目的:构建羟基喜树碱耐药结肠癌细胞株SW1116/HCPT细胞的酵母双杂交cDNA文库.方法:用TRIzol从SW1116/HCPT细胞提取总RNA,采用CLONTECH SMART(switching mechanism at 5' end of RNA transcript)技术和同源重组(homologous recombina...目的:构建羟基喜树碱耐药结肠癌细胞株SW1116/HCPT细胞的酵母双杂交cDNA文库.方法:用TRIzol从SW1116/HCPT细胞提取总RNA,采用CLONTECH SMART(switching mechanism at 5' end of RNA transcript)技术和同源重组(homologous recombination)的方法构建SW1116/HCPT细胞的cDNA文库.结果:提取的RNA的A260/A280为1.98,甲醛变性琼脂糖凝胶电泳示出28SrRNA、18SrRNA及5SrRNA3条特异性带,28S与18S带浓度及亮度比值约为2.文库dscDNAsmear较长分布在0.1-5kb,成长瀑布条带,而且在接近1kb的区域有密集的带子,为高丰度表达基因.依据生长菌落计数,共获得(1.2-1.9)×106个转化子,重组率达93.7%,文库插入片段大小为0.2-5kb.结论:成功构建了SW1116/HCPT cDNA文库.展开更多
辣椒素类物质是辣椒属植物特有的一种次生代谢产物,特异地在辣椒果实胎座中合成,且具有明显的发育阶段特异性。辣椒素合成途径相关基因主要在辣椒花后16 d胎座发育至绿熟期转录,然而转录调控的精细分子机制尚不太清楚。用已知参与调控...辣椒素类物质是辣椒属植物特有的一种次生代谢产物,特异地在辣椒果实胎座中合成,且具有明显的发育阶段特异性。辣椒素合成途径相关基因主要在辣椒花后16 d胎座发育至绿熟期转录,然而转录调控的精细分子机制尚不太清楚。用已知参与调控辣椒素生物合成的转录因子为诱饵,采用酵母双杂交技术从辣椒素合成阶段酵母cDNA文库中筛选与其互作的蛋白,有利于明确辣椒素生物合成的转录调控机制。因此,获得辣椒素合成阶段的高质量酵母cDNA文库是关键。本研究采用辣椒‘59’自交系花后16 d(DPA)的RNA作为模板,之后用SMART c DNA/均一化c DNA文库技术(Switching mechanism at 5’end of the RNA transcript)与同源重组的方法将‘59’自交系的胎座cDNA文库转化酵母感受态Y187。通过对收集的文库进行鉴定发现,滴度达到3.63×10~7CFU/m L,插入片段大约250~2000 bp,文库满足酵母双杂交要求。辣椒胎座高质量cDNA文库的构建为后续辣椒素生物合成的转录调控网络研究提供了基础。展开更多
asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell...asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells. Compared with asy gene, overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.展开更多
文摘目的:构建羟基喜树碱耐药结肠癌细胞株SW1116/HCPT细胞的酵母双杂交cDNA文库.方法:用TRIzol从SW1116/HCPT细胞提取总RNA,采用CLONTECH SMART(switching mechanism at 5' end of RNA transcript)技术和同源重组(homologous recombination)的方法构建SW1116/HCPT细胞的cDNA文库.结果:提取的RNA的A260/A280为1.98,甲醛变性琼脂糖凝胶电泳示出28SrRNA、18SrRNA及5SrRNA3条特异性带,28S与18S带浓度及亮度比值约为2.文库dscDNAsmear较长分布在0.1-5kb,成长瀑布条带,而且在接近1kb的区域有密集的带子,为高丰度表达基因.依据生长菌落计数,共获得(1.2-1.9)×106个转化子,重组率达93.7%,文库插入片段大小为0.2-5kb.结论:成功构建了SW1116/HCPT cDNA文库.
文摘辣椒素类物质是辣椒属植物特有的一种次生代谢产物,特异地在辣椒果实胎座中合成,且具有明显的发育阶段特异性。辣椒素合成途径相关基因主要在辣椒花后16 d胎座发育至绿熟期转录,然而转录调控的精细分子机制尚不太清楚。用已知参与调控辣椒素生物合成的转录因子为诱饵,采用酵母双杂交技术从辣椒素合成阶段酵母cDNA文库中筛选与其互作的蛋白,有利于明确辣椒素生物合成的转录调控机制。因此,获得辣椒素合成阶段的高质量酵母cDNA文库是关键。本研究采用辣椒‘59’自交系花后16 d(DPA)的RNA作为模板,之后用SMART c DNA/均一化c DNA文库技术(Switching mechanism at 5’end of the RNA transcript)与同源重组的方法将‘59’自交系的胎座cDNA文库转化酵母感受态Y187。通过对收集的文库进行鉴定发现,滴度达到3.63×10~7CFU/m L,插入片段大约250~2000 bp,文库满足酵母双杂交要求。辣椒胎座高质量cDNA文库的构建为后续辣椒素生物合成的转录调控网络研究提供了基础。
文摘asy gene is a novel apoptosis-inducing gene, but its mechanism is unclear. To investigate the mechanism of asy inducing apoptosis, a novel gene encoding ASY interacting protein (asyip) is isolated from human lung cell line (WI-38) cDNA library with yeast two-hybrid system. The asyip gene is constitutively expressed as two mRNA transcripts with the size of 1.8 and 2.7 kb in various human tissues at different levels. Sequence analysis of full-length cDNA reveals that the two alternative transcripts of asyip gene contain common 5' end and different 3' end, and share a common open reading frame encoding a polypeptide of 236 amino acids. Two protein kinase C phosphorylation sites and two casein kinase II phosphorylation sites are found in ASYIP amino acid sequence. Two highly hydrophobic regions encoding potentially two transmembrane domains are present. The ASYIP protein contains a C-terminal endoplasmic reticulum retrieval signal (Lys-Lys-Lys-Ala-Glu). Immunoprecipitation assay confirmed the interaction of ASY and ASYIP in mammalian cells. Compared with asy gene, overexpression of asyip gene can inhibit growth of tumor cell Saos2 and induce cell apoptosis with a low efficiency.
