MOLECULAR-CLONING OF THE PAPILLARY RENAL-CELL CARCINOMA-ASSOCIATED TRANSLOCATION (X-1)(P11-Q21) BREAKPOINT

A combination of Southern blot analysis on a panel of tumor-derived so matic cell hybrids and fluorescence in situ hybridization techniques w as used to map YACs, cosmids and DNA markers from the Xp11.2 region re lative to the X chromosome breakpoint of the renal cell carcinoma-asso ciated t(X;1)(p11;q21). The position of the breakpoint could be determ ined as follows: n-OATL2-DXS146-DXS255-SYP-t(X;1)-TFE3-OATL1-Xpter. fl uorescence in situ hybridization experiments using TFE3-containing YAC s and cosmids revealed split signals indicating that the corresponding DNA inserts span the breakpoint region. Subsequent Southern blot anal ysis showed that a 2.3-kb EcoRI fragment which is present in all TFE3 cosmids identified, hybridizes to aberrant restriction fragments in th ree independent t(X;1)-positive renal cell carcinoma DNAs. The breakpo ints in these tumors are not the same, but map within a region of appr oximately 6.5 kb. Through preparative gel electrophoresis an (X;1) chi maeric 4.4-kb EcoRI fragment could be isolated with encompasses the br eakpoint region present on der(X). Preliminary characterization of thi s fragment revealed the presence of a 150-bp region with a strong homo logy to the 5' end of the mouse TFE3 cDNA in the X-chromosome part, an d a 48-bp segment in the chromosome 1-derived part identical to the 5' end of a known EST (accession number R93849). These observations sugg est that a fusion gene is formed between the two corresponding genes i n t(X;1)(p11;q21)-positive papillary renal cell carcinomas.