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Production of transgenic lines of roughest in Drosophila melanogaster and its phenotypic characterization

Grant number: 16/24547-7
Support type:Scholarships in Brazil - Scientific Initiation
Effective date (Start): March 01, 2017
Effective date (End): February 28, 2018
Field of knowledge:Biological Sciences - Morphology - Cytology and Cell Biology
Principal Investigator:Ricardo Guelerman Pinheiro Ramos
Grantee:Vitor Santana Soares Rodrigues
Home Institution: Faculdade de Medicina de Ribeirão Preto (FMRP). Universidade de São Paulo (USP). Ribeirão Preto , SP, Brazil

Abstract

The roughest (rst) gene has a pleiotropic character, and its involvement is described in several processes of the Drosophila melanogaster development, such as axonal targeting, salivary gland histolysis, embryonic musculature formation and differentiation of pigmentary cells during the development process of the compound eye. The protein encoded by this locus is transmembrane, unipasso, with five immunoglobulin domains in the extracellular portion (509 amino acids) and one cytoplasmic tail (208 amino acids) containing several phosphorylable serine and threonine residues and with functionally important subdomains - as a region rich in glutamines known as the opa-like domain. In our laboratory transgenic lines of D. melanogaster containing truncated portions of Roughest under control of heat shock promoters (hsp70) and Glass-multimer-reporter (expression restricted to the compound eye) were constructed. It was observed that the overexpression of constructs containing only the extracellular domain of the protein led to defects in histolysis of the larval salivary gland and the compound eye, block in the cellularisation of blastoderm and interference in the midline embryonic formation of the central nervous system. However, generalized expression and interference in the integral development process, in the case of hsp70 promoter-mediated expression, and GMR promoter expression specificity, hinder a finer dissection of the role of different portions of the protein in the development of the insect. In order to circumvent this problem and to elucidate the effects produced by truncated versions of the intracellular domain of Rst in Drosophila melanogaster tissues, the UAS / Gal4 bipartite expression system (Ptashne, 1988) will be used: a cDNA containing the complete rst ORF (CDNA-HB3, Ramos et al., 1993) will be digested to obtain different coding regions of specific portions of the protein, and subsequently cloned into pUAST vector, for expression under UAS promoter control. After this, microinjections will be performed on Drosophila melanogaster embryos, to obtain transformant animals. Initial analyzes of the development of different tissues, such as adult ovary and larval salivary gland, will then be performed under the effect of tissue-specific overexpression (mediated by a driver gene linked to the expression of the UAS activator, GAL4) from the Roughest portions. (AU)