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PubMed1994

Molecular characterisation of the human apo(a)-plasminogen gene family clustered on the telomeric region of chromosome 6 (6q26-27).

The genes coding for apo(a) and plasminogen belong to a family of related genes sharing several structural sequences like leader, kringle, and protease domains. YAC cloning has allowed to understand that all these genes are clustered within 400 Kb of genomic DNA on the telomeric region of chromosome 6 (6q26-27). We have now characterized the two remaining members of the apo(a) and plasminogen gene cluster. One of them was found to contain a leader highly homologous to that of apo(a) and plasminogen, followed by several kringle IV-like units, kringle V and protease domains although no tail sequences could be detected. This apo(a)-like gene was found to be expressed at the RNA level in liver although an in-frame stop codon was detected in one of its kringle units. The other member of the cluster besides the leader shows a plasminogen tail-like domain whose sequences contain a frameshift resulting in a stop codon; another mutation, destroying a consensus splicing site, has been found in a large intron separating the exon coding for the leader from the one encoding the tail-like sequences. The structural organisation of this cluster suggests that new arrangements of these four genes will be a likely finding.

Apolipoproteins A↗
PubMed2004

Mutations within the protein Z-dependent protease inhibitor gene are associated with venous thromboembolic disease: a new form of thrombophilia.

Protein Z-dependent protease inhibitor (ZPI) is a serpin that inhibits the activated coagulation factors X and XI. The precise physiological significance of ZPI in the control of haemostasis is unknown although a deficiency of ZPI may be predicted to alter this balance. The coding region of the ZPI gene was screened for mutations using denaturing high-performance liquid chromatography. 16 mutations/polymorphisms within the coding region of ZPI were identified including two mutations, which generated stop codons at residues R67 and W303. We observed nonsense mutations within the ZPI gene in 4.4% of thrombosis patients (n = 250) compared with 0.8% of controls (n = 250). The difference in distribution of stop codon mutations between thrombosis patients and controls was significant (P = 0.02) with an odds ratio of 5.7 (95% confidence interval, 1.25-26.0). Our results suggest an association between ZPI deficiency and venous thrombosis and we propose that ZPI deficiency is potentially a new form of thrombophilia.

Adult↗
PubMed2000

Sequence variation in the porA gene of a clone of Neisseria meningitidis during epidemic spread.

The ET-15 clone within the electrophoretic type (ET)-37 complex of Neisseria meningitidis was first detected in Canada in 1986 and has since been associated with outbreaks of meningococcal disease in many parts of the world. While the majority of the strains of the ET-37 complex are serosubtype P1.5,2, serosubtype determination of ET-15 strains may often be incomplete, with either only one or none of the two variable regions (VRs) of the serosubtype PorA outer membrane protein reacting with monoclonal antibodies. DNA sequence analysis of the porA gene from ET-15 strains with one or both unidentified serosubtype determinants was undertaken to identify the genetic basis of the lack of reaction with the monoclonal antibodies. Fourteen different porA alleles were identified among 38 ET-15 strains from various geographic origins. The sequences corresponding to subtypes P1.5a,10d, P1.5,2, P1.5,10d, P1.5a,10k, and P1.5a,10a were identified in 18, 11, 2, 2, and 1 isolate, respectively. Of the remaining four strains, which all were nonserosubtypeable, two had a stop codon within the VR1 and the VR2, respectively, while in the other two the porA gene was interrupted by the insertion element, IS1301. Of the strains with P1.5,2 sequence, one had a stop codon between the VR1 and VR2, one had a four-amino-acid deletion outside the VR2, and another showed no expression of PorA on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Our results reveal that numerous genetic events have occurred in the porA gene of the ET-15 clone in the short time of its epidemic spread. The magnitude of microevolutionary mechanisms available in meningococci and the remarkable genetic flexibility of these bacteria need to be considered in relation to PorA vaccine development.

Amino Acid Sequence↗
PubMed2006

Comparative genomics of transcriptional regulation in yeasts and its application to identification of a candidate alpha-isopropylmalate transporter.

Conservation rates in non-protein-coding regions of five yeast genomes of the genus Saccharomyces were analyzed using multiple whole-genome alignments. This analysis confirmed previously shown decrease in conservation rates observed immediately upstream of the translation start point and downstream of the stop-codon. Further, there was a sharp conservation peak in the upstream regions likely related to the core promoter (-35 bp to +35 bp around TSS) and a conservation peak downstream of the stop-codon whose function is not yet clear. Regulation of leucine and methionine biosynthesis controlled by the global regulator Gcn4p and pathway-specific regulators was analyzed in detail. A candidate alpha-isopropylmalate carrier, YOR271cp, was identified based on conservation of Leu3p binding sites, analysis of ChIP-chip data, protein localization and sequence similarity.

Chromosome Mapping↗
PubMed2004

Interaction of two classes of release factors from Euplotes octocarinatus.

Translation termination on the ribosome is an essential process for cell viability. This process is maintained by two classes of peptide release factors (RF1/RF2, RF3 and eRF1, eRF3 in prokaryotes and eukaryotes, respectively). In protozoa ciliates Euplotes octocarinatus, an unicellular eukaryotes, universal stop codon UGA is reassigned for cysteine suggesting the specificity of evolution of translation termination system. We cloned two classes of release factors from Euplotes octocarinatus previously. In this paper, three in-frame stop codons UGA in Eo-eRF3 gene were mutated mediated by PCR site directed mutagenesis method. The interaction between eRF1 and eRF3 from E. octocarinatus was assayed in vivo using Yeast Two-hybrid System, which has an advantage of highly sensitivity. The results showed that the eRF1 x eRF3 complex was formed in living cells to function in the process of translation termination, differing from that in prokaryotes in which RF1/RF2 and RF3 function separately. The evolution of translation termination of life-form was analyzed using phylogenetic tree of amino acids sequences of RFs (32 (e) RF1s and 24 (e) RF3s) obtained from GenBank. Two classes of RFs are useful information in analysis of evolution of life-form and further elucidation of mechanism of translation termination of protein synthesis on ribosome.

Animals↗
PubMed1991

Gene structure of mammalian acetylcholinesterase. Alternative exons dictate tissue-specific expression.

The genes encoding mouse and human acetylcholinesterases have been cloned from genomic and cosmid libraries. Restriction analysis and a comparison of sequence with the cDNAs have defined the exon-intron boundaries. In mammals, three invariant exons encode the signal peptide and the amino-terminal 535 amino acids common to all forms of the enzyme whereas alternative exon usage of the next exon accounts for the structural divergence in the carboxyl termini of the catalytic subunits. mRNA protection studies show that the cDNA encoding the hydrophilic catalytic subunits represents the dominant mRNA species in mammalian brain and muscle whereas divergent mRNA species are evident in cells of hematopoietic origin (bone marrow cells and a erythroleukemia cell line). Analyses of mRNA species in these cells and the genomic sequence have enabled us to define two alternative exons in addition to the one found in the cDNAs; they encode unique carboxyl-terminal sequences. One mRNA consists of a direct extension through the intervening sequence between the common exon and the 3' exon deduced from the cDNA. This sequence encodes a subunit lacking the cysteine critical to oligomer formation. Another mRNA results from a splice that encodes a stretch of hydrophobic amino acids immediately upstream of a stop codon. This exon, when spliced to the upstream invariant exons, should encode glycophospholipid-linked species of the enzyme. Homologous sequence, identity of exon-intron junctions, and identity of position of the stop codon are seen for this region in mouse and human. Polymerase chain reactions carried out across the expected intron region and mRNA protection studies show that this splice occurs in mouse bone marrow and erythroleukemia cells yielding the appropriate cDNA.

Acetylcholinesterase↗
PubMed1993

Sequence analysis of 22 kDa-like alpha-coixin genes and their comparison with homologous zein and kafirin genes reveals highly conserved protein structure and regulatory elements.

Several genomic and cDNA clones encoding the 22 kDa-like alpha-coixin, the alpha-prolamin of Coix seeds, were isolated and sequenced. Three contiguous 22 kDa-like alpha-coixin genes designated alpha-3A, alpha-3B and alpha-3C were found in the 15 kb alpha-3 genomic clone. The alpha-3A and alpha-3C genes presented in-frame stop codons at position +652. The two genes with truncated ORFs are flanking the alpha-3B gene, suggesting that the three alpha-coixin genes may have arisen by tandem duplication and that the stop codon was introduced before the duplication. Comparison of the deduced amino acid sequences of alpha-coixin clones with the published sequences of 22 kDa alpha-zein and 22 kDa-like alpha-kafirin revealed a highly conserved protein structure. The protein consists of an N-terminus, containing the signal peptide, followed by ten highly conserved tandem repeats of 15-20 amino acids flanked by polyglutamines, and a short C-terminus. The difference between the 22 kDa-like alpha-prolamins and the 19 kDa alpha-zein lies in the fact that the 19 kDa protein is exactly one repeat motif shorter than the 22 kDa proteins. Several putative regulatory sequences common to the zein and kafirin genes were identified within both the 5' and 3' flanking regions of alpha-3B. Nucleotide sequences that match the consensus TATA, CATC and the ca. -300 prolamin box are present at conserved positions in alpha-3B relative to zein and kafirin genes. Two putative Opaque-2 boxes are present in alpha-3B that occupies approximately the same positions as those identified for the 22 kDa alpha-zein and alpha-kafirin genes. Southern hybridization, using a fragment of a maize Opaque-2 cDNA clone as a probe, confirmed the presence of Opaque-2 homologous sequences in the Coix and sorghum genomes. The overall results suggest that the structural and regulatory genes involved in the expression of the 22 kDa-like alpha-prolamin genes of Coix, sorghum and maize, originated from a common ancestor, and that variations were introduced in the structural and regulatory sequences after species separation.

Amino Acid Sequence↗
PubMed2005

Mutation in the 5' alternatively spliced region of the XNP/ATR-X gene causes Chudley-Lowry syndrome.

The Chudley-Lowry syndrome (ChLS, MIM 309490) is an X-linked recessive condition characterized by moderate to severe mental retardation, short stature, mild obesity, hypogonadism, and distinctive facial features characterized by depressed nasal bridge, anteverted nares, inverted-V-shaped upper lip, and macrostomia. The original Chudley-Lowry family consists of three affected males in two generations. Linkage analysis had localized the gene to a large interval, Xp21-Xq26 and an obligate carrier was demonstrated to have highly skewed X inactivation. The combination of the clinical phenotype, consistent with that of the patients with ATR-X syndrome, the skewed X-inactivation pattern in a carrier female, as well as the mapping interval including band Xq13.3, prompted us to consider the XNP/ATR-X gene being involved in this syndrome. Using RT-PCR analysis, we screened the entire XNP/ATR-X gene and found a mutation in exon 2 (c.109C > T) giving rise to a stop codon at position 37 (p.R37X). Western blot and immunocytochemical analyses using a specific monoclonal antibody directed against XNP/ATR-X showed the protein to be present in lymphoblastoid cells from one affected male, despite the premature stop codon. To explain these discordant results, we further analyzed the 5' region of the XNP/ATR-X gene and found three alternative transcripts, which differ in the presence or absence of exon 2, and the length of exon 1. Our data suggest that ChLS is allelic to the ATR-X syndrome with its less severe phenotype being due to the presence of some XNP/ATR-X protein.

Alternative Splicing↗
PubMed1990

Molecular basis of argininemia. Identification of two discrete frame-shift deletions in the liver-type arginase gene.

Argininemia results from a deficiency of arginase (EC 3.5.3.1), the last enzyme of the urea cycle in the liver. We examined the molecular basis for argininemia by constructing a genomic library followed by cloning and DNA sequencing. Discrete mutations were found on two alleles from the patient, a product of a nonconsanguineous marriage. There was a four-base deletion at protein-coding region 262-265 or 263-266 in exon 3 that would lead to a reading-frame shift after amino acid residue 87 and make a new stop codon at residue 132. The other was a one-base deletion at 77 or 78 in exon 2 that would lead to a reading-frame shift after residue 26 and make a stop codon at residue 31. For confirmation, genomic DNAs from the patient and from her parents were amplified by the polymerase chain reaction method. The patient was shown to be a compound heterozygote, inheriting an allele with the four-base deletion from the father and the other allele with the one-base deletion from the mother. These data seem to be the first evidence of a case of argininemia caused by two different deletion mutations.

Amino Acid Metabolism, Inborn Errors↗
PubMed1993

Residues added to the carboxyl terminus of the Yersinia pseudotuberculosis invasin protein interfere with recognition by integrin receptors.

Escherichia coli strains encoding the Yersinia pseudotuberculosis invasin protein are efficiently internalized by mammalian cells. Bacterial uptake into cultured cell lines became defective, however, if invasin was altered by fusion of its carboxyl terminus to E. coli alkaline phosphatase or by the addition of two hydrophobic amino acids to its carboxyl-terminal end. Probing with anti-invasin monoclonal antibodies revealed that the amino-terminal end of invasin was properly localized on the bacterial cell surface in strains encoding invasin with 2 additional amino acids, whereas the carboxyl terminus was not accessible to the monoclonal antibody. Therefore, the 2 additional amino acids interfered with the folding or orientation of the carboxyl terminus in the outer membrane. Alkylation experiments in the absence of reduction indicated that this defect was not caused by a gross inability to form a critical disulfide bond. Revertants were selected from a strain encoding this mutant protein by enriching for organisms able to enter cultured mammalian cells. The vast majority of revertants that were isolated following this enrichment contained a stop codon at the usual position found in the wild type inv gene. The most efficient of the remaining revertants resulted in the introduction of a glycine residue at the site of the wild type stop codon, presumably restoring proper conformation of the carboxyl-terminal region.

ATP-Binding Cassette Transporters↗
PubMed2001

A sequence required for -1 ribosomal frameshifting located four kilobases downstream of the frameshift site.

Programmed ribosomal frameshifting allows one mRNA to encode regulate expression of, multiple open reading frames (ORFs). The polymerase encoded by ORF 2 of Barley yellow dwarf virus (BYDV) is expressed via minus one (-1) frameshifting from the overlapping ORF 1. Previously, this appeared to be mediated by a 116 nt RNA sequence that contains canonical -1 frameshift signals including a shifty heptanucleotide followed by a highly structured region. However, unlike known -1 frameshift signals, the reporter system required the zero frame stop codon and did not require a consensus shifty site for expression of the -1 ORF. In contrast, full-length viral RNA required a functional shifty site for frameshifting in wheat germ extract, while the stop codon was not required. Increasing translation initiation efficiency by addition of a 5' cap on the naturally uncapped viral RNA, decreased the frameshift rate. Unlike any other known RNA, a region four kilobases downstream of the frameshift site was required for frameshifting. This included an essential 55 base tract followed by a 179 base tract that contributed to full frameshifting. The effects of most mutations on frameshifting correlated with the ability of viral RNA to replicate in oat protoplasts, indicating that the wheat germ extract accurately reflected control of BYDV RNA translation in the infected cell. However, the overall frameshift rate appeared to be higher in infected cells, based on immunodetection of viral proteins. These findings show that use of short recoding sequences out of context in reporter constructs may overlook distant signals. Most importantly, the remarkably long-distance interaction reported here suggests the presence of a novel structure that can facilitate ribosomal frameshifting.

3' Untranslated Regions↗
PubMed1989

Translation of phage f1 gene VII occurs from an inherently defective initiation site made functional by coupling.

Expression of the filamentous phage f1 gene VII is shown to be translationally coupled to that of the upstream gene V. Fusions of the gene VII initiation site to the lacZ coding region were used to determine that initiation at the VII site is completely dependent on the process of translation having proceeded up to a stop codon immediately upstream from the VII site. Coupled expression from the VII site was found to be inefficient, proportional to the level of upstream translation, and very sensitive to the distance from the functional upstream stop codon. Independent expression from the VII site was not observed, even in a deletion series designed to remove potentially masking RNA structure. On the basis of the VII site's dissimilarity to ribosome binding site sequences and its properties overall, we suggest that it inherently lacks the features required for independent recognition by ribosomes, and acquires the ability to initiate synthesis of gene VII protein by virtue of the coupling process.

Base Sequence↗
PubMed1994

Molecular basis of nanomelia, a heritable chondrodystrophy of chicken.

Nanomelia is a recessively inherited connective tissue disorder of chicken affecting cartilage development. Other investigators have demonstrated that it involves low aggrecan production and diminished aggrecan mRNA levels. Based on genetic linkage studies showing a high likelihood that the mutation responsible for the nanomelic phenotype lay within the aggrecan gene, a series of experiments was performed to define the molecular basis of the trait. Aggrecan mRNA was present in the nucleus of the nanomelic chondrocyte but greatly reduced in the cytoplasmic compartment, a finding suggestive of a premature stop codon within the aggrecan transcript. Since no defect in mRNA splicing could be demonstrated by ribonucleasease protection studies, direct DNA sequencing was initiated by polymerase chain reaction of the mRNA and of genomic DNA. A stop codon was demonstrated at codon 1513, which is located in the eighth repeat of the chondroitin sulfate 2 domain of the large tenth exon. The mutation creates a unique BasBI restriction site which readily distinguishes the mutant and wild-type alleles.

Aggrecans↗
PubMed1997

smg mutants affect the expression of alternatively spliced SR protein mRNAs in Caenorhabditis elegans.

The expression of alternatively spliced mRNAs from genes is an ubiquitous phenomenon in metazoa. A screen for trans-acting factors that alter the expression of alternatively spliced mRNAs reveals that the smg genes of Caenorhabditis elegans participate in this process. smg genes have been proposed to function in degradation of nonsense mutant mRNAs. Here we show that smg genes affect normal gene expression by modulating the levels of alternatively spliced SRp20 and SRp30b mRNAs. These SR genes contain alternatively spliced exons that introduce upstream stop codons. The effect of smg genes on SR transcripts is specific, because the gene encoding the catalytic subunit of the cAMP-dependent protein kinase, which also contains an alternatively spliced exon that introduces upstream stop codon, is not effected in a smg background. These results suggest that the levels of alternatively spliced mRNAs may, in part, be regulated by alternative mRNA stability.

Alternative Splicing↗
PubMed2004

HLA DQA1 genes generate multiple transcripts by alternative splicing and polyadenylation of the 3' untranslated region.

Regulation of the human leucocyte antigen (HLA) class II genes expression is an important field in immunology, because these molecules play a crucial role in the function of the immune system. HLA DQ genes expression is a complex phenomenon regulated at both transcriptional and post-transcriptional levels. In this study, we have investigated the post-transcriptional mechanisms accounting for alleles-dependent length polymorphism of DQA1 mRNA. We have first sequenced the genomic DNA encoding the 3' untranslated region (UTR) of DQA1 *0101, *0102, *0103, *0201, *0301, *0401, and *0501 alleles. We have identified two competing splicing sites: a unique splicing donor site AG/GTA located 20 nucleotides downstream from the stop codon associated to two spliced acceptor sequences, approximately 165 and approximately 370 nucleotides downstream. In addition, three polyadenylation signals have been identified, respectively, at approximately 475, approximately 795, and approximately 855 nucleotides downstream from the stop codon. Subsequently, we have analyzed mRNAs derived from DQA1 alleles in homozygous B lymphoblastoid cell lines by reverse transcriptase-polymerase chain reaction. We show that allele-dependent length polymorphism of DQA1 mRNA-3' UTR results from a combination of differential splicing and alternative polyadenylations. Four mRNA isoforms (two spliced variant cleaved at two distinct polyadenylation sites) were detected in DQA1 *0101, *0102, and *0103 homozygous cell lines, and six mRNA species (three spliced variant cleaved at two polyadenylation-sequence signal) were generated by the other four alleles. Possible advantages for cells to generate multiple transcripts previously undetected are discussed.

Alleles↗
PubMed2006

Posttranscriptional control of the Salmonella enterica flagellar hook protein FlgE.

Previous work suggested that the FlgE (flagellar hook subunit) protein in Salmonella enterica serovar Typhimurium was posttranscriptionally regulated in response to the stage of flagellar assembly. Specifically, the FlgE protein could be detected in flagellar mutants defective at the stages of assembly before or after rod assembly but not in rod assembly mutants, yet flgE mRNA levels were unaffected. To elucidate posttranscriptional mechanisms involved in the coupling of flgE gene expression to hook assembly, the RNA sequences at the 5' and 3' ends of the flgE-containing mRNA processed from the large flgBCDEFGHIJKL operon were determined by rapid amplification of cDNA ends, and secretion of the FlgE protein in different flagellar assembly mutant strains was analyzed. The sequences 5' and 3' of the flgE gene where RNA processing occurred was within 15 bases upstream of the flgD stop codon and at bases 145 to 147 downstream of the flgF start codon, respectively. The ribosome binding site of the flgD gene was found to be inhibitory to flgE translation in strains deleted for the upstream flgD gene, unless the region 15 bases upstream of the flgD stop codon was present. Secretion of FlgE into the periplasm was monitored using beta-lactamase (Bla) fusions as a periplasm-specific reporter, which conferred resistance to ampicillin when FlgE-Bla was secreted into the periplasm. Using this assay, we found that the effect of rod assembly mutants on FlgE levels was due to FlgE turnover in the periplasm and that the FliE rod component protein was required for efficient FlgE-Bla secretion.

Bacterial Proteins↗
PubMed2005

Inhibition of human type i gonadotropin-releasing hormone receptor (GnRHR) function by expression of a human type II GnRHR gene fragment.

Humans possess only one functional GnRH receptor, the type I GnRH receptor (GnRHR-I). A type II GnRH receptor (GnRHR-II) gene homolog exists, but it is disrupted by a frame shift and premature stop codon, suggesting that a conventional receptor is not translated from this gene. However, the gene remains transcriptionally active and displays alternative splicing. We identified a putative translational start site 117 bp downstream of the premature stop codon. Use of this start codon encodes a protein (designated as the GnRHR-II-reliquum) corresponding to the domains from the cytoplasmic end of transmembrane domain-5 to the carboxyl terminus of the putative full-length receptor. Immunocytochemistry revealed that GnRHR-II-reliquum expression appeared to be localized throughout the cytoplasm. Transient cotransfection of GnRHR-I and GnRHR-II-reliquum constructs into COS-7 cells resulted in reduced expression of the GnRHR-I at the cell surface and impaired signaling via the GnRHR-I as revealed by reduction of GnRH-induced inositol phosphate accumulation. This inhibitory effect was specific and dependent on the degree of GnRHR-II-reliquum coexpressed. Immunoblot analysis revealed that the total cell GnRHR-I complement, i.e. both cell-surface and nascent intracellular receptors, was markedly reduced by coexpression of the GnRHR-II-reliquum. Treatments with cell-permeable agents that blocked either de novo protein synthesis (cycloheximide) or proteinase-mediated degradation (leupeptin and phenylmethylsulfonyl fluoride) failed to alter the inhibitory effect of GnRHR-II-reliquum coexpression, suggesting that the inhibitory effect is exerted at the nucleus/endoplasmic reticulum or Golgi apparatus level, possibly by perturbing normal processing of GnRHR-I from these sites. We suggest that the GnRHR-II-reliquum plays a modulatory role in GnRHR-I expression.

Amino Acid Sequence↗
PubMed1995

Rapid screening of open reading frames by protein synthesis with an in vitro transcription and translation assay.

The analysis of open reading frames (ORFs) to predict full-length or truncated proteins in genes is conventionally achieved by DNA sequencing. This method becomes labor-intensive when a large number of specimens or when large genes are to be examined. To circumvent this problem, we used an in vitro transcription and translation (TT) assay to identify full-length or truncated proteins in PCR-amplified genes. A total of 47 nef genes from the simian immunodeficiency virus (SIV) were cloned from 13 SIV-infected monkeys and were screened for ORFs by using the TT assay. Of these 47 genes, 20 had an intact ORF and 27 had premature stop codons at variable positions in the nef gene. All 20 nef genes with intact ORFs produced full-length proteins, while truncated proteins of different sizes were synthesized from all 27 nef genes with premature stop codons. In addition, we validated a simplified TT protocol that allows the direct screening of ORFs from transformed bacterial colonies, thus eliminating the need for plasmid preparations. By being rapid, simple and cost-effective, this technique should be widely applicable to examine the integrity of the ORF of any gene.

Animals↗