CREATION OF A DELETION SERIES OF MOUSE YAGS COVERING A 500 KB REGION AROUND XIST

Two mouse YACs, PA-2 and PA-3, contain the Xist gene and are 460 kb an d 3.3 Mb long respectively. While PA-2 is non-chimeric, PA-3 contains a substantial proportion of non-contiguous DNA. As a prerequisite to f unctional studies of the role of this region in X inactivation, we hav e created a deletion series of YACs that are spaced at approximately 5 0 kb intervals and were able to eliminate the unwanted chimeric sequen ces in YAC PA-3. For this purpose, we have constructed mouse B1 fragme ntation vectors based on those described for human Alu fragmentation. Having created this series of YAC deletion derivatives, we were able t o eliminate efficiently the 10 - 15% aberrant YACs that arise during t he course of a fragmentation experiment by assessing their marker cont ent. The overlap and the opposite orientation of the two YAC inserts p ermitted the creation of deletions on both sides of the 500 kb region around Xist. The use of this series of YACs in a biological assay will help us define the extent of the sequences necessary to bring about X chromosome inactivation.