CLONING AND SEQUENCE-ANALYSIS OF HOMEOBOX TRANSCRIPTION FACTOR CDNAS WITH AN INOSINE-CONTAINING PROBE

Much effort has been directed toward the isolation and characterizatio n of homeobox cDNAs from numerous cell types because they encode trans cription factors important to many cellular processes, including patte rn formation in the embryo, cell growth and cell differentiation. Many novel homeobox cDNAs have been isolated by screening libraries by hyb ridization with degenerate oligonucleotides designed from conserved am ino acid sequences in the third helix of the homeodomain. However, the degeneracy of the genetic code necessitates that these oligonucleotid es be highly degenerate, often precluding their use as sequencing prim ers to rapidly determine clone identity. Here we describe a screening protocol for homeobox cDNAs that utilizes a short oligonucleotide prob e with inosine residues incorporated at positions of maximum codon deg eneracy. This probe specifically hybridizes to many classes of homeobo x transcription factor cDNAs, but its primary advantage is that it als o serves as an effective sequencing primer which allows the investigat or to rapidly determine whether the clones encode a protein of interes t. In a screen of 500 000 plaques of a rat aorta cDNA library by this method, we identified 13 positive plaques of which 12 were found to co ntain homeobox cDNAs representing 5 distinct genes, and using this pro be, it was possible to obtain initial high-quality sequence informatio n from every clone isolated that contained a homeodomain.