The Key Involvement of Poly (ADP-Ribosylation) in Defense against Toxic Agents in Molecular Biology Studies
Abstract
The program has 3 interrelated aims. The first stage involves recombinant construction of polymerase cDNA into a family of selected expression vectors which in most cases will possess an inducible promoter. During the first year, the cDNA was inserted in both sense and antisense orientations and also site-directed mutants. We were also concerned with construction (I-D) and expression of site-directed mutants and potential inhibitory peptides in vivo in order to eventually modulate the activity of poly(ADP-Rib) polymerase in cells, upon induction during DNA repair. Various expression vectors will be stably transfected into eukaryotic cells generally by co-transfection with a selectable gene. We expect various levels of overexpression and underexpression of POLY(ADP-Rib) polymerase. In the case of the site-directed mutants and the inhibitory peptides (IID), we anticipate cells with reduced capacity for ADP- ribosylation. Biochemical and molecular biological characterizations of the gene products of the various transfected cells are proposed prior to cytotoxicity or DNA repair analysis. These will include: Southern analysis to confirm integrated copies of the cDNA; both Northern and primer extension analysis of cellular mRNA to confirm that upon induction actual expression of the foreign gene occurs; and immunoprecipitation of poly(ADP-Rib) polymerase in vivo after induction. Mutagenicity.
Document Details
- Document Type
- Technical Report
- Publication Date
- Nov 15, 1989
- Accession Number
- ADA217017
Entities
People
- Mark E. Smulson
Organizations
- Georgetown University