Cloning of cDNA from induced K562 cells which can activate globin gene expression

Abstract

A cDNA library from induced K562 cells was constructed and differentially screened for the isolation of clones encoding trans-acting factors which increase globin gene expression. The current study assumes that induced K562 cells contain transcriptionally active factors specific for globin genes which are absent or present only at very low levels in uninduced K562 cells. Upon screening the recombinant library, 75 cDNA clones hybridized specifically with cDNA probes from induced K562 cells and hybridized only slightly or not at all with cDNA probes from uninduced K562 cells and HL-60 cells, or from globin genes. Forty-five of the cDNA clones were full length complements to the corresponding RNA. To screen for trans-acting factors which can activate globin gene expression, the cDNA clones were inserted into a eukaryotic expression vector and co-transfected into HeLa cells with another vector containing the epsilon globin promoter 5' to the bacterial CAT (chloramphenicol acetyl transferase) reporter gene. Partial screening of the 45 full length clones revealed one cDNA (clone #17) that was able to increase CAT activity (driven by the epsilon-globin promoter) by about 2.5 times. This cDNA is 522 nucleotides in length and contains a long open reading frame. Examination with known DNA sequences indicates greater than 95% homology with the ferritin heavy chain.