The specific transcription of human globin genes may involve a complex interaction of a variety of factors including trans-acting factors, none of which has thus far been completely characterized. The K562 human erythroleukemia cell line can serve as a model for the study of globin gene expression. The goal of the present study is trying to clone and characterize such factors. The current study assumes that induced K562 cells contain transcriptional factors specific for embryonic and fetal globin genes, which are absent or present only at very low levels in uninduced K562 cells. For isolation of cDNA clones encoding the trans-acting factors, a cDNA library from mRNA of induced K562 cells, consisting of 450,000 independent recombinants, was constructed and 150,000 recombinants has been differentially screened. Seventy-five cDNA clones were found to hybridize only with cDNA probes from induced K562 cellular RNA and further examined by hybridization with Northern blot of induced and uninduced K562 cellular RNA. Forty-five cDNA clones have shown full length complements to the corresponding RNA. To determine wnether some of the 45 cDNA encode trans-acting factors required to activate epsilon- or gamma-globin gene promoter, those cDNA have been inserted into Okayama-Berg expression vector and co- transfected into HeLa cells with another expression vector which contains epsilon promoter or gamma promoter and CAT gene or human growth hormone gene. One cDNA (No #17) has shown to be able to increase CAT gene expression about 2.5 times. This cDNA has been sequenced and is 522 nucleotides in length and contains an open reading from of 282 nucleotides. A search of the NBRF protein database revealed that this protein is not homologous to any known protein sequences. It is possible that the #17 cDNA encodes a protein or a part of a protein unknown before and involved in activation of epsilon-globin gene promoter. We are now continuing to examine the remaining cDNA clones using the same strategy.
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