GENOTYPE CHARACTERIZATION OF THE Haematobia irritans (DIPTERA: MUSCIDAE) FROM BRAZIL, DOMINICAN REPUBLIC AND COLOMBIA BASED ON RANDOMLY AMPLIFIED POLYMORPHIC DNA (RAPD) ANALYSIS

Blood-sucking flies are important parasites in animal production systems, especially regarding confinement conditions. Haematobia irritans, the horn fly, is one of the most troublesome species within bovine production systems, due to the intense stress imposed to the animals. H. irritans is one of the parasites of cattle that cause significant economic losses in many parts of the world, including South America. In the present work, Brazilian, Colombian and Dominican Republic populations of this species were studied by Random Amplified Polymorphic DNA(RAPD) to assess basically genetic variability between populations. Fifteen different decamer random primers were employed in the genomic DNA amplification, yielding 196 fragments in the three H. irritans populations. Among h. irritans samples, that from Colombia produced the smallest numbers of polymorphic hands. This high genetic homogeneity may be ascribed to its geographic origin, which causes high isolation, low gene flow, unlike the other American populations, from Brazil and Dominican Republic. Molecular marker fragments, which its produced exclusive bands, detected in every sample enabled the population origin to be characterized, but they are also potentially useful for further approaches such as the putative origin of Brazilian, Colombian and Dominican Republic populations of horn fly from South America. Similarity indices produced by chemo metric analysis showed the closest relationships between flies from Brazil and Dominican Republic, while flies from Colombia showed the greatest genotypic differentiation relative to the others populations.

An oligonucleotide primer set for PCR amplification of the complete honey bee mitochondrial genome

Mitochondrial DNA markers have been widely used to address population and evolutionary questions in the honey bee Apis mellifera. Most of the polymorphic markers are restricted to few mitochondrial regions. Here we describe a set of 24 oligonucleotides that allow PCR amplification of the entire mitochondrial genome of the honey bee A. mellifera in 12 amplicons. These fragments have important applications for the study of mitochondrial genes in different subspecies of A. mellifera and as heterospecific probes to characterize mitochondrial genomes in other bee species.

Biotype stability of Candida albicans isolates after culture storage determined by randomly amplified polymorphic DNA and phenotypical methods

P>Typing methods to evaluate isolates in relation to their phenotypical and molecular characteristics are essential in epidemiological studies. In this study, Candida albicans biotypes were determined before and after storage in order to verify their stability. Twenty C. albicans isolates were typed by Randomly Amplified Polymorphic DNA (RAPD), production of phospholipase and proteinase exoenzymes (enzymotyping) and morphotyping before and after 180 days of storage in Sabouraud dextrose agar (SDA) and sterilised distilled water. Before the storage, 19 RAPD patterns, two enzymotypes and eight morphotypes were identified. The fragment patterns obtained by RAPD, on the one hand, were not significantly altered after storage. On the other hand, the majority of the isolates changed their enzymotype and morphotype after storage. RAPD typing provided the better discriminatory index (DI) among isolates (DI = 0.995) and maintained the profile identified, thereby confirming its utility in epidemiological surveys. Based on the low reproducibility observed after storage in SDA and distilled water by morphotyping (DI = 0.853) and enzymotyping (DI = 0.521), the use of these techniques is not recommended on stored isolates.

Analysis of five polymorphic DNA markers for indirect genetic diagnosis of haemophilia A in the Brazilian population

Hemophilia A is an X-linked, inherited, bleeding disorder caused by the partial or total inactivity of the coagulation factor VIII (FVIII). Due to difficulties in the direct recognition of the disease-associated mutation in the F8 gene, indirect diagnosis using polymorphic markers located inside or close to the gene is used as an alternative for determining the segregation of the mutant gene within families and thus for detecting carrier individuals and/or assisting in prenatal diagnosis. This study characterizes the allelic and haplotype frequencies, genetic diversity, population differentiation and linkage disequilibrium of five microsatellites (F8Int1, F8Int13, F8Int22, F8Int25.3 and IKBKG) in samples of healthy individuals from Sao Paulo, Rio Grande do Sul and Pernambuco and of patients from Sao Paulo with haemophilia A to determine the degree of informativeness of these microsatellites for diagnostic purposes. The interpopulational diversity parameters highlight the differences among the analyzed population samples. Regional differences in allelic frequencies must be taken into account when conducting indirect diagnosis of haemophilia A. With the exception of IKBKG, all of the microsatellites presented high heterozygosity levels. Using the markers described, diagnosis was possible in 10 of 11 families. The F8Int22, F8Int1, F8Int13, F8Int25.3 and IKBKG microsatellites were informative in seven, six, five and two of the cases, respectively, demonstrating the effectiveness of using these microsatellites in prenatal diagnosis and in carrier identification in the Brazilian population.

Efficiency of the DNA repair and polymorphisms of the XRCC1, XRCC3 and XRCC4 DNA repair genes in systemic lupus erythematosus

Impaired DNA repair efficiency in systematic lupus erythematosus (SLE) patients has been reported ill some studies, mainly regarding the repair of oxidative damage, but little is known about repair kinetics towards primarily single-stranded DNA breaks. In the present study, we aimed to investigate: (a) the efficiency of SLE peripheral blood leucocytes in repairing DNA damage induced by ionizing radiation and (b) the association of DNA repair gene (XRCC1 Arg399Gln, XRCC3 Thr241Met and XRCC4 Ile401Thr) polymorphisms in SLE patients, considering the whole group, or stratified sub-groups according to clinical and laboratory features. A total of 163 SLE patients and 125 healthy control were studied. The kinetics of DNA strand break repair was evaluated by the comet assay, and genotyping for DNA repair genes was performed by PCR-RFLP. Compared with controls. SLE leucocytes exhibited decreased efficiency of DNA repair evaluated at 30 min following irradiation. A significant association with DNA repair gene polymorphisms was not observed for the whole group of SLE patients; however, the XRCC1Arg399Gln polymorphism was associated with the presence of anti-dsDNA antibody. The concomitance of two DNA repair polymorphic sites was associated with the presence of neuropsychiatric manifestations and antiphospholipid antibody syndrome. Taken together, these results indicated that SLE leucocytes repair less efficiently the radiation-induced DNA damage, and DNA repair polymorphic sites may predispose to the development of particular clinical and laboratory features. Lupus (2008) 17, 988-995.

Segmental amplification of MLL gene associated with high expression of AURKA and AURKB genes in a case of acute monoblastic leukemia with complex karyotype

We report a case of acute monoblastic leukemia showing a jumping translocation with the MLL gene in a 17-year-old male. Classic cytogenetic and spectral karyotyping revealed a complex karyotype, and fluorescence in situ hybridization (FISH) demonstrated amplification of the MLL gene followed by translocation to chromosomes 15q, 17q, and 19q. In addition, molecular analyses showed a high expression of AURKA and AURKB genes. It is already known that overexpression of Aurora kinases is associated with chromosomal instability and poor prognosis. The formation of jumping translocations is a rare cytogenetic event and there is evidence pointing toward preferential involvement of the heterochromatin region of donor chromosomes and the telomere ends of recipient chromosomes. Jumping translocation with the MLL gene rearrangement is an uncommon phenomenon reported in leukemia cytogenetics. (C) 2010 Elsevier Inc. All rights reserved.

Species determination of Brazilian mammals implicated in the epidemiology of rabies based on the control region of mitochondrial DNA

Identification of animals that are decomposing or have been run over or burnt and cannot be visually identified is a problem in the surveillance and control of infectious diseases. Many of these animals are wild and represent a valuable source of information for epidemiologic research as they may be carriers of an infectious agent. This article discusses the results obtained using a method for identifying mammals genetically by sequencing their mitochondrial DNA control region. Fourteen species were analyzed and identified. These included the main reservoirs and transmitters of rabies virus, namely, canids, chiroptera and primates. The results prove that this method of genetic identification is both efficient and simple and that it can be used in the surveillance of infectious diseases which includes mammals in their epidemiologic cycle, such as rabies.

Helminth Coinfection Does Not Affect Therapeutic Effect of a DNA Vaccine in Mice Harboring Tuberculosis

Background: Helminthiasis and tuberculosis (TB) coincide geographically and there is much interest in exploring how concurrent worm infections might alter immune responses against bacilli and might necessitate altered therapeutic approaches. A DNA vaccine that codifies heat shock protein Hsp65 from M. leprae (DNAhsp65) has been used in therapy during experimental tuberculosis. This study focused on the impact of the co-existence of worms and TB on the therapeutic effects of DNAhsp65. Methodology/Principal Findings: Mice were infected with Toxocara canis or with Schistosoma mansoni, followed by coinfection with M. tuberculosis and treatment with DNAhsp65. While T. canis infection did not increase vulnerability to pulmonary TB, S. mansoni enhanced susceptibility to TB as shown by higher numbers of bacteria in the lungs and spleen, which was associated with an increase in Th2 and regulatory cytokines. However, in coinfected mice, the therapeutic effect of DNAhsp65 was not abrogated, as indicated by colony forming units and analysis of histopathological changes. In vitro studies indicated that Hsp65-specific IFN-gamma production was correlated with vaccine-induced protection in coinfected mice. Moreover, in S. mansoni-coinfected mice, DNA treatment inhibited in vivo TGF-beta and IL-10 production, which could be associated with long-term protection. Conclusions/Significance: We have demonstrated that the therapeutic effects of DNAhsp65 in experimental TB infection are persistent in the presence of an unrelated Th2 immune response induced by helminth infections.

Development of a DNA-dosimeter system for monitoring the effects of solar-ultraviolet radiation

Solar radiation sustains and affects all life forms on Earth. In recent years, the increase in environmental levels of solar-UV radiation due to depletion of the stratospheric ozone layer, as a result of anthropogenic emission of destructive chemicals, has highlighted serious issues of social concern. This becomes still more dramatic in tropical and subtropical regions, where the intensity of solar radiation is higher. To better understand the impact of the harmful effects of solar-UV radiation on the DNA molecule, we developed a reliable biological monitoring system based on the exposure of plasmid DNA to artificial UV lamps and sunlight. The determination and quanti. cation of different types of UV photoproducts were performed through the use of specific DNA repair enzymes and antibodies. As expected, a significant number of CPDs and 6-4PPs was observed when the DNA-dosimeter system was exposed to increasing doses of UVB radiation. Moreover, CPDs could also be clearly detected in plasmid DNA when this system was exposed to either UVA or directly to sunlight. Interestingly, although less abundant, 6-4PPs and oxidative DNA damage were also generated after exposure to both UVA and sunlight. These results confirm the genotoxic potential of sunlight, reveal that UVA may also produce CPDs and 6-4PPs directly in naked DNA and demonstrate the applicability of a DNA-dosimeter system for monitoring the biological effects of solar-UV radiation.

Random perturbations of stochastic processes with unbounded variable length memory

We consider binary infinite order stochastic chains perturbed by a random noise. This means that at each time step, the value assumed by the chain can be randomly and independently flipped with a small fixed probability. We show that the transition probabilities of the perturbed chain are uniformly close to the corresponding transition probabilities of the original chain. As a consequence, in the case of stochastic chains with unbounded but otherwise finite variable length memory, we show that it is possible to recover the context tree of the original chain, using a suitable version of the algorithm Context, provided that the noise is small enough.

Exploring the random genesis of co-occurrence networks

Using the network random generation models from Gustedt (2009)[23], we simulate and analyze several characteristics (such as the number of components, the degree distribution and the clustering coefficient) of the generated networks. This is done for a variety of distributions (fixed value, Bernoulli, Poisson, binomial) that are used to control the parameters of the generation process. These parameters are in particular the size of newly appearing sets of objects, the number of contexts in which new elements appear initially, the number of objects that are shared with `parent` contexts, and, the time period inside which a context may serve as a parent context (aging). The results show that these models allow to fine-tune the generation process such that the graphs adopt properties as can be found in real world graphs. (C) 2011 Elsevier B.V. All rights reserved.

Heteroplasmy in Hair: Study of Mitochondrial DNA Third Hypervariable Region in Hair and Blood Samples

Mitochondrial DNA (mtDNA) analysis has proved useful for forensic identification especially in cases where nuclear DNA is not available, such as with hair evidence. Heteroplasmy, the presence of more than one type of mtDNA in one individual, is a common situation often reported in the first and second mtDNA hypervariable regions (HV1/HV2), particularly in hair samples. However, there is no data about heteroplasmy frequency in the third mtDNA hypervariable region (HV3). To investigate possible heteroplasmy hotspots, HV3 from hair and blood samples of 100 individuals were sequenced and compared. No point heteroplasmy was observed, but length heteroplasmy was, both in C-stretch and CA repeat. To observe which CA ""alleles"" were present in each tissue, PCR products were cloned and re-sequenced. However, no variation among CA alleles was observed. Regarding forensic practice, we conclude that point heteroplasmy in HV3 is not as frequent as in the HV1/HV2.

Genetic interactions of the Ashergillus nidulans atmA(ATM) homolog with different components of the DNA damage response pathway

Ataxia telangiectasia mutated (ATM) is a phosphatidyl-3-kinase-related protein kinase that functions as a central regulator of the DNA damage response in eukaryotic cells. In humans, mutations in ATM cause the devastating neurodegenerative disease ataxia telangiectasia. Previously, we characterized the homolog of ATM (AtmA) in the filamentous fungus Aspergillus nidulans. In addition to its expected role in the DNA damage response, we found that AtmA is also required for polarized hyphal growth. Here, we extended these studies by investigating which components of the DNA damage response pathway are interacting with AtmA. The AtmA(ATM) loss of function caused synthetic lethality when combined with mutation in UvsB(ATR). Our results suggest that AtmA and UvsB are interacting and they are probably partially redundant in terms of DNA damage sensing and/or repairing and polar growth. We identified and inactivated A. nidulans chkA(CHK1) and chkB(CHK2) genes. These genes are also redundantly involved in A. nidulans DNA damage response. We constructed several combinations of double mutants for Delta atmA, Delta uvsB, Delta chkA, and Delta chkB. We observed a complex genetic relationship with these mutations during the DNA replication checkpoint and DNA damage response. Finally, we observed epistatic and synergistic interactions between AtmA, and bimE(APCI), ankA(WEE1) and the cdc2-related kinase npkA, at S-phase checkpoint and in response to DNA-damaging agents.

High-Resolution Genomic Mapping Reveals Consistent Amplification of the Fibroblast Growth Factor Receptor Substrate 2 Gene in Well-Differentiated and Dedifferentiated Liposarcoma

Well-differentiated liposarcoma (WDLS) is one of the most common malignant mesenchymal tumors and dedifferentiated liposarcoma (DDLS) is a malignant tumor consisting of both WDLS and a transformed nonlipogenic sarcomatous component. Cytogenetically, WDLS is characterized by the presence of ring or giant rod chromosomes containing several amplified genes, including MDM2, TSPAN31 CDK4, and others mainly derived from chromosome bands 12q13-15. However, the 12q13-15 amplicon is large and discontinuous. The focus of this study was to identify novel critical genes that are consistently amplified in primary (nonrecurrent) WDLS and with potential relevance for future targeted therapy. Using a high-resolution (5.0 kb) ""single nucleotide polymorphism""/copy number variation microarray to screen the whole genome in a series of primary WDLS, two consistently amplified areas were found on chromosome 12: one region containing the MDM2 and CPM genes, and another region containing the FRS2 gene. Based on these findings, we further validated FRS2 amplification in both WDLS and DDLS. Fluorescence in situ hybridization confirmed FRS2 amplification in all WDLS and DDLS tested (n = 57). Real time PCR showed FRS2 mRNA transcriptional upregulation in WDLS (n = 19) and DDLS (n = 13) but not in lipoma (n = 5) and normal fat (n = 9). Immunoblotting revealed high expression levels of phospho-FRS2 at 1436 and slightly overexpression of total FRS2 protein in liposarcoma but not in normal fat or preadipocytes. Considering the critical role of FRS2 in mediating fibroblast growth factor receptor signaling, our findings indicate that FRS2 signaling should be further investigated as a potential therapeutic target for liposarcoma. (C) 2011 Wiley-Liss, Inc.

Cloning, expression and purification of a glycosylated form of the DNA-binding protein Dps from Salmonella enterica Typhimurium

Dps, found in many eubacterial and archaebacterial species, appears to protect cells from oxidative stress and/or nutrient-limited environment. Dps has been shown to accumulate during the stationary phase, to bind to DNA non-specifically, and to form a crystalline structure that compacts and protects the chromosome. Our previous results have indicated that Dps is glycosylated at least for a certain period of the bacterial cell physiology and this glycosylation is thought to be orchestrated by some factors not yet understood, explaining our difficulties in standardizing the Dps purification process. In the present work, the open reading frame of the dps gene, together with all the upstream regulatory elements, were cloned into a PCR cloning vector. As a result, the expression of dps was also controlled by the plasmid system introduced in the bacterial cell. The gene was then over-expressed regardless of the growth phase of the culture and a glycosylated fraction was purified to homogeneity by lectin-immobilized chromatography assay. Unlike the high level expression of Dps in Salmonella cells, less than 1% of the recombinant protein was purified by affinity chromatography using jacalin column. Sequencing and mass spectrometry data confirmed the identity of the dps gene and the protein, respectively. In spite of the low level of purification of the jacalin-binding Dps, this work shall aid further investigations into the mechanism of Dps glycosylation. (C) 2008 Elsevier Inc. All rights reserved.