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PubMed1993

The translational termination signal database.

The Translational Termination Database (TransTerm) consists of the immediate context sequences around the natural termination codons from 45 organisms, and summary tables. The influence of termination codon context on their effectivness as stop signals has been widely documented. The SPECIES--TRI.DAT table shows trinucleotide stop codon usage in each organism and for comparison the occurrence of these sequences in the noncoding region. The SPECIES--TETRA.DAT table contains is a similar table of tetranucleotide stop signal usage. The database is available from EMBL.

Animals↗
PubMed1999

Rett syndrome and beyond: recurrent spontaneous and familial MECP2 mutations at CpG hotspots.

Rett syndrome (RTT) is a neurodevelopmental disorder characterized by loss of acquired skills after a period of normal development in infant girls. The responsible gene, encoding methyl-CpG binding protein 2 (MeCP2), was recently discovered. Here we explore the spectrum of phenotypes resulting from MECP2 mutations. Both nonsense (R168X and R255X) and missense (R106W and R306C) mutations have been found, with multiple recurrences. R168X mutations were identified in six unrelated sporadic cases, as well as in two affected sisters and their normal mother. The missense mutations were de novo and affect conserved domains of MeCP2. All of the nucleotide substitutions involve C-->T transitions at CpG hotspots. A single nucleotide deletion, at codon 137, that creates a L138X stop codon within the methyl-binding domain was found in an individual with features of RTT and incontinentia pigmenti. An 806delG deletion causing a V288X stop in the transcription-repression domain was identified in a woman with motor-coordination problems, mild learning disability, and skewed X inactivation; in her sister and daughter, who were affected with classic RTT; and in her hemizygous son, who died from congenital encephalopathy. Thus, some males with RTT-causing MECP2 mutations may survive to birth, and female heterozygotes with favorably skewed X-inactivation patterns may have little or no involvement. Therefore, MECP2 mutations are not limited to RTT and may be implicated in a much broader phenotypic spectrum.

Alleles↗
PubMed2000

Hepatitis B virus X gene variability in French-born patients with chronic hepatitis and hepatocellular carcinoma.

The polymorphism of the hepatitis B virus (HBV) X gene from patients born in Lorraine has been studied in serum samples from 22 HBV infected patients, 14 presenting with chronic hepatitis and 8 with hepatocellular carcinoma (HCC). Subtypes adw and ayw represented 21 of the 22 sequenced isolates. The sequence of the X gene of HBV strains from these patients differed from the ones of Far East origin by A to T(1678) and G to A(1759) changes for subtype ayw and C to T(1792) for adw. The expression of the preC region, as indicated by the detection of HBe antigen (HBeAg), was not observed in 11 patients. In 6 patients (3 HCC and 3 non HCC), the absence of HBeAg could be related to a stop codon at position 28. For the 5 remaining patients, the precore stop mutation at codon 28 was not evidenced but 3 out these 5 patients had mutations at nt 1764 and nt 1766 in the promoter of the preC/C gene. These two mutations were also observed in 2 patients with HBeAg, indicating that they are not implicated in the suppression of expression of this gene. Independently of the serotype, two main differences were noted between aminoacid (aa) sequences of chronic hepatitis and HCC related strains: first, twice as many aa changes were found in HCC patients than in chronic hepatitis B carriers (mean of aa changes per patient 4.1 vs. 2.0) and second, we found apparition of polar aa in HCC patients. Most mutations already described in patients from the Far East with HCC have been found in strains of patients from Lorraine. The changes K130M and V131I, considered as "hot spot mutations," were found in strains of HCC patients carrying an ayw subtype of the HBV genome but not in the ones of chronically infected patients. In contrast, strains of the adw subtype had these two changes in the two groups of patients. However when considering the 22 sequenced genes, these hot spot mutations were associated with HCC (P = 0.034).

Adolescent↗
PubMed2005

Beta-thalassemia due to a novel nonsense mutation at codon 37 (TGG-->TAG) found in an Afghanistani family.

We have identified and characterized a novel beta-thalassemic mutation in an Afghanistanifamily. The molecular pathology consists of a single base substitution (TGG-->TAG) at codon 37 of the beta-globin gene, giving rise to a stop codon (TAG). Premature stop of translation results in a truncated protein and usually the phenotype of beta-thalassemia (thal) major in homozygous individuals. However, this was not the case in our proband, who was homozygous for the codon 37 mutation. He presented with the phenotype of thalassemia intermedia with a hemoglobin (Hb) level of 8.1 g/dL and no previous history of blood transfusions. High performance liquid chromatography (HPLC) analysis showed exclusively Hb F except for a Hb A2 level within normal limits. Subsequent analysis demonstrated homozygosity for the XmnI Ggamma polymorphism and heterozygosity for a deletional alpha-thal (alphaalpha/-alpha(-3.7)). These findings might, at least partly, explain the beta-thal intermedia phenotype observed in the proband.

Adult↗
PubMed2001

A double mutation in a patient with X-linked myotubular myopathy.

In this report a double mutation was identified in a patient with X-linked myotubular myopathy. The mutations present in the patient were a C-->T substitution of nucleotide 163, which led to an Arg 55 stop codon (nonsense mutation), and an "A" insertion at nucleotide 440, which caused a shift of the reading frame and a premature stop at codon 153 (frameshift mutation). The nonsense mutation was heterozygously present in the mother but not identified in the father or in normal controls. The frameshift mutation was not identified in either parent or normal controls (de novo mutation). These mutations are predicted to truncate the myotubularin protein.

Codon, Nonsense↗
PubMed1997

Molecular cloning and sequencing of cDNA encoding for a novel testis-specific antigen.

cDNA encoding for a sperm antigen, designated NZ-1, was cloned and sequenced from murine testis cDNA-lambda gt11 expression library using antibodies to human sperm surface antigens belonging to 14-18 kD molecular region. These sperm antigens are involved in zona pellucida binding and have tyrosine phyosphorylation activity. Computer generated translation analysis of 1395-bp cDNA yielded an open reading frame (ORF) of 152 aa with first ATG, Met start codon at nt 32 and the stop codon TGA at nt 487. The translated protein has a calculated molecular weight of 17.9 kD and a potential tyrosine phosphorylation site at aa 46-54, besides at least two O-linked glycosylation sites. The hydropathy plot generated from the deduced aa sequence indicated it to be a membrane-anchored peptide with a hydrophobic NH2-terminus that is characteristic of a signal peptide. Extensive computer search in the GenBank, NBRF, and Swiss sequence banks, indicating it to be a novel protein. Northern blot analysis indicated testis-specific expression of NZ-1 antigen. The NZ-1 cDNA was subcloned into pGEX-1 lambda T vector and expressed in glutathione-S-transferase gene fusion system to obtain the recombinant protein. The recombinant protein specifically reacted with the original antibodies raised against the native 14-18 kD sperm proteins. These findings suggest that the sperm-specific recombinant NZ-1 may find applications in the development of a contraceptive vaccine, and in studying the normal and abnormal sperm function and the signal transduction mechanism.

Amino Acid Sequence↗
PubMed2000

Rapid determination of COL2A1 mutations in individuals with Stickler syndrome: analysis of potential premature termination codons.

Stickler syndrome is one of the milder phenotypes resulting from mutations in the gene that encodes type-II collagen, COL2A1. All COL2A1 mutations known to cause Stickler syndrome result in the formation of a premature termination codon within the type-II collagen gene. COL2A1 has 10 in-frame CGA codons, which can mutate to TGA STOP codons via a methylation-deamination mechanism. We have analyzed these sites in genomic DNA from a panel of 40 Stickler syndrome patients to test the hypothesis that mutations that cause Stickler syndrome preferentially occur at these bases. Polymerase chain reaction (PCR) amplification of genomic DNA containing each of the in-frame CGA codons was done by one of two methods: either using primers that amplify DNA that includes the CGA codon, or using allele-specific primers that either amplify normal sequence containing a CGA codon or amplify a mutant sequence containing a TGA codon. Analysis of PCR products by restriction endonuclease digestion or sequencing demonstrated the presence of a normal or mutated codon. TGA mutations were identified in eight patients, at five of the 10 in-frame CGA codons. The identification of these mutations in eight of 40 patients demonstrates that these sites are common sites for mutations in individuals with Stickler syndrome and, we propose, should be analyzed as a first step in the search for mutations that result in this disorder.

Alleles↗
PubMed1995

Hb Lulu Island (alpha 2 beta 2 107[G9]Gly-->Asp)-beta zero- thalassemia (codon 15; TGG-->TAG), a form of thalassemia intermedia.

Hb Lulu Island [beta 107(G9)Gly-->Asp] was discovered in an East Indian female who carried a common beta zero-thalassemia allele, i.e., codon 15, TGG-->TAG (is a stop codon) in trans. Both abnormalities were detected through sequencing of the amplified beta-globin genes and were confirmed by hybridization with 32P-labeled probes. Hb Lulu Island is mildly unstable with a borderline decrease in oxygen affinity; its instability is less severe than that of Hb Burke or beta 107(G9)Gly-->Arg. The compound heterozygosity expresses as a thalassemia intermedia with moderate anemia, a variable need for blood transfusions, Heinz body formation, and a red cell morphology which is typical for such a condition. The level of HbA2 was greatly increased (6.5-7.0%) as was the delta chain level (12% of total non-alpha) probably because of the instability of Hb Lulu Island and the decreased ability of the beta x chain to form dimers with the normal alpha chain.

Adult↗
PubMed2001

An extended Escherichia coli "selenocysteine insertion sequence" (SECIS) as a multifunctional RNA structure.

The genetic code, once thought to be rigid, has been found to permit several alternatives in its reading. Interesting alternative relates to the function of the UGA codon. Usually, it acts as a stop codon, but it can also direct the incorporation of the amino acid selenocysteine into a polypeptide. UGA-directed selenocysteine incorporation requires a cis-acting mRNA element called the "selenocysteine insertion sequence" (SECIS) that can form a stem-loop RNA structure. Here we discuss our investigation on the E. coli SECIS. This includes the follows: 1) The nature of the minimal E. coli SECIS. We found that in E. coli only the upper-stem and loop of 17 nucleotides of the SECIS is necessary for selenocysteine incorporation on the condition that it is located in the proper distance from the UGA [34]; 2) The upper stem and loop structure carries a bulged U residue that is required for selenocysteine incorporation [34] because of its interaction with SelB; and 3) We described an extended fdhF SECIS that includes the information for an additional function: The prevention of UGA readthrough under conditions of selenium deficiency [35]. This information is contained in a short mRNA region consisting of a single C residue adjacent to the UGA on its downstream side, and an additional segment consisting of the six nucleotides immediately upstream from it. These two regions act independently and additively and probably through different mechanisms. The single C residue acts as itself; the upstream region acts at the level of the two amino acids, arginine and valine, for which it codes. These two codons at the 5' side of the UGA correspond to the ribosomal E and P sites. Finally, we present a model for the E. coli fdhF SECIS as a multifunctional RNA structure containing three functional elements. Depending on the availability of selenium the SECIS enables one of two alternatives for the translational machinery: Either selenocysteine incorporation into a polypeptide or termination of the polypeptide chain.

Amino Acid Sequence↗
PubMed2006

A p21/WAF1 mutation favors the appearance of drug resistance to paclitaxel in human noncancerous epithelial mammary cells.

We investigated the mechanisms responsible for paclitaxel resistance in HME-1 cells (human mammary epithelial cells immortalized with hTERT). These cells were exposed to paclitaxel (10 pM for 7 days) and 20 cellular surviving populations (PSP) were obtained. PSP demonstrated high levels of resistance to paclitaxel cytotoxicity as compared with HME-1 cells. Activation of mdr-1 gene expression was observed in 2 PSP. Protein expression analysis using a C-terminal targeted antibody showed that 13 PSP were negative for p21/WAF1 expression after ionizing radiation (6 Gy) or doxorubicin (100 nM) treatment. Sequencing of the 3 exons of the CDKN1A gene revealed that 13 PSP contained a point mutation in exon 2. This mutation consisted in a T insertion at codon 104 leading to a premature STOP codon appearance. Mismatch amplification mutation assay and RFLP-PCR confirmed the presence of the mutation in 16 PSP. Western blot using an N-terminal targeted antibody demonstrated that the C-terminal-truncated p21/WAF1 protein (14 kDa) was indeed expressed in the 13 PSP. Our data suggest that p21/WAF1 inactivation may confer a strong resistance to paclitaxel in noncancerous breast epithelial cells harboring a p21/WAF1 mutant.

Amino Acid Sequence↗
PubMed2006

Nuclease sensitive element binding protein 1 associates with the selenocysteine insertion sequence and functions in mammalian selenoprotein translation.

Biosynthesis of selenium-containing proteins requires insertion of the unusual amino acid selenocysteine by alternative translation of a UGA codon, which ordinarily serves as a stop codon. In eukaryotes, selenoprotein translation depends upon one or more selenocysteine insertion sequence (SECIS) elements located in the 3'-untranslated region of the mRNA, as well as several SECIS-binding proteins. Our laboratory has previously identified nuclease sensitive element binding protein 1 (NSEP1) as another SECIS-binding protein, but evidence has been presented both for and against its role in SECIS binding in vivo and in selenoprotein translation. Our current studies sought to resolve this controversy, first by investigating whether NSEP1 interacts closely with SECIS elements within intact cells. After reversible in vivo cross-linking and ribonucleoprotein immunoprecipitation, mRNAs encoding two glutathione peroxidase family members co-precipitated with NSEP1 in both human and rat cell lines. Co-immunoprecipitation of an epitope-tagged GPX1 construct depended upon an intact SECIS element in its 3'-untranslated region. To test the functional importance of this interaction on selenoprotein translation, we used small inhibitory RNAs to reduce the NSEP1 content of tissue culture cells and then examined the effect of that reduction on the activity of a SECIS-dependent luciferase reporter gene for which expression depends upon readthrough of a UGA codon. Co-transfection of small inhibitory RNAs directed against NSEP1 decreased its expression by approximately 50% and significantly reduced luciferase activity. These studies demonstrate that NSEP1 is an authentic SECIS binding protein that is structurally associated with the selenoprotein translation complex and functionally involved in the translation of selenoproteins in mammalian cells.

Animals↗
PubMed2002

Prenatal diagnosis in two families with autosomal, p47(phox)-deficient chronic granulomatous disease due to a novel point mutation in NCF1.

OBJECTIVE: Prenatal diagnosis was required in two unrelated families with chronic granulomatous disease (CGD) patients who lacked expression of p47(phox) protein; thus a search for mutations in NCF1 was undertaken. METHODS: Gene scanning was applied to establish the relative number of coding and pseudo-NCF1 genes. PCRs specific for coding NCF1 cDNA and coding NCF1 exon-7 genomic DNA were devised. RESULTS: The normal 1:2 ratio of coding and pseudo-NCF1 genes was found in the patients. Sequencing of the RT-PCR product specific for mRNA from the coding NCF1 genes revealed a novel homozygous G579A mutation in both patients, changing the TGG codon for Trp193 into the TAG stop codon. This mutation was confirmed in genomic DNA. The parents of both patients were found to be heterozygotes for this mutation. In the chorionic villus DNA of the first family a heterozygous G579A mutation was found. Postpartum, functional NADPH oxidase tests were normal. In the second family, this mutation was present in homozygous form in the chorionic villus DNA. Following termination of the pregnancy, the diagnosis of p47(phox)-deficient CGD was confirmed on DNA extracted from fetal blood. CONCLUSION: This is the first report of prenatal diagnosis in p47(phox)-deficient CGD.

Adolescent↗
PubMed1993

DUMPS cattle carry a point mutation in the uridine monophosphate synthase gene.

Deficiency of uridine monophosphate synthase (DUMPS) is a monogenic autosomal recessive disorder in cattle, resulting in early embryonic death of homozygous offspring. To identify the mutation responsible for DUMPS, liver RNA from identified, DUMPS heterozygous animals from the Holstein and Red Holstein breeds was reverse transcribed. Amplification of cDNA with sequence-specific primers and subsequent sequencing of the PCR products revealed a mutation (C-->T) with the loss of an AvaI site at codon 405, resulting in a premature stop codon with a truncated C-terminal catalytic subunit of the protein. A direct DNA test based on PCR was developed and subsequently tested on 102 animals. Complete concurrence of deficiency of UMPS and the presence of the described point mutation in heterozygous animals was observed, thus confirming this point mutation as the basic defect in DUMPS cattle.

Animals↗
PubMed1995

carP, involved in pyrimidine regulation of the Escherichia coli carbamoylphosphate synthetase operon encodes a sequence-specific DNA-binding protein identical to XerB and PepA, also required for resolution of ColEI multimers.

The carP gene involved in pyrimidine-specific regulation of the upstream P1 promoter of the Escherichia coli carAB operon has been cloned in vivo on a mini-Mu replicon, sequenced and shown to be identical to the xerB (pepA) gene encoding aminopeptidase A, a protein also involved in the Xer-mediated site-specific recombination at ColEI cer. The trans-dominant allele carP6 was cloned as well and shown to bear a single G-->A transition that converts the TGG codon (Trp473) into a TAG amber stop codon. The truncated mutant protein, missing the 31 C-terminal amino acid residues, was shown to be partially active; in the multicopy state the carP6 allele can restore pyrimidine repressibility of the carAB promoter P1. The trans-dominant character of the single copy carP6 allele was found to be suppressed in the presence of multiple copies of the wild-type gene. The carP (pepA) control region was sequenced and transcription shown to be initiated at three promoters, the most upstream one of which was shown to be subject to negative autoregulation. The aminopeptidase activity of CarP (PepA) was found to be dispensable for its role in pyrimidine-mediated repression of carAB transcription. CarP (PepA) was shown to be a sequence-specific DNA-binding protein that does not require, at least not in vitro, any pyrimidine cofactor to bind to the DNA. Mobility-shift and DNase I footprinting experiments have revealed a specific binding of purified CarP (PepA) to two sites in each one of the control regions of the E. coli and Salmonella typhimurium carAB operons and to a single site in the carP (pepA) control region. We propose that integration host factor and CarP/PepA-induced structural modifications in the carAB control region cause conformational changes required to assemble a pyrimidine-specific nucleo-protein regulatory complex.

Amino Acid Sequence↗
PubMed1998

Temporal expression of the AcMNPV lef-4 gene and subcellular localization of the protein.

Autographa californica nucleopolyhedrovirus (AcMNPV) lef-4 gene [ORF 90; Ayres et al. (1994) Virology 202, 586-605] is involved in both late and very late gene expression [Passarelli and Miller (1993) Virology 197, 704-714]. The transcriptional properties of this gene have been analyzed. It is transcribed as a single 1.6-kb mRNA and transcripts were first detected 3 h postinfection (pi). The extremities of the transcript have been mapped by primer extension and 3' RACE-PCR to positions -56 from the translation start codon and +96 downstream of the stop codon. A rabbit polyclonal antiserum has been raised against an internal polypeptide of LEF-4. A 55-kDa protein was observed by Western blot analysis from 5 h pi. LEF-4 localizes preferentially in the nucleus of infected cells and is associated with the virogenic stroma.

Amino Acid Sequence↗
PubMed2004

Cloning of the gene encoding urease subunit A in Helicobacter pylori.

The gene encoding urease subunit A (ureA) of Helicobacter pylori (H. pylori) was cloned from H. pylori isolate by polymerase chain reaction (PCR). Sterile distilled water instead of DNA served as negative control. The nucleotide sequence of the amplified product was determined. Homologous analysis of the ureA against that reported by Clayton CL and the GenBank and SwissProt databases were performed with the BLAST program at the Genome Net through the Internet. 0.8 kb PCR product was amplified from all H. pylori clinical isolators. The nucleotide sequence of the ureA was determined. The nucleotide sequence of the ureA began with ATG as the initiation codon and terminated in TAA as stop codon. The coding regions had a 44% G + C content. The DNA sequence was 98% homologous to that reported by Clayton CL (688 out of 702 residues were identical). The derived amino-acid sequences of the ureA were 99% homologous to that reported by Clayton CL (232 out of 234 residues were identical). The nucleotide sequence and the predicted protein showed significant homology to ureA of H. pylori in the NCBI Entrez database.

Base Sequence↗
PubMed2001

Cloning of the cel9A gene and characterization of its gene product from marine bacterium Pseudomonas sp. SK38.

The yellow-pigmented bacterial strain causing green spot rot and death of layer was isolated from Porphyra dentata. This strain has been identified as Pseudomonas sp., harboring agarase, xylanase, and protease activity, as well as carboxymethyl-cellulase (CMCase). Using genomic DNA from the Pseudomonas sp. SK38 digested with Sau3AI and ligated into pBluescript II KS+, we isolated a cel gene encoding a CMCase in Pseudomonas sp. SK38. A 4.5-kb fragment was subcloned into pKR400. The structure of the cel9A gene consists of an open reading frame of 1,521 bp starting with a GTG start codon and ending with a TAG stop codon. It thus encodes 506 amino acid residues of a protein with a calculated molecular weight of 52,636 daltons plus a signal peptide of 22 amino acids. The deduced amino acid sequence of the cel9A protein is similar to the same protein of Clostridium thermocellum. It contains, in particular, the two conserved regions of the glycoside hydrolase family 9. The apparent molecular mass of the Cel9A protein is 52 kDa as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme is most active at pH 6-7 and an optimal temperature of around 30 degrees C.

Amino Acid Sequence↗
PubMed1997

Analysis of the non-sense mutants of varicella-zoster virus thymidine kinase.

Three non-sense mutants of varicella-zoster virus (VZV) thymidine kinase (TK) gene, VZTK325, VZTK278 and VZTK224, were isolated. The mutants had a single nucleotide substitution at codons 326, 279 and 225, which changed the codon of TGG (tryptophan) to the stop codon TGA. The wild type (WT) and mutant TKs were expressed in E. coli cells and their characteristics were evaluated. VZTK224 lost TK activity, but VZTK325 and VZTK278 maintained 74.8% and 21.2% of the WT TK activity. On the other hand, all mutants lost the thymidylate kinase activity. Moreover, VZTK325 and VZTK278 polypeptides were heat-labile. These data suggest that the carboxy-terminal portion of herpesvirus TK plays an important role in the stable folding of TK and thymidylate kinase activity.

Heating↗