SEARCH · PubMed Health

Results for “start codons”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 649 records · Page 36Linked to original sources

PubMed1986

Construction and characterisation of a series of multi-copy promoter-probe plasmid vectors for Streptomyces using the aminoglycoside phosphotransferase gene from Tn5 as indicator.

Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans. An efficient transcriptional terminator from E. coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters. A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions. Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised. The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts. Some derivatives contain a polylinker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors.

Cloning, Molecular↗

PubMed1997

Molecular characterisation of the pifC gene encoding translation initiation factor 3, which is required for normal photosynthetic complex formation in Rhodobacter sphaeroides NCIB 8253.

In order to determine whether translation initiation events play a selective role in regulating the expression of photosynthetic complexes in the photosynthetic bacterium Rhodobacter sphaeroides, we have undertaken an initial study to investigate the potential role of translation initiation factor IF3, which also behaves as a pleiotropic regulatory factor in some bacteria. Following the isolation and purification of a 24-kDa IF3-like protein (PifC) from R. sphaeroides, we used nested PCR to clone and characterise the encoding gene, pifC (photosynthesis-affecting initiation factor). The 545-bp pifC encodes a protein exhibiting 60% identity (78.6% similarity) with the Escherichia coli IF3 (InfC) protein and, in common with all other IF3 genes identified to date, pifC possesses a rare initiation codon (AUA). Furthermore, in common with IF3, PifC was shown here to perform a discriminatory function towards CUG start codons, confirming its role and function as an IF3 in R. sphaeroides. Insertion of a kanamycin resistance cassette into the 5' end of pifC resulted in a viable phenotype which exhibits growth rates similar to wild type but which possesses reduced bacteriochlorophyll and photosynthetic complexes in semi-aerobic cultures. It is shown here that the mutant is still able to produce a PifC protein but that it possesses reduced IF3 activity. This may account for the viable nature of the mutant strain, and may indicate that the effect of the mutation on photosynthesis can be more severe than shown in the present study. The mechanisms by which PifC may exert its selective regulatory effect on photosynthesis expression are discussed.

Amino Acid Sequence↗

PubMed2007

A UspA2H-negative variant of Moraxella catarrhalis strain O46E has a deletion in a homopolymeric nucleotide repeat common to uspA2H genes.

Moraxella catarrhalis strains can express either a UspA2 protein or a UspA2H protein. The latter protein is encoded by a gene that possesses a homopolymeric nucleotide tract containing eight adenine (A) residues [i.e., a poly(A) tract] which is located near the 5' end. A spontaneous UspA2H-negative variant of M. catarrhalis strain O46E, designated O46E.U2V, was found to have a uspA2H poly(A) tract that contained seven A residues. Northern blot analysis of total RNA from the O46E parent strain revealed a readily detectable uspA2H mRNA transcript, whereas little or no uspA2H transcript was detectable in total RNA from the UspA2H-negative variant O46E.U2V. The 5' end of the uspA2H genes from both the O46E parent strain and the O46E.U2V variant were ligated to a promoterless lacZ gene to prepare translational fusions for use as reporter constructs. The level of beta-galactosidase activity expressed by the fusion construct containing eight A residues in its poly(A) tract was 200-fold greater than that obtained with the construct that had seven A residues. Site-directed mutagenesis of the 5' end of the uspA2H gene confirmed that translation was initiated at a GTG codon located 21 nucleotides (nt) upstream of the poly(A) tract. Primer extension analysis determined that the transcriptional start site of the uspA2H gene was located 291 nt upstream from the GTG translational start codon. This poly(A) tract was also found to be present in the uspA2H genes of other M. catarrhalis strains.

Amino Acid Sequence↗

PubMed2006

A functional dlt operon, encoding proteins required for incorporation of d-alanine in teichoic acids in gram-positive bacteria, confers resistance to cationic antimicrobial peptides in Streptococcus pneumoniae.

Streptococcus pneumoniae is one of the few species within the group of low-G +C gram-positive bacteria reported to contain no d-alanine in teichoic acids, although the dltABCD operon encoding proteins responsible for d-alanylation is present in the genomes of two S. pneumoniae strains, the laboratory strain R6 and the clinical isolate TIGR4. The annotation of dltA in R6 predicts a protein, d-alanine-d-alanyl carrier protein ligase (Dcl), that is shorter at the amino terminus than all other Dcl proteins. Translation of dltA could also start upstream of the annotated TTG start codon at a GTG, resulting in the premature termination of dltA translation at a stop codon. Applying a novel integrative translation probe plasmid with Escherichia coli 'lacZ as a reporter, we could demonstrate that dltA translation starts at the upstream GTG. Consequently, S. pneumoniae R6 is a dltA mutant, whereas S. pneumoniae D39, the parental strain of R6, and Rx, another derivative of D39, contained intact dltA genes. Repair of the stop codon in dltA of R6 and insertional inactivation of dltA in D39 and Rx yielded pairs of dltA-deficient and dltA-proficient strains. Subsequent phenotypic analysis showed that dltA inactivation resulted in enhanced sensitivity to the cationic antimicrobial peptides nisin and gallidermin, a phenotype fully consistent with those of dltA mutants of other gram-positive bacteria. In addition, mild alkaline hydrolysis of heat-inactivated whole cells released d-alanine from dltA-proficient strains, but not from dltA mutants. The results of our study suggest that, as in many other low-G+C gram-positive bacteria, teichoic acids of S. pneumoniae contain d-alanine residues in order to protect this human pathogen against the actions of cationic antimicrobial peptides.

Alanine↗

PubMed2003

Functional split and crosslinking of the membrane domain of the beta subunit of proton-translocating transhydrogenase from Escherichia coli.

Proton pumping nicotinamide nucleotide transhydrogenase from Escherichia coli contains an alpha subunit with the NAD(H)-binding domain I and a beta subunit with the NADP(H)-binding domain III. The membrane domain (domain II) harbors the proton channel and is made up of the hydrophobic parts of the alpha and beta subunits. The interface in domain II between the alpha and the beta subunits has previously been investigated by cross-linking loops connecting the four transmembrane helices in the alpha subunit and loops connecting the nine transmembrane helices in the beta subunit. However, to investigate the organization of the nine transmembrane helices in the beta subunit, a split was introduced by creating a stop codon in the loop connecting transmembrane helices 9 and 10 by a single mutagenesis step, utilizing an existing downstream start codon. The resulting enzyme was composed of the wild-type alpha subunit and the two new peptides beta1 and beta2. As compared to other split membrane proteins, the new transhydrogenase was remarkably active and catalyzed activities for the reduction of 3-acetylpyridine-NAD(+) by NADPH, the cyclic reduction of 3-acetylpyridine-NAD(+) by NADH (mediated by bound NADP(H)), and proton pumping, amounting to about 50-107% of the corresponding wild-type activities. These high activities suggest that the alpha subunit was normally folded, followed by a concerted folding of beta1 + beta2. Cross-linking of a betaS105C-betaS237C double cysteine mutant in the functional split cysteine-free background, followed by SDS-PAGE analysis, showed that helices 9, 13, and 14 were in close proximity. This is the first time that cross-linking between helices in the same beta subunit has been demonstrated.

Catalysis↗

PubMed1997

Cloning of the human phospholipase C-gamma1 promoter and identification of a DR6-type vitamin D-responsive element.

The 5'-flanking region of the human phospholipase C-gamma1 gene was isolated from a human P1 genomic DNA library. The S1-nuclease mapping and primer extension analysis revealed that there is a single transcriptional start site located at 135 bases upstream from the translation start codon in the human phospholipase C-gamma1 gene. DNA sequence analysis showed that the sequence around the transcriptional start site is very GC-rich and has no TATA box. The fragment +135 to -877 in the 5'-flanking region of the human phospholipase C-gamma1 gene was subcloned into a luciferase reporter vector. The chimeric gene produced a high level of luciferase activity and responded to 1,25-(OH)2D3 in transiently transfected human keratinocytes. Deletion and mutation studies of the fragment +135 to -877 demonstrated a vitamin D-responsive element that contains a motif arranged as two direct repeats separated by 6 bases (DR6), AGGTCAgaccacTGGACA, located between -786 and -803 base pairs. Incubation of the oligonucleotide containing the DR6 with keratinocyte nuclear extracts produced a specific protein-DNA complex that shifted to a higher molecular weight form upon the addition of an antibody specific to the 1,25-(OH)2D3 receptor. Therefore, the 5'-flanking region of the human phospholipase C-gamma1 gene confers promoter activity and contains a DR6-type vitamin D-responsive element that mediates, at least in part, the enhanced expression of this gene in human keratinocytes by 1, 25-(OH)2D3.

Base Sequence↗

PubMed1989

Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched-chain-oxoacid dehydrogenase of Pseudomonas putida.

The production of two lipoamide dehydrogenases by Pseudomonas is so far unique. One, LPD-val, is the specific E3 component of the branched-chain-oxoacid dehydrogenase and the second, LPD-glc, is the E3 component of 2-oxoglutarate dehydrogenase and the L-factor of the glycine oxidation system. The objective of the present research was to determine the nucleotide sequence of the structural gene for LPD-val in order to compare its deduced amino acid structure with that of other redox-active disulfide flavoproteins. Northern blots using mRNA isolated from P. putida grown in media with branched-chain amino acids identified a transcript of 6.2 kb which is long enough to encode all the structural genes for the complex. The nucleotide sequence of the structural gene for LPD-val, lpdV, was determined and consists of 459 codons plus the stop codon. The open reading frame begins two bases after the stop codon for the E2 subunit and is composed of 66.3% G + C. Codon usage is characteristic of moderately strongly expressed genes. There is a ribosome-binding site preceding the ATG start codon and a strong candidate for a rho-independent terminator at the 3' end of the reading frame. The Mr of the protein encoded is 48,164 and when the Mr of FAD is added, the total Mr is 48,949, which is very close to the value of 49,000 obtained by SDS-polyacrylamide gel electrophoresis. Similarity comparisons of LPD-val with sequences of three other lipoamide dehydrogenases showed that LPD-val was somewhat more distantly related. It is probable that the lipoamide dehydrogenases and the glutathione and mercuric reductases evolved from a common ancestral flavoprotein.

3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide)↗

PubMed1996

Cloning and transcriptional analysis of two threonine biosynthetic genes from Lactococcus lactis MG1614.

Two genes, hom and thrB, involved in threonine biosynthesis in Lactococcus lactis MG1614, were cloned and sequenced. These genes, which encode homoserine dehydrogenase and homoserine kinase, were initially identified by the homology of their gene products with known homoserine dehydrogenases and homoserine kinases from other organisms. The identification was supported by construction of a mutant containing a deletion in hom and thrB that was unable to grow in a defined medium lacking threonine. Transcriptional analysis showed that the two genes were located in a bicistronic operon with the order 5' hom-thrB 3' and that transcription started 66 bp upstream of the translational start codon of the hom gene. A putative -10 promoter region (TATAAT) was located 6 bp upstream of the transcriptional start point, but no putative -35 region was identified. A DNA fragment covering 155 bp upstream of the hom translational start site was functional in pAK80, an L. lactis promoter probe vector. In addition, transcriptional studies showed no threonine-dependent regulation of hom-thrB transcription.

Amino Acid Sequence↗

PubMed1985

Identification of the promoter of the Pseudomonas gene coding for carboxypeptidase G2.

A 213-bp region of noncoding DNA upstream of the ATG start codon of the Pseudomonas carboxypeptidase G2 (CPG2) structural gene has been shown to contain the CPG2 promoter. The mRNA start point (+1) on the DNA sequence has been identified by mapping the 5' end of the CPG2 transcript. The identified promoter region contains a -10 region (TATAAG) that closely resembles the Escherichia coli consensus sequence (TATAAT), but has no easily recognisable -35 region. The lack of homology in the -35 region explains why this particular pseudomonad gene is poorly expressed in E. coli. Similar sequence differences may turn out to be the cause of the generally observed inefficient expression of Pseudomonas genes in E. coli. The promoter region also carries a sequence (CTGGCACTCGAATTGCT) that closely matches the consensus nif promoter sequence (CTGGPyAPyPuNNNNTTGCA) of Klebsiella pneumoniae and Rhizobium, and a similar sequence (ATGGCATGGCGGTTGCT) found in the promoter region of the xylABC operon of the TOL plasmid of Pseudomonas putida.

Base Sequence↗

PubMed1997

Functional importance of RNA interactions in selection of translation initiation codons.

RNA base pairing between the initiation codon and anticodon loop of initiator tRNA is essential but not sufficient for the selection of the 'correct' mRNA translational start site by ribosomes. In prokaryotes, additional RNA interactions between small ribosomal subunit RNA and mRNA sequences just upstream of the start codon can efficiently direct the ribosome to the initiation site. Although there is presently no proof for a similar important ribosomal RNA interaction in eukaryotes, the 5' non-coding regions of their mRNAs and 'consensus sequences' surrounding initiation codons have been shown to be strong determinants for initiation-site selection, but the exact mechanisms are not yet understood. Intramolecular base pairing in mRNA and participation of translation initiation factors can strongly influence the formation of mRNA-small ribosomal subunit-initiator tRNA complexes and modulate translational activities in both prokaryotes and eukaryotes. Only recently has it been appreciated that alternative mechanisms may also contribute to the selection of initiation codons in all organisms. Although direct proof is currently lacking, there is accumulating evidence that additional cis-acting mRNA elements and trans-acting proteins may form specific 'bridging' interactions with ribosomes during translation initiation.

Base Sequence↗

PubMed1988

Translational coupling between the ilvD and ilvA genes of Escherichia coli.

The hypothesis that translation of the ilvD and ilvA genes of Escherichia coli may be linked has been examined in strains in which lacZ-ilvD protein fusions are translated in all three reading frames with respect to ilvD. In these strains, the nucleotide sequence was altered to obtain premature termination of ilvD translation, and in one strain translation termination of ilvD DNA occurred two bases downstream of the ilvA initiation codon. In the wild-type strain, the ilvD translation termination site was located two bases upstream of the ilvA start codon. In each of the mutant strains, expression of ilvA, as determined by the level of threonine deaminase activity, was strikingly lower than in the wild-type strain. The data suggest that expression of ilvD and ilvA is translationally coupled. By inserting a promoterless cat gene downstream of ilvA, it was shown that the differences in enzyme activity were not the result of differences in the amount of ilvA mRNA produced.

DNA Mutational Analysis↗

PubMed1991

Molecular cloning of a cDNA encoding the amino end of the mammalian multifunctional protein CAD and analysis of the 5'-flanking region of the CAD gene.

CAD is a 243-kDa multidomain polypeptide which catalyzes the first three steps in mammalian de novo pyrimidine biosynthesis. The largest cDNA clone obtained thus far, pCAD142 (Shigesada, K., Stark, G.R., Maley, J. A., Niswander, L. A., and Davidson, J. N. (1985) Mol. Cell. Biol. 5, 1735), lacks the 5' end of the mRNA which encodes the amino terminus of CAD. To clone this missing segment, a synthetic oligonucleotide complementary to pCAD142 and poly(A)+ RNA template, isolated from a Syrian hamster cell line which overproduces the CAD mRNA, were used for cDNA synthesis. The resulting clone pKB11, which has a 1369-base pair (bp) cDNA insert, overlapping pCAD142 by 781 bp, was identified by hybridization methods and sequence analysis and found to contain the entire cDNA sequence for the amino end of the CAD polypeptide. The deduced amino acid sequence is homologous to seven carbamyl phosphate synthetases. Primer extension, oligonucleotide-directed RNase H digestion, and RNA sequencing indicated that pKB11 extends to within 68 bases of the 5' end of the CAD mRNA. This conclusion was confirmed by Northern blotting analysis of the 5'-flanking region of CAD gene. The probable 3' end of an unidentified gene which codes for a 1-kilobase (kb) transcript was identified immediately upstream of the CAD gene. Northern analysis using probes complementary to the region between the CAD and the 1-kb genes detected the presence of a small transcript of less than 300 nucleotides. The sequence revealed three potential translation initiation sites raising the possibility of more than one CAD translation product. The major translation start codon was identified as the first ATG in pKB11 by sequence homology, in vitro transcription and translation, and protein studies. Starting from this ATG within pKB11, the clone encodes a 143-residue domain of unknown function. This study completes the determination of the primary structure of the CAD polypeptide. The CAD mRNA is 7.5 kb in length and has 6675 bp of coding sequence and about 200 bp and 600 bp of untranslated sequence at the 5' and 3' ends, respectively.

Amino Acid Sequence↗

PubMed1990

The gene family encoding eggshell proteins of Schistosoma japonicum.

The four closely related genes encoding eggshell proteins in the human parasite Schistosoma japonicum are described. A cDNA and a genomic DNA library were constructed and members of the eggshell protein gene family isolated. The four genes in this family do not contain introns, and differ in organization and nucleotide sequence from the related set of genes in Schistosoma mansoni and Schistosoma haematobium. The coding sequences of two of the S. japonicum genes and their flanking regions were determined. Transcription start sites for these genes were shown by primer extension analysis to occur 47 and 50 nucleotides in front of the start codon. A female-specific component in nuclear extracts binds to a DNA fragment containing conserved sequences upstream of the transcription start sites. The deduced protein sequences of 207 and 212 amino acids are composed of 50% glycine with continuous glycine regions as long as 11 residues. In vitro translations of male and female RNAs revealed female-specific translation products, the sizes of which were consistent with the eggshell proteins.

Amino Acid Sequence↗

PubMed2000

The hormone-sensitive lipase gene is transcribed from at least five alternative first exons in mouse adipose tissue.

Hormone-sensitive lipase (HSL) mediates triglyceride hydrolysis in adipocytes, in which its expression varies with physiological stress and is controlled posttranslationally and transcriptionally. We sequenced the mouse HSL gene for 8.2 kb upstream of the translation start codon and studied the steady-state HSL mRNA levels in mouse adipose tissue. In 50 clones derived from primer extension and PCR of mouse adipose cDNA, we found five distinct 5' extremities that correspond to distinct exons in genomic DNA. Exon A is located approximately 7 kb 5' to the HSL translation start site. Exons B. C, and D are clustered 1.5-2 kb upstream, and the previously described exon 1 is immediately upstream and contiguous with the previously described HSL translation start site. Exon A is located -7 kb upstream and contains an in-frame methionine codon that could potentially generate another HSL isoform with 43 additional N-terminal residues. cDNA clones containing the newly described exons suggested that each exon has several transcription start sites but that all splice to an acceptor site located 20 nt upstream of the translation initiation codon in exon 1. HSL transcription in mouse adipose tissue originates from multiple sites in the 7-kb region between exon A and exon 1, with peaks at exon C (50-70% of HSL transcripts), exon 1 (5-30%), and exon A (approximately 10%). There are multiple potential transcription factor-binding elements upstream of each exon, suggesting the possibility of differential transcriptional regulation of HSL in different tissues and under various physiologic conditions.

Adipose Tissue↗

PubMed1993

The fdx gene encoding the [2Fe--2S] ferredoxin of Halobacterium salinarium (H. halobium).

The gene encoding the [2Fe--2S] ferredoxin (fdx gene) was isolated from Halobacterium salinarium using two oligonucleotides deduced from the ferredoxin sequence as probes. Cosmid DNAs exhibiting hybridization were isolated, the fdx gene was localized to smaller subfragments and the nucleotide sequence determined. The 390 bp coding sequence is located in the halobacterial FI-DNA and transcribed as a 440 nucleotide mRNA. S1 mapping indicated that the 5' terminus of the mRNA maps immediately upstream of the ATG start codon. The promoter box A, centred around position -25 (5' AC-TATG 3'), and box B (TG) elements at the start of the transcript resemble the sequences of a typical archaeal promoter. The restriction pattern of an approximately 50 kb region surrounding the fdx gene is conserved in various Halobacterium species. The halobacterial ferredoxin and the major gas vesicle protein GvpA exhibit up to 70% similarity to their respective counterparts in cyanobacteria suggesting lateral gene transfer between the organisms. These similarities prompted a more detailed investigation of the relative positions of the genes in the halobacterial genome.

Amino Acid Sequence↗

PubMed2001

Sp1 and ETS family transcription factors regulate the mouse Mta2 gene expression.

Dynamic changes in chromatin structure through nucleosome remodelling and core histone tail acetylation play important roles in transcriptional regulation. The purification and functional characterization of a nucleosome remodelling and histone deacetylase complex, NuRD, has suggested that nucleosome remodelling and core histone tail modification are potentially linked processes. MTA2, a component of the NuRD complex, plays an important role in regulating histone deacetylase activity of the NuRD complex. Similar to the candidate metastasis associated protein MTA1, an elevated level of MTA2 correlates with cellular proliferation. To understand the regulation of Mta2 transcription, we characterized the mouse Mta2 gene and its transcriptional regulatory elements. We found that MTA2 is encoded by 18 exons that span 10 kb. Primer extension analysis identified a major transcriptional start site locates 259 base pairs upstream of the ATG translational start codon. Transient transfection studies localized its promoter, lacking a canonical TATA box, to within 60 base pairs upstream of the transcriptional start site. Gel-mobility-shift and mutagenesis studies revealed that Sp1 and ETS elements play important roles in regulating Mta2 transcription. Information concerning the regulation of the Mta2 gene expression will be useful in understanding the regulation of NuRD histone deacetylase activity, which in turn will help in our general understanding of the transcriptional repression mechanism.

3T3 Cells↗

PubMed2006

AUG sequences are required to sustain nonsense-codon-mediated suppression of splicing.

More than 90% of human genes are rich in intronic latent 5' splice sites whose utilization in pre-mRNA splicing would introduce in-frame stop codons into the resultant mRNAs. We have therefore hypothesized that suppression of splicing (SOS) at latent 5' splice sites regulates alternative 5' splice site selection in a way that prevents the production of toxic nonsense mRNAs and verified this idea by showing that the removal of such in-frame stop codons is sufficient to activate latent splicing. Splicing control by SOS requires recognition of the mRNA reading frame, presumably recognizing the start codon sequence. Here we show that AUG sequences are indeed essential for SOS. Although protein translation does not seem to be required for SOS, the first AUG is shown here to be necessary but not sufficient. We further show that latent splicing can be elicited upon treatment with pactamycin-a drug known to block translation by its ability to recognize an RNA fold-but not by treatment with other drugs that inhibit translation through other mechanisms. The effect of pactamycin on SOS is dependent neither on steady-state translation nor on the pioneer round of translation. This effect is found for both transfected and endogenous genes, indicating that SOS is a natural mechanism.

Animals↗

PubMed1995

Identification of two novel polymorphisms and a rare deletion variant in the human dopamine D4 receptor gene.

We report two novel polymorphisms and a rare deletion variant in the human dopaine D4 receptor gene. The two polymorphisms are characterized by single base pair substitutions, namely a G-->C transversion changing codon 11 from GGG (encoding Gly) to CGG (encoding Arg) and a C-->T transition in position -11 upstream from the start codon. The Arg11 variant occurs at a frequency of about 1% and the C-->T transition at a frequency of about 7% in German control subjects (n = 148). Allele frequencies observed in patients suffering from schizophrenia (n = 256) and bipolar affective disorder (n = 99) were similar. The deletion variant is characterized by a 21 bp deletion affecting codons 36 to 42 coding for amino acids Ala-Ala-Leu-Val-Gly-Gly-Val located in the first transmembrane domain of the dopamine D4 receptor. The mutation was identified in a single individual suffering from obsessive-compulsive disorder and panic disorder. We were unable to detect the deletion in patients with schizophrenia and bipolar affective disorder, nor in healthy controls.

Adult↗