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At least 1,441 records · Page 80Linked to original sources
PubMed1999

Structural studies of the RNA pseudoknot required for readthrough of the gag-termination codon of murine leukemia virus.

Retroviruses, such as murine leukemia virus (MuLV), whose gag and pol genes are in the same reading frame but separated by a UAG stop codon, require that 5-10 % of ribosomes decode the UAG as an amino acid and continue translation to synthesize the Gag-Pol fusion polyprotein. A specific pseudoknot located eight nucleotides 3' of the UAG is required for this redefinition of the UAG stop codon. The structural probing and mutagenic analyses presented here provide evidence that loop I of the pseudoknot is one nucleotide, stem II has seven base-pairs, and the nucleotides 3' of stem II are important for function. Stem II is more resistant to single-strand-specific probes than stem I. Sequences upstream of the UAG codon allow formation of two competing structures, a stem-loop and the pseudoknot.

Aldehydes↗
PubMed2001

Gene function analysis by amber stop codon suppression: CMBF is a nuclear protein that supports growth and development of Dictyostelium amoebae.

The C-module-binding factor, CMBF, is a nuclear DNA-binding protein which was originally identified through its specific binding to a promoter element within the retrotransposable element TRE5-A of Dictyostelium discoideum AX2 cells. In order to analyse putative physiological functions of CMBF for the TRE5-A-hosting D. discoideum cells, we used a novel strategy to create mutant cell lines which stably underexpressed functional CMBF. An amber (UAG) translation stop codon was introduced into the chromosomal copy of the CMBF-encoding gene (cbfA), and an amber suppressor tRNA gene was expressed in the same mutant cells. Due to the low efficiency of translation stop codon suppression in this system all recovered cell lines expressed <20 % of wild-type CMBF levels. The mutant cell lines displayed strong growth phenotypes when plated on their natural food source, bacteria. We show evidence that growth reduction was due to impaired phagocytosis of bacteria in the mutants. All obtained mutants showed a strong developmental defect which was defined by the formation of very small fruiting bodies. The strength of the developmental phenotype appeared to depend upon the residual CMBF levels maintained in the mutants. We propose that CMBF is a general transcription regulator which supports the normal expression of several genes required for the maintenance of high proliferation rates of D. discoideum amoebae as well as proper aggregation and development. Our results demonstrate that amber stop codon suppression may be a useful strategy to stably underexpress proteins whose coding genes cannot be successfully disrupted by homologous recombination.

Active Transport, Cell Nucleus↗
PubMed1995

Selection, mutations and codon usage in a bacterial model.

We present a statistical model of bacterial evolution based on the coupling between codon usage and tRNA abundance. Such a model interprets this aspect of the evolutionary process as a balance between the codon homogenization effect due to mutation process and the improvement of the translation phase due to natural selection. We develop a thermodynamical description of the asymptotic state of the model. The analysis of naturally occurring sequences shows that the effect of natural selection on codon bias affects genes whose products are largely required at maximal growth rate conditions or undergo rapid transient increases.

Bacteria↗
PubMed1997

Effects of codon usage and vector-host combinations on the expression of spinach plastocyanin in Escherichia coli.

Spinach plastocyanin has been expressed in Escherichia coli and exported to the periplasmic space. The effects of codon usage, expression system, growth length, and temperature on expression levels in LB medium were investigated. A stretch of codons, rare in E. coli, was identified and replaced with highly expressed codons, increasing the yield by at least 20%. Plastocyanin was more efficiently expressed under the T7 promoter than under the lac promoter. Maximum yields were obtained at 37 degrees C when growing the cells for 16 h after induction. The optimized expression system produced 38 mg holoprotein per liter culture. In this system it was also possible to express plastocyanin in minimal medium, at a yield of 10 mg per liter. N-terminal sequencing and mass spectrometry showed that plastocyanin was correctly processed. The expressed plastocyanin was purified to homogeneity, as shown by an A278/A597 ratio of 1.0, and together with amino acid analysis and the determination of oxidized and total copper contents, both the absorption coefficients for epsilon 278 and for epsilon 597 were determined to be 4700 M-1 cm-1.

Amino Acid Sequence↗
PubMed2000

High-level periplasmic expression in Escherichia coli using a eukaryotic signal peptide: importance of codon usage at the 5' end of the coding sequence.

We investigated the ability of signal peptides of eukaryotic origin (human, mouse, and yeast) to efficiently direct model proteins to the Escherichia coli periplasm. These were compared against a well-characterized prokaryotic signal peptide-OmpA. Surprisingly, eukaryotic signal peptides can work very efficiently in E. coli, but require optimization of codon usage by codon-based mutagenesis of the signal peptide coding region. Analysis of the 5' of periplasmic and cytoplasmic E. coli genes shows some codon usage differences.

Amino Acid Sequence↗
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↗
PubMed1996

Natural populations of woodchuck hepatitis virus contain variant precore and core sequences including a premature stop codon in the epsilon motif.

We have determined a consensus sequence and the type and the frequency of spontaneous sequence variations in the woodchuck hepatitis virus (WHV) precore gene and the 5' region of the core gene in 101 serum samples from 53 naturally WHV-infected woodchucks by polymerase chain reaction sequencing. Twenty of the 53 woodchucks were found to have variant sequences. Ten patterns of variant sequences were identified in these 20 animals. WHV sequences from 4 woodchucks had 1 nucleotide change, 3 had 2 nucleotide changes and 3 had 3 nucleotide changes. The nucleotide changes were not randomly distributed, but were limited to only 8 sites. Four sites were in the epsilon motif of the precore gene and four were in the 5' region of the core gene. Sixteen of the 53 (30%) woodchucks had precore sequence variants. All altered sites were analogous to previously described mutations in hepatitis B virus. There was a nucleotide change at nucleotide 2016 in codon 29 of the precore region that produced a stop codon in 4 animals. This site is analogous to a common hepatitis B virus e antigen mutation. The sequence from the initial blood samples from 3 of 4 animals with this stop codon producing variant appeared to be the consensus sequence; however, in later samples the variant occurred as a mixed infection with the consensus sequence. The mixed infections were chronic and the proportion of the variant sequence was maintained or increased in the course of infection. In the fourth animal only the variant was found and it persisted for over 14 months of infection. WHV appears to be a valuable model for the study of the structure and function of the hepadnavirus precore region.

Animals↗
PubMed1998

Varicella-zoster virus ORF61 deletion mutants replicate in cell culture, but a mutant with stop codons in ORF61 reverts to wild-type virus.

Varicella-zoster virus (VZV) ORF61 encodes a phosphoprotein that transactivates VZV promoters. Transfection of cells with cosmid DNAs, including a cosmid with a large deletion in ORF61, resulted in a VZV ORF61 deletion mutant that was impaired for growth in vitro and could be partially complemented by growth in neuroblastoma or osteosarcoma cell lines. Cells infected with the VZV ORF61 deletion mutant expressed normal levels of an immediate-early VZV protein, but had reduced levels of a late protein and showed abnormal syncytia. Carboxy terminal truncation mutants of VZV ORF61 protein have a transrepressing phenotype and inhibit the infectivity of cotransfected wild-type viral DNA. Transfection of cells with cosmid DNAs, including a cosmid with stop codons that should result in an ORF61 truncation mutant expressing a transrepressing protein that retains the RING finger domain, resulted in a viral genome which reverted back to the wild-type sequence. BAL-31 exonuclease was used to produce deletions at the site of the stop codons in ORF61 of the cosmid, resulting in loss of the RING finger domain. Transfection of tissue culture cells with the ORF61 BAL-31 deletion mutants and other cosmid DNAs yielded viable viruses. Thus, while deletion mutants lacking the RING finger domain of ORF61 replicate in cell culture, a mutant with stop codons that retains this domain could not be propagated and reverted to wild-type virus.

Codon, Terminator↗
PubMed2000

Translation initiation of a bicistronic mRNA of Borna disease virus: a 16-kDa phosphoprotein is initiated at an internal start codon.

We examined translational initiation of a bicistronic 0.8-kb mRNA of Borna disease virus (BDV) using a cDNA clone of the mRNA. Upon transfection with the clone, COS-7 cells produced a 16-kDa protein (P'), in addition to the previously identified products of BDV, 24- (P) and 14.5-kDa proteins. The 16-kDa product was detected by anti-P monoclonal antibody and was shown to exist in BDV-infected cell lines as well as in infected animal brain cells. Transient expression analysis of mutated cDNA clones encoding the BDV 0.8-kb mRNA revealed that the 16-kDa protein was initiated at the second AUG codon on the same open reading frame of the P protein. The mutational analysis also demonstrated that the first AUG within the 0.8-kb mRNA is not optimal, although the signal contains a better Kozak's motif. These results demonstrated the presence of three functional AUG codons in the smallest mRNA of BDV and also suggested that a leaky scanning mechanism is involved in translational initiation at AUG codons downstream of the bicistronic mRNA of BDV. Furthermore, the 16-kDa protein was located in the BDV-specific nuclear foci and was found to associate with the other viral proteins in BDV-infected cells, demonstrating an important role of the novel identified BDV protein in viral replication.

Amino Acid Sequence↗
PubMed2002

Codon modified human papillomavirus type 16 E7 DNA vaccine enhances cytotoxic T-lymphocyte induction and anti-tumour activity.

Polynucleotide immunisation with the E7 gene of human papillomavirus (HPV) type 16 induces only moderate levels of immune response, which may in part be due to limitation in E7 gene expression influenced by biased HPV codon usage. Here we compare for expression and immunogenicity polynucleotide expression plasmids encoding wild-type (pWE7) or synthetic codon optimised (pHE7) HPV16 E7 DNA. Cos-1 cells transfected with pHE7 expressed higher levels of E7 protein than similar cells transfected with pW7. C57BL/6 mice and F1 (C57x FVB) E7 transgenic mice immunised intradermally with E7 plasmids produced high levels of anti-E7 antibody. pHE7 induced a significantly stronger E7-specific cytotoxic T-lymphocyte response than pWE7 and 100% tumour protection in C57BL/6 mice, but neither vaccine induced CTL in partially E7 tolerant K14E7 transgenic mice. The data indicate that immunogenicity of an E7 polynucleotide vaccine can be enhanced by codon modification. However, this may be insufficient for priming E7 responses in animals with split tolerance to E7 as a consequence of expression of E7 in somatic cells.

Animals↗
PubMed2002

Intraepithelial DNA immunisation with a plasmid encoding a codon optimised COPV E1 gene sequence, but not the wild-type gene sequence completely protects against mucosal challenge with infectious COPV in beagles.

DNA plasmids encoding the open reading frames of canine oral papillomavirus (COPV) nonstructural early genes E1, E2, or E7 protein were delivered into both oral mucosal and cutaneous epithelial sites in beagle dogs using particle-mediated immunotherapeutic delivery (PMID) technology. Control dogs were vaccinated with plasmid encoding either hepatitis B virus surface antigen (HBVs) or COPV L1. Using a prophylactic immunisation protocol, a priming dose of plasmid DNA was followed by a booster dose 6 weeks later. Four weeks after boost, all dogs were challenged with infectious COPV particles. Following viral challenge, as shown previously (M. A. Stanley et al., 2001, Vaccine 19, 2783-2792), mucosal papillomas developed in the negative-control HBVs vaccinated dogs, but all animals in the COPV L1 group were fully protected from disease development. In the early gene-vaccinated groups five of six in the E1-vaccinated dogs, two of six in E2-vaccinated dogs, and three of six in the E7-vaccinated beagles developed oral papillomas. Compared to the HBVs negative-control group the oral papillomas that did develop in the early-gene vaccinated beagles were significantly smaller, shorter in duration, and fewer in number. Taken together the disease burden was markedly reduced and this was statistically significant. In a second experiment one group of animals was vaccinated with plasmid encoding the wild-type COPV E1 gene, and a separate group was vaccinated with plasmid encoding a synthetic codon-optimised COPV E1 gene sequence. None of the codon-optimised E1-vaccinated animals developed papillomas at any challenge site. However, all animals vaccinated with wild-type E1 had papillomas. These data suggest that immunisation by PMID with papillomavirus early genes can significantly impact upon subsequent disease development and that full protection can be achieved using improved vectors encoding codon-optimised gene sequences perhaps emphasizing the importance of antigen load in the generation of protective responses to papillomavirus proteins.

Animals↗
PubMed1990

Analysis of leaky viral translation termination codons in vivo by transient expression of improved beta-glucuronidase vectors.

Plant RNA viruses commonly exploit leaky translation termination signals in order to express internal protein coding regions. As a first step to elucidate the mechanism(s) by which ribosomes bypass leaky stop codons in vivo, we have devised a system in which readthrough is coupled to the transient expression of beta-glucuronidase (GUS) in tobacco protoplasts. GUS vectors that contain the stop codons and surrounding nucleotides from the readthrough regions of several different RNA viruses were constructed and the plasmids were tested for the ability to direct transient GUS expression. These studies indicated that ribosomes bypass the leaky termination sites at efficiencies ranging from essentially 0 to ca. 5% depending upon the viral sequence. The results suggest that the efficiency of readthrough is determined by the sequence surrounding the stop codon. We describe improved GUS expression vectors and optimized transfection conditions which made it possible to assay low-level translational events.

Base Sequence↗
PubMed1991

Amber codon suppression: the in vivo and in vitro analysis of two C-hordein genes from barley.

A 1420 bp genomic fragment (lambda-hor1-17) encompassing a Hor-1 gene encoding a C-hordein polypeptide is presented. The deduced amino acid sequence is 261 residues long. It comprises a 20 amino acid signal peptide, unique NH2- and COOH-terminal regions and a coding region comprised of pentapeptide (PQQPY) and octapeptide (PQQPFPQQ) repeat motifs. The 431 bp of 5' non-coding region contains a 'TATA box' at -105, a 'CACA box' (-181 to -201) and a -300 prolamin element. In the 3' non-coding region there are two putative polyadenylation signals located 88 and 142 bp downstream of the stop codon. The structure of lambda-hor1-17 is compared with that of another gene (lambda-hor1-14) encoding a C-hordein polypeptide, which contains an amber codon interrupting the ORF. A functional assay in which the 5' non-coding regions of the two genes were fused to the beta-glucuronidase (GUS) gene demonstrated that both genes were transcriptionally active and that circa 430 bp of the C-hordein promoters were sufficient to drive the expression of the GUS gene in developing barley endosperms. It also demonstrated that both promoters had transcriptional efficiencies comparable with that of the 35S CaMV promoter. The in vitro translation of the coding region of lambda-hor1-14 in the wheat germ system showed that the premature stop codon could be partially suppressed. The suppression was also demonstrated in a transient expression assay in vivo using isolated barley endosperms.

Amino Acid Sequence↗
PubMed1994

Codon usage patterns among genes for lepidopteran hemolymph proteins.

Patterns in codon usage were examined for the coding regions of the 23 known lepidopteran hemolymph proteins. Coding triplets are GC rich at the third position and a significant linear relationship between GC content of silent and nonsilent (replacement) sites was demonstrated. Intron GC content was significantly lower than in coding regions and no relationship between intron GC content and the same at silent and nonsilent sites was found. Though hemolymph proteins are all produced by the same tissue--fat body--significantly less bias was observed when all moth sequences were pooled than when sequences of the two major species were analyzed separately, as predicted by the genome hypothesis. In cases where no statistically significant bias was observed, polar or acidic/basic amino acids were almost exclusively involved. Calculation of codon adaptation indices (CAI) was of limited value in quantifying the degree of codon bias and probably reflects the complexity of multicellular-organism life cycles and the changing patterns of gene expression over different developmental stages.

Animals↗
PubMed1995

Kinetics of synonymous codon change for an amino acid of arbitrary degeneracy.

The kinetics of synonymous codon change and species divergence is described in a matrix formalism that is equally applicable to all levels of codon degeneracy and all levels of codon or nucleotide bias. Based on the formalism it is possible to calculate the sum of all synonymous substitution rate constants from the observed sequence differences between two species. This sum, the relaxation rate, is equivalent to the Log Det transformation that has recently been proposed as a new measure of evolutionary distance (Lockhardt et al. Mol. Biol. Evol. 11(4):605-612, 1994). The relationship between this measure and the average number of base changes per site (Ks) is discussed. The formalism is tested on some sets of simulated sequence divergence data.

Amino Acids↗
PubMed1995

Detection of a germline mutation at codon 918 of the RET proto-oncogene in French MEN 2B families.

Multiple endocrine neoplasia type 2A (MEN 2A), type 2B (MEN 2B), and familial medullary thyroid carcinoma (FMTC) are three dominantly inherited disorders linked to the same disease locus on chromosome 10. Two types of germline mutation of the RET proto-onco-gene, which codes for a transmembrane tyrosine kinase, are associated with MEN 2. Missense mutations at cysteine residues in the extra-cytoplasmic domain are exclusively associated with MEN 2A and FMTC. In MEN 2B patients, a single point mutation at codon 918 has recently been characterized, leading to the replacement of a methionine by a threonine within the RET tyrosine kinase domain. We now report the identification of a mutation at codon 918 in the germline of 16 patients out of 18 unrelated MEN 2B families analyzed. In these families we have been able to demonstrate that, in five cases, the mutation arose de novo, and that, in one kindred, it was coinherited with the disease. These results indicate that a unique mutation at codon 918 of the RET gene is the most prevalent genetic defect causing MEN 2B, but also that rare MEN 2B cases are associated with different mutations yet to be defined.

Adolescent↗
PubMed1990

Functional analysis of the 5' regulatory region and the UUG translation initiation codon of the Arthrobacter oxidans 6-hydroxy-D-nicotine oxidase gene.

A functional analysis of the Arthrobacter oxidans 6-hydroxy-D-nicotine oxidase (6-HDNO) gene promoter (-35 region TTGACA and -10 region TATCAAT) and the UUG translation start codon was performed using site-directed mutagenesis. Deletion of the C residue from the -10 promoter region or mutations introduced upstream of the -10 region resulted in an increased 6-HDNO expression in Escherichia coli cells in vivo and in both E. coli and A. oxidans coupled transcription-translation systems in vitro. From the identical behaviour of 6-HDNO promoter mutants in the heterologous and homologous systems, it is concluded that A. oxidans harbours an RNA polymerase functionally homologous to the E. coli sigma 70 and Bacillus subtilis sigma 43 polymerases. Replacement of the TTG codon (UUG translation initiation codon) with ATG led to a 3.7-fold increase in 6-HDNO expression in E. coli. This effect was less pronounced at higher promoter strengths, from 3.7 in the case of the 6-HDNO wild-type promoter, to 2.5 in the case of the consensus -10 region and to 1.7 in the case of the tac promoter. A double point mutation introduced close to the ribosome binding site resulted in almost the same increase in 6-HDNO expression (3.1-fold) as the TTG-to-ATG exchange. The failure of cAMP to stimulate 6-HDNO expression in the A. oxidans system indicated that expression of this gene in stationary phase cells is not regulated by cAMP-catabolite repressore protein-mediated mechanism of catabolite repression.(ABSTRACT TRUNCATED AT 250 WORDS)

Arthrobacter↗
PubMed1980

"Two out of three" codon reading leading to mistranslation in vivo.

Strains of Escherichia coli were starved for asparagine or lysine in order to increase the in vivo level of mistranslation. In a relA strain, asparagine starvation increased the error frequency in elongation factor Tu to 0.12 mistake per asparagine codon, while with lysine starvation in the same strain the error frequency per lysine codon was 0.008. The pattern of isoelectric point changes in the altered protein produced is consistent with third position misreading in the AAN codon group. This high level of mistranslation is not seen in streptomycin resistant (rpsL) strains or in most relA+ strains.

Asparagine↗