The purpose of this project is to use thymidine kinase as a selective vector for the transfer of purified eucaryotic genes to cells in culture. The thymidine kinase gene from Herpes Simplex virus Type 1 (HSV-1) has been shown by others to reside on a 3.4 kb fragment obtained from a Bam H-1 restriction enzyme digestion of total viral DNA. This DNA fragment is capable of integration with the cellular DNA to transform mouse L cells deficient in thymidine kinase (LMTK minus) to LMTK plus. The change of phenotype has been shown to be due to the viral enzyme (1). This proposal will study methods of increasing the transformation frequency of the thymidine kinase gene gy 1) comparing the efficiency of the linear versus the circular form of the DNA in transformation; 2) test to see if the presence of cellular DNA, covalently linked to the viral thymidine kinase gene, will increase its rate of incorporation and expression; 3) use of a temperature sensitive mutant of thymidine kinase to select for an increase in the copy number of the viral DNA in the cell. When an efficient system of transfer is developed, the thymidine kinase gene will be linked to the cloned nuclear ovalbumin gene and transferred to LMTK minus cells. Cells which become TK plus will be assayed, by DNA hybridization, for the presence of the non-selected ovalbumin gene. The expression of the ovalbumin gene in positive cells will be studied both transcriptionally and translationally.
NIH ExPorter
NIH ExPorter