Cloning and sequence analysis of homeobox transcription factor cDNAs with an inosine-containing probe

Much effort has been directed toward the isolation and characterization of homeobox cDNAs from numerous cell types because they encode transcription factors important to many cellular processes, including pattern formation in the embryo, cell growth and cell differentiation. Many novel homeobox cDNAs have been isolated by screening libraries by hybridization with degenerate oligonucleotides designed from conserved amino acid sequences in the third helix of the homeodomain. However, the degeneracy of the genetic code necessitates that these oligonucleotides be highly degenerate, often precluding their use as sequencing primers to rapidly determine clone identity. Here we describe a screening protocol for homeobox cDNAs that utilizes a short oligonucleotide probe with inosine residues incorporated at positions of maximum codon degeneracy. This probe specifically hybridizes to many classes of homeobox transcription factor cDNAs, but its primary advantage is that it also serves as an effective sequencing primer, which allows the investigator to rapidly determine whether the clones encode a protein of interest. 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 contain homeobox cDNAs representing 5 distinct genes, and, using this probe, it was possible to obtain initial high-quality sequence information from every clone isolated that contained a homeodomain.