IDENTIFICATION OF NEW GENES EXPRESSED IN A HUMAN ERYTHROLEUKEMIA CELL-LINE

We cloned novel cDNAs from MB02 human erythroleukemia cells using PCR based approaches: a) Differential display by means of RT-PCR using one 5' primer CTTGATTGCC and four different 3' primers (T(12)AA, T(12)CA, T(12)GA, and T(12)AT). Ninety-three percent of the differential clone s which were reamplified and sequenced were cDNAs of previously uniden tified genes. b) Cloning using degenerate TFIIIA-like zinc finger doma in specific oligonucleotide, Of the 54 clones sequenced, 20 contained two or more zinc finger sequences. Ten of these clones were new zinc f inger cDNAs and one belonged to a known zinc finger gene (ZFP7), c) Cl oning using degenerate tyrosine kinase(TK) domain-specific oligonucleo tides corresponding to the highly conserved amino acid sequences IHRDL AA and DVWSFG, Of the 28 cDNA clones sequenced, 7 were JAK1 TK, one wa s atk TK, one was tec TK. The remaining sequences belonged to new EST' s or to ribosomal genes. d) Cloning using degenerate POU domain-specif ic oligonucleotides corresponding to sequence FK(QV)RRIK of the POU-sp ecific domain and to sequence VWFCN(RQ)R of the POU-homeodomain. Sixte en clones were sequenced and all but one were identical with the Oct-1 transcriptional factor, Differential display RT-PCR and PCR-based cDN A cloning using degenerate primers for zinc finger motifs yielded the largest number of new genes expressed in MB02 cells.