High resolution restriction mapping of YACs using chromosome fragmentation.

The large insert size of yeast artificial chromosomes (YACs; 1) has made them ideal for the analysis of complex genomes. YACs can be restriction mapped with rare cutting enzymes but this does not provide the resolution required to investigate gene structure (such as locating exons and introns). Detailed mapping normally requires sub-cloning into cosmid or phage vectors. Here we describe a method which generates high resolution restriction maps of YACs without the need for sub-cloning. Chromosome fragmentation (2) was used to produce a set of nested deletion mutants of a 200 kb YAC. Partial digestion with EcoRI and indirect end-labelling was used to map the end of each deletion mutant. The overlapping restriction maps were then aligned to produce a high resolution map of the YAC. A 200 kb telomeric YAC clone (yIgH6; 3) containing a YAC vector arm at one end (pTYAC1; 4) and a human telomere repeat sequence at the other was isolated by complementation of telomere function in yeast strain AB1380 (MATa ade2-1 can1-100 lys2-1 trp1 ura3-52 his5). Deletion mutants were created using the Alu fragmentation vectors, BP108 and