Structural analysis of the Ppo-155 β-YAC tranferred into MEL cells by lipofection. (A) Diagram of thePpo-155 β-YAC. The 144-kb I-Ppo-I fragment used to assess YAC integrity is shown. This YAC contains an intact β-locus with 39 kb of DNA upstream of the 5′HS5 of the LCR and 23 kb of DNA downstream of 3′HS1. The HS sites of the LCR and the 3′HS1 are displayed as arrows. The location of the I-Ppo-I sites are shown as straight lines next to the vector sequences (open boxes). YAC-vector sequences: TRP1 and LYS2, YAC selectable markers for tryptophane and lysine prototrophy, respectively; ARS1, yeast autonomous replicating sequence; CEN1, centromere; PGK-Neo, selectable marker for G418 resistance. (B) Structural analysis of the MEL(Ppo-155 β-YAC) clones 15 and 17; I-Ppo-I digestion, PFGE, and capillary transfer were performed as described in Materials and Methods. The blot was cut into strips and each strip was hybridized with one of the three probes indicated by the dotted lines between the YAC diagram and the autoradiograph (ie, HS5, ^Aγ-globin, and DF-10). The strips were realigned to reconstruct the original membrane, and a hybridization profile for each I-PpoI fragment was obtained. Fragments with a positive hybridization signal in all three lanes were considered as intact. Notice that clone 15 has two fragmented copies, whereas clone 17 has two intact copies at 140 and 280 kb and a third that extends from ^Aγ to the 3′ end of the β globin locus. (C) Graphic representation of the β-YACs in the MEL(β-YAC) clones according to their hybridization profile. Solid lines represent intact β globin loci, whereas the dotted lines indicate lack of hybridization and fragmented β-YAC copies.