RESUMO
Discovered in the 1940s by Selman Waksman, the aminoglycoside antibiotic streptomycin is clinically important in the treatment of tuberculosis worldwide. However, strains of Mycobacterium tuberculosis and other pathogenic bacteria have become resistant to streptomycin. One mechanism by which this can occur is through the action of phosphotransferases that attach a phosphate group to position 6 of the streptidine ring of streptomycin, thereby inactivating it. Two such phosphotransferases are APH(6)-Ia from producer strain Streptomyces griseus, and APH(6)-Id found in animal, plant and human pathogenic isolates. Here, we report the subcloning and expression in Escherichia coli of soluble recombinant APH(6)-Ia and Id enzymes. Sequencing of aph(6)-Ia revealed a one-nucleotide disagreement with the published sequence, such that the amino acid at position 262 is an alanine instead of a serine. The sequence of aph(6)-Id is identical to that of the gene found in transposon Tn5393 of plant pathogen Erwinia amylovora. The successful expression of soluble forms of these enzymes now paves the way for experiments to study their structure and function by using site-directed mutagenesis.
Assuntos
Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Streptomyces griseus/enzimologia , Sequência de Aminoácidos , Sequência de Bases , Western Blotting , Clonagem Molecular , Primers do DNA , Elementos de DNA Transponíveis/genética , Erwinia amylovora/genética , Escherichia coli , Dados de Sequência Molecular , Estrutura Molecular , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Análise de Sequência de DNARESUMO
Three separate calmodulin (CaM) genes (I, II and III) encoding an identical CaM protein but differing in the 5'- and 3'-untranslated regions of each of the three mRNAs are present and highly conserved in all mammals (so far examined). Primers complementary to the 3'- untranslated region (3'UTR) of each of the three mRNAs occurring in human, rat and mouse were synthesized and used to amplify regions of the 3'UTR from genomic DNA isolated from cetaceans, specifically from the bottled-nosed dolphin (Tursiops truncates), the pygmy sperm whale (Kogia breviceps) and the humpback whale (Megaptera novaeangliae). Using several primers and PCR conditions, the three CaM genes were identified in all three species by this method with one exception. The sequenced regions of the 3'UTRs of the three genes of the cetaceans exhibited a high percentage identity when compared to the corresponding regions of these three CaM mRNAs isolated from humans (85-96%). These partial sequences of the 3'UTR regions and the corresponding regions for humans, rats and mice that were available from the database were aligned and a phylogenetic tree was constructed. The three CaM genes from all species showed a close phylogenetic relationship based on these 3'UTR sequences. Such high conservation of the 3'UTRs suggests a specialized and significant function for this region in mammals.