ISOLATION AND CHARACTERIZATION OF TARGET SEQUENCES OF THE CHICKEN CDXA HOMEOBOX GENE

The DNA binding specificity of the chicken homeodomain protein CDXA wa s studied. Using a CDXA-glutathione-S-tranferase fusion protein, DNA f ragments containing the binding site for this protein were isolated. T he sources of DNA were oligonucleotides with random sequence and chick en genomic DNA. The DNA fragments isolated were sequenced and tested i n DNA binding assays. Sequencing revealed that most DNA fragments are AT rich which is a common feature of homeodomain binding sites. By ele ctrophoretic mobility shift assays it was shown that the different tar get sequences isolated bind to the CDXA protein with different affinit ies. The specific sequences bound by the CDXA protein in the genomic f ragments isolated, were determined by DNase I footprinting. From the f ootprinted sequences, the CDXA consensus binding site was determined. The CDXA protein binds the consensus sequence A, A/T, T, A/T, A, T, A/ G. The CAUDAL binding site in the ftz promoter is also included in thi s consensus sequence. When tested, some of the genomic target sequence s were capable of enhancing the transcriptional activity of reporter p lasmids when introduced into CDXA expressing cells. This study determi ned the DNA sequence specificity of the CDXA protein and it also shows that this protein can further activate transcription in cells in cult ure.