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PubMed1987

Translational regulation of SV40 early mRNA defines a new viral protein.

SP6-initiated in vitro transcripts, representing the three major classes of early SV40 mRNAs, early-early (EE) and two late-early (LE) transcripts, were assayed by in vitro translation to compare their relative efficiencies for synthesis of the SV40 T antigens. The presence of one or two potential AUG initiator codons in the leader sequences of the LE RNAs inhibits efficient translation from the downstream T-antigen initiator AUG. In vitro translation of the capped form of the shorter SV40 LE RNA resulted in the synthesis of a 2.7-kd protein. In vivo pulse labeling of SV40-infected CV-1 cells demonstrated the accumulation of a peptide of similar size at late times after lytic infection, indicating that it is an authentic viral protein encoded by the early leader sequence of SV40.

Animals↗

PubMed1998

The low level expression of chloramphenicol acetyltransferase (CAT) mRNA in Escherichia coli is not dependent on either Shine-Dalgarno or the downstream boxes in the CAT gene.

Recent studies have shown that the canonical Shine-Dalgarno (SD)-anti-SD interaction is dispensable for the initiation of translation of certain mRNAs in Escherichia coli. Alternative non-SD sequences (located upstream from the initiation codon) and also downstream sequences ("downstream boxes") complementary to 16S rRNA were found to be involved in the initiation of translation of mRNAs devoid of either SD or any leader sequences. In this study the chloramphenicol acetyltransferase (CAT) gene was modified to remove the 5' terminal non-translated region and/or the two potential downstream boxes in the CAT gene. Thus a series of ten CAT gene constructs was created and expressed in E. coli under a strong constitutive promoter. The results showed that CAT mRNAs devoid of both leader sequence nucleotides and the two downstream boxes in the CAT gene remained active in vivo and produced CAT protein in sufficient amounts for survival of the transformed cells at chloramphenicol concentrations up to 20-30 micrograms/ml.

5' Untranslated Regions↗

PubMed1978

Nucleotide sequence at the 5' extremity of tobacco-mosaic-virus RNA. 1. The noncoding region (nucleotides 1-68).

The sequence of the 5' noncoding region of tobacco mosaic virus RNA has been determined. The noncoding region is 68 nucleotides long and is unusual in that it contains no internal guanosine residues. The long T1 oligonucleotide containing the guanosine-free tract was isolated from a T1 ribonuclease digest of tobacco mosaic virus RNA and sequenced by labelling techniques in vitro using polynucleotide kinase. The guanosine-free tract is terminated by the first potential initiation codon in the RNA molecule and several lines of evidence suggest that this AUG triplet is operational in initiating viral protein synthesis (see following paper). The 5'-noncoding region cannot base-pair extensively with the 3'-terminal sequence of 18-S ribosomal RNA from rabbit reticulocytes.

Base Sequence↗

PubMed1986

Origin of transfer of IncF plasmids and nucleotide sequences of the type II oriT, traM, and traY alleles from ColB4-K98 and the type IV traY allele from R100-1.

The complete nucleotide sequences of the ColB4-K98 (ColB4) plasmid transfer genes oriT, traM, and traY as well as the traY gene of R100-1 are presented and compared with the corresponding regions from the conjugative plasmids F, R1, and R100. The sequence encoding the oriT nick sites and surrounding inverted repeats identified in F was conserved in ColB4. The adenine-thymine-rich sequence following these nick sites was conserved in R1 and ColB4 but differed in F and R100, indicating that this region may serve as the recognition site for the traY protein. A series of direct repeats unique to the ColB4 plasmid was found in the region of dyad symmetry following this AT-rich region. This area also encodes 21-base-pair direct repeats which are homologous to those in F and R100. The traM gene product may bind in this region. Overlapping and following these repeats is the promoter(s) for the traM protein. The traM protein from ColB4 is similar to the equivalent products from F, R1, and R100. The traY protein from ColB4 is highly homologous to the R1 traY gene product, while the predicted R100-1 traY product differs at several positions. These differences presumably define the different alleles of traM and traY previously identified for IncF plasmids by genetic criteria. The translational start codons of the ColB4 and R100-1 traY genes are GUG and UUG, respectively, two examples of rare initiator codon usage.

Alleles↗

PubMed1993

Menaquinone (vitamin K2) biosynthesis: cloning, nucleotide sequence, and expression of the menC gene from Escherichia coli.

The benzenoid aromatic compound o-succinylbenzoic acid is formed by dehydration of the prearomatic compound 2-succinyl-6-hydroxy-2,4-cyclohexadiene-1-carboxylic acid by the enzyme o-succinylbenzoate synthase, encoded by the menC gene. A 1.3-kb PstI-PvuII fragment was found to complement the menC mutation. The complete nucleotide sequence of this fragment revealed a single open reading frame of 954 bp capable of encoding a 35-kDa protein. A consensus sequence for a ribosomal binding site but no promoter consensus sequences were found. However, the first base of the initiating codon of this open reading frame overlaps the upstream menB gene termination codon, suggesting an operon-like organization for these genes. Consistent with this suggestion, the menB promoter can initiate transcription of the menC gene.

Amino Acid Sequence↗

PubMed1983

Complete sequence of the Rous sarcoma virus env gene: identification of structural and functional regions of its product.

The amino-terminal amino acid sequences of gp85 and gp37, the envelope glycoproteins of Rous sarcoma virus (RSV), were determined. Alignment of these sequences with the amino acid sequence predicted from the complete nucleotide sequence of the Prague strain of RSV, subgroup C (PR-C), has allowed us to delineate the env gene-coding region of this virus. The coding sequences for gp85 and gp37 have been placed in an open reading frame that extends from nucleotide 5045 to nucleotide 6862 and predict sizes of 341 amino acids (36,962 molecular weight) for gp85 and 198 amino acids (21,566 molecular weight) for gp37. Carbohydrate makes a significant contribution to the observed molecular weights of these polypeptides--the amino acid sequence contains 14 potential glycosylation sites (Asn-X-Ser/Thr) in gp85 and two in gp37. Experiments aimed at estimating the number of carbohydrate side chains yielded results consistent with most or all of these sites being occupied. Although an initiation codon is located early (codon 4) in the open reading frame, it is likely that splicing yields an mRNA on which translation initiates at the same AUG as that of the gag gene to produce a nascent polypeptide in which gp85 is preceded by a 62-amino-acid-long leader peptide. This leader contains the hydrophobic sequence (signal sequence) necessary for translocation across the endoplasmic reticulum and is completely removed from the env gene product during translation. The polyprotein precursor, Pr95env, is cleaved to gp85 and gp37 at the carboxyl side of the basic sequence:-Arg-Arg-Lys-Arg-. gp85 is attached through a disulphide linkage to gp37, and although the positions of the cysteines involved in this linkage are not known, the presence of a 27-amino-acid-long hydrophobic region at the carboxy-terminus of gp37 is consistent with its role as a membrane anchor for the viral glycoprotein complex. The location of host range variable regions with respect to the possible tertiary structure of the complex is discussed.

Amino Acid Sequence↗

PubMed1990

Bovine papillomavirus E2 repressor mutant displays a high-copy-number phenotype and enhanced transforming activity.

The methionine codon at bovine papillomavirus type 1 nucleotide 3091 was mutated to determine whether it may serve as an initiation codon for an E2 transcriptional repressor protein and to determine the role of the repressor in the biological activities of the virus. A series of transient expression experiments with CV1 cells documented that the mutation reduced expression of repressor activity from the viral genome and resulted in increased expression of the E5 transforming gene. Viral genomes containing the mutation displayed enhanced transforming activity in several assays in mouse C127 cells, including focus formation, colony formation in agarose, and tumorigenicity. In transformed cells, the mutant viral DNA was maintained as a plasmid with approximately 500 genomes per cell, whereas the wild-type copy number was approximately 75. These results indicate that the wild-type bovine papillomavirus type 1 genome encodes an E2 repressor protein that moderates the viral transforming activity and allows maintenance of the viral DNA at a relatively low copy number.

Animals↗

PubMed1990

Nucleotide sequence of messenger RNA encoding human isovaleryl-coenzyme A dehydrogenase and its expression in isovaleric acidemia fibroblasts.

Isovaleric acidemia (IVA) is caused by a genetic deficiency of isovaleryl-CoA dehydrogenase (IVD). At least five distinct variant IVD alleles are known. We isolated five overlapping IVD cDNA clones from a human placenta cDNA library. They covered the entire coding region, except the initiation codon, and 587 bp in the 3'-noncoding region plus the poly(A) tail. The structure of the initiation site was identified by the study of genomic DNA and by the sequence comparison with rat IVD. Human IVD shared 89.6, 35.8, and 31.6% identical amino acid residues with rat IVD and human short and medium chain acyl-CoA dehydrogenases, respectively. In the Northern blot analysis of normal human liver and fibroblast poly(A)+ RNA, three mRNA species of different sizes (4.6, 3.8, and 2.1 kb) hybridized to IVD cDNA. Three mRNA species with similar sizes were also detected in five IVA fibroblast lines of different genotypes (variants 1, 1 X 2, 2, 3, and 5), suggesting that these variants are each due to a point mutation or small deletion.

Amino Acid Sequence↗

PubMed2005

Suppression of carbonic anhydrase III mRNA level by an aryl hydrocarbon receptor ligand in primary cultured hepatocytes of rat.

The effect of an aryl hydrocarbon receptor (AhR) ligand on the carbonic anhydrase III (CAIII) mRNA level was studied using primary cultured hepatocytes of rats. CAIII gene which is highly suppressible by dioxins in vivo, was also suppressible in primary cultured hepatocytes of rats by an AhR ligand, 3-methylchlanthrene (3MC). The suppression of CAIII by 3MC was observed in a dose-dependent fashion. The suppression was marked at 10 microM MC. It is likely that AhR is involved in the suppression of the CAIII gene. The transcriptional regulation region of rat CAIII gene was cloned by polymerase chain reaction on the basis of the similarity to the mouse and human CAIII genes. A 1.5 kb section upstream of rat CAIII was sequenced and the transcription initiation site of this gene was mapped to 58 bases upstream of the initiation codon. A xenobiotic responsive element (XRE)-like sequence was found at -555 to -549 bp of the transcription initiation site. The location of XRE-like element was conserved between rats and mice those CAIIIs in liver were shown as dioxins-suppressible. Although the roles of the XRE have not been clarified, these results suggest that the AhR ligands could elicit the suppressive effect on hepatic CAIII and the effect on the factors from extrahepatic tissues is not required for the suppression.

Animals↗

PubMed1992

Rapid molecular diagnosis of hemoglobin variants by RT-PCR of reticulocyte mRNA and direct sequencing.

We have developed a rapid and simple approach for the molecular characterization of hemoglobin variants by a one-step reverse transcription-polymerase chain reaction of reticulocyte mRNA and direct sequencing of the product. This method can selectively amplify the alpha 1- or alpha 2-globin gene or the beta-globin gene transcript. The amino acid substitution of Hb G-Taichung is due to a G----C mutation at codon 74 of the alpha 1-globin gene, that of Hb J-Meinung to a G----A substitution at codon 56 of the beta-globin gene, and that of Hb Kaohsiung (or New York) to a T----A substitution at codon 113 of the beta-globin gene. The amplified segment encompassed the sequence from upstream of the initial codon behind the Cap site to downstream of the terminal codon before the polyadenylation addition signal. Hence, all hemoglobin variants should be able to be characterized by this approach.

Base Sequence↗

PubMed1989

An efficient and site-specific gene trimming method.

Trimming a DNA strand into a precisely determined fragment can be carried out efficiently by an improved method involving a site-specific trim-primer and a single-stranded DNA template which is generated from a multifunctional vector, pTZ18R, and linearized by using an Eco RI-pTZ18R splinter. A complementary DNA strand is synthesized by DNA polymerase I (Klenow), and the 3'-end of the template upstream from the annealed primer is trimmed by subsequent T4 DNA polymerase reaction. An ATG translation initiator codon or a termination codon can be incorporated into the trim-primer, providing versatility to this single-stranded DNA-initiated gene trimming method that can be applied to subcloning and expression of any DNA fragment with known terminal sequences.

Biotechnology↗

PubMedDate not supplied

Mutation that affects pepN translation initiation in Escherichia coli.

Mutants altered in their expression of the hybrid pepN-lacZ gene have been selected for resistance to p-nitrophenyl-beta-D-thiogalactopyranoside (a bacteriostatic compound that enters the cells via lac permease). A unique mutation decreasing the level of pepN expression to 9% of that of the wild type has been studied in detail. This mutation controls in cis the expression of the pepN gene. The pepN region from a pepN-lacZ gene fusion has been cloned and sequenced. Comparison of the mutant and wild type sequences indicates that the mutation lies between the Shine-Dalgarno sequence (AGGT) and the initiation codon (AUG). This mutation is a T----C transition which might allow the formation of a stable secondary structure in the region of translation initiation thus decreasing the level of pepN expression.

Aminopeptidases↗

PubMed1994

Identification and analysis of the site of -1 ribosomal frameshifting in red clover necrotic mosaic virus.

The genomic RNA-1 of red clover necrotic mosaic dianthovirus (RCNMV) contains the heptanucleotide GGAUUUU that precedes the termination codon of the 5' proximal p27 open reading frame (ORF). This heptanucleotide is followed by a sequence with the potential to form a stable, complex secondary structure. Translation of RNA-1 is postulated to utilize a -1 ribosomal frameshifting mechanism to express the 88-kDa viral RNA polymerase. Using site-directed mutagenesis together with cell-free translation to monitor frameshifting and a biological assay of the mutants in plants, we establish the role of the GGAUUUU as the site where -1 ribosomal frameshifting occurs. The frameshifting signal sequence conforms to the simultaneous slippage model. Stop codons flanking the shifty signal are not required for frameshifting but the p27 ORF termination codon is necessary for maintaining optimal infectivity of the virus. Mutations abolishing the RCNMV RNA-1 internal p57 ORF initiation codon did not affect infectivity of the virus, suggesting that this cistron is only expressed in vivo as an 88-kDa ribosomal frameshifting product. Shifty heptanucleotide signals from a number of animal retroviruses and RNA plant viruses facilitate RCNMV frameshifting in vitro. However, only a limited number of the heterologous shifty heptanucleotides were functional in plant cells. We suggest that specific shifty tRNA populations in the cell facilitate viral -1 ribosomal frameshifting. This analysis also suggests that the slippery sequence requirements are not identical in mammalian and in plant systems.

Base Sequence↗

PubMed2005

Analysis of the guinea-pig estrogen-regulated gec1/GABARAPL1 gene promoter and identification of a functional ERE in the first exon.

The gec1/GABARAPL1 (GABA(A)-receptor-associated protein like-1) gene has been identified as an early estrogen-regulated gene in guinea-pig cultured endometrial glandular epithelial cells (GEC). Guinea-pig and human gec1/GABARAPL1 proteins share 87% identity with GABARAP, which acts as a protein linker between microtubules and the GABA(A) receptor. To investigate the molecular mechanisms regulating gec1/GABARAPL1 gene expression, the 1.5-kbp region upstream of the translation initiation codon of the guinea-pig gec1/GABARAPL1 gene was cloned. A 300-bp fragment encompassing a pyrimidine-rich initiator element (INR) and the transcription start site (+1) was sufficient to initiate transcription. Transfection and gel shift experiments showed that a sequence located at +36/+50 in the first exon permitted induction of expression of this gene by estradiol acting via ERalpha. This sequence (GGGTCAACGTGACGT) differs only by one base pair from the consensus estrogen response element ERE (GGGTCAACGTGACCT). It can be concluded that the ERE located in the first exon encoding the 5'-untranslated region is sufficient for E2 activation of gec1/GABARAPL1 transcription.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Molecular characterisation of a hsp70 gene from the filarial parasite Setaria digitata.

The filarial parasite Setaria digitata is the causative agent of cerebrospinal nematodiasis in its abnormal hosts such as sheep, goats and horses, and therefore is of significant veterinary importance. Since very little is currently known about the biology of this parasite at molecular level, we have cloned and characterised a hsp70 gene, the first gene to be reported from this parasite. The genomic clone isolated contained sequences from two hsp70 genes. One gene, hsp70-2, was completely sequenced and found to contain nine introns ranging in size from 78 to 195 bp. The region upstream of the initiation codon contained a putative TATA box, two CAAT box elements and three heat-shock elements. A putative transcription initiation site was also identified. The 5' untranslated region contained a splice acceptor sequence. The gene was typically AT rich, having a GC content of 44.5%. The deduced aa sequence potentially encoded a cytosolic protein of 645 aa, which had three consecutive repeats of a tetrapeptide motif, GGMP, at the carboxyl end. The gene appeared to be constitutively transcribed and was not significantly enhanced in response to heat shock in adult worms. Another hsp70 gene (hsp70-1) was located further upstream, arranged in direct tandem with hsp70-2. Southern blot analysis revealed the presence of two or three additional hsp70-related genes in the S. digitata genome.

Amino Acid Sequence↗

PubMed2001

Enhanced activity of cloned hamster TERT gene promoter in transformed cells.

In 7,12-dimethylbenz[a]anthracene-treated hamster pouch epithelial cells, telomerase activity increased within 1 week of treatment and reached a 6-7-fold increase within 3 weeks. To investigate this phenomenon, we have cloned and sequenced the hamster telomerase catalytic subunit (hamTERT) promoter. Transient transfection with different genomic segments upstream of the ATG translation initiation codon linked to the luciferase reporter gene mapped the core promoter within a 250 bp region. Three major transcription initiation sites and several minor sites were found between -42 and -140 bp relative to the ATG site. Like the human and murine TERT promoters, the hamTERT promoter lacks TATA and CAT boxes and all three promoters share similar regulatory factor binding sites. DNase I footprint analysis revealed six protected regions which contain sequences homologous with known transcription factor binding sites. Three protein binding regions (I, II, and III) were essential for the promoter activity. Regions I and III bound to Sp1 and Sp3 transcriptional factors, whereas region II bound to an unknown factor. Transient transfection of a promoter-luciferase plasmid into Drosophila SL2 cells showed that Sp1 and Sp3 regulated the hamster TERT promoter in a concentration-dependent and synergistic manner. Telomerase activity showed a 2-4-fold and 8-10-fold increase in immortalized cells and tumor cells, respectively, but hamTERT expression was only increased 1.7-fold and 2.4-fold, respectively, in the same cells.

9,10-Dimethyl-1,2-benzanthracene↗

PubMed1998

Initiation factors of protein biosynthesis in bacteria and their structural relationship to elongation and termination factors.

Initiation of protein biosynthesis in bacteria requires three initiation factors: initiation factor 1, initiation factor 2 and initiation factor 3. The mechanism by which initiation factors form the 70S initiation complex with initiator fMet-tRNA(fMet) interacting with the initiation codon in the ribosomal P site and the second mRNA codon exposed in the A site is not yet understood. Here, we present a model for the function of initiation factors 1 and 2 that is based on the analysis of sequence homologies, biochemical evidence and the present knowledge of the three-dimensional structures of translation factors and ribosomes. The model predicts that initiation factors 1 and 2 interact with the ribosomal A site mimicking the structure of the elongation factor G. We present data that extend the mimicry hypothesis to initiation factors 1 and 2, originally postulated for the aminoacyl-tRNA x elongation factor Tu x GTP ternary complex, elongation factor G and release factors.

Amino Acid Sequence↗

PubMed1998

Genomic organization and functional characterization of the chemokine receptor CXCR4, a major entry co-receptor for human immunodeficiency virus type 1.

CXCR4 is both a chemokine receptor and entry co-receptor for T-cell line-adapted human immunodeficiency virus type 1. The genomic organization and promoter function for the entire transcription unit of CXCR4 were determined. The gene contains 2 exons of 103 and 1563 base pairs (bp) interrupted by a 2132-bp intron precisely between codons 5 and 6 of the coding sequences. A transcription start site was identified 88 bp upstream of the initiation codon, and a polyadenylate addition site was identified 22 bp 3' to a polyadenylation signal. Transient expression assays defined a minimal promoter at positions -114 to +43 relative to the transcription start site. This region contains a TATA box, a nuclear respiratory factor-1 (NRF-1) site, and two GC boxes. Specific factor binding to the NRF-1 site and GC boxes were demonstrated by gel mobility shifts and DNase I footprinting. Site-directed mutagenesis showed that the NRF-1 site is crucial for promoter activity providing the first evidence for the regulation of a signal transduction gene by NRF-1. Sequences between -691 and -191 repress CXCR4 promoter activity. Further study of these regulatory elements will be important to understanding how CXCR4 functions as both a chemokine receptor and human immunodeficiency virus type 1 entry co-receptor.

Amino Acid Sequence↗