Cloning of the entire FLI1 gene, disrupted by the Ewing's sarcoma translocation breakpoint on 11q24, in a yeast artificial chromosome.

FLI1 (Friend leukemia virus integration 1), a member of the Ets gene family, is disrupted on 11q24 by the Ewing's Sarcoma (ES) and Peripheral Neuroepithelioma (PNE) t(11;22)(q24;q12) translocation. ES and PNE are Primitive Neuroectodermal Tumors (PNETs) and the consistent translocation t(11;22)(q24;q12) can be used for differential diagnosis. In PNETs the 3' part of human FLI1 is translocated from 11q24 to 22q12, where it is juxtaposed to the 5' end of the Ewing's Sarcoma gene (EWS). A fusion transcript, resulting in a chimeric protein, is generated. Here, we present the isolation and detailed characterization of a 250-kb colinear YAC, B45C11, which encompasses the ES and PNE breakpoint on 11q24, as shown by FISSH on ES and PNE chromosomes and interphase nuclei. This YAC represents a new reagent for potential use in rapid differential diagnosis by FISSH on tumor biopsies and on paraffin embedded samples, particularly when DNA and/or RNA are not available for molecular analysis. YAC B45C11, which spans 250 kb of contiguous DNA around the ES and PNE breakpoint, contains the entire FLI1 gene. Three potential HpaII-tiny-fragment (HTF) islands are revealed within the YAC. One of these islands appears to be associated with the 5' end of FLI1, which extends over approximately 120 kb of DNA on 11q24. In addition, we demonstrate that YAC B45C11 contains other transcribed sequences in addition to FLI1, by "cross-species" Northern blot hybridizations, which suggests the presence of additional genes in the immediate vicinity of the ES breakpoint on 11q24.