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Characterisation of putative gene CDK7 and its possible role in endocycles and larval development of Rhynchosciara americana

Grant number: 09/11276-1
Support type:Scholarships in Brazil - Master
Effective date (Start): August 01, 2010
Effective date (End): April 30, 2012
Field of knowledge:Biological Sciences - Biochemistry - Molecular Biology
Principal researcher:Fábio Siviero
Grantee:Adam Arai Martens
Home Institution: Instituto de Ciências Biomédicas (ICB). Universidade de São Paulo (USP). São Paulo , SP, Brazil


This work aims to characterize a gene of a putative cyclin-dependent kinase and analyze its possible role in the endocycles present in different tissues of Rhynchosciara Americana. Interestingly, a transcript corresponding to this gene was identified in a cDNA library from salivary glands, which was constructed with mRNAs present during the period in which the last of politene replicative cycle and the onset of gene amplification of different DNA puffs. However, we identified only two CDKs (Cdk7 and Cdc2) and a cyclin a message, and its involvement in cases of endoreplication are still unclear, making the characterization and study of gene and its role extremely interesting.In order to characterize the putative gene Cdk7 is intended to clone portions 5 'and 3' of its messenger through RACE and use the cDNA as a probe to perform a screening of a genomic library constructed in bacteriophage. The genomic clone will be used for chromosomal localization of this gene by fluorescent in situ hybridization (FISH) and sequencing. The expression profile will be determined by RT-PCR in real time in the salivary gland and other tissues at different stages of development and complemented by western blots, using a specific antibody against Rhynchosciara Cdk7. Imunoblots analysis of 2D electrophoresis of cell extracts will be performed to determine other information about Cdk7, such as possible isoforms and post-translational modifications in different tissues or developmental stages.

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