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At least 505 records · Page 28Linked to original sources
PubMed1989

Nucleotide sequence determination of the DNA region coding for Bacillus stearothermophilus glyceraldehyde-3-phosphate dehydrogenase and of the flanking DNA regions required for its expression in Escherichia coli.

The complete nucleotide sequence of a 3541-base pairs (bp) DNA fragment from Bacillus stearothermophilus able to complement an Escherichia coli glyceraldehyde-3-phosphate-dehydrogenase (GAPDH) mutant (gapD-) has been determined. The B. stearothermophilus gap gene consists of a 1005-bp open reading frame commencing with an ATG start codon and ending with a TAA stop codon. Upstream from the start codon is a strong Shine-Dalgarno sequence typical of Gram-positive bacteria. Only one putative RNA polymerase recognition signal (-35 and -10 regions) is found 1153 bp upstream from the ATG start codon. In vivo utilization of this signal is in agreement with the study of gene expression from different subclones of the original fragment. 57 bp downstream from the TAA stop codon is a structure resembling Rho-independent transcription termination signals. Although B. stearothermophilus GAPDH-coding gene is highly expressed in E. coli, it contains several rare codons for E. coli. The predicted amino acid sequence of the GAPDH enzyme presents several differences with the amino acid sequence previously determined from the protein and is in better agreement with published crystallographic data.

Amino Acid Sequence↗
PubMed1998

Novel compound heterozygous laminina2-chain gene (LAMA2) mutations in congenital muscular dystrophy. Mutations in brief no. 159. Online.

The laminina2-chain gene (LAMA2) encodes a basal lamina protein, laminina2, known to be deficient in one form of congenital muscular dystrophy (CMD). In a laminina2 deficient-CMD patient, we screened the entire LAMA2 cDNA (953bp) by reverse transcriptase polymerase chain reaction combined with single strand conformational polymorphism analysis. Direct sequencing of aberrant conformers in this patient revealed two loss-of-function mutations, consistent with autosomal recessive inheritance. The patient had two novel heterozygous mutations: 1) an exon 4 nonsense mutation caused by a G-->A substitution at cDNA position 547, changing the TGG codon for tryptophan into a TGA stop codon (W166X) in the N-terminus domain VI;ii) an exon 54 frameshift mutation due to a deletion of nucleotide 'C' at cDNA position 7707 (S2553Y), resulting in a premature stop codon (V2587X) in exon 55 in the globular G domain of laminina2 at the C-terminus. These mutations cause a disruption of the open reading frame of LAMA2. The absence of laminina2 observed in the patient's muscle biopsy could result from diminished levels of the LAMA2 transcript. Alternatively, the mutations might lead to translation of a truncated laminina2. By either mechanism the phenotype of congenital muscular dystrophy is believed to be the result of disruption of linkage between the extracellular matrix and the dystrophin glycoprotein complex.

DNA Mutational Analysis↗
PubMed2000

Frameshift mutations in rdxA and metronidazole resistance in North American Helicobacter pylori isolates.

In Helicobacter pylori, the oxygen-insensitive nitroreductase RdxA is likely to activate metronidazole (Mtz) by reduction and formation of cytotoxic intermediates. Mutations in rdxA have been associated with Mtz resistance in H. pylori. In vitro Mtz susceptibilities of 17 randomly selected H. pylori isolates were determined by the agar dilution method. DNA sequence analysis of rdxA alleles of eight susceptible isolates (MIC range: 0.25-1.0 mg/L) and nine resistant isolates (MIC range: 16-256 mg/L) showed that six of nine Mtz-resistant H. pylori isolates contained insertion or deletion mutations ('indel' mutations). One isolate contained a substitution mutation at codon position 148 that resulted in the introduction of a premature stop codon. Creation of stop codons within the rdxA coding sequence by either frameshift or substitution mutations resulted in premature translation termination and expression of putatively truncated RdxA polypeptides.

Anti-Bacterial Agents↗
PubMed1999

Recognition of yeast mRNAs as "nonsense containing" leads to both inhibition of mRNA translation and mRNA degradation: implications for the control of mRNA decapping.

A critical step in the degradation of many eukaryotic mRNAs is a decapping reaction that exposes the transcript to 5' to 3' exonucleolytic degradation. The dual role of the cap structure as a target of mRNA degradation and as the site of assembly of translation initiation factors has led to the hypothesis that the rate of decapping would be specified by the status of the cap binding complex. This model makes the prediction that signals that promote mRNA decapping should also alter translation. To test this hypothesis, we examined the decapping triggered by premature termination codons to determine whether there is a down-regulation of translation when mRNAs were recognized as "nonsense containing." We constructed an mRNA containing a premature stop codon in which we could measure the levels of both the mRNA and the polypeptide encoded upstream of the premature stop codon. Using this system, we analyzed the effects of premature stop codons on the levels of protein being produced per mRNA. In addition, by using alterations either in cis or in trans that inactivate different steps in the recognition and degradation of nonsense-containing mRNAs, we demonstrated that the recognition of a nonsense codon led to a decrease in the translational efficiency of the mRNA. These observations argue that the signal from a premature termination codon impinges on the translation machinery and suggest that decapping is a consequence of the change in translational status of the mRNA.

Blotting, Northern↗
PubMed2004

Cellular and molecular studies of B cells exhibiting reverse somatic mutation throughout life.

Somatic mutation of immunoglobulin (Ig) genes plays an important role in generating antibody diversity. The frequency of somatic mutation appears to vary throughout life. However, this process has been difficult to study in vivo because the DNA in and around rearranged V genes undergoes random mutation, causing silent or replacement mutations. Therefore, we have developed a transgenic mouse model for studying the frequency of B cells exhibiting mutation in young and old mice. The system is based on a reporter transgene (HuG-X) that encodes a chimeric Ig heavy chain composed of a murine VDJ segment and a human IgG1 constant region. The VDJ has been mutated to contain a TAG stop codon in the D segment. Therefore, the transgene is transcribed but not translated. Point mutation of the stop codon results in expression of the chimeric H chain, which is readily detected as human IgG1 expression. In vivo, we found that the transgene undergoes spontaneous reverse somatic mutation at a low frequency. Treatment of HuG-X mice with anti-IgD greatly increases the frequency of somatic mutation. The observed mutation frequency in anti-IgD-treated mice increases with age until adulthood, then plateaux and finally declines in aged mice. The mutations in the stop codon were associated with increased double-stranded DNA breaks (DSB) within and around the TAG site. Our results demonstrate that the rate of frequency of spontaneous reverse mutation is very low in vivo, yet it is significantly increased after stimulation with anti-IgD antibodies. The frequency of point mutation is age dependent and correlates with increased DSB.

Animals↗
PubMed2002

Alternative splicing of an insect sodium channel gene generates pharmacologically distinct sodium channels.

Alternative splicing is a major mechanism by which potassium and calcium channels increase functional diversity in animals. Extensive alternative splicing of the para sodium channel gene and developmental regulation of alternative splicing have been reported in Drosophila species. Alternative splicing has also been observed for several mammalian voltage-gated sodium channel genes. However, the functional significance of alternative splicing of sodium channels has not been demonstrated. In this study, we identified three mutually exclusive alternative exons encoding part of segments 3 and 4 of domain III in the German cockroach sodium channel gene, para(CSMA). The splice site is conserved in the mouse, fish, and human Na(v)1.6 sodium channel genes, suggesting an ancient origin. One of the alternative exons possesses a stop codon, which would generate a truncated protein with only the first two domains. The splicing variant containing the stop codon is detected only in the PNS, whereas the other two full-size variants were detected in both the PNS and CNS. When expressed in Xenopus oocytes, the two splicing variants produced robust sodium currents, but with different gating properties, whereas the splicing variant with the stop codon did not produce any detectable sodium current. Furthermore, these two functional splicing variants exhibited a striking difference in sensitivity to a pyrethroid insecticide, deltamethrin. Exon swapping partially reversed the channel sensitivity to deltamethrin. Our results therefore provide the first evidence that alternative splicing of a sodium channel gene produces pharmacologically distinct channels.

Alternative Splicing↗
PubMed1997

Physical mapping and structural analysis of new gene families RT1.S and Rps2r in the grc region of the rat major histocompatibility complex.

Five new genes were identified in the growth and reproduction complex (grc) region: RT1.S1, RT1.S2, Rps2r1, Rps2r2, and Rps2r3. The class Ib RT1.S1 and RT1.S2 genes have five distinct exons (1, 4, 5, 6, 7) similar to other class I major histocompatibility complex genes but the conventional exons 2 and 3 are absent. The genes are 97% similar, have CAAT and TATA boxes much upstream of the conventional position, obey the GT/AG rule in their exon-intron boundaries, and are transcribed at a low level in thymus and testis but not in the liver and spleen. The region between exon 1 and exon 4 was analyzed by obtaining transcripts by reverse transcription-polymerase chain reaction (RT-PCR) amplification which revealed the presence of four alternatively spliced mRNA transcripts of RT1.S1: 1) S1-1 (clones 14 and 16) has no exon between exons 1 and 4; 2) S1-2 (clones 7 and 8) has an exon of 45 nucleotides that can translate into 15 amino acids; 3) S1-3 (clone 5) has an exon of 42 nucleotides with a stop codon; and 4) S1-4 (clone 10) has two exons of 42 and 38 nucleotides, respectively, with stop codons. Only one RT1.S2 mRNA transcript was obtained, and it has an exon of 45 nucleotides between exon 1 and exon 4 which can form a peptide identical to the S1-2 isoform for that region. The 45 nucleotide exon between exon 1 and exon 4 was unique for RT1.S1 and RT1.S2 and only matched a sequence in the RT1.O intron region (nucleotides 2905 - 2949). The three ribosomal-protein-S2-related (Rps2r) genes are 94% - 98% similar; they are related to the genes encoding ribosomal protein S2 of the black rat and the LLRep3 genes of the mouse and the human and to the genes encoding Saccharomyces cerevisiae S4, Escherichia coli S5, and other members of prokaryote S5 family. The Rps2r1 gene is located just outside of the grc region. The Rps2r2 and Rps2r3 genes are in the grc and have multiple stop codons in their genomic sequences. The Rps2r1 mRNA transcript was identified by RT-PCR in the thymus and testis but not in the liver and spleen.

Amino Acid Sequence↗
PubMed2003

Structure of the Escherichia coli ribosomal termination complex with release factor 2.

Termination of protein synthesis occurs when the messenger RNA presents a stop codon in the ribosomal aminoacyl (A) site. Class I release factor proteins (RF1 or RF2) are believed to recognize stop codons via tripeptide motifs, leading to release of the completed polypeptide chain from its covalent attachment to transfer RNA in the ribosomal peptidyl (P) site. Class I RFs possess a conserved GGQ amino-acid motif that is thought to be involved directly in protein-transfer-RNA bond hydrolysis. Crystal structures of bacterial and eukaryotic class I RFs have been determined, but the mechanism of stop codon recognition and peptidyl-tRNA hydrolysis remains unclear. Here we present the structure of the Escherichia coli ribosome in a post-termination complex with RF2, obtained by single-particle cryo-electron microscopy (cryo-EM). Fitting the known 70S and RF2 structures into the electron density map reveals that RF2 adopts a different conformation on the ribosome when compared with the crystal structure of the isolated protein. The amino-terminal helical domain of RF2 contacts the factor-binding site of the ribosome, the 'SPF' loop of the protein is situated close to the mRNA, and the GGQ-containing domain of RF2 interacts with the peptidyl-transferase centre (PTC). By connecting the ribosomal decoding centre with the PTC, RF2 functionally mimics a tRNA molecule in the A site. Translational termination in eukaryotes is likely to be based on a similar mechanism.

Binding Sites↗
PubMed1989

Deletion/frameshift mutation in the alpha 1-antitrypsin null allele, PI*QObolton.

The most common deficiency allele of the protease inhibitor (PI) alpha 1-antitrypsin (alpha 1AT) is PI*Z. Other rare deficiency alleles of alpha 1AT are of two types: those producing low but detectable amounts of alpha 1AT (less than 20% of normal serum concentrations), and null alleles producing less than 1% of normal alpha 1AT and therefore not detectable by routine quantitative methods. We have previously used DNA polymorphisms and family data to determine heterozygosity in an individual producing low levels of serum alpha 1AT (12% of normal) of PI type Mmalton. By DNA analysis we observed the typical haplotype associated with PI*Mmalton and a unique null haplotype associated with the allele PI*QObolton. The QObolton allele produces no detectable serum alpha 1AT. We have cloned and sequenced the QObolton allele from a phage genomic library. Deletion of a single cytosine residue near the active site of alpha 1 AT in exon V results in a frameshift causing an in-frame stop codon downstream of the deletion. This stop codon leads to premature termination of protein translation at amino acid 373, resulting in a truncated protein. The truncated protein is predicted to have an altered carboxy terminus (amino acids 363-372) and will lack structurally important amino acids.

Alleles↗
PubMed1996

A relationship between GC content and coding-sequence length.

Since base composition of translational stop codons (TAG, TAA, and TGA) is biased toward a low G+C content, a differential density for these termination signals is expected in random DNA sequences of different base compositions. The expected length of reading frames (DNA segments of sense codons flanked by in-phase stop codons) in random sequences is thus a function of GC content. The analysis of DNA sequences from several genome databases stratified according to GC content reveals that the longest coding sequences-exons in vertebrates and genes in prokaryotes-are GC-rich, while the shortest ones are GC-poor. Exon lengthening in GC-rich vertebrate regions does not result, however, in longer vertebrate proteins, perhaps because of the lower number of exons in the genes located in these regions. The effects on coding-sequence lengths constitute a new evolutionary meaning for compositional variations in DNA GC content.

Animals↗
PubMed1997

Evidence for multiple functional copies of the male sex-determining locus, Sry, in African murine rodents.

Southern hybridization data suggest that the male sex-determining locus, Sry, is often duplicated in rodents. Here we explore DNA sequence evolution of orthologous and paralogous copies of Sry isolated from six species of African murines. PCR amplification followed by direct sequencing revealed from two to four copies of Sry per species. All copies include a long open reading frame, with a stop codon that coincides closely with the stop codon of the house mouse, Mus musculus, a species known to have a single copy of Sry. A phylogenetic analysis suggests that there are at least seven paralogous copies of Sry in this group of rodents. Putative orthologues are identical; sequence divergence among putative paralogues ranges from 1 to 8% (excluding the CAG repeat), with much lower levels of divergence in the high-mobility group (HMG-box) region than in the C-terminal region. A high proportion of nucleotide substitutions in both regions result in amino-acid replacement. The long open reading frame, conserved HMG-box, and pattern of evolution of the putative paralogues suggest that they are functional.

Amino Acid Sequence↗
PubMed2005

In vitro selection of phage RB69 RegA RNA binding sites yields UAA triplets.

The SELEX method of in vitro selection was used to isolate RNAs that bind the RB69 RegA translational repressor protein immobilized on Ni-NTA agarose. After five rounds of SELEX, the pool of selected RNA displayed striking sequence uniformity: UAAUAAUAAUAAUA was clearly enriched in the 14 nucleotides that underwent selection. Individual, cloned molecules displayed a repeating (UAA) sequence, with only two RNAs having a 3' AUG. Removing the 3' AUG slightly reduced binding in gel shift assays, moving the AUG 5' proximal of the (UAA) slightly improved binding, but (UAA)4 alone still bound the purified protein. Dissociation constants showed that RNA shortened to (UAA)3 and (UAA)2 also retained binding, whereas cytosine clearly prevented binding by RB69 RegA. Scanning of RB69 gene starts and ends with an RB69 RegA SELEX information weight matrix yielded 21 sequences as potential RegA sites. One site, on the mRNA for the pentameric (4:1) phage gp44/62 DNA polymerase clamp loader complex, has the RB69 gene 44 stop codon and 3'-adjacent gene 62 initiation codon in a sequence (GAAAUAAUAUG) that is similar to in vitro selected RNA and was shown to bind RB69 RegA. Sequences between the Shine-Dalgarno and initiation codon, which frequently contain a UAA stop codon of a 5'-adjacent gene, appear to be preferred RB69 RegA binding sites.

Binding Sites↗
PubMed2003

Highly abundant pea LTR retrotransposon Ogre is constitutively transcribed and partially spliced.

We have isolated and characterized a novel giant retroelement, named Ogre, which is over 22 kb long and makes up at least 5% of the pea (Pisum sativum L.) genome. This element can be classified as a Ty3/gypsy-like LTR retrotransposon based on the presence of long terminal repeats (LTRs) and the order of the domains coding for typical retrotransposon proteins. In addition to its extreme length, it has several features which make it unique among the retroelements described so far: (1) the sequences coding for gag and prot proteins are separated from the rt/rh-int domains by several stop codons; (2) the region containing these stop codons is removed from the element transcripts by splicing which results in reconstitution of the complete gag-pol coding sequence; (3) only a part of the transcripts is spliced which probably determines the ratio of translated proteins; (4) the element contains an extra ORF located upstream the gag-pol coding sequences, potentially coding for a protein of 546-562 amino acids with unknown function. The transcriptional activity of the Ogre elements has been detected in all organs tested (leaves, roots, flowers) as well as in wounded leaves and protoplasts. Considering this retroelement's constitutive expression and observed high mutual similarity of the element genomic sequences, it is possible to speculate about its recent amplification in the genomes of pea and other legume plants.

Alternative Splicing↗
PubMed2002

Epsilon-tubulin is an essential component of the centriole.

Centrioles and basal bodies are cylinders composed of nine triplet microtubule blades that play essential roles in the centrosome and in flagellar assembly. Chlamydomonas cells with the bld2-1 mutation fail to assemble doublet and triplet microtubules and have defects in cleavage furrow placement and meiosis. Using positional cloning, we have walked 720 kb and identified a 13.2-kb fragment that contains epsilon-tubulin and rescues the Bld2 defects. The bld2-1 allele has a premature stop codon and intragenic revertants replace the stop codon with glutamine, glutamate, or lysine. Polyclonal antibodies to epsilon-tubulin show peripheral labeling of full-length basal bodies and centrioles. Thus, epsilon-tubulin is encoded by the BLD2 allele and epsilon-tubulin plays a role in basal body/centriole morphogenesis.

Amino Acid Sequence↗
PubMed2000

Genetic heterogeneity and spectrum of mutations of the PRKAR1A gene in patients with the carney complex.

Carney complex (CNC) is an autosomal dominant multiple neoplasia syndrome, which has been linked to loci on 2p16 and 17q22-24. We recently reported that PRKAR1A, which codes for the type 1A regulatory subunit of protein kinase A (PKA), is a tumor suppressor gene on chromosome 17 that is mutated in some CNC families. To evaluate the spectrum of PRKAR1A mutations, we identified its genomic structure and screened for mutations in 54 CNC kindreds (34 families and 20 patients with sporadic disease). Fourteen families were informative for linkage analysis: four of four families that mapped to 17q had PRKAR1A mutations, whereas there were no mutations found in seven families exhibiting at least one recombination with 17q. In six of the latter, CNC mapped to 2p16. PRKAR1A mutations were also found in 12 of 20 non-informative families and 7 of 20 sporadic cases. Altogether, 15 distinct PRKAR1A mutations were identified in 22 of 54 kindreds (40.7%). In 14 mutations, the sequence change was predicted to lead to a premature stop codon; one altered the initiator ATG codon. Mutant mRNAs containing a premature stop codon were unstable, as a result of nonsense-mediated mRNA decay. Accordingly, the predicted truncated PRKAR1A protein products were absent in these cells. We conclude that (i) genetic heterogeneity exists in CNC; and (ii) all of the CNC alleles on 17q are functionally null mutations of PRKAR1A. CNC is the first human disease recognized to be caused by mutations of the PKA holoenzyme, a critical component of cellular signaling.

Amino Acid Sequence↗
PubMed1993

Characterization of a noncontiguous gene deletion of the growth hormone receptor in Laron's syndrome.

Noncontiguous deletion of the GH receptor (GHR) gene has been described as a molecular defect causing Laron's syndrome (LS). The abnormal allele (GHR Del-3,5,6) lacks exons 3, 5, and 6. Exon 4 is retained on variant-sized restriction fragments. We studied DNA from 10 additional LS subjects of Jewish-oriental origin and found this allele in 3 samples. All subjects with this complex deletion allele were from Iran or Iraq and were not clinically distinguishable from other individuals with LS. Multiple restriction enzyme digests and Southern blotting with GHR exon 4-specific probes demonstrated that the allele retains genomic material for at least 9.8 kilobases 5' and 1.8 kilobases 3' to exon 4. Reverse transcription of mRNA, polymerase chain reaction amplification, and cDNA sequencing showed that exon 4 is syntenic and colinear with the other GHR exons. Normal splice sites were used, but the predicted protein was severely truncated due to a frameshift and a premature stop codon in exon 7. The premature stop codon terminated the protein after amino acid 53 and led to loss of the transmembrane and intracellular portions of the receptor. This mutant GHR gene results clinically in severe GH insensitivity.

Alleles↗
PubMed2001

DNA diversity of the wla gene cluster among serotype HS:19 and non-HS:19 Campylobacter jejuni strains.

Campylobacter jejuni infection is an important trigger of Guillain-Barré syndrome (GBS), and serotype HS:19 strains are over-represented among GBS-associated isolates. Structures in C. jejuni lipooligosaccharide (LOS) resemble human gangliosides, suggesting that molecular mimicry could be important in triggering the neural injury. We assessed the genetic diversity among 36 C. jejuni serotype HS:19 and non-HS:19 strains by analysis of PCR-based restriction fragment length polymorphism (RFLP) patterns of 12 LOS biosynthesis-related genes (wla cluster). PCR amplification revealed that the size, order, and direction of each wla gene was identical among all strains tested. However, an additional ORF, located between wlaI and wlaK, was detected in 28 of the 36 isolates examined, and nucleotide sequence analysis revealed that the gene was identical to orfE in C. jejuni strain NCTC 11168. An inverted repeat motif was found downstream of the wlaI stop codon and upstream of the orfE stop codon, an organization allowing pairing of repeated sequences that could lead to deletion of the internal segment. Digestion of the PCR products with restriction endonuclease DdeI or AluI and cluster analysis of RFLP banding patterns showed that all HS:19 strains were closely related and distinct from non-HS:19 strains, consistent with earlier analyses, suggesting that HS:19 strains represent a highly clonal population. RFLP analysis of wla genes also may be useful for epidemiological studies.

Base Sequence↗
PubMed1996

Identification of 19 protein S gene mutations in patients with phenotypic protein S deficiency and thrombosis. Protein S Study Group.

Protein S is a protein C-dependent and independent inhibitor of the coagulation cascade. Deficiency of protein S is an established risk factor for venous thromboembolism. We have used a strategy of specific amplification of the coding regions and intron/exon boundaries of the active protein S gene (PROS1) and direct single-strand solid phase sequencing, to seek mutations in 35 individuals with phenotypic protein S deficiency. Nineteen point mutations (16 novel) in 19 probands (or relatives of probands) with venous thromboembolism are reported here. Fifteen of the 19 mutations were expected to be causal and included 10 missense mutations (Lys9Glu, Glu26Ala, Gly54Glu, Cys145Tyr, Cys200Ser, Ser283Pro, Gly340Asp, Cys408Ser, Ser460Pro, and Cys625Arg). Three of the 15 mutations resulted in premature stop codons (delete T 635 producing a stop codon at position 126, Lys368stop and Tyr595stop) and two were at intron/exon boundaries (+1 G to A in intron d and +3 A to C in intron j). Of the remaining four mutations, three were within intronic sequence and one was a silent mutation within the coding region and did not alter amino acid composition. In two of the 10 missense mutations, reduced plasma protein S activity compared with antigen level suggested the presence of variant (type II) protein S.

Alleles↗