Cloning of the entire FL11 gene, disrupted by the Ewing’s Sarcoma translocation breakpoint on 11q24, in a yeast artificial chromosome

Abstract

FLU (Friend leukemia virus integration 1), a member of the Ets gene family, is disrupted on llq24 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 FLU 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 FLU 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 FLU, which extends over approximately 120 kb of DNA on 1 lq24. In addition, we demonstrate that YAC B45C11 contains other transcribed sequences in addition to FLU, by “cross-species” Northern blot hybridizations, which suggests the presence of additional genes in the immediate vicinity of the ES breakpoint on 1 lq24.

The role of Fli-1 in normal cell function and malignant transformation

“…Pulse-®eld gel analysis has localized the Fli-1 gene within 240 kb of the Ets-1 locus on mouse chromosome 9 and on human chromosome 11q23, suggesting that these two ets genes arose by gene duplication from a common ancestral gene (Ben-David et al, 1991a;Watson et al, 1992). Human Fli-1 contains nine exons which extend over approximately 120 kb (Selleri et al, 1994). A comparison of the amino acid sequences of Fli-1 revealed an 81% homology to the ets-related protein Erg-2 (Prasad et al, 1992).…”

Molecular Cytogenetics in Ewing Tumors: Diagnostic and Prognostic Information

“…To ascertain the involvement of the FLI1 gene at 11q24, cosmids flanking or overlapping the breakpoint region EWS-R2 were hybridized in different studies [18][19][20][21][22]. Also, yeast artificial chromosome (YAC) probes are available for 3.2 Mb of DNA within band 11q24 including the entire FLI1 gene, thus providing new DNA probes for detection of rearrangements involving this chromosomal region [23,24]. Simultaneous hybridization of probes located proximally to the EWS breakpoint region on chromosome 22 and distally to the FLI1 gene on chromosome 11 identifies the fused breakpoint regions of the classical t(11;22)(q24;q12).…”

ETS-1 induces increased expression of erythroid markers in the pluripotent erythroleukemic cell lines K562 and HEL

“…1,2,[11][12][13] The human homologs of many of these genes including FLI-1, PU.1, ETS-1, ETS-2, ELK-1 and K562, HEL, and CRL9078 (PA317) cells were purchased from ELK-2, have been mapped to chromosomal regions known to the American Type Culture Collection; CRE cells were be involved in translocations characteristic of specific leukeobtained from Dr R Mulligan; K562 and HEL were cultured mias, lymphomas, and other tumors. [14][15][16][17][18][19][20][21][22][23][24][25] in RPMI medium supplemented with 10% fetal bovine serum Many of the ETS genes are known to be expressed in hema-(FBS) plus antibiotics (Atlanta Biological, Atlanta, GA, USA); topoietic tissues and cell lines. [26][27][28][29][30][31] It was demonstrated that CRE and PA317 cells were cultured in Dulbecco's MEM supthe PU.1 gene product is essential for the normal development plemented with antibiotics and 8% calf serum or 10% FBS, of myeloid and B lymphoid lineages, 32 while inactivation of respectively.…”