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Functional and molecular alterations of proteins of the erythrocyte membrane in hereditary spherocytosis and cloning of the band 3 gene in the Caiman crocodilus yacare (Pantanal crocodile) reptile


The aim of this project is to investigate molecular alterations and the gene regulation of band 3, ankirin and b-spectrin in hereditary spherocytosis in about 50 patients, in whom the protein characterization was already realized. This study will be put into practice through the searching of mutations and polymorphisms in all of the gene mentioned, including the promoter region of the band 3 and ankirin gene, as both could be involved in the physiopathology of the defect. For the detection of the molecular alterations SSCP and sequencing techniques will be used. The transcription will be evaluated by RT-PCR. There is a great possibility of our detecting new mutation in the promoters of ankirin or band 3 and, in this case, we will perform experiences in vitro to characterize the effects of the mutation in the promoter activity using genetic construction and luciferase experiments, and in the promoter link with transcription factors, through studies on DNA mobility in polyacrilamide gel and footprinting. The investigation of the renal alterations of patients with hereditary spherocytosis and the deficiency of band 3, is also the aim of this study, as this protein is also expressed in intercalated cells of the distal tube and the collecting duct, responsible for the reabsorption of bicarbonate and acid secretion. it should be pointed out that, both erythrocyte and renal band 3 are isoforms of the same gene that due to an alternative promoter in intron 3, differ in the transcription, in the kidney, of a mrna, with an absence of the three first exons. The renal studies are performed by urinary acidification tests and by the obtention of bicarbonature measures, urinary pH, tritratable acidity, and basal amonia excretion and after stimulation with furosemide. We will also study the countertransport of sodium and lithium in red cells of patients with hereditary spherocytosis, as this study is an indirect measure of the Na+/H+ countertransport, also involved in the erythrocyte acid-basic homeostasis and possibly renal. Finally, we will perform the cloning of band 3 cDNA of the reptile Caiman crocodillus yacare with the objective of obtaining the amino-acid sequence, as the acid-basic homeostasis of these species differ from man s, mainly in respect to the Cl-/HCO3- Exchange. (AU)

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