DNA methylation levels analysis in four tissues of sea cucumber Apostichopus japonicus based on fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) during aestivation.

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Zhao, Ye; Chen, Muyan; Storey, Kenneth B; Sun, Lina; Yang, Hongsheng

2015-03-01

DNA methylation plays an important role in regulating transcriptional change in response to environmental stimuli. In the present study, DNA methylation levels of tissues of the sea cucumber Apostichopus japonicus were analyzed by the fluorescence-labeled methylation-sensitive amplified polymorphism (F-MSAP) technique over three stages of the aestivation cycle. Overall, a total of 26,963 fragments were amplified including 9112 methylated fragments among four sea cucumber tissues using 18 pairs of selective primers. Results indicated an average DNA methylation level of 33.79% for A. japonicus. The incidence of DNA methylation was different across tissue types in the non-aestivation stage: intestine (30.16%), respiratory tree (27.61%), muscle (27.94%) and body wall (56.25%). Our results show that hypermethylation accompanied deep-aestivation in A. japonicus, which suggests that DNA methylation may have an important role in regulating global transcriptional suppression during aestivation. Further analysis indicated that the main DNA modification sites were focused on intestine and respiratory tree tissues and that full-methylation but not hemi-methylation levels exhibited significant increases in the deep-aestivation stage. Copyright © 2014 Elsevier Inc. All rights reserved.

Methylation-sensitive amplified polymorphism analysis of Verticillium wilt-stressed cotton (Gossypium).

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Wang, W; Zhang, M; Chen, H D; Cai, X X; Xu, M L; Lei, K Y; Niu, J H; Deng, L; Liu, J; Ge, Z J; Yu, S X; Wang, B H

2016-10-06

In this study, a methylation-sensitive amplification polymorphism analysis system was used to analyze DNA methylation level in three cotton accessions. Two disease-sensitive near-isogenic lines, PD94042 and IL41, and one disease-resistant Gossypium mustelinum accession were exposed to Verticillium wilt, to investigate molecular disease resistance mechanisms in cotton. We observed multiple different DNA methylation types across the three accessions following Verticillium wilt exposure. These included hypomethylation, hypermethylation, and other patterns. In general, the global DNA methylation level was significantly increased in the disease-resistant accession G. mustelinum following disease exposure. In contrast, there was no significant difference in the disease-sensitive accession PD94042, and a significant decrease was observed in IL41. Our results suggest that disease-resistant cotton might employ a mechanism to increase methylation level in response to disease stress. The differing methylation patterns, together with the increase in global DNA methylation level, might play important roles in tolerance to Verticillium wilt in cotton. Through cloning and analysis of differently methylated DNA sequences, we were also able to identify several genes that may contribute to disease resistance in cotton. Our results revealed the effect of DNA methylation on cotton disease resistance, and also identified genes that played important roles, which may shed light on the future cotton disease-resistant molecular breeding.

Patterns of cytosine methylation in an elite rice hybrid and its parental lines, detected by a methylation-sensitive amplification polymorphism technique.

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Xiong, L Z; Xu, C G; Saghai Maroof, M A; Zhang, Q

1999-04-01

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. In this study, we assessed the extent and pattern of cytosine methylation in the rice genome, using the technique of methylation-sensitive amplified polymorphism (MSAP), which is a modification of the amplified fragment length polymorphism (AFLP) method that makes use of the differential sensitivity of a pair of isoschizomers to cytosine methylation. The tissues assayed included seedlings and flag leaves of an elite rice hybrid, Shanyou 63, and the parental lines Zhenshan 97 and Minghui 63. In all, 1076 fragments, each representing a recognition site cleaved by either or both of the isoschizomers, were amplified using 16 pairs of selective primers. A total of 195 sites were found to be methylated at cytosines in one or both parents, and the two parents showed approximately the same overall degree of methylation (16.3%), as revealed by the incidence of differential digestion by the isoschizomers. Four classes of patterns were identified in a comparative assay of cytosine methylation in the parents and hybrid; increased methylation was detected in the hybrid compared to the parents at some of the recognition sites, while decreased methylation in the hybrid was detected at other sites. A small proportion of the sites was found to be differentially methylated in seedlings and flag leaves; DNA from young seedlings was methylated to a greater extent than that from flag leaves. Almost all of the methylation patterns detected by MSAP could be confirmed by Southern analysis using the isolated amplified fragments as probes. The results clearly demonstrate that the MSAP technique is highly efficient for large-scale detection of cytosine methylation in the rice genome. We believe that the technique can be adapted for use in other plant species.

Analysis of DNA Methylation of Gracilariopsis lemaneiformis Under Temperature Stress Using the Methylation Sensitive Amplification Polymorphism (MSAP) Technique

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Peng, Chong; Sui, Zhenghong; Zhou, Wei; Hu, Yiyi; Mi, Ping; Jiang, Minjie; Li, Xiaodong; Ruan, Xudong

2018-06-01

Gracilariopsis lemaneiformis is an economically important agarophyte, which contains high quality gel and shows a high growth rate. Wild population of G. lemaneiformis displayed resident divergence, though with a low genetic diversity as was revealed by amplified fragment length polymorphism (AFLP) and simple sequence repeat (SSR) analyses. In addition, different strains of G. lemaneiformis are diverse in morphology. The highly inconsistence between genetic background and physiological characteristics recommends strongly to the regulation at epigenetic level. In this study, the DNA methylation change in G. lemaneiformis among different generation branches and under different temperature stresses was assessed using methylation sensitive amplified polymorphism (MSAP) technique. It was shown that DNA methylation level among different generation branches was diverse. The full and total methylated DNA level was the lowest in the second generation branch and the highest in the third generation. The total methylation level was 61.11%, 60.88% and 64.12% at 15°C, 22°C and 26°C, respectively. Compared with the control group (22°C), the fully methylated and totally methylated ratios were increased in both experiment groups (15°C and 26°C). All of the cytosine methylation/demethylation transform (CMDT) was further analyzed. High temperature treatment could induce more CMDT than low temperature treatment did.

Genetic diversity analysis of Jatropha curcas L. (Euphorbiaceae) based on methylation-sensitive amplification polymorphism.

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Kanchanaketu, T; Sangduen, N; Toojinda, T; Hongtrakul, V

2012-04-13

Genetic analysis of 56 samples of Jatropha curcas L. collected from Thailand and other countries was performed using the methylation-sensitive amplification polymorphism (MSAP) technique. Nine primer combinations were used to generate MSAP fingerprints. When the data were interpreted as amplified fragment length polymorphism (AFLP) markers, 471 markers were scored. All 56 samples were classified into three major groups: γ-irradiated, non-toxic and toxic accessions. Genetic similarity among the samples was extremely high, ranging from 0.95 to 1.00, which indicated very low genetic diversity in this species. The MSAP fingerprint was further analyzed for DNA methylation polymorphisms. The results revealed differences in the DNA methylation level among the samples. However, the samples collected from saline areas and some species hybrids showed specific DNA methylation patterns. AFLP data were used, together with methylation-sensitive AFLP (MS-AFLP) data, to construct a phylogenetic tree, resulting in higher efficiency to distinguish the samples. This combined analysis separated samples previously grouped in the AFLP analysis. This analysis also distinguished some hybrids. Principal component analysis was also performed; the results confirmed the separation in the phylogenetic tree. Some polymorphic bands, involving both nucleotide and DNA methylation polymorphism, that differed between toxic and non-toxic samples were identified, cloned and sequenced. BLAST analysis of these fragments revealed differences in DNA methylation in some known genes and nucleotide polymorphism in chloroplast DNA. We conclude that MSAP is a powerful technique for the study of genetic diversity for organisms that have a narrow genetic base.

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

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Hui Xia

Full Text Available 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.

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

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Xia, Hui; Huang, Weixia; 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.

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

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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.

Search for methylation-sensitive amplification polymorphisms in mutant figs

OpenAIRE

Rodrigues, M. G F; Martins, A. B G [UNESP; Bertoni, B. W.; Figueira, A.; Giuliatti, S.

2013-01-01

Fig (Ficus carica) breeding programs that use conventional approaches to develop new cultivars are rare, owing to limited genetic variability and the difficulty in obtaining plants via gamete fusion. Cytosine methylation in plants leads to gene repression, thereby affecting transcription without changing the DNA sequence. Previous studies using random amplification of polymorphic DNA and amplified fragment length polymorphism markers revealed no polymorphisms among select fig mutants that ori...

Comparative analysis of DNA methylation polymorphism in drought sensitive (HPKC2) and tolerant (HPK4) genotypes of horse Gram (Macrotyloma uniflorum).

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Bhardwaj, Jyoti; Mahajan, Monika; Yadav, Sudesh Kumar

2013-08-01

DNA methylation is known as an epigenetic modification that affects gene expression in plants. Variation in CpG methylation behavior was studied in two natural horse gram (Macrotyloma uniflorum [Lam.] Verdc.) genotypes, HPKC2 (drought-sensitive) and HPK4 (drought-tolerant). The methylation pattern in both genotypes was studied through methylation-sensitive amplified polymorphism. The results revealed that methylation was higher in HPKC2 (10.1%) than in HPK4 (8.6%). Sequencing demonstrated sequence homology with the DRE binding factor (cbf1), the POZ/BTB protein, and the Ty1-copia retrotransposon among some of the polymorphic fragments showing alteration in methylation behavior. Differences in DNA methylation patterns could explain the differential drought tolerance and the epigenetic signature of these two horse gram genotypes.

A relative quantitative Methylation-Sensitive Amplified Polymorphism (MSAP) method for the analysis of abiotic stress.

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Bednarek, Piotr T; Orłowska, Renata; Niedziela, Agnieszka

2017-04-21

We present a new methylation-sensitive amplified polymorphism (MSAP) approach for the evaluation of relative quantitative characteristics such as demethylation, de novo methylation, and preservation of methylation status of CCGG sequences, which are recognized by the isoschizomers HpaII and MspI. We applied the technique to analyze aluminum (Al)-tolerant and non-tolerant control and Al-stressed inbred triticale lines. The approach is based on detailed analysis of events affecting HpaII and MspI restriction sites in control and stressed samples, and takes advantage of molecular marker profiles generated by EcoRI/HpaII and EcoRI/MspI MSAP platforms. Five Al-tolerant and five non-tolerant triticale lines were exposed to aluminum stress using the physiologicaltest. Total genomic DNA was isolated from root tips of all tolerant and non-tolerant lines before and after Al stress following metAFLP and MSAP approaches. Based on codes reflecting events affecting cytosines within a given restriction site recognized by HpaII and MspI in control and stressed samples demethylation (DM), de novo methylation (DNM), preservation of methylated sites (MSP), and preservation of nonmethylatedsites (NMSP) were evaluated. MSAP profiles were used for Agglomerative hierarchicalclustering (AHC) based on Squared Euclidean distance and Ward's Agglomeration method whereas MSAP characteristics for ANOVA. Relative quantitative MSAP analysis revealed that both Al-tolerant and non-tolerant triticale lines subjected to Al stress underwent demethylation, with demethylation of CG predominating over CHG. The rate of de novo methylation in the CG context was ~3-fold lower than demethylation, whereas de novo methylation of CHG was observed only in Al-tolerant lines. Our relative quantitative MSAP approach, based on methylation events affecting cytosines within HpaII-MspI recognition sequences, was capable of quantifying de novo methylation, demethylation, methylation, and non-methylated status in control

Different DNA methylation patterns detected by the Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR technique among various cell types of bulls

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Carroll Bernie

2010-03-01

Full Text Available Abstract Background The purpose of this study was to apply an arbitrarily primed methylation sensitive polymerase chain reaction (PCR assay called Amplified Methylation Polymorphism Polymerase Chain Reaction (AMP PCR to investigate the methylation profiles of somatic and germ cells obtained from Holstein bulls. Methods Genomic DNA was extracted from sperm, leukocytes and fibroblasts obtained from three bulls and digested with a methylation sensitive endonuclease (HpaII. The native genomic and enzyme treated DNA samples were used as templates in an arbitrarily primed-PCR assay with 30 sets of single short oligonucleotide primer. The PCR products were separated on silver stained denaturing polyacrylamide gels. Three types of PCR markers; digestion resistant-, digestion sensitive-, and digestion dependent markers, were analyzed based on the presence/absence polymorphism of the markers between the two templates. Results Approximately 1,000 PCR markers per sample were produced from 27 sets of primer and most of them (>90% were digestion resistant markers. The highest percentage of digestion resistant markers was found in leukocytic DNA (94.8% and the lowest in fibroblastic DNA (92.3%, P ≤ 0.05. Spermatozoa contained a higher number of digestion sensitive markers when compared with the others (3.6% vs. 2.2% and 2.6% in leukocytes and fibroblasts respectively, P ≤ 0.05. Conclusions The powerfulness of the AMP PCR assay was the generation of methylation-associated markers without any prior knowledge of the genomic sequence. The data obtained from different primers provided an overview of genome wide DNA methylation content in different cell types. By using this technique, we found that DNA methylation profile is tissue-specific. Male germ cells were hypomethylated at the HpaII locations when compared with somatic cells, while the chromatin of the well-characterized somatic cells was heavily methylated when compared with that of the versatile somatic

Analysis of DNA methylation in Arabidopsis thaliana based on methylation-sensitive AFLP markers.

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Cervera, M T; Ruiz-García, L; Martínez-Zapater, J M

2002-12-01

AFLP analysis using restriction enzyme isoschizomers that differ in their sensitivity to methylation of their recognition sites has been used to analyse the methylation state of anonymous CCGG sequences in Arabidopsis thaliana. The technique was modified to improve the quality of fingerprints and to visualise larger numbers of scorable fragments. Sequencing of amplified fragments indicated that detection was generally associated with non-methylation of the cytosine to which the isoschizomer is sensitive. Comparison of EcoRI/ HpaII and EcoRI/ MspI patterns in different ecotypes revealed that 35-43% of CCGG sites were differentially digested by the isoschizomers. Interestingly, the pattern of digestion among different plants belonging to the same ecotype is highly conserved, with the rate of intra-ecotype methylation-sensitive polymorphisms being less than 1%. However, pairwise comparisons of methylation patterns between samples belonging to different ecotypes revealed differences in up to 34% of the methylation-sensitive polymorphisms. The lack of correlation between inter-ecotype similarity matrices based on methylation-insensitive or methylation-sensitive polymorphisms suggests that whatever the mechanisms regulating methylation may be, they are not related to nucleotide sequence variation.

Infraspecific DNA methylation polymorphism in cotton (Gossypium hirsutum L.).

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Keyte, Anna L; Percifield, Ryan; Liu, Bao; Wendel, Jonathan F

2006-01-01

Cytosine methylation is important in the epigenetic regulation of gene expression and development in plants and has been implicated in silencing duplicate genes after polyploid formation in several plant groups. Relatively little information exists, however, on levels and patterns of methylation polymorphism (MP) at homologous loci within species. Here we explored the levels and patterns of methylation-polymorphism diversity at CCGG sites within allotetraploid cotton, Gossypium hirsutum, using a methylation-sensitive amplified fragment length polymorphism screen and a selected set of 20 G. hirsutum accessions for which we have information on genetic polymorphism levels and relationships. Methylation and MP exist at high levels within G. hirsutum: of 150 HpaII/MspI sites surveyed, 48 were methylated at the inner cytosine (32%) and 32 of these were polymorphic (67%). Both these values are higher than comparable measures of genetic diversity using restriction fragment length polymorphisms. The high percentage of methylation-polymorphic sites and potential relationship to gene expression underscore the potential significance of MP within and among populations. We speculate that biased correlation of methylation-polymorphic sites and genes in cotton may be a consequence of polyploidy and the attendant doubling of all genes.

Methylation-sensitive amplification polymorphism in date palms (Phoenix dactylifera L.) and their off-shoots.

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Fang, J-G; Chao, C T

2007-07-01

DNA methylation plays an important role in the regulation of gene expression in eukaryotes. In this study, the extent and patterns of DNA methylation were assessed in date palm mother-plants and their off-shoots using the methylation-sensitive amplified polymorphism (MSAP) technique. Three types of bands were generated using 12 pairs of primers. Type I were present in both ECOR I + HPA II and ECOR I + MSP I lanes; type II were present in ECOR I + HPA II lanes, but not in ECOR I + MSP I lanes; and type III bands were present in ECOR I + MSP I lanes, but not in ECOR I + HPA II lanes. The total numbers of these three types of bands were 782, 55, and 34, respectively. Among these three types of bands, the polymorphic bands were, respectively, 37, 10, and 0. The distribution of polymorphic bands among mother-plants and off-shoots suggests the methylation variation was present in both the mother-plants and off-shoots. Forty- four out of these 47 polymorphic bands show clear difference between mother-plant and off-shoots: 38 were present only in off-shoots and 6 in both mother-plants and off-shoots. Compared to methylation status in mother-plants, the methylation variation during off-shoot growth of date palm can be characterized as a process involving primarily de-methylation. Hypomethylation of DNA in off-shoots, compared with mother-plants, reflects the marked expression of this molecular feature, which may be related to gene expression during off-shoot development. The methylation or de-methylation status of specific loci in the mother-plants and their off-shoots were probably random events.

Methylation-sensitive amplification polymorphism analysis of fat and muscle tissues in pigs.

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Ma, J D; Li, M Z; Zhou, S L; Zhou, C W; Li, X W

2012-09-26

DNA methylation may be involved in regulating the expression of protein-coding genes, resulting in different fat and muscle phenotypes. Using a methylation-sensitive amplified polymorphism approach, we obtained 7423 bands by selective amplification of genomic DNA from six different fat depots and two heterogeneous muscle types from Duroc/Landrace/Yorkshire cross-bred pigs. The degrees of DNA methylation, determined by the percentages of hemi- and fully methylated sites relative to the total number of CCGG sites, were similar in male and female pigs for each specific tissue [χ(2) test; P (two-tailed) > 0.05]. Gender bias was therefore ignored. There were significant differences in the degree of DNA methylation among the eight tissue types [χ(2) test; P(total) (two-tailed) = 0.009]. However, similar degrees of methylation were observed among the six fat depots [χ(2) test; P(fat) (two-tailed) = 0.24 > 0.05]and between the two muscle types [χ(2) test; P(muscle) (two-tailed) = 0.76 > 0.05]. We conclude that the degree of DNA methylation differs between porcine fat and muscle tissue, but that the methylation status of a particular tissue type is similar, despite being deposited at different body sites.

Search for methylation-sensitive amplification polymorphisms associated with the mantled variant phenotype in oil palm (Elaeis guineensis Jacq).

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Jaligot, E; Beulé, T; Baurens, F-C; Billotte, N; Rival, A

2004-02-01

The methylation-sensitive amplification polymorphism (MSAP) technique has been employed on somatic embryo-derived oil palms (Elaeis guineensis Jacq.) to identify methylation polymorphisms correlated with the "mantled" somaclonal variation. The variant phenotype displays an unstable feminization of male organs in both male and female flowers. Using MSAP, the methylation status of CCGG sites was compared in three normal versus three mantled regenerants sampled in clonal populations obtained through somatic embryogenesis from four genotypically distinct mother palms. Overall, 64 selective primer combinations were used and they have amplified 23 markers exhibiting a differential methylation pattern between the two phenotypes. Our results indicate that CCGG sites are poorly affected by the considerable decrease in global DNA methylation that has been previously associated with the mantled phenotype. Each of the 23 markers isolated in the present study could discriminate between the two phenotypes only when they were from the same genetic origin. This result hampers at the moment the direct use of MSAP markers for the early detection of variants, even though valuable information on putative target sequences will be obtained from a further characterization of these polymorphic markers.

Methylation-Sensitive Amplification Length Polymorphism (MS-AFLP) Microarrays for Epigenetic Analysis of Human Genomes.

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Alonso, Sergio; Suzuki, Koichi; Yamamoto, Fumiichiro; Perucho, Manuel

2018-01-01

Somatic, and in a minor scale also germ line, epigenetic aberrations are fundamental to carcinogenesis, cancer progression, and tumor phenotype. DNA methylation is the most extensively studied and arguably the best understood epigenetic mechanisms that become altered in cancer. Both somatic loss of methylation (hypomethylation) and gain of methylation (hypermethylation) are found in the genome of malignant cells. In general, the cancer cell epigenome is globally hypomethylated, while some regions-typically gene-associated CpG islands-become hypermethylated. Given the profound impact that DNA methylation exerts on the transcriptional profile and genomic stability of cancer cells, its characterization is essential to fully understand the complexity of cancer biology, improve tumor classification, and ultimately advance cancer patient management and treatment. A plethora of methods have been devised to analyze and quantify DNA methylation alterations. Several of the early-developed methods relied on the use of methylation-sensitive restriction enzymes, whose activity depends on the methylation status of their recognition sequences. Among these techniques, methylation-sensitive amplification length polymorphism (MS-AFLP) was developed in the early 2000s, and successfully adapted from its original gel electrophoresis fingerprinting format to a microarray format that notably increased its throughput and allowed the quantification of the methylation changes. This array-based platform interrogates over 9500 independent loci putatively amplified by the MS-AFLP technique, corresponding to the NotI sites mapped throughout the human genome.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)-A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes.

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Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare . However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

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Karolina Chwialkowska

2017-11-01

Full Text Available Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq. We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

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Chwialkowska, Karolina; Korotko, Urszula; Kosinska, Joanna; Szarejko, Iwona; Kwasniewski, Miroslaw

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing DNA methylation changes in plants. This method involves gel-based visualization of PCR fragments from selectively amplified DNA that are cleaved using methylation-sensitive restriction enzymes. In this study, we developed and validated a new method based on the conventional MSAP approach called Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq). We improved the MSAP-based approach by replacing the conventional separation of amplicons on polyacrylamide gels with direct, high-throughput sequencing using Next Generation Sequencing (NGS) and automated data analysis. MSAP-Seq allows for global sequence-based identification of changes in DNA methylation. This technique was validated in Hordeum vulgare. However, MSAP-Seq can be straightforwardly implemented in different plant species, including crops with large, complex and highly repetitive genomes. The incorporation of high-throughput sequencing into MSAP-Seq enables parallel and direct analysis of DNA methylation in hundreds of thousands of sites across the genome. MSAP-Seq provides direct genomic localization of changes and enables quantitative evaluation. We have shown that the MSAP-Seq method specifically targets gene-containing regions and that a single analysis can cover three-quarters of all genes in large genomes. Moreover, MSAP-Seq's simplicity, cost effectiveness, and high-multiplexing capability make this method highly affordable. Therefore, MSAP-Seq can be used for DNA methylation analysis in crop

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance*

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Zhao, Jie-hong; Zhang, Ji-shun; Wang, Yi; Wang, Ren-gang; Wu, Chun; Fan, Long-jiang; Ren, Xue-liang

2011-01-01

DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits. PMID:22042659

DNA methylation polymorphism in flue-cured tobacco and candidate markers for tobacco mosaic virus resistance.

Science.gov (United States)

Zhao, Jie-hong; Zhang, Ji-shun; Wang, Yi; Wang, Ren-gang; Wu, Chun; Fan, Long-jiang; Ren, Xue-liang

2011-11-01

DNA methylation plays an important role in the epigenetic regulation of gene expression during plant growth, development, and polyploidization. However, there is still no distinct evidence in tobacco regarding the distribution of the methylation pattern and whether it contributes to qualitative characteristics. We studied the levels and patterns of methylation polymorphism at CCGG sites in 48 accessions of allotetraploid flue-cured tobacco, Nicotiana tabacum, using a methylation-sensitive amplified polymorphism (MSAP) technique. The results showed that methylation existed at a high level among tobacco accessions, among which 49.3% sites were methylated and 69.9% allelic sites were polymorphic. A cluster analysis revealed distinct patterns of geography-specific groups. In addition, three polymorphic sites significantly related to tobacco mosaic virus (TMV) resistance were explored. This suggests that tobacco breeders should pay more attention to epigenetic traits.

Analysis of DNA methylation of perennial ryegrass under drought using the methylation-sensitive amplification polymorphism (MSAP) technique.

Science.gov (United States)

Tang, Xiao-Mei; Tao, Xiang; Wang, Yan; Ma, Dong-Wei; Li, Dan; Yang, Hong; Ma, Xin-Rong

2014-12-01

Perennial ryegrass (Lolium perenne), an excellent grass for forage and turf, is widespread in temperate regions. Drought is an important factor that limits its growth, distribution, and yield. DNA methylation affects gene expression and plays an important role in adaptation to adverse environments. In this study, the DNA methylation changes in perennial ryegrass under drought stress were assessed using methylation-sensitive amplified polymorphism (MSAP). After 15 days of drought stress treatment, the plant height was less than half of the control, and the leaves were smaller and darker. Genome-wide, a total of 652 CCGG sites were detected by MSAP. The total methylation level was 57.67 and 47.39 % in the control and drought treatment, respectively, indicating a decrease of 10.28 % due to drought exposure. Fifteen differentially displayed DNA fragments in MSAP profiles were cloned for sequencing analysis. The results showed that most of the genes involved in stress responses. The relative expression levels revealed that three demethylated fragments were up-regulated. The expression of a predicted retrotransposon increased significantly, changing from hypermethylation to non-methylation. Although the extent of methylation in two other genes decreased, the sites of methylation remained, and the expression increased only slightly. All of these results suggested that drought stress decreased the total DNA methylation level in perennial ryegrass and demethylation up-regulated related gene expressions and that the extent of methylation was negatively correlated with expression. Overall, the induced epigenetic changes in genome probably are an important regulatory mechanism for acclimating perennial ryegrass to drought and possibly other environmental stresses.

Analysis of DNA methylation related to rice adult plant resistance to bacterial blight based on methylation-sensitive AFLP (MSAP) analysis.

Science.gov (United States)

Sha, A H; Lin, X H; Huang, J B; Zhang, D P

2005-07-01

DNA methylation is known to play an important role in the regulation of gene expression in eukaryotes. The rice cultivar Wase Aikoku 3 becomes resistant to the blight pathogen Xanthomonas oryzae pv. oryzae at the adult stage. Using methylation-sensitive amplified polymorphism (MSAP) analysis, we compared the patterns of cytosine methylation in seedlings and adult plants of the rice cultivar Wase Aikoku 3 that had been inoculated with the pathogen Xanthomonas oryzae pv. oryzae, subjected to mock inoculation or left untreated. In all, 2000 DNA fragments, each representing a recognition site cleaved by either or both of two isoschizomers, were amplified using 60 pairs of selective primers. A total of 380 sites were found to be methylated. Of these, 45 showed differential cytosine methylation among the seedlings and adult plants subjected to different treatments, and overall levels of methylation were higher in adult plants than in seedlings. All polymorphic fragments were sequenced, and six showed homology to genes that code for products of known function. Northern analysis of three fragments indicated that their expression varied with methylation pattern, with hypermethylation being correlated with repression of transcription, as expected. The results suggest that significant differences in cytosine methylation exist between seedlings and adult plants, and that hypermethylation or hypomethylation of specific genes may be involved in the development of adult plant resistance (APR) in rice plants.

DNA methylation polymorphism in a set of elite rice cultivars and its possible contribution to inter-cultivar differential gene expression.

Science.gov (United States)

Wang, Yongming; Lin, Xiuyun; Dong, Bo; Wang, Yingdian; Liu, Bao

2004-01-01

RAPD (randomly amplified polymorphic DNA) and ISSR (inter-simple sequence repeat) fingerprinting on HpaII/MspI-digested genomic DNA of nine elite japonica rice cultivars implies inter-cultivar DNA methylation polymorphism. Using both DNA fragments isolated from RAPD or ISSR gels and selected low-copy sequences as probes, methylation-sensitive Southern blot analysis confirms the existence of extensive DNA methylation polymorphism in both genes and DNA repeats among the rice cultivars. The cultivar-specific methylation patterns are stably maintained, and can be used as reliable molecular markers. Transcriptional analysis of four selected sequences (RdRP, AC9, HSP90 and MMR) on leaves and roots from normal and 5-azacytidine-treated seedlings of three representative cultivars shows an association between the transcriptional activity of one of the genes, the mismatch repair (MMR) gene, and its CG methylation patterns.

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

Science.gov (United States)

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

2017-04-01

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.

Methylation Sensitive Amplification Polymorphism Sequencing (MSAP-Seq)—A Method for High-Throughput Analysis of Differentially Methylated CCGG Sites in Plants with Large Genomes

OpenAIRE

Karolina Chwialkowska; Urszula Korotko; Joanna Kosinska; Iwona Szarejko; Miroslaw Kwasniewski

2017-01-01

Epigenetic mechanisms, including histone modifications and DNA methylation, mutually regulate chromatin structure, maintain genome integrity, and affect gene expression and transposon mobility. Variations in DNA methylation within plant populations, as well as methylation in response to internal and external factors, are of increasing interest, especially in the crop research field. Methylation Sensitive Amplification Polymorphism (MSAP) is one of the most commonly used methods for assessing ...

How to interpret Methylation Sensitive Amplified Polymorphism (MSAP) profiles?

Czech Academy of Sciences Publication Activity Database

Fulneček, Jaroslav; Kovařík, Aleš

2014-01-01

Roč. 15, JAN 2014 (2014) ISSN 1471-2156 R&D Projects: GA ČR(CZ) GBP501/12/G090; GA ČR(CZ) GA13-10057S; GA ČR(CZ) GA206/09/1751 Institutional support: RVO:68081707 Keywords : MSAP * DNA methylation * Methylcytosine Subject RIV: BO - Biophysics Impact factor: 2.397, year: 2014

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

Science.gov (United States)

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.

Effect of different light quality on DNA methylation variation for brown ...

African Journals Online (AJOL)

DNA methylation plays an important role in regulating gene expression during plant development. We studied the effects of different light quality on DNA methylation patterns of brown cotton (Gossypium hirstum) by using the methylation sensitive amplified polymorphism (MSAP). We selected 66 pairs of MSAP selective ...

Analysis of DNA methylation variation in sibling tobacco ( Nicotiana ...

African Journals Online (AJOL)

Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) analysis were used to investigate the genome of two sibling tobacco cultivars, Yunyan85 and Yunyan87, their parent K326 and the other tobacco cultivar NC89. AFLP analysis indicated that, the genome primary ...

A DNA microarray-based methylation-sensitive (MS)-AFLP hybridization method for genetic and epigenetic analyses.

Science.gov (United States)

Yamamoto, F; Yamamoto, M

2004-07-01

We previously developed a PCR-based DNA fingerprinting technique named the Methylation Sensitive (MS)-AFLP method, which permits comparative genome-wide scanning of methylation status with a manageable number of fingerprinting experiments. The technique uses the methylation sensitive restriction enzyme NotI in the context of the existing Amplified Fragment Length Polymorphism (AFLP) method. Here we report the successful conversion of this gel electrophoresis-based DNA fingerprinting technique into a DNA microarray hybridization technique (DNA Microarray MS-AFLP). By performing a total of 30 (15 x 2 reciprocal labeling) DNA Microarray MS-AFLP hybridization experiments on genomic DNA from two breast and three prostate cancer cell lines in all pairwise combinations, and Southern hybridization experiments using more than 100 different probes, we have demonstrated that the DNA Microarray MS-AFLP is a reliable method for genetic and epigenetic analyses. No statistically significant differences were observed in the number of differences between the breast-prostate hybridization experiments and the breast-breast or prostate-prostate comparisons.

Methylated site display (MSD)-AFLP, a sensitive and affordable method for analysis of CpG methylation profiles.

Science.gov (United States)

Aiba, Toshiki; Saito, Toshiyuki; Hayashi, Akiko; Sato, Shinji; Yunokawa, Harunobu; Maruyama, Toru; Fujibuchi, Wataru; Kurita, Hisaka; Tohyama, Chiharu; Ohsako, Seiichiroh

2017-03-09

It has been pointed out that environmental factors or chemicals can cause diseases that are developmental in origin. To detect abnormal epigenetic alterations in DNA methylation, convenient and cost-effective methods are required for such research, in which multiple samples are processed simultaneously. We here present methylated site display (MSD), a unique technique for the preparation of DNA libraries. By combining it with amplified fragment length polymorphism (AFLP) analysis, we developed a new method, MSD-AFLP. Methylated site display libraries consist of only DNAs derived from DNA fragments that are CpG methylated at the 5' end in the original genomic DNA sample. To test the effectiveness of this method, CpG methylation levels in liver, kidney, and hippocampal tissues of mice were compared to examine if MSD-AFLP can detect subtle differences in the levels of tissue-specific differentially methylated CpGs. As a result, many CpG sites suspected to be tissue-specific differentially methylated were detected. Nucleotide sequences adjacent to these methyl-CpG sites were identified and we determined the methylation level by methylation-sensitive restriction endonuclease (MSRE)-PCR analysis to confirm the accuracy of AFLP analysis. The differences of the methylation level among tissues were almost identical among these methods. By MSD-AFLP analysis, we detected many CpGs showing less than 5% statistically significant tissue-specific difference and less than 10% degree of variability. Additionally, MSD-AFLP analysis could be used to identify CpG methylation sites in other organisms including humans. MSD-AFLP analysis can potentially be used to measure slight changes in CpG methylation level. Regarding the remarkable precision, sensitivity, and throughput of MSD-AFLP analysis studies, this method will be advantageous in a variety of epigenetics-based research.

DNA damage and genetic methylation changes caused by Cd in Arabidopsis thaliana seedlings.

Science.gov (United States)

Li, Zhaoling; Liu, Zhihong; Chen, Ruijuan; Li, Xiaojun; Tai, Peidong; Gong, Zongqiang; Jia, Chunyun; Liu, Wan

2015-09-01

Amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MASP) techniques are sensitive to deoxyribonucleic acid (DNA) damage and genetic methylation, respectively. Using these 2 techniques, Arabidopsis thaliana cultured with 0 mg/L (control), 0.5 mg/L, 1.5 mg/L, and 5.0 mg/L Cd(2+) for 16 d was used to analyze the DNA damage and methylation changes as a result of cadmium (Cd). The DNA was amplified by 14 AFLP primer pairs and 13 MSAP primer combinations. In the AFLP experiment, 62 polymorphic sites were found in the patterns of 11 primer combinations and a total of 1116 fragments were obtained in these patterns. There were no polymorphic bands in the remaining 3 pairs. The proportions of polymorphic sites in the 0.5-mg/L Cd(2+) and 5.0-mg/L Cd(2+) treatments were significantly different. Seven polymorphic fragments were then separated and successfully sequenced, yielding 6 nucleobase substitutions and 1 nucleobase deletion. Similarly, in the MSAP experiment, the MSAP% and number of demethylated-type bands were unchanged after Cd treatment, but the number of methylated-type bands was increased significantly in the 5.0-mg/L Cd(2+) treatment group, a finding that may be associated with the AFLP results. The polymorphic bands were also sequenced and the functions of their homologous genes were determined. The DNA damage and methylation changes may be the primary cause of certain pathology changes as a result of Cd uptake in plants. © 2015 SETAC.

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

Science.gov (United States)

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.

Complementary DNA-amplified fragment length polymorphism ...

African Journals Online (AJOL)

Complementary DNA-amplified fragment length polymorphism (AFLP-cDNA) analysis of differential gene expression from the xerophyte Ammopiptanthus mongolicus in response to cold, drought and cold together with drought.

Conversion of the random amplified polymorphic DNA (RAPD ...

African Journals Online (AJOL)

Conversion of the random amplified polymorphic DNA (RAPD) marker UBC#116 linked to Fusarium crown and root rot resistance gene (Frl) into a co-dominant sequence characterized amplified region (SCAR) marker for marker-assisted selection of tomato.

Amplified fragment length polymorphism and pulsed field gel electrophoresis for subspecies differentiation of Serpulina pilosicoli

DEFF Research Database (Denmark)

Møller, Kristian; Jensen, Tim Kåre; Boye, Mette

1999-01-01

Pulsed field gel electrophoresis (PFGE) and amplified fragment length polymorphism (AFLP) were compared for their ability to differentiate between 50 porcine Serpulina pilosicoli isolates. Both techniques were highly sensitive, dividing the isolates into 36 and 38 groups, respectively. Due...

Evaluation of genetic diversity in Chinese kale (Brassica oleracea L. var. alboglabra Bailey) by using rapid amplified polymorphic DNA and sequence-related amplified polymorphism markers.

Science.gov (United States)

Zhang, J; Zhang, L G

2014-02-14

Chinese kale is an original Chinese vegetable of the Cruciferae family. To select suitable parents for hybrid breeding, we thoroughly analyzed the genetic diversity of Chinese kale. Random amplified polymorphic DNA (RAPD) and sequence-related amplified polymorphism (SRAP) molecular markers were used to evaluate the genetic diversity across 21 Chinese kale accessions from AVRDC and Guangzhou in China. A total of 104 bands were detected by 11 RAPD primers, of which 66 (63.5%) were polymorphic, and 229 polymorphic bands (68.4%) were observed in 335 bands amplified by 17 SRAP primer combinations. The dendrogram showed the grouping of the 21 accessions into 4 main clusters based on RAPD data, and into 6 clusters based on SRAP and combined data (RAPD + SRAP). The clustering of accessions based on SRAP data was consistent with petal colors. The Mantel test indicated a poor fit for the RAPD and SRAP data (r = 0.16). These results have an important implication for Chinese kale germplasm characterization and improvement.

Efficiency of random amplified polymorphic DNA (RAPD) and inter ...

African Journals Online (AJOL)

Efficiency of random amplified polymorphic DNA (RAPD) and inter-simple sequence repeats (ISSR) markers for genotype fingerprinting and genetic diversity studies in canola ( ) ... The number of amplified fragments with RAPD primers ranged from 8 to 21, with the size of amplicons ranging from 162 to 3154 bp.

Genetic and epigenetic diversity and structure of Phragmites australis from local habitats of the Songnen Prairie using amplified fragment length polymorphism markers.

Science.gov (United States)

Qiu, T; Jiang, L L; Yang, Y F

2016-08-19

The genetic and epigenetic diversity and structure of naturally occurring Phragmites australis populations occupying two different habitats on a small spatial scale in the Songnen Prairie in northeastern China were investigated by assessing amplified fragment length polymorphisms (AFLPs) and methylation-sensitive amplified polymorphisms (MSAPs) through fluorescent capillary detection. The two groups of P. australis were located in a seasonal waterlogged low-lying and alkalized meadow with a pH of 8-8.5 and in an alkaline patch without accumulated rainwater and with a pH greater than 10. These groups showed high levels of genetic diversity at the habitat level based on the percentage of polymorphic bands (90.32, 82.56%), Nei's gene diversity index (0.262, 0.248), and the Shannon diversity index (0.407, 0.383). Although little is known about the between-habitat genetic differentiation of P. australis on a small spatial scale, our results implied significant genetic differentiation between habitats. Extensive epigenetic diversity within habitats, along with clear differentiation, was found. Specifically, the former habitat (Habitat 1, designated H1) harbored higher levels of genetic and epigenetic diversity than the latter (Habitat 2, designated H2), and population-level diversity was also high. This study represents one of few attempts to predict habitat-based genetic differentiation of reeds on a small scale. These assessments of genetic and epigenetic variation are integral aspects of molecular ecological studies on P. australis. Possible causes for within- and between-habitat genetic and epigenetic variations are discussed.

Extensive genetic and DNA methylation variation contribute to heterosis in triploid loquat hybrids.

Science.gov (United States)

Liu, Chao; Wang, Mingbo; Wang, Lingli; Guo, Qigao; Liang, Guolu

2018-04-24

We aim to overcome the unclear origin of the loquat and elucidate the heterosis mechanism of the triploid loquat. Here we investigated the genetic and epigenetic variations between the triploid plant and its parental lines using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified fragment length polymorphism (MSAP) analyses. We show that in addition to genetic variations, extensive DNA methylation variation occurred during the formation process of triploid loquat, with the triploid hybrid having increased DNA methylation compared to the parents. Furthermore, a correlation existed between genetic variation and DNA methylation remodeling, suggesting that genome instability may lead to DNA methylation variation or vice versa. Sequence analysis of the MSAP bands revealed that over 53% of them overlap with protein-coding genes, which may indicate a functional role of the differential DNA methylation in gene regulation and hence heterosis phenotypes. Consistent with this, the genetic and epigenetic alterations were associated closely to the heterosis phenotypes of triploid loquat, and this association varied for different traits. Our results suggested that the formation of triploid is accompanied by extensive genetic and DNA methylation variation, and these changes contribute to the heterosis phenotypes of the triploid loquats from the two cross lines.

induced by cadmium using random amplified polymorphic DNA

African Journals Online (AJOL)

darya

2013-04-17

Apr 17, 2013 ... metallurgy, painting, plastic production, etc., and is being released into the biosphere, and ...... aquatic macrophytes: Random amplified polymorphic DNA analysis and identification of ... ecotoxicology. Toxicol. Ecotoxicol.

Genomic change, retrotransposon mobilization and extensive cytosine methylation alteration in Brassica napus introgressions from two intertribal hybridizations.

Science.gov (United States)

Zhang, Xueli; Ge, Xianhong; Shao, Yujiao; Sun, Genlou; Li, Zaiyun

2013-01-01

Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP), sequence-specific amplification polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4-39.8%) and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed.

Random amplified polymorphic DNA based genetic characterization ...

African Journals Online (AJOL)

Random amplified polymorphic DNA based genetic characterization of four important species of Bamboo, found in Raigad district, Maharashtra State, India. ... Bambusoideae are differentiated from other members of the family by the presence of petiolate blades with parallel venation and stamens are three, four, six or more, ...

Molecular markers. Amplified fragment length polymorphism

Directory of Open Access Journals (Sweden)

Pržulj Novo

2005-01-01

Full Text Available Amplified Fragment Length Polymorphism molecular markers (AFLPs has been developed combining procedures of RFLPs and RAPDs molekular markers, i.e. the first step is restriction digestion of the genomic DNA that is followed by selective amplification of the restricted fragments. The advantage of the AFLP technique is that it allows rapid generation of a large number of reproducible markers. The reproducibility of AFLPs markers is assured by the use of restriction site-specific adapters and adapter-specific primers for PCR reaction. Only fragments containing the restriction site sequence plus the additional nucleotides will be amplified and the more selected nucleotides added on the primer sequence the fewer the number of fragments amplified by PCR. The amplified products are normally separated on a sequencing gel and visualized after exposure to X-ray film or by using fluorescent labeled primers. AFLP shave proven to be extremely proficient in revealing diversity at below the species level. A disadvantage of AFLP technique is that AFLPs are essentially a dominant marker system and not able to identify heterozygotes.

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

Science.gov (United States)

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.

Genomic change, retrotransposon mobilization and extensive cytosine methylation alteration in Brassica napus introgressions from two intertribal hybridizations.

Directory of Open Access Journals (Sweden)

Xueli Zhang

Full Text Available Hybridization and introgression represent important means for the transfer and/or de novo origination of traits and play an important role in facilitating speciation and plant breeding. Two sets of introgression lines in Brassica napus L. were previously established by its intertribal hybridizations with two wild species and long-term selection. In this study, the methods of amplified fragment length polymorphisms (AFLP, sequence-specific amplification polymorphism (SSAP and methylation-sensitive amplified polymorphism (MSAP were used to determine their genomic change, retrotransposon mobilization and cytosine methylation alteration in these lines. The genomic change revealed by the loss or gain of AFLP bands occurred for ∼10% of the total bands amplified in the two sets of introgressions, while no bands specific for wild species were detected. The new and absent SSAP bands appeared for 9 out of 11 retrotransposons analyzed, with low frequency of new bands and their total percentage of about 5% in both sets. MSAP analysis indicated that methylation changes were common in these lines (33.4-39.8% and the hypermethylation was more frequent than hypomethylation. Our results suggested that certain extents of genetic and epigenetic alterations were induced by hybridization and alien DNA introgression. The cryptic mechanism of these changes and potential application of these lines in breeding were also discussed.

Random amplified polymorphic DNA (RAPD) markers reveal genetic ...

African Journals Online (AJOL)

The present study evaluated genetic variability of superior bael genotypes collected from different parts of Andaman Islands, India using fruit characters and random amplified polymorphic DNA (RAPD) markers. Genomic DNA extracted from leaf material using cetyl trimethyl ammonium bromide (CTAB) method was ...

Inheritance of restriction fragment length polymorphisms, random amplified polymorphic DNAs and isozymes in coastal Douglas-fir

Science.gov (United States)

K.D. Jermstad; A.M. Reem; J.R. Henifin; N.C. Wheeler; D.B Neale

1994-01-01

A total of 225 new genetic loci [151 restriction fragment length polymorphisms (RFLP) and 74 random amplified polymorphic DNAs (RAPD)] in coastal Douglas- fir [Pseudotsuga menziesii (Mirb.) Franco var. menziesii] have been identified using a three-generation outbred pedigree. The Mendelian inheritance of 16 RFLP loci and 29...

Heritable alteration of DNA methylation induced by whole-chromosome aneuploidy in wheat.

Science.gov (United States)

Gao, Lihong; Diarso, Moussa; Zhang, Ai; Zhang, Huakun; Dong, Yuzhu; Liu, Lixia; Lv, Zhenling; Liu, Bao

2016-01-01

Aneuploidy causes changes in gene expression and phenotypes in all organisms studied. A previous study in the model plant Arabidopsis thaliana showed that aneuploidy-generated phenotypic changes can be inherited to euploid progenies and implicated an epigenetic underpinning of the heritable variations. Based on an analysis by amplified fragment length polymorphism and methylation-sensitive amplified fragment length polymorphism markers, we found that although genetic changes at the nucleotide sequence level were negligible, extensive changes in cytosine DNA methylation patterns occurred in all studied homeologous group 1 whole-chromosome aneuploid lines of common wheat (Triticum aestivum), with monosomic 1A showing the greatest amount of methylation changes. The changed methylation patterns were inherited by euploid progenies derived from the aneuploid parents. The aneuploidy-induced DNA methylation alterations and their heritability were verified at selected loci by bisulfite sequencing. Our data have provided empirical evidence supporting earlier suggestions that heritability of aneuploidy-generated, but aneuploidy-independent, phenotypic variations may have an epigenetic basis. That at least one type of aneuploidy - monosomic 1A - was able to cause significant epigenetic divergence of the aneuploid plants and their euploid progenies also lends support to recent suggestions that aneuploidy may have played an important and protracted role in polyploid genome evolution. © 2015 The Authors. New Phytologist © 2015 New Phytologist Trust.

DNA methylation alteration is a major consequence of genome doubling in autotetraploid Brassica rapa

Directory of Open Access Journals (Sweden)

Xu Yanhao

2017-01-01

Full Text Available Polyploids are typically classified as autopolyploids or allopolyploids based on the origin of their chromosome sets. Autopolyploidy is much more common than traditionally believed. Allopolyploidization, accompanied by genomic and transcriptomic changes, has been well investigated. In this study, genetic, DNA methylation and gene expression changes in autotetraploid Brassica rapa were investigated. No genetic alteration was detected using an amplified fragment length polymorphism (AFLP approach. Using a cDNA-AFLP approach, approximately 0.58% of fragments showed changes in gene expression in autotetraploid B. rapa. The methylation-sensitive amplification polymorphism (MSAP analysis showed that approximately 1.7% of the fragments underwent DNA methylation changes upon genome doubling, with hypermethylation and demethylation changes equally affected. Fragments displaying changes in gene expression and methylation status were isolated and then sequenced and characterized, respectively. This study showed that variation in cytosine methylation is a major consequence of genome doubling in autotetraploid Brassica rapa.

Detection of somaclonal variation by random amplified polymorphic ...

African Journals Online (AJOL)

Detection of somaclonal variation by random amplified polymorphic DNA analysis during micropropagation of Phalaenopsis bellina (Rchb.f.) Christenson. ... Among the primers used, P 16 produced the highest number of bands (29), while primer OPU 10 produced the lowest number (15). The range of similarity coefficient ...

Application of random amplified polymorphic DNA (RAPD) markers ...

African Journals Online (AJOL)

The random amplified polymorphic DNA (RAPD) technique has been widely applied to identify different varieties of plants for molecular breeding. However, application of RAPD markers to identify parthenogenesis in plants has not been reported. In this investigation, we used pedigree and RAPD markers to differentiate ...

( Quercus spp. ) using random amplified polymorphic DNA (RAPD)

African Journals Online (AJOL)

Quercus is one of the most important woody genera of the Northern hemisphere and considered as one of the main forest tree species in Iran. In this study, genetic relationships in the genus Quercus, using random amplified polymorphic DNA (RAPD) was examined. Five species, including: Quercus robur, Quercus ...

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

OpenAIRE

Xia, Hui; Huang, Weixia; 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 investig...

Sensitive electrochemical assaying of DNA methyltransferase activity based on mimic-hybridization chain reaction amplified strategy.

Science.gov (United States)

Zhang, Linqun; Liu, Yuanjian; Li, Ying; Zhao, Yuewu; Wei, Wei; Liu, Songqin

2016-08-24

A mimic-hybridization chain reaction (mimic-HCR) amplified strategy was proposed for sensitive electrochemically detection of DNA methylation and methyltransferase (MTase) activity In the presence of methylated DNA, DNA-gold nanoparticles (DNA-AuNPs) were captured on the electrode by sandwich-type assembly. It then triggered mimic-HCR of two hairpin probes to produce many long double-helix chains for numerous hexaammineruthenium (III) chloride ([Ru(NH3)6](3+), RuHex) inserting. As a result, the signal for electrochemically detection of DNA MTase activity could be amplified. If DNA was non-methylated, however, the sandwich-type assembly would not form because the short double-stranded DNAs (dsDNA) on the Au electrode could be cleaved and digested by restriction endonuclease HpaII (HapII) and exonuclease III (Exo III), resulting in the signal decrement. Based on this, an electrochemical approach for detection of M.SssI MTase activity with high sensitivity was developed. The linear range for M.SssI MTase activity was from 0.05 U mL(-1) to 10 U mL(-1), with a detection limit down to 0.03 U mL(-1). Moreover, this detecting strategy held great promise as an easy-to-use and highly sensitive method for other MTase activity and inhibition detection by exchanging the corresponding DNA sequence. Copyright © 2016 Elsevier B.V. All rights reserved.

Cytosine methylation alteration in natural populations of Leymus chinensis induced by multiple abiotic stresses.

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Yingjie Yu

Full Text Available 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

Cytosine Methylation Alteration in Natural Populations of Leymus chinensis Induced by Multiple Abiotic Stresses

Science.gov (United States)

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

Identification of exotic genetic components and DNA methylation pattern analysis of three cotton introgression lines from Gossypium bickii.

Science.gov (United States)

He, Shou-Pu; Sun, Jun-Ling; Zhang, Chao; Du, Xiong-Ming

2011-01-01

The impact of alien DNA fragments on plant genome has been studied in many species. However, little is known about the introgression lines of Gossypium. To study the consequences of introgression in Gossypium, we investigated 2000 genomic and 800 epigenetic sites in three typical cotton introgression lines, as well as their cultivar (Gossypium hirsutum) and wild parents (Gossypium bickii), by amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP). The results demonstrate that an average of 0.5% of exotic DNA segments from wild cotton is transmitted into the genome of each introgression line, with the addition of other forms of genetic variation. In total, an average of 0.7% of genetic variation sites is identified in introgression lines. Simultaneously, the overall cytosine methylation level in each introgression line is very close to that of the upland cotton parent (an average of 22.6%). Further dividing patterns reveal that both hypomethylation and hypermethylation occurred in introgression lines in comparison with the upland cotton parent. Sequencing of nine methylation polymorphism fragments showed that most (7 of 9) of the methylation alternations occurred in the noncoding sequences. The molecular evidence of introgression from wild cotton into introgression lines in our study is identified by AFLP. Moreover, the causes of petal variation in introgression lines are discussed.

Molecular Identification of Date Palm Cultivars Using Random Amplified Polymorphic DNA (RAPD) Markers.

Science.gov (United States)

Al-Khalifah, Nasser S; Shanavaskhan, A E

2017-01-01

Ambiguity in the total number of date palm cultivars across the world is pointing toward the necessity for an enumerative study using standard morphological and molecular markers. Among molecular markers, DNA markers are more suitable and ubiquitous to most applications. They are highly polymorphic in nature, frequently occurring in genomes, easy to access, and highly reproducible. Various molecular markers such as restriction fragment length polymorphism (RFLP), amplified fragment length polymorphism (AFLP), simple sequence repeats (SSR), inter-simple sequence repeats (ISSR), and random amplified polymorphic DNA (RAPD) markers have been successfully used as efficient tools for analysis of genetic variation in date palm. This chapter explains a stepwise protocol for extracting total genomic DNA from date palm leaves. A user-friendly protocol for RAPD analysis and a table showing the primers used in different molecular techniques that produce polymorphisms in date palm are also provided.

Analysis of DNA methylation in various swine tissues.

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Chun Yang

Full Text Available 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.

Analysis of mutation/rearrangement frequencies and methylation patterns at a given DNA locus using restriction fragment length polymorphism.

Science.gov (United States)

Boyko, Alex; Kovalchuk, Igor

2010-01-01

Restriction fragment length polymorphism (RFLP) is a difference in DNA sequences of organisms belonging to the same species. RFLPs are typically detected as DNA fragments of different lengths after digestion with various restriction endonucleases. The comparison of RFLPs allows investigators to analyze the frequency of occurrence of mutations, such as point mutations, deletions, insertions, and gross chromosomal rearrangements, in the progeny of stressed plants. The assay involves restriction enzyme digestion of DNA followed by hybridization of digested DNA using a radioactively or enzymatically labeled probe. Since DNA can be digested with methylation sensitive enzymes, the assay can also be used to analyze a methylation pattern of a particular locus. Here, we describe RFLP analysis using methylation-insensitive and methylation-sensitive enzymes.

Genetic and DNA methylation changes in cotton (Gossypium genotypes and tissues.

Directory of Open Access Journals (Sweden)

Kenji Osabe

Full Text Available In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP assays including a methylation insensitive enzyme (BsiSI, and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC. DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP.

Genetic and DNA methylation changes in cotton (Gossypium) genotypes and tissues.

Science.gov (United States)

Osabe, Kenji; Clement, Jenny D; Bedon, Frank; Pettolino, Filomena A; Ziolkowski, Lisa; Llewellyn, Danny J; Finnegan, E Jean; Wilson, Iain W

2014-01-01

In plants, epigenetic regulation is important in normal development and in modulating some agronomic traits. The potential contribution of DNA methylation mediated gene regulation to phenotypic diversity and development in cotton was investigated between cotton genotypes and various tissues. DNA methylation diversity, genetic diversity, and changes in methylation context were investigated using methylation-sensitive amplified polymorphism (MSAP) assays including a methylation insensitive enzyme (BsiSI), and the total DNA methylation level was measured by high-performance liquid chromatography (HPLC). DNA methylation diversity was greater than the genetic diversity in the selected cotton genotypes and significantly different levels of DNA methylation were identified between tissues, including fibre. The higher DNA methylation diversity (CHG methylation being more diverse than CG methylation) in cotton genotypes suggest epigenetic regulation may be important for cotton, and the change in DNA methylation between fibre and other tissues hints that some genes may be epigenetically regulated for fibre development. The novel approach using BsiSI allowed direct comparison between genetic and epigenetic diversity, and also measured CC methylation level that cannot be detected by conventional MSAP.

Genome-wide DNA methylation alterations of Alternanthera philoxeroides in natural and manipulated habitats: implications for epigenetic regulation of rapid responses to environmental fluctuation and phenotypic variation.

Science.gov (United States)

Gao, Lexuan; Geng, Yupeng; Li, Bo; Chen, Jiakuan; Yang, Ji

2010-11-01

Alternanthera philoxeroides (alligator weed) is an invasive weed that can colonize both aquatic and terrestrial habitats. Individuals growing in different habitats exhibit extensive phenotypic variation but little genetic differentiation in its introduced range. The mechanisms underpinning the wide range of phenotypic variation and rapid adaptation to novel and changing environments remain uncharacterized. In this study, we examined the epigenetic variation and its correlation with phenotypic variation in plants exposed to natural and manipulated environmental variability. Genome-wide methylation profiling using methylation-sensitive amplified fragment length polymorphism (MSAP) revealed considerable DNA methylation polymorphisms within and between natural populations. Plants of different source populations not only underwent significant morphological changes in common garden environments, but also underwent a genome-wide epigenetic reprogramming in response to different treatments. Methylation alterations associated with response to different water availability were detected in 78.2% (169/216) of common garden induced polymorphic sites, demonstrating the environmental sensitivity and flexibility of the epigenetic regulatory system. These data provide evidence of the correlation between epigenetic reprogramming and the reversible phenotypic response of alligator weed to particular environmental factors. © 2010 Blackwell Publishing Ltd.

Population structure of Salmonella investigated by amplified fragment length polymorphism

DEFF Research Database (Denmark)

Torpdahl, M.; Ahrens, Peter

2004-01-01

Aims: This study was undertaken to investigate the usefulness of amplified fragment length polymorphism (AFLP) in determining the population structure of Salmonella. Methods and Results: A total of 89 strains were subjected to AFLP analysis using the enzymes BglII and BspDI, a combination...... that is novel in Salmonella. Both species S. bongori and S. enterica and all subsp. of S. enterica were represented with emphasis on S. enterica subsp. enterica using a local strain collection and strains from the Salmonella Reference Collection B (SARB). The amplified fragments were used in a band...

Differential DNA Methylation Patterns Are Related to Phellogen Origin and Quality of Quercus suber Cork.

Science.gov (United States)

Inácio, Vera; Barros, Pedro M; Costa, Augusta; Roussado, Cristóvão; Gonçalves, Elsa; Costa, Rita; Graça, José; Oliveira, M Margarida; Morais-Cecílio, Leonor

2017-01-01

DNA methylation is thought to influence Quercus suber cork quality, which is the main constraint for its economic valorisation. However, a deep knowledge of the cytosine methylation patterns disclosing the epigenetic variability of trees with different cork quality types is totally missing. This study investigates the hypothesis that variations in DNA methylation contribute to differences in cork cellular characteristics directly related to original or traumatic phellogen activity. We used MSAPs (Methylation Sensitive Amplified Polymorphism) to assess DNA methylation patterns of cork and leaf tissues of Q. suber adult trees growing in three cork oak stands. The relationship between the detected polymorphisms and the diversity of cork quality traits was explored by a marker-trait analysis focusing on the most relevant quality characteristics. Populations differed widely in cork quality, but only slightly in degree of epigenetic differentiation. Four MSAP markers (1.3% of the total) were significantly associated with the most noteworthy quality traits: wood inclusions (nails) and porosity. This evidence supports the potential role of cytosine methylation in the modulation of differential phellogen activity either involved in localized cell death or in pore production, resulting in different cork qualities. Although, the underlying basis of the methylation polymorphism of loci affecting cork quality traits remain unclear, the disclosure of markers statistically associated with cork quality strengthens the potential role of DNA methylation in the regulation of these traits, namely at the phellogen level.

High-resolution genomic fingerprinting of Campylobacter jejuni and Campylobacter coli by analysis of amplified fragment length polymorphisms

DEFF Research Database (Denmark)

Kokotovic, Branko; On, Stephen L.W.

1999-01-01

A method for high-resolution genomic fingerprinting of the enteric pathogens Campylobacter jejuni and Campylobacter coli, based on the determination of amplified fragment length polymorphism, is described. The potential of this method for molecular epidemiological studies of these species...... is evaluated with 50 type, reference, and well-characterised field strains. Amplified fragment length polymorphism fingerprints comprised over 60 bands detected in the size range 35-500 bp. Groups of outbreak strains, replicate subcultures, and 'genetically identical' strains from humans, poultry and cattle......, proved indistinguishable by amplified fragment length polymorphism fingerprinting, but were differentiated fi-om unrelated isolates. Previously unknown relationships between three hippurate-negative C. jejuni strains, and two C. coil var, hyoilei strains, were identified. These relationships corresponded...

Analysis of methylated patterns and quality-related genes in tobacco (Nicotiana tabacum) cultivars.

Science.gov (United States)

Jiao, Junna; Jia, Yanlong; Lv, Zhuangwei; Sun, Chuanfei; Gao, Lijie; Yan, Xiaoxiao; Cui, Liusu; Tang, Zongxiang; Yan, Benju

2014-08-01

Methylation-sensitive amplified polymorphism was used in this study to investigate epigenetic information of four tobacco cultivars: Yunyan 85, NC89, K326, and Yunyan 87. The DNA fragments with methylated information were cloned by reamplified PCR and sequenced. The results of Blast alignments showed that the genes with methylation information included chitinase, nitrate reductase, chloroplast DNA, mitochondrial DNA, ornithine decarboxylase, ribulose carboxylase, and promoter sequences. Homologous comparison in three cloned gene sequences (nitrate reductase, ornithine decarboxylase, and ribulose decarboxylase) indicated that geographic factors had significant influence on the whole genome methylation. Introns also contained different information in different tobacco cultivars. These findings suggest that synthetic mechanisms for tobacco aromatic components could be affected by different environmental factors leading to variation of noncoding regions in the genome, which finally results in different fragrance and taste in different tobacco cultivars.

Genetic diversity of Actinobacillus pleuropneumoniae assessed by amplified fragment length polymorphism analysis

DEFF Research Database (Denmark)

Kokotovic, Branko; Angen, Øystein

2007-01-01

Amplified fragment length polymorphism (AFLP) was evaluated as a method for genotypic characterization and subtyping within the bacterial species Actinobacillus pleuropneumoniae. A total of 155 isolates of A. pleuropneumoniae, representing the serotypic variation described to occur within...

Salinity induced differential methylation patterns in contrasting cultivars of foxtail millet (Setaria italica L.).

Science.gov (United States)

Pandey, Garima; Yadav, Chandra Bhan; Sahu, Pranav Pankaj; Muthamilarasan, Mehanathan; Prasad, Manoj

2017-05-01

Genome-wide methylation analysis of foxtail millet cultivars contrastingly differing in salinity tolerance revealed DNA demethylation events occurring in tolerant cultivar under salinity stress, eventually modulating the expression of stress-responsive genes. Reduced productivity and significant yield loss are the adverse effects of environmental conditions on physiological and biochemical pathways in crop plants. In this context, understanding the epigenetic machinery underlying the tolerance traits in a naturally stress tolerant crop is imperative. Foxtail millet (Setaria italica) is known for its better tolerance to abiotic stresses compared to other cereal crops. In the present study, methylation-sensitive amplified polymorphism (MSAP) technique was used to quantify the salt-induced methylation changes in two foxtail millet cultivars contrastingly differing in their tolerance levels to salt stress. The study highlighted that the DNA methylation level was significantly reduced in tolerant cultivar compared to sensitive cultivar. A total of 86 polymorphic MSAP fragments were identified, sequenced and functionally annotated. These fragments showed sequence similarity to several genes including ABC transporter, WRKY transcription factor, serine threonine-protein phosphatase, disease resistance, oxidoreductases, cell wall-related enzymes and retrotransposon and transposase like proteins, suggesting salt stress-induced methylation in these genes. Among these, four genes were chosen for expression profiling which showed differential expression pattern between both cultivars of foxtail millet. Altogether, the study infers that salinity stress induces genome-wide DNA demethylation, which in turn, modulates expression of corresponding genes.

ALIS-FLP: Amplified ligation selected fragment-length polymorphism method for microbial genotyping

DEFF Research Database (Denmark)

Brillowska-Dabrowska, A.; Wianecka, M.; Dabrowski, Slawomir

2008-01-01

A DNA fingerprinting method known as ALIS-FLP (amplified ligation selected fragment-length polymorphism) has been developed for selective and specific amplification of restriction fragments from TspRI restriction endonuclease digested genomic DNA. The method is similar to AFLP, but differs...

The Role of DNA Methylation in Xylogenesis in Different Tissues of Poplar

Directory of Open Access Journals (Sweden)

Qingshi Wang

2016-07-01

Full Text Available 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.

Sequence-Related Amplified Polymorphism (SRAP Markers: A Potential Resource for Studies in Plant Molecular Biology

Directory of Open Access Journals (Sweden)

Daniel W. H. Robarts

2014-07-01

Full Text Available In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR, random-amplified polymorphic DNA (RAPD, and amplified fragment length polymorphism (AFLP to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use. highly variable marker with inherent biological significance.

Genetic variability of cultivated cowpea in Benin assessed by random amplified polymorphic DNA

NARCIS (Netherlands)

Zannou, A.; Kossou, D.K.; Ahanchédé, A.; Zoundjihékpon, J.; Agbicodo, E.; Struik, P.C.; Sanni, A.

2008-01-01

Characterization of genetic diversity among cultivated cowpea [Vigna unguiculata (L.) Walp.] varieties is important to optimize the use of available genetic resources by farmers, local communities, researchers and breeders. Random amplified polymorphic DNA (RAPD) markers were used to evaluate the

Sequence-related amplified polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology1

Science.gov (United States)

Robarts, Daniel W. H.; Wolfe, Andrea D.

2014-01-01

In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use, highly variable marker with inherent biological significance. PMID:25202637

Sequence-related amplified polymorphism (SRAP) markers: A potential resource for studies in plant molecular biology(1.).

Science.gov (United States)

Robarts, Daniel W H; Wolfe, Andrea D

2014-07-01

In the past few decades, many investigations in the field of plant biology have employed selectively neutral, multilocus, dominant markers such as inter-simple sequence repeat (ISSR), random-amplified polymorphic DNA (RAPD), and amplified fragment length polymorphism (AFLP) to address hypotheses at lower taxonomic levels. More recently, sequence-related amplified polymorphism (SRAP) markers have been developed, which are used to amplify coding regions of DNA with primers targeting open reading frames. These markers have proven to be robust and highly variable, on par with AFLP, and are attained through a significantly less technically demanding process. SRAP markers have been used primarily for agronomic and horticultural purposes, developing quantitative trait loci in advanced hybrids and assessing genetic diversity of large germplasm collections. Here, we suggest that SRAP markers should be employed for research addressing hypotheses in plant systematics, biogeography, conservation, ecology, and beyond. We provide an overview of the SRAP literature to date, review descriptive statistics of SRAP markers in a subset of 171 publications, and present relevant case studies to demonstrate the applicability of SRAP markers to the diverse field of plant biology. Results of these selected works indicate that SRAP markers have the potential to enhance the current suite of molecular tools in a diversity of fields by providing an easy-to-use, highly variable marker with inherent biological significance.

[Recent advances of amplified fragment length polymorphism and its applications in forensic botany].

Science.gov (United States)

Li, Cheng-Tao; Li, Li

2008-10-01

Amplified fragment length polymorphism (AFLP) is a new molecular marker to detect genomic polymorphism. This new technology has advantages of high resolution, good stability, and reproducibility. Great achievements have been derived in recent years in AFLP related technologies with several AFLP expanded methodologies available. AFLP technology has been widely used in the fields of plant, animal, and microbes. It has become one of the hotspots in Forensic Botany. This review focuses on the recent advances of AFLP and its applications in forensic biology.

Assessing the germplasm of Laminaria (phaeophyceae) with random amplified polymorphic DNA (RAPD) method

Science.gov (United States)

He, Yingjun; Zou, Yuping; Wang, Xiaodong; Zheng, Zhiguo; Zhang, Daming; Duan, Delin

2003-06-01

Eighteen gametophytes including L. japonica, L. ochotensis and L. longissima, were verified with random amplified polymorphic DNA (RAPD) technique. Eighteen ten-base primers were chosen from 100 primers selected for final amplification test. Among the total of 205 bands amplified, 181 (88.3%) were polymorphic. The genetic distance among different strains ranged from 0.072 to 0.391. The dendrogram constructed by unweighted pair-group method with arithmetic (UPGMA) method showed that the female and male gametophytes of the same cell lines could be grouped in pairs respectively. It indicated that RAPD analysis could be used not only to distinguish different strains of Laminaria, but also to distinguish male and female gametophyte within the same cell lines. There is ambiguous systematic relationship if judged merely by the present data. It seems that the use of RAPD marker is limited to elucidation of the phylogenetic relationship among the species of Laminaria.

Transgenerational epigenetics: Inheritance of global cytosine methylation and methylation-related epigenetic markers in the shrub Lavandula latifolia.

Science.gov (United States)

Herrera, Carlos M; Alonso, Conchita; Medrano, Mónica; Pérez, Ricardo; Bazaga, Pilar

2018-04-01

The ecological and evolutionary significance of natural epigenetic variation (i.e., not based on DNA sequence variants) variation will depend critically on whether epigenetic states are transmitted from parents to offspring, but little is known on epigenetic inheritance in nonmodel plants. We present a quantitative analysis of transgenerational transmission of global DNA cytosine methylation (= proportion of all genomic cytosines that are methylated) and individual epigenetic markers (= methylation status of anonymous MSAP markers) in the shrub Lavandula latifolia. Methods based on parent-offspring correlations and parental variance component estimation were applied to epigenetic features of field-growing plants ('maternal parents') and greenhouse-grown progenies. Transmission of genetic markers (AFLP) was also assessed for reference. Maternal parents differed significantly in global DNA cytosine methylation (range = 21.7-36.7%). Greenhouse-grown maternal families differed significantly in global methylation, and their differences were significantly related to maternal origin. Methylation-sensitive amplified polymorphism (MSAP) markers exhibited significant transgenerational transmission, as denoted by significant maternal variance component of marker scores in greenhouse families and significant mother-offspring correlations of marker scores. Although transmission-related measurements for global methylation and MSAP markers were quantitatively lower than those for AFLP markers taken as reference, this study has revealed extensive transgenerational transmission of genome-wide global cytosine methylation and anonymous epigenetic markers in L. latifolia. Similarity of results for global cytosine methylation and epigenetic markers lends robustness to this conclusion, and stresses the value of considering both types of information in epigenetic studies of nonmodel plants. © 2018 Botanical Society of America.

Arsenic methylation capacity in relation to nutrient intake and genetic polymorphisms in one-carbon metabolism.

Science.gov (United States)

Gamboa-Loira, Brenda; Hernández-Alcaraz, César; Gandolfi, A Jay; Cebrián, Mariano E; Burguete-García, Ana; García-Martínez, Angélica; López-Carrillo, Lizbeth

2018-07-01

Nutrients and genetic polymorphisms participating in one-carbon metabolism may explain interindividual differences in inorganic arsenic (iAs) methylation capacity, which in turn may account for variations in susceptibility to iAs-induced diseases. 1) To evaluate the association between polymorphisms in five one-carbon metabolism genes (FOLH1 c.223 T > C, MTHFD1 c.1958 G > A, MTHFR c.665 C > T, MTR c.2756 A > G, and MTRR c.66 A > G) and iAs methylation capacity; 2) To assess if previously reported associations between nutrient intake and iAs methylation capacity are modified by those polymorphisms. Women (n = 1027) exposed to iAs in Northern Mexico were interviewed. Blood and urine samples were collected. Nutrient dietary intake was estimated using a validated food frequency questionnaire. iAs methylation capacity was calculated from urinary iAs species (iAs, monomethylarsonic acid [MMA] and dimethylarsinic acid [DMA]) measured by high performance liquid chromatography (HPLC-ICP-MS). One polymorphism in each of the five genes evaluated was genotyped by allelic discrimination. Multivariable linear regression models were used to evaluate if genetic polymorphisms modified the associations between iAs methylation capacity parameters and nutrient intake. The median (min-max) concentration of total arsenic (TAs) was 20.2 (1.3-2776.0) µg/g creatinine in the study population. Significant interactions for iAs metabolism were only found with FOLH1 c.223 T > C polymorphism and vitamin B12 intake, so that CT and CC genotype carriers had significantly lower %iAs, and higher DMA/iAs with an increased vitamin B12 intake, as compared to carriers of wild-type TT. Differences in dietary nutrient intake and genetic variants in one-carbon metabolism may jointly influence iAs methylation capacity. Confirmation of these interactions in other populations is warranted. Copyright © 2018 Elsevier Inc. All rights reserved.

Analysis of Different Ploidy and Parent–Offspring Genomic DNA Methylation in the Loach Misgurnus anguillicaudatus

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He Zhou

2016-08-01

Full Text Available 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.

Analysis of Different Ploidy and Parent–Offspring Genomic DNA Methylation in the Loach Misgurnus anguillicaudatus

Science.gov (United States)

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

AMPLIFIED FRAGMENT LENGTH POLYMORPHISM ANALYSIS OF MYCOBACTERIUM AVIUM COMPLEX ISOLATES RECOVERED FROM SOUTHERN CALIFORNIA

Science.gov (United States)

Fine-scale genotyping methods are necessary in order to identify possible sources of human exposure to opportunistic pathogens belonging to the Mycobacterium avium complex (MAC). In this study, amplified fragment length polymorphism (AFLP) analysis was evaluated for fingerprintin...

Genetic polymorphisms of PPAR gamma, arsenic methylation capacity and breast cancer risk in Mexican women.

Science.gov (United States)

Pineda-Belmontes, Cristina P; Hernández-Ramírez, Raúl U; Hernández-Alcaraz, César; Cebrián, Mariano E; López-Carrillo, Lizbeth

2016-04-01

To evaluate whether the presence of polymorphisms of peroxisome proliferator-activated receptor gamma PPARγ (Pro 1 2Ala) and PPARGC1B (Ala203Pro) modifies the association between the inorganic arsenic (iAs) methylation capacity and breast cancer (BC). Mexican women were interviewed, and blood and urine samples were collected from them (cases/controls= 197/220). The concentration of urinary arsenic species and the polymorphisms of interest were determined by high-performance liquid chromatography with inductively coupled plasma mass spectrometry (HPLC-ICP-MS) and polymerase chain reaction (PCR), respectively. In women with a high %MMA (urinary monomethyl arsenic) and high primary methylation ratio (PM = MMA/iAs), the risk of BC was increased (odds ratio [OR]%MMA T3 vs.T1= 3.60: 95% confidence interval [CI] 2.02-6.41, ORPMI T3 vs.T1= 3.47: 95%CI 1.95-6.17), which was maintained after adjusting for polymorphisms. No significant interactions were observed between the polymorphisms and the arsenic variables on the risk of BC. Pro 12Ala and Ala203Pro polymorphisms did not modify the association between the iAs methylation capacity and BC.

Methylation-Sensitive High Resolution Melting (MS-HRM).

Science.gov (United States)

Hussmann, Dianna; Hansen, Lise Lotte

2018-01-01

Methylation-Sensitive High Resolution Melting (MS-HRM) is an in-tube, PCR-based method to detect methylation levels at specific loci of interest. A unique primer design facilitates a high sensitivity of the assays enabling detection of down to 0.1-1% methylated alleles in an unmethylated background.Primers for MS-HRM assays are designed to be complementary to the methylated allele, and a specific annealing temperature enables these primers to anneal both to the methylated and the unmethylated alleles thereby increasing the sensitivity of the assays. Bisulfite treatment of the DNA prior to performing MS-HRM ensures a different base composition between methylated and unmethylated DNA, which is used to separate the resulting amplicons by high resolution melting.The high sensitivity of MS-HRM has proven useful for detecting cancer biomarkers in a noninvasive manner in urine from bladder cancer patients, in stool from colorectal cancer patients, and in buccal mucosa from breast cancer patients. MS-HRM is a fast method to diagnose imprinted diseases and to clinically validate results from whole-epigenome studies. The ability to detect few copies of methylated DNA makes MS-HRM a key player in the quest for establishing links between environmental exposure, epigenetic changes, and disease.

Comparative Analysis of Human and Canine Campylobacter upsaliensis Isolates by Amplified Fragment Length Polymorphism

DEFF Research Database (Denmark)

Damborg, Peter; Guardabassi, Luca; Pedersen, Karl

2008-01-01

Human (n = 33) and canine (n = 53) Campylobacter upsaliensis isolates from seven countries were genotyped by a new amplified fragment length polymorphism method. We observed 100% typeability and high overall diversity. The majority of human strains (23/33) clustered separately from canine strains...

Genotyping and genetic diversity of Arcobacter butzleri by amplified fragment length polymorphism (AFLP) analysis

DEFF Research Database (Denmark)

On, Stephen L.W.; Atabay, H.I.; Amisu, K.O.

2004-01-01

Aims: To investigate the potential of amplified fragment length polymorphism (AFLP) profiling for genotyping Arcobacter butzleri and to obtain further data on the genetic diversity of this organism. Methods and Results: Seventy-three isolates of Danish, British, Turkish, Swedish, Nigerian and Nor...

Use of MSAP markers to analyse the effects of salt stress on DNA methylation in rapeseed (Brassica napus var. oleifera.

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Gianpiero Marconi

Full Text Available 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

Acute and chronic gregarisation are associated with distinct DNA methylation fingerprints in desert locusts.

Science.gov (United States)

Mallon, Eamonn B; Amarasinghe, Harindra E; Ott, Swidbert R

2016-10-18

Desert locusts (Schistocerca gregaria) show a dramatic form of socially induced phenotypic plasticity known as phase polyphenism. In the absence of conspecifics, locusts occur in a shy and cryptic solitarious phase. Crowding with conspecifics drives a behavioural transformation towards gregariousness that occurs within hours and is followed by changes in physiology, colouration and morphology, resulting in the full gregarious phase syndrome. We analysed methylation-sensitive amplified fragment length polymorphisms (MS-AFLP) to compare the effect of acute and chronic crowding on DNA methylation in the central nervous system. We find that crowd-reared and solitary-reared locusts show markedly different neural MS-AFLP fingerprints. However, crowding for a day resulted in neural MS-AFLP fingerprints that were clearly distinct from both crowd-reared and uncrowded solitary-reared locusts. Our results indicate that changes in DNA methylation associated with behavioural gregarisation proceed through intermediate states that are not simply partial realisations of the endpoint states.

Molecular typing of Borrelia burgdorferi sensu lato by randomly amplified polymorphic DNA fingerprinting analysis

NARCIS (Netherlands)

Wang, G.; van Dam, A. P.; Spanjaard, L.; Dankert, J.

1998-01-01

To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA

[Study of androgen receptor and phosphoglycerate kinase gene polymorphism in major cellular components of the so-called pulmonary sclerosing hemangioma].

Science.gov (United States)

Qi, Feng-jie; Zhang, Xiu-wei; Zhang, Yong-xing; Dai, Shun-dong; Wang, En-hua

2006-05-01

To study the clonality of polygonal cells and surface cuboidal cells in the so-called pulmonary sclerosing hemangioma (PSH). 17 female surgically resected PSH were found. The polygonal cells and surface cuboidal cells of the 17 PSH cases were microdissected from routine hematoxylin and eosin-stained sections. Genomic DNA was extracted, pretreated through incubation with methylation-sensitive restrictive endonuclease HhaI or HpaII, and amplified by nested polymerase chain reaction for X chromosome-linked androgen receptor (AR) and phosphoglycerate kinase (PGK) genes. The length polymorphism of AR gene was demonstrated by denaturing polyacrylamide gel electrophoresis and silver staining. The PGK gene products were treated with Bst XI and resolved on agarose gel. Amongst the 17 female cases of PSH, 15 samples were successfully amplified for AR and PGK genes. The rates of polymorphism were 53% (8/15) and 27% (4/15) for AR and PGK genes respectively. Polygonal cells and surface cuboidal cells of 10 cases which were suitable for clonality study, showed the same loss of alleles (clonality ratio = 0) or unbalanced methylation pattern (clonality ratio < 0.25). The polygonal cells and surface cuboidal cells in PSH demonstrate patterns of monoclonal proliferation, indicating that both represent true neoplastic cells.

Localization of the cochlear amplifier in living sensitive ears.

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Tianying Ren

Full Text Available BACKGROUND: To detect soft sounds, the mammalian cochlea increases its sensitivity by amplifying incoming sounds up to one thousand times. Although the cochlear amplifier is thought to be a local cellular process at an area basal to the response peak on the spiral basilar membrane, its location has not been demonstrated experimentally. METHODOLOGY AND PRINCIPAL FINDINGS: Using a sensitive laser interferometer to measure sub-nanometer vibrations at two locations along the basilar membrane in sensitive gerbil cochleae, here we show that the cochlea can boost soft sound-induced vibrations as much as 50 dB/mm at an area proximal to the response peak on the basilar membrane. The observed amplification works maximally at low sound levels and at frequencies immediately below the peak-response frequency of the measured apical location. The amplification decreases more than 65 dB/mm as sound levels increases. CONCLUSIONS AND SIGNIFICANCE: We conclude that the cochlea amplifier resides at a small longitudinal region basal to the response peak in the sensitive cochlea. These data provides critical information for advancing our knowledge on cochlear mechanisms responsible for the remarkable hearing sensitivity, frequency selectivity and dynamic range.

A two primers random amplified polymorphic DNA procedure to obtain polymerase chain reaction fingerprints of bacterial species.

Science.gov (United States)

Rivas, R; Velázquez, E; Valverde, A; Mateos, P F; Martínez-Molina, E

2001-04-01

Polymerase chain reation (PCR) fingerprints are used to characterize and recognize bacteria and are generally obtained using universal primers that generate an array of DNA amplicons, which can be separated by electrophoresis. Universal primers 8F and 1491 R have been used to amplify specifically 16S rDNA. We have used these primers at an annealing temperature of 50 degrees C. Agarose gel electrophoresis of PCR products revealed several bands. The band pattern of each bacterial species was different and the strains belonging to the same species shared an identical pattern. The patterns obtained did not show variations with plasmid DNA content or the growth stage of the bacteria. The peculiarity of the randomly amplified polymorphic DNA (RAPD) described in this work lies in the use of two large primers (proximately 20 nt) to obtain the pattern, since normally a only smaller primer is used, and in the new application for the primers used to amplify 16S rDNA. This new procedure, called two primers (TP)-RAPD fingerprinting, is thus rapid, sensitive, reliable, highly reproducible and suitable for experiments with a large number of microorganisms, and can be applied to bacterial taxonomy, ecological studies and for the detection of new bacterial species.

Estimation of genetic variability among elite wheat genotypes using random amplified polymorphic DNA (RAPD) analysis

International Nuclear Information System (INIS)

BIBI, S.; Khan, I.A.; Naqvi, M.H.; Siddiqui, M.A.; Yasmeen, S.; Seema, M.

2012-01-01

Twenty four wheat varieties/lines were assessed through RAPD for genetic diversity. Of forty primers, thirteen were able to amplify the genomic DNA and yielded 269 polymorphic bands. The percentage of the polymorphic loci was 86.22%. Nei's genetic diversity (h) ranged from 0.248 to 0.393, with an average of 0.330. Shanon's index ranged from 0.382 to 0.567, with an average of 0.487. The proportion of genetic variation among the populations ( Ds) accounted for 28.58 % of the whole genetic diversity. The level of gene flow (Nm) was 1.25. Some specific RAPD bands were also identified, variety C-591, and QM-4531 contain a specific segment of 4.9 kbp. Whereas SARC-1 and PKV-1600 amplified a specific DNA segment with primer A-09. Marvi-2000 contains two specific segments of 3.2 kb and 200 bp amplified with primer B-07. Genetically most similar genotypes were C-591 and Pasban-90 (76%) and most dissimilar genotypes were Rawal-87 and Khirman (36.1%). On the basis of results, 24 wheat varieties under study could be divided into 'two' groups and five clusters 'A' to 'E. (author)

DNA Methylation Modulates Nociceptive Sensitization after Incision.

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Yuan Sun

Full Text Available DNA methylation is a key epigenetic mechanism controlling DNA accessibility and gene expression. Blockade of DNA methylation can significantly affect pain behaviors implicated in neuropathic and inflammatory pain. However, the role of DNA methylation with regard to postoperative pain has not yet been explored. In this study we sought to investigate the role of DNA methylation in modulating incisional pain and identify possible targets under DNA methylation and contributing to incisional pain. DNA methyltranferase (DNMT inhibitor 5-Aza-2'-deoxycytidine significantly reduced incision-induced mechanical allodynia and thermal sensitivity. Aza-2'-deoxycytidine also reduced hindpaw swelling after incision, suggesting an anti-inflammatory effect. Global DNA methylation and DNMT3b expression were increased in skin after incision, but none of DNMT1, DNMT3a or DNMT3b was altered in spinal cord or DRG. The expression of proopiomelanocortin Pomc encoding β-endorphin and Oprm1 encoding the mu-opioid receptor were upregulated peripherally after incision; moreover, Oprm1 expression was further increased under DNMT inhibitor treatment. Finally, local peripheral injection of the opioid receptor antagonist naloxone significantly exacerbated incision-induced mechanical hypersensitivity. These results suggest that DNA methylation is functionally relevant to incisional nociceptive sensitization, and that mu-opioid receptor signaling might be one methylation regulated pathway controlling sensitization after incision.

MethylMeter(®): bisulfite-free quantitative and sensitive DNA methylation profiling and mutation detection in FFPE samples.

Science.gov (United States)

McCarthy, David; Pulverer, Walter; Weinhaeusel, Andreas; Diago, Oscar R; Hogan, Daniel J; Ostertag, Derek; Hanna, Michelle M

2016-06-01

Development of a sensitive method for DNA methylation profiling and associated mutation detection in clinical samples. Formalin-fixed and paraffin-embedded tumors received by clinical laboratories often contain insufficient DNA for analysis with bisulfite or methylation sensitive restriction enzymes-based methods. To increase sensitivity, methyl-CpG DNA capture and Coupled Abscription PCR Signaling detection were combined in a new assay, MethylMeter(®). Gliomas were analyzed for MGMT methylation, glioma CpG island methylator phenotype and IDH1 R132H. MethylMeter had 100% assay success rate measuring all five biomarkers in formalin-fixed and paraffin-embedded tissue. MGMT methylation results were supported by survival and mRNA expression data. MethylMeter is a sensitive and quantitative method for multitarget DNA methylation profiling and associated mutation detection. The MethylMeter-based GliomaSTRAT assay measures methylation of four targets and one mutation to simultaneously grade gliomas and predict their response to temozolomide. This information is clinically valuable in management of gliomas.

Genomic variations of Mycoplasma capricolum subsp capripneumoniae detected by amplified fragment length polymorphism (AFLP) analysis

DEFF Research Database (Denmark)

Kokotovic, Branko; Bolske, G.; Ahrens, Peter

2000-01-01

The genetic diversity of Mycoplasma capricolum subsp. capripneumoniae strains based on determination of amplified fragment length polymorphisms (AFLP) is described. AFLP fingerprints of 38 strains derived from different countries in Africa and the Middle East consisted of over 100 bands in the size...

Comparative analyses of genetic/epigenetic diversities and structures in a wild barley species (Hordeum brevisubulatum) using MSAP, SSAP and AFLP.

Science.gov (United States)

Shan, X H; Li, Y D; Liu, X M; Wu, Y; Zhang, M Z; Guo, W L; Liu, B; Yuan, Y P

2012-08-17

We analyzed genetic diversity and population genetic structure of four artificial populations of wild barley (Hordeum brevisubulatum); 96 plants collected from the Songnen Prairie in northeastern China were analyzed using amplified fragment length polymorphism (AFLP), specific-sequence amplified polymorphism (SSAP) and methylation-sensitive amplified polymorphism (MSAP) markers. Indices of (epi-)genetic diversity, (epi-)genetic distance, gene flow, genotype frequency, cluster analysis, PCA analysis and AMOVA analysis generated from MSAP, AFLP and SSAP markers had the same trend. We found a high level of correlation in the artificial populations between MSAP, SSAP and AFLP markers by the Mantel test (r > 0.8). This is incongruent with previous findings showing that there is virtually no correlation between DNA methylation polymorphism and classical genetic variation; the high level of genetic polymorphism could be a result of epigenetic regulation. We compared our results with data from natural populations. The population diversity of the artificial populations was lower. However, different from what was found using AFLP and SSAP, based on MSAP results the methylation polymorphism of the artificial populations was not significantly reduced. This leads us to suggest that the DNA methylation pattern change in H. brevisubulatum populations is not only related to DNA sequence variation, but is also regulated by other controlling systems.

Amplified-fragment length polymorphism fingerprinting of Mycoplasma species

DEFF Research Database (Denmark)

Kokotovic, Branko; Friis, N.F.; Jensen, J.S.

1999-01-01

Amplified-fragment length polymorphism (AFLP) is a whole-genome fingerprinting method based on selective amplification of restriction fragments. The potential of the method for the characterization of mycoplasmas was investigated in a total of 50 strains of human and animal origin, including...... Mycoplasma genitalium (n = 11), Mycoplasma pneumoniae (n = 5), Mycoplasma hominis (n = 5), Mycoplasma hyopneunmoniae (n = 9), Myco plasma flocculare (n = 5), Mycoplasma hyosynoviae (n = 10), and Mycoplasma dispar (n = 5), AFLP templates were prepared by the digestion of mycoplasmal DNA with BglII and Mfe...... to discriminate the analyzed strains at species and intraspecies levels as well, Each of the tested Mycoplasma species developed a banding pattern entirely different from those obtained from other species under analysis, Subtle intraspecies genomic differences were detected among strains of all of the Mycoplasma...

Genomic Relatedness of Chlamydia Isolates Determined by Amplified Fragment Length Polymorphism Analysis

OpenAIRE

Meijer, Adam; Morré, Servaas A.; Van Den Brule, Adriaan J. C.; Savelkoul, Paul H. M.; Ossewaarde, Jacobus M.

1999-01-01

The genomic relatedness of 19 Chlamydia pneumoniae isolates (17 from respiratory origin and 2 from atherosclerotic origin), 21 Chlamydia trachomatis isolates (all serovars from the human biovar, an isolate from the mouse biovar, and a porcine isolate), 6 Chlamydia psittaci isolates (5 avian isolates and 1 feline isolate), and 1 Chlamydia pecorum isolate was studied by analyzing genomic amplified fragment length polymorphism (AFLP) fingerprints. The AFLP procedure was adapted from a previously...

Resistor-less charge sensitive amplifier for semiconductor detectors

Energy Technology Data Exchange (ETDEWEB)

Pelczar, K., E-mail: krzysztof.pelczar@doctoral.uj.edu.pl; Panas, K.; Zuzel, G.

2016-11-01

A new concept of a Charge Sensitive Amplifier without a high-value resistor in the feedback loop is presented. Basic spectroscopic parameters of the amplifier coupled to a coaxial High Purity Germanium detector (HPGe) are discussed. The amplifier signal input is realized with an n-channel J-FET transistor. The feedback capacitor is discharged continuously by the second, forward biased n-channel J-FET, driven by an RC low–pass filter. Both the analog—with a standard spectroscopy amplifier and a multi-channel analyzer—and the digital—by applying a Flash Analog to Digital Converter—signal readouts were tested. The achieved resolution in the analog and the digital readouts was 0.17% and 0.21%, respectively, at the Full Width at Half Maximum of the registered {sup 60}Co 1332.5 keV gamma line.

Polymorphism and methylation of the MC4R gene in obese and non-obese dogs.

Science.gov (United States)

Mankowska, Monika; Nowacka-Woszuk, Joanna; Graczyk, Aneta; Ciazynska, Paulina; Stachowiak, Monika; Switonski, Marek

2017-08-01

The dog is considered to be a useful biomedical model for human diseases and disorders, including obesity. One of the numerous genes associated with human polygenic obesity is MC4R, encoding the melanocortin 4 receptor. The aim of our study was to analyze polymorphisms and methylation of the canine MC4R in relation to adiposity. Altogether 270 dogs representing four breeds predisposed to obesity: Labrador Retriever (n = 187), Golden Retriever (n = 38), Beagle (n = 28) and Cocker Spaniel (n = 17), were studied. The dogs were classified into three groups: lean, overweight and obese, according to the 5-point Body Condition Score (BCS) scale. In the cohort of Labradors a complete phenotypic data (age, sex, neutering status, body weight and BCS) were collected for 127 dogs. The entire coding sequence as well as 5' and 3'-flanking regions of the studied gene were sequenced and six polymorphic sites were reported. Genotype frequencies differed considerably between breeds and Labrador Retrievers appeared to be the less polymorphic. Moreover, distribution of some polymorphic variants differed significantly (P C, c.868C>T and c.*33C>G) and Beagles (c.-435T>C and c.637G>T). On the contrary, in Labradors no association between the studied polymorphisms and BCS or body weight was observed. Methylation analysis, using bisulfite DNA conversion followed by Sanger sequencing, was carried out for 12 dogs with BCS = 3 and 12 dogs with BCS = 5. Two intragenic CpG islands, containing 19 cytosines, were analyzed and the methylation profile did not differ significantly between lean and obese animals. We conclude that an association of the MC4R gene polymorphism with dog obesity or body weight is unlikely, in spite of the fact that some associations were found in small cohorts of Beagles and Golden Retrievers. Also methylation level of this gene is not related with dog adiposity.

Identification and DNA fingerprinting of Legionella strains by randomly amplified polymorphic DNA analysis.

OpenAIRE

Bansal, N S; McDonell, F

1997-01-01

The randomly amplified polymorphic DNA (RAPD) technique was used in the development of a fingerprinting (typing) and identification protocol for Legionella strains. Twenty decamer random oligonucleotide primers were screened for their discriminatory abilities. Two candidate primers were selected. By using a combination of these primers, RAPD analysis allowed for the differentiation between all different species, between the serogroups, and further differentiation between subtypes of the same ...

Evaluation of genetic diversity in jackfruit (Artocarpus heterophyllus Lam.) based on amplified fragment length polymorphism markers.

Science.gov (United States)

Shyamalamma, S; Chandra, S B C; Hegde, M; Naryanswamy, P

2008-07-22

Artocarpus heterophyllus Lam., commonly called jackfruit, is a medium-sized evergreen tree that bears high yields of the largest known edible fruit. Yet, it has been little explored commercially due to wide variation in fruit quality. The genetic diversity and genetic relatedness of 50 jackfruit accessions were studied using amplified fragment length polymorphism markers. Of 16 primer pairs evaluated, eight were selected for screening of genotypes based on the number and quality of polymorphic fragments produced. These primer combinations produced 5976 bands, 1267 (22%) of which were polymorphic. Among the jackfruit accessions, the similarity coefficient ranged from 0.137 to 0.978; the accessions also shared a large number of monomorphic fragments (78%). Cluster analysis and principal component analysis grouped all jackfruit genotypes into three major clusters. Cluster I included the genotypes grown in a jackfruit region of Karnataka, called Tamaka, with very dry conditions; cluster II contained the genotypes collected from locations having medium to heavy rainfall in Karnataka; cluster III grouped the genotypes in distant locations with different environmental conditions. Strong coincidence of these amplified fragment length polymorphism-based groupings with geographical localities as well as morphological characters was observed. We found moderate genetic diversity in these jackfruit accessions. This information should be useful for tree breeding programs, as part of our effort to popularize jackfruit as a commercial crop.

Meristem micropropagation of cassava (Manihot esculenta) evokes genome-wide changes in DNA methylation.

Science.gov (United States)

Kitimu, Shedrack R; Taylor, Julian; March, Timothy J; Tairo, Fred; Wilkinson, Mike J; Rodríguez López, Carlos M

2015-01-01

There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta) cultivars. Principal Co-ordinate Analysis of profiles generated by methylation-sensitive amplified polymorphism revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype by sequencing identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologs have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops.

Detection of differentially methylated regions of irradiated fig tree selections

Energy Technology Data Exchange (ETDEWEB)

Rodrigues, Maria Gabriela Fontanetti; Soares, Murilo Racy; Gimenez, Daniele Fernanda Jovino; Fonseca, Larissa Fernanda Simielli; Torrieri, Erico; Ramos, Ester Silveira; Giuliatti, Silvana, E-mail: mariagabriela@dracena.unesp.br [Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Dracena, SP (Brazil). Faculdade de Agricultura e Ciencias Tecnologicas; Universidade de Sao Paulo (FMRP/USP), Ribeirao Preto, SP (Brazil). Dept. de Genetica; Universidade Estadual Paulista Julio de Mesquita Filho (UNESP), Jaboticabal, SP (Brazil). Faculdade de Agricultura e Ciencias Veterinarias

2017-07-15

Fig tree (Ficus carica L.) breeding programs using conventional methods, such as directed crosses, to obtain new cultivars, are unworkable in many countries, including Brazil. Consequently, genetic breeding through mutagenesis has emerged as an important line of research that can improve this crop, and be a significant source of information about this species and assist in the implementation of propagation projects and appropriate management. The aim of this study was to verify the existence of epigenetic variability attributable to DNA methylation in irradiated fig selections when compared both to each other and to the main commercial cultivar, 'Roxo-de-Valinhos', which had previously used methylation-sensitive amplified polymorphism (MSAP) and DNA sequencing to detect the position of polymorphic regions, analyzable by bioinformatic tools. The sequencing of DNA, isolated from the differentially methylated sites, makes it possible to observe different patterns of methylation by sequencing the treated DNA with sodium bisulfite in the coding regions of regulatory genes active in the development, and fruit ripening stages. Furthermore, they have been found in the mitochondrial DNA of treatments which regulate the supply of energy in Adenosine triphosphate (ATP) form in plants. Closely related to their development, they justify the different phenotypes found in both fruit and plant growth that have suffered stress due to exposure to gamma radiation. Thus, future studies on gene expression in treatments have emerged as an extremely important strategy for understanding these complex regulatory systems, which may lead to the identification of genes of agricultural interest for the fig tree crop, and allow for manipulation and subsequent propagation of improved crops for commercial purposes. (author)

Detection of differentially methylated regions of irradiated fig tree selections

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Maria Gabriela Fontanetti Rodrigues

Full Text Available ABSTRACT Fig tree (Ficus carica L. breeding programs using conventional methods, such as directed crosses, to obtain new cultivars, are unworkable in many countries, including Brazil. Consequently, genetic breeding through mutagenesis has emerged as an important line of research that can improve this crop, and be a significant source of information about this species and assist in the implementation of propagation projects and appropriate management. The aim of this study was to verify the existence of epigenetic variability attributable to DNA methylation in irradiated fig selections when compared both to each other and to the main commercial cultivar, “Roxo-de-Valinhos”, which had previously used methylation-sensitive amplified polymorphism (MSAP and DNA sequencing to detect the position of polymorphic regions, analyzable by bioinformatic tools. The sequencing of DNA, isolated from the differentially methylated sites, makes it possible to observe different patterns of methylation by sequencing the treated DNA with sodium bisulfite in the coding regions of regulatory genes active in the development, and fruit ripening stages. Furthermore, they have been found in the mitochondrial DNA of treatments which regulate the supply of energy in Adenosine triphosphate (ATP form in plants. Closely related to their development, they justify the different phenotypes found in both fruit and plant growth that have suffered stress due to exposure to gamma radiation. Thus, future studies on gene expression in treatments have emerged as an extremely important strategy for understanding these complex regulatory systems, which may lead to the identification of genes of agricultural interest for the fig tree crop, and allow for manipulation and subsequent propagation of improved crops for commercial purposes.

Detection of differentially methylated regions of irradiated fig tree selections

International Nuclear Information System (INIS)

Rodrigues, Maria Gabriela Fontanetti; Soares, Murilo Racy; Gimenez, Daniele Fernanda Jovino; Fonseca, Larissa Fernanda Simielli; Torrieri, Erico; Ramos, Ester Silveira; Giuliatti, Silvana; Universidade de Sao Paulo; Universidade Estadual Paulista Julio de Mesquita Filho

2017-01-01

Fig tree (Ficus carica L.) breeding programs using conventional methods, such as directed crosses, to obtain new cultivars, are unworkable in many countries, including Brazil. Consequently, genetic breeding through mutagenesis has emerged as an important line of research that can improve this crop, and be a significant source of information about this species and assist in the implementation of propagation projects and appropriate management. The aim of this study was to verify the existence of epigenetic variability attributable to DNA methylation in irradiated fig selections when compared both to each other and to the main commercial cultivar, 'Roxo-de-Valinhos', which had previously used methylation-sensitive amplified polymorphism (MSAP) and DNA sequencing to detect the position of polymorphic regions, analyzable by bioinformatic tools. The sequencing of DNA, isolated from the differentially methylated sites, makes it possible to observe different patterns of methylation by sequencing the treated DNA with sodium bisulfite in the coding regions of regulatory genes active in the development, and fruit ripening stages. Furthermore, they have been found in the mitochondrial DNA of treatments which regulate the supply of energy in Adenosine triphosphate (ATP) form in plants. Closely related to their development, they justify the different phenotypes found in both fruit and plant growth that have suffered stress due to exposure to gamma radiation. Thus, future studies on gene expression in treatments have emerged as an extremely important strategy for understanding these complex regulatory systems, which may lead to the identification of genes of agricultural interest for the fig tree crop, and allow for manipulation and subsequent propagation of improved crops for commercial purposes. (author)

Combination of methylated-DNA precipitation and methylation-sensitive restriction enzymes (COMPARE-MS) for the rapid, sensitive and quantitative detection of DNA methylation.

Science.gov (United States)

Yegnasubramanian, Srinivasan; Lin, Xiaohui; Haffner, Michael C; DeMarzo, Angelo M; Nelson, William G

2006-02-09

Hypermethylation of CpG island (CGI) sequences is a nearly universal somatic genome alteration in cancer. Rapid and sensitive detection of DNA hypermethylation would aid in cancer diagnosis and risk stratification. We present a novel technique, called COMPARE-MS, that can rapidly and quantitatively detect CGI hypermethylation with high sensitivity and specificity in hundreds of samples simultaneously. To quantitate CGI hypermethylation, COMPARE-MS uses real-time PCR of DNA that was first digested by methylation-sensitive restriction enzymes and then precipitated by methyl-binding domain polypeptides immobilized on a magnetic solid matrix. We show that COMPARE-MS could detect five genome equivalents of methylated CGIs in a 1000- to 10,000-fold excess of unmethylated DNA. COMPARE-MS was used to rapidly quantitate hypermethylation at multiple CGIs in >155 prostate tissues, including benign and malignant prostate specimens, and prostate cell lines. This analysis showed that GSTP1, MDR1 and PTGS2 CGI hypermethylation as determined by COMPARE-MS could differentiate between malignant and benign prostate with sensitivities >95% and specificities approaching 100%. This novel technology could significantly improve our ability to detect CGI hypermethylation.

High sensitivity amplifier/discriminator for PWC's

International Nuclear Information System (INIS)

Hansen, S.

1983-01-01

The facility support group at Fermilab is designing and building a general purpose beam chamber for use in several locations at the laboratory. This pwc has 128 wires per plane spaced 1 mm apart. An initial production of 25 signal planes is anticipated. In proportional chambers, the size of the signal depends exponentially on the charge stored per unit of length along the anode wire. As the wire spacing decreases, the capacitance per unit length decreases, thereby requiring increased applied voltage to restore the necessary charge per unit length. In practical terms, this phenomenon is responsible for difficulties in constructing chambers with less than 2 mm wire spacing. 1 mm chambers, therefore, are frequently operated very near to their breakdown point and/or a high gain gas containing organic compounds such as magic gas is used. This argon/iso-butane mixture has three drawbacks: it is explosive when exposed to the air, it leaves a residue on the wires after extended use and is costly. An amplifier with higher sensitivity would reduce the problems associated with operating chambers with small wire spacings and allow them to be run a safe margin below their breakdown voltage even with an inorganic gas mixture such as argon/CO2, this eliminating the need to use magic gas. Described here is a low cost amplifier with a usable threshold of less than 0.5 μA. Data on the performance of this amplifier/discriminator in operation on a prototype beam chamber are given. This data shows the advantages of the high sensitivity of this design

Application of fluorescent amplified fragment length polymorphism for comparison of human and animal isolates of Yersinia enterocolitica

DEFF Research Database (Denmark)

Fearnley, C.; On, S.L.W.; Kokotovic, Branko

2005-01-01

An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y enterocolitica strains according...

The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis.

Science.gov (United States)

Wang, Dan; Zhao, Jieyu; Bai, Yan; Ao, You; Guo, Changhong

2017-08-10

Gametocidal (Gc) chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP) technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS-3C and CS-3C3C, 48.68% and 48.65%, respectively) were significantly increased when compared to common wheat CS (41.31%) and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted-repeat transposable elements), LTR-retrotransposon WIS-1p and retrotransposon Gypsy , respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.

The Variation Analysis of DNA Methylation in Wheat Carrying Gametocidal Chromosome 3C from Aegilops triuncialis

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Dan Wang

2017-08-01

Full Text Available Gametocidal (Gc chromosomes can ensure their preferential transmission by killing the gametes without themselves through causing chromosome breakage and therefore have been exploited as an effective tool for genetic breeding. However, to date very little is known about the molecular mechanism of Gc action. In this study, we used methylation-sensitive amplified polymorphism (MSAP technique to assess the extent and pattern of cytosine methylation alterations at the whole genome level between two lines of wheat Gc addition line and their common wheat parent. The results indicated that the overall levels of cytosine methylation of two studied Gc addition lines (CS–3C and CS–3C3C, 48.68% and 48.65%, respectively were significantly increased when compared to common wheat CS (41.31% and no matter fully methylated or hemimethylated rates enhanced in Gc addition lines. A set of 30 isolated fragments that showed different DNA methylation or demethylation patterns between the three lines were sequenced and the results indicated that 8 fragments showed significant homology to known sequences, of which three were homologous to MITE transposon (Miniature inverted–repeat transposable elements, LTR-retrotransposon WIS-1p and retrotransposon Gypsy, respectively. Overall, our results showed that DNA methylation could play a role in the Gc action.

DNA methylation and genetic diversity analysis of genus Cycas in ...

African Journals Online (AJOL)

10 Cycas species as well as one subspecies localized in Thailand were studied using the methylation sensitive amplification polymorphism (MSAP) technique. 11 MSAP primer combinations were used and 720 MSAP bands were generated. The percentages of DNA methylation estimated from MSAP fingerprints were in ...

Genetic diversity of edible mushroom pleurotus spp. revealed by randomly amplified polymorphic dna fingerprinting

International Nuclear Information System (INIS)

Khan, N. A.; Awan, F. S.; Khan, A. I.; Waseem, M.

2017-01-01

The Oyster mushroom (Pleurotus) cultivation is a profitable agribusiness and having high significance due its nutritive and therapeutic value. Due to deficient knowledge on Pleurotus mushroom genetics seven strains of Oyster mushroom, two local and five exotic were studied for their genetic diversity through RAPD markers. It was clear from similarity matrix that similarity index ranges from 45 to 72%. The cluster analysis of combined data set of all the markers resulted in three major clades, while isolate P-17 remains ungrouped and shown to be the most diverse strain of the seven. During amplification of genomic DNA yielded 70 fragments that could be scored, of which 41 were polymorphic, with an average of 2.73 polymorphic fragments per primer. Number of amplified fragments with random primers ranged from three to six. Polymorphism ranged from 0% to 83.33%, with an overall 58% polymorphism. The allele frequency of RAPD primers ranged from 0.71 to 1.00 while the polymorphic information content highest for the primer GL-C-20 (0.29) followed by the primers GL A-20 and GL C-16 that is zero, indicating medium level of polymorphism among the strains of Oyster mushroom. The objective of the study was to characterize Pleurotus strains collected from different origins and to find out the variability at molecular level. (author)

Amplified Fragment Length Polymorphism Diversity in Cephalosporium maydis from Egypt.

Science.gov (United States)

Saleh, Amgad A; Zeller, Kurt A; Ismael, Abou-Serie M; Fahmy, Zeinab M; El-Assiuty, Elhamy M; Leslie, John F

2003-07-01

ABSTRACT Cephalosporium maydis, the causal agent of late wilt of maize, was first described in Egypt in the 1960s, where it can cause yield losses of up to 40% in susceptible plantings. We characterized 866 isolates of C. maydis collected from 14 governates in Egypt, 7 in the Nile River Delta and 7 in southern (Middle and Upper) Egypt, with amplified fragment length polymorphism (AFLP) markers. The four AFLP primer-pair combinations generated 68 bands, 25 of which were polymorphic, resulting in 52 clonal haplotypes that clustered the 866 isolates into four phylogenetic lineages. Three lineages were found in both the Nile River Delta and southern Egypt. Lineage IV, the most diverse group (20 haplotypes), was recovered only from governates in the Nile River Delta. In some locations, one lineage dominated (up to 98% of the isolates recovered) and, from some fields, only a single haplotype was recovered. Under field conditions in Egypt, there is no evidence that C. maydis reproduces sexually. The nonuniform geographic distribution of the pathogen lineages within the country could be due to differences in climate or in the farming system, because host material differs in susceptibility and C. maydis lineages differ in pathogenicity.

Genome-wide methylation study of diploid and triploid brown trout (Salmo trutta L.).

Science.gov (United States)

Covelo-Soto, L; Leunda, P M; Pérez-Figueroa, A; Morán, P

2015-06-01

The induction of triploidization in fish is a very common practice in aquaculture. Although triploidization has been applied successfully in many salmonid species, little is known about the epigenetic mechanisms implicated in the maintenance of the normal functions of the new polyploid genome. By means of methylation-sensitive amplified polymorphism (MSAP) techniques, genome-wide methylation changes associated with triploidization were assessed in DNA samples obtained from diploid and triploid siblings of brown trout (Salmo trutta). Simple comparative body measurements showed that the triploid trout used in the study were statistically bigger, however, not heavier than their diploid counterparts. The statistical analysis of the MSAP data showed no significant differences between diploid and triploid brown trout in respect to brain, gill, heart, liver, kidney or muscle samples. Nonetheless, local analysis pointed to the possibility of differences in connection with concrete loci. This is the first study that has investigated DNA methylation alterations associated with triploidization in brown trout. Our results set the basis for new studies to be undertaken and provide a new approach concerning triploidization effects of the salmonid genome while also contributing to the better understanding of the genome-wide methylation processes. © 2015 Stichting International Foundation for Animal Genetics.

Epigenetic variability in the genetically uniform forest tree species Pinus pinea L.

Science.gov (United States)

Sáez-Laguna, Enrique; Guevara, María-Ángeles; Díaz, Luis-Manuel; Sánchez-Gómez, David; Collada, Carmen; Aranda, Ismael; Cervera, María-Teresa

2014-01-01

There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP) technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments). Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP) data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.

Epigenetic variability in the genetically uniform forest tree species Pinus pinea L.

Directory of Open Access Journals (Sweden)

Enrique Sáez-Laguna

Full Text Available There is an increasing interest in understanding the role of epigenetic variability in forest species and how it may contribute to their rapid adaptation to changing environments. In this study we have conducted a genome-wide analysis of cytosine methylation pattern in Pinus pinea, a species characterized by very low levels of genetic variation and a remarkable degree of phenotypic plasticity. DNA methylation profiles of different vegetatively propagated trees from representative natural Spanish populations of P. pinea were analyzed with the Methylation Sensitive Amplified Polymorphism (MSAP technique. A high degree of cytosine methylation was detected (64.36% of all scored DNA fragments. Furthermore, high levels of epigenetic variation were observed among the studied individuals. This high epigenetic variation found in P. pinea contrasted with the lack of genetic variation based on Amplified Fragment Length Polymorphism (AFLP data. In this manner, variable epigenetic markers clearly discriminate individuals and differentiates two well represented populations while the lack of genetic variation revealed with the AFLP markers fail to differentiate at both, individual or population levels. In addition, the use of different replicated trees allowed identifying common polymorphic methylation sensitive MSAP markers among replicates of a given propagated tree. This set of MSAPs allowed discrimination of the 70% of the analyzed trees.

Genetic and epigenetic status of triple exotic consanguinity cotton introgression lines.

Science.gov (United States)

He, S P; Sun, J L; Du, X M

2011-10-03

Introgression lines are some of the most important germplasm for breeding applications and other research conducted on cotton crops. The DNA methylation level among 10 introgression lines of cotton (Gossypium hirsutum) and three exotic parental species (G. arboreum, G. thurberi and G. barbadense) were assessed by methylation-sensitive amplified polymorphism (MSAP) technology. The methylation level in the introgression lines ranged from 33.3 to 51.5%. However, the lines PD0111 and PD0113 had the lowest methylation level (34.6 and 33.3%, respectively) due to demethylation of most non-coding sequences. Amplified fragment length polymorphism (AFLP) was used to evaluate the genetic polymorphism in the cotton introgression lines. A high degree of polymorphism was observed in all introgression lines (mean 47.2%) based on AFLP and MSAP analyses. This confirmed the effects of genetic improvement on cotton introgression lines. The low methylation varieties, PD0111 and PD0113 (introgression lines), clustered outside of the introgression lines based on MSAP data, which was incongruent with an AFLP-based dendrogram. This phenomenon could be caused by environmental changes or introgression of exotic DNA fragments.

Meristem micropropagation of cassava (Manihot esculenta evokes genome-wide changes in DNA methylation

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Shedrack Reuben Kitimu

2015-08-01

Full Text Available There is great interest in the phenotypic, genetic and epigenetic changes associated with plant in vitro culture known as somaclonal variation. In vitro propagation systems that are based on the use of microcuttings or meristem cultures are considered analogous to clonal cuttings and so widely viewed to be largely free from such somaclonal effects. In this study, we surveyed for epigenetic changes during propagation by meristem culture and by field cuttings in five cassava (Manihot esculenta cultivars. Principal Co-ordinate Analysis of profiles generated by Methylation Sensitive Amplified Polymorphism (MSAP revealed clear divergence between samples taken from field-grown cuttings and those recovered from meristem culture. There was also good separation between the tissues of field samples but this effect was less distinct among the meristem culture materials. Application of methylation-sensitive Genotype By Sequencing (msGBS identified 105 candidate epimarks that distinguish between field cutting and meristem culture samples. Cross referencing the sequences of these epimarks to the draft cassava genome revealed 102 sites associated with genes whose homologues have been implicated in a range of fundamental biological processes including cell differentiation, development, sugar metabolism, DNA methylation, stress response, photosynthesis, and transposon activation. We explore the relevance of these findings for the selection of micropropagation systems for use on this and other crops.

Modeling of Yb3+-sensitized Er3+-doped silica waveguide amplifiers

DEFF Research Database (Denmark)

Lester, Christian; Bjarklev, Anders Overgaard; Rasmussen, Thomas

1995-01-01

A model for Yb3+-sensitized Er3+-doped silica waveguide amplifiers is described and numerically investigated in the small-signal regime. The amplified spontaneous emission in the ytterbium-band and the quenching process between excited erbium ions are included in the model. For pump wavelengths...

Random amplified polymorphic DNA markers of the Brassica alboglabra chromosome of a B. campestris-alboglabra addition line

DEFF Research Database (Denmark)

Bagger Jørgensen, Rikke; Chen, B.Y.; Cheng, B.F.

1996-01-01

The alien C-genome chromosome in a Brassica campestris-alboglabra monosomic addition line was characterized by random amplified polymorphic DNA (RAPD) analysis. The alien chromosome carried three loci, E(c), W-c and Lap-1C, controlling synthesis of erucic acid, white flower colour and a fast...

Genomic relations among 31 species of Mammillaria haworth (Cactaceae) using random amplified polymorphic DNA.

Science.gov (United States)

Mattagajasingh, Ilwola; Mukherjee, Arup Kumar; Das, Premananda

2006-01-01

Thirty-one species of Mammillaria were selected to study the molecular phylogeny using random amplified polymorphic DNA (RAPD) markers. High amount of mucilage (gelling polysaccharides) present in Mammillaria was a major obstacle in isolating good quality genomic DNA. The CTAB (cetyl trimethyl ammonium bromide) method was modified to obtain good quality genomic DNA. Twenty-two random decamer primers resulted in 621 bands, all of which were polymorphic. The similarity matrix value varied from 0.109 to 0.622 indicating wide variability among the studied species. The dendrogram obtained from the unweighted pair group method using arithmetic averages (UPGMA) analysis revealed that some of the species did not follow the conventional classification. The present work shows the usefulness of RAPD markers for genetic characterization to establish phylogenetic relations among Mammillaria species.

The role of DNA methylation on Octopus vulgaris development and their perspectives

Directory of Open Access Journals (Sweden)

Eva eDíaz-Freije

2014-02-01

Full Text Available 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 change 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 any significant effect was revealed in the AMOVA performed. Our observations highlight the importance of epigenetic mechanism in the first developmental steps of the common octopus and open 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 aquaculture.

Epigenetic differentiation and relationship to adaptive genetic divergence in discrete populations of the violet Viola cazorlensis.

Science.gov (United States)

Herrera, Carlos M; Bazaga, Pilar

2010-08-01

*In plants, epigenetic variations based on DNA methylation are often heritable and could influence the course of evolution. Before this hypothesis can be assessed, fundamental questions about epigenetic variation remain to be addressed in a real-world context, including its magnitude, structuring within and among natural populations, and autonomy in relation to the genetic context. *Extent and patterns of cytosine methylation, and the relationship to adaptive genetic divergence between populations, were investigated for wild populations of the southern Spanish violet Viola cazorlensis (Violaceae) using the methylation-sensitive amplified polymorphism (MSAP) technique, a modification of the amplified fragment length polymorphism method (AFLP) based on the differential sensitivity of isoschizomeric restriction enzymes to site-specific cytosine methylation. *The genome of V. cazorlensis plants exhibited extensive levels of methylation, and methylation-based epigenetic variation was structured into distinct between- and within- population components. Epigenetic differentiation of populations was correlated with adaptive genetic divergence revealed by a Bayesian population-genomic analysis of AFLP data. Significant associations existed at the individual genome level between adaptive AFLP loci and the methylation state of methylation-susceptible MSAP loci. *Population-specific, divergent patterns of correlated selection on epigenetic and genetic individual variation could account for the coordinated epigenetic-genetic adaptive population differentiation revealed by this study.

Charge sensitive amplifies. The state of arts

Energy Technology Data Exchange (ETDEWEB)

Mori, Kunishiro [Clear Pulse Co., Tokyo (Japan)

1996-07-01

In the radiation detectors, signals are essentially brought with charges produced by radiation, then it is naturally the best way to use a charge sensitive amplifier (CSA) system to extract those signals. The CSA is thought to be the best amplifier suitable to almost all the radiation detectors, if neglecting economical points of view. The CSA has been only applied to special fields like radiation detection because the concept of `charges` is not so universal against the concepts of `voltage` and `current`. The CSA, however, is low in noise and a high speed amplifier and may be applicable not only to radiation measurement but also piezoelectric devices and also bolometers. In this article, noise in the CSA, basic circuit on the CSA, concepts of `equivalent noise charge` (ENC), a method for the ENC, and importance of the `open-loop gain` in the CSA to achieve better performance of it and how to realize in a practical CSA were described. And, characteristics on a counting rate of the CSA, various circuit used in the CSA, and CSAs which are commercially available at present and special purpose CSAs were also introduced. (G.K.)

A low noise charge sensitive amplifier for use in vacuum photo diode readout

International Nuclear Information System (INIS)

Stephenson, R.

1982-08-01

The amplifier described consists of a charge sensitive pre-amplifier optimised for low noise with low values of input shunt capacitance, and a shaping amplifier providing both differentiation and integration. Amplifier gain is adjustable up to a maximum of approximately 100 μV/electron with a rise time of 2 μS to the peak of the output voltage, and with an open circuit input noise level of 150 electrons RMS. (author)

Optical Sensitivity of a Monolithic Integrated InP PIN-HEMT-HBT Transimpedance Amplifier

OpenAIRE

Matiss, A.; Janssen, G.; Bertenburg, R. M.; Brockerhoff, W.; Tegude, F.J.

2004-01-01

To improve sensitivity of optical receivers, a special integration concept is chosen that includes a pinphotodiode, high-electron mobility transistors (HEMT) and heterostructure bipolar transistors (HBT) on a single substrate. This work focuses on the optimization of the amplifier design to achieve lowest input noise currents of a transimpedance amplifier, and thus highest receiver sensitivity. The respective advantages of the components used are investigated with respect...

AFLP and AMP Fingerprints as Markers to Evaluate Genetic Differences between Medicago truncatula Line Jemalong and 2HA, a New Line Produced by in vitro Culture Selection

Directory of Open Access Journals (Sweden)

R.R. Irwanto

2008-09-01

Full Text Available A new line, Medicago truncatula cv. Jemalong 2HA (herein known as 2HA has been developed via repetitive regeneration and selection of M. truncatula cv. Jemalong. During somatic embryogenesis, 2HA produces 500 times more embryos than its progenitor, Jemalong. It is interesting to study if those two lines are isogenic or has genetic differences. The main objectives of the study was to evaluate the genotypic differences between Jemalong and 2HA also to evaluate the methylation event in 2HA utilized two DNA fingerprinting techniques, i.e AFLP fingerprints (Amplified Length of Polymorphism and AMP (Amplified Methylation Polymorphism. The results showed that AFLP analysis using eight primers combinations could not detect any differences between Jemalong and 2HA. However, using AMP methylation sensitive primers it could detect 15 polymorphisms out of 840 markers. These results lead to a conclusion that Jemalong and 2HA are isogenic lines. 2HA may have higher regeneration capacities due to methylation process which occurs during the production of 2HA through repetitive regeneration cycles.

Genetic polymorphisms in one-carbon metabolism: associations with CpG island methylator phenotype (CIMP) in colon cancer and the modifying effects of diet.

Science.gov (United States)

Curtin, Karen; Slattery, Martha L; Ulrich, Cornelia M; Bigler, Jeannette; Levin, Theodore R; Wolff, Roger K; Albertsen, Hans; Potter, John D; Samowitz, Wade S

2007-08-01

This study investigated associations between CpG island methylator phenotype (CIMP) colon cancer and genetic polymorphisms relevant to one-carbon metabolism and thus, potentially the provision of methyl groups and risk of colon cancer. Data from a large, population-based case-control study (916 incident colon cancer cases and 1,972 matched controls) were used. Candidate polymorphisms in methylenetetrahydrofolate reductase (MTHFR), thymidylate synthase (TS), transcobalamin II (TCNII), methionine synthase (MTR), reduced folate carrier (RFC), methylenetetrahydrofolate dehydrogenase 1 (MTHFD1), dihydrofolate reductase (DHFR) and alcohol dehydrogenase 3 (ADH3) were evaluated. CIMP- or CIMP+ phenotype was based on five CpG island markers: MINT1, MINT2, MINT31, p16 and MLH1. The influence of specific dietary factors (folate, methionine, vitamin B(12) and alcohol) on these associations was also analyzed. We hypothesized that polymorphisms involved in the provision of methyl groups would be associated with CIMP+ tumors (two or more of five markers methylated), potentially modified by diet. Few associations specific to CIMP+ tumors were observed overall, which does not support the hypothesis that the provision of methyl groups is important in defining a methylator phenotype. However, our data suggest that genetic polymorphisms in MTHFR 1,298A > C, interacting with diet, may be involved in the development of highly CpG-methylated colon cancers. AC and CC genotypes in conjunction with a high-risk dietary pattern (low folate and methionine intake and high alcohol use) were associated with CIMP+ (OR = 2.1, 95% CI = 1.3-3.4 versus AA/high risk; P-interaction = 0.03). These results provide only limited support for a role of polymorphisms in one-carbon metabolism in the etiology of CIMP colon cancer.

Active rc filter permits easy trade-off of amplifier gain and sensitivity to gain

Science.gov (United States)

Kerwin, W. J.; Shaffer, C. V.

1968-01-01

Passive RC network was designed with zeros of transmission in the right half of the complex frequency plane in the feedback loop of a simple negative-gain amplifier. The proper positioning provides any desired trade-off between amplifier gain and sensitivity to amplifier gain.

Does epigenetic polymorphism contribute to phenotypic variances in Jatropha curcas L.?

Directory of Open Access Journals (Sweden)

Bui Ha TN

2010-11-01

Full Text Available Abstract Background There is a growing interest in Jatropha curcas L. (jatropha as a biodiesel feedstock plant. Variations in its morphology and seed productivity have been well documented. However, there is the lack of systematic comparative evaluation of distinct collections under same climate and agronomic practices. With the several reports on low genetic diversity in jatropha collections, there is uncertainty on genetic contribution to jatropha morphology. Result In this study, five populations of jatropha plants collected from China (CN, Indonesia (MD, Suriname (SU, Tanzania (AF and India (TN were planted in one farm under the same agronomic practices. Their agronomic traits (branching pattern, height, diameter of canopy, time to first flowering, dormancy, accumulated seed yield and oil content were observed and tracked for two years. Significant variations were found for all the agronomic traits studied. Genetic diversity and epigenetic diversity were evaluated using florescence Amplified Fragment Length Polymorphism (fAFLP and methylation sensitive florescence AFLP (MfAFLP methods. Very low level of genetic diversity was detected (polymorphic band Conclusion Our study confirmed climate and practice independent differences in agronomic performance among jatropha collections. Such agronomic trait variations, however, were matched by very low genetic diversity and medium level but significant epigenetic diversity. Significant difference in inner cytosine and double cytosine methylation at CCGG sites was also found among populations. Most epigenetic differential markers can be inherited as epialleles following Mendelian segregation. These results suggest possible involvement of epigenetics in jatropha development.

Reset charge sensitive amplifier for NaI(Tl) gamma-ray spectrometer

International Nuclear Information System (INIS)

Zeng, Guoqiang; Tan, Chengjun; Li, Qiang; Ge, Liangquan; Liu, Xiyao; Luo, Qun

2015-01-01

The time constant of the output signal of the front-end readout circuit of a traditional gamma-ray spectrometer with a NaI(Tl)+PMT structure is affected by temperature, measurement environment and the signal transmission cable, so it is difficult to get a good resolution spectrum, especially at higher counting rates. In this paper, a reset charge sensitive amplifier (RCSA) is designed for the gamma-ray spectrometer with a NaI(Tl)+PMT structure. The designed RCSA outputs a step signal, thus enabling the acquisition of double-exponential signals with a stable time constant by using the next stage of a CR differentiating circuit. The designed RCSA is mainly composed of a basic amplifying circuit, a reset circuit and a dark current compensation circuit. It provides the output step signal through the integration of the PMT output charge signal. When the amplitude of the step signal exceeds a preset voltage threshold, it triggers the reset circuit to generate a reset pulse (about 5 µs pulse width) to reset the output signal. Experimental results demonstrated that the designed RCSA achieves a charge sensitivity of 4.26×10 10 V/C, with a zero capacitance noise of 51.09 fC and a noise slope of 1.98 fC/pF. Supported by the digital shaping algorithm of the digital multi-channel analyzer (DMCA), it can maintain good energy resolution with high counting rates up to 150 kcps and with a temperature range from −19 °C to 50 °C. - Highlights: • A new reset type charge sensitive amplifier for gamma-ray spectrometer based on a photomultiplier tube is proposed. • Reset circuit formed by constant current source output a fixed width pulse to reset charge sensitive amplifier. • Photomultiplier tube dark current compensation circuit could increase the pulse through rate by decreasing reset frequency. • This amplifier outputs a step function signal that could match next stage circuit easily

Higher-order organisation of extremely amplified, potentially functional and massively methylated 5S rDNA in European pikes (Esox sp.).

Science.gov (United States)

Symonová, Radka; Ocalewicz, Konrad; Kirtiklis, Lech; Delmastro, Giovanni Battista; Pelikánová, Šárka; Garcia, Sonia; Kovařík, Aleš

2017-05-18

Pikes represent an important genus (Esox) harbouring a pre-duplication karyotype (2n = 2x = 50) of economically important salmonid pseudopolyploids. Here, we have characterized the 5S ribosomal RNA genes (rDNA) in Esox lucius and its closely related E. cisalpinus using cytogenetic, molecular and genomic approaches. Intragenomic homogeneity and copy number estimation was carried out using Illumina reads. The higher-order structure of rDNA arrays was investigated by the analysis of long PacBio reads. Position of loci on chromosomes was determined by FISH. DNA methylation was analysed by methylation-sensitive restriction enzymes. The 5S rDNA loci occupy exclusively (peri)centromeric regions on 30-38 acrocentric chromosomes in both E. lucius and E. cisalpinus. The large number of loci is accompanied by extreme amplification of genes (>20,000 copies), which is to the best of our knowledge one of the highest copy number of rRNA genes in animals ever reported. Conserved secondary structures of predicted 5S rRNAs indicate that most of the amplified genes are potentially functional. Only few SNPs were found in genic regions indicating their high homogeneity while intergenic spacers were more heterogeneous and several families were identified. Analysis of 10-30 kb-long molecules sequenced by the PacBio technology (containing about 40% of total 5S rDNA) revealed that the vast majority (96%) of genes are organised in large several kilobase-long blocks. Dispersed genes or short tandems were less common (4%). The adjacent 5S blocks were directly linked, separated by intervening DNA and even inverted. The 5S units differing in the intergenic spacers formed both homogeneous and heterogeneous (mixed) blocks indicating variable degree of homogenisation between the loci. Both E. lucius and E. cisalpinus 5S rDNA was heavily methylated at CG dinucleotides. Extreme amplification of 5S rRNA genes in the Esox genome occurred in the absence of significant pseudogenisation

Extinction Ratio and Gain Optimization of Dual- Pump Degenerate-Idler Phase Sensitive Amplifiers

DEFF Research Database (Denmark)

Kang, Ning; Lund-Hansen, Toke; Seoane, Jorge

2011-01-01

Numerical optimization of dual-pump degenerateidler phase sensitive amplifiers is performed for Al-doped and standard highly nonlinear fibers. Design considerations for operating the PSAs at an optimum combination of gain and extinction ratio are discussed.......Numerical optimization of dual-pump degenerateidler phase sensitive amplifiers is performed for Al-doped and standard highly nonlinear fibers. Design considerations for operating the PSAs at an optimum combination of gain and extinction ratio are discussed....

Charge-sensitive poly-silicon TFT amplifiers for a-Si:H pixel particle detectors

International Nuclear Information System (INIS)

Cho, G.; Perez-Mendez, V.; Hack, M.; Lewis, A.

1992-04-01

Prototype charge-sensitive poly-Si TFT amplifiers have been made for the amplification of signals (from an a-Si:H pixel diode used as an ionizing particle detector). They consist of a charge-sensitive gain stage, a voltage gain stage and a source follower output stage. The gain-bandwidth product of the amplifier is ∼ 300 MHz. When the amplifier is connected to a pixel detector of 0.2 pF, it gives a charge-to-voltage gain of ∼ 0.02 mV/electrons with a pulse rise time less than 100 nsec. An equivalent noise charge of the front-end TFT is ∼ 1000 electrons for a shaping time of 1 μsec

Glucose Tolerance, MTHFR C677T and NOS3 G894T Polymorphisms, and Global DNA Methylation in Mixed Ancestry African Individuals

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Tandi E. Matsha

2016-01-01

Full Text Available The aim of this study is to quantify global DNA methylation and investigate the relationship with diabetes status and polymorphisms in MTHFR C677T and NOS3 G894T genes in mixed ancestry subjects from South Africa. Global DNA methylation was measured, and MTHFR rs1801133 and NOS3 rs1799983 polymorphisms were genotyped using high throughput real-time polymerase chain reaction and direct DNA sequencing. Of the 564 participants, 158 (28% individuals had T2DM of which 97 (17.2% were screen-detected cases. Another 119 (21.1% had prediabetes, that is, impaired fasting glucose, impaired glucose tolerance, or the combination of both, and the remainder 287 (50.9% had normal glucose tolerance. Global DNA methylation was significantly higher in prediabetes and screen-detected diabetes than in normal glucose tolerance (both p≤0.033 and in screen-detected diabetes compared to known diabetes on treatment (p=0.019. There was no difference in global DNA methylation between known diabetes on treatment and normal glucose tolerance (p>0.999. In multivariable linear regression analysis, only NOS3 was associated with increasing global DNA methylation (β=0.943; 95% CI: 0.286 to 1.560. The association of global DNA methylation with screen-detected diabetes but not treated diabetes suggests that glucose control agents to some extent may be reversing DNA methylation. The association between NOS3 rs1799983 polymorphisms and DNA methylation suggests gene-epigenetic mechanisms through which vascular diabetes complications develop despite adequate metabolic control.

Glucose Tolerance, MTHFR C677T and NOS3 G894T Polymorphisms, and Global DNA Methylation in Mixed Ancestry African Individuals

Science.gov (United States)

Mutize, Tinashe; Erasmus, Rajiv T.

2016-01-01

The aim of this study is to quantify global DNA methylation and investigate the relationship with diabetes status and polymorphisms in MTHFR C677T and NOS3 G894T genes in mixed ancestry subjects from South Africa. Global DNA methylation was measured, and MTHFR rs1801133 and NOS3 rs1799983 polymorphisms were genotyped using high throughput real-time polymerase chain reaction and direct DNA sequencing. Of the 564 participants, 158 (28%) individuals had T2DM of which 97 (17.2%) were screen-detected cases. Another 119 (21.1%) had prediabetes, that is, impaired fasting glucose, impaired glucose tolerance, or the combination of both, and the remainder 287 (50.9%) had normal glucose tolerance. Global DNA methylation was significantly higher in prediabetes and screen-detected diabetes than in normal glucose tolerance (both p ≤ 0.033) and in screen-detected diabetes compared to known diabetes on treatment (p = 0.019). There was no difference in global DNA methylation between known diabetes on treatment and normal glucose tolerance (p > 0.999). In multivariable linear regression analysis, only NOS3 was associated with increasing global DNA methylation (β = 0.943; 95% CI: 0.286 to 1.560). The association of global DNA methylation with screen-detected diabetes but not treated diabetes suggests that glucose control agents to some extent may be reversing DNA methylation. The association between NOS3 rs1799983 polymorphisms and DNA methylation suggests gene-epigenetic mechanisms through which vascular diabetes complications develop despite adequate metabolic control. PMID:27990443

msgbsR: An R package for analysing methylation-sensitive restriction enzyme sequencing data.

Science.gov (United States)

Mayne, Benjamin T; Leemaqz, Shalem Y; Buckberry, Sam; Rodriguez Lopez, Carlos M; Roberts, Claire T; Bianco-Miotto, Tina; Breen, James

2018-02-01

Genotyping-by-sequencing (GBS) or restriction-site associated DNA marker sequencing (RAD-seq) is a practical and cost-effective method for analysing large genomes from high diversity species. This method of sequencing, coupled with methylation-sensitive enzymes (often referred to as methylation-sensitive restriction enzyme sequencing or MRE-seq), is an effective tool to study DNA methylation in parts of the genome that are inaccessible in other sequencing techniques or are not annotated in microarray technologies. Current software tools do not fulfil all methylation-sensitive restriction sequencing assays for determining differences in DNA methylation between samples. To fill this computational need, we present msgbsR, an R package that contains tools for the analysis of methylation-sensitive restriction enzyme sequencing experiments. msgbsR can be used to identify and quantify read counts at methylated sites directly from alignment files (BAM files) and enables verification of restriction enzyme cut sites with the correct recognition sequence of the individual enzyme. In addition, msgbsR assesses DNA methylation based on read coverage, similar to RNA sequencing experiments, rather than methylation proportion and is a useful tool in analysing differential methylation on large populations. The package is fully documented and available freely online as a Bioconductor package ( https://bioconductor.org/packages/release/bioc/html/msgbsR.html ).

Genetic analysis of 430 Chinese Cynodon dactylon accessions using sequence-related amplified polymorphism markers.

Science.gov (United States)

Huang, Chunqiong; Liu, Guodao; Bai, Changjun; Wang, Wenqiang

2014-10-21

Although Cynodon dactylon (C. dactylon) is widely distributed in China, information on its genetic diversity within the germplasm pool is limited. The objective of this study was to reveal the genetic variation and relationships of 430 C. dactylon accessions collected from 22 Chinese provinces using sequence-related amplified polymorphism (SRAP) markers. Fifteen primer pairs were used to amplify specific C. dactylon genomic sequences. A total of 481 SRAP fragments were generated, with fragment sizes ranging from 260-1800 base pairs (bp). Genetic similarity coefficients (GSC) among the 430 accessions averaged 0.72 and ranged from 0.53-0.96. Cluster analysis conducted by two methods, namely the unweighted pair-group method with arithmetic averages (UPGMA) and principle coordinate analysis (PCoA), separated the accessions into eight distinct groups. Our findings verify that Chinese C. dactylon germplasms have rich genetic diversity, which is an excellent basis for C. dactylon breeding for new cultivars.

Methylation changes associated with early maturation stages in the Atlantic salmon

Directory of Open Access Journals (Sweden)

Pérez-Figueroa Andrés

2011-10-01

Full Text Available Abstract Background Early maturation in the Atlantic salmon is an interesting subject for numerous research lines. Prior to sea migration, parr can reach sexual maturation and successfully fertilize adult female eggs during the reproductive season. These individuals are known as precocious parr, mature parr or "sneakers". Reasons for early maturation are unknown and this transitory stage is usually considered to be a threshold trait. Here, we compare methylation patterns between mature and immature salmon parr from two different rivers in order to infer if such methylation differences may be related to their maturation condition. First we analyzed genetic differences between rivers by means of AFLPs. Then, we compared the DNA methylation differences between mature and immature parrs, using a Methylation-Sensitive Amplified Polymorphism (MSAP, which is a modification of the AFLPs method by making use of the differential sensitivity of a pair of restriction enzymes isoschizomeres to cytosine methylation. The tissues essayed included brain, liver and gonads. Results AFLPs statistical analysis showed that there was no significant differentiation between rivers or a significant differentiation between maturation states in each river. MSAP statistical analysis showed that among the three tissues sampled, the gonads had the highest number of significant single-locus variation among populations with 74 loci followed by brain with 70 and finally liver with only 12. Principal components analysis (PCA of the MSAP profiles revealed different profiles among different tissues (liver, brain and testis clearly separating maturation states in the testis tissue when compared to the liver. Conclusions Our results reveal that genetically-similar mature and immature salmon parr present high levels of DNA methylation variation in two of the three analyzed tissues. We hypothesize that early maturation may be mostly mediated by epigenetic processes rather than by

Morphological analysis and DNA methylation in Conyza bonariensis L. cronquist (Asteraceae phenotypes

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Juliana Maria de Paula

2017-08-01

Full Text Available ABSTRACT The species Conyza bonariensis (L. cause losses in agriculture due to their invasive capacity and resistance to herbicides like glyphosate. The species of this genus exhibit phenotypic plasticity, which complicates their identification and characterization. Thus, experiments were performed with 2 extreme C. bonariensis phenotypes (called broad leaf and narrow leaf in greenhouse conditions and in the laboratory, in order to verify if the morphological differences among these phenotypes are a genetic character or result from environmental effects. In addition to the comparative morphological analysis, assessment of DNA methylation profile was performed to detect the occurrence, or not, of differences in the epigenetic level. The morphological characteristics evaluated were length, width, shape, margin and leaves indument; plant height and stem indument; the number of capitula, flowers and seeds. The Methylation Sensitive Amplified Polymorphism technique was used to investigate the methylation levels. The morphological differences of phenotypes supposed to be C. bonariensis are probably genetic in origin and not the result of environmental effects, since, after 6 crop cycles in a greenhouse under the same environmental conditions, these phenotypes remained with the same morphological characteristics and seed production in relation to the original phenotypes found in the collection site. The different phenotypes did not show differences corresponding to DNA methylation patterns that could indicate an epigenetic effect as the cause of the differences between the 2 phenotypes. The results of morphological analysis and methylation probably indicate that maybe they are individuals of populations from different taxa not registered yet in the literature.

Sensitive determination of pyrrolizidine alkaloids in Tussilago farfara L. by field-amplified, sample-stacking, sweeping micellar electrokinetic chromatography.

Science.gov (United States)

Cao, Kun; Xu, Yi; Mu, Xiuni; Zhang, Qing; Wang, Renjie; Lv, Junjiang

2016-11-01

Pyrrolizidine alkaloids are the toxic components in Tussilago farfara L. Due to the lack of standard substances for quantitative analysis and traces of pyrrolizidine alkaloids in total alkaloids, the full quality control of Tussilago farfara L has been limited. In this study, we aimed to solve the difficulty of determination of pyrrolizidine alkaloids and identify more components in the total alkaloids. An on-line preconcentration method has been applied to improve determining sensitivity of pyrrolizidine alkaloids in Tussilago farfara L. in which included field-amplified sample stacking and sweeping in micellar electrokinetic capillary chromatography. The main parameters that affected separation and stacking efficiency were investigated in details. Under the optimal conditions, the sensitivity enhancement factors obtained by the developed method for the analytes were from 15- to 12-fold, the limits of detection of senkirkine and senecionine were 2∼5 μg/L. Senkirkine and senecionine have been detected in alkaloids (c) of Tussilago farfara L, along ferulic acid methyl ester and methyl caffeate. The developed method was also applied to the analysis of acid extraction (a) of Tussilago farfara L, and senkirkine could be detected directly. The results indicated that the developed method is feasible for the analysis of pyrrolizidine alkaloids in Tussilago farfara L with good recoveries. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Highly sensitive electrochemical detection of methyl salicylate using electroactive gold nanoparticles.

Science.gov (United States)

Umasankar, Yogeswaran; Ramasamy, Ramaraja P

2013-11-07

Electrochemical sensing of methyl salicylate, a key plant volatile has been achieved using a gold nanoparticle (AuNP) modified screen printed carbon electrode (SPCE). The electrochemical response of planar gold electrodes, SPCE and AuNP-SPCE in alkaline electrolyte in the presence and absence of methyl salicylate were studied to understand the amperometric response of various electrochemical reactions. The reaction mechanism includes hydrolysis of methyl salicylate and the oxidation of negative species. The electrochemical responses were recorded using cyclic voltammetry and differential pulse voltammetry techniques, where the results showed characteristic signals for methyl salicylate oxidation. Among the examined electrodes, AuNP-SPCE possessed three fold better sensitivity than planar gold and 35 times better sensitivity than SPCE (at 0.5 V). The methyl salicylate sensing by AuNP-SPCE possessed 95% of its methyl salicylate response. The electroanalytical results of soybean extract showed that AuNP-SPCE can be employed for the determination of methyl salicylate in real samples.

Relation of polymorphism of arsenic metabolism genes to arsenic methylation capacity and developmental delay in preschool children in Taiwan

Energy Technology Data Exchange (ETDEWEB)

Hsieh, Ru-Lan [Department of Physical Medicine and Rehabilitation, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan (China); Department of Physical Medicine and Rehabilitation, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Su, Chien-Tien [Department of Family Medicine, Taipei Medical University Hospital, Taipei, Taiwan (China); School of Public Health, College of Public Health, Taipei Medical University, Taipei, Taiwan (China); Shiue, Horng-Sheng [Department of Chinese Medicine, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, Taiwan (China); Chen, Wei-Jen; Huang, Shiau-Rung [School of Public Health, College of Public Health, Taipei Medical University, Taipei, Taiwan (China); Lin, Ying-Chin [Department of Family Medicine, Shuang Ho Hospital, Taipei Medical University, Taipei, Taiwan (China); Department of Health Examination, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan (China); Division of Family Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan (China); Lin, Ming-I; Mu, Shu-Chi [Department of Pediatrics, Shin Kong Wu Ho-Su Memorial Hospital, Taipei, Taiwan (China); Chen, Ray-Jade [Department of Digestive Surgery, Taipei Medical University Hospital, Taipei, Taiwan (China); Hsueh, Yu-Mei, E-mail: ymhsueh@tmu.edu.tw [Department of Family Medicine, Taipei Medical University Hospital, Taipei, Taiwan (China); Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China)

2017-04-15

Inefficient arsenic methylation capacity has been associated with developmental delay in children. The present study was designed to explore whether polymorphisms and haplotypes of arsenic methyltransferase (AS3MT), glutathione-S-transferase omegas (GSTOs), and purine nucleoside phosphorylase (PNP) affect arsenic methylation capacity and developmental delay. A case-control study was conducted from August 2010 to March 2014. All participants were recruited from the Shin Kong Wu Ho-Su Memorial Teaching Hospital. In total, 179 children with developmental delay and 88 children without delay were recruited. Urinary arsenic species, including arsenite (As{sup III}), arsenate (As{sup V}), monomethylarsonic acid (MMA{sup V}), and dimethylarsinic acid (DMA{sup V}) were measured using a high-performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. The polymorphisms of AS3MT, GSTO, and PNP were performed using the Sequenom MassARRAY platform with iPLEX Gold chemistry. Polymorphisms of AS3MT genes were found to affect susceptibility to developmental delay in children, but GSTO and PNP polymorphisms were not. Participants with AS3MT rs3740392 A/G + G/G genotype, compared with AS3MT rs3740392 A/A genotype, had a significantly lower secondary methylation index. This may result in an increased OR for developmental delay. Participants with the AS3MT high-risk haplotype had a significantly higher OR than those with AS3MT low-risk haplotypes [OR and 95% CI, 1.59 (1.08–2.34)]. This is the first study to show a joint dose-response effect of this AS3MT high-risk haplotype and inefficient arsenic methylation capacity on developmental delay. Our data provide evidence that AS3MT genes are related to developmental delay and may partially influence arsenic methylation capacity. - Highlights: • AS3MT genotypes were found to affect susceptibility to developmental delay. • AS3MT rs3740392 A/G and G/G genotype had a significantly low SMI (DMA

Relation of polymorphism of arsenic metabolism genes to arsenic methylation capacity and developmental delay in preschool children in Taiwan

International Nuclear Information System (INIS)

Hsieh, Ru-Lan; Su, Chien-Tien; Shiue, Horng-Sheng; Chen, Wei-Jen; Huang, Shiau-Rung; Lin, Ying-Chin; Lin, Ming-I; Mu, Shu-Chi; Chen, Ray-Jade; Hsueh, Yu-Mei

2017-01-01

Inefficient arsenic methylation capacity has been associated with developmental delay in children. The present study was designed to explore whether polymorphisms and haplotypes of arsenic methyltransferase (AS3MT), glutathione-S-transferase omegas (GSTOs), and purine nucleoside phosphorylase (PNP) affect arsenic methylation capacity and developmental delay. A case-control study was conducted from August 2010 to March 2014. All participants were recruited from the Shin Kong Wu Ho-Su Memorial Teaching Hospital. In total, 179 children with developmental delay and 88 children without delay were recruited. Urinary arsenic species, including arsenite (As III ), arsenate (As V ), monomethylarsonic acid (MMA V ), and dimethylarsinic acid (DMA V ) were measured using a high-performance liquid chromatography-linked hydride generator and atomic absorption spectrometry. The polymorphisms of AS3MT, GSTO, and PNP were performed using the Sequenom MassARRAY platform with iPLEX Gold chemistry. Polymorphisms of AS3MT genes were found to affect susceptibility to developmental delay in children, but GSTO and PNP polymorphisms were not. Participants with AS3MT rs3740392 A/G + G/G genotype, compared with AS3MT rs3740392 A/A genotype, had a significantly lower secondary methylation index. This may result in an increased OR for developmental delay. Participants with the AS3MT high-risk haplotype had a significantly higher OR than those with AS3MT low-risk haplotypes [OR and 95% CI, 1.59 (1.08–2.34)]. This is the first study to show a joint dose-response effect of this AS3MT high-risk haplotype and inefficient arsenic methylation capacity on developmental delay. Our data provide evidence that AS3MT genes are related to developmental delay and may partially influence arsenic methylation capacity. - Highlights: • AS3MT genotypes were found to affect susceptibility to developmental delay. • AS3MT rs3740392 A/G and G/G genotype had a significantly low SMI (DMA/MMA) index. • AS3MT

using random amplified polymorphic DNA (RAPD)

African Journals Online (AJOL)

To study the pattern of genetic diversity in 45 genotypes of common bean, 19 RAPD primers were used. Of 253 bands produced, 236 bands (94.22%) were polymorphic in which maximum number (20 polymorphic bands) were observed in the profiles of the primer OPB-07. Highest PIC value (0.79) was observed for the ...

Composting oily sludges: Characterizing microflora using randomly amplified polymorphic DNA

International Nuclear Information System (INIS)

Persson, A.; Quednau, M.; Ahrne, S.

1995-01-01

Laboratory-scale composts in which oily sludge was composted under mesophilic conditions with amendments such as peat, bark, and fresh or decomposed horse manure, were studied with respect to basic parameters such as oil degradation, respirometry, and bacterial numbers. Further, an attempt was made to characterize a part of the bacterial flora using randomly amplified polymorphic DNA (RAPD). The compost based on decomposed horse manure showed the greatest reduction of oil (85%). Comparison with a killed control indicated that microbial degradation actually had occurred. However, a substantial part of the oil was stabilized rather than totally broken down. Volatiles, on the contrary, accounted for a rather small percentage (5%) of the observed reduction. RAPD indicated that a selection had taken place and that the dominating microbial flora during the active degradation of oil were not the same as the ones dominating the different basic materials. The stabilized compost, on the other hand, had bacterial flora with similarities to the ones found in peat and bark

Genotyping of human and porcine Yersinia enterocolitica, Yersinia intertmedia, and Yersinia bercovieri strains from Switzerland by amplified fragment length polymorphism analysis

DEFF Research Database (Denmark)

Kuehni-Boghenbor, Kathrin; On, Stephen L.W.; Kokotovic, Branko

2006-01-01

In this study, 231 strains of Yersinia enterocolitica, 25 strains of Y. intermedia, and 10 strains of Y. bercovieri from human and porcine sources (including reference strains) were analyzed using amplified fragment length polymorphism (AFLP), a whole-genome fingerprinting method for subtyping...

Random amplified polymorphic DNA (RAPD analysis of Lutzomyia longipalpis laboratory populations

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DiaS Edelberto S.

1998-01-01

Full Text Available The phlebotomine sand fly Lutzomyia longipalpis has been incriminated as a vector of American visceral leishmaniasis, caused by Leishmania chagasi. However, some evidence has been accumulated suggesting that it may exist in nature not as a single but as a species complex. Our goal was to compare four laboratory reference populations of L. longipalpis from distinct geographic regions at the molecular level by RAPD-PCR. We screened genomic DNA for polymorphic sites by PCR amplification with decamer single primers of arbitrary nucleotide sequences. One primer distinguished one population (Marajó Island, Pará State, Brazil from the other three (Lapinha Cave, Minas Gerais State, Brazil; Melgar, Tolima Department, Colombia and Liberia, Guanacaste Province, Costa Rica. The population-specific and the conserved RAPD-PCR amplified fragments were cloned and shown to differ only in number of internal repeats.

Genetic relationship among nine Rhododendron species in Qinling mountains, China using amplified fragment length polymorphism markers

International Nuclear Information System (INIS)

Zhao, B.; Zheng, X.Z.

2015-01-01

Genetic relationships of nine species of Rhododendron in the Qinling Mountains were evaluated using amplified fragment length polymorphism (AFLP) markers. A total of 440 amplification products were obtained using nine selected AFLP markers, of which 421 (95.40%) showed polymorphism. With these polymorphic products, a dendrogram was constructed using the unweighted pair-group method with arithmetic mean (UPGMA). R. calophytum, R. hypoglaucum and R. clementinae, belonging to Subgen Hymenanthes, gathered together, and the species derived from Subgen Rhododendron and Subgen Tsutsusi formed another two groups. R. tsinlingense, R. purdomii, R. Taibaiense and R. capitatum (Subsect. Lapponica), and R. concinnum (Subsect. Triflora) were clustered as one group, but they belong to difference subsect. and R. purdomii and R. Taibaiense showed the closest genetic distance, but both species differed greatly in morphological characteristics.These results showed that the genetic relationships among nine Rhododendron species, determined by AFLP markers, were partially related to their taxonomic position, geography distribution and morphological classification. The present study will benefit the identification and conservation of Rhododendron, and the development of new Rhododendron cultivar. (author)

Changes in DNA methylation fingerprint of Quercus ilex trees in response to experimental field drought simulating projected climate change.

Science.gov (United States)

Rico, L; Ogaya, R; Barbeta, A; Peñuelas, J

2014-03-01

Rapid genetic changes in plants have been reported in response to current climate change. We assessed the capacity of trees in a natural forest to produce rapid acclimation responses based on epigenetic modifications. We analysed natural populations of Quercus ilex, the dominant tree species of Mediterranean forests, using the methylation-sensitive amplified polymorphism (MSAP) technique to assess patterns and levels of methylation in individuals from unstressed forest plots and from plots experimentally exposed to drought for 12 years at levels projected for the coming decades. The percentage of hypermethylated loci increased, and the percentage of fully methylated loci clearly decreased in plants exposed to drought. Multivariate analyses exploring the status of methylation at MSAP loci also showed clear differentiation depending on stress. The PCA scores for the MSAP profiles clearly separated the genetic from the epigenetic structure, and also significantly separated the samples within each group in response to drought. Changes in DNA methylation highlight the large capacity of plants to rapidly acclimate to changing environmental conditions, including trees with long life spans, and our results demonstrate those changes. These changes, although unable to prevent the decreased growth and higher mortality associated with this experimental drought, occurred together with a dampening in such decreases as the long-term treatment progressed. © 2013 German Botanical Society and The Royal Botanical Society of the Netherlands.

Epigenetic Diversity of Clonal White Poplar (Populus alba L. Populations: Could Methylation Support the Success of Vegetative Reproduction Strategy?

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Francesco Guarino

Full Text Available The widespread poplar populations of Sardinia are vegetatively propagated and live in different natural environments forming large monoclonal stands. The main goals of the present study were: i to investigate/measure the epigenetic diversity of the poplar populations by determining their DNA methylation status; ii to assess if and how methylation status influences population clustering; iii to shed light on the changes that occur in the epigenome of ramets of the same poplar clone. To these purposes, 83 white poplar trees were sampled at different locations on the island of Sardinia. Methylation sensitive amplified polymorphism analysis was carried out on the genomic DNA extracted from leaves at the same juvenile stage. The study showed that the genetic biodiversity of poplars is quite limited but it is counterbalanced by epigenetic inter-population molecular variability. The comparison between MspI and HpaII DNA fragmentation profiles revealed that environmental conditions strongly influence hemi-methylation of the inner cytosine. The variable epigenetic status of Sardinian white poplars revealed a decreased number of population clusters. Landscape genetics analyses clearly demonstrated that ramets of the same clone were differentially methylated in relation to their geographic position. Therefore, our data support the notion that studies on plant biodiversity should no longer be restricted to genetic aspects, especially in the case of vegetatively propagated plant species.

Characterization of Mycoplasma hyosynoviae strains by amplified fragment length polymorphism analysis, pulsed-field gel electrophoresis and 16S ribosomal DNA sequencing

DEFF Research Database (Denmark)

Kokotovic, Branko; Friis, N.F.; Ahrens, Peter

2002-01-01

, were investigated by analysis of amplified fragment length polymorphisms of the Bgl II and Mfe I restriction sites and by pulsed-field gel electrophoresis of a Bss HII digest of chromosomal DNA. Both methods allowed unambiguous differentiation of the analysed strains and showed similar discriminatory...

Does DNA methylation regulate metamorphosis? The case of the sea lamprey (Petromyzon marinus) as an example.

Science.gov (United States)

Covelo-Soto, Lara; Saura, María; Morán, Paloma

2015-07-01

Lampreys represent one of the most ancient vertebrate lineages enclosing a special interest for genetic and epigenetic studies. The sea lamprey (Petromyzon marinus) is an anadromous species that experiences metamorphosis all the way up to the adult stage. Although representing a gradual process, metamorphosis in this species involves dramatic conversions with regard to physiological together with structural body changes preparing individuals for a marine and parasitic life; in consequence, multiple gene expression modifications are expected. The implications of thyroid hormones and HOX gene expression changes have previously been reported in this species and also in other vertebrate species. Nonetheless, information lacks on how these genes are regulated in lampreys. We here report about the existence of methylation pattern differences between the adult and the larvae sea lamprey life cycle stages making use of the Methylation-Sensitive Amplified Polymorphism (MSAP) technique. Differentially methylated fragment sequencing allowed to establish homologous identities with HOX genes involved in morphogenesis, along with genes related to the water balance and to the osmotic homoeostasis, all associated to a marine environment adaptation. These results provide evidences revealing that DNA methylation plays a role in the epigenetic regulation of the P. marinus post-natal development representing a starting point for future studies. To the best of our knowledge, this is the first study which detects DNA methylation changes associated with metamorphosis in lampreys. Copyright © 2015 Elsevier Inc. All rights reserved.

Reduced DNA methylation of FKBP5 in Cushing's syndrome.

Science.gov (United States)

Resmini, Eugenia; Santos, Alicia; Aulinas, Anna; Webb, Susan M; Vives-Gilabert, Yolanda; Cox, Olivia; Wand, Gary; Lee, Richard S

2016-12-01

FKBP5 encodes a co-chaperone of HSP90 protein that regulates intracellular glucocorticoid receptor sensitivity. When it is bound to the glucocorticoid receptor complex, cortisol binds with lower affinity to glucocorticoid receptor. Cushing's syndrome is associated with memory deficits, smaller hippocampal volumes, and wide range of cognitive impairments. We aimed at evaluating blood DNA methylation of FKBP5 and its relationship with memory and hippocampal volumes in Cushing's syndrome patients. Polymorphism rs1360780 in FKBP5 has also been assessed to determine whether genetic variations can also govern CpG methylation. Thirty-two Cushing's syndrome patients and 32 matched controls underwent memory tests, 3-Tesla MRI of the brain, and DNA extraction from total leukocytes. DNA samples were bisulfite treated, PCR amplified, and pyrosequenced to assess a total of 41CpG-dinucleotides in the introns 1, 2, 5, and 7 of FKBP5. Significantly lower intronic FKBP5 DNA methylation in CS patients compared to controls was observed in ten CpG-dinucleotides. DNA methylation at these CpGs correlated with left and right HV (Intron-2-Region-2-CpG-3: LHV, r = 0.73, p = 0.02; RHV, r = 0.58, p = 0.03). Cured and active CS patients showed both lower methylation of intron 2 (92.37, 91.8, and 93.34 %, respectively, p = 0.03 for both) and of intron 7 (77.08, 73.74, and 79.71 %, respectively, p = 0.02 and p < 0.01) than controls. Twenty-two subjects had the CC genotype, 34 had the TC genotype, and eight had the TT genotype. Lower average DNA methylation in intron 7 was observed in the TT subjects compared to CC (72.5vs. 79.5 %, p = 0.02) and to TC (72.5 vs. 79.0 %, p = 0.03). Our data demonstrate, for the first time, a reduction of intronic DNA methylation of FKBP5 in CS patients.

Effects of temperature and salinity on survival, growth and DNA methylation of juvenile Pacific abalone, Haliotis discus hannai Ino

Science.gov (United States)

Kong, Ning; Liu, Xiao; Li, Junyuan; Mu, Wendan; Lian, Jianwu; Xue, Yanjie; Li, Qi

2017-09-01

Temperature and salinity are two of the most potent abiotic factors influencing marine mollusks. In this study, we investigated the individual and combined effects of temperature and salinity on the survival and growth of juvenile Pacific abalone, Haliotis discus hannai Ino, and also examined the DNA methylation alteration that may underpin the phenotypic variation of abalone exposed to different rearing conditions. The single-factor data showed that the suitable ranges of temperature and salinity were 16-28°C at a constant salinity of 32, and 24-40 at a constant temperature of 20°C, respectively. The two-factor data indicated that both survival and growth were significantly affected by temperature, salinity and their interaction. The optimal temperature-salinity combination for juveniles was 23-25°C and 30-36. To explore environment-induced DNA methylation alteration, the methylation-sensitive amplified polymorphism (MSAP) technique was used to analyze the genomic methylation profiles of abalone reared in optimal and adverse conditions. Neither temperature nor salinity induced evident changes in the global methylation level, but 67 and 63 differentially methylated loci were identified in temperature and salinity treatments, respectively. The between-group eigen analysis also showed that both temperature and salinity could induce epigenetic differentiation in H. discus hannai Ino. The results of our study provide optimal rearing conditions for juvenile H. discus hannai Ino, and represent the first step toward revealing the epigenetic regulatory mechanism of abalone in response to thermal and salt stresses.

Rapid analysis of heterogeneously methylated DNA using digital methylation-sensitive high resolution melting: application to the CDKN2B (p15) gene

DEFF Research Database (Denmark)

Candiloro, Ida Lm; Mikeska, Thomas; Hokland, Peter

2008-01-01

ABSTRACT: BACKGROUND: Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (dMS-HRM) t......ABSTRACT: BACKGROUND: Methylation-sensitive high resolution melting (MS-HRM) methodology is able to recognise heterogeneously methylated sequences by their characteristic melting profiles. To further analyse heterogeneously methylated sequences, we adopted a digital approach to MS-HRM (d......MS-HRM) that involves the amplification of single templates after limiting dilution to quantify and to determine the degree of methylation. We used this approach to study methylation of the CDKN2B (p15) cell cycle progression inhibitor gene which is inactivated by DNA methylation in haematological malignancies...... the methylated alleles and assess the degree of methylation. Direct sequencing of selected dMS-HRM products was used to determine the exact DNA methylation pattern and confirmed the degree of methylation estimated by dMS-HRM. CONCLUSION: dMS-HRM is a powerful technique for the analysis of methylation in CDKN2B...

Genetic differentiation of Octopus minor (Mollusca, Cephalopoda) off the northern coast of China as revealed by amplified fragment length polymorphisms.

Science.gov (United States)

Yang, J M; Sun, G H; Zheng, X D; Ren, L H; Wang, W J; Li, G R; Sun, B C

2015-12-02

Octopus minor (Sasaki, 1920) is an economically important cephalopod that is found in the northern coastal waters of China. In this study, we investigated genetic differentiation in fishery populations using amplified fragment length polymorphisms (AFLPs). A total of 150 individuals were collected from five locations: Dalian (DL), Yan-tai (YT), Qingdao (QD), Lianyungang (LY), and Zhoushan (ZS), and 243 reproducible bands were amplified using five AFLP primer combinations. The percentage of polymorphic bands ranged from 53.33 to 76.08%. Nei's genetic identity ranged from 0.9139 to 0.9713, and the genetic distance ranged from 0.0291 to 0.0900. A phylogenetic tree was constructed using the unweighted pair group method with arithmetic mean, based on the genetic distance. The DL and YT populations originated from one clade, while the QD, LY, and ZS populations originated from another. The results indicate that the O. minor stock consisted of two genetic populations with an overall significantly analogous FST value (0.1088, P octopus fisheries, so that this marine resource can be conserved for its long-term utilization.

Analysis of RET promoter CpG island methylation using methylation-specific PCR (MSP), pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM): impact on stage II colon cancer patient outcome.

Science.gov (United States)

Draht, Muriel X G; Smits, Kim M; Jooste, Valérie; Tournier, Benjamin; Vervoort, Martijn; Ramaekers, Chantal; Chapusot, Caroline; Weijenberg, Matty P; van Engeland, Manon; Melotte, Veerle

2016-01-01

Already since the 1990s, promoter CpG island methylation markers have been considered promising diagnostic, prognostic, and predictive cancer biomarkers. However, so far, only a limited number of DNA methylation markers have been introduced into clinical practice. One reason why the vast majority of methylation markers do not translate into clinical applications is lack of independent validation of methylation markers, often caused by differences in methylation analysis techniques. We recently described RET promoter CpG island methylation as a potential prognostic marker in stage II colorectal cancer (CRC) patients of two independent series. In the current study, we analyzed the RET promoter CpG island methylation of 241 stage II colon cancer patients by direct methylation-specific PCR (MSP), nested-MSP, pyrosequencing, and methylation-sensitive high-resolution melting (MS-HRM). All primers were designed as close as possible to the same genomic region. In order to investigate the effect of different DNA methylation assays on patient outcome, we assessed the clinical sensitivity and specificity as well as the association of RET methylation with overall survival for three and five years of follow-up. Using direct-MSP and nested-MSP, 12.0 % (25/209) and 29.6 % (71/240) of the patients showed RET promoter CpG island methylation. Methylation frequencies detected by pyrosequencing were related to the threshold for positivity that defined RET methylation. Methylation frequencies obtained by pyrosequencing (threshold for positivity at 20 %) and MS-HRM were 13.3 % (32/240) and 13.8 % (33/239), respectively. The pyrosequencing threshold for positivity of 20 % showed the best correlation with MS-HRM and direct-MSP results. Nested-MSP detected RET promoter CpG island methylation in deceased patients with a higher sensitivity (33.1 %) compared to direct-MSP (10.7 %), pyrosequencing (14.4 %), and MS-HRM (15.4 %). While RET methylation frequencies detected by nested

Epigenetic variation, inheritance, and parent-of-origin effects of cytosine methylation in maize (Zea mays).

Science.gov (United States)

Lauria, Massimiliano; Piccinini, Sara; Pirona, Raul; Lund, Gertrud; Viotti, Angelo; Motto, Mario

2014-03-01

Pure epigenetic variation, or epigenetic variation that is independent of genetic context, may provide a mechanism for phenotypic variation in the absence of DNA mutations. To estimate the extent of pure epigenetic variation within and across generations and to identify the DNA regions targeted, a group of eight plants derived from a highly inbred line of maize (Zea mays) was analyzed by the methylation-sensitive amplified polymorphism (MSAP) technique. We found that cytosine methylation (mC) differences among individuals accounted for up to 7.4% of CCGG sites investigated by MSAP. Of the differentially methylated fragments (DMFs) identified in the S0 generation, ∼12% were meiotically inherited for at least six generations. We show that meiotically heritable mC variation was consistently generated for an average of 0.5% CCGG sites per generation and that it largely occurred somatically. We provide evidence that mC variation can be established and inherited in a parent-of-origin manner, given that the paternal lineage is more prone to both forward and reverse mC changes. The molecular characterization of selected DMFs revealed that the variation was largely determined by CG methylation changes that map within gene regions. The expression analysis of genes overlapping with DMFs did not reveal an obvious correlation between mC variation and transcription, reinforcing the idea that the primary function of gene-body methylation is not to control gene expression. Because this study focuses on epigenetic variation in field-grown plants, the data presented herein pertain to spontaneous epigenetic changes of the maize genome in a natural context.

Development of high sensitivity transimpedance amplifier module for self powered neutron detectors

International Nuclear Information System (INIS)

Khan, T.K.; Tamboli, P.K.; Antony, J.; Balasubramanian, R.; Agilandaeswari, K.; Pramanik, M.

2010-01-01

This paper describes design and development of a Transimpedance Amplifier for amplification of very low current from in core Self Powered Neutron Detectors (SPND). Measurement of neutron flux is very important for operation, control and protection of Nuclear Power Plant (NPP). SPND is used to measure Reactor incore flux/power. Based on sensitivity of emitter material used in SPND, pitch length and neutron flux (power level); the current output from SPND varies from few pA to few μA. The described amplifier is suitable to use for this current range. The amplifier provides a very high gain using a resistive T network feedback topology. The amplifier is designed in two stages using ultra low bias current FET OPAMPs. Design of Transimpedance amplifier is carefully done to include ultra low input bias current, low offset voltage and noise. The amplifier has in built test facility for calibration and on line test facility for measurement of insulation resistance (IR). The amplifier module has on board isolated DC-DC converter circuit complying MIL/STD/461C/D which generate isolated +/-15V and +12V supply to provide parameter to parameter ground isolation and independence among each module/signal.The output from the amplifier is 0V to 6V for 0 to 150%FP. The design is simulated in computer and amplifier used at TAPS-3 was modified as per new design and has been tested at TAPS-3 site. The amplifier performed satisfactorily. The results showed that the IR measurement technique adopted in the design can tolerate lower IR of SPND in existing design. (author)

Genomic diversity among Danish field strains of Mycoplasma hyosynoviae assessed by amplified fragment length polymorphism analysis

DEFF Research Database (Denmark)

Kokotovic, Branko; Friis, Niels F.; Nielsen, Elisabeth O.

2002-01-01

Genomic diversity among strains of Mycoplasma hyosynoviae isolated in Denmark was assessed by using amplified fragment length polymorphism (AFLP) analysis. Ninety-six strains, obtained from different specimens and geographical locations during 30 years and the type strain of M. hyosynoviae S16(T......) were concurrently examined for variance in BglII-MfeI and EcoRI-Csp6I-A AFLP markers. A total of 56 different genomic fingerprints having an overall similarity between 77 and 96% were detected. No correlation between AFLP variability and period of isolation or anatomical site of isolation could...

Identification of three randomly amplified polymorphic DNA-polymerase chain reaction markers for distinguishing Asian and North American Gypsy Moths (Lepidoptera: Lymantriidae)

Science.gov (United States)

David E. Schreiber; Karen J. Garner; James M. Slavicek

1997-01-01

Gypsy moths originating in Asia have recently been introduced into North America, making it necessary to develop markers for distinguishing the Asian strain from the established North American population. We have identified 3 randomly amplified polymorphic DNA-polymerase chain reaction generated (RAPD-PCR) markers which are specific for either Asian or North American...

Production and genetic analysis of resynthesized Brassica napus from a B. rapa landrace from the Qinghai-Tibet Plateau and B. alboglabra.

Science.gov (United States)

Liu, H D; Zhao, Z G; Du, D Z; Deng, C R; Fu, G

2016-01-08

This study aimed to reveal the genetic and epigenetic variations involved in a resynthesized Brassica napus (AACC) generated from a hybridization between a B. rapa (AA) landrace and B. alboglabra (CC). Amplified fragment length polymorphism (AFLP), methylation-sensitive amplified polymorphism, and the cDNA-AFLP technique were performed to detect changes between different generations at the genome, methylation, and transcription levels. We obtained 30 lines of resynthesized B. napus with a mean 1000-seed weight of over 7.50 g. All of the lines were self-compatible, probably because both parents were self-compatible. At the genome level, the S0 generation had the lowest frequency of variations (0.18%) and the S3 generation had the highest (6.07%). The main variation pattern was the elimination of amplified restriction fragments on the CC genome from the S0 to the S4 generations. At the methylation level, we found three loci that exhibited altered methylation patterns on the parental A genome; the variance rate was 1.35%. At the transcription level, we detected 43.77% reverse mutations and 37.56% deletion mutations that mainly occurred on the A and C genomes, respectively, in the S3 generation. Our results highlight the genetic variations that occur during the diploidization of resynthesized B. napus.

Association of polymorphism in TP53 gene with susceptibility and radiation sensitivity of non-small-cell-lung cancer in Chinese population

International Nuclear Information System (INIS)

Shao Guoguang; Liu Linlin; Xu Chuanjie

2005-01-01

Objective: To study the association of polymorphism in TP53 gene with the susceptibility and radiation sensitivity of non-small-cell-lung cancer (NSCLC) of the population in the North of China. Methods: Using RFLP-PCR assays, TP53 genotypes were detected by amplifying DNA fragments with sequence specific primers and digested by FnuD II enzyme in 88 patients with NSCLC as well as 112 healthy controls. Results: The C/C allele frequency was significantly higher in NSCLC patients than that in the healthy controls (χ 2 =5.65, P=0.017). The C/C genotype frequency was significantly higher in NSCLC patients than that of the healthy controls (χ 2 =9.33, P=0.0023). The risk of C/C homozygotes in NSCLC patients was about 2.7 times against G/G homozygotes with odds ratio of 2.43(95% CI=1.32-4.51). Conclusion: In the population in the North of China, TP53 C/C genotype is closely associated with the susceptibility of NSCLC. There is no significant relationship between the polymorphism in TP53 gene and radiation sensitivity in NSCLC. (authors)

Genetic relatedness of artichoke (Cynara scolymus L.) hybrids using random amplified polymorphic DNA (RAPD) fingerprinting.

Science.gov (United States)

Sharaf-Eldin, M A; Al-Tamimi, A; Alam, P; Elkholy, S F; Jordan, J R

2015-12-28

The artichoke (Cynara scolymus L.) is an important food and medicinal crop that is cultivated in Mediterranean countries. Morphological characteristics, such as head shape and diameter, leaf shape, and bract shape, are mainly affected by environmental conditions. A molecular marker approach was used to analyze the degree of polymorphism between artichoke hybrid lines. The degree of genetic difference among three artichoke hybrids was evaluated using random amplified polymorphic DNA-PCR (RAPD-PCR). In this study, the DNA fingerprints of three artichoke lines (A13-010, A11-018, and A12-179) were generated, and a total of 10 decamer primers were applied for RAPD-PCR analyses. Polymorphism  (16.66 to 62.50%) was identified using eight arbitrary decamers and total genomic DNA extracted from the hybrids. Of the 59 loci detected, there were 25 polymorphic and 34 monomorphic loci. Jaccard's similarity index (JSI) ranged between 1.0 and 0.84. Based on the unweighted pair group method with arithmetic mean (UPGMA) similarity matrix and dendrogram, the results indicated that two hybrids (A13-010 and A11-018) were closely related to each other, and the A12-179 line showed more divergence. When identifying correct accessions, consideration of the genetic variation and genetic relationships among the genotypes are required. The RAPD-PCR fingerprinting of artichoke lines clearly showed that it is possible to analyze the RAPD patterns for correlation between genetic means and differences or resemblance between close accessions (A13-010 and A11- 018) at the genomic level.

Chilling- and Freezing-Induced Alterations in Cytosine Methylation and Its Association with the Cold Tolerance of an Alpine Subnival Plant, Chorispora bungeana.

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Yuan Song

Full Text Available Chilling (0-18°C and freezing (<0°C are two distinct types of cold stresses. Epigenetic regulation can play an important role in plant adaptation to abiotic stresses. However, it is not yet clear whether and how epigenetic modification (i.e., DNA methylation mediates the adaptation to cold stresses in nature (e.g., in alpine regions. Especially, whether the adaptation to chilling and freezing is involved in differential epigenetic regulations in plants is largely unknown. Chorispora bungeana is an alpine subnival plant that is distributed in the freeze-thaw tundra in Asia, where chilling and freezing frequently fluctuate daily (24 h. To disentangle how C. bungeana copes with these intricate cold stresses through epigenetic modifications, plants of C. bungeana were treated at 4°C (chilling and -4°C (freezing over five periods of time (0-24 h. Methylation-sensitive amplified fragment-length polymorphism markers were used to investigate the variation in DNA methylation of C. bungeana in response to chilling and freezing. It was found that the alterations in DNA methylation of C. bungeana largely occurred over the period of chilling and freezing. Moreover, chilling and freezing appeared to gradually induce distinct DNA methylation variations, as the treatment went on (e.g., after 12 h. Forty-three cold-induced polymorphic fragments were randomly selected and further analyzed, and three of the cloned fragments were homologous to genes encoding alcohol dehydrogenase, UDP-glucosyltransferase and polygalacturonase-inhibiting protein. These candidate genes verified the existence of different expressive patterns between chilling and freezing. Our results showed that C. bungeana responded to cold stresses rapidly through the alterations of DNA methylation, and that chilling and freezing induced different DNA methylation changes. Therefore, we conclude that epigenetic modifications can potentially serve as a rapid and flexible mechanism for C. bungeana

Differentiation in a geographical mosaic of plants coevolving with ants: phylogeny of the Leonardoxa africana complex (Fabaceae: Caesalpinioideae) using amplified fragment length polymorphism markers.

Science.gov (United States)

Brouat, C; McKey, D; Douzery, E J P

2004-05-01

Comprising four allopatric subspecies that exhibit various grades of ant-plant interactions, from diffuse to obligate and symbiotic associations, the Leonardoxa africana complex (Fabaceae, Caesalpinioideae) provides a good opportunity to investigate the evolutionary history of ant-plant mutualisms. A previous study of the L. africana complex based on chloroplast DNA noncoding sequences revealed a lack of congruence between clades suggested by morphological and plastid characters. In this study, we analysed phylogenetic relationships within the L. africana complex using a Bayesian probability approach on amplified fragment length polymorphism markers. The results reported permit partial validation of the four subspecies of L. africana previously defined by morphological and ecological markers. Incongruences between phylogenies based on chloroplast DNA and amplified fragment length polymorphism markers are discussed in the light of morphological and ecological data, and confronted with hypotheses of convergence, lineage sorting and introgression.

Validation of methylation-sensitive high-resolution melting (MS-HRM) for the detection of stool DNA methylation in colorectal neoplasms.

Science.gov (United States)

Xiao, Zhujun; Li, Bingsheng; Wang, Guozhen; Zhu, Weisi; Wang, Zhongqiu; Lin, Jinfeng; Xu, Angao; Wang, Xinying

2014-04-20

Methylation-sensitive high-resolution melting (MS-HRM) is a new technique for assaying DNA methylation, but its feasibility for assaying stool in patients with colorectal cancer (CRC) is unknown. First, the MS-HRM and methylation-specific PCR (MSP) detection limits were tested. Second, the methylation statuses of SFRP2 and VIM were analyzed in stool samples by MS-HRM, and in matching tumor and normal colon tissues via bisulfite sequencing PCR (BSP). Third, a case-control study evaluated the diagnostic sensitivity and specificity of MS-HRM relative to results obtained with MSP and the fecal immunochemical test (FIT). Finally, the linearity and reproducibility of MS-HRM were assessed. The detection limits of MS-HRM and MSP were 1% and 5%, respectively. The diagnostic sensitivities of MS-HRM (87.3%, 55/63) in stool and BSP in matching tumor tissue (92.1%, 58/63) were highly consistent (κ=0.744). The MS-HRM assay detected 92.5% (37/40) methylation in CRCs, 94.4% (34/36) in advanced adenomas, and 8.8% (5/57) in normal controls. The results of MS-HRM analysis were stable and reliable and showed fairly good linearity for both SFRP2 (PHRM shows potential for CRC screening. Copyright © 2014 Elsevier B.V. All rights reserved.

A Novel Phase-Locking-Free Phase Sensitive Amplifier based Regenerator

DEFF Research Database (Denmark)

Kjøller, Niels-Kristian; Røge, Kasper Meldgaard; Guan, Pengyu

2016-01-01

We propose a scheme for phase regeneration of optical binary phase-shift keying (BPSK) data signals based on phase sensitive amplification without active phase-locking. A delay interferometer (DI) is used to convert a BPSK signal impaired by noise to an amplitude modulated signal followed by cross......-locked pumps. As a result, active phase-stabilization is avoided. A proof-of-principle experiment is carried out with a dual-pump degenerate phase sensitive amplifier (PSA), demonstrating regeneration for a 10 Gb/s non-return-to-zero differential BPSK (NRZ-DPSK) data signal degraded by a sinusoidal phase...

Analysis of genetic diversity of Tunisian pistachio (Pistacia vera L.) using sequence-related amplified polymorphism (SRAP) markers.

Science.gov (United States)

Guenni, K; Aouadi, M; Chatti, K; Salhi-Hannachi, A

2016-10-17

Sequence-related amplified polymorphism (SRAP) markers preferentially amplify open reading frames and were used to study the genetic diversity of Tunisian pistachio. In the present study, 43 Pistacia vera accessions were screened using seven SRAP primer pairs. A total of 78 markers was revealed (95.12%) with an average polymorphic information content of 0.850. The results suggest that there is strong genetic differentiation, which characterizes the local resources (G ST = 0.307). High gene flow (N m = 1.127) among groups was explained by the exchange of plant material among regions. Analysis of molecular variance revealed significant differences within groups and showed that 73.88% of the total genetic diversity occurred within groups, whereas the remaining 26.12% occurred among groups. Bayesian clustering and principal component analysis identified three pools, El Guettar, Pollenizers, and the rest of the pistachios belonging to the Gabès, Kasserine, and Sfax localities. Bayesian analysis revealed that El Guettar and male genotypes were assigned with more than 80% probability. The BayeScan method proposed that locus 59 (F13-R9) could be used in the development of sex-linked SCAR markers from SRAP since it is a commonly detected locus in comparisons involving the Pollenizers group. This is the first application of SRAP markers for the assessment of genetic diversity in Tunisian germplasm of P. vera. Such information will be useful to define conservation strategies and improvement programs for this species.

AS3MT, GSTO, and PNP polymorphisms: impact on arsenic methylation and implications for disease susceptibility.

Science.gov (United States)

Antonelli, Ray; Shao, Kan; Thomas, David J; Sams, Reeder; Cowden, John

2014-07-01

Oral exposure to inorganic arsenic (iAs) is associated with adverse health effects. Epidemiological studies suggest differences in susceptibility to these health effects, possibly due to genotypic variation. Genetic polymorphisms in iAs metabolism could lead to increased susceptibility by altering urinary iAs metabolite concentrations. To examine the impact of genotypic polymorphisms on iAs metabolism. We screened 360 publications from PubMed and Web of Science for data on urinary mono- and dimethylated arsenic (MMA and DMA) percentages and polymorphic genes encoding proteins that are hypothesized to play roles in arsenic metabolism. The genes we examined were arsenic (+3) methyltransferase (AS3MT), glutathione-s-transferase omega (GSTO), and purine nucleoside phosphorylase (PNP). Relevant data were pooled to determine which polymorphisms are associated across studies with changes in urinary metabolite concentration. In our review, AS3MT polymorphisms rs3740390, rs11191439, and rs11191453 were associated with statistically significant changes in percent urinary MMA. Studies of GSTO polymorphisms did not indicate statistically significant associations with methylation, and there are insufficient data on PNP polymorphisms to evaluate their impact on metabolism. Collectively, these data support the hypothesis that AS3MT polymorphisms alter in vivo metabolite concentrations. Preliminary evidence suggests that AS3MT genetic polymorphisms may impact disease susceptibility. GSTO polymorphisms were not associated with iAs-associated health outcomes. Additional data are needed to evaluate the association between PNP polymorphisms and iAs-associated health outcomes. Delineation of these relationships may inform iAs mode(s) of action and the approach for evaluating low-dose health effects for iAs. Genotype impacts urinary iAs metabolite concentrations and may be a potential mechanism for iAs-related disease susceptibility. Published by Elsevier Inc.

Current feedback operational amplifiers as fast charge sensitive preamplifiers for photomultiplier read out

Energy Technology Data Exchange (ETDEWEB)

Giachero, A; Gotti, C; Maino, M; Pessina, G, E-mail: claudio.gotti@mib.infn.it [INFN - Sezione di Milano-Bicocca, I-20126, Milano (Italy)

2011-05-01

Fast charge sensitive preamplifiers were built using commercial current feedback operational amplifiers for fast read out of charge pulses from a photomultiplier tube. Current feedback opamps prove to be particularly well suited for this application where the charge from the detector is large, of the order of one million electrons, and high timing resolution is required. A proper circuit arrangement allows very fast signals, with rise times down to one nanosecond, while keeping the amplifier stable. After a review of current feedback circuit topology and stability constraints, we provide a 'recipe' to build stable and very fast charge sensitive preamplifiers from any current feedback opamp by adding just a few external components. The noise performance of the circuit topology has been evaluated and is reported in terms of equivalent noise charge.

Overexpression of Human-Derived DNMT3A Induced Intergenerational Inheritance of Active DNA Methylation Changes in Rat Sperm

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Xiaoguo Zheng

2017-12-01

Full Text Available DNA methylation is the major focus of studies on paternal epigenetic inheritance in mammals, but most previous studies about inheritable DNA methylation changes are passively induced by environmental factors. However, it is unclear whether the active changes mediated by variations in DNA methyltransferase activity are heritable. Here, we established human-derived DNMT3A (hDNMT3A transgenic rats to study the effect of hDNMT3A overexpression on the DNA methylation pattern of rat sperm and to investigate whether this actively altered DNA methylation status is inheritable. Our results revealed that hDNMT3A was overexpressed in the testis of transgenic rats and induced genome-wide alterations in the DNA methylation pattern of rat sperm. Among 5438 reliable loci identified with 64 primer-pair combinations using a methylation-sensitive amplification polymorphism method, 28.01% showed altered amplified band types. Among these amplicons altered loci, 68.42% showed an altered DNA methylation status in the offspring of transgenic rats compared with wild-type rats. Further analysis based on loci which had identical DNA methylation status in all three biological replicates revealed that overexpression of hDNMT3A in paternal testis induced hypermethylation in sperm of both genotype-negative and genotype-positive offspring. Among the differentially methylated loci, 34.26% occurred in both positive and negative offspring of transgenic rats, indicating intergenerational inheritance of active DNA methylation changes in the absence of hDNM3A transmission. Furthermore, 75.07% of the inheritable loci were hyper-methylated while the remaining were hypomethylated. Distribution analysis revealed that the DNA methylation variations mainly occurred in introns and intergenic regions. Functional analysis revealed that genes related to differentially methylated loci were involved in a wide range of functions. Finally, this study demonstrated that active DNA methylation

DNA methyl transferase (DNMT gene polymorphisms could be a primary event in epigenetic susceptibility to schizophrenia.

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Koramannil Radha Saradalekshmi

Full Text Available DNA methylation has been implicated in the etiopathology of various complex disorders. DNA methyltransferases are involved in maintaining and establishing new methylation patterns. The aim of the present study was to investigate the inherent genetic variations within DNA methyltransferase genes in predisposing to susceptibility to schizophrenia. We screened for polymorphisms in DNA methyltransferases, DNMT1, DNMT3A, DNMT3B and DNMT3L in 330 schizophrenia patients and 302 healthy controls for association with Schizophrenia in south Indian population. These polymorphisms were also tested for subgroup analysis with patient's gender, age of onset and family history. DNMT1 rs2114724 (genotype P = .004, allele P = 0.022 and rs2228611 (genotype P = 0.004, allele P = 0.022 were found to be significantly associated at genotypic and allelic level with Schizophrenia in South Indian population. DNMT3B rs2424932 genotype (P = 0.023 and allele (P = 0.0063 increased the risk of developing schizophrenia in males but not in females. DNMT3B rs1569686 (genotype P = 0.027, allele P = 0.033 was found to be associated with early onset of schizophrenia and also with family history and early onset (genotype P = 0.009. DNMT3L rs2070565 (genotype P = 0.007, allele P = 0.0026 confers an increased risk of developing schizophrenia at an early age in individuals with family history. In-silico prediction indicated functional relevance of these SNPs in regulating the gene. These observations might be crucial in addressing and understanding the genetic control of methylation level differences from ethnic viewpoint. Functional significance of genotype variations within the DNMTs indeed suggest that the genetic nature of methyltransferases should be considered while addressing epigenetic events mediated by methylation in Schizophrenia.

Genetic Variation among Isolates of Sarcocystis neurona, the Agent of Protozoal Myeloencephalitis, as Revealed by Amplified Fragment Length Polymorphism Markers

OpenAIRE

Elsheikha, H. M.; Schott, H. C.; Mansfield, L. S.

2006-01-01

Sarcocystis neurona causes serious neurological disease in horses and other vertebrates in the Americas. Based on epidemiological data, this parasite has recently emerged. Here, the genetic diversity of Sarcocystis neurona was evaluated using the amplified fragment length polymorphism (AFLP) method. Fifteen S. neurona taxa from different regions collected over the last 10 years were used; six isolates were from clinically diseased horses, eight isolates were from wild-caught opossums (Didelph...

Cadmium-induced genomic instability in Arabidopsis: Molecular toxicological biomarkers for early diagnosis of cadmium stress.

Science.gov (United States)

Wang, Hetong; He, Lei; Song, Jie; Cui, Weina; Zhang, Yanzhao; Jia, Chunyun; Francis, Dennis; Rogers, Hilary J; Sun, Lizong; Tai, Peidong; Hui, Xiujuan; Yang, Yuesuo; Liu, Wan

2016-05-01

Microsatellite instability (MSI) analysis, random-amplified polymorphic DNA (RAPD), and methylation-sensitive arbitrarily primed PCR (MSAP-PCR) are methods to evaluate the toxicity of environmental pollutants in stress-treated plants and human cancer cells. Here, we evaluate these techniques to screen for genetic and epigenetic alterations of Arabidopsis plantlets exposed to 0-5.0 mg L(-1) cadmium (Cd) for 15 d. There was a substantial increase in RAPD polymorphism of 24.5, and in genomic methylation polymorphism of 30.5-34.5 at CpG and of 14.5-20 at CHG sites under Cd stress of 5.0 mg L(-1) by RAPD and of 0.25-5.0 mg L(-1) by MSAP-PCR, respectively. However, only a tiny increase of 1.5 loci by RAPD occurred under Cd stress of 4.0 mg L(-1), and an additional high dose (8.0 mg L(-1)) resulted in one repeat by MSI analysis. MSAP-PCR detected the most significant epigenetic modifications in plantlets exposed to Cd stress, and the patterns of hypermethylation and polymorphisms were consistent with inverted U-shaped dose responses. The presence of genomic methylation polymorphism in Cd-treated seedlings, prior to the onset of RAPD polymorphism, MSI and obvious growth effects, suggests that these altered DNA methylation loci are the most sensitive biomarkers for early diagnosis and risk assessment of genotoxic effects of Cd pollution in ecotoxicology. Copyright © 2016 Elsevier Ltd. All rights reserved.

Immune sensitization against epidermal antigens in polymorphous light eruption

International Nuclear Information System (INIS)

Gonzalez-Amaro, R.; Baranda, L.; Salazar-Gonzalez, J.F.; Abud-Mendoza, C.; Moncada, B.

1991-01-01

To get further insight into the pathogenesis of polymorphous light eruption, we studied nine patients with polymorphous light eruption and six healthy persons. Two skin biopsy specimens were obtained from each person, one from previously ultraviolet light-irradiated skin and another one from unirradiated skin. An epidermal cell suspension, skin homogenate, or both were prepared from each specimen. Autologous cultures were made with peripheral blood mononuclear cells combined with irradiated or unirradiated skin homogenate and peripheral blood mononuclear cells combined with irradiated or unirradiated epidermal cell suspension. Cell proliferation was assessed by 3H-thymidine incorporation assay. The response of peripheral blood mononuclear cells to unirradiated epidermal cells or unirradiated skin homogenate was similar in both patients and controls. However, peripheral blood mononuclear cells from patients with polymorphous light eruption showed a significantly increased proliferative response to both irradiated epidermal cells and irradiated skin homogenate. Our results indicate that ultraviolet light increases the stimulatory capability of polymorphous light eruption epidermal cells in a unidirectional mixed culture with autologous peripheral blood mononuclear cells. This suggests that an immune sensitization against autologous ultraviolet light-modified skin antigens occurs in polymorphous light eruption

Identification of sequence-related amplified polymorphism markers linked to the red leaf trait in ornamental kale (Brassica oleracea L. var. acephala).

Science.gov (United States)

Wang, Y S; Liu, Z Y; Li, Y F; Zhang, Y; Yang, X F; Feng, H

2013-04-02

Artistic diversiform leaf color is an important agronomic trait that affects the market value of ornamental kale. In the present study, genetic analysis showed that a single-dominant gene, Re (red leaf), determines the red leaf trait in ornamental kale. An F2 population consisting of 500 individuals from the cross of a red leaf double-haploid line 'D05' with a white leaf double-haploid line 'D10' was analyzed for the red leaf trait. By combining bulked segregant analysis and sequence-related amplified polymorphism technology, we identified 3 markers linked to the Re/re locus. A genetic map of the Re locus was constructed using these sequence-related amplified polymorphism markers. Two of the markers, Me8Em4 and Me8Em17, were located on one side of Re/re at distances of 2.2 and 6.4 cM, whereas the other marker, Me9Em11, was located on the other side of Re/re at a distance of 3.7 cM. These markers could be helpful for the subsequent cloning of the red trait gene and marker-assisted selection in ornamental kale breeding programs.

Morphological changes of Paulownia seedlings infected phytoplasmas reveal the genes associated with witches' broom through AFLP and MSAP.

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Xibing Cao

Full Text Available Paulownia witches' broom (PaWB caused by phytoplasma might result in devastating damage to the growth and wood production of Paulownia. To study the effect of phytoplasma on DNA sequence and to discover the genes related to PaWB occurrence, DNA polymorphisms and DNA methylation levels and patterns in PaWB seedlings, the ones treated with various concentration of methyl methane sulfonate (MMS and healthy seedlings were investigated with amplified fragment length polymorphism (AFLP and methylation-sensitive amplification polymorphism (MSAP. Our results indicated that PaWB seedlings recovered a normal morphology, similar to healthy seedlings, after treatment with more than 20 mg · L-1 MMS; Phytoplasma infection did not change the Paulownia genomic DNA sequence at AFLP level, but changed the global DNA methylation levels and patterns; Genes related to PaWB were discovered through MSAP and validated using quantitative real-time PCR (qRT-PCR. These results implied that changes of DNA methylation levels and patterns were closely related to the morphological changes of seedlings infected with phytoplasmas.

GCH1-polymorphism and pain sensitivity among women with provoked vestibulodynia

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Heddini Ulrika

2012-09-01

Full Text Available Abstract Background Provoked vestibulodynia (PVD is a pain disorder localized in the vestibular mucosa. It is the most common cause of dyspareunia among young women and it is associated with general pain hypersensitivity and other chronic pain conditions. Polymorphism in the guanosine triphosphate cyclohydrolase (GCH1 gene has been found to influence general pain sensitivity and the risk of developing a longstanding pain condition. The aim of this study was to investigate GCH1-polymorphism in women with PVD and healthy controls, in correlation to pain sensitivity. Results We found no correlation between the previously defined pain-protective GCH1-SNP combination and the diagnosis of PVD. Nor any correlation with pain sensitivity measured as pressure pain thresholds on the arm, leg and in the vestibule, coital pain scored on a visual analog scale and prevalence of other bodily pain conditions among women with PVD (n = 98 and healthy controls (n = 102. However, among patients with current treatment (n = 36, there was a significant interaction effect of GCH1-gene polymorphism and hormonal contraceptive (HC therapy on coital pain (p = 0.04 as well as on pressure pain thresholds on the arm (p = 0.04. PVD patients carrying the specified SNP combination and using HCs had higher pain sensitivity compared to non-carriers. In non-HC-users, carriers had lower pain sensitivity. Conclusions The results of this study gave no support to the hypothesis that polymorphism in the GCH1-gene contributes to the etiology of PVD. However, among patients currently receiving treatment an interaction effect of the defined SNP combination and use of hormonal contraceptives on pain sensitivity was found. This finding offers a possible explanation to the clinically known fact that some PVD patients improve after cessation of hormonal contraceptives, indicating that PVD patients carrying the defined SNP combination of GCH1 would benefit from this

Blood DNA methylation biomarkers predict clinical reactivity in food-sensitized infants.

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Martino, David; Dang, Thanh; Sexton-Oates, Alexandra; Prescott, Susan; Tang, Mimi L K; Dharmage, Shyamali; Gurrin, Lyle; Koplin, Jennifer; Ponsonby, Anne-Louise; Allen, Katrina J; Saffery, Richard

2015-05-01

The diagnosis of food allergy (FA) can be challenging because approximately half of food-sensitized patients are asymptomatic. Current diagnostic tests are excellent makers of sensitization but poor predictors of clinical reactivity. Thus oral food challenges (OFCs) are required to determine a patient's risk of reactivity. We sought to discover genomic biomarkers of clinical FA with utility for predicting food challenge outcomes. Genome-wide DNA methylation (DNAm) profiling was performed on blood mononuclear cells from volunteers who had undergone objective OFCs, concurrent skin prick tests, and specific IgE tests. Fifty-eight food-sensitized patients (aged 11-15 months) were assessed, half of whom were clinically reactive. Thirteen nonallergic control subjects were also assessed. Reproducibility was assessed in an additional 48 samples by using methylation data from an independent population of patients with clinical FA. Using a supervised learning approach, we discovered a DNAm signature of 96 CpG sites that predict clinical outcomes. Diagnostic scores were derived from these 96 methylation sites, and cutoffs were determined in a sensitivity analysis. Methylation biomarkers outperformed allergen-specific IgE and skin prick tests for predicting OFC outcomes. FA status was correctly predicted in the replication cohort with an accuracy of 79.2%. DNAm biomarkers with clinical utility for predicting food challenge outcomes are readily detectable in blood. The development of this technology in detailed follow-up studies will yield highly innovative diagnostic assays. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

First characterization of Candida albicans by random amplified polymorphic DNA method in Nicaragua and comparison of the diagnosis methods for vaginal candidiasis in Nicaraguan women

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Bello Martha Darce

2002-01-01

Full Text Available A total of 106 women with vaginitis in Nicaragua were studied. The positive rate for the identification of Candida species was 41% (44 positive cultures out of 106 women with vaginitis. The sensitivity of microscopic examination of wet mount with the potassium hydroxide (KOH was 61% and 70% with Gram's stain when using the culture of vaginal fluid as gold standard for diagnosis of candidiasis. Among the 44 positives cultures, isolated species of yeast from vaginal swabs were C. albicans (59%, C. tropicalis (23%, C. glabrata (14% and C. krusei (4%. This study reports the first characterization of 26 C. albicans stocks from Nicaragua by the random amplified polymorphic DNA method. The genetic analysis in this small C. albicans population showed the existence of linkage disequilibrium, which is consistent with the hypothesis that C. albicans undergoes a clonal propagation.

DNA polymorphism of butyrophilin gene by PCR-RFLP technique ...

African Journals Online (AJOL)

We used the polymerase chain reaction-restriction fragment length polymorphism (PCRRFLP) technique to screen for DNA polymorphism in 109 cattle. In all cattle, we amplified an 863 fragment consisting of part of exon 8. The amplified fragment digested with HaeIII restriction endonuclease and subjected to electrophoretic ...

Application of Amplified Fragment Length Polymorphism Fingerprinting for Taxonomy and Identification of the Soft Rot Bacteria Erwinia carotovora and Erwinia chrysanthemi

OpenAIRE

Avrova, Anna O.; Hyman, Lizbeth J.; Toth, Rachel L.; Toth, Ian K.

2002-01-01

The soft rot bacteria Erwinia carotovora and Erwinia chrysanthemi are important pathogens of potato and other crops. However, the taxonomy of these pathogens, particularly at subspecies level, is unclear. An investigation using amplified fragment length polymorphism (AFLP) fingerprinting was undertaken to determine the taxonomic relationships within this group based on their genetic relatedness. Following cluster analysis on the similarity matrices derived from the AFLP gels, four clusters (c...

Analysis of three polymorphisms in Bidayuh ethnic of Sarawak ...

African Journals Online (AJOL)

Insertion/deletion polymorphism of YAP (DYS287), M96 and M120 polymorphisms in Bidayuh ethnic populations of Sarawak, Malaysia were analyzed in this study. Genomic DNA was extracted from 180 buccal samples and amplified by Hot-Start PCR method. The amplified PCR products were separated by using 2% ...

Association Between Genetic Polymorphisms and Pain Sensitivity in Patients with Hip Osteoarthritis

DEFF Research Database (Denmark)

Olesen, Anne E; Nielsen, Lecia M; Feddersen, Søren

2018-01-01

, kappa, and delta opioid receptor genes (OPRM1, OPRK1, and OPRD1) and the catechol-O-methyltransferase gene (COMT) influenced the pain phenotype in patients with osteoarthritis. METHODS: The frequencies of 17 polymorphisms were examined. Pain sensitivity was assessed preoperatively by (1) hip rotation......BACKGROUND: Factors such as age, gender, and genetic polymorphisms may explain individual differences in pain phenotype. Genetic associations with pain sensitivity have previously been investigated in osteoarthritis patients, with a focus on the P2X7, TRPV1, and TACR1 genes. However, other genes...... may play a role as well. Osteoarthritis is a common joint disease, and many patients suffering from this disease are thought to have increased sensitivity to noxious stimuli resulting from sensitization in the nociceptive system. The aim of this study was to investigate if genetic variants of mu...

Wide bandwidth transimpedance amplifier for extremely high sensitivity continuous measurements.

Science.gov (United States)

Ferrari, Giorgio; Sampietro, Marco

2007-09-01

This article presents a wide bandwidth transimpedance amplifier based on the series of an integrator and a differentiator stage, having an additional feedback loop to discharge the standing current from the device under test (DUT) to ensure an unlimited measuring time opportunity when compared to switched discharge configurations while maintaining a large signal amplification over the full bandwidth. The amplifier shows a flat response from 0.6 Hz to 1.4 MHz, the capability to operate with leakage currents from the DUT as high as tens of nanoamperes, and rail-to-rail dynamic range for sinusoidal current signals independent of the DUT leakage current. Also available is a monitor output of the stationary current to track experimental slow drifts. The circuit is ideal for noise spectral and impedance measurements of nanodevices and biomolecules when in the presence of a physiological medium and in all cases where high sensitivity current measurements are requested such as in scanning probe microscopy systems.

Inter simple sequence repeats (ISSR) and random amplified ...

African Journals Online (AJOL)

21 of 30 random amplified polymorphic DNA (RAPD) primers produced 220 reproducible bands with average of 10.47 bands per primer and 80.12% of polymorphism. OPR02 primer showed the highest number of effective allele (Ne), Shannon index (I) and genetic diversity (H). Some of the cultivars had specific bands, ...

Genetic diversity of Santalum album using random amplified ...

African Journals Online (AJOL)

In the present study Random Amplified Polymorphic DNA (RAPD) technique was used to accesses the genetic diversity among 30 accessions of S. album collected from different parts of South India. A total of 248 polymorphic amplicons were obtained from 30 primers. The value of Jaccard coefficient ranged from 0.66 to ...

Assessment of genetic and epigenetic variation in hop plants regenerated from sequential subcultures of organogenic calli.

Science.gov (United States)

Peredo, Elena L; Revilla, M Angeles; Arroyo-García, Rosa

2006-10-01

Organogenic calli induced from internodal segments were subcultured three times. Regenerated plants obtained from each subculture were analysed by molecular methods. No major genetic rearrangements were detected in the callus-derived plants since none of the amplified fragment-length polymorphism (AFLP) loci were found to be polymorphic. However, epigenetic changes due to a demethylation process were detected by methylation-sensitive amplified polymorphism (MSAP) technique. The results allowed inference of the possible relationship among the plants derived from different calli subcultures and the in vitro control. The plants recovered from the first and second callus subcultures clustered with the in vitro control pools in the phenogram while the regenerants from the third callus subculture showed the highest genetic distance with the controls. This is the first study reporting data about the genetic stability of callus-derived Humulus lupulus L. plants.

Characterization of in vitro haploid and doubled haploid Chrysanthemum morifolium plants via unfertilized ovule culture for phenotypical traits and DNA methylation pattern.

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Haibin eWang

2014-12-01

Full Text Available Chrysanthemum is one of important ornamental species in the world. Its highly heterozygous state complicates molecular analysis, so it is of interest to derive haploid forms. A total of 2,579 non-fertilized chrysanthemum ovules pollinated by Argyranthemum frutescens were cultured in vitro to isolate haploid progeny. One single regenerant emerged from each of three of the 105 calli produced. Chromosome counts and microsatellite fingerprinting showed that only one of the regenerants was a true haploid. Nine doubled haploid derivatives were subsequently generated by colchicine treatment of 80 in vitro cultured haploid nodal segments. Morphological screening showed that the haploid plant was shorter than the doubled haploids, and developed smaller leaves, flowers and stomata. An in vitro pollen germination test showed that few of the haploid's pollen were able to germinate and those which did so were abnormal. Both the haploid and the doubled haploids produced yellow flowers, whereas those of the maternal parental cultivar were mauve. Methylation-sensitive amplification polymorphism (MSAP profiling was further used to detect alterations in cytosine methylation caused by the haploidization and/or the chromosome doubling processes. While 52.2% of the resulting amplified fragments were cytosine methylated in the maternal parent's genome, the corresponding proportions for the haploid's and doubled haploids' genomes were, respectively, 47.0% and 51.7%, demonstrating a reduction in global cytosine methylation caused by haploidization and a partial recovery following chromosome doubling.

Genotyping of Campylobacter jejuni from broiler carcasses and slaughterhouse environment by amplified fragment length polymorphism.

Science.gov (United States)

Johnsen, G; Kruse, H; Hofshagen, M

2006-12-01

We examined the occurrence and diversity of Campylobacter jejuni on broiler carcasses during slaughter of an infected flock and in the slaughterhouse environment during slaughter and postdisinfection before a new production run. During the slaughter of a known C. jejuni infected broiler flock, samples were taken from broiler carcasses at 7 different stages during the process. Thirty-seven sites in the slaughterhouse environment were sampled both during process and postdisinfection. The samples were analyzed for C. jejuni, and genetic fingerprinting was performed using amplified fragment length polymorphism. All carcass samples were positive. Of the environmental samples collected during slaughter, 89% were positive; 100% of those from the arrival, stunning, scalding, defeathering, and evisceration facilities and 67% of those from the cooling and sorting facilities. Postdisinfection, 41% of the samples were positive; 71% of those from the arrival and stunning area, 60% of those from the scalding and defeathering area, and 20% of those from the evisceration, cooling, and sorting area. The C. jejuni isolates (n = 60) recovered were grouped into 4 different amplified fragment length polymorphism clones with a similarity index of 95% or greater. All isolates obtained from the flock and 94% of the isolates obtained from the environment during slaughtering belonged to clone A, whereas 1 environmental isolate belonged to each of the clones B and C. Isolates from clones A, B, and D were present postdisinfection. Only clone B was detected on flocks slaughtered during the previous week. The high level and continuous presence of Campylobacter in the environment constitutes a risk for transmission to negative carcasses. In Norway, where above 96% of the broiler flocks are Campylobacter-negative, this aspect is of special importance. The ability of Campylobacter to remain in the slaughterhouse environment through washing and disinfection is associated with constructional

Identification and characterization of some aromatic rice mutants using amplified fragment length polymorphism (AFLP) technique

International Nuclear Information System (INIS)

Fahmy, E.M.; Sobieh, S. E. S.; Ayaad, M. H.; El-Gohary, A. A.; Rownak, A.

2012-12-01

Accurate identifying of the genotypes is considered one of the most important mechanisms used in the recording or the protection of plant varieties. The investigation was conducted at the experimental form belonging to the egyptian Atomic Energy Authority, Inshas. The aim was to evaluate grain quality characteristics and molecular genetic variation using Amplified Fragment Length Polymorphism (AFLP) technique among six rice genotypes, Egyptian Jasmine aromatic rice cultivar and five aromatic rice mutants in (M3 mutagenic generation). Two mutation (Egy22 and Egy24) were selected from irradiated Sakha 102 population with 200 and 400Gy of gamma rays in the M2 generation, respectively, and three mutations ( Egy32, Egy33, and Egy34) were selected from irradiated Sakha 103 population with 200, 300, 400Gy of gamma rays in the M2 generation, respectively. The obtained results showed that the strong aroma was obtained for mutant Egy22 as compared with Egyptian Jasmine rice cultivar (moderate aroma). Seven primer combinations were used through six rice genotypes on the molecular level using AFLP marker. The size of AFLP Fragments Were Ranged from 51- 494bp. The total number of amplified bands was 997 band among them 919 polymorphic bans representing 92.2%. The highest similarity index (89%) was observed between Egyptian Jasmine and Egy32 followed by (82%) observed between Egyptian Jasmine and Egy34. On the other hand, the lowest similarity index was (48%) between Egyptian Jasmine and Egy24. In six rice genotypes, Egy24 produced the highest number of the AFLP makers giving 49 unique markers (23 positive and 26 negative), then Egy22 showed 23 unique markers (27 positive and 6 negative) while Egy33 was characterized by 17 unique markers (12 positive and 5 negative). At last Egyptian Jasmine was discriminated by the lowest number of markets, 10 (6 positive and 4 negative). The study further confirmed that AFLP technique was able to differentiate rice genotypes by a higher number

Random amplified polymorphic DNA analysis of Anopheles nuneztovari (Diptera: Culicidae from Western and Northeastern Colombia

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Carmen Elisa Posso

2003-06-01

Full Text Available Random amplified polymorphic DNA (RAPD markers were used to analyze 119 DNA samples of three Colombian Anopheles nuneztovari populations to study genetic variation and structure. Genetic diversity, estimated from heterozygosity, averaged 0.34. Genetic flow was greater between the two populations located in Western Colombia (F ST: 0.035; Nm: 6.8 but lower between these two and the northeastern population (F ST: 0.08; Nm: 2.8. According to molecular variance analysis, the genetic distance between populations was significant (phiST 0.1131, P < 0.001. The variation among individuals within populations (phiST 0.8869, P < 0.001was also significant, suggesting a greater degree of population subdivision, not considered in this study. Both the parameters evaluated and the genetic flow suggest that Colombian An. nuneztovari populations are co-specific.

Evolution of finger millet: evidence from random amplified polymorphic DNA.

Science.gov (United States)

Hilu, K W

1995-04-01

Finger millet (Eleusine coracana ssp. coracana) is an annual tetraploid member of a predominantly African genus. The crop is believed to have been domesticated from the tetraploid E. coracana ssp. africana. Cytogenetic and isozyme data point to the allopolyploid nature of the species and molecular information has shown E. indica to be one of the genomic donors. A recent isozyme study questioned the proposed phylogenetic relationship between finger millet and its direct ancestor subspecies africana. An approach using random amplified polymorphic DNA (RAPD) was employed in this study to examine genetic diversity and to evaluate hypotheses concerning the evolution of domesticated and wild annual species of Eleusine. Unlike previous molecular approaches, the RAPD study revealed genetic diversity in the crop. The pattern of genetic variation was loosely correlated to geographic distribution. The allotetraploid nature of the crop was confirmed and molecular markers that can possibly identify the other genomic donor were proposed. Genotypes of subspecies africana did not group closely with those of the crop but showed higher affinities to E. indica, reflecting the pattern of similarity revealed by the isozyme study. The multiple origin of subspecies africana could explain the discrepancy between the isozyme-RAPD evidence and previous information. The RAPD study showed the close genetic affinity of E. tristachya to the E. coracana--E. indica group and understood the distinctness of E. multiflora.

Genotyping via Sequence Related Amplified Polymorphism Markers in Fusarium culmorum

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Işıl Melis Zümrüt

2018-02-01

Full Text Available Fusarium culmorum is predominating causal agent of head blight (HB and root rot (RR in cereals worldwide. Since F. culmorum has a great level of genetic diversity and the parasexual stage is assumed for this phytopathogen, characterization of isolates from different regions is significant step in food safety and controlling the HB. In this study, it was aimed to characterize totally 37 F. culmorum isolates from Turkey via sequence related amplified polymorphism (SRAP marker based genotyping. MAT-1/MAT-2 type assay was also used in order to reveal intraspecific variation in F. culmorum. MAT-1 and MAT-2 specific primer pairs for mating assays resulted in 210 and 260 bp bands, respectively. 11 of isolates were belonged to MAT-1 type whereas 19 samples were of MAT-2. Remaining 7 samples yielded both amplicons. Totally 9 SRAP primer sets yielded amplicons from all isolates. Genetic similarity values were ranged from 39 to 94.7%. Total band number was 127 and PCR product sizes were in the range of 0.1-2.5 kb. Amplicon numbers for individuals were ranged from 1 to 16. According to data obtained from current study, SRAP based genotyping is powerful tool for supporting the data obtained from investigations including phenotypic and agro-ecological characteristics. Findings showed that SRAP-based markers could be useful in F. culmorum characterization.

Chilling-Mediated DNA Methylation Changes during Dormancy and Its Release Reveal the Importance of Epigenetic Regulation during Winter Dormancy in Apple (Malus x domestica Borkh.).

Science.gov (United States)

Kumar, Gulshan; Rattan, Usha Kumari; Singh, Anil Kumar

2016-01-01

Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover, significant association

Chilling-Mediated DNA Methylation Changes during Dormancy and Its Release Reveal the Importance of Epigenetic Regulation during Winter Dormancy in Apple (Malus x domestica Borkh..

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Gulshan Kumar

Full Text Available Winter dormancy is a well known mechanism adopted by temperate plants, to mitigate the chilling temperature of winters. However, acquisition of sufficient chilling during winter dormancy ensures the normal phenological traits in subsequent growing period. Thus, low temperature appears to play crucial roles in growth and development of temperate plants. Apple, being an important temperate fruit crop, also requires sufficient chilling to release winter dormancy and normal phenological traits, which are often associated with yield and quality of fruits. DNA cytosine methylation is one of the important epigenetic modifications which remarkably affect the gene expression during various developmental and adaptive processes. In present study, methylation sensitive amplified polymorphism was employed to assess the changes in cytosine methylation during dormancy, active growth and fruit set in apple, under differential chilling conditions. Under high chill conditions, total methylation was decreased from 27.2% in dormant bud to 21.0% in fruit set stage, while no significant reduction was found under low chill conditions. Moreover, the demethylation was found to be decreased, while methylation increased from dormant bud to fruit set stage under low chill as compared to high chill conditions. In addition, RNA-Seq analysis showed high expression of DNA methyltransferases and histone methyltransferases during dormancy and fruit set, and low expression of DNA glcosylases during active growth under low chill conditions, which was in accordance with changes in methylation patterns. The RNA-Seq data of 47 genes associated with MSAP fragments involved in cellular metabolism, stress response, antioxidant system and transcriptional regulation showed correlation between methylation and their expression. Similarly, bisulfite sequencing and qRT-PCR analysis of selected genes also showed correlation between gene body methylation and gene expression. Moreover

A combined HM-PCR/SNuPE method for high sensitive detection of rare DNA methylation

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Tierling Sascha

2010-06-01

Full Text Available Abstract Background DNA methylation changes are widely used as early molecular markers in cancer detection. Sensitive detection and classification of rare methylation changes in DNA extracted from circulating body fluids or complex tissue samples is crucial for the understanding of tumor etiology, clinical diagnosis and treatment. In this paper, we describe a combined method to monitor the presence of methylated tumor DNA in an excess of unmethylated background DNA of non-tumorous cells. The method combines heavy methyl-PCR, which favors preferential amplification of methylated marker sequence from bisulfite-treated DNA with a methylation-specific single nucleotide primer extension monitored by ion-pair, reversed-phase, high-performance liquid chromatography separation. Results This combined method allows detection of 14 pg (that is, four to five genomic copies of methylated chromosomal DNA in a 2000-fold excess (that is, 50 ng of unmethylated chromosomal background, with an analytical sensitivity of > 90%. We outline a detailed protocol for the combined assay on two examples of known cancer markers (SEPT9 and TMEFF2 and discuss general aspects of assay design and data interpretation. Finally, we provide an application example for rapid testing on tumor methylation in plasma DNA derived from a small cohort of patients with colorectal cancer. Conclusion The method allows unambiguous detection of rare DNA methylation, for example in body fluid or DNA isolates from cells or tissues, with very high sensitivity and accuracy. The application combines standard technologies and can easily be adapted to any target region of interest. It does not require costly reagents and can be used for routine screening of many samples.

16 channel WDM regeneration in a single phase-sensitive amplifier through optical Fourier transformation

DEFF Research Database (Denmark)

Guan, Pengyu; Da Ros, Francesco; Lillieholm, Mads

2016-01-01

We demonstrate simultaneous phase regeneration of 16-WDM DPSK channels using optical Fourier transformation and a single phase-sensitive amplifier. The BERs of 16-WDM×10-Gbit/s phase noise degraded DPSK signals are improved by 0.4-1.3 orders of magnitude...

Epigenetic Variance, Performing Cooperative Structure with Genetics, Is Associated with Leaf Shape Traits in Widely Distributed Populations of Ornamental Tree Prunus mume

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Kaifeng Ma

2018-01-01

Full Text Available Increasing evidence shows that epigenetics plays an important role in phenotypic variance. However, little is known about epigenetic variation in the important ornamental tree Prunus mume. We used amplified fragment length polymorphism (AFLP and methylation-sensitive amplified polymorphism (MSAP techniques, and association analysis and sequencing to investigate epigenetic variation and its relationships with genetic variance, environment factors, and traits. By performing leaf sampling, the relative total methylation level (29.80% was detected in 96 accessions of P. mume. And the relative hemi-methylation level (15.77% was higher than the relative full methylation level (14.03%. The epigenetic diversity (I∗ = 0.575, h∗ = 0.393 was higher than the genetic diversity (I = 0.484, h = 0.319. The cultivated population displayed greater epigenetic diversity than the wild populations in both southwest and southeast China. We found that epigenetic variance and genetic variance, and environmental factors performed cooperative structures, respectively. In particular, leaf length, width and area were positively correlated with relative full methylation level and total methylation level, indicating that the DNA methylation level played a role in trait variation. In total, 203 AFLP and 423 MSAP associated markers were detected and 68 of them were sequenced. Homologous analysis and functional prediction suggested that the candidate marker-linked genes were essential for leaf morphology development and metabolism, implying that these markers play critical roles in the establishment of leaf length, width, area, and ratio of length to width.

Epigenetic Variance, Performing Cooperative Structure with Genetics, Is Associated with Leaf Shape Traits in Widely Distributed Populations of Ornamental Tree Prunus mume.

Science.gov (United States)

Ma, Kaifeng; Sun, Lidan; Cheng, Tangren; Pan, Huitang; Wang, Jia; Zhang, Qixiang

2018-01-01

Increasing evidence shows that epigenetics plays an important role in phenotypic variance. However, little is known about epigenetic variation in the important ornamental tree Prunus mume . We used amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques, and association analysis and sequencing to investigate epigenetic variation and its relationships with genetic variance, environment factors, and traits. By performing leaf sampling, the relative total methylation level (29.80%) was detected in 96 accessions of P . mume . And the relative hemi-methylation level (15.77%) was higher than the relative full methylation level (14.03%). The epigenetic diversity ( I ∗ = 0.575, h ∗ = 0.393) was higher than the genetic diversity ( I = 0.484, h = 0.319). The cultivated population displayed greater epigenetic diversity than the wild populations in both southwest and southeast China. We found that epigenetic variance and genetic variance, and environmental factors performed cooperative structures, respectively. In particular, leaf length, width and area were positively correlated with relative full methylation level and total methylation level, indicating that the DNA methylation level played a role in trait variation. In total, 203 AFLP and 423 MSAP associated markers were detected and 68 of them were sequenced. Homologous analysis and functional prediction suggested that the candidate marker-linked genes were essential for leaf morphology development and metabolism, implying that these markers play critical roles in the establishment of leaf length, width, area, and ratio of length to width.

Genetic variations of Lansium domesticum Corr. accessions from Java, Sumatra and Ceram based on Random Amplified Polymorphic DNA fingerprints

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KUSUMADEWI SRI YULITA

2011-07-01

Full Text Available Yulita KS (2011 Genetic variations of Lansium domesticum Corr. accessions from Java, Bengkulu and Ceram based on Random Amplified Polymorphic DNA fingerprints. Biodiversitas 12: 125-130. Duku (Lansium domesticum Corr. is one of popular tropical fruits in SE Asia. The spesies has three varieties, known as duku, langsat and kokosan; and duku is the most popular one for being the sweetiest fruit. Indonesia has several local varieties of duku, such as duku Condet, duku Sumber and duku Palembang. This present study aimed to assess genetic diversity of 47 accessions of duku from Java, Sumatra, and Ceram based on RAPD fingerprints. Ten RAPD’s primers were initially screened and five were selected for the analysis. These five primers (OPA 7, 13, 18, OPB 7, and OPN 12 generated 53 scorable bands with an average of 10.6 polymorphic fragment per primer. Percentage of polymorphism ranged from 16.89% (OPA 7 and OPN 12 to 24.54% (OPB 7 with an average of 20.16% polymorphism. OPB 7 at 450 bp was exclusively possessed by accession 20 (Java, OPA 18 at 500 bp was by accession 6 (Java, 550 bp by 3 clones from Bengkulu. While OPN 12 at 300 bp and OPA 13 at 450 bp were shared among the accessions. Clustering analysis was performed based on RAPD profiles using the UPGMA method. The range of genetic similarity value among accessions was 0.02-0.65 suggesting high variation of gene pool existed among accessions.

Development of cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers from the chloroplast genome of Glycyrrhiza species.

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Jo, Ick-Hyun; Sung, Jwakyung; Hong, Chi-Eun; Raveendar, Sebastin; Bang, Kyong-Hwan; Chung, Jong-Wook

2018-05-01

Licorice ( Glycyrrhiza glabra ) is an important medicinal crop often used as health foods or medicine worldwide. The molecular genetics of licorice is under scarce owing to lack of molecular markers. Here, we have developed cleaved amplified polymorphic sequence (CAPS) and high-resolution melting (HRM) markers based on single nucleotide polymorphisms (SNP) by comparing the chloroplast genomes of two Glycyrrhiza species ( G. glabra and G. lepidota ). The CAPS and HRM markers were tested for diversity analysis with 24 Glycyrrhiza accessions. The restriction profiles generated with CAPS markers classified the accessions (2-4 genotypes) and melting curves (2-3) were obtained from the HRM markers. The number of alleles and major allele frequency were 2-6 and 0.31-0.92, respectively. The genetic distance and polymorphism information content values were 0.16-0.76 and 0.15-0.72, respectively. The phylogenetic relationships among the 24 accessions were estimated using a dendrogram, which classified them into four clades. Except clade III, the remaining three clades included the same species, confirming interspecies genetic correlation. These 18 CAPS and HRM markers might be helpful for genetic diversity assessment and rapid identification of licorice species.

γ-radiation induces cellular sensitivity and aberrant methylation in human tumor cell lines.

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Kumar, Ashok; Rai, Padmalatha S; Upadhya, Raghavendra; Vishwanatha; Prasada, K Shama; Rao, B S Satish; Satyamoorthy, Kapettu

2011-11-01

Ionizing radiation induces cellular damage through both direct and indirect mechanisms, which may include effects from epigenetic changes. The purpose of this study was to determine the effect of ionizing radiation on DNA methylation patterns that may be associated with altered gene expression. Sixteen human tumor cell lines originating from various cancers were initially tested for radiation sensitivity by irradiating them with γ-radiation in vitro and subsequently, radiation sensitive and resistant cell lines were treated with different doses of a demethylating agent, 5-Aza-2'-Deoxycytidine (5-aza-dC) and a chromatin modifier, Trichostatin-A (TSA). Survival of these cell lines was measured using 3-(4, 5-Dimethylthiazol- 2-yl)-2, 5-diphenyltetrazolium (MTT) and clonogenic assays. The effect of radiation on global DNA methylation was measured using reverse phase high performance liquid chromatography (RP-HPLC). The transcription response of methylated gene promoters, from cyclin-dependent kinase inhibitor 2A (p16(INK4a)) and ataxia telangiectasia mutated (ATM) genes, to radiation was measured using a luciferase reporter assay. γ-radiation resistant (SiHa and MDAMB453) and sensitive (SaOS2 and WM115) tumor cell lines were examined for the relationship between radiation sensitivity and DNA methylation. Treatment of cells with 5-aza-dC and TSA prior to irradiation enhanced DNA strand breaks, G2/M phase arrest, apoptosis and cell death. Exposure to γ-radiation led to global demethylation in a time-dependent manner in tumor cells in relation to resistance and sensitivity to radiation with concomitant activation of p16(INK4a) and ATM gene promoters. These results provide important information on alterations in DNA methylation as one of the determinants of radiation effects, which may be associated with altered gene expression. Our results may help in delineating the mechanisms of radiation resistance in tumor cells, which can influence diagnosis, prognosis and

Molecular Diversity of Antagonistic Streptomyces spp. against Botrytis allii, the agent of onion gray mold using Random Amplified Polymorphic DNA (RAPD Markers

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M. Jorjandi

2014-08-01

Full Text Available As an aim in sustainable agriculture, biological control of plant diseases has received intensive attention mainly as a response to public concern about the use of chemical fungicides in the environment. Soil Actinomycetes particularly Streptomyces spp. enhance soil fertility and have antagonistic activity against wide range of plant pathogens. To investigate for biocontrol means against the pathogen, 30 isolates of Actinomycetes have been isolated from agricultural soils of Kerman province of Iran and assayed for antagonistic activity against Botrytis allii, the agent of onion gray mold. RAPD DNA analysis has been used to determine the relatedness of active and non-active isolates based on their RAPD-PCR fingerprints. PCR amplifiable DNA samples have been isolated using the CTAB method and amplified fragments have been obtained from 5 random 10-mer primers. Different DNA fingerprinting patterns have been obtained for all of the isolates. Electrophoretic and cluster analysis of the amplification products has revealed incidence of polymorphism among the isolates. A total of 138 bands, ranging in size from 150-2800 bp, have been amplified from primers which 63.7% of the observed bands have been polymorphic. Genetic distances among different varieties have been analyzed with a UPGMA (Unweighted pair-group method, arithmetic average-derived dendrogram. Resulting dendrogram has showed from 0.65 to 0.91 similarities among varieties and divided the isolates into five major groups. Isolates which haven’t had any antagonistic activity against B. allii have been separated into a group and other isolates classified into four groups. The results indicate that RAPD is an efficient method for discriminating and studying genetic diversity of Streptomyces isolates.

Methylation of the ATM promoter in glioma cells alters ionizing radiation sensitivity

International Nuclear Information System (INIS)

Roy, Kanaklata; Wang, Lilin; Makrigiorgos, G. Mike; Price, Brendan D.

2006-01-01

Glioblastomas are among the malignancies most resistant to radiation therapy. In contrast, cells lacking the ATM protein are highly sensitive to ionizing radiation. The relationship between ATM protein expression and radiosensitivity in 3 glioma cell lines was examined. T98G cells exhibited normal levels of ATM protein, whereas U118 and U87 cells had significantly lower levels of ATM and increased (>2-fold) sensitivity to ionizing radiation compared to T98G cells. The ATM promoter was methylated in U87 cells. Demethylation by azacytidine treatment increased ATM protein levels in the U87 cells and decreased their radiosensitivity. In contrast, the ATM promoter in U118 cells was not methylated. Further, expression of exogenous ATM did not significantly alter the radiosensitivity of U118 cells. ATM expression is therefore heterogeneous in the glioma cells examined. In conclusion, methylation of the ATM promoter may account for the variable radiosensitivity and heterogeneous ATM expression in a fraction of glioma cells

Comparison of Ribotyping, Randomly Amplified Polymorphic DNA Analysis, and Pulsed-Field Gel Electrophoresis in Typing of Lactobacillus rhamnosus and L. casei Strains

OpenAIRE

Tynkkynen, Soile; Satokari, Reetta; Saarela, Maria; Mattila-Sandholm, Tiina; Saxelin, Maija

1999-01-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 33...

Spectral sensitivities and color signals in a polymorphic damselfly.

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Shao-chang Huang

Full Text Available Animal communication relies on conspicuous signals and compatible signal perception abilities. Good signal perception abilities are particularly important for polymorphic animals where mate choice can be a challenge. Behavioral studies suggest that polymorphic damselflies use their varying body colorations and/or color patterns as communication signal for mate choice and to control mating frequencies. However, solid evidence for this hypothesis combining physiological with spectral and behavioral data is scarce. We investigated this question in the Australian common blue tail damselfly, Ischnura heterosticta, which has pronounced female-limited polymorphism: andromorphs have a male-like blue coloration and gynomorphs display green/grey colors. We measured body color reflectance and investigated the visual capacities of each morph, showing that I. heterosticta have at least three types of photoreceptors sensitive to UV, blue, and green wavelength, and that this visual perception ability enables them to detect the spectral properties of the color signals emitted from the various color morphs in both males and females. We further demonstrate that different color morphs can be discriminated against each other and the vegetation based on color contrast. Finally, these findings were supported by field observations of natural mating pairs showing that mating partners are indeed chosen based on their body coloration. Our study provides the first comprehensive evidence for the function of body coloration on mate choice in polymorphic damselflies.

Methyl methacrylate and respiratory sensitization: A Critical review

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Borak, Jonathan; Fields, Cheryl; Andrews, Larry S; Pemberton, Mark A

2011-01-01

Methyl methacrylate (MMA) is a respiratory irritant and dermal sensitizer that has been associated with occupational asthma in a small number of case reports. Those reports have raised concern that it might be a respiratory sensitizer. To better understand that possibility, we reviewed the in silico, in chemico, in vitro, and in vivo toxicology literature, and also epidemiologic and occupational medicine reports related to the respiratory effects of MMA. Numerous in silico and in chemico studies indicate that MMA is unlikely to be a respiratory sensitizer. The few in vitro studies suggest that MMA has generally weak effects. In vivo studies have documented contact skin sensitization, nonspecific cytotoxicity, and weakly positive responses on local lymph node assay; guinea pig and mouse inhalation sensitization tests have not been performed. Cohort and cross-sectional worker studies reported irritation of eyes, nose, and upper respiratory tract associated with short-term peaks exposures, but little evidence for respiratory sensitization or asthma. Nineteen case reports described asthma, laryngitis, or hypersensitivity pneumonitis in MMA-exposed workers; however, exposures were either not well described or involved mixtures containing more reactive respiratory sensitizers and irritants.The weight of evidence, both experimental and observational, argues that MMA is not a respiratory sensitizer. PMID:21401327

Novel fluorescent sequence-related amplified polymorphism(FSRAP markers for the construction of a genetic linkage map of wheat(Triticum aestivum L.

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Zhao Lingbo

2017-01-01

Full Text Available Novel fluorescent sequence-related amplified polymorphism (FSRAP markers were developed based on the SRAP molecular marker. Then, the FSRAP markers were used to construct the genetic map of a wheat (Triticum aestivumL. recombinant inbred line population derived from a Chuanmai 42×Chuannong 16 cross. Reproducibility and polymorphism tests indicated that the FSRAP markers have repeatability and better reflect the polymorphism of wheat varieties compared with SRAP markers. A total of 430 polymorphic loci between Chuanmai 42 and Chuannong 16 were detected with 189 FSRAP primer combinations. A total of 281 FSARP markers and 39 SSR markers re classified into 20 linkage groups. The maps spanned a total length of 2499.3cM with an average distance of 7.81cM between markers. A total of 201 markers were mapped on the B genome and covered a distance of 1013cM. On the A genome, 84 markers were mapped and covered a distance of 849.6cM. On the D genome, however, only 35 markers were mapped and covered a distance of 636.7cM. No FSRAP markers were distributed on the 7D chromosome. The results of the present study revealed that the novel FSRAP markers can be used to generate dense, uniform genetic maps of wheat.

Application of randomly amplified polymorphic DNA (RAPD ...

African Journals Online (AJOL)

Jane

2011-10-10

Oct 10, 2011 ... and T7-010 based on functional markers according to He et al. (2007). These primers were constructed by Invitrogen GmbH,. Karlsruhe, Germany, and used to amplify the polyphenol oxidases genes. The sequences of these primers were as follows: T3-001: 5`-CCA TTA ACC CTC ACT AAA GGG ACC GTA ...

Restriction fragment length polymorphism (RFLP) analysis of PCR products amplified from 18S ribosomal RNA gene of Trypanosoma congolense

International Nuclear Information System (INIS)

Osanyo, A.; Majiwa, P.W.

2006-01-01

Oligonucleotide primers were designed from the conserved nucleotide sequences of 18S ribosomal RNA (18S rRNA) gene of protozoans: Trypanosoma brucei, Leishmania donovani, Triponema aequale and Lagenidium gigantum. The primers were used in polymerace chain reaction (PCR) to generate PCR products of approximately 1 Kb using genomic DNA from T. brucei and the four genotypic groups of T. congolense as template. The five PCR products so produced were digested with several restriction enzymes and hybridized to a DNA probe made from T. brucei PCR product of the same 18S rRNA gene region. Most restriction enzyme digests revealed polymorphism with respect to the location of their recognition sites on the five PCR products. The restriction fragment length polymorphism (RFLP) pattern observed indicate that the 18S rRNA gene sequences of trypanosomes: T. brucei and the four genotypes of T.congolence group are heterogeneous. The results further demonstrate that the region that was amplified can be used in specific identification of trypanosomes species and subspecies.(author)

Mass amplifying probe for sensitive fluorescence anisotropy detection of small molecules in complex biological samples.

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Cui, Liang; Zou, Yuan; Lin, Ninghang; Zhu, Zhi; Jenkins, Gareth; Yang, Chaoyong James

2012-07-03

Fluorescence anisotropy (FA) is a reliable and excellent choice for fluorescence sensing. One of the key factors influencing the FA value for any molecule is the molar mass of the molecule being measured. As a result, the FA method with functional nucleic acid aptamers has been limited to macromolecules such as proteins and is generally not applicable for the analysis of small molecules because their molecular masses are relatively too small to produce observable FA value changes. We report here a molecular mass amplifying strategy to construct anisotropy aptamer probes for small molecules. The probe is designed in such a way that only when a target molecule binds to the probe does it activate its binding ability to an anisotropy amplifier (a high molecular mass molecule such as protein), thus significantly increasing the molecular mass and FA value of the probe/target complex. Specifically, a mass amplifying probe (MAP) consists of a targeting aptamer domain against a target molecule and molecular mass amplifying aptamer domain for the amplifier protein. The probe is initially rendered inactive by a small blocking strand partially complementary to both target aptamer and amplifier protein aptamer so that the mass amplifying aptamer domain would not bind to the amplifier protein unless the probe has been activated by the target. In this way, we prepared two probes that constitute a target (ATP and cocaine respectively) aptamer, a thrombin (as the mass amplifier) aptamer, and a fluorophore. Both probes worked well against their corresponding small molecule targets, and the detection limits for ATP and cocaine were 0.5 μM and 0.8 μM, respectively. More importantly, because FA is less affected by environmental interferences, ATP in cell media and cocaine in urine were directly detected without any tedious sample pretreatment. Our results established that our molecular mass amplifying strategy can be used to design aptamer probes for rapid, sensitive, and selective

Experimental implementation of a nonlinear beamsplitter based on a phase-sensitive parametric amplifier

Energy Technology Data Exchange (ETDEWEB)

Fang, Yami; Feng, Jingliang; Cao, Leiming; Wang, Yaxian; Jing, Jietai, E-mail: jtjing@phy.ecnu.edu.cn [State Key Laboratory of Precision Spectroscopy, East China Normal University, Shanghai 200062 (China)

2016-03-28

Beamsplitters have played an important role in quantum optics experiments. They are often used to split and combine two beams, especially in the construct of an interferometer. In this letter, we experimentally implement a nonlinear beamsplitter using a phase-sensitive parametric amplifier, which is based on four-wave mixing in hot rubidium vapor. Here we show that, despite the different frequencies of the two input beams, the output ports of the nonlinear beamsplitter exhibit interference phenomena. We make measurements of the interference fringe visibility and study how various parameters, such as the intensity gain of the amplifier, the intensity ratio of the two input beams, and the one and two photon detunings, affect the behavior of the nonlinear beamsplitter. It may find potential applications in quantum metrology and quantum information processing.

Investigation of the Sensitivity of Transmission Raman Spectroscopy for Polymorph Detection in Pharmaceutical Tablets.

Science.gov (United States)

Feng, Hanzhou; Bondi, Robert W; Anderson, Carl A; Drennen, James K; Igne, Benoît

2017-08-01

Polymorph detection is critical for ensuring pharmaceutical product quality in drug substances exhibiting polymorphism. Conventional analytical techniques such as X-ray powder diffraction and solid-state nuclear magnetic resonance are utilized primarily for characterizing the presence and identity of specific polymorphs in a sample. These techniques have encountered challenges in analyzing the constitution of polymorphs in the presence of other components commonly found in pharmaceutical dosage forms. Laborious sample preparation procedures are usually required to achieve satisfactory data interpretability. There is a need for alternative techniques capable of probing pharmaceutical dosage forms rapidly and nondestructively, which is dictated by the practical requirements of applications such as quality monitoring on production lines or when quantifying product shelf lifetime. The sensitivity of transmission Raman spectroscopy for detecting polymorphs in final tablet cores was investigated in this work. Carbamazepine was chosen as a model drug, polymorph form III is the commercial form, whereas form I is an undesired polymorph that requires effective detection. The concentration of form I in a direct compression tablet formulation containing 20% w/w of carbamazepine, 74.00% w/w of fillers (mannitol and microcrystalline cellulose), and 6% w/w of croscarmellose sodium, silicon dioxide, and magnesium stearate was estimated using transmission Raman spectroscopy. Quantitative models were generated and optimized using multivariate regression and data preprocessing. Prediction uncertainty was estimated for each validation sample by accounting for all the main variables contributing to the prediction. Multivariate detection limits were calculated based on statistical hypothesis testing. The transmission Raman spectroscopic model had an absolute prediction error of 0.241% w/w for the independent validation set. The method detection limit was estimated at 1.31% w/w. The

Rapid Genetic and Epigenetic Alterations under Intergeneric Genomic Shock in Newly Synthesized Chrysanthemum morifolium × Leucanthemum paludosum Hybrids (Asteraceae)

Science.gov (United States)

Wang, Haibin; Jiang, Jiafu; Chen, Sumei; Qi, Xiangyu; Fang, Weimin; Guan, Zhiyong; Teng, Nianjun; Liao, Yuan; Chen, Fadi

2014-01-01

The Asteraceae family is at the forefront of the evolution due to frequent hybridization. Hybridization is associated with the induction of widespread genetic and epigenetic changes and has played an important role in the evolution of many plant taxa. We attempted the intergeneric cross Chrysanthemum morifolium × Leucanthemum paludosum. To obtain the success in cross, we have to turn to ovule rescue. DNA profiling of the amphihaploid and amphidiploid was investigated using amplified fragment length polymorphism, sequence-related amplified polymorphism, start codon targeted polymorphism, and methylation-sensitive amplification polymorphism (MSAP). Hybridization induced rapid changes at the genetic and the epigenetic levels. The genetic changes mainly involved loss of parental fragments and gaining of novel fragments, and some eliminated sequences possibly from the noncoding region of L. paludosum. The MSAP analysis indicated that the level of DNA methylation was lower in the amphiploid (∼45%) than in the parental lines (51.5–50.6%), whereas it increased after amphidiploid formation. Events associated with intergeneric genomic shock were a feature of C. morifolium × L. paludosum hybrid, given that the genetic relationship between the parental species is relatively distant. Our results provide genetic and epigenetic evidence for understanding genomic shock in wide crosses between species in Asteraceae and suggest a need to expand our current evolutionary framework to encompass a genetic/epigenetic dimension when seeking to understand wide crosses. PMID:24407856

Rapid genetic and epigenetic alterations under intergeneric genomic shock in newly synthesized Chrysanthemum morifolium x Leucanthemum paludosum hybrids (Asteraceae).

Science.gov (United States)

Wang, Haibin; Jiang, Jiafu; Chen, Sumei; Qi, Xiangyu; Fang, Weimin; Guan, Zhiyong; Teng, Nianjun; Liao, Yuan; Chen, Fadi

2014-01-01

The Asteraceae family is at the forefront of the evolution due to frequent hybridization. Hybridization is associated with the induction of widespread genetic and epigenetic changes and has played an important role in the evolution of many plant taxa. We attempted the intergeneric cross Chrysanthemum morifolium × Leucanthemum paludosum. To obtain the success in cross, we have to turn to ovule rescue. DNA profiling of the amphihaploid and amphidiploid was investigated using amplified fragment length polymorphism, sequence-related amplified polymorphism, start codon targeted polymorphism, and methylation-sensitive amplification polymorphism (MSAP). Hybridization induced rapid changes at the genetic and the epigenetic levels. The genetic changes mainly involved loss of parental fragments and gaining of novel fragments, and some eliminated sequences possibly from the noncoding region of L. paludosum. The MSAP analysis indicated that the level of DNA methylation was lower in the amphiploid (∼45%) than in the parental lines (51.5-50.6%), whereas it increased after amphidiploid formation. Events associated with intergeneric genomic shock were a feature of C. morifolium × L. paludosum hybrid, given that the genetic relationship between the parental species is relatively distant. Our results provide genetic and epigenetic evidence for understanding genomic shock in wide crosses between species in Asteraceae and suggest a need to expand our current evolutionary framework to encompass a genetic/epigenetic dimension when seeking to understand wide crosses.

Assessment of genetic and epigenetic variation during long-term Taxus cell culture.

Science.gov (United States)

Fu, Chunhua; Li, Liqin; Wu, Wenjuan; Li, Maoteng; Yu, Xiaoqing; Yu, Longjiang

2012-07-01

Gradual loss of secondary metabolite production is a common obstacle in the development of a large-scale plant cell production system. In this study, cell morphology, paclitaxel (Taxol®) biosynthetic ability, and genetic and epigenetic variations in the long-term culture of Taxus media cv Hicksii cells were assessed over a 5-year period to evaluate the mechanisms of the loss of secondary metabolites biosynthesis capacity in Taxus cell. The results revealed that morphological variations, gradual loss of paclitaxel yield and decreased transcriptional level of paclitaxel biosynthesis key genes occurred during long-term subculture. Genetic and epigenetic variations in these cultures were also studied at different times during culture using amplified fragment-length polymorphism (AFLP), methylation-sensitive amplified polymorphism (MSAP), and high-performance liquid chromatography (HPLC) analyses. A total of 32 primer combinations were used in AFLP amplification, and none of the AFLP loci were found to be polymorphic, thus no major genetic rearrangements were detected in any of the tested samples. However, results from both MSAP and HPLC indicated that there was a higher level of DNA methylation in the low-paclitaxel yielding cell line after long-term culture. Based on these results, we proposed that accumulation of paclitaxel in Taxus cell cultures might be regulated by DNA methylation. To our knowledge, this is the first report of increased methylation with the prolongation of culture time in Taxus cell culture. It provides substantial clues for exploring the gradual loss of the taxol biosynthesis capacity of Taxus cell lines during long-term subculture. DNA methylation maybe involved in the regulation of paclitaxel biosynthesis in Taxus cell culture.

Optimization of the alignment sensitivity and energy stability of the NIF regenerative amplifier cavity

International Nuclear Information System (INIS)

Hopps, N. W.

1998-01-01

The work to improve the energy stability of the regenerative amplifier ('regen') for the National Ignition Facility is described. This includes a fast feed-forward system, designed to regulate the output energy of the regen by monitoring how quickly a pulse builds up over many round trips. Shot-to-shot energy fluctuations of all elements prior to (and including) the regen may be compensated for in this way, at the expense of a loss of approximately 50%. Also included is a detailed study into the alignment sensitivity of the regen cavity, with the goal of quantifying the effect of misalignment on the output energy. This is done by calculating the displacement of the eigenmode by augmenting the cavity ABCD matrix with the misalignment matrix elements, E, F. In this way, cavity misalignment issues due to thermal loading of the gain medium are investigated. Alternative cavity designs, which reduce the alignment sensitivity and therefore the energy drift over periods of continuous operation, are considered. Alterations to the amplifier head design are also considered

Epigenetic changes detected in micropropagated hop plants.

Science.gov (United States)

Peredo, Elena L; Arroyo-García, Rosa; Revilla, M Angeles

2009-07-01

Micropropagation is a widely used technique in hops (Humulus lupulus L.). However, to the best of our knowledge, the genetic and epigenetic stability of the microplants has never been tested before. In the present study, two hop accessions were established in vitro and micropropagated for 2 years. The genetic and epigenetic stability of the in vitro plants was analyzed with several molecular techniques: random amplified DNA polymorphism (RAPD), retrotransposon microsatellite amplified polymorphism (REMAP), and methylation-sensitive amplification polymorphism (MSAP). No genetic variation among control and treated plants was found, even after 12 cycles of micropropagation. Epigenetic variation was detected, first, when field and in vitro samples were compared. Nearly a 30% of the detected fragments presented the same pattern of alterations in all the vitroplants. Second, lower levels of epigenetic variation were detected among plants from the different subcultures. Part of this detected variation seemed to be accumulated along the 12 sequential subcultures tested.

Amplified Fragment Length Polymorphism Fingerprinting for Identification of a Core Group of Neisseria gonorrhoeae Transmitters in the Population Attending a Clinic for Treatment of Sexually Transmitted Diseases in Amsterdam, The Netherlands

OpenAIRE

Spaargaren, Joke; Stoof, Jeroen; Fennema, Han; Coutinho, Roel; Savelkoul, Paul

2001-01-01

Amplified fragment length polymorphism analysis seems well suited for studying the epidemiology of isolates of Neisseria gonorrhoeae obtained from patients attending the Sexually Transmitted Disease Outpatient Clinic in Amsterdam, The Netherlands. It shows potential to identify the core group of transmitters.

Genetic and Epigenetic Changes in Oilseed Rape (Brassica napus L.) Extracted from Intergeneric Allopolyploid and Additions with Orychophragmus.

Science.gov (United States)

Gautam, Mayank; Dang, Yanwei; Ge, Xianhong; Shao, Yujiao; Li, Zaiyun

2016-01-01

Allopolyploidization with the merger of the genomes from different species has been shown to be associated with genetic and epigenetic changes. But the maintenance of such alterations related to one parental species after the genome is extracted from the allopolyploid remains to be detected. In this study, the genome of Brassica napus L. (2n = 38, genomes AACC) was extracted from its intergeneric allohexaploid (2n = 62, genomes AACCOO) with another crucifer Orychophragmus violaceus (2n = 24, genome OO), by backcrossing and development of alien addition lines. B. napus-type plants identified in the self-pollinated progenies of nine monosomic additions were analyzed by the methods of amplified fragment length polymorphism, sequence-specific amplified polymorphism, and methylation-sensitive amplified polymorphism. They showed modifications to certain extents in genomic components (loss and gain of DNA segments and transposons, introgression of alien DNA segments) and DNA methylation, compared with B. napus donor. The significant differences in the changes between the B. napus types extracted from these additions likely resulted from the different effects of individual alien chromosomes. Particularly, the additions which harbored the O. violaceus chromosome carrying dominant rRNA genes over those of B. napus tended to result in the development of plants which showed fewer changes, suggesting a role of the expression levels of alien rRNA genes in genomic stability. These results provided new cues for the genetic alterations in one parental genome that are maintained even after the genome becomes independent.

Distress of ostracism: oxytocin receptor gene polymorphism confers sensitivity to social exclusion.

Science.gov (United States)

McQuaid, Robyn J; McInnis, Opal A; Matheson, Kimberly; Anisman, Hymie

2015-08-01

A single-nucleotide polymorphism on the oxytocin receptor gene (OXTR), rs53576, involving a guanine (G) to adenine (A) substitution has been associated with altered prosocial features. Specifically, individuals with the GG genotype (i.e. the absence of the polymorphism) display beneficial traits including enhanced trust, empathy and self-esteem. However, because G carriers might also be more socially sensitive, this may render them more vulnerable to the adverse effects of a negative social stressor. The current investigation, conducted among 128 white female undergraduate students, demonstrated that relative to individuals with AA genotype, G carriers were more emotionally sensitive (lower self-esteem) in response to social ostracism promoted through an on-line ball tossing game (Cyberball). Furthermore, GG individuals also exhibited altered blood pressure and cortisol levels following rejection, effects not apparent among A carriers. The data support the view that the presence of the G allele not only promotes prosocial behaviors but also favors sensitivity to a negative social stressor. © The Author (2015). Published by Oxford University Press. For Permissions, please email: journals.permissions@oup.com.

Identification, genetic localization, and allelic diversity of selectively amplified microsatellite polymorphic loci in lettuce and wild relatives (Lactuca spp.).

Science.gov (United States)

Witsenboer, H; Michelmore, R W; Vogel, J

1997-12-01

Selectively amplified microsatellite polymorphic locus (SAMPL) analysis is a method of amplifying microsatellite loci using generic PCR primers. SAMPL analysis uses one AFLP primer in combination with a primer complementary to microsatellite sequences. SAMPL primers based on compound microsatellite sequences provided the clearest amplification patterns. We explored the potential of SAMPL analysis in lettuce to detect PCR-based codominant microsatellite markers. Fifty-eight SAMPLs were identified and placed on the genetic map. Seventeen were codominant. SAMPLs were dispersed with RFLP markers on 11 of the 12 main linkage groups in lettuce, indicating that they have a similar genomic distribution. Some but not all fragments amplified by SAMPL analysis were confirmed to contain microsatellite sequences by Southern hybridization. Forty-five cultivars of lettuce and five wild species of Lactuca were analyzed to determine the allelic diversity for codominant SAMPLs. From 3 to 11 putative alleles were found for each SAMPL; 2-6 alleles were found within Lactuca sativa and 1-3 alleles were found among the crisphead genotypes, the most genetically homogeneous plant type of L. sativa. This allelic diversity is greater than that found for RFLP markers. Numerous new alleles were observed in the wild species; however, there were frequent null alleles. Therefore, SAMPL analysis is more applicable to intraspecific than to interspecific comparisons. A phenetic analysis based on SAMPLs resulted in a dendrogram similar to those based on RFLP and AFLP markers.

A simple and sensitive fluorescent sensor for methyl parathion based on L-tyrosine methyl ester functionalized carbon dots.

Science.gov (United States)

Hou, Juying; Dong, Jing; Zhu, Haishuang; Teng, Xue; Ai, Shiyun; Mang, Minglin

2015-06-15

In this paper, a simple and sensitive fluorescent sensor for methyl parathion is developed based on L-tyrosine methyl ester functionalized carbon dots (Tyr-CDs) and tyrosinase system. The carbon dots are obtained by simple hydrothermal reaction using citric acid as carbon resource and L-tyrosine methyl ester as modification reagent. The carbon dots are characterized by transmission electron microscope, high resolution transmission electron microscopy, X-ray diffraction spectrum, Fourier transform infrared spectroscopy, and X-ray photoelectron spectroscopy. The carbon dots show strong and stable photoluminescence with a quantum yield of 3.8%. Tyrosinase can catalyze the oxidation of tyrosine methyl ester on the surface of carbon dots to corresponding quinone products, which can quench the fluorescence of carbon dots. When organophosphorus pesticides (OPs) are introduced in system, they can decrease the enzyme activity, thus decrease the fluorescence quenching rate. Methyl parathion, as a model of OPs, was detected. Experimental results show that the enzyme inhibition rate is proportional to the logarithm of the methyl parathion concentration in the range 1.0×10(-10)-1.0×10(-4) M with the detection limit (S/N=3) of 4.8×10(-11) M. This determination method shows a low detection limit, wide linear range, good selectivity and high reproducibility. This sensing system has been successfully used for the analysis of cabbage, milk and fruit juice samples. Copyright © 2014 Elsevier B.V. All rights reserved.

Decoherence of odd compass states in the phase-sensitive amplifying/dissipating environment

Energy Technology Data Exchange (ETDEWEB)

Dodonov, V.V., E-mail: vdodonov@fis.unb.br [Instituto de Física, Universidade de Brasília, Caixa Postal 04455, 70919-970 Brasília, DF (Brazil); Valverde, C. [Unidade de Ciências Exatas e Tecnológicas, Universidade Estadual de Goiás, BR 153, km 98, 75001-970 Anápolis, GO (Brazil); Universidade Paulista, BR 153, km 7, 74845-090 Goiânia, GO (Brazil); Souza, L.S. [Unidade de Ciências Exatas e Tecnológicas, Universidade Estadual de Goiás, BR 153, km 98, 75001-970 Anápolis, GO (Brazil); Baseia, B. [Instituto de Física, Universidade Federal de Goiás, 74.690-900 Goiânia, GO (Brazil); Departamento de Física, Universidade Federal da Paraíba, 58.051-970 João Pessoa, PB (Brazil)

2016-08-15

We study the evolution of odd compass states (specific superpositions of four coherent states), governed by the standard master equation with phase-sensitive amplifying/attenuating terms, in the presence of a Hamiltonian describing a parametric degenerate linear amplifier. Explicit expressions for the time-dependent Wigner function are obtained. The time of disappearance of the so called “sub-Planck structures” is calculated using the negative value of the Wigner function at the origin of phase space. It is shown that this value rapidly decreases during a short “conventional interference degradation time” (CIDT), which is inversely proportional to the size of quantum superposition, provided the anti-Hermitian terms in the master equation are of the same order (or stronger) as the Hermitian ones (governing the parametric amplification). The CIDT is compared with the final positivization time (FPT), when the Wigner function becomes positive. It appears that the FPT does not depend on the size of superpositions, moreover, it can be much bigger in the amplifying media than in the attenuating ones. Paradoxically, strengthening the Hamiltonian part results in decreasing the CIDT, so that the CIDT almost does not depend on the size of superpositions in the asymptotical case of very weak reservoir coupling. We also analyze the evolution of the Mandel factor, showing that for some sets of parameters this factor remains significantly negative, even when the Wigner function becomes positive.

Chemical exchange-sensitive spin-lock MRI of glucose analog 3-O-methyl-d-glucose in normal and ischemic brain.

Science.gov (United States)

Jin, Tao; Mehrens, Hunter; Wang, Ping; Kim, Seong-Gi

2018-05-01

Glucose transport is important for understanding brain glucose metabolism. We studied glucose transport with a presumably non-toxic and non-metabolizable glucose analog, 3-O-methyl-d-glucose, using a chemical exchange-sensitive spin-lock MRI technique at 9.4 Tesla. 3-O-methyl-d-glucose showed comparable chemical exchange properties with d-glucose and 2-deoxy-d-glucose in phantoms, and higher and lower chemical exchange-sensitive spin-lock sensitivity than Glc and 2-deoxy-d-glucose in in vivo experiments, respectively. The changes of the spin-lattice relaxation rate in the rotating frame (Δ R 1 ρ) in normal rat brain peaked at ∼15 min after the intravenous injection of 1 g/kg 3-O-methyl-d-glucose and almost maintained a plateau for >1 h. Doses up to 4 g/kg 3-O-methyl-d-glucose were linearly correlated with Δ R 1 ρ. In rats with focal ischemic stroke, chemical exchange-sensitive spin-lock with 3-O-methyl-d-glucose injection at 1 h after stroke onset showed reduced Δ R 1 ρ in the ischemic core but higher Δ R 1 ρ in the peri-core region compared to normal tissue, which progressed into the ischemic core at 3 h after stroke onset. This suggests that the hyper-chemical exchange-sensitive spin-lock region observed at 1 h is the ischemic penumbra at-risk of infarct. In summary, 3-O-methyl-d-glucose-chemical exchange-sensitive spin-lock can be a sensitive MRI technique to probe the glucose transport in normal and ischemic brains.

High-Resolution Amplified Fragment Length Polymorphism Typing of Lactococcus lactis Strains Enables Identification of Genetic Markers for Subspecies-Related Phenotypes▿

Science.gov (United States)

Kütahya, Oylum Erkus; Starrenburg, Marjo J. C.; Rademaker, Jan L. W.; Klaassen, Corné H. W.; van Hylckama Vlieg, Johan E. T.; Smid, Eddy J.; Kleerebezem, Michiel

2011-01-01

A high-resolution amplified fragment length polymorphism (AFLP) methodology was developed to achieve the delineation of closely related Lactococcus lactis strains. The differentiation depth of 24 enzyme-primer-nucleotide combinations was experimentally evaluated to maximize the number of polymorphisms. The resolution depth was confirmed by performing diversity analysis on 82 L. lactis strains, including both closely and distantly related strains with dairy and nondairy origins. Strains clustered into two main genomic lineages of L. lactis subsp. lactis and L. lactis subsp. cremoris type-strain-like genotypes and a third novel genomic lineage rooted from the L. lactis subsp. lactis genomic lineage. Cluster differentiation was highly correlated with small-subunit rRNA homology and multilocus sequence analysis (MLSA) studies. Additionally, the selected enzyme-primer combination generated L. lactis subsp. cremoris phenotype-specific fragments irrespective of the genotype. These phenotype-specific markers allowed the differentiation of L. lactis subsp. lactis phenotype from L. lactis subsp. cremoris phenotype strains within the same L. lactis subsp. cremoris type-strain-like genomic lineage, illustrating the potential of AFLP for the generation of phenotype-linked genetic markers. PMID:21666014

Stable MSAP markers for the distinction of Vitis vinifera cv Pinot noir clones.

Science.gov (United States)

Ocaña, Juan; Walter, Bernard; Schellenbaum, Paul

2013-11-01

Grapevine is one of the most economically important fruit crops. Molecular markers have been used to study grapevine diversity. For instance, simple sequence repeats are a powerful tool for identification of grapevine cultivars, while amplified fragment length polymorphisms have shown their usefulness in intra-varietal diversity studies. Other techniques such as sequence-specific amplified polymorphism are based on the presence of mobile elements in the genome, but their detection lies upon their activity. Relevant attention has been drawn toward epigenetic sources of variation. In this study, a set of Vitis vinifera cv Pinot noir clones were analyzed using the methylation-sensitive amplified polymorphism technique with isoschizomers MspI and HpaII. Nine out of fourteen selective primer combinations were informative and generated two types of polymorphic fragments which were categorized as "stable" and "unstable." In total, 23 stable fragments were detected and they discriminated 92.5 % of the studied clones. Detected stable polymorphisms were either common to several clones, restricted to a few clones or unique to a single clone. The identification of these stable epigenetic markers will be useful in clonal diversity studies. We highlight the relevance of stable epigenetic variation in V. vinifera clones and analyze at which level these markers could be applicable for the development of forthright techniques for clonal distinction.

Skin score correlates with global DNA methylation and GSTO1 A140D polymorphism in arsenic-affected population of Eastern India.

Science.gov (United States)

Majumder, Moumita; Dasgupta, Uma B; Guha Mazumder, D N; Das, Nilansu

2017-07-01

Arsenic is a potent environmental toxicant causing serious public health concerns in India, Bangladesh and other parts of the world. Gene- and promoter-specific hypermethylation has been reported in different arsenic-exposed cell lines, whereas whole genome DNA methylation study suggested genomic hypo- and hypermethylation after arsenic exposure in in vitro and in vivo studies. Along with other characteristic biomarkers, arsenic toxicity leads to typical skin lesions. The present study demonstrates significant correlation between severities of skin manifestations with their whole genome DNA methylation status as well as with a particular polymorphism (Ala 140 Asp) status in arsenic metabolizing enzyme Glutathione S-transferase Omega-1 (GSTO1) in arsenic-exposed population of the district of Nadia, West Bengal, India.

Estimating Genetic Conformism of Korean Mulberry Cultivars Using Random Amplified Polymorphic DNA and Inter-Simple Sequence Repeat Profiling

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Sunirmal Sheet

2018-03-01

Full Text Available Apart from being fed to silkworms in sericulture, the ecologically important Mulberry plant has been used for traditional medicine in Asian countries as well as in manufacturing wine, food, and beverages. Germplasm analysis among Mulberry cultivars originating from South Korea is crucial in the plant breeding program for cultivar development. Hence, the genetic deviations and relations among 8 Morus alba plants, and one Morus lhou plant, of different cultivars collected from South Korea were investigated using 10 random amplified polymorphic DNA (RAPD and 10 inter-simple sequence repeat (ISSR markers in the present study. The ISSR markers exhibited a higher polymorphism (63.42% among mulberry genotypes in comparison to RAPD markers. Furthermore, the similarity coefficient was estimated for both markers and found to be varying between 0.183 and 0.814 for combined pooled data of ISSR and RAPD. The phenogram drawn using the UPGMA cluster method based on combined pooled data of RAPD and ISSR markers divided the nine mulberry genotypes into two divergent major groups and the two individual independent accessions. The distant relationship between Dae-Saug (SM1 and SangchonJo Sang Saeng (SM5 offers a possibility of utilizing them in mulberry cultivar improvement of Morus species of South Korea.

Identification of genes related to Paulownia witches' broom by AFLP and MSAP.

Science.gov (United States)

Cao, Xibing; Fan, Guoqiang; Deng, Minjie; Zhao, Zhenli; Dong, Yanpeng

2014-08-21

DNA methylation is believed to play important roles in regulating gene expression in plant growth and development. Paulownia witches' broom (PaWB) infection has been reported to be related to gene expression changes in paulownia plantlets. To determine whether DNA methylation is associated with gene expression changes in response to phytoplasma, we investigated variations in genomic DNA sequence and methylation in PaWB plantlets treated with methyl methane sulfonate (MMS) using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) techniques, respectively. The results indicated that PaWB seedings recovered a normal morphology after treatment with more than 15 mg·L(-1) MMS. PaWB infection did not cause changes of the paulownia DNA sequence at the AFLP level; However, DNA methylation levels and patterns were altered. Quantitative real-time PCR (qRT-PCR) showed that three of the methylated genes were up-regulated and three were down-regulated in the MMS-treated PaWB plantlets that had regained healthy morphology. These six genes might be involved in transcriptional regulation, plant defense, signal transduction and energy. The possible roles of these genes in PaWB are discussed. The results showed that changes of DNA methylation altered gene expression levels, and that MSAP might help identify genes related to PaWB.

Identification of Genes Related to Paulownia Witches’ Broom by AFLP and MSAP

Science.gov (United States)

Cao, Xibing; Fan, Guoqiang; Deng, Minjie; Zhao, Zhenli; Dong, Yanpeng

2014-01-01

DNA methylation is believed to play important roles in regulating gene expression in plant growth and development. Paulownia witches’ broom (PaWB) infection has been reported to be related to gene expression changes in paulownia plantlets. To determine whether DNA methylation is associated with gene expression changes in response to phytoplasma, we investigated variations in genomic DNA sequence and methylation in PaWB plantlets treated with methyl methane sulfonate (MMS) using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplification polymorphism (MSAP) techniques, respectively. The results indicated that PaWB seedings recovered a normal morphology after treatment with more than 15 mg·L−1 MMS. PaWB infection did not cause changes of the paulownia DNA sequence at the AFLP level; However, DNA methylation levels and patterns were altered. Quantitative real-time PCR (qRT-PCR) showed that three of the methylated genes were up-regulated and three were down-regulated in the MMS-treated PaWB plantlets that had regained healthy morphology. These six genes might be involved in transcriptional regulation, plant defense, signal transduction and energy. The possible roles of these genes in PaWB are discussed. The results showed that changes of DNA methylation altered gene expression levels, and that MSAP might help identify genes related to PaWB. PMID:25196603

Milk: an epigenetic amplifier of FTO-mediated transcription? Implications for Western diseases.

Science.gov (United States)

Melnik, Bodo C

2015-12-21

Single-nucleotide polymorphisms within intron 1 of the FTO (fat mass and obesity-associated) gene are associated with enhanced FTO expression, increased body weight, obesity and type 2 diabetes mellitus (T2DM). The N (6) -methyladenosine (m(6)A) demethylase FTO plays a pivotal regulatory role for postnatal growth and energy expenditure. The purpose of this review is to provide translational evidence that links milk signaling with FTO-activated transcription of the milk recipient. FTO-dependent demethylation of m(6)A regulates mRNA splicing required for adipogenesis, increases the stability of mRNAs, and affects microRNA (miRNA) expression and miRNA biosynthesis. FTO senses branched-chain amino acids (BCAAs) and activates the nutrient sensitive kinase mechanistic target of rapamycin complex 1 (mTORC1), which plays a key role in translation. Milk provides abundant BCAAs and glutamine, critical components increasing FTO expression. CpG hypomethylation in the first intron of FTO has recently been associated with T2DM. CpG methylation is generally associated with gene silencing. In contrast, CpG demethylation generally increases transcription. DNA de novo methylation of CpG sites is facilitated by DNA methyltransferases (DNMT) 3A and 3B, whereas DNA maintenance methylation is controlled by DNMT1. MiRNA-29s target all DNMTs and thus reduce DNA CpG methylation. Cow´s milk provides substantial amounts of exosomal miRNA-29s that reach the systemic circulation and target mRNAs of the milk recipient. Via DNMT suppression, milk exosomal miRNA-29s may reduce the magnitude of FTO methylation, thereby epigenetically increasing FTO expression in the milk consumer. High lactation performance with increased milk yield has recently been associated with excessive miRNA-29 expression of dairy cow mammary epithelial cells (DCMECs). Notably, the galactopoietic hormone prolactin upregulates the transcription factor STAT3, which induces miRNA-29 expression. In a retrovirus-like manner

Modulation of DNA methylation levels sensitizes doxorubicin-resistant breast adenocarcinoma cells to radiation-induced apoptosis

Energy Technology Data Exchange (ETDEWEB)

Luzhna, Lidia [Department of Biological Sciences, University of Lethbridge, AB, Canada T1K 3M4 (Canada); Kovalchuk, Olga, E-mail: olga.kovalchuk@uleth.ca [Department of Biological Sciences, University of Lethbridge, AB, Canada T1K 3M4 (Canada)

2010-02-05

Chemoresistant tumors often fail to respond to other cytotoxic treatments such as radiation therapy. The mechanisms of chemo- and radiotherapy cross resistance are not fully understood and are believed to be epigenetic in nature. We hypothesize that MCF-7 cells and their doxorubicin-resistant variant MCF-7/DOX cells may exhibit different responses to ionizing radiation due to their dissimilar epigenetic status. Similar to previous studies, we found that MCF-7/DOX cells harbor much lower levels of global DNA methylation than MCF-7 cells. Furthermore, we found that MCF-7/DOX cells had lower background apoptosis levels and were less responsive to radiation than MCF-7 cells. Decreased radiation responsiveness correlated to significant global DNA hypomethylation in MCF-7/DOX cells. Here, for the first time, we show that the radiation resistance of MCF-7/DOX cells can be reversed by an epigenetic treatment - the application of methyl-donor SAM. SAM-mediated reversal of DNA methylation led to elevated radiation sensitivity in MCF-7/DOX cells. Contrarily, application of SAM on the radiation sensitive and higher methylated MCF-7 cells resulted in a decrease in their radiation responsiveness. This data suggests that a fine balance of DNA methylation is needed to insure proper radiation and drug responsiveness.

Application of amplified fragment length polymorphism (AFLPs) for ...

African Journals Online (AJOL)

Uapaca kirkiana Muell. Årg is a dioecious fruit tree species for priority domestication in Southern Africa. It reaches reproductive maturity in eight to ten years with male plants making up 50% of breeding populations. Early identification of sex of seedlings is a prerequisite for selection and tree improvement. The amplified ...

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

Energy Technology Data Exchange (ETDEWEB)

Wu, Chia-Chang [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Department of Urology, Taipei Medical University—Shuang Ho Hospital, Taipei, Taiwan (China); Huang, Yung-Kai [School of Oral Hygiene, College of Oral Medicine, Taipei Medical University, Taipei, Taiwan (China); Chung, Chi-Jung [Department of Health Risk Management, College of Public Health, China Medical University and Hospital, Taichung, Taiwan (China); Department of Medical Research, China Medical University Hospital, Taichung, Taiwan (China); Huang, Chao-Yuan; Pu, Yeong-Shiau [Department of Urology, National Taiwan University Hospital, College of Medicine National Taiwan University, Taipei, Taiwan (China); Shiue, Horng-Sheng [Department of Chinese Medicine, Chang Gung Memorial Hospital, Taipei, Taiwan (China); Lai, Li-An [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Lin, Ying-Chin [Department of Family Medicine, Shung Ho Hospital, Taipei Medical University, Taipei, Taiwan (China); Department of Health Examination, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan (China); Su, Chien-Tien [Department of Family Medicine, Taipei Medical University Hospital, Taipei, Taiwan (China); Hsueh, Yu-Mei, E-mail: ymhsueh@tmu.edu.tw [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China)

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.

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

International Nuclear Information System (INIS)

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

2013-01-01

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

Genetic alterations of hepatocellular carcinoma by random amplified polymorphic DNA analysis and cloning sequencing of tumor differential DNA fragment

Science.gov (United States)

Xian, Zhi-Hong; Cong, Wen-Ming; Zhang, Shu-Hui; Wu, Meng-Chao

2005-01-01

AIM: To study the genetic alterations and their association with clinicopathological characteristics of hepatocellular carcinoma (HCC), and to find the tumor related DNA fragments. METHODS: DNA isolated from tumors and corresponding noncancerous liver tissues of 56 HCC patients was amplified by random amplified polymorphic DNA (RAPD) with 10 random 10-mer arbitrary primers. The RAPD bands showing obvious differences in tumor tissue DNA corresponding to that of normal tissue were separated, purified, cloned and sequenced. DNA sequences were analyzed and compared with GenBank data. RESULTS: A total of 56 cases of HCC were demonstrated to have genetic alterations, which were detected by at least one primer. The detestability of genetic alterations ranged from 20% to 70% in each case, and 17.9% to 50% in each primer. Serum HBV infection, tumor size, histological grade, tumor capsule, as well as tumor intrahepatic metastasis, might be correlated with genetic alterations on certain primers. A band with a higher intensity of 480 bp or so amplified fragments in tumor DNA relative to normal DNA could be seen in 27 of 56 tumor samples using primer 4. Sequence analysis of these fragments showed 91% homology with Homo sapiens double homeobox protein DUX10 gene. CONCLUSION: Genetic alterations are a frequent event in HCC, and tumor related DNA fragments have been found in this study, which may be associated with hepatocarcin-ogenesis. RAPD is an effective method for the identification and analysis of genetic alterations in HCC, and may provide new information for further evaluating the molecular mechanism of hepatocarcinogenesis. PMID:15996039

Genetic variability of Amorphophallus muelleri Blume in Java based on Random Amplified Polymorphic DNA

Directory of Open Access Journals (Sweden)

DIYAH MARTANTI

2008-10-01

Full Text Available Amorphophallus muelleri Blume (Araceae is valued for its glucomanan content for use in food industry (healthy diet food, paper industry, pharmacy and cosmetics. The species is triploid (2n=3x=39 and the seed is developed apomictically. The present research is aimed to identify genetic variability of six population of A. muelleri from Java (consisted of 50 accessions using random amplified polymorphic DNA (RAPD. The six populations of the species are: East Java: (1 Silo-Jember, (2 Saradan-Madiun, (3 IPB (cultivated, from Saradan-Madiun, (4 Panti-Jember, (5 Probolinggo; and Central Java: (6 Cilacap. The results showed that five RAPD primers generated 42 scorable bands of which 29 (69.05% were polymorphic. Size of the bands varied from 300bp to 1.5kbp. The 50 accessions of A. muelleri were divided into two main clusters, some of them were grouped based on their populations, and some others were not. The range of individual genetic dissimilarity was from 0.02 to 0.36. The results showed that among six populations investigated, Saradan population showed the highest levels of genetic variation with mean values of na = 1.500+ 0.5061, ne = 1.3174 + 0.3841, PLP = 50% and He = 0, 0.1832+0.2054, whereas Silo-Jember population showed the lowest levels of genetic variation with mean values na = 1.2619+ 0.4450, ne = 1.1890 + 0.3507, PLP = 26.19% and He = 0.1048+0.1887. Efforts to conserve, domesticate, cultivate and improve genetically should be based on the genetic properties of each population and individual within population, especially Saradan population which has the highest levels of genetic variation, need more attention for its conservation.

Quantum-Limited Directional Amplifiers with Optomechanics

Science.gov (United States)

Malz, Daniel; Tóth, László D.; Bernier, Nathan R.; Feofanov, Alexey K.; Kippenberg, Tobias J.; Nunnenkamp, Andreas

2018-01-01

Directional amplifiers are an important resource in quantum-information processing, as they protect sensitive quantum systems from excess noise. Here, we propose an implementation of phase-preserving and phase-sensitive directional amplifiers for microwave signals in an electromechanical setup comprising two microwave cavities and two mechanical resonators. We show that both can reach their respective quantum limits on added noise. In the reverse direction, they emit thermal noise stemming from the mechanical resonators; we discuss how this noise can be suppressed, a crucial aspect for technological applications. The isolation bandwidth in both is of the order of the mechanical linewidth divided by the amplitude gain. We derive the bandwidth and gain-bandwidth product for both and find that the phase-sensitive amplifier has an unlimited gain-bandwidth product. Our study represents an important step toward flexible, on-chip integrated nonreciprocal amplifiers of microwave signals.

Development of a sequence characterized amplified region (SCAR ...

African Journals Online (AJOL)

The varieties of rice are difficult to be distinguished because of their similar morphological characters, therefore rice variety identification or differentiation is very important. The authentication by using molecular marker or DNA fingerprinting is the most accurate method. In this study, random amplified polymorphic DNA ...

Randomly amplified polymorphic-DNA analysis for detecting genotoxic effects of Boron on maize (Zea mays L.).

Science.gov (United States)

Sakcali, M Serdal; Kekec, Guzin; Uzonur, Irem; Alpsoy, Lokman; Tombuloglu, Huseyin

2015-08-01

This study was carried out to investigate the genotoxic effect of boron (B) on maize using randomly amplified polymorphic DNA (RAPD) method. Experimental design was conducted under 0, 5, 10, 25, 50, 100, 125, and 150 ppm B exposures, and physiological changes have revealed a sharp decrease in root growth rates from 28% to 85%, starting from 25 ppm to 150 ppm, respectively. RAPD-polymerase chain reaction (PCR) analysis shows that DNA alterations are clearly observed from beginning to 100 ppm. B-induced inhibition in root growth had a positive correlation with DNA alterations. Total soluble protein, root and stem lengths, and B content analysis in root and leaves encourage these results as a consequence. These preliminary findings reveal that B causes chromosomal aberration and genotoxic effects on maize. Meanwhile, usage of RAPD-PCR technique is a suitable biomarker to detect genotoxic effect of B on maize and other crops for the future. © The Author(s) 2013.

Genome-Wide Single-Nucleotide Polymorphisms Discovery and High-Density Genetic Map Construction in Cauliflower Using Specific-Locus Amplified Fragment Sequencing

Science.gov (United States)

Zhao, Zhenqing; Gu, Honghui; Sheng, Xiaoguang; Yu, Huifang; Wang, Jiansheng; Huang, Long; Wang, Dan

2016-01-01

Molecular markers and genetic maps play an important role in plant genomics and breeding studies. Cauliflower is an important and distinctive vegetable; however, very few molecular resources have been reported for this species. In this study, a novel, specific-locus amplified fragment (SLAF) sequencing strategy was employed for large-scale single nucleotide polymorphism (SNP) discovery and high-density genetic map construction in a double-haploid, segregating population of cauliflower. A total of 12.47 Gb raw data containing 77.92 M pair-end reads were obtained after processing and 6815 polymorphic SLAFs between the two parents were detected. The average sequencing depths reached 52.66-fold for the female parent and 49.35-fold for the male parent. Subsequently, these polymorphic SLAFs were used to genotype the population and further filtered based on several criteria to construct a genetic linkage map of cauliflower. Finally, 1776 high-quality SLAF markers, including 2741 SNPs, constituted the linkage map with average data integrity of 95.68%. The final map spanned a total genetic length of 890.01 cM with an average marker interval of 0.50 cM, and covered 364.9 Mb of the reference genome. The markers and genetic map developed in this study could provide an important foundation not only for comparative genomics studies within Brassica oleracea species but also for quantitative trait loci identification and molecular breeding of cauliflower. PMID:27047515

Carriage of a Tumor Necrosis Factor Polymorphism Amplifies the Cytotoxic T-Lymphocyte Antigen 4 Attributed Risk of Primary Biliary Cirrhosis: Evidence for a Gene–Gene Interaction

Science.gov (United States)

Juran, Brian D.; Atkinson, Elizabeth J.; Larson, Joseph J.; Schlicht, Erik M.; Liu, Xiangdong; Heathcote, E. Jenny; Hirschfield, Gideon M.; Siminovitch, Katherine A.; Lazaridis, Konstantinos N.

2010-01-01

Common genetic variants significantly influence complex diseases such as primary biliary cirrhosis (PBC). We recently reported an association between PBC and a single nucleotide polymorphism (rs231725) of the immunoreceptor gene cytotoxic T-lymphocyte antigen 4 (CTLA4). We hypothesized that PBC risk attributed to this polymorphism might be increased by propensity to an overly robust inflammatory response. Thus, we examined its potential interaction with the commonly studied −308AG promoter polymorphism (rs1800629) of the tumor necrosis factor (TNF) gene for which the variant TNF2A allele causes increased TNF production. The polymorphisms were genotyped in 866 PBC patients and 761 controls from independent US and Canadian registries; the effects of individual single nucleotide polymorphisms (SNPs) and their interaction on PBC risk was assessed by logistic regression. The reported association of PBC with the CTLA4 “A/A” genotype was replicated in the Canadian cohort and significant for PBC risk in the combined data (odds ratio [OR], 1.68; P = 0.0005). TNF2A allele frequency was elevated in PBC patients, but only reached borderline significance using the combined data (OR, 1.21; P = 0.042). Analysis showed that TNF2A carriage was significantly increased in CTLA4 “A/A” PBC patients compared with CTLA4 “A/A” controls (39.7% versus 16.5%, P = 0.0004); no apparent increase of TNF2A carriage was noted in CTLA4 “A/G” or “G/G” individuals. Finally, interaction under a logistic model was highly significant, as TNF2A carriage in combination with the CTLA4 “A/A” genotype was present in 6.5% of PBC patients, compared with 1.7% of controls (OR, 3.98; P < 0.0001). Conclusion TNF2A amplifies the CTLA4 rs231725 “A/A” genotype risk for PBC. Although the mechanisms remain unclear, the premise that deficiency in T-cell regulation resulting in an increased risk of PBC is amplified by overexpression of an important proinflammatory cytokine provides a basis

Genetic and epigenetic changes in oilseed rape (Brassica napus L. extracted from intergeneric allopolyploid and additions with Orychophragmus

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Mayank eGautam

2016-04-01

Full Text Available ABSTRACT Allopolyploidization with the merger of the genomes from different species has been shown to be associated with genetic and epigenetic changes. But the maintenance of such alterations related to one parental species after the genome is extracted from the allopolyploid remains to be detected. In this study, the genome of Brassica napus L. (2n=38, genomes AACC was extracted from its intergeneric allohexaploid (2n=62, genomes AACCOO with another crucifer Orychophragmus violaceus (2n=24, genome OO, by backcrossing and development of alien addition lines. B. napus-type plants identified in the self-pollinated progenies of nine monosomic additions were analyzed by the methods of amplified fragment length polymorphism (AFLP, sequence-specific amplified polymorphism (SSAP, and methylation-sensitive amplified polymorphism (MSAP. They showed modifications to certain extents in genomic components (loss and gain of DNA segments and transposons, introgression of alien DNA segments and DNA methylation, compared with B. napus donor. The significant differences in the changes between the B. napus types extracted from these additions likely resulted from the different effects of individual alien chromosomes. Particularly, the additions which harbored the O. violaceus chromosome carrying dominant rRNA genes over those of B. napus tended to result in the development of plants which showed fewer changes, suggesting a role of the expression levels of alien rRNA genes in genomic stability. These results provided new cues for the genetic alterations in one parental genome that are maintained even after the genome becomes independent.

Methyl-Analyzer--whole genome DNA methylation profiling.

Science.gov (United States)

Xin, Yurong; Ge, Yongchao; Haghighi, Fatemeh G

2011-08-15

Methyl-Analyzer is a python package that analyzes genome-wide DNA methylation data produced by the Methyl-MAPS (methylation mapping analysis by paired-end sequencing) method. Methyl-MAPS is an enzymatic-based method that uses both methylation-sensitive and -dependent enzymes covering >80% of CpG dinucleotides within mammalian genomes. It combines enzymatic-based approaches with high-throughput next-generation sequencing technology to provide whole genome DNA methylation profiles. Methyl-Analyzer processes and integrates sequencing reads from methylated and unmethylated compartments and estimates CpG methylation probabilities at single base resolution. Methyl-Analyzer is available at http://github.com/epigenomics/methylmaps. Sample dataset is available for download at http://epigenomicspub.columbia.edu/methylanalyzer_data.html. fgh3@columbia.edu Supplementary data are available at Bioinformatics online.

Allele-Specific DNA Methylation Detection by Pyrosequencing®

DEFF Research Database (Denmark)

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......-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....

Genetic variation among isolates of Sarcocystis neurona, the agent of protozoal myeloencephalitis, as revealed by amplified fragment length polymorphism markers.

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Elsheikha, H M; Schott, H C; Mansfield, L S

2006-06-01

Sarcocystis neurona causes serious neurological disease in horses and other vertebrates in the Americas. Based on epidemiological data, this parasite has recently emerged. Here, the genetic diversity of Sarcocystis neurona was evaluated using the amplified fragment length polymorphism (AFLP) method. Fifteen S. neurona taxa from different regions collected over the last 10 years were used; six isolates were from clinically diseased horses, eight isolates were from wild-caught opossums (Didelphis virginiana), and one isolate was from a cowbird (Molothrus ater). Additionally, four outgroup taxa were also fingerprinted. Nine primer pairs were used to generate AFLP patterns, with a total number of amplified fragments ranging from 30 to 60, depending on the isolate and primers tested. Based on the presence/absence of amplified AFLP fragments and pairwise similarity values, all the S. neurona isolates tested were clustered in one monophyletic group. No significant correlation could be found between genomic similarity and host origin of the S. neurona isolates. AFLP revealed significant intraspecific genetic variations, and S. neurona appeared as a highly variable species. Furthermore, linkage disequilibrium analysis suggested that S. neurona populations within Michigan have an intermediate type of population structure that includes characteristics of both clonal and panamictic population structures. AFLP is a reliable molecular technique that has provided one of the most informative approaches to ascertain phylogenetic relationships in S. neurona and its closest relatives, allowing them to be clustered by relative similarity using band matching and unweighted pair group method with arithmetic mean analysis, which may be applicable to other related protozoal species.

Quantitative DNA methylation analysis of candidate genes in cervical cancer.

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Erin M Siegel

Full Text Available Aberrant DNA methylation has been observed in cervical cancer; however, most studies have used non-quantitative approaches to measure DNA methylation. The objective of this study was to quantify methylation within a select panel of genes previously identified as targets for epigenetic silencing in cervical cancer and to identify genes with elevated methylation that can distinguish cancer from normal cervical tissues. We identified 49 women with invasive squamous cell cancer of the cervix and 22 women with normal cytology specimens. Bisulfite-modified genomic DNA was amplified and quantitative pyrosequencing completed for 10 genes (APC, CCNA, CDH1, CDH13, WIF1, TIMP3, DAPK1, RARB, FHIT, and SLIT2. A Methylation Index was calculated as the mean percent methylation across all CpG sites analyzed per gene (~4-9 CpG site per sequence. A binary cut-point was defined at >15% methylation. Sensitivity, specificity and area under ROC curve (AUC of methylation in individual genes or a panel was examined. The median methylation index was significantly higher in cases compared to controls in 8 genes, whereas there was no difference in median methylation for 2 genes. Compared to HPV and age, the combination of DNA methylation level of DAPK1, SLIT2, WIF1 and RARB with HPV and age significantly improved the AUC from 0.79 to 0.99 (95% CI: 0.97-1.00, p-value = 0.003. Pyrosequencing analysis confirmed that several genes are common targets for aberrant methylation in cervical cancer and DNA methylation level of four genes appears to increase specificity to identify cancer compared to HPV detection alone. Alterations in DNA methylation of specific genes in cervical cancers, such as DAPK1, RARB, WIF1, and SLIT2, may also occur early in cervical carcinogenesis and should be evaluated.

Association of 5, 10- methylenetetrahydrofolate reductase C677T polymorphism in susceptibility to tropical chronic pancreatitis in north Indian population.

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Singh, S; Choudhuri, G; Kumar, R; Agarwal, S

2012-12-22

MTHFR is a key enzyme in folate metabolism that catalyzes the conversion of 5, 10—methlenetetrahydrofolate (5, 10— methylene THF) to 5—methyltetrahydrofolate (5—methyl THF), a predominant circulatory form of folate and methyl donor for the remethylation of homocysteine to methionine. Some studies have shown that C667T polymorphism increases the risk of pancreatic cancer. Since MTHFR is involved in methylation, inflammation and protection against oxidative stress, the processes especially important for pancreatic homeostasis. The altered enzyme activity could play a role in pancreatic injury. The role of MTHFR C677T polymorphism in chronic pancreatitis has been explored by conducting a hospital based; case—control study involving 100 patients radiologically confirmed chronic pancreatitis and 329 healthy controls. All samples were analyzed for MTHFR C677T polymorphism using PCR—RFLP method. Restriction enzyme Hinf I was used to digest the 198 bp amplified product. The frequency of the MTHFR was 57.3%, 34.1% and 8.5% among cases compared with 87.2%,11.2% and 1.5% of controls for CC, CT and TT genotypes, respectively. The T Allele frequency was found significantly higher in patients than in controls. A significant association with T allele was observed with p—value (< 0.0001) odds ratio 4.475 and (95% CI=2.961—7.046). It could be predisposing to the traditional risk factors such as diabetes, dietary, alcohal and smoking habit that are known to be associated with chronic pancreatitis. Additionally it was observed that smoking increases the risk of chronic pancreatitis by 4.1 times. The T allele frequency of MTHFR (C667T) was found to be a significant risk factor for chronic pancreatitis playing a crucial role in altered folate metabolsim.

Feedback regulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 via ATM/Chk2 pathway contributes to the resistance of MCF-7 breast cancer cells to cisplatin.

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Lv, Juan; Qian, Ying; Ni, Xiaoyan; Xu, Xiuping; Dong, Xuejun

2017-03-01

The methyl methanesulfonate and ultraviolet-sensitive gene clone 81 protein is a structure-specific nuclease that plays important roles in DNA replication and repair. Knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 has been found to sensitize cancer cells to chemotherapy. However, the underlying molecular mechanism is not well understood. We found that methyl methanesulfonate and ultraviolet-sensitive gene clone 81 was upregulated and the ATM/Chk2 pathway was activated at the same time when MCF-7 cells were treated with cisplatin. By using lentivirus targeting methyl methanesulfonate and ultraviolet-sensitive gene clone 81 gene, we showed that knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 enhanced cell apoptosis and inhibited cell proliferation in MCF-7 cells under cisplatin treatment. Abrogation of ATM/Chk2 pathway inhibited cell viability in MCF-7 cells in response to cisplatin. Importantly, we revealed that ATM/Chk2 was required for the upregulation of methyl methanesulfonate and ultraviolet-sensitive gene clone 81, and knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 resulted in inactivation of ATM/Chk2 pathway in response to cisplatin. Meanwhile, knockdown of methyl methanesulfonate and ultraviolet-sensitive gene clone 81 activated the p53/Bcl-2 pathway in response to cisplatin. These data suggest that the ATM/Chk2 may promote the repair of DNA damage caused by cisplatin by sustaining methyl methanesulfonate and ultraviolet-sensitive gene clone 81, and the double-strand breaks generated by methyl methanesulfonate and ultraviolet-sensitive gene clone 81 may activate the ATM/Chk2 pathway in turn, which provide a novel mechanism of how methyl methanesulfonate and ultraviolet-sensitive gene clone 81 modulates DNA damage response and repair.

A parallel and sensitive software tool for methylation analysis on multicore platforms.

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Tárraga, Joaquín; Pérez, Mariano; Orduña, Juan M; Duato, José; Medina, Ignacio; Dopazo, Joaquín

2015-10-01

DNA methylation analysis suffers from very long processing time, as the advent of Next-Generation Sequencers has shifted the bottleneck of genomic studies from the sequencers that obtain the DNA samples to the software that performs the analysis of these samples. The existing software for methylation analysis does not seem to scale efficiently neither with the size of the dataset nor with the length of the reads to be analyzed. As it is expected that the sequencers will provide longer and longer reads in the near future, efficient and scalable methylation software should be developed. We present a new software tool, called HPG-Methyl, which efficiently maps bisulphite sequencing reads on DNA, analyzing DNA methylation. The strategy used by this software consists of leveraging the speed of the Burrows-Wheeler Transform to map a large number of DNA fragments (reads) rapidly, as well as the accuracy of the Smith-Waterman algorithm, which is exclusively employed to deal with the most ambiguous and shortest reads. Experimental results on platforms with Intel multicore processors show that HPG-Methyl significantly outperforms in both execution time and sensitivity state-of-the-art software such as Bismark, BS-Seeker or BSMAP, particularly for long bisulphite reads. Software in the form of C libraries and functions, together with instructions to compile and execute this software. Available by sftp to anonymous@clariano.uv.es (password 'anonymous'). juan.orduna@uv.es or jdopazo@cipf.es. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Relationship Between ABCB1 Polymorphisms and Cold Pain Sensitivity Among Healthy Opioid-naive Malay Males.

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Zahari, Zalina; Lee, Chee Siong; Ibrahim, Muslih Abdulkarim; Musa, Nurfadhlina; Mohd Yasin, Mohd Azhar; Lee, Yeong Yeh; Tan, Soo Choon; Mohamad, Nasir; Ismail, Rusli

2017-09-01

Endogenous and exogenous opioids are substrates of the permeability glycoprotein (P-gp) efflux transporter, which is encoded by the ABCB1 (MDR1) gene. Genetic polymorphisms of ABCB1 may contribute to interindividual differences in pain modulation and analgesic responses. We investigated the relationship between ABCB1 polymorphisms and cold pain sensitivity among healthy males. Cold pain responses, including pain threshold and pain tolerance, were measured using the cold-pressor test (CPT). DNA was extracted from whole blood and genotyped for ABCB1 polymorphisms, including c.1236C>T (rs1128503), c.2677G>T/A (rs2032582), and c.3435C>T (rs1045642), using the allelic discrimination real-time polymerase chain reaction. A total of 152 participants were recruited in this observational study. Frequencies of mutated allele for c.1236C>T, c.2677G>T/A, and c.3435C>T polymorphisms were 56.6%, 49.7%, and 43.4%, respectively. Our results revealed an association of the CGC/CGC diplotype (c.1236C>T, c.2677G>T/A, and c.3435C>T) with cold pain sensitivity. Participants with the CGC/CGC diplotype had 90% and 72% higher cold pain thresholds (87.62 seconds vs. 46.19 seconds, P = 0.010) and cold pain tolerances (97.24 seconds vs. 56.54 seconds, P = 0.021), respectively, when compared with those without the diplotype. The CGC/CGC diplotype of ABCB1 polymorphisms was associated with variability in cold pain threshold and pain tolerance in healthy males. © 2016 World Institute of Pain.

Identification of Genes Related to Paulownia Witches’ Broom by AFLP and MSAP

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Xibing Cao

2014-08-01

Full Text Available DNA methylation is believed to play important roles in regulating gene expression in plant growth and development. Paulownia witches’ broom (PaWB infection has been reported to be related to gene expression changes in paulownia plantlets. To determine whether DNA methylation is associated with gene expression changes in response to phytoplasma, we investigated variations in genomic DNA sequence and methylation in PaWB plantlets treated with methyl methane sulfonate (MMS using amplified fragment length polymorphism (AFLP and methylation-sensitive amplification polymorphism (MSAP techniques, respectively. The results indicated that PaWB seedings recovered a normal morphology after treatment with more than 15 mg·L−1 MMS. PaWB infection did not cause changes of the paulownia DNA sequence at the AFLP level; However, DNA methylation levels and patterns were altered. Quantitative real-time PCR (qRT-PCR showed that three of the methylated genes were up-regulated and three were down-regulated in the MMS-treated PaWB plantlets that had regained healthy morphology. These six genes might be involved in transcriptional regulation, plant defense, signal transduction and energy. The possible roles of these genes in PaWB are discussed. The results showed that changes of DNA methylation altered gene expression levels, and that MSAP might help identify genes related to PaWB.

RAPD cluster analysis and chlorate sensitivity of some Indian isolates of Macrophomina phaseolina from sorghum and their relationships with pathogenicity.

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Das, I K; Fakrudin, B; Arora, D K

2008-01-01

Charcoal rot caused by Macrophomina phaseolina is an economically important disease in sorghum grown during the post rainy season in India. Variations in random amplified polymorphic DNA (RAPD) polymorphisms, chlorate sensitivity and pathogenicity were studied among sorghum isolates of M. phaseolina collected from different parts of India. RAPD data based on 14 random primers of Kit A and C (OPA and OPC) on 20 isolates showed a high degree of polymorphism (98.1%) in different isolates. UPGMA dendrogram on RAPD data produced 7 clusters at the level of 37% similarity. Isolates from the same locations showed a tendency to group closer, substantiating closer genetic relatedness. Sorghum infecting Macrophomina isolates showed a mixed response for sensitivity to potassium chlorate (120 mM). Chlorate-resistant isolates were predominant (>65% of the isolates) over sensitive isolates. Chlorate-sensitive isolates were found to be genetically closer among them than the resistant ones. For the first time it was shown that chlorate sensitivity in Macrophomina had some relations with charcoal rot severity in sorghum.

Linkage of DNA Methylation Quantitative Trait Loci to Human Cancer Risk

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Holger Heyn

2014-04-01

Full Text Available Epigenetic regulation and, in particular, DNA methylation have been linked to the underlying genetic sequence. DNA methylation quantitative trait loci (meQTL have been identified through significant associations between the genetic and epigenetic codes in physiological and pathological contexts. We propose that interrogating the interplay between polymorphic alleles and DNA methylation is a powerful method for improving our interpretation of risk alleles identified in genome-wide association studies that otherwise lack mechanistic explanation. We integrated patient cancer risk genotype data and genome-scale DNA methylation profiles of 3,649 primary human tumors, representing 13 solid cancer types. We provide a comprehensive meQTL catalog containing DNA methylation associations for 21% of interrogated cancer risk polymorphisms. Differentially methylated loci harbor previously reported and as-yet-unidentified cancer genes. We suggest that such regulation at the DNA level can provide a considerable amount of new information about the biology of cancer-risk alleles.

The role of glutathione S-transferase and claudin-1 gene polymorphisms in contact sensitization

DEFF Research Database (Denmark)

Ross-Hansen, K; Linneberg, A; Johansen, J D

2013-01-01

BACKGROUND: Contact sensitization is frequent in the general population and arises from excessive or repeated skin exposure to chemicals and metals. However, little is known about its genetic susceptibility. OBJECTIVES: To determine the role of polymorphisms of glutathione S-transferase (GST) genes...

Amplified OTDR Systems for Multipoint Corrosion Monitoring

Science.gov (United States)

Nascimento, Jehan F.; Silva, Marcionilo J.; Coêlho, Isnaldo J. S.; Cipriano, Eliel; Martins-Filho, Joaquim F.

2012-01-01

We present two configurations of an amplified fiber-optic-based corrosion sensor using the optical time domain reflectometry (OTDR) technique as the interrogation method. The sensor system is multipoint, self-referenced, has no moving parts and can measure the corrosion rate several kilometers away from the OTDR equipment. The first OTDR monitoring system employs a remotely pumped in-line EDFA and it is used to evaluate the increase in system reach compared to a non-amplified configuration. The other amplified monitoring system uses an EDFA in booster configuration and we perform corrosion measurements and evaluations of system sensitivity to amplifier gain variations. Our experimental results obtained under controlled laboratory conditions show the advantages of the amplified system in terms of longer system reach with better spatial resolution, and also that the corrosion measurements obtained from our system are not sensitive to 3 dB gain variations. PMID:22737017

Assessment of intercentre reproducibility and epidemiological concordance of Legionella pneumophila serogroup 1 genotyping by amplified fragment length polymorphism analysis

DEFF Research Database (Denmark)

Fry, N K; Bangsborg, Jette Marie; Bernander, S

2000-01-01

The aims of this work were to assess (i) the intercentre reproducibility and epidemiological concordance of amplified fragment length polymorphism analysis for epidemiological typing of Legionella pneumophila serogroup 1, and (ii) the suitability of the method for standardisation and implementation...... by members of the European Working Group on Legionella Infections. Fifty coded isolates comprising two panels of well-characterised strains, a "reproducibility" panel (n=20) and an "epidemiologically related" panel (n=30), were sent to 13 centres in 12 European countries. Analysis was undertaken in each...... using gel analysis software yielded R=1.00 and E=1.00, with 12, 13 or 14 types. This method can be used as a simple, rapid screening tool for epidemiological typing of isolates of Legionella pneumophila serogroup 1. Results demonstrate that the method can be highly reproducible (R=1...

Sensitivity enhancement of chemically amplified resists and performance study using EUV interference lithography

Science.gov (United States)

Buitrago, Elizabeth; Nagahara, Seiji; Yildirim, Oktay; Nakagawa, Hisashi; Tagawa, Seiichi; Meeuwissen, Marieke; Nagai, Tomoki; Naruoka, Takehiko; Verspaget, Coen; Hoefnagels, Rik; Rispens, Gijsbert; Shiraishi, Gosuke; Terashita, Yuichi; Minekawa, Yukie; Yoshihara, Kosuke; Oshima, Akihiro; Vockenhuber, Michaela; Ekinci, Yasin

2016-03-01

Extreme ultraviolet lithography (EUVL, λ = 13.5 nm) is the most promising candidate to manufacture electronic devices for future technology nodes in the semiconductor industry. Nonetheless, EUVL still faces many technological challenges as it moves toward high-volume manufacturing (HVM). A key bottleneck from the tool design and performance point of view has been the development of an efficient, high power EUV light source for high throughput production. Consequently, there has been extensive research on different methodologies to enhance EUV resist sensitivity. Resist performance is measured in terms of its ultimate printing resolution, line width roughness (LWR), sensitivity (S or best energy BE) and exposure latitude (EL). However, there are well-known fundamental trade-off relationships (LRS trade-off) among these parameters for chemically amplified resists (CARs). Here we present early proof-of-principle results for a multi-exposure lithography process that has the potential for high sensitivity enhancement without compromising other important performance characteristics by the use of a Photosensitized Chemically Amplified Resist (PSCAR). With this method, we seek to increase the sensitivity by combining a first EUV pattern exposure with a second UV flood exposure (λ = 365 nm) and the use of a PSCAR. In addition, we have evaluated over 50 different state-of-the-art EUV CARs. Among these, we have identified several promising candidates that simultaneously meet sensitivity, LWR and EL high performance requirements with the aim of resolving line space (L/S) features for the 7 and 5 nm logic node (16 nm and 13 nm half-pitch HP, respectively) for HVM. Several CARs were additionally found to be well resolved down to 12 nm and 11 nm HP with minimal pattern collapse and bridging, a remarkable feat for CARs. Finally, the performance of two negative tone state-of-the-art alternative resist platforms previously investigated was compared to the CAR performance at and

Epigenetic patterns newly established after interspecific hybridization in natural populations of Solanum

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Cara, Nicolás; Marfil, Carlos F; Masuelli, Ricardo W

2013-01-01

Interspecific hybridization is known for triggering genetic and epigenetic changes, such as modifications on DNA methylation patterns and impact on phenotypic plasticity and ecological adaptation. Wild potatoes (Solanum, section Petota) are adapted to multiple habitats along the Andes, and natural hybridizations have proven to be a common feature among species of this group. Solanum × rechei, a recently formed hybrid that grows sympatrically with the parental species S. kurtzianum and S. microdontum, represents an ideal model for studying the ecologically and evolutionary importance of hybridization in generating of epigenetic variability. Genetic and epigenetic variability and their correlation with morphological variation were investigated in wild and ex situ conserved populations of these three wild potato species using amplified fragment length polymorphism (AFLP) and methylation-sensitive amplified polymorphism (MSAP) techniques. We observed that novel methylation patterns doubled the number of novel genetic patterns in the hybrid and that the morphological variability measured on 30 characters had a higher correlation with the epigenetic than with the genetic variability. Statistical comparison of methylation levels suggested that the interspecific hybridization induces genome demethylation in the hybrids. A Bayesian analysis of the genetic data reveled the hybrid nature of S. × rechei, with genotypes displaying high levels of admixture with the parental species, while the epigenetic information assigned S. × rechei to its own cluster with low admixture. These findings suggested that after the hybridization event, a novel epigenetic pattern was rapidly established, which might influence the phenotypic plasticity and adaptation of the hybrid to new environments. PMID:24198938

Charge-sensitive and shaping amplifier microassemblies for dosimetry and spectrometry on CZT-detectors

International Nuclear Information System (INIS)

Perevertaylo, V.L.; Zaitsevsky, I.L.; Tarasenko, L.I.; Perevertaylo, A.V.; Shkirenko, E.A.

2012-01-01

Developments of new spectrometric channel electronics on the basis of microassemblies, which allowed to reduce the noise and increase of signal-to-noise ratio, lowering power consumption and dimensions. The complete line of front-end electronics for CZT detectors implemented as micro-assemblies is described, the design concept, operation details and application features of charge sensitive amplifier and shaping amplifier microassemblies are discussed, and the results obtained when registering low energy X-ray spectra are shown. It has a high energy resolution δE at the level of the leading companies. For direct detection with silicon p-i-n-diode new electronic channel can resolve 241 Am peaks up to 8 keV with a resolution of about 2 keV at room temperature. New electronics is universal and can be used with different semiconductor detectors - Si, CdZnTe, Scintillator-photodiode, as shown in the spectra. Low power consumption and reduced dimensions allows the using in portable equipment. Manufacturability of micro assembly opens up the possibility for mass production and low cost opens up the possibility to supply them with detectors as S tart kit f or the construction of radiometric and spectrometric devices

MLH1-93 G/a polymorphism is associated with MLH1 promoter methylation and protein loss in dysplastic sessile serrated adenomas with BRAFV600E mutation.

Science.gov (United States)

Fennell, Lochlan J; Jamieson, Saara; McKeone, Diane; Corish, Tracie; Rohdmann, Megan; Furner, Tori; Bettington, Mark; Liu, Cheng; Kawamata, Futoshi; Bond, Catherine; Van De Pols, Jolieke; Leggett, Barbara; Whitehall, Vicki

2018-01-05

Sessile serrated adenomas with BRAF mutation progress rapidly to cancer following the development of dysplasia (SSAD). Approximately 75% of SSADs methylate the mismatch repair gene MLH1, develop mismatch repair deficiency and the resultant cancers have a good prognosis. The remaining SSADs and BRAF mutant traditional serrated adenomas (TSA) develop into microsatellite stable cancers with a poor prognosis. The reason for this dichotomy is unknown. In this study, we assessed the genotypic frequency of the MLH1-93 polymorphism rs1800734 in SSADs and TSAs to determine if the uncommon variant A allele predisposes to MLH1 promoter hypermethylation. We performed genotyping for the MLH1-93 polymorphism, quantitative methylation specific PCR, and MLH1 immunohistochemistry on 124 SSAD, 128 TSA, 203 BRAF mutant CRCs and 147 control subjects with normal colonoscopy. The minor A allele was significantly associated with a dose dependent increase in methylation at the MLH1 promoter in SSADs (p = 0.022). The AA genotype was only observed in SSADs with MLH1 loss. The A allele was also overrepresented in BRAF mutant cancers with MLH1 loss. Only one of the TSAs showed loss of MLH1 and the overall genotype distribution in TSAs did not differ from controls. The MLH1-93 AA genotype is significantly associated with promoter hypermethylation and MLH1 loss in the context of SSADs. BRAF mutant microsatellite stable colorectal cancers with the AA genotype most likely arise in TSAs since the A allele does not predispose to methylation in this context.

Assessment of genetic and epigenetic changes in virus-free garlic (Allium sativum L.) plants obtained by meristem culture followed by in vitro propagation.

Science.gov (United States)

Gimenez, Magalí Diana; Yañez-Santos, Anahí Mara; Paz, Rosalía Cristina; Quiroga, Mariana Paola; Marfil, Carlos Federico; Conci, Vilma Cecilia; García-Lampasona, Sandra Claudia

2016-01-01

This is the first report assessing epigenetic variation in garlic. High genetic and epigenetic polymorphism during in vitro culture was detected.Sequencing of MSAP fragments revealed homology with ESTs. Garlic (Allium sativum) is a worldwide crop of economic importance susceptible to viral infections that can cause significant yield losses. Meristem tissue culture is the most employed method to sanitize elite cultivars.Often the virus-free garlic plants obtained are multiplied in vitro (micro propagation). However, it was reported that micro-propagation frequently produces somaclonal variation at the phenotypic level, which is an undesirable trait when breeders are seeking to maintain varietal stability. We employed amplification fragment length polymorphism and methylation sensitive amplified polymorphism (MSAP) methodologies to assess genetic and epigenetic modifications in two culture systems: virus-free plants obtained by meristem culture followed by in vitro multiplication and field culture. Our results suggest that garlic exhibits genetic and epigenetic polymorphism under field growing conditions. However, during in vitro culture system both kinds of polymorphisms intensify indicating that this system induces somaclonal variation. Furthermore, while genetic changes accumulated along the time of in vitro culture, epigenetic polymorphism reached the major variation at 6 months and then stabilize, being demethylation and CG methylation the principal conversions.Cloning and sequencing differentially methylated MSAP fragments allowed us to identify coding and unknown sequences of A. sativum, including sequences belonging to LTR Gypsy retrotransposons. Together, our results highlight that main changes occur in the initial 6 months of micro propagation. For the best of our knowledge, this is the first report on epigenetic assessment in garlic.

Population structure of a lodgepole pine (Pinus contorta) and jack pine (P. banksiana) complex as revealed by random amplified polymorphic DNA.

Science.gov (United States)

Ye, Terrance Z; Yang, Rong-Cai; Yeh, Francis C

2002-06-01

We studied the population structure of a lodgepole (Pinus contorta Dougl.) and jack pine (Pinus banksiana Lamb.) complex in west central Alberta and neighboring areas by assessing random amplified polymorphic DNA (RAPD) variability in 23 lodgepole pine, 9 jack pine, and 8 putative hybrid populations. Of 200 random primers screened, 10 that amplified 39 sharp and reproducible RAPDs were chosen for the study. None of the 39 RAPDs were unique to the parental species. RAPD diversity ranged from 0.085 to 0.190 among populations and averaged 0.143 for lodgepole pine, 0.156 for jack pine, 0.152 for hybrids, and 0.148 for all 40 populations. The estimated population differentiation based on G(ST) was 0.168 for hybrids, 0.162 for lodgepole pine, 0.155 for jack pine, and 0.247 across all 40 populations. Cluster analysis of genetic distances generally separated jack pine from lodgepole pine and hybrids, but no division could be identified that further separated lodgepole pine from hybrids. The observed weak to mild trend of "introgression by distance" in the complex and neighbouring areas was consistent with the view that introgressive hybridization between lodgepole and jack pines within and outside the hybrid zone may have been through secondary contact and primary intergradation, respectively.

DNA methylation and transcriptomic changes in response to different lights and stresses in 7B-1 male-sterile tomato.

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Vahid Omidvar

Full Text Available We reported earlier that 7B-1 mutant in tomato (Solanum lycopersicum L., cv. Rutgers, an ABA overproducer, is defective in blue light (B signaling leading to B-specific resistance to abiotic and biotic stresses. Using a methylation-sensitive amplified polymorphism (MSAP assay, a number of genes were identified, which were differentially methylated between 7B-1 and its wild type (WT seedlings in white (W, blue (B, red (R lights and dark (D or in response to exogenous ABA and mannitol-induced stresses. The genomic methylation level was almost similar in different lights between 7B-1 and WT seedlings, while significant differences were observed in response to stresses in D, but not B. Using a cDNA-AFLP assay, several transcripts were identified, which were differentially regulated between 7B-1 and WT by B or D or in response to stresses. Blue light receptors cryptochrome 1 and 2 (CRY1 and CRY2 and phototropin 1 and 2 (PHOT1 and PHOT2 were not affected by the 7B-1 mutation at the transcriptional level, instead the mutation had likely affected downstream components of the light signaling pathway. 5-azacytidine (5-azaC induced DNA hypomethylation, inhibited stem elongation and differentially regulated the expression of a number of genes in 7B-1. In addition, it was shown that mir167 and mir390 were tightly linked to auxin signaling pathway in 5-azaC-treated 7B-1 seedlings via the regulation of auxin-response factor (ARF transcripts. Our data showed that DNA methylation remodeling is an active epigenetic response to different lights and stresses in 7B-1 and WT, and highlighted the differences in epigenetic and transcriptional regulation of light and stress responses between 7B-1 and WT. Furthermore, it shed lights on the crosstalk between DNA hypomethylation and miRNA regulation of ARFs expression. This information could also be used as a benchmark for future studies of male-sterility in other crops.

Development of an optimized random amplified polymorphic DNA protocol for fingerprinting of Klebsiella pneumoniae.

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Ashayeri-Panah, M; Eftekhar, F; Feizabadi, M M

2012-04-01

To develop an optimized random amplified polymorphic DNA (RAPD) protocol for fingerprinting clinical isolates of Klebsiella pneumoniae. Employing factorial design of experiments, repeatable amplification patterns were obtained for 54 nosocomial isolates using 1 μmol 1(-1) primer, 4 mmol 1(-1) MgCl(2), 0·4 mmol 1(-1) dNTPs, 2·5 U Taq DNA polymerase and 90 ng DNA template in a total volume of 25 μl. The optimum thermocycling program was: initial denaturation at 94°C for 4 min followed by 50 cycles of 1 min at 94°C, 2 min at 34°C, 2 min at 72°C and a final extension at 72°C for 10 min. The optimized RAPD protocol was highly discriminatory (Simpson's diversity index, 0·982), and all isolates were typable with repeatable patterns (Pearson's similarity coefficient ≈ 100%). Seven main clusters were obtained on a similarity level of 70% and 32 distinct clusters on a similarity level of 85%, reflecting the heterogeneity of the isolates. Systematic optimization of RAPD generated reliable DNA fingerprints for nosocomial isolates of K. pneumoniae. This is the first report on RAPD optimization based on factorial design of experiments for discrimination of K. pneumoniae. © 2012 The Authors. Letters in Applied Microbiology © 2012 The Society for Applied Microbiology.

Sensitivity enhancement of chemically amplified resists and performance study using extreme ultraviolet interference lithography

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Buitrago, Elizabeth; Nagahara, Seiji; Yildirim, Oktay; Nakagawa, Hisashi; Tagawa, Seiichi; Meeuwissen, Marieke; Nagai, Tomoki; Naruoka, Takehiko; Verspaget, Coen; Hoefnagels, Rik; Rispens, Gijsbert; Shiraishi, Gosuke; Terashita, Yuichi; Minekawa, Yukie; Yoshihara, Kosuke; Oshima, Akihiro; Vockenhuber, Michaela; Ekinci, Yasin

2016-07-01

Extreme ultraviolet lithography (EUVL, λ=13.5 nm) is the most promising candidate to manufacture electronic devices for future technology nodes in the semiconductor industry. Nonetheless, EUVL still faces many technological challenges as it moves toward high-volume manufacturing (HVM). A key bottleneck from the tool design and performance point of view has been the development of an efficient, high-power EUV light source for high throughput production. Consequently, there has been extensive research on different methodologies to enhance EUV resist sensitivity. Resist performance is measured in terms of its ultimate printing resolution, line width roughness (LWR), sensitivity [S or best energy (BE)], and exposure latitude (EL). However, there are well-known fundamental trade-off relationships (line width roughness, resolution and sensitivity trade-off) among these parameters for chemically amplified resists (CARs). We present early proof-of-principle results for a multiexposure lithography process that has the potential for high sensitivity enhancement without compromising other important performance characteristics by the use of a "Photosensitized Chemically Amplified Resist™" (PSCAR™). With this method, we seek to increase the sensitivity by combining a first EUV pattern exposure with a second UV-flood exposure (λ=365 nm) and the use of a PSCAR. In addition, we have evaluated over 50 different state-of-the-art EUV CARs. Among these, we have identified several promising candidates that simultaneously meet sensitivity, LWR, and EL high-performance requirements with the aim of resolving line space (L/S) features for the 7- and 5-nm logic node [16- and 13-nm half-pitch (HP), respectively] for HVM. Several CARs were additionally found to be well resolved down to 12- and 11-nm HP with minimal pattern collapse and bridging, a remarkable feat for CARs. Finally, the performance of two negative tone state-of-the-art alternative resist platforms previously investigated

Characterization of Grain Amaranth (Amaranthus spp. Germplasm in South West Nigeria Using Morphological, Nutritional, and Random Amplified Polymorphic DNA (RAPD Analysis

Directory of Open Access Journals (Sweden)

Pamela E. Akin-Idowu

2016-01-01

Full Text Available Efficient utilization of plant genetic resources for nutrition and crop improvement requires systematic understanding of the important traits. Amaranthus species are distributed worldwide with an interesting diversity of landraces and cultivars whose leaves and seeds are consumed. Despite their potential to enhance food security and economic livelihoods, grain amaranth breeding to improve nutritional quality and adoption by farmers in sub-Saharan Africa is scanty. This study assessed the variation among 29 grain amaranth accessions using 27 phenotypic (10 morphological and 17 nutritional characters and 16 random amplified polymorphic DNA (RAPD primers. Multivariate analysis of phenotypic characters showed the first four principal components contributing 57.53% of observed variability, while cluster analysis yielded five groups at 87.5% similarity coefficient. RAPD primers generated a total of 193 amplicons with an average of 12.06 amplicons per primer, 81% of which were polymorphic. Genetic similarities based on Jaccard’s coefficient ranged from 0.61 to 0.88. The RAPD-based unweighted pair group method with arithmetic mean dendrogram grouped the accessions into nine clusters, with the same species clustering together. RAPD primers distinguished the accessions more effectively than phenotypic markers. Accessions in the different clusters as obtained can be exploited for heterotic gain in desired nutritional traits.

Genetic and Epigenetic Alterations of Brassica nigra Introgression Lines from Somatic Hybridization: A Resource for Cauliflower Improvement.

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Wang, Gui-Xiang; Lv, Jing; Zhang, Jie; Han, Shuo; Zong, Mei; Guo, Ning; Zeng, Xing-Ying; Zhang, Yue-Yun; Wang, You-Ping; Liu, Fan

2016-01-01

Broad phenotypic variations were obtained previously in derivatives from the asymmetric somatic hybridization of cauliflower "Korso" (Brassica oleracea var. botrytis, 2n = 18, CC genome) and black mustard "G1/1" (Brassica nigra, 2n = 16, BB genome). However, the mechanisms underlying these variations were unknown. In this study, 28 putative introgression lines (ILs) were pre-selected according to a series of morphological (leaf shape and color, plant height and branching, curd features, and flower traits) and physiological (black rot/club root resistance) characters. Multi-color fluorescence in situ hybridization revealed that these plants contained 18 chromosomes derived from "Korso." Molecular marker (65 simple sequence repeats and 77 amplified fragment length polymorphisms) analysis identified the presence of "G1/1" DNA segments (average 7.5%). Additionally, DNA profiling revealed many genetic and epigenetic differences among the ILs, including sequence alterations, deletions, and variation in patterns of cytosine methylation. The frequency of fragments lost (5.1%) was higher than presence of novel bands (1.4%), and the presence of fragments specific to Brassica carinata (BBCC 2n = 34) were common (average 15.5%). Methylation-sensitive amplified polymorphism analysis indicated that methylation changes were common and that hypermethylation (12.4%) was more frequent than hypomethylation (4.8%). Our results suggested that asymmetric somatic hybridization and alien DNA introgression induced genetic and epigenetic alterations. Thus, these ILs represent an important, novel germplasm resource for cauliflower improvement that can be mined for diverse traits of interest to breeders and researchers.

Epigenetic Differentiation of Natural Populations of Lilium bosniacum Associated with Contrasting Habitat Conditions.

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Zoldoš, Vlatka; Biruš, Ivan; Muratovic, Edina; Šatovic, Zlatko; Vojta, Aleksandar; Robin, Odile; Pustahija, Fatima; Bogunic, Faruk; Vicic Bockor, Vedrana; Siljak-Yakovlev, Sonja

2018-01-01

Epigenetic variation in natural populations with contrasting habitats might be an important element, in addition to the genetic variation, in plant adaptation to environmental stress. Here, we assessed genetic, epigenetic, and cytogenetic structure of the three Lilium bosniacum populations growing on distinct habitats. One population was growing under habitual ecological conditions for this species and the other two were growing under stress associated with high altitude and serpentine soil. Amplified fragment length polymorphism and methylation-sensitive amplification polymorphism analyses revealed that the three populations did not differentiate genetically, but were clearly separated in three distinct clusters according to DNA methylation profiles. Principal coordinate analysis showed that overall epigenetic variation was closely related to habitat conditions. A new methylation-sensitive amplification polymorphism scoring approach allowed identification of mainly unmethylated (φST = 0.190) and fully CpG methylated (φST = 0.118) subepiloci playing a role in overall population differentiation, in comparison with hemimethylated sites (φST = 0.073). In addition, unusual rDNA repatterning and the presence of B chromosomes bearing 5S rDNA loci were recorded in the population growing on serpentine soil, suggesting dynamic chromosome rearrangements probably linked to global genome demethylation, which might have reactivated some mobile elements. We discuss our results considering our earlier data on morphology and leaf anatomy of several L. bosniacum populations, and suggest a possible role of epigenetics as a key element in population differentiation associated with environmental stress in these particular lily populations. © The Author(s) 2018. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

Polymorphic sequence-characterized codominant loci in the chestnut blight fungus, Cryphonectria parasitica

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J. E. Davis; Thomas L. Kubisiak; M. G. Milgroom

2005-01-01

Studies on the population biology of the chestnut blight fungus, Cryphonectria parasitica, have previously been carried out with dominant restriction fragment length polymorphism (RFLP) fingerprinting markers. In this study, we described the development of 11 condominant markers from randomly amplified polymorphic DNAs (RAPDs). RAPD fragments were...

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

Energy Technology Data Exchange (ETDEWEB)

Chiang, Chien-I [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Huang, Ya-Li [Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China); Chen, Wei-Jen [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Shiue, Horng-Sheng [Department of Chinese Medicine, Chang Gung Memorial Hospital and Chang Gung University College of Medicine, Taoyuan, Taiwan (China); Huang, Chao-Yuan; Pu, Yeong-Shiau [Department of Urology, National Taiwan University Hospital, College of Medicine National Taiwan University, Taipei, Taiwan (China); Lin, Ying-Chin [Department of Family Medicine, Shung Ho Hospital, Taipei Medical University, New Taipei, Taiwan (China); Department of Health Examination, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan (China); Division of Family Medicine, School of Medicine, Taipei Medical University, Taipei, Taiwan (China); Hsueh, Yu-Mei, E-mail: ymhsueh@tmu.edu.tw [School of Public Health, College of Public Health and Nutrition, Taipei Medical University, Taipei, Taiwan (China); Department of Public Health, School of Medicine, College of Medicine, Taipei Medical University, Taipei, Taiwan (China)

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

Divergent methylation pattern in adult stage between two forms of Tetranychus urticae (Acari: Tetranychidae).

Science.gov (United States)

Yang, Si-Xia; Guo, Chao; Zhao, Xiu-Ting; Sun, Jing-Tao; Hong, Xiao-Yue

2017-02-19

The two-spotted spider mite, Tetranychus urticae Koch has two forms: green form and red form. Understanding the molecular basis of how these two forms established without divergent genetic background is an intriguing area. As a well-known epigenetic process, DNA methylation has particularly important roles in gene regulation and developmental variation across diverse organisms that do not alter genetic background. Here, to investigate whether DNA methylation could be associated with different phenotypic consequences in the two forms of T. urticae, we surveyed the genome-wide cytosine methylation status and expression level of DNA methyltransferase 3 (Tudnmt3) throughout their entire life cycle. Methylation-sensitive amplification polymorphism (MSAP) analyses of 585 loci revealed variable methylation patterns in the different developmental stages. In particular, principal coordinates analysis (PCoA) indicates a significant epigenetic differentiation between female adults of the two forms. The gene expression of Tudnmt3 was detected in all examined developmental stages, which was significantly different in the adult stage of the two forms. Together, our results reveal the epigenetic distance between the two forms of T. urticae, suggesting that DNA methylation might be implicated in different developmental demands, and contribute to different phenotypes in the adult stage of these two forms. © 2017 Institute of Zoology, Chinese Academy of Sciences.

Analysis of the background noise of field effect transistors in MOS complementary technology and application in the construction of a current-sensitive integrated amplifier

International Nuclear Information System (INIS)

Beuville, E.

1989-10-01

A low noise amplifier for use in high energy physics is developed. The origin and the mechanisms of the noise in MOSFET transistors is carried out with the aim of minimizing such effects in amplifiers. The research is applied in the construction of a current-sensitive integrated amplifier. The time scale continuous filtering principle is used and allows the detection of particles arriving in the counter in a random distribution. The rules which must be taken into account in the construction of an analog integrated circuit are shown [fr

Salt sensitivity of blood pressure is associated with polymorphisms in the sodium-bicarbonate cotransporter.

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Carey, Robert M; Schoeffel, Cynthia D; Gildea, John J; Jones, John E; McGrath, Helen E; Gordon, Lindsay N; Park, Min Jeong; Sobota, Rafal S; Underwood, Patricia C; Williams, Jonathan; Sun, Bei; Raby, Benjamin; Lasky-Su, Jessica; Hopkins, Paul N; Adler, Gail K; Williams, Scott M; Jose, Pedro A; Felder, Robin A

2012-11-01

Previous studies have demonstrated that single nucleotide polymorphisms (SNPs) of the sodium-bicarbonate co-transporter gene (SLC4A5) are associated with hypertension. We tested the hypothesis that SNPs in SLC4A5 are associated with salt sensitivity of blood pressure in 185 whites consuming an isocaloric constant diet with a randomized order of 7 days of low Na(+) (10 mmol/d) and 7 days of high Na(+) (300 mmol/d) intake. Salt sensitivity was defined as a ≥ 7-mm Hg increase in mean arterial pressure during a randomized transition between high and low Na(+) diet. A total of 35 polymorphisms in 17 candidate genes were assayed, 25 of which were tested for association. Association analyses with salt sensitivity revealed 3 variants that associated with salt sensitivity, 2 in SLC4A5 (P<0.001) and 1 in GRK4 (P=0.020). Of these, 2 SNPs in SLC4A5 (rs7571842 and rs10177833) demonstrated highly significant results and large effects sizes, using logistic regression. These 2 SNPs had P values of 1.0 × 10(-4) and 3.1 × 10(-4) with odds ratios of 0.221 and 0.221 in unadjusted regression models, respectively, with the G allele at both sites conferring protection. These SNPs remained significant after adjusting for body mass index and age (P=8.9 × 10(-5) and 2.6 × 10(-4) and odds ratios 0.210 and 0.286, respectively). Furthermore, the association of these SNPs with salt sensitivity was replicated in a second hypertensive population. Meta-analysis demonstrated significant associations of both SNPs with salt sensitivity (rs7571842 [P=1.2 × 10(-5)]; rs1017783 [P=1.1 × 10(-4)]). In conclusion, SLC4A5 variants are strongly associated with salt sensitivity of blood pressure in 2 separate white populations.

Amplified fragment length polymorphism fingerprinting of Pseudomonas strains from a poultry processing plant.

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Geornaras, I; Kunene, N F; von Holy, A; Hastings, J W

1999-09-01

Molecular typing has been used previously to identify and trace dissemination of pathogenic and spoilage bacteria associated with food processing. Amplified fragment length polymorphism (AFLP) is a novel DNA fingerprinting technique which is considered highly reproducible and has high discriminatory power. This technique was used to fingerprint 88 Pseudomonas fluorescens and Pseudomonas putida strains that were previously isolated from plate counts of carcasses at six processing stages and various equipment surfaces and environmental sources of a poultry abattoir. Clustering of the AFLP patterns revealed a high level of diversity among the strains. Six clusters (clusters I through VI) were delineated at an arbitrary Dice coefficient level of 0.65; clusters III (31 strains) and IV (28 strains) were the largest clusters. More than one-half (52.3%) of the strains obtained from carcass samples, which may have represented the resident carcass population, grouped together in cluster III. By contrast, 43.2% of the strains from most of the equipment surfaces and environmental sources grouped together in cluster IV. In most cases, the clusters in which carcass strains from processing stages grouped corresponded to the clusters in which strains from the associated equipment surfaces and/or environmental sources were found. This provided evidence that there was cross-contamination between carcasses and the abattoir environment at the DNA level. The AFLP data also showed that strains were being disseminated from the beginning to the end of the poultry processing operation, since many strains associated with carcasses at the packaging stage were members of the same clusters as strains obtained from carcasses after the defeathering stage.

Development of cleaved amplified polymorphic sequence markers and a CAPS-based genetic linkage map in watermelon (Citrullus lanatus [Thunb.] Matsum. and Nakai) constructed using whole-genome re-sequencing data.

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Liu, Shi; Gao, Peng; Zhu, Qianglong; Luan, Feishi; Davis, Angela R; Wang, Xiaolu

2016-03-01

Cleaved amplified polymorphic sequence (CAPS) markers are useful tools for detecting single nucleotide polymorphisms (SNPs). This study detected and converted SNP sites into CAPS markers based on high-throughput re-sequencing data in watermelon, for linkage map construction and quantitative trait locus (QTL) analysis. Two inbred lines, Cream of Saskatchewan (COS) and LSW-177 had been re-sequenced and analyzed by Perl self-compiled script for CAPS marker development. 88.7% and 78.5% of the assembled sequences of the two parental materials could map to the reference watermelon genome, respectively. Comparative assembled genome data analysis provided 225,693 and 19,268 SNPs and indels between the two materials. 532 pairs of CAPS markers were designed with 16 restriction enzymes, among which 271 pairs of primers gave distinct bands of the expected length and polymorphic bands, via PCR and enzyme digestion, with a polymorphic rate of 50.94%. Using the new CAPS markers, an initial CAPS-based genetic linkage map was constructed with the F2 population, spanning 1836.51 cM with 11 linkage groups and 301 markers. 12 QTLs were detected related to fruit flesh color, length, width, shape index, and brix content. These newly CAPS markers will be a valuable resource for breeding programs and genetic studies of watermelon.

Association between UGT2B7 gene polymorphisms and fentanyl sensitivity in patients undergoing painful orthognathic surgery

Science.gov (United States)

Muraoka, Wataru; Nishizawa, Daisuke; Fukuda, Kenichi; Kasai, Shinya; Hasegawa, Junko; Wajima, Koichi; Nakagawa, Taneaki

2016-01-01

Background Fentanyl is often used instead of morphine for the treatment of pain because it has fewer side effects. The metabolism of morphine by glucuronidation is known to be influenced by polymorphisms of the UGT2B7 gene. Some metabolic products of fentanyl are reportedly metabolized by glucuronate conjugation. The genes that are involved in the metabolic pathway of fentanyl may also influence fentanyl sensitivity. We analyzed associations between fentanyl sensitivity and polymorphisms of the UGT2B7 gene to clarify the hereditary determinants of individual differences in fentanyl sensitivity. Results This study examined whether single-nucleotide polymorphisms (SNPs) of the UGT2B7 gene affect cold pain sensitivity and the analgesic effects of fentanyl, evaluated by a standardized pain test and fentanyl requirements in healthy Japanese subjects who underwent uniform surgical procedures. The rs7439366 SNP of UGT2B7 is reportedly associated with the metabolism and analgesic effects of morphine. We found that this SNP is also associated with the analgesic effects of fentanyl in the cold pressor-induced pain test. It suggested that the C allele of the rs7439366 SNP may enhance analgesic efficacy. Two SNPs of UGT2B7, rs4587017 and rs1002849, were also found to be novel SNPs that may influence the analgesic effects of fentanyl in the cold pressor-induced pain test. Conclusions Fentanyl sensitivity for cold pressor-induced pain was associated with the rs7439366, rs4587017, and rs1002849 SNPs of the UGT2B7 gene. Our findings may provide valuable information for achieving satisfactory pain control and open to new avenues for personalized pain treatment. PMID:28256933

DNA Methylation Alterations at 5'-CCGG Sites in the Interspecific and Intraspecific Hybridizations Derived from Brassica rapa and B. napus.

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Wanshan Xiong

Full Text Available 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.

A modular positive feedback-based gene amplifier

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Bhalerao Kaustubh D

2010-02-01

Full Text Available Abstract Background Positive feedback is a common mechanism used in the regulation of many gene circuits as it can amplify the response to inducers and also generate binary outputs and hysteresis. In the context of electrical circuit design, positive feedback is often considered in the design of amplifiers. Similar approaches, therefore, may be used for the design of amplifiers in synthetic gene circuits with applications, for example, in cell-based sensors. Results We developed a modular positive feedback circuit that can function as a genetic signal amplifier, heightening the sensitivity to inducer signals as well as increasing maximum expression levels without the need for an external cofactor. The design utilizes a constitutively active, autoinducer-independent variant of the quorum-sensing regulator LuxR. We experimentally tested the ability of the positive feedback module to separately amplify the output of a one-component tetracycline sensor and a two-component aspartate sensor. In each case, the positive feedback module amplified the response to the respective inducers, both with regards to the dynamic range and sensitivity. Conclusions The advantage of our design is that the actual feedback mechanism depends only on a single gene and does not require any other modulation. Furthermore, this circuit can amplify any transcriptional signal, not just one encoded within the circuit or tuned by an external inducer. As our design is modular, it can potentially be used as a component in the design of more complex synthetic gene circuits.

Molecular Diversity Analysis of Two in vitro and Irradiated Potato Varieties Expressed by Random Amplified Polymorphic DNA

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Ayman El-FIKI

2018-03-01

Full Text Available Potato buds cvs. ҅Valor’ and ‘Spunta’ were cultured in vitro on MS solid medium with 0.2 mg -1 BAP. The resulting plantlets were irradiated with gamma radiation doses 10, 20, 30, 40 and 50 Gy. Irradiated segments were transferred onto fresh MS with BAP and plantlets survival percentage was calculated after eight weeks. Gamma radiation caused the death of 3.8% to 81% in cv. ҅Spunta’ and 2.9% to 83.9% in cv. ҅Valor’. Microtubers produced from irradiated plantlets were decreased with increasing gamma radiation doses, with notable changes in shape, size and numbers. The proline contents in irradiated plantlets were steady increase with gamma radiation doses. The genomic DNA of the two cultivars and ten radiation treatments was amplified with 10 RAPD primers that generated 53 polymorphic bands. The highest number of genetic identity was 0.9672 showed between irradiated plantlets with 20 and 30 Gy in cv. ҅Valor’. However, the highest genetic distance was 0.3995 observed between irradiated plantlets with dose 20 Gy in cv. ҅Valor’ and 30 Gy in cv. ҅Spunta’. The dendrogram generated by cluster analysis distinguished the irradiated plantlets genetically.

Application of Fluorescent Amplified Fragment Length Polymorphism for Comparison of Human and Animal Isolates of Yersinia enterocolitica

Science.gov (United States)

Fearnley, Catherine; On, Stephen L. W.; Kokotovic, Branko; Manning, Georgina; Cheasty, Tom; Newell, Diane G.

2005-01-01

An amplified fragment length polymorphism (AFLP) method, developed to genotype Yersinia enterocolitica, has been used to investigate 70 representative strains isolated from humans, pigs, sheep, and cattle in the United Kingdom. AFLP primarily distinguished Y. enterocolitica strains according to their biotype, with strains dividing into two distinct clusters: cluster A, comprising largely the putatively pathogenic biotypes (BT2 to -4), and cluster B, comprising the putatively nonpathogenic biotype 1A strains and a single BT1B isolate. Within these two clusters, subclusters formed largely on the basis of serotype. However, AFLP profiles also allowed differentiation of strains within these serotype-related subclusters, indicating the high discriminatory power of the technique for Y. enterocolitica. Investigation of the relationship between strain AFLP profile and host confirmed that pigs are, and provides further proof that sheep may be, potential sources of human infection with putatively pathogenic strains. However, the results suggest that some strains causing human disease do not come from veterinary sources identifiable at this time. The distribution of some BT1A isolates within cluster A raises questions about the relationship between virulence potential and biotype. PMID:16151073

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes.

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Kotoka Masuyama

Full Text Available Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted "bidirectional analysis," which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples.

Two-section semiconductor optical amplifier used as an efficient channel dropping node

DEFF Research Database (Denmark)

Jørgensen, Carsten; Storkfelt, Niels; Durhuus, T.

1992-01-01

High responsivity in a two-section semiconductor optical amplifier/detector, serving as a channel dropping mode is described. A simple receiver constructed using a 50 Ω amplifier with a sensitivity of -30.2 dBm at 140 Mb/s is demonstrated......High responsivity in a two-section semiconductor optical amplifier/detector, serving as a channel dropping mode is described. A simple receiver constructed using a 50 Ω amplifier with a sensitivity of -30.2 dBm at 140 Mb/s is demonstrated...

Classicalization times of parametrically amplified 'Schroedinger cat' states coupled to phase-sensitive reservoirs

International Nuclear Information System (INIS)

Dodonov, V.V.; Valverde, C.; Souza, L.S.; Baseia, B.

2011-01-01

The exact Wigner function of a parametrically excited quantum oscillator in a phase-sensitive amplifying/attenuating reservoir is found for initial even/odd coherent states. Studying the evolution of negativity of the Wigner function we show the difference between the 'initial positivization time' (IPT), which is inversely proportional to the square of the initial size of the superposition, and the 'final positivization time' (FPT), which does not depend on this size. Both these times can be made arbitrarily long in maximally squeezed high-temperature reservoirs. Besides, we find the conditions when some (small) squeezing can exist even after the Wigner function becomes totally positive. -- Highlights: → We study parametric excitation of a quantum oscillator in phase-sensitive baths. → Exact time-dependent Wigner function for initial even/odd coherent states is found. → The evolution of negativity of Wigner function is compared with the squeezing dynamics. → The difference between initial and final 'classicalization times' is emphasized. → Both these times can be arbitrarily long for rigged reservoirs at infinite temperature.

Nuclear magnetic resonance experiments with dc SQUID amplifiers

International Nuclear Information System (INIS)

Heaney, M.B.

1990-11-01

The development and fabrication of dc SQUIDs (Superconducting QUantum Interference Devices) with Nb/Al 2 O 3 /Nb Josephson junctions is described. A theory of the dc SQUID as a radio-frequency amplifier is presented, with an optimization strategy that accounts for the loading and noise contributions of the postamplifier and maximizes the signal-to-noise ratio of the total system. The high sensitivity of the dc SQUID is extended to high field NMR. A dc SQUID is used as a tuned radio-frequency amplifier to detect pulsed nuclear magnetic resonance at 32 MHz from a metal film in a 3.5 Tesla static field. A total system noise temperature of 11 K has been achieved, at a bath temperature of 4.2 K. The minimum number of nuclear Bohr magnetons observable from a free precession signal after a single pulse is about 2 x 10 17 in a bandwidth of 25 kHz. In a separate experiment, a dc SQUID is used as a rf amplifier in a NQR experiment to observe a new resonance response mechanism. The net electric polarization of a NaClO 3 crystal due to the precessing electric quadrupole moments of the Cl nuclei is detected at 30 MHz. The sensitivity of NMR and NQR spectrometers using dc SQUID amplifiers is compared to the sensitivity of spectrometers using conventional rf amplifiers. A SQUID-based spectrometer has a voltage sensitivity which is comparable to the best achieved by a FET-based spectrometer, at these temperatures and operating frequencies

Genet-specific DNA methylation probabilities detected in a spatial epigenetic analysis of a clonal plant population.

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Kiwako S Araki

Full Text Available In sessile organisms such as plants, spatial genetic structures of populations show long-lasting patterns. These structures have been analyzed across diverse taxa to understand the processes that determine the genetic makeup of organismal populations. For many sessile organisms that mainly propagate via clonal spread, epigenetic status can vary between clonal individuals in the absence of genetic changes. However, fewer previous studies have explored the epigenetic properties in comparison to the genetic properties of natural plant populations. Here, we report the simultaneous evaluation of the spatial structure of genetic and epigenetic variation in a natural population of the clonal plant Cardamine leucantha. We applied a hierarchical Bayesian model to evaluate the effects of membership of a genet (a group of individuals clonally derived from a single seed and vegetation cover on the epigenetic variation between ramets (clonal plants that are physiologically independent individuals. We sampled 332 ramets in a 20 m × 20 m study plot that contained 137 genets (identified using eight SSR markers. We detected epigenetic variation in DNA methylation at 24 methylation-sensitive amplified fragment length polymorphism (MS-AFLP loci. There were significant genet effects at all 24 MS-AFLP loci in the distribution of subepiloci. Vegetation cover had no statistically significant effect on variation in the majority of MS-AFLP loci. The spatial aggregation of epigenetic variation is therefore largely explained by the aggregation of ramets that belong to the same genets. By applying hierarchical Bayesian analyses, we successfully identified a number of genet-specific changes in epigenetic status within a natural plant population in a complex context, where genotypes and environmental factors are unevenly distributed. This finding suggests that it requires further studies on the spatial epigenetic structure of natural populations of diverse organisms

Genet-specific DNA methylation probabilities detected in a spatial epigenetic analysis of a clonal plant population.

Science.gov (United States)

Araki, Kiwako S; Kubo, Takuya; Kudoh, Hiroshi

2017-01-01

In sessile organisms such as plants, spatial genetic structures of populations show long-lasting patterns. These structures have been analyzed across diverse taxa to understand the processes that determine the genetic makeup of organismal populations. For many sessile organisms that mainly propagate via clonal spread, epigenetic status can vary between clonal individuals in the absence of genetic changes. However, fewer previous studies have explored the epigenetic properties in comparison to the genetic properties of natural plant populations. Here, we report the simultaneous evaluation of the spatial structure of genetic and epigenetic variation in a natural population of the clonal plant Cardamine leucantha. We applied a hierarchical Bayesian model to evaluate the effects of membership of a genet (a group of individuals clonally derived from a single seed) and vegetation cover on the epigenetic variation between ramets (clonal plants that are physiologically independent individuals). We sampled 332 ramets in a 20 m × 20 m study plot that contained 137 genets (identified using eight SSR markers). We detected epigenetic variation in DNA methylation at 24 methylation-sensitive amplified fragment length polymorphism (MS-AFLP) loci. There were significant genet effects at all 24 MS-AFLP loci in the distribution of subepiloci. Vegetation cover had no statistically significant effect on variation in the majority of MS-AFLP loci. The spatial aggregation of epigenetic variation is therefore largely explained by the aggregation of ramets that belong to the same genets. By applying hierarchical Bayesian analyses, we successfully identified a number of genet-specific changes in epigenetic status within a natural plant population in a complex context, where genotypes and environmental factors are unevenly distributed. This finding suggests that it requires further studies on the spatial epigenetic structure of natural populations of diverse organisms, particularly for

Analysis of isolates within species of anuran trypanosomes using random amplified polymorphic DNA.

Science.gov (United States)

Lun, Z R; Desser, S S

1996-01-01

A total of 20 decamer primers were used to generate random applied polymorphic DNA (RAPD) markers from 5 isolates of Trypanosoma fallisi, 3 isolates of T. ranarum, 2 isolates of T. rotatorium, and 2 isolates of T. rotatorium-like trypanosomes in addition to 2 species from the American Type Culture Collection, T. chattoni (ATCC 50294) and Trypanosoma sp. (ATCC 50295). A slight polymorphism was observed among the four isolates of T. fallisi obtained form American toads, Bufo americanus, collected in Algonquin Park, Ontario, Canada, and an isolate obtained from the same species of host collected in Marquette, Michigan, United States, and produced similarity coefficients ranging from 80.7% to 96.9%. Pronounced polymorphism was recorded among the three isolates of T. ranarum from bullfrogs, Rana catesbeiana, collected in Ontario, Canada, and in Maryland, United States, and from a Northern leopard frog, R. pipiens, collected in Minnesota (USA). The similarity coefficients ranged from 54.7% to 59.5%, suggesting that alleles of these isolates were conserved over a wide geographic range. The high degree of polymorphism observed in two isolates of T. rotatorium from a bullfrog collected in Ontario and two isolates of a T. rotatorium-like parasite from the green frog R. clamitans, collected in Louisiana (USA) suggests that they are different species. These results reflect the high similarity among isolates from the same geographic location and the pronounced polymorphism apparent among isolates from distant geographic locations.

Designation of the European Working Group on Legionella Infection (EWGLI) amplified fragment length polymorphism types of Legionella pneumophila serogroup 1 and results of intercentre proficiency testing Using a standard protocol

DEFF Research Database (Denmark)

Fry, N K; Bangsborg, Jette Marie; Bergmans, A

2002-01-01

The utility of amplified fragment length polymorphism (AFLP) analysis as a genotyping method for the epidemiological typing of Legionella pneumophila serogroup 1 has been previously demonstrated. This study (i). reports recommendations for the designation of the European Working Group on Legionella...... centres), and 92% (7 centres) to 100% (7 centres). The AFLP method as described is robust and rapid and allows the genotypic comparison of isolates of Legionella pneumophila between different testing centres without the need for exchange of the strains studied....

DYE-SENSITIZED PHOTOPOLYMERIZATION OF METHYL METHACRYLATE INITIATED BY COUMARIN DYE/IODONIUM SALT SYSTEM

Institute of Scientific and Technical Information of China (English)

Fang Gao; Yong-yuan Yang

1999-01-01

The photosensitive initiating system composed of 7-diethylamino-3-(2'-benzimidazolyl)coumarin dye (DEDC) and diphenyliodonium hexafluorophosphate (DIHP) which act as the sensitizer and the initiator respectively, can be used to initiate the polymerization of methyl methacrylate (MMA). The results showed that when exposed to visible light, coumarin dye/iodonium salt undergoes quick electron transfer from DEDC to DIHP and free radicals are produced. The visible light photoinduced reaction between DEDC and DIHP is mainly through the excited singlet state of DEDC and thus it is a little sensitive to O2. The influence of concentration of DEDC, DIHP and MMA on the rate of photopolymerization of MMA was also investigated.

A high-throughput and sensitive method to measure Global DNA Methylation: Application in Lung Cancer

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Mamaev Sergey

2008-08-01

Full Text Available Abstract Background Genome-wide changes in DNA methylation are an epigenetic phenomenon that can lead to the development of disease. The study of global DNA methylation utilizes technology that requires both expensive equipment and highly specialized skill sets. Methods We have designed and developed an assay, CpGlobal, which is easy-to-use, does not utilize PCR, radioactivity and expensive equipment. CpGlobal utilizes methyl-sensitive restriction enzymes, HRP Neutravidin to detect the biotinylated nucleotides incorporated in an end-fill reaction and a luminometer to measure the chemiluminescence. The assay shows high accuracy and reproducibility in measuring global DNA methylation. Furthermore, CpGlobal correlates significantly with High Performance Capillary Electrophoresis (HPCE, a gold standard technology. We have applied the technology to understand the role of global DNA methylation in the natural history of lung cancer. World-wide, it is the leading cause of death attributed to any cancer. The survival rate is 15% over 5 years due to the lack of any clinical symptoms until the disease has progressed to a stage where cure is limited. Results Through the use of cell lines and paired normal/tumor samples from patients with non-small cell lung cancer (NSCLC we show that global DNA hypomethylation is highly associated with the progression of the tumor. In addition, the results provide the first indication that the normal part of the lung from a cancer patient has already experienced a loss of methylation compared to a normal individual. Conclusion By detecting these changes in global DNA methylation, CpGlobal may have a role as a barometer for the onset and development of lung cancer.

Diet folate, DNA methylation and genetic polymorphisms of MTHFR C677T in association with the prognosis of esophageal squamous cell carcinoma

International Nuclear Information System (INIS)

Lu, Cheng; Xie, Hui; Wang, Fengliang; Shen, Hongbing; Wang, Jianming

2011-01-01

Folic acid may affect the development of human cancers. However, few studies have evaluated the consumption of diet folate in the prognosis of patients with esophageal squamous cell carcinoma (ESCC). One hundred and twenty five ESCC patients underwent esophagectomy between January 2005 and March 2006 in the Yangzhong People's Hospital were recruited and followed up. The effects of diet folate, aberrant DNA methylation of selected genes and methylenetetrahydrofolate reductase (MTHFR) C677T genetic polymorphisms on the prognosis of ESCC were evaluated by using Cox proportional hazard regression models. Our analysis showed an inverse association between diet folate intake and the risk of death after esophagectomy. The median survival time was 3.06 years for low or moderate folate consumption and over 4.59 years for high folate consumption. After adjusting for potential confounders, the hazard ratios (95% confidence interval) [HRs (95% CI)] were 0.72 (0.36-1.46) for moderate and 0.39 (0.20-0.78) for high folate intake, respectively (P for trend = 0.007). This preventive effect was more evident in patients carrying MTHFR 677CC genotype. No significant relation was observed between aberrant DNA methylation of P16, MGMT and hMLH1 gene, as well as MTHFR C677T genetic polymorphisms and the prognosis of ESCC. Our research indicated that diet folate intake may have benefits on the prognosis of ESCC after esophagectomy. From a practical viewpoint, the findings of our study help to establish practical intervention and surveillance strategies for managements of ESCC patients and can finally decrease the disease burden

Detection of Non-Amplified Genomic DNA

CERN Document Server

Corradini, Roberto

2012-01-01

This book offers a state-of-the-art overview on non amplified DNA detection methods and provides chemists, biochemists, biotechnologists and material scientists with an introduction to these methods. In fact all these fields have dedicated resources to the problem of nucleic acid detection, each contributing with their own specific methods and concepts. This book will explain the basic principles of the different non amplified DNA detection methods available, highlighting their respective advantages and limitations. The importance of non-amplified DNA sequencing technologies will be also discussed. Non-amplified DNA detection can be achieved by adopting different techniques. Such techniques have allowed the commercialization of innovative platforms for DNA detection that are expected to break into the DNA diagnostics market. The enhanced sensitivity required for the detection of non amplified genomic DNA has prompted new strategies that can achieve ultrasensitivity by combining specific materials with specifi...

Genetic and epigenetic alterations of Brassica nigra introgression lines from somatic hybridization: a resource for cauliflower improvement

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Guixiang Wang

2016-08-01

Full Text Available Broad phenotypic variations were obtained previously in derivatives from the asymmetric somatic hybridization of cauliflower ‘Korso’ (Brassica oleracea var. botrytis, 2n = 18, CC genome and black mustard ‘G1/1’ (Brassica nigra, 2n = 16, BB genome. However, the mechanisms underlying these variations were unknown. In this study, 28 putative introgression lines (ILs were pre-selected according to a series of morphological (leaf shape and color, plant height and branching, curd features, and flower traits and physiological (black rot/club root resistance characters. Multi-color fluorescence in situ hybridization revealed that these plants contained 18 chromosomes derived from ‘Korso’. Molecular marker (65 simple sequence repeats and 77 amplified fragment length polymorphisms analysis identified the presence of ‘G1/1’ DNA segments (average 7.5%. Additionally, DNA profiling revealed many genetic and epigenetic differences among the ILs, including sequence alterations, deletions, and variation in patterns of cytosine methylation. The frequency of fragments lost (5.1% was significantly higher than presence of novel bands (1.4%, and the presence of fragments specific to B. carinata (BBCC 2n = 34 were common (average 15.5%. Methylation-sensitive amplified polymorphism analysis indicated that methylation changes were common and that hypermethylation (12.4% was more frequent than hypomethylation (4.8%. Our results suggested that asymmetric somatic hybridization and alien DNA introgression induced genetic and epigenetic alterations. Thus, these ILs represent an important, novel germplasm resource for cauliflower improvement that can be mined for diverse traits of interest to breeders and researchers.

Novel Bacteriocinogenic Lactobacillus plantarum Strains and Their Differentiation by Sequence Analysis of 16S rDNA, 16S-23S and 23S-5S Intergenic Spacer Regions and Randomly Amplified Polymorphic DNA Analysis

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Morteza Shojaei Moghadam

2010-01-01

Full Text Available Six strains of bacteriocinogenic Lactobacillus plantarum (TL1, RG11, RS5, UL4, RG14 and RI11 isolated from Malaysian foods were investigated for their structural bacteriocin genes. A new combination of plantaricin EF and plantaricin W bacteriocin structural genes was successfully amplified from all studied strains, suggesting that they were novel bacteriocin-producing L. plantarum strains. A four-base pair variable region was detected in the short 16S-23S intergenic spacer regions of the studied strains by a comparative analysis with 17 L. plantarum strains deposited in the GenBank, implying they were new genotypes. The studied L. plantarum strains were subsequently differentiated into four groups on the basis of the detected four-base pair variable region of the short 16S-23S intergenic spacer region. Further analysis of the DNA sequence of 23S-5S intergenic spacer region revealed only one type of 23S-5S intergenic spacer region present in the studied strains, indicating it was highly conserved among the studied L. plantarum strains. Three randomly amplified polymorphic DNA experiments using three different combinations of arbitrary primers successfully differentiated the studied L. plantarum strains from each other, confirming they were different strains. In conclusion, the studied L. plantarum strains were shown to be novel bacteriocin producers and high level of strain discrimination could be achieved with a combination of randomly amplified polymorphic DNA analysis and the analysis of the variable region of short 16S-23S intergenic spacer region present in L. plantarum strains.

Virulence properties and random amplification of polymorphic DNA ...

African Journals Online (AJOL)

Genotypic and phenotypic characterization as well as studies on the virulence factors of Candida albicans isolates obtained from oral cavity of patients was carried out using random amplified polymorphic DNA (RAPD) fingerprinting and epithelial cells adherence assay, respectively. RAPD patterns revealed the presence of ...

DNA methylation analysis of allotetraploid hybrids of red crucian carp (Carassius auratus red var. and common carp (Cyprinus carpio L..

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Jun Xiao

Full Text Available Hybridization and polyploidization may lead to divergence in adaptation and boost speciation in angiosperms and some lower animals. Epigenetic change plays a significant role in the formation and adaptation of polyploidy. Studies of the effects of methylation on genomic recombination and gene expression in allopolyploid plants have achieved good progress. However, relevant advances in polyploid animals have been relatively slower. In the present study, we used the bisexual, fertile, genetically stable allotetraploid generated by hybridization of Carassius auratus red var. and Cyprinus carpio L. to investigate cytosine methylation level using methylation-sensitive amplification polymorphism (MSAP analysis. We observed 38.31% of the methylation changes in the allotetraploid compared with the parents at 355 randomly selected CCGG sites. In terms of methylation status, these results indicate that the level of methylation modification in the allotetraploid may have increased relative to that in the parents. We also found that the major methylation changes were hypermethylation on some genomic fragments and genes related to metabolism or cell cycle regulation. These results provide circumstantial evidence that DNA methylation might be related to the gene expression and phenotype variation in allotetraploid hybrids. Our study partly fulfils the need for epigenetic research in polyploid animals, and provides evidence for the epigenetic regulation of allopolyploids.

Ames and random amplified polymorphic DNA tests for the validation of the mutagenic and/or genotoxic potential of the drinking water disinfection by-products chloroform and bromoform.

Science.gov (United States)

Khallef, Messaouda; Cenkci, Süleyman; Akyil, Dilek; Özkara, Arzu; Konuk, Muhsin; Benouareth, Djamel Eddine

2018-01-28

Chloroform and Bromoform are two abundant trihalomethanes found in Algerian drinking water. The investigation of the mutagenic hazard of these disinfection by-products was studied by Ames test as prokaryotic bioassay to show their mutagenic effects. For this, Salmonella typhimurium TA98 and TA100 strains were employed. Both chloroform and bromoform showed a direct mutagenic effect since the number of revertant colonies gradually increase in dose-dependent manner with all concentrations tested with the two bacterial strains and these were both in the absence and presence of S9 metabolic activation. The genotoxic hazard was also studied by random amplified polymorphic DNA test on the root cells of Allium cepa as eukaryotic bioassay. DNA extracted from the roots of the onion were incubated at different concentrations of chloroform and bromoform and then amplified by polymerase chain reaction. This was based on demonstrating a major effect of disappearance of bands compared to roots incubated in the negative control (distilled water). The results showed that these two compounds affected genomic DNA by breaks although by mutations.

Synthesis and evaluation of translocator 18 kDa protein (TSPO) positron emission tomography (PET) radioligands with low binding sensitivity to human single nucleotide polymorphism rs6971.

Science.gov (United States)

Zanotti-Fregonara, Paolo; Zhang, Yi; Jenko, Kimberly J; Gladding, Robert L; Zoghbi, Sami S; Fujita, Masahiro; Sbardella, Gianluca; Castellano, Sabrina; Taliani, Sabrina; Martini, Claudia; Innis, Robert B; Da Settimo, Federico; Pike, Victor W

2014-10-15

The imaging of translocator 18 kDa protein (TSPO) in living human brain with radioligands by positron emission tomography (PET) has become an important means for the study of neuroinflammatory conditions occurring in several neuropsychiatric disorders. The widely used prototypical PET radioligand [(11)C](R)-PK 11195 ([(11)C](R)-1; [N-methyl-(11)C](R)-N-sec-butyl-1-(2-chlorophenyl)-N-methylisoquinoline-3-carboxamide) gives a low PET signal and is difficult to quantify, whereas later generation radioligands have binding sensitivity to a human single nucleotide polymorphism (SNP) rs6971, which imposes limitations on their utility for comparative quantitative PET studies of normal and diseased subjects. Recently, azaisosteres of 1 have been developed with improved drug-like properties, including enhanced TSPO affinity accompanied by moderated lipophilicity. Here we selected three of these new ligands (7-9) for labeling with carbon-11 and for evaluation in monkey as candidate PET radioligands for imaging brain TSPO. Each radioligand was readily prepared by (11)C-methylation of an N-desmethyl precursor and was found to give a high proportion of TSPO-specific binding in monkey brain. One of these radioligands, [(11)C]7, the direct 4-azaisostere of 1, presents many radioligand properties that are superior to those reported for [(11)C]1, including higher affinity, lower lipophilicity, and stable quantifiable PET signal. Importantly, 7 was also found to show very low sensitivity to the human SNP rs6971 in vitro. Therefore, [(11)C]7 now warrants evaluation in human subjects with PET to assess its utility for imaging TSPO in human brain, irrespective of subject genotype.

Gene expression profiling of a Zn-tolerant and a Zn-sensitive Suillus luteus isolate exposed to increased external zinc concentrations

OpenAIRE

MULLER, Ludo; Craciun, A. R.; RUYTINX, Joske; LAMBAERTS, Marc; Verbruggen, N.; VANGRONSVELD, Jaco; COLPAERT, Jan

2007-01-01

Complementary DNA (cDNA)-amplified fragment-length polymorphism (AFLP) was applied to analyze transcript profiles of a Zn-tolerant and a Zn-sensitive isolate of the ectomycorrhizal basidiomycete Suillus luteus, both cultured with and without increased external zinc concentrations. From the obtained transcript profiles that covered approximately 2% of the total expected complement of genes in S. luteus, 144 nonredundant, differentially expressed transcript-derived fragments (TDFs), falling in ...

Sex Determination from Fragmented and Degenerated DNA by Amplified Product-Length Polymorphism Bidirectional SNP Analysis of Amelogenin and SRY Genes

Science.gov (United States)

Masuyama, Kotoka; Shojo, Hideki; Nakanishi, Hiroaki; Inokuchi, Shota; Adachi, Noboru

2017-01-01

Sex determination is important in archeology and anthropology for the study of past societies, cultures, and human activities. Sex determination is also one of the most important components of individual identification in criminal investigations. We developed a new method of sex determination by detecting a single-nucleotide polymorphism in the amelogenin gene using amplified product-length polymorphisms in combination with sex-determining region Y analysis. We particularly focused on the most common types of postmortem DNA damage in ancient and forensic samples: fragmentation and nucleotide modification resulting from deamination. Amplicon size was designed to be less than 60 bp to make the method more useful for analyzing degraded DNA samples. All DNA samples collected from eight Japanese individuals (four male, four female) were evaluated correctly using our method. The detection limit for accurate sex determination was determined to be 20 pg of DNA. We compared our new method with commercial short tandem repeat analysis kits using DNA samples artificially fragmented by ultraviolet irradiation. Our novel method was the most robust for highly fragmented DNA samples. To deal with allelic dropout resulting from deamination, we adopted “bidirectional analysis,” which analyzed samples from both sense and antisense strands. This new method was applied to 14 Jomon individuals (3500-year-old bone samples) whose sex had been identified morphologically. We could correctly identify the sex of 11 out of 14 individuals. These results show that our method is reliable for the sex determination of highly degenerated samples. PMID:28052096

Polymorphisms of three genes (ACE, AGT and CYP11B2) in the renin-angiotensin-aldosterone system are not associated with blood pressure salt sensitivity: A systematic meta-analysis.

Science.gov (United States)

Sun, Jiahong; Zhao, Min; Miao, Song; Xi, Bo

2016-01-01

Many studies have suggested that polymorphisms of three key genes (ACE, AGT and CYP11B2) in the renin-angiotensin-aldosterone system (RAAS) play important roles in the development of blood pressure (BP) salt sensitivity, but they have revealed inconsistent results. Thus, we performed a meta-analysis to clarify the association. PubMed and Embase databases were searched for eligible published articles. Fixed- or random-effect models were used to pool odds ratios and 95% confidence intervals based on whether there was significant heterogeneity between studies. In total, seven studies [237 salt-sensitive (SS) cases and 251 salt-resistant (SR) controls] for ACE gene I/D polymorphism, three studies (130 SS cases and 221 SR controls) for AGT gene M235T polymorphism and three studies (113 SS cases and 218 SR controls) for CYP11B2 gene C344T polymorphism were included in this meta-analysis. The results showed that there was no significant association between polymorphisms of these three polymorphisms in the RAAS and BP salt sensitivity under three genetic models (all p > 0.05). The meta-analysis suggested that three polymorphisms (ACE gene I/D, AGT gene M235T, CYP11B2 gene C344T) in the RAAS have no significant effect on BP salt sensitivity.

DNA methylation and histone deacetylation regulating insulin sensitivity due to chronic cold exposure.

Science.gov (United States)

Wang, Xiaoqing; Wang, Lai; Sun, Yizheng; Li, Ruiping; Deng, Jinbo; Deng, Jiexin

2017-02-01

In this study, we investigated the causal relationship between chronic cold exposure and insulin resistance and the mechanisms of how DNA methylation and histone deacetylation regulate cold-reduced insulin resistance. 46 adult male mice from postnatal day 90-180 were randomly assigned to control group and cold-exposure group. Mice in cold-exposure group were placed at temperature from -1 to 4 °C for 30 days to mimic chronic cold environment. Then, fasting blood glucose, blood insulin level and insulin resistance index were measured with enzymatic methods. Immunofluorescent labeling was carried out to visualize the insulin receptor substrate 2 (IRS2), Obese receptor (Ob-R, a leptin receptor), voltage-dependent anion channel protein 1 (VDAC1), cytochrome C (cytC), 5-methylcytosine (5-mC) positive cells in hippocampal CA1 area. Furthermore, the expressions of some proteins mentioned above were detected with Western blot. The results showed: ① Chronic cold exposure could reduce the insulin resistance index (P cold-exposure group than in control group with both immunohistochemical staining and Western blot (P cold exposure increased DNA methylation and histone deacetylation in the pyramidal cells of CA1 area and led to an increase in the expression of histone deacetylase 1 (HDAC1) and DNA methylation relative enzymes (P cold exposure can improve insulin sensitivity, with the involvement of DNA methylation, histone deacetylation and the regulation of mitochondrial energy metabolism. These epigenetic modifications probably form the basic mechanism of cold-reduced insulin resistance. Copyright © 2016 Elsevier Inc. All rights reserved.

Evaluation of a reverse-hybridization StripAssay for the detection of genetic polymorphisms leading to acenocoumarol sensitivity.

Science.gov (United States)

Gialeraki, Argyri; Markatos, Christos; Grouzi, Elisabeth; Merkouri, Efrosyni; Travlou, Anthi; Politou, Marianna

2010-04-01

Acenocoumarol is mainly catabolized by CYP2C9 isoform of cytochrome P450 (CYP) liver complex and exerts its anticoagulant effect through the inhibition of Vitamin K Epoxide Reductase (VKOR). The most important genetic polymorphisms which lead to an impaired enzymatic activity and therefore predispose to acenocoumarol sensitivity, are considered to be CYP2C9*2 (Arg144Cys), CYP2C9*3 (Ile359Leu) and VKORC1-1639G>A, respectively. In this study we compared the results of the PGXThrombo StripAssay kit (ViennaLab Diagnostics,Vienna, Austria) with direct DNA sequencing and in house Restriction Fragment Length Polymorphisms (RFLP) for the detection of the aforementioned Single Nucleotide Polymorphisms (SNPs). The reverse hybridization StripAssay was found to be equally effective with RFLP and direct DNA sequencing for the detection of CYP2C9*2 and CYP2C9*3 polymorphisms, respectively. The comparison of the RFLP reference method with the reverse hybridization StripAssay for the detection of VKORC1-1639 G>A polymorphism showed that the reverse hybridization StripAsssay might misclassify some A/A homozygotes as heterozygotes. Optimization of the hybridization procedures may eliminate the extra low signal band observed in some samples at the reverse hybridization StripAssay and improve its diagnostic value.

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

OpenAIRE

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

2016-01-01

Abstract 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...

AFLP and MS-AFLP analysis of the variation within saffron crocus (Crocus sativus L. germplasm.

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Matteo Busconi

Full Text Available The presence and extent of genetic variation in saffron crocus are still debated, as testified by several contradictory articles providing contrasting results about the monomorphism or less of the species. Remarkably, phenotypic variations have been frequently observed in the field, such variations are usually unstable and can change from one growing season to another. Considering that gene expression can be influenced both by genetic and epigenetic changes, epigenetics could be a plausible cause of the alternative phenotypes. In order to obtain new insights into this issue, we carried out a molecular marker analysis of 112 accessions from the World Saffron and Crocus Collection. The accessions were grown for at least three years in the same open field conditions. The same samples were analysed using Amplified Fragment Length Polymorphism (AFLP and Methyl Sensitive AFLP in order to search for variation at the genetic (DNA sequence and epigenetic (cytosine methylation level. While the genetic variability was low (4.23% polymorphic peaks and twelve (12 effective different genotypes, the methyl sensitive analysis showed the presence of high epigenetic variability (33.57% polymorphic peaks and twenty eight (28 different effective epigenotypes. The pattern obtained by Factorial Correspondence Analysis of AFLP and, in particular, of MS-AFLP data was consistent with the geographical provenance of the accessions. Very interestingly, by focusing on Spanish accessions, it was observed that the distribution of the accessions in the Factorial Correspondence Analysis is not random but tends to reflect the geographical origin. Two clearly defined clusters grouping accessions from the West (Toledo and Ciudad Real and accessions from the East (Cuenca and Teruel were clearly recognised.

Individual Variations in Inorganic Arsenic Metabolism Associated with AS3MT Genetic Polymorphisms

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Haruo Takeshita

2011-04-01

Full Text Available Individual variations in inorganic arsenic metabolism may influence the toxic effects. Arsenic (+3 oxidation state methyltransferase (AS3MT that can catalyze the transfer of a methyl group from S-adenosyl-L-methionine (AdoMet to trivalent arsenical, may play a role in arsenic metabolism in humans. Since the genetic polymorphisms of AS3MT gene may be associated with the susceptibility to inorganic arsenic toxicity, relationships of several single nucleotide polymorphisms (SNPs in AS3MT with inorganic arsenic metabolism have been investigated. Here, we summarize our recent findings and other previous studies on the inorganic arsenic metabolism and AS3MT genetic polymorphisms in humans. Results of genotype dependent differences in arsenic metabolism for most of SNPs in AS3MT were Inconsistent throughout the studies. Nevertheless, two SNPs, AS3MT 12390 (rs3740393 and 14458 (rs11191439 were consistently related to arsenic methylation regardless of the populations examined for the analysis. Thus, these SNPs may be useful indicators to predict the arsenic metabolism via methylation pathways.

Note: A high dynamic range, linear response transimpedance amplifier.

Science.gov (United States)

Eckel, S; Sushkov, A O; Lamoreaux, S K

2012-02-01

We have built a high dynamic range (nine decade) transimpedance amplifier with a linear response. The amplifier uses junction-gate field effect transistors (JFETs) to switch between three different resistors in the feedback of a low input bias current operational amplifier. This allows for the creation of multiple outputs, each with a linear response and a different transimpedance gain. The overall bandwidth of the transimpedance amplifier is set by the bandwidth of the most sensitive range. For our application, we demonstrate a three-stage amplifier with transimpedance gains of approximately 10(9)Ω, 3 × 10(7)Ω, and 10(4)Ω with a bandwidth of 100 Hz.

Atypical DNA methylation of genes encoding cysteine-rich peptides in Arabidopsis thaliana

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You Wanhui

2012-04-01

Full Text Available Abstract Background In plants, transposons and non-protein-coding repeats are epigenetically silenced by CG and non-CG methylation. This pattern of methylation is mediated in part by small RNAs and two specialized RNA polymerases, termed Pol IV and Pol V, in a process called RNA-directed DNA methylation. By contrast, many protein-coding genes transcribed by Pol II contain in their gene bodies exclusively CG methylation that is independent of small RNAs and Pol IV/Pol V activities. It is unclear how the different methylation machineries distinguish between transposons and genes. Here we report on a group of atypical genes that display in their coding region a transposon-like methylation pattern, which is associated with gene silencing in sporophytic tissues. Results We performed a methylation-sensitive amplification polymorphism analysis to search for targets of RNA-directed DNA methylation in Arabidopsis thaliana and identified several members of a gene family encoding cysteine-rich peptides (CRPs. In leaves, the CRP genes are silent and their coding regions contain dense, transposon-like methylation in CG, CHG and CHH contexts, which depends partly on the Pol IV/Pol V pathway and small RNAs. Methylation in the coding region is reduced, however, in the synergid cells of the female gametophyte, where the CRP genes are specifically expressed. Further demonstrating that expressed CRP genes lack gene body methylation, a CRP4-GFP fusion gene under the control of the constitutive 35 S promoter remains unmethylated in leaves and is transcribed to produce a translatable mRNA. By contrast, a CRP4-GFP fusion gene under the control of a CRP4 promoter fragment acquires CG and non-CG methylation in the CRP coding region in leaves similar to the silent endogenous CRP4 gene. Conclusions Unlike CG methylation in gene bodies, which does not dramatically affect Pol II transcription, combined CG and non-CG methylation in CRP coding regions is likely to

A 3-plex methylation assay combined with the FGFR3 mutation assay sensitively detects recurrent bladder cancer in voided urine

DEFF Research Database (Denmark)

Kandimalla, Raju; Masius, Roy; Beukers, Willemien

2013-01-01

is to determine the sensitivity and specificity of a urine assay for the diagnosis of recurrences in patients with a previous primary NMIBC G1/G2 by using cystoscopy as the reference standard. Experimental Design: We selected eight CpG islands (CGI) methylated in bladder cancer from our earlier genome-wide study......Purpose: DNA methylation is associated with bladder cancer and these modifications could serve as useful biomarkers. FGFR3 mutations are present in 60% to 70% of non–muscle invasive bladder cancer (NMIBC). Low-grade bladder cancer recurs in more than 50% of patients. The aim of this study......, and nonmalignant urines (n = 130). Results: The 3-plex assay identified recurrent bladder cancer in voided urine with a sensitivity of 74% in the validation set. In combination with the FGFR3 mutation assay, a sensitivity of 79% was reached (specificity of 77%). Sensitivity of FGFR3 and cytology was 52% and 57...

Molecular Typing of Borrelia burgdorferi Sensu Lato by Randomly Amplified Polymorphic DNA Fingerprinting Analysis

Science.gov (United States)

Wang, Guiqing; van Dam, Alje P.; Spanjaard, Lodewijk; Dankert, Jacob

1998-01-01

To study whether pathogenic clusters of Borrelia burgdorferi sensu lato strains occur, we typed 136 isolates, cultured from specimens from patients (n = 49) with various clinical entities and from ticks (n = 83) or dogs (n = 4) from different geographic regions, by randomly amplified polymorphic DNA (RAPD) fingerprinting with four arbitrary primers. The RAPD patterns were reproducible up to the 95% similarity level as shown in duplicate experiments. In these experiments the purified DNAs prepared on different days, from different colonies, and after various passages were used as templates. With an intergroup difference of 55%, the 136 strains could be divided into seven genetic clusters. Six clusters comprised and corresponded to the established species B. burgdorferi sensu stricto (n = 23), Borrelia garinii (n = 39), Borrelia afzelii (n = 59), Borrelia japonica (n = 1), Borrelia valaisiana (n = 12), and genomic group DN127 (n = 1). One strain from a patient with erythema migrans (EM) did not belong to any of the species or genomic groups known up to now. The RAPD types of B. burgdorferi sensu stricto, B. garinii, and B. afzelii isolates, which may give rise to human Lyme borreliosis (LB), were associated with their geographic origins. A high degree of genetic diversity was observed among the 39 B. garinii strains, and six subgroups could be recognized. One of these comprised eight isolates from patients with disseminated LB only and no tick isolates. B. afzelii strains from patients with EM or acrodermatitis chronica atrophicans were not clustered in particular branches. Our study showed that RAPD analysis is a powerful tool for discriminating different Borrelia species as well as Borrelia isolates within species. PMID:9508310

Assessing genetic divergence in interspecific hybrids of Aechmea gomosepala and A. recurvata var. recurvata using inflorescence characteristics and sequence-related amplified polymorphism markers.

Science.gov (United States)

Zhang, F; Ge, Y Y; Wang, W Y; Shen, X L; Yu, X Y

2012-12-03

Conventional hybridization and selection techniques have aided the development of new ornamental crop cultivars. However, little information is available on the genetic divergence of bromeliad hybrids. In the present study, we investigated the genetic variability in interspecific hybrids of Aechmea gomosepala and A. recurvata var. recurvata using inflorescence characteristics and sequence-related amplified polymorphism (SRAP) markers. The morphological analysis showed that the putative hybrids were intermediate between both parental species with respect to inflorescence characteristics. The 16 SRAP primer combinations yield 265 bands, among which 154 (57.72%) were polymorphic. The genetic similarity was an average of 0.59 and ranged from 0.21 to 0.87, indicating moderate genetic divergence among the hybrids. The unweighted pair group method with arithmetic average (UPGMA)-based cluster analysis distinguished the hybrids from their parents with a genetic distance coefficient of 0.54. The cophenetic correlation was 0.93, indicating a good fit between the dendrogram and the original distance matrix. The two-dimensional plot from the principal coordinate analysis showed that the hybrids were intermediately dispersed between both parents, corresponding to the results of the UPGMA cluster and the morphological analysis. These results suggest that SRAP markers could help to identify breeders, characterize F(1) hybrids of bromeliads at an early stage, and expedite genetic improvement of bromeliad cultivars.

Changes in DNA Methylation Pattern at Two Seedling Stages in Water Saving and Drought-Resistant Rice Variety after Drought Stress Domestication

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Xiao-guo ZHENG

2014-09-01

Full Text Available Recent studies revealed that DNA methylation plays an important role in plant growth and development. In this study, a water-saving and drought-resistant rice variety Huhan 3 was subjected to drought stress from tillering to grain-filling stages in six successive growth cycles. The variations in DNA methylation pattern between the original generation (G0 and the sixth generation (G6 were analyzed by using methylation sensitive amplification polymorphism method. The results revealed that the methylated loci accounted for 34.3% to 34.8% of the total loci. Among these methylated loci, 83.1% to 84.8% were full- and hyper-methylated and 15.2% to 16.9% were hemi-methylated. The DNA methylation level decreased from the three-leaf to four-leaf stages in Huhan 3. Differentially methylated loci (DML between generations or/and between different developmental stages accounted for 4.0% of the total loci, most of which were only related to plant development (57.9%. Compared to G0, the DNA methylation pattern of G6 changed after drought domestication, at the three-leaf stage, de-methylation accounting for 59.1%, while at the four-leaf stage, re-methylation for 47.9%. Genome-wide alternations of DNA methylation were observed between the two seedling stages, and DML mainly occurred on the gene's promoter and exon region. The genes related to DML involved in a wide range of functional biology and participated in many important biological processes.

Synthesis and characterization of a thermo-sensitive poly( N-methyl acryloylglycine methyl ester) used as a drug release carrier

Science.gov (United States)

Deng, Kui-Lin; Zhong, Hai-Bin; Jiao, Yi-Suo; Fan, Ting; Qiao, Xiao; Zhang, Peng-Fei; Ren, Xiao-Bo

2010-06-01

In this article, poly( N-methyl acryloylglycine methyl ester) (PNMAME) was prepared as a novel thermosensitive material with a lower critical solution temperature (LCST) at around 49.5°C. The chemical structures of the monomer NMAME and PNMAME were characterized by 1H NMR and IR measurements. The LCST was investigated systematically as a function of PNMAME concentration, inorganic salt solution and pH value. The results indicated that LCST of PNMAME was obviously dependent on PNMAME concentration and pH. The LCST was increased with a decrease in pH value and PNMAME concentration. To obtain a thermo-sensitive hydrogel with the phase transition temperature close to human body temperature, the copolymerization was conducted between NMAME and N-acryloylglycine ethyl ester (NAGEE). The release behavior of caffeine was evaluated at different temperatures and contents of cross-linkers ( N, N-methylenebis(acrylamide) (NMBA)). The increase of cross-linker content led to a decrease in the release rate of caffeine due to higher crossing density in the hydrogel network. In addition, a faster release of caffeine from the hydrogel with 3% NMBA at 37°C was found in contrast to that at 18°C.

The influence of L-opsin gene polymorphisms and neural ageing on spatio-chromatic contrast sensitivity in 20-71 year olds.

Science.gov (United States)

Dees, Elise W; Gilson, Stuart J; Neitz, Maureen; Baraas, Rigmor C

2015-11-01

Chromatic contrast sensitivity may be a more sensitive measure of an individual's visual function than achromatic contrast sensitivity. Here, the first aim was to quantify individual- and age-related variations in chromatic contrast sensitivity to a range of spatial frequencies for stimuli along two complementary directions in color space. The second aim was to examine whether polymorphisms at specific amino acid residues of the L- and M-opsin genes (OPN1LW and OPN1MW) known to affect spectral tuning of the photoreceptors could influence spatio-chromatic contrast sensitivity. Chromatic contrast sensitivity functions were measured in 50 healthy individuals (20-71 years) employing a novel pseudo-isochromatic grating stimulus. The spatio-chromatic contrast sensitivity functions were found to be low pass for all subjects, independent of age and color vision. The results revealed a senescent decline in spatio-chromatic contrast sensitivity. There were considerable between-individual differences in sensitivity within each age decade for individuals 49 years old or younger, and age did not predict sensitivity for these age decades alone. Forty-six subjects (including a color deficient male and eight female carriers) were genotyped for L- and M-opsin genes. The Ser180Ala polymorphisms on the L-opsin gene were found to influence the subject's color discrimination and their sensitivity to spatio-chromatic patterns. The results expose the significant role of neural and genetic factors in the deterioration of visual function with increasing age. Copyright © 2015 Elsevier Ltd. All rights reserved.

Low Loss Nanostructured Polymers for Chip-scale Waveguide Amplifiers.

Science.gov (United States)

Chen, George F R; Zhao, Xinyu; Sun, Yang; He, Chaobin; Tan, Mei Chee; Tan, Dawn T H

2017-06-13

On-chip waveguide amplifiers offer higher gain in small device sizes and better integration with photonic devices than the commonly available fiber amplifiers. However, on-chip amplifiers have yet to make its way into the mainstream due to the limited availability of materials with ideal light guiding and amplification properties. A low-loss nanostructured on-chip channel polymeric waveguide amplifier was designed, characterized, fabricated and its gain experimentally measured at telecommunication wavelength. The active polymeric waveguide core comprises of NaYF 4 :Yb,Er,Ce core-shell nanocrystals dispersed within a SU8 polymer, where the nanoparticle interfacial characteristics were tailored using hydrolyzed polyhedral oligomeric silsesquioxane-graft-poly(methyl methacrylate) to improve particle dispersion. Both the enhanced IR emission intensity from our nanocrystals using a tri-dopant scheme and the reduced scattering losses from our excellent particle dispersion at a high solid loading of 6.0 vol% contributed to the outstanding optical performance of our polymeric waveguide. We achieved one of the highest reported gain of 6.6 dB/cm using a relatively low coupled pump power of 80 mW. These polymeric waveguide amplifiers offer greater promise for integrated optical circuits due to their processability and integration advantages which will play a key role in the emerging areas of flexible communication and optoelectronic devices.

Transgenerational variations in DNA methylation induced by drought stress in two rice varieties with distinguished difference to drought resistance.

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Xiaoguo Zheng

Full Text Available Adverse environmental conditions have large impacts on plant growth and crop production. One of the crucial mechanisms that plants use in variable and stressful natural environments is gene expression modulation through epigenetic modification. In this study, two rice varieties with different drought resistance levels were cultivated under drought stress from tilling stage to seed filling stage for six successive generations. The variations in DNA methylation of the original generation (G0 and the sixth generation (G6 of these two varieties in normal condition (CK and under drought stress (DT at seedling stage were assessed by using Methylation Sensitive Amplification Polymorphism (MSAP method. The results revealed that drought stress had a cumulative effect on the DNA methylation pattern of both varieties, but these two varieties had different responses to drought stress in DNA methylation. The DNA methylation levels of II-32B (sensitive and Huhan-3 (resistant were around 39% and 32%, respectively. Genome-wide DNA methylation variations among generations or treatments accounted for around 13.1% of total MSAP loci in II-32B, but was only approximately 1.3% in Huhan-3. In II-32B, 27.6% of total differentially methylated loci (DML were directly induced by drought stress and 3.2% of total DML stably transmitted their changed DNA methylation status to the next generation. In Huhan-3, the numbers were 48.8% and 29.8%, respectively. Therefore, entrainment had greater effect on Huhan-3 than on II-32B. Sequence analysis revealed that the DML were widely distributed on all 12 rice chromosomes and that it mainly occurred on the gene's promoter and exon region. Some genes with DML respond to environmental stresses. The inheritance of epigenetic variations induced by drought stress may provide a new way to develop drought resistant rice varieties.

Glucose tolerance, insulin sensitivity and insulin release in European non-diabetic carriers of a polymorphism upstream of CDKN2A and CDKN2B

DEFF Research Database (Denmark)

Hribal, M L; Presta, I; Procopio, T

2011-01-01

The aim of this study was to investigate the association of the rs10811661 polymorphism near the CDKN2B/CDKN2A genes with glucose tolerance, insulin sensitivity and insulin release in three samples of white people with European ancestry.......The aim of this study was to investigate the association of the rs10811661 polymorphism near the CDKN2B/CDKN2A genes with glucose tolerance, insulin sensitivity and insulin release in three samples of white people with European ancestry....

Tissue-specific and cation/anion-specific DNA methylation variations occurred in C. virgata in response to salinity stress.

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Xiang Gao

Full Text Available Salinity is a widespread environmental problem limiting productivity and growth of plants. Halophytes which can adapt and resist certain salt stress have various mechanisms to defend the higher salinity and alkalinity, and epigenetic mechanisms especially DNA methylation may play important roles in plant adaptability and plasticity. In this study, we aimed to investigate the different influences of various single salts (NaCl, Na2SO4, NaHCO3, Na2CO3 and their mixed salts on halophyte Chloris. virgata from the DNA methylation prospective, and discover the underlying relationships between specific DNA methylation variations and specific cations/anions through the methylation-sensitive amplification polymorphism analysis. The results showed that the effects on DNA methylation variations of single salts were ranked as follows: Na2CO3> NaHCO3> Na2SO4> NaCl, and their mixed salts exerted tissue-specific effects on C. virgata seedlings. Eight types of DNA methylation variations were detected and defined in C. virgata according to the specific cations/anions existed in stressful solutions; in addition, mix-specific and higher pH-specific bands were the main type in leaves and roots independently. These findings suggested that mixed salts were not the simple combination of single salts. Furthermore, not only single salts but also mixed salts showed tissue-specific and cations/anions-specific DNA methylation variations.

Tissue-specific and cation/anion-specific DNA methylation variations occurred in C. virgata in response to salinity stress.

Science.gov (United States)

Gao, Xiang; Cao, Donghui; Liu, Jie; Wang, Xiaoping; Geng, Shujuan; Liu, Bao; Shi, Decheng

2013-01-01

Salinity is a widespread environmental problem limiting productivity and growth of plants. Halophytes which can adapt and resist certain salt stress have various mechanisms to defend the higher salinity and alkalinity, and epigenetic mechanisms especially DNA methylation may play important roles in plant adaptability and plasticity. In this study, we aimed to investigate the different influences of various single salts (NaCl, Na2SO4, NaHCO3, Na2CO3) and their mixed salts on halophyte Chloris. virgata from the DNA methylation prospective, and discover the underlying relationships between specific DNA methylation variations and specific cations/anions through the methylation-sensitive amplification polymorphism analysis. The results showed that the effects on DNA methylation variations of single salts were ranked as follows: Na2CO3> NaHCO3> Na2SO4> NaCl, and their mixed salts exerted tissue-specific effects on C. virgata seedlings. Eight types of DNA methylation variations were detected and defined in C. virgata according to the specific cations/anions existed in stressful solutions; in addition, mix-specific and higher pH-specific bands were the main type in leaves and roots independently. These findings suggested that mixed salts were not the simple combination of single salts. Furthermore, not only single salts but also mixed salts showed tissue-specific and cations/anions-specific DNA methylation variations.

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

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Pooja H. Gupta

2015-05-01

Full Text Available 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.

Tetranucleotide repeat polymorphism at the human prostatic acid phosphatase (ACPP) gene

Energy Technology Data Exchange (ETDEWEB)

Polymeropoulos, M H; Xiao, Hong; Rath, D S; Merril, C R [National Inst. of Mental Health Neuroscience Center, Washington, DC (United States)

1991-09-11

The polymorphic (AAAT){sub n} repeat begins at base pair 2342 of the human prostatic acid phosphatase gene on chromosome 3q21-qter. The polymorphism can be typed using the polymerase chain reaction (PCR) as described previously. The predicted length of the amplified sequence was 275 bp. Co-dominant segregation was observed in two informative families. The human prostatic acid phosphatase gene has been assigned to chromosome 3q21-qter.

Radiation hardening techniques for rare-earth based optical fibers and amplifiers

International Nuclear Information System (INIS)

Girard, Sylvain; Marcandella, Claude; Vivona, Marilena; Prudenzano, Luciano Mescia F.; Laurent, Arnaud; Robin, Thierry; Cadier, Benoit; Pinsard, Emmanuel; Ouerdane, Youcef; Boukenter, Aziz; Cannas, Marco; Boscaino, Roberto

2012-01-01

Er/Yb doped fibers and amplifiers have been shown to be very radiation sensitive, limiting their integration in space. We present an approach including successive hardening techniques to enhance their radiation tolerance. The efficiency of our approach is demonstrated by comparing the radiation responses of optical amplifiers made with same lengths of different rare-earth doped fibers and exposed to gamma-rays. Previous studies indicated that such amplifiers suffered significant degradation for doses exceeding 10 krad. Applying our techniques significantly enhances the amplifier radiation resistance, resulting in a very limited degradation up to 50 krad. Our optimization techniques concern the fiber composition, some possible pre-treatments and the interest of simulation tools used to harden by design the amplifiers. We showed that adding cerium inside the fiber phospho-silicate-based core strongly decreases the fiber radiation sensitivity compared to the standard fiber. For both fibers, a pre-treatment with hydrogen permits to enhance again the fiber resistance. Furthermore, simulations tools can also be used to improve the tolerance of the fiber amplifier by helping identifying the best amplifier configuration for operation in the radiative environment. (authors)

Amplified fragment length polymorphism mapping of quantitative trait loci for malaria parasite susceptibility in the yellow fever mosquito Aedes aegypti.

Science.gov (United States)

Zhong, Daibin; Menge, David M; Temu, Emmanuel A; Chen, Hong; Yan, Guiyun

2006-07-01

The yellow fever mosquito Aedes aegypti has been the subject of extensive genetic research due to its medical importance and the ease with which it can be manipulated in the laboratory. A molecular genetic linkage map was constructed using 148 amplified fragment length polymorphism (AFLP) and six single-strand conformation polymorphism (SSCP) markers. Eighteen AFLP primer combinations were used to genotype two reciprocal F2 segregating populations. Each primer combination generated an average of 8.2 AFLP markers eligible for linkage mapping. The length of the integrated map was 180.9 cM, giving an average marker resolution of 1.2 cM. Composite interval mapping revealed a total of six QTL significantly affecting Plasmodium susceptibility in the two reciprocal crosses of Ae. aegypti. Two common QTL on linkage group 2 were identified in both crosses that had similar effects on the phenotype, and four QTL were unique to each cross. In one cross, the four main QTL accounted for 64% of the total phenotypic variance, and digenic epistasis explained 11.8% of the variance. In the second cross, the four main QTL explained 66% of the variance, and digenic epistasis accounted for 16% of the variance. The actions of these QTL were either dominance or underdominance. Our results indicated that at least three new QTL were mapped on chromosomes 1 and 3. The polygenic nature of susceptibility to P. gallinaceum and epistasis are important factors for significant variation within or among mosquito strains. The new map provides additional information useful for further genetic investigation, such as identification of new genes and positional cloning.

Amplified fragment length polymorphism analysis to assess crossover interference and homozygosity in gynogenetic diploid Pacific abalone (Haliotis discus hannai).

Science.gov (United States)

Nie, H-T; Li, Q; Kong, L-F

2014-06-01

Recombination analysis in gynogenetic diploids is a powerful tool for assessing the degree of inbreeding, investigating crossover events and understanding chiasma interference during meiosis. To estimate the marker-centromere recombination rate, the inheritance pattern of 654 amplified fragment length polymorphism (AFLP) markers was examined in the 72-h veliger larvae of two meiogynogenetic diploid families in the Pacific abalone (Haliotis discus hannai). The second-division segregation frequency (y) of the AFLP loci ranged from 0.00 to 0.96, with 23.9% of loci showing y-values higher than 0.67, evidencing the existence of interference. The average recombination frequency across the 654 AFLP loci was 0.45, allowing estimation of the fixation index of 0.55, indicating that meiotic gynogenesis could provide an effective means of rapid inbreeding in the Pacific abalone. The AFLP loci have a small proportion (4.4%) of y-values greater than 0.90, suggesting that a relatively low or intermediate degree of chiasma interference occurred in the abalone chromosomes. The information obtained in this study will enhance our understanding of the abalone genome and will be useful for genetic studies in the species. © 2014 Stichting International Foundation for Animal Genetics.

Markers and mapping revisited: finding your gene.

Science.gov (United States)

Jones, Neil; Ougham, Helen; Thomas, Howard; Pasakinskiene, Izolda

2009-01-01

This paper is an update of our earlier review (Jones et al., 1997, Markers and mapping: we are all geneticists now. New Phytologist 137: 165-177), which dealt with the genetics of mapping, in terms of recombination as the basis of the procedure, and covered some of the first generation of markers, including restriction fragment length polymorphisms (RFLPs), random amplified polymorphic DNA (RAPDs), simple sequence repeats (SSRs) and quantitative trait loci (QTLs). In the intervening decade there have been numerous developments in marker science with many new systems becoming available, which are herein described: cleavage amplification polymorphism (CAP), sequence-specific amplification polymorphism (S-SAP), inter-simple sequence repeat (ISSR), sequence tagged site (STS), sequence characterized amplification region (SCAR), selective amplification of microsatellite polymorphic loci (SAMPL), single nucleotide polymorphism (SNP), expressed sequence tag (EST), sequence-related amplified polymorphism (SRAP), target region amplification polymorphism (TRAP), microarrays, diversity arrays technology (DArT), single-strand conformation polymorphism (SSCP), denaturing gradient gel electrophoresis (DGGE), temperature gradient gel electrophoresis (TGGE) and methylation-sensitive PCR. In addition there has been an explosion of knowledge and databases in the area of genomics and bioinformatics. The number of flowering plant ESTs is c. 19 million and counting, with all the opportunity that this provides for gene-hunting, while the survey of bioinformatics and computer resources points to a rapid growth point for future activities in unravelling and applying the burst of new information on plant genomes. A case study is presented on tracking down a specific gene (stay-green (SGR), a post-transcriptional senescence regulator) using the full suite of mapping tools and comparative mapping resources. We end with a brief speculation on how genome analysis may progress into the future of

MethPrimer: designing primers for methylation PCRs.

Science.gov (United States)

Li, Long-Cheng; Dahiya, Rajvir

2002-11-01

DNA methylation is an epigenetic mechanism of gene regulation. Bisulfite- conversion-based PCR methods, such as bisulfite sequencing PCR (BSP) and methylation specific PCR (MSP), remain the most commonly used techniques for methylation mapping. Existing primer design programs developed for standard PCR cannot handle primer design for bisulfite-conversion-based PCRs due to changes in DNA sequence context caused by bisulfite treatment and many special constraints both on the primers and the region to be amplified for such experiments. Therefore, the present study was designed to develop a program for such applications. MethPrimer, based on Primer 3, is a program for designing PCR primers for methylation mapping. It first takes a DNA sequence as its input and searches the sequence for potential CpG islands. Primers are then picked around the predicted CpG islands or around regions specified by users. MethPrimer can design primers for BSP and MSP. Results of primer selection are delivered through a web browser in text and in graphic view.

Comparison of ribotyping, randomly amplified polymorphic DNA analysis, and pulsed-field gel electrophoresis in typing of Lactobacillus rhamnosus and L. casei strains.

Science.gov (United States)

Tynkkynen, S; Satokari, R; Saarela, M; Mattila-Sandholm, T; Saxelin, M

1999-09-01

A total of 24 strains, biochemically identified as members of the Lactobacillus casei group, were identified by PCR with species-specific primers. The same set of strains was typed by randomly amplified polymorphic DNA (RAPD) analysis, ribotyping, and pulsed-field gel electrophoresis (PFGE) in order to compare the discriminatory power of the methods. Species-specific primers for L. rhamnosus and L. casei identified the type strain L. rhamnosus ATCC 7469 and the neotype strain L. casei ATCC 334, respectively, but did not give any signal with the recently revived species L. zeae, which contains the type strain ATCC 15820 and the strain ATCC 393, which was previously classified as L. casei. Our results are in accordance with the suggested new classification of the L. casei group. Altogether, 21 of the 24 strains studied were identified with the species-specific primers. In strain typing, PFGE was the most discriminatory method, revealing 17 genotypes for the 24 strains studied. Ribotyping and RAPD analysis yielded 15 and 12 genotypes, respectively.

Instability of chromosome number and DNA methylation variation induced by hybridization and amphidiploid formation between Raphanus sativus L. and Brassica alboglabra Bailey

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Wang Yanjie

2010-09-01

Full Text Available Abstract Background Distant hybridization can result genome duplication and allopolyploid formation which may play a significant role in the origin and evolution of many plant species. It is unclear how the two or more divergent genomes coordinate in one nucleus with a single parental cytoplasm within allopolyploids. We used cytological and molecular methods to investigate the genetic and epigenetic instabilities associated with the process of distant hybridization and allopolyploid formation, measuring changes in chromosome number and DNA methylation across multiple generations. Results F1 plants from intergeneric hybridization between Raphanus sativus L. (2n = 18, RR and Brassica alboglabra Bailey (2n = 18, CC were obtained by hand crosses and subsequent embryo rescue. Random amplification of polymorphic DNA (RAPD markers were used to identify the F1 hybrid plants. The RAPD data indicated that the hybrids produced specific bands similar to those of parents and new bands that were not present in either parent. Chromosome number variation of somatic cells from allotetraploids in the F4 to F10 generations showed that intensive genetic changes occurred in the early generations of distant hybridization, leading to the formation of mixopolyploids with different chromosome numbers. DNA methylation variation was revealed using MSAP (methylation-sensitive amplification polymorphism, which showed that cytosine methylation patterns changed markedly in the process of hybridization and amphidiploid formation. Differences in cytosine methylation levels demonstrated an epigenetic instability of the allopolyploid of Raphanobrassica between the genetically stable and unstable generations. Conclusions Our results showed that chromosome instability occurred in the early generations of allopolyploidy and then the plants were reverted to largely euploidy in later generations. During this process, DNA methylation changed markedly. These results suggest that

RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

International Nuclear Information System (INIS)

Wang Tiegu; Huang Qunce; Feng Weisen

2007-01-01

Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning

RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

Science.gov (United States)

Wang, Tiegu; Huang, Qunce; Feng, Weisen

2007-10-01

Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.

RAPD and SSR Polymorphisms in Mutant Lines of Transgenic Wheat Mediated by Low Energy Ion Beam

Energy Technology Data Exchange (ETDEWEB)

Tiegu, Wang [Henan Provincial Key Laboratory of Ion Beam Bio-Engineering, Zhengzhou University, Zhengzhou 450052 (China); Qunce, Huang [Henan Provincial Key Laboratory of Ion Beam Bio-Engineering, Zhengzhou University, Zhengzhou 450052 (China); Weisen, Feng [Luoyang Institute of Agricultural Science, Luoyang 471022 (China)

2007-10-15

Two types of markers-random amplified polymorphic DNA (RAPD) and simple sequence repeat DNA (SSR)-have been used to characterize the genetic diversity among nine mutant lines of transgenic wheat intermediated by low energy ion beam and their four receptor cultivars. The objectives of this study were to analyze RAPD-based and SSR-based genetic variance among transgenic wheat lines and with their receptors, and to find specific genetic markers of special traits of transgenic wheat lines. 170 RAPD primers were amplified to 733 fragments in all the experimental materials. There were 121 polymorphic fragments out of the 733 fragments with a ratio of polymorphic fragments of 16.5%. 29 SSR primer pairs were amplified to 83 fragments in all the experiment materials. There were 57 polymorphic fragments out of the 83 fragments with a ratio of polymorphic fragments of 68.7%. The dendrograms were prepared based on a genetic distance matrix using the UPGMA (Unweighted Pair-group Method with Arithmetic averaging) algorithm, which corresponded well to the results of the wheat pedigree analysis and separated the 13 genotypes into four groups. Association analysis between RAPD and SSR markers with the special traits of transgenic wheat mutant lines discovered that three RAPD markers, s1, opt-16, and f14, were significantly associated with the muticate trait, while three SSR markers, Rht8 (Xgwm261), Rht-B1b, and Rht-D1b, highly associated with the dwarf trait. These markers will be useful for marker-assistant breeding and can be used as candidate markers for further gene mapping and cloning.

Evidence of transmission of Mycobacterium tuberculosis by random amplified polymorphic DNA (RAPD) fingerprinting in Taipei City, Taiwan.

Science.gov (United States)

Harn, H J; Shen, K L; Ho, L I; Yu, K W; Liu, G C; Yueh, K C; Lee, J H

1997-01-01

AIMS: To determine, by strain identification of Mycobacterium tuberculosis, whether transmission has occurred between individuals or whether new strains are present. METHODS: A rapid protocol for random amplified polymorphic DNA (RAPD) analysis was developed. This protocol was applied to 64 strains of M tuberculosis that had been confirmed by culture and microbiological methods. RESULTS: There are five groups of M tuberculosis prevalent in Taipei city, Taiwan. The major types are groups I and III. Groups I and II had been prevalent until the end of last year when, according to our group analysis, they had been eradicated. However, group III was continuously present from the middle of 1995 to the middle of 1996, and group IV was present at the end of both years, which indicated that both groups were transmitted continuously. These clustered strains had demographic characteristics consistent with a finding of transmission tuberculosis. Also, there were 13 of 64 strains with unique RAPD fingerprints that were inferred to be due primarily to the reactivation of infection. In the drug resistance analysis, the major type represented included group III and part of group IV. CONCLUSIONS: Our preliminary data imply, not only that the prevalence of M tuberculosis in Taipei city is due to transmission rather than reactivation, but that drug resistance also may play a role in tuberculosis transmission. Images PMID:9378819

A lower degree of PBMC L1 methylation in women with lower folate status may explain the MTHFR C677T polymorphism associated higher risk of CIN in the US post folic acid fortification era.

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Suguna Badiga

Full Text Available Studies in populations unexposed to folic acid (FA fortification have demonstrated that MTHFR C677T polymorphism is associated with increased risk of higher grades of cervical intraepithelial neoplasia (CIN 2+. However, it is unknown whether exposure to higher folate as a result of the FA fortification program has altered the association between MTHFR C677T and risk of CIN, or the mechanisms involved with such alterations. The current study investigated the following in a FA fortified population: 1 The association between MTHFR C677T polymorphism and risk of CIN 2+; 2 The modifying effects of plasma folate concentrations on this association; and 3 The modifying effects of plasma folate on the association between the polymorphism and degree of methylation of long interspersed nucleotide elements (L1s, in peripheral blood mononuclear cell (PBMC DNA, a documented biomarker of CIN risk.The study included 457 US women diagnosed with either CIN 2+ (cases or ≤ CIN 1 (non-cases. Unconditional logistic regression models were used to test the associations after adjusting for relevant risk factors for CIN.The 677CT/TT MTHFR genotypes were not associated with the risk of CIN 2+. Women with CT/TT genotype with lower folate, however, were more likely to be diagnosed with CIN 2+ compared to women with CT/TT genotype with higher folate (OR = 2.41, P = 0.030. Women with CT/TT genotype with lower folate were less likely to have a higher degree of PBMC L1 methylation compared to women with CT/TT genotype with higher folate (OR = 0.28, P = 0.017.This study provides the first evidence that the MTHFR 677CT/TT genotype-associated lower degree of PBMC L1 methylation increases the risk of CIN 2+ in women in the US post-FA fortification era. Thus, even in the post-FA fortification era, not all women have adequate folate status to overcome MTHFR 677CT/TT genotype-associated lower degree of L1 methylation.

Oxytocin and Social Sensitivity: Gene Polymorphisms in Relation to Depressive Symptoms and Suicidal Ideation

Directory of Open Access Journals (Sweden)

Robyn Jane McQuaid

2016-07-01

Full Text Available Although the neuropeptide oxytocin has been associated with enhanced prosocial behaviors, it has also been linked to aggression and mental health disorders. Thus, it was suggested that oxytocin might act by increasing the salience of social stimuli, irrespective of whether these are positive or negative, thus increasing vulnerability to negative mental health outcomes. The current study (N = 243, conducted among White university students, examined the relation of trauma, depressive symptoms including suicidal ideation in relation to a single nucleotide polymorphism (SNP within the oxytocin receptor gene (OXTR, rs53576, and a SNP on the CD38 gene that controls oxytocin release, rs3796863. Individuals with the polymorphism on both alleles (AA genotype of the CD38 SNP had previously been linked to elevated plasma oxytocin levels. Consistent with the social sensitivity perspective, however, in the current study, individuals carrying the AA genotype displayed elevated feelings of alienation from parents and peers as well as increased levels of suicidal ideation. Moreover, they tended to report elevated depressive symptoms compared to CC homozygotes. It was also observed that the CD38 genotype moderated the relation between trauma and suicidal ideation scores, such that high levels of trauma were associated with elevated suicidal ideation among all CD38 genotypes, but this relationship was stronger among individuals with the AA genotype. In contrast, there was no relationship between the OXTR SNP, rs53576, depression or suicidal ideation. These findings support a social sensitivity hypothesis of oxytocin, wherein the AA genotype of the CD38 SNP, which has been considered the ‘protective allele’ was associated with increased sensitivity and susceptibility to disturbed social relations and suicidal ideation.

‘‘Blind'' mapping of genic DNA sequence polymorphisms in Lolium perenne L. by high resolution melting curve analysis

DEFF Research Database (Denmark)

Studer, Bruno; Jensen, Louise Bach; Fiil, Alice

2009-01-01

High resolution melting curve analysis (HRM) measures dissociation of double stranded DNA of a PCR product amplified in the presence of a saturating fluorescence dye. Recently, HRM proved successful to genotype DNA sequence polymorphisms such as SSRs and SNPs based on the shape of the melting...... curves. In this study, HRM was used for simultaneous screening and genotyping of genic DNA sequence polymorphisms identified in the Lolium perenne F2 mapping population VrnA. Melting profiles of PCR products amplified from previously published gene loci and from a novel gene putatively involved...

BDNF Val66Met Polymorphism Influences Visuomotor Associative Learning and the Sensitivity to Action Observation

Science.gov (United States)

Taschereau-Dumouchel, Vincent; Hétu, Sébastien; Michon, Pierre-Emmanuel; Vachon-Presseau, Etienne; Massicotte, Elsa; De Beaumont, Louis; Fecteau, Shirley; Poirier, Judes; Mercier, Catherine; Chagnon, Yvon C.; Jackson, Philip L.

2016-01-01

Motor representations in the human mirror neuron system are tuned to respond to specific observed actions. This ability is widely believed to be influenced by genetic factors, but no study has reported a genetic variant affecting this system so far. One possibility is that genetic variants might interact with visuomotor associative learning to configure the system to respond to novel observed actions. In this perspective, we conducted a candidate gene study on the Brain-derived neurotrophic factor (BDNF) Val66Met polymorphism, a genetic variant linked to motor learning in regions of the mirror neuron system, and tested the effect of this polymorphism on motor facilitation and visuomotor associative learning. In a single-pulse TMS study carried on 16 Met (Val/Met and Met/Met) and 16 Val/Val participants selected from a large pool of healthy volunteers, Met participants showed significantly less muscle-specific corticospinal sensitivity during action observation, as well as reduced visuomotor associative learning, compared to Val homozygotes. These results are the first evidence of a genetic variant tuning sensitivity to action observation and bring to light the importance of considering the intricate relation between genetics and associative learning in order to further understand the origin and function of the human mirror neuron system. PMID:27703276

AFLP genetic polymorphism in wild barley (Hordeum spontaneum) populations in Israel

NARCIS (Netherlands)

Turpeinen, T.; Vanhala, T.; Nevo, E.; Nissila, E.

2003-01-01

The genetic diversity produced by the amplified fragment length polymorphism (AFLP) method was studied in 94 genotypes of wild barley, Hordeum spontaneum (C. Koch) Thell., originating from ten ecologically and geographically different locations in Israel. Eight primer pairs produced 204 discernible

Amplifier channel for a fission fragment semiconductor detector

International Nuclear Information System (INIS)

Tyurin, G.P.

1981-01-01

To compensate the decrease of the transformation coefficient of fission fragment semiconductor detector (SCD) developed is a special amplification channel with controlled transfer coefficient. The block diagram of the channel is presented, the main functional units of which are as follows: preamplifying head with charge-sensitive and timing preamplifiers, linear amplifier and the circuit of spectrum position stabilization, which includes a differential discriminator, integrator and reference signal generator. The amplification channel is made in the CAMAC standard and has the following specifications: dinamical input capacitance of charge-sensitive amplifier c=10000 n PHI, signal amplitude at output of the linear amplifier at energy of fission fragments of 120 MeV has negative polarity and is equal to 5 V. Pulse amplitude change at SCD sensitivity decrease to 50% constitutes not more than 1%. Timing preamplifier has the gain factor at voltage of K=80 at front duration of 3.5 nc. Time resolution of the amplification channel is not worse than 1 nc. Dimensions of preamplifying head are 40x40x15 mm. The amplification channel permitted to use SCD for long-term measurements of fission fragment spectra [ru

Low noise amplifier for ZnS(Ag) scintillation chamber

International Nuclear Information System (INIS)

Do Hoang Cuong

1998-01-01

A new pulse amplifier that can be used with standard photomultiplier tubes coupled with Zn(Ag) scintillation chamber is presented. The amplifier based on an IC operational amplifier LF 356N consists of a low-noise charge sensitive preamplifier and pulse shaping circuits for optimization of signal to noise ratio. Temperature instability is ≤ 0.05%/ o C. Dynamic range for linear output signals is equal +7 V. The presented amplifier is used in a measuring head for 0.17 L Lucas chambers developed in Department of Nuclear Instruments and Methods of the INCT in laboratory investigations aimed to develop methods and instruments for measurement of radon concentration in the air. The amplifier can also be employed for measurement of ionizing radiation by means of other scintillators coupled to PM tube. The amplifier is followed by a pulse discriminator with adjustable discrimination level. The amplifier output signal and discriminator output pulses are fed to external devices. (author)

Amplified fluorescent aptasensor through catalytic recycling for highly sensitive detection of ochratoxin A.

Science.gov (United States)

Wei, Yin; Zhang, Ji; Wang, Xu; Duan, Yixiang

2015-03-15

This paper describes a novel approach utilizing nano-graphite-aptamer hybrid and DNase I for the amplified detection of ochratoxin A (OTA) for the first time. Nano-graphite can effectively quench the fluorescence of carboxyfluorescein (FAM) labeled OTA specific aptamer due to their strong π-π; stacking interactions; while upon OTA addition, it will bind with aptamer to fold into an OTA-aptamerG-quadruplex structure, which does not adsorb on the surface of nano-graphite and thus retains the dye fluorescence. Meanwhile, the G-quadruplex structure can be cleaved by DNase I, and in such case OTA is delivered from the complex. The released OTA then binds other FAM-labeled aptamers on the nano-graphite surface, and touches off another target recycling, resulting in the successive release of dye-labeled aptamers from the nano-graphite, which leads to significant amplification of the signal. Under the optimized conditions, the present amplified sensing system exhibits high sensitivity toward OTA with a limit of detection of 20nM (practical measurement), which is about 100-fold higher than that of traditional unamplified homogeneous assay. Our developed method also showed high selectivity against other interference molecules and can be applied for the detection of OTA in real red wine samples. The proposed assay is simple, cost-effective, and might open a door for the development of new assays for other biomolecules. This aptasensor is of great practical importance in food safety and could be widely extended to the detection of other toxins by replacing the sequence of the recognition aptamer. Copyright © 2014 Elsevier B.V. All rights reserved.

A self-amplified transistor immunosensor under dual gate operation: highly sensitive detection of hepatitis B surface antigen

Science.gov (United States)

Lee, I.-K.; Jeun, M.; Jang, H.-J.; Cho, W.-J.; Lee, K. H.

2015-10-01

Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor based on a self-amplified transistor under dual gate operation (immuno-DG ISFET) for the detection of hepatitis B surface antigen. To address the challenges in current ISFET-based immunosensors, we have enhanced the sensitivity of an immunosensor by precisely tailoring the nanostructure of the transistor. In the pH sensing test, the immuno-DG ISFET showed superior sensitivity (2085.53 mV per pH) to both standard ISFET under single gate operation (58.88 mV per pH) and DG ISFET with a non-tailored transistor (381.14 mV per pH). Moreover, concerning the detection of hepatitis B surface antigens (HBsAg) using the immuno-DG ISFET, we have successfully detected trace amounts of HBsAg (22.5 fg mL-1) in a non-diluted 1× PBS medium with a high sensitivity of 690 mV. Our results demonstrate that the proposed immuno-DG ISFET can be a biosensor platform for practical use in the diagnosis of various diseases.Ion-sensitive field-effect transistors (ISFETs), although they have attracted considerable attention as effective immunosensors, have still not been adopted for practical applications owing to several problems: (1) the poor sensitivity caused by the short Debye screening length in media with high ion concentration, (2) time-consuming preconditioning processes for achieving the highly-diluted media, and (3) the low durability caused by undesirable ions such as sodium chloride in the media. Here, we propose a highly sensitive immunosensor

Detection of genetic variability in Basmati and non-Basmati rice varieties and their radiation induced mutants through random amplified polymorphic DNA (RAPD)

Energy Technology Data Exchange (ETDEWEB)

Farooq, S; Iqbal, N; Arif, M [Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad (Pakistan)

1998-03-01

Random Amplified Polymorphic DNA (RAPDs) markers were utilized to detect polymorphism between pure lines and commercially available Basmati rice varieties to assess variation which may be helpful in quality control and varietal identification (Basmati-370 and derived radiation induced mutants), differentiation of mutants and parents, and identification of RAPD markers co-segregating with important agronomic traits including plant height, days to flower and grain quality. Basmati varieties were distinguished from non-Basmati varieties with the help of five diagnostic markers which will be useful for detecting mixing of non-Basmati and Basmati rices, currently a serious marketing problem. Different Basmati cultivars were identified with the help of diagnostic RAPD markers which can be used in quality control as well as for ``fingerprinting`` of cultivars. Different radiation induced mutants were also successfully distinguished from the parents on the basis of variety specific and mutant specific markers which will be useful for varietal identification. In addition to this, other markers were also identified which can differentiate mutants from each other and are being, used for the fingerprinting of different mutants, particularly the dwarf mutants having similar appearance but different parentage. For identification of RAPD markers co-segregating with plant height and days to flower, 50 F{sub 2} plants and four F{sub 3} families were studied from a reciprocal cross made between Kashmir Basmati (tall and early) and Basmati-198 (dwarf and late). Segregating bands were observed within these populations, and indicating the possible use of RAPD markers for tagging gene(s) of agronomic importance in rice. (author). 38 refs, 6 figs, 3 tabs.

Detection of genetic variability in Basmati and non-Basmati rice varieties and their radiation induced mutants through random amplified polymorphic DNA (RAPD)

International Nuclear Information System (INIS)

Farooq, S.; Iqbal, N.; Arif, M.

1998-01-01

Random Amplified Polymorphic DNA (RAPDs) markers were utilized to detect polymorphism between pure lines and commercially available Basmati rice varieties to assess variation which may be helpful in quality control and varietal identification (Basmati-370 and derived radiation induced mutants), differentiation of mutants and parents, and identification of RAPD markers co-segregating with important agronomic traits including plant height, days to flower and grain quality. Basmati varieties were distinguished from non-Basmati varieties with the help of five diagnostic markers which will be useful for detecting mixing of non-Basmati and Basmati rices, currently a serious marketing problem. Different Basmati cultivars were identified with the help of diagnostic RAPD markers which can be used in quality control as well as for ''fingerprinting'' of cultivars. Different radiation induced mutants were also successfully distinguished from the parents on the basis of variety specific and mutant specific markers which will be useful for varietal identification. In addition to this, other markers were also identified which can differentiate mutants from each other and are being, used for the fingerprinting of different mutants, particularly the dwarf mutants having similar appearance but different parentage. For identification of RAPD markers co-segregating with plant height and days to flower, 50 F 2 plants and four F 3 families were studied from a reciprocal cross made between Kashmir Basmati (tall and early) and Basmati-198 (dwarf and late). Segregating bands were observed within these populations, and indicating the possible use of RAPD markers for tagging gene(s) of agronomic importance in rice. (author)

DNA methylation-based variation between human populations.

Science.gov (United States)

Kader, Farzeen; Ghai, Meenu

2017-02-01

Several studies have proved that DNA methylation affects regulation of gene expression and development. Epigenome-wide studies have reported variation in methylation patterns between populations, including Caucasians, non-Caucasians (Blacks), Hispanics, Arabs, and numerous populations of the African continent. Not only has DNA methylation differences shown to impact externally visible characteristics, but is also a potential biomarker for underlying racial health disparities between human populations. Ethnicity-related methylation differences set their mark during early embryonic development. Genetic variations, such as single-nucleotide polymorphisms and environmental factors, such as age, dietary folate, socioeconomic status, and smoking, impacts DNA methylation levels, which reciprocally impacts expression of phenotypes. Studies show that it is necessary to address these external influences when attempting to differentiate between populations since the relative impacts of these factors on the human methylome remain uncertain. The present review summarises several reported attempts to establish the contribution of differential DNA methylation to natural human variation, and shows that DNA methylation could represent new opportunities for risk stratification and prevention of several diseases amongst populations world-wide. Variation of methylation patterns between human populations is an exciting prospect which inspires further valuable research to apply the concept in routine medical and forensic casework. However, trans-generational inheritance needs to be quantified to decipher the proportion of variation contributed by DNA methylation. The future holds thorough evaluation of the epigenome to understand quantification, heritability, and the effect of DNA methylation on phenotypes. In addition, methylation profiling of the same ethnic groups across geographical locations will shed light on conserved methylation differences in populations.

Molecular profiles of Venezuelan isolates of Trypanosoma sp. by random amplified polymorphic DNA method.

Science.gov (United States)

Perrone, T M; Gonzatti, M I; Villamizar, G; Escalante, A; Aso, P M

2009-05-12

Nine Trypanosoma sp. Venezuelan isolates, initially presumed to be T. evansi, were collected from three different hosts, capybara (Apure state), horse (Apure state) and donkey (Guarico state) and compared by the random amplification polymorphic DNA technique (RAPD). Thirty-one to 46 reproducible fragments were obtained with 12 of the 40 primers that were used. Most of the primers detected molecular profiles with few polymorphisms between the seven horse, capybara and donkey isolates. Quantitative analyses of the RAPD profiles of these isolates revealed a high degree of genetic conservation with similarity coefficients between 85.7% and 98.5%. Ten of the primers generated polymorphic RAPD profiles with two of the three Trypanosoma sp. horse isolates, namely TeAp-N/D1 and TeGu-N/D1. The similarity coefficient between these two isolates and the rest, ranged from 57.9% to 68.4% and the corresponding dendrogram clustered TeAp-N/D1 and Te Gu-N/D1 in a genetically distinct group.

relationship of status of polymorphic rapd bands with genotypic

African Journals Online (AJOL)

jen

Department of Plant Breeding and Genetics, College of Agriculture, Orissa University of Agriculture and. Technology .... Amplified Polymorphic DNA) have been ... DNA quantification was done by visualising under UV light after electrophoresis on 0.8% (w/v) agarose gel. The. DNA was again diluted in TE buffer to 5 μg μl-1.

Detection of rifampin resistance patterns in Mycobacterium tuberculosis strains isolated in Iran by polymerase chain reaction-single-strand conformation polymorphism and direct sequencing methods

Directory of Open Access Journals (Sweden)

Bahram Nasr Isfahani

2006-09-01

Full Text Available Mutations in the rpoB locus confer conformational changes leading to defective binding of rifampin (RIF to rpoB and consequently resistance in Mycobacterium tuberculosis. Polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP was established as a rapid screening test for the detection of mutations in the rpoB gene, and direct sequencing has been unambiguously applied to characterize mutations. A total of 37 of Iranian isolates of M. tuberculosis, 16 sensitive and 21 resistant to RIF, were used in this study. A 193-bp region of the rpoB gene was amplified and PCR-SSCP patterns were determined by electrophoresis in 10% acrylamide gel and silver staining. Also, 21 samples of 193-bp rpoB amplicons with different PCR-SSCP patterns from RIFr and 10 from RIFs were sequenced. Seven distinguishable PCR-SSCP patterns were recognized in the 21 Iranian RIFr strains, while 15 out of 16 RIFs isolates demonstrated PCR-SSCP banding patterns similar to that of sensitive standard strain H37Rv. However one of the sensitive isolates demonstrated a different pattern. There were seen six different mutations in the amplified region of rpoB gene: codon 516(GAC/GTC, 523(GGG/GGT, 526(CAC/TAC, 531(TCG/TTG, 511(CTG/TTG, and 512(AGC/TCG. This study demonstrated the high specificity (93.8% and sensitivity (95.2% of PCR-SSCP method for detection of mutation in rpoB gene; 85.7% of RIFr strains showed a single mutation and 14.3% had no mutations. Three strains showed mutations caused polymorphism. Our data support the common notion that rifampin resistance genotypes are generally present mutations in codons 531 and 526, most frequently found in M. tuberculosis populations regardless of geographic origin.

Fiber Amplifiers

DEFF Research Database (Denmark)

Rottwitt, Karsten

2017-01-01

The chapter provides a discussion of optical fiber amplifiers and through three sections provides a detailed treatment of three types of optical fiber amplifiers, erbium doped fiber amplifiers (EDFA), Raman amplifiers, and parametric amplifiers. Each section comprises the fundamentals including...... the basic physics and relevant in-depth theoretical modeling, amplifiers characteristics and performance data as a function of specific operation parameters. Typical applications in fiber optic communication systems and the improvement achievable through the use of fiber amplifiers are illustrated....

Identification of a panel of sensitive and specific DNA methylation markers for lung adenocarcinoma

Directory of Open Access Journals (Sweden)

Hagen Jeffrey A

2007-10-01

Full Text Available Abstract Background Lung cancer is the number one cancer killer of both men and women in the United States. Three quarters of lung cancer patients are diagnosed with regionally or distantly disseminated disease; their 5-year survival is only 15%. DNA hypermethylation at promoter CpG islands shows great promise as a cancer-specific marker that would complement visual lung cancer screening tools such as spiral CT, improving early detection. In lung cancer patients, such hypermethylation is detectable in a variety of samples ranging from tumor material to blood and sputum. To date the penetrance of DNA methylation at any single locus has been too low to provide great clinical sensitivity. We used the real-time PCR-based method MethyLight to examine DNA methylation quantitatively at twenty-eight loci in 51 primary human lung adenocarcinomas, 38 adjacent non-tumor lung samples, and 11 lung samples from non-lung cancer patients. Results We identified thirteen loci showing significant differential DNA methylation levels between tumor and non-tumor lung; eight of these show highly significant hypermethylation in adenocarcinoma: CDH13, CDKN2A EX2, CDX2, HOXA1, OPCML, RASSF1, SFPR1, and TWIST1 (p-value Conclusion The identification of eight CpG island loci showing highly significant hypermethylation in lung adenocarcinoma provides strong candidates for evaluation in patient remote media such as plasma and sputum. The four most highly ranked loci, CDKN2A EX2, CDX2, HOXA1 and OPCML, which show significant DNA methylation even in stage IA tumor samples, merit further investigation as some of the most promising lung adenocarcinoma markers identified to date.

Factor VII R353Q genetic polymorphism is associated with altered warfarin sensitivity among CYP2C9 *1/*1 carriers.

Science.gov (United States)

Mlynarsky, Liat; Bejarano-Achache, Idit; Muszkat, Mordechai; Caraco, Yoseph

2012-05-01

Warfarin responsiveness is characterized by marked interindividual variability. A major portion of this variability is attributed to CYP2C9 and VKORC1 polymorphisms, but almost 50% is still unaccounted for. This paper reports the first prospective study on the association between factor VII R353Q polymorphism and warfarin responsiveness during induction. Genotyping for factor VII R353Q and 323D/I polymorphisms was performed in a cohort consisting of 374 patients (198 CYP2C9*1/*1) treated with warfarin who were prospectively followed from warfarin initiation. Compared with *1/*1-R/R and *1/*1-R/Q genotype carriers, *1/*1-Q/Q homozygotes achieved higher International Normalized Ratio (INR) values while consuming lower warfarin doses. The greater sensitivity was illustrated by 82.1% higher Warfarin Sensitivity Index During Induction (WSIDI) (0.14 ± 0.11 vs. 0.08 ± 0.50 mg⁻¹ Mann-Whitney, P = 0.043). Multiple regression analysis consisting of both genetic and nongenetic factors explained 26% of WSIDI variability, with R353Q genetic polymorphism having a modest yet significant effect and accounting for 1.7% of the overall variability. Moreover, the incidence of overanticoagulation (i.e., INR > 4) was 6.94-fold higher among *1/*1-Q/Q vs. *1/*1-R/R&R/Q carriers during warfarin induction (Pearson chi-square, P = 0.005). These findings were not accounted for by a chance difference in the distribution of VKORC1 genotypes. Analysis of these parameters among the entire cohort, including CYP2C9*2 and CYP2C9*3 variant allele carriers, did not reach statistical significance. Warfarin responsiveness during induction was unrelated to factor VII 323D/I genetic polymorphism. The response to warfarin during induction is influenced by factor VII R353Q polymorphism. The prospective use of this polymorphism, along with CYP2C9 and VKORC1, may enhance the accuracy of warfarin loading. However, the impact of R353Q polymorphism on overall warfarin response is subtle, and it is therefore

AFLPMax: a user-friendly application for computing the optimal number of amplified fragment length polymorphism markers needed in phylogenetic reconstruction.

Science.gov (United States)

García-Pereira, M J; Quesada, H; Caballero, A; Carvajal-Rodríguez, A

2012-05-01

Amplified fragment length polymorphisms (AFLPs) are widely used for phylogenetic inference especially in non-model species. Frequently, trees obtained with other nuclear or mitochondrial markers or with morphological information need additional resolution, increased branch support, or independent data sources (i.e. unlinked loci). In such cases, the use of AFLPs is a quick and cheap option. Computer simulation has shown that dominant AFLP markers lead to less accurate tree topologies than bi-allelic codominant markers such as SNPs, but this difference becomes negligible for shallow trees when using AFLP data sets that include a sufficiently large number of characters. Thus, determining how many AFLP characters are required to recover a given phylogeny is a key issue regarding the appropriateness of AFLPs for phylogenetic reconstruction. Here, we present a user-friendly, java-based graphical interface, AFLPMax, which executes an automatic pipeline of different programs providing the user with the optimal number of AFLP characters needed to recover a given phylogeny with high accuracy and support. Executables for Windows, linux and MacOS X operating systems, source code and user manual are available from: http://webs.uvigo.es/acraaj/AFLPMax.htm. © 2012 Blackwell Publishing Ltd.

A novel bi-enzyme electrochemical biosensor for selective and sensitive determination of methyl salicylate.

Science.gov (United States)

Fang, Yi; Umasankar, Yogeswaran; Ramasamy, Ramaraja P

2016-07-15

An amperometric sensor based on a bi-enzyme modified electrode was fabricated to detect methyl salicylate, a volatile organic compound released by pathogen-infected plants via systemic response. The detection is based on cascadic conversion reactions that result in an amperometric electrochemical signal. The bi-enzyme electrode is made of alcohol oxidase and horseradish peroxidase enzymes immobilized on to a carbon nanotube matrix through a molecular tethering method. Methyl salicylate undergoes hydrolysis to form methanol, which is consumed by alcohol oxidase to form formaldehyde while simultaneously reducing oxygen to hydrogen peroxide. The hydrogen peroxide will be further reduced to water by horseradish peroxidase, which results in an amperometric signal via direct electron transfer. The bi-enzyme biosensor was evaluated by cyclic voltammetry and constant potential amperometry using hydrolyzed methyl salicylate as the analyte. The sensitivity of the bi-enzyme biosensor as determined by cyclic voltammetry and constant potential amperometry were 112.37 and 282.82μAcm(-2)mM(-1) respectively, and the corresponding limits of detection were 22.95 and 0.98μM respectively. Constant potential amperometry was also used to evaluate durability, repeatability and interference from other compounds. Wintergreen oil was used for real sample study to establish the application of the bi-enzyme sensor for selective determination of plant pathogen infections. Copyright © 2016 Elsevier B.V. All rights reserved.

Sensitive pre-amplifier to load for Pin diodes

International Nuclear Information System (INIS)

Jacobo V, R. Y.; Hernandez D, V.; Ramirez J, F. J.

2013-10-01

The electronic instrumentation is indispensable for the measurement and characterization of the radiation. By means of this essential characteristics of the radiation are determined, as activity and their energy components. The nuclear instrumentation is based on the technical characteristics of the radiation detectors and the electronic devices associates (amplifiers, ana logical and digital converters, multichannel analyzers, etc.) The radiation detectors are very important instruments in fields as the nuclear physics, medicine, radiological protection, industry and in other fields, since they are the only method to capture the radiation and to be able to quantify it in precise form. To detect radiation diverse detector types are used, as the semiconductor type, inside them are the photodiodes type Pin. In this work the results that were obtained of the design, simulation, construction and tests of a preamplifier that was designed starting from a photodiode type Pin are presented. The system was designed and simulated with a program for electronic circuits, in this were carried out many tests being obtained a compact design and achieving the best necessary characteristics for its optimization. With the results of the simulation phase the electronics phase was built, which was couples to a spectroscopic amplifier and a multichannel analyzer. The total of the system was evaluated analyzing its performance before a triple source of alphas. Of the tests phase we find that the system allows obtaining, in a multichannel analyzer, the pulses height spectrum, with a good resolution and with this was calibrated the multichannel analyzer

Solid state solvation effect and reduced amplified spontaneous emission threshold value of glass forming DCM derivative in PMMA films

Energy Technology Data Exchange (ETDEWEB)

Vembris, Aivars, E-mail: aivars.vembris@cfi.lu.lv [Institute of Solid State Physics, University of Latvia, 8 Kengaraga Street, Riga LV 1063 (Latvia); Zarins, Elmars; Kokars, Valdis [Institute of Applied Chemistry, Riga Technical University, 14/24 Azenes Street, Riga LV 1048 (Latvia)

2015-02-15

Molecule crystallization is one of the limitations for obtaining high-gain organic laser systems. One of the examples is well known red laser dye 4-(dicyanomethylene)-2-methyl-6-(p-dimethylaminostyryl)-4H-pyran (DCM). The lowest threshold value of amplified spontaneous emission was achieved by doping 2 wt% of DCM molecule in tris-(8-hydroxy quinoline) aluminum (Alq{sub 3}) matrix. Further increase of the DCM dye concentration makes the system less efficient as its threshold value increases. It is due to large intermolecular interaction, which induces photoluminescence quenching. Compounds with reduced intermolecular interaction could be prospective in organic laser systems due to higher possible doping. In this work photoluminescence and amplified spontaneous emission properties of modified DCM molecule in poly(methyl methacrylate) (PMMA) matrix were investigated. Bulky trityloxyethyl groups were attached to the donor part of DCM. These groups increase intermolecular distance wherewith reduce photoluminescence quenching. More than one order of magnitude lower excitation threshold energy of the amplified spontaneous emission was achieved in doped polymer films with investigated compound in comparison to doped polymer with DCM. It means that the investigated compound is more perspective as a laser material compared to the previously studied. In addition, amplified spontaneous emission maximum could be tuned within 15 nm by changing concentration from 0.1 wt% to 10 wt% DWK-1 in PMMA matrix due to solid state solvation effect. - Highlights: • Bulky groups attached to DCM dye reduce photoluminescence quenching. • Amplified spontaneous emission is in red spectral region. • Amplified spontaneous emission spectra were tuned by 15 nm. • Amplified spontaneous emission threshold value was reduced by one order of magnitude.

Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease.

Science.gov (United States)

Chiba, Hirofumi; Kakuta, Yoichi; Kinouchi, Yoshitaka; Kawai, Yosuke; Watanabe, Kazuhiro; Nagao, Munenori; Naito, Takeo; Onodera, Motoyuki; Moroi, Rintaro; Kuroha, Masatake; Kanazawa, Yoshitake; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Negoro, Kenichi; Nagasaki, Masao; Unno, Michiaki; Shimosegawa, Tooru

2018-01-01

Inflammatory bowel disease (IBD) has an unknown etiology; however, accumulating evidence suggests that IBD is a multifactorial disease influenced by a combination of genetic and environmental factors. The influence of genetic variants on DNA methylation in cis and cis effects on expression have been demonstrated. We hypothesized that IBD susceptibility single-nucleotide polymorphisms (SNPs) regulate susceptibility gene expressions in cis by regulating DNA methylation around SNPs. For this, we determined cis-regulated allele-specific DNA methylation (ASM) around IBD susceptibility genes in CD4+ effector/memory T cells (Tem) in lamina propria mononuclear cells (LPMCs) in patients with IBD and examined the association between the ASM SNP genotype and neighboring susceptibility gene expressions. CD4+ effector/memory T cells (Tem) were isolated from LPMCs in 15 Japanese IBD patients (ten Crohn's disease [CD] and five ulcerative colitis [UC] patients). ASM analysis was performed by methylation-sensitive SNP array analysis. We defined ASM as a changing average relative allele score ([Formula: see text]) >0.1 after digestion by methylation-sensitive restriction enzymes. Among SNPs showing [Formula: see text] >0.1, we extracted the probes located on tag-SNPs of 200 IBD susceptibility loci and around IBD susceptibility genes as candidate ASM SNPs. To validate ASM, bisulfite-pyrosequencing was performed. Transcriptome analysis was examined in 11 IBD patients (seven CD and four UC patients). The relation between rs36221701 genotype and neighboring gene expressions were analyzed. We extracted six candidate ASM SNPs around IBD susceptibility genes. The top of [Formula: see text] (0.23) was rs1130368 located on HLA-DQB1. ASM around rs36221701 ([Formula: see text] = 0.14) located near SMAD3 was validated using bisulfite pyrosequencing. The SMAD3 expression was significantly associated with the rs36221701 genotype (p = 0.016). We confirmed the existence of cis-regulated ASM around

Allele-specific DNA methylation of disease susceptibility genes in Japanese patients with inflammatory bowel disease

Science.gov (United States)

Chiba, Hirofumi; Kakuta, Yoichi; Kinouchi, Yoshitaka; Kawai, Yosuke; Watanabe, Kazuhiro; Nagao, Munenori; Naito, Takeo; Onodera, Motoyuki; Moroi, Rintaro; Kuroha, Masatake; Kanazawa, Yoshitake; Kimura, Tomoya; Shiga, Hisashi; Endo, Katsuya; Negoro, Kenichi; Nagasaki, Masao; Unno, Michiaki; Shimosegawa, Tooru

2018-01-01

Background Inflammatory bowel disease (IBD) has an unknown etiology; however, accumulating evidence suggests that IBD is a multifactorial disease influenced by a combination of genetic and environmental factors. The influence of genetic variants on DNA methylation in cis and cis effects on expression have been demonstrated. We hypothesized that IBD susceptibility single-nucleotide polymorphisms (SNPs) regulate susceptibility gene expressions in cis by regulating DNA methylation around SNPs. For this, we determined cis-regulated allele-specific DNA methylation (ASM) around IBD susceptibility genes in CD4+ effector/memory T cells (Tem) in lamina propria mononuclear cells (LPMCs) in patients with IBD and examined the association between the ASM SNP genotype and neighboring susceptibility gene expressions. Methods CD4+ effector/memory T cells (Tem) were isolated from LPMCs in 15 Japanese IBD patients (ten Crohn's disease [CD] and five ulcerative colitis [UC] patients). ASM analysis was performed by methylation-sensitive SNP array analysis. We defined ASM as a changing average relative allele score (ΔRAS¯) >0.1 after digestion by methylation-sensitive restriction enzymes. Among SNPs showing ΔRAS¯ >0.1, we extracted the probes located on tag-SNPs of 200 IBD susceptibility loci and around IBD susceptibility genes as candidate ASM SNPs. To validate ASM, bisulfite-pyrosequencing was performed. Transcriptome analysis was examined in 11 IBD patients (seven CD and four UC patients). The relation between rs36221701 genotype and neighboring gene expressions were analyzed. Results We extracted six candidate ASM SNPs around IBD susceptibility genes. The top of ΔRAS¯ (0.23) was rs1130368 located on HLA-DQB1. ASM around rs36221701 (ΔRAS¯ = 0.14) located near SMAD3 was validated using bisulfite pyrosequencing. The SMAD3 expression was significantly associated with the rs36221701 genotype (p = 0.016). Conclusions We confirmed the existence of cis-regulated ASM around IBD

Automated sequence- and stereo-specific assignment of methyl-labeled proteins by paramagnetic relaxation and methyl-methyl nuclear overhauser enhancement spectroscopy

Energy Technology Data Exchange (ETDEWEB)

Venditti, Vincenzo; Fawzi, Nicolas L.; Clore, G. Marius, E-mail: mariusc@mail.nih.gov [National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Laboratory of Chemical Physics (United States)

2011-11-15

Methyl-transverse relaxation optimized spectroscopy is rapidly becoming the preferred NMR technique for probing structure and dynamics of very large proteins up to {approx}1 MDa in molecular size. Data interpretation, however, necessitates assignment of methyl groups which still presents a very challenging and time-consuming process. Here we demonstrate that, in combination with a known 3D structure, paramagnetic relaxation enhancement (PRE), induced by nitroxide spin-labels incorporated at only a few surface-exposed engineered cysteines, provides fast, straightforward and robust access to methyl group resonance assignments, including stereoassignments for the methyl groups of leucine and valine. Neither prior assignments, including backbone assignments, for the protein, nor experiments that transfer magnetization between methyl groups and the protein backbone, are required. PRE-derived assignments are refined by 4D methyl-methyl nuclear Overhauser enhancement data, eliminating ambiguities and errors that may arise due to the high sensitivity of PREs to the potential presence of sparsely-populated transient states.

Calpastatin ( CAST ) gene polymorphism in Kajli, Lohi and Thalli ...

African Journals Online (AJOL)

Restriction fragment length polymorphisms (RFLPs) in the amplified fragments were studied using Msp1 restriction enzyme. Frequencies of MM, MN and NN genotypes were found to be 77, 20 and 3% in Lohi breed and 68, 26 and 6% in Kajli breed respectively. In Thalli sheep, only the MM (80%) and MN (20%) genotypes ...

Association of PPARG Pro12Ala polymorphism with insulin sensitivity and body mass index in patients with polycystic ovary syndrome.

Science.gov (United States)

Baldani, Dinka Pavicic; Skrgatic, Lana; Cerne, Jasmina Z; Ferk, Polonca; Simunic, Velimir; Gersak, Ksenija

2014-03-01

Insulin resistance is one of the key factors in the pathogenesis of polycystic ovary syndrome (PCOS). The peroxisome proliferator-activated receptor gamma (PPARG) plays a role in the regulation of insulin sensitivity. The aim of the present study was to establish a possible association of the PPARG Pro12Ala polymorphism with PCOS and its effect on family and personal history, as well as on the metabolic and endocrine parameters in PCOS patients. A total of 151 PCOS patients and 179 healthy women of reproductive age were enrolled. History, body mass index (BMI), waist-to-hip ratio and the presence of phenotypic hyperandrogenism were recorded. Hormonal, metabolic and biochemical profiles were assessed. A molecular analysis for the genetic polymorphism was performed. One third (29.8%) of the PCOS patients were found to be carriers of at least one variant of the Ala allele (X/Ala), while 70.2% carried two wild-type Pro alleles (Pro/Pro), with an equal distribution observed in the control group. The PCOS patients carrying the X/Ala alleles exhibited lower serum fasting insulin levels, homeostatic model assessment of insulin resistance (HOMA-IR) and BMI compared to Pro/Pro carriers. This finding was significant only in the lean PCOS group. The polymorphic genotype exerted no effect on history, hormonal and clinical hyperandrogenism, lipid status or C-reactive protein, leptin, adiponectin, resistin and ghrelin serum levels in women with PCOS. In conclusion, although the PPARG Pro12Ala polymorphism is not a major determinant of PCOS in the Croatian population, it may exert a positive effect on insulin sensitivity and BMI. As these associations were recorded exclusively in the lean group of patients with PCOS, this polymorphism potentially contributes to a protective role against hyperinsulinemia and obesity.

Identification of Differentially Methylated Sites with Weak Methylation Effects

Directory of Open Access Journals (Sweden)

Hong Tran

2018-02-01

Full Text Available Deoxyribonucleic acid (DNA methylation is an epigenetic alteration crucial for regulating stress responses. Identifying large-scale DNA methylation at single nucleotide resolution is made possible by whole genome bisulfite sequencing. An essential task following the generation of bisulfite sequencing data is to detect differentially methylated cytosines (DMCs among treatments. Most statistical methods for DMC detection do not consider the dependency of methylation patterns across the genome, thus possibly inflating type I error. Furthermore, small sample sizes and weak methylation effects among different phenotype categories make it difficult for these statistical methods to accurately detect DMCs. To address these issues, the wavelet-based functional mixed model (WFMM was introduced to detect DMCs. To further examine the performance of WFMM in detecting weak differential methylation events, we used both simulated and empirical data and compare WFMM performance to a popular DMC detection tool methylKit. Analyses of simulated data that replicated the effects of the herbicide glyphosate on DNA methylation in Arabidopsis thaliana show that WFMM results in higher sensitivity and specificity in detecting DMCs compared to methylKit, especially when the methylation differences among phenotype groups are small. Moreover, the performance of WFMM is robust with respect to small sample sizes, making it particularly attractive considering the current high costs of bisulfite sequencing. Analysis of empirical Arabidopsis thaliana data under varying glyphosate dosages, and the analysis of monozygotic (MZ twins who have different pain sensitivities—both datasets have weak methylation effects of <1%—show that WFMM can identify more relevant DMCs related to the phenotype of interest than methylKit. Differentially methylated regions (DMRs are genomic regions with different DNA methylation status across biological samples. DMRs and DMCs are essentially the same

Designing and development of a synchronous analogic amplifier

International Nuclear Information System (INIS)

De la Hoz, E.; Ortiz, A.

1989-01-01

A large number of electronic systems used in the measurement and analysis of weak signals involve a ''lock-in'' amplifier, because it provides an effective method for the recovery of signals buried below a considerable high level of noise. The use of the ''lock-in'' amplifier in our institutions is very limited due to the high expense and the lack of warranties for its maintenance. The purpose of the present work is to design and build a low cost ''lock-in'' amplifier using components available in the local market. Its optimization has allowed us to increase the sensitivity and linearity of the systems, making it very versatile for dedicated applications

KARAKTERISTIK GENETIK Kappaphycus alvarezii SEHAT DAN TERINFEKSI PENYAKIT ICE-ICE DENGAN METODE Amplified Fragment Length Polymorphism (AFLP

Directory of Open Access Journals (Sweden)

Emma Suryati

2013-03-01

Full Text Available Infeksi penyakit ice-ice pada Kappaphycus alvarezii seringkali menyebabkan penurunan produksi yang sangat signifikan. K. alvarezii merupakan alga merah penghasil karaginan yang memiliki nilai ekonomi tinggi dan banyak dimanfaatkan dalam berbagai industri, seperti farmasi, makanan, stabilizer, dan kosmetik. Perbaikan genetik sangat diperlukan untuk meningkatkan produksi. Penelitian ini bertujuan untuk mengetahui karakteristik kemiripan genetik K. alvarezii sehat dan terinfeksi penyakit dari Balai Penelitian dan Pengembangan Budidaya Air Payau (BPPBAP, Maros dengan metode Amplified Fragment Length Polymorphism (AFLP. Pada penelitian ini juga dianalisis K. alvarezii asal Bone (BNE, Gorontalo (GRL, Tambalang (TMB, dan Kendari (KND sebagai kontrol rumput laut sehat. Metode AFLP menggunakan enzim restriksi Psti dan Mset, preamplifikasi dan amplifikasi selektif diawali dengan isolsi DNA, uji genimoc DNA, restriksi dan ligasi. Hasil yang diperoleh menunjukkan penggunaan marker AFLP dengan primer forward P11 dan primer reverse M48, M49 dan M50 terhadap K. alvarezii yang berasal dari Takalar (TKL, dan Mataram (MTR, tanpa infeksi (sehat dan terinfeksi penyakit Takalar ice (TKL+, Mataram ice (MTR+, serta K. alvarezii kontrol (BNE, (GRL, (TMB, dan (KND menghasilkan 519 fragmen dalam 122 lokus dengan ukuran 50 - ~370 pb. Kemiripan genetik K. alvarezii yang terinfeksi penyakit ice-ice lebih rendah jika dibandingkan dengan yang sehat. Kemiripan genetik K. alvarezii dari Takalar sehat (TKL dan terinfeksi ice-ice (TKL+ adalah 0,8176 dan MTR-MTR+ adalah 0,8033.

Detection of new single nucleotide polymorphisms by means of real ...

Indian Academy of Sciences (India)

Unknown

amplified millions to billions of times by means of a PCR before the PCR product ... Keywords. Single nucleotide polymorphism; real time PCR; DNA melting curve analysis. ... VAL158MET SNP and alcoholism and to test for interac- tions between the .... indicate a heterozygote sample (VAL/MET genotype). The curve with ...

Detection of DNA polymorphisms in Dendrobium Sonia White mutant lines

International Nuclear Information System (INIS)

Affrida Abu Hassan; Putri Noor Faizah Megat Mohd Tahir; Zaiton Ahmad; Mohd Nazir Basiran

2006-01-01

Dendrobium Sonia white mutant lines were obtained through gamma ray induced mutation of purple flower Dendrobium Sonia at dosage 35 Gy. Amplified Fragment Length Polymorphism (AFLP) technique was used to compare genomic variations in these mutant lines with the control. Our objectives were to detect polymorphic fragments from these mutants to provide useful information on genes involving in flower colour expression. AFLP is a PCR based DNA fingerprinting technique. It involves digestion of DNA with restriction enzymes, ligation of adapter and selective amplification using primer with one (pre-amplification) and three (selective amplification) arbitrary nucleotides. A total number of 20 primer combinations have been tested and 7 produced clear fingerprint patterns. Of these, 13 polymorphic bands have been successfully isolate and cloned. (Author)

A Carbon Nanotube-based NEMS Parametric Amplifier for Enhanced Radio Wave Detection and Electronic Signal Amplification

Energy Technology Data Exchange (ETDEWEB)

Aleman, B J; Sussman, A; Zettl, A [Physics Department, University of California, Berkeley, CA 94720 (United States); Mickelson, W, E-mail: azettl@berkeley.edu [Center of Integrated Nanomechanical Systems, University of California, Berkeley, CA 94720 (United States)

2011-07-20

We propose a scheme for a parametric amplifier based on a single suspended carbon nanotube field-emitter. This novel electromechanical nanotube device acts as a phase-sensitive, variable-gain, band-pass-filtering amplifier for electronic signal processing and, at the same time, can operate as a variable-sensitivity, tuneable detector and transducer of radio frequency electromagnetic waves. The amplifier can exhibit infinite gain at pumping voltages much less than 10 Volts. Additionally, the amplifier's low overhead power consumption (10-1000 nW) make it exceptionally attractive for ultra-low-power applications.

A Carbon Nanotube-based NEMS Parametric Amplifier for Enhanced Radio Wave Detection and Electronic Signal Amplification

International Nuclear Information System (INIS)

Aleman, B J; Sussman, A; Zettl, A; Mickelson, W

2011-01-01

We propose a scheme for a parametric amplifier based on a single suspended carbon nanotube field-emitter. This novel electromechanical nanotube device acts as a phase-sensitive, variable-gain, band-pass-filtering amplifier for electronic signal processing and, at the same time, can operate as a variable-sensitivity, tuneable detector and transducer of radio frequency electromagnetic waves. The amplifier can exhibit infinite gain at pumping voltages much less than 10 Volts. Additionally, the amplifier's low overhead power consumption (10-1000 nW) make it exceptionally attractive for ultra-low-power applications.

Null association of maternal MTHFR A1298C polymorphism with ...

African Journals Online (AJOL)

Background: Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme of folate/homocysteine pathway and is essential for DNA synthesis and methylation. MTHFR gene polymorphisms have been reported as risk factors for congenital defects and several metabolic and neurological disorders. Several studies ...

Spectroscopic amplifier for pin diode

International Nuclear Information System (INIS)

Alonso M, M. S.; Hernandez D, V. M.; Vega C, H. R.

2014-10-01

The photodiode remains the basic choice for the photo-detection and is widely used in optical communications, medical diagnostics and field of corpuscular radiation. In detecting radiation it has been used for monitoring radon and its progeny and inexpensive spectrometric systems. The development of a spectroscopic amplifier for Pin diode is presented which has the following characteristics: canceler Pole-Zero (P/Z) with a time constant of 8 μs; constant gain of 57, suitable for the acquisition system; 4th integrator Gaussian order to waveform change of exponential input to semi-Gaussian output and finally a stage of baseline restorer which prevents Dc signal contribution to the next stage. The operational amplifier used is the TLE2074 of BiFET technology of Texas Instruments with 10 MHz bandwidth, 25 V/μs of slew rate and a noise floor of 17 nv/(Hz)1/2. The integrated circuit has 4 operational amplifiers and in is contained the total of spectroscopic amplifier that is the goal of electronic design. The results show like the exponential input signal is converted to semi-Gaussian, modifying only the amplitude according to the specifications in the design. The total system is formed by the detector, which is the Pin diode, a sensitive preamplifier to the load, the spectroscopic amplifier that is what is presented and finally a pulse height analyzer (Mca) which is where the spectrum is shown. (Author)

[Study on association of CTLA4 gene polymorphism with Grave's disease in Guangxi Zhuang nationality population].

Science.gov (United States)

Liang, Xing-huan; Qin, Ying-fen; Ma, Yan; Xie, Xin-rong; Xie, Kai-qing; Luo, Zuo-jie

2006-06-01

To investigate the relationship between the polymorphic (AT)n repeats in 3ountranslated region of exon 4 of CTLA4 gene [CTLA4(AT)n] and Graveso disease (GD) in Zhuang nationality population of Guangxi province. The studied groups comprised 48 patients with GD and 44 normal controls. Amplification of target DNA was carried out by polymerase chain reaction (PCR). The amplified products were run by 8% polyacrylamide gel electrophoresis, and then followed by 0.1% silver staining. Some of amplified products were sequenced directly. Nineteen alleles of CTLA4 gene microsatellite polymorphism were found in Guangxi Zhuang nationality individuals. The 106 bp long allele was apparently increased in patients with GD of Zhuang nationality but not in healthy controls (Pdisease in Zhuang nationality population of Guangxi province. CTLA4(AT)n 106 bp may be the susceptible gene in GD patients of Zhuang nationality in Guangxi; 19 alleles of CTLA4 gene microsatellite polymorphism were found in Guangxi Zhuang nationality individuals.

Herbivore-Induced DNA Demethylation Changes Floral Signalling and Attractiveness to Pollinators in Brassica rapa.

Directory of Open Access Journals (Sweden)

Roman T Kellenberger

Full Text Available Plants have to fine-tune their signals to optimise the trade-off between herbivore deterrence and pollinator attraction. An important mechanism in mediating plant-insect interactions is the regulation of gene expression via DNA methylation. However, the effect of herbivore-induced DNA methylation changes on pollinator-relevant plant signalling has not been systematically investigated. Here, we assessed the impact of foliar herbivory on DNA methylation and floral traits in the model crop plant Brassica rapa. Methylation-sensitive amplified fragment length polymorphism (MSAP analysis showed that leaf damage by the caterpillar Pieris brassicae was associated with genome-wide methylation changes in both leaves and flowers of B. rapa as well as a downturn in flower number, morphology and scent. A comparison to plants with jasmonic acid-induced defence showed similar demethylation patterns in leaves, but both the floral methylome and phenotype differed significantly from P. brassicae infested plants. Standardised genome-wide demethylation with 5-azacytidine in five different B. rapa full-sib groups further resulted in a genotype-specific downturn of floral morphology and scent, which significantly reduced the attractiveness of the plants to the pollinator bee Bombus terrestris. These results suggest that DNA methylation plays an important role in adjusting plant signalling in response to changing insect communities.

Deletion and aberrant CpG island methylation of Caspase 8 gene in medulloblastoma.

Science.gov (United States)

Gonzalez-Gomez, Pilar; Bello, M Josefa; Inda, M Mar; Alonso, M Eva; Arjona, Dolores; Amiñoso, Cinthia; Lopez-Marin, Isabel; de Campos, Jose M; Sarasa, Jose L; Castresana, Javier S; Rey, Juan A

2004-09-01

Aberrant methylation of promoter CpG islands in human genes is an alternative genetic inactivation mechanism that contributes to the development of human tumors. Nevertheless, few studies have analyzed methylation in medulloblastomas. We determined the frequency of aberrant CpG island methylation for Caspase 8 (CASP8) in a group of 24 medulloblastomas arising in 8 adult and 16 pediatric patients. Complete methylation of CASP8 was found in 15 tumors (62%) and one case displayed hemimethylation. Three samples amplified neither of the two primer sets for methylated or unmethylated alleles, suggesting that genomic deletion occurred in the 5' flanking region of CASP8. Our findings suggest that methylation commonly contributes to CASP8 silencing in medulloblastomas and that homozygous deletion or severe sequence changes involving the promoter region may be another mechanism leading to CASP8 inactivation in this neoplasm.

Characterization of polymorphic microsatellites for the invasive grass Microstegium vimineum (Poaceae).

Science.gov (United States)

Novy, Ari; Flory, S Luke; Honig, Joshua A; Bonos, Stacy; Hartman, Jean Marie

2012-02-01

Microsatellite markers were developed for the invasive plant Microstegium vimineum (Poaceae) to assess its population structure and to facilitate tracking of invasion expansion. Using 454 sequencing, 11 polymorphic and six monomorphic microsatellite primer sets were developed for M. vimineum. The primer sets were tested on individuals sampled from six populations in the United States and China. The polymorphic primers amplified di-, tri-, and tetranucleotide repeats with three to 10 alleles per locus. These markers will be useful for a variety of applications including tracking of invasion dynamics and population genetics studies.

Continuous-variable Measurement-device-independent Quantum Relay Network with Phase-sensitive Amplifiers

Science.gov (United States)

Li, Fei; Zhao, Wei; Guo, Ying

2018-01-01

Continuous-variable (CV) measurement-device-independent (MDI) quantum cryptography is now heading towards solving the practical problem of implementing scalable quantum networks. In this paper, we show that a solution can come from deploying an optical amplifier in the CV-MDI system, aiming to establish a high-rate quantum network. We suggest an improved CV-MDI protocol using the EPR states coupled with optical amplifiers. It can implement a practical quantum network scheme, where the legal participants create the secret correlations by using EPR states connecting to an untrusted relay via insecure links and applying the multi-entangled Greenberger-Horne-Zeilinger (GHZ) state analysis at relay station. Despite the possibility that the relay could be completely tampered with and imperfect links are subject to the powerful attacks, the legal participants are still able to extract a secret key from network communication. The numerical simulation indicates that the quantum network communication can be achieved in an asymmetric scenario, fulfilling the demands of a practical quantum network. Furthermore, we show that the use of optical amplifiers can compensate the inherent imperfections and improve the secret key rate of the CV-MDI system.

Genetic relationship and diversity in a sesame (Sesamum indicum L. germplasm collection using amplified fragment length polymorphism (AFLP

Directory of Open Access Journals (Sweden)

Karlovsky Petr

2006-02-01

Full Text Available Abstract Background Sesame is an important oil crop in tropical and subtropical areas. Despite its nutritional value and historic and cultural importance, the research on sesame has been scarce, particularly as far as its genetic diversity is concerned. The aims of the present study were to clarify genetic relationships among 32 sesame accessions from the Venezuelan Germplasm Collection, which represents genotypes from five diversity centres (India, Africa, China-Korea-Japan, Central Asia and Western Asia, and to determine the association between geographical origin and genetic diversity using amplified fragment length polymorphism (AFLP. Results Large genetic variability was found within the germplasm collection. A total of 457 AFLP markers were recorded, 93 % of them being polymorphic. The Jaccard similarity coefficient ranged from 0.38 to 0.85 between pairs of accessions. The UPGMA dendrogram grouped 25 of 32 accessions in two robust clusters, but it has not revealed any association between genotype and geographical origin. Indian, African and Chinese-Korean-Japanese accessions were distributed throughout the dendrogram. A similar pattern was obtained using principal coordinates analysis. Genetic diversity studies considering five groups of accessions according to the geographic origin detected that only 20 % of the total diversity was due to diversity among groups using Nei's coefficient of population differentiation. Similarly, only 5% of the total diversity was attributed to differences among groups by the analysis of molecular variance (AMOVA. This small but significant difference was explained by the fact that the Central Asia group had a lower genetic variation than the other diversity centres studied. Conclusion We found that our sesame collection was genetically very variable and did not show an association between geographical origin and AFLP patterns. This result suggests that there was considerable gene flow among diversity centres

Tissue culture-induced genetic and epigenetic variation in triticale (× Triticosecale spp. Wittmack ex A. Camus 1927) regenerants.

Science.gov (United States)

Machczyńska, Joanna; Zimny, Janusz; Bednarek, Piotr Tomasz

2015-10-01

Plant regeneration via in vitro culture can induce genetic and epigenetic variation; however, the extent of such changes in triticale is not yet understood. In the present study, metAFLP, a variation of methylation-sensitive amplified fragment length polymorphism analysis, was used to investigate tissue culture-induced variation in triticale regenerants derived from four distinct genotypes using androgenesis and somatic embryogenesis. The metAFLP technique enabled identification of both sequence and DNA methylation pattern changes in a single experiment. Moreover, it was possible to quantify subtle effects such as sequence variation, demethylation, and de novo methylation, which affected 19, 5.5, 4.5% of sites, respectively. Comparison of variation in different genotypes and with different in vitro regeneration approaches demonstrated that both the culture technique and genetic background of donor plants affected tissue culture-induced variation. The results showed that the metAFLP approach could be used for quantification of tissue culture-induced variation and provided direct evidence that in vitro plant regeneration could cause genetic and epigenetic variation.

Evaluation of insulin-like growth factor-I gene polymorphism in ...

African Journals Online (AJOL)

This study aimed to detect the genetic polymorphism of IGF-1 in different Egyptian sheep and goat breeds. The amplified fragments at 320-bp were digested with HaeIII endonuclease and the results show the presence of three different genotypes: CC (15.71%), CG (29.29%) and GG (55.0%). The nucleotide sequence ...

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

Science.gov (United States)

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...

X-ray structural studies and physicochemical characterization of (E)-6-(3,4-dimethoxyphenyl)-1-ethyl-4-mesitylimino-3-methyl- 3,4-dihydro-2(1H)-pyrimidinone polymorphs.

Science.gov (United States)

Miyamae, A; Kitamura, S; Tada, T; Koda, S; Yasuda, T

1991-10-01

The polymorphism of (E)-6-(3,4-dimethoxyphenyl)-1-ethyl-4-mesitylimino-3-methyl-3,4-di hydro- 2(1 H)-pyrimidinone (FK664; 1) was characterized by using X-ray powder diffractometry, differential scanning calorimetry (DSC), and IR spectroscopy. Structures of two polymorphs (Forms A and B) were determined by X-ray crystallographic analysis. Form A crystallized in the monoclinic space group P2(1)/c, with a = 13.504(2), b = 6.733(1), c = 24.910(8) A, beta = 96.55(4) degrees, z = 4, and dcal = 1.203 g/cm3, while Form B crystallized in the same space group, with a = 8.067(2), b = 15.128(4), c = 18.657(4) A, beta = 102.34(3) degrees, z = 4, and dcal = 1.216 g/cm3. The conformational features of 1 were very similar between the two polymorphs. Compound 1, in both crystal forms, took an energetically reasonable conformation in three rigid planes, such as 2-pyrimidone, trimethylphenyl, and dimethoxyphenyl rings, but the molecules were packed in different ways between the two polymorphs. In the Form B crystal, a short contact was possible, to form pi-pi interactions between two dimethoxyphenyl groups related with the inversion center in the crystal lattice; this interaction seems to contribute to stabilizing the crystal structure of Form B. Both Forms A and B showed only one endothermic peak due to fusion at 115 and 140 degrees C, respectively, on the DSC thermograms; therefore, it is suggested that there are no transition points between the two polymorphs. The heats of fusion obtained from the DSC thermograms were 33.2(2) kJ/mol for Form A and 36.8(1) kJ/mol for Form B.(ABSTRACT TRUNCATED AT 250 WORDS)

Genotyping of the 19-bp insertion/deletion polymorphism in the 5' flank of beta-hydroxylase gene by dissociation analysis of allele-specific PCR products

DEFF Research Database (Denmark)

Rasmussen, Henrik Berg; Werge, Thomas

2005-01-01

The 19-bp insertion/deletion polymorphism in the 5' flank of the dopamine beta-hydroxylase (DBH) gene has been associated with psychiatric disorders. We have developed a simple, reliable and inexpensive closed-tube assay for genotyping of this polymorphism based upon T(m) determination of amplified...... and a conventional approach based upon agarose gel electrophoresis of amplified fragments revealed complete concordance between the two procedures. The insights obtained in this study may be utilized to develop assays based upon dissociation analysis of PCR products for genotyping of other insertion...