Estrutura genômica de uma população de suínos base Landrace

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Identification of Aspergillus flavus isolates as potential biocontrol agents of aflatoxin contamination in crops

Aspergillus flavus, a haploid organism found worldwide in a variety of crops, including maize, cottonseed, almond, pistachio, and peanut, causes substantial and recurrent worldwide economic liabilities. This filamentous fungus produces aflatoxins (AFLs) B1 and B2, which are among the most carcinogenic compounds from nature, acutely hepatotoxic and immunosuppressive. Recent efforts to reduce AFL contamination in crops have focused on the use of nonaflatoxigenic A. flavus strains as biological control agents. Such agents are applied to soil to competitively exclude native AFL strains from crops and thereby reduce AFL contamination. Because the possibility of genetic recombination in A. flavus could influence the stability of biocontrol strains with the production of novel AFL phenotypes, this article assesses the diversity of vegetative compatibility reactions in isolates of A. flavus to identify heterokaryon self-incompatible (HSI) strains among nonaflatoxigenic isolates, which would be used as biological controls of AFL contamination in crops. Nitrate nonutilizing (nit) mutants were recovered from 25 A. flavus isolates, and based on vegetative complementation between nit mutants and on the microscopic examination of the number of hyphal fusions, five nonaflatoxigenic (6, 7, 9 to 11) and two nontoxigenic (8 and 12) isolates of A. flavus were phenotypically characterized as HSI. Because the number of hyphal fusions is reduced in HSI strains, impairing both heterokaryon formation and the genetic exchanges with aflatoxigenic strains, the HSI isolates characterized here, especially isolates 8 and 12, are potential agents for reducing AFL contamination in crops

Polymorphisms, linkage and mapping of four enzyme loci in the fish genus Xiphophorus (Poeciliidae).

Electrophoretic variants at four additional enzyme loci--two esterases (Est-2, Est-3), retinal lactate dehydrogenase (LDH-1) and mannose phosphate isomerase (MPI)--among three species and four subspecies of fish of the genus Xiphophorus were observed. Electrophoretic patterns in F1 hybrid heterozygotes confirmed the monomeric structures of MPI and the esterase and the tetrametric structure of LDH in these fishes. Variant alleles of all four loci displayed normal Mendelian segregation in backcross and F2 hybrids. Recombination data from backcross hybrids mapped with Haldane's mapping function indicate the four loci to be linked as Est-2--0.43--Est3--0.26--LDH-1--0.19--MPI. Significant interference was detected and apparently concentrated in the Est-3 to MPI region. No significant sex-specific differences in recombination were observed. This group (designated linkage group II) was shown to assort independently from the three loci of linkage group I (adenosine deaminase, glucose-6-phosphate dehydrogenase, and 6-phosphogluconate dehydrogenase) and from glyceraldehyde-3-phosphate dehydrogenase and two isocitrate dehydrogenase loci. Evidence for conservation of the linkage group, at least in part, in other vertebrate species is presented.

Crossover isomer bias is the primary sequence-dependent property of immobilized Holliday junctions

Recombination of genes is essential to the evolution of genetic diversity, the segregation of chromosomes during cell division, and certain DNA repair processes. The Holliday junction, a four-arm, four-strand branched DNA crossover structure, is formed as a transient intermediate during genetic recombination and repair processes in the cell. The recognition and subsequent resolution of Holliday junctions into parental or recombined products appear to be critically dependent on their three-dimensional structure. Complementary NMR and time-resolved fluorescence resonance energy transfer experiments on immobilized four-arm DNA junctions reported here indicate that the Holliday junction cannot be viewed as a static structure but rather as an equilibrium mixture of two conformational isomers. Furthermore, the distribution between the two possible crossover isomers was found to depend on the sequence in a manner that was not anticipated on the basis of previous low-resolution experiments.

High-resolution mapping and isolation of a yeast artificial chromosome contig containing fw2.2: A major fruit weight quantitative trait locus in tomato

A high-resolution physical and genetic map of a major fruit weight quantitative trait locus (QTL), fw2.2, has been constructed for a region of tomato chromosome 2. Using an F2 nearly isogenic line mapping population (3472 individuals) derived from Lycopersicon esculentum (domesticated tomato) × Lycopersicon pennellii (wild tomato), fw2.2 has been placed near TG91 and TG167, which have an interval distance of 0.13 ± 0.03 centimorgan. The physical distance between TG91 and TG167 was estimated to be ≤ 150 kb by pulsed-field gel electrophoresis of tomato DNA. A physical contig composed of six yeast artificial chromosomes (YACs) and encompassing fw2.2 was isolated. No rearrangements or chimerisms were detected within the YAC contig based on restriction fragment length polymorphism analysis using YAC-end sequences and anchored molecular markers from the high-resolution map. Based on genetic recombination events, fw2.2 could be narrowed down to a region less than 150 kb between molecular markers TG91 and HSF24 and included within two YACs: YAC264 (210 kb) and YAC355 (300 kb). This marks the first time, to our knowledge, that a QTL has been mapped with such precision and delimited to a segment of cloned DNA. The fact that the phenotypic effect of the fw2.2 QTL can be mapped to a small interval suggests that the action of this QTL is likely due to a single gene. The development of the high-resolution genetic map, in combination with the physical YAC contig, suggests that the gene responsible for this QTL and other QTLs in plants can be isolated using a positional cloning strategy. The cloning of fw2.2 will likely lead to a better understanding of the molecular biology of fruit development and to the genetic engineering of fruit size characteristics.

Branch migration during Rad51-promoted strand exchange proceeds in either direction

The Saccharomyces cerevisiae Rad51 protein is important for genetic recombination and repair of DNA double-strand breaks in vivo and can promote strand exchange between linear double-stranded DNA and circular single-stranded DNA in vitro. However, unlike Escherichia coli RecA, Rad51 requires an overhanging complementary 3′ or 5′ end to initiate strand exchange; given that fact, we previously surmised that the fully exchanged molecules resulted from branch migration in either direction depending on which type of end initiated the joint molecule. Our present experiments confirm that branch migration proceeds in either direction, the polarity depending on whether a 3′ or 5′ end initiates the joint molecules. Furthermore, heteroduplex DNA is formed rapidly, first at the overhanging end of the linear double-stranded DNA’s complementary strand and then more slowly by progressive lengthening of the heteroduplex region until strand exchange is complete. Although joint molecule formation occurs equally efficiently when initiated with a 3′ or 5′ overhanging end, branch migration proceeds more rapidly when it is initiated by an overhanging 3′ end, i.e., in the 5′ to 3′ direction with respect to the single-stranded DNA.

Evidence for clonal propagation in natural isolates of Plasmodium falciparum from Venezuela

We have analyzed 75 isolates of Plasmodium falciparum, collected in Venezuela during both the dry (November) and rainy (May–July) seasons, with a range of genetic markers including antigen genes and 14 random amplified polymorphic DNA (RAPD) primers. Thirteen P. falciparum stocks from Kenya and four other Plasmodium species are included in the analysis for comparison. Cross-hybridization shows that the 14 RAPD primers reveal 14 separate regions of the parasite's genome. The P. falciparum isolates are a monophyletic clade, significantly different from the other Plasmodium species. We identify three RAPD characters that could be useful as “tags” for rapid species identification. The Venezuelan genotypes fall into two discrete genetic subdivisions associated with either the dry or the rainy season; the isolates collected in the rainy season exhibit greater genetic diversity. There is significant linkage disequilibrium in each seasonal subsample and in the full sample. In contrast, no linkage disequilibrium is detected in the African sample. These results support the hypothesis that the population structure of P. falciparum in Venezuela, but not in Africa, is predominantly clonal. However, the impact of genetic recombination on Venezuelan P. falciparum seems higher than in parasitic species with long-term clonal evolution like Trypanosoma cruzi, the agent of Chagas' disease. The genetic structure of the Venezuelan samples is similar to that of Escherichia coli, a bacterium that propagates clonally, with occasional genetic recombination.

Historical overview: Searching for replication help in all of the rec places

For several decades, research into the mechanisms of genetic recombination proceeded without a complete understanding of its cellular function or its place in DNA metabolism. Many lines of research recently have coalesced to reveal a thorough integration of most aspects of DNA metabolism, including recombination. In bacteria, the primary function of homologous genetic recombination is the repair of stalled or collapsed replication forks. Recombinational DNA repair of replication forks is a surprisingly common process, even under normal growth conditions. The new results feature multiple pathways for repair and the involvement of many enzymatic systems. The long-recognized integration of replication and recombination in the DNA metabolism of bacteriophage T4 has moved into the spotlight with its clear mechanistic precedents. In eukaryotes, a similar integration of replication and recombination is seen in meiotic recombination as well as in the repair of replication forks and double-strand breaks generated by environmental abuse. Basic mechanisms for replication fork repair can now inform continued research into other aspects of recombination. This overview attempts to trace the history of the search for recombination function in bacteria and their bacteriophages, as well as some of the parallel paths taken in eukaryotic recombination research.

DNA strand annealing is promoted by the yeast Rad52 protein.

The Saccharomyces cerevisiae RAD52 gene plays a pivotal role in genetic recombination. Here we demonstrate that yeast Rad52 is a DNA binding protein. To show that the interaction between Rad52 and DNA is direct and not mediated by other yeast proteins and to facilitate protein purification, a recombinant expression system was developed. The recombinant protein can bind both single- and double-stranded DNA and the addition of either Mg2+ or ATP does not enhance the binding of single-stranded DNA. Furthermore, a DNA binding domain was found in the evolutionary conserved N terminus of the protein. More importantly, we show that the protein stimulates DNA annealing even in the presence of a large excess of nonhomologous DNA. Rad52-promoted annealing follows second-order kinetics and the rate is 3500-fold faster than that of the spontaneous reaction. How this annealing activity relates to the genetic phenotype associated with rad52 mutant cells is discussed.

Resolution of Holliday junctions by eukaryotic DNA topoisomerase I.

The Holliday junction, a key intermediate in both homologous and site-specific recombination, is generated by the reciprocal exchange of single strands between two DNA duplexes. Resolution of the junctions can occur in two directions with respect to flanking markers, either restoring the parental DNA configuration or generating a genetic crossover. Recombination can be regulated, in principle, by factors that influence the directionality of the resolution step. We demonstrate that the vaccinia virus DNA topoisomerase, a eukaryotic type I enzyme, catalyzes resolution of synthetic Holliday junctions in vitro. The mechanism entails concerted transesterifications at two recognition sites, 5'-CCCTT decreases, that are opposed within a partially mobile four-way junction. Cruciforms are resolved unidirectionally and with high efficiency into two linear duplexes. These findings suggest a model whereby type I topoisomerases may either promote or suppress genetic recombination in vivo.

Sex and the single cell: meiosis in yeast.

Recent studies of Saccharomyces cerevisiae have significantly advanced our understanding of the molecular mechanisms of meiotic chromosome behavior. Structural components of the synaptonemal complex have been identified and studies of mutants defective in synapsis have provided insight into the role of the synaptonemal complex in homolog pairing, genetic recombination, crossover interference, and meiotic chromosome segregation. There is compelling evidence that most or all meiotic recombination events initiate with double-strand breaks. Several intermediates in the double-strand break repair pathway have been characterized and mutants blocked at different steps in the pathway have been identified. With the application of genetic, molecular, cytological, and biochemical methods in a single organism, we can expect an increasingly comprehensive and unified view of the meiotic process.

RuvC protein resolves Holliday junctions via cleavage of the continuous (noncrossover) strands.

The RuvC protein of Escherichia coli resolves Holliday junctions during genetic recombination and the postreplicational repair of DNA damage. Using synthetic Holliday junctions that are constrained to adopt defined isomeric configurations, we show that resolution occurs by symmetric cleavage of the continuous (noncrossing) pair of DNA strands. This result contrasts with that observed with phage T4 endonuclease VII, which cleaves the pair of crossing strands. In the presence of RuvC, the pair of continuous strands (i.e., the target strands for cleavage) exhibit a hypersensitivity to hydroxyl radicals. These results indicate that the continuous strands are distorted within the RuvC/Holliday junction complex and that RuvC-mediated resolution events require protein-directed structural changes to the four-way junction.

Análise de metabólitos e proteínas totais em folhas de Eucalyptus grandis durante a infecção por Puccinia psidii

Os mecanismos moleculares envolvidos na resistência de plantas contra patógenos são um tema bastante discutido no meio acadêmico, sendo o objetivo maior dos estudos a diminuição das perdas de produtividade provocadas por doenças em plantações do mundo todo. Muitos modelos de interação patógeno-hospedeiro foram propostos e desenvolvidos priorizando plantas e culturas de rápido desenvolvimento com ciclo de vida curto. Espécies de ciclo longo, porém, devem lidar durante anos - ao menos até a idade reprodutiva - contra o ataque de bactérias, fungos e vírus, sem contar, nesse meio tempo, com recombinações genéticas e mutações que tornariam possível o escape contra as moléstias causadas por microrganismos. Assim, como alternativa aos modelos usuais, o presente trabalho estudou um diferente par de antagonistas: Eucalyptus grandis e Puccinia psidii. Apesar da contribuição de programas de melhoramento genético, o patossistema E. grandis X P. psidii ainda é pouco descrito no nível molecular, havendo poucos estudos sobre os processos e as moléculas que agem de forma a conferir resistência às plantas. Assim, buscando o melhor entendimento da relação entre E. grandis X P. psidii, o presente trabalho estudou a mudança dos perfis de proteínas e metabólitos secundários ocorrida nos tecidos foliares de plantas resistentes e susceptíveis durante a infecção pelo patógeno, com o auxílio da técnica de cromatografia líquida acoplada à espectrometria de massas. Os resultados obtidos indicam que as plantas resistentes percebem a presença do patógeno logo nas primeiras horas pós-infecção, produzindo proteínas ligadas à imunidade (HSP90, ILITYHIA, LRR Kinase, NB-ARC disease resistance protein). Essa percepção desencadeia a produção de proteínas de parede celular e de resposta oxidativa, além de modificar o metabolismo primário e secundário. As plantas susceptíveis, por outro lado, têm o metabolismo subvertido, produzindo proteínas responsáveis pelo afrouxamento da parede celular, beneficiando a absorção de nutrientes, crescimento e propagação de P. psidii. No trabalho também são propostos metabólitos biomarcadores de resistência, moléculas biomarcadoras de resposta imune e sinais da infecção por patógeno em E. grandis.

Gene therapy for human patients : information for the general public.

Mode of access: Internet.

Identification of two evolutionarily conserved and functional regulatory elements in intron 2 of the human BRCA1 gene

Cross-species comparative genomics is a powerful strategy for identifying functional regulatory elements within noncoding DNA. In this paper, comparative analysis of human and mouse intronic sequences in the breast cancer susceptibility gene (BRCA1) revealed two evolutionarily conserved noncoding sequences (CNS) in intron 2, 5 kb downstream of the core BRCA1 promoter. The functionality of these elements was examined using homologous-recombination-based mutagenesis of reporter gene-tagged cosmids incorporating these regions and flanking sequences from the BRCA1 locus. This showed that CNS-1 and CNS-2 have differential transcriptional regulatory activity in epithelial cell lines. Mutation of CNS-1 significantly reduced reporter gene expression to 30% of control levels. Conversely mutation of CNS-2 increased expression to 200% of control levels. Regulation is at the level of transcription and shows promoter specificity. Both elements also specifically bind nuclear proteins in vitro. These studies demonstrate that the combination of comparative genomics and functional analysis is a successful strategy to identify novel regulatory elements and provide the first direct evidence that conserved noncoding sequences in BRCA1 regulate gene expression. (c) 2005 Elsevier Inc. All rights reserved.