968 resultados para DNA Restriction Enzymes


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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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The Bola-DRB3 gene participates in the development of the immune response and is highly polymorphic. For these reasons, it has been a candidate gene in studies of the genetic basis of disease resistance and in population genetic analysis. South American native cattle breeds have been widely replaced by improved exotic breeds leading to a loss of genetic resources. In particular South American native breeds have high levels of fertility and disease resistance. This work describes genetic variability in the BoLA-DRB3 gene in native (Caracu, Pantaneiro, Argentinean Creole) and exotic (Holstein, Jersey, Nelore, Gir) cattle breeds in Brazil and Argentina. PCR-RFLP alleles were identified by combining the restriction patterns for the BoLA-DRB3.2 locus obtained with RsaI, BstY, and HaeIII restriction enzymes. Allelic frequencies and deviations from the Hardy-Weinberg equilibrium were also calculated. Analysis of the 24 BoLA-DRB3 PCR-RFLP alleles identified showed differences in the allele distributions among breeds.

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Despite advances in vaccine development and therapy, bacterial meningitis (BM) remains a major cause of death and long-term neurological disabilities. As part of the host inflammatory response to the invading pathogen, factors such as reactive oxygen species are generated, which may damage DNA and trigger the overactivation of DNA repair mechanisms. It is conceivable that the individual susceptibility and outcome of BM may be in part determined by non synonymous polymorphisms that may alter the function of crucial BER DNA repair enzymes as PARP-1, OGG-1 and APE-1. These enzymes, in addition to their important DNA repair function, also perform role of inflammatory regulators. In this work was investigated the non synonymous SNPs APE-1 Asn148Glu, OGG-1 Ser326Cys,PARP-1 Val762Ala, PARP-1 Pro882Leu and PARP-1 Cys908Tyr in patients with bacterial meningitis (BM), chronic meningitis (CM), aseptic meningitis (AM) and not infected (controls). As results we found increased frequency of variant alleles of PARP-1 Val762Ala (P = 0.005) and APE-1 Asn148Glu (P=0.018) in BM patients, APE-1 Asn148Glu in AM patients (P = 0.012) and decrease in the frequency of the variant allele OGG-1 Ser326Cys in patients with CM (P = 0.013), regarding the allelic frequencies in the controls. A major incidence of individuals heterozygous and/ or polymorphic homozygous in BM for PARP-1 Val762Ala (P= 0.0399, OD 4.2, 95% IC 1.213 -14.545) and PARP-1 Val762Ala/ APE-1 Asn148Glu (P = 0.0238, OD 11.111, 95% IC 1.274 - 96.914) was observed related to what was expected in a not infected population. It was also observed a major incidence of combined SNPs in the BM patients compared with the control group (P=0.0281), giving evidences that SNPs can cause some susceptibility to the disease. This combined effect of SNPs seems to regulate the principal cytokines and other factors related to BM inflammatory response and point the importance of DNA repair not only to repair activity when DNA is damaged, but to others essential functions to human organism balance.

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The objective of this study was to identify DNA polymorphisms at the genes leptin, β-lactoglobulin and pituitary-specific transcription factor in three genetic groups of Holstein x Guzerat dairy cows and investigate the relationship between their genotypes and the composition and quality of milk of dairy cows. Samples were collected in August 2009, being 113 blood samples from lactating crossbred cows and 58 milk samples. For analysis of DNA polymorphisms blood samples were collected, analyzed later in the Genetic Laboratory affiliated to the Zootechny Institute of São Paulo and individual milk samples were collected according to standards established by the laboratory of Management Program of Northeast Dairy Herds (PROGEN), at Federal Rural University of Pernambuco (UFRPE) for analysis of milk composition and quality. The characterization of genotypes was performed by PCR-RFLP, for which were designed specific primers for each studied gene and restriction enzymes Kpn2I, HaeIII and HinfI that cut the DNA of the following genes: leptin, β-lactoglobulin and a PIT, respectively. The leptin estimate genotypic frequence were CC 0.112, TT 0.225 and CT 0.661, for β-lactoglobulin were AA 0.136, AB 0.323 and BB 0.539, and for PIT were ++ 0.655, -- 0.311 and +- 0.032. The results show that the population is in Hardy-Weinberg disequilibrium for leptin, β-lactoglobulin and a PIT due to excess of heterozygotes in the population, however, as these genes are associated with the milk production it is considered that the animals have genetic potential for milk production in the Brazilian semi-arid conditions. Through the characterization of the studied herd there were not found implications of the polymorphism of leptin, β-lactoglobulin and PIT in the composition and quality of milk from cows in the different genetic groups 1/2, 3/4 and 7/8 Holstein x Guzerat. Key words: β-lactoglobulin, crossbred cows, leptin, PCR-RFLP, PIT1, semi-arid.

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Haplotypes linked to the βS gene represent patterns of DNA polymorphisms along chromosome 11 of individuals bearing the βS gene. Analysis of haplotypes, in addition to serving as an important source for anthropological studies about the ethnic origin of a population, contributes to a better understanding of the variations in clinical severity of sickle cell anemia. The aim of the present study was to determine βS gene haplotypes in a group of patients with sickle cell anemia treated at the Dalton Barbosa Cunha Hematology Center (Hemonorte) in Natal, Brazil and the Oncology and Hematology Center in Mossoró, Brazil. Blood samples were obtained from 53 non-related patients (27 males and 26 females), aged between 3 months and 61 years (mean age: 16.9 ± 12.1 years). Laboratory analyses consisted of the following: erythrogram, reticulocyte count, hemoglobin electrophoresis at alkaline pH, measurement of hemoglobin A2 and Fetal hemoglobin, solubility test and molecular analysis to determine βS gene haplotypes. DNA samples were extracted by illustra blood genomicPrep Mini Spin kit and βS gene haplotypes were determined by PCR-RFLP, using Xmn I, Hind III, Hinc II and Hinf I restriction enzymes for analysis of six polymorphic restriction sites in the beta cluster. Of 106 βS chromosomes studied, 75.5% were Central African Republic (CAR) haplotype, 11.3% Benin (BEN) and 6.6% Cameroon (CAM). The atypical haplotypes had a frequency of 6.6%. More than half the patients (58.5%) were identified as CAR/CAR genotype carriers, 16.9% heterozygous CAR/BEN, 13.2% CAR/CAM and 1.9% BEN/BEN. Patients with atypical haplotype in one or two chromosomes accounted for 9.5% (CAR/Atp, BEN/Atp and Atp/Atp). The genotype groups showed no statistically significant difference (p < 0.05) in their laboratory parameters. This is the first study related to βS haplotypes conducted in state of Rio Grande do Norte and the higher frequency of Cameroon halotype found, compared to other Brazilian states, suggests the existence of a peculiarity of African origin

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Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

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We have determined the copy number and the presence of full-size hobo transposable elements in eight Brasilian strains of Drosophila melanogaster. Genomic DNA was digested with AvaII and XhoI restriction enzymes, respectively, and probed with a 963 bp sequence of the hobo element. Variable numbers of full-sized and defective elements were detected in all strains. The range of the copy number was 22.13 +/- 4.52. Blots showed the presence of a 2.6 kb fragment, corresponding to the complete element, in all strains exception of one and the 1.0 kb sequence, correponding to the Th1 and Th2 repressor elements. There was neither association among copy numbers of hobo elements and latitude nor the mean annual temperatures in the original geographical region of each strain.

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The milk is an important food because it contents Conjugated Linoleic Acids (CIA). These fatty acids are synthesized in mammary gland under action of the enzyme Stearoyl CoA-Desaturase (SCD) and have showed some positive effects in human disease prevention and treatments. A variation of CLA in milk fat exists and can be partially explained by the different levels of expression of SCD. The aim was to study part of the encoding regions of SCD's gene using PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism). Genomic DNA was extracted from lactating Murrah females. After this, PCR reactions were made by using primers Z (sic) (sic) D1 that encloses exon I, II and intron I. The fragments amplified are composed by 938 pb. Then, RFLP techniques were applied in the fragments using the restriction enzymes Pst I and Sma I. The enzyme Pst I has generated fragments of 788pb and 150bp and the Sma I has generated fragments of 693pb and 245pb. All the animals showed the same migration standard for both enzymes, characterizing a genetic monomorphism for this region of SCD gene. The analysis determined that there aren't genetic differences between these animals in the studied regions by using Pst I and Sma I enzymes.

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Genetic variation within and among accessions of the genus Arachis representing sections Extranervosae, Caulorrhizae, Heteranthae, and Triseminatae was evaluated using RFLP and RAPD markers. RAPD markers revealed a higher level of genetic diversity than did RFLP markers, both within and among the species evaluated. Phenograms based on various band-matching algorithms revealed three major clusters of similarity among the sections evaluated. The first group included the species from section Extranervosae, the second group consisted of sections Triseminatae, Caulorrhizae, and Heteranthae, and the third group consisted of one accession of Arachis hypogaea, which had been included as a representative of section Arachis. The phenograms obtained from the RAPD and RFLP data were similar but not identical. Arachis pietrarellii, assayed only by RAPD, showed a high degree of genetic similarity with Arachis villosulicarpa. This observation supported the hypothesis that these two species are closely related. It was also shown that accession V 7786, previously considered to be Arachis sp. aff. pietrarellii, and assayed using both RFLPs and RAPDs, was possibly a new species from section Extranervosae, but very distinct from A. pietrarellii.

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Advances in DNA technology have created biotechnological tools that can be used in animal selection and new strategies for increasing herd productivity and quality. The objective of the present work was to associate the genotypes of leptin gene exon 2 polymorphisms with productive traits in Nellore cattle. Blood was collected from Nellore males and PCR-RFLP reactions were performed with the restriction enzymes ClaI and Kpn2I. The gene frequencies resulting from digestion by ClaI were 0.60 and 0.40 for allele A and T, respectively; the genotypic frequencies were AA = 0.20 and AT = 0.80. The gene frequencies from digestion by Kpn2I were 0.81 for allele C and 0.194 for allele T; the genotypic frequencies were CC = 0.62 and CT = 0.38. The populations in both cases were not in Hardy-Weinberg equilibrium (p > 0.05), and the TT genotype was not found. Significant associations were noted between leptin gene exon 2 polymorphisms and five productive traits in Nellore cattle: carcass fat distribution, the intensity of red muscle coloration, pH, marbling, and post-slaughter fat thickness. © 2013 Copyright Taylor and Francis Group, LLC.

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

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Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)