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.
|Zeng, M; Martsen, E O; Lapeyre, J N (1998) Re-amplification of short primer-generated bands from RAPD and methylation-sensitive restriction fingerprinting by discrimination primers. Biotechniques 24:402-3, 406|
|Martsen, E O; Zeng, M; Lapeyre, J N (1997) Fast plasmid DNA sequencing using a thermal cycler and high temperature alkali denaturation. Biotechniques 22:420-2|
|Marini 3rd, F C; Pan, B F; Nelson, J A et al. (1996) The drug verapamil inhibits bystander killing but not cell suicide in thymidine kinase-ganciclovir prodrug-activated gene therapy. Cancer Gene Ther 3:405-12|
|Zeng, M; Huang, B R; Lapeyre, J N (1996) Cloning strategies using a third irrelevant enzyme site (TIES) to overcome certain cloning problems. Biotechniques 20:68-72, 74, 76 passim|
|Marini 3rd, F C; Nelson, J A; Lapeyre, J N (1995) Assessment of bystander effect potency produced by intratumoral implantation of HSVtk-expressing cells using surrogate marker secretion to monitor tumor growth kinetics. Gene Ther 2:655-9|
|Marini 3rd, F C; Cannon, J P; Belmont, J W et al. (1995) In vivo marking of spontaneous or vaccine-induced fibrosarcomas in the domestic house cat, using an adenoviral vector containing a bifunctional fusion protein, GAL-TEK. Hum Gene Ther 6:1215-23|