Figure 1.

Isolation of a targeted human chromosome region as circular or linear YACs by yeast transformation. (A) A scheme to rescue the targeted chromosome region as a series of circular YACs. Competent yeast spheroplasts are transformed with genomic DNA isolated from cells containing a targeted chromosome. The targeting vector (open box) incorporated into a specific region of a human chromosome contains a yeast centromere, CEN, a yeast origin of replication, ARSH4, a yeast selectable marker, TRP1, and a mammalian selectable marker, Zeo^R. The position and orientation of several repeats (SINE or LINE) located near the end of the targeted chromosomal fragment are shown. The presence of multiple repeated sequences in the targeted chromosomal fragment provides many opportunities for homologous recombination. Recombination between inverted repeats leads to the establishment of series of different size circular YACs. Arrows indicate the orientation of the repeats in the targeted fragment. (B) A scheme to isolate the targeted chromosome region by recombination with a YAC fragmentation vector. Yeast spheroplasts are transformed with genomic DNA along with a YAC fragmentation vector marked by the HIS3 gene. This vector contains a yeast telomere (TEL) on one end and an Alu sequence at the other end. Recombination between the Alu sequence in the vector and an Alu sequence in the targeted chromosome fragment leads to the establishment of a linear semi-YAC (TEL–HIS3–CEN–ARS–TRP1) with one telomere. Because there are many yeast-like telomere sequences in the fragment (shown by small arrows) a series of different size linear YACs are generated from the semi-YAC by healing of the broken end. The position and orientation of several Alu sequences within the targeted fragment are shown.