ANTISENSE RNA AND DNA INHIBITION OF KI-RAS TRANSLATION

已观察到21 kDa膜蛋白(P21)的过度生产。 几种哺乳动物肿瘤，并与ras基因扩增相关。在……里面 正在研究的实验系统，Y1小鼠肾上腺癌，有 大约30个拷贝的明显正常的Ki-ras基因和一个 相应地高水平的p21。这项研究的主要目标是 开发专门抑制p21合成的系统并测试 这种抑制作用可以逆转转化后的表型。 具体目标如下：(1)优化全基因转录 长度反义Ki-ras序列(来自 初步实验)，并证实这些杂交 以特定的方式与Y1ki-ras mRNA结合，抑制p21的翻译。(2) 构建额外的转录部分反义ras基因的抗ras载体 RNA某些区域特异的Ki-ras序列，并检测其 在体外和培养细胞中抑制p21的有效性。(3) 构建针对不同区域的序列特异性反义ras寡核苷酸 Ki-ras信使核糖核酸及其对p21的体外抑制作用 在培养的细胞中。(4)研究p21减少的后果 通过评估与其相关的Y1细胞的特性 转型。其中包括增长率、增长因子的变化 要求、核型(如DM或HSR缺失)、恶性程度和 在裸鼠体内诱发肿瘤的能力。(五)确定政策的效力 7反义杂交子(Ki-ras质粒构建物或寡核苷酸) P21在其他细胞系中的翻译，包括那些过表达的细胞 未扩增的Ki-ras基因和具有突变的ras基因 吉恩。

Overproduction of a 21 Kda membrane protein (p21) has been observe in several mammalian tumors and is correlated with ras gene amplification. In the experimental system under study, the Y1 mouse adrenal carcinoma, there are approximately 30 copies of an apparently normal Ki-ras gene and a correspondingly high level of p21. The main goals of this study are to develop systems to specifically inhibit p21 synthesis and to test whether such inhibition can produce a reversal of the transformed phenotype. Specific goals are as follows: (1) Optimize the transcription of full- length antisense Ki-ras sequences (from the plasmid pIFN-ras described in the preliminary experiments) in Y1 cells and confirm that these hybridize in a specific manner with Y1 Ki-ras mRNA and inhibit p21 translation. (2) Construct additional anti-ras plasmids which transcribe partial antisense Ki-ras sequences specific for certain areas of the mRNA and test their effectiveness on p21 inhibition both in vitro and in cultured cells. (3) Construct sequence specific anti-ras oligonucleotides to various regions of Ki-ras mRNA and test their effectiveness on p21 inhibition both in vitro and in cultured cells. (4) Examine the consequences of p21 reduction through assessment of properties of Y1 cells associated with their transformation. Among these would be changes in growth rate, growth factor requirements, karyotype (e.g., loss of DMs or HSR) degree of malignancy and ability to induce tumors in nude mice. (5) Determine the effectiveness of 7antisense hybridons (Ki-ras plasmid constructs or oligonucleotides) on the translation of p21 in other cell lines including those with overexpressed Ki-ras mRNA but an unamplified Ki-ras gene and those with a mutated ras gene.