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Hwang, Sue Yun,Kim, Seung Hoon,Hwang, Sung Hee,Cho, Chul Soo,Kim, Ho Youn The Korean Society for Integrative Biology 2001 Korean journal of biological sciences Vol.5 No.2
A key aspect of genomic research in the “post-genome era”is to associate sequence variations with heritable phenotypes. The most common variations in the human genome are single nucleotide polymorphisms (SNPs) that occur approximately once in every 500 to 1,000 bases. Although analyzing the phenotypic outcome of these SNPs is crucial to facilitate large-scale association studies of genetic diseases, detection of SNPs from an extended number of human DNA samples is often difficult, labor-intensive and time-consuming. Recent development in SNP detection methods using DNA microarrays and mass spectrophotometry has allowed automated high throughput analyses, but such equipments are not accessible to many scientists. In this study, we demonstrate that a simple PCR-based method using primers with a mismatched base at the 3'-end provides a fast and easy tool to identify known SNPs from human genomic DNA in a regular molecular biology laboratory. Results from this PCR amplification of specific alleles (PASA) analysis efficiently and accurately typed the Q576R polymorphism of human IL4 receptor from the genomic DNAs of 29 Koreans, including 9 samples whose genotype could not be discerned by the conventiona1 PCR-SSCP (single strand conformation polymorphism) method. Given the increasing attention to disease-associated polymorphisms in genomic research, this alternative technique will be very useful to identify SNPs in large-scale population studies.
Hwang, Sung Chul,Jhon, Deok-Young,Rhee, Sue Goo 아주대학교 의과학연구소 1996 아주의학 Vol.1 No.1
During the purification of phospholipase C (PLC)-rl which was over-expressed in HeLa cells transfected with a recombinant vaccinia virus carrying a complete cDNA sequence for PLC-γ₁ we noticed the presence of a heat-stable activator in crude cytosolic extract of HeLa cells which markedly stimulated phosphatidylinositol (PI) hydrolysis, when reconstituted with purified PLC-γ₁. Moreover, this putative factor was also found to be present in bovine brain cytosol. Subsequently, based on its ability to stimulate PI hydrolysis of PLC-γ₁ as an assay, this activation factor was purified to homogeneity from bovine brain cytosol. It was purified by heat treatment, trichloroacetic acid precipitation, and successive chromatographic steps on DEAE-5PW, phenyl-5PW, and heparin-5PW HPLC columns. The purified protein, as seen on SDS-PAGE, consisted of 4 or 5 closely spaced bands of apparent molecular weight between 48 and 62 kDa. However, on gel filtration chromatography on TSK-G3000SW HPLC, the estimated molecular weight was revealed to be approximately 350 kDa. The purified activator was identified as a microtubule-associated protein tau by electroelution from an SDS-polyacrylamide gel, partial peptide mapping by Staphylococcal V_(8) protease, and amino acid sequencing of cyanogen bromide cleaved peptides. The identity of the activator was re-confirmed by an immunoblotring using a specific anti-lau monoclonal antibody. In addition, reconstituting PLC-γ₁ with tau protein purified by an alternative method described elsewhere^ resulted in the same magnitude of activation. Tau protein mediated activation of PLC isozymes was calcium dependent. Isozyme specific activation of PLCs in 0.1% deoxycholate substrate appeared to be preferential toward PI hydrolysis. The magnitude of activation of PLC isozymes in PI substrate were PLC-γ₁ > PLC-r2 > PLC-δ₁ > PLC-β₁ in decreasing order. Approximately 200 nM concentration of tau-protein produced 15.5-, 5.4-, 4.2-, and 1.6-fold increase in activity of these enzymes, respectively, in the presence of 1 mM free calcium ion.