[Gene disruption in Salmonella typhimurim by modified Lamda Red disruption system]

There are many techniques to knock out directed genes in bacteria, some of which have been described in Salmonella species. In this study, a combination of SOEing PCR method and the lamda Red disruption system were used to disrupt phoP gene in wild type and standard strains of Salmonella typhimurium. Three standards PCR and one fusion PCR reactions were performed to construct a linear DNA including upstream and downstream of phoP gene and Kanamycin cassette. As a template plasmid, we used pKD4 which carries kanamycin gene flanked by FRT [FLP recognition target] sites. The resulting construct was electroporated into prepared competent cells of S. typhimurium. The transformants colonies related to the standard strain appeared on the LB-Km-agar plates after incubation, but there was no colony on LB-Km-agar plates corresponding to the wild type strain. The failure in transformation of the wild type strain may be because of inflexibility of the lamda Red disruption system in this strain or its unique restriction-modification system. However, by this construct we are able to generate phoP mutant in many of the Salmonella species due to high homology of the phoP gene which exists in different species

Construction of a recombinant vector for site-directed mutagenesis in Salmonella typhimurium

Among all common techniques in site directed mutagenesis, Lambda Red recombinase system has been widely used to knock out chromosomal genes in bacteria. In this method, there is always the risk of DNA Linear digestion by host's restriction enzymes that leads to the low frequency of recombination. To overcome this, we constructed a recombinant vector to disrupt phoP gene in Salmonella typhimurium. The SOEing PCR method and restriction enzymes were used to construct the vector. The resulting plasmid, pTAAZ92, contains a Kanamycin cassette with two long homologous arms flanking of the phoP gene. After electrotransformation of the pTAAZ92 into the Salmonella typhimurium, the phoP gene is replaced by the Kanamycin cassette through homologous recombination. According to the high homology of the phoP gene in many of Salmonella species the pTAAZ92 can be used to disrupt the phoP gene in most of these species

[Report of a novel mutation in RB1 gene from an Iranian retinoblastoma patient and its effect on splicing pattern of mRNA]

Mutations in RB1 gene may lead to retinoblastoma which is the most common solid intraocular tumor in under-six year old children. To date, a wide spectrum of the mutations has been reported in the splicing of RB1 which either affect splicing sequences or splicing regulatory elements. This report introduces a new mutation in RB1and its influence on the splicing of mRNA. Case report: In the present survey, mutation analysis was done in an Iranian patient with sporadic unilateral retinoblastoma using direct sequencing and MLPA. Also, RB1 gene splicing pattern was analyzed by RT-PCR method. As a result, a same-sense nucleotide change [g.70 320C>T] was found near the 5' end of exon 12. This alteration disrupts the consensus sequence of an exonic splicing enhancer and changes the binding site of SC-35 protein. Structural analysis of cDNA in this patient showed the disruption of normal splicing pattern and the skipping of exon 12 from the RB1 transcript. Based on these findings, it may be reasonable to conclude that the above nucleotide change could be a pathogenic mutation. Also, for the first time we report an evidence for the presence of an exonic splicing enhancer in the exon 12 of the RB1 gene