SELECTION AND ISOLATION OF RADIATION INDUCIBLE GENES

这个应用程序的目标是通过药物识别和分离 选择介导的基因，其活性由电离诱导 辐射(IR)，目的是为这些基因和 界定对其发起人的监管。这是第一步 确定分子和生化功能的执行 未鉴定的辐射调节蛋白(RMP)是由 并将有助于阐明许多鲜为人知的放射学结局- 哺乳动物细胞中的点。人类基因组将被饱和 逆转录病毒载体介导的小于2微米启动子的随机整合 含有剪接受体和IVS的酵母重组酶基因(FLPase) 在大量HT1080细胞中增强稳定性的领先者。这 标记还允许电离辐射的正显性选择 放射治疗中的辐射剂量诱导(IRI)基因 转新霉素/潮霉素触发器的细胞 显性选择基因。这种分离IRI基因的方法是一种自旋- 从之前描述的使用FACS的基因标签方法 B-半乳糖苷酶辐射诱导表达产物的分选方案 重组逆转录病毒载体pros-BGAL-pgkeno的随机整合 无启动子的LacZ指示基因，一个可供选择的新标记 带有G418的整合子，以及一个用于挽救遗传区的supF标记。 药物选择方法消除了背景和零的问题 细胞爬过多个FACS屏幕，阻碍了对 实际IRI基因的互补脱氧核糖核酸文库 与世隔绝。申请人使用该基因标签进行的初步实验 这种方法表明，大约存在100-200个IRI基因。到目前为止，其他 方法只定义或分离了大约12个诱导或分离的基因 参与电离辐射反应。Aim I的目标是 识别，然后分离，IRI cDNA，将被测序和 特色化的。AIM II的目标是使用IRI cdna探针来 从基因组文库中分离IRI基因，包括上游调控因子 用于表征传感器的响应元件的区域 感应效应。

The goal of this application is to identify and isolate by a drug selection-mediated approach genes whose activity is induced by ionizing radiation (IR) with the purpose of generating probes for these genes and defining the regulation of their promoters. This is a first step in determining the molecular and biochemical functions carried out by uncharacterized radiation modulated proteins (RMPS) that are induced by radiation and will help elucidate many poorly understood radiologic end- points in mammalian cells. The human genome will be saturated by retroviral vector-mediated random integration of a promoter less 2 micron yeast recombinase gene (FLPase) containing a splice acceptor and IVS leader for enhanced stability in a large population of HT1080 cells. This marker also permits positive dominant selection of ionizing radiation induced (IRI) genes induced by radiation doses employed in radiotherapy in cells that have been transfected with a neomycin/hygromycin flip-flop dominant selection gene. This approach to isolating IRI genes is a spin- off of a previously described gene tagging approach that employed a FACS protocol to sort radiation induced expressors of B-galactosidase after random integration of pROS-Bgal-pgkneo, a retroviral vector that encodes the promoterless lacZ indicator gene, a neor marker to select for integrants with G418, and a supF marker for rescue of the genetic region. The drug selection approach eliminates the problem of background and null cells creeping through multiple FACS screens that hampers the screening of complementary deoxyribonucleic acid (cDNA) libraries for actual IRI gene isolation. The applicant's preliminary experiments using this gene tagging approach suggest the existence of about 100-200 IRI genes. So far, other approaches have only defined or isolated about a dozen genes induced or involved in ionizing radiation response. The goal of Aim I is to identify, then isolate, IRI cDNAs, which will be sequenced and characterized. The goal of Aim II is to use the IRI cDNA probes to isolate IRI genes from a genomic library, including the upstream regulator regions for characterization of the response elements transducing the inductive effect.