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Abstract: Karyotype, mapping of nucleolar and 5S rRNA genes and distribution of constitutive heterochromatin supposedly AT-rich were characterized on two isolate populations of Salminus brasiliensis, the biggest characid fish, and three population of Salminus hilarii. The diploid number 2n =50 and the karyotype formulae (10M+20SM+20ST/A) were the same to Salminus species studied. The position of 18S rDNA cluster identified by FISH coincide with chromomycin A3 labeling (CMA+) in the long arm telomeric portion of sixth pair. Subtle differences for the disposal of the 5S rRNA gene in the chromosome of the Salminus are presented. The distribution of the constitutive heterochromatins and DA/DAPI+ bands are described. [Copyright &y& Elsevier]

Abstract: The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502Da. The enzymatic C24-methylation gave a K mapp of 38μM and k catapp of 0.14min−1. Quite unexpectedly, “plant” cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-13C]- or [24-2H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of 1H and 13CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Δ24(25)-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (K i 14nM) and 26,27-dehydrolanosterol (K i 54μM and k inact of 0.24min−1) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC50, 30nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C1-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT. [Copyright &y& Elsevier]