Southern blot analysis of a geneticin-resistant HeLa cell line stably
transformed by Agrobacterium. (A)
Organization of the pNeo plasmid. The T-DNA region of pNeo with the
restriction sites used for the Southern blot analysis is shown. P, H,
E, B, and EO indicate PstI, BamHI,
EcoRI, HindIII, or
Eco0109I restriction enzyme sites, respectively. LB and
RB indicate left and right T-DNA borders, respectively. Thin lines
indicate the length of the entire T-DNA and distances of each
restriction site from RB in base pairs. The location of the neomycin
resistance (neo) gene is shown. The pNeo backbone (6 kb, not shown)
outside of the T-DNA region is that of the parental pPZP221 vector
(ref. 15; GenBank accession no. U10490). (B) Wild-type
untransformed cell line. (C) Stably transformed cell
line. Lanes 1 to 6 show digestions with SalI,
PstI, BamHI, EcoRI,
HindIII, or Eco0109I restriction enzymes,
respectively. The blots were hybridized with a 740-bp radiolabeled
probe corresponding to the neomycin resistance gene of pNeo (see
A). The sizes of the restriction-fragment bands were
calculated based on standards and are indicated on right in kilobase
pairs (kb).