AMPLIFIKASI DAN KARAKTERISASI FRAGMEN 618 PASANG BASA DNA LOKUS

• Main Author: Mulyani, Sri
• Format: Theses
• Language: Indonesia
• Online Access: https://digilib.itb.ac.id/gdl/view/2865
• Institution: Institut Teknologi Bandung

<b>ABSTRACT:</b><br> <br /> <br /> <br /> <br /> Salmonella tyhimurium is a bacterial pathogen capable of causing a typhoid-like disease in mice which has served as a model system for investigations on human typhoid caused by Salmonella typhi. Presently, molecular mechanism of S. typhi pathogenesis is still an interesting study, because not all the genes causing the pathogenity have been known. There was a presumption that the molecular mechanism of pathogenesis is related to genes that were expressed in vivo but were not expressed in vitro. Employing In Vivo Expression Technology (WET), Mekalanos group discovered five ivi (in vivo-induced) genes of Salmonella typhimurium. One of them, has homology with the nucleotide sequence of carAB operon of E. coli K-12 (GenBank version 72), whose genes encode the two subunits of carbacmoyl phosphate synthetase.</p> In S. typhimurium, this enzyme is encoded by the genes in the pyrA locus. Information about nucleotide sequence of the pyrA locus is very important for investigating the S. typhi genes that are expressed only in vivo. This research was aimed to amplify a 618 bp fragment of S. typhimurium pyrA locus by PCR technology using CA-1 primer and CA-2 primer which were designed based on the nucleotide sequence of E. coli K12 carAB operon. Then the PCR products were cloned into the pMOSBlue T-vector, followed by sequencing of the cloned fragment employing dideoxy-Sanger method. We obtained 582 nucleotide sequence of the 618 bp fragment of S. typhimurium pyrA locus.</p> The first twenty nucleotides were a CA-1 primer molecule, and the remaining 562 nucleotides were compared with the nucleotide sequence of E. coli K12 carAB operon (GenBank version 72) and S. typhimurium LT2 carAB operon (GenBank version 90). The comparison of the obtained sequence with E. coil K12 carAB operon showed that 496 nucleotides were homologous, 5 nucleotides were ambiguous, and 61 nucleotides were not homologous (transitions: 42; transversions: 19). The comparison of the obtained sequence with S. typhimurium LT2 carAB operon showed that 554 nucleotides were homologous, 5 nucleotides were ambiguous, and 3 nucleotides were not homologous (transitions: 2; transversion: 1).