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Genic expression regulation in microorganisms


This research project aims at investigating the molecular aspects related to the control of gene expression during cell differentiation, heat shock and other stress conditions utilizing as model systems: the gram-negative bacterium Caulobacter crescentus and the chytridiomycete Blastocladiella emersonii. In Caulobacter crescentus, we intend to investigate a putative homologue of the gene hspR (CC0081), which in other bacteria encodes a repressor that controls the expression of several heat shock genes. The HspR protein represses gene expression by binding to an inverted repeat present in the regulatory region of the genes, denominated HAIR (HspR associated inverted repeat). For these studies we will construct a null mutant in gen CC0081, whose phenotype will be analyzed with respect to different kinds of environmental stresses. Another objective of this project deals with the characterization of 5 ECF (extracytoplasmic function) sigma factors from Caulobacter: SigF, SigU, SigT, SigE and SigR. Concerning SigF, which we recently showed to be involved in the response to hydrogen peroxide in stationary phase cells, we will investigate the molecular mechanisms controlling its activity. Preliminary results indicate that SigF is post-transcriptionally regulated during stationary phase. In addition, gene CC3252, which forms with SigF a probable operon, will be investigated as a possible anti-sigma factor. SigU and SigT, whose expression is induced by osmotic stress, will be better characterized, looking for Caulobacter genes that are regulated by these sigma factors. Null mutants of SigE and SigR will be obtained and their phenotypes analyzed, to uncover the role of these sigma factors in the control of gene expression in this bacterium. We also propose to characterize expressed sequence tags (ESTs) from Blastocladiella emersonii exposed to heat shock or Cadmium. In the case of exposure to Cadmium, very little is known with respect to the primary targets of this heavy metal. With the use of Blastocladiella cDNA microarrays, recently constructed in our laboratory, we intend to analyse the global gene expression of this fungus during its two differentiation phases: the sporulation and the germination. We also intend to investigate B. emersonii genes expressed under different nutritional growth conditions. One of the objectives of the project is to characterize cyclin dependent kinases (Cdks) in B. emersonii, and to investigate their expression during the sporulation and germination stages. Taking into account preliminary results, we intend to characterize two kinases with non conserved PST AIRE motif (cyclin dependent kinases non PST AIRE) found in B. emersonii. In addition, we intend to screen different B. emersonii cDNA libraries looking for sequences encoding other putative Cdks for their characterization. Mapping and sequencing of the mitochondrial genome of Blastocladiella, aiming a phylogenetic study, will be also carried out during this project. (AU)

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