Endonucleases HpaII Research Articles

Wheat plant DNA methylation pattern changes at chronic seed γ irradiation

Alterations of DNA methylation patterns of two wheat sorts Al’batros odesskii and Donetskaya-48 whose seeds were irradiated with a low dose rate (3 × 10−7 Gy/s) for 4 months have been studied. Six restriction endonucleases were used in the experiments. Primary distinction in DNA methylation patterns of the studied sorts has been demonstrated. Chronic irradiation resulted in an increase of the methylation level on the recognion site for GluI and Sou3AI and in a decrease of this index in recognion sites for endonucleases GlaI and HpaII. The alterations of the methylation level in recognition sites for restrictases MboI and MspI were not found. The considerable increase of chromosome aberration level at the same dose of chronic irradiation has been shown. The role of DNA methylation pattern changes in development of irradiation damage and organism protective reactions is discussed.

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.

Identification of Aspergillus tubingensis strains responsible for OTA contamination in grapes and wine based on the acyl transferase domain of a polyketide synthase gene

SummaryRestriction digestion analysis of the acyl transferase (AT) domain sequences of a polyketide synthase (PKS) gene was tested as a rapid method to identify isolates of Aspergillus tubingensis from grapes. Restriction endonuclease digestion of PKS products using the endonucleases BccI, HaeIII, HpaII, MboI and TaqI distinguished five types of restriction fragment length polymorphism (RFLP). Ochratoxigenic isolates were only identified within RFLP‐types I and III. The RFLP assay is proposed as a rapid and easy method to identify A. tubingensis isolates from grapes. Amino acid sequences of AT domains from representative A. tubingensis isolates of the RFLP types obtained were aligned and analysed using phylogenetic methods. A comparison was also made with reference strains of Aspergillus section Nigri. Most of the A. tubingensis strains clustered into two distinct groups Gr1 and Gr2 with the exception of two isolates that remained unclustered. These results support the intraspeficific variability within A. tubingensis species reported using other techniques.

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.

A Phytoplasma Associated with an Outbreak of an Unusual Disease of Chrysanthemum in China in 2008.

In August of 2008, a disease of chrysanthemum (Dendranthema morifolium (Ramat.) Tzvel) caused losses of 70 to 80% in one of the largest chrysanthemum gardens in Yangling, Shanxi Province, China. Chrysanthemum plants in nearby areas also were affected to various degrees. Symptoms included flattened stems, shortening of internodes, yellowing of leaf margins, root death, and dwarfing of plants. Affected plants eventually collapsed. On the basis of these symptoms, a phytoplasma was suspected. Total nucleic acids were extracted from 0.5 g of phloem tissue from stems of eight symptomatic and eight asymptomatic plants by the cetyltrimethylammoniumbromide (CTAB) method (1). To amplify phytoplasma DNA, primer pairs R16mF2/R16mR1, followed by R16F2n/R16R1 (2), were used in a nested PCR. A final amplicon product (1.2 kb) was obtained from all symptomatic plants but not from asymptomatic ones. Restriction fragment length polymorphism (RFLP) analyses of R16F2n/R16R1 amplicons with MseI, AluI, HhaI, HaeIII, KpnI, RsaI, and HpaII endonucleases indicated that all symptomatic plants, but none of the asymptomatic plants, contained a phytoplasma strain of group 16SrI, subgroup B (3). A search of rDNA sequences in GenBank revealed a similarity (>99%) to aster yellow phytoplasma, 16SrI group, thereby confirming strain identity based on RFLP analysis. These results indicate the disease of chrysanthemum is associated with a phytoplasma related to the aster yellow phytoplasma group. Sequences were deposited in GenBank (Accession No. FJ543467). A vector of this phytoplasma in chrysanthemum has not been identified.

Phytoplasma associated with a witches’ broom disease of Gleditsia sinensis (Fabaceae) newly reported in China

Gleditsia sinensis (Chinese honeylocust) is widely distributed in China due to its important use in traditional Chinese medicine. In August of 2008, G. sinensis trees in Shanxi Province, exhibited symptoms of witches’ broom, stunting, smaller leaves, and yellowing gradually becoming browning: a phytoplasma infection was suspected. Total DNA was extracted from 0·5 g of phloem tissue from leaf midribs and stems of eight plants with, and eight plants without symptoms (Angelini et al., 2001). DNA was analyzed for phytoplasma by a nested PCR assay using phytoplasma universal 16S rDNA primers R16mF1/R16mR1 and R16F2n/R16R2 (Gundersen & Lee, 1996). PCR products of 1·4 and 1·2 kb, respectively, were produced from all plants with symptoms, but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analyses of R16F2n/R16R2 amplicons with AluI, HhaI, TaqI, EcoRI, HaeIII, RsaI and HpaII endonucleases, indicated that all plants with symptoms contained a phytoplasma strain of group 16SrV (‘Candidatus Phytoplasma ulmi’), subgroup B (16SrV-B). The G. sinensis phytoplasma 16S rDNA sequence (GenBank Accession No. FJ457095) scored the highest identity (99%) with those of group 16SrV, thereby confirming strain identity based on RFLP. Phytoplasmas of 16SrV have previously reported on jujube (Zhu et al., 1997) and paper mulberry (Liu et al., 2004) causing witches’ broom in China. This is the first report of a ‘Candidatus Phytoplasma ulmi’-related strain affecting G. sinensis in China.

Occurrence of an aster yellows (16SrI) group phytoplasma associated with a leaf roll disease of shinyleaf yellowhorn in China

Shinyleaf yellowhorn (Xanthoceras sorbifolia), family Sapindaceae, is a common ornamental and afforestation plant widely grown in northwest China because it is well adapted to cold, arid and barren environments. There have been no previous reports of phytoplasma infections in this species. During a survey in 2008, 9% of shinyleaf yellowhorn plants in the ornamental gardens of Northwest A&F University displayed symptoms resembling phytoplasma infection. Affected plants showed leaf roll, as well as little leaf symptoms, which greatly reduced the ornamental value of the plants. On the basis of symptoms and transmission electron microscopy observations, nested PCR assays were used to verify association of phytoplasma with the disease. Total nucleic acids were extracted from the leaf veins of eight plants with, and six plants without symptoms, using the modified CTAB method (Angelini et al., 2001). Using phytoplasma universal primer pair R16mF2/R16mR1 followed by R16F2n/ R16R2, PCR products of 1·4-kb and a 1·2-kb respectively, were amplified only from plants showing symptoms. After sequencing of the 16S rDNA fragment from one sample (GenBank Accession No. FJ459913), a BLAST search determined that the shinyleaf yellowhorn leaf roll (SYLR) phytoplasma shared closest homology with 16SrI-D aster yellows group members (Lee et al., 1998). Furthermore, phylogenetic analysis of 16S rDNA sequences revealed SYLR phytoplasma to be most similar (99·68%) to paulownia witches’ broom phytoplasma (PaWB, FJ263621). No differences were detected between the phytoplasmas from the eight affected shinyleaf yellowhorn plants by restriction fragment length polymorphism (RFLP) analysis of nested rDNA products (1·2 kb) using endonucleases MseI, TaqI, RsaI, AluI, HpaII, and EcoRI. This is the first report of an aster yellows (16SrI) group phytoplasma associated with leaf roll disease of shinyleaf yellowhorn in China. The authors thank the 111 project from Education Ministry of China, No.B07049, and National Natural Science Foundation of China (30871625) for their support.

Fas Expression in Metastatic Osteosarcoma Cells Is Not Regulated by CpG Island Methylation

Fas expression in osteosarcoma (OS) cells is inversely correlated with the metastatic potential of OS to the lung. The purpose of this study was to determine whether loss of Fas expression in metastatic OS cells is secondary to DNA methylation of CpG islands in the Fas gene. SAOS-2 cells have high levels of Fas expression and do not form lung metastases when injected intravenously, whereas LM7 cells have low levels of Fas expression and do produce lung metastases. Using the endonucleases HpaII and MspI and a polymerase chain reaction-based methylation assay, we found that all four CpG sites in the CCGG sequence in the Fas promoter region were unmethylated in both SAOS-2 and LM7 cells. We performed detailed analysis of the 28 and 46 CpG sites in the Fas promoter and first intron region, respectively, using bisulfite-modified genomic DNA sequencing. More than 99.8% of the examined CpG sites were unmethylated and there was no difference of CpG methylation in SAOS-2 and LM7 cells as well as LM7 metastatic lung tumor tissue samples. Treatment of LM7 cells and another OS cell line, DLM8 with low levels of Fas expression, with demethylation agent, 5-azadeoxycitidine (AzadC), did not change the Fas expression and did not increase sensitivity of AzadC-treated cells to Fas ligand (FasL) treatment. In conclusion, our data indicate that decreased Fas expression in OS cells is not secondary to DNA methylation of CpG islands in the Fas gene and that Fas expression cannot be increased by using demethylation agents.

Study of heterogeneity of Coxiella burnettii strains by analysis of groEL gene restriction fragment length polymorphism

Restriction fragment length polymorphism was used for evaluation of genetic heterogeneity of Coxiella burnettii strains. A 594-b. p. fragment of groEL gene was cleaved with MN1I, HpaII, and Hin6I endonucleases. Genetic homogeneity of Coxiella burnettii strains from the Russian collection and their relation to the group of European and North American strains were detected.

Occurrence of a Clover Proliferation (16SrVI) Group Phytoplasma Associated with Little Leaf Disease of Portulaca grandiflora in India.

Portulaca grandiflora (family Portulacaceae), commonly known as moss rose purslane, is a popular ornamental plant widely grown in temperate climates because it blooms all summer. Portulaca is also used for medicinal purposes since it is rich in vitamins A, B1, and C and has antimicrobial and cytotoxic activity. Since March 2005, 30 to 50% of P. grandiflora plants in the ornamental gardens as well as in pots at the Central Institute of Medicinal and Aromatic Plants, Lucknow, India have displayed symptoms resembling phytoplasma infection. Disease symptoms start as a typical bud proliferation, downward curling, and diminishing size of leaves, followed by overall stunted growth and yellowing of the whole plant from April to June. Some plants also formed rosettes and a proliferation of axillary shoots resulting in a witches'-broom appearance. Typical pleomorphic bodies, mostly spherical to oval, ranging from 340 to 1,100 nm were observed only in sieve elements of infected plants by transmission electron microscopy (TEM). On the basis of symptoms, TEM observations, PCR, and response to antibiotic treatment, the causal organism was identified as phytoplasma (1). Total genomic DNA from healthy and infected plants was extracted with the CTAB buffer method (2). Of 27 suspected samples screened by PCR, 23 were phytoplasma positive. Presence of phytoplasmas in plants was demonstrated by a nested PCR assay employing primer pair P1/P6 followed by R16F2n/R16R2 that generated rDNA products of 1.5 and 1.2 kb, respectively, only from symptomatic plants. No differences among phytoplasmas in Portulaca plants were detected by restriction fragment length polymorphism (RFLP) analysis of nested rDNA (1.2 kb) products using endonucleases BamHI, RsaI, AluI, HpaII, and EcoRI. Comparative analysis of RFLP patterns with those derived from reference phytoplasmas tentatively identified the Portulaca little leaf (PLL) phytoplasma as a member of 16S rDNA RFLP group 16SrVI (3). A nested PCR product (1.25 kb) was cloned with a TOPO TA cloning kit (Invitrogen, Carlsbad, CA) and sequenced. The sequence was deposited in the GenBank database (Accession No. EF651786). Sequence analysis revealed the PLL phytoplasma to be most similar (98%) to Indian brinjal little leaf (Accession No. EF186820) and 'Candidatus Phytoplasma trifolii' (Accession No. AY390261), two 16SrVI group phytoplasmas previously reported from India and Canada, respectively. The status of PLL (EF651786) was also verified by in silico RFLP analysis (4) of the F2n/R2 sequence of six closely related strains (Accession Nos. AF228052, AY390261, AY270156, AY409070, AY409069, and EF186820) of the 16SrVI group using 17 restriction enzymes (AluI, BamHI, BfaI, BsfUI, DraI, EcoRI, HaeIII, HhaI, HinfI, HpaI, HpaII, KpnI, MseI, Sau3AI, RsaI, SspI, and TaqI). In silico restriction digestion and virtual gel plotting showed similar patterns for all enzymes. To our knowledge, this is the first report of a 16SrVI group phytoplasma infecting Portulaca plants in India.

First Report of 'Candidatus Phytoplasma trifolii'-Related Strain Associated with Safflower Phyllody Disease in Iran.

During a survey in 2003, safflower plants (Carthamus tinctorius) with phyllody symptoms were observed in production fields in several districts of Fars and Yazd provinces in Iran. Affected plants showed floral virescence, phyllody, proliferation of axillary buds, and little leaf symptoms. Incidence of the disease was less than 10%. Direct and nested PCR assays were used to verify association of phytoplasma with the disease. Total DNA was extracted from fresh, fine roots of eight phyllody-affected safflower plants and one symptomless plant. With phytoplasma universal primer pair P1/P7 (5'-AAGAGTTTGATCCTGGCTCAGGATT-3'/5'-CGTCCTTCATCGGCTCTT-3'), target DNA fragments of approximately 1.8 kb were amplified by direct PCR from phyllody-affected plants and Iranian cabbage yellows (ICY) phytoplasma used as a positive control. Reamplification of P1/P7 products with 16S rRNA gene primer pair R16F2n/R16R2 (5'-GAAACGACTGCTAAGACTGG-3'/5'-TGACGGGCGGTGTGTACAAACCCCG-3') yielded fragments of the expected size (1.2 kb) from all eight diseased plants and the ICY-positive control. No products were amplified from the symptomless plant by either assay. R16F2n/R16R2 products were subjected to restriction fragment length polymorphism (RFLP) analysis by separate digestion with AluI, HaeIII, HhaI, HinfI, HpaII, MseI, RsaI, Sau3AI, or TaqI endonuclease. Comparison of resulting RFLP patterns with published patterns of other phytoplasmas (2) tentatively identified safflower phyllody (SP) phytoplasma as a member of clover proliferation group 16SrVI, subgroup C. HhaI digests also differentiated SP from ICY phytoplasma, a previously reported subgroup 16SrVI-A strain (3). After sequencing of the 16S rDNA fragment (GenBank Accession No. DQ88948), a BLAST search determined that SP phytoplasma shared closest homology with 16SrVI group members ('Candidatus Phytoplasma trifolii') and related strains (4). Furthermore, phylogenetic analysis of 16S rDNA sequences revealed SP phytoplasma to be most similar (99.7%) to brinjal little leaf (BLL) phytoplasma (GenBank Accession No. X83431). Analysis of putative restriction sites in 16S rRNA gene sequences revealed that SP and BLL shared identical restriction profiles and that both differed from the 'Ca. Phytoplasma trifolii' reference strain (GenBank Accession No. AY390261) because of the absence of a single HhaI site and the presence of an additional MseI site. Although safflower phyllody disease has been previously reported in Israel, the associated phytoplasma was classified as a strain of the aster yellows subgroup 16SrI-B (1). To our knowledge, this is the first report of safflower as a host of a 'Ca. Phytoplasma trifolii'-related strain.

Genome-wide mapping and characterization of hypomethylated sites in human tissues and breast cancer cell lines

We have developed a method for mapping unmethylated sites in the human genome based on the resistance of TspRI-digested ends to ExoIII nuclease degradation. Digestion with TspRI and methylation-sensitive restriction endonuclease HpaII, followed by ExoIII and single-strand DNA nuclease allowed removal of DNA fragments containing unmethylated HpaII sites. We then used array comparative genomic hybridization (CGH) to map the sequences depleted by these procedures in human genomes derived from five human tissues, a primary breast tumor, and two breast tumor cell lines. Analysis of methylation patterns of the normal tissue genomes indicates that the hypomethylated sites are enriched in the 5' end of widely expressed genes, including promoter, first exon, and first intron. In contrast, genomes of the MCF-7 and MDA-MB-231 cell lines show extensive hypomethylation in the intragenic and intergenic regions whereas the primary tumor exhibits a pattern between those of the normal tissue and the cell lines. A striking characteristic of tumor cell lines is the presence of megabase-sized hypomethylated zones. These hypomethylated zones are associated with large genes, fragile sites, evolutionary breakpoints, chromosomal rearrangement breakpoints, tumor suppressor genes, and with regions containing tissue-specific gene clusters or with gene-poor regions containing novel tissue-specific genes. Correlation with microarray analysis shows that genes with a hypomethylated sequence 2 kb up- or downstream of the transcription start site are highly expressed, whereas genes with extensive intragenic and 3' untranslated region (UTR) hypomethylation are silenced. The method described herein can be used for large-scale screening of changes in the methylation pattern in the genome of interest.

First Report of Yucca Phyllody Associated with 16SrI-A Phytoplasmas in Texas.

Buckley's yucca (Yucca constricta Buckl.) is a native flowering perennial plant widely distributed in Texas and northeast Mexico. It is also grown as an ornamental plant in its native range as well as in other dry regions in the United States and Mexico. In 2006, during an extended drought, Buckley's yucca plants sporadically exhibited phyllody and abnormal bud proliferation on the inflorescence in Uvalde County in southwestern Texas. Symptoms resembled those caused by phytoplasmal infection. Samples from four symptomatic and two asymptomatic yucca plants were collected. Total nucleic acid was extracted from abnormal bud tissue. To assess the etiological aspect of the disease nested PCR using phytoplasma specific primer pair P1/16S-SR or P1/P7 followed by R16F2n/R16R2n was employed for the detection of putative phytoplasmas (2). An amplicon of approximately 1.2 kb was amplified from all four symptomatic yucca plants but not from asymptomatic plants. Restriction fragment length polymorphism (RFLP) patterns of 16S rDNA digested singly with AluI, KpnI, HpaII, MseI, HhaI, and RsaI endonucleases indicated that affected yucca plants were infected by a phytoplasma belonging to aster yellows group 16SrI ('Candidatus Phytoplasma asteris'), subgroup 16SrI-A (1). Nucleotide sequence analysis of cloned 16S rDNA (GenBank Accession No. EF190067) confirmed the results on the basis of RFLP analyses. Yucca phyllody has not been reported elsewhere. This disease appears to be newly emerging in Texas with only a few affected plants. To our knowledge, this is the first report of 16SrI-A phytoplasma infecting a Yucca sp.

P.1.23 Asenapine: preclinical evidence for clinical effects in Schizophrenia

Higher plant DNA is methylated at CG and CNG targets. In this study we have investigated the tobacco methylation system in tissue culture using the methylation inhibitors 5-azacytidine (5-azaC), dihydroxypropyladenine (DHPA) and ethionine (Ethi), and methylation-sensitive restriction endonucleases HpaII, MspI, HhaI, EcoRII, ScrFI, and Fnu4HI. Surprisingly, CAG/CTG sequences, contrary to CG doublets and CCG/CGG triplets, appeared to be refractory to the inhibitory effect of 5-azaC. Thus 5-azaC cannot be considered a general inhibitor of DNA methylation in tobacco cells. On the other hand, DHPA, the inhibitor of S-adenosylhomocysteine (SAH) hydrolase, and Ethi caused hypomethylation of both CAG/CTG and CCG/CGG triplets but not of the CG doublets. The sensitivity of triplet-specific methylation to the inhibition of SAH hydrolase suggests the possibility that plant-specific DNA methylation at CNG targets might be modulated by alterations of the SAH/S-Adenosyhnethionine ratio in plant cells.

First Report of a "Candidatus Phytoplasma australiense"-Related Strain in Lucerne (Medicago sativa) in Australia.

Australian lucerne yellows (ALuY), a phytoplasma-associated disease, is a major problem in Australia that causes the pasture seed industry millions of dollars in losses annually (3). Samples were collected from lucerne (Medicago sativa L.) plants exhibiting symptoms indicative of ALuY (4) in a seed lucerne paddock (cv CW 5558) at Griffith, southwestern New South Wales (NSW), Australia, in November 2005 and again in January 2006. Samples were kept at 4°C and processed within 36 h of collection. Total DNA was extracted from approximately 0.3 g of leaf midribs and petioles of each plant sample and used as template in a nested PCR assay with phytoplasma universal primer pairs P1/P7 and fU5/m23sr. PCR products resulting from the first amplification were diluted (1:30) with sterile distilled water (SDW) before reamplification with fU5/m23sr. DNA of Australian tomato big bud (TBB) phytoplasma and SDW were used as positive and negative assay controls, respectively. Ten of fifteen plant samples collected in November tested positive for phytoplasma DNA. Restriction digestion profiles of nested PCR amplicons with HpaII endonuclease were the same for all symptomatic plants but differed from the control. Phytoplasma identity was determined by sequencing two nested PCR products that yielded identical sequences. One was deposited in the GenBank database (Accession No. DQ786394). BLAST analysis of the latter sequence revealed a >99.6% similarity with "Candidatus Phytoplasma australiense" (L76865) and related strains papaya dieback (Y10095), phormium yellow leaf (U43570), strawberry green petal (AJ243044), and strawberry lethal yellows (AJ243045). Direct PCR with primers FP 5'-GCATGTCGCGGTGAATAC-3' and RY 5'-TGAGCTATAGGCCCTTAATC-3' designed to specifically amplify DNA of "Ca. P. australiense" detected the phytoplasma in 8 of 40 samples collected in January. Whether this phytoplasma is the etiological agent solely responsible for ALuY is currently under investigation. "Ca. P. asteris" and stolbur group (16SrXII) phytoplasmas have been reported in lucerne in the United States (2) and Italy (1), respectively. Within the stolbur group 16SrXII, "Ca. P. australiense" and stolbur phytoplasma are regarded as separate species and both are distinct from "Ca. P. asteris", a group 16SrI strain. To our knowledge, this is the first report of a "Ca. P. australiense" related strain in lucerne.

Physicochemical Characterization of El Tor Vibriophage S20

Objective:To characterize Vibrio cholerae El Tor typing phage S20 (ATCC No. 51352-B3). Methods and Results: The phage has a hexagonal head and a short tail. Cryo-electron microscopy and three-dimensional image reconstruction showed that the phage head has icosahedral symmetry. The phage has two major structural polypeptides of 50 and 42 kDa. Adsorption of the phage to its host followed a biphasic kinetics and its intracellular growth is characterized by a latent period of 12 min and a burst size of around 60 particles per infected cell. The phage was found to be stable at a pH range 5.0–9.0 and moderately thermotolerant and highly UV sensitive. Phage genome comprises a 40.7 ± 1.5-kb linear DNA molecule with random circular permutation and terminal redundancy. The restriction endonucleases AccI, HpaII, HaeIII, HindIII, EcoRV, HincII, DraI and XmnI cut vibriophage S20 DNA. Conclusion: Vibriophage S20, which belongs to Podoviridae, has an icosahedral head and the genome, which is double-stranded linear DNA, has random circular permutation and terminal redundancy.

Clonal analysis of palmar fibromatosis: a study whether palmar fibromatosis is a real tumor

BackgroundPalmar fibromatosis that arises in the palmar soft tissue is characterized by infiltrative growth with a tendency toward local recurrence but does not metastasize. This study investigated the clonality of this process in twelve female patients, each with a single lesion, by examining the pattern of X-chromosome inactivation.MethodsHematoxylin and eosin stained sections of formalin-fixed, paraffin-embedded tissues were microdissected by laser capture microdissection to obtain the proliferative spindle cells. Tumor cells were isolated from the sections of rectum adenocarcinoma, and used for positive control. The genomic DNAs was extracted with phenol-chloroform, digested with a methylation-sensitive restriction endonuclease HpaII, and amplified by polymerase chain reaction (PCR), using primers targeted to a highly polymorphic short tandem repeat (STR) of the human androgen receptor gene (HUMARA).ResultsAmong the twelve samples, three samples failed amplification, one sample showed homozygosity which was not suitable for further analysis, eight samples were successfully amplified, and showed a random X chromosome inactivation pattern, suggesting polyclonality of these lesions.ConclusionThe current findings suggest that palmar fibromatosis is a reactive proliferation rather than a clonal neoplasm.

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

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.

Clonality study of palmar fibromatosis

To study the clonality of palmar fibromatosis by molecular genetic analysis of X chromosome inactivation pattern at a polymorphic site of human androgen receptor gene (HUMARA). Twelve female cases of palmar fibromatosis were enrolled into this study. Hematoxylin and eosin-stained sections of paraffin-embedded, formalin-fixed tissues were microdissected by laser capture microdissection technology in order to obtain the proliferative spindle cells. Tumor cells isolated from rectal adenocarcinoma in a female patient were used as positive control. The genomic DNAs were extracted with phenol and chloroform, digested with methylation-sensitive restriction endonuclease HpaII, and amplified by polymerase chain reaction (PCR) using primers targeted to a highly polymorphic short tandem repeat of HUMARA. The amplimers were separated on vertical 8% non-denaturing polyacrylamide gels and the patterns were visualized with ethidium bromide stain. The methodology for clonality analysis was validated in the positive control using rectal adenocarcinoma cells. Among the 12 cases studied, PCR amplification failed in 3 samples and 1 sample showed homozygosity which was not suitable for further analysis. Eight samples were successfully amplified and showed a random X chromosome inactivation pattern, suggesting polyclonality in these lesions. Palmar fibromatosis is a polyclonal condition and should be considered as a form of non-neoplasmic fibroblastic proliferation.

Impact of Cytosine 5-Halogens on the Interaction of DNA with Restriction Endonucleases and Methyltransferase

Growing evidence from both prokaryotes and eukaryotes indicates that pyrimidine 5-methyl groups can have profound biological consequences that are mediated by the affinity of DNA-protein interactions. The presence of the 5-methyl group could potentially create a steric block preventing the binding of some proteins whereas the affinity of many other proteins is substantially increased by pyrimidine methylation. In this paper, we have constructed a series of oligonucleotides containing cytosine and a series of 5-substituted cytosine analogues including all halogens. This set of oligonucleotides has been used to probe the relationship between the size of the substituent and its capacity to modulate cleavage by the methylation-sensitive restriction endonucleases MspI and HpaII. Additionally, we have examined the impact of the halogen substitution on the corresponding bacterial methyltransferase (M.HpaII). We observed that MspI cleavage is only subtly affected by substituted cytosine analogues at the inner position of the CCGG recognition site. In contrast, HpaII cleaves cytosine-containing oligonucleotides completely whereas 5-fluorocytosine-containing oligonucleotides are cleaved at a reduced rate. The presence of the larger halogens Cl, Br, or I as well as a methyl group completely prevents cleavage by HpaII. These data suggest that the steric wall is encountered by HpaII slightly beyond the fluorine substituent, at about 2.65 A from the pyrimidine C5-position. It is known that 5-fluorocytosine in an oligonucleotide can form a covalent irreversible suicide complex with either prokaryotic or eukaryotic methyltransferases. Kinetic data reported here suggest that the 5-fluorocytosine-containing oligonucleotide can also inhibit M.HpaII by formation of a reversible, noncovalent complex. Our results indicate that although a 5-Cl substituent has electronic properties similar to 5-F, 5-chlorocytosine duplexes neither form a complex with M.HpaII nor inhibit enzymatic methylation. Emerging data suggest that halogenation of cytosine can occur in DNA in vivo from inflammation-mediated reactive molecules. The results reported here suggest that the inadvertent halogenation of cytosine residues in DNA could alter the affinity of sequence-specific DNA-binding proteins.