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PubMed1996

Accumulation of a mRNA decay intermediate by ribosomal pausing at a stop codon.

A RNA fragment which is protected from degradation by ribosome pausing at a stop codon has been identified in growing Escherichia coli. The fragment is 261 nt long and corresponds to the 3'-end of the mRNA expressed from a semi-synthetic model gene. The 5'-end of the RNA fragment, denoted rpRNA (ribosomal pause RNA), is located 13 bases upstream of the stop codon. In vivo decay of the complete mRNA and accumulation of rpRNA are dependent on the nature of the stop codon and its codon context. The data indicate that the rpRNA fragment arises from interrupted decay of the S3A'mRNA in the 5'-->m3'direction, in connection with a ribosomal pause at the stop codon. RF-2 decoding of UGA is less efficient than RF-1 decoding of UAG in identical codon contexts, as judged from rpRNA steady-state levels. The half-life of UGA-containing rpRNAs is at least 5 min, indicating that ribosomal pausing can be a major factor in stabilising downstream regions of messenger RNAs.

Base Sequence↗
PubMed1997

Eukaryotic release factor 1 (eRF1) abolishes readthrough and competes with suppressor tRNAs at all three termination codons in messenger RNA.

It is known from experiments with bacteria and eukaryotic viruses that readthrough of termination codons located within the open reading frame (ORF) of mRNAs depends on the availability of suppressor tRNA(s) and the efficiency of termination in cells. Consequently, the yield of readthrough products can be used as a measure of the activity of polypeptide chain release factor(s) (RF), key components of the translation termination machinery. Readthrough of the UAG codon located at the end of the ORF encoding the coat protein of beet necrotic yellow vein furovirus is required for virus replication. Constructs harbouring this suppressible UAG codon and derivatives containing a UGA or UAA codon in place of the UAG codon have been used in translation experiments in vitro in the absence or presence of human suppressor tRNAs. Readthrough can be virtually abolished by addition of bacterially-expressed eukaryotic RF1 (eRF1). Thus, eRF1 is functional towards all three termination codons located in a natural mRNA and efficiently competes in vitro with endogenous and exogenous suppressor tRNA(s) at the ribosomal A site. These results are consistent with a crucial role of eRF1 in translation termination and forms the essence of an in vitro assay for RF activity based on the abolishment of readthrough by eRF1.

Animals↗
PubMed1999

Proteome composition and codon usage in spirochaetes: species-specific and DNA strand-specific mutational biases.

The genomes of the spirochaetes Borrelia burgdorferi and Treponema pallidum show strong strand-specific skews in nucleotide composition, with the leading strand in replication being richer in G and T than the lagging strand in both species. This mutation bias results in codon usage and amino acid composition patterns that are significantly different between genes encoded on the two strands, in both species. There are also substantial differences between the species, with T.pallidum having a much higher G+C content than B. burgdorferi. These changes in amino acid and codon compositions represent neutral sequence change that has been caused by strong strand- and species-specific mutation pressures. Genes that have been relocated between the leading and lagging strands since B. burgdorferi and T.pallidum diverged from a common ancestor now show codon and amino acid compositions typical of their current locations. There is no evidence that translational selection operates on codon usage in highly expressed genes in these species, and the primary influence on codon usage is whether a gene is transcribed in the same direction as replication, or opposite to it. The dnaA gene in both species has codon usage patterns distinctive of a lagging strand gene, indicating that the origin of replication lies downstream of this gene, possibly within dnaN. Our findings strongly suggest that gene-finding algorithms that ignore variability within the genome may be flawed.

Amino Acids↗
PubMed2001

Five-base codons for incorporation of nonnatural amino acids into proteins.

Extension of the genetic code for the introduction of nonnatural amino acids into proteins was examined by using five-base codon-anticodon pairs. A streptavidin mRNA containing a CGGUA codon at the Tyr54 position and a tRNA(UACCG) chemically aminoacylated with a nonnatural amino acid were added to an Escherichia coli in vitro translation system. Western blot analysis indicated that the CGGUA codon is decoded by the aminoacyl-tRNA containing the UACCG anticodon. HPLC analysis of the tryptic fragment of the translation product revealed that the nonnatural amino acid was incorporated corresponding to the CGGUA codon without affecting the reading frame adjacent to the CGGUA codon. Another 15 five-base codons CGGN(1)N(2), where N(1) and N(2) indicate one of four nucleotides, were also successfully decoded by aminoacyl-tRNAs containing the complementary five-base anticodons. These results provide a novel strategy for nonnatural mutagenesis as well as a novel insight into the mechanism of frameshift suppression.

Amino Acid Sequence↗
PubMed2002

Convergence and constraint in eukaryotic release factor 1 (eRF1) domain 1: the evolution of stop codon specificity.

Class 1 release factor in eukaryotes (eRF1) recognizes stop codons and promotes peptide release from the ribosome. The 'molecular mimicry' hypothesis suggests that domain 1 of eRF1 is analogous to the tRNA anticodon stem-loop. Recent studies strongly support this hypothesis and several models for specific interactions between stop codons and residues in domain 1 have been proposed. In this study we have sequenced and identified novel eRF1 sequences across a wide diversity of eukaryotes and re-evaluated the codon-binding site by bioinformatic analyses of a large eRF1 dataset. Analyses of the eRF1 structure combined with estimates of evolutionary rates at amino acid sites allow us to define the residues that are under structural (i.e. those involved in intramolecular interactions) versus non-structural selective constraints. Furthermore, we have re-assessed convergent substitutions in the ciliate variant code eRF1s using maximum likelihood-based phylogenetic approaches. Our results favor the model proposed by Bertram et al. that stop codons bind to three 'cavities' on the protein surface, although we suggest that the stop codon may bind in the opposite orientation to the original model. We assess the feasibility of this alternative binding orientation with a triplet stop codon and the eRF1 domain 1 structures using molecular modeling techniques.

Amino Acid Sequence↗
PubMed1979

The preferential codon usages in variable and constant regions of immunoglobulin genes are quite distinct from each other.

The pattern of codon utilization in the variable and constant regions of immunoglobulin genes are compared. It is shown that, in these regions, codon utilizations are quite distinct from one another: For most degenerate codons, there is a selective bias that prefers C and/or G ending codons to U and/or A ending codons in the constant region compared with the bias in the variable region. This would strongly suggest that, in immunoglobulin genes, the bias in code word usage is determined by other factors than those concerning with the translational mechanism such as tRNA availability and codon-anticodon interaction. A possibility is also suggested that this differance of code word usage between them is due to the existence of secondary structure in the constant region but not in the variable region.

Anticodon↗
PubMed2002

Use and misuse of correspondence analysis in codon usage studies.

Correspondence analysis has frequently been used for codon usage studies but this method is often misused. Because amino acid composition exerts constraints on codon usage, it is common to use tables containing relative codon frequencies (or ratios of frequencies) instead of simple codon counts to get rid of these amino acid biases. The problem is that some important properties of correspondence analysis, such as rows weighting, are lost in the process. Moreover, the use of relative measures sometimes introduces other biases and often diminishes the quantity of information to analyse, occasionally resulting in interpretation errors. For instance, in the case of an organism such as Borrelia burgdorferi, the use of relative measures led to the conclusion that there was no translational selection, while analyses based on codon counts show that there is a possibility of a selective effect at that level. In this paper, we expose these problems and we propose alternative strategies to correspondence analysis for studying codon usage biases when amino acid composition effects must be removed.

Bacillus subtilis↗
PubMed1999

Nucleotide sequences of animal mitochondrial tRNAs(Met) possibly recognizing both AUG and AUA codons.

To elucidate the role of modified nucleosides of tRNA in mitochondrial translation systems, especially with regard to their codon recognition, we purified mitochondrial tRNAs(Met) isolated from liver of frog, chicken and rat, and determined their nucleotide sequences. All of these tRNAs(Met) were found to possess 5-formylcytidine in the first letter of the anticodon, which is known to be prerequisite for bovine mt tRNA(Met) to decode AUA codon as well as AUG codon. These tRNA possesses two pseudeuridines in similar positions, and only chicken tRNA(Met) had ribothymidine at the first position of the T-loop, which is always found in the usual tRNAs. Considering that AUA codon is used as five times frequently as AUG codon in these animal mitochondrial genomes, it is deduced that 5-formylcytidine at the wobble position is essential for the recognition of both AUA and AUG codons.

Animals↗
PubMed2002

Alternative CUG codon usage (Ser for Leu) in Pichia farinosa and the effect of a mutated killer gene in Saccharomyces cerevisiae.

The halotolerant yeast Pichia farinosa KK1 strain produces a killer toxin termed SMKT (salt-mediated killer toxin). Mass spectrometry and Edman sequencing of peptides from the mature SMKT and secreted protoxin demonstrate that positions specified by the CUG codon contain unmodified serine (Ser) in P.farinosa. In order to express the authentic SMK1 product in Saccharomyces cerevisiae, which uses the universal genetic code, the three CUG codons corresponding to Ser87, Ser137 and Ser206 in the SMK1 gene were changed to universal Ser codons by site-directed mutagenesis. The expression of the modified SMK1 gene with universal Ser codons was lethal in S.cerevisiae, as well as that of the unmodified SMK1 gene with the CUG codons. The secretion of protoxin with the authentic amino acid sequence from the modified SMK1 was significantly increased, whereas the transcription level of SMK1 was not affected in the presence or absence of CUG codon. Our results provide the first in vivo evidence that non-universal decoding of CUG is used in a hemiascomycetous yeast, P.farinosa.

Amino Acid Substitution↗
PubMed1997

A two-step enriched-nested PCR technique enhances sensitivity for detection of codon 12 K-ras mutations in pancreatic adenocarcinoma.

Mutations at codon 12 of the K-ras gene have been detected in pancreatic adenocarcinomas by a variety of techniques. A few of these techniques are very sensitive, identifying the mutations in 96-100% of cases. However, these sensitive techniques are labor intensive, utilizing multistep processing and radioactive material. Much simpler techniques, involving nonradioactive single-step polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) have been employed to detect K-ras mutations at codon 12 in pancreatic adenocarcinomas. However, the low sensitivity of these single-step PCR/ RFLP techniques is unacceptable. A simple and nonradio-active PCR/RFLP-based method for detection of K-ras codon 12 mutations in formalin-fixed, paraffin-embedded tissue sections of pancreatic adenocarcinoma is described and compared to the traditional PCR technique. K-ras gene mutations at codon 12 were detected by a modified two-step enrich-nested PCR (EN-PCR)/RFLP method, and their existence was confirmed by direct DNA sequencing analysis of the product. When the two-step EN-PCR/RFLP technique was compared to the single-step PCR/RFLP method, K-ras codon 12 mutations were detected in 100% of pancreatic adenocarcinomas (15/15) with the EN-PCR/RFLP method, while half as many (9/15) were detected with the single-step PCR/RFLP method. This study demonstrates that the sensitivity of the simple two-step EN-PCR/RFLP technique is comparable to that of the more complex methods for detecting K-ras mutations at codon 12 in formalin-fixed, paraffin-embedded tissue sections of pancreatic adenocarcinoma and its sensitivity is superior to that of the single-step technique.

Base Sequence↗
PubMed2000

Reassessment of K-ras mutations at codon 12 by direct PCR and sequencing from tissue microdissection in human pancreatic adenocarcinomas.

K-ras mutations at codon 12 have been detected in almost all pancreatic adenocarcinomas by highly sensitive assays. We reassessed the K-ras mutation status by direct polymerase chain reaction (PCR) and sequencing from tissue microdissection without DNA extraction in 10 pancreatic adenocarcinomas, and also assessed the K-ras and DPC4 genes in nine pancreatic cancer cell lines. Eight pancreatic adenocarcinomas were found to harbor K-ras mutations at codon 12 of either GTT or GAT, five of which were inferred to harbor amplified mutant alleles. Mutations at the sites other than codon 12 were found in seven of 70 clones (seven of 9,380 bases) by the TA cloning analysis, suggesting that artifactual mutations at the first or second base of codon 12 before and during PCR could occur at a frequency of approximately 10(-3), enough for highly sensitive assays to detect. Two cell lines without K-ras mutations at codon 12 were found to have homozygous deletions at the DPC4 gene. Thus the K-ras mutation status was demonstrated to be correctly determined by just direct sequencing from tissue microdissection. All possible mutations or multiple mutations at K-ras codon 12 that have been reported in pancreatic adenocarcinomas might include artifacts or mutations without a selective advantage. In addition, we must be very cautious about contamination.

Adenocarcinoma↗
PubMed2000

Absence of translationally selected synonymous codon usage bias in Helicobacter pylori.

Synonymous codon usage in the complete genome of Helicobacter pylori was investigated. The moderate A+T-richness of the genome (G+C=39 mol%) is reflected in the overall synonymous codon usage but the frequencies of some codons cannot be explained by simple mutational biases. A low level of heterogeneity among genes was observed, but this does not appear to be due to varying mutational bias or translational selection. Some of the heterogeneity was due to amino acid composition variation among the encoded proteins, and some may be attributable to recent acquisition of genes from other species. Since Hel. pylori codon usage is not dominated by biased mutation patterns, the absence of evidence for translationally mediated selection among synonymous codons is striking. This has implications with regard to the life history of this species, and in particular suggests that Hel. pylori strains are not subject to periods of competitive exponential growth. Despite the lack of selected codon usage, base composition immediately after the translation initiation site is skewed, consistent with selection against secondary structure formation in this region.

Codon↗
PubMed1990

Frequent glycine-to-aspartic acid mutations at codon 12 of c-Ki-ras gene in human pancreatic cancer in Japanese.

Point mutations at codons 12 and 13 of c-Ki-ras gene were analyzed in human pancreatic cancer. DNAs obtained from sample tissues were amplified by means of polymerase chain reaction and were analyzed by dot blot hybridization assays with oligonucleotide probes appropriate for detecting mutations at these codons. Out of 38 evaluated cases, point mutations at codon 12 were found in 35 cases; these mutations resulted in changes of the coded amino acid to aspartic acid in 24 cases, to valine in 9 cases, to arginine in 2 cases and to cysteine in one case. In one case, a glycine-to-aspartic acid mutation was found at codon 13. In two cases, two distinct mutations were simultaneously present. The frequency pattern of mutations at codon 12 was somewhat different from those given in two previous reports on the similar analysis of pancreatic cancers in European countries. This may indicate the presence of possible genetic or non-genetic factors in determining preferential mutational patterns at these particular codons.

Adult↗
PubMed1993

Codon 12 Harvey-ras mutations are rare events in non-melanoma human skin cancer.

ras mutations have been reported as an early event in some human malignancies and in the mouse skin model of multistep carcinogenesis; early studies in human non-melanoma skin cancers have reported variable rates of ras mutations. A recent study, however, has reported a high frequency of activating mutations of the Harvey-ras proto-oncogene in non-melanoma skin cancers, and the site specificity of the mutation at the second position of codon 12 prompted us to re-examine the importance of Ha-ras codon 12 mutations as an early event in the development of these tumours, using a combination of PCR and restriction fragment polymorphism of codon 12 of the Ha-ras gene. Dilution experiments confirmed that the method was sensitive and capable of detecting mutations at this codon when only 4% of the total alleles are mutated. We were surprised to find no mutations in the 40 basal cell carcinomas, 12 squamous cell carcinomas and 12 cases of Bowen's disease studied. We conclude that Ha-ras codon 12 mutations are rare events in human non-melanoma skin cancer in the U.K. The marked differences in the frequency of codon 12 Ha-ras mutations in published studies may relate to either technical artefacts, or differences in the molecular epidemiology between areas of low and high sun exposure.

Base Sequence↗
PubMedDate not supplied

Codon usage in Tetrahymena and other ciliates.

Codon usage in ciliates was examined by analyzing the coding regions of 22 ciliate genes corresponding to a total of 26,142 nucleotides (8,714 codons). It was found that Tetrahymena, Paramecium and the hypotrichs (Oxytricha and Stylonychia) differed in which synonymous codons were used most frequently by their genes. In fact, the codon choices in highly expressed Tetrahymena genes were more similar to those of yeast genes than those of Paramecium genes. The ciliates do not appear to have unusually strong biases in codon usage frequency when compared to other protists such as yeast. The analysis of the Tetrahymena genes indicated that genes which are highly expressed during normal cell growth have a stronger bias towards using the "preferred" codons than those expressed at lower levels during growth or for brief periods during processes such as conjugation. This conforms to what is found in other protists.

Amino Acids↗
PubMedDate not supplied

Codon usage in Giardia lamblia.

A codon usage table for the intestinal parasite Giardia lamblia was generated by analysis of the nucleotide sequences of eight genes comprising 3,135 codons. Codon usage revealed a biased use of synonymous codons with a preference for NNC codons (42.1%). The codon usage of G. lamblia more closely resembles that of the prokaryote Halobacterium halobium (correlation coefficient r = 0.73) rather than that of other eukaryotic protozoans, i.e. Trypanosoma brucei (r = 0.434) and Plasmodium falciparum (r = -0.31). These observations are consistent with the view that G. lamblia represents the first line of descent from the ancestral cells that first took on eukaryotic features.

Animals↗
PubMed2000

Ki-ras codon 12 point and P53 mutations: a molecular examination of the main tumor, liver, portal vein, peripheral arterial blood and para-aortic lymph node in pancreatic cancer.

OBJECTIVE: Frequent P53 mutations and Ki-ras codon 12 point mutations have been reported in pancreatic cancer. Pancreatic cancer often recurs in the liver and/or lymph nodes shortly after a surgical resection. The purpose of this study is to elucidate the occurrence of microcirculating cancer cells and micrometastasis in pancreatic cancer. METHODS: P53 mutations and Ki-ras codon 12 point mutations were examined in the main tumor, liver, portal vein, and peripheral arterial blood, and para-aortic lymph nodes of patients with pancreatic cancer using molecular examinations. RESULTS: P53 mutations in the main tumor were present in nine (29%) of 31 patients with pancreatic cancer, whereas a Ki-ras codon 12 point mutation was evident in 18 (62%) of 29 examined patients. The peripheral arterial and portal vein blood and liver were positive for gene abnormalities in one (5%) of 21, in none (0%) of 19, and in one (1%) of 20, respectively. A P53 mutation in the main tumor was evident in none (0%) of seven stage I or II carcinomas and in nine (38%) of 24 stage III or IV cases, whereas a Ki-ras codon 12 point mutation was present in four (67%) of six stage I or II cases and in 14 (61%) of 23 stage III or IV cases. In addition, 15 (71%) of 21 patients with gene abnormalities (Ki-ras codon 12 point and/or p53 mutation) in the main tumor showed lymph node metastasis at surgery, whereas five (42%) of 12 without gene abnormalities did not demonstrate lymph node metastasis. Two (29%) of six patients with gene abnormalities in the main tumor and without metastatic disease at surgery developed liver metastasis within 6 months after surgery, whereas all five (100%) without the gene abnormalities and metastatic disease at surgery did not develop the metastasis, with the sensitivity being 100%, specificity 44%, the predictive value of the positive test 36%, and the predictive value of the negative test 100%. Two patients who had gene abnormalities in the para-aortic lymph node were free from histopathological metastasis and these two patients developed para-aortic lymph node metastasis within 6 months after surgery. CONCLUSIONS: A molecular examination of Ki-ras codon 12 and p53 mutations therefore enables us to predict, to some degree, the occurrence of liver and lymph node metastasis in pancreatic carcinoma.

Aged↗
PubMed2001

Variants other than aspartic acid at codon 69 of the human immunodeficiency virus type 1 reverse transcriptase gene affect susceptibility to nuleoside analogs.

The T69D mutation in the human immunodeficiency virus type 1 reverse transcriptase (RT) gene has been associated with reduced susceptibility to dideoxycytosine (ddC); however, several other mutations at codon 69 have been observed in antiretroviral drug-treated patients. The Stanford HIV RT and Protease Sequence Database was interrogated and showed that 23% of patients treated with nucleoside RT inhibitors (NRTI) had mutations at codon 69. These variants included T69N, -S, -A, -G, -E, -I, and -K mutations that were present in patients treated with NRTI but not in drug-naive patients. Treatment history information showed that a substantial percentage of these codon 69 changes occurred in patients administered non-ddC-containing regimens. Different and specific patterns of other RT gene mutations were associated with the various codon 69 mutations. Drug susceptibility assays showed that viral constructs containing codon 69 variants could have reduced susceptibility to ddC and other RT inhibitors. These results suggest that the T69D mutation is not the only codon 69 variant associated with drug resistance and that ddC is not the only drug affected.

Codon↗