It has been demonstrated that it is possible to express site-specific bacterial methylase and endonuclease genes in eukaryotic cells. In addition the expression of these genes appears to have no deleterious effect on the host cell. In initial studies to express the PaeR7 methylase in mouse L cells, the levels of methylase produced varied greatly and were insufficient to protect the host cell genomes from the activities of the cognate endonucleases. The problems that need to be solved now in this proposal, are (1) the incomplete methylation of cell genome by PaeR7 and (2) the inefficient restriction of infecting viral DNA. In order to increase methylase and endonuclease activity, methods such as multiple rounds of transformation, selection of alternative promoters, gene amplification will be tried. Hopefully, a stable expression of the bacterial restriction/modification system in animal cells will be achieved. The ultimate goal of the project is to establish this system in cultured mammalian cells which would render them more resistant to viral infection. Dr. Gingeras is eminently qualified to carry out this work. Support if possible with a very good priority.
