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An ACG codon, which replaces the AUG codon used to initiate the synthesis of bacteriophage T7 gene 0.3 protein, was shown to function as a low-efficiency initiation codon in a wheat germ cell-free protein-synthesizing system.
The gene for Wilson disease (WD) has been cloned as a P type copper transporter gene (ATP7B). To elucidate the possible genetic mechanism for the diversity of clinical manifestations, we characterised 22 Taiwanese families with WD by microsatellite haplotyping of close DNA markers D13S314-D13S301-D13S316. We also screened for mutations of codon 778 in the transmembrane region. There were at least 15 haplotypes within eight broad subgroups observed among 44 WD chromosomes. Among the 22 unrelated patients, we found that six patients (27%) carried a codon 778 mutation. Nucleotide sequence analysis showed there were two different mutations: the previously reported Arg778Leu mutation in four patients and Arg778Gln, a new mutation, in two patients. The two different mutations of the same codon occurred in two distinct microsatellite haplotypes.
A man was studied with sporadic Creutzfeldt-Jakob disease (sCJD) who had serial cortical syndromes evolving over 15 months without significant ataxia, prominent myoclonus, or periodic complexes on EEG examinations. This clinical phenotype correlated with a predominantly cortical and striatal distribution of lesions and accumulation of protease resistant prion protein with relative sparing of the brainstem or cerebellum. No amyloid plaques were seen and prion protein (PrP) immunohistochemistry only demonstrated very faint granular deposits in the cerebral cortex. Molecular analysis showed homozygosity for valine at codon 129 in the prion protein gene (PRNP) and protease resistant prion protein type 1 deposition. The comparison of molecular and clinicopathological features of the present case with those previously reported in sCJD, indicates that valine homozygosity at codon 129 and type 1 protease resistant prion protein are associated with a distinct phenotypic variant of sCJD. The data also support the view that the PRNP codon 129 polymorphism and the physicochemical properties of the protease resistant prion protein are major determinants of phenotypic variability in sCJD.
The translational stop signal and polypeptide release factor (RF) complexed with Escherichia coli ribosomes have been shown to be in close physical contact by site-directed photochemical cross-linking experiments. The RF has a protease-sensitive site in a highly conserved exposed loop that is proposed to interact with the peptidyltransferase center of the ribosome. Loss of peptidyl-tRNA hydrolysis activity and enhanced codon-ribosome binding by the cleaved RF is consistent with a model whereby the RF spans the decoding and peptidyltransferase centers of the ribosome with domains of the RF linked by conformational coupling. The cross-link between the stop signal and RF at the ribosomal decoding site is influenced by the base following the termination codon. This base determines the efficiency with which the stop signal is decoded by the RF in both mammalian and bacterial systems in vivo. The wide range of efficiencies correlates with the frequency with which the signals occur at natural termination sites, with rarely used weak signals often found at recoding sites and strong signals found in highly expressed genes. Stop signals are found at some recoding sites in viruses where -1 frame-shifting occurs, but the generally accepted mechanism of simultaneous slippage from the A and P sites does not explain their presence here. The HIV-1 gag-pol-1 frame shifting site has been used to show that stop signals significantly influence frame-shifting efficiency on prokaryotic ribosomes by a RF-mediated mechanism. These data can be explained by an E/P site simultaneous slippage mechanism whereby the stop codon actually enters the ribosomal A site and can influence the event.
The mechanism of elongation factor Tu (EF-Tu) catalyzed aminoacyl-tRNA (aa-tRNA) binding to the A site of the ribosome was studied. Two types of complexes of EF-Tu with GTP and aa-tRNA, EF-Tu.GTP-aa-tRNA (ternary) and (EF-Tu.GTP)2.aa-tRNA (quinternary), can be formed in vitro depending on the conditions. On interaction with the ribosomal A site, generally only one molecule of GTP is hydrolysed per aa-tRNA bound and peptide bond formed. The second GTP molecule from the quinternary complex is hydrolyzed only during translation of an oligo(U) tract in the presence of EF-G. The first step in the interaction between the ribosome and the ternary complex is the codon-independent formation of an initial complex. In the absence of codon recognition, the aa-tRNA-EF-Tu complex does not enter further steps of A site binding and remains in the initial binding state. Despite the rapid formation of the initial complex, the rate constant of GTP hydrolysis in the noncognate complex is four orders of magnitude lower compared with the cognate complex. This, together with the results of time-resolved fluorescence measurements, suggests that codon recognition by the ternary complex on the ribosome initiates a series of structural rearrangements that result in a conformational change of EF-Tu, presumably involving the effector region, which, in turn, triggers GTP hydrolysis and the subsequent steps of A site binding.
Nonsteroidal anti-inflammatory drug (NSAID) use reduces the risk of colorectal cancer by 40-50%. Previous studies suggest that effective inhibition of colorectal cancer by NSAIDs may be dependent on the presence or absence of a K-ras mutation. This study was aimed at determining the relationship between inhibition of colorectal cancer by sulindac and sulindac sulfone and the presence of activating K-ras mutations in the 1,2-dimethylhydrazine dihydrochloride rat model. Sulindac (20 mg x kg(-1) x day(-1)), sulindac sulfone (40 mg x kg(-1) x day(-1)), or vehicle was administered orally to male Sprague-Dawley rats for a 4-wk period beginning 20 wk after tumor induction. Tumor number and volume were measured before treatment by laparotomy and colonoscopy and again after treatment. Sulindac and sulindac sulfone treatment significantly reduced the number and volume of colorectal tumors compared with control rats. For K-ras (codon 12) mutation detection, frozen tumor tissue was collected at the endpoint. We found K-ras codon 12 mutations in 11 of 21 (52%) control tumors. The proportion of tumors with K-ras mutations in the sulindac-treated group [5 of 8 (62%); odds ratio = 1.51 (95% confidence interval = 0.29, 8.33)] and the proportion of sulindac sulfone-treated tumors [9 of 14 (64%); odds ratio = 1.63 (95% confidence interval = 0.41, 6.66)] were not significantly different from controls. Tumor inhibition did not correlate with K-ras (codon 12) mutation status, which suggests that the mechanism of inhibition of rat colorectal cancer by sulindac and sulindac sulfone is independent of K-ras mutation.
Mutations in p53 are common events during carcinogenesis and have been suggested to affect sensitivity to chemotherapy. Recently, the common polymorphism at codon 72, resulting in either an arginine (72R) or proline (72P) residue, was shown to differentially affect the response to anticancer drugs. Here, we have generated isogenic lung cancer cell lines to evaluate the effect of six p53 hotspot mutations (R175H, G245S, R248W, R249S, R273H, and R282W) in conjunction with the codon 72 polymorphism, for their response to a variety of anticancer drugs, either alone or in combination. The data indicate that 72R mutations do not confer general resistance to cisplatin, etoposide, gemcitabine, vinblastine, and taxol. For doxorubicin, cells expressing 249-72R were more resistant than the 249-72P cells. Combined treatment with cisplatin + etoposide resulted in an additive effect in cells expressing most 72R and 72P mutations, except for the 175-72R cells which were refractory to combined treatment. However, combined treatment with cisplatin + gemcitabine resulted in the absence of an additive effect in cells expressing the 273-72R and 282-72R mutants, unlike their 72P counterparts. Nonetheless, all p53 mutants (72R or 72P) equally inhibited p73-mediated transcriptional activity in lung cancer cells, suggesting that the selective resistance conferred by some 72R mutants to certain drugs is probably due to other p73-independent effects of these mutants. Together, the data show that the status of codon 72 polymorphism and p53 mutations can be used as a means for prediction of treatment response, although variables for each cancer type requires detailed evaluation.
BACKGROUND: The major house dust mite allergen Der p 1 is associated with allergic diseases such as asthma. Production of recombinant Der p 1 was previously attempted, but with limited success. The present study describes the expression of recombinant (rec) ProDer p 1, a recombinant precursor form of Der p 1, in CHO cells. METHODS: As optimization of the codon usage may allow successful overexpression of protein in mammalian cells, a synthetic gene encoding ProDer p 1 was designed on the basis of the codon usage frequently found in highly expressed human genes. Gene synthesis was accomplished from a set of 14 mutually priming overlapping oligonucleotides and after two runs of polymerase chain reaction. RESULTS: COS cells transiently transfected with the synthetic ProDer p 1 gene produced up to 5--10 times as much ProDer p 1 compared with the expression level obtained after transfection with the authentic gene. To stably express the recombinant allergen, CHO-K1 cells were transfected with the ProDer p 1 synthetic gene, and one amplified recombinant clone produced up to 30 mg of recProDer p 1 per liter in the culture medium before purification. recProDer p 1 was secreted as an enzymatically inactive single-chain molecule presenting three glycosylated immunoreactive forms of 41, 38 and 36 kD. When examined with respect to direct binding, recProDer p 1 and natural Der p 1 displayed very similar IgE reactivities. However, IgE inhibition and histamine release assays showed a much higher reactivity to natural Der p 1 compared to recProDer p 1. CONCLUSIONS: These data indicated that codon optimization represents an attractive strategy for high-level production of allergen in mammalian cells.
To determine the phenotype of a Japanese family in which retinitis pigmentosa cosegregates with a rhodopsin gene mutation, i.e. an asparagine-to-serine change at codon 15 (Asn-15-Ser), 5 affected and 5 unaffected members of one pedigree underwent several ophthalmic examinations as well as Ganzfeld electroretinography (ERG) and multifocal ERG. Genomic DNA samples were analyzed by PCR amplification, sequencing and restriction enzyme digestion. A codon 15 rhodopsin gene mutation (Asn-15-Ser) was found in all affected members. The region of pigmentary degeneration was localized in the lower hemiretina, and visual field defects corresponded to the retinal pigmentary changes. Scotopic ERG amplitudes, rather than photopic ERG amplitudes, were reduced. Multifocal ERG revealed a low magnitude of response density, even for the upper hemiretina, which showed no bony corpuscle pigmentation. Visual function in sectorial retinitis pigmentosa associated with rhodopsin gene codon 15 mutation is on the basis of the rod-cone dystrophy, regardless of differences in phenotypic expression.
The region located downstream of the initiation codon constitutes part of the translation initiation signal, significantly affecting the level of protein expression in E. coli. In order to determine its influence on translation initiation, we inserted random 12-base sequences downstream of the initiation codon of the lacZ gene. A total of 119 random clones showing higher beta-galactosidase activities than the control lacZ gene were isolated and subsequently sequenced. Analysis of these clones revealed that their insertion sequences are strikingly rich in A and T, but poor in G, with no consensus sequences among them. Toeprinting experiments and polysome profile analysis confirmed that the A/T-rich sequences enhance translation at the level of initiation. Collectively, the present data demonstrate that A/T richness of the region following the initiation codon plays a significant role in E. coli gene expression.
alpha-globin is encoded by two adjacent genes, alpha 1 and alpha 2. Recent evidence suggests that these genes are not equally expressed and that the alpha 2-globin gene encodes the majority of alpha-globin. This finding would predict that a thalassemic mutation of the alpha 2-globin gene would result in a more severe loss of alpha-chain synthesis than a similar mutation in the alpha 1-globin gene. In a previous study we described a nondeletion alpha-thalassemia defect in the alpha 2-globin gene resulting from an AUG----ACG initiation codon mutation. In the present study we describe a different initiation codon mutation, AUG----GUG, present in the alpha 1-globin gene. The alpha 1- and alpha 2-globin gene initiation codon mutations result in similarly lowered levels of encoded mRNA. Despite the similarity of these two mutations, the alpha 2 mutant results in a more severe loss of alpha-globin synthesis and a more severe clinical alpha-thalassemia phenotype than the corresponding alpha 1-globin gene mutation. This difference reflects the dominant role of alpha 2-globin gene in overall alpha-globin synthesis.
Mutations of the androgen receptor that impair the action of 5 alpha-dihydrotestosterone and testosterone result in abnormal male sexual development. The definition of the organization of the androgen receptor gene has permitted us to examine its structure in nine patients with androgen resistance that exhibit absent 5 alpha-dihydrotestosterone binding in cultured fibroblasts (receptor-negative androgen resistance). Using labeled probes specific for each individual coding exon, we find no gross rearrangements, insertions, or deletions of the androgen receptor gene in these patients. To analyze the genetic defect in these receptor-negative patients, we used the polymerase chain reaction to amplify each individual exon of the androgen receptor gene in nine affected patients. In all patients, the size of each amplified exon segment was identical to that in normal individuals. The nucleotide sequence of the entire coding region of the androgen receptor was determined in one of these patients. A single nucleotide substitution was identified that results in a premature termination codon in exon 6 at amino acid 794. S1 nuclease protection assays demonstrated that normal levels of androgen receptor mRNA are present in skin fibroblasts of this patient. Transfection of a mutated androgen receptor cDNA containing a termination codon at position 794 into eukaryotic cells resulted in formation of a normal amount of receptor protein, as indicated by immunoblotting, but the expressed protein does not bind 5 alpha-dihydrotestosterone. These findings suggest that the presence of a premature termination codon at amino acid 794 of the androgen receptor is the cause of androgen resistance in this patient.
AIMS: A case-control study was performed to investigate the relationship between cervical cancer and TP53 polymorphism at codon 72 in young black African women from The Gambia. MATERIALS AND METHODS: The TP53 polymorphism at codon 72 was examined by PCR amplification and SSCP analysis in 40 patients with primary cervical cancer and in 20 healthy women of the same age and from the same geographical area. The occurrence of TP53 polymorphism in combination with the HPV-16 E6 genotype (assayed by PCR) was evaluated. RESULTS: The distribution of TP53 genotypes in cervical cancer patients and in the control group was not statistically different (p = 0.45) and homozygosity for argine at residue 72 was not associated with cervical cancer (odds ratio: 1.24; 95% confidence interval 0.21-9.16). Similarly, a different genotype distribution, cervical cancer and presence of HPV-16 E6 were not observed. CONCLUSIONS: These results cannot rule out an association between TP53 polymorphism at codon 72, HPV infection and the etiology of cervical cancer in this population sample.
Activating point mutations in codons 12, 13, or 61 of the K-ras and N-ras genes have been reported to occur in up to 40% of patients with multiple myeloma at presentation. In a study of 34 presentation myeloma cases using a sensitive polymerase chain reaction-restriction fragment length polymorphism strategy on enriched tumor cell populations, the present study detected N-ras codon 61 mutation-positive cells in all patients. Quantitative plaque hybridization using allele-specific oligonucleotide probes showed that in the majority of patients, ras mutation-positive cells comprise only a subpopulation of the total malignant plasma cell compartment (range, 12%-100%). Using clonospecific point mutations in the 5' untranslated region of the BCL6 gene to quantitate clonal B cells in FACS-sorted bone marrow populations from 2 patients, the representation of ras mutation-positive cells was independent of immunophenotype. These observations imply that mutational activation of N-ras codon 61 is a mandatory event in the pathogenesis of multiple myeloma; such mutations provide a marker of intraclonal heterogeneity that may originate at an earlier ontologic stage than immunophenotypic diversification of the malignant B cell clone.
BACKGROUND: The moss Physcomitrella patens is an emerging plant model system due to its high rate of homologous recombination, haploidy, simple body plan, physiological properties as well as phylogenetic position. Available EST data was clustered and assembled, and provided the basis for a genome-wide analysis of protein encoding genes. RESULTS: We have clustered and assembled Physcomitrella patens EST and CDS data in order to represent the transcriptome of this non-seed plant. Clustering of the publicly available data and subsequent prediction resulted in a total of 19,081 non-redundant ORF. Of these putative transcripts, approximately 30% have a homolog in both rice and Arabidopsis transcriptome. More than 130 transcripts are not present in seed plants but can be found in other kingdoms. These potential "retained genes" might have been lost during seed plant evolution. Functional annotation of these genes reveals unequal distribution among taxonomic groups and intriguing putative functions such as cytotoxicity and nucleic acid repair. Whereas introns in the moss are larger on average than in the seed plant Arabidopsis thaliana, position and amount of introns are approximately the same. Contrary to Arabidopsis, where CDS contain on average 44% G/C, in Physcomitrella the average G/C content is 50%. Interestingly, moss orthologs of Arabidopsis genes show a significant drift of codon fraction usage, towards the seed plant. While averaged codon bias is the same in Physcomitrella and Arabidopsis, the distribution pattern is different, with 15% of moss genes being unbiased. Species-specific, sensitive and selective splice site prediction for Physcomitrella has been developed using a dataset of 368 donor and acceptor sites, utilizing a support vector machine. The prediction accuracy is better than those achieved with tools trained on Arabidopsis data. CONCLUSION: Analysis of the moss transcriptome displays differences in gene structure, codon and splice site usage in comparison with the seed plant Arabidopsis. Putative retained genes exhibit possible functions that might explain the peculiar physiological properties of mosses. Both the transcriptome representation (including a BLAST and retrieval service) and splice site prediction have been made available on http://www.cosmoss.org, setting the basis for assembly and annotation of the Physcomitrella genome, of which draft shotgun sequences will become available in 2005.
The aim of the study is to investigate whether the functional p53 codon 72 polymorphism is associated with susceptibility to SLE and its clinical features. A polymerase chain reaction of genomic DNA-restriction fragment length polymorphism was used to determine genotypes of the p53 codon 72 in 90 SLE patients and 114 healthy controls. Clinical/serological manifestations were analysed in each patient and correlated with the genotypes. The OR of the association of the Pro allele with SLE was 1.70 (95% CI, 1.15-2.53, P = 0.0079) and the OR of the Pro/Pro (a recessive model) was significantly increased (OR = 2.58, 95% CI = 1.24-5.39, P = 0.0093). The Armitage's trend test indicated a significant dosage effect of the Pro allele for SLE (OR = 1.73, chi-square = 7.08, P = 0.0078). However, there was no significant association of the polymorphism with clinical/serological manifestations studied here. In conclusion, our finding suggests the functional p53 codon 72 polymorphism may be associated with SLE susceptibility, suggesting individuals who carry the Pro allele may have a higher risk to SLE susceptibility than those with the Arg allele. Further studies for replications are needed to confirm that the p53 polymorphism contributes to SLE.
PURPOSE: The aim of this study was to investigate the prognostic importance of codon 12 K-ras mutations in patients with early-stage non-small-cell lung cancer (NSCLC). PATIENTS AND METHODS: We identified 260 patients with surgically resected stage I (n = 193) and stage II (n = 67) NSCLC with at least a 5-year follow-up. We performed polymerase chain reaction analysis of DNA obtained from paraffin-embedded NSCLC tissue, using mutation-specific probes for codon 12 K-ras. RESULTS: K-ras mutations were detected in 35 of 213 assessable specimens (16.4%). K-ras mutations were detected in 27 of 93 adenocarcinomas (29.0%), one of 61 squamous cell carcinomas (1.6%), five of 39 large-cell carcinomas (12.8%), and two of 20 adenosquamous carcinomas (10%) (P = .001). G to T transversions accounted for 71% of the mutations. There was no statistically significant difference in overall survival for all patients with K-ras mutations (median survival, 39 months) compared with patients without K-ras mutations (median survival, 53 months; P = .33). There was no statistically significant difference in overall or disease-free survival for subgroups with stage I disease, adenocarcinoma, or non-squamous cell carcinoma or for specific amino acid substitutions. The median survival time for stage II patients with K-ras mutations was 13 months, compared with 38 months for patients without K-ras mutations (P = .03). CONCLUSION: Codon 12 K-ras mutations were more common in adenocarcinomas than in squamous cell carcinomas. For the subgroup with stage II NSCLC, there was a statistically significant adverse effect on survival for the presence of K-ras mutations. However, when the entire group was considered, the presence of K-ras mutations was not of prognostic significance in this cohort of patients with resected early-stage NSCLC.
PURPOSE: To explore the prognostic value of mutations in c-KIT and PDGFR-alpha genes with respect to relapse-free survival (RFS) in patients with gastrointestinal stromal tumors (GIST). We have investigated the prognostic relevance of the type and position of the mutations, in addition to other clinicopathologic factors, in a large series of patients with GIST. METHODS: For this study, 162 patients were selected according to the following criteria: completely resected tumors with negative margins attended between 1994 and 2001; no metastasis at diagnosis; tumor larger than 2 cm, c-KIT-positive immunostaining; and no other primary tumors. RESULTS: The median follow-up was 42 months for patients free of recurrence. Mutations were detected in 96 tumors (60%): 82 cases involving c-KIT and 14 cases involving PDFGR-alpha. Univariate analysis demonstrated the following as poor prognostic factors for RFS: tumors larger than 10 cm (P < .0001); mitotic count higher than 10 mitoses per 50 high-power fields (P < .0001); high risk index (P < .0001); intestinal GIST location (P = .0041); high cellularity (P < .0001); tumor necrosis (P < .0001); deletions affecting exon 11 (P = .0007); and deletions affecting codons 557 to 558 (P < .0001). After the multivariate analysis, only the high risk index (relative risk [RR], 12.36), high cellularity (RR, 3.97), and deletions affecting codons 557 to 558 of c-KIT (RR, 2.57) corresponded to independent prognostic factors for RFS in GIST patients. CONCLUSION: Deletions affecting codons 557 to 558 are relevant for the prognosis of RFS in GIST patients. This critical genetic alteration should be considered to be a new prognostic stratification variable for randomized trials exploring imatinib mesylate in the adjuvant setting in GIST patients.
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