Figure 2.

Analysis of β^s YAC DNA and β^s YAC transgenic mice. A shows the products of DdeI digestion of a 534-bp fragment after PCR amplification of DNA from six yeast clones identified by genetic screening. The fragment was digested with DdeI and fractionated on a 5% acrylamide gel. Normal restriction fragments are 49, 88, 180, and 201 bp in length. The codon 6 A-to-T transversion results in the loss of a DdeI site and the appearance of a 381 (180+201)-bp fragment. Lanes 1–6: DdeI-restricted DNA from six independent yeast clones containing the β-globin YAC with the β^s mutation. Lane 7: DdeI-restricted PCR-amplified DNA from an individual with sickle trait (A/S). The normal restriction fragments as well as the 381-bp mutant fragment are observed. Two additional bands present in this lane are the products of partial digestion. Lane 8: Undigested PCR-amplified yeast DNA demonstrating the 534-bp full-length fragment. Lanes M: φx174 HaeIII marker DNA ladder. (B) Southern analysis of EcoRI-restricted genomic DNA from transgenic line β^s.32 (lanes 1 and 6) and from four independent transgenic lines carrying intact copies of the wild-type β-globin YAC sequences (lanes 2–5). The correct fragments hybridizing with the γ- (pGγ) and β-globin genes, LCR-hypersensitive site 3 (PaH III), upstream LCR sequences (3.3R1), and sequences 20 kb downstream of the β-globin gene (RK29) are present in all of the lanes.