Predictive Modeling of Loss-of-Heterozygosity Events in Yeast

During mitotic cell cycles, DNA experiences many types of endogenous and exogenous damaging agents that could potentially cause double strand breaks (DSB). In S. cerevisiae, DSBs are primarily repaired by mitotic recombination and as a result, could lead to loss-of-heterozygosity (LOH). Genetic recombination can happen in both meiosis and mitosis. While genome-wide distribution of meiotic recombination events has been intensively studied, mitotic recombination events have not been mapped unbiasedly throughout the genome until recently. Methods for selecting mitotic crossovers and mapping the positions of crossovers have recently been developed in our lab. Our current approach uses a diploid yeast strain that is heterozygous for about 55,000 SNPs, and employs SNP-Microarrays to map LOH events throughout the genome. These methods allow us to examine selected crossovers and unselected mitotic recombination events (crossover, noncrossover and BIR) at about 1 kb resolution across the genome. Using this method, we generated maps of spontaneous and UV-induced LOH events. In this study, we explore machine learning and variable selection techniques to build a predictive model for where the LOH events occur in the genome.

Randomly from the yeast genome, we simulated control tracts resembling the LOH tracts in terms of tract lengths and locations with respect to single-nucleotide-polymorphism positions. We then extracted roughly 1,100 features such as base compositions, histone modifications, presence of tandem repeats etc. and train classifiers to distinguish control tracts and LOH tracts. We found interesting features of good predictive values. We also found that with the current repertoire of features, the prediction is generally better for spontaneous LOH events than UV-induced LOH events.

Autosomal dominant retinitis pigmentosa (ADRP):localization of an ADRP gene to the long arm of chromosome 3

Members of a large pedigree of Irish origin presenting with early onset Type I autosomal dominant retinitis pigmentosa (ADRP) have been typed for D3S47 (C17), a polymorphic marker from the long arm of chromosome 3. Significant, tight linkage of ADRP to D3S47, with a lod score of 14.7 maximizing at 0.00 recombination, has been obtained, hence localizing the ADRP gene (RP1) segregating in this pedigree to 3q.

Development of techniques for testing the effect of the E1 region on adenovirus integration /

Adenoviral vectors are currently the most widely used gene therapeutic vectors, but their inability to integrate into host chromosomal DNA shortened their transgene expression and limited their use in clinical trials. In this project, we initially planned to develop a technique to test the effect of the early region 1 (E1) on adenovirus integration by comparing the integration efficiencies between an E1-deleted adenoviral vector (SubE1) and an Elcontaining vector (SubE3). However, we did not harvest any SubE3 virus, even if we repeated the transfection and successfully rescued the SubE1 virus (2/4 transfections generated viruses) and positive control virus (6/6). The failure of rescuing SubE3 could be caused by the instability of the genomic plasmid pFG173, as it had frequent intemal deletions when we were purifying It. Therefore, we developed techniques to test the effect of E1 on homologous recombination (HR) since literature suggested that adenovirus integration is initiated by HR. We attempted to silence the E1 in 293 cells by transfecting E1A/B-specific small interfering RNA (siRNA). However, no silenced phenotype was observed, even if we varied the concentrations of E1A/B siRNA (from 30 nM to 270 nM) and checked the silencing effects at different time points (48, 72, 96 h). One possible explanation would be that the E1A/B siRNA sequences are not potent enough to Induce the silenced phenotype. For evaluating HR efficiencies, an HR assay system based on bacterial transfonmatJon was designed. We constmcted two plasmids ( designated as pUC19-dl1 and pUC19-dl2) containing different defective lacZa cassettes (forming white colonies after transformation) that can generate a functional lacZa cassette (forming blue colonies) through HR after transfecting into 293 cells. The HR efficiencies would be expressed as the percentages of the blue colonies among all the colonies. Unfortunately, after transfonnation of plasmid isolated from 293 cells, no colony was found, even at a transformation efficiency of 1.8x10^ colonies/pg pUC19, suggesting the sensitivity of this system was low. To enhance the sensitivity, PCR was used. We designed a set of primers that can only amplify the recombinant plasmid fomied through HR. Therefore, the HR efficiencies among different treatments can be evaluated by the amplification results, and this system could be used to test the effect of E1 region on adenovirus integration. In addition, to our knowledge there was no previous studies using PCR/ Realtime PCR to evaluate HR efficiency, so this system also provides a PCR-based method to carry out the HR assays.

Haplotype analysis suggest common founders in carriers of the recurrent BRCA2 mutation, 3398delAAAAG, in French Canadian hereditary breast and/ovarian cancer families

Affiliation: Faculté de médicine, Université de Montréal

Identification and characterization of the transcriptional targets of the WNT/β-catenin signaling pathway in granulosa cells

Les Wnts représentent une famille de glycoprotéines de signalisation qui sont connues pour les nombreux rôles qu'ils jouent durant le développement embryonnaire et dans la cancerogénèse. Plusieurs Wnts, leurs récepteurs (Fzd) et d'autres composants des voies de signalisation des Wnt sont exprimés dans l’ovaire postnatal, et il a été démontré que l’expression de certains de ces gènes est régulée pendant le développement et l'ovulation/luteinization folliculaires. Toutefois, leurs rôles physiologiques dans l’ovaire demeurent mal définis. Pour étudier le rôle de WNT4 dans le développement folliculaire, nous avons entrepris d’identifier ses cibles transcriptionnels dans les cellules de la granulosa. Pour ce faire, nous avons employé la souris Catnbflox(ex3)/flox(ex3), chez laquelle une activation constitutive de la voie de Wnt/β-catenin a lieu suite à l’action de la recombinare Cre. Des cellules de la granulosa de ces souris ont été mises en culture et infectées avec un adenovirus pour causer la surexpression de WNT4 ou l’expression de Cre. L’ARN a alors été extrait de ces cellules et analysé par micro-puce. Les résultats ont démontré qu’une forte proportion des gènes induits par WNT4 étaient des gènes impliqués dans la réponse cellulaire au stress. Presque tous gènes induits par WNT4 ont également été induits par Cre, indiquant que WNT4 signale via la voie Wnt/β-catenin dans ces cellules. Nos résultats suggèrent donc que WNT4 favorise la survie des follicules par l’induction de gènes de réponse au stress dans les cellules de la granulosa, augmentant ainsi la résistance cellulaire à l'apoptose.

An eight-parent multiparent advanced generation inter-cross population for winter-sown wheat: creation, properties, and validation

MAGIC populations represent one of a new generation of crop genetic mapping resources combining high genetic recombination and diversity. We describe the creation and validation of an eight-parent MAGIC population consisting of 1091 F7 lines of winter-sown wheat (Triticum aestivum L.). Analyses based on genotypes from a 90,000-single nucleotide polymorphism (SNP) array find the population to be well-suited as a platform for fine-mapping quantitative trait loci (QTL) and gene isolation. Patterns of linkage disequilibrium (LD) show the population to be highly recombined; genetic marker diversity among the founders was 74% of that captured in a larger set of 64 wheat varieties, and 54% of SNPs segregating among the 64 lines also segregated among the eight founder lines. In contrast, a commonly used reference bi-parental population had only 54% of the diversity of the 64 varieties with 27% of SNPs segregating. We demonstrate the potential of this MAGIC resource by identifying a highly diagnostic marker for the morphological character "awn presence/absence" and independently validate it in an association-mapping panel. These analyses show this large, diverse, and highly recombined MAGIC population to be a powerful resource for the genetic dissection of target traits in wheat, and it is well-placed to efficiently exploit ongoing advances in phenomics and genomics. Genetic marker and trait data, together with instructions for access to seed, are available at http://www.niab.com/MAGIC/.

Effects of selective logging on the mating system and pollen dispersal of Hymenaea courbaril L. (Leguminosae) in the Eastern Brazilian Amazon as revealed by microsatellite analysis

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Tuberculose e o estudo molecular da sua epidemiologia

Systems that can distinguish epidemiologically-related Mycobacterium tuberculosis strains from unrelated ones are extremely valuable. Molecular biology techniques have allowed a great deal of information to be acquired about the infectious disease tuberculosis (TB) that was very hard or impossible to obtain by conventional epidemiology. A typing method based on bacterial DNA genome differences, known as RFLP (restriction fragment length polymorphism), is widely used to discriminate strains in the epidemiologic study of TB. However, RFLP is laborious and there is a tendency to replace it by other methods. Thus, other DNA sequences have been employed as epidemiological markers, as in Spoligotyping, a fast technique based on PCR followed by differential hybridization of amplified products. The polymorphism observed among different isolates is probably the product of strain-dependent recombination. MIRU (mycobacterial interspersed repetitive unit) typing is a reproducible and fast assay, involving the generation of genotypes based on the study of 12 loci containing VNTRs (variable-number tandem repeats) in strains of the M. tuberculosis complex. It compares strains from different geographic areas and allows the movement of individual lineages to be tracked, as in RFLP. This approach enables a greater number of isolates to be analyzed, leading to the identification of a larger number of foci of transmission within the population and thus to improved ways of slowing the progress of the disease.

Report of a chimeric origin of transposable elements in a bovine-coding gene

Despite the wide distribution of transposable elements (TEs) in mammalian genomes, part of their evolutionary significance remains to be discovered. Today there is a substantial amount of evidence showing that TEs are involved in the generation of new exons in different species. In the present study, we searched 22,805 genes and reported the occurrence of TE-cassettes in coding sequences of 542 cow genes using the RepeatMasker program. Despite the significant number (542) of genes with TE insertions in exons only 14 (2.6%) of them were translated into protein, which we characterized as chimeric genes. From these chimeric genes, only the FAST kinase domains 3 (FASTKD3) gene, present on chromosome BTA 20, is a functional gene and showed evidence of the exaptation event. The genome sequence analysis showed that the last exon coding sequence of bovine FASTKD3 is ∼85% similar to the ART2A retrotransposon sequence. In addition, comparison among FASTKD3 proteins shows that the last exon is very divergent from those of Homo sapiens, Pan troglodytes and Canis familiares. We suggest that the gene structure of bovine FASTKD3 gene could have originated by several ectopic recombinations between TE copies. Additionally, the absence of TE sequences in all other species analyzed suggests that the TE insertion is clade-specific, mainly in the ruminant lineage. ©FUNPEC-RP.

Thelytokous parthenogenesis in the fungus-gardening ant Mycocepurus smithii (Hymenoptera: Formicidae)

The general prevalence of sexual reproduction over asexual reproduction among organisms testifies to the evolutionary benefits of recombination, such as accelerated adaptation to changing environments and elimination of deleterious mutations. Documented instances of asexual reproduction in groups otherwise dominated by sexual reproduction challenge evolutionary biologists to understand the special circumstances that might confer an advantage to asexual reproductive strategies. Here we report one such instance of asexual reproduction in the ants. We present evidence for obligate thelytoky in the asexual fungus-gardening ant, Mycocepurus smithii, in which queens produce female offspring from unfertilized eggs, workers are sterile, and males appear to be completely absent. Obligate thelytoky is implicated by reproductive physiology of queens, lack of males, absence of mating behavior, and natural history observations. An obligate thelytoky hypothesis is further supported by the absence of evidence indicating sexual reproduction or genetic recombination across the species' extensive distribution range (Mexico-Argentina). Potential conflicting evidence for sexual reproduction in this species derives from three Mycocepurus males reported in the literature, previously regarded as possible males of M. smithii. However, we show here that these specimens represent males of the congeneric species M. obsoletus, and not males of M. smithii. Mycocepurus smithii is unique among ants and among eusocial Hymenoptera, in that males seem to be completely absent and only queens (and not workers) produce diploid offspring via thelytoky. Because colonies consisting only of females can be propagated consecutively in the laboratory, M. smithii could be an adequate study organism a) to test hypotheses of the population-genetic advantages and disadvantages of asexual reproduction in a social organism and b) inform kin conflict theory. For a Portuguese translation of the abstract, please see Abstract S1. © 2009 Rabeling et al.

Análise de variabilidade genética em populações segregantes de soja

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Identificação e caracterização de um gene cry recombinante de Bacillus thuringiensis var. londrina

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Estudo biotecnológico, citogenético e molecular em espécies de orquídeas endêmicas da flora brasileira

Pós-graduação em Biociências - FCLAS

Phyllosticta citricarpa: diversidade genética temporal em pomares de citrus sinensis e sensibilidade a fungicidas

Pós-graduação em Agronomia (Genética e Melhoramento de Plantas) - FCAV

Molecular study on endemic cattleya species from brazilian flora

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