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At least 523 records · Page 29Linked to original sources
PubMed1994

Carboxypeptidase Taq, a thermostable zinc enzyme, from Thermus aquaticus YT-1: molecular cloning, sequencing, and expression of the encoding gene in Escherichia coli.

The gene for carboxypeptidase Taq, a thermostable metallo-carboxypeptidase from Thermus aquaticus YT-1, was cloned and sequenced. The gene comprised an open reading frame of 1,536 base pairs with a GTG initiation codon and a TGA termination codon, which encodes a protein of 56,210 Da consisting of 511 amino acid residues. The GTG initiation codon of the gene was replaced with ATG by site-directed mutagenesis, and then the gene was expressed in Escherichia coli. The enzyme purified from E. coli cells showed the same properties as those of carboxypeptidase Taq prepared from T. aquaticus cells. Analysis for metal ions bound to the enzyme found that one molecule of the enzyme contains one tightly bound zinc ion. Comparison of the entire sequence showed that the enzyme has no obvious sequence similarity to any other metallo-peptidases. However, a His-Glu-X-X-His sequence, which is a conserved sequence in the active site of zinc-dependent endopeptidases and aminopeptidases, was found at positions 276 to 280 of the enzyme. These findings suggest that carboxypeptidase Taq is a novel type of zinc-dependent metallocarboxypeptidase.

Amino Acid Sequence↗
PubMed2001

Mutations in the EDA gene in three unrelated families reveal no apparent correlation between phenotype and genotype in the patients with an X-linked anhidrotic ectodermal dysplasia.

Anhidrotic ectodermal dysplasia (EDA) is caused by mutations in the EDA gene encoding ectodysplasin A, a member of the TNF ligand superfamily involved in the communication between the cells. The structure of the EDA gene was investigated in three patients exhibiting clinical symptoms of EDA in an attempt to correlate the molecular findings with the phenotype of the patients. Genomic DNA was analyzed by single stranded conformation polymorphism (SSCP) followed by direct sequencing. In one of the patients, as well as in his heterozygous mother and sister, a single T insertion was evidenced in exon 3 between nucleotides 713 and 714 that changed Lys codon (AAA) into a termination codon TAA (Lys158Ter). In the other patient, A1321T transversion was demonstrated. The same mutation was found in his heterozygous mother and resulted in a change of Ileu360Asn that might generate an additional glycosylation site. In the third patient an A1285G transition was revealed. This mutation that originated de novo was localized in a region that is highly conserved in TNF ligand family and caused substitution of Ala349Thr. Localization of the mutations in the extracellular domain of ectodysplasin A suggested that the primary cause of EDA is a defect in communication between the cells responsible for the development of skin appendages. Despite a different character and localization of the mutations, no apparent correlation between phenotype and genotype of the patients was evidenced. Some differences in the patients' phenotype were observed.

Child↗
PubMed2000

Cloning and Overexpression of the Mitomycin C Resistance Gene (mcr) from Streptoverticillium caespitosum.

Streptoverticillium caespitosum ATCC27422 is a major producer of an anti-cancer drug, mitomycin C. A 6.6 kb DNA fragment containing the mitomycin C resistance gene (mcr) was isolated from ATCC27422 by shotgun method in order to learn the molecular mechanism of mitomycin C resistance. By constructing a series of subclones from this 6.6 kb DNA fragment, the mitomycin C resistance gene was localized on a 3.1 kb DNA fragment. Sequence analysis revealed that the open reading frame of mcr gene was 1 347 bp in size, encoding 448 amino acids with ATG as initiation codon and TGA as termination codon. The mcr gene was specifically expressed under the control of T7 promoter in E.coli, and the resistance to mitomycin C in the transformant was over 100-fold higher than that in wild-type strain. The overexpression of mcr gene in E.coli is very helpful for the further research about the molecular mechanism of drug resistance.

Journal Article↗
PubMedDate not supplied

Novel mutations of the AGXT gene causing primary hyperoxaluria type 1.

BACKGROUND: Primary hyperoxaluria type 1 (PH1), an inherited cause of nephrolithiasis, is due to a functional defect of the liver-specific peroxisomal enzyme alanine:glyoxylate aminotransferase (AGT). A definitive PH1 diagnosis can be established by analyzing AGT activity in liver tissue or mutation analysis of the AGXT gene. METHODS: The molecular basis of PH1 in three Chinese patients, two with adult-onset and one with childhood-onset recurrent nephrolithiasis, was established by analyzing the entire AGXT gene. RESULTS: Three novel mutations (c2T>C, c817insAG and c844C>T) and two previously reported mutations (c33insC and 679-IVS6+2delAAgt) were identified. c2T>C converts the initiation codon from ATG to ACG, which predicts significant reduction, if not complete abolition, of protein translation. c817insAG leads to a frameshift and changes the amino acid sequence after codon 274. c844C>T changes glutamine at codon 282 to a termination codon, resulting in protein truncation. CONCLUSIONS: This is the first report describing AGXT gene mutations in Chinese patients with PH1. AGXT genotypes cannot fully explain the clinical heterogeneity of PH1, and other factors involved in disease pathogenesis remain to be identified. Our experience emphasizes the importance of excluding PH1 in patients with recurrent nephrolithiasis to avoid delay or inappropriate management.

Adult↗
PubMed2005

Mutation analysis of PAX6 gene in a large Chinese family with aniridia.

BACKGROUND: Mutations in PAX6 gene have been shown to be the genetic cause of aniridia, which is a severe panocular eye disease characterised by iris hypoplasia. However, there is no study to do genetic analysis of aniridia, although there are several case reports in China. Here, we describe a mutation analysis of PAX6 in a large Chinese family with aniridia. METHODS: Genomic DNA from venous blood samples was prepared. Haplotype analysis was performed with two genetic markers (D11S904 and D11S935). Fourteen exons of the PAX6 gene were amplified from genomic DNA. Polymerase chain reaction (PCR) products of each exon were analysed by single strand conformational polymorphism (SSCP). The PCR products having an abnormal pattern were sequenced to confirm the mutation. RESULTS: Significant evidence for allele sharing in affected patients was detected suggesting that PAX6 mutation links to aniridia in this family. An extra band corresponding to exon 9 in PAX6 was found by single strand conformational polymorphism analysis in all the aniridia patients in this family, but not detected in the unaffected members. A mutation of C to T was detected by sequencing at the nucleotide 1080 that converts the Arg codon (CGA) to the termination codon (TGA). CONCLUSIONS: Aniridia is caused by a nonsense mutation of PAX6 gene in the large Chinese kindred. Genetic test is important to prevent the transmission of aniridia to their offsprings in the kindred by prenatal diagnosis.

Aniridia↗
PubMed1995

The Wiskott-Aldrich syndrome and X-linked congenital thrombocytopenia are caused by mutations of the same gene.

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive disorder characterized by thrombocytopenia, small platelets, eczema, recurrent infections, and immunodeficiency. Besides the classic WAS phenotype, there is a group of patients with congenital X-linked thrombocytopenia (XLT) who have small platelets but only transient eczema, if any, and minimal immune deficiency. Because the gene responsible for WAS has been sequenced, it was possible to correlate the WAS phenotypes with WAS gene mutations. Using a fingerprinting screening technique, we determined the approximate location of the mutation in 13 unrelated WAS patients with mild to severe clinical symptoms. Direct sequence analysis of cDNA and genomic DNA obtained from patient-derived cell lines showed 12 unique mutations distributed throughout the WAS gene, including insertions, deletions, and point mutations resulting in amino acid substitutions, termination, exon skipping, or splicing defects. Of 4 unrelated patients with the XLT phenotype, 3 had missense mutations affecting exon 2 and 1 had a splice-site mutation affecting exon 9. Patients with classic WAS had more complex mutations, resulting in termination codons, frameshift, and early termination. These findings provide direct evidence that XLT and WAS are caused by mutations of the same gene and suggest that severe clinical phenotypes are associated with complex mutations.

Base Sequence↗
PubMed1986

Molecular cloning of cDNA for human prothymosin alpha.

A cDNA library was constructed from human spleen mRNA and screened for clones containing cDNAs coding for prothymosin alpha. A clone containing a 503-base-pair insert including the entire coding sequence for the translated portion of the mRNA was isolated. The deduced amino acid sequence confirms and completes the partial sequence of human prothymosin alpha determined by protein sequencing methods. The presence of an initiator codon immediately preceding the codon for the NH2-terminal serine residue and of a terminator codon immediately following the codon for Asp-109, the COOH-terminal residue, suggests that prothymosin alpha is synthesized without formation of a larger precursor polypeptide. Analysis of the 5' sequence preceding the initiator methionine codon excluded the presence of a signal peptide in the translated sequence.

Amino Acid Sequence↗
PubMed1998

Molecular basis of type III hyperlipoproteinemia in Germany.

Type III hyperlipoproteinemia (HLP) is usually associated with homozygosity for apolipoprotein (apo) E2 (Arg112 --> Cys, Arg158 --> Cys). This common apo E isoform is defective in its binding to lipoprotein receptors. However, other rare mutations in the apo epsilon gene may also, in part dominantly, predispose to the disease. In order to assess the prevalence of rare apo E variants and mutations in the apo epsilon gene in Germany, we examined apo epsilon genotypes by restriction isotyping (RI) and apo E phenotypes by isoelectric focusing (IEF) in 107 German patients with type III HLP. Concordance between apo epsilon genotype and apo E phenotype was observed in 101 subjects (94.4%). Six individuals (5.6%) had genotypes and phenotypes other than E2/2. One subject was apparently homozygous for apo E2 by IEF, but heterozygous for epsilon3/2 by RI. Sequencing of the apo epsilon gene disclosed a hitherto undescribed point mutation (TGG --> TGA) at the third position of the codon for amino acid 20 (Trp), introducing a premature termination codon. This is the first study demonstrating that in the German population type III HLP is mainly associated with homozygosity for apo E2 (Arg112 --> Cys, Arg158 --> Cys) and that discrepancies between apo epsilon genotype and apo E phenotype are rare in this genetic condition.

Adult↗
PubMed2006

Titration and conditional knockdown of the prfB gene in Escherichia coli: effects on growth and overproduction of the recombinant mammalian selenoprotein thioredoxin reductase.

Release factor 2 (RF2), encoded by the prfB gene in Escherichia coli, catalyzes translational termination at UGA and UAA codons. Termination at UGA competes with selenocysteine (Sec) incorporation at Sec-dedicated UGA codons, and RF2 thereby counteracts expression of selenoproteins. prfB is an essential gene in E. coli and can therefore not be removed in order to increase yield of recombinant selenoproteins. We therefore constructed an E. coli strain with the endogenous chromosomal promoter of prfB replaced with the titratable P(BAD) promoter. Knockdown of prfB expression gave a bacteriostatic effect, while two- to sevenfold overexpression of RF2 resulted in a slightly lowered growth rate in late exponential phase. In a turbidostatic fermentor system the simultaneous impact of prfB knockdown on growth and recombinant selenoprotein expression was subsequently studied, using production of mammalian thioredoxin reductase as model system. This showed that lowering the levels of RF2 correlated directly with increasing Sec incorporation specificity, while also affecting total selenoprotein yield concomitant with a lower growth rate. This study thus demonstrates that expression of prfB can be titrated through targeted exchange of the native promoter with a P(BAD)-promoter and that knockdown of RF2 can result in almost full efficiency of Sec incorporation at the cost of lower total selenoprotein yield.

Amino Acid Sequence↗
PubMed2004

Novel mutations in the EXT1 gene in two consanguineous families affected with multiple hereditary exostoses (familial osteochondromatosis).

Multiple hereditary exostoses (HME) is an autosomal dominant developmental disorder exhibiting multiple osteocartilaginous bone tumors that generally arise near the ends of growing long bones. Here, we report two large consanguineous families from Pakistan, who display the typical features of HME. Affected individuals also show a previously unreported feature--bilateral overriding of single toes. Analysis using microsatellite markers for each of the known EXT loci, EXT1, EXT2, and EXT3 showed linkage to EXT1. In the first family, mutation analysis of the EXT1 gene revealed that affected individuals were heterozygous for an in-frame G-to-C transversion at the conserved splice donor site in intron 1. This mutation is predicted to disrupt splicing of the first intron and produce a frameshift that leads to a premature termination codon. In the second family, an insertion of an A in exon 8 is predicted to produce a frameshift at codon 555 followed by a premature termination, a further 10 codons downstream. In both families, an increased number of affected male subjects were observed. In affected females in family 2, phenotypic variability and incomplete penetrance were noted.

Base Sequence↗
PubMed2002

Transterm: a database of mRNAs and translational control elements.

Transterm is a database that facilitates studies of translation and the translational control of protein synthesis. It contains a curated collection of elements in mRNAs that control translation, and biologically relevant mRNA regions extracted from GenBank. It is organised largely on a taxonomic basis with files and summaries for each species. Global patterns that may affect translation in particular species, for example bias in the context of initiation codons (Kozak's consensus or Shine-Dalgarno sequences) or termination codons, can be detected in the consensus and information content bias summaries. Several types of access are provided via a web browser interface. Transterm defined elements may be matched in a user's sequence or in the database. Alternatively, elements can be entered by the user to search specific sections of the database (for example, coding regions or 3' flanking regions or the 3'-UTRs) or the user's sequence. Each Transterm defined element has an associated biological description with references. The database is accessible at http://uther.otago.ac.nz/Transterm.html.

Animals↗
PubMed1991

Molecular characterization of the nisin resistance region of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3.

The nisin resistance determinant of Lactococcus lactis subsp. lactis biovar diacetylactis DRC3 was localized onto a 1.3-kb EcoRI-NdeI fragment by subcloning and interrupting the NdeI site by cloning random NdeI fragments into it; the nisin resistance determinant was then sequenced. The nucleotide sequence revealed a large open reading frame containing 318 codons. Putative transcription and translation signal sequences were located directly upstream from the initiation codon. Immediately downstream of the termination codon was a palindromic region resembling a rho-independent termination sequence. This 957-nucleotide open reading frame and its associated transcription and translation signal sequences were cloned into plasmid-free L. lactis subsp. lactis LM0230 and conferred an MIC of 160 IU of nisin per ml. This level of nisin resistance is equivalent to that of the initial nisin-resistant subclone, pFM011, used for further subcloning in this study. The inferred amino acid sequence would result in a protein with a molecular mass of 35,035 Da. This value was in agreement with the molecular mass of a protein detected after in vitro transcription and translation of DNA encoding the nisin resistance gene, nsr. This protein contained a hydrophobic region at the N terminus that was predicted to be membrane associated but did not contain a typical signal sequence cleavage site. No significant homology was detected when the DNA sequence of the nsr gene and the amino acid sequence of its putative product were compared with other available sequences. When subjected to Southern hybridization, a 1.2-kb DraI fragment encoding the nsr gene did not hybridize with the genomic DNA of the nisin-producing strain L. lactis subsp. lactis 11454.

Amino Acid Sequence↗
PubMed2002

Stop codons preceded by rare arginine codons are efficient determinants of SsrA tagging in Escherichia coli.

The SsrA or tmRNA quality control system intervenes when ribosomes stall on mRNAs and directs the addition of a C-terminal peptide tag that targets the modified polypeptide for degradation. Although hundreds of SsrA-tagged proteins can be detected in cells when degradation is prevented, most of these species have not been identified. Consequently, the mRNA sequence determinants that cause ribosome stalling and SsrA tagging are poorly understood. SsrA tagging of Escherichia coli ribokinase occurs at three specific sites at or near the C terminus of this protein. The sites of tagging correspond to ribosome stalling at the termination codon and at rare AGG codons encoding Arg-307 and Arg-309, the antepenultimate and C-terminal residues of E. coli ribokinase. Mutational analyses and studies of the effects of overexpressing the tRNA that decodes AGG reveal that the combination of a rare arginine codon at the C terminus and the adjacent inefficient UGA termination codon act to recruit the SsrA-tagging system, presumably by slowing the rate of translation elongation and termination.

Amino Acid Sequence↗
PubMed1980

Structure of a split yeast gene: complete nucleotide sequence of the actin gene in Saccharomyces cerevisiae.

The complete nucleotide sequence of the actin gene from Saccharomyces cerevisiae has been determined. The coding region is interrupted by a 304-base-pair intervening sequence that is located within the triplet coding for amino acid 4. DNA sequences of the intron-exon junctions are similar to those found in higher eukaryotes and can be aligned such that the intron starts with the dinucleotide 5'-G-T-3' and ends with 5'-A-G-3'. Regions fo homology within the sequences upstream from the initiation codon and those following the termination codon have been detected between the yeast iso-1-cytochrome c gene and the actin gene. As deduced from the nucleotide sequence, yeast actin has 374 amino acid residues. Its primary structure, especially the NH2-terminal third of the protein, is highly conserved during evolution.

Actins↗
PubMed1993

Effect of the relative position of the UGA codon to the unique secondary structure in the fdhF mRNA on its decoding by selenocysteinyl tRNA in Escherichia coli.

The fdhF mRNA for formate dehydrogenase H of Escherichia coli contains a UGA codon at position 140. This termination codon is decoded by selenocysteinyl tRNA (the selC product) with the aid of its own specific elongation factor, SelB. For this decoding, a unique secondary structure immediately downstream of the UGA codon has been shown to be essential (Zinoni, F., Heider, J., and Böck, A. (1990) Proc. Natl. Acad. Sci. U. S. A. 87, 4660-4664). We examined the positional effect of the UGA codon relative to the secondary structure on its decoding using a fdhF-lacZ fusion gene. When the UGA codon was separated by one codon (position -1) from the secondary structure, the UGA decoding, as measured by the beta-galactosidase activity, dropped to approximately 76% of the normal level but was still almost as fully dependent upon selC and selenium in the culture medium as in the case of the UGA codon in the normal position (position 0). However, when the UGA codon was separated by two codons (position -2), the decoding level further dropped to 20% of the normal level, and in addition, became dependent only on selC but independent of selenium. When the UGA codon was further separated by three codons (position -3), the decoding level of UGA (-3) became higher than the decoding of UGA (-2) and was completely independent from selC and selenium, indicating that the UGA codon was nonspecifically suppressed. A similar nonspecific suppression was observed for the UGA codon at position -4, but at a lower level. When two UGA codons were tandemly placed at positions 0 and -1, they were still able to be decoded at 17% of the normal level in a selC- and selenium-dependent manner. In the absence of the SelB function, the decoding level of UGA(0) dropped to 1.6% of the normal level, whereas the UGA(-1) decoding dropped to 7.5%. These results indicate that the UGA codon at position 0 is not only most effectively decoded by selenocysteinyl tRNA but also tightly blocked from its nonspecific suppression in the absence of any components required for the decoding.

Base Sequence↗
PubMed2005

Acquisition of a stable mutation in metY allows efficient initiation from an amber codon in Escherichia coli.

Escherichia coli strains harbouring elongator tRNAs that insert amino acids in response to a termination codon during elongation have been generated for various applications. Additionally, it was shown that expression of an initiator tRNA containing a CUA anticodon from a multicopy plasmid in E. coli resulted in initiation from an amber codon. Even though the initiation-based system remedies toxicity-related drawbacks, its usefulness has remained limited for want of a strain with a chromosomally encoded initiator tRNA 'suppressor'. E. coli K strains possess four initiator tRNA genes: the metZ, metW and metV genes, located at a single locus, encode tRNA(1)(fMet), and a distantly located metY gene encodes a variant, tRNA(2)(fMet). In this study, a stable strain of E. coli K-12 that affords efficient initiation from an amber initiation codon was isolated. Genetic analysis revealed that the metY gene in this strain acquired mutations to encode tRNA(2)(fMet) with a CUA anticodon (a U35A36 mutation). The acquisition of the mutations depended on the presence of a plasmid-borne copy of the mutant metY and recA(+) host background. The mutations were observed when the plasmid-borne gene encoded tRNA(2)(fMet) (U35A36) with additional changes in the acceptor stem (G72; G72G73) but not in the anticodon stem (U29C30A31/U35A36/psi39G40A41). The usefulness of this strain, and a possible role for multiple tRNA(1)(fMet) genes in E. coli in safeguarding their intactness, are discussed.

Anticodon↗
PubMed1996

Restriction isotyping of the premature termination variant of lipoprotein lipase in Alberta Hutterites.

OBJECTIVE: Lipoprotein lipase (LPL) plays a pivotal role in lipoprotein metabolism. A relatively common LPL variant results from a C --> G transversion in exon 9, which creates a premature termination codon (S447X) and results in a truncated LPL molecule lacking the C-terminal dipeptide Ser-Gly. We wished to determine the functional relevance of this variant. DESIGN AND METHODS: We used Mn/l restriction digestion of amplified genomic DNA to genotype Alberta Hutterites for the S447X variant. We tested for association with biochemical phenotypes. RESULTS: Complete linkage disequilibrium between alleles of an LPL genomic variant in intron 6 and LPL S447X was detected. However, as a single independent variable, LPL S447X genotype was not significantly associated with variation in any dependent biochemical variable in the Hutterites. CONCLUSIONS: Restriction isotyping for S447X permits large-scale screening of individuals to identify linkage relationships between this marker and other DNA variations of LPL and to study associations with clinical phenotypes.

Alberta↗