Figure 4.
Identification of transfected and endogenous ras DNAs in G418-resistant C3H cells transfected with proto-Ha-ras or v-Ha-ras under human FN or MN promoters. About 20 μg cellular DNAs were digested with BamHI (A) or XbaI (B), resolved on a 0.8% agarose gel, transferred to positively charged nylon membrane by the alkaline transfer method (55), and hybridized with 32P-labeled ras DNA from HaSV (see text). The positions of HindIII-resistant fragments of lambda DNA were used as molecular weight standards and are indicated between the two filters in A and on each side of the two filters in B. (A, Left) Lane 1, untransfected C3H; lane 2, pFN-SV40/proto-rasm-transformed C3H clone; lane 3, pMN-SV40/proto-rasm-transformed C3H clone; lane 4, pFN/proto-rasm-transfected, untransformed C3H clone; lane 5, rare pFN/proto-rasm-transformed C3H clone; and lane 6, pMN/proto-rasm-transfected, untransformed normal C3H clone. (A, Right) Lane 1, untransfected C3H; lanes 2–4, pFN-SV40/proto-rasm-transfected, untransformed C3H clones 1–3; and lanes 5–7, pMN-SV40/proto-rasm-transfected, untransformed C3H clones 1–3. The 3.4-kb BamHI-resistant ras fragment represents the genomic proto-Ha-ras-1 gene of C3H mice. The 4.07-kb (A, Left, lanes 3 and and A, Right, lanes 4–7) and 3.81-kb (A, Left, lanes 2, 4 and 5 and A, Right, lanes 2–4) BamHI-resistant ras fragments are diagnostic of the plasmids pFN-SV40/proto-rasm and pMN-SV40/proto-rasm, respectively (see Fig. 1). (B) Lane 1, untransfected C3H; lane 2,: pLTR/v-ras (pHa#8)-transformed C3H; lane 3, untransfected C3H; and lane 4, pLTR/proto-rasm-transformed C3H. The ≈12-kb XbaI fragment represents the genomic mouse proto-Ha-ras-1 gene cut with XbaI. The 2.2-kb XbaI-resistant ras fragment (lane 2) is diagnostic of the pLTR/v-ras plasmid; and the 3.66-kb and 1.4-kb XbaI-resistant ras fragments (lane 4) are diagnostic of the pLTR/proto-rasm plasmid.