Home About us Contact
|
Home About us Contact | ||
|
|
||
Start Codon (start + codon)
Selected AbstractsExpression 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] The human complement C9 gene: structural analysis of the 5, gene region and genetic polymorphism studiesINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 5 2001K. Witzel-Schlömp Summary C9 is the last of the human complement components creating the membrane attack complex. The single chain serum protein is encoded by a gene located on chromosome 5p13 that is composed of 11 exons. With the aid of inverse PCR, the hitherto unknown regions flanking exon 1 and the 3, part of exon 11 (3,UTR) have been sequenced. A computer-based analysis of the 300-bp region located just upstream of the AUG start codon showed homologies to known DNA modules which affect the transcriptional regulation of certain genes. The most striking of these is a sequence that may substitute the missing TATA box in initiating C9 transcription. In the 3,UTR, three successive polyadenylation signals were found. Although the C9 protein is invariant, four different single nucleotide polymorphisms (SNPs) have been observed at the DNA level by exon-specific PCR and direct sequencing. None of them changes the amino acid composition of the mature protein. Due to a C , T transition in exon 1 at cDNA position 17, the fifth amino acid of the leader peptide may be either an arginine or a tryptophane. Using either PCR/RFLP analysis (exons 1 and 11) or allele-specific PCR (intron 1 and exon 4), each polymorphism can be characterized without sequencing. All of the exon 1, intron 1 and exon 11 variants could be detected in small population samples of European, Thai or South American Indian origin. In contrast, the exon 4 C variant was observed only once in a European. The first three SNPs can be combined to designate eight different ,C9 alleles'. Of these, six have actually be found. These data provide strong evidence that several mutation and recombination events occurred in the course of C9 gene evolution. [source] Transcriptional analysis of the gdhA gene in Streptococcus thermophilusJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2009C. Lazzi Abstract Aims:, To study the transcriptional analysis of glutamate dehydrogenase gene, involved in the amino acid conversion to aroma compound in Streptococcus thermophilus. Methods and Results:, Analysis of the gdhA gene nucleotide sequence of S. thermophilus CNRZ1066 revealed that the coding region is 1353 nucleotides long. The deduced amino acids sequence exhibits the putative GDH active site and some conserved domains characteristic of family I of hexameric GDHs. Phylogenetic analysis revealed that the gdh gene of S. thermophilus clustered with the orthologues of other streptococci such as Streptococcus mutans, Streptococcus agalactiae and Streptococcus infantarius. Studying the structural organization of the gdhA locus the amino acid similarity of GDHs was higher than 87%, but the locus organization was not conserved. A dominant transcript of approximately 1·4 kbp was revealed by Northern blot hybridization, suggesting that gdhA mRNA is monocystronic. Primer extension showed that transcription start point of gdhA was localized 43 bp upstream of the potential start codon (ATG). Conclusions:, The gdhA represents a monocistronic operon highly conserved in phylogenetic-related bacteria. Significance and Impact of the Study:, A deeper knowledge of gdh transcriptional mechanisms could lead to develop S. thermophilus industrial starter cultures with optimized aromatic properties. [source] A HYPOTHESIS FOR IMPORT OF THE NUCLEAR-ENCODED PsaE PROTEIN OF PAULINELLA CHROMATOPHORA (CERCOZOA, RHIZARIA) INTO ITS CYANOBACTERIAL ENDOSYMBIONTS/PLASTIDS VIA THE ENDOMEMBRANE SYSTEM,JOURNAL OF PHYCOLOGY, Issue 5 2010Mackiewicz The cyanobacterial endosymbionts of Paulinella chromatophora can shed new light on the process of plastid acquisition. Their genome is devoid of many essential genes, suggesting gene transfer to the host nucleus and protein import back into the endosymbionts/plastids. Strong evidence for such gene transfer is provided by the psaE gene, which encodes a PSI component that was efficiently transferred to the Paulinella nucleus. It remains unclear, however, how this protein is imported into the endosymbionts/plastids. We reanalyzed the sequence of Paulinella psaE and identified four potential non-AUG translation initiation codons upstream of the previously proposed start codon. Interestingly, the longest polypeptide, starting from the first UUG, contains a clearly identifiable signal peptide with very high (90%) predictability. We also found several downstream hairpin structures that could enhance translation initiation from the alternative codon. These results strongly suggest that the PsaE protein is targeted to the outer membrane of Paulinella endosymbionts/plastids via the endomembrane system. On the basis of presence of respective bacterial homologs in the Paulinella endosymbiont/plastid genome, we discuss further trafficking of PsaE through the peptidoglycan wall and the inner envelope membrane. It is possible that other nuclear-encoded proteins of P. chromatophora also carry signal peptides, but, alternatively, some may be equipped with transit peptides. If this is true, Paulinella endosymbionts/plastids would possess two distinct targeting systems, one cotranslational and the second posttranslational, as has been found in higher plant plastids. Considering the endomembrane system-mediated import pathway, we also discuss homology of the membranes surrounding Paulinella endosymbionts/plastids. [source] A single-nucleotide polymorphism in the 5,-untranslated region of the hPER2 gene is associated with diurnal preferenceJOURNAL OF SLEEP RESEARCH, Issue 3 2005JAYSHAN D. CARPEN Summary The PERIOD2 (PER2) gene is a key component of the molecular mechanism that generates circadian rhythms in mammals. A missense mutation in the human PER2 gene has previously been linked to advanced sleep phase syndrome (ASPS). We have investigated three other single-nucleotide polymorphisms in the hPER2 gene, one downstream of the transcription start site (C,1228T), one in exon 2 in the 5,-untranslated region (5,-UTR) (C111G), and one missense mutation (G3853A) causing a glycine to glutamine substitution in the predicted protein. Subjects selected from a group of 484 volunteers for extreme morning or evening preference, or intermediate diurnal preference were genotyped with regard to the three polymorphisms (n = 35 for each group). Whereas allele frequencies for the other two polymorphisms did not differ significantly between any of the groups, the 111G allele frequency was significantly higher in subjects with extreme morning preference (0.14) than in subjects with extreme evening preference (0.03) (Fisher's exact test, two-sided P value = 0.031, odds ratio = 5.67). No significant difference in 111G allele frequency was observed between either of these groups and subjects with intermediate diurnal preference. Computer prediction indicated that the C111G polymorphism, which occurs 12 bases upstream from the translation start codon, might alter the secondary structure of the transcript. The PER2 111G allele associates with morning preference and is a potential candidate allele for ASPS. [source] Hepatitis B viral DNA is methylated in liver tissuesJOURNAL OF VIRAL HEPATITIS, Issue 2 2008P. Vivekanandan Summary., The mechanisms that regulate hepatitis B virus (HBV) replication within the liver are poorly understood. Given that methylation of CpG islands regulates gene expression in human tissues, we sought to identify CpG islands in HBV-DNA and to determine if they are methylated in human tissues. In silico analysis demonstrated three CpG islands in HBV genotype A sequences, two of which were of particular interest because of their proximity to the HBV surface gene start codon (island 1) and to the enhancer 1/X gene promoter region (island 2). Human sera with intact virions that were largely unmethylated were used to transfect HepG2 cells and HBV-DNA became partially methylated at both islands 1 and 2 by day 6 following exposure of HepG2 to virus. Examination of three additional human sera and 10 liver tissues showed no methylation in sera but tissues showed methylation of island 1 in six of 10 cases and of island 2 in five of 10 cases. The cell line Hep3B, with integrated HBV, showed complete methylation of island 1 but no methylation of island 2. In conclusion, HBV-DNA can be methylated in human tissues and methylation may play an important role in regulation of HBV gene expression. [source] Regulation of catalase,peroxidase (KatG) expression, isoniazid sensitivity and virulence by furA of Mycobacterium tuberculosisMOLECULAR MICROBIOLOGY, Issue 4 2001Alexander S. Pym Mycobacterium tuberculosis has two genes for ferric uptake regulator orthologues, one of which, furA, is situated immediately upstream of katG encoding catalase,peroxidase, a major virulence factor that also activates the prodrug isoniazid. This association suggested that furA might regulate katG and other genes involved in pathogenesis. Transcript mapping showed katG to be expressed from a strong promoter, with consensus ,10 and ,35 elements, preceding furA. No promoter activity was demonstrated downstream of the furA start codon, using different gene reporter systems, indicating that furA and katG are co-transcribed from a common regulatory region. The respective roles of these two genes in the isoniazid susceptibility and virulence of M. tuberculosis were assessed by combinatorial complementation of a ,(furA,katG) strain that is heavily attenuated in a mouse model of tuberculosis. In the absence of furA, katG was upregulated, cells became hypersensitive to isoniazid, and full virulence was restored, indicating that furA regulates the transcription of both genes. When furA alone was introduced into the ,(furA,katG) mutant, survival in mouse lungs was moderately increased, suggesting that FurA could regulate genes, other than katG, that are involved in pathogenesis. These do not include the oxidative stress genes ahpC and sodA, or those for siderophore production. [source] A Rox1-independent hypoxic pathway in yeast.MOLECULAR MICROBIOLOGY, Issue 4 2000Antagonistic action of the repressor Ord, activator Yap1 for hypoxic expression of the SRP1/TIR1 gene Hypoxic SRP1/TIR1 gene expression depends on the absence of haem but is independent of Rox1-mediated repression. We have found a new hypoxic pathway involving an antagonistic interaction between the Ixr1/Ord1 repressor and the Yap1 factor, a transcriptional activator involved in oxidative stress response. Here, we show that Ord1 repressed SRP1 gene expression under normoxia and hypoxia, whereas Yap1 activated it. Ord1 and Yap1 have been shown to bind the SRP1 promoter in a region extending from ,299 to ,156 bp upstream of the start codon. A typical AP-1 responsive element lying from ,247 to ,240 bp allows Yap1 binding. Internal deletion of sequences within the SRP1 promoter were introduced. Two regions were characterized at positions ,299/,251 and ,218/,156 that, once removed, resulted in a constitutive expression of SRP1 in a wild-type strain under normoxic conditions. Deletion of both these two sequences allowed the bypass of YAP1 requirement in a ,yap1 strain, whereas these two internal deletions did not yield increased expression in a ,ord1 strain compared with the full-length promoter. Both a single ,ord1 mutant and a doubly disrupted ,yap1 ,ord1 strain yielded normoxic constitutive SRP1 expression and increased hypoxic SRP1 induction, thereby demonstrating that ord1 is epistatic to yap1. Thus, Yap1 is not directly involved in SRP1 induction by hypoxia, but is necessary to counteract the Ord1 effect. [source] Friedreich's ataxia with chorea and myoclonus caused by a compound heterozygosity for a novel deletion and the trinucleotide GAA expansionMOVEMENT DISORDERS, Issue 3 2002Danqing Zhu PhD Abstract Friedreich's ataxia (FRDA) is the most common hereditary ataxia, affecting about 1 in 50,000 individuals. It is caused by mutations in the frataxin gene; 98% of cases have homozygous expansions of a GAA trinucleotide in intron 1 of the frataxin gene. The remaining 2% of patients are compound heterozygotes, who have a GAA repeat expansion in one allele and a point mutation in the other allele. FRDA patients with point mutation have been suggested to have atypical clinical features. We present a case of compound heterozygotes in a FRDA patient who has a deletion of one T in the start codon (ATG) of the frataxin gene and a GAA repeat expansion in the other allele. The patient presented with chorea and subsequently developed FRDA symptoms. The disease in this case is the result of both a failure of initiation of translation and the effect of the expansion. This novel mutation extends the range of point mutations seen in FRDA patients, and also broadens the spectrum of FRDA genotype associated with chorea. © 2002 Movement Disorder Society [source] Mammalian reovirus core protein,µ2 initiates at the first start codon and is acetylatedRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 24 2002Magdalena I. Swanson Mammalian reovirus is an enteric virus that contains a double-stranded RNA genome. The genome consists of ten RNA segments that encode eight structural and three non-structural proteins. The structural proteins form a double-layered structure. The innermost layer, called the core, consists of five proteins (,1, ,2, ,3, µ2, and ,2). Protein ,3 is the RNA-dependent RNA polymerase (RdRp) and µ2 is thought to be an RdRp cofactor. Translation of most reovirus proteins is known to commence at the first start codon. However, the translation initiation site of the viral core protein,µ2, encoded by the M1 RNA segment, has been in dispute. Although the theoretical molecular weight of µ2 is 83 267,Da the actual molecular weight is unknown because,µ2 runs aberrantly in SDS-PAGE and has resisted characterization by Edman degradation, indicating that the amino terminus is post-translationally modified. In this study, we used proteolysis coupled with MALDI-Qq-TOFMS to determine that translation of µ2 initiates at the first AUG codon, that its actual molecular weight approximates the theoretical value of 83,kDa, that the amino terminal methionine residue is removed, and that the next amino acid (alanine) is post-translationally acetylated. Copyright © 2002 John Wiley & Sons, Ltd. [source] Visualization of aligned genomic open reading frame data,BIOCHEMISTRY AND MOLECULAR BIOLOGY EDUCATION, Issue 1 2003Alan P. Boyle Abstract Students can better appreciate the value of genomic data if they are asked to use the data themselves. However, in general the enormous volume of data involved makes detailed examination difficult. Here we present a web site that allows students to study one particular aspect of sequenced genomes. They are able to align the open reading frames (ORFs) of any available genome that is of reasonable size. The ORFs may be aligned using either the start codon or the stop codon as the starting points. Results will readily show the presence of common ribosome binding sites as well as reveal interesting order within the ORFs that is nonexistent outside of them. Students will be able to ask various questions involving comparisons of genomes and see the results presented in both a tabular and graphic format. An example problem is presented under "Results." [source] Overexpression, purification and preliminary X-ray diffraction analysis of the controller protein C.Csp231I from Citrobacter sp.ACTA CRYSTALLOGRAPHICA SECTION F (ELECTRONIC), Issue 9 2009RFL23 Restriction,modification controller proteins play an essential role in regulating the temporal expression of restriction,modification genes. The controller protein C.Csp231I represents a new class of controller proteins. The gene was sublconed to allow overexpression in Escherichia coli. The protein was purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. The crystals diffracted to 2.0,Å resolution and belonged to space group P21. An electrophoretic mobility-shift assay provided evidence of strong binding of C.Csp231I to a sequence located upstream of the csp231IC start codon. [source] Novel sequence insertion in a Mâori patient with transfusion-dependent , -thalassaemiaBRITISH JOURNAL OF HAEMATOLOGY, Issue 3 2005Hilary A. Blacklock Summary Although , -thalassaemia is common throughout the world, it has not been previously described in Polynesia. We report a novel sequence insertion where homozygosity for the defect results in transfusion-dependent anaemia. The repeated 45 base pair (bp) insertion causes duplication of the start codon and consequent transcription from the original initiation code would be predicted to lead to the production of an irrelevant seven-residue peptide, while residual translation from the novel initiation site would result in diminished yields of , -globin and consequent clinical ,+ -thalassaemia. [source] High prevalence of blaCTX-M extended-spectrum ,-lactamase genes in Escherichia coli isolates from pets and emergence of CTX-M-64 in ChinaCLINICAL MICROBIOLOGY AND INFECTION, Issue 9 2010Y. Sun Clin Microbiol Infect 2010; 16: 1475,1481 Abstract As a cause of community-acquired infections, extended-spectrum ,-lactamase (ESBL)-producing Escherichia coli constitute an emerging public-health concern. Few data on the molecular epidemiology of ESBL-producing E. coli isolates from pets are available in China. Detection and characterization of ESBL genes (blaCTX-M, blaSHV and blaTEM) was conducted among 240 E. coli isolates recovered from healthy and sick pets in South China from 2007 to 2008. The clonal relatedness of ESBL-producing E. coli isolates was assessed by pulsed field gel electrophoresis. ESBL-encoding genes were identified in 97 (40.4%) of the 240 isolates and 96 (40.0%) of them harbored CTX-M. The most common CTX-M types were CTX-M-14 (n = 45) and CTX-M-55 (n = 24). The recently reported CTX-M-64 was identified in three isolates. Isolates producing CTX-M-27, -15, -65, -24, -3 and -9 were also identified. Ten isolates carried two or three CTX-M types, with the combination of CTX-M-14 and CTX-M-55 being the most frequent (n = 6). ISEcp1 was identified in the upstream region of 93 out of the 107 blaCTX-M genes (86.9%). The sequence of the spacer region (45 bp) between ISEcp1 and the start codon of all blaCTX-M-55 genes (except four) was identical to that of blaCTX-M-64. No major clonal relatedness was observed among these CTX-M producers. It is suggested that the horizontal transfer of blaCTX-M genes, mediated by mobile elements, contributes to their dissemination among E. coli isolates from pets. Our finding of high prevalence of ESBL in E. coli of companion animal origin illustrates the importance of molecular surveillance in tracking CTX-M-producing E. coli strains in pets. [source] Association of the , nucleotide with codon bias, amino acid usage and expressivity: differences between Bacillus subtilis and Escherichia coliAPMIS, Issue 10 2003ANDERS FUGLSANG By measuring the non-randomness in Shine-Dalgarno regions it was recently shown that the compositional non-randomness peaks approximately 10 nucleotides upstream of the start codons. This position, termed the , position, was furthermore shown to be associated with certain characteristics of the gene/protein and start codon usage. This raises the question whether codon usage in general is associated with the , position. In this study, the connection between the , nucleotide and general codon usage, both gene-wide and at the level of individual amino acids, was studied in Eschericia coli and Bacillus subtilis. E. coli but not B. subtilis shows a strong general association between the , position and codon usage bias. In both species, the genes with higher expressivity show stronger conservation in the Shine-Dalgarno region compared to the genes with lower expressivity. [source] Dinoflagellate mitochondrial genomes: stretching the rules of molecular biologyBIOESSAYS, Issue 2 2009Ross F. Waller Abstract Mitochondrial genomes represent relict bacterial genomes derived from a progenitor ,-proteobacterium that gave rise to all mitochondria through an ancient endosymbiosis. Evolution has massively reduced these genomes, yet despite relative simplicity their organization and expression has developed considerable novelty throughout eukaryotic evolution. Few organisms have reengineered their mitochondrial genomes as thoroughly as the protist lineage of dinoflagellates. Recent work reveals dinoflagellate mitochondrial genomes as likely the most gene-impoverished of any free-living eukaryote, encoding only two to three proteins. The organization and expression of these genomes, however, is far from the simplicity their gene content would suggest. Gene duplication, fragmentation, and scrambling have resulted in an inflated and complex genome organization. Extensive RNA editing then recodes gene transcripts, and trans-splicing is required to assemble full-length transcripts for at least one fragmented gene. Even after these processes, messenger RNAs (mRNAs) lack canonical start codons and most transcripts have abandoned stop codons altogether. [source] | |||||||||||||||||||||||||