Produced DNA Fragments Research Articles

Whole genome phylogeny for 21Drosophilaspecies using predicted 2b-RAD fragments

Type IIB restriction endonucleases are site-specific endonucleases that cut both strands of double-stranded DNA upstream and downstream of their recognition sequences. These restriction enzymes have recognition sequences that are generally interrupted and range from 5 to 7 bases long. They produce DNA fragments which are uniformly small, ranging from 21 to 33 base pairs in length (without cohesive ends). The fragments are generated from throughout the entire length of a genomic DNA providing an excellent fractional representation of the genome. In this study we simulated restriction enzyme digestions on 21 sequenced genomes of various Drosophila species using the predicted targets of 16 Type IIB restriction enzymes to effectively produce a large and arbitrary selection of loci from these genomes. The fragments were then used to compare organisms and to calculate the distance between genomes in pair-wise combination by counting the number of shared fragments between the two genomes. Phylogenetic trees were then generated for each enzyme using this distance measure and the consensus was calculated. The consensus tree obtained agrees well with the currently accepted tree for the Drosophila species. We conclude that multi-locus sub-genomic representation combined with next generation sequencing, especially for individuals and species without previous genome characterization, can accelerate studies of comparative genomics and the building of accurate phylogenetic trees.

Cloning and Expression of Serotype-2 Simian Betaretrovirus Reverse Transcriptase Gene Isolated from Indonesian Cynomolgus Monkey in Escherichia coli

In this study, we isolated the simian betaretrovirus serotype-2 (SRV-2) reverse transcriptase (RT) gene from infected Indonesian cynomolgus monkey (Macaca fascicularis). The gene was then cloned in Escherichia coli expression system. The SRV-2 RT gene is located between nucleotides 3284-4925 in the polyprotein (Pol) region encodes 547 amino acids. Analysis of expression using SDS-PAGE and western blot techniques showed a specific band of 64.9 kDa, indicating SRV-2 RT recombinant enzyme. Purification of SRV-2 RT recombinant enzyme produced 312 μg mL-1 protein with 7.1 U μL-1 enzyme activities. Application of this recombinant enzyme in reverse transcription-PCR (RT-PCR) of β-globin and β-actin genes produced DNA fragments of 206 and 350 bp, indicating amplification of β-globin and β-actin genes, respectively. Therefore, the expressed SRV-2 RT enzyme was proven to be functional, although the activity was low.

VALIDATION OF PCR-RFLP TESTING METHOD TO DETECT PORCINE CONTAMINATION IN CHICKEN NUGGET

PCR-RFLP technique to detect porcine contamination in chicken nugget has been developed and validated in this research. Various concentrations of pork were fortified during preparation of the nugget. DNA was then isolated from the nugget followed by PCR employed primers which targeted a 359 bp cytB gene fragment of mitochondrial DNA. For RFLP, the PCR product was digested by means of BamHI and BseDI enzymes. Cutting DNA fragments from nugget containing pork using BseDI enzyme produced DNA fragment with size 228 and 131 bp, while cutting with BamHI enzyme produce DNA fragments with sizes 244 and 115 bp. All of these fragments were not present in RFLP analysis of pork-free nugget. The method shows good specificity and precision and could detect porcine contamination in the nugget up to 5%. The method has been applied to test commercial nugget. Four brand of Halal-labeled commercial nugget as well as four brand of non labeled one gave negative porcine contamination.

Confirmation of the presence of the Citrus leprosis virus C (CiLV-C) in Southern Mexico

Citrus leprosis was detected in sweet oranges in Chiapas, Mexico, in 2005 based on symptoms. The disease was soon after observed in sweet orange orchards at Huimanguillo and Cunduacan, state of Tabasco. Leaf samples of leprosis-affected Valencia or Hamlin sweet oranges were collected and subjected to ultrastructural examination and molecular detection of CiLV-C by RT-PCR. Cytopathic effects typical of CiLV-C infection as presence of short, baciliform particles in the endoplasmic reticulum cisternae and electron dense, vacuolated viroplasma in the cytoplasm, as well as hypertrophy of some spongy parenchyma cells were observed in the tissues of the leaf lesions. RT-PCR using specific primers for CiLV-C produced DNA fragments of the expected size. Brevipalpus mites were present in these orchards and identified as B. phoenicis, the known vector for CiLV-C. Transmission tests with these mites confirmed their role as leprosis vectors to sweet orange but not to lemon, Volkamerian lemon, C35 citrange and Carrizo citrange. These data confirm the presence of CiLV-C in Tabasco, Mexico and that the citrus leprosis found in Chiapas was caused by CiLV-C.

Combination of cytochrome b heteroduplex-assay and sequencing for identification of triatomine blood meals

The identification of blood meals in vectors contributes greatly to the understanding of interactions between vectors, microorganisms and hosts. The aim of the current work was to complement the validation of cytochrome b (Cytb) heteroduplex assay (HDA) previously described, and to add the sequencing of the Cytb gene of some samples for the identification of blood meals in triatomines. Experimental feedings of reared triatomines helped to clarify the sensitivity of the HDA. Moreover, the sequencing coupled with the HDA, allowed the assessment of the technique’s taxonomic level of discrimination. The primers used to produce DNA fragments of Cytb genes for HDA had a very high sensitivity for vertebrate DNAs, rather similar for mammals, birds and reptiles. However, the formation of heteroduplex depended on blood meal’s quality rather than its quantity; a correlation was observed between blood meals’ color and the positivity of HDA. HDA electrophoresis profiles were reproducible, and allowed the discrimination of blood origins at the species level. However, in some cases, intraspecific variability of Cytb gene generated different HDA profiles. The HDA based on comparison of electrophoresis profiles is a very useful tool for screening large samples to determine blood origins; the subsequent sequencing of PCR products of Cytb corresponding to different HDA profiles allowed the identification of species whatever the biotope in which the vectors were captured.

Genotoxicity of the copper antineoplastic coordination complexes casiopeinas®

Casiopeinas is the generic name of a group of coordination complexes with a central copper atom bound to organic ligands, designed to be an alternative to cancer therapy. Indeed, some of these compounds can reduce implanted tumors in mice. Casiopeinas were expressly designed to interact with the genetic material, so the aim of the present work is to determine if these compounds have genotoxic activity. The results indicate that casiopeinas produce DNA fragmentation and base oxidation and suggest that their mode of action is related to reactive oxygen species (ROS) generation after copper reduction.

Capsaicin induces apoptosis in SCC‐4 human tongue cancer cells through mitochondria‐dependent and ‐independent pathways

Although there have been advances in the fields of surgery, radiotherapy, and chemotherapy of tongue cancer, the cure rates are still not substantially satisfactory. Capsaicin (trans-8-methyl-N-vanillyl-6-nonenamide) is the major pungent ingredient of hot chili pepper and has been reported to have an antitumor effect on many human cancer cell types. The molecular mechanisms of the antitumor effect of capsaicin are not yet completely understood. Herein, we investigated whether capsaicin induces apoptosis in human tongue cancer cells. Capsaicin decreased the percentage of viable cells in a dose-dependent manner in human tongue cancer SCC-4 cells. In addition, capsaicin produced DNA fragmentation, decreased the DNA contents (sub-G1 phase), and induced G0/G1 phase arrest in SCC-4 cells. We demonstrated that capsaicin-induced apoptosis is associated with an increase in reactive oxygen species and Ca²⁺ generations and a disruption of the mitochondrial transmenbrane potential (ΔΨ(m)). Treatment with capsaicin induced a dramatic increase in caspase-3 and -9 activities, as assessed by flow cytometric methods. A possible mechanism of capsaicin-induced apoptosis is involved in the activation of caspase-3 (one of the apoptosis-executing enzyme). Confocal laser microscope examination also showed that capsaicin induced the releases of AIF, ATF-4, and GADD153 from mitochondria of SCC-4 cells.

A Highly Toxic Cellular Prion Protein Induces a Novel, Nonapoptotic Form of Neuronal Death

Several different deletions within the N-terminal tail of the prion protein (PrP) induce massive neuronal death when expressed in transgenic mice. This toxicity is dose-dependently suppressed by coexpression of full-length PrP, suggesting that it results from subversion of a normal physiological activity of cellular PrP. We performed a combined biochemical and morphological analysis of Tg(DeltaCR) mice, which express PrP carrying a 21-aa deletion (residues 105-125) within a highly conserved region of the protein. Death of cerebellar granule neurons in Tg(DeltaCR) mice is not accompanied by activation of either caspase-3 or caspase-8 or by increased levels of the autophagy marker, LC3-II. In electron micrographs, degenerating granule neurons displayed a unique morphology characterized by heterogeneous condensation of the nuclear matrix without formation of discrete chromatin masses typical of neuronal apoptosis. Our data demonstrate that perturbations in PrP functional activity induce a novel, nonapoptotic, nonautophagic form of neuronal death whose morphological features are reminiscent of those associated with excitotoxic stress.

In vitro evaluation of baseline and induced DNA damage in human sperm exposed to benzo[a]pyrene or its metabolite benzo[a]pyrene-7,8-diol-9,10-epoxide, using the comet assay

Exposure to genotoxins may compromise DNA integrity in male reproductive cells, putting future progeny at risk for developmental defects and diseases. To study the usefulness of sperm DNA damage as a biomarker for genotoxic exposure, we have investigated cellular and molecular changes induced by benzo[a]pyrene (B[a]P) in human sperm in vitro, and results have been compared for smokers and non-smokers. Sperm DNA obtained from five smokers was indeed more fragmented than sperm of six non-smokers (mean % Tail DNA 26.5 and 48.8, respectively), as assessed by the alkaline comet assay (P < 0.05). B[a]P-related DNA adducts were detected at increased levels in smokers as determined by immunostaining. Direct exposure of mature sperm cells to B[a]P (10 or 25 μM) caused moderate increases in DNA fragmentation which was independent of addition of human liver S9 mix for enzymatic activation of B[a]P, suggesting some unknown metabolism of B[a]P in ejaculates. In vitro exposure of samples to various doses of B[a]P (with or without S9) did not reveal any significant differences in sensitivity to DNA fragmentation between smokers and non-smokers. Incubations with the proximate metabolite benzo[a]pyrene-r-7,t-8-dihydrodiol-t9,10-epoxide (BPDE) produced DNA fragmentation in a dose-dependent manner (20 or 50 μM), but only when formamidopyrimidine DNA glycosylase treatment was included in the comet assay. These levels of DNA fragmentation were, however, low in relation to very high amounts of BPDE–DNA adducts as measured with 32P postlabelling. We conclude that sperm DNA damage may be useful as a biomarker of direct exposure of sperm using the comet assay adapted to sperm, and as such the method may be applicable to cohort studies. Although the sensitivity is relatively low, DNA damage induced in earlier stages of spermatogenesis may be detected with higher efficiencies.

Death mechanisms in epithelial cells following rotavirus infection, exposure to inactivated rotavirus or genome transfection

Intestinal epithelial cell death following rotavirus infection is associated with villus atrophy and gastroenteritis. Roles for both apoptosis and necrosis in cytocidal activity within rotavirus-infected epithelial cells have been proposed. Additionally, inactivated rotavirus has been reported to induce diarrhoea in infant mice. We further examined the death mechanisms induced in epithelial cell lines following rotavirus infection or inactivated rotavirus exposure. Monolayer integrity changes in MA104, HT-29 and partially differentiated Caco-2 cells following inactivated rotavirus exposure or RRV or CRW-8 rotavirus infection paralleled cell metabolic activity and viability reductions. MA104 cell exposure to rotavirus dsRNA also altered monolayer integrity. Inactivated rotaviruses induced delayed cell function losses that were unrelated to apoptosis. Phosphatidylserine externalization, indicating early apoptosis, occurred in MA104 and HT-29 but not in partially differentiated Caco-2 cells by 11 h after infection. Rotavirus activation of phosphatidylinositol 3-kinase partially protected MA104 and HT-29 cells from early apoptosis. In contrast, activation of the stress-activated protein kinase JNK by rotavirus did not influence apoptosis induction in these cells. RRV infection produced DNA fragmentation, indicating late-stage apoptosis, in fully differentiated Caco-2 cells only. These studies show that the apoptosis initiation and cell death mechanism induced by rotavirus infection depend on cell type and degree of differentiation. Early stage apoptosis resulting from rotavirus infection is probably counter-balanced by virus-induced phosphatidylinositol 3-kinase activation. The ability of inactivated rotaviruses and rotavirus dsRNA to perturb monolayer integrity supports a potential role for these rotavirus components in disease pathogenesis.

CONSTRUCTION OF CHROMOSOME #1 SPECIFIC LIBRARY OF LILIUM TIGRINUM

Metaphase chromosome #1 of Lilium tigrinum was microdissected and microcloned. DOP-PCR and linker-mediated PCR were used to amplify microdissected L. tigrinum chr. #1. DOP-PCR and linker-mediated PCR yielded products ranging in size from 100 to 3000 bp, with predominant fragments at 300 to 2000 bp. When two methods were compared, linker-mediated PCR gave rise to a little bigger fragment. Southern hybridization with DIG-labeled genomic DNA confirmed that the products from both methods were amplified from the L. tigrinum genome. Cloning using size-fractionated DNA gave recombinant clones larger than 500 bp. DOP-PCR gave larger recombinant clones than linker-mediated PCR. The insert size was approximately from 300 to 2000 bp. DOP-PCR produced DNA fragments ranging in size from 400 to 2000 bp, and linker-mediated PCR from 300 to 1200 bp, with predominant fragment at 500 to 1000 bp. When randomly selected 30 clones were sequenced, only two clones from DOP-PCR showed homology with Lilium henryi or L. philadelphicum and others were identified as unknown unique sequences. On the other hand, 8 out of 15 clones from linker-mediated PCR showed homology with microsatellite region of other plant species. Based on the results obtained in this experiment, DOP-PCR was more suitable for finding unknown unique sequences than linker-mediated PCR.

Loss of the Gata1 Gene IE Exon Leads to Variant Transcript Expression and the Production of a GATA1 Protein Lacking the N-terminal Domain

GATA1 is essential for the differentiation of erythroid cells and megakaryocytes. The Gata1 gene is composed of multiple untranslated first exons and five common coding exons. The erythroid first exon (IE exon) is important for Gata1 gene expression in hematopoietic lineages. Because previous IE exon knockdown analyses resulted in embryonic lethality, less is understood about the contribution of the IE exon to adult hematopoiesis. Here, we achieved specific deletion of the floxed IE exon in adulthood using an inducible Cre expression system. In this conditional knock-out mouse line, the Gata1 mRNA level was significantly down-regulated in the megakaryocyte lineage, resulting in thrombocytopenia with a marked proliferation of megakaryocytes. By contrast, in the erythroid lineage, Gata1 mRNA was expressed abundantly utilizing alternative first exons. Especially, the IEb/c and newly identified IEd exons were transcribed at a level comparable with that of the IE exon in control mice. Surprisingly, in the IE-null mouse, these transcripts failed to produce full-length GATA1 protein, but instead yielded GATA1 lacking the N-terminal domain inefficiently. With low level expression of the short form of GATA1, IE-null mice showed severe anemia with skewed erythroid maturation. Notably, the hematological phenotypes of adult IE-null mice substantially differ from those observed in mice harboring conditional ablation of the entire Gata1 gene. The present study demonstrates that the IE exon is instrumental to adult erythropoiesis by regulating the proper level of transcription and selecting the correct transcription start site of the Gata1 gene.

First comparative phenetic studies of the polymorphic species of Coccinella undecimpunctata Linnaeus, using morphometric and RAPD approaches in Egypt

In this study, numerical phenetic method and RAPD-PCR technique were carried out on the species Coccinella undecimpunctata L. and its 13 aberrations which were collected from 10 localities in Egypt. The morphometric analysis and phenotypic features were determined for the aberrations to reveal the phenetic relationships among them. The resulted phenogram showed high degree of variations and affinities (Similarity coeffeciant ranged from 44.7 to 85.7%). RAPD fingerprint profiles were generated by using 5 random primers on genomic DNA to evaluate their phenetic relationships and to investigate the molecular markers among the aberrations genotypes. The similarity coefficient of the produced DNA fragments ranged from 40% to 93.75%. Cluster analysis based on both morphometric and RAPD data showed that the 14 morphs are grouped into 9 clusters against 8 clusters respectively. In addition, PCA plot allowed differentiating three groups from morphometric data against four groups from RAPD data.

Rapid assessment of the effect of ciprofloxacin on chromosomal DNA from Escherichia coli using an in situ DNA fragmentation assay

BackgroundFluoroquinolones are extensively used antibiotics that induce DNA double-strand breaks (DSBs) by trapping DNA gyrase and topoisomerase IV on DNA. This effect is usually evaluated using biochemical or molecular procedures, but these are not effective at the single-cell level. We assessed ciprofloxacin (CIP)-induced chromosomal DNA breakage in single-cell Escherichia coli by direct visualization of the DNA fragments that diffused from the nucleoid obtained after bacterial lysis in an agarose microgel on a slide.ResultsExposing the E. coli strain TG1 to CIP starting at a minimum inhibitory concentration (MIC) of 0.012 μg/ml and at increasing doses for 40 min increased the DNA fragmentation progressively. DNA damage started to be detectable at the MIC dose. At a dose of 1 μg/ml of CIP, DNA damage was visualized clearly immediately after processing, and the DNA fragmentation increased progressively with the antibiotic incubation time. The level of DNA damage was much higher when the bacteria were taken from liquid LB broth than from solid LB agar. CIP treatment produced a progressively slower rate of DNA damage in bacteria in the stationary phase than in the exponentially growing phase. Removing the antibiotic after the 40 min incubation resulted in progressive DSB repair activity with time. The magnitude of DNA repair was inversely related to CIP dose and was noticeable after incubation with CIP at 0.1 μg/ml but scarce after 10 μg/ml. The repair activity was not strictly related to viability. Four E. coli strains with identified mechanisms of reduced sensitivity to CIP were assessed using this procedure and produced DNA fragmentation levels that were inversely related to MIC dose, except those with very high MIC dose.ConclusionThis procedure for determining DNA fragmentation is a simple and rapid test for studying and evaluating the effect of quinolones.

Role of alpha-tocopherol in counteracting DNA damage induced by Ochratoxin A in primary porcine fibroblasts

Ochratoxin A is a mycotoxin responsible for disease states in both humans and animals. OTA mechanisms of action are numerous, including lipid peroxidation. Oxidative damage results in the modification of macromolecules (i.e. DNA), cell death and tissue injure. Several strategies, such as the use of antioxidants, have been used to reduce OTA cytotoxicity. The aim of this study was to evaluate the role of alpha-toco in counteracting DNA damage induced by OTA in cell cultures. Primary porcine fibroblasts, pherol isolated from embryo and from ear, were incubated for 24h with several concentrations of OTA in order to detect DNA fragmentation. OTA produced DNA fragmentation in a concentration dependent manner in both primary cell cultures. The pre-treatment with alpha-tocopherol caused the reduction of DNA fragmentation in both primary cell cultures, after 24h of incubation with OTA. In particular, when OTA was added at 10 μg/ml in embryo fibroblasts, alpha-tocopherol at the concentrations of 1 nM was significantly (P<0.05) able to reduce DNA fragmentation by 16%. In ear fibroblast cultures, alpha-tocopherol at the 1nM concentration was significantly (P<0.05) able to reduce DNA fragmentation by 15.23% in the presence of 5 μg/ml of OTA.

Detection and Characterization of a Distinct Isolate of Tomato yellow fruit ring virus from Potato.

A distinct isolate (TY-PF36) of Tomato yellow fruit ring virus (TYFRV) was obtained from potato (Solanum tuberosum) in Iran. Chlorosis and necrotic spots on leaves associated with leaf and stem necrosis symptoms appear on the affected plants. Of 32 plant species and cultivars mechanically inoculated, 24 were susceptible to the virus isolate. The isolate strongly reacted with TYFRV antibodies in enzyme-linked immunosorbent assay (ELISA), but not with the specific antibodies of other tospoviruses tested. TYFRV-specific N gene primers described previously, however, failed to produce DNA fragments from the total RNA extracts of the infected plants in reverse transcription-polymerase chain reaction (RT-PCR). Nucleotide sequencing of the complete N gene and partial L gene of this isolate revealed considerable differences to those reported for TYFRV with identities ranging from 83.9 to 84.2% and 84.9 to 85.4%, respectively. Two specific primers were designed for detecting TY-PF36 using RT-PCR; TY-PF36 was detected in symptomatic field samples of potato, peanut, soybean, and two weed species, Datura metel and D. stramonium.

NK314, a novel topoisomerase II inhibitor, induces rapid DNA double-strand breaks and exhibits superior antitumor effects against tumors resistant to other topoisomerase II inhibitors

NK314 is a novel synthetic benzo[ c]phenanthridine alkaloid that shows strong antitumor activity. It inhibited topoisomerase II activity and stabilized topoisomerase II-DNA cleavable complexes. The DNA breaks occurred within 1 h after treatment with NK314 even without digestion of topoisomerase II by proteinase K, whereas etoposide required digestion of the enzyme protein in cleavable complex to detect DNA breaks. Pretreatment with topoisomerase II catalytic inhibitors, ICRF-193 and suramin, reduced both cleavable complex-mediated DNA breaks and proteinase K-independent DNA breaks, but protease inhibitors and nuclease inhibitors only decreased the latter. These results indicate that NK314 might affect topoisomerase II in the different manner from cleavable complex formation and activate intracellular proteinase and nuclease to produce DNA fragmentation. As a result of this unique mechanism of DNA breakage, NK314 showed substantial growth inhibition of topoisomerase II inhibitor-resistant tumors.

A crystalline compound (BM-ANF1) from the Indian toad ( Bufo melanostictus, Schneider) skin extract, induced antiproliferation and apoptosis in leukemic and hepatoma cell line involving cell cycle proteins

In our earlier communication, it was reported that Indian toad ( Bufo melanostictus) skin extract (TSE) possesses antiproliferative and apoptogenic activity in U937 and K562 cells [Giri et al., 2006. Antiproliferative, cytotoxic and apoptogenic activity of Indian toad ( Bufo melanostictus, Schneider) skin extract in U937 and K562 cells. Toxicon 48 (4), 388–400]. In the present study, a compound (BM-ANF1) has been isolated from the TSE by alumina gel column chromatography, crystallized and evaluated for its antiproliferative and apoptogenic activity in U937, K562 and HepG2 cells. BM-ANF1 produced dose-dependent inhibition of U937, K562 and HepG2 cell growth. The antiproliferative activity was reflected by the MTT assay and demonstrated by the reduced expression of proliferative cell nuclear antigen (PCNA). Flow-cytometric analysis showed that BM-ANF1 arrested the cell cycle at G1 phase and enhanced annexin-V binding in U937 and K562 cells. Scanning electron microscopic and fluorescent microscopic analysis of U937 and K562 cells revealed the apoptogenic nature of the compound. Alkaline comet assay showed that BM-ANF1 produced DNA fragmentation. The dose-dependent expression of caspase 3 indicated that the apoptogenic properties of BM-ANF1 were mediated through the activation of downstream effector nucleases in the cancer cells. The increased expression of p53 and moderate expression of p21 Cip1/p27 Kip1 due to BM-ANF1 treatment in HepG2 cells supported that the apoptogenic activity of BM-ANF1 was mediated through p53 tumor-suppressor gene expression followed by the expression of p21 Cip1 and p27 Kip1 and it was likely to be linked with cell cycle arrest at G1 phase in cancer cells. From the present study, it may be suggested that the crystalline compound, BM-ANF1, was antiproliferative and apoptogenic in human leukemic and hepatoma cells.

DNA Fragmentation Induced in Human Fibroblasts by Accelerated56Fe Ions of Differing Energies

DNA fragmentation was studied in the fragment size range 0.023-5.7 Mbp after irradiation of human fibroblasts with iron-ion beams of four different energies, i.e., 200 MeV/nucleon, 500 MeV/nucleon, 1 GeV/nucleon and 5 GeV/nucleon, with gamma rays used as the reference radiation. The double-strand break (DSB) yield (and thus the RBE for DNA DSB induction) of the four iron-ion beams, which have LETs ranging from 135 to 442 keV/mum, does not vary greatly as a function of LET. As a consequence, the variation of the cross section for DSB induction mainly reflects the variation in LET. However, when the fragmentation spectra were analyzed with a simple theoretical tool that we recently introduced, the results showed that spatially correlated DSBs, which are absent after gamma irradiation, increased markedly with LET for the iron-ion beams. This occurred because iron ions produce DNA fragments smaller than 0.75 Mbp with a higher probability than gamma rays (a probability that increases with LET). These sizes include those expected from fragmentation of the chromatin loops with Mbp dimensions. This result does not exclude a correlation at distances smaller than the lower size analyzed here, i.e. 23 kbp. Moreover, the DSB correlation is dependent on dose, decreasing when dose increases; this can be explained with the argument that at increasing dose there is an increasing fraction of fragments produced by DSBs caused by separate, uncorrelated tracks.

Diversidade de Phytophthora parasitica isolados de Citrus usando seqüências de nucleotídeos da região ITS-5.8S rDNA

Realizou-se estudo para caracterização e verificação da diversidade genética de Phytophthora parasitica, agente causador da gomose dos citros. Quatorze isolados de Phytophthora parasitica, provenientes do Estado de São Paulo, foram seqüenciados a partir das regiões internas transcritas (ITS1 e ITS2) do gene 5.8S. Obtiveram-se seqüências de 812 pb a 860 pb que foram comparadas com seqüências de outras espécies de Phytophthora spp depositadas no NCBI. Foram feitos estudos filogenéticos, utilizando-se o método "neighbor-joining" com 1000 "bootstrap" e construído o dendrograma mais representativo. Obtiveram-se os resultados de 98,88% a 100% de similaridade genética entre os 14 isolados paulistas, e 99,5% a 98,8% entre estes e a seqüência de P. nicotianae (gi| 8927482) obtida do GenBank NCBI.