SELECTION AND ISOLATION OF RADIATION INDUCIBLE GENES

该应用程序的目标是通过药物识别和分离 选择介导的途径基因，其活性由电离诱导 辐射（IR），目的是产生这些基因的探针， 明确其发起人的监管。 这是第一步， 确定分子和生物化学功能， 未表征的辐射调制蛋白（RMPS），由 辐射，并将有助于阐明许多鲜为人知的放射学结束， 点在哺乳动物细胞中。 人类基因组将被 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（FLX 2） 增强大量HT 1080细胞稳定性的领导者。 这 标记还允许电离辐射的正显性选择 诱导（IRI）基因诱导的放射治疗中使用的辐射剂量， 已经用新霉素/潮霉素触发器转染的细胞 显性选择基因这种分离IRI基因的方法是一种自旋- 之前描述的基因标记方法，采用流式细胞仪， 筛选辐射诱导的B-半乳糖苷酶表达子的方案 随机整合pROS-Bgal-pgkneo，一种逆转录病毒载体， 无启动子lacZ指示基因，一个新的选择标记， 与G418的整合体，和用于拯救遗传区域的supF标记。 药物选择方法消除了背景和空值的问题 细胞爬行通过多个流式细胞仪屏幕，阻碍了筛选 实际IRI基因的互补脱氧核糖核酸（cDNA）文库 隔离申请人使用该基因标记的初步实验 研究表明，IRI基因约有100-200个。 到目前为止，其他 方法只定义或分离了大约12个诱导或 参与电离辐射反应。 目标一的目标是 鉴定，然后分离IRI cDNA，将其测序， 表征了 Aim II的目标是使用IRI cDNA探针， 从基因组文库中分离IRI基因，包括上游调节因子 用于表征转导所述信号的应答元件的区域 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.