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Flanking Regions (flanking + regions)
Selected AbstractsRapid assessment of the sex of codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) eggs and larvaeJOURNAL OF APPLIED ENTOMOLOGY, Issue 4 2009I. Fuková Abstract Two different methods were tested to identify the sex of the early developmental stages of the codling moth Cydia pomonella (Linnaeus) (Lepidoptera: Tortricidae) with a WZ/ZZ (female/male) sex chromosome system. First, it was shown that the sex of all larval stages can be easily determined by the presence or absence of sex chromatin, which is formed by the female-specific W chromosome in interphase nuclei. This trait can also be used to identify the sex of newly hatched larvae but it does require care and accuracy. Secondly, a new sexing technique was developed based on a molecular marker of the codling moth W chromosome. Flanking regions of an earlier described W-specific sequence (CpW2) were isolated and sequenced and a 2.74 kb sequence (CpW2- EcoRI), specific for the W chromosome, was obtained. Several PCR tests were conducted, which confirmed that the CpW2- EcoRI sequence is a reliable marker for the sex identification in codling moth samples of different geographical origin. In addition, a fragment of a codling moth gene, period (Cpper) was isolated and sequenced. Results of southern hybridization of the Cpper probe with female and male genomic DNA suggested that the Cpper gene is located on the Z chromosome. Then a multiplex PCR assay was developed, which co-amplified the CpW2- EcoRI sequence to identify the W chromosome and the Z-linked Cpper sequence, which served as a positive control of accurate processing of tested samples. The multiplex PCR provides an easy and rapid identification of the sex of embryos and early larval instars of the codling moth. [source] Myosin Va phosphorylated on Ser1650 is found in nuclear speckles and redistributes to nucleoli upon inhibition of transcriptionCYTOSKELETON, Issue 6 2008Maria Cristina S. Pranchevicius Abstract Nuclear actin and nuclear myosins have been implicated in the regulation of gene expression in vertebrate cells. Myosin V is a class of actin-based motor proteins involved in cytoplasmic vesicle transport and anchorage, spindle-pole alignment and mRNA translocation. In this study, myosin-Va, phosphorylated on a conserved serine in the tail domain (phospho-ser1650 MVa), was localized to subnuclear compartments. A monoclonal antibody, 9E6, raised against a peptide corresponding to phosphoserine1650 and flanking regions of the murine myosin Va sequence, was immunoreactive to myosin Va heavy chain in cellular and nuclear extracts of HeLa cells, PC12 cells and B16-F10 melanocytes. Immunofluorescence microscopy with this antibody revealed discrete irregular spots within the nucleoplasm that colocalized with SC35, a splicing factor that earmarks nuclear speckles. Phospho-ser1650 MVa was not detected in other nuclear compartments, such as condensed chromatin, Cajal bodies, gems and perinucleolar caps. Although nucleoli also were not labeled by 9E6 under normal conditions, inhibition of transcription in HeLa cells by actinomycin D caused the redistribution of phospho-ser1650 MVa to nucleoli, as well as separating a fraction of phospho-ser1650 MVa from SC35 into near-neighboring particles. These observations indicate a novel role for myosin Va in nuclear compartmentalization and offer a new lead towards the understanding of actomyosin-based gene regulation. Cell Motil. Cytoskeleton 2008. © 2008 Wiley-Liss, Inc. [source] Transcription of individual tRNAGly1 genes from within a multigene family is regulated by transcription factor TFIIIBFEBS JOURNAL, Issue 20 2005Akhila Parthasarthy Members of a multigene family from the silkworm Bombyx mori have been classified based on their transcriptions in homologous nuclear extracts, into three groups of highly, moderately and poorly transcribed genes. Because all these gene copies have identical coding sequences and consequently identical promoter elements (the A and B boxes), the flanking sequences modulate their expression levels. Here we demonstrate the interaction of transcription factor TFIIIB with these genes and its role in regulating differential transcriptions. The binding of TFIIIB to the poorly transcribed gene -6,7 was less stable compared with binding of TFIIIB to the highly expressed copy, -1. The presence of a 5, upstream TATA sequence closer to the coding region in -6,7 suggested that the initial binding of TFIIIC to the A and B boxes sterically hindered anchoring of TFIIIB via direct interactions, leading to lower stability of TFIIIC,B-DNA complexes. Also, the multiple TATATAA sequences present in the flanking regions of this poorly transcribed gene successfully competed for TFIIIB reducing transcription. The transcription level could be enhanced to some extent by supplementation of TFIIIB but not by TATA box binding protein. The poor transcription of -6,7 was thus attributed both to the formation of a less stable transcription complex and the sequestration of TFIIIB. Availability of the transcription factor TFIIIB in excess could serve as a general mechanism to initiate transcription from all the individual members of the gene family as per the developmental needs within the tissue. [source] Expression of the gene and processed pseudogenes encoding the human and rabbit translationally controlled tumour protein (TCTP)FEBS JOURNAL, Issue 17 2000Holger Thiele In humans and rabbits, the TPT1 gene encoding the translationally controlled tumour protein TCTP generates two mRNAs (TCTP mRNA1 and TCTP mRNA2) which differ in the length of their 3, untranslated regions. The distribution of these mRNAs was investigated in 10 rabbit and 50 human tissues. They were transcribed in all tissues investigated, but differed considerably in their quantity and ratio of expression. This indicates an extensive transcriptional control and involvement of tissue-specific factors. In the rabbit genome numerous processed, intronless pseudogenes were detected. Four, corresponding to both types of mRNAs, were sequenced and analysed in detail; all displayed only few mutations and were either preserved completely in the original amino acid sequence of the intron containing gene, or contained only minor mutations in the coding region which did not interrupt the open reading frame. In the mRNA population of rabbit reticulocytes two additional TCTP RNAs of the TCTP mRNA2 type were detected, which have the characteristics of pseudogene transcripts. Pseudogene transcription was supported further by CAT reporter gene assays showing substantial promoter activity of 5,-flanking regions of two TPT1 pseudogenes. [source] A common cortactin gene variation confers differential susceptibility to severe asthmaGENETIC EPIDEMIOLOGY, Issue 8 2008Shwu-Fan Ma Abstract Genomic regions with replicated linkage to asthma-related phenotypes likely harbor multiple susceptibility loci with relatively minor effects on disease susceptibility. The 11q13 chromosomal region has repeatedly been linked to asthma with five genes residing in this region with reported replicated associations. Cortactin, an actin-binding protein encoded by the CTTN gene in 11q13, constitutes a key regulator of cytoskeletal dynamics and contractile cell machinery, events facilitated by interaction with myosin light chain kinase; encoded by MYLK, a gene we recently reported as associated with severe asthma in African Americans. To evaluate potential association of CTTN gene variation with asthma susceptibility, CTTN exons and flanking regions were re-sequenced in 48 non-asthmatic multiethnic samples, leading to selection of nine tagging polymorphisms for case-control association studies in individuals of European and African descent. After ancestry adjustments, an intronic variant (rs3802780) was significantly associated with severe asthma (odds ratio [OR]: 1.71; 95% confidence interval [CI]: 1.20,2.43; p=0.003) in a joint analysis. Further analyses evidenced independent and additive effects of CTTN and MYLK risk variants for severe asthma susceptibility in African Americans (accumulated OR: 2.93, 95% CI: 1.40,6.13, p=0.004). These data suggest that CTTN gene variation may contribute to severe asthma and that the combined effects of CTTN and MYLK risk polymorphisms may further increase susceptibility to severe asthma in African Americans harboring both genetic variants. Genet. Epidemiol. 2008. © 2008 Wiley-Liss, Inc. [source] Gene conversion causing human inherited disease: Evidence for involvement of non-B-DNA-forming sequences and recombination-promoting motifs in DNA breakage and repair,HUMAN MUTATION, Issue 8 2009Nadia Chuzhanova Abstract A variety of DNA sequence motifs including inverted repeats, minisatellites, and the , recombination hotspot, have been reported in association with gene conversion in human genes causing inherited disease. However, no methodical statistically based analysis has been performed to formalize these observations. We have performed an in silico analysis of the DNA sequence tracts involved in 27 nonoverlapping gene conversion events in 19 different genes reported in the context of inherited disease. We found that gene conversion events tend to occur within (C+G)- and CpG-rich regions and that sequences with the potential to form non-B-DNA structures, and which may be involved in the generation of double-strand breaks that could, in turn, serve to promote gene conversion, occur disproportionately within maximal converted tracts and/or short flanking regions. Maximal converted tracts were also found to be enriched (P<0.01) in a truncated version of the ,-element (a TGGTGG motif), immunoglobulin heavy chain class switch repeats, translin target sites and several novel motifs including (or overlapping) the classical meiotic recombination hotspot, CCTCCCCT. Finally, gene conversions tend to occur in genomic regions that have the potential to fold into stable hairpin conformations. These findings support the concept that recombination-inducing motifs, in association with alternative DNA conformations, can promote recombination in the human genome. Hum Mutat 30:1,10, 2009. © 2009 Wiley-Liss, Inc. [source] Association of IL4R gene polymorphisms with asthma in Chinese populations,,HUMAN MUTATION, Issue 10 2007Haibing Zhang Abstract Cytokines, having central functions in immunological and inflammatory processes, are expected to play important roles in the pathogenesis of various diseases, such as asthma. Genetic polymorphisms of those cytokine and cytokine receptor genes are the focus of genetic association studies. In an effort to identify gene(s) whose variant(s) are associated with asthma, we screened all exons and their flanking regions, as well as the promoter region (1.5kb) of eight genes, including IL4, IL13, IL12B, IL5, IL3, IL9, CD14 and IL4R. We identified 42 single nucleotide polymorphisms, 15 of which were novel. Then, we examined the genetic effects of 30 single nucleotide polymorphisms in eight cytokine and cytokine receptor genes on asthma in a Chinese asthma cohort (n,=,537). Genetic association analysis of polymorphisms revealed that six polymorphisms (c.899-2C>A, c.1199A>C, c.1242G>T, c.1291T>C, c.1299T>C, c.1507T>C) in the IL4R gene, three of which resulted in an amino-acid change, showed significant association with the risk of asthma (P,=,0.00023). Further analysis indicates that these six polymorphisms segregated in strong linkage disequilibrium. The genetic association of IL4R with asthma might provide valuable insights into the pathogenesis of asthma. © 2007 Wiley-Liss, Inc. [source] Single nucleotide polymorphisms in succinate dehydrogenase subunits and citrate synthase genes: association results for impaired spermatogenesisINTERNATIONAL JOURNAL OF ANDROLOGY, Issue 3 2007Sandra Bonache Abstract Evaluation of the possible implication of the SDHA, SDHB, SDHC, SDHD and CS genes in non-obstructive male infertility was performed on the basis that sperm concentration in the ejaculate has been previously correlated with nuclear-encoded mitochondrial enzyme activities (the four subunits of succinate dehydrogenase/complex II of the respiratory chain and citrate synthase). We performed an exhaustive analysis of the five genes for the presence of sequence variants that could be associated with impairment of sperm production. blastn searches in the genomic sequence NCBI database evidenced the presence of highly homologous sequences elsewhere on the genome that can interfere with polymerase chain reaction experiments. Therefore, a careful design of the analytical strategy to search for sequence variants was performed. In this report, we provide primer sequences that allowed selective amplification of coding and immediate flanking regions of the five genes. Fifty-five sequence variations in the five genes were identified in infertile and normozoospermic fertile individuals as controls and only one of them (SDHA c.456+32G>A) showed significant genotype association with impairment of sperm production. Moreover, new single nucleotide polymorphisms identified should be useful in future association studies for other human diseases related to nuclear-encoded genes, leading to mitochondrial respiratory chain activity impairment revealing the physiological role of these genes. [source] SiPep: a system for the prediction of tissue-specific minor histocompatibility antigensINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 4 2006M. Halling-Brown Summary Approximately 50 years ago it was found that inbred strains of mice were able to reject tumours and skin grafts from major histocompatibility complex (MHC) identical donors. They proposed that additional transplantation antigens must exist outside the MHC. These were described as minor histocompatibility antigens (mHAgs). Since then, related studies in humans have identified 16 human mHAgs. The aim of this work is to increase the number of known mHAgs by prediction of candidate minor histocompatibility loci by identifying coding single nucleotide polymorphisms (SNPs) where the amino acid variation lies within an MHC-binding peptide and alters the ability of that peptide to bind. We have developed an algorithm called SiPep which uses peptide sequences derived from the flanking regions of known non-synonymous SNPs, various MHC-binding and proteolytic cleavage evaluation methods and protein expression data to predict mHAgs. We have processed 45094 SNPs using the SiPep algorithm and have stored the results in a database called SNPBinder. The facilities to process submitted proteins through the SiPep algorithm as well as the SNPBinder database are available to the public. A set of peptides that are predicted as possible mHAgs by the SiPep algorithm have been tested using refolding assays and gel filtration and the results are presented in this paper. The SiPep tools and SNPBinder database are available free of charge via the internet. An HTML interface providing search facilities can be found at the following address: http://www.sipep.org/. [source] Rapid denaturing high-performance liquid chromatography (DHPLC) for mutation scanning of the transforming growth factor ,3 gene using a novel proof-reading polymeraseINTERNATIONAL JOURNAL OF IMMUNOGENETICS, Issue 5 2003A. Bayat Summary We have utilized a novel variation on the conventional denaturing high-performance liquid chromatography (DHPLC) technology, which we term rapid DHPLC, combining changes in instrumentation, cartridge technology and analysis conditions to enable significant increases in throughput to be achieved. In addition, the use of a novel proof-reading polymerase for sample amplification with a low misincorporation rate enables simplification of the DHPLC patterns and hence enhanced mutation detection recognition. This scheme for increasing DHPLC throughput has been tested by scanning the transforming growth factor (TGF) ,3 gene for the presence of mutations for which there is limited published or on-line data available regarding the presence of gene polymorphisms. TGF, isoforms have multiple roles in cell division, growth, proliferation, transformation and differentiation. TGF,3 is a TGF, cytokine isoform, and has an important role in embryogenesis, cell differentiation and wound healing. The TGF,3 gene consists of seven exons and six introns spanning 43 000 bp of the human genome on chromosome 14q23,24. The rapid DHPLC approach enabled scanning of all seven exons and part of the promoter region (1000 bp upstream from exon 1 in the 5,-flanking regions) of the TGF,3 gene in 95 Caucasian individuals in only 8 days, in comparison to the 17 days it would have previously taken. Mutations were clearly identified in the promoter region of the TGF,3 gene but were absent from the exonic regions. Understanding the genetic variations affecting the TGF,3 gene is important as this molecule has multiple regulatory functions on a variety of cell types. [source] Genetic variation in the RANKL/RANK/OPG signaling pathway is associated with bone turnover and bone mineral density in menJOURNAL OF BONE AND MINERAL RESEARCH, Issue 8 2010Delnaz Roshandel Abstract The aim of this study was to determine if single-nucleotide polymorphisms (SNPs) in RANKL, RANK, and OPG influence bone turnover and bone mineral density (BMD) in men. Pairwise tag SNPs (r2,,,0.8) were selected for RANKL, RANK, and OPG and their 10-kb flanking regions. Selected tag SNPs plus five SNPs near RANKL and OPG, associated with BMD in published genome-wide association studies (GWAS), were genotyped in 2653 men aged 40 to 79 years of age recruited for participation in a population-based study of male aging, the European Male Ageing Study (EMAS). N-terminal propeptide of type I procollagen (PINP) and C-terminal cross-linked telopeptide of type I collagen (CTX-I) serum levels were measured in all men. BMD at the calcaneus was estimated by quantitative ultrasound (QUS) in all men. Lumbar spine and total-hip areal BMD (BMDa) was measured by dual-energy X-ray absorptiometry (DXA) in a subsample of 620 men. Multiple OPG, RANK, and RANKL SNPs were associated with bone turnover markers. We also identified a number of SNPs associated with BMD, including rs2073618 in OPG and rs9594759 near RANKL. The minor allele of rs2073618 (C) was associated with higher levels of both PINP (,,=,1.83, p,=,.004) and CTX-I (,,=,17.59, p,=,4.74,×,10,4), and lower lumbar spine BMDa (,,=,,0.02, p,=,.026). The minor allele of rs9594759 (C) was associated with lower PINP (,,=,,1.84, p,=,.003) and CTX-I (,,=,,27.02, p,=,6.06,×,10,8) and higher ultrasound BMD at the calcaneus (,,=,0.01, p,=,.037). Our findings suggest that genetic variation in the RANKL/RANK/OPG signaling pathway influences bone turnover and BMD in European men. © 2010 American Society for Bone and Mineral Research [source] Modular changes of cis-regulatory elements from two functional Pit1 genes in the duplicated genome of Cyprinus carpioJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 3 2006G. Kausel Abstract The pituitary-specific transcription factor Pit1 is involved in its own regulation and in a network of transcriptional regulation of hypothalamo-hypophyseal factors including prolactin (PRL) and growth hormone (GH). In the ectotherm teleost Cyprinus carpio, Pit1 plays an important role in regulation of the adaptive response to seasonal environmental changes. Two Pit1 genes exist in carp, a tetraploid vertebrate and transcripts of both genes were detected by RT-PCR analysis. Powerful comparative analyses of the 5,-flanking regions revealed copy specific changes comprising modular functional units in the naturally evolved promoters. These include the precise replacement of four nucleotides around the transcription start site embedded in completely conserved regions extending upstream of the TATA-box, an additional transcription factor binding site in the 5,-UTR of gene-I and, instead, duplication of a 9 bp element in gene-II. Binding of nuclear factors was assessed by electro mobility shift assays using extracts from rat pituitary cells and carp pituitary. Binding was confirmed at one conserved Pit1, one conserved CREB and one consensus MTF1. Interestingly, two functional Pit1 sites and one putative MTF1 binding site are unique to the Pit1 gene-I. In situ hybridization experiments revealed that the expression of gene-I in winter carp was significantly stronger than that of gene-II. Our data suggest that the specific control elements identified in the proximal regulatory region are physiologically relevant for the function of the duplicated Pit1 genes in carp and highlight modular changes in the architecture of two Pit1 genes that evolved for at least 12 MYA in the same organism. J. Cell. Biochem. 99: 905,921, 2006. © 2006 Wiley-Liss, Inc. [source] Inhibition of DNA topoisomerase II may trigger illegitimate recombination in living cells: Experiments with a model systemJOURNAL OF CELLULAR BIOCHEMISTRY, Issue 2 2006Olga N. Umanskaya Abstract We have developed a plasmid test system to study recombination in vitro and in mammalian cells in vivo, and to analyze the possible role of DNA topoisomerase II. The system is based on a plasmid construct containing an inducible marker gene ccdB ("killer" (KIL) gene) whose product is lethal for bacterial cells, flanked by two different potentially recombinogenic elements. The plasmids were subjected to recombinogenic conditions in vitro or in vivo after transient transfection into COS-1 cells, and subsequently transformed into E. coli which was then grown in the presence of the ccdB gene inducer. Hence, all viable colonies contained recombinant plasmids since only recombination between the flanking regions could remove the KIL gene. Thus, it was possible to detect recombination events and to estimate their frequency. We found that the frequency of topoisomerase II-mediated recombination in vivo is significantly higher than in a minimal in vitro system. The presence of VM-26, an inhibitor of the religation step of the topoisomerase II reaction, increased the recombination frequency by 60%. We propose that cleavable complexes of topoisomerase II are either not religated, triggering error-prone repair of the DNA breaks, or are incorrectly religated resulting in strand exchange. We also studied the influence of sequences known to contain preferential breakpoints for recombination in vivo after chemotherapy with topoisomerase II-targeting drugs, but no preferential stimulation of recombination by these sequences was detected in this non-chromosomal context. J. Cell. Biochem. 99: 598,608, 2006. © 2006 Wiley-Liss, Inc. [source] NFATc1 and TNF, expression in giant cell lesions of the jawsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 3 2010Fabrício Rezende Amaral J Oral Pathol Med (2010) 39: 269,274 Background:, Activation mutations of SH3BP2 gene have been demonstrated in cherubism and central giant cell lesion (CGCL). In the present study we first attempted to investigate the SH3BP2 gene in peripheral giant cell lesion (PGCL). The effect of SH3BP2 gene mutations on the transcription of the downstream genes nuclear factor of activated T cells (NFATc1) and the cytokine tumor necrosis factor-, (TNF-,) was also investigated together with the immunolocalization of NFATc1 protein in a set of cases of PGCL, CGCL and cherubism with and without SH3BP2 mutation. Method:, Fresh samples of five PGCL, five CGCL and one cherubism cases were included in this study. One of the samples of CGCL presented a somatic heterozygous mutation c.1442A>T in exon 11. The cherubism case showed a heterozygotic substitution c.320C>T in both blood and lesion. These mutations were previously published. All coding and flanking regions of the SH3BP2 gene were sequenced in the cases of PGCL. The real-time polymerase chain reaction (RT-PCR) was performed to analyze the transcription of NFATc1 and TNF-, genes. The immunohistochemical analysis of the NFATc1 protein was also performed. Results:, No SH3BP2 gene mutation was found in PGCL. The RT-PCR showed increased expression of NFATc1 and decreased transcription of TNF-, in all the samples. The immunohistochemical analysis of the NFATc1 protein showed a predominant nuclear staining in the multinucleated giant cells. Conclusion:, The development of giant cells lesions of the jaws and cherubism are possibly mediated by overexpression of NFAT in the nucleus of the multinucleated cells. [source] Acetaminophen UDP-glucuronosyltransferase in ferrets: species and gender differences, and sequence analysis of ferret UGT1A6JOURNAL OF VETERINARY PHARMACOLOGY & THERAPEUTICS, Issue 6 2001M. H. Court The principal objective of this study was to determine whether ferrets glucuronidate acetaminophen more slowly compared with other species, and if so investigate the molecular basis for the difference. Acetaminophen-UDP-glucuronosyltransferase (UGT) activities were measured using hepatic microsomes from eight ferrets, four humans, four cats, four dogs, rat, mouse, cow, horse, monkey, pig and rabbit. Gender differences between male and female ferret livers were explored using enzyme kinetic analysis. Immunoblotting of microsomal proteins was also performed using UGT-specific antibodies. Finally, the exon 1 region of UGT1A6, a major acetaminophen-UGT, was sequenced. Glucuronidation of acetaminophen was relatively slow in ferret livers compared with livers from all other species except cat. Gender differences were also apparent, with intrinsic clearance (Vmax/Km) values significantly higher in male compared with female ferret livers. Furthermore, Vmax values correlated with densitometric measurements of two protein bands identified with a UGT1A subfamily-specific antibody. No deleterious mutations were identified in the exon 1 or flanking regions of the ferret UGT1A6 gene. In conclusion, like cats, ferret livers glucuronidate acetaminophen relatively slowly. However, unlike cats, in which UGT1A6 is encoded by a pseudogene and dysfunctional, there are no defects in the ferret UGT1A6 gene which could account for the low activity. [source] A highly efficient gene-targeting system for Aspergillus parasiticusLETTERS IN APPLIED MICROBIOLOGY, Issue 5 2008P.-K. Chang Abstract Aims:, To establish a system that greatly increases the gene-targeting frequency in Aspergillus parasiticus. Methods and Results:, The ku70 gene, a gene of the nonhomologous end-joining (NHEJ) pathway, was replaced by the nitrate reductase gene (niaD) in A. parasiticus RHN1 that accumulates O -methylsterigmatocystin (OMST). The NHEJ-deficient strain, RH,ku70, produced conidia, sclerotia and OMST normally. It had identical sensitivity as RHN1 to the DNA-topoisomerase I complex inhibitor, camptothecin, and the DNA-damaging agent, melphalan. For targeting an aflatoxin biosynthetic pathway gene, adhA, partial restriction enzyme recognition sequences in its flanking regions were manipulated to create homologous ends for integration. Using a linearized DNA fragment that contained Aspergillus oryzae pyrithiamine resistance gene (ptr) marker the adhA -targeting frequency in RH,ku70 reached 96%. Conclusions:, The homologous recombination pathway is primarily responsible for repair of DNA damages in A. parasiticus. The NHEJ-deficient RH,ku70, easy creation of homologous ends for integration, and the ptr -based selection form a highly efficient gene-targeting system. It substantially reduces the time and workload necessary to obtain knockout strains for functional studies. Significance and Impact of the Study:, The developed system not only streamlines targeted gene replacement and disruption but also can be used to target specific chromosomal locations like promoters or intergenic regions. It will expedite the progresses in the functional genomic studies of A. parasiticus and Aspergilllus flavus. [source] Development and characterization of polymorphic markers for the sap-stain fungus Ophiostoma quercusMOLECULAR ECOLOGY RESOURCES, Issue 1 2009J. W. GROBBELAAR Abstract Eight polymorphic markers were developed from South African isolates of Ophiostoma quercus. The genome was screened for repeat regions using the fast isolation by amplified fragment length polymorphism of sequences containing repeats protocol and 20 de novo primer pairs flanking putative microsatellite regions were designed. Eight loci were optimized and their polymorphisms evaluated by sequencing. The repeat and flanking regions were highly polymorphic containing both indels and base-pair substitutions revealing a total of 46 alleles in 14 isolates and an average heterozygosity of 0.68. Substantial sequence variability makes these markers useful for genotyping populations in order to calculate diversity and monitor global movement of O. quercus. [source] Development of novel polymerase chain reaction (PCR) based microsatellite markers in Armillaria gallica by cross-species amplification and species-specific cloningMOLECULAR ECOLOGY RESOURCES, Issue 2 2002J.-B. Lefrancois Abstract Cross-species PCR amplification of Armillaria mellea group taxa with previously reported A. ostoyae microsatellite markers, indicative of flanking sequence conservation, was exploited for the species-specific isolation of simple sequence repeat (SSR) motifs from A. gallica. Six SSR motifs were sequence characterized from cloned PCR fragments generated with primers previously developed from A. ostoyae. Five novel primer pairs, designed from motif flanking regions, allowed for improved, efficient amplification in this species. One original A. ostoyae primer pair was used directly. Polymorphims were observed at wide geographical levels only. Relative cross-species amplification intensities generally supported the currently accepted molecular phylogeny of this group. [source] The vanG glycopeptide resistance operon from Enterococcus faecalis revisitedMOLECULAR MICROBIOLOGY, Issue 3 2003Florence Depardieu Summary Acquired VanG-type resistance to vancomycin (MIC = 16 µg ml,1) but susceptibility to teicoplanin in Enterococcus faecalis BM4518 and WCH9 is due to the inducible synthesis of peptidoglycan precursors ending in d -alanine- d -serine. The vanG cluster, assigned to a chromosomal location, was composed of genes recruited from various van operons. The 3, end encoded VanG, a d -Ala:d -Ser ligase, VanXYG, a putative bifunctional d,d -peptidase and VanTG, a serine racemase: VanG and VanTG were implicated in the synthesis of d -Ala:d -Ser as in VanC- and VanE-type strains. Upstream from the structural genes for these proteins were vanWG with unknown function and vanYG containing a frameshift mutation which resulted in premature termination of the encoded protein and accounted for the lack of UDP-MurNAc-tetrapeptide in the cytoplasm. Without the frameshift mutation, VanYG had homology with Zn2+ dependent d,d -carboxypeptidases. The 5, end of the gene cluster contained three genes vanUG, vanRG and vanSG encoding a putative regulatory system, which were co-transcribed constitutively from the PYG promoter, whereas transcription of vanYG,WG,G,XYG,TG was inducible and initiated from the PYG promoter. Transfer of VanG-type glycopeptide resistance to E. faecalis JH2-2 was associated with the movement, from chromosome to chromosome, of genetic elements of c. 240 kb carrying also ermB -encoded erythromycin resistance. Sequence determination of the flanking regions of the vanG cluster in donor and transconjugants revealed the same 4 bp direct repeats and 22 bp imperfect inverted repeats that delineated the large element. [source] Disruption of msl3 abolishes the synthesis of mycolipanoic and mycolipenic acids required for polyacyltrehalose synthesis in Mycobacterium tuberculosis H37Rv and causes cell aggregationMOLECULAR MICROBIOLOGY, Issue 5 2002Vinod S. Dubey Summary Cell wall lipids of Mycobacterium tuberculosis containing multiple methylbranched fatty acids play critical roles in pathogenesis and thus offer targets for new antimycobacterial drugs. Mycocerosic acid synthase gene (mas) encodes the enzyme that produces one class of such acids. Seven mas -like genes (msls) were identified in the genome. One of them, msl3, originally annotated as two separate genes, pks 3 and pks 4, is now shown to constitute a single open reading frame, which encodes a 220.3 kDa protein. Msl3 was disrupted using a phage mediated delivery system and the gene replacement in the mutant was confirmed by polymerase chain reaction analysis of the flanking regions of the introduced disrupted gene and by Southern analysis. Biochemical analysis showed that the msl3 mutant does not produce mycolipanoic acids and mycolipenic (phthienoic) acids, the major constituents of polyacyl trehaloses and thus lacks this cell wall lipid, but synthesizes all of the other classes of lipids. The absence of the major acyl chains that anchor the surface-exposed acyltrehaloses causes a novel growth morphology; the cells stick to each other, most probably via the intercellular interaction between the exposed hydrophobic cell surfaces, manifesting a bead-like growth morphology without affecting the overall growth rate. [source] Mapping of IS6110 flanking regions in clinical isolates of Mycobacterium tuberculosis demonstrates genome plasticityMOLECULAR MICROBIOLOGY, Issue 6 2000R. M. Warren Southern hybridization was used in combination with IS6110 insertion-locus-specific probes in a comparative study to determine the structure of chromosomal domains flanking IS6110 elements in clinical isolates of Mycobacterium tuberculosis. The resulting restriction fragment length polymorphism (RFLP) data demonstrated three mutational mechanisms responsible for the polymorphisms observed: IS6110 insertion, chromosomal mutation and deletion. The frequency of IS6110 insertion within many of the chromosomal regions demonstrates that preferential integration regions are common in M. tuberculosis. Mapping the IS6110 insertion positions and chromosomal deletions in relation to the M. tuberculosis H37Rv and M. bovis BCG genome sequences reveals numerous disruptions of predicted open reading frames (ORFs). A phylogenetic tree, based on the mutational data, showed a number of independently evolving lineages of M. tuberculosis, while analysis of the mutational events occurring at each branch point suggests both divergent and convergent evolution. A significant positive correlation was demonstrated between the mutation rate and the frequency of occurrence of different isolates in families of strains, suggesting that evolution may impact on strain ,fitness' or that strain proliferation may increase the chance of mutation. We conclude that the genome of clinical isolates of M. tuberculosis continues to evolve. [source] Microsatellite variability and its use in the characterization of cultivated clones of Hevea brasiliensisPLANT BREEDING, Issue 1 2005T. Saha Abstract Microsatellite markers were developed and evaluated in Hevea brasiliensis, an important crop species producing natural rubber of commercial utility. Of eight microsatellite markers, four were found to be highly informative, amplifying a total of 19 alleles when evaluated against 27 cultivated Hevea clones/genotypes. Power of discrimination of the microsatellite loci was in the range of 0.62-0.89, with a mean of 0.76 indicating these microsatellites could be valuable genetic markers for diversity characterization. A combination of four microsatellite markers was successfully used to discriminate uniquely all the 27 Hevea clones and some clone-specific allelic profiles were generated. Cross-species amplification of the markers developed in H. brasiliensis had also been demonstrated with two other Hevea species, H. benthamiana and H. spruceana, indicating a high degree of sequence homology at the flanking regions. Sequence analysis of the repeat region at the 3,-UTR of the hydroxymethylglutaryl-coenzyme A reductase gene, containing clusters of AG repeats in 15 clones, revealed the existence of two alleles based on the repeat length polymorphisms. Homozygosity as well as heterozygosity for both the alleles had also been detected among the clones. Frequency of homozygotes for the smaller allele (allele-1) was found to be lower than the larger allele (allele-2) among the primary clones of H. brasiliensis. [source] Characterization of the porcine AMPK alpha 2 catalytic subunitgene (PRKAA2): genomic structure, polymorphism detection and association studyANIMAL GENETICS, Issue 2 2010L. Lin Summary AMP-activated protein kinase (AMPK), known as a key regulator of cellular energy homeostasis, plays an important role in regulation of glucose and lipid metabolism, and protein synthesis in mammals. The characterization of porcine PRKAA2 encoding the alpha 2 catalytic subunit of AMPK is reported in this study. PRKAA2 was assigned to porcine chromosome 6q by analysis of radiation hybrids (IMpRH panel), and its genomic structure was determined by BAC sequencing. PRKAA2 spans more than 62 kb and consists of nine exons and eight introns. A total of 25 polymorphisms were identified by re-sequencing approximately 7 kb, including all the exons, exon,intron boundaries and 5, and 3, gene flanking regions using twelve founder animals of a Mangalitsa × Piétrain intercross. Neither of two single nucleotide polymorphisms (SNPs) found in the coding region caused an amino acid substitution. Two SNPs (NM_214266.1: c.236+142A>G and NM_214266.1: c.630C>T) in PRKAA2 were genotyped in the Mangalitsa × Piétrain F2 cross (n = 589) and two commercial populations [Piétrain (n = 1173) and German Landrace (n = 536)] and evaluated for association with traits of interest (muscle development and fat deposition). Single SNP and haplotype analyses revealed weak associations between the PRKAA2 genotypes and loin muscle area in the investigated populations. [source] Characterization and genetic analysis of bovine ,s1 -casein I variantANIMAL GENETICS, Issue 4 2009G. Lühken Summary The aim of this study was to identify the molecular genetic origin underlying the I variant of ,s1 -casein and to develop a DNA-based test for this polymorphism as a tool for genetic analyses independent of milk sample testing. All coding exons and flanking regions of the ,s1 -casein gene were sequenced in DNA samples from cattle of known ,s1 -casein genotypes (BI, CI, II, CC), determined by isoelectric focusing of milk samples. A nucleotide substitution (A>T) in exon 11 (g.19836A>T) leads to the exchange of Glu with Asp at amino acid position 84 of the mature protein (p.Glu84Asp) and perfectly co-segregated with the presence of the ,s1 -casein I variant in the milk of the analysed animals. Genotyping of a total of 680 DNA samples from 31 Bos taurus and Bos indicus cattle breeds and from Bos grunniens, Bison bison and Bison bonasus by restriction fragment length polymorphism analysis revealed the occurrence of Asp at position 84 at low frequencies in Bos taurus and Bos indicus breeds and established its origin from the ,s1 -casein C variant (p.Glu192Gly). Ten different intragenic haplotypes in the gene region from intron 8 to intron 12 were observed by sequencing, of which two occurred in Bison bison and one in Bison bonasus only. Using available casein gene complex information, an association of Asp at position 84 to ,-casein A2 and ,-casein B was shown in the Bos indicus breed Banyo Gudali. Taken together, we can postulate that the ,s1 -casein variant I is caused by a non-synonymous nucleotide substitution in exon 11 of the gene and that it originated within Bos indicus and spread to Bos taurus subsequently. [source] Microsatellite flanking regions: a SNP mine in South African abalone (Haliotis midae)ANIMAL GENETICS, Issue 3 2008C. Rhode No abstract is available for this article. [source] Nine porcine microsatellite loci tested for size homoplasy in genetically diverse breedsANIMAL GENETICS, Issue 3 2005T. Peischl Summary Kind and probability of homoplasy across allelic microsatellite fragments can be investigated using DNA of genetically diverse pig breeds. In this study, nine microsatellite loci (SW1897, SW2427, SW489, SW957, TNFB, IFNG, SW2410, SW2019 and S0215) were analysed using DNA samples of pigs from Vietnam (Indigenous breeds Co, Meo, Muong Khuong, Tap Na) and Germany (European Wild Boar, Piétrain). In a total of 39 sequences, 20 differences within isomorphic alleles were observed in comparison with the respective reference sequences. They affected five of the nine tested microsatellite loci. The majority (18) of SNPs occurred in the 5,-flanking regions of the microsatellite repeats, 10 were found in the 3,-flanking regions and only one SNP occurred within the repeat of the Wild Boar sequence of SW2427. The compound microsatellites IFNG and S0215 were unaffected by size homoplasy (SH) within our material. We conclude that the fragment length analysis of microsatellites is a reliable tool for intraspecific phylogenetic studies because SH rates within a species were low. [source] A microsatellite linkage map for Atlantic salmon (Salmo salar)ANIMAL GENETICS, Issue 2 2004J. Gilbey Summary A linkage map of the Atlantic salmon is described here consisting of 15 linkage groups containing 50 microsatellite loci with a 14 additional unlinked markers (including three allozymes). The map shows the largest sex-specific recombination rate differences so far found in any vertebrate species (3.92:1 female:male). Homologies with previous linkage mapping studies of Atlantic salmon and rainbow trout are described. An in silico search of the Genbank database carried out using the microsatellites used in the mapping process identified significant matches between the flanking regions of the microsatellite SS11 and the calcium-binding mitochondrial carrier protein, ,Aralar1'. [source] Simultaneous detection of pathogens causing francisellosis, furunculosis and vibriosis in Atlantic cod by multiplex polymerase chain reactionAQUACULTURE RESEARCH, Issue 10 2010Amod Kulkarni Abstract An assay for the simultaneous detection of three major bacterial pathogens of Atlantic cod with multiplex polymerase chain reaction (mPCR) was developed. Primers targeting the flanking regions of genes groEL, gyrB and amiB were standardized to diagnose francisellosis, furunculosis and vibriosis respectively. The detection limit of Francisella piscicida, Aeromonas salmonicida and Vibrio anguillarum in the mPCR assay ranged from 10 ng to 100 pg of bacterial DNA mL,1, 10 ,g to 50 ng of bacterial DNA mL,1 and 1 ,g to 100 ng of bacterial DNA mL,1 respectively. These primers were found to be specific in the determination of the respective genes in the individual pathogen without cross-reaction, thus making the assay an efficient tool to detect the presence of these pathogens simultaneously or individually. [source] Metabolic characteristics of an isocitrate dehydrogenase defective derivative of escherichia coli BL21(DE3)BIOTECHNOLOGY & BIOENGINEERING, Issue 6 2003Miho Aoshima Abstract A 7.8 kb fragment containing isocitrate dehydrogenase (ICDH) gene and its flanking regions was cloned from Escherichia coli BL21(DE3) and sequenced. Unlike the case of the K-12 strain, the e14 element was not found. The nucleotide divergence between these two strains was about 2%. Using the cloned fragment, ICDH defective mutant strain, MA1935, was generated from BL21(DE3). Although MA1935 accumulated citrate, citrate synthase activity was not repressed but was rather high. In addition, isocitrate lyase was not highly induced at the stationary phase. MA1935 was shown to be a good host strain for ICDH gene expression. © 2003 Wiley Periodicals Inc. Biotechnol Bioeng84: 732,737, 2003. [source] ,2 -Adrenergic Receptor Promoter Haplotype Influences Spirometric Response During an Acute Asthma ExacerbationCLINICAL AND TRANSLATIONAL SCIENCE, Issue 2 2008Paul E. Moore M.D. Abstract Genetic variants in the ,2 -adrenergic receptor (ADRB2) coding block have been associated with different parameters of asthma severity, but there is no consensus on which variants are most important. Our objective was to determine whether the genetic variants in the 5,- or 3,-flanking regions of ADRB2 impact the response to therapy. DNA was obtained initially from 72 adults hospitalized for an asthma exacerbation. We sequenced a 5,000 bp region of the ADRB2 gene that spanned the flanking regions and identified 31 single nucleotide polymorphisms (SNPs). Nonresponders to asthma therapy were defined as patients whose forced expiratory volume in 1 second (FEV1) worsened by >10% at 24 hours after admission. We then evaluated the relationship between the 19 common SNPs and response to asthma-specific therapy during acute disease exacerbations. Our results showed a significant association between nonresponders and a haplotype of five promoter SNPs in a nearly complete linkage disequilibrium. An analysis of the promoter and coding block polymorphisms in an extended cohort of 99 patients confirmed that promoter haplotype was the genetic component most strongly associated with asthmatic nonresponders, which was statistically significant among whites (p < 0.05). An identification of this promoter haplotype may provide an alternate explanation for the variation in the asthma responses observed with ADRB2 coding block polymorphisms. [source] | ||||||||||||||||