Restriction Enzymes HpaII Research Articles

First Report of a Group 16SrII Phytoplasma Associated with Witches'-Broom of Eggplant in Oman.

Eggplant (Solanum melongena L.) belongs to the family Solanaceae and is an important vegetable cash crop grown in most parts of Oman. In February 2010, plants showing phyllody symptoms and proliferation of shoots resembling those caused by phytoplasma infection were observed at Khasab, 500 km north of Muscat. Total genomic DNA was extracted from healthy and two symptomatic plants with a modified (CTAB) buffer method (2) and analyzed by direct and nested PCR with universal phytoplasma 16S rDNA primers P1/P7 and R16F2n/ R16R2, respectively. PCR amplifications from all infected plants yielded an expected product of 1.8 kb with P1/P7 primers and a 1.2-kb fragment with nested PCR, while no products were evident with DNA from healthy plants. Restriction fragment length polymorphism (RFLP) profiles of the 1.2-kb nested PCR products of two eggplant phyllody phytoplasma and five phytoplasma control strains belonging to different groups used as positive control were generated with the restriction endonucleases RsaI, AluI, Tru9I, T-HB8I, and HpaII. The eggplant phytoplasma DNA yielded patterns similar to alfalfa witches'-broom phytoplasma (GenBank Accession No. AF438413) belonging to subgroup 16SrII-D, which has been recorded in Oman (1). The DNA sequence of the 1.8-kb direct PCR product was deposited in GenBank (Accession No. HQ423156). Sequence homology results using BLAST revealed that the eggplant phyllody phytoplasma shared >99% sequence identity with Scaevola witches'-broom phytoplasma (Accession No. AB257291.1), eggplant phyllody phytoplasma (Accession No. FN257482.1), and alfalfa witches'-broom phytoplasma (Accession No. AY169323). The RFLP and BLAST results of 16S rRNA gene sequences confirm that eggplant phyllody phytoplasma is similar to the alfalfa phytoplasma belonging to subgroup 16SrII-D. To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group causing witches'-broom disease on eggplant in Oman.

A Sida sp. Is a New Host for "Candidatus Phytoplasma brasiliense" in Brazil.

Sida is a genus of flowering herbs in the family Malvaceae, which includes several species that are weeds in Brazil. Plants of a Sida sp. exhibiting symptoms characterized by stunting, chlorosis, small leaves, and witches'-broom, indicative of infection by phytoplasmas, were found in a field previously cultivated with tomato, located in the region of Campinas, State of São Paulo, in December 2008. To demonstrate the presence of phytoplasmas in diseased tissues, DNA was extracted from shoots and leaves from three symptomatic and eight asymptomatic plants. Nested PCR was performed using primers P1/Tint followed by primer pair R16F2n/R16R2 (1). DNA fragments of 1.2 kb, corresponding to 16S rDNA, were amplified only for DNA from two symptomatic samples. Phytoplasma identification was initially carried out by restriction fragment length polymorphism (RFLP) analysis through digesting the PCR products with the restriction enzymes AluI, HhaI, HaeIII, HpaII, MseI, and RsaI. The two phytoplasma isolates found to be infecting a Sida sp. showed identical RFLP patterns, which were indistinguishable from the phytoplasma previously reported in association with hibiscus (Hibiscus rosa-sinensis) witches'-broom in Brazil (2). Nucleotide sequence alignment revealed that 16S rDNA of both phytoplasma isolates found in a Sida sp. (GenBank Accession No. HQ230579) shared 99.9% sequence similarity with 16S rDNA from hibiscus witches'-broom phytoplasma (HibWB) (GenBank Accession No. AF147708). HibWB is the representative of the 16SrXV group and it was proposed as a putative species nominated "Candidatus Phytoplasma brasiliense" (2). The disease is frequently observed in hibiscus plants used as ornamentals in the states of São Paulo (4) and Rio de Janeiro (2). "Ca. Phytoplasma brasiliense" has only been reported in Brazil to be infecting hibiscus (2,4) and periwinkle (Catharanthus roseus) (3). The presence of a phytoplasma belonging to group 16SrXV in a Sida sp. expands its natural host range. The role of this weed as a potential source of inoculum for crops should be investigated.

First Report of Bituminaria Witches'-Broom in Australia Caused by a 16SrII Phytoplasma

Bituminaria bituminosa (L.) Stirt. is a perennial legume known as Arabian pea that is used as a forage in arid areas and for stabilization of degraded soils. It is widely distributed in the Mediterranean Basin with wider adaptation across the Canary Islands (4). In July 2010, during a survey for phytoplasma, some Canary Island B. bituminosa plants with typical phytoplasma symptoms, including stunted growth with small leaves, shortened internodes, and bushy growth, were found in seed multiplication nurseries at Medina, Perth, Western Australia (115°48.5'E; 32°13.2'S). Two samples from plants with clear disease symptoms and two visibly healthy plants were collected and total DNA was extracted with the Illustra DNA extraction kit Phytopure (GE Healthcare) according to the manufacturer's instructions. Direct and nested PCR were used to test the presence of phytoplasma 16S rDNA in samples with universal primers P1/P7 and R16F2n/R16R2, respectively (1,3). The PCR amplifications from all diseased samples yielded an expected product of 1.8 kb by direct and 1.2 kb by nested PCR, but not from the healthy plant samples. The direct PCR product was used as a template DNA in sequencing and the DNA sequence was deposited in the NCBI GenBank (Accession No. HQ404357). Sequence homology analysis indicated there was a perfect match between the two isolates. BLAST search of the NCBI GenBank revealed that B. bituminosa phytoplasma shares >99% sequence identity with Crotalaria witches'-broom phytoplasma (Accession No. EU650181.1), pear decline phytoplasma (Accession No. EF656453.1), and Scaevola witches'-broom phytoplasma (Accession No. AB257291.1). On the basis of BLAST analyses of 16S rRNA gene sequences, B. bituminosa phytoplasma in Western Australia appears to belong to the peanut witches'-broom group (16SrII-D) of phytoplasma. Restriction fragment length polymorphism analysis was also performed on nested PCR products of two samples of B. bituminosa phytoplasma by separate digestion with HaeIII, Hind6I, HpaII, MboI, RsaI, Tru9I, and T-HB8I restriction enzymes. Samples yielded patterns similar to alfalfa witches'-broom phytoplasma (Accession No. AF438413) belonging to subgroup 16SrII-D (2). To our knowledge, this is the first report of a phytoplasma of the 16SrII-D group infecting B. bituminosa in Australia and should be referred to as "Bituminaria witches'-broom phytoplasma" (BiWB). This report also indicates that the occurrence of the phytoplasma in B. bituminosa may be widespread in the Canary Islands and other species of Bituminaria might be susceptible to infection by Bituminaria witches'-broom phytoplasma.

Bacterial leaf spot of coffee caused by Pseudomonas syringae pv. tabaci in Brazil

Coffee (Coffea arabica) is widely planted and an economically important crop in Brazil both for domestic consumption and export. During 2006 a new disease affected coffee seedlings cv. Catuai in a commercial nursery located in Arandu county, State of São Paulo. Disease incidence was estimated to be about 1–2% of seedlings. Initially small brown lesions were observed on leaves and then becoming black and angular; alternatively, there were irregular lesions surrounded by a large yellow halo, which sometimes coalesced. These symptoms were similar to those caused by Pseudomonas syringae pv. garcae or Burkholderia andropogonis, pathogens of coffee (Amaral et al., 1956; Rodrigues-Neto et al., 1981). From diseased tissues fluorescent pseudomonad bacteria were isolated on King’s B medium. Colonies were creamy white, rounded, with irregular margins. The isolates selected (IBSBF 2240, 2241 and 2249) were positive for levan production and tobacco hypersensitivity; and negative for oxidase, protopectinase and arginine dihydrolase (LOPAT group 1a) (Lelliott et al., 1966). Also, the bacterial strains produced acid from D-mannitol, inositol, D-sorbitol and erytritol but not from adonitol. D(-) tartrate and L-lactate were not utilised. Ice nucleation was negative. For comparison, the type and reference strains of P. syringae pv. garcae (IBSBF 248P), P. syringae pv. syringae (IBSBF 281T) and P. syringae pv. tabaci (IBSBF 1972P) were included in the identification assays. Biochemical tests were applied according to Braun-Kiewnick & Sands (2001) and molecular tests were based on PCR-RFLP of the hrp L gene of P. syringae pv. syringae. Pathogenicity of the three strains was confirmed by spraying bacterial suspensions (approximately 108 cfu mL−1) on healthy leaves of coffee seedlings previously wounded with a sterile needle and then covered with transparent plastic bags for 3 days. Control plants were treated with sterile distilled water. After 2 weeks, lesions developed only on inoculated leaves and identical bacteria to those inoculated were re-isolated. In molecular tests, the hrp gene was employed using the primers pshrp 1F (5′-CTCAGA/GGCGTTCATC/TCA-3′) and pshrp 2R (5′-TCAGGCC/TAACGGGTCA/TATCT-3′). In PCR-RFLP using AluI, HaeIII, HinfI, HpaII and TaqI restriction endonucleases, the isolated strains showed identical profiles with P. syringae pv. tabaci. Based on biochemical and molecular tests the pathogen was identified as P. syringae pv. tabaci. This is the first report of this species causing disease on coffee.

Genome organization of major tandem repeats in the hard tick, Ixodes scapularis

In spite of the global medical and veterinary importance of Ixodid ticks, relatively little is known about their genome organization. To address this, we developed the first fluorescence in situ hybridization (FISH)-based chromosome markers in the Lyme disease vector, Ixodes scapularis. Shotgun genomic DNA (gDNA) sequences were used to identify three major tandem repeat families which were localized to specific heterochromatic regions of I. scapularis chromosomes prepared from the mitotic cell line ISE18. Together, these repeats were estimated to contribute approximately 159 Mb (8%) of the 2.1 Gb (haploid) I. scapularis genome. The relative arrangement of each tandem repeat family and the nucleolar organizing regions was determined by rehybridization to individual chromosome spreads, which was useful to distinguish different chromosomes in the ISE18 karyotype. Long stretches (>20 kb) of tandem repeat-containing gDNA were resistant to digestion by the methylation-sensitive restriction enzyme HpaII and localized to the presumed peri-centromeric regions of the chromosomes. A telomeric probe based on the arthropod-conserved (TTAGG)(n) tandemly repetitive motif was localized to the termini of each I. scapularis chromosome. Localization of these markers produced the first link between DNA sequences and major structural features of I. scapularis chromosomes and thereby provided the framework for a FISH-based physical map.

Assessing the DNA methylation status of single cells with the comet assay

The comet assay (single cell gel electrophoresis) is a cost-effective, sensitive, and simple technique that is traditionally used for analyzing and quantifying DNA damage in individual cells. The aim of this study was to determine whether the comet assay could be modified to detect changes in the levels of DNA methylation in single cells. We used the difference in methylation sensitivity of the isoschizomeric restriction endonucleases HpaII and MspI to demonstrate the feasibility of the comet assay to measure the global DNA methylation level of individual cells. The results were verified with the well-established cytosine extension assay. We were able to show variations in DNA methylation after treatment of cultured cells with 5-azacytidine and succinylacetone, an accumulating metabolite in human tyrosinemia type I.

Lactobacillus species in supragingival plaque in subjects with hyposalivation

The aim of the study was to analyse the frequency of different Lactobacillus species in relation to the pH-lowering potential of the plaque. Methods Ten subjects with radiation-induced hyposalivation (RT group) and 10 subjects with primary Sjögren's syndrome (pSS group) and matched controls with normal salivary secretion were included. Lactobacillus species were selected from their growth on Rogosa agar and identified by PCR and restriction fragment length polymorphism using HaeIII and HpaII restriction enzymes. Results Lactobacilli were isolated from 14 subjects (6 RT, 3 pSS, 5 controls). The most prevalent species were Lactobacillus fermentum (7 subjects), Lactobacillus casei (7 subjects) and Lactobacillus rhamnosus (6 subjects). L. fermentum and L. casei were the most prevalent species in anterior sites and L. rhamnosus and L. fermentum in posterior sites. In anterior sites, hyposalivated subjects with high Lactobacillus counts had an increased plaque acidogenicity compared to those with low counts. In posterior sites, subjects with high Lactobacillus counts had a lower final pH compared with those with low counts. Conclusion There were large intra- and inter-individual variations in frequencies of Lactobacillus species and Lactobacillus counts, but no specific species could be related to plaque acidogenicity.

Association Between T102C Polymorphism of Serotonin 2A Receptor Gene and Urinary Incontinence in Older Women

BackgroundThe bladder's regulatory function is influenced by central serotonergic modulation. T102C polymorphism of the serotonin 2A (5-HT2A) receptor gene is associated with urinary incontinence (UI) that has been reported by...

COTTON VIRESCENCE PHYTOPLASMA AND ITS WEED RESERVOIR IN MALI

A phytoplasma associated with cotton virescence disease was identified in several cotton plants showing virescence, stunting, yellowing and reddening in the cotton-growing areas of Yanfolila and Kolondjeba, in the Bougouni Region (south west Mali). The agent was detected by PCR with phytoplasma universal primer pairs and identified as a 16SrII-C subgroup member by RFLP of PCR fragments separately digested with MseI, AluI, HhaI and HpaII restriction enzymes. The same phytoplasma was found in Sida cordifolia, a weed that can act as a reservoir. The spatial distribution of infected plants in a field was aggregated along the rows. A higher number of infected plants was present on the border rows, suggesting that primary infections are probably more important than secondary spread from cotton to cotton. The incidence of the disease in Mali cotton-growing areas fluctuates, but in some fields up to 50% of the plants were infected. We observed more infected plants in organic cotton. This can be due to the use of neem as insecticide, which is ineffective against leafhopper adults that spread the disease.

5-Lipoxygenase and epigenetic DNA methylation in aging cultures of cerebellar granule cells

5-Lipoxygenase (5-Lox), an enzyme involved in the metabolism of arachidonic acid participates in the modulation of the proliferation and differentiation of neural stem cells and cerebellar granule cell (CGC) precursors. Since epigenetic mechanisms including DNA methylation regulate 5-LOX expression and have been suggested as possible modulators of stem cell differentiation and aging, using primary cultures of mouse CGC (1, 5, 10, 14, 30 days in vitro; DIV), we studied DNA methylation patterns of the 5-LOX promoter and 5-LOX mRNA levels. We also measured the mRNA and protein content of the DNA methyltransferases DNMT1 and DNMT3a. 5-LOX, DNMT1, and DNMT3a mRNA levels were measured by real-time PCR. We observed that 5-LOX expression and the expression of maintenance DNMT1 is maximal at 1 DIV (proliferating neuronal precursors), whereas the expression of the de novo DNA methyltransferase DNMT3a mRNA increased in aging cultures. We analyzed the methylation status of the 5-LOX promoter using the methylation-sensitive restriction endonucleases AciI, BstUI, HpaII, and HinP1I, which digest unmethylated CpGs while leaving methylated CpGs intact. The 5-LOX DNA methylation increased with the age of the cells. Taken together, our data show that as cultured CGC mature and age in vitro, a decrease in 5-LOX mRNA content is accompanied by an increase in the methylation of the gene DNA. In addition, an increase in DNMT3a but not DNMT1 expression accompanies an increase of 5-LOX methylation during in vitro maturation.

Candidatus Phytoplasma brasiliense associado ao superbrotamento do hibisco (Hibiscus rosa-sinensis L.) no Estado de São Paulo

Plantas de hibisco com superbrotamento e definhamento seguido de morte têm sido observadas nos municípios de São Paulo, Campinas e Piracicaba. Como os sintomas são sugestivos daqueles induzidos por fitoplasmas, o presente trabalho buscou identificar o possível fitoplasma associado com a doença. Assim, 14 plantas sintomáticas de hibisco foram coletadas em Piracicaba (SP) e submetidas ao PCR duplo com os primers P1/Tint-R16F2n/R2 e ao exame em microscópio eletrônico de transmissão. A identificação foi realizada por análise de RFLP com as enzimas de restrição BfaI, DraI, HaeIII, HhaI, HpaII, MboI, MseI, RsaI e TaqI. Testes de transmissão foram conduzidos com enxertia de ramos e uso de Cuscuta subinclusa. Os resultados de nested-PCR revelaram a presença consistente de fitoplasmas em todas as plantas sintomáticas e foram confirmados pela observação de corpúsculos pleomórficos no floema, através da microscopia eletrônica. A análise de RFLP mostrou que o fitoplasma encontrado em hibisco pertence ao grupo 16SrXV, o mesmo grupo do Candidatus Phytoplasma brasiliense. O fitoplasma foi transmitido de planta doente para sadia, tanto pela enxertia como pela C. subinclusa, demonstrando ser o agente do superbrotamento do hibisco.

Distinct DNA Methylation Profiles Between Adenocarcinoma and Squamous Cell Carcinoma of Human Uterine Cervix

Alterations in DNA methylation offer unique prospects as tumor markers. The big limitation in cervical cancer research is that it is too hard to obtain the pure normal tissue from a cervical cancer mass. So, we first profile type-specific DNA methylation of major two types of human uterine cervical cancer, adenocarcinoma (ACA) and squamous cell carcinoma (SCC), to establish a precise source of marker research. To assess the DNA methylation status of promoter regions in human uterine cervical ACAs and SCCs, fresh frozen tissues were obtained from bulky tumor masses to minimize the contamination from normal tissues and two array platforms using digestion with methylation-sensitive restriction-enzyme HpaII, ligation, and PCR were performed: an array of 11,994 (approximately 1.5 kb) PCR products from 10,445 promoter regions, and an array of 355,264 oligonucleotides for 18,212 HpaII fragments in 12,617 promoter regions. Loci near 21 genes showed significant differences between six ACA and four SCC from the analysis of two array data. Real-time PCR-based validation was performed on 13 loci using other nearby candidate methylation targets in the same promoter. Methylation patterns of 11 of 13 linked loci concurred with the microarray results. Four loci were further studied using tissues from additional patients (23 ACA and 24 SCC). Hypermethylation of loci in PAK6 and NOGOR most strongly correlated with ACA. Therefore, we have identified the 21 genes with differential methylation pattern between ACA and SCC and, furthermore, we found that PAK6 and NOGOR could be useful markers of ACA to be distinct from SCC.

First Report of a Group 16SrIII-B Phytoplasma Associated with Decline of China Tree in Brazil.

China tree (Melia azedarach L.), originally from Asia, is an exotic deciduous species in Brazil and is used as an ornamental shade tree in the southern region of the country. Since 2005, plants displaying yellowing, little leaves, witches' broom, and decline have been observed in the State of Rio Grande do Sul. In the streets and avenues of the capital city of Porto Alegre, there are approximately 173 tree species and China tree (6.57% of all trees) is among the top 10 (80,000 China trees and most are symptomatic). Plants with those symptoms are very distinctive and have been found also in the cities of Livramento, Rio Grande, Santa Maria, and Vacaria, places located in seashore areas, and along highways everywhere in the state. The high incidence seems to be related to drought during the last few years. These symptoms are typical of a disease identified by yellowing or decline of China tree associated with phytoplasma and previously reported in the neighboring countries of Argentina, Paraguay, and Bolivia (2). To demonstrate the presence of phytoplasma in diseased trees and to confirm its identity, total DNA was extracted from China tree leaf midribs collected from 10 symptomatic and three asymptomatic plants. Nested PCR was performed with the P1/P7 primer pair in the primary PCR to amplify a 1.8-kb fragment encompassing the 16S rRNA gene, the 16S-23S spacer region, and the 5' end of the 23S rRNA gene, while the secondary PCR was primed by the R16F2n/R16R2 primer pair to amplify a 1.2-kb fragment of the 16S rRNA gene from the 1.8-kb fragment (3,4). DNA fragments of 1.2 kb amplified from nested PCR were analyzed by restriction fragment length polymorphism with restriction enzymes AluI, HhaI, HpaII, KpnI, MboI, MseI, and RsaI, revealing identical profiles for each amplicon and demonstrating that a phytoplasma belonging to group 16SrIII, subgroup B (16SrIII-B) (1) was associated consistently with all symptomatic plants. BLAST analysis revealed 99% identity among these cloned 1.2-kb sequences and representative sequences of phytoplasmas affiliated with group 16SrIII (GenBank Accession Nos. AY081817 and AF147706). A majority consensus sequence representing the phytoplasma found in China trees was selected and deposited in GenBank (Accession No. FJ404775). These results were confirmed by observation with transmission electron microscopy of pleomorphic bodies 400 to 2,000 nm in diameter in the phloem sieve tubes of all symptomatic trees. No phytoplasma was detected or visualized in asymptomatic samples. These results corroborate those from studies conducted in neighboring countries that demonstrated the association between phytoplasmas of group 16SrIII and decline of China trees (1). In conclusion, the current study revealed that a phytoplasma affiliated with group 16SrIII-B is associated with the decline of China tree in Brazil, a disease previously described based solely on symptoms (2). The incidence and severity of the disease are enough to prevent further use of these trees as landscape plants in southern Brazil.

Epigenetic Alteration of the Metallothionein 1E Gene in Human Endometrial Carcinomas

Aberrant expression of metallothioneins (MTs) has been observed in several human tumors. In our microarray analysis, MT-1E was found to have much lower expression in endometrial cancer cells as compared with other types of cancer cells generated from the cervix, ovary or prostate. The result was confirmed by quantitative RT-PCR analysis of the MT-1E levels in individual cancer cells. Treatment of endometrial cancer cells with 5-azacytidine could reactivate MT-1E expression. We further analyzed the DNA methylation status of the promoter region of MT-1E using methylation-sensitive restriction enzymes HhaI and HpaII, followed by PCR. Promoter hypermethylation was detected in 42.4% (53/125) of the endometrial carcinoma samples, whilst none of the 38 normal tissues or hyperplasia samples were methylated. The mRNA levels of MT-1E were significantly lower in the methylation-positive than in the methylation-negative samples. Endometrial carcinoma samples with low MT-1E expression coincidently had low levels of estrogen receptor-α expression and vice versa. This phenomenon was not observed in the expression pattern between estrogen receptor-β and MT-1E. There was no significant correlation between MT-1E methylation and any clinical parameters. In conclusion, a high frequency of cancer-specific hypermethylation of MT-1E was found in endometrial carcinomas. Its functional consequence in the development of endometrial cancer warrants further investigation.

New plant hosts for group 16SrII, ‘Candidatus Phytoplasma aurantifolia’, in India

Lettuce (Lactuca sativa), carrot (Daucus carota) and French bean (Phaseolus vulgaris) are important staple food crops in India. Symptoms of leaf yellowing, chlorosis, and little leaf were recently observed in these species at the Vegetable Research Center, GBPUA&T, Uttarakhand, Pantnagar, India. Plants of Amaranthus sp. growing in adjacent hedges also exhibited leaf yellowing symptoms. Three leaf samples of each plant species with symptoms were collected, including healthy looking plants. Total DNA was extracted and indexed in a nested PCR assay with universal primers R16mF2/R1 and fU5/rU3 that target the phytoplasma 16S rRNA. PCR amplicons (880 bp) were produced in all samples with, but not without symptoms. PCR products were subjected to RFLP analyses with RsaI and HpaII restriction enzymes, and further purified, cloned (pGEM-T Easy Vector, Promega), and sequenced in both directions using M13 forward and reverse sequencing primers (http://www.dnaseq.co.uk). The 16S rRNA sequences were compared with those of reference strains from GenBank using blast, and aligned with clustal W (Thompson et al., 1994) to produce a phylogenetic tree using the neighbour-joining method and 1000 replicates with mega 3·1 (Kumar et al., 2004). RFLP profiles were all similar to those of group 16SrII, ‘CandidatusPhytoplasma aurantifolia’. Sequences of phytoplasmas (GenBank) identified in lettuce (EU362630), carrot (EU362628), French bean (EU362629) and Amaranthus sp. (EU362627) shared 99% identity to each other, and to a member of group 16SrII, ‘CandidatusPhytoplasma aurantifolia’ (EU099570). Phylogeny supported RFLP results and sequence comparisons, and indicates that the Pantnagar phytoplasmas cluster in the 16SrII group branch more closely related to the cactus witches’ broom phytoplasma. ‘CandidatusPhytoplasma aurantifolia’ has been previously reported in acid lime in India (Ghosh et al., 1999) and chickpea (Ghanekar et al., 1988), but this is the first record of a 16SrII phytoplasma isolate in lettuce, carrot, French bean, and Amaranthussp. in this country. Work in the UK was done under Defra licence No. PHF 174D/5185(08/2005). Sequencing was done by the Sequencing Service (School of Life Sciences, University of Dundee, Scotland, http://www.dnaseq.co.uk) using Applied Biosystems Big-Dye Ver 3·1 chemistry on an Applied Biosystems model 3730 automated capillary DNA sequencer.

NA-Seq: A Discovery Tool for the Analysis of Chromatin Structure and Dynamics during Differentiation

It is well established that epigenetic modulation of genome accessibility in chromatin occurs during biological processes. Here we describe a method based on restriction enzymes and next-generation sequencing for identifying accessible DNA elements using a small amount of starting material, and use it to examine myeloid differentiation of primary human CD34+ cells. The accessibility of several classes of cis-regulatory elements was a predictive marker of in vivo DNA binding by transcription factors, and was associated with distinct patterns of histone posttranslational modifications. We also mapped large chromosomal domains with differential accessibility in progenitors and maturing cells. Accessibility became restricted during differentiation, correlating with a decreased number of expressed genes and loss of regulatory potential. Our data suggest that a permissive chromatin structure in multipotent cells is progressively and selectively closed during differentiation, and illustrate the use of our method for the identification of functional cis-regulatory elements.

Detection of DNA Hypomethylation Mediated Floral Induction in <i>Longan</i> and <i>Spinach</i> Using the HAT-RAPD Technique

In this study, the HAT-RAPD technique was used to detect DNA methylation in the four plant species, rice (Oryza sativa), petunia (Petunia hybrida), spinach (Spinacea oleracea L.) and longan (Dimocarpus longan Lour.), which were treated using 5-azacytidine, potassium chlorate (KClO3) and a low temperature induction. Rice and petunia were chosen to be induced because in these species it is known that 5-azacytidine is able to induce hypomethylation in their genomes leading to morphological changes, in particular floral induction, in the developing plants. Using the HAT-RAPD technique, DNA methylation was detected using the restriction enzymes HpaII and MspI in rice and petunia (as expected) and in spinach, but was found to be absent in longan, which suggests that floral induction in longan is undetectable in longan.

Random X chromosome methylation patterns in the carriers with clinical phenotypic expressions of X-linked recessive bulbospinal neuronopathy.

We report the unusual phenotypic expression in 2 female carriers of a family with X-linked recessive bulbospinal neuronopathy (X-BSN). We analyze the methylation pattern of the androgen receptor (AR) gene to inspect the possibility of non-random X chromosome inactivation to be the underlying mechanism. Twenty-three members in 3 generations of a Taiwanese family were examined and studied for genomic DNA analysis. We analyzed the sequence of the first exon of the AR gene to identify the numbers of CAG repeats, and to determine the methylation pattern by using the restriction enzymes HpaII and HhaI. There were 3 probands and 5 carriers and 2 of the carriers manifested clinical symptoms. Sequence analysis revealed that the numbers of trinucleotide repeats ranged from 42 to 45 in one allele of the X-chromosome in the 5 female carriers. The restriction pattern of the HpaII and HhaI recognizable sites of the X-chromosome indicated a random methylation. Our data suggest that molecular genetic studies are important in confirming the diagnosis of X-BSN and early detection of female carriers, and the random or non-random methylation pattern of the X-chromosome is not a determining factor for partial expression of the abnormal AR gene in some carriers.

In silico enhanced restriction enzyme based methylation analysis of the human glioblastoma genome using Agilent 244K CpG Island microarrays

Genome wide methylation profiling of gliomas is likely to provide important clues to improving treatment outcomes. Restriction enzyme based approaches have been widely utilized for methylation profiling of cancer genomes and will continue to have importance in combination with higher density microarrays. With the availability of the human genome sequence and microarray probe sequences, these approaches can be readily characterized and optimized via in silico modeling. We adapted the previously described HpaII/MspI based Methylation Sensitive Restriction Enzyme (MSRE) assay for use with two-color Agilent 244K CpG island microarrays. In this assay, fragmented genomic DNA is digested in separate reactions with isoschizomeric HpaII (methylation-sensitive) and MspI (methylation-insensitive) restriction enzymes. Using in silico hybridization, we found that genomic fragmentation with BfaI was superior to MseI, providing a maximum effective coverage of 22,362 CpG islands in the human genome. In addition, we confirmed the presence of an internal control group of fragments lacking HpaII/MspI sites which enable separation of methylated and unmethylated fragments. We used this method on genomic DNA isolated from normal brain, U87MG cells, and a glioblastoma patient tumor sample and confirmed selected differentially methylated CpG islands using bisulfite sequencing. Along with additional validation points, we performed a receiver operating characteristics (ROC) analysis to determine the optimal threshold (p ≤ 0.001). Based on this threshold, we identified ∼2,400 CpG islands common to all three samples and 145 CpG islands unique to glioblastoma. These data provide general guidance to individuals seeking to maximize effective coverage using restriction enzyme based methylation profiling approaches.

CXCL12 rs1801157 polymorphism in patients with breast cancer, Hodgkin's lymphoma, and non-Hodgkin's lymphoma.

Chemokines and their receptors regulate the trafficking of immune cells during their development, inflammation, and tissue repair. The single-nucleotide polymorphism (SNP) rs1801157 (previously known as CXCL12-A/ stromal cell-derived factor-1 (SDF1)-3'A) in CXCL12/SDF1 gene was assessed in breast cancer, Hodgkin's lymphoma (HL), and non-Hodgkin's lymphoma (NHL), since the chemokine CXCL12, previously known as SDF1, and its receptor CXCR4 regulate leukocyte trafficking and many essential biological processes, including tumor growth, angiogenesis, and metastasis of different types of tumors. Genotyping was performed by PCR-RFLP (polymerase chain reaction followed by restriction fragment length polymorphism) using a restriction enzyme HpaII cleavage. No significant difference was observed in genotype distribution between breast cancer patients (GG: 57.3%; GA: 39.8%; AA: 2.9%) and healthy female controls (GG: 62.9%; GA: 33%; AA: 4.1%) nor between HL patients (GG: 61.1%; GA:27.8%; AA: 11.1%) and healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%), whereas a significant difference was observed in genotype distribution between NHL patients (GG: 51.4%; GA: 47.1%; AA: 1.5%) and healthy controls (GG: 65.6%; GA: 28.9%; AA: 5.5%). Further studies will be necessary to elucidate the cancer chemokine network. However, this study suggests that CXCL12 rs1801157 polymorphism may have important implications in the pathogenesis of NHL.