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Sample records for methylation polymorphism polymerase

  1. Methylation-sensitive polymerase chain reaction.

    PubMed

    Moore, Hannah R; Meehan, Richard R; Young, Lorraine E

    2006-01-01

    Here, we describe a robust and reproducible methylation-sensitive polymerase chain reaction (MS-PCR) method to detect the percentage methylation in repeat sequences of individual pre-implantation ovine embryos produced by different embryo technologies. This method allows the comparison of embryos produced by nuclear transfer with other production and embryo culture methods, accounting for the heterogeneity between embryos within a single treatment. DNA extracted from single embryos is digested with a methylation-sensitive restriction enzyme to determine the percentage methylation after PCR amplification in comparison with an undigested control. The undigested control represents 100% methylation because methylation-sensitive enzymes do not cut methylated DNA, allowing the entire sample to be amplified by PCR. Image analysis quantification of the digested subsample PCR product on an ethidium bromide-stained agarose gel is proportional to the amount of methylated DNA in each embryo. By comparing quadruplicate values obtained for each embryo against a standard curve, we are able to ensure the validity of our results for each individual embryo. Compared with bisulphite sequencing methods, the method described is rapid, inexpensive, and relatively high-throughput. PMID:16761730

  2. Nested methylation-specific polymerase chain reaction cancer detection method

    DOEpatents

    Belinsky, Steven A.; Palmisano, William A.

    2007-05-08

    A molecular marker-based method for monitoring and detecting cancer in humans. Aberrant methylation of gene promoters is a marker for cancer risk in humans. A two-stage, or "nested" polymerase chain reaction method is disclosed for detecting methylated DNA sequences at sufficiently high levels of sensitivity to permit cancer screening in biological fluid samples, such as sputum, obtained non-invasively. The method is for detecting the aberrant methylation of the p16 gene, O 6-methylguanine-DNA methyltransferase gene, Death-associated protein kinase gene, RAS-associated family 1 gene, or other gene promoters. The method offers a potentially powerful approach to population-based screening for the detection of lung and other cancers.

  3. Effect of MTHFR Gene Polymorphism Impact on Atherosclerosis via Genome-Wide Methylation

    PubMed Central

    Lin, Xuefeng; Zhang, Wei; Lu, Qun; Lei, Xinjun; Wang, Tingzhong; Han, Xuanmao; Ma, Aiqun

    2016-01-01

    Background Atherosclerosis seriously threats human health. Homocysteine is an independent risk factor closely related to DNA methylation. MTHFR C667T loci polymorphism is closely associated with homocysteine level. This study aimed to investigate the relationship among MTHFR C667T loci polymorphism, genome-wide methylation, and atherosclerosis. Material/Methods Blood sample was collected from 105 patients with coronary atherosclerosis and 105 healthy controls. Pyrosequencing methylation was used to detect LINE-1 methylation level. Polymerase chain reaction-restriction enzyme fragment length polymorphism (PCR-RFLP) was used to test MTHFR. Results LINE-1 methylation level in the patient group was significantly lower than in the controls (t=5.007, P<0.001). MTHFR C667T genotype distribution presented marked differences in the 2 groups. TT genotype carriers had significantly increased risk of atherosclerosis (OR=3.56, P=0.009). Three different genotypes of MTHFR C667T loci showed different LINE-1 methylation level between the 2 groups (P<0.01). LINE-1 methylation level in TT and CT genotype carriers was obviously lower than in CC genotype carriers (P<0.05). Conclusions MTHFR C667T loci polymorphism may affect atherosclerosis by regulating genome methylation level. PMID:26828698

  4. Expression, polymorphism and methylation pattern of interleukin-6 in periodontal tissues.

    PubMed

    Stefani, Florença Abdanur; Viana, Michelle Beatriz; Dupim, Ana Carolina; Brito, João Artur Ricieri; Gomez, Ricardo Santiago; da Costa, José Eustáquio; Moreira, Paula Rocha

    2013-07-01

    Periodontitis is considered an inflammatory disorder of bacterial etiology that results in periodontal tissue destruction, as a result of complex interactions between periodontal pathogens, host and immune response. Genetic and epigenetic mechanisms may modulate the individual response since it is able to influence the gene expression. The aim of this study was to evaluate the impact of -174 G/C polymorphism and the methylation status of the promoter region of IL-6 gene on the expression of IL-6 in gingival samples from individuals with chronic periodontitis. Gingival biopsies were collected from 21 patients with chronic periodontitis and 21 controls. Histologic sections stained by hematoxylin-eosin were used for histopathological evaluation. The IL-6 gene expression was assessed by quantitative real-time PCR. The polymorphism IL-6 -174 C/G was studied by polymerase chain reaction (PCR) amplification and restriction endonuclease digestion (HspII). Methylation-specific polymerase chain reaction was used to verify the DNA methylation pattern. The number of inflammatory cells in tissue fragments from individuals with chronic periodontitis was higher than in the control group and the inflammatory infiltrate was predominantly mononuclear. The expression of IL-6 was higher in the group with periodontitis. In polymorphism assay, no statistical difference in the distribution of genotypes and alleles in both groups were observed. The most of samples were partially methylated. No difference was observed in methylation pattern from two different regions of the IL-6 gene among groups. The high expression of IL-6 is an important factor related to chronic periodontitis, but was not associated with methylation status or the -174 (G/C) genetic polymorphism, suggesting that other mechanisms are involved in this gene transcription regulation.

  5. Pervasive polymorphic imprinted methylation in the human placenta

    PubMed Central

    Hanna, Courtney W.; Peñaherrera, Maria S.; Saadeh, Heba; Andrews, Simon; McFadden, Deborah E.; Kelsey, Gavin; Robinson, Wendy P.

    2016-01-01

    The maternal and paternal copies of the genome are both required for mammalian development, and this is primarily due to imprinted genes, those that are monoallelically expressed based on parent-of-origin. Typically, this pattern of expression is regulated by differentially methylated regions (DMRs) that are established in the germline and maintained after fertilization. There are a large number of germline DMRs that have not yet been associated with imprinting, and their function in development is unknown. In this study, we developed a genome-wide approach to identify novel imprinted DMRs in the human placenta and investigated the dynamics of these imprinted DMRs during development in somatic and extraembryonic tissues. DNA methylation was evaluated using the Illumina HumanMethylation450 array in 134 human tissue samples, publicly available reduced representation bisulfite sequencing in the human embryo and germ cells, and targeted bisulfite sequencing in term placentas. Forty-three known and 101 novel imprinted DMRs were identified in the human placenta by comparing methylation between diandric and digynic triploid conceptions in addition to female and male gametes. Seventy-two novel DMRs showed a pattern consistent with placental-specific imprinting, and this monoallelic methylation was entirely maternal in origin. Strikingly, these DMRs exhibited polymorphic imprinted methylation between placental samples. These data suggest that imprinting in human development is far more extensive and dynamic than previously reported and that the placenta preferentially maintains maternal germline-derived DNA methylation. PMID:26769960

  6. High-speed droplet-allele-specific polymerase chain reaction for genotyping of single nucleotide polymorphisms.

    PubMed

    Matsuda, Kazuyuki; Honda, Takayuki

    2015-01-01

    Single nucleotide alternations such as single nucleotide polymorphisms (SNPs) or single nucleotide mutations are useful genetic markers for molecular diagnosis, prognosis, drug response, and predisposition to diseases. Rapid identification of SNPs or mutations is clinically important, especially for determining drug responses and selection of molecular-targeted therapy. Here, we describe a rapid genotyping assay based on the allele-specific polymerase chain reaction (AS-PCR) by using our droplet-PCR machine (droplet-AS-PCR).

  7. Genetic polymorphism of toll-like receptors 4 gene by polymerase chain reaction-restriction fragment length polymorphisms, polymerase chain reaction-single-strand conformational polymorphism to correlate with mastitic cows

    PubMed Central

    Gupta, Pooja H.; Patel, Nirmal A.; Rank, D. N.; Joshi, C. G.

    2015-01-01

    Aim: An attempt has been made to study the toll-like receptors 4 (TLR4) gene polymorphism from cattle DNA to correlate with mastitis cows. Materials and Methods: In present investigation, two fragments of TLR4 gene named T4CRBR1 and T4CRBR2 of a 316 bp and 382 bp were amplified by polymerase chain reaction (PCR), respectively from Kankrej (22) and Triple cross (24) cattle. The genetic polymorphisms in the two populations were detected by a single-strand conformational polymorphism in the first locus and by digesting the fragments with restriction endonuclease Alu I in the second one. Results: Results showed that both alleles (A and B) of two loci were found in all the two populations and the value of polymorphism information content indicated that these were highly polymorphic. Statistical results of χ2 test indicated that two polymorphism sites in the two populations fit with Hardy–Weinberg equilibrium (p<0.05). Meanwhile, the effect of polymorphism of TLR4 gene on the somatic cell score (SCS) indicated the cattle with allele a in T4CRBR1 showed lower SCS than that of allele B (p<0.05). Thus, the allele A might play an important role in mastitis resistance in cows. Conclusion: The relationship between the bovine mastitis trait and the polymorphism of TLR4 gene indicated that the bovine TLR4 gene may play an important role in mastitis resistance. PMID:27047144

  8. Folic acid, polymorphism of methyl-group metabolism genes, and DNA methylation in relation to GI carcinogenesis.

    PubMed

    Fang, Jing Yuan; Xiao, Shu Dong

    2003-01-01

    DNA methylation is the main epigenetic modification after replication in humans. DNA (cytosine-5)-methyltransferase (DNMT) catalyzes the transfer of a methyl group from S-adenosyl-L-methionine (SAM) to C5 of cytosine within CpG dinucleotide sequences in the genomic DNA of higher eukaryotes. There is considerable evidence that aberrant DNA methylation plays an integral role in carcinogenesis. Folic acid or folate is crucial for normal DNA synthesis and can regulate DNA methylation, and through this, it affects cellular SAM levels. Folate deficiency results in DNA hypomethylation. Epidemiological studies have indicated that folic acid protects against gastrointestinal (GI) cancers. Methylene-tetrahydrofolate reductase (MTHFR) and methionine synthase (MS) are the enzymes involved in folate metabolism and are thought to influence DNA methylation. MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level. Two common MTHFR polymorphisms, 677CT (or 677TT) and A1298C, and an MS polymorphism, A-->G at 2756, have been identified. Most studies support an inverse association between folate status and the rate of colorectal adenomas and carcinomas. During human GI carcinogenesis, MTHFR is highly polymorphic, and the variant genotypes result in decreased MTHFR enzyme activity and lower plasma folate level, as well as aberrant methylation.

  9. The CAG repeat polymorphism of mitochondrial polymerase gamma (POLG) is associated with male infertility in Tunisia.

    PubMed

    Baklouti-Gargouri, S; Ghorbel, M; Chakroun, N; Sellami, A; Fakhfakh, F; Ammar-Keskes, L

    2012-05-01

    Male fertility largely depends on sperm quality, which may be affected by environmental and genetic factors. Recent data emphasised the implication of the polymorphism of mitochondrial DNA polymerase gamma (POLG) CAG repeats in male infertility. In this report, we explored a possible role of the (POLG) gene polymorphism in male infertility in Tunisian men. The polymorphic CAG repeat in the nuclear POLG gene was studied in 339 male subjects (216 patients with infertility (69 azoospermic, 115 oligoasthenoteratospermic and 32 normospermic) and 123 fertile) after DNA amplification by PCR, followed by genotyping using an automatic sequencer. The heterozygous and the homozygous mutant genotypes (10/ ≠ 10 and ≠ 10/ ≠ 10) were significantly more frequent among infertile patients than among fertile controls (11.2% versus 1.6%, P = 1.3 × 10(-3) and 4.6% versus 0.8%, P = 4.2 × 10(-7) respectively). We also found a significant difference between the frequencies of 10/ ≠ 10 genotype in azoospermic (4.4%) and in oligoasthenoteratospermic (15.6%) infertile patients (P = 2.6 × 10(-2) ). However, the homozygous mutant genotype (≠ 10/ ≠ 10) was seen at similar frequencies in azoospermic, normospermic and oligoasthenospermic men (4.4%, 3.1% and 5.2% respectively). Under our conditions, the findings showed an association between POLG CAG repeat polymorphism and male infertility in Tunisian population.

  10. Detection of short tandem repeat polymorphisms from human nails using direct polymerase chain reaction method.

    PubMed

    Tie, Jian; Uchigasaki, Seisaku

    2014-11-01

    Human nail is an important forensic material for parental testing and individual identification in large-scale disasters. Detection of STR polymorphism from hard tissues generally requires DNA purification, which is technically complicated and time consuming. In the present study, we attempted to detect STR polymorphisms from untreated human nail samples by direct PCR amplification method using the primer mixture supplied with the GenePrint® SilverSTR® III System or the AmpFℓSTR® Identifiler® PCR Amplification Kit, and Tks Gflex DNA polymerase known to be effective for amplification from crude samples. A nail fragment measuring approximately 1.5 mm in breadth and 0.5 mm in length was placed directly into a PCR tube, and various PCR conditions were tested. The PCR products were analyzed by denaturing acrylamide gel electrophoresis or CE. Multiple STR polymorphisms were detected successfully. This method that detects STR polymorphisms not only from fresh human fingernails, but also from old nail fragments stored at room temperature for up to 10 years is expected to become a novel DNA analytical method in forensic medicine and genetic studies.

  11. Glutamine methylation in histone H2A is an RNA-polymerase-I-dedicated modification

    NASA Astrophysics Data System (ADS)

    Tessarz, Peter; Santos-Rosa, Helena; Robson, Sam C.; Sylvestersen, Kathrine B.; Nelson, Christopher J.; Nielsen, Michael L.; Kouzarides, Tony

    2014-01-01

    Nucleosomes are decorated with numerous post-translational modifications capable of influencing many DNA processes. Here we describe a new class of histone modification, methylation of glutamine, occurring on yeast histone H2A at position 105 (Q105) and human H2A at Q104. We identify Nop1 as the methyltransferase in yeast and demonstrate that fibrillarin is the orthologue enzyme in human cells. Glutamine methylation of H2A is restricted to the nucleolus. Global analysis in yeast, using an H2AQ105me-specific antibody, shows that this modification is exclusively enriched over the 35S ribosomal DNA transcriptional unit. We show that the Q105 residue is part of the binding site for the histone chaperone FACT (facilitator of chromatin transcription) complex. Methylation of Q105 or its substitution to alanine disrupts binding to FACT in vitro. A yeast strain mutated at Q105 shows reduced histone incorporation and increased transcription at the ribosomal DNA locus. These features are phenocopied by mutations in FACT complex components. Together these data identify glutamine methylation of H2A as the first histone epigenetic mark dedicated to a specific RNA polymerase and define its function as a regulator of FACT interaction with nucleosomes.

  12. [Analysis of genomic DNA methylation level in radish under cadmium stress by methylation-sensitive amplified polymorphism technique].

    PubMed

    Yang, Jin-Lan; Liu, Li-Wang; Gong, Yi-Qin; Huang, Dan-Qiong; Wang, Feng; He, Ling-Li

    2007-06-01

    The level of cytosine methylation induced by cadmium in radish (Raphanus sativus L.) genome was analysed using the technique of methylation-sensitive amplified polymorphism (MSAP). The MSAP ratios in radish seedling exposed to cadmium chloride at the concentration of 50, 250 and 500 mg/L were 37%, 43% and 51%, respectively, and the control was 34%; the full methylation levels (C(m)CGG in double strands) were at 23%, 25% and 27%, respectively, while the control was 22%. The level of increase in MSAP and full methylation indicated that de novo methylation occurred in some 5'-CCGG sites under Cd stress. There was significant positive correlation between increase of total DNA methylation level and CdCl(2) concentration. Four types of MSAP patterns: de novo methylation, de-methylation, atypical pattern and no changes of methylation pattern were identified among CdCl(2) treatments and the control. DNA methylation alteration in plants treated with CdCl(2) was mainly through de novo methylation.

  13. Polymorphism of inflammatory genes and arsenic methylation capacity are associated with urothelial carcinoma

    SciTech Connect

    Wu, Chia-Chang; Huang, Yung-Kai; Chung, Chi-Jung; Huang, Chao-Yuan; Pu, Yeong-Shiau; Shiue, Horng-Sheng; Lai, Li-An; Lin, Ying-Chin; Su, Chien-Tien; Hsueh, Yu-Mei

    2013-10-01

    Chronic exposure to arsenic can generate reactive oxidative species, which can induce certain proinflammatory cytokines such as tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6) and interleukin-8 (IL-8). TNF-α, IL-6 and IL-8 have been shown to be involved in the development and progression of various cancers, including bladder cancer. This study aimed to investigate the joint effect of the polymorphism of TNF-α − 308 G/A, IL-6 − 174 G/C, IL-8 − 251 T/A and urinary arsenic profiles on urothelial carcinoma (UC) risk. This study evaluated 300 pathologically-confirmed cases of UC and 594 cancer-free controls. Urinary arsenic species were detected using high-performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. The polymorphism of TNF-α − 308 G/A, IL-6 − 174 G/C and IL-8 − 251 T/A was determined using polymerase chain reaction-restriction fragment length polymorphism. The joint effects on UC risk were estimated by odds ratios and 95% confidence intervals using unconditional logistic regression. We found that the TNF-α − 308 A/A and IL-8 − 251 T/T polymorphisms were significantly associated with UC. Moreover, significant dose–response joint effect of TNF-α − 308 A/A or IL-8 − 251 T/T genotypes and arsenic methylation indices were seen to affect UC risk. The present results also showed a significant increase in UC risk in subjects with the IL-8 − 251 T/T genotype for each SD increase in urinary total arsenic and MMA%. In contrast, a significant decrease in UC risk was found in subjects who carried the IL-8 − 251 T/T genotype for each SD increase in DMA%. - Highlights: • Joint effect of the TNF-α -308 A/A genotype and urinary total arsenic affected UC. • Joint effect of the IL-8 -251 T/T genotype and urinary total arsenic affected UC. • Urinary total arsenic level, TNF-α -308 A/A and IL-8 -251 T/T genotype affected UC.

  14. Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells

    PubMed Central

    Dias, João D; Rito, Tiago; Torlai Triglia, Elena; Kukalev, Alexander; Ferrai, Carmelo; Chotalia, Mita; Brookes, Emily; Kimura, Hiroshi; Pombo, Ana

    2015-01-01

    Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels. DOI: http://dx.doi.org/10.7554/eLife.11215.001 PMID:26687004

  15. A Germline Polymorphism of DNA Polymerase Beta Induces Genomic Instability and Cellular Transformation

    PubMed Central

    Keh, Agnes; Sweasy, Joann B.

    2012-01-01

    Several germline single nucleotide polymorphisms (SNPs) have been identified in the POLB gene, but little is known about their cellular and biochemical impact. DNA Polymerase β (Pol β), encoded by the POLB gene, is the main gap-filling polymerase involved in base excision repair (BER), a pathway that protects the genome from the consequences of oxidative DNA damage. In this study we tested the hypothesis that expression of the POLB germline coding SNP (rs3136797) in mammalian cells could induce a cancerous phenotype. Expression of this SNP in both human and mouse cells induced double-strand breaks, chromosomal aberrations, and cellular transformation. Following treatment with an alkylating agent, cells expressing this coding SNP accumulated BER intermediate substrates, including single-strand and double-strand breaks. The rs3136797 SNP encodes the P242R variant Pol β protein and biochemical analysis showed that P242R protein had a slower catalytic rate than WT, although P242R binds DNA similarly to WT. Our results suggest that people who carry the rs3136797 germline SNP may be at an increased risk for cancer susceptibility. PMID:23144635

  16. Molecular identification of Amazonian stingless bees using polymerase chain reaction single-strand conformation polymorphism.

    PubMed

    Souza, M T; Carvalho-Zilse, G A

    2014-07-25

    In countries containing a mega diversity of wildlife, such as Brazil, identifying and characterizing biological diversity is a continuous process for the scientific community, even in face of technological and scientific advances. This activity demands initiatives for the taxonomic identification of highly diverse groups, such as stingless bees, including molecular analysis strategies. This type of bee is distributed in all of the Brazilian states, with the highest species diversity being found in the State of Amazônia. However, the estimated number of species diverges among taxonomists. These bees are considered the main pollinators in the Amazon rainforest, in which they obtain food and shelter; however, their persistence is constantly threatened by deforestation pressure. Hence, it is important to classify the number and abundance of bee specie, to measure their decline and implement meaningful, priority conservation strategies. This study aims to maximize the implementation of more direct, economic and successful techniques for the taxonomic identification of stingless bees. Specifically, the genes 16S rRNA and COI from mitochondrial DNA were used as molecular markers to differentiate 9 species of Amazonian stingless bees based on DNA polymorphism, using the polymerase chain reaction-single-strand conformation polymorphism technique. We registered different, exclusive SSCP haplotypes for both genes in all species analyzed. These results demonstrate that SSCP is a simple and cost-effective technique that is applicable to the molecular identification of stingless bee species.

  17. Analysis of polymorphism in the bovine casein genes by use of the polymerase chain reaction.

    PubMed

    Pinder, S J; Perry, B N; Skidmore, C J; Savva, D

    1991-01-01

    Methods have been devised for detecting polymorphisms in the bovine beta- and kappa-casein genes using the polymerase chain reaction (PCR) followed either by restriction enzyme digestion (to reveal a restriction fragment length polymorphism (RFLP] or by hybridization of an allele-specific oligonucleotide. These methods, as well as being faster and more sensitive than traditional RFLP methods, are of more general applicability since they can detect any change in DNA sequence. They require only a small sample of blood or semen and are applicable to animals of any age or sex. These methods make possible large-scale screening and thus selection for alleles at these loci. Typing of blood DNA can give erroneous results when the animal concerned is a twin; however, this can be overcome by retesting using milk or semen. Analysis of the kappa-casein genotype of Holstein-Friesian bulls gives frequencies for the A and B alleles of 0.80 and 0.20 respectively. Selection in favour of the B allele, which is superior for cheese production, could thus have a large effect. The A3 and B alleles at the beta-casein locus have been shown to be rare in the Holstein-Friesian population. Linkage disequilibrium exists between beta-casein B and kappa-casein B.

  18. Molecular identification of Amazonian stingless bees using polymerase chain reaction single-strand conformation polymorphism.

    PubMed

    Souza, M T; Carvalho-Zilse, G A

    2014-01-01

    In countries containing a mega diversity of wildlife, such as Brazil, identifying and characterizing biological diversity is a continuous process for the scientific community, even in face of technological and scientific advances. This activity demands initiatives for the taxonomic identification of highly diverse groups, such as stingless bees, including molecular analysis strategies. This type of bee is distributed in all of the Brazilian states, with the highest species diversity being found in the State of Amazônia. However, the estimated number of species diverges among taxonomists. These bees are considered the main pollinators in the Amazon rainforest, in which they obtain food and shelter; however, their persistence is constantly threatened by deforestation pressure. Hence, it is important to classify the number and abundance of bee specie, to measure their decline and implement meaningful, priority conservation strategies. This study aims to maximize the implementation of more direct, economic and successful techniques for the taxonomic identification of stingless bees. Specifically, the genes 16S rRNA and COI from mitochondrial DNA were used as molecular markers to differentiate 9 species of Amazonian stingless bees based on DNA polymorphism, using the polymerase chain reaction-single-strand conformation polymorphism technique. We registered different, exclusive SSCP haplotypes for both genes in all species analyzed. These results demonstrate that SSCP is a simple and cost-effective technique that is applicable to the molecular identification of stingless bee species. PMID:25117306

  19. Association Between Single Nucleotide Polymorphisms in DNA Polymerase Kappa Gene and Breast Cancer Risk in Chinese Han Population

    PubMed Central

    Dai, Zhi-Jun; Liu, Xing-Han; Ma, Yun-Feng; Kang, Hua-Feng; Jin, Tian-Bo; Dai, Zhi-Ming; Guan, Hai-Tao; Wang, Meng; Liu, Kang; Dai, Cong; Yang, Xue-Wen; Wang, Xi-Jing

    2016-01-01

    Abstract DNA polymerases are responsible for ensuring stability of the genome and avoiding genotoxicity caused by a variety of factors during DNA replication. Consequently, these proteins have been associated with an increased cancer risk. DNA polymerase kappa (POLK) is a specialized DNA polymerase involved in translesion DNA synthesis (TLS) that allows DNA synthesis over the damaged DNA. Recently, some studies investigated relationships between POLK polymorphisms and cancer risk, but the role of POLK genetic variants in breast cancer (BC) remains to be defined. In this study, we aimed to evaluate the effects of POLK polymorphisms on BC risk. We used the Sequenom MassARRAY method to genotype 3 single nucleotide polymorphisms (SNPs) in POLK (rs3213801, rs10077427, and rs5744533), in order to determine the genotypes of 560 BC patients and 583 controls. The association of genotypes and BC was assessed by computing the odds ratio (OR) and 95% confidence intervals (95% CIs) from logistic regression analyses. We found a statistically significant difference between patient and control groups in the POLK rs10077427 genotypic groups, excluding the recessive model. A positive correlation was also found between positive progesterone receptor (PR) status, higher Ki67 index, and rs10077427 polymorphism. For rs5744533 polymorphism, the codominant, dominant, and allele models frequencies were significantly higher in BC patients compared to healthy controls. Furthermore, our results indicated that rs5744533 SNP has a protective role in the postmenopausal women. However, we failed to find any associations between rs3213801 polymorphism and susceptibility to BC. Our results indicate that POLK polymorphisms may influence the risk of developing BC, and, because of this, may serve as a prognostic biomarker among Chinese women. PMID:26765445

  20. A polymerase chain reaction-based method for constructing a linear vector with site-specific DNA methylation.

    PubMed

    Arakawa, Toshiya; Ohta, Tohru; Abiko, Yoshihiro; Okayama, Miki; Mizoguchi, Itaru; Takuma, Taishin

    2011-09-15

    DNA methylation is an important epigenetic modification that leads to a wide variety of biological functions, including transcription, growth and development, and diseases associated with altered gene expression such as cancers. However, tools to insert site-specific methylation into DNA for analyzing epigenetic functions are limited. Here we describe a novel polymerase chain reaction (PCR)-based approach to provide site-specific DNA methylation at any site, including CpG or CpNpG islands. This method is simple and versatile, and it consists of four steps to construct the DNA methylation vector: (I) design and synthesis of methylated primers, (II) PCR amplification, (III) isolation of single-stranded DNA, and (IV) annealing and ligation of isolated single-stranded DNAs. First we produced and validated a linear green fluorescence protein (GFP) vector by this method. Next we applied this method to introduce methyl groups into the promoter of the cyclooxygenase-2 (COX-2) gene and found that site-specific DNA methylation at the CRE element significantly altered COX-2 gene expression. These results demonstrate that this PCR-based approach is useful for the analysis of biological functions that depend on DNA methylation. PMID:21669180

  1. PHF8 Targets Histone Methylation and RNA Polymerase II To Activate Transcription▿ †

    PubMed Central

    Fortschegger, Klaus; de Graaf, Petra; Outchkourov, Nikolay S.; van Schaik, Frederik M. A.; Timmers, H. T. Marc; Shiekhattar, Ramin

    2010-01-01

    Mutations in PHF8 are associated with X-linked mental retardation and cleft lip/cleft palate. PHF8 contains a plant homeodomain (PHD) in its N terminus and is a member of a family of JmjC domain-containing proteins. While PHDs can act as methyl lysine recognition motifs, JmjC domains can catalyze lysine demethylation. Here, we show that PHF8 is a histone demethylase that removes repressive histone H3 dimethyl lysine 9 marks. Our biochemical analysis revealed specific association of the PHF8 PHD with histone H3 trimethylated at lysine 4 (H3K4me3). Chromatin immunoprecipitation followed by high-throughput sequencing indicated that PHF8 is enriched at the transcription start sites of many active or poised genes, mirroring the presence of RNA polymerase II (RNAPII) and of H3K4me3-bearing nucleosomes. We show that PHF8 can act as a transcriptional coactivator and that its activation function largely depends on binding of the PHD to H3K4me3. Furthermore, we present evidence for direct interaction of PHF8 with the C-terminal domain of RNAPII. Importantly, a PHF8 disease mutant was defective in demethylation and in coactivation. This is the first demonstration of a chromatin-modifying enzyme that is globally recruited to promoters through its association with H3K4me3 and RNAPII. PMID:20421419

  2. Direct and site-specific quantification of RNA 2'-O-methylation by PCR with an engineered DNA polymerase.

    PubMed

    Aschenbrenner, Joos; Marx, Andreas

    2016-05-01

    Methylation of the 2'-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2'-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2'-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2'-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR. PMID:27016740

  3. Direct and site-specific quantification of RNA 2′-O-methylation by PCR with an engineered DNA polymerase

    PubMed Central

    Aschenbrenner, Joos; Marx, Andreas

    2016-01-01

    Methylation of the 2′-hydroxyl-group of ribonucleotides is found in all major classes of RNA in eukaryotes and is one of the most abundant posttranscriptional modifications of stable RNAs. In spite of intense studies, the multiple functions of RNA 2′-O-methylation are still not understood. One major obstacle in the field are the technical demanding detection methods, which are typically laborious and do not always deliver unambiguous results. We present a thermostable KlenTaq DNA polymerase variant with significant reverse transcription activity that is able to discriminate 2′-O-methylated from unmethylated RNAs. The engineered enzyme catalyzes DNA synthesis from DNA as well as RNA templates and enables expeditious quantification of 2′-O-methylation of individual nucleotides directly from total RNA extracts by a simple qRT-PCR. PMID:27016740

  4. Mutagenicity Assessment of Organophosphates using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Assay

    PubMed Central

    Bhinder, Preety; Chaudhry, Asha

    2013-01-01

    Objectives: In this study we have evaluated the mutagenicity of organophosphate pesticides acephate, chlorpyrifos, and profenofos using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay with the mosquito Culex quinquefasciatus taken as an experimental model. Materials and Methods: Second instar larvae were treated with LC20 of each pesticide for 24 h and mutations induced in the sequence of mitochondrial COII gene (690bp) were studied from restriction patterns generated with AluI, PacI, and PsiI restriction endonucleases. Results: Variations in the number and size of digested fragments were recorded from treated individuals compared with controls showing that the restriction enzymes created a cut at different locations. In addition, sequences of COII gene from control and treated individuals were also used to confirm the RFLP patterns. From the sequence alignment data, it was found that mutations caused the destruction and generation of restriction sites in the gene sequence of treated individuals. Conclusion: This study indicates that all the three pesticides had potential to induce mutations in the normal sequence of COII gene and also advocates the use of PCR-RFLP assay as an efficient, rapid, and sensitive technique to detect mutagenicity of pesticides. PMID:24403735

  5. [Relationship between Arsenic (+3 Oxidation State) Methyltransferase Genetic Polymorphisms and Methylation Capacity of Inorganic Arsenic].

    PubMed

    Agusa, Tetsuro; Kunito, Takashi; Minh Tue, Nguyen; Thi Mai Lan, Vi; Binh Minh, Tu; Thi Kim Trang, Pham; Fujihara, Junko; Takeshita, Haruo; Takahashi, Shin; Hung Viet, Pham; Tanabe, Shinsuke; Iwata, Hisato

    2015-01-01

    Arsenic metabolism affects the susceptibility of humans to arsenic toxicity; therefore, clarification of the factors associated with individual variations in arsenic metabolism is an important task. Genetic polymorphisms such as single nucleotide polymorphisms (SNPs) in arsenic (+3 oxidation state) methyltransferase (AS3MT), which can methylate arsenic compounds using S-adenosyl-l-methionine (AdoMet), have been reported to modify arsenic methylation. In this review, we summarize studies conducted by us in Vietnam and by others on the association of AS3MT genetic polymorphisms with arsenic metabolism as well as human health effects. Most of the SNPs in AS3MT showed inconsistent results in terms of genotype-dependent differences in arsenic metabolism among the studies. However, AS3MT 12390 (rs3740393) and 14458 (rs11191439) were consistently related to arsenic methylation regardless of the study population: AS3MT 12390 (rs3740393) affected the second step of methylation of arsenic, whereas 14458 (rs11191439) affected the first methylation step.

  6. [Relationship between Arsenic (+3 Oxidation State) Methyltransferase Genetic Polymorphisms and Methylation Capacity of Inorganic Arsenic].

    PubMed

    Agusa, Tetsuro; Kunito, Takashi; Minh Tue, Nguyen; Thi Mai Lan, Vi; Binh Minh, Tu; Thi Kim Trang, Pham; Fujihara, Junko; Takeshita, Haruo; Takahashi, Shin; Hung Viet, Pham; Tanabe, Shinsuke; Iwata, Hisato

    2015-01-01

    Arsenic metabolism affects the susceptibility of humans to arsenic toxicity; therefore, clarification of the factors associated with individual variations in arsenic metabolism is an important task. Genetic polymorphisms such as single nucleotide polymorphisms (SNPs) in arsenic (+3 oxidation state) methyltransferase (AS3MT), which can methylate arsenic compounds using S-adenosyl-l-methionine (AdoMet), have been reported to modify arsenic methylation. In this review, we summarize studies conducted by us in Vietnam and by others on the association of AS3MT genetic polymorphisms with arsenic metabolism as well as human health effects. Most of the SNPs in AS3MT showed inconsistent results in terms of genotype-dependent differences in arsenic metabolism among the studies. However, AS3MT 12390 (rs3740393) and 14458 (rs11191439) were consistently related to arsenic methylation regardless of the study population: AS3MT 12390 (rs3740393) affected the second step of methylation of arsenic, whereas 14458 (rs11191439) affected the first methylation step. PMID:26411936

  7. DNA Methyltransferase Candidate Polymorphisms, Imprinting Methylation, and Birth Outcome

    PubMed Central

    Haggarty, Paul; Hoad, Gwen; Horgan, Graham W.; Campbell, Doris M.

    2013-01-01

    Background Birth weight and prematurity are important obstetric outcomes linked to lifelong health. We studied a large birth cohort to look for evidence of epigenetic involvement in birth outcomes. Methods We investigated the association between birth weight, length, placental weight and duration of gestation and four candidate variants in 1,236 mothers and 1,073 newborns; DNMT1 (rs2162560), DNMT3A (rs734693), DNMT3B (rs2424913) and DNMT3L (rs7354779). We measured methylation of LINE1 and the imprinted genes, PEG3, SNRPN, and IGF2, in cord blood. Results The minor DNMT3L allele in the baby was associated with higher birth weight (+54 95% CI 10,99 g; p = 0.016), birth length (+0.23 95% CI 0.04,0.42 cm; p = 0.017), placental weight, (+18 95% CI 3,33 g; p = 0.017), and reduced risk of being in the lowest birth weight decile (p = 0.018) or requiring neonatal care (p = 0.039). The DNMT3B minor allele in the mother was associated with an increased risk of prematurity (p = 0.001). Placental size was related to PEG3 (p<0.001) and IGF2 (p<0.001) methylation. Birth weight was related to LINE1 and IGF2 methylation but only at p = 0.052. The risk of requiring neonatal treatment was related to LINE1 (p = 0.010) and SNRPN (p = 0.001) methylation. PEG3 methylation was influenced by baby DNMT3A genotype (p = 0.012) and LINE1 by baby 3B genotype (p = 0.044). Maternal DNMT3L genotype was related to IGF2 methylation in the cord blood but this effect was only seen in carriers of the minor frequency allele (p = 0.050). Conclusions The results here suggest that epigenetic processes are linked birth outcome and health in early life. Our emerging understanding of the role of epigenetics in health and biological function across the lifecourse suggests that these early epigenetic events could have longer term implications. PMID:23922667

  8. AP endonuclease knockdown enhances methyl methanesulfonate hypersensitivity of DNA polymerase β knockout mouse embryonic fibroblasts.

    PubMed

    Yamamoto, Ryohei; Umetsu, Makio; Yamamoto, Mizuki; Matsuyama, Satoshi; Takenaka, Shigeo; Ide, Hiroshi; Kubo, Kihei

    2015-05-01

    Apurinic/apyrimidinic (AP) endonuclease (Apex) is required for base excision repair (BER), which is the major mechanism of repair for small DNA lesions such as alkylated bases. Apex incises the DNA strand at an AP site to leave 3'-OH and 5'-deoxyribose phosphate (5'-dRp) termini. DNA polymerase β (PolB) plays a dominant role in single nucleotide (Sn-) BER by incorporating a nucleotide and removing 5'-dRp. Methyl methanesulfonate (MMS)-induced damage is repaired by Sn-BER, and thus mouse embryonic fibroblasts (MEFs) deficient in PolB show significantly increased sensitivity to MMS. However, the survival curve for PolB-knockout MEFs (PolBKOs) has a shoulder, and increased sensitivity is only apparent at relatively high MMS concentrations. In this study, we prepared Apex-knockdown/PolB-knockout MEFs (AKDBKOs) to examine whether BER is related to the apparent resistance of PolBKOs at low MMS concentrations. The viability of PolBKOs immediately after MMS treatment was significantly lower than that of wild-type MEFs, but there was essentially no effect of Apex-knockdown on cell viability in the presence or absence of PolB. In contrast, relative counts of MEFs after repair were decreased by Apex knockdown. Parental PolBKOs showed especially high sensitivity at >1.5 mM MMS, suggesting that PolBKOs have another repair mechanism in addition to PolB-dependent Sn-BER, and that the back-up mechanism is unable to repair damage induced by high MMS concentrations. Interestingly, AKDBKOs were hypersensitive to MMS in a relative cell growth assay, suggesting that MMS-induced damage in PolB-knockout MEFs is repaired by Apex-dependent repair mechanisms, presumably including long-patch BER.

  9. Genetic relationships among Aedes aegypti (Diptera: Culicidae) populations from Argentina using random amplified polymorphic DNA polymerase chain reaction markers.

    PubMed

    de Sousa, G B; Blanco, A; Gardenal, C N

    2001-05-01

    Random amplified polymorphic DNA polymerase chain reaction (RAPD-PCR) polymorphism was analyzed in five Aedes aegypti (L.) populations from Argentina and one from Puerto Rico to estimate levels of intraspecific polymorphism and genetic relatedness. Allele frequencies were estimated assuming that RAPD products segregate as dominants and that genotype frequencies at those loci are in Hardy-Weinberg equilibrium. Mean expected heterozygosity (He) was 0.350; F(ST) values were significant at all loci except one, supporting the usefulness of the fragments used here to discriminate among populations. Rogers' genetic similarity between samples ranged from 0.806 to 0.621. The population from Puerto Rico was the most different from the Argentina populations. Considering that Ae. aegypti eggs, larvae, and pupae can be transported easily, relationships among the Argentinian populations may reflect the routes and intensity of commercial transit. PMID:11372960

  10. Amplification refractory mutation system polymerase chain reaction versus optimized polymerase chain reaction restriction-fragment length polymorphism for apolipoprotein E genotyping of majorly depressed patients.

    PubMed

    You, Hongmin; Chen, Jin; Zhou, Jingjing; Huang, Hua; Pan, Junxi; Wang, Ziye; Lv, Lin; Zhang, Lujun; Li, Juan; Qin, Bin; Yang, Yongtao; Xie, Peng

    2015-11-01

    Major depressive disorder (MDD) is a prevalent, debilitating mood disorder that has been associated with several genetic polymorphisms. One such polymorphism, namely that of apolipoprotein E (APOE), has three allelic forms (ε2, ε3 and ε4) that encode for six unique isoforms of the APOE protein. A growing number of techniques have been developed for APOE genotyping; however, not all polymerase chain reaction (PCR)‑based genotyping techniques are equally accurate or cost‑effective. In order to find a more accurate and cost‑effective APOE genotyping method for MDD screening in large populations, the present study comparatively evaluated two genotyping methods, amplification refractory mutation system PCR (ARMS‑PCR) and optimized PCR restriction‑fragment length polymorphism (PCR‑RFLP), in blood samples taken from a population of 708 MDD patients. Although either of the two methods were able to detect all six unique APOE genotypes, comparisons of the two methods with Sanger sequencing demonstrated that ARMS‑PCR (94%) was significantly more accurate than optimized PCR‑RFLP (82%). ARMS‑PCR should prove useful in quickly verifying ambiguous results obtained by other APOE genotyping methods and can be cost-effectively performed in the setting of a small laboratory or a population-based screening program.

  11. DNA Methylation and Methylation Polymorphism in Genetically Stable In vitro Regenerates of Jatropha curcas L. Using Methylation-Sensitive AFLP Markers.

    PubMed

    Rathore, Mangal S; Jha, Bhavanath

    2016-03-01

    The present investigation aimed to evaluate the degree and pattern of DNA methylation using methylation-sensitive AFLP (MS-AFLP) markers in genetically stable in vitro regenerates of Jatropha curcas L.. The genetically stable in vitro regenerates were raised through direct organogenesis via enhanced axillary shoot bud proliferation (Protocol-1) and in vitro-derived leaf regeneration (Protocol-2). Ten selective combinations of MS-AFLP primers produced 462 and 477 MS-AFLP bands in Protocol-1 (P-1) and Protocol-2 (P-2) regenerates, respectively. In P-1 regenerates, 15.8-31.17 % DNA was found methylated with an average of 25.24 %. In P-2 regenerates, 15.93-32.7 % DNA was found methylated with an average of 24.11 %. Using MS-AFLP in P-1 and P-2 regenerates, 11.52-25.53 % and 13.33-25.47 % polymorphism in methylated DNA was reported, respectively. Compared to the mother plant, P-1 regenerates showed hyper-methylation while P-2 showed hypo-methylation. The results clearly indicated alternation in degree and pattern of DNA methylation; hence, epigenetic instability in the genetically stable in vitro regenerates of J. curcas, developed so far using two different regeneration systems and explants of two different origins. The homologous nucleotide fragments in genomes of P-1 and P-2 regenerates showing methylation re-patterning might be involved in immediate adaptive responses and developmental processes through differential regulation of transcriptome under in vitro conditions. PMID:26588922

  12. On-chip detection of a single nucleotide polymorphism without polymerase amplification

    PubMed Central

    Han, Jinhee; Tan, Matthew; Sudheendra, Lakshmana; Weiss, Robert H.; Kennedy, Ian M.

    2014-01-01

    A nanoparticle-assembled photonic crystal (PC) array was used to detect single nucleotide polymorphism (SNP). The assay platform with PC nanostructure enhanced the fluorescent signal from nanoparticle-hybridized DNA complexes due to phase matching of excitation and emission. Nanoparticles coupled with probe DNA were trapped into nanowells in an array by using an electrophoretic particle entrapment system. The PC/DNA assay platform was able to identify a 1 base pair (bp) difference in synthesized nucleotide sequences that mimicked the mutation seen in a feline model of human autosomal dominant polycystic kidney disease (PKD) with a sensitivity of 0.9 fg/mL (50 aM)-sensitivity, which corresponds to 30 oligos/array. The reliability of the PC/DNA assay platform to detect SNP in a real sample was demonstrated by using genomic DNA (gDNA) extracted from the urine and blood of two PKD− wild type and three PKD positive cats. The standard curves for PKD positive (PKD+) and negative (PKD−) DNA were created using two feline-urine samples. An additional three urine samples were analyzed in a similar fashion and showed satisfactory agreement with the standard curve, confirming the presence of the mutation in affected urine. The limit of detection (LOD) was 0.005 ng/mL which corresponds to 6 fg per array for gDNA in urine and blood. The PC system demonstrated the ability to detect a number of genome equivalents for the PKD SNP that was very similar to the results reported with real time polymerase chain reaction (PCR). The favorable comparison with quantitative PCR suggests that the PC technology may find application well beyond the detection of the PKD SNP, into areas where a simple, cheap and portable nucleic acid analysis is desirable. PMID:25580203

  13. Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis.

    PubMed

    Santos, M S; Souza, E S; S Junior, R M; Talhari, S; Souza, J V B

    2010-08-01

    Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas--FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods.

  14. Identification of fungemia agents using the polymerase chain reaction and restriction fragment length polymorphism analysis.

    PubMed

    Santos, M S; Souza, E S; S Junior, R M; Talhari, S; Souza, J V B

    2010-08-01

    Prompt and specific identification of fungemia agents is important in order to define clinical treatment. However, in most cases conventional culture identification can be considered to be time-consuming and not without errors. The aim of the present study was to identify the following fungemia agents: Candida albicans, Candida parapsilosis, Candida tropicalis, Candida glabrata, Cryptococcus neoformans, Cryptococcus gattii, and Histoplasma capsulatum using the polymerase chain reaction and restriction fragment length polymorphism analysis (PCR/RFLP). More specifically: a) to evaluate 3 different amplification regions, b) to investigate 3 different restriction enzymes, and c) to use the best PCR/RFLP procedure to indentify 60 fungemia agents from a culture collection. All 3 pairs of primers (ITS1/ITS4, NL4/ITS5 and Primer1/Primer2) were able to amplify DNA from the reference strains. However, the size of these PCR products did not permit the identification of all the species studied. Three restriction enzymes were used to digest the PCR products: HaeIII, Ddel and Bfal. Among the combinations of pairs of primers and restriction enzymes, only one (primer pair NL4/ITS5 and restriction enzyme Ddel) produced a specific RFLP pattern for each microorganism studied. Sixty cultures of fungemia agents (selected from the culture collection of Fundação de Medicina Tropical do Amazonas--FMTAM) were correctly identified by PCR/RFLP using the prime pair NL4/ITS5 and Ddel. We conclude that the method proved to be both simple and reproducible, and may offer potential advantages over phenotyping methods. PMID:20640387

  15. Effects of As2O3 on DNA methylation, genomic instability, and LTR retrotransposon polymorphism in Zea mays.

    PubMed

    Erturk, Filiz Aygun; Aydin, Murat; Sigmaz, Burcu; Taspinar, M Sinan; Arslan, Esra; Agar, Guleray; Yagci, Semra

    2015-12-01

    Arsenic is a well-known toxic substance on the living organisms. However, limited efforts have been made to study its DNA methylation, genomic instability, and long terminal repeat (LTR) retrotransposon polymorphism causing properties in different crops. In the present study, effects of As2O3 (arsenic trioxide) on LTR retrotransposon polymorphism and DNA methylation as well as DNA damage in Zea mays seedlings were investigated. The results showed that all of arsenic doses caused a decreasing genomic template stability (GTS) and an increasing Random Amplified Polymorphic DNAs (RAPDs) profile changes (DNA damage). In addition, increasing DNA methylation and LTR retrotransposon polymorphism characterized a model to explain the epigenetically changes in the gene expression were also found. The results of this experiment have clearly shown that arsenic has epigenetic effect as well as its genotoxic effect. Especially, the increasing of polymorphism of some LTR retrotransposon under arsenic stress may be a part of the defense system against the stress.

  16. Methylenetetrahydrofolate reductase (MTHFR) polymorphisms and promoter methylation in cervical oncogenic lesions and cancer

    PubMed Central

    Botezatu, Anca; Socolov, Demetra; Iancu, Iulia V; Huica, Irina; Plesa, Adriana; Ungureanu, Carmen; Anton, Gabriela

    2013-01-01

    The aim of this study was to investigate the role of methylenetetrahydrofolate reductase (MTHFR) polymorphisms and MTHFR methylation pattern in cervical lesions development among women from Romania, a country with high prevalence of human papillomavirus (HPV) cervical infections. To achieve this goal, blood samples and cervical cytology specimens (n = 77)/tumour tissue specimens (n = 23) were investigated. As control, blood and negative cytological smears (n = 50) were used. A statistically significant association was found between T allele of C677T polymorphism and cervical lesions, heterozygote women presenting a threefold increased risk (normal/cervical lesions and tumours: wild homozygote 34/41 (0.68/0.41), heterozygote 14/51 (0.28/0.51), mutant homozygote 2/8 (0.04/0.08); OR = 3.081, P = 0.0035). Using χ square test for the control group, the HPV-negative and HPV-positive patients with cervix lesions, a significant correlation between viral infection and T allele of C677T polymorphism (P = 0.0287) was found. The MTHFR promoter was methylated in all HGSIL and tumour samples, significant differences being noted between HPV-positive samples, control group and cases of cervical dysplastic lesions without HPV DNA (P < 0. 0001) and between samples from patients with high-risk (hr)HPV versus low-risk (lr)HPV (P = 0.0026). No correlations between polymorphisms and methylation were observed. In Romania, individuals carrying T allele are susceptible for cervical lesions. MTHFR promoter methylation is associated with cervical severity lesions and with hrHPV. PMID:23444906

  17. Four Polymorphs of Methyl Paraben: Structural Relationships and Relative Energy Differences

    PubMed Central

    2013-01-01

    Four polymorphic forms of methyl paraben (methyl 4-hydroxybenzoate, 1), denoted 1-I (melting point 126 °C), 1-III (109 °C), 1-107 (107 °C), and 1-112 (112 °C), have been investigated by thermomicroscopy, infrared spectroscopy, and X-ray crystallography. The crystal structures of the metastable forms 1-III, 1-107, and 1-112 have been determined. All polymorphs contain the same O–H···O=C connected catemer motif, but the geometry of the resulting H-bonded chain is different in each form. The Z′ = 3 structure of 1-I (stable form; space group Cc) contains local symmetry elements. The crystal packing of each of the four known crystal structures of 1 was compared with the crystal structures of 12 chemical analogues. Close two-dimensional relationships exist between 1-112 and a form of methyl 4-aminobenzoate and between 1-107 and dimethyl terephthalate. The lattice energies of the four methyl paraben structures have been calculated with a range of methods based on ab initio electronic calculations on either the crystal or single molecule. This shows that the differences in the induction energy of the different hydrogen-bonded chain geometries have a significant effect on relative lattice energies, but that conformational energy, repulsion, dispersion, and electrostatic also contribute. PMID:23505337

  18. METHYLATION OF ARSENIC BY RECOMBINANT HUMAN AS3MT/287M AND AS3MT/287T POLYMORPHS

    EPA Science Inventory

    Arsenic (+3 oxidation state) methyltransferase (AS3MT) is the key enzyme in the pathway for methylation of inorganic arsenic (iAs). AS3MT polymorphism is, in part, responsible for interindividual differences in iAs metabolism. AS3MT/M287T polymorphism that is found in ~ 10% of C...

  19. Fibrillarin methylates H2A in RNA polymerase I trans-active promoters in Brassica oleracea

    PubMed Central

    Loza-Muller, Lloyd; Rodríguez-Corona, Ulises; Sobol, Margarita; Rodríguez-Zapata, Luis C.; Hozak, Pavel; Castano, Enrique

    2015-01-01

    Fibrillarin is a well conserved methyltransferase involved in several if not all of the more than 100 methylations sites in rRNA which are essential for proper ribosome function. It is mainly localized in the nucleoli and Cajal bodies inside the cell nucleus where it exerts most of its functions. In plants, fibrillarin binds directly the guide RNA together with Nop56, Nop58, and 15.5ka proteins to form a snoRNP complex that selects the sites to be methylated in pre-processing of ribosomal RNA. Recently, the yeast counterpart NOP1 was found to methylate histone H2A in the nucleolar regions. Here we show that plant fibrillarin can also methylate histone H2A. In Brassica floral meristem cells the methylated histone H2A is mainly localized in the nucleolus but unlike yeast or human cells it also localize in the periphery of the nucleus. In specialized transport cells the pattern is altered and it exhibits a more diffuse staining in the nucleus for methylated histone H2A as well as for fibrillarin. Here we also show that plant fibrillarin is capable of interacting with H2A and carry out its methylation in the rDNA promoter. PMID:26594224

  20. Arsenic methylation, GSTO1 polymorphisms, and metabolic syndrome in an arseniasis endemic area of southwestern Taiwan.

    PubMed

    Chen, Jein-Wen; Wang, Shu-Li; Wang, Ya-Hui; Sun, Chien-Wen; Huang, Yeou-Lih; Chen, Chien-Jen; Li, Wan-Fen

    2012-07-01

    Previous studies have shown that hair arsenic (As) levels are associated with an increased prevalence of metabolic syndrome (MetS), which is a strong predictor for type 2 diabetes. The objective of this study was to evaluate whether urinary arsenic methylation is related to MetS in an arseniasis endemic area of southwestern Taiwan, taking genetic factors into account. Subjects were from a community-based cohort recruited in 1990 from three villages in Putai Township. In 2002-2003, we successfully followed 247 subjects and measured their urinary arsenic species including inorganic arsenic, monomethylarsonic acid (MMA) and dimethylarsinic acid (DMA), as well as the coding region polymorphisms of three genes known to involve in arsenic methylation. Results showed that subjects of MetS had a history of consuming well water of higher arsenic concentration as compared to those without MetS. We also found a significant association between urinary arsenic species and risk for MetS, where the odds ratio of MetS was increased with decreasing proportion of MMA and low rate of primary methylation (defined as MMA/inorganic As). The increased risk associated with low primary methylation rate was further modified by the GSTO1 A140D polymorphism, with the D allele carriers showing a slightly higher risk for MetS. Our results suggest that a low MMA% is associated with increased risk for MetS among As-exposed subjects and the genetic polymorphism of GSTO1, an enzyme responsible for the reduction of pentavalent arsenic species, may also play a modest modification role.

  1. Genetic divergence between Mexican Opuntia accessions inferred by polymerase chain reaction-restriction fragment length polymorphism analysis.

    PubMed

    Samah, S; Valadez-Moctezuma, E; Peláez-Luna, K S; Morales-Manzano, S; Meza-Carrera, P; Cid-Contreras, R C

    2016-01-01

    Molecular methods are powerful tools in characterizing and determining relationships between plants. The aim of this study was to study genetic divergence between 103 accessions of Mexican Opuntia. To accomplish this, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of three chloroplast intergenic spacers (atpB-rbcL, trnL-trnF, and psbA-trnH), one chloroplast gene (ycf1), two nuclear genes (ppc and PhyC), and one mitochondrial gene (cox3) was conducted. The amplified products from all the samples had very similar molecular sizes, and there were only very small differences between the undigested PCR amplicons for all regions, with the exception of ppc. We obtained 5850 bp from the seven regions, and 136 fragments were detected with eight enzymes, 37 of which (27.2%) were polymorphic. We found that 40% of the fragments from the chloroplast regions were polymorphic, 9.8% of the bands detected in the nuclear genes were polymorphic, and 20% of the bands in the mitochondrial locus were polymorphic. trnL-trnF and psbA-trnH were the most variable regions. The Nei and Li/Dice distance was very short, and ranged from 0 to 0.12; indeed, 77 of the 103 genotypes had the same genetic profile. All the xoconostle accessions (acidic fruits) were grouped together without being separated from three genotypes of prickly pear (sweet fruits). We assume that the genetic divergence between prickly pears and xoconostles is very low, and question the number of Opuntia species currently considered in Mexico. PMID:27323120

  2. Genetic divergence between Mexican Opuntia accessions inferred by polymerase chain reaction-restriction fragment length polymorphism analysis.

    PubMed

    Samah, S; Valadez-Moctezuma, E; Peláez-Luna, K S; Morales-Manzano, S; Meza-Carrera, P; Cid-Contreras, R C

    2016-06-03

    Molecular methods are powerful tools in characterizing and determining relationships between plants. The aim of this study was to study genetic divergence between 103 accessions of Mexican Opuntia. To accomplish this, polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis of three chloroplast intergenic spacers (atpB-rbcL, trnL-trnF, and psbA-trnH), one chloroplast gene (ycf1), two nuclear genes (ppc and PhyC), and one mitochondrial gene (cox3) was conducted. The amplified products from all the samples had very similar molecular sizes, and there were only very small differences between the undigested PCR amplicons for all regions, with the exception of ppc. We obtained 5850 bp from the seven regions, and 136 fragments were detected with eight enzymes, 37 of which (27.2%) were polymorphic. We found that 40% of the fragments from the chloroplast regions were polymorphic, 9.8% of the bands detected in the nuclear genes were polymorphic, and 20% of the bands in the mitochondrial locus were polymorphic. trnL-trnF and psbA-trnH were the most variable regions. The Nei and Li/Dice distance was very short, and ranged from 0 to 0.12; indeed, 77 of the 103 genotypes had the same genetic profile. All the xoconostle accessions (acidic fruits) were grouped together without being separated from three genotypes of prickly pear (sweet fruits). We assume that the genetic divergence between prickly pears and xoconostles is very low, and question the number of Opuntia species currently considered in Mexico.

  3. Rifampicin-resistance, rpoB polymorphism and RNA polymerase genetic engineering.

    PubMed

    Alifano, Pietro; Palumbo, Carla; Pasanisi, Daniela; Talà, Adelfia

    2015-05-20

    Following its introduction in 1967, rifampicin has become a mainstay of therapy in the treatment of tuberculosis, leprosy and many other widespread diseases. Its potent antibacterial activity is due to specific inhibition of bacterial RNA polymerase. However, resistance to rifampicin was reported shortly after its introduction in the medical practice. Studies in the model organism Escherichia coli helped to define the molecular mechanism of rifampicin-resistance demonstrating that resistance is mostly due to chromosomal mutations in rpoB gene encoding the RNA polymerase β chain. These studies also revealed the amazing potential of the molecular genetics to elucidate the structure-function relationships in bacterial RNA polymerase. The scope of this paper is to illustrate how rifampicin-resistance has been recently exploited to better understand the regulatory mechanisms that control bacterial cell physiology and virulence, and how this information has been used to maneuver, on a global scale, gene expression in bacteria of industrial interest. In particular, we reviewed recent literature regarding: (i) the effects of rpoB mutations conferring rifampicin-resistance on transcription dynamics, bacterial fitness, physiology, metabolism and virulence; (ii) the occurrence in nature of "mutant-type" or duplicated rifampicin-resistant RNA polymerases; and (iii) the RNA polymerase genetic engineering method for strain improvement and drug discovery.

  4. Taxonomic and ecological discrimination of Fagaceae species based on internal transcribed spacer polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Coutinho, João Paulo; Carvalho, Ana; Lima-Brito, José

    2014-11-26

    The internal transcribed spacer (ITS) of ribosomal DNA has been used to confirm taxonomic classifications and define phylogenies in several plant species following sequencing or polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) techniques. In this study, co-dominant ITS PCR-RFLP molecular markers were produced in 30 Fagaceae individuals belonging to the Castanea, Fagus and Quercus genera in order to assess the potential of this technique for taxonomic discrimination and determination of phylogenies. The complete ITS region (ITS1-5.8S rRNA-ITS2) was amplified in most of the Fagaceae individuals as a single fragment of ∼700 bp. The ITS amplified products were digested with nine restriction enzymes, but only four (HaeIII, HpaII, TaqI and Sau96I) produced polymorphic/discriminative patterns. The total expected heterozygosity (HE) was 20.31 % and the gene diversity (I), 32.97 %. The ITS polymorphism was higher within the Quercus genus (85.3 %). The ITS PCR-RFLP markers clustered the Fagaceae species according to genus or infrageneric group (in the case of Quercus sp. individuals). Five oaks did not cluster in line with the adopted infrageneric classification, but three of these were grouped according to their actual ecological distributions. The ITS PCR-RFLP markers indicated their potential for phylogenetic studies since all Fagaceae individuals were discriminated according to genus, and most of the oaks were clustered according to infrageneric group or ecological area.

  5. Identification of polymorphic and off-target probe binding sites on the Illumina Infinium MethylationEPIC BeadChip.

    PubMed

    McCartney, Daniel L; Walker, Rosie M; Morris, Stewart W; McIntosh, Andrew M; Porteous, David J; Evans, Kathryn L

    2016-09-01

    Genome-wide analysis of DNA methylation has now become a relatively inexpensive technique thanks to array-based methylation profiling technologies. The recently developed Illumina Infinium MethylationEPIC BeadChip interrogates methylation at over 850,000 sites across the human genome, covering 99% of RefSeq genes. This array supersedes the widely used Infinium HumanMethylation450 BeadChip, which has permitted insights into the relationship between DNA methylation and a wide range of conditions and traits. Previous research has identified issues with certain probes on both the HumanMethylation450 BeadChip and its predecessor, the Infinium HumanMethylation27 BeadChip, which were predicted to affect array performance. These issues concerned probe-binding specificity and the presence of polymorphisms at target sites. Using in silico methods, we have identified probes on the Infinium MethylationEPIC BeadChip that are predicted to (i) measure methylation at polymorphic sites and (ii) hybridise to multiple genomic regions. We intend these resources to be used for quality control procedures when analysing data derived from this platform. PMID:27330998

  6. Modulation of the W748S mutation in DNA polymerase γ by the E1143G polymorphism in mitochondrial disorders

    PubMed Central

    Chan, Sherine S.L.; Longley, Matthew J.; Copeland, William C.

    2007-01-01

    DNA polymerase gamma (pol γ) is required for replication and repair of mitochondrial DNA. Over 80 mutations in POLG, the gene encoding the catalytic subunit of pol γ, have been linked with disease. The W748S mutation in POLG is the most common mutation in ataxia-neuropathy spectrum disorders and is generally found in cis with the common E1143G polymorphism. It has been unclear whether E1143G participates in the disease process. We investigated the biochemical consequences of pol γ proteins containing W748S or E1143G, or both. W748S pol γ exhibited low DNA polymerase activity, low processivity and a severe DNA-binding defect. However, interactions between the catalytic and accessory subunits were normal. Despite the benefits derived from binding with the accessory subunit, catalytic activities did not reach wild-type (WT) levels. Also, nucleotide selectivity decreased 2.1-fold compared with WT. Surprisingly, pol γ containing only E1143G was 1.4-fold more active than WT, and this increased polymerase activity could be due to higher thermal stability for E1143G pol γ. The E1143G substitution partially rescued the deleterious effects of the W748S mutation, as DNA binding, catalytic activity and fidelity values were intermediate for W748S-E1143G. However, W748S-E1143G had a notably lower change in enthalpy for protein folding than W748S alone. We suggest that when E1143G is in cis with other pathogenic mutations, it can modulate the effects of these mutations. For W748S-E1143G pol γ, the benefits bestowed by E1143G include increased DNA binding and polymerase activity; however, E1143G was somewhat detrimental to protein stability. PMID:17088268

  7. Analysis of ancient DNA from coprolites: a perspective with random amplified polymorphic DNA-polymerase chain reaction approach.

    PubMed

    Iñiguez, Alena M; Araújo, Adauto; Ferreira, Luiz Fernando; Vicente, Ana Carolina P

    2003-01-01

    The aim of this work was to determine approaches that would improve the quality of ancient DNA (aDNA) present in coprolites to enhance the possibility of success in retrieving specific sequence targets. We worked with coprolites from South American archaeological sites in Brazil and Chile dating up to 7,000 years ago. Using established protocols for aDNA extraction we obtained samples showing high degradation as usually happens with this kind of material. The reconstructive polymerization pretreatment was essential to overcome the DNA degradation and the serial dilutions helped with to prevent polymerase chain reaction (PCR) inhibitors. Moreover, the random amplified polymorphic DNA-PCR has been shown to be a reliable technique for further experiments to recover specific aDNA sequences.

  8. A new measurement approach of ionizing radiation in irradiated trout (Oncorhynchus mykiss) by Randomly Polymorphic DNA-Polymerase Chain Reaction.

    PubMed

    Şakalar, Ergün; Mol, Sühendan

    2016-05-01

    Trout (Oncorhynchus mykiss) were irradiated at doses of 0.250, 0.500, 1, 3, 5, 7 and 9 kGy in gamma cell. DNAs were extracted from the irradiated samples before and after storage. 1ERP primers were designed, and RAPD-PCR (Randomly Polymorphic DNA-Polymerase Chain Reaction) was applied to make randomly amplifications on the DNA of the irradiated samples. Agarose gel profiles of irradiated fish were obtained to determine change of band profiles. In addition, DNA fragmentation occurring in each dose was determined by comet assay for the verification of methodology developed in this study. The molecular methodology was developed to estimate ionizing radiation (IR) level in irradiated fish. This methodology allows the analysis of the trout irradiated up to the dose limit of around 0.5 kGy and stored for a period of three months. PMID:27407216

  9. Leishmania spp. identification by polymerase chain reaction-restriction fragment length polymorphism analysis and its applications in French Guiana.

    PubMed

    Simon, Stéphane; Veron, Vincent; Carme, Bernard

    2010-02-01

    Leishmania (Viannia) guyanensis was for many years the only species commonly identified in French Guiana, but precise species identifications were quite rare. We describe a new restriction fragment length polymorphism-polymerase chain reaction technique using a 615-bp fragment of the RNA polymerase II gene and 2 restriction enzymes, TspRI and HgaI. Seven reference strains (Leishmania (Leishmania) amazonensis, Leishmania (Viannia) lainsoni, Leishmania (Viannia) braziliensis, L. (V.) guyanensis, Leishmania (Viannia) naiffi, Leishmania (Leishmania) major, Leishmania (Leishmania) infantum) and 112 clinical samples from positive lesions were used for the development of the technique. The rates of positive species identification were 85.7% for punch skin biopsy specimens, 93.1% for positive Giemsa-stained smears, and 100% for positive culture supernatants. In the framework of cutaneous leishmaniasis species surveillance for the 2006 to 2008 period, parasite identification was carried out for 199 samples from different patients. The prevalence of the various Leishmania spp. was 84.4% for L. (V.) guyanensis, 8.0% for L. (V.) braziliensis, 5.0% for L. (L.) amazonensis, and 2.6% for L. (V.) lainsoni. L. (V.) braziliensis seems to be locally an emerging pathogen.

  10. Haplotyping using a combination of polymerase chain reaction-single-strand conformational polymorphism analysis and haplotype-specific PCR amplification.

    PubMed

    Zhou, Huitong; Li, Shaobin; Liu, Xiu; Wang, Jiqing; Luo, Yuzhu; Hickford, Jon G H

    2014-12-01

    A single nucleotide polymorphism (SNP) may have an impact on phenotype, but it may also be influenced by multiple SNPs within a gene; hence, the haplotype or phase of multiple SNPs needs to be known. Various methods for haplotyping SNPs have been proposed, but a simple and cost-effective method is currently unavailable. Here we describe a haplotyping approach using two simple techniques: polymerase chain reaction-single-strand conformational polymorphism (PCR-SSCP) and haplotype-specific PCR. In this approach, individual regions of a gene are analyzed by PCR-SSCP to identify variation that defines sub-haplotypes, and then extended haplotypes are assembled from the sub-haplotypes either directly or with the additional use of haplotype-specific PCR amplification. We demonstrate the utility of this approach by haplotyping ovine FABP4 across two variable regions that contain seven SNPs and one indel. The simplicity of this approach makes it suitable for large-scale studies and/or diagnostic screening.

  11. Identification of roots of woody species using polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP) analysis

    PubMed

    Bobowski; Hole; Wolf; Bryant

    1999-03-01

    Within the last two decades, substantial progress has been made in understanding seed-bank dynamics and the contribution of the soil seed bank to a postdisturbance plant community. There has been relatively little progress, however, in understanding perennial bud-bank dynamics and the contribution of the soil bud bank to secondary succession. This lack of information is due primarily to the inability to reliably identify roots, rhizomes and lignotubers that lie dormant beneath the soil surface. This investigation addressed the issue of identification of below-ground woody structures. The first objective was to develop a method that used molecular tools to identify woody plant species from subsoil tissue samples. The second objective was to develop a key in which molecular markers served as criteria for the identification and differentiation of selected tree and shrub species common to the mountains of northeast Oregon and southeast Washington. Application of restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified rbcL appears to be a reliable method to identify and differentiate 15 plants to the genus level. Two restriction enzymes, DpnII and HhaI, provided restriction site polymorphisms in the PCR product. The fragment number and length were used to develop an identification key. However, plants not analysed in this 'exploratory key' might share the same banding patterns, resulting in a false identification of unknowns. PMID:10199009

  12. Polymorphism, recombination and alternative unscrambling in the DNA polymerase alpha gene of the ciliate Stylonychia lemnae (Alveolata; class Spirotrichea).

    PubMed Central

    Ardell, David H; Lozupone, Catherine A; Landweber, Laura F

    2003-01-01

    DNA polymerase alpha is the most highly scrambled gene known in stichotrichous ciliates. In its hereditary micronuclear form, it is broken into >40 pieces on two loci at least 3 kb apart. Scrambled genes must be reassembled through developmental DNA rearrangements to yield functioning macronuclear genes, but the mechanism and accuracy of this process are unknown. We describe the first analysis of DNA polymorphism in the macronuclear version of any scrambled gene. Six functional haplotypes obtained from five Eurasian strains of Stylonychia lemnae were highly polymorphic compared to Drosophila genes. Another incompletely unscrambled haplotype was interrupted by frameshift and nonsense mutations but contained more silent mutations than expected by allelic inactivation. In our sample, nucleotide diversity and recombination signals were unexpectedly high within a region encompassing the boundary of the two micronuclear loci. From this and other evidence we infer that both members of a long repeat at the ends of the loci provide alternative substrates for unscrambling in this region. Incongruent genealogies and recombination patterns were also consistent with separation of the two loci by a large genetic distance. Our results suggest that ciliate developmental DNA rearrangements may be more probabilistic and error prone than previously appreciated and constitute a potential source of macronuclear variation. From this perspective we introduce the nonsense-suppression hypothesis for the evolution of ciliate altered genetic codes. We also introduce methods and software to calculate the likelihood of hemizygosity in ciliate haplotype samples and to correct for multiple comparisons in sliding-window analyses of Tajima's D. PMID:14704164

  13. Association between polymorphisms in arsenic metabolism genes and urinary arsenic methylation profiles in girls and boys chronically exposed to arsenic.

    PubMed

    Recio-Vega, Rogelio; González-Cortes, Tania; Olivas-Calderón, Edgar; Clark Lantz, R; Jay Gandolfi, A; Michel-Ramirez, Gladis

    2016-08-01

    Disease manifestations or susceptibilities often differ among individuals exposed to the same concentrations of arsenic (As). These differences have been associated with several factors including As metabolism, sex, age, genetic variants, nutritional status, smoking, and others. This study evaluated the associations between four As metabolism-related gene polymorphisms/null genotypes with urinary As methylation profiles in girls and boys chronically exposed to As. In a total of 332 children aged 6-12 years, the frequency of AS3MT, GSTO1, GSTT1, and GSTM1 polymorphisms/null genotypes and As urinary metabolites were measured. The results revealed that total As and monomethyl metabolites of As (MMA) levels were higher in boys than in girls. No differences in the frequency of the evaluated polymorphisms were found between girls and boys. In AS3MT-Met287Thr carriers, %MMA levels were higher and second methylation levels (defined as dimethylarsinic acid divided by MMA) were lower. In children with the GSTM1 null genotype, second methylation levels were higher. In boys, a positive association between the AS3MT-Met287Thr polymorphism with %MMA and between the GSTO1-Glu155del and As(v) was found; whereas, a negative relationship was identified between AS3MT-Met287Thr and second methylation profiles. In girls, a positive association was found between the GSTO1-Ala140Asp polymorphism with second methylation levels. In conclusion, our data indicate that gender, high As exposure levels, and polymorphisms in the evaluated genes negatively influenced As metabolism. Environ. Mol. Mutagen. 57:516-525, 2016. © 2016 Wiley Periodicals, Inc.

  14. Adaptive Epigenetic Differentiation between Upland and Lowland Rice Ecotypes Revealed by Methylation-Sensitive Amplified Polymorphism

    PubMed Central

    Xiong, Jie; Tao, Tao; Zheng, Xiaoguo; Wei, Haibin; Yue, Yunxia; Chen, Liang; Luo, Lijun

    2016-01-01

    The stress-induced epimutations could be inherited over generations and play important roles in plant adaption to stressful environments. Upland rice has been domesticated in water-limited environments for thousands of years and accumulated drought-induced epimutations of DNA methylation, making it epigenetically differentiated from lowland rice. To study the epigenetic differentiation between upland and lowland rice ecotypes on their drought-resistances, the epigenetic variation was investigated in 180 rice landraces under both normal and osmotic conditions via methylation-sensitive amplified polymorphism (MSAP) technique. Great alterations (52.9~54.3% of total individual-locus combinations) of DNA methylation are recorded when rice encountering the osmotic stress. Although the general level of epigenetic differentiation was very low, considerable level of ΦST (0.134~0.187) was detected on the highly divergent epiloci (HDE). The HDE detected in normal condition tended to stay at low levels in upland rice, particularly the ones de-methylated in responses to osmotic stress. Three out of four selected HDE genes differentially expressed between upland and lowland rice under normal or stressed conditions. Moreover, once a gene at HDE was up-/down-regulated in responses to the osmotic stress, its expression under the normal condition was higher/lower in upland rice. This result suggested expressions of genes at the HDE in upland rice might be more adaptive to the osmotic stress. The epigenetic divergence and its influence on the gene expression should contribute to the higher drought-resistance in upland rice as it is domesticated in the water-limited environment. PMID:27380174

  15. DNA fragment length polymorphism analysis of Mycobacterium tuberculosis isolates by arbitrarily primed polymerase chain reaction.

    PubMed

    Palittapongarnpim, P; Chomyc, S; Fanning, A; Kunimoto, D

    1993-04-01

    Strain identification of Mycobacterium tuberculosis would prove whether transmission had occurred between individuals. A method to characterize strains of M. tuberculosis has been developed utilizing polymerase chain reaction (PCR). Purified chromosomal DNA of cultured clinical samples of M. tuberculosis were subjected to PCR using short (10-12 nucleotide) oligonucleotide primers. PCR products visualized after agarose gel electrophoresis and ethidium bromide staining demonstrated that different strains of M. tuberculosis give different banding patterns. This technique was used to confirm the relationship between cases of tuberculosis in several clusters, prove the lack of relationship between 2 isolates with the same antibiotic-resistance pattern, confirm a suspected mislabeling event, and suggest the source of infection in a case of tuberculous meningitis. This method is rapid and simple and does not require radioactive probes.

  16. Association Between Single Nucleotide Polymorphisms in DNA Polymerase Kappa Gene and Breast Cancer Risk in Chinese Han Population: A STROBE-Compliant Observational Study.

    PubMed

    Dai, Zhi-Jun; Liu, Xing-Han; Ma, Yun-Feng; Kang, Hua-Feng; Jin, Tian-Bo; Dai, Zhi-Ming; Guan, Hai-Tao; Wang, Meng; Liu, Kang; Dai, Cong; Yang, Xue-Wen; Wang, Xi-Jing

    2016-01-01

    DNA polymerases are responsible for ensuring stability of the genome and avoiding genotoxicity caused by a variety of factors during DNA replication. Consequently, these proteins have been associated with an increased cancer risk. DNA polymerase kappa (POLK) is a specialized DNA polymerase involved in translesion DNA synthesis (TLS) that allows DNA synthesis over the damaged DNA. Recently, some studies investigated relationships between POLK polymorphisms and cancer risk, but the role of POLK genetic variants in breast cancer (BC) remains to be defined. In this study, we aimed to evaluate the effects of POLK polymorphisms on BC risk.We used the Sequenom MassARRAY method to genotype 3 single nucleotide polymorphisms (SNPs) in POLK (rs3213801, rs10077427, and rs5744533), in order to determine the genotypes of 560 BC patients and 583 controls. The association of genotypes and BC was assessed by computing the odds ratio (OR) and 95% confidence intervals (95% CIs) from logistic regression analyses.We found a statistically significant difference between patient and control groups in the POLK rs10077427 genotypic groups, excluding the recessive model. A positive correlation was also found between positive progesterone receptor (PR) status, higher Ki67 index, and rs10077427 polymorphism. For rs5744533 polymorphism, the codominant, dominant, and allele models frequencies were significantly higher in BC patients compared to healthy controls. Furthermore, our results indicated that rs5744533 SNP has a protective role in the postmenopausal women. However, we failed to find any associations between rs3213801 polymorphism and susceptibility to BC.Our results indicate that POLK polymorphisms may influence the risk of developing BC, and, because of this, may serve as a prognostic biomarker among Chinese women. PMID:26765445

  17. Calmodulin Polymerase Chain Reaction-Restriction Fragment Length Polymorphism for Leishmania Identification and Typing.

    PubMed

    Miranda, Aracelis; Samudio, Franklyn; González, Kadir; Saldaña, Azael; Brandão, Adeilton; Calzada, Jose E

    2016-08-01

    A precise identification of Leishmania species involved in human infections has epidemiological and clinical importance. Herein, we describe a preliminary validation of a restriction fragment length polymorphism assay, based on the calmodulin intergenic spacer region, as a tool for detecting and typing Leishmania species. After calmodulin amplification, the enzyme HaeIII yielded a clear distinction between reference strains of Leishmania mexicana, Leishmania amazonensis, Leishmania infantum, Leishmania lainsoni, and the rest of the Viannia reference species analyzed. The closely related Viannia species: Leishmania braziliensis, Leishmania panamensis, and Leishmania guyanensis, are separated in a subsequent digestion step with different restriction enzymes. We have developed a more accessible molecular protocol for Leishmania identification/typing based on the exploitation of part of the calmodulin gene. This methodology has the potential to become an additional tool for Leishmania species characterization and taxonomy.

  18. Pdx-1 links histone H3-Lys-4 methylation to RNA polymerase II elongation during activation of insulin transcription.

    PubMed

    Francis, Joshua; Chakrabarti, Swarup K; Garmey, James C; Mirmira, Raghavendra G

    2005-10-28

    Expression of the insulin gene is nearly exclusive to the beta cells of the pancreatic islets. Although the sequence-specific transcription factors that regulate insulin expression have been well studied, the interrelationship between these factors, chromatin structure, and transcriptional elongation by RNA polymerase II (pol II) has remained undefined. In this regard, recent studies have begun to establish a role for the methylation of histone H3 in the initiation or elongation of transcription by pol II. To determine a role for the transcriptional activator Pdx-1 in the maintenance of chromatin structure and pol II recruitment at the insulin gene, we performed small interfering RNA-mediated knockdown of Pdx-1 in betaTC3 cells and subsequently studied histone modifications and pol II recruitment by chromatin immunoprecipitation. We demonstrated here that the 50% fall in insulin transcription following knockdown of Pdx-1 is accompanied by a 60% fall in dimethylated histone H3-Lys-4 at the insulin promoter. H3-Lys-4 methylation at the insulin promoter may be mediated, at least partially, by the methyltransferase Set9. Immunohistochemical analysis revealed that Set9 is expressed in an islet-enriched pattern in the pancreas, similar to the pattern of Pdx-1 expression. The recruitment of Set9 to the insulin gene appears to be a consequence of its direct interaction with Pdx-1, and small interfering RNA-mediated knockdown of Set9 attenuates insulin transcription. Pdx-1 knockdown was also associated with an overall shift in the recruitment of pol II isoforms to the insulin gene, from an elongation isoform (Ser(P)-2) to an initiation isoform (Ser(P)-5). Our findings therefore suggest a model whereby Pdx-1 plays a novel role in linking H3-Lys-4 dimethylation and pol II elongation to insulin transcription.

  19. Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation-sensitive amplified polymorphism.

    PubMed

    Kim, Nam-Soo; Im, Min-Ji; Nkongolo, Kabwe

    2016-08-01

    Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal-contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation-sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal-contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal-contaminated site compared to uncontaminated populations. Other genotypes from a different metal-contaminated site within the same region appear to be recalcitrant to metal-induced DNA alterations even ≥30 years of tree life exposure to nickel and copper. MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal-contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed. PMID:27547351

  20. Determination of DNA methylation associated with Acer rubrum (red maple) adaptation to metals: analysis of global DNA modifications and methylation-sensitive amplified polymorphism.

    PubMed

    Kim, Nam-Soo; Im, Min-Ji; Nkongolo, Kabwe

    2016-08-01

    Red maple (Acer rubum), a common deciduous tree species in Northern Ontario, has shown resistance to soil metal contamination. Previous reports have indicated that this plant does not accumulate metals in its tissue. However, low level of nickel and copper corresponding to the bioavailable levels in contaminated soils in Northern Ontario causes severe physiological damages. No differentiation between metal-contaminated and uncontaminated populations has been reported based on genetic analyses. The main objective of this study was to assess whether DNA methylation is involved in A. rubrum adaptation to soil metal contamination. Global cytosine and methylation-sensitive amplified polymorphism (MSAP) analyses were carried out in A. rubrum populations from metal-contaminated and uncontaminated sites. The global modified cytosine ratios in genomic DNA revealed a significant decrease in cytosine methylation in genotypes from a metal-contaminated site compared to uncontaminated populations. Other genotypes from a different metal-contaminated site within the same region appear to be recalcitrant to metal-induced DNA alterations even ≥30 years of tree life exposure to nickel and copper. MSAP analysis showed a high level of polymorphisms in both uncontaminated (77%) and metal-contaminated (72%) populations. Overall, 205 CCGG loci were identified in which 127 were methylated in either outer or inner cytosine. No differentiation among populations was established based on several genetic parameters tested. The variations for nonmethylated and methylated loci were compared by analysis of molecular variance (AMOVA). For methylated loci, molecular variance among and within populations was 1.5% and 13.2%, respectively. These values were low (0.6% for among populations and 5.8% for within populations) for unmethylated loci. Metal contamination is seen to affect methylation of cytosine residues in CCGG motifs in the A. rubrum populations that were analyzed.

  1. Approach to molecular characterization of different strains of Fasciola hepatica using random amplified polymorphic DNA polymerase chain reaction.

    PubMed

    Scarcella, S; Miranda-Miranda, E; Solana, M V; Solana, H

    2015-04-01

    The aim of the present study was to genetically characterize Fasciola hepatica strains from diverse ecogeographical regions (America and Europe), susceptible and resistant to Triclabendazole, using the random amplified polymorphic DNA fragments (RAPDs-PCR) technique to elucidate genetic variability between the different isolates. Ten different oligonucleotide primers of 10 bases with GC content varying from 50-70% were used. A polymerase chain reaction (PCR) was carried out in 25 μl of total volume. Duplicate PCR reactions on each individual template DNA were performed to test the reproducibility of the individual DNA bands. The size of the RAPD-PCR fragments was determined by the reciprocal plot between the delay factors (Rf) versus the logarithm of molecular weight ladder. The phenogram obtained showed three main clusters, the major of which contained European Strains (Cullompton and Sligo) showing a genetic distance of 27.2 between them. The American strains (Cedive and Cajamarca) on the other hand formed each their distinctive group but clearly maintaining a closer genetic relationship among them than that to their European counterparts, with which showed a distance of 33.8 and 37.8, respectively. This polymorphism would give this species enhanced adaptability against the host, as well as the environment. The existence of genetically different populations of F. hepatica could allow, against any selection pressure, natural or artificial (for use fasciolicides products and/or control measures), one or more populations of F. hepatica to be able to survive and create resistance or adaptability to such selective pressure. PMID:25595655

  2. Approach to molecular characterization of different strains of Fasciola hepatica using random amplified polymorphic DNA polymerase chain reaction.

    PubMed

    Scarcella, S; Miranda-Miranda, E; Solana, M V; Solana, H

    2015-04-01

    The aim of the present study was to genetically characterize Fasciola hepatica strains from diverse ecogeographical regions (America and Europe), susceptible and resistant to Triclabendazole, using the random amplified polymorphic DNA fragments (RAPDs-PCR) technique to elucidate genetic variability between the different isolates. Ten different oligonucleotide primers of 10 bases with GC content varying from 50-70% were used. A polymerase chain reaction (PCR) was carried out in 25 μl of total volume. Duplicate PCR reactions on each individual template DNA were performed to test the reproducibility of the individual DNA bands. The size of the RAPD-PCR fragments was determined by the reciprocal plot between the delay factors (Rf) versus the logarithm of molecular weight ladder. The phenogram obtained showed three main clusters, the major of which contained European Strains (Cullompton and Sligo) showing a genetic distance of 27.2 between them. The American strains (Cedive and Cajamarca) on the other hand formed each their distinctive group but clearly maintaining a closer genetic relationship among them than that to their European counterparts, with which showed a distance of 33.8 and 37.8, respectively. This polymorphism would give this species enhanced adaptability against the host, as well as the environment. The existence of genetically different populations of F. hepatica could allow, against any selection pressure, natural or artificial (for use fasciolicides products and/or control measures), one or more populations of F. hepatica to be able to survive and create resistance or adaptability to such selective pressure.

  3. Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism

    PubMed Central

    Bounamous, Azzedine; Lehrter, Véronique; Hadj-Henni, Leila; Delecolle, Jean-Claude; Depaquit, Jérôme

    2014-01-01

    A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin. PMID:24936911

  4. Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry.

    PubMed

    Miyaguchi, Hajime

    2016-09-20

    An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 ± 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard.

  5. Polymerase chain reaction-restriction fragment length polymorphism method for differentiation of uropathogenic specific protein gene types.

    PubMed

    Lai, Yun Mei; Zaw, Myo Thura; Shamsudin, Shamsul Bahari; Lin, Zaw

    2016-08-01

    The putative pathogenicity island (PAI) containing the uropathogenic specific protein (usp) gene and three small open reading frames (orfU1, orfU2, and orfU3) encoding 98, 97, and 96 amino acid proteins is widely distributed among uropathogenic Escherichia coli (UPEC) strains. This PAI was designated as PAIusp. Sequencing analysis of PAIusp has revealed that the usp gene can be divided into two types - uspI and uspII - based on sequence variation at the 3' terminal region and the number and position of orfUs differ from strain to strain. Based on usp gene types and orfU sequential patterns, PAIusp can be divided into four subtypes. Subtyping of PAIusp is a useful method to characterize UPEC strains. In this study, we developed a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method to differentiate usp gene types. This method could correctly identify the usp gene type in usp-positive UPEC strains in our laboratory.

  6. Limits of a rapid identification of common Mediterranean sandflies using polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Bounamous, Azzedine; Lehrter, Véronique; Hadj-Henni, Leila; Delecolle, Jean-Claude; Depaquit, Jérôme

    2014-07-01

    A total of 131 phlebotomine Algerian sandflies have been processed in the present study. They belong to the species Phlebotomus bergeroti, Phlebotomus alexandri, Phlebotomus sergenti, Phlebotomus chabaudi, Phlebotomus riouxi, Phlebotomus perniciosus, Phlebotomus longicuspis, Phlebotomus perfiliewi, Phlebotomus ariasi, Phlebotomus chadlii, Sergentomyia fallax, Sergentomyia minuta, Sergentomyia antennata, Sergentomyia schwetzi, Sergentomyia clydei, Sergentomyia christophersi and Grassomyia dreyfussi. They have been characterised by sequencing of a part of the cytochrome b (cyt b), t RNA serine and NADH1 on the one hand and of the cytochrome C oxidase I of the mitochondrial DNA (mtDNA) on the other hand. Our study highlights two sympatric populations within P. sergenti in the area of its type-locality and new haplotypes of P. perniciosus and P. longicuspis without recording the specimens called lcx previously found in North Africa. We tried to use a polymerase chain reaction-restriction fragment length polymorphism method based on a combined double digestion of each marker. These method is not interesting to identify sandflies all over the Mediterranean Basin.

  7. Improved Polymerase Chain Reaction-restriction Fragment Length Polymorphism Genotyping of Toxic Pufferfish by Liquid Chromatography/Mass Spectrometry.

    PubMed

    Miyaguchi, Hajime

    2016-01-01

    An improved version of a polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) method for genotyping toxic pufferfish species by liquid chromatography/electrospray ionization mass spectrometry (LC/ESI-MS) is described. DNA extraction is carried out using a silica membrane-based DNA extraction kit. After the PCR amplification using a detergent-free PCR buffer, restriction enzymes are added to the solution without purifying the reaction solution. A reverse-phase silica monolith column and a Fourier transform high resolution mass spectrometer having a modified Kingdon trap analyzer are employed for separation and detection, respectively. The mobile phase, consisting of 400 mM 1,1,1,3,3,3-hexafluoro-2-propanol, 15 mM triethylamine (pH 7.9) and methanol, is delivered at a flow rate of 0.4 ml/min. The cycle time for LC/ESI-MS analysis is 8 min including equilibration of the column. Deconvolution software having an isotope distribution model of the oligonucleotide is used to calculate the corresponding monoisotopic mass from the mass spectrum. For analysis of oligonucleotides (range 26-79 nucleotides), mass accuracy was 0.62 ± 0.74 ppm (n = 280) and excellent accuracy and precision were sustained for 180 hr without use of a lock mass standard. PMID:27684516

  8. Analysis of DNA methylation level by methylation-sensitive amplification polymorphism in half smooth tongue sole (Cynoglossus semilaevis) subjected to salinity stress

    NASA Astrophysics Data System (ADS)

    Li, Siping; He, Feng; Wen, Haishen; Li, Jifang; Si, Yufeng; Liu, Mingyuan; He, Huiwen; Huang, Zhengju

    2016-09-01

    Increasingly arisen environmental constraints may contribute to heritable phenotypic variation including methylation changes, which can help the animals with development, growth and survival. In this study, we assessed the DNA methylation levels in three tissues (gonad, kidney and gill) of half smooth tongue sole under the salinity stress. The methylation-sensitive amplification polymorphism (MSAP) technique was applied to illustrate the regulation of epigenetic mechanism in environmental stimuli. Fish were subjected to 15 salinity treatment for 7 and 60 days, respectively. A total of 11259 fragments were amplified with 8 pairs of selective primers. The levels of methylated DNA in different tissues of females and males without salinity stress were analyzed, which were 32.76% and 47.32% in gonad; 38.13% and 37.69% in kidney; 37.58% and 34.96% in gill, respectively. In addition, the significant difference was observed in gonad between females and males, indicating that discrepant regulation in gonadal development and differentiation may involve sex-related genes. Further analysis showed that total and hemi-methylation were significantly decreased under 15 salinity for 7 days, probably resulting in up-regulating salt-tolerance genes expression to adjust salt changing. With the adjustment for 60 days, total and hemi-methylation prominently went back to its normal levels to obtain equilibrium. Particularly, full methylation levels were steady along with salinity stress to maintain the stability of gene expression. Additionally, the data showed that gonads in females and gills in males were superior in adaptability. As a result, DNA methylation regulates tissue- specific epiloci, and may respond to salinity stress by regulating gene expression to maintain animal survival and activity.

  9. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism

    PubMed Central

    Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Morano, Annalisa; Zuchegna, Candida; Romano, Antonella; Muller, Mark T.; Gottesman, Max E.; Porcellini, Antonio; Avvedimento, Enrico V.

    2016-01-01

    We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5′ and 3′ ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells. PMID:27629060

  10. DNA damage and Repair Modify DNA methylation and Chromatin Domain of the Targeted Locus: Mechanism of allele methylation polymorphism.

    PubMed

    Russo, Giusi; Landi, Rosaria; Pezone, Antonio; Morano, Annalisa; Zuchegna, Candida; Romano, Antonella; Muller, Mark T; Gottesman, Max E; Porcellini, Antonio; Avvedimento, Enrico V

    2016-01-01

    We characterize the changes in chromatin structure, DNA methylation and transcription during and after homologous DNA repair (HR). We find that HR modifies the DNA methylation pattern of the repaired segment. HR also alters local histone H3 methylation as well chromatin structure by inducing DNA-chromatin loops connecting the 5' and 3' ends of the repaired gene. During a two-week period after repair, transcription-associated demethylation promoted by Base Excision Repair enzymes further modifies methylation of the repaired DNA. Subsequently, the repaired genes display stable but diverse methylation profiles. These profiles govern the levels of expression in each clone. Our data argue that DNA methylation and chromatin remodelling induced by HR may be a source of permanent variation of gene expression in somatic cells. PMID:27629060

  11. Detection of HLA-DRB1 microchimerism using nested polymerase chain reaction and single-strand conformation polymorphism analysis.

    PubMed

    Song, Eun Young; Chung, Hye Yoon; Joo, Shin Young; Roh, Eun Youn; Seong, Moon-Woo; Shin, Yunsu; Park, Myoung Hee

    2012-03-01

    For the detection of microchimerism, molecular methods detecting donor-specific HLA-DRB1 alleles in the recipient are most commonly used. Nested polymerase chain reaction sequence specific primer (nested PCR-SSP) methods widely used to increase the sensitivity of detection have been reported to give frequent false-positive reactions. We have developed a new method combining nested PCR with single-strand conformation polymorphism analysis (nested PCR-SSCP) and tested the 1 to 0.00001% level of microchimerism for 27 different HLA-DRB1 alleles. For most (26/27) of the HLA-DRB1 alleles tested, this method could detect 0.01 to 0.001% of microchimerism and its sensitivity was equal to or better than that of nested PCR-SSP tested in parallel. Its specificity was verified by visualizing particular DRB1-specific SSCP bands under test. Nested PCR-SSP indicated frequent false-positive reactions, mainly caused by nonspecific amplification of DRB3/B4/B5 alleles present in the major (recipient) DNAs. We have compared a real-time quantitative PCR for non-human leukocyte antigen (HLA) target (insertion/deletion marker) using a commercial kit (AlleleSEQR Chimerism assay), and its microchimerism detection sensitivity (around 0.1%) was 1 step (10 times) lower than that of nested PCR-SSP or -SSCP methods for HLA-DRB1 alleles. We validated that the newly designed nested PCR-SSCP affords good sensitivity and specificity and may be useful for studying microchimerism in clinical settings.

  12. Rapid detection of clarithromycin resistant Helicobacter pylori strains in Spanish patients by polymerase chain reaction-restriction fragment length polymorphism

    PubMed Central

    Agudo, Sonia; Pérez-Pérez, Guillermo; Alarcón, Teresa; López-Brea, Manuel

    2014-01-01

    Introduction The aim of this study was to characterize the mutations types present in the 23S rRNA gene related to H. pylori clarithromycin-resistance strains in Spain and evaluate a novel PCR-RFLP method for detection of the most frequent point mutation in our population. Methods Gastric biopsies were obtained by endoscopy from patients with gastric symptoms. H. pylori was cultured according to standard microbiological procedures and clarithromycin resistance was determined by E-test. DNA extraction was performed by NucliSens platform with the NucliSens magnetic extraction reagents (bioMérieux) according to the manufacturer instructions. Analyses for point mutations in 23S rRNA gene strains were performed by sequence analysis of amplified polymerase chain reaction products. Restriction fragment length polymorphism was performed using BsaI enzyme to detect restriction sites that correspond to the mutation (A2143G). Result We found 42 out of 118 (35.6%) strains resistant to clarithromycin by E-test. E-test results were confirmed for the presence of point mutation in 34 (88.1%) of these strains. Mutation A2143G was found in 85.3% of the strains. Analyses with the restriction enzyme BsaI was able to confirm the presence of A2143G mutation. There were 8 H. pylori strains resistant to clarithromycin by E-test but without any point mutation in the 23 rRNA gene. Conclusion We conclude that PCR-RFLP is a reliable method to detect clarithromycin-resistance H. pylori strains in countries with a high prevalence of clarithromycin-resistance as Spain It may be useful before choosing regimens of H. pylori eradication. PMID:21412667

  13. Polymorphic tandem repeats within gene promoters act as modifiers of gene expression and DNA methylation in humans

    PubMed Central

    Quilez, Javier; Guilmatre, Audrey; Garg, Paras; Highnam, Gareth; Gymrek, Melissa; Erlich, Yaniv; Joshi, Ricky S.; Mittelman, David; Sharp, Andrew J.

    2016-01-01

    Despite representing an important source of genetic variation, tandem repeats (TRs) remain poorly studied due to technical difficulties. We hypothesized that TRs can operate as expression (eQTLs) and methylation (mQTLs) quantitative trait loci. To test this we analyzed the effect of variation at 4849 promoter-associated TRs, genotyped in 120 individuals, on neighboring gene expression and DNA methylation. Polymorphic promoter TRs were associated with increased variance in local gene expression and DNA methylation, suggesting functional consequences related to TR variation. We identified >100 TRs associated with expression/methylation levels of adjacent genes. These potential eQTL/mQTL TRs were enriched for overlaps with transcription factor binding and DNaseI hypersensitivity sites, providing a rationale for their effects. Moreover, we showed that most TR variants are poorly tagged by nearby single nucleotide polymorphisms (SNPs) markers, indicating that many functional TR variants are not effectively assayed by SNP-based approaches. Our study assigns biological significance to TR variations in the human genome, and suggests that a significant fraction of TR variations exert functional effects via alterations of local gene expression or epigenetics. We conclude that targeted studies that focus on genotyping TR variants are required to fully ascertain functional variation in the genome. PMID:27060133

  14. Polymorphic tandem repeats within gene promoters act as modifiers of gene expression and DNA methylation in humans.

    PubMed

    Quilez, Javier; Guilmatre, Audrey; Garg, Paras; Highnam, Gareth; Gymrek, Melissa; Erlich, Yaniv; Joshi, Ricky S; Mittelman, David; Sharp, Andrew J

    2016-05-01

    Despite representing an important source of genetic variation, tandem repeats (TRs) remain poorly studied due to technical difficulties. We hypothesized that TRs can operate as expression (eQTLs) and methylation (mQTLs) quantitative trait loci. To test this we analyzed the effect of variation at 4849 promoter-associated TRs, genotyped in 120 individuals, on neighboring gene expression and DNA methylation. Polymorphic promoter TRs were associated with increased variance in local gene expression and DNA methylation, suggesting functional consequences related to TR variation. We identified >100 TRs associated with expression/methylation levels of adjacent genes. These potential eQTL/mQTL TRs were enriched for overlaps with transcription factor binding and DNaseI hypersensitivity sites, providing a rationale for their effects. Moreover, we showed that most TR variants are poorly tagged by nearby single nucleotide polymorphisms (SNPs) markers, indicating that many functional TR variants are not effectively assayed by SNP-based approaches. Our study assigns biological significance to TR variations in the human genome, and suggests that a significant fraction of TR variations exert functional effects via alterations of local gene expression or epigenetics. We conclude that targeted studies that focus on genotyping TR variants are required to fully ascertain functional variation in the genome. PMID:27060133

  15. Crystal structure of the tetra-gonal polymorph of bis-(1-ethyl-3-methyl-imidazolium) tetra-bromido-cadmate.

    PubMed

    Đorđević, Tamara; Gerger, Sabrina; Karanović, Ljiljana

    2016-07-01

    Both unique Cd atoms in the tetra-gonal polymorph of bis-(1-ethyl-3-methyl-imidazolium) tetra-bromido-cadmate, (C6H11N2)2[CdBr4], occupy special positions (site symmetry -4). The crystal structure consists of isolated tetra-hedral [CdBr4](2-) anions which are surrounded by 1-ethyl-3-methyl-imidazolium cations. The methyl and ethyl side chains of the cations show positional disorder in a 0.590 (11):0.410 (11) ratio. In the crystal, (C6H11N2)(+) cations display three weak C-H⋯Br hydrogen-bond inter-actions through the imidazolium ring H atoms with the Br(-) ligands of the surrounding complex anions. The alkyl groups of the side chains are not involved in hydrogen bonding. PMID:27555953

  16. The histone methyltransferase KMT2B is required for RNA polymerase II association and protection from DNA methylation at the MagohB CpG island promoter.

    PubMed

    Ladopoulos, Vasileios; Hofemeister, Helmut; Hoogenkamp, Maarten; Riggs, Arthur D; Stewart, A Francis; Bonifer, Constanze

    2013-04-01

    KMT2B (MLL2/WBP7) is a member of the MLL subfamily of H3K4-specific histone lysine methyltransferases (KMT2) and is vital for normal embryonic development in the mouse. To gain insight into the molecular mechanism underlying KMT2B function, we focused on MagohB, which is controlled by a CpG island promoter. We show that in cells lacking Mll2-the gene encoding KMT2B-the MagohB promoter resides in inaccessible chromatin and is methylated. To dissect the molecular events leading to the establishment of silencing, we performed kinetic studies in Mll2-conditional-knockout embryonic stem cells. KMT2B depletion was followed by the loss of the active chromatin marks and progressive loss of RNA polymerase II binding with a concomitant downregulation of MagohB expression. Once the active chromatin marks were lost, the MagohB promoter was rapidly methylated. We demonstrate that in the presence of KMT2B, neither transcription elongation nor RNA polymerase II binding is required to maintain H3K4 trimethylation at the MagohB promoter and protect it from DNA methylation. Reexpression of KMT2B was sufficient to reinstate an active MagohB promoter. Our study provides a paradigm for the idea that KMT2 proteins are crucial components for establishing and maintaining the transcriptionally active and unmethylated state of CpG island promoters. PMID:23358417

  17. [Application of polymerase chain reaction-restriction fragment length polymorphism and lab-on-a-chip technology to the identification of fish species from Bohai Bay].

    PubMed

    Li, Xiao; Qu, Yanyan; Zhang, Piqiao; Zhang, Jin; Zhang, Lihua; Huang, Daliang; Zhang, Yukui

    2011-07-01

    Nine representative fish species from Bohai Bay were identified by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and lab-on-a-chip technology. The nine fish species were Pseudosciaena polyactis, Lateolabrax japonicus, Scomberomorus niphonius, Pneumatophorus japonicus, Pseudopleuronectes yokohamae, Collichthys lucidus, Sebastes schlegeli, Cleisthenes herzensteini and Saurida elongata. The 464 bp fragment of mitochondrial cytochrome b gene was amplified and further digested by Dde I , Hae III and Nla III. The digested DNA fragments were analyzed by microfluidic capillary electrophoresis. The nine fish species were fully discriminated. The results demonstrated that the proposed method is efficient, precise and fast in fish species identification.

  18. Cross talk between poly(ADP-ribose) polymerase 1 methylation and oxidative stress involved in the toxic effect of anatase titanium dioxide nanoparticles.

    PubMed

    Bai, Wenlin; Chen, Yujiao; Gao, Ai

    2015-01-01

    Given the tremendous growth in the application of titanium dioxide nanoparticles (TNPs), concerns about the potential health hazards of TNPs to humans have been raised. Poly(ADP-ribose) polymerase 1 (PARP-1), a highly conserved DNA-binding protein, is involved in many molecular and cellular processes. Limited data demonstrated that certain nanomaterials induced the aberrant hypermethylation of PARP-1. However, the mechanism involved in TNP-induced PARP-1 abnormal methylation has not been studied. A549 cells were incubated with anatase TNPs (22.1 nm) for 24 hours pretreatment with or without methyltransferase inhibitor 5-aza-2'-deoxycytidine and the reactive oxygen species (ROS) scavenger α-lipoic acid to assess the possible role of methylation and ROS in the toxic effect of TNPs. After TNPs characterization, a battery of assays was performed to evaluate the toxic effect of TNPs, PARP-1 methylation status, and oxidative damage. Results showed that TNPs decreased the cell viability in a dose-dependent manner, in accordance with the increase of lactate dehydrogenase activity, which indicated membrane damage of cells. Similar to the high level of PARP-1 methylation, the generation of ROS was significantly increased after exposure to TNPs for 24 hours. Furthermore, α-lipoic acid decreased TNP-induced ROS generation and then attenuated TNP-triggered PARP-1 hypermethylation. Meanwhile, 5-aza-2'-deoxycytidine simultaneously decreased the ROS generation induced by TNPs, resulting in the decline of PARP-1 methylation. In summary, TNPs triggered the aberrant hypermethylation of the PARP-1 promoter and there was a cross talk between oxidative stress and PARP-1 methylation in the toxic effect of TNPs.

  19. Cross talk between poly(ADP-ribose) polymerase 1 methylation and oxidative stress involved in the toxic effect of anatase titanium dioxide nanoparticles

    PubMed Central

    Bai, Wenlin; Chen, Yujiao; Gao, Ai

    2015-01-01

    Given the tremendous growth in the application of titanium dioxide nanoparticles (TNPs), concerns about the potential health hazards of TNPs to humans have been raised. Poly(ADP-ribose) polymerase 1 (PARP-1), a highly conserved DNA-binding protein, is involved in many molecular and cellular processes. Limited data demonstrated that certain nanomaterials induced the aberrant hypermethylation of PARP-1. However, the mechanism involved in TNP-induced PARP-1 abnormal methylation has not been studied. A549 cells were incubated with anatase TNPs (22.1 nm) for 24 hours pretreatment with or without methyltransferase inhibitor 5-aza-2′-deoxycytidine and the reactive oxygen species (ROS) scavenger α-lipoic acid to assess the possible role of methylation and ROS in the toxic effect of TNPs. After TNPs characterization, a battery of assays was performed to evaluate the toxic effect of TNPs, PARP-1 methylation status, and oxidative damage. Results showed that TNPs decreased the cell viability in a dose-dependent manner, in accordance with the increase of lactate dehydrogenase activity, which indicated membrane damage of cells. Similar to the high level of PARP-1 methylation, the generation of ROS was significantly increased after exposure to TNPs for 24 hours. Furthermore, α-lipoic acid decreased TNP-induced ROS generation and then attenuated TNP-triggered PARP-1 hypermethylation. Meanwhile, 5-aza-2′-deoxycytidine simultaneously decreased the ROS generation induced by TNPs, resulting in the decline of PARP-1 methylation. In summary, TNPs triggered the aberrant hypermethylation of the PARP-1 promoter and there was a cross talk between oxidative stress and PARP-1 methylation in the toxic effect of TNPs. PMID:26366077

  20. Global Analysis of H3K4 Methylation Defines MLL Family Member Targets and Points to a Role for MLL1-Mediated H3K4 Methylation in the Regulation of Transcriptional Initiation by RNA Polymerase II ▿ †

    PubMed Central

    Wang, Pengfei; Lin, Chengqi; Smith, Edwin R.; Guo, Hong; Sanderson, Brian W.; Wu, Min; Gogol, Madelaine; Alexander, Tara; Seidel, Christopher; Wiedemann, Leanne M.; Ge, Kai; Krumlauf, Robb; Shilatifard, Ali

    2009-01-01

    A common landmark of activated genes is the presence of trimethylation on lysine 4 of histone H3 (H3K4) at promoter regions. Set1/COMPASS was the founding member and is the only H3K4 methylase in Saccharomyces cerevisiae; however, in mammals, at least six H3K4 methylases, Set1A and Set1B and MLL1 to MLL4, are found in COMPASS-like complexes capable of methylating H3K4. To gain further insight into the different roles and functional targets for the H3K4 methylases, we have undertaken a genome-wide analysis of H3K4 methylation patterns in wild-type Mll1+/+ and Mll1−/− mouse embryonic fibroblasts (MEFs). We found that Mll1 is required for the H3K4 trimethylation of less than 5% of promoters carrying this modification. Many of these genes, which include developmental regulators such as Hox genes, show decreased levels of RNA polymerase II recruitment and expression concomitant with the loss of H3K4 methylation. Although Mll1 is only required for the methylation of a subset of Hox genes, menin, a component of the Mll1 and Mll2 complexes, is required for the overwhelming majority of H3K4 methylation at Hox loci. However, the loss of MLL3/MLL4 and/or the Set1 complexes has little to no effect on the H3K4 methylation of Hox loci or their expression levels in these MEFs. Together these data provide insight into the redundancy and specialization of COMPASS-like complexes in mammals and provide evidence for a possible role for Mll1-mediated H3K4 methylation in the regulation of transcriptional initiation. PMID:19703992

  1. Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2'-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases.

    PubMed

    Jin, Zhinan; Tucker, Kathryn; Lin, Xiaoyan; Kao, C Cheng; Shaw, Ken; Tan, Hua; Symons, Julian; Behera, Ishani; Rajwanshi, Vivek K; Dyatkina, Natalia; Wang, Guangyi; Beigelman, Leo; Deval, Jerome

    2015-12-01

    Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2'-C-methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2'-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities.

  2. Biochemical Evaluation of the Inhibition Properties of Favipiravir and 2′-C-Methyl-Cytidine Triphosphates against Human and Mouse Norovirus RNA Polymerases

    PubMed Central

    Tucker, Kathryn; Lin, Xiaoyan; Kao, C. Cheng; Shaw, Ken; Tan, Hua; Symons, Julian; Behera, Ishani; Rajwanshi, Vivek K.; Dyatkina, Natalia; Wang, Guangyi; Beigelman, Leo

    2015-01-01

    Norovirus (NoV) is a positive-sense single-stranded RNA virus that causes acute gastroenteritis and is responsible for 200,000 deaths per year worldwide. No effective vaccine or treatment is available. Recent studies have shown that the nucleoside analogs favipiravir (T-705) and 2′-C-methyl-cytidine (2CM-C) inhibit NoV replication in vitro and in animal models, but their precise mechanism of action is unknown. We evaluated the molecular interactions between nucleoside triphosphates and NoV RNA-dependent RNA polymerase (NoVpol), the enzyme responsible for replication and transcription of NoV genomic RNA. We found that T-705 ribonucleoside triphosphate (RTP) and 2CM-C triphosphate (2CM-CTP) equally inhibited human and mouse NoVpol activities at concentrations resulting in 50% of maximum inhibition (IC50s) in the low micromolar range. 2CM-CTP inhibited the viral polymerases by competing directly with natural CTP during primer elongation, whereas T-705 RTP competed mostly with ATP and GTP at the initiation and elongation steps. Incorporation of 2CM-CTP into viral RNA blocked subsequent RNA synthesis, whereas T-705 RTP did not cause immediate chain termination of NoVpol. 2CM-CTP and T-705 RTP displayed low levels of enzyme selectivity, as they were both recognized as substrates by human mitochondrial RNA polymerase. The level of discrimination by the human enzyme was increased with a novel analog of T-705 RTP containing a 2′-C-methyl substitution. Collectively, our data suggest that 2CM-C inhibits replication of NoV by acting as a classic chain terminator, while T-705 may inhibit the virus by multiple mechanisms of action. Understanding the precise mechanism of action of anti-NoV compounds could provide a rational basis for optimizing their inhibition potencies and selectivities. PMID:26392512

  3. Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme.

    PubMed

    Naselli, Flores; Catanzaro, Irene; Bellavia, Daniele; Perez, Alessandro; Sposito, Laura; Caradonna, Fabio

    2014-02-15

    Different individuals possess slightly different genetic information and show genetically-determined differences in several enzyme activities due to genetic variability. Following an integrated approach, we studied the polymorphisms and methylation of sites contained in the 5' flanking region of the metabolizing enzyme CYP2E1 in correlation to its expression in both tumor and non-neoplastic liver cell lines, since to date little is known about the influence of these (epi)genetic elements in basal conditions and under induction by the specific inductor and a demethylating agent. In treated cells, reduced DNA methylation, assessed both at genomic and gene level, was not consistently associated with the increase of enzyme expression. Interestingly, the Rsa/Pst haplotype differentially influenced CYP2E1 enzyme expression. In addition, regarding the Variable Number of Tandem Repeats polymorphism, cells with A4/A4 genotype showed a greater expression inhibition (ranging from 20% to 30%) compared with others carrying the A2/A2 one, while those cells bringing A2/A3 genotype showed an increase of expression (of 25%, about). Finally, we demonstrated for the first time that the A2 and A3 CYP2E1 alleles play a more important role in the expression of the enzyme, compared with other (epi)genetic factors, since they are binding sites for trans-acting proteins. PMID:24333271

  4. Role and importance of polymorphisms with respect to DNA methylation for the expression of CYP2E1 enzyme.

    PubMed

    Naselli, Flores; Catanzaro, Irene; Bellavia, Daniele; Perez, Alessandro; Sposito, Laura; Caradonna, Fabio

    2014-02-15

    Different individuals possess slightly different genetic information and show genetically-determined differences in several enzyme activities due to genetic variability. Following an integrated approach, we studied the polymorphisms and methylation of sites contained in the 5' flanking region of the metabolizing enzyme CYP2E1 in correlation to its expression in both tumor and non-neoplastic liver cell lines, since to date little is known about the influence of these (epi)genetic elements in basal conditions and under induction by the specific inductor and a demethylating agent. In treated cells, reduced DNA methylation, assessed both at genomic and gene level, was not consistently associated with the increase of enzyme expression. Interestingly, the Rsa/Pst haplotype differentially influenced CYP2E1 enzyme expression. In addition, regarding the Variable Number of Tandem Repeats polymorphism, cells with A4/A4 genotype showed a greater expression inhibition (ranging from 20% to 30%) compared with others carrying the A2/A2 one, while those cells bringing A2/A3 genotype showed an increase of expression (of 25%, about). Finally, we demonstrated for the first time that the A2 and A3 CYP2E1 alleles play a more important role in the expression of the enzyme, compared with other (epi)genetic factors, since they are binding sites for trans-acting proteins.

  5. Characterization and polymerase chain reaction (PCR) detection of an Alu deletion polymorphism in total linkage disequilibrium with myotonic dystrophy

    SciTech Connect

    Mahadevan, M.S. ); Foitzik, M.A. ); Surh, L.C.; Korneluk, R.G. Univ. of Ottawa )

    1993-02-01

    The mutation causing myotonic dystrophy has been identified as an unstable trinucleotide CRG repeat located in the 3[prime] untranslated region of a gene putatively encoding a serine-threonine protein kinase. The mutation has been reported to be in total linkage disequilibrium with an insertion/deletion polymorphism located within the kinase gene. To determine the nature of this polymorphism, we have sequenced this genomic fragment and have found that the sequence of this region consists of five consecutive Alu repeats. Further analysis suggests that the smaller of two alleles is actually due to a proposed deletion event that resulted in the loss of an equivalent of three Alu repeats. We have developed a PCR-based assay to detect this polymorphism, the closest, distal marker to the DM mutation. 12 refs., 2 figs.

  6. Inter- and Intraspecific Identification of the New World Screwworm Using Random Amplified Polymorphic DNA-Polymerase Chain Reaction

    Technology Transfer Automated Retrieval System (TEKTRAN)

    New World screwworms (NWS), Cochliomyia hominivorax (Coquerel), are one of the most important arthropod pests of livestock in the Western Hemisphere. Early instars are very difficult to distinguish morphologically from several closely related blow fly species. Random amplified polymorphic DNA polyme...

  7. Examination of meat components in commercial dog and cat feed by using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) technique.

    PubMed

    Wang, Hsien-Chi; Lee, Shu-Hwae; Chang, Tien-Jye; Wong, Min-Liang

    2004-07-01

    It has been shown that certain slow neurological diseases such as bovine spongiform encephalopathy (also known as "mad cow" disease) could be transmitted through contaminated food intake by animals; therefore, the examination of meat components in commercial feeds is important for the control of the disease in public health. The combination of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) technique was applied to examine the meat components in dog and cat commercial feeds. The partial nucleotide sequence (359 bp) of animal mitochondrial cytochrome b (cytb, CYT) gene was amplified by PCR and then digested with restriction enzyme Alu I or Mbo I. In this work, eight brands of commercial dog and cat feeds available in Taiwan were examined. All brands of dog feeds that were tested contained meat from four different animals (cattle, pig, goat and chicken). In cat feeds, the chicken meat was found in five out of eight brands. PMID:15297759

  8. [Species identification of grouper and snapper in Taiwan Strait using polymerase chain reaction-restriction fragment length polymorphism analysis and lab-on-a-chip system].

    PubMed

    Chen, Shuangya; Zhang, Jin; Chen, Weiling; Xu, Dunming; Zhou, Yu

    2011-07-01

    Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and lab-on-a-chip system were used to identify grouper and snapper species in Taiwan Strait. A fragment of 464 bp length of mitochondrial cytochrome b gene was amplified by PCR and the products were digested with restriction enzymes Dde I , Hae III and NLa III, individually. The fragments generated after digestion were further resolved on the DNA Chip. Eight grouper species and five snapper species were successfully identified. The results demonstrated that PCR-RFLP analysis and lab-on-a-chip system provide a fast, easy, automated, and reliable analysis approach. This approach is potential for the purpose of fish adulteration control.

  9. Polymerase chain reaction-restriction fragment length polymorphism method to distinguish three mealybug groups within the Planococcus citri-P. minor species complex (Hemiptera: Coccoidea: Pseudococcidae).

    PubMed

    Rung, A; Miller, D R; Scheffer, S J

    2009-02-01

    The mealybug species Planococcus citri (Risso) and Planococcus minor (Maskell) (Hemiptera: Coccoidea: Pseudococcidae) have special significance to U.S. quarantine and U.S. agriculture. Commonly intercepted at U.S. ports-of-entry, they are difficult to identify based on morphological characters. This study presents a molecular method for distinguishing P. citri, P. minor, and a genetically distinct group that is morphologically identical to P. citri, from Hawaii. This method uses polymerase chain reaction (PCR) followed by restriction fragment polymorphism analysis (RFLP) using the restriction enzymes BspH1, BsmH1, and HpH1. The resulting band patterns can be visualized in a 2% agarose gel and are sufficient to differentiate between the three entities mentioned above. PCR-RFLP diagnostics can be used for all life stages and is cheaper and faster than DNA sequencing.

  10. Examination of meat components in commercial dog and cat feed by using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) technique.

    PubMed

    Wang, Hsien-Chi; Lee, Shu-Hwae; Chang, Tien-Jye; Wong, Min-Liang

    2004-07-01

    It has been shown that certain slow neurological diseases such as bovine spongiform encephalopathy (also known as "mad cow" disease) could be transmitted through contaminated food intake by animals; therefore, the examination of meat components in commercial feeds is important for the control of the disease in public health. The combination of polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) technique was applied to examine the meat components in dog and cat commercial feeds. The partial nucleotide sequence (359 bp) of animal mitochondrial cytochrome b (cytb, CYT) gene was amplified by PCR and then digested with restriction enzyme Alu I or Mbo I. In this work, eight brands of commercial dog and cat feeds available in Taiwan were examined. All brands of dog feeds that were tested contained meat from four different animals (cattle, pig, goat and chicken). In cat feeds, the chicken meat was found in five out of eight brands.

  11. EGFR gene methylation is not involved in Royalactin controlled phenotypic polymorphism in honey bees

    PubMed Central

    Kucharski, R.; Foret, S.; Maleszka, R.

    2015-01-01

    The 2011 highly publicised Nature paper by Kamakura on honeybee phenotypic dimorphism, (also using Drosophila as an experimental surrogate), claims that a single protein in royal jelly, Royalactin, essentially acts as a master “on-off” switch in development via the epidermal growth factor receptor (AmEGFR), to seal the fate of queen or worker. One mechanism proposed in that study as important for the action of Royalactin is differential amegfr methylation in alternate organismal outcomes. According to the author differential methylation of amegfr was experimentally confirmed and shown in a supportive figure. Here we have conducted an extensive analysis of the honeybee egfr locus and show that this gene is never methylated. We discuss several lines of evidence casting serious doubts on the amegfr methylation result in the 2011 paper and consider possible origins of the author’s statement. In a broader context, we discuss the implication of our findings for contrasting context-dependent regulation of EGFR in three insect species, Apis mellifera, D. melanogaster and the carpenter ant, Camponotus floridanus, and argue that more adequate methylation data scrutiny measures are needed to avoid unwarranted conclusions. PMID:26358539

  12. Double Gene Targeting Multiplex Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Assay Discriminates Beef, Buffalo, and Pork Substitution in Frankfurter Products.

    PubMed

    Hossain, M A Motalib; Ali, Md Eaqub; Abd Hamid, Sharifah Bee; Asing; Mustafa, Shuhaimi; Mohd Desa, Mohd Nasir; Zaidul, I S M

    2016-08-17

    Beef, buffalo, and pork adulteration in the food chain is an emerging and sensitive issue. Current molecular techniques to authenticate these species depend on polymerase chain reaction (PCR) assays involving long and single targets which break down under natural decomposition and/or processing treatments. This novel multiplex polymerase chain reaction-restriction fragment length polymorphism assay targeted two different gene sites for each of the bovine, buffalo, and porcine materials. This authentication ensured better security, first through a complementation approach because it is highly unlikely that both sites will be missing under compromised states, and second through molecular fingerprints. Mitochondrial cytochrome b and ND5 genes were targeted, and all targets (73, 90, 106, 120, 138, and 146 bp) were stable under extreme boiling and autoclaving treatments. Target specificity and authenticity were ensured through cross-amplification reaction and restriction digestion of PCR products with AluI, EciI, FatI, and CviKI-1 enzymes. A survey of Malaysian frankfurter products revealed rampant substitution of beef with buffalo but purity in porcine materials.

  13. Novel polymerase chain reaction-restriction fragment length polymorphism assay to determine internal transcribed spacer-2 group in the Chagas disease vector, Triatoma dimidiata (Latreille, 1811).

    PubMed

    Richards, Bethany; Rúa, Nicholas M de la; Monroy, Carlota; Stevens, Lori; Dorn, Patricia L

    2013-06-01

    Triatoma dimidiata is the most important Chagas disease insect vector in Central America as this species is primarily responsible for Trypanosoma cruzi transmission to humans, the protozoan parasite that causes Chagas disease. T. dimidiata sensu lato is a genetically diverse assemblage of taxa and effective vector control requires a clear understanding of the geographic distribution and epidemiological importance of its taxa. The nuclear ribosomal internal transcribed spacer 2 (ITS-2) is frequently used to infer the systematics of triatomines. However, oftentimes amplification and sequencing of ITS-2 fails, likely due to both the large polymerase chain reaction (PCR) product and polymerase slippage near the 5' end. To overcome these challenges we have designed new primers that amplify only the 3'-most 200 base pairs of ITS-2. This region distinguishes the ITS-2 group for 100% of known T. dimidiata haplotypes. Furthermore, we have developed a PCR-restriction fragment length polymorphism (RFLP) approach to determine the ITS-2 group, greatly reducing, but not eliminating, the number of amplified products that need to be sequenced. Although there are limitations with this new PCR-RFLP approach, its use will help with understanding the geographic distribution of T. dimidiata taxa and can facilitate other studies characterising the taxa, e.g. their ecology, evolution and epidemiological importance, thus improving vector control.

  14. Double Gene Targeting Multiplex Polymerase Chain Reaction-Restriction Fragment Length Polymorphism Assay Discriminates Beef, Buffalo, and Pork Substitution in Frankfurter Products.

    PubMed

    Hossain, M A Motalib; Ali, Md Eaqub; Abd Hamid, Sharifah Bee; Asing; Mustafa, Shuhaimi; Mohd Desa, Mohd Nasir; Zaidul, I S M

    2016-08-17

    Beef, buffalo, and pork adulteration in the food chain is an emerging and sensitive issue. Current molecular techniques to authenticate these species depend on polymerase chain reaction (PCR) assays involving long and single targets which break down under natural decomposition and/or processing treatments. This novel multiplex polymerase chain reaction-restriction fragment length polymorphism assay targeted two different gene sites for each of the bovine, buffalo, and porcine materials. This authentication ensured better security, first through a complementation approach because it is highly unlikely that both sites will be missing under compromised states, and second through molecular fingerprints. Mitochondrial cytochrome b and ND5 genes were targeted, and all targets (73, 90, 106, 120, 138, and 146 bp) were stable under extreme boiling and autoclaving treatments. Target specificity and authenticity were ensured through cross-amplification reaction and restriction digestion of PCR products with AluI, EciI, FatI, and CviKI-1 enzymes. A survey of Malaysian frankfurter products revealed rampant substitution of beef with buffalo but purity in porcine materials. PMID:27501408

  15. Methylation of Arsenic by Recombinant Human Wild-Type Arsenic (+3 Oxidation State) Methyltransferase and its Methionine 287 Threonine (M287T) Polymorph

    EPA Science Inventory

    ABSTRACT Arsenic (+3 oxidation state) methyltransferase (AS3MT) is the key enzyme in the pathway for methylation of arsenicals. A common polymorphism in the AS3MT gene that replaces a threonyl residue in position 287 with a methionyl residue (AS3MT/M287T) occurs at a frequency...

  16. Single-nucleotide polymorphisms in DNA bypass polymerase genes and association with breast cancer and breast cancer subtypes among African Americans and Whites

    PubMed Central

    Family, Leila; Bensen, Jeannette T.; Troester, Melissa A.; Wu, Michael C.; Anders, Carey K.; Olshan, Andrew F.

    2015-01-01

    DNA damage recognition and repair is a complex system of genes focused on maintaining genomic stability. Recently, there has been a focus on how breast cancer susceptibility relates to genetic variation in the DNA bypass polymerases pathway. Race-stratified and subtype-specific logistic regression models were used to estimate odds ratios (ORs) and 95 % confidence intervals (CIs) for the association between 22 single-nucleotide polymorphisms (SNPs) in seven bypass polymerase genes and breast cancer risk in the Carolina Breast Cancer Study, a population-based, case–control study (1,972 cases and 1,776 controls). We used SNP-set kernel association test (SKAT) to evaluate the multi-gene, multi-locus (combined) SNP effects within bypass polymerase genes. We found similar ORs for breast cancer with three POLQ SNPs (rs487848 AG/AA vs. GG; OR = 1.31, 95 % CI 1.03–1.68 for Whites and OR = 1.22, 95 % CI 1.00–1.49 for African Americans), (rs532411 CT/TT vs. CC; OR = 1.31, 95 % CI 1.02–1.66 for Whites and OR = 1.22, 95 % CI 1.00–1.48 for African Americans), and (rs3218634 CG/CC vs. GG; OR = 1.29, 95 % CI 1.02–1.65 for Whites). These three SNPs are in high linkage disequilibrium in both races. Tumor subtype analysis showed the same SNPs to be associated with increased risk of Luminal breast cancer. SKAT analysis showed no significant combined SNP effects. These results suggest that variants in the POLQ gene may be associated with the risk of Luminal breast cancer. PMID:25417172

  17. Molecular variation analysis of Aspergillus flavus using polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer rDNA region

    PubMed Central

    Zarrin, Majid; Erfaninejad, Maryam

    2016-01-01

    Aspergillus flavus is the second most common disease-causing species of Aspergillus in humans. The fungus is frequently associated with life-threatening infections in immunocompromised hosts. The primary aim of the present study was to analyze the genetic variability among different isolates of A. flavus using polymerase chain reaction (PCR)-based restriction fragment length polymorphism (RFLP). A total of 62 A. flavus isolates were tested in the study. Molecular variability was searched for by analysis of the PCR amplification of the internal transcribed spacer (ITS) regions of ribosomal DNA using restriction enzymes. PCR using primers for ITS1 and ITS4 resulted in a product of ~600 bp. Amplicons were subjected to digestion with restriction endonucleases EcoRI, HaeIII and TaqI. Digestion of the PCR products using these restriction enzymes produced different patterns of fragments among the isolates, with different sizes and numbers of fragments, revealing genetic variability. In conclusion, ITS-RFLP is a useful molecular tool in screening for nucleotide polymorphisms among A. flavus isolates. PMID:27588085

  18. Analysis of the rDNA internal transcribed spacer region of the Fusarium species by polymerase chain reaction-restriction fragment length polymorphism

    PubMed Central

    ZARRIN, MAJID; GANJ, FARZANEH; FARAMARZI, SAMA

    2016-01-01

    The Fusarium species are a widely spread phytopathogen identified in an extensive variety of hosts. The Fusarium genus is one of the most heterogeneous fungi and is difficult to classify. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis is a useful method in detection of DNA polymorphism in objective sequences. The aim of the present study was to identify the phylogenetic associations and usefulness of the internal transcribed spacer (ITS) region as a genetic marker within the most clinically important strain of the Fusarium species. A total of 50 strains of Fusarium spp. were used in the study, including environmental, clinical and reference isolates. The primers ITS1 and ITS4 were used in the study. Two restriction enzymes, HaeIII and SmaI, were assessed for the digestion of PCR products. A PCR product of ~550-base pairs was generated for each Fusarium species. The digested products with HaeIII and SmaI demonstrated that the bands generated for the medically significant Fusarium species, including F. solani, F. oxysporum, F. verticillidea, F. proliferatum and F. fujikuri, have different restriction enzyme patterns. In conclusion, it appears that the PCR-RFLP method used in the present study produces a sufficient restriction profile for differentiation of the most medically significant Fusarium species. PMID:27073635

  19. Screening for JH1 genetic defect carriers in Jersey cattle by a polymerase chain reaction and restriction fragment length polymorphism assay.

    PubMed

    Zhang, Yi; Guo, Gang; Huang, Hetian; Lu, Lu; Wang, Lijie; Fang, Lingzhao; Liu, Lin; Wang, Yachun; Zhang, Shengli

    2015-09-01

    An autosomal recessive genetic defect termed JH1 has been associated with early embryonic loss in the Jersey cattle breed. The genetic basis has been identified as a cytosine to thymine mutation in the CWC15 gene that changes an amino acid from arginine to a stop code. To screen for JH1 carriers in an imported Jersey population in China, a method based on a polymerase chain reaction amplification followed by a restriction fragment length polymorphism assay (PCR-RFLP) was developed for the accurate diagnosis of the JH1 allele. A total of 449 randomly chosen cows were examined with the PCR-RFLP assay, and 31 were identified as JH1 carriers, corresponding to a carrier frequency of 6.9%. The PCR-RFLP method was validated by DNA sequencing of 8 positive and 13 negative samples, with all 21 samples giving the expected DNA sequence. In addition, 3 negative and 3 positive samples were confirmed by a commercial microarray-based single nucleotide polymorphism assay. Finally, samples from 9 bulls in the United States of known status were correctly identified as carriers (5 bulls) or noncarriers (4 bulls). As the JH1 defect has most likely spread worldwide, implementing routine screening is necessary to avoid the risk of carrier-to-carrier matings and to gradually eradicate the deleterious gene.

  20. A preliminary assessment of genetic differentiation of Triatoma dimidiata (Hemiptera: Reduviidae) in Guatemala by random amplification of polymorphic DNA-polymerase chain reaction.

    PubMed

    Calderón, Claudia I; Dorn, Patricia L; Melgar, Sergio; Chávez, Juan José; Rodas, Antonieta; Rosales, Regina; Monroy, Carlota M

    2004-09-01

    The population genetics of Triatoma dimidiata (Latreille, 1811) from five different provinces in Guatemala, including three sylvan and three domestic populations, was investigated by random amplification of polymorphic DNA-polymerase chain reaction. There is a high degree of genetic variation in all of the T. dimidiata populations as evidenced by high levels of average expected heterozygosity and polymorphism. Domestic populations are more closely related to each other (D = 0.05-0.085, Nei's genetic distance) than are the sylvan (D = 0.121-0.189). Within the limited sample size of three populations, there was a correlation with geographic and genetic distance for the domestic populations, but not for the sylvan. Surprisingly, one of the sylvan populations was genetically very similar to the domestic populations. The FST demonstrated a high degree of differentiation at the country-wide level (FST = 0.175) and a moderate degree of differentiation within the sylvan (FST = 0.135) or domestic (FST = 0.097) populations. Although these results demonstrated that gene flow is limited between different provinces in Guatemala, hierarchical analysis showed that barriers between the Atlantic and Pacific drainage slopes were not biologically significant limiters of gene flow.

  1. A duplicated region is responsible for the poly(ADP-ribose) polymerase polymorphism, on chromosome 13, associated with a predisposition to cancer

    SciTech Connect

    Lyn, D.; Cherney, B.W.; Lupold, S.; Smulson, M. ); Lalande, M. Harvard Medical School, Boston, MA ); Berenson, J.R.; Lichtenstein, A. Veterans Administration Medical Center, Los Angeles, CA ); Bhatia, K.G. )

    1993-01-01

    The poly(ADP-ribose) polymerase (PADPRP) gene (13q33-qter) depicts a two-allele (A/B) polymorphism. In the noncancer population, the frequency of the B allele is higher among blacks than among whites. Since the incidence of multiple myeloma and prostate and lung cancer is higher in the US black population, the authors have analyzed the B-allele frequency in germ-line DNA to determine whether the PADPRP gene correlates with a polymorphic susceptibility to these diseases. For multiple myeloma and prostate cancer, an increased frequency of the B allele appeared to be striking only in black patients. In contrast, the distribution of the B allele in germ-line DNA did not differ among white patients with these diseases, when compared with the control group. An elevated B-allele frequency was also found in germ-line DNA in blacks with colon cancer. These observations suggest that the PADPRP polymorphism may provide a valid marker for a predisposition to these cancers in black individuals. To determine the genomic structure of the polymorphic PADPRP sequences, a 2.68-kb HindIII clone was isolated and sequenced from a chromosome 13-enriched library. Sequence analysis of this clone (A allele) revealed a close sequence similarity (91.8%) to PADPRP cDNA (1q42) and an absence of introns, suggesting that the gene on 13q exists as a processed pseudogene. A 193-bp conserved duplicated region within the A allele was identified as the source of the polymorphism. The nucleotide differences between the PADPRP gene on chromosome 13 and related PADPRP genes were exploited to develop oligonucleotides that can detect the difference between the A/B genotypes in a PCR. This PCR assay offers the opportunity for analyzing additional black cancer patients, to determine how the PADPRP processed pseudogene or an unidentified gene that cosegregates with the PADPRP gene might be involved with the development of malignancy. 16 refs., 6 figs., 1 tab.

  2. Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance.

    PubMed

    Chen, Z J; Pikaard, C S

    1997-08-15

    Nucleolar dominance is an epigenetic phenomenon that describes nucleolus formation around rRNA genes inherited from only one progenitor of an interspecific hybrid or allopolyploid. The phenomenon is widespread, occurring in plants, insects, amphibians, and mammals, yet its molecular basis remains unclear. We have demonstrated nucleolar dominance in three allotetraploids of the plant genus Brassica. In Brassica napus, accurately initiated pre-rRNA transcripts from one progenitor, Brassica rapa are detected readily, whereas transcripts from the approximately 3000 rRNA genes inherited from the other progenitor, Brassica oleracea, are undetectable. Nuclear run-on confirmed that dominance is controlled at the level of transcription. Growth of B. napus seedlings on 5-aza-2'-deoxycytidine to inhibit cytosine methylation caused the normally silent, under-dominant B. oleracea rRNA genes to become expressed to high levels. The histone deacetylase inhibitors sodium butyrate and trichostatin A also derepressed silent rRNA genes. These results reveal an enforcement mechanism for nucleolar dominance in which DNA methylation and histone modifications combine to regulate rRNA gene loci spanning tens of megabase pairs of DNA.

  3. Epigenetic silencing of RNA polymerase I transcription: a role for DNA methylation and histone modification in nucleolar dominance

    PubMed Central

    Chen, Z. Jeffrey; Pikaard, Craig S.

    1997-01-01

    Nucleolar dominance is an epigenetic phenomenon that describes nucleolus formation around rRNA genes inherited from only one progenitor of an interspecific hybrid or allopolyploid. The phenomenon is widespread, occurring in plants, insects, amphibians, and mammals, yet its molecular basis remains unclear. We have demonstrated nucleolar dominance in three allotetraploids of the plant genus Brassica. In Brassica napus, accurately initiated pre-rRNA transcripts from one progenitor, Brassica rapa are detected readily, whereas transcripts from the ∼3000 rRNA genes inherited from the other progenitor, Brassica oleracea, are undetectable. Nuclear run-on confirmed that dominance is controlled at the level of transcription. Growth of B. napus seedlings on 5-aza-2′-deoxycytidine to inhibit cytosine methylation caused the normally silent, under-dominant B. oleracea rRNA genes to become expressed to high levels. The histone deacetylase inhibitors sodium butyrate and trichostatin A also de-epressed silent rRNA genes. These results reveal an enforcement mechanism for nucleolar dominance in which DNA methylation and histone modifications combine to regulate rRNA gene loci spanning tens of megabase pairs of DNA. PMID:9284051

  4. XRCC1 Arg194Trp and Arg399Gln polymorphisms and arsenic methylation capacity are associated with urothelial carcinoma.

    PubMed

    Chiang, Chien-I; Huang, Ya-Li; Chen, Wei-Jen; Shiue, Horng-Sheng; Huang, Chao-Yuan; Pu, Yeong-Shiau; Lin, Ying-Chin; Hsueh, Yu-Mei

    2014-09-15

    The association between DNA repair gene polymorphisms and bladder cancer has been widely studied. However, few studies have examined the correlation between urothelial carcinoma (UC) and arsenic or its metabolites. The aim of this study was to examine the association between polymorphisms of the DNA repair genes, XRCC1 Arg194Trp, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln, with urinary arsenic profiles and UC. To this end, we conducted a hospital-based case-control study with 324 UC patients and 647 age- and gender-matched non-cancer controls. Genomic DNA was used to examine the genotype of XRCC1 Arg194Trp, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln by PCR-restriction fragment length polymorphism analysis (PCR-RFLP). Urinary arsenic profiles were measured by high performance liquid chromatography (HPLC) linked with hydride generator and atomic absorption spectrometry. The XRCC1 399 Gln/Gln and 194 Arg/Trp and Trp/Trp genotypes were significantly related to UC, and the odds ratio (OR) and 95% confidence interval (95%CI) were 1.68 (1.03-2.75) and 0.66 (0.48-0.90), respectively. Participants with higher total urinary arsenic levels, a higher percentage of inorganic arsenic (InAs%) and a lower percentage of dimethylarsinic acid (DMA%) had a higher OR of UC. Participants carrying XRCC1 risk diplotypes G-C/G-C, A-C/A-C, and A-T/G-T, and who had higher total arsenic levels, higher InAs%, or lower DMA% compared to those with other XRCC1 diplotypes had a higher OR of UC. Our results suggest that the XRCC1 399 Gln/Gln and 194 Arg/Arg DNA repair genes play an important role in poor arsenic methylation capacity, thereby increasing the risk of UC in non-obvious arsenic exposure areas.

  5. NRPD4, a Protein Related to the RPB4 Subunit of RNA Polymerase II, is a Component of RNA Polymerases IV and V and is Required for RNA-directed DNA methylation

    SciTech Connect

    He, Xin-Jian; Hsu, Yi-Feng; Pontes, Olga; Zhu, Jianhua; Lu, Jian; Bressan, Ray A.; Pikaard, Craig S.; Wang, Co-Shine; Zhu, Jian-Kang

    2009-01-01

    RNA-directed DNA methylation (RdDM) is an RNAi-based mechanism for establishing transcriptional gene silencing in plants. The plant-specific RNA polymerases IV and V are required for the generation of 24-nucleotide (nt) siRNAs and for guiding sequence-specific DNA methylation by the siRNAs, respectively. However, unlike the extensively studied multisubunit Pol II, our current knowledge about Pol IV and Pol V is restricted to only the two largest subunits NRPD1a/NRPD1 and NRPD1b/NRPE1 and the one second-largest subunit NRPD2a. It is unclear whether other subunits may be required for the functioning of Pol IV and Pol V in RdDM. From a genetic screen for second-site suppressors of the DNA demethylase mutant ros1, we identified a new component (referred to as RDM2) as well as seven known components (NRPD1, NRPE1, NRPD2a, AGO4, HEN1, DRD1, and HDA6) of the RdDM pathway. The differential effects of the mutations on two mechanistically distinct transcriptional silencing reporters suggest that RDM2, NRPD1, NRPE1, NRPD2a, HEN1, and DRD1 function only in the siRNA-dependent pathway of transcriptional silencing, whereas HDA6 and AGO4 have roles in both siRNA-dependent and -independent pathways of transcriptional silencing. In the rdm2 mutants, DNA methylation and siRNA accumulation were reduced substantially at loci previously identified as endogenous targets of Pol IV and Pol V, including 5S rDNA, MEA-ISR, AtSN1, AtGP1, and AtMU1. The amino acid sequence of RDM2 is similar to that of RPB4 subunit of Pol II, but we show evidence that RDM2 has diverged significantly from RPB4 and cannot function in Pol II. An association of RDM2 with both NRPD1 and NRPE1 was observed by coimmunoprecipitation and coimmunolocalization assays. Our results show that RDM2/NRPD4/NRPE4 is a new component of the RdDM pathway in Arabidopsis and that it functions as part of Pol IV and Pol V.

  6. XRCC1 Arg194Trp and Arg399Gln polymorphisms and arsenic methylation capacity are associated with urothelial carcinoma

    SciTech Connect

    Chiang, Chien-I; Huang, Ya-Li; Chen, Wei-Jen; Shiue, Horng-Sheng; Huang, Chao-Yuan; Pu, Yeong-Shiau; Lin, Ying-Chin; Hsueh, Yu-Mei

    2014-09-15

    The association between DNA repair gene polymorphisms and bladder cancer has been widely studied. However, few studies have examined the correlation between urothelial carcinoma (UC) and arsenic or its metabolites. The aim of this study was to examine the association between polymorphisms of the DNA repair genes, XRCC1 Arg194Trp, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln, with urinary arsenic profiles and UC. To this end, we conducted a hospital-based case–control study with 324 UC patients and 647 age- and gender-matched non-cancer controls. Genomic DNA was used to examine the genotype of XRCC1 Arg194Trp, XRCC1 Arg399Gln, XRCC3 Thr241Met, and XPD Lys751Gln by PCR-restriction fragment length polymorphism analysis (PCR-RFLP). Urinary arsenic profiles were measured by high performance liquid chromatography (HPLC) linked with hydride generator and atomic absorption spectrometry. The XRCC1 399 Gln/Gln and 194 Arg/Trp and Trp/Trp genotypes were significantly related to UC, and the odds ratio (OR) and 95% confidence interval (95%CI) were 1.68 (1.03–2.75) and 0.66 (0.48–0.90), respectively. Participants with higher total urinary arsenic levels, a higher percentage of inorganic arsenic (InAs%) and a lower percentage of dimethylarsinic acid (DMA%) had a higher OR of UC. Participants carrying XRCC1 risk diplotypes G-C/G-C, A-C/A-C, and A-T/G-T, and who had higher total arsenic levels, higher InAs%, or lower DMA% compared to those with other XRCC1 diplotypes had a higher OR of UC. Our results suggest that the XRCC1 399 Gln/Gln and 194 Arg/Arg DNA repair genes play an important role in poor arsenic methylation capacity, thereby increasing the risk of UC in non-obvious arsenic exposure areas. - Highlights: • The XRCC1 399Gln/Gln genotype was significantly associated with increased OR of UC. • The XRCC1 194 Arg/Trp and Trp/Trp genotype had a significantly decreased OR of UC. • Combined effect of the XRCC1 genotypes and poor arsenic methylation capacity on

  7. Simultaneous Analysis of SEPT9 Promoter Methylation Status, Micronuclei Frequency, and Folate-Related Gene Polymorphisms: The Potential for a Novel Blood-Based Colorectal Cancer Biomarker.

    PubMed

    Ravegnini, Gloria; Zolezzi Moraga, Juan Manuel; Maffei, Francesca; Musti, Muriel; Zenesini, Corrado; Simeon, Vittorio; Sammarini, Giulia; Festi, Davide; Hrelia, Patrizia; Angelini, Sabrina

    2015-12-01

    One challenge in colorectal cancer (CRC) is identifying novel biomarkers to be introduced in screening programs. The present study investigated the promoter methylation status of the SEPT9 gene in peripheral blood samples of subjects' positive fecal occult blood test (FOBT). In order to add new insights, we investigated the association between SEPT9 promoter methylation and micronuclei frequency, and polymorphisms in the folate-related pathway genes. SEPT9 promoter methylation, micronuclei frequency, and genotypes were evaluated on 74 individuals' FOBT positive. Individuals were subjected to a colonoscopy that provided written informed consent for study participation. SEPT9 promoter methylation status was significantly lower in the CRC group than controls (p = 0.0006). In contrast, the CaCo2 cell-line, analyzed as a tissue specific model of colon adenocarcinoma, showed a significantly higher percentage of SEPT9 promoter methylation compared to the CRC group (p < 0.0001). Linear regression analysis showed an inverse correlation between micronuclei frequency and the decrease in the methylation levels of SEPT9 promoter region among CRC patients (β = -0.926, p = 0.0001). With regard to genotype analysis, we showed the involvement of the DHFR polymorphism (rs70991108) in SEPT9 promoter methylation level in CRC patients only. In particular, the presence of at least one 19 bp del allele significantly correlates with decreased SEPT9 promoter methylation, compared to the 19 bp ins/ins genotype (p = 0.007). While remaining aware of the strengths and limitations of the study, this represents the first evidence of a novel approach for the early detection of CRC, using SEPT9 promoter methylation, micronuclei frequency and genotypes, with the potential to improve CRC risk assessment.

  8. Simultaneous Analysis of SEPT9 Promoter Methylation Status, Micronuclei Frequency, and Folate-Related Gene Polymorphisms: The Potential for a Novel Blood-Based Colorectal Cancer Biomarker

    PubMed Central

    Ravegnini, Gloria; Zolezzi Moraga, Juan Manuel; Maffei, Francesca; Musti, Muriel; Zenesini, Corrado; Simeon, Vittorio; Sammarini, Giulia; Festi, Davide; Hrelia, Patrizia; Angelini, Sabrina

    2015-01-01

    One challenge in colorectal cancer (CRC) is identifying novel biomarkers to be introduced in screening programs. The present study investigated the promoter methylation status of the SEPT9 gene in peripheral blood samples of subjects’ positive fecal occult blood test (FOBT). In order to add new insights, we investigated the association between SEPT9 promoter methylation and micronuclei frequency, and polymorphisms in the folate-related pathway genes. SEPT9 promoter methylation, micronuclei frequency, and genotypes were evaluated on 74 individuals’ FOBT positive. Individuals were subjected to a colonoscopy that provided written informed consent for study participation. SEPT9 promoter methylation status was significantly lower in the CRC group than controls (p = 0.0006). In contrast, the CaCo2 cell-line, analyzed as a tissue specific model of colon adenocarcinoma, showed a significantly higher percentage of SEPT9 promoter methylation compared to the CRC group (p < 0.0001). Linear regression analysis showed an inverse correlation between micronuclei frequency and the decrease in the methylation levels of SEPT9 promoter region among CRC patients (β = −0.926, p = 0.0001). With regard to genotype analysis, we showed the involvement of the DHFR polymorphism (rs70991108) in SEPT9 promoter methylation level in CRC patients only. In particular, the presence of at least one 19 bp del allele significantly correlates with decreased SEPT9 promoter methylation, compared to the 19 bp ins/ins genotype (p = 0.007). While remaining aware of the strengths and limitations of the study, this represents the first evidence of a novel approach for the early detection of CRC, using SEPT9 promoter methylation, micronuclei frequency and genotypes, with the potential to improve CRC risk assessment. PMID:26633373

  9. Simultaneous Analysis of SEPT9 Promoter Methylation Status, Micronuclei Frequency, and Folate-Related Gene Polymorphisms: The Potential for a Novel Blood-Based Colorectal Cancer Biomarker.

    PubMed

    Ravegnini, Gloria; Zolezzi Moraga, Juan Manuel; Maffei, Francesca; Musti, Muriel; Zenesini, Corrado; Simeon, Vittorio; Sammarini, Giulia; Festi, Davide; Hrelia, Patrizia; Angelini, Sabrina

    2015-01-01

    One challenge in colorectal cancer (CRC) is identifying novel biomarkers to be introduced in screening programs. The present study investigated the promoter methylation status of the SEPT9 gene in peripheral blood samples of subjects' positive fecal occult blood test (FOBT). In order to add new insights, we investigated the association between SEPT9 promoter methylation and micronuclei frequency, and polymorphisms in the folate-related pathway genes. SEPT9 promoter methylation, micronuclei frequency, and genotypes were evaluated on 74 individuals' FOBT positive. Individuals were subjected to a colonoscopy that provided written informed consent for study participation. SEPT9 promoter methylation status was significantly lower in the CRC group than controls (p = 0.0006). In contrast, the CaCo2 cell-line, analyzed as a tissue specific model of colon adenocarcinoma, showed a significantly higher percentage of SEPT9 promoter methylation compared to the CRC group (p < 0.0001). Linear regression analysis showed an inverse correlation between micronuclei frequency and the decrease in the methylation levels of SEPT9 promoter region among CRC patients (β = -0.926, p = 0.0001). With regard to genotype analysis, we showed the involvement of the DHFR polymorphism (rs70991108) in SEPT9 promoter methylation level in CRC patients only. In particular, the presence of at least one 19 bp del allele significantly correlates with decreased SEPT9 promoter methylation, compared to the 19 bp ins/ins genotype (p = 0.007). While remaining aware of the strengths and limitations of the study, this represents the first evidence of a novel approach for the early detection of CRC, using SEPT9 promoter methylation, micronuclei frequency and genotypes, with the potential to improve CRC risk assessment. PMID:26633373

  10. GSTP1 methylation and polymorphism increase the risk of breast cancer and the effects of diet and lifestyle in breast cancer patients.

    PubMed

    Saxena, Anubha; Dhillon, Varinderpal S; Shahid, Mohammad; Khalil, Hesham Saleh; Rani, Madhu; Prasad DAS, Trinath; Hedau, Suresh; Hussain, Arif; Naqvi, Raza Ali; Deo, S V S; Shukla, N K; DAS, B C; Husain, Syed Akhtar

    2012-12-01

    Glutathione S-transferases (GSTs) are an important group of isoenzymes that play an essential role in the detoxification of carcinogens. Polymorphism at exon 5 of the GST π family decreases the catalytic activity and affects the detoxification ability of the enzyme, GSTP1. GSTP1 promoter hypermethylation and loss of expression are frequently observed in various types of carcinoma. We hypothesized that somatic epigenetic modification in homozygous mutants increases the degree to which breast cancer risk is affected by lifestyle factors and dietary habits. The present study used tumor biopsies and blood samples from 215 breast cancer patients and 215 blood samples from healthy donors. GSTP1 polymorphism was studied using PCR-restriction fragment length polymorphism, methylation using methylation-specific PCR and loss of expression using immunohistochemistry and western blotting. No significant increase was observed in the breast cancer risk of individuals with the mutant (Val) allele [odds ratio (OR), 1.48; 95% confidence interval (CI), 0.97-2.26 for heterozygotes; OR, 1.42; 95% CI, 0.86-2.42 homozygous mutants]. GSTP1 promoter hypermethylation was detected in one-third of tumor biopsies (74/215) and was found to be associated with a loss of expression. Genotype and tumor methylation associations were not observed. Estrogen (ER) and progesterone (PR) receptor-positive tumors had a higher methylation frequency. GSTP1 polymorphism was not associated with increased promoter hypermethylation. The results suggest that GSTP1 methylation is a major event in breast carcinogenesis and may act as a tumor-specific biomarker.

  11. Polymorphism analysis of Chinese Theileria sergenti using allele-specific polymerase chain reaction of the major piroplasm surface protein gene.

    PubMed

    Liu, Ai Hong; Guan, Gui Quan; Liu, Jun Long; Liu, Zhi Jie; Leblanc, Neil; Li, You Quan; Gao, Jin Liang; Ma, Mi Ling; Niu, Qing Li; Ren, Qiao Yun; Bai, Qi; Yin, Hong; Luo, Jian Xun

    2011-02-01

    Theileria sergenti is a tick-borne parasite found in many parts of the world. The major piroplasm surface protein (MPSP), a conserved protein in all Theileria species, has been used as a marker for epidemiological and phylogenetic studies of benign Theileria species. In this study, Chinese species of T. sergenti were characterized by allele-specific polymerase chain reaction (PCR) and DNA sequence analysis of the MPSP gene. Using universal or allele-specific primer sets for PCR amplification of the MPSP gene, 98 of 288 cattle blood samples, collected from 6 provinces in China, were found to be positive. Among the positive samples, only 3 allelic MPSP gene types (Chitose [C]-, Ikeda [I]-, and buffeli [B]-type) were successfully amplified. Moreover, the results revealed that the majority of the parasites sampled in this study were C- and I-type (prevalence of 84 and 69%, respectively), whereas the B-type was less common (prevalence of 36%). Co-infections with C-, I-, and B-type T. sergenti also were found. An additional known allele, Thai-type, was not detected. Phylogenetic analysis based on the MPSP gene sequences, including 3 standard stocks generated in the laboratory ( T. sergenti Wenchuan, T. sergenti Ningxian, and T. sergenti Liaoyang), revealed that the isolates of Chinese sergenti were comprised of at least 4 allelic MPSP gene types, i.e., C-, I-, B1-, and B2-type, and these parasites with 6 MPSP types 1-5 and 7 were present in China.

  12. Determination of locust bean gum and guar gum by polymerase chain reaction and restriction fragment length polymorphism analysis.

    PubMed

    Meyer, K; Rosa, C; Hischenhuber, C; Meyer, R

    2001-01-01

    A polymerase chain reaction (PCR) was developed to differentiate the thickening agents locust bean gum (LBG) and the cheaper guar gum in finished food products. Universal primers for amplification of the intergenic spacer region between trnL 3' (UAA) exon and trnF (GAA) gene in the chloroplast (cp) genome and subsequent restriction analysis were applied to differentiate guar gum and LBG. The presence of <5% (w/w) guar gum powder added to LBG powder was detectable. Based on data obtained from sequencing this intergenic spacer region, a second PCR method for the specific detection of guar gum DNA was also developed. This assay detected guar gum powder in LBG in amounts as low as 1% (w/w). Both methods successfully detected guar gum and/or LBG in ice cream stabilizers and in foodstuffs, such as dairy products, ice cream, dry seasoning mixes, a finished roasting sauce, and a fruit jelly product, but not in products with highly degraded DNA, such as tomato ketchup and sterilized chocolate cream. Both methods detected guar gum and LBG in ice cream and fresh cheese at levels <0.1%. PMID:11234856

  13. Risk of childhood asthma is associated with CpG-site polymorphisms, regional DNA methylation and mRNA levels at the GSDMB/ORMDL3 locus

    PubMed Central

    Acevedo, Nathalie; Reinius, Lovisa E.; Greco, Dario; Gref, Anna; Orsmark-Pietras, Christina; Persson, Helena; Pershagen, Göran; Hedlin, Gunilla; Melén, Erik; Scheynius, Annika; Kere, Juha; Söderhäll, Cilla

    2015-01-01

    Single-nucleotide polymorphisms (SNPs) in GSDMB (Gasdermin B) and ORMDL3 (ORMDL sphingolipid biosynthesis regulator 3) are strongly associated with childhood asthma, but the molecular alterations contributing to disease remain unknown. We investigated the effects of asthma-associated SNPs on DNA methylation and mRNA levels of GSDMB and ORMDL3. Genetic association between GSDMB/ORMDL3 and physician-diagnosed childhood asthma was confirmed in the Swedish birth-cohort BAMSE. CpG-site SNPs (rs7216389 and rs4065275) showed differences in DNA methylation depending on carrier status of the risk alleles, and were significantly associated with methylation levels in two CpG sites in the 5′ UTR (untranslated region) of ORMDL3. In the Swedish Search study, we found significant differences in DNA methylation between asthmatics and controls in five CpG sites; after adjusting for lymphocyte and neutrophil cell counts, three remained significant: one in IKZF3 [IKAROS family zinc finger 3 (Aiolos); cg16293631] and two in the CpG island (CGI) of ORMDL3 (cg02305874 and cg16638648). Also, cg16293631 and cg02305874 correlated with mRNA levels of ORMDL3. The association between methylation and asthma was independent of the genotype in rs7216389, rs4065275 and rs12603332. Both SNPs and CpG sites showed significant associations with ORMDL3 mRNA levels. SNPs influenced expression independently of methylation, and the residual association between methylation and expression was not mediated by these SNPs. We found a differentially methylated region in the CGI shore of ORMDL3 with six CpG sites less methylated in CD8+ T-cells. In summary, this study supports that there are differences in DNA methylation at this locus between asthmatics and controls; and both SNPs and CpG sites are independently associated with ORMDL3 expression. PMID:25256354

  14. Identification of blood meal sources of Lutzomyia longipalpis using polymerase chain reaction-restriction fragment length polymorphism analysis of the cytochrome B gene.

    PubMed

    Soares, Vítor Yamashiro Rocha; Silva, Jailthon Carlos da; Silva, Kleverton Ribeiro da; Pires e Cruz, Maria do Socorro; Santos, Marcos Pérsio Dantas; Ribolla, Paulo Eduardo Martins; Alonso, Diego Peres; Coelho, Luiz Felipe Leomil; Costa, Dorcas Lamounier; Costa, Carlos Henrique Nery

    2014-06-01

    An analysis of the dietary content of haematophagous insects can provide important information about the transmission networks of certain zoonoses. The present study evaluated the potential of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis of the mitochondrial cytochrome B (cytb) gene to differentiate between vertebrate species that were identified as possible sources of sandfly meals. The complete cytb gene sequences of 11 vertebrate species available in the National Center for Biotechnology Information database were digested with Aci I, Alu I, Hae III and Rsa I restriction enzymes in silico using Restriction Mapper software. The cytb gene fragment (358 bp) was amplified from tissue samples of vertebrate species and the dietary contents of sandflies and digested with restriction enzymes. Vertebrate species presented a restriction fragment profile that differed from that of other species, with the exception of Canis familiaris and Cerdocyon thous. The 358 bp fragment was identified in 76 sandflies. Of these, 10 were evaluated using the restriction enzymes and the food sources were predicted for four: Homo sapiens (1), Bos taurus (1) and Equus caballus (2). Thus, the PCR-RFLP technique could be a potential method for identifying the food sources of arthropods. However, some points must be clarified regarding the applicability of the method, such as the extent of DNA degradation through intestinal digestion, the potential for multiple sources of blood meals and the need for greater knowledge regarding intraspecific variations in mtDNA.

  15. Identification of Pork Contamination in Meatballs of Indonesia Local Market Using Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis.

    PubMed

    Erwanto, Yuny; Abidin, Mohammad Zainal; Sugiyono, Eko Yasin Prasetyo Muslim; Rohman, Abdul

    2014-10-01

    This research applied and evaluated a polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) using cytochrome b gene to detect pork contamination in meatballs from local markets in Surabaya and Yogyakarta regions, Indonesia. To confirm the effectiveness and specificity of this fragment, thirty nine DNA samples from different meatball shops were isolated and amplified, and then the PCR amplicon was digested by BseDI restriction enzyme to detect the presence of pork in meatballs. BseDI restriction enzyme was able to cleave porcine cytochrome b gene into two fragments (131 bp and 228 bp). Testing the meatballs from the local market showed that nine of twenty meatball shops in Yogyakarta region were detected to have pork contamination, but there was no pork contamination in meatball shops in Surabaya region. In conclusion, specific PCR amplification of cytochrome b gen and cleaved by BseDI restriction enzymes seems to be a powerful technique for the identification of pork presence in meatball because of its simplicity, specificity and sensitivity. Furthermore, pork contamination intended for commercial products of sausage, nugget, steak and meat burger can be checked. The procedure is also much cheaper than other methods based on PCR, immunodiffusion and other techniques that need expensive equipment.

  16. Authentication of anglerfish species (Lophius spp) by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically informative nucleotide sequencing (FINS) methodologies.

    PubMed

    Espiñeira, Montserrat; González-Lavín, Nerea; Vieites, Juan M; Santaclara, Francisco J

    2008-11-26

    Lophius represents the most important genus of the family Lophiidae from a commercial point of view. The main marketing formats of the species included in this genus are tails and cheeks, making impossible the species identification on the basis of their morphological characters. In the present study, two methods based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis of DNA sequences [forensically informative nucleotide sequencing (FINS)] were developed to differentiate the seven species contained in the genus Lophius. In both cases, the molecular marker studied was the cytochrome oxidase subunit I gene (COI). The RFLP analysis of the PCR products digested with the endonuclease Mbo I generated species-specific restriction profiles, and the phylogenetic analysis showing a neighbor-joining tree with independent nodes was strongly supported for all of the studied species. These methods were applied to 40 commercial samples, allowing us to detect the samples incorrectly labeled. The fraudulent labeling ratio was higher in processed products (68.75%) than whole fish (31.25%). The species subjected to mislabeling were L. budegassa (68.75%), L. vomerinus (18.75%), and L. piscatorius (12.5%). Therefore, both methodologies can be independently used to authenticate the species belonging to the genus Lophius, being useful to check the fulfillment of labeling regulations of seafood products and to verify the correct traceability of commercial trade and the control of fisheries. PMID:18975961

  17. Authentication of anglerfish species (Lophius spp) by means of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically informative nucleotide sequencing (FINS) methodologies.

    PubMed

    Espiñeira, Montserrat; González-Lavín, Nerea; Vieites, Juan M; Santaclara, Francisco J

    2008-11-26

    Lophius represents the most important genus of the family Lophiidae from a commercial point of view. The main marketing formats of the species included in this genus are tails and cheeks, making impossible the species identification on the basis of their morphological characters. In the present study, two methods based on the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and phylogenetic analysis of DNA sequences [forensically informative nucleotide sequencing (FINS)] were developed to differentiate the seven species contained in the genus Lophius. In both cases, the molecular marker studied was the cytochrome oxidase subunit I gene (COI). The RFLP analysis of the PCR products digested with the endonuclease Mbo I generated species-specific restriction profiles, and the phylogenetic analysis showing a neighbor-joining tree with independent nodes was strongly supported for all of the studied species. These methods were applied to 40 commercial samples, allowing us to detect the samples incorrectly labeled. The fraudulent labeling ratio was higher in processed products (68.75%) than whole fish (31.25%). The species subjected to mislabeling were L. budegassa (68.75%), L. vomerinus (18.75%), and L. piscatorius (12.5%). Therefore, both methodologies can be independently used to authenticate the species belonging to the genus Lophius, being useful to check the fulfillment of labeling regulations of seafood products and to verify the correct traceability of commercial trade and the control of fisheries.

  18. Identification of Echinococcus granulosus strains using polymerase chain reaction-restriction fragment length polymorphism amongst livestock in Moroto district, Uganda.

    PubMed

    Chamai, Martin; Omadang, Leonard; Erume, Joseph; Ocaido, Michael; Oba, Peter; Othieno, Emmanuel; Bonaventure, Straton; Kitibwa, Annah

    2016-01-01

    A descriptive study was conducted to identify the different strains of Echinococcus granulosus occurring in livestock in Moroto district, Uganda. Echinococcus cysts from 104 domestic animals, including cattle, sheep, goats and camels, were taken and examined by microscopy, polymerase chain reaction with restriction fragment length polymorphism and Sanger DNA sequencing. Echinococcus granulosus genotypes or strains were identified through use of Bioinformatics tools: BioEdit, BLAST and MEGA6. The major finding of this study was the existence of a limited number of E. granulosus genotypes from cattle, goats, sheep and camels. The most predominant genotype was G1 (96.05%), corresponding to the common sheep strain. To a limited extent (3.95%), the study revealed the existence of Echinococcus canadensis G6/7 in three (n = 3) of the E. granulosus-positive samples. No other strains of E. granulosus were identified. It was concluded that the common sheep strain of Echinococcus sensu stricto and G6/7 of E. canadensis were responsible for echinococcal disease in Moroto district, Uganda. PMID:27543147

  19. Evaluation of a new efficient procedure for single-nucleotide polymorphism genotyping: tetra-primer amplification refractory mutation system-polymerase chain reaction.

    PubMed

    Okayama, Naoko; Fujimura, Kozue; Nakamura, Junji; Suehiro, Yutaka; Hamanaka, Yuichiro; Hinoda, Yuji

    2004-01-01

    Tetra-primer amplification refractory mutation system-polymerase chain reaction (ARMS-PCR) is a new efficient method for single-nucleotide polymorphism (SNP) genotyping. To determine the optimal conditions for ARMS-PCR we attempted to genotype ten SNPs. DNA was extracted from the peripheral blood of 168 unrelated healthy Japanese volunteers. Two problems inhibited uniform efficiency of the amplification of three bands. The first problem was the lower amplification efficiency of the shorter and allele-specific products compared with the largest product. This phenomenon was overcome by increasing the relative concentration of the inner primers. The second problem was non-specific amplification of the shorter products. To reduce the amplification of these non-specific bands, adjusting any one of the following PCR conditions was effective: i) reducing the ratio of the inner primer concentration relative to that of the outer primers; ii) increasing the annealing temperature for the initial 5-10 cycles; iii) hot start PCR. With these procedures all ten of the SNPs were successfully genotyped. Our present data may be useful in the further application of tetra-primer ARMS-PCR to SNP genotyping.

  20. Identification of Echinococcus granulosus strains using polymerase chain reaction-restriction fragment length polymorphism amongst livestock in Moroto district, Uganda.

    PubMed

    Chamai, Martin; Omadang, Leonard; Erume, Joseph; Ocaido, Michael; Oba, Peter; Othieno, Emmanuel; Bonaventure, Straton; Kitibwa, Annah

    2016-07-29

    A descriptive study was conducted to identify the different strains of Echinococcus granulosus occurring in livestock in Moroto district, Uganda. Echinococcus cysts from 104 domestic animals, including cattle, sheep, goats and camels, were taken and examined by microscopy, polymerase chain reaction with restriction fragment length polymorphism and Sanger DNA sequencing. Echinococcus granulosus genotypes or strains were identified through use of Bioinformatics tools: BioEdit, BLAST and MEGA6. The major finding of this study was the existence of a limited number of E. granulosus genotypes from cattle, goats, sheep and camels. The most predominant genotype was G1 (96.05%), corresponding to the common sheep strain. To a limited extent (3.95%), the study revealed the existence of Echinococcus canadensis G6/7 in three (n = 3) of the E. granulosus-positive samples. No other strains of E. granulosus were identified. It was concluded that the common sheep strain of Echinococcus sensu stricto and G6/7 of E. canadensis were responsible for echinococcal disease in Moroto district, Uganda.

  1. Polymerase chain reaction-restriction fragment length polymorphism assays to distinguish Liriomyza huidobrensis (Diptera: Agromyzidae) from associated species on lettuce cropping systems in Italy.

    PubMed

    Masetti, Antonio; Luchetti, Andrea; Mantovani, Barbara; Burgio, Giovanni

    2006-08-01

    The pea leafminer, Liriomyza huidobrensis (Blanchard) (Diptera: Agromyzidae), is a serious insect pest infesting open field lettuce plantings in northern Italy. In these cropping systems, it coexists with several other agromyzid species that have negligible economic importance on open field vegetables. The rapid detection of L. huidobrensis is crucial for effective management strategies, but the identification of agromyzids to species can be very difficult at adult as well at immature stages. In this study, a polymerase chain reaction (PCR)-restriction fragment length polymorphism assay is proposed to separate L. huidobrensis from Liriomyza bryoniae (Kaltenbach), Liriomyza trifolii (Burgess), and Chromatomyia horticola (Goureau), which usually occur in the same lettuce plantings. An approximately 1,031-bp region of the mitochondrial genome encompassing the 3' region of cytochrome oxidase I, the whole leucine tRNA, and all of the cytochrome oxidase II was amplified by PCR and digested using the enzymes PvuII and SnaBI separately. Both endonucleases cut the amplicons of L. huidobrensis in two fragments, whereas the original band was not cleaved in the other analyzed species. The presence of Dacnusa spp. DNA does not bias the assay, because the PCR conditions and the primer set here described do not amplify any tract of this endoparasitic wasp genome. PMID:16937681

  2. Crystal structure of the tetra­gonal polymorph of bis­(1-ethyl-3-methyl­imidazolium) tetra­bromido­cadmate

    PubMed Central

    Đorđević, Tamara; Gerger, Sabrina; Karanović, Ljiljana

    2016-01-01

    Both unique Cd atoms in the tetra­gonal polymorph of bis­(1-ethyl-3-methyl­imidazolium) tetra­bromido­cadmate, (C6H11N2)2[CdBr4], occupy special positions (site symmetry -4). The crystal structure consists of isolated tetra­hedral [CdBr4]2− anions which are surrounded by 1-ethyl-3-methyl­imidazolium cations. The methyl and ethyl side chains of the cations show positional disorder in a 0.590 (11):0.410 (11) ratio. In the crystal, (C6H11N2)+ cations display three weak C—H⋯Br hydrogen-bond inter­actions through the imidazolium ring H atoms with the Br− ligands of the surrounding complex anions. The alkyl groups of the side chains are not involved in hydrogen bonding. PMID:27555953

  3. Polymorphisms in arsenic metabolism genes, urinary arsenic methylation profile and cancer.

    PubMed

    Chung, Chi-Jung; Hsueh, Yu-Mei; Bai, Chyi-Huey; Huang, Yung-Kai; Huang, Ya-Li; Yang, Mo-Hsiung; Chen, Chien-Jen

    2009-11-01

    Arsenic-metabolism-related genes can regulate the arsenic methylation process and may influence susceptibility to cancer. We evaluated the roles of arsenic metabolism genes on urinary arsenic profiles of repeated measurement with 15-year follow-up (1988-2004) through general linear model (GLM) and assessed the effect of the changed extent of urinary arsenic profiles on cancer risk. Questionnaire information and blood samples and two urines (1988 and 2004) were collected from 208 subjects in an arseniasis hyperendemic area in Taiwan. Profiles for concentrations of urinary arsenic were determined using HPLC-HG-AAS. The relative proportion of each arsenic species was calculated by dividing the concentration of each arsenic species by the total arsenic concentration. Genotyping was done using the 5' nuclease allelic discrimination (Taqman) assay. The incidence of cancer was identified through linking to the National Cancer Registry Systems. The Cox proportional hazards model and survival curves were used in the analyses. After a 15-year follow-up, baseline monomethylarsonic acid percentage (MMA%) and change in MMA% exhibited a significant dose-response relationship with cancer risk. Individuals with a higher baseline MMA% and a lower change in MMA% had the earliest cancer incidence (statistically significant). Through GLM, significant gene effects of arsenic (+3 oxidation state)-methyltransferase (AS3MT) on MMA%, dimethylarsinic acid percentage (DMA%) and DMA/MMA, purine nucleoside phosphorylase (PNP) on DMA% and glutathione S-transferase omega 2 (GSTO2) on inorganic arsenics (InAs%) were found. Our results show that MMA% might be a potential predictor of cancer risk. The change in MMA% was linked to individual cancer susceptibility related to AS3MT rs3740393.

  4. Molecular typing of Iranian mycobacteria isolates by polymerase chain reaction-restriction fragment length polymorphism analysis of 360-bp rpoB gene

    PubMed Central

    Hadifar, Shima; Moghim, Sharareh; Fazeli, Hossein; GhasemianSafaei, Hajieh; Havaei, Seyed Asghar; Farid, Fariba; Esfahani, Bahram Nasr

    2015-01-01

    Background: Diagnosis and typing of Mycobacterium genus provides basic tools for investigating the epidemiology and pathogenesis of this group of bacteria. Polymerase chain reaction (PCR)-restriction fragment length polymorphism analysis (PRA) is an accurate method providing diagnosis and typing of species of mycobacteria. The present study is conducted by the purpose of determining restriction fragment profiles of common types of mycobacteria by PRA method of rpoB gene in this geographical region. Materials and Methods: Totally 60 clinical and environmental isolates from February to October, 2013 were collected and subcultured and identified by phenotypic methods. A 360 bp fragment of the rpoB gene amplified by PCR and products were digested by MspI and HaeIII enzymes. Results: In the present study, of all mycobacteria isolates identified by PRA method, 13 isolates (21.66%) were Mycobacterium tuberculosis, 34 isolates (56.66%) were rapidly growing Nontuberculosis Mycobacteria (NTM) that including 26 clinical isolates (43.33%) and 8 environmental isolates (13.33%), 11 isolates (18.33%) were clinical slowly growing NTM. among the clinical NTM isolates, Mycobacterium fortuitum Type I with the frequency of 57.77% was the most prevalent type isolates. Furthermore, an unrecorded of the PRA pattern of Mycobacterium conceptionense (HeaIII: 120/90/80, MspI: 120/105/80) was found. This study demonstrated that the PRA method was high discriminatory power for identification and typing of mycobacteria species and was able to identify 96.6% of all isolates. Conclusion: Based on the result of this study, rpoB gene could be a potentially useful tool for identification and investigation of molecular epidemiology of mycobacterial species. PMID:26380237

  5. Analysis of p53 gene mutations in human gliomas by polymerase chain reaction-based single-strand conformation polymorphism and DNA sequencing.

    PubMed

    Sarkar, F H; Kupsky, W J; Li, Y W; Sreepathi, P

    1994-03-01

    Mutations in the p53 gene have been recognized in brain tumors, and clonal expansion of p53 mutant cells has been shown to be associated with glioma progression. However, studies on the p53 gene have been limited by the need for frozen tissues. We have developed a method utilizing polymerase chain reaction (PCR) for the direct analysis of p53 mutation by single-strand conformation polymorphism (SSCP) and by direct DNA sequencing of the p53 gene using a single 10-microns paraffin-embedded tissue section. We applied this method to screen for p53 gene mutations in exons 5-8 in human gliomas utilizing paraffin-embedded tissues. Twenty paraffin blocks containing tumor were selected from surgical specimens from 17 different adult patients. Tumors included six anaplastic astrocytomas (AAs), nine glioblastomas (GBs), and two mixed malignant gliomas (MMGs). The tissue section on the stained glass slide was used to guide microdissection of an unstained adjacent tissue section to ensure > 90% of the tumor cell population for p53 mutational analysis. Simultaneously, microdissection of the tissue was also carried out to obtain normal tissue from adjacent areas as a control. Mutations in the p53 gene were identified in 3 of 17 (18%) patients by PCR-SSCP analysis and subsequently confirmed by PCR-based DNA sequencing. Mutations in exon 5 resulting in amino acid substitution were found in one thalamic AA (codon 158, CGC > CTT: Arg > Leu) and one cerebral hemispheric GB (codon 151, CCG > CTG: Pro > Leu).(ABSTRACT TRUNCATED AT 250 WORDS)

  6. Matrilineage differentiation of the genus Tetragonisca using mitochondrial DNA markers and the polymerase chain reaction-restriction fragment length polymorphism technique.

    PubMed

    Santos, S A; Bronzato, A R; Moreira, B M T; Araujo, K F; Ronqui, L; Mangolin, C A; Toledo, V A A; Ruvolo-Takasusuki, M C C

    2015-10-21

    The Meliponinae are important pollinators of plant species, and one of the most managed species is Tetragonisca angustula. Initially, two subspecies were identified in T. angustula: T. angustula angustula and T. angustula fiebrigi. Subsequently, T. a. fiebrigi was considered a species, based on the coloration of its mesepisternum. The objective of the present study was to obtain genetic markers that could differentiate the two species by amplifying regions of mitochondrial DNA and conducting polymerase chain reaction-restriction fragment length polymorphism analysis. Worker bees were collected in three Brazilian states: Paraná (Maringá, Altônia, and Foz do Iguaçu), São Paulo (Dracena, São Carlos, and Santa Cruz do Rio Pardo), and Rondônia (Ariquemes). Ten pairs of insect heterologous primers were tested and four were used (primer pair 1, ND2 and COI; primer pair 2, COI; primer pair 8, 16S and 12S; and primer pair 9, COII). For the restriction analysis, 13 enzymes were tested: EcoRI, EcoRV, HindIII, HinfI, RsaI, PstI, XbaI, HaeIII, ClaI, XhoI, BglII, PvuII, and ScaI. Markers were obtained (primer pair 8 cleaved with EcoRV and XbaI and primer pair 9 cleaved with HaeIII, RsaI, and XbaI) that enabled matrilineage identification in the nests studied, which confirmed that hybridization could occur between both Tetragonisca species. The beginning of speciation was probably recent, and secondary contact has resulted in crosses between T. angustula females and T. fiebrigi males. Because of this hybridization, it would be appropriate to consider them as two subspecies of T. angustula.

  7. Natural Polymorphisms Conferring Resistance to HCV Protease and Polymerase Inhibitors in Treatment-Naïve HIV/HCV Co-Infected Patients in China

    PubMed Central

    Wang, Charles; Hu, Fengyu; Ning, Chuanyi; Lan, Yun; Tang, Xiaoping; Tucker, Joseph D.; Cai, Weiping

    2016-01-01

    Background The advent of direct-acting agents (DAAs) has improved treatment of HCV in HIV co-infection, but may be limited by primary drug resistance. This study reports the prevalence of natural polymorphisms conferring resistance to NS3/4A protease inhibitors and NS5B polymerase inhibitors in treatment-naïve HIV/HCV co-infected individuals in China. Methods Population based NS3/4A sequencing was completed for 778 treatment-naïve HIV/HCV co-infected patients from twelve provinces. NS3 sequences were amplified by nested PCR using in-house primers for genotypes 1–6. NS5B sequencing was completed for genotyping in 350 sequences. Resistance-associated variants (RAVs) were identified in positions associated with HCV resistance. Results Overall, 72.8% (566/778) of all HCV sequences had at least one RAV associated with HCV NS3/4A protease inhibitor resistance. Variants were found in 3.6% (7/193) of genotype 1, 100% (23/23) of genotype 2, 100% (237/237) of genotype 3 and 92% (299/325) of genotype 6 sequences. The Q80K variant was present in 98.4% of genotype 6a sequences. High-level RAVs were rare, occurring in only 0.8% of patients. 93% (64/69) patients with genotype 1b also carried the C316N variant associated with NS5B low-level resistance. Conclusions The low frequency of high-level RAVs associated with primary HCV DAA resistance among all genotypes in HIV/HCV co-infected patients is encouraging. Further phenotypic studies and clinical research are needed. PMID:27341031

  8. Quantitative real-time polymerase chain reaction (qRT-PCR) restriction fragment length polymorphism (RFLP) method for monitoring highly conserved transgene expression during gene therapy.

    PubMed

    Bruzzone, Carol M; Belcher, John D; Schuld, Nathan J; Newman, Kristal A; Vineyard, Julie; Nguyen, Julia; Chen, Chunsheng; Beckman, Joan D; Steer, Clifford J; Vercellotti, Gregory M

    2008-12-01

    Evaluation of the transfer efficiency of a rat heme oxygenase-1 (HO-1) transgene into mice requires differentiation of rat and mouse HO-1. However, rat and mouse HO-1 have 94% homology; antibodies and enzyme activity cannot adequately distinguish HO-1. We designed a quantitative real-time polymerase chain reaction (qRT-PCR) method to monitor HO-1 transcription relative to a housekeeping gene, GAPDH. The ratio of rat and mouse HO-1 mRNA could be estimated through restriction fragment length polymorphism (RFLP) analysis of the PCR products. In vitro, murine AML12 hepatocytes were transfected with rat HO-1. After 40 h, the total HO-1 mRNA was enriched 2-fold relative to control cells, and rat HO-1 comprised 84% of HO-1 cDNA. In vivo, the rat HO-1 transgene was cloned into a Sleeping Beauty transposase (SB-Tn) construct and was injected hydrodynamically into a mouse model of sickle cell disease (SCD). After 21 days, there was a 32% enrichment of HO-1 mRNA relative to control mice and the rat transgene comprised 88% of HO-1 cDNA. After 21 days, HO-1 protein expression in liver was increased 2.5-fold. In summary, qRT-PCR RFLP is a useful and reliable method to differentiate the transgene from host gene transcription, especially when the host and transgene protein are identical or highly homologous. This method has translational applications to the design, delivery, and monitoring of gene-therapy vectors. PMID:19059164

  9. Close association of predominant genotype of herpes simplex virus type 1 with eczema herpeticum analyzed using restriction fragment length polymorphism of polymerase chain reaction.

    PubMed

    Yoshida, Masami; Umene, Kenichi

    2003-04-01

    Herpes simplex virus type 1 (HSV-1) strains belonging to the same genotype can possibly share biological properties and clinical manifestations common to the genotype. We classified previously 66 HSV-1 strains into 35 genotypes (F1-F35) using restriction fragment length polymorphism (RFLP) and F1 and F35 genotypes were revealed to be predominant [Arch. Virol. 13 (1993) 29]. It was found later that the F35 genotype seemed to be closely associated with eczema herpeticum [J. Med. Virol. 49 (1996) 329]. In the present study, a convenient method was developed for classification of two predominant genotypes by RFLP of polymerase chain reaction (RFLP-PCR). Using this method, genotypes of 21 strains isolated from eczema herpeticum were analyzed; seven of 21 strains (33.3%) were of F1 and five of 21 (23.8%) were of F35. Genotypes of 19 strains isolated from facial herpes other than eczema herpeticum were as follows; six of 19 (31.6%) strains were of F1 and one of 19 (5.3%) were of F35. Thus, strains belonging to F35 were appear to have been isolated more frequently from eczema herpeticum (5/21) than from facial herpes (1/19). These ratios showed a statistically significant difference. These results support the hypothesis that F35 strains is clearly associated with eczema herpeticum, in agreement with previous study. This is the first report of PCR-based approach for classification of HSV-1 strains into genotypes seeking an association of a genotype with clinical manifestation.

  10. Prevalence of Borrelia burgdorferi species and identification of Borrelia valaisiana in questing Ixodes ricinus in the Lyon region of France as determined by polymerase chain reaction-restriction fragment length polymorphism.

    PubMed

    Quessada, T; Martial-Convert, F; Arnaud, S; Leudet De La Vallee, H; Gilot, B; Pichot, J

    2003-03-01

    Many cases of Lyme borreliosis have been reported over the years in the region of Lyon, France. The identification and prevalence of Borrelia burgdorferi sensu lato in Ixodes ricinus were investigated by polymerase chain reaction (PCR) of the flagellin gene and restriction fragment length polymorphism (RFLP) analysis. Questing Ixodes ricinus larvae, nymphs and adults were collected by the flagging method from deciduous forests in four areas in the Lyon region of France between October 1994 and September 1995 and in June 1998. The overall prevalence of Borrelia burgdorferi sensu lato was 13.2% (91/688). No significant differences in prevalence were observed between the different stages and sex of the ixodids or between collection areas. The majority of infections were simple infections (82.4%; 75/91), most of which were due to Borrelia afzelii (41.4%), while coinfections (12.1%) were predominantly (54.5%) a combination of Borrelia valaisiana and Borrelia garinii. No tick was infected with more than two borrelial species, nor was Borrelia lusitaniae identified. The Borrelia valaisiana species was detected for the first time in France, confirming its widespread presence in Europe. This study confirms that the surroundings of Lyon are risk areas for contracting Lyme disease and that no particular clinical manifestations predominate due to the heterogeneous distribution of Borrelia genospecies. Moreover, the polymerase chain reaction restriction fragment length polymorphism analysis is a rapid and easy method for genotyping of Borrelia species.

  11. DNA methylation and genetic polymorphisms of the Leptin gene interact to influence lung function outcomes and asthma at 18 years of age

    PubMed Central

    Mukherjee, Nandini; Lockett, Gabrielle A; Merid, Simon K; Melén, Erik; Pershagen, Göran; Holloway, John W; Arshad, Syed Hasan; Ewart, Susan; Zhang, Hongmei; Karmaus, Wilfried

    2016-01-01

    The leptin gene (LEP) plays a regulatory role in satiety, inflammation, and allergy. Prior findings linking leptin to asthma motivated us to investigate whether DNA methylation (DNA-M) of CpG (cytosine-phosphate-guanine) sites in concert with single nucleotide polymorphisms (SNPs) of LEP can explain the risk of asthma and lung function. Methylation of CpG sites was assessed using the Illumina Infinium Human Methylation 450 beadchip in blood samples collected from 10- and 18-year-old boys and girls from the Isle of Wight (IOW) birth cohort (UK). Four LEP SNPs were genotyped. Linear and log linear models were used for the analysis, adjusting for false discovery rate (FDR). The analyses were repeated in the BAMSE cohort (Sweden). In the IOW study, the interaction of cg00666422 and rs11763517 (CT vs TT and CC) was associated with FEV1 (FDR-adjusted p-value: 0.03), FEV1/FVC ratio (FDR-adjusted p-value: 0.0096), and FEF25-75% (FDR-adjusted p-value: 0.00048) such that they decreased with increasing DNA-M. The interaction of the same CpG-SNP pair was also associated with increased risk of asthma at age 18. We replicated the findings for FEV1/FVC and FEF25-75% in a smaller sample of 34 participants at age 10. Regarding the BAMSE cohort, although, the interaction of cg00666422 and rs11763517 on lung function were not significant, the direction of the effect was the same as in IOW cohort. Thus, penetrance of LEP genotype seems to be modified by methylation at cg00666422 and is linked to airway obstruction and asthma. PMID:27186323

  12. Molecular diagnosis of Prader-Willi syndrome: Parent-of-origin dependent methylation sites and non-isotopic detection of (CA){sub n} dinucleotide repeat polymorphisms

    SciTech Connect

    Lerer, I.; Meiner, V.; Pashut-Lavon, I.; Abeliovich, D.

    1994-08-01

    We describe our experience in the molecular diagnosis of 22 patients suspected of Prader-Willi syndrome (PWS) using a DNA probe PW71 (D15S63) which detects a parent-of-origin specific methylated site in the PWS critical region. The cause of the syndrome was determined as deletion or uniparental disomy according to the segregation of (CA){sub n} dinucleotide repeat polymorphisms of the PWS/AS region and more distal markers of chromosome 15. In 10 patients the clinical diagnosis was confirmed by the segregation of (CA){sub n}, probably due to paternal microdeletion in the PWs critical region which did not include the loci D15S97, D15S113, GABRB3, and GABRA5. This case demonstrates the advantage of the DNA probe PW71 in the diagnosis of PWS. 31 refs., 2 figs., 3 tabs.

  13. Polymorphism and methylation patterns in Agave tequilana Weber var. 'Azul' plants propagated asexually by three different methods.

    PubMed

    Díaz-Martínez, Miriam; Nava-Cedillo, Alejandro; Guzmán-López, José Alfredo; Escobar-Guzmán, Rocío; Simpson, June

    2012-04-01

    Genetic variation in three forms of asexually propagated Agave tequilana Weber var. 'Azul' plants namely offsets, bulbils and in vitro cultured individuals was studied by AFLP analysis. Low levels of variation were observed between mother plants and offsets and a higher level between mother plant and bulbils. Families obtained from commercial plantations showed lower levels of variation in comparison to families grown as ornamentals. No variation was observed between the original explant and four generations of in vitro cultured plants. Epigenetic variation was also studied by analyzing changes in methylation patterns between mother plants and offspring in each form of asexual reproduction. Offsets and bulbils showed an overall decrease in methylation whereas in vitro cultured plants showed patterns specific to each generation: Generations 1 and 4 showed overall demethylation whereas Generations 2 and 3 showed increased methylation. Analysis of ESTs associated with transposable elements revealed higher proportions of ESTs from Ty1-copia-like, Gypsy and CACTA transposable elements in cDNA libraries obtained from pluripotent tissue suggesting a possible correlation between methylation patterns, expression of transposable element associated genes and somaclonal variation. PMID:22325895

  14. Polymorphism and methylation patterns in Agave tequilana Weber var. 'Azul' plants propagated asexually by three different methods.

    PubMed

    Díaz-Martínez, Miriam; Nava-Cedillo, Alejandro; Guzmán-López, José Alfredo; Escobar-Guzmán, Rocío; Simpson, June

    2012-04-01

    Genetic variation in three forms of asexually propagated Agave tequilana Weber var. 'Azul' plants namely offsets, bulbils and in vitro cultured individuals was studied by AFLP analysis. Low levels of variation were observed between mother plants and offsets and a higher level between mother plant and bulbils. Families obtained from commercial plantations showed lower levels of variation in comparison to families grown as ornamentals. No variation was observed between the original explant and four generations of in vitro cultured plants. Epigenetic variation was also studied by analyzing changes in methylation patterns between mother plants and offspring in each form of asexual reproduction. Offsets and bulbils showed an overall decrease in methylation whereas in vitro cultured plants showed patterns specific to each generation: Generations 1 and 4 showed overall demethylation whereas Generations 2 and 3 showed increased methylation. Analysis of ESTs associated with transposable elements revealed higher proportions of ESTs from Ty1-copia-like, Gypsy and CACTA transposable elements in cDNA libraries obtained from pluripotent tissue suggesting a possible correlation between methylation patterns, expression of transposable element associated genes and somaclonal variation.

  15. IFNG rs1861494 Polymorphism is Associated with IBD Disease Severity and Functional Changes in both IFNG Methylation and Protein Secretion

    PubMed Central

    Gonsky, Rivkah; Deem, Richard L; Landers, Carol J; Haritunians, Talin; Yang, Shaohong; Targan, Stephan R

    2015-01-01

    Background Mucosal expression of IFN-γ plays a pivotal role in IBD pathogenesis and IBD-risk regions flank IFNG. The conserved IFNG rs1861494 T/C, introduces a new CpG methylation site, and is associated with disease severity and lack of therapeutic response in other infectious and immune mediated disorders, and is in linkage-disequilibrium with a UC disease severity region. It seems likely that CpG-altering SNPs modify methylation and gene expression. This study evaluated the association between rs1861494 and clinical, serologic and methylation patterns in IBD patients. Methods Peripheral T cells of UC and CD patients were genotyped for rs1861494 and analyzed for allele-specific and IFNG promoter methylation. Serum ANCA and IFN-γ secretion were measured by ELISA and nucleo-protein complex formation by EMSA. Results IFNG rs1861494 T allele carriage in IBD patients was associated with enhanced secretion of IFN-γ. T allele carriage was associated in UC with high levels of ANCA and faster progression to colectomy. In CD, it was associated with complicated disease involving a stricturing/penetrating phenotype. Likewise, IFNG rs1861494 displayed genotype specific modulation of DNA methylation and transcription factor complex formation. Conclusions This study reports the first association of IFNG rs1861494 T allele with enhanced IFN-γ secretion and known IBD clinical parameters indicative of more aggressive disease, as well as serological markers associated with treatment resistance to anti-TNF therapy in IBD patients. These data may be useful prognostically as predictors of early response to anti-TNF therapy to identify IBD patients for improved personalized therapeutics. PMID:25171510

  16. Isolation of Coxiella burnetii by a centrifugation shell-vial assay from ticks collected in Cyprus: detection by nested polymerase chain reaction (PCR) and by PCR-restriction fragment length polymorphism analyses.

    PubMed

    Spyridaki, Ioanna; Psaroulaki, Anna; Loukaides, Fidias; Antoniou, Maria; Hadjichristodolou, Christos; Tselentis, Yannis

    2002-01-01

    Ticks are the principal vectors and reservoirs of Coxiella burnetii. The identification of isolates is necessary for understanding the clinical diversity of Q fever in different geographic areas. This is the first report of isolation of C. burnetii from ticks by the shell-vial assay and by nested polymerase chain reaction (PCR) assay for the detection of this pathogen in ticks. Of 141 ticks collected in Cyprus (Rhipicephalus sanguineus and Hyalloma spp.), 10% were found to be infected with C. burnetii. Three ticks were positive by hemolymph test, and 11 triturated ticks were positive by nested PCR. Three isolates were obtained by the centrifugation shell-vial technique. Analysis by PCR, then restriction fragment length polymorphism showed that the 3 Cyprus isolates had identical restriction profiles to reference strains Nine Mile and Q212. The methods described are useful in studying the epidemiology and ecology of C. burnetii. PMID:12135275

  17. Short report: A new polymerase chain reaction-restriction fragment length polymorphism method to identify Anopheles arabiensis from An. gambiae and its two molecular forms from degraded DNA templates or museum samples.

    PubMed

    Santolamazza, Federica; Della Torre, Alessandra; Caccone, Adalgisa

    2004-06-01

    We present a polymerase chain reaction-restriction fragment length polymorphism method to simultaneously distinguish the two Anopheles gambiae M and S molecular forms and Anopheles arabiensis. This method uses different diagnostic sites than previously published methods, and it is based on the amplification of a smaller ribosomal DNA fragment. We have tested this protocol in a variety of samples from different geographic regions and various ages of preservation to ascertain the robustness of this protocol over a wide geographic window and on DNA templates of poor quality. This procedure is as efficient as previous ones in discriminating An. arabiensis from the two taxa in An gambiae s.s. However, it performs better than others on poor quality templates such as the ones from museum collections, and poorly stored field collected material. However, it must be noted that it does not allow the simultaneous discrimination of all the species in the An. gambiae complex.

  18. Methylation of arsenic by recombinant human wild-type arsenic (+ 3 oxidation state) methyltransferase and its methionine 287 threonine (M287T) polymorph: Role of glutathione

    SciTech Connect

    Ding, Lan; Saunders, R. Jesse; Drobná, Zuzana; Walton, Felecia S.; Xun, Pencheng; Thomas, David J.; Stýblo, Miroslav

    2012-10-01

    Arsenic (+ 3 oxidation state) methyltransferase (AS3MT) is the key enzyme in the pathway for methylation of arsenicals. A common polymorphism in the AS3MT gene that replaces a threonyl residue in position 287 with a methionyl residue (AS3MT/M287T) occurs at a frequency of about 10% among populations worldwide. Here, we compared catalytic properties of recombinant human wild-type (wt) AS3MT and AS3MT/M287T in reaction mixtures containing S-adenosylmethionine, arsenite (iAs{sup III}) or methylarsonous acid (MAs{sup III}) as substrates and endogenous or synthetic reductants, including glutathione (GSH), a thioredoxin reductase (TR)/thioredoxin (Trx)/NADPH reducing system, or tris (2-carboxyethyl) phosphine hydrochloride (TCEP). With either TR/Trx/NADPH or TCEP, wtAS3MT or AS3MT/M287T catalyzed conversion of iAs{sup III} to MAs{sup III}, methylarsonic acid (MAs{sup V}), dimethylarsinous acid (DMAs{sup III}), and dimethylarsinic acid (DMAs{sup V}); MAs{sup III} was converted to DMAs{sup III} and DMAs{sup V}. Although neither enzyme required GSH to support methylation of iAs{sup III} or MAs{sup III}, addition of 1 mM GSH decreased K{sub m} and increased V{sub max} estimates for either substrate in reaction mixtures containing TR/Trx/NADPH. Without GSH, V{sub max} and K{sub m} values were significantly lower for AS3MT/M287T than for wtAS3MT. In the presence of 1 mM GSH, significantly more DMAs{sup III} was produced from iAs{sup III} in reactions catalyzed by the M287T variant than in wtAS3MT-catalyzed reactions. Thus, 1 mM GSH modulates AS3MT activity, increasing both methylation rates and yield of DMAs{sup III}. AS3MT genotype exemplified by differences in regulation of wtAS3MT and AS3MT/M287T-catalyzed reactions by GSH may contribute to differences in the phenotype for arsenic methylation and, ultimately, to differences in the disease susceptibility in individuals chronically exposed to inorganic arsenic. -- Highlights: ► Human AS3MT and AS3MT(M287T) require a dithiol

  19. Excitotoxicity in the Lung: N-Methyl-D-Aspartate-Induced, Nitric Oxide-Dependent, Pulmonary Edema is Attenuated by Vasoactive Intestinal Peptide and by Inhibitors of Poly(ADP-Ribose) Polymerase

    NASA Astrophysics Data System (ADS)

    Said, Sami I.; Berisha, Hasan I.; Pakbaz, Hedayatollah

    1996-05-01

    Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the ``adult respiratory distress syndrome,'' and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase.

  20. Detailed polymorphism study on cytomegalovirus DNA polymerase gene to reveal the most suitable genomic targets for quantitative Real-time PCR.

    PubMed

    Bilenoğlu, Onur; Altındiş, Mustafa; Öz, Ersoy; Yücel-Öz, Yeliz; İrigül-Sönmez, Öykü; Ünal, Can Bora

    2015-01-01

    The human cytomegalovirus (HCMV) is an important human pathogen primarily affecting immunocompromised patients, like transplant recipients or HIV- infected individuals. Early diagnosis of cytomegalovirus (CMV) infection in high-risk patients is essential in order to start preemptive treatments. pol (UL54) gene encoding for HCMV viral DNA polymerase is a well-defined target for HCMV detection in clinical samples and identifying most highly conserved regions for primer design remains crucial. Though real-time polymerase chain reaction (qPCR) is a rapid and sensitive method for HCMV detection, failure to detect some HCMV strains due to primer and target mismatches have led the researchers to explore more sensitive and reliable methods. Hence, to understand the broader diversity of the pol mutations in HCMV and to specify the most suitable region for primer-probe design to be used in qPCR assay, we studied both nucleotide and amino acid heterogeneities in 60 HCMV positive samples that were collected to represent national mutational prevalence of pol gene of HCMV in Turkey. The test was designed with a new set of primers- probe for HCMV detection and quantification based on the sequencing data which revealed the most conserved region on the pol gene. Statistical probit analysis was applied on qPCR studies which revealed a 95% detection limit of 100 copies/mL. In addition, linearity, reproducibility, and precision of the new test were assessed for diagnostic purposes. PMID:26295291

  1. Association Study of N-Methyl-D-Aspartate Receptor Subunit 2B (GRIN2B) Polymorphisms and Schizophrenia Symptoms in the Han Chinese Population.

    PubMed

    Yang, Yongfeng; Li, Wenqiang; Zhang, Hongxing; Yang, Ge; Wang, Xiujuan; Ding, Minli; Jiang, Tianzi; Lv, Luxian

    2015-01-01

    Schizophrenia (SZ) is a common and complex psychiatric disorder that has a significant genetic component. The glutamatergic system is the major excitatory neurotransmitter system in the central nervous system, and is mediated by N-methyl-D-aspartate (NMDA) receptors. Disturbances in this system have been hypothesized to play a major role in SZ pathogenesis. Several studies have revealed that the NMDA receptor subunit 2B (GRIN2B) potentially associates with SZ and its psychiatric symptoms. In this study, we performed a case-control study to identify polymorphisms of the GRIN2B gene that may confer susceptibility to SZ in the Han Chinese population. Thirty-four single nucleotide polymorphisms (SNPs) were genotyped in 528 paranoid SZ patients and 528 control subjects. A significant association was observed in allele and genotype between SZ and controls at rs2098469 (χ2 = 8.425 and 4.994; p = 0.025 and 0.014, respectively). Significant associations were found in the allele at rs12319804 (χ2 = 4.436; p = 0.035), as well as in the genotype at rs12820037 and rs7298664 between SZ and controls (χ2 = 11.162 and 38.204; p = 0.003 and 4.27×10(-8), respectively). After applying the Bonferroni correction, rs7298664 still had significant genotype associations with SZ (p = 1.71×10(-7)). In addition, rs2098469 genotype and allele frequencies, and 12820037 allele frequencies were nominally associated with SZ. Three haplotypes, CGA (rs10845849-rs12319804-rs10845851), CC (rs12582848-rs7952915), and AAGAC (rs2041986-rs11055665-rs7314376-rs7297101-rs2098469), had significant differences between SZ and controls (χ2 = 4.324, 4.582, and 4.492; p = 0.037, 0.032, and 0.034, respectively). In addition, three SNPs, rs2098469, rs12820037, and rs7298664, were significantly associated with cognition factors PANSS subscores in SZ (F = 16.799, 7.112, and 13.357; p = 0.000, 0.017, and 0.000, respectively). In conclusion, our study provides novel evidence for an association between GRIN2B

  2. Association Study of N-Methyl-D-Aspartate Receptor Subunit 2B (GRIN2B) Polymorphisms and Schizophrenia Symptoms in the Han Chinese Population

    PubMed Central

    Zhang, Hongxing; Yang, Ge; Wang, Xiujuan; Ding, Minli; Jiang, Tianzi; Lv, Luxian

    2015-01-01

    Schizophrenia (SZ) is a common and complex psychiatric disorder that has a significant genetic component. The glutamatergic system is the major excitatory neurotransmitter system in the central nervous system, and is mediated by N-methyl-D-aspartate (NMDA) receptors. Disturbances in this system have been hypothesized to play a major role in SZ pathogenesis. Several studies have revealed that the NMDA receptor subunit 2B (GRIN2B) potentially associates with SZ and its psychiatric symptoms. In this study, we performed a case–control study to identify polymorphisms of the GRIN2B gene that may confer susceptibility to SZ in the Han Chinese population. Thirty-four single nucleotide polymorphisms (SNPs) were genotyped in 528 paranoid SZ patients and 528 control subjects. A significant association was observed in allele and genotype between SZ and controls at rs2098469 (χ2 = 8.425 and 4.994; p = 0.025 and 0.014, respectively). Significant associations were found in the allele at rs12319804 (χ2 = 4.436; p = 0.035), as well as in the genotype at rs12820037 and rs7298664 between SZ and controls (χ2 = 11.162 and 38.204; p = 0.003 and 4.27×10-8, respectively). After applying the Bonferroni correction, rs7298664 still had significant genotype associations with SZ (p = 1.71×10-7). In addition, rs2098469 genotype and allele frequencies, and 12820037 allele frequencies were nominally associated with SZ. Three haplotypes, CGA (rs10845849—rs12319804—rs10845851), CC (rs12582848—rs7952915), and AAGAC (rs2041986—rs11055665—rs7314376—rs7297101—rs2098469), had significant differences between SZ and controls (χ2 = 4.324, 4.582, and 4.492; p = 0.037, 0.032, and 0.034, respectively). In addition, three SNPs, rs2098469, rs12820037, and rs7298664, were significantly associated with cognition factors PANSS subscores in SZ (F = 16.799, 7.112, and 13.357; p = 0.000, 0.017, and 0.000, respectively). In conclusion, our study provides novel evidence for an association between

  3. Trypanosome cdc2-related kinase 9 controls spliced leader RNA cap4 methylation and phosphorylation of RNA polymerase II subunit RPB1.

    PubMed

    Badjatia, Nitika; Ambrósio, Daniela L; Lee, Ju Huck; Günzl, Arthur

    2013-05-01

    Conserved from yeast to mammals, phosphorylation of the heptad repeat sequence Tyr(1)-Ser(2)-Pro(3)-Thr(4)-Ser(5)-Pro(6)-Ser(7) in the carboxy-terminal domain (CTD) of the largest RNA polymerase II (RNA Pol II) subunit, RPB1, mediates the enzyme's promoter escape and binding of RNA-processing factors, such as the m(7)G capping enzymes. The first critical step, Ser(5) phosphorylation, is carried out by cyclin-dependent kinase 7 (CDK7), a subunit of the basal transcription factor TFIIH. Many early-diverged protists, such as the lethal human parasite Trypanosoma brucei, however, lack the heptad repeats and, apparently, a CDK7 ortholog. Accordingly, characterization of trypanosome TFIIH did not identify a kinase component. The T. brucei CTD, however, is phosphorylated and essential for transcription. Here we show that silencing the expression of T. brucei cdc2-related kinase 9 (CRK9) leads to a loss of RPB1 phosphorylation. Surprisingly, this event did not impair RNA Pol II transcription or cotranscriptional m(7)G capping. Instead, we observed that CRK9 silencing led to a block of spliced leader (SL) trans splicing, an essential step in trypanosome mRNA maturation, that was caused by hypomethylation of the SL RNA's unique cap4.

  4. Giardia duodenalis in Damascus, Syria: Identification of Giardia genotypes in a sample of human fecal isolates using polymerase chain reaction and restriction fragment length polymorphism analyzing method.

    PubMed

    Skhal, Dania; Aboualchamat, Ghalia; Al Nahhas, Samar

    2016-02-01

    Giardia duodenalis is a common gastrointestinal parasite that infects humans and many other mammals. It is most prevalent in many developing and industrialized countries. G. duodenalis is considered to be a complex species. While no morphological distinction among different assemblages exist, it can be genetically differentiated into eight major assemblages: A to H. The aim of this study was to determine the genetic heterogeneity of G. duodenalis in human isolates (a study conducted for the first time in Syria). 40 fecal samples were collected from three different hospitals during the hot summer season of 2014. Extraction of genomic DNA from all Giardia positive samples (based on a microscopic examination) was performed using QIAamp DNA Stool Mini Kit. β-giardin gene was used to differentiate between different Giardia assemblages. The 514 bp fragment was amplified using the Polymerase Chain Reaction method, followed by digestion in HaeIII restriction enzyme. Our result showed that genotype A was more frequent than genotype B, 27/40 (67.5%); 4/40 (10%) respectively. A mixed genotype of A+B was only detected in 9 isolates (22.5%). This is the first molecular study performed on G. duodenalis isolates in Syria in order to discriminate among the different genotypes. Further expanded studies using more genes are needed to detect and identify the Giardia parasite at the level of assemblage and sub-assemblage.

  5. Male rats fed methyl- and folate-deficient diets with or without niacin develop hepatic carcinomas associated with decreased tissue NAD concentrations and altered poly(ADP-ribose) polymerase activity.

    PubMed

    Henning, S M; Swendseid, M E; Coulson, W F

    1997-01-01

    Folate is an essential cofactor in the generation of endogenous methionine, and there is evidence that folate deficiency exacerbates the effects of a diet low in choline and methionine, including alterations in poly(ADP-ribose) polymerase (PARP) activity, an enzyme associated with DNA replication and repair. Because PARP requires NAD as its substrate, we postulated that a deficiency of both folate and niacin would enhance the development of liver cancer in rats fed a diet deficient in methionine and choline. In two experiments, rats were fed choline- and folate-deficient, low methionine diets containing either 12 or 8% casein (12% MCFD, 8% MCFD) or 6% casein and 6% gelatin with niacin (MCFD) or without niacin (MCFND) and were compared with folate-supplemented controls. Liver NAD concentrations were lower in all methyl-deficient rats after 2-17 mo. At 17 mo, NAD concentrations in other tissues of rats fed these diets were also lower than in controls. Compared with control values, liver PARP activity was enhanced in rats fed the 12% MCFD diet but was lower in MCFND-fed rats following a further reduction in liver NAD concentration. These changes in PARP activity associated with lower NAD concentrations may slow DNA repair and enhance DNA damage. Only rats fed the MCFD and MCFND diets developed hepatocarcinomas after 12-17 mo. In Experiment 2, hepatocarcinomas were found in 100% of rats fed the MCFD and MCFND diets. These preliminary results indicate that folic acid deficiency enhances tumor development. Because tumors developed in 100% of the MCFD-fed rats and because tissue concentrations of NAD in these animals were also low, further studies are needed to clearly define the role of niacin in methyl-deficient rats.

  6. Genotypic characterization of Indian isolates of infectious bursal disease virus strains by reverse transcription-polymerase chain reaction combined with restriction fragment length polymorphism analysis.

    PubMed

    Priyadharsini, C V; Senthilkumar, T M A; Raja, P; Kumanan, K

    2016-03-01

    The reverse transcription PCR (RT-PCR) combined with restriction fragment length polymorphism (RFLP) is used for the differentiation of classical virulent (cv), virulent (v) and very virulent (vv) strains of infectious bursal disease virus (IBDV) isolates from chicken bursal tissues in southern states of India. In the present study, six different isolates (MB11, HY12, PY12, BGE14, VCN14 and NKL14) of IBDV strains were subjected for genotyping along with vaccine virus (Georgia, intermediate strain) using RT-PCR for amplification of a 743 bp sequence in the hypervariable region of VP2 gene followed by restriction enzyme digestion with 5 different restriction enzymes (BspMI, SacI, HhaI, StuI and SspI). The RT-PCR products obtained from vvIBDV strains were digested by SspI enzyme except PY12, BGE14 and MB11 isolates. The SacI digested the isolate MB11, PY12 and the vaccine strain, but it did not cleave the very virulent isolates of IBDV. HhaI cleaved all the isolates with different restriction profile patterns. StuI digested all the vvIBDV isolates and BspMI was not able to differentiate field isolates from vaccine strain. Though RT-PCR combined with RFLP is a genotypic method, further confirmation of serotypes to distinguish the vvIBDV from cvIBDV has to be carried out using pathogenicity studies.

  7. The Effect of Poly(ADP-ribose) Polymerase-1 Gene 3′Untranslated Region Polymorphism in Colorectal Cancer Risk among Saudi Cohort

    PubMed Central

    Alhadheq, Abdullah M.; Purusottapatnam Shaik, Jilani; Alamri, Abdullah; Aljebreen, Abdulrahman M.; Alharbi, Othman; Almadi, Majid A.; Alhadeq, Faten; Azzam, Nahla A.; Alanazi, Mohammad; Bazzi, Mohammad D.

    2016-01-01

    Background. DNA repair systems are essential for each cell to repair and maintain the genome integrity. Base excision repair pathway is one of the crucial pathways to maintain genome integrity and PARP-1 plays a key role in BER pathway. The purpose of this study is to evaluate the association between polymorphisms in PARP-1 3′untranslated region (3′UTR) SNP rs8679 and its expression in colorectal cancer. Methods. Genotyping and gene expression were performed using TaqMan assays. The effects of age, gender, and tumor location were evaluated in cases and controls regarding the genotyping results. Resulting data was analyzed using SPSS software. Results and Conclusions. Genotyping analysis for SNP rs8679 showed decreased susceptibility to colorectal cancer at heterozygous TC allele and at minor allele C. Further this protective association was also observed in younger age patients (≤57), in female patients, and also in patients with tumors located at colon and rectum. PARP-1 expression levels are significantly different in colorectal cancer compared to matched normal tissue. Our findings proved that the upregulation of PARP-1 is associated with tumor progression and poor prognosis in Saudi patients with colorectal cancer, suggesting that PARP-1 can be novel and valuable signatures for predicting the clinical outcome of patients with colorectal cancer. PMID:27746584

  8. Relative frequency of 4 major strain types of Mycobacterium avium ssp. paratuberculosis in Canadian dairy herds using a novel single nucleotide polymorphism-based polymerase chain reaction.

    PubMed

    Ahlstrom, Christina; Barkema, Herman W; De Buck, Jeroen

    2016-10-01

    Johne's disease is a worldwide concern, as it causes huge economic losses. The etiological agent, Mycobacterium avium ssp. paratuberculosis (MAP), has limited genetic diversity, impeding efforts to understand transmission and distribution of strain types. Whole-genome sequencing was previously performed on a representative set of MAP isolates from Canadian dairy herds and 9 divergent clades were identified. Four clades were of particular interest, as they were either MAP types rarely reported in North America, or they represented a substantial proportion of isolates recovered from dairy farms in Canada. One clade included type I/III isolates, whereas the remaining clades included type II isolates. Variant sites in the MAP genome are often separated by thousands of base pairs, limiting use of single nucleotide polymorphism (SNP)-based genotyping on a single genomic region. Therefore, a SNP-PCR assay was developed to facilitate interrogation of 5 SNP in 2 distant regions of the genome, linking them together in a single PCR reaction for subsequent Sanger sequencing. This high-throughput assay enabled discrimination of 602 MAP isolates from 264 herds (from all 10 provinces). More than 1 isolate was cultured from 133 herds, 14 of which included multiple subtypes. A previously identified dominant type included 87% of isolates, whereas the Bison type was more widespread than previously reported. The latter type and isolates from a second clade of interest were overrepresented in Québec and Saskatchewan, respectively. In conclusion, the distribution and relative frequency of MAP subtypes within Canadian dairy herds were assessed using a novel SNP-based typing assay. These findings will contribute to understanding the clinical relevance and transmission dynamics of MAP in this population and elsewhere. PMID:27497900

  9. Differentiation of canine distemper virus isolates in fur animals from various vaccine strains by reverse transcription-polymerase chain reaction-restriction fragment length polymorphism according to phylogenetic relations in china

    PubMed Central

    2011-01-01

    In order to effectively identify the vaccine and field strains of Canine distemper virus (CDV), a new differential diagnostic test has been developed based on reverse transcription-polymerase chain reaction (RT-PCR) and restriction fragment length polymorphism (RFLP). We selected an 829 bp fragment of the nucleoprotein (N) gene of CDV. By RFLP analysis using BamHI, field isolates were distinguishable from the vaccine strains. Two fragments were obtained from the vaccine strains by RT-PCR-RFLP analysis while three were observed in the field strains. An 829 nucleotide region of the CDV N gene was analyzed in 19 CDV field strains isolated from minks, raccoon dogs and foxes in China between 2005 and 2007. The results suggest this method is precise, accurate and efficient. It was also determined that three different genotypes exist in CDV field strains in fur animal herds of the north of China, most of which belong to Asian type. Mutated field strains, JSY06-R1, JSY06-R2 and JDH07-F1 also exist in Northern China, but are most closely related to the standard virulent strain A75/17, designated in Arctic and America-2 genetype in the present study, respectively. PMID:21352564

  10. The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA

    PubMed Central

    Barbedo, Leonardo Silva; Figueiredo-Carvalho, Maria Helena Galdino; Muniz, Mauro de Medeiros; Zancopé-Oliveira, Rosely Maria

    2016-01-01

    Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, andCandida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories. PMID:27074256

  11. The identification and differentiation of the Candida parapsilosis complex species by polymerase chain reaction-restriction fragment length polymorphism of the internal transcribed spacer region of the rDNA.

    PubMed

    Barbedo, Leonardo Silva; Figueiredo-Carvalho, Maria Helena Galdino; Muniz, Mauro de Medeiros; Zancopé-Oliveira, Rosely Maria

    2016-04-01

    Currently, it is accepted that there are three species that were formerly grouped under Candida parapsilosis: C. para- psilosis sensu stricto, Candida orthopsilosis, and Candida metapsilosis. In fact, the antifungal susceptibility profiles and distinct virulence attributes demonstrate the differences in these nosocomial pathogens. An accurate, fast, and economical identification of fungal species has been the main goal in mycology. In the present study, we searched sequences that were available in the GenBank database in order to identify the complete sequence for the internal transcribed spacer (ITS)1-5.8S-ITS2 region, which is comprised of the forward and reverse primers ITS1 and ITS4. Subsequently, an in silico polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) was performed to differentiate the C. parapsilosis complex species. Ninety-eight clinical isolates from patients with fungaemia were submitted for analysis, where 59 isolates were identified as C. parapsilosis sensu stricto, 37 were identified as C. orthopsilosis, and two were identified as C. metapsilosis. PCR-RFLP quickly and accurately identified C. parapsilosis complex species, making this method an alternative and routine identification system for use in clinical mycology laboratories.

  12. The Septic Shock-associated IL-10 -1082 A>G Polymorphism Mediates Allele-specific Transcription via Poly ADP-ribose Polymerase 1 in Macrophages Engulfing Apoptotic Cells

    PubMed Central

    Kang, Xiaoyan; Kim, Ha-Jeong; Ramirez, Michelle; Salameh, Sarah; Ma, Xiaojing

    2013-01-01

    The biallelic Interleukin-10 single nucleotide polymorphism (SNP) at -1082 of the promoter region linked to individual variation in cytokine inducibility has been strongly implicated in several pathological conditions including the development of, and outcomes in, septic shock during pneumococcal infection, acute respiratory distress syndrome, and cardiac dysfunction. However, the molecular basis of the SNP-mediated variable IL-10 production levels has not been explored. Here we report that the -1082G>A alleles in the promoter region of the human IL-10 gene physically interact with a nuclear protein in an allele-specific manner that results in different levels of IL-10 transcription. This protein has been identified as poly ADP-ribose polymerase 1 (PARP-1). We show that PARP-1 acts as a transcription repressor, and its DNA-binding activity is strongly regulated in macrophages that engulf apoptotic cells but not stimulated with lippopolysaccharides. These findings unveil a novel role of PARP-1 in the regulation of IL-10 production in an allele-dependent way, which determines individual susceptibility to sepsis-induced inflammatory pathology and the immunological sequelae in a physiological process where clearance of infection-induced apoptotic cells by professional phagocytes triggers the cytokine synthesis. PMID:20181890

  13. HCV NS5b RNA-dependent RNA polymerase inhibitors: from alpha,gamma-diketoacids to 4,5-dihydroxypyrimidine- or 3-methyl-5-hydroxypyrimidinonecarboxylic acids. Design and synthesis.

    PubMed

    Summa, Vincenzo; Petrocchi, Alessia; Matassa, Victor G; Taliani, Marina; Laufer, Ralph; De Francesco, Raffaele; Altamura, Sergio; Pace, Paola

    2004-10-21

    A new class of the HCV NS5b RNA-dependent RNA polymerase inhibitors, the dihyroxypyrimidinecarboxylic acid derivative, was designed from a diketoacid and meconic acid derivative discovered by screening. Mechanism of action and essential moieties required for activity were identified. The corresponding N-methylpyrimidinone was also prepared; both classes are novel, reversible, and selective inhibitors of the HCV NS5b polymerase with improved druglike characteristics.

  14. Crystal structure of a second polymorph of tricarbon­yl(N-methyl­pyridine-2-carboxamide-κ2 N 1,O)(thio­cyanato-κN)rhenium(I)

    PubMed Central

    Lyczko, Krzysztof

    2016-01-01

    A new polymorph of the title compound, [Re(NCS)(C7H8N2O)(CO)3], crystallizing in the space group P21/n, has been obtained and structurally characterized by the experiment and DFT calculations. In this complex, the rhenium(I) cation is octa­hedrally coordinated by three carbonyl groups in a facial configuration, the N,O-bidentate N-methyl­pyridine-2-carboxamide ligand and the N-bonded thio­cyanate anion. Neighbouring mol­ecules are linked into a three-dimensional network by inter­molecular N—H⋯S and C—H⋯S inter­actions. PMID:27746925

  15. T7-RNA Polymerase

    NASA Technical Reports Server (NTRS)

    1997-01-01

    T7-RNA Polymerase grown on STS-81. Structure-Function Relationships of RNA Polymerase: DNA-dependent RNA polymerase is the key enzyme responsible for the biosynthesis of RNA, a process known as transcription. Principal Investigator's include Dr. Dan Carter, Dr. B.C. Wang, and Dr. John Rose of New Century Pharmaceuticals.

  16. DNA polymerases and cancer

    PubMed Central

    Lange, Sabine S.; Takata, Kei-ichi; Wood, Richard D.

    2013-01-01

    There are fifteen different DNA polymerases encoded in mammalian genomes, which are specialized for replication, repair or the tolerance of DNA damage. New evidence is emerging for lesion-specific and tissue-specific functions of DNA polymerases. Many point mutations that occur in cancer cells arise from the error-generating activities of DNA polymerases. However, the ability of some of these enzymes to bypass DNA damage may actually defend against chromosome instability in cells and at least one DNA polymerase, POLζ, is a suppressor of spontaneous tumorigenesis. Because DNA polymerases can help cancer cells tolerate DNA damage, some of these enzymes may be viable targets for therapeutic strategies. PMID:21258395

  17. [Relationship of MTHFR gene polymorphisms with infertility].

    PubMed

    Guo, Kai-min; Tian, Run-hui; Wang, Hong-liang

    2016-02-01

    The folate metabolic pathway plays important roles in cellular physiology by participating in nucleotide synthesis, DNA repair and methylation, and maintenance and stability of the genome. Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme involved in folate metabolism. Polymorphisms of MTHFR may change the level of homocysteine and affect DNA synthesis and methylation, leading to an increased oxidative stress and disturbed methylation reactions and consequently affecting reproductive function. This article presents an overview on MTHFR gene polymorphisms, proposing that multicentered, large-sample and long-term prospective studies are needed to reveal the relationship between MTHFR gene polymorphisms and infertility.

  18. A second monoclinic polymorph of {bis­[5-methyl-3-(trifluoro­meth­yl)pyrazol-1-yl]borato}{tris­[5-methyl-3-(trifluoro­meth­yl)pyrazol-1-yl]borato}cobalt(II): a structure containing a B—H⋯Co agostic inter­action

    PubMed Central

    Stibrany, Robert T.; Potenza, Joseph A.

    2011-01-01

    The title compound, [Co(C10H10BF6N4)(C15H13BF9N6)], is a polymorph of the previously reported neutral cobalt(II) complex [Stibrany & Potenza (2010 ▶). Acta Cryst. E66, m506–m507], which contains one each of the monoanionic ligands, bis­[5-methyl-3-(trifluoro­meth­yl)pyrazol-1-yl]borate (Bp) and tris­[5-methyl-3-(trifluoro­meth­yl)pyrazol-1-yl]borate (Tp). A distorted octahedral coordination geometry of the CoII atom results from ligation of an H atom, which is part of an agostic B—H⋯Co inter­action [H⋯Co = 2.12 (3) Å], and by five imine N atoms, two from a Bp ligand and three from a Tp ligand. Weak intra- and inter­molecular C—F⋯π inter­actions with F⋯centroid distances ranging from 3.025 (4) to 3.605 (4) Å are observed. PMID:21836897

  19. DNA polymerase profiling.

    PubMed

    Summerer, Daniel

    2008-01-01

    We report a simple homogeneous fluorescence assay for quantification of DNA polymerase function in high throughput. The fluorescence signal is generated by the DNA polymerase triggering opening of a molecular beacon extension of the template strand. A resulting distance alteration is reported by fluorescence resonance energy transfer between two dyes introduced into the molecular beacon stem. We describe real-time reaction profiling of two model DNA polymerases. We demonstrate kinetic characterization, rapid optimization of reaction conditions, and inhibitor profiling using the presented assay. Furthermore, to supersede purification steps in screening procedures of DNA polymerase mutant libraries, detection of enzymatic activity in bacterial expression lysates is described.

  20. Crystal structure of a new monoclinic polymorph of N-(4-methyl­phen­yl)-3-nitro­pyridin-2-amine

    PubMed Central

    Aznan, Aina Mardia Akhmad; Abdullah, Zanariah; Lee, Vannajan Sanghiran; Tiekink, Edward R. T.

    2014-01-01

    The title compound, C12H11N3O2, is a second monoclinic polymorph (P21, with Z′ = 4) of the previously reported monoclinic (P21/c, with Z′ = 2) form [Akhmad Aznan et al. (2010 ▶). Acta Cryst. E66, o2400]. Four independent mol­ecules comprise the asymmetric unit, which have the common features of a syn disposition of the pyridine N atom and the toluene ring, and an intra­molecular amine–nitro N—H⋯O hydrogen bond. The differences between mol­ecules relate to the dihedral angles between the rings which range from 2.92 (19) to 26.24 (19)°. The geometry-optimized structure [B3LYP level of theory and 6–311 g+(d,p) basis set] has the same features except that the entire mol­ecule is planar. In the crystal, the three-dimensional architecture is consolidated by a combination of C—H⋯O, C—H⋯π, nitro-N—O⋯π and π–π inter­actions [inter-centroid distances = 3.649 (2)–3.916 (2) Å]. PMID:25249854

  1. Crystal structure of a new monoclinic polymorph of 2,4-di­hydroxy­benzaldehyde 4-methyl­thio­semi­carbazone

    PubMed Central

    Salam, M. A.; Hussein, Mouayed A.; Tiekink, Edward R. T.

    2015-01-01

    The title compound, C9H11N3O2S, is a second monoclinic (P21/c) polymorph of the previously reported Cc form [Tan et al. (2008b ▸). Acta Cryst. E64, o2224]. The mol­ecule is non-planar, with the dihedral angle between the N3CS residue (r.m.s. deviation = 0.0816 Å) and the benzene ring being 21.36 (4)°. The conformation about the C=N bond [1.292 (2) Å] is E, the two N-bound H atoms are anti, and the inner hy­droxy O-bound and outer amide N-bound H atoms form intra­molecular hydrogen bonds to the imine N atom. Crucially, the H atom of the outer hy­droxy group is approximately syn to the H atom of the benzene C atom connecting the two C atoms bearing the hy­droxy substituents. This arrangement enables the formation of supra­molecular tubes aligned along [010] and sustained by N—H⋯O, O—H⋯S and N—H⋯S hydrogen bonds; the tubes pack with no specific inter­actions between them. While the mol­ecular structure in the Cc form is comparable, the H atom of the outer hy­droxy group is approximately anti, rather than syn. This different orientation leads to the formation a three-dimensional architecture based on N—H⋯O and O—H⋯S hydrogen bonds. PMID:25705451

  2. RNA Polymerases of Maize: Nuclear RNA Polymerases*

    PubMed Central

    Strain, Gustave C.; Mullinix, Kathleen P.; Bogorad, Lawrence

    1971-01-01

    Two DNA-dependent RNA polymerases of nuclear origin have been purified from leaves of Zea mays. The two enzymes can be separated on DEAE-cellulose columns. Enzymes I and II are eluted with 0.08 and 0.20 M (NH4)2SO4, respectively. Both enzymes prefer maize nuclear DNA as a template; they are also more active in the presence of Mg++ than Mn++ and are inhibited by (NH4)2-SO4 or KCl. Neither enzyme is inhibited by rifamycin SV. Enzyme II is strongly inhibited by α-amanitin, whereas enzyme I is not significantly affected. Their ability to use native and denatured DNA as templates varies according to the extent and method of purification of the polymerase. Furthermore, enzyme II can be resolved by DEAE-chromatography or glycerol-gradient centrifugation into two components, one of which prefers native DNA, while the other prefers denatured DNA. PMID:5288239

  3. A new and improved method based on polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) for the determination of A1298C mutation in the methylenetetrahydrofolate reductase (MTHFR) gene.

    PubMed

    Machnik, Grzegorz; Zapala, Malgorzata; Pelc, Ewa; Gasecka-Czapla, Monika; Kaczmarczyk, Grzegorz; Okopien, Boguslaw

    2013-01-01

    Intracellular folate homeostasis and metabolism is regulated by numerous genes. Among them, 5,10-methylenetetrahydrofolate reductase (MTHFR) is of special interest because of its involvement in regulation of the homocysteine level in the body as a result of folate metabolism. Moreover, some studies demonstrated that the homocysteine plasma level in individuals may be influenced by polymorphisms present in the MTHFR gene. Two common, clinically relevant mutations have been described: MTHFR C677T and MTHFR A1298C. Although several laboratory techniques allow genotyping of both polymorphisms, PCR-RFLP analysis is simple to perform, relatively cheap, and thus one of the most utilized. In the case of A1298C, the PCR-RFLP technique that utilizes MboII endonuclease class II requires an acrylamide gel electrophoresis, since agarose gel electrophoresis is unable to resolve short deoxyribonucleic acid (DNA) fragments after restriction digestion. Agarose gel electrophoresis is commonly preferred over that of acrylamide. To resolve this inconvenience, a novel PCR-RFLP, AjuI-based method to genotype A1298C alleles has been developed that can be performed on standard agarose gel.

  4. The expanding polymerase universe.

    PubMed

    Goodman, M F; Tippin, B

    2000-11-01

    Over the past year, the number of known prokaryotic and eukaryotic DNA polymerases has exploded. Many of these newly discovered enzymes copy aberrant bases in the DNA template over which 'respectable' polymerases fear to tread. The next step is to unravel their functions, which are thought to range from error-prone copying of DNA lesions, somatic hypermutation and avoidance of skin cancer, to restarting stalled replication forks and repairing double-stranded DNA breaks.

  5. Methylation matters

    PubMed Central

    Costello, J.; Plass, C.

    2001-01-01

    DNA methylation is not just for basic scientists any more. There is a growing awareness in the medical field that having the correct pattern of genomic methylation is essential for healthy cells and organs. If methylation patterns are not properly established or maintained, disorders as diverse as mental retardation, immune deficiency, and sporadic or inherited cancers may follow. Through inappropriate silencing of growth regulating genes and simultaneous destabilisation of whole chromosomes, methylation defects help create a chaotic state from which cancer cells evolve. Methylation defects are present in cells before the onset of obvious malignancy and therefore cannot be explained simply as a consequence of a deregulated cancer cell. Researchers are now able to detect with exquisite sensitivity the cells harbouring methylation defects, sometimes months or years before the time when cancer is clinically detectable. Furthermore, aberrant methylation of specific genes has been directly linked with the tumour response to chemotherapy and patient survival. Advances in our ability to observe the methylation status of the entire cancer cell genome have led us to the unmistakable conclusion that methylation abnormalities are far more prevalent than expected. This methylomics approach permits the integration of an ever growing repertoire of methylation defects with the genetic alterations catalogued from tumours over the past two decades. Here we discuss the current knowledge of DNA methylation in normal cells and disease states, and how this relates directly to our current understanding of the mechanisms by which tumours arise.


Keywords: methylation; cancer PMID:11333864

  6. Methylene tetrahydrofolate reductase (MTHFR) gene polymorphisms in chronic myeloid leukemia: an Egyptian study.

    PubMed

    Khorshied, Mervat Mamdooh; Shaheen, Iman Abdel Mohsen; Abu Khalil, Reham E; Sheir, Rania Elsayed

    2014-01-01

    Methylenetetrahydrofolate reductase (MTHFR) gene plays a pivotal role in folate metabolism. Several genetic variations in MTHFR gene as MTHFR-C677T and MTHFR-A1298C result in decreased MTHFR activity, which could influence efficient DNA methylation and explain susceptibility to different cancers. The etiology of chronic myeloid leukemia (CML) is obscure and little is known about individual's susceptibility to CML. In order to assess the influence of these genetic polymorphisms on the susceptibility to CML and its effect on the course of the disease among Egyptians, we performed an age-gender-ethnic matched case-control study. The study included 97 CML patients and 130 healthy controls. Genotyping of MTHFR-C677T and -A1298C was performed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. The results showed no statistical difference in the distribution of MTHFR-C677T and -A1298C polymorphic genotypes between CML patients and controls. The frequency of MTHFR 677-TT homozygous variant was significantly higher in patients with accelerated/blastic transformation phase when compared to those in the chronic phase of the disease. In conclusion, our study revealed that MTHFR-C677T and -A1298C polymorphisms could not be considered as genetic risk factors for CML in Egyptians. However, MTHFR 677-TT homozygous variant might be considered as a molecular predictor for disease progression.

  7. Polymorphisms of glutathione S-transferase and methylenetetrahydrofolate reductase genes in Moldavian patients with ulcerative colitis: Genotype-phenotype correlation

    PubMed Central

    Varzari, Alexander; Deyneko, Igor V.; Tudor, Elena; Turcan, Svetlana

    2015-01-01

    Background Glutathione S-transferases (GSTM1, GSTT1, and GSTP1) and methylenetetrahydrofolate reductase (MTHFR) are important enzymes for protection against oxidative stress. In addition, MTHFR has an essential role in DNA synthesis, repair, and methylation. Their polymorphisms have been implicated in the pathogenesis of ulcerative colitis (UC). The aim of the present study was to investigate the role of selected polymorphisms in these genes in the development of UC in the Moldavian population. Methods In a case-control study including 128 UC patients and 136 healthy individuals, GSTM1 and GSTT1 genotypes (polymorphic deletions) were determined using multiplex polymerase chain reaction (PCR). The GSTP1 rs1695 (Ile105Val), MTHFR rs1801133 (C677T), and MTHFR rs1801131 (A1298C) polymorphisms were studied with restriction fragment length polymorphism (RFLP) analysis. Genotype–phenotype correlations were examined using logistic regression analysis. Results None of the genotypes, either alone or in combination, showed a strong association with UC. The case-only sub-phenotypic association analysis showed an association of the MTHFR rs1801133 polymorphism with the extent of UC under co-dominant (p corrected = 0.040) and recessive (p corrected = 0.020; OR = 0.15; CI = 0.04–0.63) genetic models. Also, an association between the MTHFR rs1801131 polymorphism and the severity of UC was reported for the over-dominant model (p corrected = 0.023; coefficient = 0.32; 95% CI = 0.10–0.54). Conclusion The GST and MTHFR genotypes do not seem to be a relevant risk factor for UC in our sample. There was, however, evidence that variants in MTHFR may influence the clinical features in UC patients. Additional larger studies investigating the relationship between GST and MTHFR polymorphisms and UC are required. PMID:26862484

  8. Polymerase chain reaction system

    DOEpatents

    Benett, William J.; Richards, James B.; Stratton, Paul L.; Hadley, Dean R.; Milanovich, Fred P.; Belgrader, Phil; Meyer, Peter L.

    2004-03-02

    A portable polymerase chain reaction DNA amplification and detection system includes one or more chamber modules. Each module supports a duplex assay of a biological sample. Each module has two parallel interrogation ports with a linear optical system. The system is capable of being handheld.

  9. DNA polymorphism identity determination using flow cytometry

    DOEpatents

    Nolan, John P.; White, P. Scott; Cai, Hong

    2001-01-01

    DNA polymorphism identity determination using flow cytometry. Primers designed to be immobilized on microspheres are allowed to anneal to the DNA strand under investigation, and are extended by either DNA polymerase using fluorescent dideoxynucleotides or ligated by DNA ligase to fluorescent reporter oligonucleotides. The fluorescence of either the dideoxynucleotide or the reporter oligonucleotide attached to the immobilized primer is measured by flow cytometry, thereby identifying the nucleotide polymorphism on the DNA strand.

  10. Methyl Iodide

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Methyl iodide (MeI, iodomethane, CH3I) was reported as a potential alternative to the stratospheric ozone-depleting fumigant methyl bromide (MeBr) in the mid-1990s (Sims et al., 1995; Ohr et al., 1996). It has since received significant research attention to determine its environmental fate and tran...

  11. Methyl chloroform

    SciTech Connect

    Wray, T.K.

    1994-04-01

    Methyl chloroform is identified as a Class 1 ozone-depleting substance under Title VI of the CAA Amendments. On Nov. 30, 1993, EPA ordered the phaseout of Class 1 ozone-depleting substances -- chlorofluorocarbons (CFCs), halons, carbon tetrachloride and methyl chloroform -- by Jan. 1, 1996. Methyl chloroform and other Class 1 substances may be used after the dead-line if sources can be found through recycling or existing inventories. Methyl chloroform is listed as a hazardous air pollutant under CAA. It also is a SARA Title III, Sec. 313 compound with a reportable quantity of 1,000 pounds. OSHA and the American Conference of Government Industrial Hygienists have set 350 ppm as the time-weighted average airborne exposure level for methyl chloroform. NIOSH lists its immediately dangerous to life or health'' concentration as 1,000 parts per million. DOT identifies the substance as a hazardous material, Class 6.1 (poison).

  12. Lack of association between MTHFR C677T polymorphism and breast cancer risk in Ahvaz, west south-Iran

    PubMed Central

    Mohammadzadeh, Ghorban; Karimi, Maryam; Bazyar, Mohammad; Hosseini, Seyed-Mohammad

    2016-01-01

    Background: Association between C677T polymorphism of the methylenetetrahydrofolate reductase (MTHFR), a key enzyme involved in folate metabolism and DNA methylation, and breast cancer risk are inconsistent. We investigated in a case-control study, possible effect of the common MTHFR C677T polymorphism on breast cancer risk in a sample of Iranian patients. Materials and Methods: The study subjects comprised of 123 breast cancer cases and 110 cancer-free control, who were matched for age and body mass index (BMI). C677T genotypes were determined by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) assay. Lipid profile was measured in all subjects by standard method. Results: The genotypes distributions (CC, CT, and TT) were 55.3, 39, and 5.7% in breast cancer cases and 51.8, 44.5, and 3.6% in controls. Chi square analysis revealed that there was no significant association between breast cancer risk and MTHFR genotypes and alleles. Additionally, no significant association was observed between C677T genotypes and biochemistry parameters. A multinomial logistic regression model with MTHFR genotypes, lipid profiles, BMI and age as covariates revealed that there is no significant association between MTHFR genotypes and risk of breast cancer, but higher values of LDL and HDL significantly increase risk of breast cancer. Conclusions: Our findings do not support the hypothesis that genetic variation in the MTHFR C677T polymorphism is implicated in the breast cancer risk in a sample of Iranian patients. PMID:27014653

  13. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase

    DOE PAGESBeta

    McInerney, Peter; Adams, Paul; Hadi, Masood Z.

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Errormore » rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu , Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition.« less

  14. Error Rate Comparison during Polymerase Chain Reaction by DNA Polymerase.

    PubMed

    McInerney, Peter; Adams, Paul; Hadi, Masood Z

    2014-01-01

    As larger-scale cloning projects become more prevalent, there is an increasing need for comparisons among high fidelity DNA polymerases used for PCR amplification. All polymerases marketed for PCR applications are tested for fidelity properties (i.e., error rate determination) by vendors, and numerous literature reports have addressed PCR enzyme fidelity. Nonetheless, it is often difficult to make direct comparisons among different enzymes due to numerous methodological and analytical differences from study to study. We have measured the error rates for 6 DNA polymerases commonly used in PCR applications, including 3 polymerases typically used for cloning applications requiring high fidelity. Error rate measurement values reported here were obtained by direct sequencing of cloned PCR products. The strategy employed here allows interrogation of error rate across a very large DNA sequence space, since 94 unique DNA targets were used as templates for PCR cloning. The six enzymes included in the study, Taq polymerase, AccuPrime-Taq High Fidelity, KOD Hot Start, cloned Pfu polymerase, Phusion Hot Start, and Pwo polymerase, we find the lowest error rates with Pfu, Phusion, and Pwo polymerases. Error rates are comparable for these 3 enzymes and are >10x lower than the error rate observed with Taq polymerase. Mutation spectra are reported, with the 3 high fidelity enzymes displaying broadly similar types of mutations. For these enzymes, transition mutations predominate, with little bias observed for type of transition. PMID:25197572

  15. DNA Methylation

    PubMed Central

    Marinus, M.G.; Løbner-Olesen, A.

    2014-01-01

    The DNA of E. coli contains 19,120 6-methyladenines and 12,045 5-methylcytosines in addition to the four regular bases and these are formed by the postreplicative action of three DNA methyltransferases. The majority of the methylated bases are formed by the Dam and Dcm methyltransferases encoded by the dam (DNA adenine methyltransferase) and dcm (DNA cytosine methyltransferase) genes. Although not essential, Dam methylation is important for strand discrimination during repair of replication errors, controlling the frequency of initiation of chromosome replication at oriC, and regulation of transcription initiation at promoters containing GATC sequences. In contrast, there is no known function for Dcm methylation although Dcm recognition sites constitute sequence motifs for Very Short Patch repair of T/G base mismatches. In certain bacteria (e.g., Vibrio cholerae, Caulobacter crescentus) adenine methylation is essential and in C. crescentus, it is important for temporal gene expression which, in turn, is required for coordinating chromosome initiation, replication and division. In practical terms, Dam and Dcm methylation can inhibit restriction enzyme cleavage; decrease transformation frequency in certain bacteria; decrease the stability of short direct repeats; are necessary for site-directed mutagenesis; and to probe eukaryotic structure and function. PMID:26442938

  16. Polymerase chain reaction

    SciTech Connect

    Arnhelm, N. ); Levenson, C.H. )

    1990-10-01

    This paper discusses the polymerase chain reaction (PCR) an in-vitro method of amplifying DNA sequences. Beginning with DNA of any origin- bacterial, viral, plant, or animal- PCR can increase the amount of a DNA sequence hundreds of millions to billions of times. The procedure can amplify a targeted sequence even when it makes up less than one part in a million of the total initial sample. PCR is an enzymatic process that is carried out in discrete cycles of amplification, each of which can double the amount of target DNA in the sample. Thus, n cycles can produce 2{sup n} times as much target as was present to begin with. This paper discusses how PCR has had an impact on molecular biology, human genetics, infectious and genetic disease diagnosis, forensic science, and evolutionary biology.

  17. Application of real-time polymerase chain reaction in the clinical genetic practice.

    PubMed

    Nagy, Bálint

    2013-03-01

    The development of polymerase chain reaction revolutionized the molecular genetics and diagnostics. Technical improvements helped to make more specific and sensitive target determinations. Introduction of real-time polymerase chain reaction makes possible several applications in clinical genetics like detection of gene mutations, single nucleotide polymorphisms, deletions, measurement of gene expressions, micro ribonucleic acids, free nucleic acids and microbial genomes. Here I discuss a few examples for specific applications in prenatal clinical genetic practice. These are the detection of microbial genomes, deletions, trisomies, mutations, single nucleotide polymorphisms and free nucleic acids. PMID:27625833

  18. Application of real-time polymerase chain reaction in the clinical genetic practice

    PubMed Central

    Nagy, Bálint

    2013-01-01

    The development of polymerase chain reaction revolutionized the molecular genetics and diagnostics. Technical improvements helped to make more specific and sensitive target determinations. Introduction of real-time polymerase chain reaction makes possible several applications in clinical genetics like detection of gene mutations, single nucleotide polymorphisms, deletions, measurement of gene expressions, micro ribonucleic acids, free nucleic acids and microbial genomes. Here I discuss a few examples for specific applications in prenatal clinical genetic practice. These are the detection of microbial genomes, deletions, trisomies, mutations, single nucleotide polymorphisms and free nucleic acids.

  19. Methyl chloride

    Integrated Risk Information System (IRIS)

    Methyl chloride ; CASRN 74 - 87 - 3 ( 07 / 17 / 2001 ) Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for

  20. Methyl acrylate

    Integrated Risk Information System (IRIS)

    Methyl acrylate ; CASRN 96 - 33 - 3 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Ef

  1. Methyl chlorocarbonate

    Integrated Risk Information System (IRIS)

    Methyl chlorocarbonate ; CASRN 79 - 22 - 1 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinog

  2. Methyl isocyanate

    Integrated Risk Information System (IRIS)

    Methyl isocyanate ; CASRN 624 - 83 - 9 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic

  3. Methyl parathion

    Integrated Risk Information System (IRIS)

    Methyl parathion ; CASRN 298 - 00 - 0 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic

  4. Methyl methacrylate

    Integrated Risk Information System (IRIS)

    Methyl methacrylate ; CASRN 80 - 62 - 6 ( 03 / 02 / 98 ) Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments f

  5. Methyl iodide

    Integrated Risk Information System (IRIS)

    Methyl iodide ; CASRN 74 - 88 - 4 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinogenic Effe

  6. Polymorphisms of the thiopurine S-methyltransferase gene among the Libyan population

    PubMed Central

    Zeglam, Hamza Ben; Benhamer, Abdrazak; Aboud, Adel; Rtemi, Haitem; Mattardi, Meftah; Saleh, Saleh Suleiman; Bashein, Abdullah; Enattah, Nabil

    2015-01-01

    Background Thiopurine S-methyltransferase (TPMT) is a cytosolic enzyme that catalyses the S-methylation of 6-mercaptopurine and azathioprine. Low activity phenotypes are correlated with polymorphism in the TPMT gene. Patients with low or undetectable TMPT activity could develop severe myelosuppression when they are treated with standard doses of thiopurine drugs. Since ethnic differences in the TPMT gene polymorphism have been demonstrated worldwide, assessing it in the Libyan population is worthwhile. Methods We investigated TPMT gene polymorphism in a total of 246 Libyan healthy adult blood donors from three different Libyan regions (Tripoli, Yefren, and Tawargha) and 50 children with acute lymphoblastic leukaemia (ALL). We used polymerase chain reaction restriction length polymorphism (PCR-RFLP) and allele-specific PCR-based assays to analyse the TPMT gene for the variants *2 c.238 G>C, *3A (c.460 G>A and c.719 A>G), *3B (c.460 G>A), and *3C (c.719 A>G). Results Our results show that the TPMT variants associated with low enzymatic activity were detected in 3.25% (8 in 246) of adult Libyan individuals and the frequency of total mutant alleles was 1.63%. Heterozygous genotypes were TPMT*3A in three subjects (0.61%) and TPMT*3C in five subjects (1.02%). No TPMT*2 and TPMT*3B allelic variants and no homozygous or compound heterozygous mutant alleles were detected. The normal allele (wild-type) was found in 98.4% of the adult individuals studied. No mutant alleles were detected among the 50 children who had ALL. Conclusions We report on the presence of the TPMT*3C and *3A mutant alleles in the Libyan population. Therefore, monitoring the patients to be treated with doses of thiopurine drugs for TPMT variants is worthwhile to avoid the development of severe myelosuppression. PMID:25819542

  7. Evaluation of a blood-specific DNA methylated region and trial for allele-specific blood identification from mixed body fluid DNA.

    PubMed

    Watanabe, Ken; Akutsu, Tomoko; Takamura, Ayari; Sakurada, Koichi

    2016-09-01

    The identification of blood samples obtained from crime scenes has been an important step in forensic investigation. Recently, a novel approach using the blood-specific methylated CpG site cg06379435 has been reported. In this study, we developed a real-time polymerase-chain-reaction-based method that can simply and rapidly quantitate the methylation ratio of cg06379435 and its neighboring CpGs and set the threshold ratios for blood identification by analyzing various body fluid samples. Blood identification using the thresholds was successfully performed in the analysis of a small amount (1ng) of DNA from blood and various aged blood samples, including 29-year-old stains. We also demonstrated a test for allele-specific blood identification from a mixed DNA sample by bisulfite sequencing analysis of these CpG sites and their neighboring single nucleotide polymorphism, rs7359943 (A/G), which is of relevance in cases where mixed samples are obtained from crime scenes. The stability of DNA methylation in aged samples and the usefulness of neighboring genetic information shown in this study suggest that DNA-methylation-based body fluid identification will play a major role in future forensic investigations. PMID:27591539

  8. Genome-wide analysis of DNA methylation in hepatoblastoma tissues

    PubMed Central

    Cui, Ximao; Liu, Baihui; Zheng, Shan; Dong, Kuiran; Dong, Rui

    2016-01-01

    DNA methylation has a crucial role in cancer biology. In the present study, a genome-wide analysis of DNA methylation in hepatoblastoma (HB) tissues was performed to verify differential methylation levels between HB and normal tissues. As alpha-fetoprotein (AFP) has a critical role in HB, AFP methylation levels were also detected using pyrosequencing. Normal and HB liver tissue samples (frozen tissue) were obtained from patients with HB. Genome-wide analysis of DNA methylation in these tissues was performed using an Infinium HumanMethylation450 BeadChip, and the results were confirmed with reverse transcription-quantitative polymerase chain reaction. The Infinium HumanMethylation450 BeadChip demonstrated distinctively less methylation in HB tissues than in non-tumor tissues. In addition, methylation enrichment was observed in positions near the transcription start site of AFP, which exhibited lower methylation levels in HB tissues than in non-tumor liver tissues. Lastly, a significant negative correlation was observed between AFP messenger RNA expression and DNA methylation percentage, using linear Pearson's R correlation coefficients. The present results demonstrate differential methylation levels between HB and normal tissues, and imply that aberrant methylation of AFP in HB could reflect HB development. Expansion of these findings could provide useful insight into HB biology. PMID:27446465

  9. Mutation of DNA Polymerase β R137Q Results in Retarded Embryo Development Due to Impaired DNA Base Excision Repair in Mice

    PubMed Central

    Pan, Feiyan; Zhao, Jing; Zhou, Ting; Kuang, Zhihui; Dai, Huifang; Wu, Huan; Sun, Hongfang; Zhou, Xiaolong; Wu, Xuping; Hu, Zhigang; He, Lingfeng; Shen, Binghui; Guo, Zhigang

    2016-01-01

    DNA polymerase β (Pol β), a key enzyme in the DNA base excision repair (BER) pathway, is pivotal in maintaining the integrity and stability of genomes. One Pol β mutation that has been identified in tumors, R137Q (arginine to glutamine substitution), has been shown to lower polymerase activity, and impair its DNA repair capacity. However, the exact functional deficiency associated with this polymorphism in living organisms is still unknown. Here, we constructed Pol β R137Q knock-in mice, and found that homozygous knock-in mouse embryos were typically small in size and had a high mortality rate (21%). These embryonic abnormalities were caused by slow cell proliferation and increased apoptosis. In R137Q knock-in mouse embryos, the BER efficiency was severely impaired, which subsequently resulted in double-strand breaks (DSBs) and chromosomal aberrations. Furthermore, R137Q mouse embryo fibroblasts (MEFs) were more sensitive to DNA-damaging reagents, such as methyl methanesulfonate (MMS) and H2O2. They displayed a higher percentage of DSBs, and were more likely to undergo apoptosis. Our results indicate that R137 is a key amino acid site that is essential for proper Pol β functioning in maintaining genomic stability and embryo development. PMID:27358192

  10. Morphological changes in gray matter volume correlate with catechol-O-methyl transferase gene Val158Met polymorphism in first-episode treatment-naïve patients with schizophrenia.

    PubMed

    Li, Ming-Li; Xiang, Bo; Li, Yin-Fei; Hu, Xun; Wang, Qiang; Guo, Wan-Jun; Lei, Wei; Huang, Chao-Hua; Zhao, Lian-Sheng; Li, Na; Ren, Hong-Yan; Wang, Hui-Yao; Ma, Xiao-Hong; Deng, Wei; Li, Tao

    2015-02-01

    The catechol-O-methyltransferase (COMT) gene is a schizophrenia susceptibility gene. A common functional polymorphism of this gene, Val158/158Met, has been proposed to influence gray matter volume (GMV). However, the effects of this polymorphism on cortical thickness/surface area in schizophrenic patients are less clear. In this study, we explored the relationship between the Val158Met polymorphism of the COMT gene and the GMV/cortical thickness/cortical surface area in 150 first-episode treatment-naïve patients with schizophrenia and 100 healthy controls. Main effects of diagnosis were found for GMV in the cerebellum and the visual, medial temporal, parietal, and middle frontal cortex. Patients with schizophrenia showed reduced GMVs in these regions. And main effects of genotype were detected for GMV in the left superior frontal gyrus. Moreover, a diagnosis × genotype interaction was found for the GMV of the left precuneus, and the effect of the COMT gene on GMV was due mainly to cortical thickness rather than cortical surface area. In addition, a pattern of increased GMV in the precuneus with increasing Met dose found in healthy controls was lost in patients with schizophrenia. These findings suggest that the COMTMet variant is associated with the disruption of dopaminergic influence on gray matter in schizophrenia, and the effect of the COMT gene on GMV in schizophrenia is mainly due to changes in cortical thickness rather than in cortical surface area. PMID:25564193

  11. Transgenerational epigenetic instability is a source of novel methylation variants.

    PubMed

    Schmitz, Robert J; Schultz, Matthew D; Lewsey, Mathew G; O'Malley, Ronan C; Urich, Mark A; Libiger, Ondrej; Schork, Nicholas J; Ecker, Joseph R

    2011-10-21

    Epigenetic information, which may affect an organism's phenotype, can be stored and stably inherited in the form of cytosine DNA methylation. Changes in DNA methylation can produce meiotically stable epialleles that affect transcription and morphology, but the rates of spontaneous gain or loss of DNA methylation are unknown. We examined spontaneously occurring variation in DNA methylation in Arabidopsis thaliana plants propagated by single-seed descent for 30 generations. We identified 114,287 CG single methylation polymorphisms and 2485 CG differentially methylated regions (DMRs), both of which show patterns of divergence compared with the ancestral state. Thus, transgenerational epigenetic variation in DNA methylation may generate new allelic states that alter transcription, providing a mechanism for phenotypic diversity in the absence of genetic mutation.

  12. [The relationship between BDNF gene polymorphisms and alcoholics in Japan].

    PubMed

    Narita, Shin; Nagahori, Kenta; Yoshihara, Eiji; Nishizawa, Daisuke; Ikeda, Kazutaka; Kawai, Atsuko; Iwahashi, Kazuhiko

    2013-12-01

    As a help of the mechanism elucidation of alcoholism, we studied the relationship between brain-derived neurotrophic factor (BDNF) rs6265, 270 C/T (ID number has not yet been determined), and rs10835210 gene polymorphisms, which are reported to be related to bipolar disorder, and alcoholics. We genotyped the three polymorphisms in the BDNF gene using polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) in 65 alcoholics and 71 healthy controls. In this study, there was no significant difference in the frequency of rs6265 and 270 C/T polymorphisms between alcoholics and controls (P > 0.05). However, there was a significant difference in the genotype frequency of rs10835210 polymorphism between alcoholics and controls (P < 0.05), in which the CA heterozygote genotype and A allele frequency was higher in alcoholics than in the controls. It suggests the possibility that the BDNF rs10835210 gene polymorphism affects the etiology of alcoholism.

  13. A monoclinic polymorph of [(Z)-N-(3-chloro-phen-yl)-O-methyl-thio-carbamato-κS](tri-phenyl-phosphane-κP)gold(I): crystal structure and Hirshfeld surface analysis.

    PubMed

    Yeo, Chien Ing; Tan, Sang Loon; Tiekink, Edward R T

    2016-08-01

    The title compound, [Au(C8H7ClNOS)(C18H15P)], is a monoclinic (P21/n, Z' = 1; form β) polymorph of the previously reported triclinic form (P-1, Z' = 1; form α) [Tadbuppa & Tiekink (2010 ▸). Acta Cryst. E66, m664]. The mol-ecular structures of both forms feature an almost linear gold(I) coordination geometry [P-Au-S = 175.62 (5)° in the title polymorph], being coordinated by thiol-ate S and phosphane P atoms, a Z conformation about the C=N bond and an intra-molecular Au⋯O contact. The major conformational difference relates to the relative orientations of the residues about the Au-S bond: the P-Au-S-C torsion angles are -8.4 (7) and 106.2 (7)° in forms α and β, respectively. The mol-ecular packing of form β features centrosymmetric aggregates sustained by aryl-C-H⋯O inter-actions, which are connected into a three-dimensional network by aryl-C-H⋯π contacts. The Hirshfeld analysis of forms α and β shows many similarities with the notable exception of the influence of C-H⋯O inter-actions in form β. PMID:27536384

  14. A monoclinic polymorph of [(Z)-N-(3-chloro­phen­yl)-O-methyl­thio­carbamato-κS](tri­phenyl­phosphane-κP)gold(I): crystal structure and Hirshfeld surface analysis

    PubMed Central

    Yeo, Chien Ing; Tan, Sang Loon; Tiekink, Edward R. T.

    2016-01-01

    The title compound, [Au(C8H7ClNOS)(C18H15P)], is a monoclinic (P21/n, Z′ = 1; form β) polymorph of the previously reported triclinic form (P-1, Z′ = 1; form α) [Tadbuppa & Tiekink (2010 ▸). Acta Cryst. E66, m664]. The mol­ecular structures of both forms feature an almost linear gold(I) coordination geometry [P—Au—S = 175.62 (5)° in the title polymorph], being coordinated by thiol­ate S and phosphane P atoms, a Z conformation about the C=N bond and an intra­molecular Au⋯O contact. The major conformational difference relates to the relative orientations of the residues about the Au—S bond: the P—Au—S—C torsion angles are −8.4 (7) and 106.2 (7)° in forms α and β, respectively. The mol­ecular packing of form β features centrosymmetric aggregates sustained by aryl-C—H⋯O inter­actions, which are connected into a three-dimensional network by aryl-C—H⋯π contacts. The Hirshfeld analysis of forms α and β shows many similarities with the notable exception of the influence of C—H⋯O inter­actions in form β. PMID:27536384

  15. Polymerase chain reaction: A molecular diagnostic tool in periodontology.

    PubMed

    Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

    2016-01-01

    This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease.

  16. Polymerase chain reaction: A molecular diagnostic tool in periodontology

    PubMed Central

    Maheaswari, Rajendran; Kshirsagar, Jaishree Tukaram; Lavanya, Nallasivam

    2016-01-01

    This review discusses the principles of polymerase chain reaction (PCR) and its application as a diagnostic tool in periodontology. The relevant MEDLINE and PubMed indexed journals were searched manually and electronically by typing PCR, applications of PCR, PCR in periodontics, polymorphism studies in periodontitis, and molecular techniques in periodontology. The searches were limited to articles in English language and the articles describing PCR process and its relation to periodontology were collected and used to prepare a concise review. PCR has now become a standard diagnostic and research tool in periodontology. Various studies reveal that its sensitivity and specificity allow it as a rapid, efficient method of detecting, identifying, and quantifying organism. Different immune and inflammatory markers can be identified at the mRNA expression level, and also the determination of genetic polymorphisms, thus providing the deeper insight into the mechanisms underlying the periodontal disease. PMID:27143822

  17. Molecular typing among beef isolates of Escherichia coli using consensus repetitive intergenic enterobacteria-polymerase chain reaction (ERIC-PCR)

    NASA Astrophysics Data System (ADS)

    Zoolkifli, Nurliyana Wan; Mutalib, Sahilah Abd

    2013-11-01

    Genomic DNA of Escherichia coli were characterized by enterobacterial repetitive intergenic consensus-Polymerase chain reaction (ERIC-PCR) and the presence of Shiga toxin gene-I (Stx1) and Shiga toxin gene-2 (Stx2). These isolates were originated from imported raw beef which are come from two countries namely Australia and India. The isolation of E. coli was conducted by using Eosin Methylene Blue Agar (EMBA). A total of 94 strains had been isolated from 30 samples of imported raw beefand 42 strains had been detected positively E. coli by doing biochemical tests. All strains had been tested and the results of biochemical tests showed that 3 strains were from Australia samples while the other 39 strains were from India samples. The biochemical tests used are Indole test, Methyl Red test, Voges-Proskauer test and Citrate test. All the 42 strains were examined for Shiga toxin (stx1 and stx2) gene detection by two pair primers which are stx2F (5'-TTCTTCGGTATCCTATTCCC-3'), stx2R (5'-ATGCATCTCTGGTCATTGTA-3'), stx1F (5'-CAGTTAATGTGGTGGCGAAG-3'), and stx1R (5'-CTGTCACAGTAACAACCGT-3'). The results showed that none of the strains are positive for Shiga toxin gene. Application of ERIC-PCR method towards E. coli had successfully shown the high diversity polymorphism in 21 different genome types of DNA with primers ERIC1R (5'- CACTTAGGGGTCCTCGAATGTA- 3') and ERIC2R (5'- AAGTAAGTGACTGGGGTGACGC- 3').

  18. [Applications of DNA methylation markers in forensic medicine].

    PubMed

    Zhao, Gui-sen; Yang, Qing-en

    2005-02-01

    DNA methylation is a post-replication modification that is predominantly found in cytosines of the dinucleotide sequence CpG. Epigenetic information is stored in the distribution of the modified base 5-methylcytosine. DNA methylation profiles represent a more chemically and biologically stable source of molecular diagnostic information than RNA or most proteins. Recent advances attest to the great promise of DNA methylation markers as powerful future tools in the clinic. In the past decade, DNA methylation analysis has been revolutionized by two technological advances--bisulphite modification of DNA and methylation-specific polymerase chain reaction (MSP). The methylation pattern of human genome is space-time specific, sex-specific, parent-of-origin specific and disease specific, providing us an alternative way to solve forensic problems.

  19. Survivin promoter polymorphism and cervical carcinogenesis

    PubMed Central

    Borbély, A A; Murvai, M; Szarka, K; Kónya, J; Gergely, L; Hernádi, Z; Veress, G

    2007-01-01

    Background Survivin, a novel member of the inhibitor of apoptosis family, plays an important role in cell cycle regulation. A common polymorphism at the survivin gene promoter (G/C at position 31) was shown to be correlated with survivin gene expression in cancer cell lines. Aim To investigate whether this polymorphism could be involved in the development of human papillomavirus (HPV)‐associated cervical carcinoma. Methods Survivin promoter polymorphism was detected in patients with cervical cancer, in patients with equivocal cytological atypia and in a control population using polymerase chain reaction (PCR‐restriction fragment length polymorphism (RFLP) and PCR‐single strand conformation polymorphism analysis. HPV was typed in patients with cervical cancer and cytological atypia using PCR‐RFLP. Results No statistically significant differences were found in the genotype distributions of the survivin promoter variants among our study groups. Conclusions The survivin promoter polymorphism at position 31 may not represent an increased risk for the development of cervical cancer, at least in the population studied here. PMID:16714396

  20. Subunit Compositions of the RNA-Silencing Enzymes Pol IV and Pol V Reveal Their Origins as Specialized Forms of RNA Polymerase II

    SciTech Connect

    Ream, Thomas S.; Haag, J. R.; Wierzbicki, A. T.; Nicora, Carrie D.; Norbeck, Angela D.; Zhu, J. K.; Hagen, G.; Guilfoyle, T. J.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2009-01-30

    In addition to RNA polymerases I, II and III, which are multi-subunit RNA polymerases found in all eukaryotes, plants have catalytic subunits for two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V (formerly Pol IVa and Pol IVb, respectively). Pol IV and Pol V play non-redundant roles in siRNA-directed DNA methylation and gene silencing pathways.

  1. Shotgun metagenomics indicates novel family A DNA polymerases predominate within marine virioplankton.

    PubMed

    Schmidt, Helen F; Sakowski, Eric G; Williamson, Shannon J; Polson, Shawn W; Wommack, K Eric

    2014-01-01

    Virioplankton have a significant role in marine ecosystems, yet we know little of the predominant biological characteristics of aquatic viruses that influence the flow of nutrients and energy through microbial communities. Family A DNA polymerases, critical to DNA replication and repair in prokaryotes, are found in many tailed bacteriophages. The essential role of DNA polymerase in viral replication makes it a useful target for connecting viral diversity with an important biological feature of viruses. Capturing the full diversity of this polymorphic gene by targeted approaches has been difficult; thus, full-length DNA polymerase genes were assembled out of virioplankton shotgun metagenomic sequence libraries (viromes). Within the viromes novel DNA polymerases were common and found in both double-stranded (ds) DNA and single-stranded (ss) DNA libraries. Finding DNA polymerase genes in ssDNA viral libraries was unexpected, as no such genes have been previously reported from ssDNA phage. Surprisingly, the most common virioplankton DNA polymerases were related to a siphovirus infecting an α-proteobacterial symbiont of a marine sponge and not the podoviral T7-like polymerases seen in many other studies. Amino acids predictive of catalytic efficiency and fidelity linked perfectly to the environmental clades, indicating that most DNA polymerase-carrying virioplankton utilize a lower efficiency, higher fidelity enzyme. Comparisons with previously reported, PCR-amplified DNA polymerase sequences indicated that the most common virioplankton metagenomic DNA polymerases formed a new group that included siphoviruses. These data indicate that slower-replicating, lytic or lysogenic phage populations rather than fast-replicating, highly lytic phages may predominate within the virioplankton. PMID:23985748

  2. Norovirus Proteinase-Polymerase and Polymerase Are Both Active Forms of RNA-Dependent RNA Polymerase

    PubMed Central

    Belliot, Gaël; Sosnovtsev, Stanislav V.; Chang, Kyeong-Ok; Babu, Vijay; Uche, Uzo; Arnold, Jamie J.; Cameron, Craig E.; Green, Kim Y.

    2005-01-01

    In vitro mapping studies of the MD145 norovirus (Caliciviridae) ORF1 polyprotein identified two stable cleavage products containing the viral RNA-dependent RNA polymerase (RdRp) domains: ProPol (a precursor comprised of both the proteinase and polymerase) and Pol (the mature polymerase). The goal of this study was to identify the active form (or forms) of the norovirus polymerase. The recombinant ProPol (expressed as Pro−Pol with an inactivated proteinase domain to prevent autocleavage) and recombinant Pol were purified after synthesis in bacteria and shown to be active RdRp enzymes. In addition, the mutant His-E1189A-ProPol protein (with active proteinase but with the natural ProPol cleavage site blocked) was active as an RdRp, confirming that the norovirus ProPol precursor could possess two enzymatic activities simultaneously. The effects of several UTP analogs on the RdRp activity of the norovirus and feline calicivirus Pro−Pol enzymes were compared and found to be similar. Our data suggest that the norovirus ProPol is a bifunctional enzyme during virus replication. The availability of this recombinant ProPol enzyme might prove useful in the development of antiviral drugs for control of the noroviruses associated with acute gastroenteritis. PMID:15681440

  3. Genomic DNA sequence and cytosine methylation changes of adult rice leaves after seeds space flight

    NASA Astrophysics Data System (ADS)

    Shi, Jinming

    In this study, cytosine methylation on CCGG site and genomic DNA sequence changes of adult leaves of rice after seeds space flight were detected by methylation-sensitive amplification polymorphism (MSAP) and Amplified fragment length polymorphism (AFLP) technique respectively. Rice seeds were planted in the trial field after 4 days space flight on the shenzhou-6 Spaceship of China. Adult leaves of space-treated rice including 8 plants chosen randomly and 2 plants with phenotypic mutation were used for AFLP and MSAP analysis. Polymorphism of both DNA sequence and cytosine methylation were detected. For MSAP analysis, the average polymorphic frequency of the on-ground controls, space-treated plants and mutants are 1.3%, 3.1% and 11% respectively. For AFLP analysis, the average polymorphic frequencies are 1.4%, 2.9%and 8%respectively. Total 27 and 22 polymorphic fragments were cloned sequenced from MSAP and AFLP analysis respectively. Nine of the 27 fragments from MSAP analysis show homology to coding sequence. For the 22 polymorphic fragments from AFLP analysis, no one shows homology to mRNA sequence and eight fragments show homology to repeat region or retrotransposon sequence. These results suggest that although both genomic DNA sequence and cytosine methylation status can be effected by space flight, the genomic region homology to the fragments from genome DNA and cytosine methylation analysis were different.

  4. Inheritance and Variation of Genomic DNA Methylation in Diploid and Triploid Pacific Oyster (Crassostrea gigas).

    PubMed

    Jiang, Qun; Li, Qi; Yu, Hong; Kong, Lingfeng

    2016-02-01

    DNA methylation is an important epigenetic mechanism that could be responsive to environmental changes indicating a potential role in natural selection and adaption. In order to evaluate an evolutionary role of DNA methylation, it is essential to first gain a better insight into inheritability. To address this question, this study investigated DNA methylation variation from parents to offspring in the Pacific oyster Crassostrea gigas using fluorescent-labeled methylation-sensitive amplified polymorphism (F-MSAP) analysis. Most of parental methylated loci were stably transmitted to offspring segregating following Medelian expectation. However, methylated loci deviated more often than non-methylated loci and offspring showed a few de novo methylated loci indicating DNA methylation changes from parents to offspring. Interestingly, some male-specific methylated loci were found in this study which might help to explore sex determination in oyster. Despite environmental stimuli, genomic stresses such as polyploidization also can induce methylation changes. This study also compared global DNA methylation level and individual methylated loci between diploid and triploid oysters. Results showed no difference in global methylation state but a few ploidy-specific loci were detected. DNA methylation variation during polyploidization was less than autonomous methylation variation from parents to offspring.

  5. Ligand 5,10,15,20-tetra(N-methyl-4-pyridyl)porphine (TMPyP4) prefers the parallel propeller-type human telomeric G-quadruplex DNA over its other polymorphs.

    PubMed

    Ali, Asfa; Bansal, Manju; Bhattacharya, Santanu

    2015-01-01

    The binding of ligand 5,10,15,20-tetra(N-methyl-4-pyridyl)porphine (TMPyP4) with telomeric and genomic G-quadruplex DNA has been extensively studied. However, a comparative study of interactions of TMPyP4 with different conformations of human telomeric G-quadruplex DNA, namely, parallel propeller-type (PP), antiparallel basket-type (AB), and mixed hybrid-type (MH) G-quadruplex DNA, has not been done. We considered all the possible binding sites in each of the G-quadruplex DNA structures and docked TMPyP4 to each one of them. The resultant most potent sites for binding were analyzed from the mean binding free energy of the complexes. Molecular dynamics simulations were then carried out, and analysis of the binding free energy of the TMPyP4-G-quadruplex complex showed that the binding of TMPyP4 with parallel propeller-type G-quadruplex DNA is preferred over the other two G-quadruplex DNA conformations. The results obtained from the change in solvent excluded surface area (SESA) and solvent accessible surface area (SASA) also support the more pronounced binding of the ligand with the parallel propeller-type G-quadruplex DNA.

  6. Population differences in the human arsenic (+3 oxidation state) methyltransferase (AS3MT) gene polymorphism detected by using genotyping method.

    PubMed

    Fujihara, Junko; Kunito, Takashi; Agusa, Tetsuro; Yasuda, Toshihiro; Iida, Reiko; Fujii, Yoshimi; Takeshita, Haruo

    2007-12-15

    Arsenic poisoning from drinking groundwater is a serious problem, particularly in developing Asian countries. Human arsenic (+3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite. Recently, a single nucleotide polymorphism (SNPs; rs17885947, M287T (T860C)) in the AS3MT gene was shown to be related to enzyme activity and considered to be related to genetic susceptibility to arsenic. In the present study, a useful genotyping method for M287T was developed using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique. Applying this method, the genotype distribution of M287T in Ovambo (n=185), Turkish (n=191), Mongolian (n=233), Korean (n=200), and Japanese (n=370) populations were investigated. The mutation frequencies in Asian populations were relatively lower than those of African and Caucasian populations, including those from previous studies: the frequencies of mutation in the Mongolian, Korean, and Japanese populations were 0.040, 0.010, and 0.010, respectively. In the course of this study, a PCR-based genotyping method that is inexpensive and does not require specialized equipment was developed. This method could be applied to a large number of residents at risk for arsenic poisoning.

  7. Population differences in the human arsenic (+ 3 oxidation state) methyltransferase (AS3MT) gene polymorphism detected by using genotyping method

    SciTech Connect

    Fujihara, Junko; Kunito, Takashi; Agusa, Tetsuro; Yasuda, Toshihiro; Iida, Reiko; Fujii, Yoshimi; Takeshita, Haruo

    2007-12-15

    Arsenic poisoning from drinking groundwater is a serious problem, particularly in developing Asian countries. Human arsenic (+ 3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite. Recently, a single nucleotide polymorphism (SNPs; rs17885947, M287T (T860C)) in the AS3MT gene was shown to be related to enzyme activity and considered to be related to genetic susceptibility to arsenic. In the present study, a useful genotyping method for M287T was developed using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique. Applying this method, the genotype distribution of M287T in Ovambo (n = 185), Turkish (n = 191), Mongolian (n = 233), Korean (n = 200), and Japanese (n = 370) populations were investigated. The mutation frequencies in Asian populations were relatively lower than those of African and Caucasian populations, including those from previous studies: the frequencies of mutation in the Mongolian, Korean, and Japanese populations were 0.040, 0.010, and 0.010, respectively. In the course of this study, a PCR-based genotyping method that is inexpensive and does not require specialized equipment was developed. This method could be applied to a large number of residents at risk for arsenic poisoning.

  8. Population differences in the human arsenic (+3 oxidation state) methyltransferase (AS3MT) gene polymorphism detected by using genotyping method.

    PubMed

    Fujihara, Junko; Kunito, Takashi; Agusa, Tetsuro; Yasuda, Toshihiro; Iida, Reiko; Fujii, Yoshimi; Takeshita, Haruo

    2007-12-15

    Arsenic poisoning from drinking groundwater is a serious problem, particularly in developing Asian countries. Human arsenic (+3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite. Recently, a single nucleotide polymorphism (SNPs; rs17885947, M287T (T860C)) in the AS3MT gene was shown to be related to enzyme activity and considered to be related to genetic susceptibility to arsenic. In the present study, a useful genotyping method for M287T was developed using the polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP) technique. Applying this method, the genotype distribution of M287T in Ovambo (n=185), Turkish (n=191), Mongolian (n=233), Korean (n=200), and Japanese (n=370) populations were investigated. The mutation frequencies in Asian populations were relatively lower than those of African and Caucasian populations, including those from previous studies: the frequencies of mutation in the Mongolian, Korean, and Japanese populations were 0.040, 0.010, and 0.010, respectively. In the course of this study, a PCR-based genotyping method that is inexpensive and does not require specialized equipment was developed. This method could be applied to a large number of residents at risk for arsenic poisoning. PMID:17889916

  9. Applicability of genetic polymorphism analysis for the diagnosis of Angelman syndrome and the correlation between language difficulties and disease phenotype.

    PubMed

    Wang, K; Li, Y T; Hou, M

    2016-01-01

    Angelman syndrome (AS) is a neurogenetic disorder caused by a defect in the expression of the maternally inherited ubiquitin protein ligase E3A (UBE3A) gene in chromosome 15. The most common genetic defects include maternal deletions in chromosome 15q11-13; however, paternal uniparental disomy and imprinting defects allow for the identification of mutations in UBE3A in 10% of patients with AS. The aim of this study was to validate the clinical features and genetic polymorphisms of AS, and to discuss the relationship between functional language lateralization and the arcuate fasciculus in the Broca's and Wernicke's areas. Six children with AS (mean age = 32.57 months) presenting characteristic behavioral patterns of AS (frequent laughter and happy demeanor, hand flapping, and hypermotor behavior) were recruited to this study. The patients underwent a clinical evaluation (clinical history, dysmorphological and neurological examinations, and psychological evaluations) and paraclinical investigations [genetic tests (fluorescence in situ hybridization and methylation polymerase chain reaction), electroencephalogram, and magnetic resonance imaging]. We conclude that AS diagnosis cannot rely solely on genetic testing for polymorphisms in UBE3A and must consider its clinical characteristics. Moreover, functional language lateralization and the arcuate fasciculus in the Broca's and Wernicke's areas were found to be closely correlated. Therefore, UBE3A gene mutation analysis combined with comprehensive clinical evaluations may be suitable for the diagnosis of AS. PMID:27323188

  10. Modeling RNA polymerase interaction in mitochondria of chordates

    PubMed Central

    2012-01-01

    Background In previous work, we introduced a concept, a mathematical model and its computer realization that describe the interaction between bacterial and phage type RNA polymerases, protein factors, DNA and RNA secondary structures during transcription, including transcription initiation and termination. The model accurately reproduces changes of gene transcription level observed in polymerase sigma-subunit knockout and heat shock experiments in plant plastids. The corresponding computer program and a user guide are available at http://lab6.iitp.ru/en/rivals. Here we apply the model to the analysis of transcription and (partially) translation processes in the mitochondria of frog, rat and human. Notably, mitochondria possess only phage-type polymerases. We consider the entire mitochondrial genome so that our model allows RNA polymerases to complete more than one circle on the DNA strand. Results Our model of RNA polymerase interaction during transcription initiation and elongation accurately reproduces experimental data obtained for plastids. Moreover, it also reproduces evidence on bulk RNA concentrations and RNA half-lives in the mitochondria of frog, human with or without the MELAS mutation, and rat with normal (euthyroid) or hyposecretion of thyroid hormone (hypothyroid). The transcription characteristics predicted by the model include: (i) the fraction of polymerases terminating at a protein-dependent terminator in both directions (the terminator polarization), (ii) the binding intensities of the regulatory protein factor (mTERF) with the termination site and, (iii) the transcription initiation intensities (initiation frequencies) of all promoters in all five conditions (frog, healthy human, human with MELAS syndrome, healthy rat, and hypothyroid rat with aberrant mtDNA methylation). Using the model, absolute levels of all gene transcription can be inferred from an arbitrary array of the three transcription characteristics, whereas, for selected genes only

  11. The methylation status of plant genomic DNA influences PCR efficiency.

    PubMed

    Kiselev, K V; Dubrovina, A S; Tyunin, A P

    2015-03-01

    During the polymerase chain reaction (PCR), which is a versatile and widely used method, certain DNA sequences are rapidly amplified through thermocycling. Although there are numerous protocols of PCR optimization for different applications, little is known about the effect of DNA modifications, such as DNA methylation, on PCR efficiency. Recent studies show that cytosine methylation alters DNA mechanical properties and suggest that DNA methylation may directly or indirectly influence the effectiveness of DNA amplification during PCR. In the present study, using plant DNA, we found that highly methylated plant DNA genomic regions were amplified with lower efficiencies compared to that for the regions methylated at a lower level. The correlation was observed when amplifying stilbene synthase (STS1, STS10) genes of Vitis amurensis, the Actin2 gene of Arabidopsis thaliana, the internal transcribed spacer (AtITS), and tRNAPro of A. thaliana. The level of DNA methylation within the analyzed DNA regions has been analyzed with bisulfite sequencing. The obtained data show that efficient PCRs of highly methylated plant DNA regions can be hampered. Proteinase K treatment of the plant DNA prior to PCR and using HotTaq DNA polymerase improved amplification of the highly methylated plant DNA regions. We suggest that increased DNA denaturation temperatures of the highly methylated DNA and contamination with DNA-binding proteins contribute to the hampered PCR amplification of highly methylated DNA. The data show that it is necessary to use current DNA purification protocols and commercial kits with caution to ensure appropriate PCR product yield and prevent bias toward unmethylated DNA amplification in PCRs.

  12. Regulation of Human RNA Polymerase III Transcription by DNMT1 and DNMT3a DNA Methyltransferases*

    PubMed Central

    Selvakumar, Tharakeswari; Gjidoda, Alison; Hovde, Stacy L.; Henry, R. William

    2012-01-01

    The human small nuclear RNA (snRNA) and small cytoplasmic RNA (scRNA) gene families encode diverse non-coding RNAs that influence cellular growth and division. Many snRNA and scRNA genes are related via their compact and yet powerful promoters that support RNA polymerase III transcription. We have utilized the human U6 snRNA gene family to examine the mechanism for regulated transcription of these potent transcription units. Analysis of nine U6 family members showed enriched CpG density within the promoters of actively transcribed loci relative to inert genes, implying a relationship between gene potency and DNA methylation. Indeed, both pharmacological inhibition of DNA methyltransferase (DNMT) activity and the forced diminution of DNMT-1, DNMT-3a, and DNMT-3b by siRNA targeting resulted in increased U6 levels in asynchronously growing MCF7 adenocarcinoma cells. In vitro transcription assays further showed that template methylation impedes U6 transcription by RNA polymerase III. Both DNMT-1 and DNMT-3a were detected at the U6-1 locus by chromatin immunoprecipitation directly linking these factors to RNA polymerase III regulation. Despite this association, the endogenous U6-1 locus was not substantially methylated in actively growing cells. However, both DNMT occupancy and low frequency methylation were correlated with increased Retinoblastoma tumor suppressor (RB) expression, suggesting that the RB status can influence specific epigenetic marks. PMID:22219193

  13. Association of Toll-like receptor 2 polymorphisms with gout

    PubMed Central

    CAI, YAN; PENG, YI-HUA; TANG, ZHONG; GUO, XIAO-LAN; QING, YU-FENG; LIANG, SU-HUA; JIANG, HONG; DANG, WANG-TAI; MA, QIANG; HE, CHENG; ZHOU, JING-GUO

    2014-01-01

    Gout is the most common autoinflammatory arthritis characterized by elevated serum urate and recurrent attacks of intra-articular crystal deposition of monosodium urate (MSU) in tissues. The pathogenesis of gout has not been fully determined, although certain genetic factors are involved in the development of gout. Accumulated data suggested that MSU crystal-induced inflammation is a paradigm of innate immunity. As Toll-like receptors (TLRs) are the underlying mechanisms of the innate immune response, the present study aimed to investigate whether TLR2 polymorphisms are associated with gout. Two single-nucleotide polymorphisms (Arg677Trp and Arg753Gln, rs5743708) in TLR2 were genotyped by polymerase chain reaction-restriction fragment length polymorphism and the −196 to −174 del polymorphism was investigated using the allele-specific polymerase chain reaction in 431 individuals (215 patients with gout and 216 healthy controls). TLR2 Arg677Trp and Arg753Gln genotyping indicated that all the positive samples were of the wild-type genotype. No significant differences in genotype (χ2=1.686, P=0.430) and allele (χ2=1.430, P=0.232) frequencies of the −196 to −174 del polymorphism between the patients with gout and the control groups was observed. Our results suggested that the TLR2 Arg677Trp, Arg753Gln and the −196 to −174 del polymorphisms were not associated with susceptibility to primary gouty arthritis. PMID:24649113

  14. The Influence of DNA Methylation on Bone Cells

    PubMed Central

    Reppe, Sjur; Datta, Harish; Gautvik, Kaare M.

    2015-01-01

    DNA methylation in eukaryotes invokes heritable alterations of the of the cytosine base in DNA without changing the underlying genomic DNA sequence. DNA methylation may be modified by environmental exposures as well as gene polymorphisms and may be a mechanistic link between environmental risk factors and the development of disease. In this review, we consider the role of DNA methylation in bone cells (osteoclasts/osteoblasts/osteocytes) and their progenitors with special focus on in vitro and ex vivo analyses. The number of studies on DNA methylation in bone cells is still somewhat limited, nevertheless it is getting increasingly clear that this type of the epigenetic changes is a critical regulator of gene expression. DNA methylation is necessary for proper development and function of bone cells and is accompanied by disease characteristic functional alterations as presently reviewed including postmenopausal osteoporosis and mechanical strain. PMID:27019613

  15. Allele-Specific DNA Methylation Detection by Pyrosequencing®.

    PubMed

    Kristensen, Lasse Sommer; Johansen, Jens Vilstrup; Grønbæk, Kirsten

    2015-01-01

    DNA methylation is an epigenetic modification that plays important roles in healthy as well as diseased cells, by influencing the transcription of genes. In spite the fact that human somatic cells are diploid, most of the currently available methods for the study of DNA methylation do not provide information on the methylation status of individual alleles of genes. This information may be of importance in many situations. In particular, in cancer both alleles of tumour suppressor genes generally need to be inactivated for a phenotypic effect to be observed. Here, we present a simple and cost-effective protocol for allele-specific DNA methylation detection based on Pyrosequencing(®) of methylation-specific PCR (MSP) products including a single nucleotide polymorphism (SNP) within the amplicon. PMID:26103906

  16. The evolutionary conservation of DNA polymerase. alpha

    SciTech Connect

    Miller, M.A.; Korn, D.; Wang, T.S.F. )

    1988-08-25

    The evolutionary conservation of DNA polymerase {alpha} was assessed by immunological and molecular genetic approaches. Four anti-human KB cell DNA polymerase {alpha} monoclonal antibodies were tested for their ability to recognize a phylogenetically broad array of eukaryotic DNA polymerases. While the single non-neutralizing antibody used in this study recognizes higher mammalian (human, simian, canine, and bovine) polymerases only, three neutralizing antibodies exhibit greater, but variable, extents of cross-reactivity among vertebrate species. Genomic Southern hybridization studies with the cDNA of the human DNA polymerase {alpha} catalytic polypeptide identify the existence of many consensus DNA sequences within the DNA polymerase genes of vertebrate, invertebrate, plant and unicellular organisms. These findings illustrate the differential evolutionary conservation of four unique epitopes on DNA sequences, presumably reflective of critical functional domains, in the DNA polymerase genes from a broad diversity of living forms.

  17. Unmasking risk loci: DNA methylation illuminates the biology of cancer predisposition: analyzing DNA methylation of transcriptional enhancers reveals missed regulatory links between cancer risk loci and genes.

    PubMed

    Aran, Dvir; Hellman, Asaf

    2014-02-01

    Paradoxically, DNA sequence polymorphisms in cancer risk loci rarely correlate with the expression of cancer genes. Therefore, the molecular mechanism underlying an individual's susceptibility to cancer has remained largely unknown. However, recent evaluations of the correlations between DNA methylation and gene expression levels across healthy and cancerous genomes have revealed enrichment of disease-related DNA methylation variations within disease-associated risk loci. Moreover, it appears that transcriptional enhancers embedded in cancer risk loci often contain DNA methylation sites that closely define the expression of prominent cancer genes, despite the lack of significant correlations between gene expression levels and the surrounding disease-associated polymorphic sequences. We suggest that DNA methylation variations may obscure the effect of co-residing risk sequence alleles. Analysis of enhancer methylation data may help to reveal the regulatory circuits underlying predisposition to cancers and other common diseases.

  18. Population-Level Immune-Mediated Adaptation in HIV-1 Polymerase during the North American Epidemic

    PubMed Central

    Kinloch, Natalie N.; MacMillan, Daniel R.; Le, Anh Q.; Cotton, Laura A.; Bangsberg, David R.; Buchbinder, Susan; Carrington, Mary; Fuchs, Jonathan; Harrigan, P. Richard; Koblin, Beryl; Kushel, Margot; Markowitz, Martin; Mayer, Kenneth; Milloy, M. J.; Schechter, Martin T.; Wagner, Theresa; Walker, Bruce D.; Carlson, Jonathan M.; Poon, Art F. Y.

    2015-01-01

    ABSTRACT Human leukocyte antigen (HLA) class I-associated polymorphisms in HIV-1 that persist upon transmission to HLA-mismatched hosts may spread in the population as the epidemic progresses. Transmission of HIV-1 sequences containing such adaptations may undermine cellular immune responses to the incoming virus in future hosts. Building upon previous work, we investigated the extent of HLA-associated polymorphism accumulation in HIV-1 polymerase (Pol) through comparative analysis of linked HIV-1/HLA class I genotypes sampled during historic (1979 to 1989; n = 338) and modern (2001 to 2011; n = 278) eras from across North America (Vancouver, BC, Canada; Boston, MA; New York, NY; and San Francisco, CA). Phylogenies inferred from historic and modern HIV-1 Pol sequences were star-like in shape, with an inferred most recent common ancestor (epidemic founder virus) sequence nearly identical to the modern North American subtype B consensus sequence. Nevertheless, modern HIV-1 Pol sequences exhibited roughly 2-fold-higher patristic (tip-to-tip) genetic distances than historic sequences, with HLA pressures likely driving ongoing diversification. Moreover, the frequencies of published HLA-associated polymorphisms in individuals lacking the selecting HLA class I allele was on average ∼2.5-fold higher in the modern than in the historic era, supporting their spread in circulation, though some remained stable in frequency during this time. Notably, polymorphisms restricted by protective HLA alleles appear to be spreading to a greater relative extent than others, though these increases are generally of modest absolute magnitude. However, despite evidence of polymorphism spread, North American hosts generally remain at relatively low risk of acquiring an HIV-1 polymerase sequence substantially preadapted to their HLA profiles, even in the present era. IMPORTANCE HLA class I-restricted cytotoxic T-lymphocyte (CTL) escape mutations in HIV-1 that persist upon transmission may

  19. DNA Methylation and Potential for Epigenetic Regulation in Pygospio elegans

    PubMed Central

    Kesäniemi, Jenni E.; Heikkinen, Liisa; Knott, K. Emily

    2016-01-01

    Transitions in developmental mode are common evolutionarily, but how and why they occur is not understood. Developmental mode describes larval phenotypes, including morphology, ecology and behavior of larvae, which typically are generalized across different species. The polychaete worm Pygospio elegans is one of few species polymorphic in developmental mode, with multiple larval phenotypes, providing a possibility to examine the potential mechanisms allowing transitions in developmental mode. We investigated the presence of DNA methylation in P. elegans, and, since maternal provisioning is a key factor determining eventual larval phenotype, we compared patterns of DNA methylation in females during oogenesis in this species. We demonstrate that intragenic CpG site DNA methylation and many relevant genes necessary for DNA methylation occur in P. elegans. Methylation-sensitive AFLP analysis showed that gravid females with offspring differing in larval developmental mode have significantly different methylation profiles and that the females with benthic larvae and non-reproductive females from the same location also differ in their epigenetic profiles. Analysis of CpG sites in transcriptome data supported our findings of DNA methylation in this species and showed that CpG observed/expected ratios differ among females gravid with embryos destined to different developmental modes. The differences in CpG site DNA methylation patterns seen among the samples suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species. PMID:27008314

  20. DNA Methylation and Potential for Epigenetic Regulation in Pygospio elegans.

    PubMed

    Kesäniemi, Jenni E; Heikkinen, Liisa; Knott, K Emily

    2016-01-01

    Transitions in developmental mode are common evolutionarily, but how and why they occur is not understood. Developmental mode describes larval phenotypes, including morphology, ecology and behavior of larvae, which typically are generalized across different species. The polychaete worm Pygospio elegans is one of few species polymorphic in developmental mode, with multiple larval phenotypes, providing a possibility to examine the potential mechanisms allowing transitions in developmental mode. We investigated the presence of DNA methylation in P. elegans, and, since maternal provisioning is a key factor determining eventual larval phenotype, we compared patterns of DNA methylation in females during oogenesis in this species. We demonstrate that intragenic CpG site DNA methylation and many relevant genes necessary for DNA methylation occur in P. elegans. Methylation-sensitive AFLP analysis showed that gravid females with offspring differing in larval developmental mode have significantly different methylation profiles and that the females with benthic larvae and non-reproductive females from the same location also differ in their epigenetic profiles. Analysis of CpG sites in transcriptome data supported our findings of DNA methylation in this species and showed that CpG observed/expected ratios differ among females gravid with embryos destined to different developmental modes. The differences in CpG site DNA methylation patterns seen among the samples suggest a potential for epigenetic regulation of gene expression (through DNA methylation) in this species. PMID:27008314

  1. A new family of polymerases related to superfamily A DNA polymerases and T7-like DNA-dependent RNA polymerases.

    PubMed

    Iyer, Lakshminarayan M; Abhiman, Saraswathi; Aravind, L

    2008-01-01

    Using sequence profile methods and structural comparisons we characterize a previously unknown family of nucleic acid polymerases in a group of mobile elements from genomes of diverse bacteria, an algal plastid and certain DNA viruses, including the recently reported Sputnik virus. Using contextual information from domain architectures and gene-neighborhoods we present evidence that they are likely to possess both primase and DNA polymerase activity, comparable to the previously reported prim-pol proteins. These newly identified polymerases help in defining the minimal functional core of superfamily A DNA polymerases and related RNA polymerases. Thus, they provide a framework to understand the emergence of both DNA and RNA polymerization activity in this class of enzymes. They also provide evidence that enigmatic DNA viruses, such as Sputnik, might have emerged from mobile elements coding these polymerases. PMID:18834537

  2. MTHFR A1298C and C677T gene polymorphisms and susceptibility to chronic myeloid leukemia in Egypt.

    PubMed

    Aly, Rabab M; Taalab, Mona M; Ghazy, Hayam F

    2014-01-01

    Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the intracellular folate metabolism which plays an important role in carcinogenesis through DNA methylation. We aimed to evaluate the association between MTHFR A1298C and C677T polymorphisms and the risks of chronic myeloid leukemia (CML). Eighty-five patients with CML and a control group containing 100 healthy, age and sex matched individuals were examined for MTHFR C677T and A1298C polymorphisms using polymerase chain reaction-restriction fragment-length (PCR-RFLP) method. The frequency of 677TT genotype in patients with CML was significantly higher compared to controls (OR=2.513, 95% CI: 0.722-4.086, P=0.025). No such association was shown for heterozygous 677CT (OR=1.010, 95% CI: 0.460-2.218, P=0.981). Moreover, for A1298C genotype, a statistically significant higher frequency of 1298CC was also detected in CML patients compared to control group (OR=1.1816, 95% CI: 0.952-3.573, P=0.036), 0.036). No such statistical significance was demonstrable for heterozygote 1298AC (OR=1.046, 95% CI: 0.740-1.759, P=0.092). In addition, patients with joint 677CT/1298AC or 677TT/1298CC genotypes showed an association with increased risk of CML (OR=1.849, 95% CI: 0.935-2.540, P=0.024; OR=1.915, 95% CI: 1.202-3.845, P=0.020 respectively). .A statistically significant increased risk of resistant to therapy was observed with 677CT and 1298AC genotypes (P=0.001, P=0.002 respectively). We conclude that both MTHFR 677TT and 1298CC polymorphisms have been associated with risk of CML and both 677CT and 1298AC genotypes are associated with higher risk of resistant to therapy. PMID:24966971

  3. MTHFR A1298C and C677T gene polymorphisms and susceptibility to chronic myeloid leukemia in Egypt.

    PubMed

    Aly, Rabab M; Taalab, Mona M; Ghazy, Hayam F

    2014-01-01

    Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme regulating the intracellular folate metabolism which plays an important role in carcinogenesis through DNA methylation. We aimed to evaluate the association between MTHFR A1298C and C677T polymorphisms and the risks of chronic myeloid leukemia (CML). Eighty-five patients with CML and a control group containing 100 healthy, age and sex matched individuals were examined for MTHFR C677T and A1298C polymorphisms using polymerase chain reaction-restriction fragment-length (PCR-RFLP) method. The frequency of 677TT genotype in patients with CML was significantly higher compared to controls (OR=2.513, 95% CI: 0.722-4.086, P=0.025). No such association was shown for heterozygous 677CT (OR=1.010, 95% CI: 0.460-2.218, P=0.981). Moreover, for A1298C genotype, a statistically significant higher frequency of 1298CC was also detected in CML patients compared to control group (OR=1.1816, 95% CI: 0.952-3.573, P=0.036), 0.036). No such statistical significance was demonstrable for heterozygote 1298AC (OR=1.046, 95% CI: 0.740-1.759, P=0.092). In addition, patients with joint 677CT/1298AC or 677TT/1298CC genotypes showed an association with increased risk of CML (OR=1.849, 95% CI: 0.935-2.540, P=0.024; OR=1.915, 95% CI: 1.202-3.845, P=0.020 respectively). .A statistically significant increased risk of resistant to therapy was observed with 677CT and 1298AC genotypes (P=0.001, P=0.002 respectively). We conclude that both MTHFR 677TT and 1298CC polymorphisms have been associated with risk of CML and both 677CT and 1298AC genotypes are associated with higher risk of resistant to therapy.

  4. MLH1 methylation screening is effective in identifying epimutation carriers

    PubMed Central

    Pineda, Marta; Mur, Pilar; Iniesta, María Dolores; Borràs, Ester; Campos, Olga; Vargas, Gardenia; Iglesias, Sílvia; Fernández, Anna; Gruber, Stephen B; Lázaro, Conxi; Brunet, Joan; Navarro, Matilde; Blanco, Ignacio; Capellá, Gabriel

    2012-01-01

    Recently, constitutional MLH1 epimutations have been identified in a subset of Lynch syndrome (LS) cases. The aim of this study was the identification of patients harboring constitutional MLH1 epimutations in a set of 34 patients with a clinical suspicion of LS, MLH1-methylated tumors and non-detected germline mutations in mismatch repair (MMR) genes. MLH1 promoter methylation was analyzed in lymphocyte DNA samples by MS-MLPA (Methylation-specific multiplex ligation-dependent probe amplification). Confirmation of MLH1 constitutional methylation was performed by MS-MCA (Methylation-specific melting curve analysis), bisulfite sequencing and pyrosequencing in different biological samples. Allelic expression was determined using heterozygous polymorphisms. Vertical transmission was evaluated by MS-MLPA and haplotype analyses. MS-MLPA analysis detected constitutional MLH1 methylation in 2 of the 34 individuals whose colorectal cancers showed MLH1 methylation (5.9%). These results were confirmed by bisulfite-based methods. Both epimutation carriers had developed metachronous early-onset LS tumors, with no family history of LS-associated cancers in their first-degree relatives. In one of the cases, the identified MLH1 constitutional methylation was monoallelic and results in MLH1 and EPM2AIP1 allele-specific transcriptional silencing. It was present in normal somatic tissues and absent in spermatozoa. The methylated MLH1 allele was maternally transmitted and methylation was reversed in a daughter who inherited the same allele. MLH1 methylation screening in lymphocyte DNA from patients with early-onset MLH1-methylated LS-associated tumors allows the identification of epimutation carriers. The present study adds further evidence to the emerging entity of soma-wide MLH1 epimutation and its heritability. PMID:22763379

  5. Archaeal DNA polymerases in biotechnology.

    PubMed

    Zhang, Likui; Kang, Manyu; Xu, Jiajun; Huang, Yanchao

    2015-08-01

    DNA polymerase (pol) is a ubiquitous enzyme that synthesizes DNA strands in all living cells. In vitro, DNA pol is used for DNA manipulation, including cloning, PCR, site-directed mutagenesis, sequencing, and several other applications. Family B archaeal DNA pols have been widely used for molecular biological methods. Biochemical and structural studies reveal that each archaeal DNA pol has different characteristics with respect to fidelity, processivity and thermostability. Due to their high fidelity and strong thermostability, family B archaeal DNA pols have the extensive application on high-fidelity PCR, DNA sequencing, and site-directed mutagenesis while family Y archaeal DNA pols have the potential for error-prone PCR and random mutagenesis because of their low fidelity and strong thermostability. This information combined with mutational analysis has been used to construct novel DNA pols with altered properties that enhance their use as biotechnological reagents. In this review, we focus on the development and use of family B archaeal DNA pols.

  6. A new polymorph of physcion.

    PubMed

    Hopf, Henning; Jones, Peter G; Goclik, Eva; Aust, Pauline; Rödiger, Johanna

    2012-08-01

    The structure of the title compound, 7-methoxy-2-methyl-4,5-dihydroxyanthracene-9,10-dione, C(16)H(12)O(5), was originally reported by Ulický et al. [Acta Cryst. (1991). C47, 1879-1881] in the space group P2(1)2(1)2(1) [polymorph (Io)]. The new polymorph, (Im), crystallizes in the space group P2(1)/c. The molecular structures are closely similar, with both -OH groups forming intramolecular hydrogen bonds to one of the neighbouring quinone O atoms, thus slightly lengthening this C=O bond; the pattern of C-C bond lengths in the ring system is consistent with some contribution from a resonance form with a negative charge at the hydrogen-bonded quinone O atom and an aromatic region around its neighbouring C atoms. The packing of (Im) is simpler than the extensively crosslinked pattern of (Io), with molecular tapes connected by classical (but three-centre) and `weak' hydrogen bonds, parallel to [201].

  7. DNA Methylation Variation Trends during the Embryonic Development of Chicken

    PubMed Central

    Li, Shizhao; Zhu, Yufei; Zhi, Lihui; Han, Xiaoying; Shen, Jing; Liu, Yanli; Yao, Junhu; Yang, Xiaojun

    2016-01-01

    The embryogenesis period is critical for epigenetic reprogramming and is thus of great significance in the research field of poultry epigenetics for elucidation of the trends in DNA methylation variations during the embryonic development of birds, particularly due to differences in embryogenesis between birds and mammals. Here, we first examined the variations in genomic DNA methylation during chicken embryogenesis through high-performance liquid chromatography using broilers as the model organism. We then identified the degree of DNA methylation of the promoters and gene bodies involved in two specific genes (IGF2 and TNF-α) using the bisulfite sequencing polymerase chain reaction method. In addition, we measured the expression levels of IGF2, TNF-α and DNA methyltransferase (DNMT) 1, 3a and 3b. Our results showed that the genomic DNA methylation levels in the liver, heart and muscle increased during embryonic development and that the methylation level of the liver was significantly higher in mid-anaphase. In both the muscle and liver, the promoter methylation levels of TNF-α first increased and then decreased, whereas the gene body methylation levels remained lower at embryonic ages E8, 11 and 14 before increasing notably at E17. The promoter methylation level of IGF2 decreased persistently, whereas the methylation levels in the gene body showed a continuous increase. No differences in the expression of TNF-α were found among E8, 11 and 14, whereas a significant increase was observed at E17. IGF2 showed increasing expression level during the examined embryonic stages. In addition, the mRNA and protein levels of DNMTs increased with increasing embryonic ages. These results suggest that chicken shows increasing genomic DNA methylation patterns during the embryonic period. Furthermore, the genomic DNA methylation levels in tissues are closely related to the genes expression levels, and gene expression may be simultaneously regulated by promoter hypomethylation

  8. Rapid electrochemiluminescence assays of polymerase chain reaction products.

    PubMed

    Kenten, J H; Casadei, J; Link, J; Lupold, S; Willey, J; Powell, M; Rees, A; Massey, R

    1991-09-01

    We demonstrate the first use of an electrochemiluminescent (ECL) label, [4-(N-succimidyloxycarbonylpropyl)-4'-methyl-2,2'- bipyridine]ruthenium(II) dihexafluorophosphate (Origen label; IGEN Inc.), in DNA probe assays. This label allows rapid (less than 25 min) quantification and detection of polymerase chain reaction (PCR)-amplified products from oncogenes, viruses, and cloned genes. For the PCR, we used labeled oligonucleotide primers complementary to human papiloma virus and the Ha-ras oncogene. These samples were followed by ECL analysis or hybridization with specific, Origen-labeled oligonucleotide probes. These studies demonstrate the speed, specificity, and effectiveness of the new ECL labels, compared with 32P, for nucleic acid probe applications. We describe formats involving conventional methodologies and a new format that requires no wash step, allowing simple and rapid sample analysis. These rapid assays also reduce PCR contamination, by requiring less sample handling. Improvements in ECL detectability are currently under investigation for use in DNA probe assays without amplification.

  9. Childhood Maltreatment and Methylation of FKBP5

    PubMed Central

    Tyrka, Audrey R.; Ridout, Kathryn K.; Parade, Stephanie H.; Paquette, Alison; Marsit, Carmen J.; Seifer, Ronald

    2016-01-01

    A growing body of evidence suggests that alterations of the stress response system may be a mechanism by which childhood maltreatment alters risk for psychopathology. FK506 binding protein 51 (FKBP5) binds to the glucocorticoid receptor and alters its ability to respond to stress signaling. The aim of the present study was to examine methylation of the FKBP5 gene (FKBP5), and the role of an FKBP5 genetic variant, in relation to childhood maltreatment in a sample of impoverished preschool-aged children. One hundred seventy-four families, including n=69 with child welfare documentation of moderate-severe maltreatment in the past six months, participated in this study. Children ranged in age from 3 to 5 years, and were racially and ethnically diverse. Structured record review and interviews in the home were used to assess a history of maltreatment, other traumas, and contextual life stressors, and a composite variable assessed the number exposures to these adversities. Methylation of two CpG sites in intron 7 of FKBP5 was measured via sodium bisulfite pyrosequencing. Maltreated children had significantly lower levels of methylation at both CpG sites (p<.05). Lifetime contextual stress exposure showed a trend for lower levels of methylation at one of the sites, and a trend for an interaction with the FKBP5 polymorphism. A composite adversity variable was associated with lower levels of methylation at one of the sites as well (p<.05). FKBP5 alters glucocorticoid receptor responsiveness and FKBP5 gene methylation may be a mechanism of the bio-behavioral effects of adverse exposures in young children. PMID:26535949

  10. DNA methylation is crucial for the early development in the Oyster C. gigas.

    PubMed

    Riviere, Guillaume; Wu, Guan-Chung; Fellous, Alexandre; Goux, Didier; Sourdaine, Pascal; Favrel, Pascal

    2013-12-01

    In vertebrates, epigenetic modifications influence gene transcription, and an appropriate DNA methylation is critical in development. Indeed, a precise temporal and spatial pattern of early gene expression is mandatory for a normal embryogenesis. However, such a regulation and its underlying mechanisms remain poorly understood in more distant organisms such as Lophotrochozoa. Thus, despite DNA in the oyster genome being methylated, the role of DNA methylation in development is unknown. To clarify this point, oyster genomic DNA was examined during early embryogenesis and found differentially methylated. Reverse transcriptase quantitative polymerase chain reaction indicated stage-specific levels of transcripts encoding DNA-methyltransferase (DNMT) and methyl-binding domain proteins. In addition, as highlighted by electronic microscopy and immunohistochemistry, the DNMT inhibitor 5-aza-cytidine induced alterations in the quantity and the localisation of methylated DNA and severe dose-dependent development alterations and was lethal after zygotic genome reinitiation. Furthermore, methyl-DNA-immunoprecipitation-quantitative polymerase chain reaction revealed that the transcription level of most of the homeobox gene orthologues examined, but not of the other early genes investigated, was inversely correlated with their specific DNA methylation. Altogether, our results demonstrate that DNA methylation influences gene expression in Crassostrea gigas and is critical for oyster development, possibly by specifically controlling the transcription level of homeobox orthologues. These findings provide evidence for the importance of epigenetic regulation of development in Lophotrochozoans and bring new insights into the early life of C. gigas, one of the most important aquaculture resources worldwide.

  11. Polymerase Gamma Disease through the Ages

    ERIC Educational Resources Information Center

    Saneto, Russell P.; Naviaux, Robert K.

    2010-01-01

    The most common group of mitochondrial disease is due to mutations within the mitochondrial DNA polymerase, polymerase gamma 1 ("POLG"). This gene product is responsible for replication and repair of the small mitochondrial DNA genome. The structure-function relationship of this gene product produces a wide variety of diseases that at times, seems…

  12. Replication dynamics in fission and budding yeasts through DNA polymerase tracking

    PubMed Central

    Vázquez, Enrique

    2015-01-01

    The dynamics of eukaryotic DNA polymerases has been difficult to establish because of the difficulty of tracking them along the chromosomes during DNA replication. Recent work has addressed this problem in the yeasts Schizosaccharomyces pombe and Saccharomyces cerevisiae through the engineering of replicative polymerases to render them prone to incorporating ribonucleotides at high rates. Their use as tracers of the passage of each polymerase has provided a picture of unprecedented resolution of the organization of replicons and replication origins in the two yeasts and has uncovered important differences between them. Additional studies have found an overlapping distribution of DNA polymorphisms and the junctions of Okazaki fragments along mononucleosomal DNA. This sequence instability is caused by the premature release of polymerase δ and the retention of non proof‐read DNA tracts replicated by polymerase α. The possible implementation of these new experimental approaches in multicellular organisms opens the door to the analysis of replication dynamics under a broad range of genetic backgrounds and physiological or pathological conditions. PMID:26293347

  13. DNA methylation: A source of random variation in natural populations.

    PubMed

    Massicotte, Rachel; Whitelaw, Emma; Angers, Bernard

    2011-04-01

    Epigenetic processes (e.g., DNA methylation) have been proposed as potentially important evolutionary mechanisms. However, before drawing conclusions about their evolutionary relevance, we need to evaluate the independence of epigenetic variation from genetic variation, as well as the extent of methylation polymorphism in nature. We evaluated these in natural populations of a clonal fish, Chrosomus eos-neogaeus, for which genetically identical individuals may be found in distinct environments. A genomic survey confirms the genetic uniformity of individuals, whereas a substantial level of inter-individual variation results in DNA methylation. Survey of the methylation status of the CpG dinucleotides of a fragment of a retrotransposon confirmed a marked difference in epiallelic composition among tissues, as well as among individuals. This study provides further evidence of epigenetic variation in the absence of genetic variation and demonstrates that this process can be a source of random variation in natural populations. PMID:21266851

  14. DNA methylation perspectives in the pathogenesis of autoimmune diseases.

    PubMed

    Sun, Bao; Hu, Lei; Luo, Zhi-Ying; Chen, Xiao-Ping; Zhou, Hong-Hao; Zhang, Wei

    2016-03-01

    DNA methylation is now widely recognized as being critical to maintain the function of immune cells. Recent studies suggest that aberrant DNA methylation levels not only can result in immune cells autoreactivity in vitro, but also are related to autoimmunity in vivo. Environmental factors and genetic polymorphisms cause abnormal methylation, which affects the expression of certain immune-related genes, being becoming hot spot of explaining the mechanism of autoimmune diseases. This paper reviews the importance of abnormal methylation during the development of common autoimmune diseases, such as systemic lupus erythematosus, rheumatoid arthritis, multiple sclerosis and type 1 diabetes, aiming at a better understanding of the pathogenesis of autoimmune diseases and providing new ideas for the treatment of these diseases. PMID:26821302

  15. A global profile of replicative polymerase usage

    PubMed Central

    Müller, Carolin A.; Miyabe, Izumi; Brooks, Tony; Retkute, Renata; Hubank, Mike; Nieduszyski, Conrad A.; Carr, Antony M.

    2014-01-01

    Three eukaryotic DNA polymerases are essential for genome replication. Polα-primase initiates each synthesis event and is rapidly replaced by processive DNA polymerases: Polε replicates the leading strand while Polδ performs lagging strand synthesis. However, it is not known whether this division of labour is maintained across the whole genome or how uniform it is within single replicons. Using S. pombe, we have developed a polymerase usage sequencing (Pu-seq) strategy to map polymerase usage genome–wide. Pu–seq provides direct replication origin location and efficiency data and indirect estimates of replication timing. We confirm that the division of labour is broadly maintained across an entire genome. However, our data suggest a subtle variability in the usage of the two polymerases within individual replicons. We propose this results from occasional leading strand initiation by Polδ followed by exchange for Polε. PMID:25664722

  16. Thermally multiplexed polymerase chain reaction.

    PubMed

    Phaneuf, Christopher R; Pak, Nikita; Saunders, D Curtis; Holst, Gregory L; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L; Jerris, Robert; Forest, Craig R

    2015-07-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously-each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  17. Thermally multiplexed polymerase chain reaction

    PubMed Central

    Phaneuf, Christopher R.; Pak, Nikita; Saunders, D. Curtis; Holst, Gregory L.; Birjiniuk, Joav; Nagpal, Nikita; Culpepper, Stephen; Popler, Emily; Shane, Andi L.; Jerris, Robert; Forest, Craig R.

    2015-01-01

    Amplification of multiple unique genetic targets using the polymerase chain reaction (PCR) is commonly required in molecular biology laboratories. Such reactions are typically performed either serially or by multiplex PCR. Serial reactions are time consuming, and multiplex PCR, while powerful and widely used, can be prone to amplification bias, PCR drift, and primer-primer interactions. We present a new thermocycling method, termed thermal multiplexing, in which a single heat source is uniformly distributed and selectively modulated for independent temperature control of an array of PCR reactions. Thermal multiplexing allows amplification of multiple targets simultaneously—each reaction segregated and performed at optimal conditions. We demonstrate the method using a microfluidic system consisting of an infrared laser thermocycler, a polymer microchip featuring 1 μl, oil-encapsulated reactions, and closed-loop pulse-width modulation control. Heat transfer modeling is used to characterize thermal performance limitations of the system. We validate the model and perform two reactions simultaneously with widely varying annealing temperatures (48 °C and 68 °C), demonstrating excellent amplification. In addition, to demonstrate microfluidic infrared PCR using clinical specimens, we successfully amplified and detected both influenza A and B from human nasopharyngeal swabs. Thermal multiplexing is scalable and applicable to challenges such as pathogen detection where patients presenting non-specific symptoms need to be efficiently screened across a viral or bacterial panel. PMID:26339317

  18. Use of RNA polymerase molecular beacon assay to measure RNA polymerase interactions with model promoter fragments.

    PubMed

    Mekler, Vladimir; Severinov, Konstantin

    2015-01-01

    RNA polymerase-promoter interactions that keep the transcription initiation complex together are complex and multipartite, and formation of the RNA polymerase-promoter complex proceeds through multiple intermediates. Short promoter fragments can be used as a tool to dissect RNA polymerase-promoter interactions and to pinpoint elements responsible for specific properties of the entire promoter complex. A recently developed fluorometric molecular beacon assay allows one to monitor the enzyme interactions with various DNA probes and quantitatively characterize partial RNA polymerase-promoter interactions. Here, we present detailed protocols for the preparation of an Escherichia coli molecular beacon and its application to study RNA polymerase interactions with model promoter fragments.

  19. Correlation of MGMT promoter methylation status with gene and protein expression levels in glioblastoma

    PubMed Central

    Uno, Miyuki; Oba-Shinjo, Sueli Mieko; Camargo, Anamaria Aranha; Moura, Ricardo Pereira; de Aguiar, Paulo Henrique; Cabrera, Hector Navarro; Begnami, Marcos; Rosemberg, Sérgio; Teixeira, Manoel Jacobsen; Marie, Suely Kazue Nagahashi

    2011-01-01

    OBJECTIVES: 1) To correlate the methylation status of the O6-methylguanine-DNA-methyltransferase (MGMT) promoter to its gene and protein expression levels in glioblastoma and 2) to determine the most reliable method for using MGMT to predict the response to adjuvant therapy in patients with glioblastoma. BACKGROUND: The MGMT gene is epigenetically silenced by promoter hypermethylation in gliomas, and this modification has emerged as a relevant predictor of therapeutic response. METHODS: Fifty-one cases of glioblastoma were analyzed for MGMT promoter methylation by methylation-specific PCR and pyrosequencing, gene expression by real time polymerase chain reaction, and protein expression by immunohistochemistry. RESULTS: MGMT promoter methylation was found in 43.1% of glioblastoma by methylation-specific PCR and 38.8% by pyrosequencing. A low level of MGMT gene expression was correlated with positive MGMT promoter methylation (p = 0.001). However, no correlation was found between promoter methylation and MGMT protein expression (p = 0.297). The mean survival time of glioblastoma patients submitted to adjuvant therapy was significantly higher among patients with MGMT promoter methylation (log rank = 0.025 by methylation-specific PCR and 0.004 by pyrosequencing), and methylation was an independent predictive factor that was associated with improved prognosis by multivariate analysis. DISCUSSION AND CONCLUSION: MGMT promoter methylation status was a more reliable predictor of susceptibility to adjuvant therapy and prognosis of glioblastoma than were MGMT protein or gene expression levels. Methylation-specific polymerase chain reaction and pyrosequencing methods were both sensitive methods for determining MGMT promoter methylation status using DNA extracted from frozen tissue. PMID:22012047

  20. DRD2 C957T polymorphism interacts with the COMT Val158Met polymorphism in human working memory ability.

    PubMed

    Xu, Haiyan; Kellendonk, Christoph B; Simpson, Eleanor H; Keilp, John G; Bruder, Gerard E; Polan, H Jonathan; Kandel, Eric R; Gilliam, T Conrad

    2007-02-01

    The C957T polymorphism in the dopamine D2 receptor (DRD2) gene and the Val158Met polymorphism in the Catechol-O-Methyl-Transferase (COMT) gene affect dopamine transmission and have been found to be associated with schizophrenia. Since DRD2 in mice and the COMT gene in humans modulate working memory, we examined the relationship and possible interaction of both polymorphisms to working memory performance in 188 healthy adults. Subjects having the DRD2 C/C allele showed the poorest performance in a word serial position test. Moreover, the effect of the C957T genotype was strengthened when interaction with the COMT Val158Met polymorphism was included in the analysis. We propose that an interaction of the DRD2 C957T and COMT Val158Met may be involved in the generation of some working memory deficits in schizophrenia.

  1. Variable DNA methylation changes during differentiation of human melanoma cells.

    PubMed

    Steigerwald, S D; Pfeifer, G P

    1988-09-01

    The DNA 5-methylcytosine content has been analyzed in the human melanoma cell line M21 at several time points after induction of differentiation by a variety of inducers. 5-Aza-2'-deoxycytidine reduces DNA methylation to about 50% of the control level and this demethylation occurs prior to the establishment of the differentiated phenotype. The DNA synthesis inhibitors cytosine arabinoside, aphidicolin, and hydroxyurea exert different effects on DNA methylation in these cells. Cytosine arabinoside induces an early DNA hypermethylation, which is however reversible and drops to the original level after 24 h. Hydroxyurea induces DNA hypermethylation after a lag period of more than 48 h and the DNA polymerase alpha inhibitor aphidicolin has no effect on the DNA methylation level. Treatment of cells with phorbol 12-myristate 13-acetate, another potent inducer of melanoma cell differentiation, does not result in a change of total DNA methylation over a period of 96 h. These results indicate that differentiation of human melanoma cells can be accompanied by variable changes of the DNA methylation pattern. These changes can be neither generally related to the differentiation process itself nor related to the effects of DNA synthesis inhibition on DNA methylation, but may more likely reflect a direct or indirect particular effect of the inducer on the DNA methylation process.

  2. H19 gene methylation status is associated with male infertility

    PubMed Central

    LI, XIAO-PING; HAO, CHAO-LIANG; WANG, QIAN; YI, XIAO-MEI; JIANG, ZHI-SHENG

    2016-01-01

    The present study investigated the H19 gene methylation status in male infertility. Between March 2013 and June 2014, semen samples were collected from 15 normal fertile males and 15 males experiencing infertility, and routine analysis and sperm morphological assessment were performed. The semen samples were subjected to density gradient centrifugation to separate the sperm fraction, and genomic DNA from the sperms was extracted and treated for bisulfite modification. Following in vitro amplification by polymerase chain reaction (PCR), the purified PCR products were cloned into pMD®18-T vectors and successful cloning was confirmed by restriction enzyme digestion. Positive clones were sequenced and the DNA methylation status was analyzed. The overall methylation rate in the normal fertile group was 100% (270/270), whereas in the infertile group the methylation rate was lower at 94.1% (525/558), revealing a statistically significant decrease in overall methylation rate in the infertile patients compared with the control group (χ2=15.12; P<0.001). The average methylation rates of CpG 1, 3 and 6 in the infertile group were statistically different from those in the normal control group (all P<0.05). The abnormal methylation of imprinted gene H19 is associated with male infertility, suggesting that H19 may serve as a biomarker for the detection of defects in human spermiogenesis. PMID:27347077

  3. Polymorphous computing fabric

    DOEpatents

    Wolinski, Christophe Czeslaw; Gokhale, Maya B.; McCabe, Kevin Peter

    2011-01-18

    Fabric-based computing systems and methods are disclosed. A fabric-based computing system can include a polymorphous computing fabric that can be customized on a per application basis and a host processor in communication with said polymorphous computing fabric. The polymorphous computing fabric includes a cellular architecture that can be highly parameterized to enable a customized synthesis of fabric instances for a variety of enhanced application performances thereof. A global memory concept can also be included that provides the host processor random access to all variables and instructions associated with the polymorphous computing fabric.

  4. Protein Affinity Chromatography with Purified Yeast DNA Polymerase α Detects Proteins that Bind to DNA Polymerase

    NASA Astrophysics Data System (ADS)

    Miles, Jeff; Formosa, Tim

    1992-02-01

    We have overexpressed the POL1 gene of the yeast Saccharomyces cerevisiae and purified the resulting DNA polymerase α polypeptide in an apparently intact form. We attached the purified DNA polymerase covalently to an agarose matrix and used this matrix to chromatograph extracts prepared from yeast cells. At least six proteins bound to the yeast DNA polymerase α matrix that did not bind to a control matrix. We speculate that these proteins might be DNA polymerase α accessory proteins. Consistent with this interpretation, one of the binding proteins, which we have named POB1 (polymerase one binding), is required for normal chromosome transmission. Mutations in this gene cause increased chromosome loss and an abnormal cell morphology, phenotypes that also occur in the presence of mutations in the yeast α or δ polymerase genes. These results suggest that the interactions detected by polymerase affinity chromatography are biologically relevant and may help to illuminate the architecture of the eukaryotic DNA replication machinery.

  5. Structural Basis for Proficient Incorporation of dTTP Opposite O[superscript 6]-Methylguanine by Human DNA Polymerase [iota

    SciTech Connect

    Pence, Matthew G.; Choi, Jeong-Yun; Egli, Martin; Guengerich, F. Peter

    2012-03-15

    O{sup 6}-Methylguanine (O{sup 6}-methylG) is highly mutagenic and is commonly found in DNA exposed to methylating agents, even physiological ones (e.g. S-adenosylmethionine). The efficiency of a truncated, catalytic DNA polymerase L core enzyme was determined for nucleoside triphosphate incorporation opposite O{sup 6}-methylG, using steady-state kinetic analyses. The results presented here corroborate previous work from this laboratory using full-length pol L, which showed that dTTP incorporation occurs with high efficiency opposite O{sup 6}-methylG. Misincorporation of dTTP opposite O{sup 6}-methylG occurred with {approx}6-fold higher efficiency than incorporation of dCTP. Crystal structures of the truncated form of pol L with O{sup 6}-methylG as the template base and incoming dCTP or dTTP were solved and showed that O{sup 6}-methylG is rotated into the syn conformation in the pol L active site and that dTTP misincorporation by pol L is the result of Hoogsteen base pairing with the adduct. Both dCTP and dTTP base paired with the Hoogsteen edge of O{sup 6}-methylG. A single, short hydrogen bond formed between the N3 atom of dTTP and the N7 atom of O{sup 6}-methylG. Protonation of the N3 atom of dCTP and bifurcation of the N3 hydrogen between the N7 and O{sup 6} atoms of O{sup 6}-methylG allow base pairing of the lesion with dCTP. We conclude that differences in the Hoogsteen hydrogen bonding between nucleotides is the main factor in the preferential selectivity of dTTP opposite O{sup 6}-methylG by human pol L, in contrast to the mispairing modes observed previously for O{sup 6}-methylG in the structures of the model DNA polymerases Sulfolobus solfataricus Dpo4 and Bacillus stearothermophilus DNA polymerase I.

  6. Classification of mutations at the HLA-A locus by use of the polymerase chain reaction

    SciTech Connect

    Joseph, G.; Grist, S.; Firgaira, F.; Turner, D.; Morley, A. )

    1993-01-01

    The authors investigated whether the polymerase chain reaction (PCR) could be used to determine the mechanism of mutation in lymphocyte clones mutated at the HLA-A locus. Three polymorphisms, at Factor XIIIA, D6S109, and intron 3 of the HLA-A gene, were used to study a series of clones previously characterized by Southern blotting (SB) at multiple loci on chromosome 6. For detection of loss of heterozygosity, the results of PCR and SB were concordant in 140 of 141 clones when polymorphism in the Factor XIIIA region was studied and in 144 of 145 clones when polymorphism in the HLA-A gene was studied. For classification of the mechanism of mutation, PCR and SB gave the same result in 88 of 92 clones (96%) when a combination of the HLA-A and Factor XIIIA polymorphisms was used and in 46 of 47 clones (98%) when a combination of the HLA-A and D6S109 polymorphisms was used. The results indicate that PCR provides a simple and reliable method for categorizing mutations at the HLA-A locus as arising from mitotic recombination, deletion, or from presumptive minor changes within the gene. Rare events such as gene conversion, nondisjunction, or large deletions extending to the telomere will be misclassified. However, such events are rare for mutations at this locus. 9 refs., 2 figs., 5 tabs.

  7. RNA polymerase gene, microorganism having said gene and the production of RNA polymerase by the use of said microorganism

    DOEpatents

    Kotani, Hirokazu; Hiraoka, Nobutsugu; Obayashi, Akira

    1991-01-01

    SP6 bacteriophage RNA polymerase is produced by cultivating a new microorganism (particularly new strains of Escherichia coli) harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene and recovering SP6 bacteriophage RNA polymerase from the culture broth. SP6 bacteriophage RNA polymerase gene is provided as are new microorganisms harboring a plasmid that carries SP6 bacteriophage RNA polymerase gene.

  8. Ribonucleic Acid Polymerase in Allomyces arbuscula

    PubMed Central

    Cain, Alice K.; Nester, Eugene W.

    1973-01-01

    Three distinct species of ribonucleic acid (RNA) polymerase were resolved from Allomyces arbuscula by diethylaminoethyl-cellulose chromatography and characterized as to ionic strength and divalent cation preference. α-Amanitin specifically inhibited enzyme II; neither rifampin nor cycloheximide had any effect on the three enzymes. RNA polymerase was isolated from three stages of the diploid life cycle: the hyphal growth stage, mycelia in the process of forming sporangia, and the mitospores. The same three enzyme species could be resolved from each stage. Thus, there is no evidence from this work that RNA polymerase plays a major role in the control of development. PMID:4728272

  9. Involvement of DNA polymerase beta overexpression in the malignant transformation induced by benzo[a]pyrene

    PubMed Central

    Zhao, Wei; Wu, Mei; Lai, Yanhao; Deng, Wenwen; Liu, Yuan; Zhang, Zunzhen

    2014-01-01

    Objective To explore the relationship between DNA polymerase β (pol β) overexpression and benzo[a]pyrene (BaP) carcinogenesis. Methods Firstly, mouse embryonic fibroblasts that express wild-type level of DNA polymerase β (pol β cell) and high level of pol β (pol β oe cell) were treated by various concentrations of BaP to determine genetic instability induced by BaP under differential expression levels of pol β. Secondly, malignant transformation of pol β cells by low concentration of BaP (20 μM) was determined by soft agar colony formation assay and transformation focus assay. Thirdly, the mRNA and protein levels of BaP-transformed pol β cells (named pol β-T cells) was measured by reverse transcriptase-polymerase chain reaction (RT-PCR) and western blot, and the genetic instability of these cells were examined by HPRT gene mutation assay and random amplified polymorphic DNA (RAPD) assay. Results Pol β cells were successfully transformed into malignant pol β-T cells by an exposure to low concentration of BaP for 6 months. Pol β-T cells exhibited increased levels of pol β gene expression, HPRT gene mutation frequency and polymorphisms of RAPD products that were comparable to those of pol β oe cells. Conclusion Pol β overexpression and its-associated genetic instability may play a key role in BaP carcinogenesis. PMID:23652152

  10. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, Stanley; Richardson, Charles

    1997-01-01

    Modified gene encoding a modified DNA polymerase wherein the modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase.

  11. DNA polymerase having modified nucleotide binding site for DNA sequencing

    DOEpatents

    Tabor, S.; Richardson, C.

    1997-03-25

    A modified gene encoding a modified DNA polymerase is disclosed. The modified polymerase incorporates dideoxynucleotides at least 20-fold better compared to the corresponding deoxynucleotides as compared with the corresponding naturally-occurring DNA polymerase. 6 figs.

  12. Allele-specific DNA methylation reinforces PEAR1 enhancer activity.

    PubMed

    Izzi, Benedetta; Pistoni, Mariaelena; Cludts, Katrien; Akkor, Pinar; Lambrechts, Diether; Verfaillie, Catherine; Verhamme, Peter; Freson, Kathleen; Hoylaerts, Marc F

    2016-08-18

    Genetic variation in the PEAR1 locus is linked to platelet reactivity and cardiovascular disease. The major G allele of rs12041331, an intronic cytosine guanine dinucleotide-single-nucleotide polymorphism (CpG-SNP), is associated with higher PEAR1 expression in platelets and endothelial cells than the minor A allele. The molecular mechanism underlying this difference remains elusive. We have characterized the histone modification profiles of the intronic region surrounding rs12041331 and identified H3K4Me1 enhancer-specific enrichment for the region that covers the CpG-SNP. Interestingly, methylation studies revealed that the CpG site is fully methylated in leukocytes of GG carriers. Nuclear protein extracts from megakaryocytes, endothelial cells, vs control HEK-293 cells show a 3-fold higher affinity for the methylated G allele compared with nonmethylated G or A alleles in a gel electrophoretic mobility shift assay. To understand the positive relationship between methylation and gene expression, we studied DNA methylation at 4 different loci of PEAR1 during in vitro megakaryopoiesis. During differentiation, the CpG-SNP remained fully methylated, while we observed rapid methylation increases at the CpG-island overlapping the first 5'-untranslated region exon, paralleling the increased PEAR1 expression. In the same region, A-allele carriers of rs12041331 showed significantly lower DNA methylation at CGI1 compared with GG homozygote. This CpG-island contains binding sites for the methylation-sensitive transcription factor CTCF, whose binding is known to play a role in enhancer activation and/or repression. In conclusion, we report the molecular characterization of the first platelet function-related CpG-SNP, a genetic predisposition that reinforces PEAR1 enhancer activity through allele-specific DNA methylation. PMID:27313330

  13. Analysis of DNA Methylation in Various Swine Tissues

    PubMed Central

    Niu, Weiping; Yang, Runjun; Zhang, Yonghong; Qiu, Zhengyan; Sun, Boxing; Zhao, Zhihui

    2011-01-01

    DNA methylation is known to play an important role in regulating gene expression during biological development and tissue differentiation in eukaryotes. In this study, we used the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) method to assess the extent and pattern of cytosine methylation in muscle, heart, liver, spleen, lung, kidney and stomach from the swine strain Laiwu, and we also examined specific methylation patterns in the seven tissues. In total, 96,371 fragments, each representing a recognition site cleaved by either or both EcoRI + HpaII and EcoRI + MspI, the HpaII and MspI are isoschizomeric enzymes, were amplified using 16 pairs of selective primers. A total of 50,094 sites were found to be methylated at cytosines in seven tissues. The incidence of DNA methylation was approximately 53.99% in muscle, 51.24% in the heart, 50.18% in the liver, 53.31% in the spleen, 51.97% in the lung, 51.15% in the kidney and 53.39% in the stomach, as revealed by the incidence of differential digestion. Additionally, differences in DNA methylation levels imply that such variations may be related to specific gene expression during tissue differentiation, growth and development. Three types of bands were generated in the F-MSAP profile, the total numbers of these three types of bands in the seven tissues were 46,277, 24,801 and 25,293, respectively. In addition, different methylation patterns were observed in seven tissues from pig, and almost all of the methylation patterns detected by F-MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrated that the F-MSAP technique can be adapted for use in large-scale DNA methylation detection in the pig genome. PMID:21283691

  14. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele).

  15. Genotyping for cytochrome P450 polymorphisms.

    PubMed

    Daly, Ann K; King, Barry P; Leathart, Julian B S

    2006-01-01

    Protocols for the extraction of DNA from human blood and for genotyping for a number of common cytochrome P450 polymorphisms using either polymerase chain reaction (PCR)-restriction fragment length polymorphism or PCR-single-strand conformational polymorphism (SSCP) analysis are described. Rapid high-throughput techniques are also available for analyses of this type, but they require access to specialized equipment and are not considered here. General guidelines for performing amplification using PCR are described together with electrophoresis protocols for analysis of restriction digests of PCR products with agarose and polyacrylamide gels including the use of polyacrylamide-based gels for SSCP analysis. Protocols for the following specific isoforms and alleles are also provided: CYP1A1 (*2B and *4 alleles), CYP2C8 (*3 and *4 alleles), CYP2C9 (*2, *3, and *11 alleles), CYP2C19 (*2 and *3 alleles), CYP2D6 (*3, *4, *5, and *6 alleles), CYP2E1 (*5A, *5B, and *6 alleles), and CYP3A5 (*3 allele). PMID:16719392

  16. DNA methylation in plants.

    PubMed

    Vanyushin, B F

    2006-01-01

    DNA in plants is highly methylated, containing 5-methylcytosine (m5C) and N6-methyladenine (m6A); m5C is located mainly in symmetrical CG and CNG sequences but it may occur also in other non-symmetrical contexts. m6A but not m5C was found in plant mitochondrial DNA. DNA methylation in plants is species-, tissue-, organelle- and age-specific. It is controlled by phytohormones and changes on seed germination, flowering and under the influence of various pathogens (viral, bacterial, fungal). DNA methylation controls plant growth and development, with particular involvement in regulation of gene expression and DNA replication. DNA replication is accompanied by the appearance of under-methylated, newly formed DNA strands including Okazaki fragments; asymmetry of strand DNA methylation disappears until the end of the cell cycle. A model for regulation of DNA replication by methylation is suggested. Cytosine DNA methylation in plants is more rich and diverse compared with animals. It is carried out by the families of specific enzymes that belong to at least three classes of DNA methyltransferases. Open reading frames (ORF) for adenine DNA methyltransferases are found in plant and animal genomes, and a first eukaryotic (plant) adenine DNA methyltransferase (wadmtase) is described; the enzyme seems to be involved in regulation of the mitochondria replication. Like in animals, DNA methylation in plants is closely associated with histone modifications and it affects binding of specific proteins to DNA and formation of respective transcription complexes in chromatin. The same gene (DRM2) in Arabidopsis thaliana is methylated both at cytosine and adenine residues; thus, at least two different, and probably interdependent, systems of DNA modification are present in plants. Plants seem to have a restriction-modification (R-M) system. RNA-directed DNA methylation has been observed in plants; it involves de novo methylation of almost all cytosine residues in a region of si

  17. Ethnic differences in five intronic polymorphisms associated with arsenic metabolism within human arsenic (+ 3 oxidation state) methyltransferase (AS3MT) gene

    SciTech Connect

    Fujihara, Junko; Fujii, Yoshimi; Agusa, Tetsuro; Kunito, Takashi; Yasuda, Toshihiro; Moritani, Tamami; Takeshita, Haruo

    2009-01-01

    Human arsenic (+ 3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite, and intronic single-nucleotide polymorphisms (SNPs: G7395A, G12390C, T14215C, T35587C, and G35991A) in the AS3MT gene were shown to be related to inter-individual variation in the arsenic metabolism. In the present study, the genotyping for these SNPs was developed using the polymerase chain reaction and restriction fragment length polymorphism technique. Applying this method, the genotype distribution among the Ovambo, Turkish, Mongolian, Korean, and Japanese populations was investigated, and our results were compared with those from other studies. G7395, G12390, T35587, and A35991 were predominant among the five populations in our study. However, a previous study in Argentina, C12390 and G35991 showed the highest allele frequency among the eight populations studied in other studies. The dominant allele of T14215C differed among populations: the T14215 allele was predominant in Argentina, the allele frequency of C14215 was higher than that of T14215 among Turks, Mongolians, Europeans, and American ancestry. In Korea and Japan, similar allele frequencies were observed in T14215 and C14215. Higher allele frequencies were observed in haplotype G7395/G12390/C14215/T35587 with frequencies of 0.40 (Turks), 0.28 (Mongolians), and 0.23 (Koreans). On the other hand, the allele frequency for G7395/G14215/T35587/A35991 was the highest among the Ovambos (0.32), and the frequency for G7395/G12390/C35587/G35991 was the highest among the Japanese (0.27). It is noteworthy that the Japanese haplotype differs from that of the Koreans and Mongolians, which indicates the importance of investigating other intronic polymorphisms in AS3MT, especially in Asians.

  18. Ethnic differences in five intronic polymorphisms associated with arsenic metabolism within human arsenic (+3 oxidation state) methyltransferase (AS3MT) gene.

    PubMed

    Fujihara, Junko; Fujii, Yoshimi; Agusa, Tetsuro; Kunito, Takashi; Yasuda, Toshihiro; Moritani, Tamami; Takeshita, Haruo

    2009-01-01

    Human arsenic (+3 oxidation state) methyltransferase (AS3MT) is known to catalyze the methylation of arsenite, and intronic single-nucleotide polymorphisms (SNPs: G7395A, G12390C, T14215C, T35587C, and G35991A) in the AS3MT gene were shown to be related to inter-individual variation in the arsenic metabolism. In the present study, the genotyping for these SNPs was developed using the polymerase chain reaction and restriction fragment length polymorphism technique. Applying this method, the genotype distribution among the Ovambo, Turkish, Mongolian, Korean, and Japanese populations was investigated, and our results were compared with those from other studies. G7395, G12390, T35587, and A35991 were predominant among the five populations in our study. However, a previous study in Argentina, C12390 and G35991 showed the highest allele frequency among the eight populations studied in other studies. The dominant allele of T14215C differed among populations: the T14215 allele was predominant in Argentina, the allele frequency of C14215 was higher than that of T14215 among Turks, Mongolians, Europeans, and American ancestry. In Korea and Japan, similar allele frequencies were observed in T14215 and C14215. Higher allele frequencies were observed in haplotype G7395/G12390/C14215/T35587 with frequencies of 0.40 (Turks), 0.28 (Mongolians), and 0.23 (Koreans). On the other hand, the allele frequency for G7395/G14215/T35587/A35991 was the highest among the Ovambos (0.32), and the frequency for G7395/G12390/C35587/G35991 was the highest among the Japanese (0.27). It is noteworthy that the Japanese haplotype differs from that of the Koreans and Mongolians, which indicates the importance of investigating other intronic polymorphisms in AS3MT, especially in Asians. PMID:18976679

  19. Structures of the Leishmania infantum polymerase beta

    PubMed Central

    Mejia, Edison; Burak, Matthew; Alonso, Ana; Larraga, Vicente; Kunkel, Thomas A.; Bebenek, Katarzyna; Garcia-Diaz, Miguel

    2014-01-01

    Protozoans of the genus Leishmania, the pathogenic agent causing leishmaniasis, encode the family X DNA polymerase Li Pol β. Here, we report the first crystal structures of Li Pol β. Our pre- and post-catalytic structures show that the polymerase adopts the common family X DNA polymerase fold. However, in contrast to other family X DNA polymerases, the dNTP-induced conformational changes in Li Pol β are much more subtle. Moreover, pre- and post-catalytic structures reveal that Li Pol β interacts with the template strand through a nonconserved, variable region known as loop3. Li Pol β Δloop3 mutants display a higher catalytic rate, catalytic efficiency and overall error rates with respect to WT Li Pol β. These results further demonstrate the subtle structural variability that exists within this family of enzymes and provides insight into how this variability underlies the substantial functional differences among their members. PMID:24666693

  20. Polymorphism of the DNA Base Excision Repair Genes in Keratoconus

    PubMed Central

    Wojcik, Katarzyna A.; Synowiec, Ewelina; Sobierajczyk, Katarzyna; Izdebska, Justyna; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.

    2014-01-01

    Keratoconus (KC) is a degenerative corneal disorder for which the exact pathogenesis is not yet known. Oxidative stress is reported to be associated with this disease. The stress may damage corneal biomolecules, including DNA, and such damage is primarily removed by base excision repair (BER). Variation in genes encoding BER components may influence the effectiveness of corneal cells to cope with oxidative stress. In the present work we genotyped 5 polymorphisms of 4 BER genes in 284 patients and 353 controls. The A/A genotype of the c.–1370T>A polymorphism of the DNA polymerase γ (POLG) gene was associated with increased occurrence of KC, while the A/T genotype was associated with decreased occurrence of KC. The A/G genotype and the A allele of the c.1196A>G polymorphism of the X-ray repair cross-complementing group 1 (XRCC1) were associated with increased, and the G/G genotype and the G allele, with decreased KC occurrence. Also, the C/T and T as well as C/C genotypes and alleles of the c.580C>T polymorphism of the same gene displayed relationship with KC occurrence. Neither the g.46438521G>C polymorphism of the Nei endonuclease VIII-like 1 (NEIL1) nor the c.2285T>C polymorphism of the poly(ADP-ribose) polymerase-1 (PARP-1) was associated with KC. In conclusion, the variability of the XRCC1 and POLG genes may play a role in KC pathogenesis and determine the risk of this disease. PMID:25356504

  1. MMP-14 and TGFβ-1 methylation in pituitary adenomas

    PubMed Central

    Ruskyte, Kornelija; Liutkevicienė, Rasa; Vilkeviciute, Alvita; Vaitkiene, Paulina; Valiulytė, Indre; Glebauskiene, Brigita; Kriauciuniene, Loresa; Zaliuniene, Dalia

    2016-01-01

    Pituitary adenoma (PA) is one of the most common abnormalities in the sellar region. Despite the fact that PA is a benign monoclonal neoplasm, it can cause serious complications, including ophthalmological, neurological and endocrinological abnormalities. Currently, the causes that increase the progression of tumors are unknown. Epigenetic silencing of the matrix metalloproteinase-14 (MMP-14) and transforming growth factor beta-1 (TGFβ-1) genes may be associated with the development of PA, since these genes are important in the processes of tumor metastasis and angiogenesis. The purpose of the present study was to determine if the methylation status of the MMP-14 and TGFβ-1 promoters is associated with PA development. In the present study, 120 tissue samples of PA were used. The methylation status of the MMP-14 and TGFβ-1 promoters was investigated by methylation specific-polymerase chain reaction. Statistical analysis was conducted to investigate the associations between the methylation status, age and gender of PA patients, PA tumoral activity, recurrence and invasiveness. The MMP-14 gene was methylated in 30.00% (17/56 functioning and 19/64 non-functioning) of patients with PA, while the TGFβ-1 gene was methylated in 13.33% (9/56 functioning and 7/64 non-functioning) of patients with PA. It was also observed that promoter methylation of MMP-14 correlated with the male gender (58.8 vs. 35.7%, P=0.022), while unmethylated (non-silenced) MMP-14 correlated with the female gender (64.3 vs. 41.7%, P=0.027). Associations between the promoter methylation status of the MMP-14 and TGFβ-1 genes and PA functioning or recurrence were not identified. The present study reveals that silencing of the MMP-14 gene correlates with patients' gender. However, MMP-14 and TGFβ-1 promoter methylation cannot be considered as a prognostic marker in PAs.

  2. MMP-14 and TGFβ-1 methylation in pituitary adenomas

    PubMed Central

    Ruskyte, Kornelija; Liutkevicienė, Rasa; Vilkeviciute, Alvita; Vaitkiene, Paulina; Valiulytė, Indre; Glebauskiene, Brigita; Kriauciuniene, Loresa; Zaliuniene, Dalia

    2016-01-01

    Pituitary adenoma (PA) is one of the most common abnormalities in the sellar region. Despite the fact that PA is a benign monoclonal neoplasm, it can cause serious complications, including ophthalmological, neurological and endocrinological abnormalities. Currently, the causes that increase the progression of tumors are unknown. Epigenetic silencing of the matrix metalloproteinase-14 (MMP-14) and transforming growth factor beta-1 (TGFβ-1) genes may be associated with the development of PA, since these genes are important in the processes of tumor metastasis and angiogenesis. The purpose of the present study was to determine if the methylation status of the MMP-14 and TGFβ-1 promoters is associated with PA development. In the present study, 120 tissue samples of PA were used. The methylation status of the MMP-14 and TGFβ-1 promoters was investigated by methylation specific-polymerase chain reaction. Statistical analysis was conducted to investigate the associations between the methylation status, age and gender of PA patients, PA tumoral activity, recurrence and invasiveness. The MMP-14 gene was methylated in 30.00% (17/56 functioning and 19/64 non-functioning) of patients with PA, while the TGFβ-1 gene was methylated in 13.33% (9/56 functioning and 7/64 non-functioning) of patients with PA. It was also observed that promoter methylation of MMP-14 correlated with the male gender (58.8 vs. 35.7%, P=0.022), while unmethylated (non-silenced) MMP-14 correlated with the female gender (64.3 vs. 41.7%, P=0.027). Associations between the promoter methylation status of the MMP-14 and TGFβ-1 genes and PA functioning or recurrence were not identified. The present study reveals that silencing of the MMP-14 gene correlates with patients' gender. However, MMP-14 and TGFβ-1 promoter methylation cannot be considered as a prognostic marker in PAs. PMID:27698891

  3. Anxiety and Methylenetetrahydrofolate Reductase Mutation Treated With S-Adenosyl Methionine and Methylated B Vitamins.

    PubMed

    Anderson, Shanna; Panka, Jacob; Rakobitsch, Robin; Tyre, Kaitlin; Pulliam, Kerry

    2016-04-01

    This case report highlights challenges faced in the clinical management of patients with methylenetetrahydrofolate reductase (MTHFR) gene mutations and the importance of precise dosage when recommending methylated B vitamins to compensate for deficiencies caused by the polymorphism or symptoms related to the polymorphism. It also underscores the importance of obtaining ongoing objective assessments of anxiety (eg, Patient Reported Outcomes Measurement Information System, or PROMIS) to help gauge patient response. PMID:27330489

  4. BIALLELIC POLYMORPHISM IN THE INTRON REGION OF B-TUBULIN GENE OF CRYPTOSPORIDIUM PARASITES

    EPA Science Inventory

    Nucleotide sequencing of polymerase chain reaction-amplified intron region of the Cryptosporidium parvum B-tubulin gene in 26 human and 15 animal isolates revealed distinct genetic polymorphism between the human and bovine genotypes. The separation of 2 genotypes of C. parvum is...

  5. Assessment of Quantitative and Allelic MGMT Methylation Patterns as a Prognostic Marker in Glioblastoma.

    PubMed

    Kristensen, Lasse S; Michaelsen, Signe R; Dyrbye, Henrik; Aslan, Derya; Grunnet, Kirsten; Christensen, Ib J; Poulsen, Hans S; Grønbæk, Kirsten; Broholm, Helle

    2016-03-01

    Methylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) gene is a predictive and prognostic marker in newly diagnosed glioblastoma patients treated with temozolomide but how MGMT methylation should be assessed to ensure optimal detection accuracy is debated. We developed a novel quantitative methylation-specific PCR (qMSP) MGMT assay capable of providing allelic methylation data and analyzed 151 glioblastomas from patients receiving standard of care treatment (Stupp protocol). The samples were also analyzed by immunohistochemistry (IHC), standard bisulfite pyrosequencing, and genotyped for the rs1690252 MGMT promoter single nucleotide polymorphism. Monoallelic methylation was observed more frequently than biallelic methylation, and some cases with monoallelic methylation expressed the MGMT protein whereas others did not. The presence of MGMT methylation was associated with better overall survival (p = 0.006; qMSP and p = 0.002; standard pyrosequencing), and the presence of the protein was associated with worse overall survival (p = 0.009). Combined analyses of qMSP and standard pyrosequencing or IHC identified additional patients who benefited from temozolomide treatment. Finally, low methylation levels were also associated with better overall survival (p = 0.061; qMSP and p = 0.02; standard pyrosequencing). These data support the use of both MGMT methylation and MGMT IHC but not allelic methylation data as prognostic markers in patients with temozolomide-treated glioblastoma. PMID:26883115

  6. DNA Methylation as Surrogate Marker For Gastric Cancer

    PubMed Central

    Oh, Jung-Hwan; Jung, Sung-Hoon; Hong, Seung-Jin; Rhyu, Mun-Gan

    2015-01-01

    Stomach cancer remains, stubbornly, highly prevalent in East Asia. Still, stomach cancer has few biomarkers by which it can be predicted. Helicobacter pylori infection, a known carcinogen of stomach cancer, usually goes undetected prior to cancer diagnosis, due to the poor mucosal environments that its related gastric atrophy causes. We propose, herein, an endoscopic-biopsy-based cancer-predicting DNA methylation marker. We semi-quantitatively examined the methylation-variable sites near the CpG-island margins by radioisotope-labeling methylation-specific polymerase chain reaction in association with H. pylori, which increases age-related over-methylation in CpG islands of gastric mucosa. These age-related methylation patterns of the transitional-CpG sites are proposed as useful surrogate markers for stomach cancer. It would be helpful for setting the optimal screening interval for high-risk subjects as well as for estimating the prognosis and the predictability for recurrence of early gastric cancer in patients having undergone endoscopic submucosal dissection. New screening-interval guidelines for gastric cancer should be suggested considering individual risk based on age, severity of atrophy, H. pylori status, and DNA methylation pattern. PMID:26473155

  7. Promoter Methylation of SFRP3 Is Frequent in Hepatocellular Carcinoma

    PubMed Central

    Shih, Yu-Lueng; Lien, Gi-Shih; Suk, Fat-Moon; Hsieh, Chung-Bao; Yan, Ming-De

    2014-01-01

    Oncogenic activation of the Wnt/β-catenin signaling pathway is common in human cancers. The secreted frizzled-related proteins (SFRPs) function as negative regulators of Wnt signaling and have important implications in carcinogenesis. Because there have been no reports about the role of SFRP3 in hepatocellular carcinoma (HCC), we investigated the level of methylation and transcription of SFRP3. Four HCC cell lines, 60 HCCs, 23 cirrhosis livers, 37 chronic hepatitis livers, and 30 control livers were prescreened for SFRP3 promoter methylation by methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing. SFRP3 promoter methylation was observed in 100%, 60%, 39.1%, 16.2%, and 0% in HCC cell lines, primary HCCs, cirrhosis livers, chronic hepatitis livers, and control livers, respectively. Demethylation treatment with 5-aza-2′-deoxycytidine in HCC cells restored or increased the SFRP3 mRNA expression. We next used quantitative MS-PCR (QMSP) to analyze the methylation level of SFRP3 in 60 HCCs and their corresponding nontumor tissues. Methylation of SFRP3 promoter region in HCCs increased significantly compared with control tissues. There is a positive correlation between promoter hypermethylation and SFRP3 mRNA downregulation. Our data suggest that promoter hypermethylation of SFRP3 is a common event in HCCs and plays an important role in regulation of SFRP3 mRNA expression. PMID:24591760

  8. Dicer-independent RNA-directed DNA methylation in Arabidopsis.

    PubMed

    Yang, Dong-Lei; Zhang, Guiping; Tang, Kai; Li, Jingwen; Yang, Lan; Huang, Huan; Zhang, Heng; Zhu, Jian-Kang

    2016-01-01

    RNA-directed DNA methylation (RdDM) is an important de novo DNA methylation pathway in plants. Small interfering RNAs (siRNAs) generated by Dicers from RNA polymerase IV (Pol IV) transcripts are thought to guide sequence-specific DNA methylation. To gain insight into the mechanism of RdDM, we performed whole-genome bisulfite sequencing of a collection of Arabidopsis mutants, including plants deficient in Pol IV (nrpd1) or Dicer (dcl1/2/3/4) activity. Unexpectedly, of the RdDM target loci that required Pol IV and/or Pol V, only 16% were fully dependent on Dicer activity. DNA methylation was partly or completely independent of Dicer activity at the remaining Pol IV- and/or Pol V-dependent loci, despite the loss of 24-nt siRNAs. Instead, DNA methylation levels correlated with the accumulation of Pol IV-dependent 25-50 nt RNAs at most loci in Dicer mutant plants. Our results suggest that RdDM in plants is largely guided by a previously unappreciated class of Dicer-independent non-coding RNAs, and that siRNAs are required to maintain DNA methylation at only a subset of loci. PMID:26642813

  9. Histoplasma capsulatum var. duboisii infection in a patient with AIDS: rapid diagnosis using polymerase chain reaction-sequencing.

    PubMed

    Pellaton, Cyril; Cavassini, Matthias; Jaton-Ogay, Katia; Carron, Pierre-Nicolas; Christen-Zaech, Stéphanie; Calandra, Thierry; Bille, Jacques; Hauser, Philippe M

    2009-05-01

    We describe an original case of disseminated infection with Histoplasma capsulatum (Hc) var. duboisii in an African patient with AIDS who migrated to Switzerland. The diagnosis of histoplasmosis was suggested using direct examination of tissues and confirmed in 24 h with a panfungal polymerase chain reaction assay. The variety duboisii of Hc was established using DNA sequencing of the polymorphic genomic region OLE. Molecular tools allow diagnosis of histoplasmosis in 24 h, which is drastically shorter than culture procedures. PMID:19304436

  10. Neural Tube Defects, Folic Acid and Methylation

    PubMed Central

    Imbard, Apolline; Benoist, Jean-François; Blom, Henk J.

    2013-01-01

    Neural tube defects (NTDs) are common complex congenital malformations resulting from failure of the neural tube closure during embryogenesis. It is established that folic acid supplementation decreases the prevalence of NTDs, which has led to national public health policies regarding folic acid. To date, animal studies have not provided sufficient information to establish the metabolic and/or genomic mechanism(s) underlying human folic acid responsiveness in NTDs. However, several lines of evidence suggest that not only folates but also choline, B12 and methylation metabolisms are involved in NTDs. Decreased B12 vitamin and increased total choline or homocysteine in maternal blood have been shown to be associated with increased NTDs risk. Several polymorphisms of genes involved in these pathways have also been implicated in risk of development of NTDs. This raises the question whether supplementation with B12 vitamin, betaine or other methylation donors in addition to folic acid periconceptional supplementation will further reduce NTD risk. The objective of this article is to review the role of methylation metabolism in the onset of neural tube defects. PMID:24048206

  11. Methyl salicylate overdose

    MedlinePlus

    Deep heating rubs overdose; Oil of wintergreen overdose ... These products contain methyl salicylate: Deep-heating creams used to relieve sore muscles and joints (Ben Gay, Icy Hot) Oil of wintergreen Solutions for vaporizers Other products may also ...

  12. ENZYMOLOGY OF ARSENIC METHYLATION

    EPA Science Inventory

    Enzymology of Arsenic Methylation

    David J. Thomas, Pharmacokinetics Branch, Experimental Toxicology Division, National
    Health and Environmental Effects Research Laboratory, Office of Research and Development, U.S. Environmental Protection Agency, Research Triangle Park...

  13. Folate: metabolism, genes, polymorphisms and the associated diseases.

    PubMed

    Nazki, Fakhira Hassan; Sameer, Aga Syed; Ganaie, Bashir Ahmad

    2014-01-01

    Folate being an important vitamin of B Complex group in our diet plays an important role not only in the synthesis of DNA but also in the maintenance of methylation reactions in the cells. Folate metabolism is influenced by several processes especially its dietary intake and the polymorphisms of the associated genes involved. Aberrant folate metabolism, therefore, affects both methylation as well as the DNA synthesis processes, both of which have been implicated in the development of various diseases. This paper reviews the current knowledge of the processes involved in folate metabolism and consequences of deviant folate metabolism, particular emphasis is given to the polymorphic genes which have been implicated in the development of various diseases in humans, like vascular diseases, Down's syndrome, neural tube defects, psychiatric disorders and cancers. PMID:24091066

  14. Exploring Genome-wide DNA Methylation Profiles Altered in Kashin-Beck Disease Using Infinium Human Methylation 450 Bead Chips.

    PubMed

    Shi, Xiao Wei; Shi, Bo Hui; Lyu, Ai Li; Zhang, Feng; Zhou, Tian Tian; Guo, Xiong

    2016-07-01

    To understand how differentially methylated genes (DMGs) might affect the pathogenesis of Kashin-Beck disease (KBD). Genome-wide methylation profiling of whole blood from 12 matched KBD and controls pairs was performed using a high-resolution Infinium 450 K methylation array. In total, 97 CpG sites were differentially methylated in KBD compared to the normal controls; of these sites, 36 sites were significantly hypermethylated (covering 22 genes) and 61 sites were significantly hypomethylated (covering 34 genes). Of these genes, 14 significant pathways were identified, the most significant P value pathway was type I diabetes mellitus pathway and pathways associated with autoimmune diseases and inflammatory diseases were included in this study. Subsequently, 4 CpG sites in HLA-DRB1 were validated using bisulfite sequencing polymerase chain reaction (BSP) in articular cartilage, and the results showed significant differences in the methylation status between KBD and controls, consistent with the results of the high-resolution array. These results suggested that differences in genome-wide DNA methylation exist between KBD and the controls, and the biological pathways support the autoimmune disease and inflammatory disease hypothesis of KBD.

  15. Exploring Genome-wide DNA Methylation Profiles Altered in Kashin-Beck Disease Using Infinium Human Methylation 450 Bead Chips.

    PubMed

    Shi, Xiao Wei; Shi, Bo Hui; Lyu, Ai Li; Zhang, Feng; Zhou, Tian Tian; Guo, Xiong

    2016-07-01

    To understand how differentially methylated genes (DMGs) might affect the pathogenesis of Kashin-Beck disease (KBD). Genome-wide methylation profiling of whole blood from 12 matched KBD and controls pairs was performed using a high-resolution Infinium 450 K methylation array. In total, 97 CpG sites were differentially methylated in KBD compared to the normal controls; of these sites, 36 sites were significantly hypermethylated (covering 22 genes) and 61 sites were significantly hypomethylated (covering 34 genes). Of these genes, 14 significant pathways were identified, the most significant P value pathway was type I diabetes mellitus pathway and pathways associated with autoimmune diseases and inflammatory diseases were included in this study. Subsequently, 4 CpG sites in HLA-DRB1 were validated using bisulfite sequencing polymerase chain reaction (BSP) in articular cartilage, and the results showed significant differences in the methylation status between KBD and controls, consistent with the results of the high-resolution array. These results suggested that differences in genome-wide DNA methylation exist between KBD and the controls, and the biological pathways support the autoimmune disease and inflammatory disease hypothesis of KBD. PMID:27554126

  16. The APOE Gene is Differentially Methylated in Alzheimer's Disease.

    PubMed

    Foraker, Jessica; Millard, Steven P; Leong, Lesley; Thomson, Zachary; Chen, Sunny; Keene, C Dirk; Bekris, Lynn M; Yu, Chang-En

    2015-01-01

    The ɛ4 allele of the human apolipoprotein E gene (APOE) is a well-proven genetic risk factor for the late onset form of Alzheimer's disease (AD). However, the biological mechanisms through which the ɛ4 allele contributes to disease pathophysiology are incompletely understood. The three common alleles of APOE, ɛ2, ɛ3 and ɛ4, are defined by two single nucleotide polymorphisms (SNPs) that reside in the coding region of exon 4, which overlaps with a well-defined CpG island (CGI). Both SNPs change not only the protein codon but also the quantity of CpG dinucleotides, primary sites for DNA methylation. Thus, we hypothesize that the presence of an ɛ4 allele changes the DNA methylation landscape of the APOE CGI and that such epigenetic alteration contributes to AD susceptibility. To explore the relationship between APOE genotype, AD risk, and DNA methylation of the APOE CGI, we applied bisulfite pyrosequencing and evaluated methylation profiles of postmortem brain from 15 AD and 10 control subjects. We observed a tissue-specific decrease in DNA methylation with AD and identified two AD-specific differentially methylated regions (DMRs), which were also associated with APOE genotype. We further demonstrated that one DMR was completely un-methylated in a sub-population of genomes, possibly due to a subset of brain cells carrying deviated APOE methylation profiles. These data suggest that the APOE CGI is differentially methylated in AD brain in a tissue- and APOE-genotype-specific manner. Such epigenetic alteration might contribute to neural cell dysfunction in AD brain. PMID:26402071

  17. Genetic Analysis of the Atrial Natriuretic Peptide Gene Polymorphisms among Essential Hypertensive Patients in Malaysia.

    PubMed

    Ghodsian, Nooshin; Ismail, Patimah; Ahmadloo, Salma; Eskandarian, Narges; Etemad, Ali

    2016-01-01

    Background. Atrial natriuretic peptide (ANP) considerably influences blood pressure regulation through water and sodium homoeostasis. Several of the studies have utilized anonymous genetic polymorphic markers and made inconsequent claims about the ANP relevant disorders. Thus, we screened Insertion/Deletion (ID) and G191A polymorphisms of ANP to discover sequence variations with potential functional significance and to specify the linkage disequilibrium pattern between polymorphisms. The relationships of detected polymorphisms with EH with or without Type 2 Diabetes Mellitus (T2DM) status were tested subsequently. Method. ANP gene polymorphisms (I/D and A191G) were specified utilizing mutagenically separated Polymerase Chain Reaction (PCR) in 320 subjects including 163 EH case subjects and 157 controls. Result. This case-control study discovered a significant association between I/D polymorphisms of ANP gene in EH patient without T2DM. However, the study determined no association between G191A polymorphisms of ANP in EH with or without T2DM. In addition, sociodemographic factors in the case and healthy subjects exhibited strong differences (P < 0.05). Conclusion. As a risk factor, ANP gene polymorphisms may affect hypertension. Despite the small sample size in this study, it is the first research assessing the ANP gene polymorphisms in both EH and T2DM patients among Malaysian population. PMID:27413750

  18. Genetic Analysis of the Atrial Natriuretic Peptide Gene Polymorphisms among Essential Hypertensive Patients in Malaysia

    PubMed Central

    Ghodsian, Nooshin; Ismail, Patimah; Ahmadloo, Salma; Eskandarian, Narges; Etemad, Ali

    2016-01-01

    Background. Atrial natriuretic peptide (ANP) considerably influences blood pressure regulation through water and sodium homoeostasis. Several of the studies have utilized anonymous genetic polymorphic markers and made inconsequent claims about the ANP relevant disorders. Thus, we screened Insertion/Deletion (ID) and G191A polymorphisms of ANP to discover sequence variations with potential functional significance and to specify the linkage disequilibrium pattern between polymorphisms. The relationships of detected polymorphisms with EH with or without Type 2 Diabetes Mellitus (T2DM) status were tested subsequently. Method. ANP gene polymorphisms (I/D and A191G) were specified utilizing mutagenically separated Polymerase Chain Reaction (PCR) in 320 subjects including 163 EH case subjects and 157 controls. Result. This case-control study discovered a significant association between I/D polymorphisms of ANP gene in EH patient without T2DM. However, the study determined no association between G191A polymorphisms of ANP in EH with or without T2DM. In addition, sociodemographic factors in the case and healthy subjects exhibited strong differences (P < 0.05). Conclusion. As a risk factor, ANP gene polymorphisms may affect hypertension. Despite the small sample size in this study, it is the first research assessing the ANP gene polymorphisms in both EH and T2DM patients among Malaysian population. PMID:27413750

  19. Mapping a mutation in Caenorhabditis elegans using a polymerase chain reaction-based approach.

    PubMed

    Myers, Edith M

    2014-01-01

    Many single nucleotide polymorphisms (SNPs) have been identified within the Caenorhabditis elegans genome. SNPs present in the genomes of two isogenic C. elegans strains have been routinely used as a tool in forward genetics to map a mutation to a particular chromosome. This article describes a laboratory exercise in which undergraduate students use molecular biological techniques to map a mutation to a chromosome using a set of SNPs. Through this multi-week exercise, students perform genetic crosses, DNA extraction, polymerase chain reaction, restriction enzyme digests, agarose gel electrophoresis, and analysis of restriction fragment length polymorphisms. Students then analyze their results to deduce the chromosomal location of the mutation. Students also use bioinformatics websites to develop hypotheses that link the genotype to the phenotype. PMID:24615818

  20. Polymerase chain reaction-based strain characterization of noncapsulate Haemophilus influenzae.

    PubMed Central

    Jordens, J Z; Leaves, N I; Anderson, E C; Slack, M P

    1993-01-01

    A polymerase chain reaction-based typing method for noncapsulate Haemophilus influenzae was developed. Randomly amplified polymorphic DNA fingerprints were generated from boiled supernatants prepared directly from bacterial colonies without the need for DNA extraction. The technique was applied to isolates obtained during putative outbreaks of chest infection and validated by comparison with sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis of outer membrane protein-enriched preparations and rRNA gene restriction analysis. There was complete concordance between the three techniques. The results show that randomly amplified polymorphic DNA analysis provides a highly discriminatory method of characterizing strains of noncapsulate H. influenzae which is eminently suitable as an epidemiological tool for the rapid investigation of outbreaks of infection. Images PMID:8263183

  1. Interleukin-18, interleukin-12B and interferon-γ gene polymorphisms in Brazilian patients with rheumatoid arthritis: a pilot study.

    PubMed

    Angelo, H D; Gomes Silva, I I F; Oliveira, R D R; Louzada-Júnior, P; Donadi, E A; Crovella, S; Maia, M M D; de Souza, P R E; Sandrin-Garcia, P

    2015-10-01

    Polymorphisms in interleukin (IL)-18, IL-12 and interferon (IFN)-γ genes are associated with different levels of cytokines expression and have been associated with rheumatoid arthritis (RA). IL-18 +105 A/C, IL-12B +1188 A/C and IFN-γ +874 T/A polymorphisms were analyzed by restriction fragment length polymorphism-polymerase chain reaction (PCR) and amplification refractory mutation system PCR from 90 RA patients and 186 healthy individuals. There were significant differences to IL-18 +105 A/C polymorphism between the RA and control groups (odds ratio = 3.77; P < 0.0001). Individual carriers of the variant allele C had a 3.77-fold increased risk of for RA (P = 0.0032). No association was observed for IL-12B and IFN-γ polymorphisms. Our finds suggest a possible role for IL-18 polymorphism in the RA susceptibility in studied population. PMID:26302971

  2. Interleukin-18, interleukin-12B and interferon-γ gene polymorphisms in Brazilian patients with rheumatoid arthritis: a pilot study.

    PubMed

    Angelo, H D; Gomes Silva, I I F; Oliveira, R D R; Louzada-Júnior, P; Donadi, E A; Crovella, S; Maia, M M D; de Souza, P R E; Sandrin-Garcia, P

    2015-10-01

    Polymorphisms in interleukin (IL)-18, IL-12 and interferon (IFN)-γ genes are associated with different levels of cytokines expression and have been associated with rheumatoid arthritis (RA). IL-18 +105 A/C, IL-12B +1188 A/C and IFN-γ +874 T/A polymorphisms were analyzed by restriction fragment length polymorphism-polymerase chain reaction (PCR) and amplification refractory mutation system PCR from 90 RA patients and 186 healthy individuals. There were significant differences to IL-18 +105 A/C polymorphism between the RA and control groups (odds ratio = 3.77; P < 0.0001). Individual carriers of the variant allele C had a 3.77-fold increased risk of for RA (P = 0.0032). No association was observed for IL-12B and IFN-γ polymorphisms. Our finds suggest a possible role for IL-18 polymorphism in the RA susceptibility in studied population.

  3. New high fidelity polymerases from Thermococcus species.

    PubMed

    Griffiths, Kate; Nayak, Sunil; Park, Kyusung; Mandelman, David; Modrell, Brett; Lee, Jun; Ng, Bernie; Gibbs, Moreland D; Bergquist, Peter L

    2007-03-01

    Two DNA polymerase genes have been isolated from Thermococcus strains, Thermococcus zilligii from New Zealand, and the other, Thermococcus 'GT', a fast-growing strain isolated from the Galapagos trench. Both genes were isolated by genomic walking PCR, a technique that does not require expression of the gene product. Phylogenetic analysis of SSU rDNA showed that the two strains were not closely related, as confirmed by an examination of the DNA polymerase sequences. Inteinless versions of each gene were generated by overlap-extension PCR and transferred into plasmid expression vectors. The proteins were produced in an Escherichia coli strain with additional copies of tRNAs corresponding to rarely used codons and purified by standard chromatographic procedures. Both enzymes were able to support PCR, but the Thermococcus 'GT' polymerase required higher concentrations of template than the enzyme from T. zilligii. Both enzymes showed 3' to 5' exonuclease activity, which was abolished in the case of T. zilligii by mutating the aspartic acid at position 141 and the glutamic acid at position 143 to alanine. Both enzymes showed a significant increase in fidelity of replication compared to the family A Thermus aquaticus DNA polymerase, in agreement with other results reported for family B polymerases with proof-reading ability. PMID:16982200

  4. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity.

    PubMed

    Lu, Gang; He, Dong; Wang, Zengchao; Ou, Shudan; Yuan, Rong; Li, Shoujun

    2016-01-01

    An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells. PMID:27258298

  5. Cloning the Horse RNA Polymerase I Promoter and Its Application to Studying Influenza Virus Polymerase Activity

    PubMed Central

    Lu, Gang; He, Dong; Wang, Zengchao; Ou, Shudan; Yuan, Rong; Li, Shoujun

    2016-01-01

    An influenza virus polymerase reconstitution assay based on the human, dog, or chicken RNA polymerase I (PolI) promoter has been developed and widely used to study the polymerase activity of the influenza virus in corresponding cell types. Although it is an important member of the influenza virus family and has been known for sixty years, no studies have been performed to clone the horse PolI promoter or to study the polymerase activity of equine influenza virus (EIV) in horse cells. In our study, the horse RNA PolI promoter was cloned from fetal equine lung cells. Using the luciferase assay, it was found that a 500 bp horse RNA PolI promoter sequence was required for efficient transcription. Then, using the developed polymerase reconstitution assay based on the horse RNA PolI promoter, the polymerase activity of two EIV strains was compared, and equine myxovirus resistance A protein was identified as having the inhibiting EIV polymerase activity function in horse cells. Our study enriches our knowledge of the RNA PolI promoter of eukaryotic species and provides a useful tool for the study of influenza virus polymerase activity in horse cells. PMID:27258298

  6. DNA polymerase alpha and beta in the California urchin.

    PubMed Central

    Racine, F M; Morris, P W

    1978-01-01

    DNA polymerase alpha and beta were identified in the urchin, Strongylocentrotus purpuratus. The DNA polymerase beta sedimented at 3.4 S, constituted 5% of total DNA polymerase activity, and was resistant to N-ethylmaleimide and high ionic strength. The polymerase alpha sedimented at 6--8 S, was inhibited by N-ethylmalemide or 0.1 M (NH4)2SO4, and was dependent upon glycerol for preservation of activity. Both the polymerases alpha and beta were nuclear associated in embryos. The DNA polymerase alpha was markedly heterogeneous on DEAE-Sephadex ion exchange and showed three modal polymerase species. These polymerase alpha species were indistinguishable by template activity assays but the DNA polymerase associated ribonucleotidyl transferase (Biochemistry 75 : 3106-3113, 1976) was found predominantly with only one of the DNA polymerase alpha species. PMID:569291

  7. RNA polymerase II mediated transcription from the polymerase III promoters in short hairpin RNA expression vector

    SciTech Connect

    Rumi, Mohammad; Ishihara, Shunji . E-mail: si360405@med.shimane-u.ac.jp; Aziz, Monowar; Kazumori, Hideaki; Ishimura, Norihisa; Yuki, Takafumi; Kadota, Chikara; Kadowaki, Yasunori; Kinoshita, Yoshikazu

    2006-01-13

    RNA polymerase III promoters of human ribonuclease P RNA component H1, human U6, and mouse U6 small nuclear RNA genes are commonly used in short hairpin RNA (shRNA) expression vectors due their precise initiation and termination sites. During transient transfection of shRNA vectors, we observed that H1 or U6 promoters also express longer transcripts enough to express several reporter genes including firefly luciferase, green fluorescent protein EGFP, and red fluorescent protein JRed. Expression of such longer transcripts was augmented by upstream RNA polymerase II enhancers and completely inhibited by downstream polyA signal sequences. Moreover, the transcription of firefly luciferase from human H1 promoter was sensitive to RNA polymerase II inhibitor {alpha}-amanitin. Our findings suggest that commonly used polymerase III promoters in shRNA vectors are also prone to RNA polymerase II mediated transcription, which may have negative impacts on their targeted use.

  8. Inhibition of Poly(ADP-Ribose) Polymerase by Nucleic Acid Metabolite 7-Methylguanine

    PubMed Central

    Nilov, D. K.; Tararov, V. I.; Kulikov, A. V.; Zakharenko, A. L.; Gushchina, I. V.; Mikhailov, S. N.; Lavrik, O. I.; Švedas, V. K.

    2016-01-01

    The ability of 7-methylguanine, a nucleic acid metabolite, to inhibit poly(ADP-ribose)polymerase-1 (PARP-1) and poly(ADP-ribose)polymerase-2 (PARP-2) has been identified in silico and studied experimentally. The amino group at position 2 and the methyl group at position 7 were shown to be important substituents for the efficient binding of purine derivatives to PARPs. The activity of both tested enzymes, PARP-1 and PARP-2, was suppressed by 7-methylguanine with IC50 values of 150 and 50 μM, respectively. At the PARP inhibitory concentration, 7-methylguanine itself was not cytotoxic, but it was able to accelerate apoptotic death of BRCA1-deficient breast cancer cells induced by cisplatin and doxorubicin, the widely used DNA-damaging chemotherapeutic agents. 7-Methylguanine possesses attractive predictable pharmacokinetics and an adverse-effect profile and may be considered as a new additive to chemotherapeutic treatment. PMID:27437145

  9. Functional Diversification of Maize RNA Polymerase IV and V subtypes via Alternative Catalytic Subunits

    SciTech Connect

    Haag, Jeremy R.; Brower-Toland, Brent; Krieger, Elysia K.; Sidorenko, Lyudmila; Nicora, Carrie D.; Norbeck, Angela D.; Irsigler, Andre; LaRue, Huachun; Brzeski, Jan; Mcginnis, Karen A.; Ivashuta, Sergey; Pasa-Tolic, Ljiljana; Chandler, Vicki L.; Pikaard, Craig S.

    2014-10-01

    Unlike nuclear multisubunit RNA polymerases I, II, and III, whose subunit compositions are conserved throughout eukaryotes, plant RNA polymerases IV and V are nonessential, Pol II-related enzymes whose subunit compositions are still evolving. Whereas Arabidopsis Pols IV and V differ from Pol II in four or five of their 12 subunits, respectively, and differ from one another in three subunits, proteomic ana- lyses show that maize Pols IV and V differ from Pol II in six subunits but differ from each other only in their largest subunits. Use of alternative catalytic second subunits, which are nonredundant for development and paramutation, yields at least two sub- types of Pol IV and three subtypes of Pol V in maize. Pol IV/Pol V associations with MOP1, RMR1, AGO121, Zm_DRD1/CHR127, SHH2a, and SHH2b extend parallels between paramutation in maize and the RNA-directed DNA methylation pathway in Arabidopsis.

  10. Relationship between TBX20 gene polymorphism and congenital heart disease.

    PubMed

    Yang, X F; Zhang, Y F; Zhao, C F; Liu, M M; Si, J P; Fang, Y F; Xing, W W; Wang, F L

    2016-01-01

    Congenital heart disease in children is a type of birth defect. Previous studies have suggested that the transcription factor, TBX20, is involved in the occurrence and development of congenital heart disease in children; however, the specific regulatory mechanisms are yet to be evaluated. Hence, this study aimed to evaluate the relationship between the TBX20 polymorphism and the occurrence and development of congenital heart disease. The TBX20 gene sequence was obtained from the NCBI database and the polymorphic locus candidate was predicted. Thereafter, the specific gene primers were designed for the restriction fragment length polymorphism-polymerase chain reaction (RFLP-PCR) of DNA extracted from the blood of 80 patients with congenital heart disease and 80 controls. The results of the PCR were subjected to correlation analysis to identify the differences between the amplicons and to determine the relationship between the TBX20 gene polymorphism and congenital heart disease. One of the single nucleotide polymorphic locus was found to be rs3999950: c.774T>C (Ala265Ala). The TC genotype frequency in the patients was higher than that in the controls, similar to that for the C locus. The odds ratio of the TC genotypes was above 1, indicating that the presence of the TC genotype increases the incidence of congenital heart diseases. Thus, rs3999950 may be associated with congenital heart disease, and TBX20 may predispose children to the defect. PMID:27323105

  11. Klotho gene polymorphisms are related to colorectal cancer susceptibility

    PubMed Central

    Liu, Chang; Cui, Wei; Wang, Li; Yan, Lei; Ruan, Xinjian; Liu, Yanfang; Jia, Xiaoyan; Zhang, Xia

    2015-01-01

    Aim: The purpose of this study was to investigate the relationship of Klotho gene G-395A and C1818T polymorphisms with colorectal cancer (CRC) susceptibility. Methods: 125 CRC patients and 125 controls were enrolled in the study. G-395A and C1818T polymorphisms were genotyped with polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technology. Haploview software was utilized to conduct linkage disequilibrium and haplotype analysis. Odds ratio (OR) and 95% confidence interval (95% CI) were used to analyze the correlation of genotypes and haplotypes with CRC susceptibility. Results: AA and GA genotypes of G-395A polymorphisms were related with CRC risk (AA: OR = 4.161, 95% CI = 1.437-12.053; GA: OR = 1.958, 95% CI = 1.133-3.385). The frequency of A allele was much higher in case group, compared with controls (31.2% vs.17.6%) and the value of OR AND 95% CI suggested that A allele served as a risk factor for CRC (OR = 2.123, 95% CI = 1.393-3.236). Haplotypes analysis indicated that A-C and A-T haplotypes were significantly associated with risk of CRC (OR = 1.822, 95% CI = 1.124-2.954; OR = 2.877, 95% CI = 1.340-6.176). Conclusion: G-395A polymorphism of Klotho gene could increase the risk of CRC. PMID:26261651

  12. [RNA polymerase as a molecular machine].

    PubMed

    Spirin, A S

    2002-01-01

    Structure and function of DNA-dependent RNA polymerase is considered in terms of a conveying molecular machine. The use of mechanical energy and mechanical devices, such as "power-stroke motor", is supposed unlikely in the conveying function of RNA polymerase, as well as other molecular machines. Brownian motion and thermal mobility of macromolecules and their parts are postulated as the only motive impulse at the molecular level. Binding of substrates and subsequent chemical reaction as the energy input may provide successive selection and fixation of alternative conformational states of the enzyme complex thus providing the directionality of the conveyance ("Brownian ratchet mechanism"). The following sequence of events "substrate binding--fixation of a certain conformational state--chemical reaction--fixation of an alternative conformational state--translocation (dissociation and downstream reassociation) of product-template duplex" is proposed as the principal scheme of the forward movement of RNA polymerase along DNA template.

  13. Error-prone polymerase activity causes multinucleotide mutations in humans

    PubMed Central

    Nielsen, Rasmus

    2014-01-01

    About 2% of human genetic polymorphisms have been hypothesized to arise via multinucleotide mutations (MNMs), complex events that generate SNPs at multiple sites in a single generation. MNMs have the potential to accelerate the pace at which single genes evolve and to confound studies of demography and selection that assume all SNPs arise independently. In this paper, we examine clustered mutations that are segregating in a set of 1092 human genomes, demonstrating that the signature of MNM becomes enriched as large numbers of individuals are sampled. We estimate the percentage of linked SNP pairs that were generated by simultaneous mutation as a function of the distance between affected sites and show that MNMs exhibit a high percentage of transversions relative to transitions, findings that are reproducible in data from multiple sequencing platforms and cannot be attributed to sequencing error. Among tandem mutations that occur simultaneously at adjacent sites, we find an especially skewed distribution of ancestral and derived alleles, with GC → AA, GA → TT, and their reverse complements making up 27% of the total. These mutations have been previously shown to dominate the spectrum of the error-prone polymerase Pol ζ, suggesting that low-fidelity DNA replication by Pol ζ is at least partly responsible for the MNMs that are segregating in the human population. We develop statistical estimates of MNM prevalence that can be used to correct phylogenetic and population genetic inferences for the presence of complex mutations. PMID:25079859

  14. Centromeric polymerase III transcription units in Chironomus pallidivittatus.

    PubMed Central

    Rovira, C; Edström, J E

    1996-01-01

    Cp1 is a polymorphic short interspersed repeat (SINE) which is distributed over the whole genome of the dipteran Chironomus pallidivittatus, and is particularly abundant in the centromeres. It contains two different sequence modules, one of which, the B module, has a polymerase III internal control region (ICR) typical for tRNA genes (A and B box). Such sequence motifs are common in SINEs and assumed to function in RNA-mediated transposition. In the present case, however, several structural features speak for another role. An investigation of the transcription of the B module shows that it encodes a 99 nt RNA species in vivo, Cp1-RNA, terminating within the module. The transcription unit is likely to have evolved from a pre-tRNA gene and the transcript has sequence similarities to non-processed pre-tRNA. Most of the in vitro transcription is eliminated by deletion or substitution mutation of an upstream TATA box, present within the B module, as well as by changing either the A or B box. The properties of the transcript suggest that it does not have a role in transposition but may have some other function, perhaps in the centromere. PMID:8649983

  15. Highly selective affinity labelling of RNA polymerase B (II) from wheat germ.

    PubMed

    Grachev, M A; Hartmann, G R; Maximova, T G; Mustaev, A A; Schäffner, A R; Sieber, H; Zaychikov, E F

    1986-05-12

    DNA-dependent RNA polymerase B (II) from wheat germ was modified by incubation with 4-[N-(beta-hydroxyethyl)-N-methyl]benzaldehyde esters of AMP, ADP or ATP, followed by reduction with NaBH4. Reaction of the modified enzyme with [alpha-32P]UTP in the presence of various DNA templates led to a highly selective affinity labelling of the subunit with Mr 140 000 by covalently linked ApU. Labelling was inhibited by 1 microgram/ml alpha-amanitin.

  16. Accounting for population stratification in DNA methylation studies.

    PubMed

    Barfield, Richard T; Almli, Lynn M; Kilaru, Varun; Smith, Alicia K; Mercer, Kristina B; Duncan, Richard; Klengel, Torsten; Mehta, Divya; Binder, Elisabeth B; Epstein, Michael P; Ressler, Kerry J; Conneely, Karen N

    2014-04-01

    DNA methylation is an important epigenetic mechanism that has been linked to complex diseases and is of great interest to researchers as a potential link between genome, environment, and disease. As the scale of DNA methylation association studies approaches that of genome-wide association studies, issues such as population stratification will need to be addressed. It is well-documented that failure to adjust for population stratification can lead to false positives in genetic association studies, but population stratification is often unaccounted for in DNA methylation studies. Here, we propose several approaches to correct for population stratification using principal components (PCs) from different subsets of genome-wide methylation data. We first illustrate the potential for confounding due to population stratification by demonstrating widespread associations between DNA methylation and race in 388 individuals (365 African American and 23 Caucasian). We subsequently evaluate the performance of our PC-based approaches and other methods in adjusting for confounding due to population stratification. Our simulations show that (1) all of the methods considered are effective at removing inflation due to population stratification, and (2) maximum power can be obtained with single-nucleotide polymorphism (SNP)-based PCs, followed by methylation-based PCs, which outperform both surrogate variable analysis and genomic control. Among our different approaches to computing methylation-based PCs, we find that PCs based on CpG sites chosen for their potential to proxy nearby SNPs can provide a powerful and computationally efficient approach to adjust for population stratification in DNA methylation studies when genome-wide SNP data are unavailable.

  17. Accounting for population stratification in DNA methylation studies.

    PubMed

    Barfield, Richard T; Almli, Lynn M; Kilaru, Varun; Smith, Alicia K; Mercer, Kristina B; Duncan, Richard; Klengel, Torsten; Mehta, Divya; Binder, Elisabeth B; Epstein, Michael P; Ressler, Kerry J; Conneely, Karen N

    2014-04-01

    DNA methylation is an important epigenetic mechanism that has been linked to complex diseases and is of great interest to researchers as a potential link between genome, environment, and disease. As the scale of DNA methylation association studies approaches that of genome-wide association studies, issues such as population stratification will need to be addressed. It is well-documented that failure to adjust for population stratification can lead to false positives in genetic association studies, but population stratification is often unaccounted for in DNA methylation studies. Here, we propose several approaches to correct for population stratification using principal components (PCs) from different subsets of genome-wide methylation data. We first illustrate the potential for confounding due to population stratification by demonstrating widespread associations between DNA methylation and race in 388 individuals (365 African American and 23 Caucasian). We subsequently evaluate the performance of our PC-based approaches and other methods in adjusting for confounding due to population stratification. Our simulations show that (1) all of the methods considered are effective at removing inflation due to population stratification, and (2) maximum power can be obtained with single-nucleotide polymorphism (SNP)-based PCs, followed by methylation-based PCs, which outperform both surrogate variable analysis and genomic control. Among our different approaches to computing methylation-based PCs, we find that PCs based on CpG sites chosen for their potential to proxy nearby SNPs can provide a powerful and computationally efficient approach to adjust for population stratification in DNA methylation studies when genome-wide SNP data are unavailable. PMID:24478250

  18. Association between HRH4 polymorphisms and ankylosing spondylitis susceptibility

    PubMed Central

    Ran, Bo; Wang, Yongcheng; Zhang, Yonggang; Mao, Keya; Wang, Yan

    2015-01-01

    Target: The purpose of the study was to investigate the association between the histamine H4 receptor (HRH4) polymorphisms and the susceptibility to ankylosing spondylitis (AS). Methods: Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to analyze the HRH4 rs8088140 and rs657132 polymorphisms. Linkage disequilibrium and haplotype analyses were conducted with Haploview software. The genotypes distributions of HRH4 polymorphisms in the control group were tested by Hardy-Weinberg equilibrium (HWE), allele, genotype and haplotype frequencies between the cases and control groups were compared by χ2 test. The controls were matched with cases by age and gender. The relative risk of AS with HRH4 polymorphisms was represented by odds ratio (OR) with 95% confidence interval (CI) calculated by χ2 test. Results: The genotypes distributions of HRH4 rs8088140, rs657132 polymorphisms in controls conformed to HWE. The frequency of rs657132 AA genotype in the case group was obviously higher than that in the control group (P=0.040), and so was the A allele (OR=2.572, 95% CI=1.475-4.486, P=0.022). The frequency differences of A-A haplotype between two groups had statistical significance (P=0.011, OR=2.071, 95% CI=1.172-3.660) through haplotype analysis, indicating A-A might be the susceptible haplotype to AS. Conclusion: The AA genotypes of HRH4 rs657132 polymorphism may be the susceptible factors for AS, and rs657132 plays a role in generation of AS. In addition, A-A haplotype in rs8088140-rs657132 is also increased the risk of AS. PMID:26823878

  19. MTHFR genetic polymorphism increases the risk of preterm delivery

    PubMed Central

    Nan, Yanrong; Li, Hongmei

    2015-01-01

    Aims: This study aimed to investigate the association between the methylene tetrahydrofolate reductase (MTHFR) gene C677T and A1298C polymorphisms and premature delivery susceptibility. Methods: With matched age and gender, 108 premature delivery pregnant women as cases and 108 healthy pregnant women as controls were recruited in this case-control study. The cases and controls had same gestational weeks. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was adopted to analyze C677T and A1298C polymorphisms of the participants. Linkage disequilibrium (LD) and haplotype analysis were conducted by Haploview software. The differences for frequencies of gene type, allele and haplotypes in cases and controls were tested by chi-square test. The relevant risk of premature delivery was represented by odds ratios (ORs) with 95% confidence intervals (95% CIs). Results: TT gene type frequency of C677T polymorphsim was higher in cases than the controls (P=0.004, OR=3.077, 95% CI=1.469-6.447), so was allele T (P=0.002, OR=1.853, 95% CI=1.265-2.716). Whereas, CC gene type of A1298C polymorphism had a lower distribution in cases than the controls (P=0.008, OR=0.095, 95% CI=0.012-0.775), so was allele C (P=0.047, OR=0.610, 95% CI=0.384-0.970). Haplotype analysis and linkage disequilibrium test conducted on the alleles of two polymorphisms in MTHFR gene, we discovered that haplotype T-A had a higher distribution in cases, which indicated that susceptible haplotype T-A was the candidate factor for premature delivery. Conclusions: Gene type TT of MTHFR C677T polymorphism might make premature delivery risk rise while gene type CC of A1298C polymorphism might have protective influence on premature delivery. PMID:26261642

  20. Role of DNA methylation in hybrid vigor in Arabidopsis thaliana

    PubMed Central

    Kawanabe, Takahiro; Ishikura, Sonoko; Miyaji, Naomi; Sasaki, Taku; Wu, Li Min; Itabashi, Etsuko; Takada, Satoko; Shimizu, Motoki; Takasaki-Yasuda, Takeshi; Osabe, Kenji; Peacock, W. James; Dennis, Elizabeth S.; Fujimoto, Ryo

    2016-01-01

    Hybrid vigor or heterosis refers to the superior performance of F1 hybrid plants over their parents. Heterosis is particularly important in the production systems of major crops. Recent studies have suggested that epigenetic regulation such as DNA methylation is involved in heterosis, but the molecular mechanism of heterosis is still unclear. To address the epigenetic contribution to heterosis in Arabidopsis thaliana, we used mutant genes that have roles in DNA methylation. Hybrids between C24 and Columbia-0 (Col) without RNA polymerase IV (Pol IV) or methyltransferase I (MET1) function did not reduce the level of biomass heterosis (as evaluated by rosette diameter). Hybrids with a mutation in decrease in dna methylation 1 (ddm1) showed a decreased heterosis level. Vegetative heterosis in the ddm1 mutant hybrid was reduced but not eliminated; a complete reduction could result if there was a change in methylation at all loci critical for generating the level of heterosis, whereas if only a proportion of the loci have methylation changes there may only be a partial reduction in heterosis. PMID:27791039

  1. Methylation of IRAK3 is a novel prognostic marker in hepatocellular carcinoma

    PubMed Central

    Kuo, Chih-Chi; Shih, Yu-Lueng; Su, Her-Young; Yan, Ming-De; Hsieh, Chung-Bao; Liu, Chin-Yu; Huang, Wei-Ting; Yu, Mu-Hsien; Lin, Ya-Wen

    2015-01-01

    AIM: To examine the methylation levels of interleukin-1 receptor-associated kinase 3 (IRAK3) and GLOXD1 and their potential clinical applications in hepatocellular carcinoma (HCC). METHODS: mRNA expression and promoter methylation of IRAK3 and GLOXD1 in HCC cells were analyzed by reverse transcription-polymerase chain reaction (RT-PCR) and methylation-specific PCR (MSP), respectively. Using pyrosequencing results, we further established a quantitative MSP (Q-MSP) system for the evaluation of IRAK3 and GLOXD1 methylation in 29 normal controls and 160 paired HCC tissues and their adjacent nontumor tissues. We also calculated Kaplan-Meier survival curves to determine the applications of gene methylation in the prognosis of HCC. RESULTS: IRAK3 and GLOXD1 expression was partially restored in several HCC cell lines after treatment with 5-aza-2′-deoxycytidine (DNA methyltransferase inhibitor; 5DAC). A partial decrease in the methylated band was also observed in the HCC cell lines after 5DAC treatment. Using GLOXD1 as an example, we found a significant correlation between the data obtained from the methylation array and from pyrosequencing. The methylation frequency of IRAK3 and GLOXD1 in HCC tissues was 46.9% and 63.8%, respectively. Methylation of IRAK3 was statistically associated with tumor stage. Moreover, HCC patients with IRAK3 methylation had a trend toward poor 3-year disease-free survival (P < 0.05). CONCLUSION: IRAK3 and GLOXD1 were frequently methylated in HCC tissues compared to normal controls and nontumor tissues. IRAK3 methylation was associated with tumor stage and poor prognosis of patients. These data suggest that IRAK3 methylation is a novel prognostic marker in HCC. PMID:25852282

  2. Age-related methylation profiles of equine blood leukocytes in the RNASEL locus.

    PubMed

    Ząbek, T; Semik, E; Szmatoła, T; Oklejewicz, B; Fornal, A; Bugno-Poniewierska, M

    2016-08-01

    Methylation profiles across three CpG islands of the RNASEL gene were determined in blood leukocyte samples of Anglo-Arabian and Hucul horses. Bisulfite sequencing revealed hypomethylated state of the RNASEL promoter coinciding with methylated CpG island placed inside the gene. Several CpG sites were identified for which the methylation state was influenced by DNA polymorphism. Two of them showed monoallelic methylation. One of the CpG sites revealed functional polymorphism. A number of partially methylated CpG sites have been observed in the promoter area of RNASEL, which were used for the comparison of breed- and age-related effects. Clone bisulfite sequencing of blood leukocyte samples collected at different ages from particular individuals of AA and HC breeds and, also, BSPCR sequencing of 50 samples of juvenile and old AA and HC horses revealed increased methylation in particular CpG sites during aging. The age-related heterogeneity of white blood cells was hypothesized as being one of the potential causes of observed variability of methylation profiles in the RNASEL promoter. PMID:26553552

  3. Quantification of regional DNA methylation by liquid chromatography/tandem mass spectrometry.

    PubMed

    Liu, Zhongfa; Wu, Jiejun; Xie, Zhiliang; Liu, Shujun; Fan-Havard, Patty; Huang, Tim H-M; Plass, Christoph; Marcucci, Guido; Chan, Kenneth K

    2009-08-15

    Promoter hypermethylation-associated tumor suppressor gene (TSG) silencing has been explored as a therapeutic target for hypomethylating agents. Promoter methylation change may serve as a pharmacodynamic endpoint for evaluation of the efficacy of these agents and predict the patient's clinical response. Here a liquid chromatography-tandem mass spectrometry (LC-MS/MS) assay has been developed for quantitative regional DNA methylation analysis using the molar ratio of 5-methyl-2'-deoxycytidine (5mdC) to 2'-deoxycytidine (2dC) in the enzymatic hydrolysate of fully methylated bisulfite-converted polymerase chain reaction (PCR) amplicons as the methylation indicator. The assay can differentiate 5% of promoter methylation level with an intraday precision ranging from 3 to 16% using two TSGs: HIN-1 and RASSF1A. This method was applied to characterize decitabine-induced promoter DNA methylation changes of these two TSGs in a breast cancer MCF-7 cell line. Promoter methylation of these TSGs was found to decrease in a dose-dependent manner. Correspondingly, the expression of these TSGs was enhanced. The sensitivity and reproducibility of the method make it a valuable tool for specific gene methylation analysis that could aid characterization of hypomethylating activity on specific genes by hypomethylating agents in a clinical setting.

  4. A movie of RNA polymerase II transcription.

    PubMed

    Cheung, Alan C M; Cramer, Patrick

    2012-06-22

    We provide here a molecular movie that captures key aspects of RNA polymerase II initiation and elongation. To create the movie, we combined structural snapshots of the initiation-elongation transition and of elongation, including nucleotide addition, translocation, pausing, proofreading, backtracking, arrest, reactivation, and inhibition. The movie reveals open questions about the mechanism of transcription and provides a useful teaching tool.

  5. Polymerase Chain Reaction for Educational Settings.

    ERIC Educational Resources Information Center

    Garrison, Stephen J.; dePamphillis, Claude

    1994-01-01

    Suggests the incorporation of the Polymerase Chain Reaction (PCR) technique into high school and college biology laboratories. Discusses the following sections: (1) current PCR applications; (2) PCR technique; (3) Manual and Machine PCR; (4) Manual PCR Preparations and Procedure; (5) Materials, Supplies, and Recipes; (6) Primer Selection; and (7)…

  6. A polymerase engineered for bisulfite sequencing

    PubMed Central

    Millar, Doug; Christova, Yonka; Holliger, Philipp

    2015-01-01

    Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains. PMID:26271989

  7. RNA polymerase and the regulation of transcription

    SciTech Connect

    Reznikoff, W.S.; Gross, C.A.; Burgess, R.R.; Record, M.T.; Dahlberg, J.E.; Wickens, M.P.

    1987-01-01

    This book consists of eight sections, each containing several papers. The section titles are: RNA Polymerases; Transcription Initiation - Bacterial; Regulation of Bacterial Transcription Initiation; Stable RNA Synthesis in Eukaryotes: Chromatin Structure; Promoters; Enhancers; and the Global Control of Eukaryotic Transcription; Specific Eukaryotic Transcription Factors; Termination of Transcription; and Short Communications.

  8. Determining Annealing Temperatures for Polymerase Chain Reaction

    ERIC Educational Resources Information Center

    Porta, Angela R.; Enners, Edward

    2012-01-01

    The polymerase chain reaction (PCR) is a common technique used in high school and undergraduate science teaching. Students often do not fully comprehend the underlying principles of the technique and how optimization of the protocol affects the outcome and analysis. In this molecular biology laboratory, students learn the steps of PCR with an…

  9. A polymerase engineered for bisulfite sequencing.

    PubMed

    Millar, Doug; Christova, Yonka; Holliger, Philipp

    2015-12-15

    Bisulfite sequencing is a key methodology in epigenetics. However, the standard workflow of bisulfite sequencing involves heat and strongly basic conditions to convert the intermediary product 5,6-dihydrouridine-6-sulfonate (dhU6S) (generated by reaction of bisulfite with deoxycytidine (dC)) to uracil (dU). These harsh conditions generally lead to sample loss and DNA damage while milder conditions may result in incomplete conversion of intermediates to uracil. Both can lead to poor recovery of bisulfite-treated DNA by the polymerase chain reaction (PCR) as either damaged DNA and/or intermediates of bisulfite treatment are poor substrate for standard DNA polymerases. Here we describe an engineered DNA polymerase (5D4) with an enhanced ability to replicate and PCR amplify bisulfite-treated DNA due to an ability to bypass both DNA lesions and bisulfite intermediates, allowing significantly milder conversion conditions and increased sensitivity in the PCR amplification of bisulfite-treated DNA. Incorporation of the 5D4 DNA polymerase into the bisulfite sequencing workflow thus promises significant sensitivity and efficiency gains.

  10. RNA polymerase II ternary transcription complexes generated in vitro.

    PubMed Central

    Ackerman, S; Bunick, D; Zandomeni, R; Weinmann, R

    1983-01-01

    Ternary transcription complexes have been formed with a HeLa cell extract, a specific DNA template, and nucleoside triphosphates. The assay depends on the formation of sarkosyl-resistant initiation complexes which contain RNA polymerase II, template DNA, and radioactive nucleoside triphosphates. Separation from the other elements in the in vitro reaction is achieved by electrophoresis in agarose - 0.25% sarkosyl gels. The mobility of the ternary complexes in this system cannot be distinguished from naked DNA. Formation of this complex is dependent on all parameters necessary for faithful in vitro transcription. Complexes are formed with both the plasmid vector and the specific adenovirus DNA insert containing a eucaryotic promoter. The formation of the complex on the eucaryotic DNA is sequence-dependent. An undecaribonucleotide predicted from the template DNA sequence remains associated with the DNA in the ternary complex and can be isolated if the chain terminator 3'-0-methyl GTP is used, or after T1 ribonuclease treatment of the RNA, or if exogenous GTP is omitted from the in vitro reaction. This oligonucleotide is not detected in association with the plasmid vector. Phosphocellulose fractionation of the extract indicates that at least one of the column fractions required for faithful runoff transcription is required for complex formation. A large molar excess of abortive initiation events was detected relative to the level of productive transcription events, indicating a 40-fold higher efficiency of transcription initiation vs. elongation. Images PMID:6193489

  11. Patterns of inheritance with RAPD molecular markers reveal novel types of polymorphism in the honey bee.

    PubMed

    Hunt, G J; Page, R E

    1992-10-01

    The polymerase chain reaction (PCR) was used to generate random amplified polymorphic DNA (RAPD) from honey bee DNA samples in order to follow the patterns of inheritance of RAPD markers in a haplodiploid insect. The genomic DNA samples from two parental bees, a haploid drone and a diploid queen, were screened for polymorphism with 68 different tennucleotide primers of random sequence. Parents were scored for the presence or absence of individual bands. An average of 6.3 bands and 1.3 polymorphisms for presence/absence were observed per primer between the parents. Thirteen of these primers were used to determine the inheritance of RAPD marker alleles in the resulting progeny and in haploid drones from a daughter queen. Four types of polymorphisms were observed. Polymorphisms for band presence/absence as well as for band brightness were inherited as dominant markers, meeting Mendelian expectations in haploid and diploid progeny. Polymorphisms for fragment-length were also observed. These segregated in a near 1∶1 ratio in drone progeny. The last type of polymorphism was manifested as a diploid-specific band. Mixing of amplification products after PCR showed that the diploid-specific band was the result of heteroduplex formation from the DNA of alternate alleles in heterozygotes. In two of the four cases of heteroduplex formation, the alternative alleles were manifested as small fragment-length polymorphisms, resulting in co-dominant markers. This is the first demonstration that a proportion of RAPD markers are not inherited in a dominant fashion.

  12. In vitro Methylation Assay to Study Protein Arginine Methylation

    PubMed Central

    Bikkavilli, Rama Kamesh; Avasarala, Sreedevi; Van Scoyk, Michelle; Karuppusamy Rathinam, Manoj Kumar; Tauler, Jordi; Borowicz, Stanley; Winn, Robert A.

    2014-01-01

    Protein arginine methylation is one of the most abundant post-translational modifications in the nucleus. Protein arginine methylation can be identified and/or determined via proteomic approaches, and/or immunoblotting with methyl-arginine specific antibodies. However, these techniques sometimes can be misleading and often provide false positive results. Most importantly, these techniques cannot provide direct evidence in support of the PRMT substrate specificity. In vitro methylation assays, on the other hand, are useful biochemical assays, which are sensitive, and consistently reveal if the identified proteins are indeed PRMT substrates. A typical in vitro methylation assay includes purified, active PRMTs, purified substrate and a radioisotope labeled methyl donor (S-adenosyl-L-[methyl-3H] methionine). Here we describe a step-by-step protocol to isolate catalytically active PRMT1, a ubiquitously expressed PRMT family member. The methyl transferase activities of the purified PRMT1 were later tested on Ras-GTPase activating protein binding protein 1 (G3BP1), a known PRMT substrate, in the presence of S-adenosyl-L-[methyl-3H] methionine as the methyl donor. This protocol can be employed not only for establishing the methylation status of novel physiological PRMT1 substrates, but also for understanding the basic mechanism of protein arginine methylation. PMID:25350748

  13. Clonality in myeloproliferative disorders: Analysis by means of polymerase chain reaction

    SciTech Connect

    Gilliland, D.G.; Blanchard, K.L.; Levy, J.; Perrin, S.; Bunn, H.F. )

    1991-08-01

    The myeloproliferative syndromes are acquired disorders of hematopoiesis that provide insights into the transition from somatic cell mutation to neoplasia. The clonal origin of specific blood cells can be assessed in patients with X chromosome-linked polymorphisms, taking advantage of random inactivation of the X chromosome. The authors have adapted the PCR for determination of clonality on as few as 100 cells, including individual colonies grown in culture. Amplifying a polymorphic portion of the X chromosome-linked phosphoglycerate kinase (PGK) gene after selective digestion of the active X chromosome with a methylation-sensitive restriction enzyme gave results fully concordant with standard Southern blotting of DNA samples form normal (polyclonal) polymorphonuclear cells (PMN) as well as clonal PMN from patients with myelodysplastic syndrome and polycythemia vera (PCV). They have used this technique to demonstrate heterogeneity of lineage involvement in patients with PCV. The same clinical phenotype may arise from clonal proliferation of different hematopoietic progenitors.

  14. Methyl isobutyl ketone (MIBK)

    Integrated Risk Information System (IRIS)

    EPA / 635 / R - 03 / 002 TOXICOLOGICAL REVIEW OF METHYL ISOBUTYL KETONE ( CAS No . 108 - 10 - 1 ) In Support of Summary Information on the Integrated Risk Information System ( IRIS ) March 2003 U.S . Environmental Protection Agency Washington DC DISCLAIMER This document has been reviewed in accordan

  15. Haloxyfop-methyl

    Integrated Risk Information System (IRIS)

    Haloxyfop - methyl ; CASRN 69806 - 40 - 2 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinoge

  16. Thiophanate-methyl

    Integrated Risk Information System (IRIS)

    Thiophanate - methyl ; CASRN 23564 - 05 - 8 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcino

  17. Chloromethyl methyl ether (CMME)

    Integrated Risk Information System (IRIS)

    Chloromethyl methyl ether ( CMME ) ; CASRN 107 - 30 - 2 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments fo

  18. Methyl ethyl ketone (MEK)

    Integrated Risk Information System (IRIS)

    Methyl ethyl ketone ( MEK ) ( CASRN 78 - 93 - 3 ) Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Nonc

  19. Pirimiphos-methyl

    Integrated Risk Information System (IRIS)

    Pirimiphos - methyl ; CASRN 29232 - 93 - 7 Human health assessment information on a chemical substance is included in the IRIS database only after a comprehensive review of toxicity data , as outlined in the IRIS assessment development process . Sections I ( Health Hazard Assessments for Noncarcinog

  20. DNA Methylation in Osteoarthritis.

    PubMed

    den Hollander, Wouter; Meulenbelt, Ingrid

    2015-12-01

    Osteoarthritis (OA) is a prevalent disease of articular joints and primarily characterized by degradation and calcification of articular cartilage. Presently, no effective treatment other than pain relief exists and patients ultimately need to undergo replacement surgery of the affected joint. During disease progression articular chondrocytes, the single cell type present in articular cartilage, show altered transcriptional profiles and undergo phenotypic changes that resemble the terminal differentiation route apparent in growth plate chondrocytes. Hence, given its prominent function in both regulating gene expression and maintaining cellular phenotypes, DNA methylation of CpG dinucleotides is intensively studied in the context of OA. An increasing number of studies have been published that employed a targeted approach on genes known to play a role in OA pathophysiology. As of such, it has become clear that OA responsive DNA methylation changes seem to mediate disease associated aberrant gene expression. Furthermore, established OA susceptibility alleles such as GDF5 and DIO2 appear to confer OA risk via DNA methylation and respective pathophysiological expression changes. In more recent years, genome wide profiling of DNA methylation in OA affected articular cartilage has emerged as a powerful tool to address the epigenetic changes in their entirety, which has resulted in the identification of putative patient subgroups as well as generic OA associated pathways. PMID:27019616

  1. Kenaf methyl esters

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Additional or alternative feedstocks are one of the major areas of interest regarding biodiesel. In this paper, for the first time, the fuel properties of kenaf (Hibiscus cannabinus L.) seed oil methyl esters are comprehensively reported. This biodiesel is also relatively unique by containing small ...

  2. Cytosine Methylation Alteration in Natural Populations of Leymus chinensis Induced by Multiple Abiotic Stresses

    PubMed Central

    Yu, Yingjie; Yang, Xuejiao; Wang, Huaying; Shi, Fengxue; Liu, Ying; Liu, Jushan; Li, Linfeng; Wang, Deli; Liu, Bao

    2013-01-01

    Background Human activity has a profound effect on the global environment and caused frequent occurrence of climatic fluctuations. To survive, plants need to adapt to the changing environmental conditions through altering their morphological and physiological traits. One known mechanism for phenotypic innovation to be achieved is environment-induced rapid yet inheritable epigenetic changes. Therefore, the use of molecular techniques to address the epigenetic mechanisms underpinning stress adaptation in plants is an important and challenging topic in biological research. In this study, we investigated the impact of warming, nitrogen (N) addition, and warming+nitrogen (N) addition stresses on the cytosine methylation status of Leymus chinensis Tzvel. at the population level by using the amplified fragment length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP) and retrotransposon based sequence-specific amplification polymorphism (SSAP) techniques. Methodology/Principal Findings Our results showed that, although the percentages of cytosine methylation changes in SSAP are significantly higher than those in MSAP, all the treatment groups showed similar alteration patterns of hypermethylation and hypomethylation. It meant that the abiotic stresses have induced the alterations in cytosine methylation patterns, and the levels of cytosine methylation changes around the transposable element are higher than the other genomic regions. In addition, the identification and analysis of differentially methylated loci (DML) indicated that the abiotic stresses have also caused targeted methylation changes at specific loci and these DML might have contributed to the capability of plants in adaptation to the abiotic stresses. Conclusions/Significance Our results demonstrated that abiotic stresses related to global warming and nitrogen deposition readily evoke alterations of cytosine methylation, and which may provide a molecular basis for rapid adaptation by

  3. Disappearing Polymorphs Revisited

    PubMed Central

    Bučar, Dejan-Krešimir; Lancaster, Robert W; Bernstein, Joel

    2015-01-01

    Nearly twenty years ago, Dunitz and Bernstein described a selection of intriguing cases of polymorphs that disappear. The inability to obtain a crystal form that has previously been prepared is indeed a frustrating and potentially serious problem for solid-state scientists. This Review discusses recent occurrences and examples of disappearing polymorphs (as well as the emergence of elusive crystal forms) to demonstrate the enduring relevance of this troublesome, but always captivating, phenomenon in solid-state research. A number of these instances have been central issues in patent litigations. This Review, therefore, also highlights the complex relationship between crystal chemistry and the law. PMID:26031248

  4. DNA Methylation and Cancer Diagnosis

    PubMed Central

    Delpu, Yannick; Cordelier, Pierre; Cho, William C.; Torrisani, Jérôme

    2013-01-01

    DNA methylation is a major epigenetic modification that is strongly involved in the physiological control of genome expression. DNA methylation patterns are largely modified in cancer cells and can therefore be used to distinguish cancer cells from normal tissues. This review describes the main technologies available for the detection and the discovery of aberrantly methylated DNA patterns. It also presents the different sources of biological samples suitable for DNA methylation studies. We discuss the interest and perspectives on the use of DNA methylation measurements for cancer diagnosis through examples of methylated genes commonly documented in the literature. The discussion leads to our consideration for why DNA methylation is not commonly used in clinical practice through an examination of the main requirements that constitute a reliable biomarker. Finally, we describe the main DNA methylation inhibitors currently used in clinical trials and those that exhibit promising results. PMID:23873296

  5. The Role of DNA Methylation in Xylogenesis in Different Tissues of Poplar.

    PubMed

    Wang, Qingshi; Ci, Dong; Li, Tong; Li, Peiwen; Song, YuePeng; Chen, Jinhui; Quan, Mingyang; Zhou, Daling; Zhang, Deqiang

    2016-01-01

    In trees, xylem tissues play a key role in the formation of woody tissues, which have important uses for pulp and timber production; also DNA methylation plays an important part in gene regulation during xylogenesis in trees. In our study, methylation-sensitive amplified polymorphism (MSAP) analysis was used to analyze the role cytosine methylation plays in wood formation in the commercially important tree species Populus tomentosa. This analysis compared the methylation patterns between xylem tissues (developing xylem and mature xylem) and non-xylem tissues (cambium, shoot apex, young leaf, mature leaf, phloem, root, male catkin, and female catkin) and found 10,316 polymorphic methylation sites. MSAP identified 132 candidate genes with the same methylation patterns in xylem tissues, including seven wood-related genes. The expression of these genes differed significantly between xylem and non-xylem tissue types (P < 0.01). This indicated that the difference of expression of specific genes with unique methylation patterns, rather than relative methylation levels between the two tissue types plays a critical role in wood biosynthesis. However, 46.2% of candidate genes with the same methylation pattern in vascular tissues (cambium, phloem, and developing xylem) did not have distinct expression patterns in xylem and non-xylem tissue. Also, bisulfite sequencing and transcriptome sequencing of MYB, NAC and FASCICLIN-LIKE AGP 13 revealed that the location of cytosine methylation in the gene might affect the expression of different transcripts from the corresponding gene. The expression of different transcripts that produce distinct proteins from a single gene might play an important role in the regulation of xylogenesis. PMID:27462332

  6. The Role of DNA Methylation in Xylogenesis in Different Tissues of Poplar

    PubMed Central

    Wang, Qingshi; Ci, Dong; Li, Tong; Li, Peiwen; Song, YuePeng; Chen, Jinhui; Quan, Mingyang; Zhou, Daling; Zhang, Deqiang

    2016-01-01

    In trees, xylem tissues play a key role in the formation of woody tissues, which have important uses for pulp and timber production; also DNA methylation plays an important part in gene regulation during xylogenesis in trees. In our study, methylation-sensitive amplified polymorphism (MSAP) analysis was used to analyze the role cytosine methylation plays in wood formation in the commercially important tree species Populus tomentosa. This analysis compared the methylation patterns between xylem tissues (developing xylem and mature xylem) and non-xylem tissues (cambium, shoot apex, young leaf, mature leaf, phloem, root, male catkin, and female catkin) and found 10,316 polymorphic methylation sites. MSAP identified 132 candidate genes with the same methylation patterns in xylem tissues, including seven wood-related genes. The expression of these genes differed significantly between xylem and non-xylem tissue types (P < 0.01). This indicated that the difference of expression of specific genes with unique methylation patterns, rather than relative methylation levels between the two tissue types plays a critical role in wood biosynthesis. However, 46.2% of candidate genes with the same methylation pattern in vascular tissues (cambium, phloem, and developing xylem) did not have distinct expression patterns in xylem and non-xylem tissue. Also, bisulfite sequencing and transcriptome sequencing of MYB, NAC and FASCICLIN-LIKE AGP 13 revealed that the location of cytosine methylation in the gene might affect the expression of different transcripts from the corresponding gene. The expression of different transcripts that produce distinct proteins from a single gene might play an important role in the regulation of xylogenesis. PMID:27462332

  7. The role of DNA methylation on Octopus vulgaris development and their perspectives

    PubMed Central

    Díaz-Freije, Eva; Gestal, Camino; Castellanos-Martínez, Sheila; Morán, Paloma

    2014-01-01

    DNA methylation is a common regulator of gene expression and development in mammalian and other vertebrate genomes. DNA methylation has been studied so far in a few bivalve mollusk species, finding a wide spectrum of levels. We focused our study in the common octopus, Octopus vulgaris, an important organism for neuroscience, physiology and ethology research as well as for human consumption. We aim to confirm the existence of DNA methylation in O. vulgaris and ultimately, if methylation plays a role in gene regulation during octopus development. We used a genome-wide approach, methylation-sensitive amplified polymorphism (MSAP), firstly in four different tissues from the same specimens from adult benthonic individuals to test whether gene expression is regulated by methylation. Secondly, we tested the hypothesis that methylation underlies development by assessing MSAP patters from paralarvae to adult developmental stages. Our data indicate that octopus genome is widely methylated since clear differences can be observed, and the methylation pattern changes with the development. The statistical analyses showed significant differences in methylation pattern between paralarvae, where higher internal cytosine methylation is observed, and the three other post-hatching stages. This suggests an important role of cytosine methylation during the first step of development, when major morphological changes take place. However, methylation seems to have little effect on gene expression during the benthonic phase, since no significant effect was revealed in the analyses of molecular variance (AMOVA) performed. Our observations highlight the importance of epigenetic mechanisms in the first developmental steps of the common octopus and opens new perspectives to overcome high mortality rate during paralarvae growth. Thus, better understanding the molecular regulation patterns could lead to new approaches that increase the efficiency of husbandry of this emergent species for

  8. Synthesis, conformational parameters and packing considerations of methyl bispyridyl ketones

    NASA Astrophysics Data System (ADS)

    Weck, Christian; Katzsch, Felix; Gruber, Tobias

    2015-10-01

    The crystal structures of two bispyridyl ketones featuring either two methyl residues or one methyl and one bromomethyl residue, respectively, are presented. In order to elucidate the influence of the substituents, a comprehensive comparison with the non-methylated mother compound has been performed. A special focus lies thereby on the relative position of the heteroatoms and their free electron pairs. The two methyl groups at the bispyridyl ketone result in two molecules in the asymmetric unit adopting rather different conformations. Due to the fast crystallization conditions and a melting point differing from the literature, a polymorph close to a local minimum in the energy hypersurface seems possible. After introducing a bromine atom to one of the two methyl groups, the molecular conformation is very similar to the unsubstituted molecule. The packing of both title compounds is dominated by weak contacts of the C-H⋯π and C-H⋯Y type (Y = O, N) and C-H⋯Br- and Br⋯π-contacts for the brominated molecule.

  9. Microbial arsenic methylation in soil and rice rhizosphere.

    PubMed

    Jia, Yan; Huang, Hai; Zhong, Min; Wang, Feng-Hua; Zhang, Li-Mei; Zhu, Yong-Guan

    2013-04-01

    Methylated arsenic (As) species are a common constituent of rice grains accounting for 10-90% of the total As. Recent studies have shown that higher plants are unlikely to methylate As in vivo suggesting that As methylation is a microbial mediated process that occurs in soils prior to plant uptake. In this study, we designed primers according to the conserved essential amino acids and structural motifs of arsenite S-adenosylmethionine methyltransferase (ArsM). We report for the first time the successful amplification of the prokaryotic arsM gene in 14 tested soils with wide ranging As concentrations. The abundance and diversity of the arsM gene in the rice rhizosphere soil and roots were analyzed using the designed primers. Results showed that microbes containing arsM genes were phylogenetically diverse, as revealed by the clone library and terminal restriction fragment length polymorphism (T-RFLP) analysis, and were branched into various phyla. Concentration of methylated As species in the soil solution was elevated in the rhizosphere soil and also by the addition of rice straw into the paddy soil, corresponding to the elevated abundance of the arsM gene in the soil. These results, together with evidence of horizontal gene transfer (HGT) of the arsM gene, suggest the genes encoding ArsM in soils are widespread. These findings demonstrate why most rice, when compared with other cereals, contains unusually high concentrations of methylated As species. PMID:23469919

  10. Association of Catechol-O-Methyltransferase (COMT) Polymorphism and Academic Achievement in a Chinese Cohort

    ERIC Educational Resources Information Center

    Yeh, Ting-Kuang; Chang, Chun-Yen; Hu, Chung-Yi; Yeh, Ting-Chi; Lin, Ming-Yeh

    2009-01-01

    Catechol-O-methyltransferase (COMT) is a methylation enzyme that catalyzes the degradation pathway and inactivation of dopamine. It is accepted widely as being involved in the modulation of dopaminergic physiology and prefrontal cortex (PFC) function. The COMT Val158Met polymorphism is associated with variation in COMT activity. COMT 158Met allele…

  11. Epigenetic and genetic influences on DNA methylation variation in maize populations.

    PubMed

    Eichten, Steven R; Briskine, Roman; Song, Jawon; Li, Qing; Swanson-Wagner, Ruth; Hermanson, Peter J; Waters, Amanda J; Starr, Evan; West, Patrick T; Tiffin, Peter; Myers, Chad L; Vaughn, Matthew W; Springer, Nathan M

    2013-08-01

    DNA methylation is a chromatin modification that is frequently associated with epigenetic regulation in plants and mammals. However, genetic changes such as transposon insertions can also lead to changes in DNA methylation. Genome-wide profiles of DNA methylation for 20 maize (Zea mays) inbred lines were used to discover differentially methylated regions (DMRs). The methylation level for each of these DMRs was also assayed in 31 additional maize or teosinte genotypes, resulting in the discovery of 1966 common DMRs and 1754 rare DMRs. Analysis of recombinant inbred lines provides evidence that the majority of DMRs are heritable. A local association scan found that nearly half of the DMRs with common variation are significantly associated with single nucleotide polymorphisms found within or near the DMR. Many of the DMRs that are significantly associated with local genetic variation are found near transposable elements that may contribute to the variation in DNA methylation. Analysis of gene expression in the same samples used for DNA methylation profiling identified over 300 genes with expression patterns that are significantly associated with DNA methylation variation. Collectively, our results suggest that DNA methylation variation is influenced by genetic and epigenetic changes that are often stably inherited and can influence the expression of nearby genes.

  12. Analysis of Different Ploidy and Parent-Offspring Genomic DNA Methylation in the Loach Misgurnus anguillicaudatus.

    PubMed

    Zhou, He; Ma, Tian-Yu; Zhang, Rui; Xu, Qi-Zheng; Shen, Fu; Qin, Yan-Jie; Xu, Wen; Wang, Yuan; Li, Ya-Juan

    2016-01-01

    In this study, we selected natural polyploidy loach (diploid, triploid and tetraploid) and hybrid F₁ generation obverse cross (4 × 2) and inverse cross (2 × 4) by diploids and tetraploids as the research model. The MSAP (methylation-sensitive amplified polymorphism) reaction system was established by our laboratory to explore methylation levels and pattern diversification features at the whole genome level of the polyploidy loach. The results showed that the total methylation and full methylation rates decreased on increased ploidy individuals; moreover, the hemimethylation rate showed no consistent pattern. Compared with diploid loach, the methylation patterns of tetraploid sites changed 68.17%, and the methylation patterns of triploid sites changed 73.05%. The proportion of hypermethylation genes is significantly higher than the proportion of demethylation genes. The methylation level of reciprocal cross F₁ generation is lower than the male diploid and higher than the female tetraploid. The hemimethylation and total methylation rate of the cross hybrid F₁ generation is significantly higher than the orthogonal F₁ generation (p < 0.01). After readjusting, the methylation pattern of genome DNA of reciprocal hybrids changed 69.59% and 72.83%, respectively. PMID:27556458

  13. Double-strand break damage and associated DNA repair genes predispose smokers to gene methylation

    PubMed Central

    Leng, Shuguang; Stidley, Christine A.; Willink, Randy; Bernauer, Amanda; Do, Kieu; Picchi, Maria A.; Sheng, Xin; Frasco, Melissa, A.; Berg, David Van Den; Gilliland, Frank D.; Zima, Christopher; Crowell, Richard E.; Belinsky, Steven A.

    2008-01-01

    Gene promoter hypermethylation in sputum is a promising biomarker for predicting lung cancer. Identifying factors that predispose smokers to methylation of multiple gene promoters in the lung could impact strategies for early detection and chemoprevention. This study evaluated the hypothesis that double-strand break repair capacity and sequence variation in genes in this pathway are associated with a high methylation index in a cohort of current and former cancer-free smokers. A 50% reduction in the mean level of double-strand break repair capacity was seen in lymphocytes from smokers with a high methylation index, defined as ≥ 3 of 8 genes methylated in sputum, compared to smokers with no genes methylated. The classification accuracy for predicting risk for methylation was 88%. Single nucleotide polymorphisms within the MRE11A, CHEK2, XRCC3, DNA-Pkc, and NBN DNA repair genes were highly associated with the methylation index. A 14.5-fold increased odds for high methylation was seen for persons with ≥ 7 risk alleles of these genes. Promoter activity of the MRE11A gene that plays a critical role in recognition of DNA damage and activation of ATM was reduced in persons with the risk allele. Collectively, ours is the first population-based study to identify double-strand break DNA repair capacity and specific genes within this pathway as critical determinants for gene methylation in sputum, that is, in turn, associated with elevated risk for lung cancer. PMID:18413776

  14. Analysis of Different Ploidy and Parent–Offspring Genomic DNA Methylation in the Loach Misgurnus anguillicaudatus

    PubMed Central

    Zhou, He; Ma, Tian-Yu; Zhang, Rui; Xu, Qi-Zheng; Shen, Fu; Qin, Yan-Jie; Xu, Wen; Wang, Yuan; Li, Ya-Juan

    2016-01-01

    In this study, we selected natural polyploidy loach (diploid, triploid and tetraploid) and hybrid F1 generation obverse cross (4 × 2) and inverse cross (2 × 4) by diploids and tetraploids as the research model. The MSAP (methylation-sensitive amplified polymorphism) reaction system was established by our laboratory to explore methylation levels and pattern diversification features at the whole genome level of the polyploidy loach. The results showed that the total methylation and full methylation rates decreased on increased ploidy individuals; moreover, the hemimethylation rate showed no consistent pattern. Compared with diploid loach, the methylation patterns of tetraploid sites changed 68.17%, and the methylation patterns of triploid sites changed 73.05%. The proportion of hypermethylation genes is significantly higher than the proportion of demethylation genes. The methylation level of reciprocal cross F1 generation is lower than the male diploid and higher than the female tetraploid. The hemimethylation and total methylation rate of the cross hybrid F1 generation is significantly higher than the orthogonal F1 generation (p < 0.01). After readjusting, the methylation pattern of genome DNA of reciprocal hybrids changed 69.59% and 72.83%, respectively. PMID:27556458

  15. Possible association of IL-4 VNTR polymorphism with susceptibility to preeclampsia.

    PubMed

    Salimi, Saeedeh; Mohammadoo-Khorasani, Milad; Yaghmaei, Minoo; Mokhtari, Mojgan; Moossavi, Maryam

    2014-01-01

    Preeclampsia (PE) is a pregnancy-specific disorder that results in maternal mortality and morbidity. Growing evidence indicated that cytokines are involved in the pathogenesis of PE and interleukin-4 VNTR polymorphism could be implicated in altering the PE risk. The aim of this study was to evaluate the possible association between IL-4 VNTR polymorphism and susceptibility to PE in Iranian population for the first time. Genetic polymorphism was evaluated in 192 PE and 186 healthy control women by polymerase chain reaction method. We found that the VNTR polymorphism of IL-4 gene has significantly increased the risk of preeclampsia (RP2/RP1 versus RP1/RP1, OR, 2.8 [95% CI, 1.7 to 8.8]; P = 0.0001 and RP2/RP2 versus RP1/RP1; P = 0.002). The results showed that carriage of IL-4 VNTR RP2 allele has positive association with preeclampsia susceptibility.

  16. Prevalence of coagulase gene polymorphism in Staphylococcus aureus isolates causing bovine mastitis.

    PubMed Central

    Aarestrup, F M; Dangler, C A; Sordillo, L M

    1995-01-01

    This study was conducted to investigate polymorphism of the coagulase gene of Staphylococcus aureus causing bovine mastitis. One hundred eighty-seven strains of S. aureus were isolated from bovine mastitic milk samples obtained from 187 different Danish dairy farms. The isolates were characterised for restriction fragment length polymorphism (RFLP) of the coagulase gene. A variable region of the coagulase gene was amplified using the polymerase chain reaction (PCR) followed by AluI restriction enzyme digestion. A total of 15 different RFLP patterns were observed. The predominant pattern was found in 35% of the isolates. The ease of analysing coagulase gene polymorphisms among a large number of strains, and the multiple distinct polymorphic patterns generated, supports the use of this technique in epidemiological investigations of bovine mastitis. The predominating variants may have predelection for causing intramammary infections. PMID:7648524

  17. Association of vitamin D receptor gene polymorphisms and Parkinson's disease in Hungarians.

    PubMed

    Török, Rita; Török, Nora; Szalardy, Levente; Plangar, Imola; Szolnoki, Zoltan; Somogyvari, Ferenc; Vecsei, Laszlo; Klivenyi, Peter

    2013-09-13

    Vitamin D receptor (VDR) gene encodes a transcription factor that influences calcium homeostasis and immunoregulation, and may play a role in neurological disorders including Parkinson's disease (PD). The investigations of the association between VDR and PD in different populations revealed various results. In a present study 100 PD patients and 109 healthy controls from the Hungarian population were genotyped for four polymorphic sites (BsmI, ApaI, FokI and TaqI) in the VDR gene. The polymorphisms were determined by polymerase chain reaction and restriction fragment length polymorphism (PCR-RFLP). Our results demonstrate an association between the FokI C allele and PD; the frequency of the C allele was significantly higher in PD patients than in controls, suggesting that this polymorphism may have a role in the development of PD in these patients.

  18. Human DNA polymerase. alpha. : Predicted functional domains and relationships with viral DNA polymerases

    SciTech Connect

    Wang, T.S.F.; Wong, S.W.; Korn, D. )

    1989-01-01

    The primary sequence of human DNA polymerase {alpha} deduced from the full-length cDNA contains regions of striking similarity to sequences in replicative DNA polymerases from Escherichia coli phages PRD1 and T4, Bacillus phage {phi}19, yeast DNA polymerase I, yeast linear plasmid pGKL1, maize S1 mitochondrial DNA, herpes family viruses, vaccinia virus, and adenovirus. The conservation of these homologous regions across this vast phylogenetic expanse indicates that these prokaryotic and eukaryotic DNA polymerases may all have evolved from a common primordial gene. Based on the sequence analysis and genetic results from yeast and herpes simplex virus studies, these consensus sequences are suggested to define potential sites that subserve essential roles in the DNA polymerase reaction. Two of these conserved regions appear to participate directly in the active site required for substrate deoxynucleotide interaction. One region toward the carboxyl-terminus has the potential to be the DNA interacting domain is predicted toward the amino-terminus. The provisional assignment of these domains can be used to identify unique or dissimilar features of functionally homologous catalytic sites in viral DBA polymerases of pathogenetic significance and thereby serve to guide more rational antiviral drug design.

  19. DNA polymerase γ and disease: what we have learned from yeast

    PubMed Central

    Lodi, Tiziana; Dallabona, Cristina; Nolli, Cecilia; Goffrini, Paola; Donnini, Claudia; Baruffini, Enrico

    2015-01-01

    Mip1 is the Saccharomyces cerevisiae DNA polymerase γ (Pol γ), which is responsible for the replication of mitochondrial DNA (mtDNA). It belongs to the family A of the DNA polymerases and it is orthologs to human POLGA. In humans, mutations in POLG(1) cause many mitochondrial pathologies, such as progressive external ophthalmoplegia (PEO), Alpers' syndrome, and ataxia-neuropathy syndrome, all of which present instability of mtDNA, which results in impaired mitochondrial function in several tissues with variable degrees of severity. In this review, we summarize the genetic and biochemical knowledge published on yeast mitochondrial DNA polymerase from 1989, when the MIP1 gene was first cloned, up until now. The role of yeast is particularly emphasized in (i) validating the pathological mutations found in human POLG and modeled in MIP1, (ii) determining the molecular defects caused by these mutations and (iii) finding the correlation between mutations/polymorphisms in POLGA and mtDNA toxicity induced by specific drugs. We also describe recent findings regarding the discovery of molecules able to rescue the phenotypic defects caused by pathological mutations in Mip1, and the construction of a model system in which the human Pol γ holoenzyme is expressed in yeast and complements the loss of Mip1. PMID:25852747

  20. 1a/1b subtype profiling of nonnucleoside polymerase inhibitors of hepatitis C virus.

    PubMed

    Nyanguile, Origène; Devogelaere, Benoit; Vijgen, Leen; Van den Broeck, Walter; Pauwels, Frederik; Cummings, Maxwell D; De Bondt, Hendrik L; Vos, Ann M; Berke, Jan M; Lenz, Oliver; Vandercruyssen, Geneviève; Vermeiren, Katrien; Mostmans, Wendy; Dehertogh, Pascale; Delouvroy, Frédéric; Vendeville, Sandrine; VanDyck, Koen; Dockx, Koen; Cleiren, Erna; Raboisson, Pierre; Simmen, Kenneth A; Fanning, Gregory C

    2010-03-01

    The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme.

  1. 1a/1b Subtype Profiling of Nonnucleoside Polymerase Inhibitors of Hepatitis C Virus ▿

    PubMed Central

    Nyanguile, Origène; Devogelaere, Benoit; Vijgen, Leen; Van den Broeck, Walter; Pauwels, Frederik; Cummings, Maxwell D.; De Bondt, Hendrik L.; Vos, Ann M.; Berke, Jan M.; Lenz, Oliver; Vandercruyssen, Geneviève; Vermeiren, Katrien; Mostmans, Wendy; Dehertogh, Pascale; Delouvroy, Frédéric; Vendeville, Sandrine; VanDyck, Koen; Dockx, Koen; Cleiren, Erna; Raboisson, Pierre; Simmen, Kenneth A.; Fanning, Gregory C.

    2010-01-01

    The RNA-dependent RNA polymerase (NS5B) of hepatitis C virus (HCV) is an unusually attractive target for drug discovery since it contains five distinct drugable sites. The success of novel antiviral therapies will require nonnucleoside inhibitors to be active in at least patients infected with HCV of subtypes 1a and 1b. Therefore, the genotypic assessment of these agents against clinical isolates derived from genotype 1-infected patients is an important prerequisite for the selection of suitable candidates for clinical development. Here we report the 1a/1b subtype profiling of polymerase inhibitors that bind at each of the four known nonnucleoside binding sites. We show that inhibition of all of the clinical isolates tested is maintained, except for inhibitors that bind at the palm-1 binding site. Subtype coverage varies across chemotypes within this class of inhibitors, and inhibition of genotype 1a improves when hydrophobic contact with the polymerase is increased. We investigated if the polymorphism of the palm-1 binding site is the sole cause of the reduced susceptibility of subtype 1a to inhibition by 1,5-benzodiazepines by using reverse genetics, X-ray crystallography, and surface plasmon resonance studies. We showed Y415F to be a key determinant in conferring resistance on subtype 1a, with this effect being mediated through an inhibitor- and enzyme-bound water molecule. Binding studies revealed that the mechanism of subtype 1a resistance is faster dissociation of the inhibitor from the enzyme. PMID:20071590

  2. Enzyme polymorphisms in Canarium

    Technology Transfer Automated Retrieval System (TEKTRAN)

    Fifty-two accessions of Canarium involving seven species, C. ovatum, C. album, C. megalanthum, C. harveyi, C. indicum, C. mehenbethene, and C. odontophyllum were studied for isozyme polymorphisms. Starch gel electrophoresis with a histidine-citrate buffer system (pH 6.5) was employed to assay six en...

  3. Polymorphous Perversity in Texts

    ERIC Educational Resources Information Center

    Johnson-Eilola, Johndan

    2012-01-01

    Here's the tricky part: If we teach ourselves and our students that texts are made to be broken apart, remixed, remade, do we lose the polymorphous perversity that brought us pleasure in the first place? Does the pleasure of transgression evaporate when the borders are opened?

  4. Polymorphism of sorbitol

    NASA Astrophysics Data System (ADS)

    Nezzal, Amale; Aerts, Luc; Verspaille, Marleen; Henderickx, Geert; Redl, Andreas

    2009-07-01

    The polymorphism of sorbitol was investigated, confirming the existence of four anhydrous crystalline phases plus the hydrate. The crystallised melt (CM), the alpha form, and the gamma form were obtained via a dry route. The CM was confirmed to be a crystalline state with a spherulite morphology. The alpha form was obtained via direct conversion from the CM, in contrast to more complicated routes previously reported, and was found to have a very high crystallinity. Gamma crystals were obtained by seeding the melt at high temperature; however, crystallinity was clearly less than for alpha crystals. Despite its lower crystallinity, the gamma polymorph was found to be the most stable of the anhydrous crystalline forms; this was confirmed by its high melting point and low hygroscopicity. In contrast, the alpha polymorph has a relatively high melting point but lacks moisture stability at high relative humidity. The hydrate form has the same resistance to moisture as the gamma form, but melts at a lower temperature. The combination of both a high melting point and high stability in the presence of water makes the gamma polymorph best suited for confectionary applications.

  5. Investigation of Uranium Polymorphs

    SciTech Connect

    Sweet, Lucas E.; Henager, Charles H.; Hu, Shenyang Y.; Johnson, Timothy J.; Meier, David E.; Peper, Shane M.; Schwantes, Jon M.

    2011-08-01

    The UO3-water system is complex and has not been fully characterized, even though these species are common throughout the nuclear fuel cycle. As an example, most production schemes for UO3 result in a mixture of up to six or more different polymorphic phases, and small differences in these conditions will affect phase genesis that ultimately result in measureable changes to the end product. As a result, this feature of the UO3-water system may be useful as a means for determining process history. This research effort attempts to better characterize the UO3-water system with a variety of optical techniques for the purpose of developing some predictive capability for estimating process history in polymorphic phases of unknown origin. Three commercially relevant preparation methods for the production of UO3 were explored. Previously unreported low temperature routes to β- and γ-UO3 were discovered. Raman and fluorescence spectroscopic libraries were established for pure and mixed polymorphic forms of UO3 in addition to the common hydrolysis products of UO3. An advantage of the sensitivity of optical fluorescence microscopy over XRD has been demonstrated. Preliminary aging studies of the α and γ forms of UO3 have been conducted. In addition, development of a 3-D phase field model used to predict phase genesis of the system was initiated. Thermodynamic and structural constants that will feed the model have been gathered from the literature for most of the UO3 polymorphic phases.

  6. Implication of alpha1-antichymotrypsin polymorphism in familial Alzheimer's disease.

    PubMed

    Nacmias, B; Marcon, G; Tedde, A; Forleo, P; Latorraca, S; Piacentini, S; Amaducci, L; Sorbi, S

    1998-03-13

    A common polymorphism in the alpha1-antichymotrypsin (ACT) gene has been shown to modify the Apolipoprotein E (ApoE) epsilon4-associated Alzheimer's disease (AD) risk identifying the combination of the ACT/AA and ApoE epsilon4/epsilon4 genotypes as a potential susceptibility marker for AD. Using the polymerase chain reaction, we analyzed the segregation of the ACT and ApoE polymorphisms in familial Alzheimer's disease (FAD) patients carrying mutations in Presenilin (PS) and APP genes and in both early onset (EO) and late onset (LO) FAD patients without known mutations. Our data suggest that ACT does not represent an additional risk factor for PS and APP mutated families. However, in LOFAD patients a high frequency of the combined ACT/AA and ApoE epsilon4/epsilon4 genotypes suggest that ACT may interact with ApoE and play a role in LOFAD. PMID:9572591

  7. Nucleolin Is Required for RNA Polymerase I Transcription In Vivo▿

    PubMed Central

    Rickards, Brenden; Flint, S. J.; Cole, Michael D.; LeRoy, Gary

    2007-01-01

    Eukaryotic genomes are packaged with histones and accessory proteins in the form of chromatin. RNA polymerases and their accessory proteins are sufficient for transcription of naked DNA, but not of chromatin, templates in vitro. In this study, we purified and identified nucleolin as a protein that allows RNA polymerase II to transcribe nucleosomal templates in vitro. As immunofluorescence confirmed that nucleolin localizes primarily to nucleoli with RNA polymerase I, we demonstrated that nucleolin allows RNA polymerase I transcription of chromatin templates in vitro. The results of chromatin immunoprecipitation experiments established that nucleolin is associated with chromatin containing rRNA genes transcribed by RNA polymerase I but not with genes transcribed by RNA polymerase II or III. Knockdown of nucleolin by RNA interference resulted in specific inhibition of RNA polymerase I transcription. We therefore propose that an important function of nucleolin is to permit RNA polymerase I to transcribe nucleolar chromatin. PMID:17130237

  8. Structural biology of bacterial RNA polymerase.

    PubMed

    Murakami, Katsuhiko S

    2015-05-11

    Since its discovery and characterization in the early 1960s (Hurwitz, J. The discovery of RNA polymerase. J. Biol. Chem. 2005, 280, 42477-42485), an enormous amount of biochemical, biophysical and genetic data has been collected on bacterial RNA polymerase (RNAP). In the late 1990s, structural information pertaining to bacterial RNAP has emerged that provided unprecedented insights into the function and mechanism of RNA transcription. In this review, I list all structures related to bacterial RNAP (as determined by X-ray crystallography and NMR methods available from the Protein Data Bank), describe their contributions to bacterial transcription research and discuss the role that small molecules play in inhibiting bacterial RNA transcription.

  9. [Analysis of the level and pattern of genomic DNA methylation in different ploidy watermelons by MSAP (Citrullus lanatus)].

    PubMed

    Wang, Chun Guo; Gu, Yu; Chen, Cheng Bin; Jiao, Ding Liang; Xue, Zhen Yi; Song, Wen Qin

    2009-04-01

    DNA methylation is one of the major epigenetic modifications. It is very important to the regulation of gene expression. In present study, an autoploidy series (2x, 3x and 4x) in watermelon (Citrullus lanatus) was constructed and MSAP (Methylation-Sensitive Amplification Polymorphism) analysis was conducted to elucidate the level and pattern of DNA methylation at CCGG sites in different ploidy watermelons. Totally, 1883 genetic loci were produced by 23 pairs of selective primers, of which 647, 655 and 581 sites were detected in diploid, autotriploid and autotetraploid, respectively. The methylation sites were 181, 150 and 159, and the corresponding total methylation ratios were 28.0%, 22.9% and 27.4% in 2x, 3x and 4x, respectively, of which the fully methylation sites were 121, 80 and 82, and the corresponding fully methylation ratios were 18.7%, 12.2% and 14.1%. Further analysis of the pattern of DNA methylation suggested that compared 4x with 2x, about half of detected sites (54.4%) shown changes of DNA methylation patterns. Similarly, compared 4x with 3x, 45.4% sites also shown changes of DNA methylation patterns. Moreover, the trend of DNA methylation adjustment mainly involved increase of DNA methylation levels in 4x. However, compared 3x with 2x or 4x, although the changes of DNA methylation pattern also widely occurred, which involved 41.6% (compared 3x with 2x) and 45.4% (compared 3x with 4x) sites, respectively, the trend of DNA methylation adjustment mainly involved decrease of DNA methylation levels in 3x. All these results indicated that DNA methylation events were widely existed in different ploidy watermelons. However, not only based on the total DNA methylation ratio or fully DNA methylation ratio, the results both implied that the DNA methylation levels were not closely associated with the autopolyploidy level in watermelon. Autotriploid watermelon shows obvious low level of DNA methylation. Analysis of DNA methylation patterns also suggested that

  10. Polymorphism of 4-bromobenzophenone.

    PubMed

    Strzhemechny, Mikhail A; Baumer, Vyacheslav N; Avdeenko, Anatoli A; Pyshkin, Oleg S; Romashkin, Roman V; Buravtseva, Lyubov M

    2007-04-01

    A combination of single-crystal and powder X-ray diffractometry was used to study the structure of two polymorphs of 4-bromobenzophenone over the temperature range from 100 to 300 K. One of the polymorphs of the title compound was known previously and its structure has been determined at room temperature [Ebbinghaus et al. (1997). Z. Kristallogr. 212, 339-340]. Two crystal growth methods were employed, one of which (a modification of the Bridgman-Stockbarger technique) resulted in single crystals of a previously unknown structure. The basic physical properties of the stable polymorph are: growth method, from 2-propanol solutions or gradient sublimation; space group, monoclinic P2(1)/c; melting point, T(m) = 355.2 K; X-ray density (at 100 K), D(x) = 1.646 g cm(-3). The same properties of the metastable polymorph (triclinic P\\overline 1 ) are: growth method, modified Bridgman-Stockbarger method; X-ray density (at 100 K), D(x) = 1.645 g cm(-3); T(m) = 354 K. Thermograms suggest that the melting of the metastable form is accompanied by at least a partial crystallization presumably into the monoclinic form; the transformation is therefore monotropic. Analysis of short distances in both polymorphs shows that numerous weak hydrogen bonds of the C-H...pi type ensure additional stabilization within the respective planes normal to the longest dimension of the molecules. The strong temperature dependence of the lattice constants and of the weak bond distances in the monoclinic form suggest that the weak bond interactions might be responsible for both the large thermal expansion within plane bc and the considerable thermal expansion anisotropy. PMID:17374940

  11. Functional Consequences of Subunit Diversity in RNA Polymerases II and V

    SciTech Connect

    Tan, Ek Han; Blevins, Todd; Ream, Thomas S.; Pikaard, Craig S.

    2012-03-01

    Multisubunit RNA polymerases IV and V (Pol IV and Pol V) evolved as specialized forms of Pol II that mediate RNA-directed DNA methylation (RdDM) and transcriptional silencing of transposons, viruses, and endogenous repeats in plants. Among the subunits common to Arabidopsis thaliana Pols II, IV, and V are 93% identical alternative ninth subunits, NRP(B/D/E)9a and NRP(B/D/E)9b. The 9a and 9b subunit variants are incompletely redundant with respect to Pol II; whereas double mutants are embryo lethal, single mutants are viable, yet phenotypically distinct. Likewise, 9a or 9b can associate with Pols IV or V but RNA-directed DNA methylation is impaired only in 9b mutants. Based on genetic and molecular tests, we attribute the defect in RdDM to impaired Pol V function. Collectively, our results reveal a role for the ninth subunit in RNA silencing and demonstrate that subunit diversity generates functionally distinct subtypes of RNA polymerases II and V.

  12. The polymerase chain reaction (PCR): general methods.

    PubMed

    Waters, Daniel L E; Shapter, Frances M

    2014-01-01

    The polymerase chain reaction (PCR) converts very low quantities of DNA into very high quantities and is the foundation of many specialized techniques of molecular biology. PCR utilizes components of the cellular machinery of mitotic cell division in vitro which respond predictably to user inputs. This chapter introduces the principles of PCR and discusses practical considerations from target sequence definition through to optimization and application.

  13. Colony Polymerase Chain Reaction with Schizosaccharomyces pombe.

    PubMed

    Murray, Johanne M; Watson, Adam T; Carr, Antony M

    2016-01-01

    When screening a large number of individual Schizosaccharomyces pombe strains by polymerase chain reaction (PCR), a rapid "colony PCR" approach may be used. Numerous colony PCR protocols are available, and fundamental to them all is that the colony must be fresh (grown overnight) and that as few cells as possible are used. In this protocol, we present three reliable methods for preparing S. pombe cells for colony PCR.

  14. DNA Methylation Screening and Analysis

    PubMed Central

    Sant, Karilyn E.; Nahar, Muna S.; Dolinoy, Dana C.

    2013-01-01

    DNA methylation is an epigenetic form of gene regulation that is universally important throughout the life course, especially during in utero and postnatal development. DNA methylation aids in cell cycle regulation and cellular differentiation processes. Previous studies have demonstrated that DNA methylation profiles may be altered by diet and the environment, and that these profiles are especially vulnerable during development. Thus, it is important to understand the role of DNA methylation in developmental governance and subsequent disease progression. A variety of molecular methods exist to assay for global, gene-specific, and epigenome-wide methylation. Here we describe these methods and discuss their relative strengths and limitations. PMID:22669678

  15. DNA Methylation Alterations at 5'-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus.

    PubMed

    Xiong, Wanshan; Li, Xiaorong; Fu, Donghui; Mei, Jiaqin; Li, Qinfei; Lu, Guanyuan; Qian, Lunwen; Fu, Yin; Disi, Joseph Onwusemu; Li, Jiana; Qian, Wei

    2013-01-01

    DNA methylation is an important regulatory mechanism for gene expression that involved in the biological processes of development and differentiation in plants. To investigate the association of DNA methylation with heterosis in Brassica, a set of intraspecific hybrids in Brassica rapa and B. napus and interspecific hybrids between B. rapa and B. napus, together with parental lines, were used to monitor alterations in cytosine methylation at 5'-CCGG sites in seedlings and buds by methylation-sensitive amplification polymorphism analysis. The methylation status of approximately a quarter of the methylation sites changed between seedlings and buds. These alterations were related closely to the genomic structure and heterozygous status among accessions. The methylation status in the majority of DNA methylation sites detected in hybrids was the same as that in at least one of the parental lines in both seedlings and buds. However, the association between patterns of cytosine methylation and heterosis varied among different traits and between tissues in hybrids of Brassica, although a few methylation loci were associated with heterosis. Our data suggest that changes in DNA methylation at 5'-CCGG sites are not associated simply with heterosis in the interspecific and intraspecific hybridizations derived from B. rapa and B. napus.

  16. Preliminary study on association of beta2-adrenergic receptor polymorphism with hypertension in hypertensive subjects attending Balok Health Centre, Kuantan.

    PubMed

    Atia, A E; Norsidah, K; Nor Zamzila, A; Rafidah Hanim, M; Samsul, D; Aznan, M A M; Rashidah, A R; Norlelawati, A T

    2012-02-01

    Polymorphisms within the beta2-adrenergic receptor (ADRB2) gene have been repeatedly linked to hypertension. Among the ADRB2 polymorphisms detected, Arg16Gly and Gln27Glu codons are considered the two most important variations. The amino acid substitution at these codons may lead to abnormal regulation of ADRB2 activity. The aim of the present study was to assess the association between ADRB2 polymorphisms and hypertension. This case-control study consisted of 100 unrelated subjects (50 hypertensive and 50 matched normal controls). Arg16Gly and the Gln27Glu polymorphisms were analyzed by polymerase chain reaction-restriction fragment length polymorphism assay. There were no significant evidence of association in allelic and genotypes distribution of Arg16Gly and Glu27Gln with blood pressure and hypertension. These findings suggest that the variation within codon 16 and 27 of ADRB2 gene were unlikely to confer genetic susceptibility for hypertension in our population samples. PMID:22582545

  17. Reticulocyte RNA-Dependent RNA Polymerase

    PubMed Central

    Downey, Kathleen M.; Byrnes, John J.; Jurmark, Bonnie S.; So, Antero G.

    1973-01-01

    A cytoplasmic, microsomal bound RNA-dependent RNA polymerase has been purified 2500-fold from rabbit reticulocyte lysates. The synthesis of RNA with the purified enzyme is absolutely dependent on the addition of an RNA template. The best template is hemoglobin messenger RNA, while bacteriophage RNA and poly(A,G) are less active, and DNA is completely inactive as a template. With poly(A,G) as a template, only UTP and CTP are incorporated into polynucleotide chains, indicating that the RNA polymerase is an RNA replicase and not a terminal transferase. With messenger RNA as a template, all four ribonucleoside triphosphates are required for maximal activity. The RNA-dependent RNA polymerase reaction is extremely sensitive to low concentrations of heme, rifamycin AF/013, and ribonuclease and resistant to actinomycin D and DNase. The discovery of RNA-directed RNA synthesis in reticulocytes offers an additional site for control of gene expression in mammalian cells and provides a possible mechanism for amplification of the expression of specific genes. PMID:4519633

  18. The role of MBL2 gene polymorphism in sepsis incidence

    PubMed Central

    Liu, Lei; Ning, Bo

    2015-01-01

    Aim: This case-control study was aimed to explore the role of mannose-binding lectin 2 (MBL2) gene rs1800450 polymorphism (codon 54 A/B, G230A) in the development of sepsis in Han Chinese. Methods: MBL2 rs1800450 polymorphism was genotyped by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). MBL serum level was detected by enzyme-linked immunosorbent assay (ELISA). Associations between rs1800450 and sepsis susceptibility was detected by Chi-square test and represented by odds ratios (ORs) and 95% confidence intervals (CIs). Correlation of rs1800450 genotypes and MBL serum level was assessed using t test. Result: Variant A allele frequency was significantly observed in cases than that in controls, indicating a significant association with the susceptibility of sepsis (OR = 1.979, 95% CI = 1.200-3.262). GA genotype also relate to the onset of sepsis (OR = 2.090, 95% CI = 1.163-3.753). MBL serum concentrations were significantly different between case and control groups (P<0.001). Meanwhile, variant allele carriers had lower serum level compared with wild homozygous (P<0.001). Conclusion: Variant A allele in MBL2 gene rs1800450 polymorphism might increase the risk of sepsis via decrease the MBL serum level. PMID:26823854

  19. Investigation of the Vitamin D Receptor Polymorphisms in Acromegaly Patients

    PubMed Central

    Ilhan, Muzaffer; Toptas-Hekimoglu, Bahar; Yaylim, Ilhan; Turgut, Seda; Turan, Saime; Karaman, Ozcan; Tasan, Ertugrul

    2015-01-01

    Objective. The genetic structural alterations in the majority of somatotroph adenomas are not clarified and the search for novel candidate genes is still a challenge. We aimed to investigate possible associations between vitamin D receptor (VDR) polymorphisms and acromegaly. Design, Patients, and Methods. 52 acromegaly patients (mean age 45.7 ± 1.9 years) and 83 controls (mean age 43.1 ± 2.6 years) were recruited to the study. VDR polymorphism was determined by polymerase chain reaction-based restriction fragment length polymorphism methods. Results. The distribution of VDR genotypes showed a significant difference in the frequencies of VDR FokI genotypes between patients and controls (P = 0.034). VDR FokI ff genotype was significantly decreased in acromegaly patients (P = 0.035) and carriers of FokI Ff genotype had a 1.5-fold increased risk for acromegaly (OR: 1.5, 95% CI: 1.07–2.1; P = 0.020). IGF1 levels after treatment were significantly higher in patients carrying the Ff genotype compared to carrying ff genotype (P = 0.0049). 25(OH)D3 levels were significantly lower in acromegaly patients (P < 0.001). Conclusions. Our study suggests that VDR FokI genotypes might affect the development of acromegaly and VDR polymorphisms may play a role in the course of acromegaly as a consequence of altering hormonal status. PMID:25839036

  20. PRODH Polymorphisms, Cortical Volumes and Thickness in Schizophrenia

    PubMed Central

    Gadelha, Ary; Santoro, Marcos L.; Noto, Cristiano; Christofolini, Denise M.; Assunção, Idaiane B.; Yamada, Karen M.; Ribeiro-dos-Santos, Ândrea K.; Santos, Sidney; Mari, Jair J.; Smith, Marília A. C.; Melaragno, Maria I.; Bressan, Rodrigo A.; Sato, João R.; Jackowski, Andrea P.; Belangero, Sintia I.

    2014-01-01

    Schizophrenia is a neurodevelopmental disorder with high heritability. Several lines of evidence indicate that the PRODH gene may be related to the disorder. Therefore, our study investigates the effects of 12 polymorphisms of PRODH on schizophrenia and its phenotypes. To further evaluate the roles of the associated variants in the disorder, we have conducted magnetic resonance imaging (MRI) scans to assess cortical volumes and thicknesses. A total of 192 patients were evaluated using the Structured Clinical Interview for DSM-IV (SCID), Positive and Negative Syndrome Scale (PANSS), Calgary Depression Scale, Global Assessment of Functioning (GAF) and Clinical Global Impression (CGI) instruments. The study included 179 controls paired by age and gender. The samples were genotyped using the real-time polymerase chain reaction (PCR), restriction fragment length polymorphism (RFLP)-PCR and Sanger sequencing methods. A sample of 138 patients and 34 healthy controls underwent MRI scans. One polymorphism was associated with schizophrenia (rs2904552), with the G-allele more frequent in patients than in controls. This polymorphism is likely functional, as predicted by PolyPhen and SIFT, but it was not associated with brain morphology in our study. In summary, we report a functional PRODH variant associated with schizophrenia that may have a neurochemical impact, altering brain function, but is not responsible for the cortical reductions found in the disorder. PMID:24498354

  1. PARP1 Val762Ala polymorphism reduces enzymatic activity

    SciTech Connect

    Wang Xiaogan; Wang Zhaoqi; Tong Weimin . E-mail: tong@iarc.fr; Shen Yan

    2007-03-02

    Poly(ADP-ribose) polymerase 1 (PARP1) modifies a variety of nuclear proteins by poly(ADP-ribosyl)ation, and plays diverse roles in molecular and cellular processes. A common PARP1 single nucleotide polymorphism (SNP) at codon 762, resulting in the substitution of alanine (Ala) for valine (Val) in the catalytic domain has been implicated in susceptibility to cancer. To characterize the functional effect of this polymorphism on PARP1, we performed in vitro enzymatic analysis on PARP1-Ala762 and PARP1-Val762. We found that PARP1-Ala762 displayed 57.2% of the activity of PARP1-Val762 for auto-poly(ADP-ribosyl)ation and 61.9% of the activity of PARP1-Val762 for trans-poly(ADP-ribosyl)ation of histone H1. The kinetic characterization revealed that the K {sub m} of PARP1-Ala762 was increased to a 1.2-fold of the K {sub m} of PARP1-Val762 for trans-poly(ADP-ribosyl)ation. Thus, the PARP1 Val762Ala polymorphism reduces the enzymatic activity of PARP1 by increasing K {sub m}. This finding suggests that different levels of poly(ADP-ribosyl)ation by PARP1 might aid in understanding Cancer risk of carriers of the PARP1 Val762Ala polymorphism.

  2. Respiratory Syncytial Virus Inhibitor AZ-27 Differentially Inhibits Different Polymerase Activities at the Promoter

    PubMed Central

    Noton, Sarah L.; Nagendra, Kartikeya; Dunn, Ewan F.; Mawhorter, Michael E.; Yu, Qin

    2015-01-01

    ABSTRACT Respiratory syncytial virus (RSV) is the leading cause of pediatric respiratory disease. RSV has an RNA-dependent RNA polymerase that transcribes and replicates the viral negative-sense RNA genome. The large polymerase subunit (L) has multiple enzymatic activities, having the capability to synthesize RNA and add and methylate a cap on each of the viral mRNAs. Previous studies (H. Xiong et al., Bioorg Med Chem Lett, 23:6789–6793, 2013, http://dx.doi.org/10.1016/j.bmcl.2013.10.018; C. L. Tiong-Yip et al., Antimicrob Agents Chemother, 58:3867–3873, 2014, http://dx.doi.org/10.1128/AAC.02540-14) had identified a small-molecule inhibitor, AZ-27, that targets the L protein. In this study, we examined the effect of AZ-27 on different aspects of RSV polymerase activity. AZ-27 was found to inhibit equally both mRNA transcription and genome replication in cell-based minigenome assays, indicating that it inhibits a step common to both of these RNA synthesis processes. Analysis in an in vitro transcription run-on assay, containing RSV nucleocapsids, showed that AZ-27 inhibits synthesis of transcripts from the 3′ end of the genome to a greater extent than those from the 5′ end, indicating that it inhibits transcription initiation. Consistent with this finding, experiments that assayed polymerase activity on the promoter showed that AZ-27 inhibited transcription and replication initiation. The RSV polymerase also can utilize the promoter sequence to perform a back-priming reaction. Interestingly, addition of AZ-27 had no effect on the addition of up to three nucleotides by back-priming but inhibited further extension of the back-primed RNA. These data provide new information regarding the mechanism of inhibition by AZ-27. They also suggest that the RSV polymerase adopts different conformations to perform its different activities at the promoter. IMPORTANCE Currently, there are no effective antiviral drugs to treat RSV infection. The RSV polymerase is an

  3. The 46359CT polymorphism of DNMT3B is associated with the risk of cervical cancer.

    PubMed

    Hernández-Sotelo, Daniel; García-Aguilar, Rubén; Castro-Coronel, Yaneth; Magaña, Jonathan J; Leyva-Vazquez, Marco Antonio; Alarcón-Romero, Luz del Carmen; López-Bayghen, Esther; Illades-Aguiar, Berenice

    2013-07-01

    Abnormal methylation is related to cancer development. Since DNMT3B is an enzyme that modulates genomic methylation, we hypothesized that genetic variants of the promoter DNMT3B may be associated with an increased risk of developing cervical cancer. Our aim was to investigate the association between -579GT and 46359CT polymorphisms of DNMT3B and cervical cancer, high-grade squamous intraepithelial lesions (HSIL), and low-grade squamous intraepithelial lesions (LSIL). Samples from 200 healthy women and 130 women with squamous intraepithelial lesions (70 with cervical cancer, 30 with HSIL, and 30 with LSIL) were analyzed. Polymorphism genotyping was performed using PCR and restriction fragment length polymorphism. The -579GT polymorphism was not associated with cervical cancer, HSIL, or LSIL. The CT genotype of 46359CT polymorphism was significantly associated with cervical cancer risk (OR 8.75, CI 1.27-374.1), whereas the TT genotype was associated with a significantly decreased risk of HSIL (OR 0.66, CI 0.01-0.32) and LSIL (OR 0.11, CI 0.026-0.45). Our results suggest that genotyping the 46359CT polymorphism in DNMT3B may help identify women who are genetically susceptible to cervical cancer development. Additional studies with larger sample sizes are necessary to confirm our findings.

  4. The Second Subunit of DNA Polymerase Delta Is Required for Genomic Stability and Epigenetic Regulation.

    PubMed

    Zhang, Jixiang; Xie, Shaojun; Cheng, Jinkui; Lai, Jinsheng; Zhu, Jian-Kang; Gong, Zhizhong

    2016-06-01

    DNA polymerase δ plays crucial roles in DNA repair and replication as well as maintaining genomic stability. However, the function of POLD2, the second small subunit of DNA polymerase δ, has not been characterized yet in Arabidopsis (Arabidopsis thaliana). During a genetic screen for release of transcriptional gene silencing, we identified a mutation in POLD2. Whole-genome bisulfite sequencing indicated that POLD2 is not involved in the regulation of DNA methylation. POLD2 genetically interacts with Ataxia Telangiectasia-mutated and Rad3-related and DNA polymerase α The pold2-1 mutant exhibits genomic instability with a high frequency of homologous recombination. It also exhibits hypersensitivity to DNA-damaging reagents and short telomere length. Whole-genome chromatin immunoprecipitation sequencing and RNA sequencing analyses suggest that pold2-1 changes H3K27me3 and H3K4me3 modifications, and these changes are correlated with the gene expression levels. Our study suggests that POLD2 is required for maintaining genome integrity and properly establishing the epigenetic markers during DNA replication to modulate gene expression. PMID:27208288

  5. Direct DNA Methylation Profiling Using Methyl Binding Domain Proteins

    PubMed Central

    Yu, Yinni; Blair, Steve; Gillespie, David; Jensen, Randy; Myszka, David G.; Badran, Ahmed H.; Ghosh, Indraneel; Chagovetz, Alexander

    2010-01-01

    Methylation of DNA is responsible for gene silencing by establishing heterochromatin structure that represses transcription, and studies have shown that cytosine methylation of CpG islands in promoter regions acts as a precursor to early cancer development. The naturally occurring methyl binding domain (MBD) proteins from mammals are known to bind to the methylated CpG dinucleotide (mCpG), and subsequently recruit other chromatin-modifying proteins to suppress transcription. Conventional methods of detection for methylated DNA involve bisulfite treatment or immunoprecipitation prior to performing an assay. We focus on proof-of-concept studies for a direct microarray-based assay using surface-bound methylated probes. The recombinant protein 1xMBD-GFP recognizes hemi-methylation and symmetric methylation of the CpG sequence of hybridized dsDNA, while displaying greater affinity for the symmetric methylation motif, as evaluated by SPR. From these studies, for symmetric mCpG, the KD for 1xMBD-GFP ranged from 106 nM to 870 nM, depending upon the proximity of the methylation site to the sensor surface. The KD values for non-symmetrical methylation motifs were consistently greater (> 2 µM), but the binding selectivity between symmetric and hemi-methylation motifs ranged from 4 to 30, with reduced selectivity for sites close to the surface or multiple sites in proximity, which we attribute to steric effects. Fitting skew normal probability density functions to our data, we estimate an accuracy of 97.5% for our method in identifying methylated CpG loci, which can be improved through optimization of probe design and surface density. PMID:20507169

  6. Human cytomegalovirus (CMV) susceptibility to currently approved antiviral drugs does not impact on CMV terminase complex polymorphism.

    PubMed

    Pilorgé, Léa; Burrel, Sonia; Aït-Arkoub, Zaïna; Agut, Henri; Boutolleau, David

    2014-11-01

    Currently approved anti-human cytomegalovirus (CMV) drugs, all targeting the viral DNA polymerase, are associated with significant toxicities and emergence of drug resistance. In this context, CMV terminase complex constitutes a promising target for novel antiviral compounds. In this study, we describe the low natural polymorphism (interstrain identity >97.7% at both nucleotide and amino acid levels) of the terminase subunits pUL56 and pUL89, and the portal protein pUL104, among 63 CMV clinical strains, and we show that the CMV resistance profile to current DNA polymerase inhibitors has no impact on the natural polymorphism of CMV terminase complex. These results support the idea that both CMV clinical strains exhibiting either susceptibility or resistance to current CMV DNA polymerase inhibitors are comparably sensitive to novel inhibitors of CMV terminase complex, such as letermovir.

  7. A Broad Requirement for TLS Polymerases η and κ, and Interacting Sumoylation and Nuclear Pore Proteins, in Lesion Bypass during C. elegans Embryogenesis

    PubMed Central

    Roerink, Sophie F.; Koole, Wouter; Stapel, L. Carine; Romeijn, Ron J.; Tijsterman, Marcel

    2012-01-01

    Translesion synthesis (TLS) polymerases are specialized DNA polymerases capable of inserting nucleotides opposite DNA lesions that escape removal by dedicated DNA repair pathways. TLS polymerases allow cells to complete DNA replication in the presence of damage, thereby preventing checkpoint activation, genome instability, and cell death. Here, we characterize functional knockouts for polh-1 and polk-1, encoding the Caenorhabditis elegans homologs of the Y-family TLS polymerases η and κ. POLH-1 acts at many different DNA lesions as it protects cells against a wide range of DNA damaging agents, including UV, γ-irradiation, cisplatin, and methyl methane sulphonate (MMS). POLK-1 acts specifically but redundantly with POLH-1 in protection against methylation damage. Importantly, both polymerases play a prominent role early in embryonic development to allow fast replication of damaged genomes. Contrary to observations in mammalian cells, we show that neither POLH-1 nor POLK-1 is required for homologous recombination (HR) repair of DNA double-strand breaks. A genome-wide RNAi screen for genes that protect the C. elegans genome against MMS–induced DNA damage identified novel components in DNA damage bypass in the early embryo. Our data suggest SUMO-mediated regulation of both POLH-1 and POLK-1, and point towards a previously unrecognized role of the nuclear pore in regulating TLS. PMID:22761594

  8. Angiotensin I - Converting Enzyme Gene Polymorphism and Activity in Patients with Ischemic Stroke

    PubMed Central

    Stankovic, Aleksandra; Asanin, Milika; Jovanovic-Markovic, Zagorka; Alavantic, Dragan; Majkic-Singh, Nada

    2011-01-01

    The possible association of ACE polymorphism with ischemic stroke (IS) was evaluated in 65 patients with IS and 330 age and BMI-matched controls. ACE genotypes were determined by polymerase chain reaction (PCR). There was no significant difference in ACE genotype/allele frequencies between case and control group (p>0.05). Patients with D allele had 4,7 times higher risk for large vessel IS than healthy persons D allele possessors. Persons with D allele had 9.2 times higher risk for large vessel disease than small vessel disease. These data suggest a possible association of ACE gene polymorphism with pathogenesis of large vessel IS.

  9. Restriction fragment length polymorphism species-specific patterns in the identification of white truffles.

    PubMed

    Bertini, L; Potenza, L; Zambonelli, A; Amicucci, A; Stocchi, V

    1998-07-15

    A molecular method for the identification of ectomycorrhizae belonging to five species of white truffle is described. The polymerase chain reaction (PCR) and universal primers were used to amplify internal transcribed spacers and 5.8S rDNA, target sequences present in a high number of copies. The amplified products were digested with restriction enzymes in order to detect interspecific polymorphisms. Species-specific restriction fragment length polymorphism patterns were determined for all five species. The use of PCR in conjunction with restriction enzymes provides a sensitive and efficient tool for use in distinguishing ectomycorrhizal species and monitoring inoculated seedlings or field mycorrhizal populations. PMID:9682488

  10. Mutability of DNA polymerase I: implications for the creation of mutant DNA polymerases.

    PubMed

    Loh, Ern; Loeb, Lawrence A

    2005-12-01

    DNA polymerases of the Family A catalyze the addition of deoxynucleotides to a primer with high efficiency, processivity, and selectivity-properties that are critical to their function both in nature and in the laboratory. These polymerases tolerate many amino acid substitutions, even in regions that are evolutionarily conserved. This tolerance can be exploited to create DNA polymerases with novel properties and altered substrate specificities, using rational design and molecular evolution. These efforts have focused mainly on the Family A DNA polymerises -Taq, E. coli Pol I, and T7 - because they are widely utilized in biotechnology today. The redesign of polymerases often requires knowledge of the function of specific residues in the protein, including those located in six evolutionarily conserved regions. The most well characterized of these are motifs A and B, which regulate the fidelity of replication and the incorporation of nucleotide analogs such as dideoxynucleotides. Regions that remain to be more thoroughly characterized are motif C, which is critical for catalysis, and motifs 1, 2 and 6, all of which bind to DNA primer or template. Several recently identified mutants with abilities to incorporate nucleotides with bulky adducts have mutations that are not located within conserved regions and warrant further study. Analysis of these mutants will help advance our understanding of how DNA polymerases select bases with high fidelity. PMID:16230053

  11. Interleukin-2 and interleukin-6 gene promoter polymorphisms, and early failure of dental implants.

    PubMed

    Campos, Maria Isabela Guimarães; Godoy dos Santos, Maria Cristina Leme; Trevilatto, Paula Cristina; Scarel-Caminaga, Raquel Mantuaneli; Bezerra, Fabio Jose; Line, Sergio Roberto Peres

    2005-12-01

    Single nucleotide polymorphisms in the promoter region of the human interleukin (IL)-2 (T-330G) and IL-6 (G-174C) genes have modified the transcriptional activity of these cytokines and are associated with several diseases. The aim of this study was to investigate the possible relationship between these single nucleotide polymorphisms and early implant failure. A sample of 74 nonsmokers was divided into 2 groups: test group comprising 34 patients (mean age 49.3 years) with >or=1 implants that failed and control group consisting of 40 patients (mean age 43.8 years) with >or=1 healthy implants. Genomic deoxyribonucleic acid from oral mucosa was amplified by polymerase chain reaction and analyzed by restriction fragment length polymorphism. Monte Carlo simulations (P < 0.05) were used to assess differences in allele and genotypes frequencies of the single nucleotide polymorphisms between the 2 groups. No significant differences were observed in the allele and genotypes distribution of both polymorphisms when the 2 groups were compared. The results indicate that polymorphisms in the IL-2 (T-330G) and IL-6 (G-174C) genes are not associated with early implant failure, suggesting that the presence of those single nucleotide polymorphisms does not constitute a genetic risk factor for implant loss in the studied population. PMID:16361891

  12. Investigation of the association between interleukin-1β polymorphism and normal tension glaucoma

    PubMed Central

    Wang, Chun Yuan; Shen, Ying-Cheng; Su, Chien-Hui; Lo, Fai-Yun; Lee, Shi-Huang; Fan, Seng-Sheen

    2007-01-01

    Purpose In normal tension glaucoma (NTG), factors other than elevated intraocular pressure are likely to have a role in the pathogenesis of optic neuropathy. The potential similarities in cellular apoptosis leading to neurodegeneration between Alzheimer's disease and NTG were shown in recent studies. The interleukin-1β (IL-1β; -511) and IL-1β (+3953) polymorphisms were found to increase risk with Alzheimer's disease. The purpose of this study was to test the hypothesis that the IL-1β polymorphism is associated with NTG in the Chinese population. Methods This is a cohort study in a Chinese population that involved 231 people with NTG and 245 healthy controls. Genomic DNA was amplified by a polymerase chain reaction, followed by the enzymatic restriction fragment length polymorphism technique. Patients and controls were genotyped for the C/T polymorphism at position -511 and +3953 of the IL-1β gene. Genotypes for NTG and control groups were compared for statistically significant differences. Results There was no significant difference in genotype frequency or allele frequency distribution of the IL-1β gene polymorphisms (position -511 and +3953) between NTG patients and the control group (p >0.3). Conclusions Our study showed no evidence for an association between the IL-1β (-511) and IL-1β (+3953) polymorphisms and NTG. The IL-1β gene polymorphisms (position -511 and +3953) may not play a key role in NTG pathogenesis in Chinese population. PMID:17563722

  13. Association of ACE Gene I/D polymorphism with migraine in Kashmiri population

    PubMed Central

    Wani, Irfan Yousuf; Sheikh, Saleem; Shah, Zafar Amin; Pandith, Arshid A.; Wani, Mushtaq; Asimi, Ravouf; Wani, Maqbool; Sheikh, Shahnawaz; Mehraj, Iqra

    2016-01-01

    Introduction: Migraine is a complex, recurrent headache disorder that is one of the most common complaints in neurology practice. The role of various genes in its pathogenesis is being studied. We did this study to see whether an association exists between ACE gene I/D polymorphism and migraine in our region. Materials and Methods: The study included 100 patients diagnosed with migraine and 121 healthy controls. The study subject were age and gender matched. The analysis was based on Polymerase Chain Reaction (PCR) and included following steps: DNA extraction from blood, PCR and Restriction Fragment Length Polymorphism (RFLP). Results: Out of 100 cases, 69 were females and 31 were males. Fifty-seven were having migraine without aura and 43 had migraine with aura. 45 of the cases had II polymorphism, 40 had ID polymorphism and 15 had DD polymorphism in ACE gene. Conclusion: We were not able to find a statistically significant association between ACE gene I/D polymorphism with migraine. The reason for difference in results between our study and other studies could be because of different ethnicity in study populations. So a continuous research is needed in this regard in order to find the genes and different polymorphism that increase the susceptibility of Kashmiri population to migraine. PMID:27011636

  14. TATA box polymorphisms in human gene promoters and associated hereditary pathologies.

    PubMed

    Savinkova, L K; Ponomarenko, M P; Ponomarenko, P M; Drachkova, I A; Lysova, M V; Arshinova, T V; Kolchanov, N A

    2009-02-01

    TATA-binding protein (TBP) is the first basal factor that recognizes and binds a TATA box on TATA-containing gene promoters transcribed by RNA polymerase II. Data available in the literature are indicative of admissible variability of the TATA box. The TATA box flanking sequences can influence TBP affinity as well as the level of basal and activated transcription. The possibility of mediated involvement in in vivo gene expression regulation of the TBP interactions with variant TATA boxes is supported by data on TATA box polymorphisms and associated human hereditary pathologies. A table containing data on TATA element polymorphisms in human gene promoters (about 40 mutations have been described), associated with particular pathologies, their short functional characteristics, and manifestation mechanisms of TATA-box SNPs is presented. Four classes of polymorphisms are considered: TATA box polymorphisms that weaken and enhance promoter, polymorphisms causing TATA box emergence and disappearance, and human virus TATA box polymorphisms. The described examples are indicative of the polymorphism-associated severe pathologies like thalassemia, the increased risk of hepatocellular carcinoma, sensitivity to H. pylori infection, oral cavity and lung cancers, arterial hypertension, etc. PMID:19267666

  15. Distribution of paraoxonase PON1 gene polymorphisms in Mexican populations. Its role in the lipid profile.

    PubMed

    Gamboa, Ricardo; Zamora, José; Rodríguez-Pérez, José Manuel; Fragoso, José Manuel; Cardoso, Guillermo; Posadas-Romero, Carlos; Vargas-Alarcón, Gilberto

    2006-02-01

    Paraoxonase gene polymorphisms (PON1-55 and PON1-192) were determined in four Mexican populations (Mestizos, Nahuas, Teenek and Mayos) belonging to different ethnic groups. The role of these polymorphisms in the lipid profile in the Mestizo group was also analyzed. PON1 polymorphisms were determined by polymerase chain reaction-restriction fragment length polymorphism. Comparison among Mexican populations showed increased frequencies of PON1-55 L allele and LL genotype and decreased frequencies of M allele and LM genotype in the three Amerindian populations when compared to Mestizos (P < 0.05). Mexicans together with Asian populations (from Japan and China) presented the highest frequencies of PON1-55 L allele (P < 0.05 when compared to Caucasian populations). Heterogeneous data were noted when PON1-192 polymorphism comparison was made. In summary, distribution frequencies of PON1 showed that Mexican populations are more related to Asians than Caucasians. This confirms previous studies with other genetic markers indicating that Native Americans have stronger genetic affinities to the Paleolithic people of North-East Asia than to other major world populations. In Mexican Mestizos, lack of correlation between PON1 polymorphisms and lipid profile was found, corroborating previous data in other populations. The present data could be helpful to understand the distribution of these polymorphisms and its role as genetic and evolutive markers in the Amerindian populations.

  16. Functional Polymorphisms of Matrix Metalloproteinases 1 and 9 Genes in Women with Spontaneous Preterm Birth

    PubMed Central

    Pleša, Ivana; Peterlin, Ana; Jan, Žiga; Tul, Nataša; Kapović, Miljenko; Ostojić, Saša; Peterlin, Borut

    2014-01-01

    Objective. The aim of this study was to investigate the association of functional MMP-1-1607 1G/2G and MMP-9-1562 C/T gene polymorphisms with spontaneous preterm birth (SPTB; preterm birth with intact membranes) in European Caucasian women, as well as the contribution of these polymorphisms to different clinical features of women with SPTB. Methods and Patients. A case-control study was conducted in 113 women with SPTB and 119 women with term delivery (control group). Genotyping of MMP-1-1607 1G/2G and MMP-9-1562 C/T gene polymorphisms was performed using the combination of polymerase chain reaction and restriction fragment length polymorphism methods. Results. There were no statistically significant differences in the distribution of neither individual nor combinations of genotype and allele frequencies of MMP-1-1607 1G/2G and MMP-9-1562 C/T polymorphisms between women with SPTB and control women. Additionally, these polymorphisms do not contribute to any of the clinical characteristics of women with SPTB, including positive and negative family history of SPTB, gestational age at delivery, and maternal age at delivery, nor fetal birth weight. Conclusion. We did not find the evidence to support the association of MMP-1-1607 1G/2G and MMP-9-1562 C/T gene polymorphisms with SPTB in European Caucasian women. PMID:25530657

  17. Association between osteoprotegerin gene polymorphisms and cardiovascular disease in type 2 diabetic patients

    PubMed Central

    Guo, Changlei; Hu, Fudong; Zhang, Shaoli; Wang, Yakun; Liu, Hengdao

    2013-01-01

    Osteoprotegerin (OPG) gene polymorphisms (T245G, T950C and G1181C) have been associated with osteoporosis and early predictors of cardiovascular disease. The aim of this study was to evaluate whether these polymorphisms contribute to cardiovascular disease (CVD) in type 2 diabetic patients. We performed a case-control study with 178 CVD subjects with diabetes and 312 diabetic patients without CVD to assess the impact of variants of the OPG gene on the risk of CVD. The OPG gene polymorphisms were analyzed by using the polymerase chain reaction (PCR) and restriction fragment length polymorphism (RFLP). There was no significant association between the T245G and G1181C polymorphisms and CVD in the additive genetic model (OR = 0.96, 95% CI 0.64–1.45, p = 0.79; OR = 1.06, 95% CI 0.81–1.39, p = 0.65, respectively). However, the C allele of the T950C polymorphism was independently associated with a risk of CVD in type 2 diabetic patients in this genetic model (OR = 1.38, 95% CI 1.07–1.80, p = 0.01). This study provides evidence that the C allele of the T950C polymorphism is associated with increased risk of CVD in diabetic patients. However, well-designed prospective studies with a larger sample size are needed to validate these results. PMID:23885198

  18. Data of expression and purification of recombinant Taq DNA polymerase.

    PubMed

    Fang, Na; Zhong, Niannian; Yang, Yueyang; Guo, Yujian; Ji, Shaoping

    2016-12-01

    Polymerase chain reaction (PCR) technique is widely used in many experimental conditions, and Taq DNA polymerase is critical in PCR process. In this article, the Taq DNA polymerase expression plasmid is reconstructed and the protein product is obtained by rapid purification, ("Rapid purification of high-activity Taq DNA polymerase" (Pluthero, 1993 [1]), "Single-step purification of a thermostable DNA polymerase expressed in Escherichia coli" (Desai and Pfaffle, 1995 [2])). Here we present the production data from protein expression and provide the analysis results of the production from two different vectors. Meanwhile, the purification data is also provided to show the purity of the protein product. PMID:27656666

  19. Clinical relevance of IL-6 gene polymorphism in severely injured patients

    PubMed Central

    Jeremić, Vasilije; Alempijević, Tamara; Mijatović, Srđan; Šijački, Ana; Dragašević, Sanja; Pavlović, Sonja; Miličić, Biljana; Krstić, Slobodan

    2014-01-01

    In polytrauma, injuries that may be surgically treated under regular circumstances due to a systemic inflammatory response become life-threatening. The inflammatory response involves a complex pattern of humoral and cellular responses and the expression of related factors is thought to be governed by genetic variations. This aim of this paper is to examine the influence of interleukin (IL) 6 single nucleotide polymorphism (SNP) -174C/G and -596G/A on the treatment outcome in severely injured patients. Forty-seven severely injured patients were included in this study. Patients were assigned an Injury Severity Score. Blood samples were drawn within 24 h after admission (designated day 1) and on subsequent days (24, 48, 72 hours and 7days) of hospitalization. The IL-6 levels were determined through ELISA technique. Polymorphisms were analyzed by a method of Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR). Among subjects with different outcomes, no statistically relevant difference was found with regards to the gene IL-6 SNP-174G/C polymorphism. More than a half of subjects who died had the SNP-174G/C polymorphism, while this polymorphism was represented in a slightly lower number in survivors. The incidence of subjects without polymorphism and those with heterozygous and homozygous gene IL-6 SNP-596G/A polymorphism did not present statistically significant variations between survivors and those who died. The levels of IL-6 over the observation period did not present any statistically relevant difference among subjects without the IL-6 SNP-174 or IL-6 SNP -596 gene polymorphism and those who had either a heterozygous or a homozygous polymorphism. PMID:24856384

  20. Association between Interleukin-18 Polymorphisms and Hepatocellular Carcinoma Occurrence and Clinical Progression.

    PubMed

    Lau, Hon-Kit; Hsieh, Ming-Ju; Yang, Shun-Fa; Wang, Hsiang-Ling; Kuo, Wu-Hsien; Lee, Hsiang-Lin; Yeh, Chao-Bin

    2016-01-01

    We investigated the association between interleukin-18 (IL-18) polymorphisms and the susceptibility and clinicopathological state of hepatocellular carcinoma (HCC). In total, 901 participants, including 559 healthy controls and 342 patients with HCC, were recruited. The allelic discrimination of -607A/C (rs1946518) and -137G/C (rs187238) polymorphisms of IL-18 was assessed through real-time polymerase chain reaction by performing the TaqMan assay. The IL-18 -137G/C polymorphism but not the -607A/C polymorphism showed a significant association with the risk of HCC. Participants carrying the IL-18 -137 polymorphism with heterozygous G/C and homozygous CC genotypes showed a 1.987-fold increase (95% CI = 1.301-3.032; p = 0.001) in the risk of HCC compared with those homozygous for wild-type G/G. The 342 patients with HCC carrying the IL-18 -137G/C polymorphism were positive for hepatitis B virus (HBV) infection with an adjusted odds ratio of 1.668. Moreover, the 142 HBV positive patients with HCC and the IL-18 -137 polymorphism were positive for at least one C genotype and showed significant vascular invasion (p = 0.018). Furthermore, the level of α-fetoprotein was high in the patients carrying the IL-18 -137 polymorphism with GC+CC alleles (p = 0.011). In conclusion, the IL-18 -137G/C polymorphism with a GC+CC genotype could be a factor that increases the risk of HCC. Furthermore, the correlation between the IL-18 -137G/C polymorphism and HCC-related HBV infection is a risk factor for vascular invasion and has a synergistic effect that can further enhance HCC prognosis.

  1. Interaction Between Polymorphisms of IFN-γ and MICA Correlated with Hepatocellular Carcinoma.

    PubMed

    Li, Hongguang; Liu, Fangfeng; Zhu, Huaqiang; Zhou, Xu; Lu, Jun; Chang, Hong; Hu, Jinhua

    2016-02-19

    BACKGROUND We explored the relationship of interferon-γ (IFN-γ) and MHC class-I chain related gene A (MICA) genes polymorphisms with hepatocellular carcinoma (HCC) risk, and tried to determine whether the interaction existed between these two genes polymorphisms on the basis of HCC. MATERIAL AND METHODS Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to detect the genotypes of the 3 single-nucleotide polymorphisms (SNPs) and to analyze the correlation of each SNP with HCC susceptibility in 120 HCC patients and 124 healthy people. The association strength between the 3 SNPs and HCC is represented with odds ratio (OR) and 95% confidence interval (95% CI). Hardy-Weinberg equilibrium (HWE) was tested by χ2 test in the control group. RESULTS GG genotype of IFN-γ rs2069727 polymorphism had apparently different distributions in case and control groups (P<0.05), and might confer increased risk of HCC (OR=3.40, 95%CI=1.23-9.38). Analysis of MICA rs2596542 polymorphism also yielded the same result (OR=2.90, 95%CI=1.10-7.67), as did their risk alleles. Specifically, the interaction between rs2596542 and rs2069705 polymorphisms increased the HCC risk by 1.41 times and between rs2596542 and rs2069727 polymorphisms the increased risk of HCC by 5.56 times. CONCLUSIONS IFN-γ rs2069727 and MICA rs2596542 polymorphisms may be related to the incidence of HCC. Interaction exists between the polymorphisms of IFN-γ and MICA, which may increase risk of HCC.

  2. Interaction Between Polymorphisms of IFN-γ and MICA Correlated with Hepatocellular Carcinoma

    PubMed Central

    Li, Hongguang; Liu, Fangfeng; Zhu, Huaqiang; Zhou, Xu; Lu, Jun; Chang, Hong; Hu, Jinhua

    2016-01-01

    Background We explored the relationship of interferon--γ (IFN-γ) and MHC class-I chain related gene A (MICA) genes polymorphisms with hepatocellular carcinoma (HCC) risk, and tried to determine whether the interaction existed between these two genes polymorphisms on the basis of HCC. Material/Methods Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method was used to detect the genotypes of the 3 single-nucleotide polymorphisms (SNPs) and to analyze the correlation of each SNP with HCC susceptibility in 120 HCC patients and 124 healthy people. The association strength between the 3 SNPs and HCC is represented with odds ratio (OR) and 95% confidence interval (95% CI). Hardy-Weinberg equilibrium (HWE) was tested by χ2 test in the control group. Results GG genotype of IFN-γ rs2069727 polymorphism had apparently different distributions in case and control groups (P<0.05), and might confer increased risk of HCC (OR=3.40, 95%CI=1.23–9.38). Analysis of MICA rs2596542 polymorphism also yielded the same result (OR=2.90, 95%CI=1.10–7.67), as did their risk alleles. Specifically, the interaction between rs2596542 and rs2069705 polymorphisms increased the HCC risk by 1.41 times and between rs2596542 and rs2069727 polymorphisms the increased risk of HCC by 5.56 times. Conclusions IFN-γ rs2069727 and MICA rs2596542 polymorphisms may be related to the incidence of HCC. Interaction exists between the polymorphisms of IFN-γ and MICA, which may increase risk of HCC. PMID:26893439

  3. Association between Interleukin-18 Polymorphisms and Hepatocellular Carcinoma Occurrence and Clinical Progression.

    PubMed

    Lau, Hon-Kit; Hsieh, Ming-Ju; Yang, Shun-Fa; Wang, Hsiang-Ling; Kuo, Wu-Hsien; Lee, Hsiang-Lin; Yeh, Chao-Bin

    2016-01-01

    We investigated the association between interleukin-18 (IL-18) polymorphisms and the susceptibility and clinicopathological state of hepatocellular carcinoma (HCC). In total, 901 participants, including 559 healthy controls and 342 patients with HCC, were recruited. The allelic discrimination of -607A/C (rs1946518) and -137G/C (rs187238) polymorphisms of IL-18 was assessed through real-time polymerase chain reaction by performing the TaqMan assay. The IL-18 -137G/C polymorphism but not the -607A/C polymorphism showed a significant association with the risk of HCC. Participants carrying the IL-18 -137 polymorphism with heterozygous G/C and homozygous CC genotypes showed a 1.987-fold increase (95% CI = 1.301-3.032; p = 0.001) in the risk of HCC compared with those homozygous for wild-type G/G. The 342 patients with HCC carrying the IL-18 -137G/C polymorphism were positive for hepatitis B virus (HBV) infection with an adjusted odds ratio of 1.668. Moreover, the 142 HBV positive patients with HCC and the IL-18 -137 polymorphism were positive for at least one C genotype and showed significant vascular invasion (p = 0.018). Furthermore, the level of α-fetoprotein was high in the patients carrying the IL-18 -137 polymorphism with GC+CC alleles (p = 0.011). In conclusion, the IL-18 -137G/C polymorphism with a GC+CC genotype could be a factor that increases the risk of HCC. Furthermore, the correlation between the IL-18 -137G/C polymorphism and HCC-related HBV infection is a risk factor for vascular invasion and has a synergistic effect that can further enhance HCC prognosis. PMID:27429592

  4. Differential methylation of the promoter and first exon of the RASSF1A gene in hepatocarcinogenesis

    PubMed Central

    Jain, Surbhi; Xie, Lijia; Boldbaatar, Batbold; Lin, Selena Y.; Hamilton, James P.; Meltzer, Stephen J.; Chen, Shun-Hua; Hu, Chi-Tan; Block, Timothy M.; Song, Wei; Su, Ying-Hsiu

    2015-01-01

    Aim Aberrant methylation of the promoter, P2, and the first exon, E1, regions of the tumor suppressor gene RASSF1A, have been associated with hepatocellular carcinoma (HCC), albeit with poor specificity. This study analyzed the methylation profiles of P1, P2 and E1 regions of the gene to identify the region of which methylation most specifically corresponds to HCC and to evaluate the potential of this methylated region as a biomarker in urine for HCC screening. Methods Bisulfite DNA sequencing and quantitative methylation-specific polymerase chain reaction assays were performed to compare methylation of the 56 CpG sites in regions P1, P2 and E1 in DNA isolated from normal, hepatitic, cirrhotic, adjacent non-HCC, and HCC liver tissue and urine samples for the characterization of hypermethylation of the RASSF1A gene as a biomarker for HCC screening. Results In tissue, comparing HCC (n = 120) with cirrhosis and hepatitis together (n = 70), methylation of P1 had an area under the receiver operating characteristics curve (AUROC) of 0.90, whereas methylation of E1 and P2 had AUROC of 0.84 and 0.72, respectively. At 90% sensitivity, specificity for P1 methylation was 72.9% versus 38.6% for E1 and 27.1% for P2. Methylated P1 DNA was detected in urine in association with cirrhosis and HCC. It had a sensitivity of 81.8% for α-fetoprotein negative HCC. Conclusion Among the three regions analyzed, methylation of P1 is the most specific for HCC and holds great promise as a DNA marker in urine for screening of cirrhosis and HCC. PMID:25382672

  5. Genome-wide DNA methylation profiling of non-small cell lung carcinomas

    PubMed Central

    2012-01-01

    Background Non-small cell lung carcinoma (NSCLC) is a complex malignancy that owing to its heterogeneity and poor prognosis poses many challenges to diagnosis, prognosis and patient treatment. DNA methylation is an important mechanism of epigenetic regulation involved in normal development and cancer. It is a very stable and specific modification and therefore in principle a very suitable marker for epigenetic phenotyping of tumors. Here we present a genome-wide DNA methylation analysis of NSCLC samples and paired lung tissues, where we combine MethylCap and next generation sequencing (MethylCap-seq) to provide comprehensive DNA methylation maps of the tumor and paired lung samples. The MethylCap-seq data were validated by bisulfite sequencing and methyl-specific polymerase chain reaction of selected regions. Results Analysis of the MethylCap-seq data revealed a strong positive correlation between replicate experiments and between paired tumor/lung samples. We identified 57 differentially methylated regions (DMRs) present in all NSCLC tumors analyzed by MethylCap-seq. While hypomethylated DMRs did not correlate to any particular functional category of genes, the hypermethylated DMRs were strongly associated with genes encoding transcriptional regulators. Furthermore, subtelomeric regions and satellite repeats were hypomethylated in the NSCLC samples. We also identified DMRs that were specific to two of the major subtypes of NSCLC, adenocarcinomas and squamous cell carcinomas. Conclusions Collectively, we provide a resource containing genome-wide DNA methylation maps of NSCLC and their paired lung tissues, and comprehensive lists of known and novel DMRs and associated genes in NSCLC. PMID:22726460

  6. Role of DNA methylation in the development of Epstein-Barr virus-associated gastric carcinoma.

    PubMed

    Saito, Mari; Nishikawa, Jun; Okada, Toshiyuki; Morishige, Akihiro; Sakai, Kouhei; Nakamura, Munetaka; Kiyotoki, Shu; Hamabe, Kouichi; Okamoto, Takeshi; Oga, Atsunori; Sasaki, Kohsuke; Suehiro, Yutaka; Hinoda, Yuji; Sakaida, Isao

    2013-01-01

    The frequencies of DNA methylation of certain tumor-related genes are higher in Epstein-Barr virus (EBV)-associated gastric carcinomas than in EBV-negative gastric carcinomas. EBV-associated gastric carcinomas have distinct clinicopathological features; however, there are no case-control studies comparing methylation frequency between EBV-associated gastric carcinomas and controls that have been adjusted according to the clinicopathological features of EBV-associated gastric carcinomas. This study evaluated 25 EBV-associated gastric carcinomas that were positive for EBV-encoded small RNA 1 (EBER-1) by in situ hybridization and 50 EBV-negative gastric carcinomas that were matched with the EBV-associated gastric carcinomas by age, sex, histology, depth of tumor invasion, and stage. Methylation status of 16 loci associated with tumor-related genes was analyzed by methylation-specific polymerase chain reaction (PCR) to identify genes in which DNA methylation specifically occurred in EBV-associated gastric carcinomas. Methylation frequencies of 12 of the 16 genes were higher in EBV-associated gastric carcinomas than in EBV-negative controls, and the frequency of methylation of 6 specific loci (MINT2, MINT31, p14, p16, p73, and RUNX3) was significantly higher in EBV-associated gastric carcinomas than in EBV-negative controls. There were no significant differences in the methylation frequencies of the other genes. The mean methylation index in EBV-associated gastric carcinomas was significantly higher than that in EBV-negative controls. DNA methylation of tumor suppressor genes that regulate the cell cycle and apoptosis specifically occurred in EBV-associated gastric carcinomas. Aberrant DNA methylation might lead to the development and progression of EBV-associated gastric carcinoma. PMID:23073987

  7. Polymorphism of phosphoric oxide

    USGS Publications Warehouse

    Hill, W.L.; Faust, G.T.; Hendricks, S.B.

    1943-01-01

    The melting points and monotropic relationship of three crystalline forms of phosphoric oxide were determined by the method of quenching. Previous vapor pressure data are discussed and interpreted to establish a pressure-temperature diagram (70 to 600??) for the one-component system. The system involves three triple points, at which solid, liquid and vapor (P4O10) coexist in equilibrium, namely: 420?? and 360 cm., 562?? and 43.7 cm. and 580?? and 55.5 cm., corresponding to the hexagonal, orthorhombic and stable polymorphs, respectively, and at least two distinct liquids, one a stable polymer of the other, which are identified with the melting of the stable form and the hexagonal modification, respectively. Indices of refraction of the polymorphs and glasses were determined. The density and the thermal, hygroscopic and structural properties of the several phases are discussed.

  8. [Influence of polymorphism's of endothelial nitric oxide synthase gene and polymorphism of NADPH oxidase gene on development of complications of arterial hypertension].

    PubMed

    Kuznetsova, T Iu; Gavrilov, D V; Dudanov, I P; Makarevich, P I; Balatskiĭ, A V; Samokhodskaia, L M; Parfenova, E V

    2008-01-01

    The aim of the study was to analyze the prevalence of polymorphism Glu298Asp of endothelial nitric oxide synthase gene and C242T p22 phox polymorphism of NADPH oxidase gene in patients with arterial hypertension (AH) and their influence on AH complications. The study included 272 AH patients, average age 50,7 years. The following analyses were performed: clinical analysis of the blood, general analysis of the urine, lipid spectrum, plasma electrolytes, creatinine, glucose, electrocardiography, echocardioscopy, examination of eye vessels, ultrasound examination of the carotid arteries, determination of microalbuminuria. The polymorphism Glu298Asp of endothelial nitric oxide synthase gene and C242T p22 phox polymorphism of NADPH oxidase gene were detected with two methods: polymerase chain reaction and restrictase reaction. The control group for Glu298Asp polymorphism detection included 102 healthy Russian donors aged 18 to 50 years. Genotypes prevalence in AH patients was as follows: GG 58,8%, GA 32,3%, AA 8,9%, and CC 48,2%, CT 44,9%, TT 6.9%. In the control group: GG 53%, GA 36%, AA 11% and CC 42%, CT 54%, TT 4%. These polymorphisms did not affect the incidence of complications, such as obliterating atherosclerosis of the lower extremity vessels, ischemic heart disease, and acute insufficiency of cerebral circulation, chronic heart failure, left ventricular hypertrophy, microalbuminuria, carotid arteries atherosclerosis. PMID:18429753

  9. Differentially methylated obligatory epialleles modulate context-dependent LAM gene expression in the honeybee Apis mellifera.

    PubMed

    Wedd, Laura; Kucharski, Robert; Maleszka, Ryszard

    2016-01-01

    Differential intragenic methylation in social insects has been hailed as a prime mover of environmentally driven organismal plasticity and even as evidence for genomic imprinting. However, very little experimental work has been done to test these ideas and to prove the validity of such claims. Here we analyze in detail differentially methylated obligatory epialleles of a conserved gene encoding lysosomal α-mannosidase (AmLAM) in the honeybee. We combined genotyping of progenies derived from colonies founded by single drone inseminated queens, ultra-deep allele-specific bisulfite DNA sequencing, and gene expression to reveal how sequence variants, DNA methylation, and transcription interrelate. We show that both methylated and non-methylated states of AmLAM follow Mendelian inheritance patterns and are strongly influenced by polymorphic changes in DNA. Increased methylation of a given allele correlates with higher levels of context-dependent AmLAM expression and appears to affect the transcription of an antisense long noncoding RNA. No evidence of allelic imbalance or imprinting involved in this process has been found. Our data suggest that by generating alternate methylation states that affect gene expression, sequence variants provide organisms with a high level of epigenetic flexibility that can be used to select appropriate responses in various contexts. This study represents the first effort to integrate DNA sequence variants, gene expression, and methylation in a social insect to advance our understanding of their relationships in the context of causality. PMID:26507253

  10. Differentially methylated obligatory epialleles modulate context-dependent LAM gene expression in the honeybee Apis mellifera

    PubMed Central

    Wedd, Laura; Kucharski, Robert; Maleszka, Ryszard

    2016-01-01

    ABSTRACT Differential intragenic methylation in social insects has been hailed as a prime mover of environmentally driven organismal plasticity and even as evidence for genomic imprinting. However, very little experimental work has been done to test these ideas and to prove the validity of such claims. Here we analyze in detail differentially methylated obligatory epialleles of a conserved gene encoding lysosomal α-mannosidase (AmLAM) in the honeybee. We combined genotyping of progenies derived from colonies founded by single drone inseminated queens, ultra-deep allele-specific bisulfite DNA sequencing, and gene expression to reveal how sequence variants, DNA methylation, and transcription interrelate. We show that both methylated and non-methylated states of AmLAM follow Mendelian inheritance patterns and are strongly influenced by polymorphic changes in DNA. Increased methylation of a given allele correlates with higher levels of context-dependent AmLAM expression and appears to affect the transcription of an antisense long noncoding RNA. No evidence of allelic imbalance or imprinting involved in this process has been found. Our data suggest that by generating alternate methylation states that affect gene expression, sequence variants provide organisms with a high level of epigenetic flexibility that can be used to select appropriate responses in various contexts. This study represents the first effort to integrate DNA sequence variants, gene expression, and methylation in a social insect to advance our understanding of their relationships in the context of causality. PMID:26507253

  11. Differentially methylated obligatory epialleles modulate context-dependent LAM gene expression in the honeybee Apis mellifera.

    PubMed

    Wedd, Laura; Kucharski, Robert; Maleszka, Ryszard

    2016-01-01

    Differential intragenic methylation in social insects has been hailed as a prime mover of environmentally driven organismal plasticity and even as evidence for genomic imprinting. However, very little experimental work has been done to test these ideas and to prove the validity of such claims. Here we analyze in detail differentially methylated obligatory epialleles of a conserved gene encoding lysosomal α-mannosidase (AmLAM) in the honeybee. We combined genotyping of progenies derived from colonies founded by single drone inseminated queens, ultra-deep allele-specific bisulfite DNA sequencing, and gene expression to reveal how sequence variants, DNA methylation, and transcription interrelate. We show that both methylated and non-methylated states of AmLAM follow Mendelian inheritance patterns and are strongly influenced by polymorphic changes in DNA. Increased methylation of a given allele correlates with higher levels of context-dependent AmLAM expression and appears to affect the transcription of an antisense long noncoding RNA. No evidence of allelic imbalance or imprinting involved in this process has been found. Our data suggest that by generating alternate methylation states that affect gene expression, sequence variants provide organisms with a high level of epigenetic flexibility that can be used to select appropriate responses in various contexts. This study represents the first effort to integrate DNA sequence variants, gene expression, and methylation in a social insect to advance our understanding of their relationships in the context of causality.

  12. [Polymorphs of clopidogrel bisulfate].

    PubMed

    Liu, Yi; Huang, Hai-Wei; Wu, Jian-Min; Shi, Ya-Qin; Yang, La-Hu

    2013-08-01

    This paper is to report the polymorphism of raw materials of clopidogrel bisulfate at home and abroad. By the analysis of Fourier transform infrared spectroscopy (FTIR) and powder X-ray diffraction (p-XRD), samples are roughly classified into two groups, except one patent material. And the differential scanning calorimeter (DSC) examination showed more detailed information for these materials. The results of the study could provide comprehensive basis for the quality evaluation of clopidogrel bisulfate. PMID:24187849

  13. Examining the Impact of Gene Variants on Histone Lysine Methylation

    PubMed Central

    Van Rechem, Capucine; Whetstine, Johnathan R.

    2015-01-01

    In recent years, there has been a boom in the amount of genome-wide sequencing data that has uncovered important and unappreciated links between certain genes, families of genes and enzymatic processes and diseases such as cancer. Such studies have highlighted the impact that chromatin modifying enzymes could have in cancer and other genetic diseases. In this review, we summarize characterized mutations and single nucleotide polymorphisms (SNPs) in histone lysine methyltransferases (KMTs), histone lysine demethylases (KDMs) and histones. We primarily focus on variants with strong disease correlations and discuss how they could impact histone lysine methylation dynamics and gene regulation. PMID:24859469

  14. Mutational clusters generated by non-processive polymerases: A case study using DNA polymerase betain vitro.

    PubMed

    García-Villada, Libertad; Drake, John W

    2010-08-01

    Available DNA mutational spectra reveal that the number of mutants with multiple mutations ("multiples") is usually greater than expected from a random distribution of mutations among mutants. These overloads imply the occurrence of non-random clusters of mutations, probably generated during episodes of low-fidelity DNA synthesis. Excess multiples have been reported not only for viruses, bacteria, and eukaryotic cells but also for the DNA polymerases of phages T4 and RB69 in vitro. In the simplest case of a purified polymerase, non-random clusters may be generated by a subfraction of phenotypic variants able to introduce more errors per cycle of DNA synthesis than the normal enzyme. According to this hypothesis, excess multiples are not expected with non-processive polymerases even if they harbor rare mutator variants. DNA polymerase beta (Pol beta) is a mammalian DNA-repair polymerase with very low processivity. Although several Pol beta mutational spectra have been described, there is conflicting evidence on whether or not excess multiples occur, with spectra based on the HSV-tk system tending to show excess multiples. Excess multiples generated by Pol beta or any of its mutants might imply that the excesses of multiples observed in numerous other systems, especially those with processive polymerases, could be artifactual. Here, the distributions of mutations generated by native and recombinant rat Pol beta and by the Pol beta(Y265C) mutator were analyzed in the M13mp2 lacZalpha system. Our results present no evidence for a significant excess of multiples over the expected numbers with any of the Pol beta enzymes tested in this system. The reported excess of Pol beta-generated multiples in the HSV-tk system may reflect a reduced efficiency of detection of base substitutions that cause weak phenotypes, which in turn may artifactually increase the frequency of multiples. PMID:20627824

  15. RNA polymerase molecular beacon as tool for studies of RNA polymerase-promoter interactions.

    PubMed

    Mekler, Vladimir; Severinov, Konstantin

    2015-09-15

    The molecular details of formation of transcription initiation complex upon the interaction of bacterial RNA polymerase (RNAP) with promoters are not completely understood. One way to address this problem is to understand how RNAP interacts with different parts of promoter DNA. A recently developed fluorometric RNAP molecular beacon assay allows one to monitor the RNAP interactions with various unlabeled DNA probes and quantitatively characterize partial RNAP-promoter interactions. This paper focuses on methodological aspects of application of this powerful assay to study the mechanism of transcription initiation complex formation by Escherichia coli RNA polymerase σ(70) holoenzyme and its regulation by bacterial and phage encoded factors.

  16. RNA polymerase molecular beacon as tool for studies of RNA polymerase - promoter interactions

    PubMed Central

    Mekler, Vladimir; Severinov, Konstantin

    2015-01-01

    The molecular details of formation of transcription initiation complex upon the interaction of bacterial RNA polymerase (RNAP) with promoters are not completely understood. One way to address this problem is to understand how RNAP interacts with different parts of promoter DNA. A recently developed fluorometric RNAP molecular beacon assay allows one to monitor the RNAP interactions with various unlabeled DNA probes and quantitatively characterize partial RNAP-promoter interactions. This paper focuses on methodological aspects of application of this powerful assay to study the mechanism of transcription initiation complex formation by Escherichia coli RNA polymerase σ70 holoenzyme and its regulation by bacterial and phage encoded factors. PMID:25956222

  17. ppGpp: magic beyond RNA polymerase.

    PubMed

    Dalebroux, Zachary D; Swanson, Michele S

    2012-02-16

    During stress, bacteria undergo extensive physiological transformations, many of which are coordinated by ppGpp. Although ppGpp is best known for enhancing cellular resilience by redirecting the RNA polymerase (RNAP) to certain genes, it also acts as a signal in many other cellular processes in bacteria. After a brief overview of ppGpp biosynthesis and its impact on promoter selection by RNAP, we discuss how bacteria exploit ppGpp to modulate the synthesis, stability or activity of proteins or regulatory RNAs that are crucial in challenging environments, using mechanisms beyond the direct regulation of RNAP activity.

  18. High-temperature effect on genes engaged in DNA methylation and affected by DNA methylation in Arabidopsis.

    PubMed

    Naydenov, Mladen; Baev, Vesselin; Apostolova, Elena; Gospodinova, Nadezhda; Sablok, Gaurav; Gozmanova, Mariyana; Yahubyan, Galina

    2015-02-01

    Along with its essential role in the maintenance of genome integrity, DNA methylation takes part in regulation of genes which are important for plant development and stress response. In plants, DNA methylation process can be directed by small RNAs in process known as RNA-directed DNA methylation (RdDM) involving two plant-specific RNA polymerases - PolIV and PolV. The aim of the present study was to investigate the effect of heat stress on the expression of genes encoding key players in DNA methylation - DNA methyltransferase (MET1, CMT3, and DRM2), the largest subunits of PoIIV and PolV (NRPD1 and NRPE1 respectively) and the DNA demethylase ROS1. We also examined the high-temperature effect on two protein-coding genes - At3g50770 and At5g43260 whose promoters contain transposon insertions and are affected by DNA-methylation, as well as on the AtSN1, a SINE-like retrotransposon. To assess the involvement of PolIV and PolV in heat stress response, the promoter methylation status and transcript levels of these genes were compared between wild type and double mutant lacking NRPD1 and NRPE1. The results demonstrate coordinated up-regulation of the DRM2, NRPD1 and NRPE1 in response to high temperature and suggest that PolIV and/or PolV might be required for the induction of DRM2 expression under heat stress. The ROS1 expression was confirmed to be suppressed in the mutant lacking active PolIV and PolV that might be a consequence of abolished DNA methylation. The increased expression of At3g50770 in response to elevated temperature correlated with reduced promoter DNA methylation, while the stress response of At5g43260 did not show inverse correlation between promoter methylation and gene expression. Our results also imply that PolIV and/or PolV could regulate gene expression under stress conditions not only through RdDM but also by acting in other regulatory processes.

  19. Five mutations of mitochondrial DNA polymerase-gamma (POLG) are not a prevalent etiology for spontaneous 46,XX primary ovarian insufficiency.

    PubMed

    Tong, Zhi-Bin; Sullivan, Shannon D; Lawless, Lindsey M; Vanderhoof, Vien; Zachman, Keith; Nelson, Lawrence M

    2010-12-01

    The study objective was to determine if mutations in mitochondrial DNA polymerase gamma (POLG) are associated with spontaneous 46,XX primary ovarian insufficiency (sPOI) using restriction fragment length polymorphism analysis of genomic DNA. Of 201 women with 46,XX sPOI analyzed, we found only one case (0.5%, 95% confidence interval 0-3%) of heterozygosity for a POLG mutation, suggesting that this is not a common genetic etiology for this form of infertility. PMID:20701905

  20. Use of MSAP markers to analyse the effects of salt stress on DNA methylation in rapeseed (Brassica napus var. oleifera).

    PubMed

    Marconi, Gianpiero; Pace, Roberta; Traini, Alessandra; Raggi, Lorenzo; Lutts, Stanley; Chiusano, Marialuisa; Guiducci, Marcello; Falcinelli, Mario; Benincasa, Paolo; Albertini, Emidio

    2013-01-01

    Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP) approach was used to assess the extent of cytosine methylation under salinity stress in salinity-tolerant (Exagone) and salinity-sensitive (Toccata) rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in Exagone and increased in Toccata. Nineteen DNA fragments showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes involved in stress responses (Lacerata and trehalose-6-phosphatase synthase S4) and were chosen to further characterization. Bisulfite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied. In particular, our data show that salinity stress influences the expression of the two stress-related genes. Moreover, we quantified the level of trehalose in Exagone shoots and found that it was correlated to TPS4 expression and, therefore, to DNA methylation. In conclusion, we found that salinity could induce genome-wide changes in DNA methylation status, and that these changes, when averaged across different genotypes and developmental stages, accounted for 16.8% of the total site-specific methylation differences

  1. Use of MSAP Markers to Analyse the Effects of Salt Stress on DNA Methylation in Rapeseed (Brassica napus var. oleifera)

    PubMed Central

    Marconi, Gianpiero; Pace, Roberta; Traini, Alessandra; Raggi, Lorenzo; Lutts, Stanley; Chiusano, Marialuisa; Guiducci, Marcello; Falcinelli, Mario; Benincasa, Paolo; Albertini, Emidio

    2013-01-01

    Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP) approach was used to assess the extent of cytosine methylation under salinity stress in salinity-tolerant (Exagone) and salinity-sensitive (Toccata) rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in Exagone and increased in Toccata. Nineteen DNA fragments showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes involved in stress responses (Lacerata and trehalose-6-phosphatase synthase S4) and were chosen to further characterization. Bisulfite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied. In particular, our data show that salinity stress influences the expression of the two stress-related genes. Moreover, we quantified the level of trehalose in Exagone shoots and found that it was correlated to TPS4 expression and, therefore, to DNA methylation. In conclusion, we found that salinity could induce genome-wide changes in DNA methylation status, and that these changes, when averaged across different genotypes and developmental stages, accounted for 16.8% of the total site-specific methylation differences

  2. Use of MSAP markers to analyse the effects of salt stress on DNA methylation in rapeseed (Brassica napus var. oleifera).

    PubMed

    Marconi, Gianpiero; Pace, Roberta; Traini, Alessandra; Raggi, Lorenzo; Lutts, Stanley; Chiusano, Marialuisa; Guiducci, Marcello; Falcinelli, Mario; Benincasa, Paolo; Albertini, Emidio

    2013-01-01

    Excessive soil salinity is a major ecological and agronomical problem, the adverse effects of which are becoming a serious issue in regions where saline water is used for irrigation. Plants can employ regulatory strategies, such as DNA methylation, to enable relatively rapid adaptation to new conditions. In this regard, cytosine methylation might play an integral role in the regulation of gene expression at both the transcriptional and post-transcriptional levels. Rapeseed, which is the most important oilseed crop in Europe, is classified as being tolerant of salinity, although cultivars can vary substantially in their levels of tolerance. In this study, the Methylation Sensitive Amplified Polymorphism (MSAP) approach was used to assess the extent of cytosine methylation under salinity stress in salinity-tolerant (Exagone) and salinity-sensitive (Toccata) rapeseed cultivars. Our data show that salinity affected the level of DNA methylation. In particular methylation decreased in Exagone and increased in Toccata. Nineteen DNA fragments showing polymorphisms related to differences in methylation were sequenced. In particular, two of these were highly similar to genes involved in stress responses (Lacerata and trehalose-6-phosphatase synthase S4) and were chosen to further characterization. Bisulfite sequencing and quantitative RT-PCR analysis of selected MSAP loci showed that cytosine methylation changes under salinity as well as gene expression varied. In particular, our data show that salinity stress influences the expression of the two stress-related genes. Moreover, we quantified the level of trehalose in Exagone shoots and found that it was correlated to TPS4 expression and, therefore, to DNA methylation. In conclusion, we found that salinity could induce genome-wide changes in DNA methylation status, and that these changes, when averaged across different genotypes and developmental stages, accounted for 16.8% of the total site-specific methylation differences

  3. DNA Methylation Assessed by SMRT Sequencing Is Linked to Mutations in Neisseria meningitidis Isolates

    PubMed Central

    Sater, Mohamad R. Abdul; Lamelas, Araceli; Wang, Guilin; Clark, Tyson A.; Röltgen, Katharina; Mane, Shrikant; Korlach, Jonas; Pluschke, Gerd; Schmid, Christoph D.

    2015-01-01

    The Gram-negative bacterium Neisseria meningitidis features extensive genetic variability. To present, proposed virulence genotypes are also detected in isolates from asymptomatic carriers, indicating more complex mechanisms underlying variable colonization modes of N. meningitidis. We applied the Single Molecule, Real-Time (SMRT) sequencing method from Pacific Biosciences to assess the genome-wide DNA modification profiles of two genetically related N. meningitidis strains, both of serogroup A. The resulting DNA methylomes revealed clear divergences, represented by the detection of shared and of strain-specific DNA methylation target motifs. The positional distribution of these methylated target sites within the genomic sequences displayed clear biases, which suggest a functional role of DNA methylation related to the regulation of genes. DNA methylation in N. meningitidis has a likely underestimated potential for variability, as evidenced by a careful analysis of the ORF status of a panel of confirmed and predicted DNA methyltransferase genes in an extended collection of N. meningitidis strains of serogroup A. Based on high coverage short sequence reads, we find phase variability as a major contributor to the variability in DNA methylation. Taking into account the phase variable loci, the inferred functional status of DNA methyltransferase genes matched the observed methylation profiles. Towards an elucidation of presently incompletely characterized functional consequences of DNA methylation in N. meningitidis, we reveal a prominent colocalization of methylated bases with Single Nucleotide Polymorphisms (SNPs) detected within our genomic sequence collection. As a novel observation we report increased mutability also at 6mA methylated nucleotides, complementing mutational hotspots previously described at 5mC methylated nucleotides. These findings suggest a more diverse role of DNA methylation and Restriction-Modification (RM) systems in the evolution of

  4. SNP-Based Quantification of Allele-Specific DNA Methylation Patterns by Pyrosequencing®.

    PubMed

    Busato, Florence; Tost, Jörg

    2015-01-01

    The analysis of allele-specific DNA methylation patterns has recently attracted much interest as loci of allele-specific DNA methylation overlap with known risk loci for complex diseases and the analysis might contribute to the fine-mapping and interpretation of non-coding genetic variants associated with complex diseases and improve the understanding between genotype and phenotype. In the presented protocol, we present a method for the analysis of DNA methylation patterns on both alleles separately using heterozygous Single Nucleotide Polymorphisms (SNPs) as anchor for allele-specific PCR amplification followed by analysis of the allele-specific DNA methylation patterns by Pyrosequencing(®). Pyrosequencing is an easy-to-handle, quantitative real-time sequencing method that is frequently used for genotyping as well as for the analysis of DNA methylation patterns. The protocol consists of three major steps: (1) identification of individuals heterozygous for a SNP in a region of interest using Pyrosequencing; (2) analysis of the DNA methylation patterns surrounding the SNP on bisulfite-treated DNA to identify regions of potential allele-specific DNA methylation; and (3) the analysis of the DNA methylation patterns associated with each of the two alleles, which are individually amplified using allele-specific PCR. The enrichment of the targeted allele is re-enforced by modification of the allele-specific primers at the allele-discriminating base with Locked Nucleic Acids (LNA). For the proof-of-principle of the developed approach, we provide assay details for three imprinted genes (IGF2, IGF2R, and PEG3) within this chapter. The mean of the DNA methylation patterns derived from the individual alleles corresponds well to the overall DNA methylation patterns and the developed approach proved more reliable compared to other protocols for allele-specific DNA methylation analysis.

  5. Solving the RNA polymerase I structural puzzle

    SciTech Connect

    Moreno-Morcillo, María; Taylor, Nicholas M. I.; Gruene, Tim; Legrand, Pierre; Rashid, Umar J.; Ruiz, Federico M.; Steuerwald, Ulrich; Müller, Christoph W.; Fernández-Tornero, Carlos

    2014-10-01

    Details of the RNA polymerase I crystal structure determination provide a framework for solution of the structures of other multi-subunit complexes. Simple crystallographic experiments are described to extract relevant biological information such as the location of the enzyme active site. Knowing the structure of multi-subunit complexes is critical to understand basic cellular functions. However, when crystals of these complexes can be obtained they rarely diffract beyond 3 Å resolution, which complicates X-ray structure determination and refinement. The crystal structure of RNA polymerase I, an essential cellular machine that synthesizes the precursor of ribosomal RNA in the nucleolus of eukaryotic cells, has recently been solved. Here, the crucial steps that were undertaken to build the atomic model of this multi-subunit enzyme are reported, emphasizing how simple crystallographic experiments can be used to extract relevant biological information. In particular, this report discusses the combination of poor molecular replacement and experimental phases, the application of multi-crystal averaging and the use of anomalous scatterers as sequence markers to guide tracing and to locate the active site. The methods outlined here will likely serve as a reference for future structural determination of large complexes at low resolution.

  6. Targeting DNA Polymerase β for Therapeutic Intervention

    PubMed Central

    Goellner, Eva M.; Svilar, David; Almeida, Karen H.; Sobol, Robert W.

    2014-01-01

    DNA damage plays a causal role in numerous disease processes. Hence, it is suggested that DNA repair proteins, which maintain the integrity of the nuclear and mitochondrial genomes, play a critical role in reducing the onset of multiple diseases, including cancer, diabetes and neurodegeneration. As the primary DNA polymerase involved in base excision repair, DNA polymerase β (Polβ) has been implicated in multiple cellular processes, including genome maintenance and telomere processing and is suggested to play a role in oncogenic transformation, cell viability following stress and the cellular response to radiation, chemotherapy and environmental genotoxicants. Therefore, Polβ inhibitors may prove to be effective in cancer treatment. However, Polβ has a complex and highly regulated role in DNA metabolism. This complicates the development of effective Polβ-specific inhibitors useful for improving chemotherapy and radiation response without impacting normal cellular function. With multiple enzymatic activities, numerous binding partners and complex modes of regulation from post-translational modifications, there are many opportunities for Polβ inhibition that have yet to be resolved. To shed light on the varying possibilities and approaches of targeting Polβ for potential therapeutic intervention, we summarize the reported small molecule inhibitors of Polβ and discuss the genetic, biochemical and chemical studies that implicate additional options for Polβ inhibition. Further, we offer suggestions on possible inhibitor combinatorial approaches and the potential for tumor specificity for Polβ-inhibitors. PMID:22122465

  7. In vitro replication slippage by DNA polymerases from thermophilic organisms.

    PubMed

    Viguera, E; Canceill, D; Ehrlich, S D

    2001-09-14

    Replication slippage of DNA polymerases is a potential source of spontaneous genetic rearrangements in prokaryotic and eukaryotic cells. Here we show that different thermostable DNA polymerases undergo replication slippage in vitro, during single-round replication of a single-stranded DNA template carrying a hairpin structure. Low-fidelity polymerases, such as Thermus aquaticus (Taq), high-fidelity polymerases, such as Pyrococcus furiosus (Pfu) and a highly thermostable polymerase from Pyrococcus abyssi (Pyra exo(-)) undergo slippage. Thermococcus litoralis DNA polymerase (Vent) is also able to slip; however, slippage can be inhibited when its strand-displacement activity is induced. Moreover, DNA polymerases that have a constitutive strand-displacement activity, such as Bacillus stearothermophilus DNA polymerase (Bst), do not slip. Polymerases that slip during single-round replication generate hairpin deletions during PCR amplification, with the exception of Vent polymerase because its strand-displacement activity is induced under these conditions. We show that these hairpin deletions occurring during PCR are due to replication slippage, and not to a previously proposed process involving polymerization across the hairpin base.

  8. Directed evolution of polymerase function by compartmentalized self-replication

    PubMed Central

    Ghadessy, Farid J.; Ong, Jennifer L.; Holliger, Philipp

    2001-01-01

    We describe compartmentalized self-replication (CSR), a strategy for the directed evolution of enzymes, especially polymerases. CSR is based on a simple feedback loop consisting of a polymerase that replicates only its own encoding gene. Compartmentalization serves to isolate individual self-replication reactions from each other. In such a system, adaptive gains directly (and proportionally) translate into genetic amplification of the encoding gene. CSR has applications in the evolution of polymerases with novel and useful properties. By using three cycles of CSR, we obtained variants of Taq DNA polymerase with 11-fold higher thermostability than the wild-type enzyme or with a >130-fold increased resistance to the potent inhibitor heparin. Insertion of an extra stage into the CSR cycle before the polymerase reaction allows its application to enzymes other than polymerases. We show that nucleoside diphosphate kinase and Taq polymerase can form such a cooperative CSR cycle based on reciprocal catalysis, whereby nucleoside diphosphate kinase produces the substrates required for the replication of its own gene. We also find that in CSR the polymerase genes themselves evolve toward more efficient replication. Thus, polymerase genes and their encoded polypeptides cooperate to maximize postselection copy number. CSR should prove useful for the directed evolution of enzymes, particularly DNA or RNA polymerases, as well as for the design and study of in vitro self-replicating systems mimicking prebiotic evolution and viral replication. PMID:11274352

  9. Purification and Characterization of Recombinant Deinococcus radiodurans RNA Polymerase.

    PubMed

    Esyunina, D M; Kulbachinskiy, A V

    2015-10-01

    The radioresistant bacterium Deinococcus radiodurans is one of the most interesting models for studies of cell stress resistance. Analysis of the mechanisms of gene expression in D. radiodurans revealed some specific features of the transcription apparatus that might play a role in cell resistance to DNA-damaging conditions. In particular, RNA polymerase from D. radiodurans forms unstable promoter complexes and during transcription elongation has a much higher rate of RNA cleavage than RNA polymerase from Escherichia coli. Analysis of the structure and functions of D. radiodurans RNA polymerase is complicated due to the absence of convenient genetic systems for making mutations in the RNA polymerase genes and difficulties with enzyme purification. In this work, we developed a system for expression of D. radiodurans RNA polymerase in E. coli cells. We obtained an expression vector encoding all core RNA polymerase subunits and defined optimal conditions for the expression and purification of the RNA polymerase. It was found that D. radiodurans RNA polymerase has much higher rates of RNA cleavage than E. coli RNA polymerase under a wide range of conditions, including variations in the concentration of catalytic magnesium ions and pH values of the reaction buffer. The expression system can be used for further studies of the RNA cleavage reaction and the mechanisms of transcription regulation in D. radiodurans, including analysis of mutant RNA polymerase variants.

  10. DNA polymerase-associated lectin (DPAL) and its binding to the galactose-containing glycoconjugate of the replication complex.

    PubMed

    Kelley, T J; St Amand, T; Groll, J M; Ray, S; Basu, S

    1999-10-01

    The highly purified DNA Pol-alpha from rat prostate tumor (PA-3) and human neuroblastoma (IMR-32) cells appeared to be inhibited by Ricin (RCA-II), and Con-A. Loss of activity (40 to 60%) of a specific form of DNA polymerase from IMR-32 was observed when the cells were treated with tunicamycin [Bhattacharya, P. and Basu, S. (1982) Proc. Natl. Acad. Sci., USA 79:1488-1492]. Binding of ConA and RCA to human recombinant DNA polymerase-alpha showed a specific labile site in the N-terminus [Hsi et al.. (1990) Nucleic Acid Res. 18:6231-6237]. The catalytic polypeptide, DNA polymerase-alpha of eukaryotic origin, was isolated from developing tissues or cultured cells as a family of 180 to 120 kDa polypeptides, perhaps derived from a single primary structure. Immunoblot analysis with a monoclonal antibody (SJK-237-71) indicated that the lower molecular weight polypeptides resulted from either proteolytic cleavage of post-translational modification after specific cleavages. Present results suggest DNA polymerase-alpha from embryonic chicken brain (ECB) contains an alpha-galactose-binding subunit which may be involved in developmental regulation of the enzyme. It was shown before that the catalytic subunit of DNA polymerase-alpha reduces from 186 kDa in 11-day-old ECB to 120 kDa in 19-day-old ECB [Ray, S. et al. Cell Growth and Differentiation 2:567-573] by the treatment with methyl-alpha-galactose. The low molecular weight DNA polymerase activity (120 kDa) can be reconstituted to high molecular weight (Mr = 186 kDa) with an alpha-galactose binding, 56kDa lectin-like protein. Polyclonal antibodies raised against the purified lectin were able to precipitate DNA. Pol-alpha as determined by immunostaining with the polymerase-alpha-specific monoclonal antibody SJK 132-20, suggesting this is a DNA polymerase associated-lectin (DPAL). RCA-II and GS-I-Sepharose 4B chromatographies resulted in significant purification of DNA-alpha and a complete separation of polymerase complex and

  11. Association of PTEN gene polymorphisms with liver cancer risk

    PubMed Central

    Li, Hong-Guang; Liu, Fang-Feng; Zhu, Hua-Qiang; Zhou, Xu; Lu, Jun; Chang, Hong; Hu, Jin-Hua

    2015-01-01

    Objective: To find out if there are any relationship between three single nucleotide polymorphisms (SNPs) of phosphatase and tensin homolog (PTEN) gene (rs1234213, rs1234220, and rs2299939) and the susceptibility of liver cancer. Methods: Genotypes of the three SNPs in the PTEN gene were achieved utilizing polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method. Comparison of genotypes and alleles distribution differences between the case and the control subjects was accomplished with χ2 test. The analysis of linkage disequilibrium (LD) and haplotypes of the three SNPs was performed using SHEsis software. We adopted odds ratios (ORs) with 95% confidence intervals (95% CIs) to show the relative risk of liver cancer. Results: TC genotype and C allele of rs1234220 polymorphism showed much more frequently in cases than in controls, reflecting that the TC genotype and the C allele may be linked to the increased risk of liver cancer (OR=2.225, 95% CI=1.178-4.204; OR=1.941, 95% CI=1.124-3.351). Rs2299939 polymorphism showed an opposite result that the GT genotype probably reduce the risk of liver cancer (OR=0.483, 95% CI=0.259-0.900). Statistical significance was not found in the distribution differences of the genotypes of rs1234213 between two groups. LD and haplotype analysis results of the three SNPs showed that the T-C-G haplotype frequency was much higher in cases than in healthy objects, which proved that the T-C-G haplotype might be a susceptibility haplotype for liver cancer (OR=3.750, 95% CI=1.396-10.077). Conclusions: PTEN gene polymorphisms might relate to liver cancer risk. PMID:26823866

  12. A Novel Approach for Mining Polymorphic Microsatellite Markers In Silico

    PubMed Central

    Hoffman, Joseph I.; Nichols, Hazel J.

    2011-01-01

    An important emerging application of high-throughput 454 sequencing is the isolation of molecular markers such as microsatellites from genomic DNA. However, few studies have developed microsatellites from cDNA despite the added potential for targeting candidate genes. Moreover, to develop microsatellites usually requires the evaluation of numerous primer pairs for polymorphism in the focal species. This can be time-consuming and wasteful, particularly for taxa with low genetic diversity where the majority of primers often yield monomorphic polymerase chain reaction (PCR) products. Transcriptome assemblies provide a convenient solution, functional annotation of transcripts allowing markers to be targeted towards candidate genes, while high sequence coverage in principle permits the assessment of variability in silico. Consequently, we evaluated fifty primer pairs designed to amplify microsatellites, primarily residing within transcripts related to immunity and growth, identified from an Antarctic fur seal (Arctocephalus gazella) transcriptome assembly. In silico visualization was used to classify each microsatellite as being either polymorphic or monomorphic and to quantify the number of distinct length variants, each taken to represent a different allele. The majority of loci (n = 36, 76.0%) yielded interpretable PCR products, 23 of which were polymorphic in a sample of 24 fur seal individuals. Loci that appeared variable in silico were significantly more likely to yield polymorphic PCR products, even after controlling for microsatellite length measured in silico. We also found a significant positive relationship between inferred and observed allele number. This study not only demonstrates the feasibility of generating modest panels of microsatellites targeted towards specific classes of gene, but also suggests that in silico microsatellite variability may provide a useful proxy for PCR product polymorphism. PMID:21853104

  13. Distribution, silencing potential and evolutionary impact of promoter DNA methylation in the human genome.

    PubMed

    Weber, Michael; Hellmann, Ines; Stadler, Michael B; Ramos, Liliana; Pääbo, Svante; Rebhan, Michael; Schübeler, Dirk

    2007-04-01

    To gain insight into the function of DNA methylation at cis-regulatory regions and its impact on gene expression, we measured methylation, RNA polymerase occupancy and histone modifications at 16,000 promoters in primary human somatic and germline cells. We find CpG-poor promoters hypermethylated in somatic cells, which does not preclude their activity. This methylation is present in male gametes and results in evolutionary loss of CpG dinucleotides, as measured by divergence between humans and primates. In contrast, strong CpG island promoters are mostly unmethylated, even when inactive. Weak CpG island promoters are distinct, as they are preferential targets for de novo methylation in somatic cells. Notably, most germline-specific genes are methylated in somatic cells, suggesting additional functional selection. These results show that promoter sequence and gene function are major predictors of promoter methylation states. Moreover, we observe that inactive unmethylated CpG island promoters show elevated levels of dimethylation of Lys4 of histone H3, suggesting that this chromatin mark may protect DNA from methylation. PMID:17334365

  14. CpG methylation increases the DNA binding of 9-aminoacridine carboxamide Pt analogues.

    PubMed

    Kava, Hieronimus W; Murray, Vincent

    2016-10-01

    This study investigated the effect of CpG methylation on the DNA binding of cisplatin analogues with an attached aminoacridine intercalator. DNA-targeted 9-aminoacridine carboxamide Pt complexes are known to bind at 5'-CpG sequences. Their binding to methylated and non-methylated 5'-CpG sequences was determined and compared with cisplatin. The damage profiles of each platinum compound were quantified via a polymerase stop assay with fluorescently labelled primers and capillary electrophoresis. Methylation at 5'-CpG was shown to significantly increase the binding intensity for the 9-aminoacridine carboxamide compounds, whereas no significant increase was found for cisplatin. 5'-CpG methylation had the largest effect on the 9-ethanolamine-acridine carboxamide Pt complex, followed by the 9-aminoacridine carboxamide Pt complex and the 7-fluoro complex. The methylation state of a cell's genome is important in maintaining normal gene expression, and is often aberrantly altered in cancer cells. An analogue of cisplatin which differentially targets methylated DNA may be able to improve its therapeutic activity, or alter its range of targets and evade the chemoresistance which hampers cisplatin efficacy in clinical use. PMID:27567075

  15. Promoter Methylation and mRNA Expression of Response Gene to Complement 32 in Breast Carcinoma

    PubMed Central

    Eskandari-Nasab, Ebrahim; Hashemi, Mohammad; Rafighdoost, Firoozeh

    2016-01-01

    Background. Response gene to complement 32 (RGC32), induced by activation of complements, has been characterized as a cell cycle regulator; however, its role in carcinogenesis is still controversial. In the present study we compared RGC32 promoter methylation patterns and mRNA expression in breast cancerous tissues and adjacent normal tissues. Materials and Methods. Sixty-three breast cancer tissues and 63 adjacent nonneoplastic tissues were included in our study. Design. Nested methylation-specific polymerase chain reaction (Nested-MSP) and quantitative PCR (qPCR) were used to determine RGC32 promoter methylation status and its mRNA expression levels, respectively. Results. RGC32 methylation pattern was not different between breast cancerous tissue and adjacent nonneoplastic tissue (OR = 2.30, 95% CI = 0.95–5.54). However, qPCR analysis displayed higher levels of RGC32 mRNA in breast cancerous tissues than in noncancerous tissues (1.073 versus 0.959; P = 0.001), irrespective of the promoter methylation status. The expression levels and promoter methylation of RGC32 were not correlated with any of patients' clinical characteristics (P > 0.05). Conclusion. Our findings confirmed upregulation of RGC32 in breast cancerous tumors, but it was not associated with promoter methylation patterns. PMID:27118972

  16. The Trypanosoma brucei DNA polymerase alpha core subunit gene is developmentally regulated and linked to a constitutively expressed open reading frame.

    PubMed Central

    Leegwater, P A; Strating, M; Murphy, N B; Kooy, R F; van der Vliet, P C; Overdulve, J P

    1991-01-01

    As an initial step towards the characterization of replicative DNA polymerases of trypanosomes, we have cloned, sequenced and examined the expression of the Trypanosoma (Trypanozoon) brucei brucei gene that encodes the DNA polymerase alpha catalytic core (pol alpha). The protein sequence contains the six conserved regions that have been recognized previously in eukaryotic and viral replicative DNA polymerases. In addition, we have identified a seventh region which appears to be conserved primarily in alpha-type DNA polymerases. The T.brucei DNA pol alpha core N-terminus is 123 and 129 amino acids smaller than that of the human and yeast homologue, respectively. The gene is separated by 386 bp from an upstream open reading frame (ORF) of 442 codons. Stable transcripts of the upstream sequence are detected in both dividing and non-dividing forms, while pol alpha transcripts are detected principally in dividing forms. Allelic copies of the T.brucei pol alpha region exhibit restriction site polymorphisms; one such sequence polymorphism affects the amino acid sequence of the T.brucei DNA pol alpha core. The T.brucei pol alpha region cross-hybridizes weakly with that of T.(Nannomonas) congolense and T.(Duttonella) vivax. Images PMID:1754381

  17. Association between TNF-α promoter polymorphism and Helicobacter pylori cagA subtype infection

    PubMed Central

    Yea, S; Yang, Y; Jang, W; Lee, Y; Bae, H; Paik, K

    2001-01-01

    Aims—To assess the importance of tumour necrosis factor α (TNF-α) promoter polymorphism in relation to infection with the cytotoxin associated gene A (cagA) subtype of Helicobacter pylori within a dyspeptic Korean population. Methods—Eighty three patients with gastric disease and 113 healthy controls were studied. The DNA from gastric biopsy specimens was analysed by H pylori specific and cagA specific polymerase chain reaction (PCR). To characterise TNF-α polymorphism at positions -308 and -238, PCR based restriction fragment length polymorphism analysis was performed. Results—Helicobacter pylori infection was closely correlated with G to A transition at position -308 of the TNF-α promoter when compared with healthy controls (odds ratio (OR), 2.912; 95% confidence interval (CI), 1.082 to 7.836; p = 0.034). Although TNF-α -308 polymorphism in patients with H pylori was not significantly different from that in patients without H pylori, the -308A polymorphism was strongly associated with H pylori cagA subtype infection when compared with the polymorphism in cagA negative H pylori infection (OR, 8.757; 95% CI, 1.413 to 54.262; p = 0.019) and healthy controls (OR, 3.683; 95% CI, 1.343 to 10.101; p = 0.011). G to A genetic change at position -238 of the TNF-α gene was not significantly associated with H pylori cagA subtype infection. In addition, genetic polymorphisms at both sites of the TNF-α promoter in patients with H pylori infection did not correlate with the severity of disease. Conclusion—TNF-α -308A polymorphism was significantly related to infection with the H pylori cagA subtype in Korean patients with gastric disease. Key Words: Helicobacter pylori • cagA • tumour necrosis factor α • polymorphism PMID:11533078

  18. Endothelial nitric oxide synthase gene polymorphism is associated with Legg-Calvé-Perthes disease

    PubMed Central

    ZHAO, YULONG; LIAO, SHIJIE; LU, RONGBIN; DANG, HAO; ZHAO, JINMIN; DING, XIAOFEI

    2016-01-01

    The aim of this study was to assess the association of 27-bp variable number tandem repeat (VNTR) polymorphism in intron 4 and G894T polymorphism in exon 7 of the endothelial nitric oxide synthase (eNOS) gene with Legg-Calvé-Perthes disease (LCPD), and to provide a scientific basis for further research into the pathogenic mechanism. A total of 80 patients with LCPD and 100 healthy subjects were recruited in this case-control study. The 27-bp VNTR and G894T polymorphisms of the eNOS gene were genotyped using polymerase chain reaction (PCR) and PCR-restriction fragment length polymorphism, respectively, followed by agarose gel electrophoresis and DNA sequencing. Allelic and genotypic frequencies were computed in the two groups and subjected to statistical analysis. For the 27-bp VNTR polymorphism, individuals with LCPD showed a higher frequency of the ab genotype [27.5 vs. 14%; odds ratio (OR), 2.33; 95% confidence interval (CI), 1.10–4.92; P=0.024]. For the G894T polymorphism, the LCPD case group showed a higher frequency of the heterozygous genotype GT than the healthy control group (35 vs. 17%; OR, 2.67; 95% CI, 1.33–5.36; P=0.005). The results indicate that these eNOS gene polymorphisms may be a risk factor for LCPD. The 27-bp VNTR polymorphism in intron 4 and G894T polymorphism in exon 7 may be involved in the etiology of LCPD. PMID:27168827

  19. Variation in cytosine methylation patterns during ploidy level conversions in Eragrostis curvula.

    PubMed

    Ochogavía, Ana C; Cervigni, Gerardo; Selva, Juan P; Echenique, Viviana C; Pessino, Silvina C

    2009-05-01

    In many species polyploidization involves rearrangements of the progenitor genomes, at both genetic and epigenetic levels. We analyzed the cytosine methylation status in a 'tetraploid-diploid-tetraploid' series of Eragrostis curvula with a common genetic background by using the MSAP (Methylation-sensitive Amplified Polymorphism) technique. Considerable levels of polymorphisms were detected during ploidy conversions. The total level of methylation observed was lower in the diploid genotype compared to the tetraploid ones. A significant proportion of the epigenetic modifications occurring during the tetraploid-diploid conversion reverted during the diploid-tetraploid one. Genetic and expression data from previous work were used to analyze correlation with methylation variation. All genetic, epigenetic and gene expression variation data correlated significantly when compared by pairs in simple Mantel tests. Dendrograms reflecting genetic, epigenetic and expression distances as well as principal coordinate analysis suggested that plants of identical ploidy levels present similar sets of data. Twelve (12) different genomic fragments displaying different methylation behavior during the ploidy conversions were isolated, sequenced and characterized.

  20. Enzymatic amplification of platelet-specific messenger RNA using the polymerase chain reaction.

    PubMed Central

    Newman, P J; Gorski, J; White, G C; Gidwitz, S; Cretney, C J; Aster, R H

    1988-01-01

    Human platelets are derived from megakaryocytes as anucleate cells, and thus contain only vestigial amounts of RNA capable of being transcribed into protein. This has greatly hampered efforts to study directly platelet-specific gene products and their associated polymorphisms. In this report, we describe direct amplification, using the polymerase chain reaction, of platelet-derived mRNA in amounts sufficient to permit detailed analysis, such as restriction mapping and nucleotide sequencing. The ability to generate large amounts of cDNA from platelet-specific mRNA sequences should make possible direct molecular characterization of normal platelet proteins, and facilitate the investigation of a wide variety of inherited platelet disorders. Images PMID:3403726

  1. G-quadruplex-generating polymerase chain reaction for visual colorimetric detection of amplicons.

    PubMed

    Bhadra, Sanchita; Codrea, Vlad; Ellington, Andrew D

    2014-01-15

    We have developed a self-reporting polymerase chain reaction (PCR) system for visual colorimetric gene detection and distinction of single nucleotide polymorphisms (SNPs). Amplification is performed using target-specific primers modified with a 5'-end tail that is complementary to a G-quadruplex deoxyribozyme-forming sequence. At end-point, G-quadruplexes are forced to fold from PCR-generated duplex DNA and then are used to colorimetrically report the successful occurrence of PCR by assaying their peroxidase activity using a chromogenic substrate. Furthermore, primer design considerations for the G-quadruplex-generating PCR system have allowed us to visually distinguish SNPs associated with Mycobacterium tuberculosis drug resistance alleles. PMID:24135653

  2. Guanine-rich sequences inhibit proofreading DNA polymerases

    PubMed Central

    Zhu, Xiao-Jing; Sun, Shuhui; Xie, Binghua; Hu, Xuemei; Zhang, Zunyi; Qiu, Mengsheng; Dai, Zhong-Min

    2016-01-01

    DNA polymerases with proofreading activity are important for accurate amplification of target DNA. Despite numerous efforts have been made to improve the proofreading DNA polymerases, they are more susceptible to be failed in PCR than non-proofreading DNA polymerases. Here we showed that proofreading DNA polymerases can be inhibited by certain primers. Further analysis showed that G-rich sequences such as GGGGG and GGGGHGG can cause PCR failure using proofreading DNA polymerases but not Taq DNA polymerase. The inhibitory effect of these G-rich sequences is caused by G-quadruplex and is dose dependent. G-rich inhibitory sequence-containing primers can be used in PCR at a lower concentration to amplify its target DNA fragment. PMID:27349576

  3. The Closing Mechanism of DNA Polymerase I at Atomic Resolution.

    PubMed

    Miller, Bill R; Beese, Lorena S; Parish, Carol A; Wu, Eugene Y

    2015-09-01

    DNA polymerases must quickly and accurately distinguish between similar nucleic acids to form Watson-Crick base pairs and avoid DNA replication errors. Deoxynucleoside triphosphate (dNTP) binding to the DNA polymerase active site induces a large conformational change that is difficult to characterize experimentally on an atomic level. Here, we report an X-ray crystal structure of DNA polymerase I bound to DNA in the open conformation with a dNTP present in the active site. We use this structure to computationally simulate the open to closed transition of DNA polymerase in the presence of a Watson-Crick base pair. Our microsecond simulations allowed us to characterize the key steps involved in active site assembly, and propose the sequence of events involved in the prechemistry steps of DNA polymerase catalysis. They also reveal new features of the polymerase mechanism, such as a conserved histidine as a potential proton acceptor from the primer 3'-hydroxyl. PMID:26211612

  4. Beta-2 adrenergic receptor gene polymorphisms Gln27Glu, Arg16Gly in patients with heart failure

    PubMed Central

    Mansur, Alfredo José; Fontes, Rosana Seleri; Canzi, Regina Airoldi; Nishimura, Raphael; Alencar, Airlane Pereira; de Lima, Antonio Carlos Pedroso; Krieger, José Eduardo; Pereira, Alexandre Costa

    2009-01-01

    Background - Beta-2 adrenergic receptor gene polymorphisms Gln27Glu, Arg16Gly and Thr164Ile were suggested to have an effect in heart failure. We evaluated these polymorphisms relative to clinical characteristics and prognosis of alarge cohort of patients with heart failure of different etiologies. Methods - We studied 501 patients with heart failure of different etiologies. Mean age was 58 years (standard deviation 14.4 years), 298 (60%) were men. Polymorphisms were identified by polymerase chain reaction-restriction fragment length polymorphism. Results - During the mean follow-up of 12.6 months (standard deviation 10.3 months), 188 (38%) patients died. Distribution of genotypes of polymorphism Arg16Gly was different relative to body mass index (χ2 = 9.797;p = 0.04). Overall the probability of survival was not significantly predicted by genotypes of Gln27Glu, Arg16Gly, or Thr164Ile. Allele and haplotype analysis also did not disclose any significant difference regarding mortality. Exploratory analysis through classification trees pointed towards a potential association between the Gln27Glu polymorphism and mortality in older individuals. Conclusion - In this study sample, we were not able to demonstrate an overall influence of polymorphisms Gln27Glu and Arg16Gly of beta-2 receptor gene on prognosis. Nevertheless, Gln27Glu polymorphism may have a potential predictive value in older individuals. PMID:19886995

  5. Genetic association of cyclooxygenase-2 gene polymorphisms with Parkinson’s disease susceptibility in Chinese Han population

    PubMed Central

    Dai, Yi; Wu, Yuquan; Li, Yansheng

    2015-01-01

    Objective: The aim of this study was to explore the genetic association of cyclooxygenase-2 (COX2) gene promoter region polymorphisms with Parkinson’s disease (PD) susceptibility in Chinese Han population. Methods: The genotyping of COX2 gene polymorphisms was conducted by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) in 122 patients with PD and 120 healthy persons. The association strength of gene polymorphism with disease was measured by odds ratio (OR) and 95% confidence interval (95% CI) calculated using χ2 test which also evaluated the Hardy-Weinberg equilibrium (HWE) of gene polymorphism in controls. The linkage disequilibrium and haplotype were also analyzed as evidence in the analysis of association. Results: On condition that the genotypes distributions of COX2 -1290A>G, -1195G>A, -765G>C in the control group all conformed to HWE, however, only the homozygous genotype AA of -1195G>A polymorphism showed an association with PD (OR=0.432, 95% CI=0.196-0.950). In addition, in haplotype analysis, G-A-C haplotype frequency in cases was significantly lower than the controls, compared with the common haplotype A-G-G (P=0.031, OR=0.375, 95% CI=0.149-0.940). Conclusions: COX2 -1195G>A polymorphism might play a protective role in the onset of PD and G-A-C haplotype in this three promoter region polymorphisms also showed a negative association. PMID:26722563

  6. Inhibition of RNA polymerase by streptolydigin: no cycling allowed.

    PubMed

    Kyzer, Scotty; Zhang, Jinwei; Landick, Robert

    2005-08-26

    Bacterial RNA polymerase is a common target for many antibiotics. In two recent papers in Cell and Molecular Cell, and describe a structural basis for inhibition of bacterial RNA polymerase by the antibiotic streptolydigin. Streptolydigin may prevent distortion of a "bridge" alpha helix postulated to occur during the nucleotide addition cycle of RNA polymerase or may block a small movement of the bridge helix that helps load nucleotide triphosphates into the active site. PMID:16122417

  7. Methods of DNA methylation detection

    NASA Technical Reports Server (NTRS)

    Maki, Wusi Chen (Inventor); Filanoski, Brian John (Inventor); Mishra, Nirankar (Inventor); Rastogi, Shiva (Inventor)

    2010-01-01

    The present invention provides for methods of DNA methylation detection. The present invention provides for methods of generating and detecting specific electronic signals that report the methylation status of targeted DNA molecules in biological samples.Two methods are described, direct and indirect detection of methylated DNA molecules in a nano transistor based device. In the direct detection, methylated target DNA molecules are captured on the sensing surface resulting in changes in the electrical properties of a nano transistor. These changes generate detectable electronic signals. In the indirect detection, antibody-DNA conjugates are used to identify methylated DNA molecules. RNA signal molecules are generated through an in vitro transcription process. These RNA molecules are captured on the sensing surface change the electrical properties of nano transistor thereby generating detectable electronic signals.

  8. DNA methylation and carcinogenesis.

    PubMed

    Lichtenstein, A V; Kisseljova, N P

    2001-03-01

    In the world of easy things truth is opposed to lie; in the world of complicated things one profound truth is opposed to another not less profound than the first. Neils Bohr The hypothesis of the exclusively genetic origin of cancer ("cancer is a disease of genes, a tumor without any damage to the genome does not exist") dominated in the oncology until recently. A considerable amount of data confirming this hypothesis was accumulated during the last quarter of the last century. It was demonstrated that the accumulation of damage of specific genes lies at the origin of a tumor and its following progression. The damage gives rise to structural changes in the respective proteins and, consequently, to inappropriate mitogenic stimulation of cells (activation of oncogenes) or to the inactivation of tumor suppressor genes that inhibit cell division, or to the combination of both (in most cases). According to an alternative (epigenetic) hypothesis that was extremely unpopular until recently, a tumor is caused not by a gene damage, but by an inappropriate function of genes ("cancer is a disease of gene regulation and differentiation"). However, recent studies led to the convergence of these hypotheses that initially seemed to be contradictory. It was established that both factors--genetic and epigenetic--lie at the origin of carcinogenesis. The relative contribution of each varies significantly in different human tumors. Suppressor genes and genes of repair are inactivated in tumors due to their damage or methylation of their promoters (in the latter case an "epimutation", an epigenetic equivalent of a mutation, occurs, producing the same functional consequences). It is becoming evident that not only the mutagens, but various factors influencing cell metabolism, notably methylation, should be considered as carcinogens.

  9. Global changes in DNA methylation in Alzheimer's disease peripheral blood mononuclear cells.

    PubMed

    Di Francesco, Andrea; Arosio, Beatrice; Falconi, Anastasia; Micioni Di Bonaventura, Maria Vittoria; Karimi, Mohsen; Mari, Daniela; Casati, Martina; Maccarrone, Mauro; D'Addario, Claudio

    2015-03-01

    Changes in epigenetic marks may help explain the late onset of Alzheimer's disease (AD). In this study we measured genome-wide DNA methylation by luminometric methylation assay, a quantitative measurement of genome-wide DNA methylation, on DNA isolated from peripheral blood mononuclear cells of 37 subjects with late-onset AD (LOAD) and 44 healthy controls (CT). We found an increase in global DNA methylation in LOAD subjects compared to CT (p=0.0122), associated with worse cognitive performances (p=0.0002). DNA hypermethylation in LOAD group was paralleled by higher DNA methyltransferase 1 (DNMT1) gene expression and protein levels. When data were stratified on the basis of the APOE polymorphisms, higher DNA methylation levels were associated with the presence of APOE ε4 allele (p=0.0043) in the global population. Among the APOE ε3 carriers, a significant increase of DNA methylation was still observed in LOAD patients compared to healthy controls (p=0.05). Our data suggest global DNA methylation in peripheral samples as a useful marker for screening individuals at risk of developing AD.

  10. Aberrant DNA methylation is a dominant mechanism in MDS progression to AML

    PubMed Central

    Jiang, Ying; Dunbar, Andrew; Gondek, Lukasz P.; Mohan, Sanjay; Rataul, Manjot; O'Keefe, Christine; Sekeres, Mikkael

    2009-01-01

    Myelodysplastic syndromes (MDSs) are clonal hematologic disorders that frequently represent an intermediate disease stage before progression to acute myeloid leukemia (AML). As such, study of MDS/AML can provide insight into the mechanisms of neoplastic evolution. In 184 patients with MDS and AML, DNA methylation microarray and high-density single nucleotide polymorphism array (SNP-A) karyotyping were used to assess the relative contributions of aberrant DNA methylation and chromosomal deletions to tumor-suppressor gene (TSG) silencing during disease progression. Aberrant methylation was seen in every sample, on average affecting 91 of 1505 CpG loci in early MDS and 179 of 1505 loci after blast transformation (refractory anemia with excess blasts [RAEB]/AML). In contrast, chromosome aberrations were seen in 79% of early MDS samples and 90% of RAEB/AML samples, and were not as widely distributed over the genome. Analysis of the most frequently aberrantly methylated genes identified FZD9 as a candidate TSG on chromosome 7. In patients with chromosome deletion at the FZD9 locus, aberrant methylation of the remaining allele was associated with the poorest clinical outcome. These results indicate that aberrant methylation can cooperate with chromosome deletions to silence TSG. However, the ubiquity, extent, and correlation with disease progression suggest that aberrant DNA methylation is the dominant mechanism for TSG silencing and clonal variation in MDS evolution to AML. PMID:18832655

  11. Polymorphic Electronic Circuits

    NASA Technical Reports Server (NTRS)

    Stoica, Adrian

    2004-01-01

    Polymorphic electronics is a nascent technological discipline that involves, among other things, designing the same circuit to perform different analog and/or digital functions under different conditions. For example, a circuit can be designed to function as an OR gate or an AND gate, depending on the temperature (see figure). Polymorphic electronics can also be considered a subset of polytronics, which is a broader technological discipline in which optical and possibly other information- processing systems could also be designed to perform multiple functions. Polytronics is an outgrowth of evolvable hardware (EHW). The basic concepts and some specific implementations of EHW were described in a number of previous NASA Tech Briefs articles. To recapitulate: The essence of EHW is to design, construct, and test a sequence of populations of circuits that function as incrementally better solutions of a given design problem through the selective, repetitive connection and/or disconnection of capacitors, transistors, amplifiers, inverters, and/or other circuit building blocks. The evolution is guided by a search-and-optimization algorithm (in particular, a genetic algorithm) that operates in the space of possible circuits to find a circuit that exhibits an acceptably close approximation of the desired functionality. The evolved circuits can be tested by computational simulation (in which case the evolution is said to be extrinsic), tested in real hardware (in which case the evolution is said to be intrinsic), or tested in random sequences of computational simulation and real hardware (in which case the evolution is said to be mixtrinsic).

  12. Role of leptin G-2548A polymorphism in age- and gender-specific development of obesity.

    PubMed

    Shahid, Adeela; Rana, Sobia; Mahmood, Saqib; Saeed, Shahid

    2015-09-01

    Leptin is involved in the regulation of food intake and energy expenditure, and therefore, is central to adipositysensing pathway. We examined the relationship of the leptin G-2548A polymorphism with obesity and obesityrelated anthropometric and metabolic parameters in a total of 394 (239 obese and 155 non-obese) subjects between 5 and 45 years of age. Body weight, height, waist circumference (WC), hip circumference (HC) and blood pressure (BP) were measured. Body mass index (BMI) and waist-to-hip ratio (WHR) were calculated. Levels of fasting blood glucose (FBG), insulin, leptin and leptin receptor were determined, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The LEP G-2548A polymorphism showed association with obesity in children and adolescents (less than or equal to 18 years of age) but not in adults. However, analysis by gender stratification revealed association with obesity in girls only. In addition, G-2548A polymorphism showed association with BMI, WC, HC, fasting blood glucose and serum leptin levels. This suggests that G-2548A polymorphism may influence the susceptibility to metabolic disturbances and obesity at an early life. Further investigation with a larger sample size is required to validate the effect of LEP G-2548A polymorphism in obese Pakistani girls. PMID:26333398

  13. Polymorphism of the Flap Endonuclease 1 Gene in Keratoconus and Fuchs Endothelial Corneal Dystrophy

    PubMed Central

    Wojcik, Katarzyna A.; Synowiec, Ewelina; Polakowski, Piotr; Głowacki, Sylwester; Izdebska, Justyna; Lloyd, Sophie; Galea, Dieter; Blasiak, Janusz; Szaflik, Jerzy; Szaflik, Jacek P.

    2014-01-01

    Oxidative stress is implicated in the pathogenesis of many diseases, including serious ocular diseases, keratoconus (KC) and Fuchs endothelial corneal dystrophy (FECD). Flap endonuclease 1 (FEN1) plays an important role in the repair of oxidative DNA damage in the base excision repair pathway. We determined the association between two single nucleotide polymorphisms (SNPs), c.–441G>A (rs174538) and g.61564299G>T (rs4246215), in the FEN1 gene and the occurrence of KC and FECD. This study involved 279 patients with KC, 225 patients with FECD and 322 control individuals. Polymerase chain reaction (PCR) and length polymorphism restriction fragment analysis (RFLP) were applied. The T/T genotype of the g.61564299G>T polymorphism was associated with an increased occurrence of KC and FECD. There was no association between the c.–441G>A polymorphism and either disease. However, the GG haplotype of both polymorphisms was observed more frequently and the GT haplotype less frequently in the KC group than the control. The AG haplotype was associated with increased FECD occurrence. Our findings suggest that the g.61564299G>T and c.–441G>A polymorphisms in the FEN1 gene may modulate the risk of keratoconus and Fuchs endothelial corneal dystrophy. PMID:25153632

  14. Genetic polymorphisms of Interleukin-18 are not associated with allograft function in kidney transplant recipients.

    PubMed

    do Nascimento, Wenna Gleyce Araújo; Cilião, Daiani Alves; Genre, Julieta; Gondim, Dikson Dibe; Alves, Renata Gomes; Hassan, Neife Deghaide; Lima, Francisco Pignataro; Pereira, Maurício Galvão; Donadi, Eduardo Antônio; de Oliveira Crispim, Janaina Cristiana

    2014-06-01

    Interleukin 18 (IL-18) is a proinflammatory cytokine that plays a role in host defense by upregulating both innate and acquired immune responses. Analysis of IL18 polymorphisms may be clinically important since their roles have been recognized in a variety of inflammatory and autoimmune disorders. However, the role of this cytokine polymorphisms in kidney transplant still remains unclear. In this study, we evaluated the associations between IL18 polymorphisms and graft function assessed by creatinine clearance in kidney transplant recipients. A total of 82 kidney transplant recipients and 183 healthy controls were enrolled, and frequencies of alleles, genotypes and haplotypes for IL18 polymorphisms were determined and compared with creatinine clearance. The -607C/A (rs1946518) and -137C/G (rs187238) variant alleles in the IL18 gene were determined by polymerase chain reaction. In our study, no significant association was found between the IL18 variants and creatinine clearance (p > 0.05). Nonetheless, polymorphism analysis revealed an increase in the frequency of the IL18 major haplotype -607C/-137G in kidney transplant patients (odds ratio 2.57, 95% confidence interval 1.45-4.55, p = 0.0014). Finally, we found that IL18 polymorphisms did not influence the renal function and that IL18 haplotype -607C/-137G seems to be associated with kidney transplant recipients.

  15. Genetic polymorphisms of Interleukin-18 are not associated with allograft function in kidney transplant recipients

    PubMed Central

    do Nascimento, Wenna Gleyce Araújo; Cilião, Daiani Alves; Genre, Julieta; Gondim, Dikson Dibe; Alves, Renata Gomes; Hassan, Neife Deghaide; Lima, Francisco Pignataro; Pereira, Maurício Galvão; Donadi, Eduardo Antônio; de Oliveira Crispim, Janaina Cristiana

    2014-01-01

    Interleukin 18 (IL-18) is a proinflammatory cytokine that plays a role in host defense by upregulating both innate and acquired immune responses. Analysis of IL18 polymorphisms may be clinically important since their roles have been recognized in a variety of inflammatory and autoimmune disorders. However, the role of this cytokine polymorphisms in kidney transplant still remains unclear. In this study, we evaluated the associations between IL18 polymorphisms and graft function assessed by creatinine clearance in kidney transplant recipients. A total of 82 kidney transplant recipients and 183 healthy controls were enrolled, and frequencies of alleles, genotypes and haplotypes for IL18 polymorphisms were determined and compared with creatinine clearance. The -607C/A (rs1946518) and -137C/G (rs187238) variant alleles in the IL18 gene were determined by polymerase chain reaction. In our study, no significant association was found between the IL18 variants and creatinine clearance (p > 0.05). Nonetheless, polymorphism analysis revealed an increase in the frequency of the IL18 major haplotype -607C/-137G in kidney transplant patients (odds ratio 2.57, 95% confidence interval 1.45–4.55, p = 0.0014). Finally, we found that IL18 polymorphisms did not influence the renal function and that IL18 haplotype -607C/-137G seems to be associated with kidney transplant recipients. PMID:25071398

  16. Role of leptin G-2548A polymorphism in age- and gender-specific development of obesity.

    PubMed

    Shahid, Adeela; Rana, Sobia; Mahmood, Saqib; Saeed, Shahid

    2015-09-01

    Leptin is involved in the regulation of food intake and energy expenditure, and therefore, is central to adipositysensing pathway. We examined the relationship of the leptin G-2548A polymorphism with obesity and obesityrelated anthropometric and metabolic parameters in a total of 394 (239 obese and 155 non-obese) subjects between 5 and 45 years of age. Body weight, height, waist circumference (WC), hip circumference (HC) and blood pressure (BP) were measured. Body mass index (BMI) and waist-to-hip ratio (WHR) were calculated. Levels of fasting blood glucose (FBG), insulin, leptin and leptin receptor were determined, and homeostasis model assessment of insulin resistance (HOMA-IR) was calculated. Genotyping was carried out by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). The LEP G-2548A polymorphism showed association with obesity in children and adolescents (less than or equal to 18 years of age) but not in adults. However, analysis by gender stratification revealed association with obesity in girls only. In addition, G-2548A polymorphism showed association with BMI, WC, HC, fasting blood glucose and serum leptin levels. This suggests that G-2548A polymorphism may influence the susceptibility to metabolic disturbances and obesity at an early life. Further investigation with a larger sample size is required to validate the effect of LEP G-2548A polymorphism in obese Pakistani girls.

  17. Association between delta-aminolevulinic acid dehydratase polymorphism and placental lead levels.

    PubMed

    Kayaaltı, Zeliha; Sert, Selda; Kaya-Akyüzlü, Dilek; Söylemez, Esma; Söylemezoğlu, Tülin

    2016-01-01

    Lead inhibits the delta-aminolevulinic acid dehydratase (ALAD) activity and results in neurotoxic aminolevulinic acid accumulation in the blood. During pregnancy, lead in the maternal blood can easily cross the placenta. The aim of this study was to determine whether the maternal ALAD G177C polymorphism (rs1800435) was related to the placental lead levels. The study population comprised 97 blood samples taken from mothers to investigate ALAD G177C polymorphism and their placentas to measure lead levels. ALAD G177C polymorphism was detected by standard polymerase chain reaction (PCR)-restriction fragment length polymorphism (RFLP) technique and atomic absorption spectrometry (AAS) equipped with a graphite furnace and Zeeman background correction system was used for lead determination. The median placental lead levels for ALAD1-1, ALAD1-2 and ALAD2-2 genotypes were 7.54 μg/kg, 11.78 μg/kg and 18.53 μg/kg, respectively. Statistically significant association was found between the maternal ALAD G177C polymorphism and placental lead levels (p<0.05). This study suggested that maternal ALAD G177C polymorphism was associated with placental lead levels.

  18. Investigation on estrogen receptor alpha gene polymorphisms in Iranian women with recurrent pregnancy loss

    PubMed Central

    Mahdavipour, Marzieh; Idali, Farah; Zarei, Saeed; Talebi, Saeed; Fatemi, Ramina; Jeddi-Tehrani, Mahmood; Pahlavan, Somayeh; Rajaei, Farzad

    2014-01-01

    Background: Recurrent pregnancy loss (RPL) is a multifactorial disorder. Environmental factors and genetics can affect pregnancy outcomes. Objective: Conflicting data suggest an association between estrogen receptor alpha (ESR1) gene polymorphisms and RPL. In this study, such association was investigated in Iranian women with RPL. Materials and Methods: In this case control study, blood samples were collected from 244 women with a history of three or more consecutive pregnancy losses and 104 healthy women with at least two live births. Using polymerase chain reaction- restriction fragment length polymorphism (PCR-RFLP), we studied -397C/T and -351A/G polymorphisms on ESR1 gene in case and control subjects. Results: The genotypic frequencies of -397C/T and -351A/G polymorphisms on ESR1were not significantly different between RPL and control groups (p=0.20 and p=0.09, respectively). A significantly negative correlation was observed between -397C/T and -351A/G (r=-0.852, p<0.001) in RPL women and complete linkage disequilibrium between the investigated polymorphisms was found (D’: 0.959; r-square= 0.758, p<0.001). Conclusion: This investigation suggests that the analyzed polymorphisms on ESR1gene are not associated with an increased risk of RPL in the studied population. PMID:25071847

  19. Altered Th17 Cytokine Expression in Helicobacter pylori Patients with TLR4 (D299G) Polymorphism.

    PubMed

    Bagheri, Nader; Azadegan-Dehkordi, Fatemeh; Rahimian, Ghorbanali; Hashemzadeh-Chaleshtori, Morteza; Rafieian-Kopaei, Mahmoud; Kheiri, Soleyman; Gholipour, Abolfazl; Shirzad, Hedayatollah

    2016-01-01

    Helicobacter pylori (H. pylori) is associated with gastric ulcer and gastric adenocarcinoma. Polymorphisms in the host genes coding for Toll-like receptors (TLRs) may influence the innate and adaptive immune response to the infection, affecting the susceptibility to H. pylori or the disease outcomes. However, the details and association with different polymorphism and clinical expression of infection remain unclear. A case-control study consisting of 58 patients with H. pylori infection and 44 H. pylori uninfection was conducted. Genomic DNA was extracted and genotypes of TLR4 Asp299Gly polymorphism were assessed through polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP). Mucosal cytokines expression in H. pylori-infected and uninfected gastric biopsies was determined by real-time PCR. The expression of IL-6, IL-17, IL-21, IL-23 and TGF-β1 was significantly higher in patients with D299G polymorphism in TLR4. But the expression of IL-18 between patients with single-nucleotide polymorphisms (SNPs) in TLR4 and patients with the wild-type allele was not significant. In H. pylori-infected patients with gastritis, SNPs in TLR4 may alter cytokine expression toward Th17 immune response in the gastric mucosa and may have increased risk for the development of peptic ulcer. PMID:26853914

  20. RUNX3 gene polymorphisms and haplotypes in Mexican patients with colorectal cancer.

    PubMed

    Suárez-Villanueva, S; Ayala-Madrigal, M L; Peregrina-Sandoval, J; Macías-Gómez, N; Ramírez-Ramírez, R; Muñiz-Mendoza, R; Moreno-Ortiz, J M; Centeno-Flores, M; Maciel-Gutiérrez, V; Cabrales, E; Gutiérrez-Angulo, M

    2015-01-01

    We analyzed a possible association between RUNX3 gene polymorphisms and haplotypes in Mexican patients with colorectal cancer (CRC). Genomic DNA samples were obtained from the peripheral blood of 176 Mexican patients with CRC at diagnosis and from 195 individuals that formed the control group. The polymorphisms were detected by polymerase chain reaction-restriction fragment length polymorphism. Association was estimated by odds ratio (OR). The haplotypes and linkage disequilibrium were established using the Arlequin v3.5 software. We found that the RUNX3 polymorphisms analyzed were in Hardy-Weinberg equilibrium. The RUNX3 rs2236852 AA genotype and A allele showed association with CRC (OR = 0.39, 95%CI = 0.21-0.73, P < 0.01; OR = 0.65, 95%CI = 0.49-0.87, P < 0.01, respectively), while the rs6672420, rs11249206, and rs760805 polymorphisms did not show significant association with CRC. The TA haplotype (SNPs rs760805 and rs2236852) showed an increased risk for CRC (OR = 2.52, 95%CI = 1.47-4.30, P < 0.001). In conclusion, we found that the AA genotype and A allele of rs2236852 polymorphism confer a decreased CRC risk, while the TA haplotype appears to increase the risk of CRC development in Mexican patients. PMID:26634516

  1. Contribution of the GSTP1 gene polymorphism to the development of osteosarcoma in a Chinese population.

    PubMed

    Qu, W R; Wu, J; Li, R

    2016-01-01

    We conducted a case-control study to investigate the associations between GSTT1, GSTM1, and GSTP1 gene polymorphisms and development of osteosarcoma in a Chinese population. Between January 2013 and February 2015, 153 patients diagnosed with osteosarcoma and 252 control subjects were enrolled in the current study from the Orthopedic Hospital of the Second Hospital of Jilin University. The GSTM1, GSTT1, and GSTP1 gene polymorphisms were detected by polymerase chain reaction coupled with restriction fragment length polymorphism analysis. As determined by a multiple-logistic regression analysis, the Val/Val genotype of GSTP1 was associated with a significantly increased risk of osteosarcoma compared to that of the Ile/Ile genotype, with an odds ratio (OR) = 3.39, and a 95% confidence interval (CI) = 1.45-8.13. Moreover, the Ile/Val+Val/Val genotype of GSTP1 was correlated with a marginally significant increased risk of osteosarcoma compared to that of the Ile/Ile genotype (OR = 1.65, 95%CI = 1.08-2.53). However, we did not find any significant associations between the GSTM1 and GSTT1 gene polymorphisms and osteosarcoma risk. In conclusion, our results suggest that the GSTP1 gene polymorphism is associated with an increased risk of osteosarcoma, whereas the GSTM1 and GSTT1 gene polymorphisms may not influence the development of this cancer. PMID:27525908

  2. ENOS-G894T polymorphism is a risk factor for essential hypertension in China.

    PubMed

    Men, Chen; Tang, Kangting; Lin, Gang; Li, Jian; Zhan, Yiyang

    2011-06-01

    Vascular endothelial cells produce nitric oxide (NO), which contributes to the regulation of blood pressure and regional blood flow. Polymorphisms of the endothelial nitric oxide synthase (eNOS) gene are associated with coronary artery disease; however, associations between polymorphism (G894T) of the eNOS gene and essential hypertension remain unclear. This study was designed to investigate the association between a eNOS-G894T polymorphism and essential hypertension (EH). A total of 190 Chinese EH patients (EH group) and 94 healthy participants (control group) were included in the study. eNOS-G894T was determined using multi-polymerase chain reaction and polymorphisms in eNOS-G894T were genotyped using gene chip technology. Patients carrying eNOS GT + TT genotypes had a higher risk of EH than those carrying the GG genotype (OR = 2.82, 95% CI: 1.05-7.60, P = 0.033). The EH group showed a significantly higher frequency of the T-allele compared with controls (OR = 3.48, 95% CI: 1.34-9.07; P = 0.007). eNOS-894T was found to be significantly associated with EH in the dominant genetic model. Thus, the study demonstrated a significant and independent association between a eNOS-G894T polymorphism and EH in the Chinese patients. The study also showed that eNOS-G894T polymorphism is a risk factor for EH in Chinese patients. PMID:21793305

  3. Molecular Mechanisms of DNA Polymerase Clamp Loaders

    NASA Astrophysics Data System (ADS)

    Kelch, Brian; Makino, Debora; Simonetta, Kyle; O'Donnell, Mike; Kuriyan, John

    Clamp loaders are ATP-driven multiprotein machines that couple ATP hydrolysis to the opening and closing of a circular protein ring around DNA. This ring-shaped clamp slides along DNA, and interacts with numerous proteins involved in DNA replication, DNA repair and cell cycle control. Recently determined structures of clamp loader complexes from prokaryotic and eukaryotic DNA polymerases have revealed exciting new details of how these complex AAA+ machines perform this essential clamp loading function. This review serves as background to John Kuriyan's lecture at the 2010 Erice School, and is not meant as a comprehensive review of the contributions of the many scientists who have advanced this field. These lecture notes are derived from recent reviews and research papers from our groups.

  4. Polymerase Chain Reaction on a Viral Nanoparticle.

    PubMed

    Carr-Smith, James; Pacheco-Gómez, Raúl; Little, Haydn A; Hicks, Matthew R; Sandhu, Sandeep; Steinke, Nadja; Smith, David J; Rodger, Alison; Goodchild, Sarah A; Lukaszewski, Roman A; Tucker, James H R; Dafforn, Timothy R

    2015-12-18

    The field of synthetic biology includes studies that aim to develop new materials and devices from biomolecules. In recent years, much work has been carried out using a range of biomolecular chassis including α-helical coiled coils, β-sheet amyloids and even viral particles. In this work, we show how hybrid bionanoparticles can be produced from a viral M13 bacteriophage scaffold through conjugation with DNA primers that can template a polymerase chain reaction (PCR). This unprecedented example of a PCR on a virus particle has been studied by flow aligned linear dichroism spectroscopy, which gives information on the structure of the product as well as a new protototype methodology for DNA detection. We propose that this demonstration of PCR on the surface of a bionanoparticle is a useful addition to ways in which hybrid assemblies may be constructed using synthetic biology.

  5. Dual phase multiplex polymerase chain reaction

    DOEpatents

    Pemov, Alexander; Bavykin, Sergei

    2008-10-07

    Highly specific and sensitive methods were developed for multiplex amplification of nucleic acids on supports such as microarrays. Based on a specific primer design, methods include five types of amplification that proceed in a reaction chamber simultaneously. These relate to four types of multiplex amplification of a target DNA on a solid support, directed by forward and reverse complex primers immobilized to the support and a fifth type--pseudo-monoplex polymerase chain reaction (PCR) of multiple targets in solution, directed by a single pair of unbound universal primers. The addition of the universal primers in the reaction mixture increases the yield over the traditional "bridge" amplification on a solid support by approximately ten times. Methods that provide multitarget amplification and detection of as little as 0.45-4.5.times.10.sup.-12 g (equivalent to 10.sup.2-10.sup.3 genomes) of a bacterial genomic DNA are disclosed.

  6. RNA Polymerase Pausing during Initial Transcription.

    PubMed

    Duchi, Diego; Bauer, David L V; Fernandez, Laurent; Evans, Geraint; Robb, Nicole; Hwang, Ling Chin; Gryte, Kristofer; Tomescu, Alexandra; Zawadzki, Pawel; Morichaud, Zakia; Brodolin, Konstantin; Kapanidis, Achillefs N

    2016-09-15

    In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA. PMID:27618490

  7. RNA Polymerase Pausing during Initial Transcription.

    PubMed

    Duchi, Diego; Bauer, David L V; Fernandez, Laurent; Evans, Geraint; Robb, Nicole; Hwang, Ling Chin; Gryte, Kristofer; Tomescu, Alexandra; Zawadzki, Pawel; Morichaud, Zakia; Brodolin, Konstantin; Kapanidis, Achillefs N

    2016-09-15

    In bacteria, RNA polymerase (RNAP) initiates transcription by synthesizing short transcripts that are either released or extended to allow RNAP to escape from the promoter. The mechanism of initial transcription is unclear due to the presence of transient intermediates and molecular heterogeneity. Here, we studied initial transcription on a lac promoter using single-molecule fluorescence observations of DNA scrunching on immobilized transcription complexes. Our work revealed a long pause ("initiation pause," ∼20 s) after synthesis of a 6-mer RNA; such pauses can serve as regulatory checkpoints. Region sigma 3.2, which contains a loop blocking the RNA exit channel, was a major pausing determinant. We also obtained evidence for RNA backtracking during abortive initial transcription and for additional pausing prior to escape. We summarized our work in a model for initial transcription, in which pausing is controlled by a complex set of determinants that modulate the transition from a 6- to a 7-nt RNA.

  8. Detection of a novel cytochrome P-450 1A2 polymorphism (F21L) in Chinese.

    PubMed

    Huang, J D; Guo, W C; Lai, M D; Guo, Y L; Lambert, G H

    1999-01-01

    Despite a wide interindividual variation of cytochrome P-450 1A2 (CYP1A2) activity, genetic polymorphism of CYP1A2 has not been reported. By amplification of exons of CYP1A2 by polymerase chain reaction in eight Chinese subjects, the polymerase chain reaction products were directly sequenced. One subject showed heterozygous C2866-->G (Phe21-->Leu) polymorphism. DNA from 157 Chinese subjects (104 polychlorinated biphenyl-exposed subjects and 53 control subjects) was screened for polymorphism by single-strand conformation polymorphism method and MboII endonuclease digestion. Only 1 of 157 samples showed another heterozygous C2866-->G mutation. The subject was previously exposed to polychlorinated biphenyl and showed a value of 3.5% in the caffeine breath test. The value is not significantly higher than the mean value of polychlorinated biphenyl-exposed subjects (3.12 +/- 0.29%, mean +/- S.E.M.). The incidence of the point mutation in these Chinese subjects is less than 1%. The prevalence of the F21L mutation in other ethnic groups and its effect on the metabolic activity of CYP1A2 remain to be further evaluated. PMID:9884316

  9. High-Throughput Polymerase Fidelity Evolution in Microfluidic Droplets

    NASA Astrophysics Data System (ADS)

    Collins, Jesse; de Paz, Alexandra; Cybulski, Ted; Bhan, Namita; Zhang, Huidan; Weitz, Dave; Tyo, Keith; Kording, Konrad

    Polymerases are technologically important as a tool in molecular biology, and are scientifically important for their role in DNA replication and inheritance. We study large numbers (at least millions) of polymerase mutants by compartmentalizing each gene in a droplet in a microfluidic device. Also in each droplet are in vitro transcription and translation proteins, such that mutant polymerases can be generated to extend their own gene along a known DNA template. Reading the resulting sequence tells us both the mutant gene sequence and the number of and particular errors that the resulting mutant polymerase made during extension. This work is supported by the NIH.

  10. Structural and functional relationships between prokaryotic and eukaryotic DNA polymerases.

    PubMed

    Bernad, A; Zaballos, A; Salas, M; Blanco, L

    1987-12-20

    The Bacillus subtilis phage luminal diameter 29 DNA polymerase, involved in protein-primed viral DNA replication, was inhibited by phosphonoacetic acid (PAA), a known inhibitor of alpha-like DNA polymerases, by decreasing the rate of elongation. Three highly conserved regions of amino acid homology, found in several viral alpha-like DNA polymerases and in the luminal diameter 29 DNA polymerase, one of them proposed to be the PAA binding site, were also found in the T4 DNA polymerase. This prokaryotic enzyme was highly sensitive to the drugs aphidicolin and the nucleotide analogues butylanilino dATP (BuAdATP) and butylphenyl dGTP (BuPdGTP), known to be specific inhibitors of eukaryotic alpha-like DNA polymerases. Two potential DNA polymerases from the linear plasmid pGKL1 from yeast and the S1 mitochondrial DNA from maize have been identified, based on the fact that they contain the three conserved regions of amino acid homology. Comparison of DNA polymerases from prokaryotic and eukaryotic origin showed extensive amino acid homology in addition to highly conserved domains. These findings reflect evolutionary relationships between hypothetically unrelated DNA polymerases.

  11. DNA replication. A familiar ring to DNA polymerase processivity.

    PubMed

    Wyman, C; Botchan, M

    1995-04-01

    Structural similarity reveals that prokaryotic and eukaryotic DNA polymerases share a mechanism for processivity--but the conservation of additional chromosomal replication mechanisms remains to be determined.

  12. Modification of RNA polymerase IIO subspecies after poliovirus infection.

    PubMed Central

    Rangel, L M; Fernandez-Tomas, C; Dahmus, M E; Gariglio, P

    1987-01-01

    Infection of HeLa cells with poliovirus results in a shutdown of host transcription. In an effort to understand the mechanism(s) that underlies this process, we analyzed the distribution of RNA polymerase IIO before and after viral infection. Analysis of free and chromatin-bound enzyme indicated that there is a significant reduction in RNA polymerase IIO following infection. This observation, together with increasing evidence that transcription is catalyzed by RNA polymerase IIO, supports the hypothesis that poliovirus-induced inhibition of host transcription occurs at the level of RNA chain initiation and involves the direct modification of RNA polymerase II. Images PMID:3029396

  13. Methyl Halide Production by Fungi

    NASA Astrophysics Data System (ADS)

    Dailey, G. D.; Varner, R. K.; Blanchard, R. O.; Sive, B. C.; Crill, P. M.

    2005-12-01

    Methyl chloride (CH3Cl), methyl bromide (CH3Br) and methyl iodide (CH3I) are methyl halide gases that contribute significant amounts of halogen radicals to the atmosphere. In an effort to better understand the global budget of methyl halides and their impact on the atmosphere, we need to identify the natural sources in addition to the known anthropogenic sources of these compounds. We are investigating the role of fungi in the production of methyl halides in the soils and wetlands in southern New Hampshire, USA. Previous research has shown that wood decay fungi and ectomycorrhizal fungi, which are within a group of fungi called basidiomycetes, emit methyl halides. In our study, measurements of headspace gas extracted from flasks containing fungi grown in culture demonstrate that a variety of fungi, including basidiomycetes and non-basidiomycetes, emit methyl halides. Our research sites include four ecosystems: an agricultural field, a temperate forest, a fresh water wetland, and coastal salt marshes. We have collected and isolated fungi at each site by culturing tissue samples of fruiting bodies and plant material, by using wood baits, and from the direct culture of soil. We compared the rates of methyl halide emissions from the fungi in the four ecosystems. In addition, we measured emissions from previously assayed fungal isolates after reintroducing them to sterilized soils that were collected from their original environments. Fungal biomass was determined by substrate-induced respiration (SIR). The emission rate by the fungus was determined by a linear regression of the concentration of methyl halide in the sample headspace over time divided by the fungal biomass.

  14. Substrate-induced DNA polymerase β activation.

    PubMed

    Beard, William A; Shock, David D; Batra, Vinod K; Prasad, Rajendra; Wilson, Samuel H

    2014-11-01

    DNA polymerases and substrates undergo conformational changes upon forming protein-ligand complexes. These conformational adjustments can hasten or deter DNA synthesis and influence substrate discrimination. From structural comparison of binary DNA and ternary DNA-dNTP complexes of DNA polymerase β, several side chains have been implicated in facilitating formation of an active ternary complex poised for chemistry. Site-directed mutagenesis of these highly conserved residues (Asp-192, Arg-258, Phe-272, Glu-295, and Tyr-296) and kinetic characterization provides insight into the role these residues play during correct and incorrect insertion as well as their role in conformational activation. The catalytic efficiencies for correct nucleotide insertion for alanine mutants were wild type ∼ R258A > F272A ∼ Y296A > E295A > D192A. Because the efficiencies for incorrect insertion were affected to about the same extent for each mutant, the effects on fidelity were modest (<5-fold). The R258A mutant exhibited an increase in the single-turnover rate of correct nucleotide insertion. This suggests that the wild-type Arg-258 side chain generates a population of non-productive ternary complexes. Structures of binary and ternary substrate complexes of the R258A mutant and a mutant associated with gastric carcinomas, E295K, provide molecular insight into intermediate structural conformations not appreciated previously. Although the R258A mutant crystal structures were similar to wild-type enzyme, the open ternary complex structure of E295K indicates that Arg-258 stabilizes a non-productive conformation of the primer terminus that would decrease catalysis. Significantly, the open E295K ternary complex binds two metal ions indicating that metal binding cannot overcome the modified interactions that have interrupted the closure of the N-subdomain. PMID:25261471

  15. A Cross-chiral RNA Polymerase Ribozyme

    PubMed Central

    Sczepanski, Jonathan T.; Joyce, Gerald F.

    2014-01-01

    Thirty years ago it was shown that the non-enzymatic, template-directed polymerization of activated mononucleotides proceeds readily in a homochiral system, but is severely inhibited by the presence of the opposing enantiomer.1 This finding poses a severe challenge for the spontaneous emergence of RNA-based life, and has led to the suggestion that either RNA was preceded by some other genetic polymer that is not subject to chiral inhibition2 or chiral symmetry was broken through chemical processes prior to the origin of RNA-based life.3,4 Once an RNA enzyme arose that could catalyze the polymerization of RNA, it would have been possible to distinguish among the two enantiomers, enabling RNA replication and RNA-based evolution to occur. It is commonly thought that the earliest RNA polymerase and its substrates would have been of the same handedness, but this is not necessarily the case. Replicating D-and L-RNA molecules may have emerged together, based on the ability of structured RNAs of one handedness to catalyze the templated polymerization of activated mononucleotides of the opposite handedness. Such a cross-chiral RNA polymerase has now been developed using in vitro evolution. The D-RNA enzyme, consisting of 83 nucleotides, catalyzes the joining of L-mono- or oligonucleotide substrates on a complementary L-RNA template, and similarly for the L-enzyme with D-substrates and a D-template. Chiral inhibition is avoided because the 106-fold rate acceleration of the enzyme only pertains to cross-chiral substrates. The enzyme's activity is sufficient to generate full-length copies of its enantiomer through the templated joining of 11 component oligonucleotides. PMID:25363769

  16. Cytoplasmic effects on DNA methylation between male sterile lines and the maintainer in wheat (Triticum aestivum L.).

    PubMed

    Ba, Qingsong; Zhang, Gaisheng; Niu, Na; Ma, Shoucai; Wang, Junwei

    2014-10-01

    Male sterile cytoplasm plays an important role in hybrid wheat, and three-line system including male sterile (A line), its maintainer (B line) and restoring (R line) has played a major role in wheat hybrid production. It is well known that DNA methylation plays an important role in gene expression regulation during biological development in wheat. However, no reports are available on DNA methylation affected by different male sterile cytoplasms in hybrid wheat. We employed a methylation-sensitive amplified polymorphism technique to characterize nuclear DNA methylation in three male sterile cytoplasms. A and B lines share the same nucleus, but have different cytoplasms which is male sterile for the A and fertile for the B. The results revealed a relationship of DNA methylation at these sites specifically with male sterile cytoplasms, as well as male sterility, since the only difference between the A lines and B line was the cytoplasm. The DNA methylation was markedly affected by male sterile cytoplasms. K-type cytoplasm affected the methylation to a much greater degree than T-type and S-type cytoplasms, as indicated by the ratio of methylated sites, ratio of fully methylated sites, and polymorphism between A lines and B line for these cytoplasms. The genetic distance between the cytoplasm and nucleus for the K-type is much greater than for the T- and S-types because the former is between Aegilops genus and Triticum genus and the latter is within Triticum genus between Triticum spelta and Triticum timopheevii species. Thus, this difference in genetic distance may be responsible for the variation in methylation that we observed.

  17. Kappa casein gene polymorphism in local Tunisian goats.

    PubMed

    Jemmali, B; Ben Gara, A; Selmi, H; Ammari, Z; Bouheni, C; Ben Larbi, M; Hammami, M; Amraoui, M; Kamoun, M; Rouissi, H; Rekik, B

    2013-12-15

    The genetic polymorphism of the goat Kappa casein was investigated in Tunisian goats. Blood samples were collected from local goat breeds. Samples of genomic DNA were obtained from leukocytes of 175 dairy goats and regions of interest in the gene were amplified by Polymerase Chain Reaction (PCR) and then evaluated in agarose gel. For a better characterization of the single nucleotide polymorphism, a PCR-Restriction Fragment Length Polymorphism was performed employing the endonuclease DNA amplification using 459 bp primers. The PCR products of primers (459 bp) digested by restriction enzyme Alw44I produced two fragments of 459 and 381 bp. The Kappa casein allelic variants in tested animals revealed different genotypes, two of them were homozygous: AA or BB, AC or BC and CC. Genotypic frequencies were 12.5, 60.5 and 27% for AA or BB, CC and AC or BC, respectively. Identification of different variants of the Kappa casein can be used for the improvement and conservation of Tunisian local goats.

  18. Association study of PTPN22 C1858T polymorphism in Trypanosoma cruzi infection.

    PubMed

    Robledo, G; González, C I; Morillo, C; Martín, J; González, A

    2007-03-01

    In this study we investigated a possible role for the single nucleotide polymorphism C1858T of the PTPN22 (protein tyrosine phosphatase nonreceptor 22) gene in determining the susceptibility to Trypanosoma cruzi infection, as well as in development of chagasic heart disease. This study included 316 patients with Chagas' disease and 520 healthy individuals from Colombia and Peru. Genotyping of PTPN22 was performed by the real-time polymerase chain reaction technology, using the TaqMan 5' allelic discrimination assay. No statistically significant differences in the frequency of PTPN22 C1858T gene polymorphism between chagasic patients and controls or between asymptomatic and cardiomyopathic individuals were observed. Our findings suggest that the PTPN22 polymorphism analyzed does not play a major role in the development of Chagas' disease in the Colombian and Peruvian populations. PMID:17493151

  19. Heat shock protein 70-hom gene polymorphism and protein expression in multiple sclerosis.

    PubMed

    Boiocchi, C; Monti, M C; Osera, C; Mallucci, G; Pistono, C; Ferraro, O E; Nosari, G; Romani, A; Cuccia, M; Govoni, S; Pascale, A; Montomoli, C; Bergamaschi, R

    2016-09-15

    Immune-mediated and neurodegenerative mechanisms are involved in multiple sclerosis (MS). Growing evidences highlight the role of HSP70 genes in the susceptibility of some neurological diseases. In this explorative study we analyzed a polymorphism (i.e. HSP70-hom rs2227956) of the gene HSPA1L, which encodes for the protein hsp70-hom. We sequenced the polymorphism by polymerase chain reaction (PCR), in 191 MS patients and 365 healthy controls. The hsp70-hom protein expression was quantified by western blotting. We reported a strong association between rs2227956 polymorphism and MS risk, which is independent from the association with HSP70-2 rs1061581, and a significant link between hsp70-hom protein expression and MS severity. PMID:27609295

  20. Subunit Compositions of the RNA-Silencing Enzymes Pol IV and Pol V Reveal Their Origins as Specialized Forms of RNA Polymerase II

    SciTech Connect

    Ream, Thomas S.; Haag, J. R.; Wierzbicki, A. T.; Nicora, Carrie D.; Norbeck, Angela D.; Zhu, Jian-Kang; Hagen, Gretchen; Guilfoyle, Thomas J.; Pasa-Tolic, Ljiljana; Pikaard, Craig S.

    2009-01-30

    In addition to RNA polymerases I, II, and III, the essential RNA polymerases present in all eukaryotes, plants have two additional nuclear RNA polymerases, abbreviated as Pol IV and Pol V, that play nonredundant roles in siRNA-directed DNA methylation and gene silencing. We show that Arabidopsis Pol IV and Pol V are composed of subunits that are paralogous or identical to the 12 subunits of Pol II. Four subunits of Pol IV are distinct from their Pol II paralogs, six subunits of Pol V are distinct from their Pol II paralogs, and four subunits differ between Pol IV and Pol V. Importantly, the subunit differences occur in key positions relative to the template entry and RNA exit paths. Our findings support the hypothesis that Pol IV and Pol V are Pol II-like enzymes that evolved specialized roles in the production of noncoding transcripts for RNA silencing and genome defense.

  1. Extrachromosomal homologous DNA recombination in plant cells is fast and is not affected by CpG methylation.

    PubMed Central

    Puchta, H; Kocher, S; Hohn, B

    1992-01-01

    Using a sensitive transient assay, we investigated extrachromosomal homologous DNA recombination (ECR) in plant cells. As the plant genome is highly C methylated, we addressed the question of whether CpG methylation has an influence on DNA recombination efficiencies. Whereas the expression level of the fully CpG-methylated DNA molecules was reduced drastically, we found no significant changes in ECR efficiencies between two partly CpG-methylated plasmids or between one fully CpG-methylated and one nonmethylated plasmid. Using a modified polymerase chain reaction analysis, we were able to detect recombination between two fully CpG-methylated plasmids. Furthermore, we characterized the kinetics of the ECR reaction. Cotransfection of plasmids carrying truncated copies of the beta-glucuronidase (GUS) gene resulted in enzyme activity with a delay of only half an hour compared with that of the plasmid carrying the functional marker gene. This indicates that the ECR reaction itself requires no more than 30 min. By polymerase chain reaction, we were able to detect the recombined GUS gene as early as 2 h after transfection. This result and the time course of the transient GUS activity indicate that ECR occurs mainly early after transfection. The biological significance of this finding is discussed, and properties of ECR and intrachromosomal recombination are compared. Images PMID:1630452

  2. Methylenetetrahydrofolate reductase C677T polymorphism is associated with increased risk of coronary artery disease in young South African Indians.

    PubMed

    Ramkaran, Prithiksha; Phulukdaree, Alisa; Khan, Sajidah; Moodley, Devapregasan; Chuturgoon, Anil A

    2015-10-15

    Methylenetetrahydrofolate reductase (MTHFR) reduces 5',10'-methylenetetrahydrofolate to 5'-methyltetrahydrofolate, and is involved in remethylation of homocysteine to methionine, two important reactions involved in folate metabolism and methylation pathways. The common MTHFR C677T single nucleotide polymorphism (SNP) (rs1801133) has been associated with raised levels of homocysteine, a well known risk factor for coronary artery disease (CAD). CAD is a major cause of mortality worldwide. The age of onset of this chronic disorder is on the decline, particularly in the Indian population. Indians in South Africa (SA) have a higher prevalence of premature CAD compared to Black South Africans. The MTHFR C677T SNP has not been investigated in the SA Indian population. The present study therefore investigated the MTHFR C677T SNP in young SA Indian males with CAD compared to young Indian and Black male controls. A total of 290 subjects were recruited into this study which included 106 CAD patients (diagnosed on angiography, mean age 37.5, range 24-45 years), 100 Indian male controls (mean age 37.5, range 28-45 years), and 84 Black male controls (mean age 36.4, range 25-45). Polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) was used to genotype CAD patients and healthy controls. Data for clinical markers were obtained from pathology reports. There was a significant association between the 677 MTHFR variant (T) allele and CAD patients compared to the healthy Indian controls (p=0.0353, OR=2.105 95% CI 1.077-4.114). Indian controls presented with a higher frequency of the variant allele compared to Black controls (7% vs. 2% respectively, p=0.0515 OR=3.086 95% CI 0.9958-9.564). The MTHFR C677T SNP did not influence levels of total cholesterol, LDL, HDL, triglycerides, fasting glucose, fasting insulin, HbA1c or hsCRP. The higher frequency of the MTHFR 677 variant allele in South African Indians may be a contributing factor to the higher

  3. Photodissociation of methyl chloride and methyl bromide in the atmosphere

    NASA Technical Reports Server (NTRS)

    Robbins, D. E.

    1976-01-01

    Methyl chloride (CH3Cl) and methyl bromide (CH3Br) have been suggested to be significant sources of the stratospheric halogens. The breakup of these compounds in the stratosphere by photodissociation or reaction with OH releases halogen atoms which catalytically destroy ozone. Experimental results are presented for ultraviolet photoabsorption cross sections of CH3Cl and CH3Br. Calculations are presented of loss rates for the methyl halides due to photodissociation and reaction with OH and of mixing ratios of these species in the stratosphere.

  4. DNA methylation pathways and their crosstalk with histone methylation

    PubMed Central

    Du, Jiamu; Johnson, Lianna M.; Jacobsen, Steven E.; Patel, Dinshaw J.

    2015-01-01

    Methylation of DNA and of histone 3 at Lys 9 (H3K9) are highly correlated with gene silencing in eukaryotes from fungi to humans. Both of these epigenetic marks need to be established at specific regions of the genome and then maintained at these sites through cell division. Protein structural domains that specifically recognize methylated DNA and methylated histones are key for targeting enzymes that catalyse these marks to appropriate genome sites. Genetic, genomic, structural and biochemical data reveal connections between these two epigenetic marks, and these domains mediate much of the crosstalk. PMID:26296162

  5. Gene Polymorphism Studies in a Teaching Laboratory

    NASA Astrophysics Data System (ADS)

    Shultz, Jeffry

    2009-02-01

    I present a laboratory procedure for illustrating transcription, post-transcriptional modification, gene conservation, and comparative genetics for use in undergraduate biology education. Students are individually assigned genes in a targeted biochemical pathway, for which they design and test polymerase chain reaction (PCR) primers. In this example, students used genes annotated for the steroid biosynthesis pathway in soybean. The authoritative Kyoto encyclopedia of genes and genomes (KEGG) interactive database and other online resources were used to design primers based first on soybean expressed sequence tags (ESTs), then on ESTs from an alternate organism if soybean sequence was unavailable. Students designed a total of 50 gene-based primer pairs (37 soybean, 13 alternative) and tested these for polymorphism state and similarity between two soybean and two pea lines. Student assessment was based on acquisition of laboratory skills and successful project completion. This simple procedure illustrates conservation of genes and is not limited to soybean or pea. Cost per student estimates are included, along with a detailed protocol and flow diagram of the procedure.

  6. Apolipoprotein E Polymorphism in Tuberculosis Patients

    NASA Astrophysics Data System (ADS)

    Naserpour Farivar, Taghi; Sharifi Moud, Batool; Sargazi, Mansur; Moeenrezakhanlou, Alireza

    In this study, we aimed to determine the significance of association between Tuberculosis and apolipoprotein E polymorphism. The apolipoprotein E genotypes were assayed in 250 tuberculosis patients by polymerase chain reaction followed by enzymatic digestion with Hha I. The results were compared with the results of the same experiments on 250 sex and age matched control peoples. Present results showed that in studied populations, prevalence of E4 genotype was lower in controls than in patients (8 v. 13.2%; OR = 1.75, p<0.05) and prevalence of E3 genotype was high in controls than in patients (86 v.51%; OR = 0.17, p<0.05). Statistically significant difference was found between patients and controls with respect to ɛ2 allele frequencies, while ɛ2 allele frequency was found to be much less prevalent in controls (6%) than in patients (35.8%; OR = 8.72, p<0.05). Also, our study revealed that there is an association between apolipoprotein E genotypes and amplitude to tuberculosis in studied populations. However, large population-based studies are needed to understand the exact role played by the locus in causing the condition.

  7. Identification of Leptospira serovars by RFLP of the RNA polymerase beta subunit gene (rpoB)

    PubMed Central

    Jung, Lenice Roteia Cardoso; Bomfim, Maria Rosa Quaresma; Kroon, Erna Geessien; Nunes, Álvaro Cantini

    2015-01-01

    Leptospires are usually classified by methods based on DNA-DNA hybridization and the conventional cross-agglutination absorption test, which uses polyclonal antibodies against lipopolysaccharides. In this study, the amplification of the rpoB gene, which encodes the beta-subunit of RNA polymerase, was used as an alternative tool to identify Leptospira. DNA extracts from sixty-eight serovars were obtained, and the hypervariable region located between 1990 and 2500-bp in the rpoB gene was amplified by polymerase chain reaction (PCR). The 600-bp amplicons of the rpoB gene were digested with the restriction endonucleases TaqI, Tru1I, Sau3AI and MslI, and the restriction fragments were separated by 6% polyacrylamide gel electrophoresis. Thirty-five fragment patters were obtained from the combined data of restriction fragment length polymorphism (PCR-RFLP) analysis and used to infer the phylogenetic relationships among the Leptospira species and serovars. The species assignments obtained were in full agreement with the established taxonomic classifications. Twenty-two serovars were effectively identified based on differences in their molecular profiles. However, the other 46 serovars remained clustered in groups that included more than one serovar of different species. This study demonstrates the value of RFLP analysis of PCR-amplified rpoB as an initial method for identifying Leptospira species and serovars. PMID:26273261

  8. RNA Polymerases of Maize. Purification and Molecular Structure of DNA-dependent RNA Polymerase II*

    PubMed Central

    Mullinix, Kathleen P.; Strain, Gustave C.; Bogorad, Lawrence

    1973-01-01

    Nuclear DNA-dependent RNA polymerase II has been purified from leaves of Zea mays by a new procedure that improves enzyme stability and thus permits more manipulation during purification. The purification procedure includes a heating step, gel filtration on Sepharose 6B and 4B, and chromatography on DEAE- and DNA-celluloses. This method of purification yields an enzyme that exhibits maximal activity when denatured DNA is used as a template. Electrophoresis of highly purified enzyme on polyacrylamide gels containing sodium dodecyl sulfate indicates that maize RNA polymerase IIa is composed of several polypeptide subunits. The most highly purified preparations contain polypeptides with molecular weights of 200,000, 160,000, 35,000, 25,000, 20,000, and 17,000. Images PMID:4525172

  9. siRNA-directed DNA Methylation in Plants.

    PubMed

    Xie, Meng; Yu, Bin

    2015-02-01

    DNA cytosine methylationis an important epigenetic process that is correlated with transgene silencing, transposon suppression, and gene imprinting. In plants, small interfering RNAs (siRNAs) can trigger DNA methylation at loci containing their homolog sequences through a process called RNA-directed DNA methylation (RdDM). In canonical RdDM, 24 nucleotide (nt) siRNAs (ra-siRNAs) will be loaded into their effector protein called ARGONAUTE 4 (AGO4) and subsequently targeted to RdDM loci through base-pairing with the non-coding transcripts produced by DNA-directed RNA Polymerase V. Then, the AGO4-ra-siRNA will recruit the DNA methyltransferase to catalyze de novo DNA methylation. Recent studies also identified non-canonical RdDM pathways that involve microRNAs or 21 nt siRNAs. These RdDM pathways are biologically important since they control responses biotic and abiotic stresses, maintain genome stability and regulate development. Here, we summarize recent pro-gresses of mechanisms governing canonical and non-canonical RdDM pathways. PMID:25937811

  10. Myc Regulation of mRNA Cap Methylation

    PubMed Central

    Cowling, Victoria H.; Cole, Michael D.

    2010-01-01

    The c-myc proto-oncogene regulates the expression of 15% to 20% of all genes, depending on the cell type, and the regulation is usually modest (1.5- to 2.0-fold). The authors discovered that in addition to regulating mRNA abundance, c-Myc regulates the formation of the 7-methylguanosine cap on many mRNAs, including transcriptional target genes and others not transcriptionally activated. Because the 7-methylguanosine cap is required for effective translation, enhanced methyl cap formation leads to increased protein production from Myc-responsive genes that exceeds the transcriptional induction. Increased cap methylation is linked to Myc-dependent enhanced activity of 2 critical kinases, TFIIH and p-TEFb, which phosphorylate the RNA polymerase II carboxy-terminal domain (CTD). Phosphorylation of the CTD recruits RNGTT and RNMT, the enzymes involved in mRNA capping, to the nascent transcript. Evidence is accumulating that enhanced cap methylation makes a significant contribution to Myc-dependent gene regulation and protein production. PMID:21170289

  11. Dynamic DNA methylation regulates neuronal intrinsic membrane excitability.

    PubMed

    Meadows, Jarrod P; Guzman-Karlsson, Mikael C; Phillips, Scott; Brown, Jordan A; Strange, Sarah K; Sweatt, J David; Hablitz, John J

    2016-01-01

    Epigenetic modifications, such as DNA cytosine methylation, contribute to the mechanisms underlying learning and memory by coordinating adaptive gene expression and neuronal plasticity. Transcription-dependent plasticity regulated by DNA methylation includes synaptic plasticity and homeostatic synaptic scaling. Memory-related plasticity also includes alterations in intrinsic membrane excitability mediated by changes in the abundance or activity of ion channels in the plasma membrane, which sets the threshold for action potential generation. We found that prolonged inhibition of DNA methyltransferase (DNMT) activity increased intrinsic membrane excitability of cultured cortical pyramidal neurons. Knockdown of the cytosine demethylase TET1 or inhibition of RNA polymerase blocked the increased membrane excitability caused by DNMT inhibition, suggesting that this effect was mediated by subsequent cytosine demethylation and de novo transcription. Prolonged DNMT inhibition blunted the medium component of the after-hyperpolarization potential, an effect that would increase neuronal excitability, and was associated with reduced expression of the genes encoding small-conductance Ca(2+)-activated K(+) (SK) channels. Furthermore, the specific SK channel blocker apamin increased neuronal excitability but was ineffective after DNMT inhibition. Our results suggested that DNMT inhibition enables transcriptional changes that culminate in decreased expression of SK channel-encoding genes and decreased activity of SK channels, thus providing a mechanism for the regulation of neuronal intrinsic membrane excitability by dynamic DNA cytosine methylation. This study has implications for human neurological and psychiatric diseases associated with dysregulated intrinsic excitability. PMID:27555660

  12. Polymorphisms in cell cycle regulatory genes, urinary arsenic profile and urothelial carcinoma

    SciTech Connect

    Chung, C.-J.; Huang, C.-J.; Pu, Y.-S.; Su, C.-T.; Huang, Y.-K.; Chen, Y.-T.; Hsueh, Y.-M.

    2008-10-15

    Introduction: Polymorphisms in p53, p21 and CCND1 could regulate the progression of the cell cycle and might increase the susceptibility to inorganic arsenic-related cancer risk. The goal of our study was to evaluate the roles of cell cycle regulatory gene polymorphisms in the carcinogenesis of arsenic-related urothelial carcinoma (UC). Methods: A hospital-based case-controlled study was conducted to explore the relationships among the urinary arsenic profile, 8-hydroxydeoxyguanosine (8-OHdG) levels, p53 codon 72, p21 codon 31 and CCND1 G870A polymorphisms and UC risk. The urinary arsenic profile was determined using high-performance liquid chromatography (HPLC) and hydride generator-atomic absorption spectrometry (HG-AAS). 8-OHdG levels were measured by high-sensitivity enzyme-linked immunosorbent assay (ELISA) kits. Genotyping was conducted using polymerase chain reaction-restriction fragment length polymerase (PCR-RFLP). Results: Subjects carrying the p21 Arg/Arg genotype had an increased UC risk (age and gender adjusted OR = 1.53; 95% CI, 1.02-2.29). However, there was no association of p53 or CCND1 polymorphisms with UC risk. Significant effects were observed in terms of a combination of the three gene polymorphisms and a cumulative exposure of cigarette smoking, along with the urinary arsenic profile on the UC risk. The higher total arsenic concentration, monomethylarsonic acid percentage (MMA%) and lower dimethylarsinic acid percentage (DMA%), possessed greater gene variant numbers, had a higher UC risk and revealed significant dose-response relationships. However, effects of urinary 8-OHdG levels combined with three gene polymorphisms did not seem to be important for UC risk. Conclusions: The results showed that the variant genotype of p21 might be a predictor of inorganic arsenic-related UC risk.

  13. Detection of Cytotoxic T-Lymphocyte Associated Antigen-4 Gene Polymorphism in Type 1 Diabetes Mellitus.

    PubMed

    Arafa, Roshdan M; Desouky, Somaya M; Emam, Sherin M; Abed, Neveen Tawfik; Mohamed, Sahar Y

    2015-01-01

    Type 1 diabetes is one of the most common chronic childhood illnesses. Interplay between genetic susceptibility and environmental factors is thought to provide the fundamental element for the disease. It has been shown that more than 40 genetic loci are associated with T1DM. Important one among these is the CTLA-4. This work aimed to detect Cytotoxic T Lymphocyte-associated antigen 4 (CTLA-4) gene polymorphism in patients with type 1 diabetes mellitus T1DM using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) to clarify its role in the susceptibility to T1DM. The study was carried out on forty unrelated Egyptian children with TIDM. Twenty unrelated healthy children were enrolled as a control group. Blood samples were collected from patients and control groups and subjected to CTLA-4 gene polymorphism analysis using polymerase chain reaction followed by restriction fragment length polymorphism (PCR-RFLP). CTLA-4 G allele and GG homozygous genotype were significantly increased in T1DM patients than in control group (P < 0.001, P = 0.002 respectively). There was significant association between the three CTLA-4 genotypes (AA, AG, GG) and diabetic complications (p = 0.002), AG and GG polymorphisms were associated with complications of diabetes with ratio 84.6% and 100% respectively. While no association was found with sex, weight, height, risk factors of diabetes or insulin treatment. It was concluded that there is a strong association between AG polymorphism and T1DM (P = 0.002). PMID:26415372

  14. Assembling long heteroduplexes by asymmetric polymerase chain reaction and annealing the resulting single-stranded DNAs.

    PubMed

    Wang, Mugui; Wei, Chuchu; Ye, Xiufen; Liu, Jianping; Zhang, Cuicui; Chen, Hao; Zhang, Xiaobo; Tu, Jumin

    2015-04-15

    We developed an effective protocol for generating high-purity heteroduplexes via annealing single-stranded DNAs (ssDNAs) derived from plasmid DNA by asymmetric polymerase chain reaction (A-PCR). With the addition of dimethyl sulfoxide, a one-step A-PCR procedure can generate ssDNAs stably at a range of reaction temperatures. Several annealing buffers can anneal two ssDNAs into heteroduplexes effectively. We further developed a simple strategy to create d(GATC) hemimethylated heteroduplexes by annealing fully methylated homoduplexes in the presence of excessive unmethylated ssDNAs. The constructed heteroduplexes have been well tested as substrates for mismatch repair in Escherichia coli and, thus, can be used in various biotechnology applications. PMID:25575760

  15. Sensitivity of Carrot Cell Cultures and RNA Polymerase II to Amatoxins 1

    PubMed Central

    Little, Michael C.; Preston, James F.

    1985-01-01

    Protoplast and cell suspension cultures of Daucus carota L. were evaluated for their sensitivity toward the three amatoxin derivatives, α-amanitin, 6′-deoxy-α-amanitin, and 6′-O-methyl-α-amanitin using inhibition of DNA synthesis to measure cell viability. Protoplasts appeared approximately 10-fold more refractory than suspension cells and α-amanitin was much less effective than the other two amatoxins, even though Ki values for isolated RNA polymerase II were similar (4-5 nanomolar). Additional studies evaluating the recoveries of all three amatoxins from cell suspension supernates indicate one basis for these differences to be the selective degradation of α-amanitin. A mechanism involving the activation of the hydroxyindole moiety of the α-amanitin is thus invoked to explain these differences and we postulate the involvement of plant oxidases in this role. PMID:16664072

  16. [Downregulation of Human Adenovirus DNA Polymerase Gene by Modified siRNAs].

    PubMed

    Nikitenko, N A; Speiseder, T; Chernolovskaya, E L; Zenkova, M A; Dobner, T; Prassolov, V S

    2016-01-01

    Human adenoviruses, in particular D8, D19, and D37, cause ocular infections. Currently, there is no available causally directed treatment, which efficiently counteracts adenoviral infectious diseases. In our previous work, we showed that gene silencing by means of RNA interference is an effective approach for downregulation of human species D adenoviruses replication. In this study, we compared the biological activity of siRNAs and their modified analogs targeting human species D adenoviruses DNA polymerase. We found that one of selectively 2'-O-methyl modified siRNAs mediates stable and long-lasting suppression of the target gene (12 days post transfection). We suppose that this siRNA can be used as a potential therapeutic agent against human species D adenoviruses.

  17. Disruption of largest subunit RNA polymerase II genes in Trypanosoma brucei.

    PubMed Central

    Chung, H M; Lee, M G; Dietrich, P; Huang, J; Van der Ploeg, L H

    1993-01-01

    Two types of largest subunit RNA polymerase II (pol II) genes (pol IIA and pol IIB), differing in 3 amino acid substitutions, are encoded in the Trypanosoma brucei (stock 427-60) genome. As a result, the alpha-amanitin-resistant transcription of the procyclic acidic repetitive protein (PARP) and variant surface glycoprotein (VSG) genes was proposed to involve a modified, alpha-amanitin-resistant form of the largest subunit of pol II. Alternatively, pol I could transcribe the PARP and VSG genes. To discriminate between these two models, we deleted the N-terminal domain (about one-third of the polypeptide), which encodes the amino acid substitutions which discriminated the pol IIA and pol IIB genes, at both pol IIB alleles. The pol IIB- trypanosomes still transcribe the PARP genes and the VSG gene promoter region in insect-form trypanosomes by alpha-amanitin-resistant RNA polymerases, while control housekeeping genes are transcribed in an alpha-amanitin-sensitive manner, presumably by pol IIA. We conclude that the alpha-amanitin-resistant transcription of protein coding genes in T. brucei is not mediated by a diverged form of the largest subunit of pol II and that the presence of both the pol IIA and pol IIB genes is not essential for trypanosome viability. This conclusion was further supported by the finding that individual trypanosome variants exhibited allelic heterogeneity for the previously identified amino acid substitutions and that various permutations of the polymorphic amino acids generate at least four different types of largest subunit pol II genes. The expression of the PARP genes and the VSG gene promoter region by alpha-amanitin-resistant RNA polymerases in the pol IIB- trypanosomes provides evidence for transcription of these genes by pol I. Images PMID:8497277

  18. The Role of DNA Methylation Changes in Radiation-Induced Bystander Effects in cranial irradiated Mice

    NASA Astrophysics Data System (ADS)

    Zhang, Meng; Sun, Yeqing; Xue, Bei; Wang, Xinwen; Wang, Jiawen

    2016-07-01

    Heavy-ion radiation could lead to bystander effect in neighboring non-hit cells by signals released from directly-irradiated cells. The exact mechanisms of radiation-induced bystander effect in distant organ remain obscure, yet accumulating evidence points to the role of DNA methylation changes in bystander effect. To identify the molecular mechanism that underlies bystander effects of heavy-ion radiation, the male Balb/c and C57BL mice were cranial exposed to 40, 200, 2000mGy dose of carbon heavy-ion radiation, while the rest of the animal body was shielded. The γH2AX foci as the DNA damage biomarker in directly irradiation organ ear and the distant organ liver were detected on 0, 1, 2, 6, 12 and 24h after radiation, respectively. Methylation-sensitive amplifcation polymorphism (MSAP) was used to monitor the level of polymorphic genomic DNA methylation changed with dose and time effects. The results show that cranial irradiated mice could induce the γH2AX foci and genomic DNA methylation changes significantly in both the directly irradiation organ ear and the distant organ liver. The percent of DNA methylation changes were time-dependent and tissue-specific. Demethylation polymorphism rate were highest separately at 1 h in 200 mGy and 6 h in 2000 mGy after irradiation in ear. The global DNA methylation changes tended to occur in the CG sites. We also found that the numbers of γH2AX foci and the genomic methylation changes of heavy-ion radiation-induced bystander effect in liver could be obvious 1 h after radiation and achieved the maximum at 6 h, while the changes could recover gradually at 12 h. The results suggest that mice head exposed to heavy-ion radiation can induce damage and methylation pattern changed in both directly radiation organ ear and distant organ liver. Moreover, our findings are important to understand the molecular mechanism of radiation induced bystander effects in vivo. Keywords: Heavy-ion radiation; Bystander effect; DNA methylation; γH2

  19. Aberrant DNA Methylation in Keratoacanthoma

    PubMed Central

    Nakagawa, Hidemi

    2016-01-01

    Background Keratoacanthoma (KA) is a self-limiting epidermal tumor for which histopathological examination sometimes suggests malignancy. Based on inconsistent clinical views, KA can be regarded as both a benign tumor and a variant of squamous cell carcinoma (SCC). Aberrant DNA methylation frequently occurs in malignant tumors but it scarcely occurs in benign tumors. Whether aberrant methylation occurs in KA has not been previously examined. Objective The aim is to elucidate whether aberrant methylation of CpG islands (CGI) containing a high density of cytosine-guanine dinucleotide (CpG) sites occurs in KA. Methods Five SCC cell lines, two cultured samples of normal human epidermal keratinocytes (NHEKs), 18 clinical SCC samples, and 21 clinical KA samples were analyzed with Infinium HumanMethylation450 BeadChips, quantitative real-time methylation-specific PCR (RT-MSP) and/or bisulfite sequencing. Results Genome-wide analyses of NHEK, KA, and SCC indicated that there was a greater number of aberrantly hypermethylated CGIs in SCC than in KA and there were aberrantly hypermethylated CGIs which are common in both. Among the common hypermethylated CGIs, RT-MSP and bisulfite sequencing targeting CGIs located on CCDC17, PVR, and MAP3K11 gene bodies also showed that methylation levels were significantly higher in KA than in normal epidermis. Statistical analyses suggested that the methylation level of CGI located on PVR in SCC might be correlated to lymph node metastasis (P = 0.013, Mann-Whitney U test) and that the methylation level of CGI in MAP3K11 in KA might be correlated to age (P = 0.031, linear regression analysis). Conclusion Aberrant DNA methylation occurs in KA. PMID:27788211

  20. Polymorphic Evolutionary Games.

    PubMed

    Fishman, Michael A

    2016-06-01

    In this paper, I present an analytical framework for polymorphic evolutionary games suitable for explicitly modeling evolutionary processes in diploid populations with sexual reproduction. The principal aspect of the proposed approach is adding diploid genetics cum sexual recombination to a traditional evolutionary game, and switching from phenotypes to haplotypes as the new game׳s pure strategies. Here, the relevant pure strategy׳s payoffs derived by summing the payoffs of all the phenotypes capable of producing gametes containing that particular haplotype weighted by the pertinent probabilities. The resulting game is structurally identical to the familiar Evolutionary Games with non-linear pure strategy payoffs (Hofbauer and Sigmund, 1998. Cambridge University Press), and can be analyzed in terms of an established analytical framework for such games. And these results can be translated into the terms of genotypic, and whence, phenotypic evolutionary stability pertinent to the original game.

  1. [DNA methylation in thyroid carcinoma].

    PubMed

    Song, Xianyun; Shang, Xiaoling; Zhang, Yutuo

    2015-03-01

    Cancer has become clear that not merely gene variations but also epigenetic modifications may contribute to it. Epigenetic changes refer to stable alterations in gene expression with unrelated to changes in the underlying genetic sequence,resulting in heritable. DNA methylation is one of the common epigenetic changes. It control the gene expression through changing DNA conformation and stability, chromatin structer, DNA-protein interaction. The reversal of dysregulated DNA methylation has emerged as a potential strategy for the treatment of thyroid carcinoma. The artical will provide an overview of how DNA methylation contribute to thyroid carcinoma dissemination,invasion and metastasis and we will summarize the latest epigenetic therapies for thyroid carcinoma.

  2. Genetic and Non-genetic Predictors of LINE-1 Methylation in Leukocyte DNA

    PubMed Central

    Tajuddin, Salman M.; Amaral, André F. S.; Rodríguez-Rodero, Sandra; Rodríguez, Ramón María; Moore, Lee E.; Tardón, Adonina; Carrato, Alfredo; García-Closas, Montserrat; Silverman, Debra T.; Jackson, Brian P.; García-Closas, Reina; Cook, Ashley L.; Cantor, Kenneth P.; Chanock, Stephen; Kogevinas, Manolis; Rothman, Nathaniel; Real, Francisco X.; Fraga, Mario F.

    2013-01-01

    Background: Altered DNA methylation has been associated with various diseases. Objective: We evaluated the association between levels of methylation in leukocyte DNA at long interspersed nuclear element 1 (LINE-1) and genetic and non-genetic characteristics of 892 control participants from the Spanish Bladder Cancer/EPICURO study. Methods: We determined LINE-1 methylation levels by pyrosequencing. Individual data included demographics, smoking status, nutrient intake, toenail concentrations of 12 trace elements, xenobiotic metabolism gene variants, and 515 polymorphisms among 24 genes in the one-carbon metabolism pathway. To assess the association between LINE-1 methylation levels (percentage of methylated cytosines) and potential determinants, we estimated beta coefficients (βs) by robust linear regression. Results: Women had lower levels of LINE-1 methylation than men (β = –0.7, p = 0.02). Persons who smoked blond tobacco showed lower methylation than nonsmokers (β = –0.7, p = 0.03). Arsenic toenail concentration was inversely associated with LINE-1 methylation (β = –3.6, p = 0.003). By contrast, iron (β = 0.002, p = 0.009) and nickel (β = 0.02, p = 0.004) were positively associated with LINE-1 methylation. Single nucleotide polymorphisms (SNPs) in DNMT3A (rs7581217-per allele, β = 0.3, p = 0.002), TCN2 (rs9606756-GG, β = 1.9, p = 0.008; rs4820887-AA, β = 4.0, p = 4.8 × 10–7; rs9621049-TT, β = 4.2, p = 4.7 × 10–9), AS3MT (rs7085104-GG, β = 0.7, p = 0.001), SLC19A1 (rs914238, TC vs. TT: β = 0.5 and CC vs. TT: β = –0.3, global p = 0.0007) and MTHFS (rs1380642, CT vs. CC: β = 0.3 and TT vs. CC; β = –0.8, global p = 0.05) were associated with LINE-1 methylation. Conclusions: We identified several characteristics, environmental factors, and common genetic variants that predicted DNA methylation among study participants. PMID:23552396

  3. Gene Polymorphisms in Chronic Periodontitis

    PubMed Central

    Laine, Marja L.; Loos, Bruno G.; Crielaard, W.

    2010-01-01

    We aimed to conduct a review of the literature for gene polymorphisms associated with chronic periodontitis (CP) susceptibility. A comprehensive search of the literature in English was performed using the keywords: periodontitis, periodontal disease, combined with the words genes, mutation, or polymorphism. Candidate gene polymorphism studies with a case-control design and reported genotype frequencies in CP patients were searched and reviewed. There is growing evidence that polymorphisms in the IL1, IL6, IL10, vitamin D receptor, and CD14 genes may be associated with CP in certain populations. However, carriage rates of the rare (R)-allele of any polymorphism varied considerably among studies and most of the studies appeared under-powered and did not correct for other risk factors. Larger cohorts, well-defined phenotypes, control for other risk factors, and analysis of multiple genes and polymorphisms within the same pathway are needed to get a more comprehensive insight into the contribution of gene polymorphisms in CP. PMID:20339487

  4. A second triclinic polymorph of azimsulfuron

    PubMed Central

    Kwon, Eunjin; Kim, Jineun; Park, Hyunjin; Kim, Tae Ho

    2016-01-01

    The title compound, C13H16N10O5S (systematic name: 1-(4,6-di­meth­oxypyrimidin-2-yl)-3-{[1-methyl-4-(2-methyl-2H-tetra­zol-5-yl)pyrazol-5-yl]sulfonyl}urea), is a second triclinic polymorph of this crystal [for the other, see: Jeon et al., (2015 ▸). Acta Cryst. E71, o470–o471]. There are two mol­ecules, A and B, in the asymmetric unit; the dihedral angles between the pyrazole ring and the tetra­zole and di­meth­oxy­pyrimidine ring planes are 72.84 (10) and 37.24 (14)°, respectively (mol­ecule A) and 84.38 (9) and 26.09 (15)°, respectively (mol­ecule B). Each mol­ecule features an intra­molecular N—H⋯N hydrogen bond. In the crystal, aromatic π–π stacking inter­actions [centroid–centroid separations = 3.9871 (16), 3.4487 (14) and 3.5455 (16) Å] link the mol­ecules into [001] chains. In addition, N—H⋯N, N—H⋯O, C—H⋯O and C—H⋯N hydrogen bonds occur, forming a three-dimensional architecture. We propose that the dimorphism results from differences in conformations and packing owing to different inter­molecular inter­actions, especially aromatic π–π stacking. PMID:27746943

  5. A DNA polymerase activity is associated with Cauliflower Mosaic Virus.

    PubMed Central

    Menissier, J; Laquel, P; Lebeurier, G; Hirth, L

    1984-01-01

    A DNA polymerase activity is found within the Cauliflower Mosaic Virus (CaMV) particle. Analysis of the reaction product reveals that the linear form of the virion DNA is preferentially labelled. The molecular weight of the DNA polymerase as determined on an "activity gel" is 76 kDa. Images PMID:6514573

  6. Human DNA polymerase beta mutations allowing efficient abasic site bypass.

    PubMed

    Gieseking, Sonja; Bergen, Konrad; Di Pasquale, Francesca; Diederichs, Kay; Welte, Wolfram; Marx, Andreas

    2011-02-01

    The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase β when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase β was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase β and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase β variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights.

  7. Human DNA Polymerase β Mutations Allowing Efficient Abasic Site Bypass*

    PubMed Central

    Gieseking, Sonja; Bergen, Konrad; Di Pasquale, Francesca; Diederichs, Kay; Welte, Wolfram; Marx, Andreas

    2011-01-01

    The DNA of every cell in the human body gets damaged more than 50,000 times a day. The most frequent damages are abasic sites. This kind of damage blocks proceeding DNA synthesis by several DNA polymerases that are involved in DNA replication and repair. The mechanistic basis for the incapability of these DNA polymerases to bypass abasic sites is not clarified. To gain insights into the mechanistic basis, we intended to identify amino acid residues that govern for the pausing of DNA polymerase β when incorporating a nucleotide opposite to abasic sites. Human DNA polymerase β was chosen because it is a well characterized DNA polymerase and serves as model enzyme for studies of DNA polymerase mechanisms. Moreover, it acts as the main gap-filling enzyme in base excision repair, and human tumor studies suggest a link between DNA polymerase β and cancer. In this study we employed high throughput screening of a library of more than 11,000 human DNA polymerase β variants. We identified two mutants that have increased ability to incorporate a nucleotide opposite to an abasic site. We found that the substitutions E232K and T233I promote incorporation opposite the lesion. In addition to this feature, the variants have an increased activity and a lower fidelity when processing nondamaged DNA. The mutations described in this work are located in well characterized regions but have not been reported before. A crystallographic structure of one of the mutants was obtained, providing structural insights. PMID:21107011

  8. A Practical Polymerase Chain Reaction Laboratory for Introductory Biology Classes.

    ERIC Educational Resources Information Center

    Bowlus, R. David; Grether, Susan C.

    1996-01-01

    Presents a polymerase chain reaction (PCR) laboratory exercise that can be performed by introductory biology students in 1 45- to 55-minute class period. Includes a general description of the polymerase chain reaction, materials needed, procedure, and details of interest to teachers. (JRH)

  9. Incorporation of reporter-labeled nucleotides by DNA polymerases.

    PubMed

    Anderson, Jon P; Angerer, Bernhard; Loeb, Lawrence A

    2005-02-01

    The incorporation of fluorescently labeled nucleotides into DNA by DNA polymerases has been used extensively for tagging genes and for labeling DNA. However, we lack studies comparing polymerase efficiencies for incorporating different fluorescently labeled nucleotides. We analyzed the incorporation of fluorescent deoxynucleoside triphosphates by 10 different DNA polymerases, representing a cross-section of DNA polymerases from families A, B, and reverse transcriptase. The substitution of one or more different reporter-labeled nucleotides for the cognate nucleotides was initially investigated by using an in vitro polymerase extension filter-binding assay with natural DNA as a template. Further analysis on longer DNA fragments containing one or more nucleotide analogs was performed using a newly developed extension cut assay. The results indicate that incorporation of fluorescent nucleotides is dependent on the DNA polymerase, fluorophore, linker between the nucleotide and the fluorophore, and position for attachment of the linker and the cognate nucleotide. Of the polymerases tested, Taq and Vent exo DNA polymerases were most efficient at incorporating a variety of fluorescently labeled nucleotides. This study suggests that it should be feasible to copy DNA with reactions mixtures that contain all four fluorescently labeled nucleotides allowing for high-density labeling of DNA. PMID:15727132

  10. The RNA Polymerase of Marine Cyanophage Syn5*

    PubMed Central

    Zhu, Bin; Tabor, Stanley; Raytcheva, Desislava A.; Hernandez, Alfredo; King, Jonathan A.; Richardson, Charles C.

    2013-01-01

    A single subunit DNA-dependent RNA polymerase was identified and purified to apparent homogeneity from cyanophage Syn5 that infects the marine cyanobacteria Synechococcus. Syn5 is homologous to bacteriophage T7 that infects Escherichia coli. Using the purified enzyme its promoter has been identified by examining transcription of segments of Syn5 DNA and sequencing the 5′-termini of the transcripts. Only two Syn5 RNAP promoters, having the sequence 5′-ATTGGGCACCCGTAA-3′, are found within the Syn5 genome. One promoter is located within the Syn5 RNA polymerase gene and the other is located close to the right genetic end of the genome. The purified enzyme and its promoter have enabled a determination of the requirements for transcription. Unlike the salt-sensitive bacteriophage T7 RNA polymerase, this marine RNA polymerase requires 160 mm potassium for maximal activity. The optimal temperature for Syn5 RNA polymerase is 24 °C, much lower than that for T7 RNA polymerase. Magnesium is required as a cofactor although some activity is observed with ferrous ions. Syn5 RNA polymerase is more efficient in utilizing low concentrations of ribonucleotides than T7 RNA polymerase. PMID:23258537

  11. Molecular Evolution of Multi-subunit RNA Polymerases: Sequence Analysis

    PubMed Central

    Lane, William J.; Darst, Seth A.

    2009-01-01

    Transcription in all cellular organisms is performed by multi-subunit, DNA-dependent RNA polymerases that synthesize RNA from DNA templates. Previous sequence and structural studies have elucidated the importance of shared regions common to all multi-subunit RNA polymerases. In addition RNA polymerases contain multiple lineage-specific domain insertions involved in protein-protein and protein-nucleic acid interactions. We have created comprehensive multiple sequence alignments using all available sequence data for the multi-subunit RNA polymerase large subunits, including the bacterial β and β′ subunits and their homologues from archaebacterial RNA polymerases, the eukaryotic RNA polymerases I, II, and III, the nuclear-cytoplasmic large double-stranded DNA Virus RNA polymerases, and plant plastid RNA polymerases. In order to overcome technical difficulties inherent to the large subunit sequences, including large sequence length, small and large lineage-specific insertions, split subunits, and fused proteins, we created an automated and customizable sequence retrieval and processing system. In addition, we used our alignments to create a more expansive set of shared sequence regions and bacterial lineage-specific domain insertions. We also analyzed the intergenic gap between the bacterial β and β′ genes. PMID:19895820

  12. Purine inhibitors of protein kinases, G proteins and polymerases

    DOEpatents

    Gray, Nathanael S.; Schultz, Peter; Kim, Sung-Hou; Meijer, Laurent

    2001-07-03

    The present invention relates to purine analogs that inhibit, inter alia, protein kinases, G-proteins and polymerases. In addition, the present invention relates to methods of using such purine analogs to inhibit protein kinases, G-proteins, polymerases and other cellular processes and to treat cellular proliferative diseases.

  13. A general strategy for expanding polymerase function by droplet microfluidics

    PubMed Central

    Larsen, Andrew C.; Dunn, Matthew R.; Hatch, Andrew; Sau, Sujay P.; Youngbull, Cody; Chaput, John C.

    2016-01-01

    Polymerases that synthesize artificial genetic polymers hold great promise for advancing future applications in synthetic biology. However, engineering natural polymerases to replicate unnatural genetic polymers is a challenging problem. Here we present droplet-based optical polymerase sorting (DrOPS) as a general strategy for expanding polymerase function that employs an optical sensor to monitor polymerase activity inside the microenvironment of a uniform synthetic compartment generated by microfluidics. We validated this approach by performing a complete cycle of encapsulation, sorting and recovery on a doped library and observed an enrichment of ∼1,200-fold for a model engineered