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Coding Regions (coding + regions)
Selected AbstractsStructural and functional differences between the promoters of independently expressed killer cell Ig-like receptorsEUROPEAN JOURNAL OF IMMUNOLOGY, Issue 7 2005Bergen, Jeroen van Abstract Killer Ig-like receptors (KIR) are important for the recognition and elimination of diseased cells by human NK cells. Myeloid leukemia patients given a hematopoietic stem cell transplantation, for example, benefit from KIR-mediated NK alloreactivity directed against the leukemia cells. To establish an effective NK cell repertoire, most KIR genes are expressed stochastically, independently of the others. However, the sequences upstream of the coding regions of these KIR genes are highly homologous to the recently identified KIR3DL1 promoter (91.1,99.6% sequence identity), suggesting that they are regulated by similar if not identical mechanisms of transcriptional activation. We investigated the effects of small sequence differences between the KIR3DL1 promoter and other KIR promoters on transcription factor binding and promoter activity. Surprisingly, electrophoretic mobility shift assays and promoter-reporter assays revealed significant structural and functional differences in the cis-acting elements of these highly homologous KIR promoters, suggesting a key role for transcription factors in independent control of expression of specific KIR loci. Thus, the KIR repertoire may be shaped by a combination of both gene-specific and stochastic mechanisms. [source] Human ameloblastin gene: genomic organization and mutation analysis in amelogenesis imperfecta patientsEUROPEAN JOURNAL OF ORAL SCIENCES, Issue 1 2001Carina Kärrman Mårdh A gene encoding the enamel protein ameloblastin (AMBN) was recently localized to a region on chromosome 4q21 containing a gene for the inherited enamel defect local hypoplastic amelogenesis imperfecta (AIH2). Ameloblastin protein is located at the Tomes processes of secretory ameloblasts and in the sheath space between rod-interrod enamel, and the AMBN gene therefore represents a viable candidate gene for local hypoplastic amelogenesis imperfecta (AI). In this study, the genomic organization of human AMBN was characterized. The gene was shown to consist of 13 exons and 12 introns. An alternatively spliced 45 bp sequence was shown not to represent a separate exon and is most likely spliced by the use of a cryptic splice site. The finding that there were no recombinations between an intragenic microsatellite and AIH2 encouraged us to evaluate this gene's potential role as a candidate gene for local hypoplastic AI. Mutation screening was performed on all 13 exons in 20 families and 8 sporadic cases with 6 different forms of AI. DNA variants were found but none that was associated exclusively with local hypoplastic AI or any of the other variants of AI in the identified Swedish families. This study excludes the coding regions and the splice sites of AMBN from a causative role in the pathogenesis of AIH2. [source] Genetic organization of A chain and B chain of ,-bungarotoxin from Taiwan banded krait (Bungarus multicinctus)FEBS JOURNAL, Issue 15 2000A chain genes, B chain genes do not share a common origin ,-Bungarotoxin, the main presynaptic neurotoxin purified from the venom of Bungarus multicinctus, consists of two dissimilar polypeptide chains, the A chain and the B chain, cross-linked by an interchain disulfide bond. In this study, A and B chain genes isolated from the liver of B. multicinctus encoded the A and B chain precursors, respectively. Analyses of the coding regions of the A and B chain genes revealed that both consist of three exons and two introns. The sequences of all exon/intron junctions agree with the GT/AG rule. However, sequence alignment and phylogenetic analysis did not support that the evolution of A and B chain genes are closely related. Comparative analysis of A chain genes with Viperinae and Crotalinae phospholipase A2 genes indicated that genetic divergence of the A chain and phospholipase A2s was in accordance with their family. Moreover, evolutionary divergence of the intron and exon regions of the A chain, as observed for phospholipase A2 genes, was not consistent. Noticeably, the transcription of A and B chain genes may be regulated under different transcription factors as revealed by analyses of their promoter sequences. In terms of the finding that A and B chains are encoded separately by different genes, this strongly supports the view that the intact ,-bungarotoxin molecules should be derived from the pairing of A and B chains after their mRNAs are translated. [source] Insight into molecular changes of the FIX protein in a series of Italian patients with haemophilia BHAEMOPHILIA, Issue 3 2006M. P. BICOCCHI Summary., Deficiency or dysfunction of factor IX FIX leads to haemophilia B (HB), an X-linked, recessive, bleeding disorder. On a molecular basis, HB is due to a heterogeneous spectrum of mutations spread throughout the F9 gene. In several instances, a cause-effect relation has been elucidated, in others predicted possibilities have been offered by crystallography inspection and by software-constructed models of the protein. The aim of this study was to contribute to the understanding of HB molecular pathology. The F9 missense mutations we identified in 21 unrelated Italian HB patients by direct sequencing of the whole F9 coding regions were inspected for the causative effect they provoked on the ensuing transcript, and on the protein structure. Each alteration was studied in order to: (i) characterize the defect on the basis of the nature of the mutation; (ii) identify the predicted defect that is induced in the gene and (iii) speculate about the potential, detrimental effects which upset the protein functionality through an idealized FIX model. The resulting data may further contribute to the comprehension of the mechanisms underlying the disease. [source] In Vitro and In Vivo Complementation of the Helicobacter pylori Arginase Mutant Using an Intergenic Chromosomal SiteHELICOBACTER, Issue 5 2006Melanie L. Langford Abstract Background:, Gene complementation strategies are important in validating the roles of genes in specific phenotypes. Complementation systems in Helicobacter pylori include shuttle vectors, which transform H. pylori at relatively low frequencies, and chromosomally based approaches. Chromosomal complementation strategies are susceptible to polar effects and disruption of other H. pylori genes, leading to unwanted pleiotropic effects. Materials and methods:, A new complementation strategy was developed for H. pylori by utilizing a suicide plasmid vector that contains fragments of an H. pylori intergenic region (hp0203,hp0204), a chloramphenicol acetyltransferase cassette (cat), and a multiple-cloning site. Genes of interest could be cloned into the intergenic plasmid and the genes integrated into H. pylori by homologous recombination into the intergenic chromosomal region without disrupting any annotated H. pylori gene. The complementation system was validated using the gene encoding arginase (rocF). Results:, A rocF mutant unable to hydrolyze or consume l -arginine regained these functions by complementation with the wild-type rocF gene. Complemented strains also had restored arginase protein as determined by Western blot analysis. The complementation system could be successfully applied to multiple H. pylori strains. The intergenic region varied in length and sequence across 17 H. pylori strains, but the flanking-3, ends of the hp0203 and hp0204 coding regions were highly conserved. Inserting a cat cassette and wild-type rocF into the intergenic region did not alter the ability of strain SS1 to colonize mice. Conclusions:, This complementation strategy should greatly facilitate genetic experiments in H. pylori. [source] BSEP and MDR3 haplotype structure in healthy Caucasians, primary biliary cirrhosis and primary sclerosing cholangitisHEPATOLOGY, Issue 3 2004Christiane Pauli-Magnus Primary biliary cirrhosis (PBC) and primary sclerosing cholangitis (PSC) are characterized by a cholestatic pattern of liver damage, also observed in hereditary or acquired dysfunction of the canalicular membrane transporters bile salt export pump (BSEP, ABCB11) and multidrug resistance protein type 3 (MDR3, ABCB4). Controversy exists whether a genetically determined dysfunction of BSEP and MDR3 plays a pathogenic role in PBC and PSC. Therefore, 149 healthy Caucasian control individuals (control group) were compared to 76 PBC and 46 PSC patients with respect to genetic variations in BSEP and MDR3. Sequencing spanned ,10,000 bp including promoter and coding regions as well as 50,350 bp of flanking intronic regions. In all, 46 and 45 variants were identified in BSEP and MDR3, respectively. No differences between the groups were detected either in the total number of variants (BSEP: control group: 37, PBC: 37, PSC: 31; and MDR3: control group: 35; PBC: 32, PSC: 30), or in the allele frequency of the common variable sites. Furthermore, there were no significant differences in haplotype distribution and linkage disequilibrium. In conclusion, this study provides an analysis of BSEP and MDR3 variant segregation and haplotype structure in a Caucasian population. Although an impact of rare variants on BSEP and MDR3 function cannot be ruled out, our data do not support a strong role of BSEP and MDR3 genetic variations in the pathogenesis of PBC and PSC. (HEPATOLOGY 2004;39:779,791.) [source] Mucopolysaccharidosis type IIID: 12 new patients and 15 novel mutations,HUMAN MUTATION, Issue 5 2010Marlies J. Valstar Abstract Mucopolysaccharidosis III D (Sanfilippo disease type D, MPS IIID) is a rare autosomal recessive lysosomal storage disorder previously described in only 20 patients. MPS IIID is caused by a deficiency of N-acetylglucosamine-6-sulphate sulphatase (GNS), one of the enzymes required for the degradation of heparan sulphate. So far only seven mutations in the GNS gene have been reported. The clinical phenotype of 12 new MPS IIID patients from 10 families was studied. Mutation analysis of GNS was performed in 16 patients (14 index cases). Clinical signs and symptoms of the MPS IIID patients appeared to be similar to previously described patients with MPS III. Early development was normal with onset of behavioral problems around the age of 4 years, followed by developmental stagnation, deterioration of verbal communication and subsequent deterioration of motor functions. Sequence analysis of the coding regions of the gene encoding GNS (GNS) resulted in the identification of 15 novel mutations: 3 missense mutations, 1 nonsense mutation, 4 splice site mutations, 3 frame shift mutations, 3 large deletions and 1 in-frame small deletion. They include the first missense mutations and a relatively high proportion of large rearrangements, which warrants the inclusion of quantitative techniques in routine mutation screening of the GNS gene. © 2010 Wiley-Liss, Inc. [source] Three novel thiopurine S-methyltransferase allelic variants (TPMT*20, *21, *22) , association with decreased enzyme function,,HUMAN MUTATION, Issue 9 2006Elke Schaeffeler Abstract The genetic polymorphism of the thiopurine S-methyltransferase, TPMT, comprises at least 21 alleles causing three distinct drug metabolism phenotypes termed normal/high, intermediate, and deficient methylators. In consequence, adverse drug reactions may occur if standard doses of thiopurines are applied routinely. Genetic prediction of the methylator phenotype as a basis for dose selection requires the extensive knowledge of single nucleotide polymorphisms occurring naturally in the population. Here we describe three novel missense variants in the TPMT gene which were associated with an intermediate red blood cell TPMT activity in three Caucasians. The following alleles were designated: TPMT*20 (c.712A>G), *21 (c.205C>G), and *22 (c.488G>C). No further genetic variations in remaining coding regions as well as the 5,flanking region of TPMT were identified. These sequence variants are present in highly conserved nucleotide positions of the TPMT gene throughout various mammalian species and in zebra fish, and are predicted to be intolerant when the functional consequences of variations were analyzed using SIFT (Sorting Intolerant From Tolerant) algorithm. In Caucasians the occurrence of these genetic variants appears to be extremely rare since none of these alleles were identified in a randomly selected control population of 1048 individuals. © 2006 Wiley-Liss, Inc. [source] BRCA1 and BRCA2 germline mutations in Korean patients with sporadic breast cancer,,HUMAN MUTATION, Issue 4 2004Jae Hong Seo Abstract In order to evaluate the role of BRCA1 and BRCA2 germline mutations in Korean patients with sporadic breast cancer, 97 patients with sporadic breast cancer were analyzed for mutations in the BRCA1 and BRCA2 coding regions, by using a combination of fluorescent-conformation sensitive gel electrophoresis (F-CSGE) and direct sequencing. Fifty-five distinct sequence variants were detected, which included three pathogenic truncating mutations, 15 missense mutations, 16 polymorphisms, and 21 intronic variants. Twenty-six of these variants have never been previously reported and may be of Korean-specific origin. Two pathogenic BRCA1 mutations (c.922_924delinsT, c.5445G>A) and one pathogenic BRCA2 mutation (c.2259delT) were observed, and two of these (BRCA1 c.5445G>A and BRCA2 c.2259delT) are novel. The total prevalence of germline pathogenic mutations in BRCA1 and/or BRCA2 in Korean sporadic breast cancer is estimated to be about 3.1%. Considering that the majority of breast cancer cases are sporadic, the present study will be helpful in the evaluation of the need for the genetic screening of germline BRCA mutations in sporadic breast cancer patients. Further study using a larger sample size is required to determine the merits of genetic diagnosis and counseling in breast cancer patients. © 2004 Wiley-Liss, Inc. [source] Isolation and molecular characterization of Musca domestica delta-9 desaturase sequencesINSECT MOLECULAR BIOLOGY, Issue 6 2002A. L. Eigenheer Abstract We have isolated fatty acyl-CoA desaturase cDNA (Mdomd9) and genomic sequences from the housefly, Musca domestica. Two ,1.66 kb cDNAs were recovered. They had identical coding regions and 3, untranslated regions (UTRs), but differed in their 5, UTRs. The open reading frame encodes a 380 amino acid (aa) protein with 82% identity to Drosophila melanogaster desat1, and significant (> 50%) identity with other insect delta-9 desaturases. Functional analyses in a yeast expression system confirmed the cDNA encodes a ,9 desaturase. Northern analysis indicated two transcripts of 1.7 and 2.9 kb that hybridized specifically to the open reading frame. PCR amplification of genomic templates revealed three intron sites that are conserved among other insect species. Southern analysis of genomic DNA indicated at least two desaturase gene copies per haploid genome. There is a high degree of polymorphism, most of which appears to be due to variable intron sequences; curiously, individual flies had varying morphs of intron II and intron III. Together, the data suggest that there are more ,9 desaturase alleles within the population studied than there are loci within the genome, and support other studies suggesting that insect fatty acyl-CoA desaturases are a dynamically evolving gene family. [source] Characterization of genomic DNA encoding cecropins from an Aedes albopictus mosquito cell lineINSECT MOLECULAR BIOLOGY, Issue 1 2002D. Sun Abstract We used cDNA probes from Aedes albopictus mosquito cecropins AalCecA, B, and C to obtain genomic DNA copies and flanking DNA. Two gene copies (AalCecA1 and A2, AalCecB1 and B2, AalCecC1 and C2) encoding each of the three mature cecropin peptides were recovered. All these genes had a similar organization, into two exons interrupted by a single short intron. AalCecA1 and AalCecA2 encode mature protein products that differ by one amino acid residue, while AalCecB1 and AalCecB2, AalCecC1 and AalCecC2 encode identical mature cecropin peptides, respectively. The AalCecB and C gene pairs each share a common intergenic region of approximately 1 kb, with the two coding regions transcribed in opposite directions. With the exception of small insertions/deletions, the intergenic spacer region was highly conserved between the B1/C1 and B2/C2 clones. In transfected cells, 0.8 kb of upstream sequence was sufficient for inducible expression of AalCecA1. Within this region, a 28 bp sequence at positions ,192 to ,165 upstream of the transcription initiation site was found to contain a potential regulatory element. In electrophoretic mobility shift assays, synthetic double-stranded DNA containing this 28 bp sequence retarded protein in cytoplasmic and nuclear extracts from C7-10 cells. [source] Molecular mechanisms of heavy metal tolerance and evolution in invertebratesINSECT SCIENCE, Issue 1 2009Thierry K. S. Janssens Abstract Following the genomics revolution, our knowledge of the molecular mechanisms underlying defenses against stress has been greatly expanded. Under strong selective pressure many animals may evolve an enhanced stress tolerance. This can be achieved by altering the structure of proteins (through mutations in the coding regions of genes) or by altering the amount of protein (through changes in transcriptional regulation). The latter type of evolution can be achieved by substitutions in the promoter of the gene of interest (cis -regulatory change) or by altering the structure or amount of transcriptional regulator proteins (trans -regulatory change). The metallothionein system is one of the best studied stress response systems in the context of heavy metals. Metallothionein expression is assumed to be regulated by metal transcription factor 1 (MTF-1); however, up to now the involvement of MTF-1 has only been proven for some vertebrates and Drosophila. Data on invertebrates such as nematodes and earthworms suggest that other mechanisms of metallothionein induction may be present. A detailed study of Cd tolerance was done for a species of soil-living springtail, Orchesella cincta. The metallothionein gene of this species is overexpressed in metal-exposed field populations. Analysis of the metallothionein promoter has demonstrated extensive polymorphisms that have a functional significance, as shown in bioreporter assays. In a study comparing 20 different populations, the frequency of a high-expresser promoter allele was positively correlated with the concentration of metals in soil, especially Cd. The springtail study shows that cis -regulatory change of genes involved in the cellular stress response may contribute to evolution of metal tolerance. [source] A high proportion of founder BRCA1 mutations in Polish breast cancer familiesINTERNATIONAL JOURNAL OF CANCER, Issue 5 2004Bohdan Górski Abstract Three mutations in BRCA1 (5382insC, C61G and 4153delA) are common in Poland and account for the majority of mutations identified to date in Polish breast and breast,ovarian cancer families. It is not known, however, to what extent these 3 founder mutations account for all of the BRCA mutations distributed throughout the country. This question has important implications for health policy and the design of epidemiologic studies. To establish the relative contributions of founder and nonfounder BRCA mutations, we established the entire spectrum of BRCA1 and BRCA2 mutations in a large set of breast,ovarian cancer families with origins in all regions of Poland. We sequenced the entire coding regions of the BRCA1 and BRCA2 genes in 100 Polish families with 3 or more cases of breast cancer and in 100 families with cases of both breast and ovarian cancer. A mutation in BRCA1 or BRCA2 was detected in 66% of breast cancer families and in 63% of breast,ovarian cancer families. Of 129 mutations, 122 (94.6%) were in BRCA1 and 7 (5.4%) were in BRCA2. Of the 122 families with BRCA1 mutations, 119 (97.5%) had a recurrent mutation (i.e., one that was seen in at least 2 families). In particular, 111 families (91.0%) carried one of the 3 common founder mutations. The mutation spectrum was not different between families with and without ovarian cancer. These findings suggest that a rapid and inexpensive assay directed at identifying the 3 common founder mutations will have a sensitivity of 86% compared to a much more costly and labor-intensive full-sequence analysis of both genes. This rapid test will facilitate large-scale national epidemiologic and clinical studies of hereditary breast cancer, potentially including studies of chemoprevention. © 2004 Wiley-Liss, Inc. [source] The interleukin-25 gene located in the inflammatory bowel disease (IBD) 4 region: no association with inflammatory bowel diseaseINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 5 2003C. Büning Summary Genetic predisposition has been suggested to play an important role in the pathogenesis of inflammatory bowel diseases (IBDs). Linkage studies have identified a Crohn's disease susceptibility locus on chromosome 14 (14q11,12; IBD4). Interleukin-25 (IL-25) is a newly identified proinflammatory cytokine that has been shown to promote Th2 responses by inducing cytokines such as IL-4, IL-5 and IL-13. The IL-25 gene is located within this susceptibility region at 14q11.2. As IBDs are characterized by an imbalance of the Th1/Th2 cytokine response, we hypothesized that genetic alterations within the IL-25 gene might contribute to IBD. First, direct sequencing of the coding regions of the IL-25 gene in 40 patients with Crohn's disease or ulcerative colitis revealed only a newly reported polymorphism (c424C/A) in exon 2. Next, the frequency of this polymorphism was further investigated in 151 patients with Crohn's disease, 111 patients with ulcerative colitis, and 119 healthy controls to determine its clinical relevance. The genotypes of the c424C/A polymorphism did not reveal any significant differences between patients with Crohn's disease or ulcerative colitis and controls. Genoytype,phenotype relations in patients with Crohn's disease showed a comparable distribution of the c424C/A polymorphism in all subgroups of the Vienna classification. In summary, our data indicate that genetic alterations in the coding regions of the IL-25 gene are unlikely to play a role in IBDs, but the c424C/A polymorphism in the IL-25 gene should be investigated for a potential association with other chronic inflammatory and inherited disorders such as autoimmune diseases. [source] Novel variants within the coding regions of the Slc11A1 gene identified in Bos taurus and Bos indicus breedsJOURNAL OF ANIMAL BREEDING AND GENETICS, Issue 1 2008R. Martínez Summary Although in the cow the genetic resistance to brucellosis has been previously attributed to the Slc11A1 gene encoding Nramp1 protein, none of the mutations described to date seems to be the cause. To be able to associate another polymorphism of the gene to brucellosis resistance, we characterized the gene and identified in different breeds of Bos taurus and Bos indicus, six new variants among a total of 11 single nucleotide mutations, of which five occurred in the coding sequence (three are missense mutations), one in the promoter region and five in introns. The allelic and genotypic frequencies calculated revealed differences (p < 0.05) among the breeds studied. [source] Type V Osteogenesis Imperfecta: A New Form of Brittle Bone Disease,JOURNAL OF BONE AND MINERAL RESEARCH, Issue 9 2000Francis H. Glorieux Abstract Osteogenesis imperfecta (OI) is commonly subdivided into four clinical types. Among these, OI type IV clearly represents a heterogeneous group of disorders. Here we describe 7 OI patients (3 girls), who would typically be classified as having OI type IV but who can be distinguished from other type IV patients. We propose to call this disease entity OI type V. These children had a history of moderate to severe increased fragility of long bones and vertebral bodies. Four patients had experienced at least one episode of hyperplastic callus formation. The family history was positive for OI in 3 patients, with an autosomal dominant pattern of inheritance. All type V patients had limitations in the range of pronation/supination in one or both forearms, associated with a radiologically apparent calcification of the interosseous membrane. Three patients had anterior dislocation of the radial head. A radiodense metaphyseal band immediately adjacent to the growth plate was a constant feature in growing patients. Lumbar spine bone mineral density was low and similar to age-matched patients with OI type IV. None of the type V patients presented blue sclerae or dentinogenesis imperfecta, but ligamentous laxity was similar to that in patients with OI type IV. Levels of biochemical markers of bone metabolism generally were within the reference range, but serum alkaline phosphatase and urinary collagen type I N-telopeptide excretion increased markedly during periods of active hyperplastic callus formation. Qualitative histology of iliac biopsy specimens showed that lamellae were arranged in an irregular fashion or had a meshlike appearance. Quantitative histomorphometry revealed decreased amounts of cortical and cancellous bone, like in OI type IV. However, in contrast to OI type IV, parameters that reflect remodeling activation on cancellous bone were mostly normal in OI type V, while parameters reflecting bone formation processes in individual remodeling sites were clearly decreased. Mutation screening of the coding regions and exon/intron boundaries of both collagen type I genes did not reveal any mutations affecting glycine codons or splice sites. In conclusion, OI type V is a new form of autosomal dominant OI, which does not appear to be associated with collagen type I mutations. The genetic defect underlying this disease remains to be elucidated. [source] Programming the genome in embryonic and somatic stem cellsJOURNAL OF CELLULAR AND MOLECULAR MEDICINE, Issue 4 2007Philippe Collas ,,Introduction ,,Epigenetic makeup of embryonic stem cells: keeping chromatin loose -,DNA methylation and gene expression -,CpG methylation profiles in mouse ESCs -,CpG methylation patterns in human ESCs -,Both active and inactive histone modification marks on developmentally regulated genes in ESCs suggest transcriptional activation potential -,A regulatory role of histone H1 in gene expression in embryonic stem cells? -,Polycomb group proteins impose a transcriptional brake on lineage-priming genes ,,The epigenetic makeup of mesenchymal stem cells reflects restricted differentiation potential -,CpG methylation patterns on lineage-specific promoters in adipose stem cells -,CpG content affects the relationship between promoter DNA methylation and transcriptional activity -,Bivalent histone modifications on potentially active genes? ,,Linking DNA methylation to histone modifications, chromatin packaging and (re)organization of the nuclear compartment ,,Perspectives: towards remodelling the stem cell epigenome? Abstract In opposition to terminally differentiated cells, stem cells can self-renew and give rise to multiple cell types. Embryonic stem cells retain the ability of the inner cell mass of blastocysts to differentiate into all cell types of the body and have acquired in culture unlimited self-renewal capacity. Somatic stem cells are found in many adult tissues, have an extensive but finite lifespan and can differentiate into a more restricted array of cell types. A growing body of evidence indicates that multi-lineage differentiation ability of stem cells can be defined by the potential for expression of lineage-specification genes. Gene expression, or as emphasized here, potential for gene expression, is largely controlled by epigenetic modifications of DNA and chromatin on genomic regulatory and coding regions. These modifications modulate chromatin organization not only on specific genes but also at the level of the whole nucleus; they can also affect timing of DNA replication. This review highlights how mechanisms by which genes are poised for transcription in undifferentiated stem cells are being uncovered through primarily the mapping of DNA methylation, histone modifications and transcription factor binding throughout the genome. The combinatorial association of epigenetic marks on developmentally regulated and lineage-specifying genes in undifferentiated cells seems to define a pluripotent state. [source] Genetic polymorphism of CYP2C8 in three Malaysian ethnics: CYP2C8*2 and CYP2C8*3 are found in Malaysian IndiansJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 5 2005Y. D. Muthiah PhD candidate Summary Background:,CYP2C8 is genetically polymorphic. Four variants, CYP2C8*2, CYP2C8*3, CYP2C8*4 and CYP2C8*5, which contain mutations in the coding regions have been reported to exhibit different enzyme activity as compared with CYP2C8*1. Objective:, To determine the allele frequency of three codon-changing variants (CYP2C8*2, CYP2C8*3 and CYP2C8*4) in the Malaysian population. Method:, Healthy unrelated volunteers from three major races in Malaysia were recruited. The study was approved by the local Research Ethics Committee. DNA was extracted using a standard protocol. A two-step multiplex PCR method was developed to detect three alleles of CYP2C8. PCR results were confirmed by subsequent direct DNA sequencing. Result:, Only the Indians showed CYP2C8 polymorphism with allele frequency of 98% for CYP2C8*1, 0·8% for CYP2C8*2 and 1·2% for CYP2C8*3. CYP2C8*4 was not detected in any of the ethnic groups. Conclusion:, To the best of our knowledge, the current study described, for the first time polymorphisms of CYP2C8 in Malaysian Indians. [source] Characteristics of HIV-1 non-B subtype infections in Northwest PolandJOURNAL OF MEDICAL VIROLOGY, Issue 8 2010osz Parczewski Abstract The number of non-B subtype HIV-1 infections in Europe has been increasing even though major regional differences have been observed. This trend was investigated in northwestern Poland using sequence and epidemiological data from a cohort of 102 HIV-1-infected patients from Szczecin, Poland. HIV-1 subtypes were defined by phylogenetic analysis of viral reverse transcriptase- and protease-partial coding regions, and results were compared with online subtyping by Standford and REGA tools. Subtype analysis using on-line subtyping methods produced varying results if compared to phylogenesis, with concordant variant assignment obtained for 98% (100/102) of sequences by Stanford and 85% (87/102) by REGA. In the population studied, non-B subtype infections comprised 21% of the infections and consisted of subtype D (57%, n,=,12), CRF01_AE (19%, n,=,4), A and C clades (9.5%, n,=,2), and the CRF13_cpx recombinant isolate (4.8%, n,=,1). Patients carrying non-B subtypes were predominantly heterosexuals with high percentage (57%) of women observed in the group. All HIV-1 non-B women were Caucasian with majority (83%) of infections acquired in Poland; however, among 12 travelers included in the study a higher proportion of non-B infections was noted (50%, P,=,0.01). Moreover, lower baseline lymphocyte CD4 counts (P,=,0.01), higher baseline HIV-1 viremia (P,=,0.08), and a more advanced stage of the disease (P,=,0.03) were observed among individuals infected with non-B subtypes. The data indicated that the proportion of HIV-1 non-B subtype infections was higher than previously reported in Poland consisting of a high subtype D prevalence. Furthermore, subtype D transmission occurred primarily between heterosexual Caucasian individuals from this region. J. Med. Virol. 82:1306,1313, 2010. © 2010 Wiley-Liss, Inc. [source] Identification of a new genotype H wild-type mumps virus strain and its molecular relatedness to other virulent and attenuated strainsJOURNAL OF MEDICAL VIROLOGY, Issue 2 2003Georgios Amexis Abstract A single clinical isolate of mumps virus designated 88-1961 was obtained from a patient hospitalized with a clinical history of upper respiratory tract infection, parotitis, severe headache, fever and lymphadenopathy. We have sequenced the full-length genome of 88-1961 and compared it against all available full-length sequences of mumps virus. Based upon its nucleotide sequence of the SH gene 88-1961 was identified as a genotype H mumps strain. The overall extent of nucleotide and amino acid differences between each individual gene and protein of 88-1961 and the full-length mumps samples showed that the missense to silent ratios were unevenly distributed. Upon evaluation of the consensus sequence of 88-1961, four positions were found to be clearly heterogeneous at the nucleotide level (NP 315C/T, NP 318C/T, F 271A/C, and HN 855C/T). Sequence analysis revealed that the amino acid sequences for the NP, M, and the L protein were the most conserved, whereas the SH protein exhibited the highest variability among the compared mumps genotypes A, B, and G. No identifying molecular patterns in the non-coding (intergenic) or coding regions of 88-1961 were found when we compared it against relatively virulent (Urabe AM9 B, Glouc1/UK96, 87-1004 and 87-1005) and non-virulent mumps strains (Jeryl Lynn and all Urabe Am9 A substrains). J. Med. Virol. 70: 284,286, 2003. © 2003 Wiley-Liss, Inc. [source] DEFINING THE MAJOR LINEAGES OF RED ALGAE (RHODOPHYTA),JOURNAL OF PHYCOLOGY, Issue 2 2006Hwan Su Yoon Previous phylogenetic studies of the Rhodophyta have provided a framework for understanding red algal phylogeny, but there still exists the need for a comprehensive analysis using a broad sampling of taxa and sufficient phylogenetic information to clearly define the major lineages. In this study, we determined 48 sequences of the PSI P700 chl a apoprotein A1 (psaA) and rbcL coding regions and established a robust red algal phylogeny to identify the major clades. The tree included most of the lineages of the Bangiophyceae (25 genera, 48 taxa). Seven well-supported lineages were identified with this analysis with the Cyanidiales having the earliest divergence and being distinct from the remaining taxa; i.e. the Porphyridiales 1,3, Bangiales, Florideophyceae, and Compsopogonales. We also analyzed data sets with fewer taxa but using seven proteins or the DNA sequence from nine genes to resolve inter-clade relationships. Based on all of these analyses, we propose that the Rhodophyta contains two new subphyla, the Cyanidiophytina with a single class, the Cyanidiophyceae, and the Rhodophytina with six classes, the Bangiophyceae, Compsopogonophyceae, Florideophyceae, Porphyridiophyceae classis nov. (which contains Porphyridium, Flintiella, and Erythrolobus), Rhodellophyceae, and Stylonematophyceae classis nov. (which contains Stylonema, Bangiopsis, Chroodactylon, Chroothece, Purpureofilum, Rhodosorus, Rhodospora, and Rufusia). We also describe a new order, Rhodellales, and a new family, Rhodellaceae (with Rhodella, Dixoniella, and Glaucosphaera). [source] Biological and Molecular Characterization of Melon-Infecting Kyuri Green Mottle Mosaic Virus in IndonesiaJOURNAL OF PHYTOPATHOLOGY, Issue 10 2005B. S. Daryono Abstract Melon (Cucumis melo L.) plants showing fruit deformation and mosaic symptoms were found in Java, Indonesia, in 2001. Leaf dips of the symptomatic melon tissue revealed rod-shaped viral particles 300 × 18 nm in size. Biological and serological data described in this study indicate that the virus belonged to the genus tobamovirus and was related to the kyuri green mottle mosaic virus (KGMMV). The genome of the virus has been completely sequenced, consisting of 6512 nucleotides and was compared in detail with KGMMV-C1 and KGMMV-Y. The sequence of their 5,- and 3,- non-coding regions (NCRs) were 91% and 94% identical to KGMMV-C1, and only 82% and 95% identical to KGMMV-Y respectively. The amino acid sequence of the shorter and longer RNA replicase components, movement protein and coat protein were 94%, 91%, 95% and 94% identical to KGMMV-C1 and 93%, 89%, 91% and 85% identical of KGMMV-Y respectively. The results from phylogenetic analysis of the coding regions revealed that KGMMV-YM is a new strain of KGMMV. This is the first report of the complete nucleotide sequence and analysis of genome organization for KGMMV isolated in anywhere in South-East Asia. [source] Production of Polyclonal Antibodies to a Recombinant Coat Protein of Potato mop-top virusJOURNAL OF PHYTOPATHOLOGY, Issue 4 2003ovská Abstract The coat protein (CP) coding regions of two Czech Potato mop-top virus (PMTV) isolates were sequenced and shown to be identical. One, the Korneta isolate CP gene, was cloned in several expression vectors. The recombinant PMTV-CP was expressed in Escherichia coli and the purified recombinant protein was used to produce PMTV-specific polyclonal antibodies. The antiserum had a titre of 1 : 2000 in an indirect enzyme-linked immunosorbent assay (ELISA) and reacted specifically in immunoblotting and IPTA- ELISA (indirect plate-trapped antigen (PTA)-ELISA). [source] Mass spectrometry strategies applied to the characterization of proline-rich peptides from secretory parotid granules of pig (Sus scrofa)JOURNAL OF SEPARATION SCIENCE, JSS, Issue 3 2008Chiara Fanali Abstract Basic proline-rich proteins (bPRPs) are a class of proteins widely present in saliva of humans and other mammals. They are synthesized as preproproteins and enzymatically cleaved into small peptides before secretion from the salivary glands. Recently, we characterized two proline-rich peptides (SP-A and SP-B) in parotid secretory granules of pig (Sus Scrofa) that are derived from three isoforms of a PRP proprotein (Swiss-Prot data bank: Q95JC9-1, Q95JC9-2 and Q95JC9-3). Together the coding regions for SP-A and SP-B, which are repeated many times, account for 52,70% of the coding regions of the PRP proproteins. This study was undertaken to identify peptides encoded by unassigned regions of the PRP proproteins. RP-HPLC-ESI-IT-MS analysis of enriched granule preparations from pig parotid glands by two different analytical strategies identified ten new proline-rich peptides derived from the three proproteins. Together with the coding regions for SP-A and SP-B already identified it was possible to assign 68,75% of the proproteins coding regions. The peptide sequences indicated a number of unusual proteolytic cleavage sites suggesting the presence of unknown proprotein convertases. [source] Evaluation of 10 plant barcodes in Bryophyta (Mosses)JOURNAL OF SYSTEMATICS EVOLUTION, Issue 1 2010Yan LIU Abstract DNA barcoding is a molecular tool that uses a standardized DNA region to identify species. Our preliminary study reported here is the first attempt to specifically focus on universality and attributes of candidate barcodes across a wide systematic range of mosses. We tested eight previously proposed plant barcoding regions (atpF-atpH, ITS2, matK, psbK-psbI, rbcL, rpoB, rpoC1, and trnH-psbA) and two popular phylogenetic markers (rps4 and trnL-trnF of cpDNA) in 49 moss species and 9 liverwort species, representing half of the orders in moss lineages. The ITS2, rbcL, rpoC1, rps4, trnH-psbA and trnL-trnF regions showed good universality, and therefore the efficacy of these loci as DNA barcodes was further evaluated in 36 mosses and 2 liverworts, each of which included two to three individuals per taxa. The five loci, viz. rbcL, rpoC1, rps4, trnH-psbA and trnL-trnF, were easy to amplify and sequence and showed significant inter-specific genetic variability, making them potentially useful DNA barcodes for mosses. The best performing single loci were the rbcL and rpoC1 coding regions. Several loci showed equivalent performance and combinations of them did not greatly increase their discrimination capacity. In addition, phylogenies generated from each of the separate regions and multi-locus combinations by using best-fit and Kimura 2-parameter models were compared, but no significant difference was found. [source] Investigation of Quantitative Trait Loci in the CCKAR Gene With Susceptibility to AlcoholismALCOHOLISM, Issue 2002Takehito Okubo Background Cholecystokinin (CCK) plays an important role in the function of the central nervous system by interacting with dopamine and other neurotransmitters. We previously reported genetic variations in the promoter and coding regions of the CCKA receptor (CCKAR), CCKBR, and CCK genes and a possible association between polymorphisms of the CCKAR gene and alcoholism. In this study, association analyses were re-examined between the polymorphisms of the promoter region of the CCKAR gene and patients with alcohol withdrawal symptoms, in addition to patients with alcoholic liver injury. Methods A total of 131 Japanese male patients with alcohol withdrawal symptoms, 70 Japanese patients with alcoholic liver injury, and 98 age-matched Japanese male controls (nonhabitual drinkers) were examined using polymerase chain reaction-based single strand conformational polymorphism and sequencing analyses. Results Significant differences between patients with hallucination and controls were found in the allele frequencies at the ,388 and ,85 loci of the CCKAR gene (p= 0.0095, p= 0.0087, respectively), but these differences were not significant after Bonferroni correction for multiple testing. In contrast, the frequency of the homozygous genotype ,85 CC was significantly higher in hallucination-positive patients than in controls (p= 0.0031) and in patients with hallucination accompanying delirium tremens than in controls (p= 0.0022), and these differences were significant after Bonferroni correction. Conclusions The data from the case control suggest that polymorphisms of the promoter region of the CCKAR gene do not play a major role in the pathogenesis of alcohol withdrawal symptoms or alcoholic liver injury. However, a significant association was found between polymorphism at the ,85 locus of the CCKAR gene and patients with hallucination, and especially patients with hallucination accompanying delirium tremens. [source] A hierarchical Bayesian model for predicting the functional consequences of amino-acid polymorphismsJOURNAL OF THE ROYAL STATISTICAL SOCIETY: SERIES C (APPLIED STATISTICS), Issue 1 2005Claudio J. Verzilli Summary., Genetic polymorphisms in deoxyribonucleic acid coding regions may have a phenotypic effect on the carrier, e.g. by influencing susceptibility to disease. Detection of deleterious mutations via association studies is hampered by the large number of candidate sites; therefore methods are needed to narrow down the search to the most promising sites. For this, a possible approach is to use structural and sequence-based information of the encoded protein to predict whether a mutation at a particular site is likely to disrupt the functionality of the protein itself. We propose a hierarchical Bayesian multivariate adaptive regression spline (BMARS) model for supervised learning in this context and assess its predictive performance by using data from mutagenesis experiments on lac repressor and lysozyme proteins. In these experiments, about 12 amino-acid substitutions were performed at each native amino-acid position and the effect on protein functionality was assessed. The training data thus consist of repeated observations at each position, which the hierarchical framework is needed to account for. The model is trained on the lac repressor data and tested on the lysozyme mutations and vice versa. In particular, we show that the hierarchical BMARS model, by allowing for the clustered nature of the data, yields lower out-of-sample misclassification rates compared with both a BMARS and a frequen-tist MARS model, a support vector machine classifier and an optimally pruned classification tree. [source] Inherited defects of coagulation Factor V: the thrombotic sideJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 1 2006H. L. VOS Summary., DNA variations in the Factor V gene have played a major role in thrombosis research ever since the discovery of Factor V Leiden. Here, all relatively common DNA variations in the coding regions of the Factor V gene are discussed. Many of them have been associated with venous thrombosis or related diseases. However, most variations have been studied separately, without taking the presence of other variations in the same gene into account. This means that their association with disease should be interpreted with caution, as it may reflect linkage with another variation. An approach in which a haplotype-based analysis of the Factor V gene is combined with in vitro assays of recombinant proteins is advocated. Finally, a possible reason for the relatively polymorphic nature of the Factor V protein is discussed. [source] Genetic variability of hepatitis C virus NS3 protein in human leukocyte antigen-A2 liver transplant recipients with recurrent hepatitis CLIVER TRANSPLANTATION, Issue 2 2004F. Xavier López-Labrador The association between the severity of chronic hepatitis C and the variability of the hepatitis C virus (HCV) genome remains controversial, but to our knowledge few data are available to date regarding T-cell epitope coding regions in transplant patients. In the current study, we identified 21 human leukocyte antigen (HLA)-A2-positive Spanish patients with chronic hepatitis C, 14 immunosuppressed liver transplant recipients, and 7 immunocompetent controls. Alanine aminotransferase, aspartate aminotransferase, viral load, and rate of fibrosis progression were determined. Genetic distances of HCV isolates and variations in epitopes of the HCV nonstructural 3 protein (NS3-1393 LIFCHSKKK and NS3-1406 KLVALGINAV) were compared between patients with slow or fast progression of fibrosis. Isolates from transplant patients with fast progression were found to be more divergent (P =.03), had a higher mean value of synonymous (dS) variations (P =.02), and some were differentiated in a phylogenetic tree, compared with isolates from patients with slow progression. The HLA-A2-restricted NS3-1406 epitope was found to be more variable (20 of 21 isolates differed from the prototype) compared with the A3-restricted NS3-1392 epitope (19% vs. 1.25% variation). A shift in the viral peptide was not detected in a subset of transplant patients, but was evident in two of three nontransplant patients with follow-up. There was no correlation noted between a particular amino acid variation and fibrosis progression (slow or fast) in either transplant or nontransplant patients. The results of the current study suggest that 1) there may be different HCV-1b strains in our geographic area, 2) immunosuppression appears to have little effect in amino acid variation at the HCV NS3-1406 epitope, and 3) variations over time might be more frequent in nonimmunosuppressed patients. (Liver Transpl 2004;10:217,227.) [source] Characterization of the 3p12.3-pcen region associated with tumor suppression in a novel ovarian cancer cell line model genetically modified by chromosome 3 fragment transferMOLECULAR CARCINOGENESIS, Issue 12 2009Neal A.L. Cody Abstract The genetic analysis of nontumorigenic radiation hybrids generated by transfer of chromosome 3 fragments into the tumorigenic OV-90 ovarian cancer cell line identified the 3p12.3-pcen region as a candidate tumor suppressor gene (TSG) locus. In the present study, polymorphic microsatellite repeat analysis of the hybrids further defined the 3p12.3-pcen interval to a 16.1 Mb common region containing 12 known or hypothetical genes: 3ptel - ROBO2-ROBO1-GBE1-CADM2-VGLL3-CHMP2B-POU1F1-HTR1F-CGGBP1-ZNF654-C3orf38-EPHA3 -3pcen. Seven of these genes, ROBO1, GBE1, VGLL3, CHMP2B, CGGBP1, ZNF654, and C3orf38, exhibited gene expression in the hybrids, placing them as top TSG candidates for further analysis. The expression of all but one (VGLL3) of these genes was also detected in the parental OV-90 cell line. Mutations were not identified in a comparative sequence analysis of the predicted protein coding regions of these candidates in OV-90 and donor normal chromosome 3 contig. However, the nondeleterious sequence variants identified in the transcribed regions distinguished parent of origin alleles for ROBO1, VGLL3, CHMP2B, and CGGBP1 and cDNA sequencing of the hybrids revealed biallelic expression of these genes. Interestingly, underexpression of VGLL3 and ZNF654 were observed in malignant ovarian tumor samples as compared with primary cultures of normal ovarian surface epithelial cells or benign ovarian tumors, and this occurred regardless of allelic content of 3p12.3-pcen. The results taken together suggest that dysregulation of VGLL3 and/or ZNF654 expression may have affected pathways important in ovarian tumorigenesis which was offset by the transfer of chromosome 3 fragments in OV-90, a cell line hemizygous for 3p. Mol. Carcinog. © 2009 Wiley-Liss, Inc. [source] | |||||||||||||||||||||||||||||||