall sequence homology of up to 35% when compared with the sequences of cyanobacteria and Cyanophora paradoxa. The results are discussed in context with the evolution of chloroplasts of the chlorophyll-a + b and -a + c lineages, respectively.

Amino Acid Sequence↗

PubMed1991

Prevalence and type of pre-C HBV mutants in anti-HBe positive carriers with chronic liver disease in a highly endemic area.

The sequence variability in the pre-C region of the hepatitis B virus (HBV) genome in the serum of 42 anti-HBe antibody positive carriers with chronic hepatitis B was studied by PCR and direct sequencing to determine prevalence and type of HBV pre-C mutants in a highly endemic area. Except for one, all patients were infected with viruses containing mutations in the pre-C region which prevent precore and e-antigen (HBeAg) expression: 33 were infected predominantly or exclusively with variants containing a stop codon; two had a mixture of wild-type and a pre-C stop codon mutant virus; three had precore variants with mutations of the pre-C initiation codon and two of them an additional stop codon; four had a frameshift mutation; and one had two stop codons. One patient was infected with viruses which contained a mutation creating an amino acid exchange which should not prevent precore and HBeAg expression. These data demonstrate that in an endemic area a higher prevalence and even broader spectrum of pre-C HBV mutants are found than has been recognized previously in anti-HBe positive patients with chronic hepatitis B.

Adult↗

PubMed1995

Role of the pyrimidine-rich tract on the translation of 'chimeric' polio-hepatitis A mRNAs with engineered 5'-terminal untranslated regions.

The 5'-terminal untranslated region (5'-UTR) of picornavirus RNA contains a series of cis-acting elements required for the internal initiation of translation, including a pyrimidine-rich tract (PRT), which in entero- and rhinoviruses is located about 20 nts upstream from a silent AUG triplet in the vicinity of the translation initiation site. In hepatitis A virus (HAV) RNA, the PRT is only 12 nts upstream from the legitimate AUG initiation codon, and a second, longer PRT in a region far removed from the translation initiation site. This 5'-distal PRT includes a 'core' sequence 80% homologous to the PRT of poliovirus RNA. A 'chimeric' polio-hepatitis A mRNA was constructed in which the sequences extending between nucleotides 45 and 156 of HAV RNA replaced the corresponding ones in poliovirus 5'-UTR. The construction was extended with poliovirus sequences up to position 1809. The recombinant mRNA so generated carried two copies of the PRT. In vitro translation in lysates of truncated poliovirus mRNAs generated a single peptide of Mr = 39 kDa, while the chimeric mRNA generated a series of short peptides as a result of fortuitous (or aberrant) initiation events. A more extensive substitution in the chimeric 5'-UTR which removed the 3'-most PRT brought by the poliovirus sequences, restored the translation at the authentic initiation site. Point mutations were engineered in the 5'-most PRT of the chimeras, and bi-cistronic plasmids were constructed in which either the parental poliovirus 5'-UTR or the chimeric ones were introduced in the intergenic region. Upon transfection of COS-1 cells, the chimeric polio-hepatitis A 5'-UTR containing two PRTs did not express the reporter gene. Removal of the 3'-distal PRT or point mutations engineered into the 5'-most PRT partially restored the transient expression of the reporter gene, consistent with the notion that a single (and only a single) functionally active PRT in a proper context is required to secure the internal initiation of translation of bi-cistronic mRNAs in vivo.

Base Sequence↗

PubMed1998

Promoter activity of the 5' flanking region of a tobacco glycolate oxidase gene in transgenic tobacco plants.

A clone harboring the 5' flanking region of a tobacco glycolate oxidase (GLO) gene was isolated from a lambda EMBL3-tobacco genomic DNA library. Primer extension analysis indicated two major transcripts with 76 and 81 bp 5' UTRs. An RT-PCR assay mapped the major mRNA transcription initiation site to thymine at position 81 upstream of the translation initiation codon. A putative TATA box spanning positions -56 to -50 upstream of the transcription initiation site was found. Promoter activity of the 5' flanking region (-3.0 kb to +82 bp) was demonstrated in tobacco plants transformed with a GLO-beta-glucuronidase (GUS) chimeric gene. Furthermore, in these transgenic plants, GUS expression patterns mimicked the expression patterns of the endogenous GLO.

Alcohol Oxidoreductases↗

PubMed1982

Nucleotide sequence of the transforming gene of simian sarcoma virus.

The sequence of the transforming region of simian sarcoma virus (SSV) has been determined by using molecularly cloned viral DNA. This region encompassed the 1.0-kilobase pair woolly monkey cell-derived insertion sequence, v-sis, and flanking simian sarcoma-associated viral (SSAV) sequences. A 675-nucleotide-long open reading frame commenced 19 nucleotides within the SSAV sequences to the left of the v-sis helper viral junction and terminated within v-sis itself. Possible promoter and acceptor splice signals were detected in helper viral sequences upstream from this open reading frame, and potential polyadenylylation sites were identified downstream both within v-sis and in helper viral sequences beyond v-sis. The recombinational event that led to the generation of SSV occurred in the middle of two functional codons, indicating that SSAV provided the regulatory elements for transcription as well as the initiation codon for translation of SSV cell-derived transforming sequences.

Cell Transformation, Viral↗

PubMed1999

Mechanism of endoplasmic reticulum retention of mutant vasopressin precursor caused by a signal peptide truncation associated with diabetes insipidus.

Autosomal dominant neurohypophyseal diabetes insipidus is caused by mutations in the gene encoding the vasopressin precursor protein, prepro-vasopressin-neurophysin II. We analyzed the molecular consequences of a mutation (DeltaG227) recently identified in a Swiss kindred that destroys the translation initiation codon. In COS-7 cells transfected with the mutant cDNA, translation was found to initiate at an alternative ATG, producing a truncated signal sequence that was functional for targeting and translocation but was not cleaved by signal peptidase. The mutant precursor was completely retained within the endoplasmic reticulum. The uncleaved signal did not affect folding of the neurophysin portion of the precursor, as determined by its protease resistance. However, formation of disulfide-linked aggregates indicated that it interfered with the formation of the disulfide bond in vasopressin, most likely by blocking its insertion into the hormone binding site of neurophysin. Preventing disulfide formation in the vasopressin nonapeptide by mutation of cysteine 6 to serine was shown to be sufficient to cause aggregation and retention. These results indicate that the DeltaG227 mutation induces translation of a truncated signal sequence that cannot be cleaved but prevents correct folding and oxidation of vasopressin, thereby causing precursor aggregation and retention in the endoplasmic reticulum.

Amino Acid Sequence↗

PubMed1982

Characterization of translational initiation sites in E. coli.

We characterize the Shine and Dalgarno sequence of 124 known gene beginnings. This information is used to make "rules" which help distinguish gene beginning from other sites in a library of over 78,000 bases of mRNA. Gene beginnings are found to have information besides the initiation codon and Shine and Dalgarno sequence which can be used to make better "rules".

Base Composition↗

PubMed1978

Bacteriophage lambda and plasmid vectors, allowing fusion of cloned genes in each of the three translational phases.

We have constructed vectors from bacteriophage lambda and from plasmid pBR322 having a single EcoRI restriction site which is immediately downstream from the lac UV5 promotor. Each vector allows the fusion of a cloned gene to the lac Z gene in a different phase relative to the translation initiation codon of the lac Z gene. These vectors were constructed through modification of the initial EcoRI restriction site by S1 endonuclease treatment and then addition of octadeoxyribonucleotides (EcoRI linkers), which shifted the restriction site by 2 or 4 nucleotides. Used in combination these vectors should allow translation of a cloned gene in any one of the three coding phases. The bacteriophages vectors are certified as B2 (EK2) safety level vectors by the French "recombinaison génétique in vitro" committee (D.G.R.S.T.).

Base Sequence↗

PubMed1987

Purification and characterization of the spoOA protein of Bacillus subtilis from an overproducing strain of Escherichia coli.

The spoOA gene of Bacillus subtilis is essential for the earliest stage of sporulation. To purify and characterize the product of the spoOA gene, we constructed a fusion plasmid in which the spoOA coding region was placed under the control of the Ptac promoter. When expression of the spoOA gene was induced in Escherichia coli cells by derepression of Ptac, the SpoOA protein constituted 15% of total cellular protein. The SpoOA protein was purified to homogeneity from these cells. We found that the NH2-terminal amino acid sequence of the purified protein was essentially the same as that of the SpoOA protein (spoOA-cat protein) from B. subtilis, and that the NH2-terminal methionine of the SpoOA protein from E. coli was formylated presumably because of insufficient amounts of the deformylating enzyme. The T signal [Ganoza, M. C., Marliere, P., Kofoid, E. C. and Louis, B. G. (1985) Proc. Natl Acad. Sci. USA 82, 4587-4591], in addition to the Shine-Dalgarno signal to determine the initiation codon of the spoOA gene, is considered to function in E. coli as well as in B. subtilis. We also found that the purified SpoOA protein had a DNA-binding activity. It was preferentially bound to the 175-bp BclI fragment of phi 105 DNA, and was released in the presence of 0.3 M KCl.

Bacillus subtilis↗

PubMed1992

Lysyl-tRNA synthetase gene of Campylobacter jejuni.

We report the cloning and complete nucleotide sequence of the Campylobacter jejuni lysyl-tRNA synthetase gene (lysS). The C. jejuni lysS gene sequence shows high homology to the two Escherichia coli lysyl-tRNA synthetase genes, lysS and lysU. The Campylobacter lysyl-tRNA synthetase protein (LysRS) shows 47.9 and 46.6% sequence identity to the E. coli enzymes encoded by the lysS and lysU genes, respectively. The LysRS encoded by the C. jejuni gene is a polypeptide of 501 amino acids with a deduced molecular weight of 57,867. The enzyme is active in E. coli. The gene is expressed from its own promoter, and the transcription start site has been mapped. The carboxyl-terminal codon of the C. jejuni lysS gene overlaps by 1 bp with the Met initiation codon of the glyA gene, which has been shown to have a promoter which is functional in E. coli (V.L. Chan and H.L. Bingham, Gene 101:51-58, 1991). C. jejuni, unlike E. coli, has only one lysyl-tRNA synthetase gene.

Amino Acid Sequence↗

PubMed1994

Regulation of the Bacillus anthracis protective antigen gene: CO2 and a trans-acting element activate transcription from one of two promoters.

The pag gene of Bacillus anthracis, located on plasmid pXO1 (185 kb), encodes protective antigen, a component of the anthrax lethal and edema toxins. Synthesis of protective antigen is enhanced during growth of the organism with elevated levels of CO2. The CO2 effect is at the level of transcription, and pXO1-encoded regulatory factors have been implicated in control of pag expression. We used a Tn917-LTV3 insertion mutant of B. anthracis in which the wild-type pag gene on pXO1 was replaced with a pag-lacZ transcriptional fusion to monitor pag promoter activity. Expression of the pag-lacZ fusion is induced five- to eightfold during growth in 5% CO2 compared with growth in air. Growth in 20% CO2 increases transcription up to 19-fold. By monitoring pag-lacZ expression in atmospheres with different O2 and CO2 concentrations, we demonstrated definitively that the CO2 effect is specific and not simply a result of increased anaerobiosis. The results of 5' end mapping of pag transcripts indicate multiple sites of transcript initiation. We have determined two major apparent start sites, designated P1 and P2, located at positions -58 and -26 relative to the translation initiation codon, respectively. Analysis of total RNA from late-log-phase cells shows comparable initiation from P1 and P2 in wild-type strains grown in aerobic conditions. However, initiation from P1 is increased approximately 10-fold in cultures grown with an elevated level (5%) of CO2. We have identified a locus on pXO1, more than 13 kb upstream from the pag gene, which enhances pag transcription. When added in trans, this locus increases the level of transcripts with 5' ends mapping to P1 but has no effect on the level of transcripts with 5' ends mapping to P2. The CO2 effect on P1 is observed only in the presence of the activator locus.

Amino Acid Sequence↗

PubMed2006

Extensive RNA editing in transcripts from the PsbB operon and RpoA gene of plastids from the enigmatic moss Takakia lepidozioides.

RNA editing is a post-transcriptional process that changes individual nucleotides in transcripts, and usually occurs in the plastids of land plants. The number of RNA editing sites in a plastid is significantly divergent in bryophytes, ranging from zero in liverworts to almost 1,000 sites in hornworts. In this study, we identified 132 RNA editing sites in the transcripts of six genes from the psbB operon and the rpoA of the moss Takakia lepidozioides. This is the highest number of RNA editing sites known in this region among land plant species. All were cytidine-to-uridine conversions. More than 91% of RNA editing occurred at the first or second codon positions, and it altered amino acid identity. Six editing sites created new translation initiation codons or stop codons. Thirty-two sites were commonly observed in the hornwort Anthoceros angustus. This finding suggests that the enigmatic bryophyte Takakia is closely related to hornworts with respect to RNA editing events.

Base Sequence↗

PubMed1992

Molecular abnormalities of a human glucose-6-phosphate dehydrogenase variant associated with undetectable enzyme activity and immunologically cross-reacting material.

Among a large number of glucose-6-phosphate dehydrogenase (G6PD) variants associated with different severity of clinical manifestations, enzyme deficiency, and kinetic abnormalities found in humans, only one variant exhibits no measurable activity and lacks an immunologically cross-reacting material in blood cells and other tissues. The mRNA content of the patient's lymphoblastoid cells was found to be normal, and the size of mRNA was also normal (i.e., approximately 2.4 kb). Western blot hybridization indicated that the patient's cells did not produce cross-reacting material. The variant mRNA was reverse transcribed and amplified by PCR. Nucleotide sequencing of the variant cDNA showed the existence of three nucleotide base changes, i.e., a C----G at nucleotide 317 (counting from adenine of the initiation codon), which should cause Ser----Cys substitution at the 106th position (counting from the initiation Met); a C----T at nucleotide 544, which induces the Arg----Trp at the 182d position; and a C----T at nucleotide 592, which induces Arg----Cys at the 198th position of the protein. The existence of three mutation sites was confirmed by sequencing of selected regions of the variant gene. No base deletion or frameshift mutation was found in the variant cDNA. No nucleotide change was detected in the extended 5' region, which included the most distal cap site. When the variant cDNA was expressed in Escherichia coli, the G6PD activity was approximately 2% of that expressed by the normal cDNA, and cross-reacting material was undetectable. However, when the variant mRNA was expressed in the in vitro translation system of rabbit reticulocytes, the variant protein was produced. These results suggest that extremely rapid in vivo degradation or precipitation of the variant enzyme induced by the three amino acid substitutions could be the major cause of the molecular deficiency.

Base Sequence↗

PubMed1995

Translation of hepatitis C virus genome.

Translation of the human hepatitis C virus (HCV) RNA genome occurs by internal ribosome entry through the 5' end (5' noncoding region) in a cap-independent fashion. The relatively long stretch of this noncoding region contains multiple initiation codons that are apparently not used for translation. Translation of the HCV polyprotein is initiated instead from an AUG located at nt 342. Using computer-assisted analysis (and subsequently substantiated by enzymatic probing), a complex secondary and tertiary structure of the 5' noncoding region (5'NCR) has been predicted. Based on an RNA folding model proposed by Brown et al. (1992), a detailed mutational analysis carried out identified the key secondary structural regions that are of functional significance in translational control. Maintenance of a helical structural element relevant to an oligopyrimidine tract is essential for internal initiation. A putative coaxial stacking or a pseudoknot structure upstream of the initiator AUG seems to be central to an internal ribosome entry site (IRES)-mediated translation of the HCV RNA genome.

Hepacivirus↗

PubMed1999

Translational control by an upstream open reading frame in the HER-2/neu transcript.

Overexpression of the HER-2 (neu, erbB-2) receptor results in cellular transformation and is associated with a variety of human cancers. Multiple mechanisms, including gene amplification and transcriptional, post-transcriptional, and translational controls contribute to the regulation of HER-2 expression. One of the components of these regulatory mechanisms is a short upstream open reading frame (uORF) in the HER-2 mRNA that represses downstream translation in a variety of cell types. Here we explore the mechanism by which this uORF exerts its inhibitory effect. As judged by comparisons of protein and mRNA abundance and by polysomal distribution analyses, the uORF represses translation of the HER-2 cistron or of a heterologous reporter gene. Despite its conservation among mammalian species, the peptide sequence of the uORF is not required for this inhibitory effect. Rather, the majority of ribosomes that load on the HER-2 mRNA most likely translate the uORF and are then unable to reinitiate at the downstream AUG codon, in part due to the short intercistronic spacing. A minority of ribosomes gain access to the HER-2 initiation codon either by leaky scanning past the upstream AUG codon or by reinitiating after having translated the uORF despite the short intercistronic region. These results suggest that the HER-2 uORF controls synthesis of this oncoprotein by limiting ribosomal access to downstream initiation sites.

Animals↗

PubMed2003

Two alternative translation mechanisms are responsible for the expression of the HCV ARFP/F/core+1 coding open reading frame.

HCV-1 produces a novel protein, known as ARFP, F, or core+1. This protein is encoded by an open reading frame (ORF) that overlaps the core gene in the +1 frame (core+1 ORF). In vitro this protein is produced by a ribosomal frameshift mechanism. However, similar studies failed to detect the ARFP/F/core+1 protein in the HCV-1a (H) isolate. To clarify this issue and to elucidate the functions of this protein, we examined the expression of the core+1 ORF by the HCV-1 and HCV-1a (H) isolates in vivo, in transfected cells. For this purpose, we carried out luciferase (LUC) tagging experiments combined with site-directed mutagenesis studies. Our results showed that the core+1-LUC chimeric protein was efficiently produced in vivo by both isolates. More importantly, neither changes in the specific 10-A residue region of HCV-1 (codons 8-11), the proposed frameshift site for the production of the ARFP/F/core+1 protein in vitro, nor the alteration of the ATG start site of the HCV polyprotein to a stop codon significantly affected the in vivo expression of the core+1 ORF. Furthermore, we showed that efficient translation initiation of the core+1 ORF is mediated by internal initiation codon(s) within the core/core+1-coding sequence, located between nucleotides 583 and 606. Collectively, our data suggest the existence of an alternative translation initiation mechanism that may result in the synthesis of a shorter form of the core+1 protein in transfected cells.

Amino Acid Sequence↗

PubMed1986

Use of a portable ribosome-binding site for maximizing expression of a eukaryotic gene in Escherichia coli.

To maximize expression of a eukaryotic gene in Escherichia coli, a series of plasmids were constructed containing various synthetic ribosome-binding sites (RBS). These sites consist of a Shine-Dalgarno (SD) region (with translation stop codons in all three reading frames) positioned at distances 5-9 nucleotides (nt) from the AUG initiator codon of the gene coding for human T-cell growth factor (TCGF or IL-2). The region encompassing the RBS through the TCGF structural gene from each of these plasmids was inserted as a 'cassette' into seven different E. coli expression vectors, and TCGF production was measured. Our results demonstrate a greater than 2000-fold range of TCGF synthesis dependent upon the promoter and the synthetic RBS used. The translational efficiency of the TCGF gene was found to be influenced by the quality of the RBS, which is in part determined by the external sequence context of this site. The synthetic RBS, containing the necessary information for the translation initiation process, readily accessible by restriction sites, should be of general usefulness in obtaining maximum expression of eukaryotic genes in E. coli.

Base Sequence↗