人U6基因POI Ⅲ启动子表达反义VEGF cDNA抑制SMMC-7721肝癌细胞VEGF表达的观察

 由 RNA聚合酶启动子在细胞内转录高浓度反义 RNA是抑制靶蛋白的一种有效手段 ,有报道 POI 启动子转录小分子 RNA存在效率不高 ,带有较多的非特异性序列等缺点 ,为了克服这些问题 ,表达反义 VEGF RNA的人 U6基因表达盒对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制作用进行了研究 .首先 PCR扩增 2 0 0 bp VEGF c DNA以正、反向插入人 U6 sn RNA.通过测序证实反向插入的正确性 .采用细胞原位杂交 ,RNA酶保护分析 ,Northern印迹来证实反义 RNA表达的情况 ,利用 RT- PCR方法研究了其对人肝癌细胞株 SMMC- 772 1 VEGF表达的抑制效果 .细胞原位杂交结果显示 U6启动子转录产物主要分布于细胞核内 ,细胞浆内亦有表达 ,RNA酶保护分析显示 U6基因 POI 启动子能高表达所需大小反义 RNA,Northern印迹结果显示脂质体 lipo-fectamine介导含 U6基因 POI 启动子的质粒转染人肝癌细胞株 SMMC- 772 1后 1 2 h即有表达且可持续表达 6 d. RT- PCR证实 U6基因 POI 启动子转录的反义 VEGF RNA能有效抑制SMMC- 772 1细胞 VEGF的 m RNA表达 .已有的研究结果揭示 POI 启动子是反义基因治疗中一种好的选择     

Molecular cloning of cDNA for the catalytic subunit of rat liver type 2A protein phosphatase, and detection of high levels of expression of the gene in normal and cancer cells

A cloned cDNA encoding a catalytic subunit of type 2A protein phosphatase from a rat liver cDNA library was obtained by use of a synthetic oligonucleotide corresponding to the tryptic peptide sequence of the purified enzyme. There was only a single amino acid difference between the deduced amino acid sequence of the clone obtained and those of the catalytic subunits, 2A alpha, of the rabbit skeletal muscle, porcine kidney and human liver enzymes, suggesting that this clone was a rat 2A alpha cDNA. On Northern blot analysis using a cDNA fragment as a probe, three mRNA species were detected in rat liver: a major mRNA of 2.0 kb and a minor one of 2.7 kb under high stringency conditions, and also a 1.1 kb mRNA under low stringency conditions. The 2A alpha gene was found to be highly expressed in various tissues of rat, especially the brain. High levels of expression of the gene were also detected in mouse NIH3T3 cells and their transformants, and in human cancer cell lines as well as a human immortalized cell line.     

Cloning and functional expression of human kynurenine 3-monooxygenase

Kynurenine 3-monooxygenase, an NADPH-dependent flavin monooxygenase, catalyses the hydroxylation of

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-kynurenine to

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-3-hydroxykynurenine. By hybridization screening using a cDNA probe encoding the entire exon 2 of Drosophila melanogaster kynurenine 3-monooxygenase, we isolated a 2.0 kb cDNA clone coding for the corresponding human liver enzyme. The deduced amino acid sequence of the human protein consists of 486 amino acids with a predicted molecular mass of 55 762 Da. Transfection of the human cDNA in HEK-293 cells resulted in the functional expression of the enzyme with kinetic properties similar to those found for the native human protein. RNA blot analysis of human tissues revealed the presence of a major mRNA species of 2.0 kb in liver, placenta and kidney.     

Molecular cloning and nucleotide sequence analysis of complementary deoxyribonucleic acid for the beta-subunit of rat follicle stimulating hormone

To provide a hybridization probe for analysis of the regulation of rat gonadotropin subunit mRNA levels, an effort was made to isolate a cloned cDNA for the beta-subunit of rat FSH (FSH beta). Using a cloned bovine FSH beta cDNA as a hybridization probe, a rat pituitary lambda gt10 cDNA library was screened and a single, strongly hybridizing clone identified. The 874 base pair cDNA insert from this clone contains the complete sequence of rat FSH beta including an amino-terminal precursor segment. Hybridization of this cloned cDNA to rat pituitary RNA demonstrated the presence of an approximately 2.0 kilobase RNA species containing FSH beta sequences. Cloned rat cDNA was also used to demonstrate that estrogen treatment of ovariectomized female rats results in decreases in mRNA concentrations for FSH beta and the beta-subunit of LH with somewhat smaller decreases in alpha-subunit mRNA concentrations. Little or no change was detected in the mRNA for the beta-subunit of TSH.     

Quantitation of specific mRNA by RNA-RNA hybridization kinetics with single-stranded riboprobes

A quantitative procedure by a solution hybridization involving RNA-RNA hybridization kinetics was developed for measurement of specific mRNA accumulated in particular tissues and cells. For quantitating mouse beta-tubulin mRNA two types of riboprobes were prepared: one was a truncated RNA covering only the coding portion of beta-tubulin cDNA and the other was a non-truncated RNA covering the vector portion as well as the coding portion. These antisense RNAs were hybridized with mouse brain total cellular RNA, yielding heat-stable hybrids. Both the truncated and non-truncated antisense RNA probes showed similar hybridization kinetics. Hybridization of the sense RNA, consisting of the beta-tubulin coding portion, with the antisense RNA probe gave standards for determining the proportion of beta-tubulin mRNA in total brain RNA. By this method, the amounts of beta-tubulin mRNA included in the brains of 10- and 50-day-old mice were quantitated to be 0.0056 and 0.0011% of total RNA, respectively.