Assignment of human X-linked genes to a zebra finch microchromosome by in situ hybridization of BAC clones

Probes and fl uorescent in situ hybridization (FISH) Zebra fi nch BAC clones were isolated from a zebra fi nch BAC library (www.genome.arizona.edu) (Luo et al., 2006). BAC clones were identifi ed by probing membranes spotted with the zebra fi nch clones with cDNAs encoding DCX (GenBank DQ189989) and SOX3 (GenBank DQ206644), and further confi rmed by probing Southern blots of the identifi ed clones after restriction digestion. Confi rmed BAC clones were: 018F09, 026M08, 048L06, 048O02, 153A11, 170M21 and 170M22 (SOX3) ; 350D21 (DCX) . The BAC clones used as probes here are 319A15 (AR; Luo et al., 2005), 350D21 (DCX) , and 048O02 (SOX3) . FISH to mitotic chromosomes prepared from zebra fi nch fi broblasts was performed as previously described (Itoh and Arnold, 2005). Hybridization of biotinylated BAC probes was detected by a series of reactions with FITC-labeled avidin (Vector Labs), biotinylated goat anti-avidin antibody (Vector Labs) and FITC-labeled avidin. Double labeling was accomplished with additional DIG labeled probes detected with sheep anti-DIG rhodamine (Roche), rabbit anti-sheep rhodamine (Chemicon) and goat anti-rabbit rhodamine (Chemicon). The chromosomes were counterstained with DAPI.