Structural And Functional Analysis Of The Chicken BRCA2 Gene
Mutations in the BRCA2 gene confer a high risk of breast and other cancers. Here the chicken BRCA2 gene, the first non-mammalian BRCA2 gene to be isolated, has been cloned and characterised. The gene spans approximately 40kb of genomic DNA and is localised to the subtelomeric region of chromosome 1q, in a region that contains other genes found on human chromosome 13. Evolutionary analysis indicates that this is the true orthologue of mammalian BRCA2. Further putative BRCA2 related genes in lower species have been discovered by sequence alignment. The cDNA for the entire 3399 amino acid chicken BRCA2 protein has also been sequenced. The amino acid sequence is poorly conserved with mammalian proteins, and overall has only 37% amino acid identity, implying that the BRCA2 gene is evolving at a rapid rate. However, certain regions are well conserved: exon 11; exons 2 and 3; a C-terminal region spanning exons 17-20 (which we have named the BLAT domain); and the nuclear localisation sequence in exon 27. Functional assays of these regions show conservation of function of these motifs. The evolutionarily divergent chicken BRCA2 sequence may be useful in reclassifying the unassigned missense variants described in the human BRCA2 gene.
The BRCA2 gene has been disrupted in the highly recombinogenic chicken cell line, DT40. Using a direct approach, double targeting of these cells generated clones partially trisomic for the BRCA2 gene, in which the wild type copy had been reduplicated, suggesting that BRCA2 is essential for viability in DT40 cells. Conditional targeting strategies yielded similar results. However, heterozygosity for a BRCA2 mutation has a distinct phenotype characterised by a reduced growth rate and heightened sensitivity to specific DNA damaging agents. The possibility that in certain cell types, tumorigenic progression might be driven directly by heterozygosity for a BRCA2 mutation is discussed.