DNA sequencing in different biological models

• Grant number: 09/08210-9
• Support Opportunities: Regular Research Grants
• Start date: October 01, 2010
• End date: September 30, 2012
• Field of knowledge: Biological Sciences - Morphology - Cytology and Cell Biology
• Principal Investigator: Estela Maris Andrade Forell Bevilacqua
• Grantee: Estela Maris Andrade Forell Bevilacqua
• Host Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil

Abstract

Aiming a solidary strategy this project gathers three research groups working on different biological models to make possible DNA sequencing procedures. This measure aims: -facilitate the interaction among our groups, enabling the active cooperation and the construction of new future integrative ideas; -it favors the exploration of new possibilities what increases the inclusion of our results into the literature; -it proposes joint technical activities among our students and technicians, optimizing thus knowledge and practicability in the execution of tasks, besides, -the obvious reduction in costs. In addition, it enlarges the repertoire of personal experiences stimulating the global development of the group members at the same time in which it reduces time of independence for acquisition of technological abilities. In this context and mainly using DNA sequencing procedures, this project intend to: 1. To clone and to characterize DNA of the NADH-oxidase subunits (gp91, p22phox, p40phox, p67phox e p47phox) of the mouse trophoblast during embryo implantation, comparatively analyzing the obtained sequences with the phagocytic neutrophil/macrophage NADH-oxidase, associated with defense mechanisms. It can be of extreme value in the comprehension of the placental barrier and embryo/fetus defense (sub-project 1). 2. To characterize messengers associated with the larval metamorphosis in Rhynchosciara americana mainly focusing on the programmed cell death process that occurs in this diptere salivary gland. This procedure will include the construction of subtractive cDNA library of larval salivary glands against prepupal, and its sequencing and characterization. Gene expression will be determined and quantified by Real Time PCR in the cells of the fat body, since this organ undergoes change in shape and functionality during metamorphosis, extending until the fly adult life. Genes will be also localized in the chromosomes through in situ hybridization (subproject 2). 3. To sequence transcriptome clones from queen ovary RNA (allowing the identification of maternal messengers associated with the embryo development) and from a mix of fat body, Malpighi, nervous ganglia, hemolymph, glands and muscles of different castes (messengers associated with different biological systems such as defense, behavior and communication). The expectation of this proposal is to identify the biggest possible quantity of transcripts. Using messengers originated from different tissues and castes we can enable in a further moment, microarray, SAGE, RACE and RT-PCR experiments. This subproject also intends to construct a gene library with high molecular weight inserts cloned in BACs from genomic DNA mechanically fragmented. This library can redundantly represent the ATTA genome. Other possible application of this approach would be make possible experiments on comparative genome, the generation of a high resolution cytogenetic map from fingerprints and in situ hybridization of specific clones and, it would be of great importance for the GAP closure in large production sequencing. This high molecular weight library (BAC) and the production of theses membranes will remain available to investigators interested in this model (subproject 3). (AU)