The Effects of Sequence Homology on Transformation of Mouse Cells By Sv40 Dna.

Abstract

Transformation is the introduction of new genetic information into animal cells by exposing them to DNA. Experiments were performed to investigate the possibility that sequence homology between transforming DNA and host chromosomal DNA facilitates transformation of mouse cells. Two recombinant SV40 DNA molecules were constructed. pRC105 is SV40 DNA cloned in pBR322. pRC101 contains pBR322, SV40, and a 4.0 kb segment of Balb/c mouse ribosomal DNA which includes sequences from the 18S and the 28S coding regions as well as the transcribed spacer region. The efficiencies of these DNAs in transforming mouse Balb/c-3T3 cells to a non-contact-inhibited, anchorage independent state were measured in st and ard assays involving colony formation in soft agar and focus formation on plastic. When the transformation efficiencies of pRC101 and pRC105 were normalized to that of SV40 DNA, the transforming DNA which carried mouse ribosomal sequences had an efficiency of about three times that of a similar recombinant (pRC105) lacking the mouse DNA, and about 1.4 times that of SV40 DNA. pRC105 DNA had only about 40% the transformation efficiency of uncloned SV40 DNA. The effect of different carrier DNAs on transformation by SV40, pRC101 and pRC105 DNAs was also tested. When mouse DNA is used as carrier, the SV40 transformation efficiency is about twice that seen when salmon sperm DNA is used, and about ten times that obtained when bacteriophage (lamda) DNA is used as carrier. The size of DNA used may explain some of the difference between mouse and salmon sperm DNA, but does not explain why (lamda) DNA results in such poor transformation efficiencies. The type of carrier DNA used was shown not to alter the transformation efficiency of pRC101 relative to pRC105, although the same alteration was seen in their absolute transformation efficiencies, as was seen for SV40 DNA. Finally, preliminary experiments were performed to determine if pRC101 DNA was integrating into homologous genomic ribosomal sequences. Neither Southern transfer analysis nor attempts to determine if SV40 sequences in pRC101 transformed cell DNA b and ed with the G-C rich ribosomal DNA produced conclusive results.