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Initiation Codon (initiation + codon)
Selected AbstractsIdentification of alternative promoter usage for the matrix Gla protein geneFEBS JOURNAL, Issue 6 2005Evidence for differential expression during early development in Xenopus laevis Recent cloning of the Xenopus laevis (Xl) matrix Gla protein (MGP) gene indicated the presence of a conserved overall structure for this gene between mammals and amphibians but identified an additional 5,-exon, not detected in mammals, flanked by a functional, calcium-sensitive promoter, 3042 bp distant from the ATG initiation codon. DNA sequence analysis identified a second TATA-like DNA motif located at the 3, end of intron 1 and adjacent to the ATG-containing second exon. This putative proximal promoter was found to direct transcription of the luciferase reporter gene in the X. laevis A6 cell line, a result confirmed by subsequent deletion mutant analysis. RT-PCR analysis of XlMGP gene expression during early development identified a different temporal expression of the two transcripts, strongly suggesting differential promoter activation under the control of either maternally inherited or developmentally induced regulatory factors. Our results provide further evidence of the usefulness of nonmammalian model systems to elucidate the complex regulation of MGP gene transcription and raise the possibility that a similar mechanism of regulation may also exist in mammals. [source] Kinetic study of sn -glycerol-1-phosphate dehydrogenase from the aerobic hyperthermophilic archaeon, Aeropyrum pernix K1FEBS JOURNAL, Issue 3 2002Jin-Suk Han A gene having high sequence homology (45,49%) with the glycerol-1-phosphate dehydrogenase gene from Methanobacterium thermoautotrophicum was cloned from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820). This gene expressed in Escherichia coli with the pET vector system consists of 1113 nucleotides with an ATG initiation codon and a TAG termination codon. The molecular mass of the purified enzyme was estimated to be 38 kDa by SDS/PAGE and 72.4 kDa by gel column chromatography, indicating presence as a dimer. The optimum reaction temperature of this enzyme was observed to be 94,96 °C at near neutral pH. This enzyme was subjected to two-substrate kinetic analysis. The enzyme showed substrate specificity for NAD(P)H- dependent dihydroxyacetone phosphate reduction and NAD+ -dependent,glycerol-1-phosphate (Gro1P) oxidation. NADP+ -dependent Gro1P oxidation was not observed with this enzyme. For the production of Gro1P in A. pernix cells, NADPH is the preferred coenzyme rather than NADH. Gro1P acted as a noncompetitive inhibitor against dihydroxyacetone phosphate and NAD(P)H. However, NAD(P)+ acted as a competitive inhibitor against NAD(P)H and as a noncompetitive inhibitor against dihydroxyacetone phosphate. This kinetic data indicates that the catalytic reaction by glycerol- 1-phosphate dehydrogenase from A. pernix follows a ordered bi,bi mechanism. [source] Expression of penicillin G acylase from the cloned pac gene of Escherichia coli ATCC11105FEBS JOURNAL, Issue 5 2001Effects of pacR, temperature The structural gene pac in Eschericia coli ATCC11105 encodes penicillin G acylase (PGA). Within the pac gene, there is a regulatory gene pacR, which is transcribed in the opposite direction. Site-directed mutagenesis was performed at base 1045 of pac by replacing a T with a C. This substitution did not alter the amino-acid sequence of PGA, but changed the translation start codon of pacR from AUG to GUG. The expression of the mutant pacR decreased dramatically and the lacZ transcriptional fusion analysis showed that GUG was an extremely poor initiation codon for pacR. The pacR mutation caused PGA expression to be constitutive rather than inductive in two strains (E. coli A56, DH10B). The pac inducer phenylacetic acid (PAA) gave significant induction of PGA production at a concentration of 0.2% in wild type, but PAA at this concentration inhibited both cell growth and PGA production in the pacR mutated strains. The temperature-dependent expression character of pac is preserved in the pacR translation-initiation mutant and the optimum temperature of PGA production was 22 °C in both wild type and mutant. At a higher temperature of 37 °C, the PGA precursor polypeptide could not be matured into subunits and formed inclusion bodies, as revealed by western blot analysis. Our investigations confirmed the hypothesis of pacR-mediated PAA induction for PGA expression and clarified the inhibitory effect of high temperature upon the post-translational processing of the PGA precursor polypeptide. [source] Ustilago maydis spermidine synthase is encoded by a chimeric gene, required for morphogenesis, and indispensable for survival in the hostFEMS YEAST RESEARCH, Issue 6 2009Laura Valdés-Santiago Abstract To analyze the role of spermidine in cell growth and differentiation of Ustilago maydis, the gene encoding spermidine synthase (Spe) was isolated using PCR. We found that the enzyme is encoded by a chimeric bifunctional gene (Spe-Sdh) that also encodes saccharopine dehydrogenase (Sdh), an enzyme involved in lysine biosynthesis. The gene contains a 5, region encoding Spe, followed, without a termination signal or a second initiation codon, by a 3, region encoding Sdh, and directs the synthesis of a single transcript that hybridizes with 3, or 5, regions' probes of the gene. The gene could not be disrupted in a wild-type strain, but only in a mutant defective in the gene encoding ornithine decarboxylase (Odc). Single spe-sdh mutants were isolated after sexual recombination in planta with a compatible wild-type strain. Mutants were auxotrophic for lysine and spermidine, but not for putrescine, and contained putrescine and spermidine, but not spermine. Putrescine in double mutants is probably synthesized from spermidine by the concerted action of polyamine acetyl transferase and polyamine oxidase. spe-sdh mutants were sensitive to stress, unable to carry out the yeast-to-mycelium dimorphic transition, and showed attenuated virulence to maize. These phenotypic alterations were reverted by complementation with the wild-type gene. [source] Erratum: Molecular bases of antithrombin deficiency: twenty-two novel mutations in the antithrombin gene,,HUMAN MUTATION, Issue 11 2006Véronique Picard Several errors are present in Table 1; most of them originated in translating our initial antithrombin gene numbering system (Olds RJ et al., Biochemistry, 1993, 32, 4216,4224) into the HGVS gene numbering system, where nucleotide +1 corresponds to the A of the ATG initiation codon of the H. sapiens antithrombin gene (SERPINC1; GenBank accession number X68793.1). This does not affect the discussion on the consequences of the mutations. Corrections are as follows, and corrections to mutations #5 and #22 apply to the Abstract (line 13) and Results and Discussion sections (lines 19 and 71). © 2006 Wiley-Liss, Inc. [source] Inhibition of synoviocytes proliferation by two types of c-myc antisense oligodeoxynucleotidesINTERNATIONAL JOURNAL OF RHEUMATIC DISEASES, Issue 2 2004Xinxin ZHAO Abstract Aim:, The c-myc proto-oncogene is over-expressed in synoviocytes from patients with rheumatoid arthritis (RA). For improving the inhibition of c-myc antisense oligodeoxynucleotides (AS ODN) on RA synoviocytes proliferation, we used two antisense sequences: one (antimyc-AUG AS ODN) targeting the initiation codon (AUG) and the next four codons on c-myc mRNA; another (antimyc-CRD AS ODN) targeting the coding region determinant (CRD) on c-myc mRNA to investigate if there was a difference on inhibiting synoviocytes proliferation. Methods:, Cultured human synoviocytes from patients with RA. The sequences were modified by phosphorothioates. Lipofectin was used as carrier. MTT assay was used to examine the inhibition of cell proliferation. Results:, Antimyc-AUG AS ODN and antimyc-CRD AS ODN both can inhibit synoviocytes proliferation dose-dependently. The maximum decrement of cell number was 40% at 2.5 µM and 48 h, 41.4% at 5 µM and 48 h, respectively. The action time of antimyc-AUG AS ODN inhibiting synoviocytes proliferation was earlier than that of antimyc-CRD AS ODN. ODN at high levels had non-sequence-specific cytotoxicity. Conclusions:, Both c-myc AS ODN are useful in inhibiting synoviocytes proliferation. [source] Antibiotics, arsenate and H2O2 induce the promoter of Staphylococcus aureus cspC gene more strongly than coldJOURNAL OF BASIC MICROBIOLOGY, Issue 2 2009Palas Kumar Chanda Abstract Proteins expressed by the bacterial cold shock genes are highly conserved at sequence level and perform various biological functions in both the cold-stressed and normal cells. To study the effects of various agents on the cold shock genes of Staphylococcus aureus, we have cloned the upstream region of cspC from S. aureus Newman and found that the above region possesses appreciable promoter (Pc) activity even at 37 °C. A reporter S. aureus strain CHANDA2, constructed by inserting the Pc - lacZ transcriptional fusion into S. aureus RN4220 genome, was found to express very low level of , -galactosidase after cold shock, indicating that low temperature induces Pc very weakly. Interestingly, transcription from Pc was induced very strongly by several antibiotics, hydrogen peroxide and arsenate salt. Cold shock proteins expressed by S. aureus are highly identical at sequence level and bear single-strand nucleic acid binding motifs. A 16 nt downstream box and a 13 nt upstream box were identified at the downstream of initiation codon and at the upstream of ribosome binding site of csp transcripts. Their roles in S. aureus cold shock gene expression have been discussed elaborately. (© 2009 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim) [source] Isolation and characterization of a novel copper-inducible metallothionein gene of a ciliate, Tetrahymena tropicalis lahorensisJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2010Raheela Chaudhry Abstract The two isoforms of copper metallothionein (CuMT) gene of a copper resistant ciliate, Tetrahymena tropicalis lahorensis (Ttl), have been isolated and characterized. The molecular cloning and nucleotide sequencing of cDNAs coding for the two CuMT isoforms revealed that TtlCuMT1 gene has 300, while TtlCuMT2 has 327 nucleotides, both with ATG as the initiation codon and TGA as the translational termination codon. TAG codes for glutamine in TtlCuMT2 gene which is peculiar to Tetrahymena. The deduced or translated TtlCuMT1 and TtlCuMT2 peptide sequences contain 100 and 108 amino acid residues including 28 and 32 cysteine residues, respectively. The amino acid sequences of TtlCuMT1 and TtlCuMT2 have special features of two and three CXCXXCXCXXCXC intragenic tandem repeats with a conserved structural pattern of cysteine, respectively. The predicted tertiary structures of these two isoforms indicate two domains. Domain I and the initial part of domain II showed >98% homology with other Tetrahymena CuMT. On the basis of the differences in the domain II, the metallothionein subfamily 7b can be divided into two groups, one (TtlCuMT1) comprising >100 amino acids and the other (TtlCuMT2) comprising <100 amino acids. This is a novel finding of the present study as no such report on this type of classification exists at the moment. TtlCuMT1 has 95%, while TtlCuMT2 has 97% resemblance with the previously reported CuMT genes of Tetrahymena spp. SDS-PAGE analysis using fluorescent probe as well as coomassie brilliant blue staining also confirmed the presence of metallothionein. J. Cell. Biochem. 110: 630,644, 2010. © 2010 Wiley-Liss, Inc. [source] A novel mechanism for control of antigenic variation in the haemagglutinin gene family of Mycoplasma synoviaeMOLECULAR MICROBIOLOGY, Issue 4 2000A. H. Noormohammadi High-frequency phase and antigenic variation of homologous lipoprotein haemagglutinins has been seen in both the major avian mycoplasma pathogens, Mycoplasma synoviae and Mycoplasma gallisepticum. The expression and, hence, antigenic variation of the pMGA gene family (encoding these lipoproteins in M. gallisepticum) is controlled by variation in the length of a trinucleotide repeat motif 5, to the promoter of each gene. However, such a mechanism was not detected in preliminary observations on M. synoviae. Thus, the basis for control of variation in the vlhA gene family (which encodes the homologous haemagglutinin in M. synoviae) was investigated to enable comparison with its homologue in M. gallisepticum and with other lipoprotein gene families in mycoplasmas. The start point of transcription was identified 119 bp upstream of the initiation codon, but features associated with control of transcription in other mycoplasma lipoprotein genes were not seen. Comparison of three copies of vlhA revealed considerable sequence divergence at the 3, end of the gene, but conservation of the 5, end. Southern blot analysis of M. synoviae genomic DNA revealed that the promoter region and part of the conserved 5, coding sequence occurred as a single copy, whereas the remainder of the coding sequence occurred as multiple copies. A 9.7 kb fragment of the genome was found to contain eight tandemly repeated regions partially homologous to vlhA, all lacking the putative promoter region and the single-copy 5, end of vlhA, but extending over one of four distinct overlapping regions of the 3, coding sequence. Examination of sequential clones of M. synoviae established that unidirectional recombination occurs between the pseudogenes and the expressed vlhA, with duplication of pseudogene sequence and loss of the corresponding region previously seen in the expressed gene. Expression of the 5, end of two variants of the vlhA gene showed that they differed in their reaction with monoclonal antibodies specific for this region. These data suggest that the control of vlhA antigenic variation in M. synoviae is achieved by multiple gene conversion events using a repertoire of coding sequences to generate a chimeric expressed gene, with the greatest potential for variation generated in the region encoding the haemagglutinin. Thus, completely distinct mechanisms have been adopted to control antigenic variation in homologous gene families. [source] Problem-based test: An in vitro experiment to analyze the genetic codeBIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 4 2010József Szeberényi Terms to be familiar with before you start to solve the test: genetic code, translation, synthetic polynucleotide, leucine, serine, filter precipitation, radioactivity measurement, template, mRNA, tRNA, rRNA, aminoacyl-tRNA synthesis, ribosomes, degeneration of the code, wobble, initiation, and elongation of protein synthesis, initiation codon [source] PROP1 gene analysis in Portuguese patients with combined pituitary hormone deficiencyCLINICAL ENDOCRINOLOGY, Issue 4 2006Manuel C. Lemos Summary Objective, Mutations of the PROP1 gene lead to combined pituitary hormone deficiency (CPHD), which is characterized by a deficiency of GH, TSH, LH/FSH, PRL and, less frequently, ACTH. This study was undertaken to investigate the molecular defect in a cohort of patients with CPHD. Design, patients and measurements, A multicentric study involving 46 cases of CPHD (17 familial cases belonging to seven kindreds and 29 sporadic cases) selected on the basis of clinical and hormonal evidence of GH deficiency, central hypothyroidism and hypogonadotrophic hypogonadism, in the absence of an identified cause of hypopituitarism. Mutations of PROP1 were investigated by DNA sequencing. Clinical, hormonal and neuroradiological data were collected at each centre. Results,PROP1 mutations were identified in all familial cases: five kindreds presented a c. 301,302delAG mutation, one kindred presented a c. 358C , T (R120C) mutation and one presented a previously unreported initiation codon mutation, c. 2T , C. Of the 29 sporadic cases, only two (6·9%) presented PROP1 germline mutations (c. 301,302delAG, in both). Phenotypic variability was observed among patients with the same mutations, particularly the presence and age of onset of hypocortisolism, the levels of PRL and the results of pituitary imaging. One patient presented a sellar mass that persisted into adulthood. Conclusions, This is the first report of a mutation in the initiation codon of the PROP1 gene and this further expands the spectrum of known mutations responsible for CPHD. The low mutation frequency observed in sporadic cases may be due to the involvement of other unidentified acquired or genetic causes. [source] Further extension of mammalian GATA-6DEVELOPMENT GROWTH & DIFFERENTIATION, Issue 9 2005Masatomo Maeda Mammalian GATA-6, which has conserved tandem zinc fingers (CVNC-X17 -CNAC)-X29 -(CXNC-X17 -CNAC), is essential for the development and specific gene regulation of the heart, gastrointestinal tract and other tissues. GATA-6 recognizes the (A/T/C)GAT(A/T)(A) sequence, and interacts with other transcriptional regulators through its zinc-finger region. The mRNA of GATA-6 uses two Met codons in frame as translational initiation codons, and produces L- and S-type GATA-6 through leaky ribosome scanning. GATA-6 is subjected to cAMP-dependent proteolysis by a proteasome in a heterologous expression system. These protein-based characteristics of GATA-6 will be helpful for the identification of target genes, together with determination of the in vivo binding sites for GATA-6 and understanding of the complex network of gene regulation mediated by GATA-6. [source] DMD exon 1 truncating point mutations: Amelioration of phenotype by alternative translation initiation in exon 6,HUMAN MUTATION, Issue 4 2009Olga L. Gurvich Abstract Mutations in the DMD gene result in two common phenotypes associated with progressive muscle weakness: the more severe Duchenne muscular dystrophy (DMD) and the milder Becker muscular dystrophy (BMD). We have previously identified a nonsense mutation (c.9G>A; p.Trp3X) within the first exon of the DMD gene, encoding the unique N-terminus of the 427-kDa muscle isoform of the dystrophin protein. Although this mutation would be expected to result in severe disease, the clinical phenotype is very mild BMD, with ambulation preserved into the seventh decade. We identify the molecular mechanism responsible for the amelioration of disease severity to be initiation of translation at two proximate AUG codons within exon 6. Analysis of large mutational data sets suggests that this may be a general mechanism of phenotypic rescue for point mutations within at least the first two exons of the DMD gene. Our results directly demonstrate, for the first time, the use of alternate translational initiation codons within the DMD gene, and suggest that dystrophin protein lacking amino acids encoded by the first five exons retains significant function. Hum Mutat 0:1,8, 2009. © 2009 Wiley-Liss, Inc. [source] | |||||||||||||