YEAST ARTIFICIAL CHROMOSOME CLONING OF 3.2 MEGABASES WITHIN CHROMOSOMAL BAND-11Q24 CLOSELY LINKING C-ETS-1 AND FLI-1 AND ENCOMPASSING THE EWING-SARCOMA BREAKPOINT

Human chromosome 11 harbors many genes of medical significance and can cer-related rearrangements. The availability of cloned DNA in cosmids and in yeast artificial chromosomes (YACs), combined with fluorescence in situ hybridization analysis, has led to the cloning of genes at si tes of chromosomal breakpoints in acute leukemias in 11q23 and in Ewin g tumors in 11q24. YAC cloning has facilitated the construction of con tigs covering large portions of chromosomes for the detailed analysis of disease gene regions. sere we have cloned in YACs approximately 3.2 Mb of DNA within band 11q24, spanning the Ewing sarcoma breakpoint. L andmark cosmids 23.2 (D11S374) and 5.8 (D11S372), shown by FISH to fla nk the breakpoint within a 1.5- to 1.8-Mb segment, were used to seed t wo YAC ''walks'' both centromeric and telomeric to the breakpoint by Y AC end cloning and screening of two total genomic YAC libraries. The c entromeric YAC contig, which consists of 23 overlapping YACs and order s 19 sequence-tagged sites (STSs), covers a minimum of 2.2 Mb and span s the Ewing sarcoma breakpoint. c-ets 1 and Fli-1, two members of the ets family, have been linked within 400 kb of intervening DNA within t his contig, which also comprises a polymorphic microsatellite, D11S912 (CA)(n), which we have localized within the Fli-1 gene. The telomeric YAC contig, which consists of 11 overlapping YACs, comprises 5 STSs a nd covers a minimum of 1 Mb distal to the breakpoint. Taken together, the two contigs, which consist of a total of 34 YACs and comprise 24 S TSs, are separated by a maximum gap of 200-400 kb and cover as a whole 3.2 Mb of DNA This represents about 70% of human chromosomal band 11q 24, which extends over approximately 4.4 Mb of DNA. (C) 1994 Academic Press, Inc.