NUSBLAT Alejandro David
congresos y reuniones científicas
Analysis of orthologs of sterol metabolism from Tetrahymena.
MARIELA L. TOMAZIC; FURLAN J; POKLEPOVICH T.J; ALEJANDRO D. NUSBLAT; NUDEL BC
Potrero de los Funes, San Luis
Congreso; SAIB - th 47 Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2011
Sociedad Argentina de Investigación en Bioquímica y Biología Molecular
Tetrahymena is a free living ciliate, that neither is auxotrophic nor synthesizes sterols de novo. Instead of sterols, it synthesizes tetrahymanol. However, the ciliate harbors orthologs of sterol metabolism, such as C-4 methyl oxidases (SMO), which are involved in C-4 demethyation process in sterol biosynthesis. Tetrahymena is able to perform sterol modifications when they are added to the culture media: C-5, C-7 and C-22 sterol desaturase and C-24 dealkylase. In order to find out whether Tetrahymena has an active SMO (SMOTt), we assayed different 4-methylsterols from plants and yeast. Wild type (WT) Tetrahymena cells were cultured with 4-methylsteros and their sterol profile was analized by GC-MS. Moreover, we have knocked-out (KO) four putative SMOTt genes and the sterol profile of the mutants was analyzed by HPLC. The preliminary results obtained suggestthat none of the substrates assayed are metabolized by WT cultures. Although phylogenetic analysis showed that orthologs from Tetrhaymena group in the same cluster that SMO, the sequence analysis the lack of some conserved motifs present in plants and yeast SMOs. On the other hand, the sterol profile of the KO mutants cultured with cholesterol or stigmasterol showed no differences with WT, indicating that the genes are clearly not belonging to the sterol desaturases nor C-24 dealkylase. However, full characterization is still necessary.