Humans undergo two developmental switches in their hemoglobin phenotype. The embryonic to fetal switch early in gestation and tne fetal to adult switch around the time of birth. The K562 human leukemia cell line expresses all globin genes other than the adult beta-globin. Previous work from this laboratory has shown that the K562 beta-globin gene functions normally in a heterologous expression system. Elucidation of the mechanism of failure of beta-globin gene expression in K562 cells may provide an insight into globin gene expression and switching in normal erythroid cells. A stable transformant system has been developed to permit the localization of sequences conferring tissue specificity to the upstream region of globin genes. It will also enable the in vivo titration of putative regulatory factor(s), thereby rigorously demonstrating the functional significance of certain sequences. The direct isolation of trans-activating gene(s) will be attempted using the strategy that led to the isolation of several oncogenes. Hybrid beta-neo plasmids, which are expressed at a low level in K562 cells, will be co-transfected with another selectable marker (the MDR gene). Selection will be for the latter, followed by the identification of clones containing non-expressing beta-neo plasmid. K562 and MEL cell genomic DNA will be transfected into these cells and activation of beta-neo sought. A fractionation and/or """"""""rescue"""""""" strategy will be employed to isolate the gene(s) of interest. Known trans-acting factors such as TAT-1 of HTLV-1, the T antigen of SV40 virus and the E1a gene product of adenovirus will be studied in stable transformants. A new cell line with a predominantly fetal phenotype has been characterized. Since no suitable human cell lines expressing beta globin are available, we will attempt to establish such lines from human bone marrow cells using oncogenes and/or origin deficient SV40 virus DNA.
