SEARCH FOR ONCOGENE MUTATIONS IN X-RAY-TRANSFORMED MOUSE 10T1 2 CELLSBY DENATURING GRADIENT GEL-ELECTROPHORESIS BLOTTING/

We have sought evidence for possible mutations within the c-myc, c-Ha- rns and c-Ki-ras loci of X-ray-transformed mouse C3H10T1/2 cell clones using the denaturing gradient gel electrophoresis (DGGE) blot techniq ue. This highly sensitive method was developed to detect any mutations (e.g. single base changes, small deletions) in genomic DNA, by measur ing differences in the melting behaviour of short DNA fragments (50-80 0 bp) obtained by digestion of genomic DNA with several specific 4 bp recognition site restriction enzymes. In this study, genomic DNAs deri ved from 23 X-ray-transformed clones were digested with several restri ction enzymes, electrophorezed on denaturing gradient gel and hybridiz ed to c-myc, c-Ha-ras and c-Ki-ras cDNA probes. No alterations in melt ing patterns were observed for any of these oncogenes as compared with DNA from 18 control, non-irradiated wild-type 10T1/2 cell clones, sug gesting that transformation was not associated with mutation of these genes nor with changes in their patterns of methylation. However, our screening of the large portion of exons 2 and 3 of c-myc as well as of exons 1 and 2 of c-Ha-ras gene cannot exclude the possibility that so me sequence differences in the high melting domains of examined fragme nts were not detected by this assay.