Purification And CDNA Cloning Of Erythroid Differentiation And Denucleation Factor(EDDF)

Being the only functional cells without nuclears in organism, the mammalian erythroblasts with unique features of the expression of hemoglobin and apperance of nuclear pyknosis and denucleation at terminal differentiaton have been considered to be a good model and ideal materials for the study of cell differentiation. Mammalian erythropoiesis is a process of proliferation of progenitor or multipotential stem cells towards differentiation along a prospective sequential gene expression program under the coordination of activation or suppression of a series of cytokines. Some of these factors play a role in the commitment to determination of hematopoietic stem cells(such as SCF, IL-3 etc.) and the others to the proliferation of early erythroid cell lineage (such as Epo, GM-CSF etc). However, it is still a moot point at present of what factor is responsible for the turnover from proliferation towards terminal differentiation, especially the regulating factor for the process of terminal differentiation.Based on the above features of terminal differentiation and the abscence of these features duringerythroleukemia oncogenesis. We have focused our research on the study of the mechanism of erythroid differentiation and oncogenesis as well as the regulation of malignancy, which include studying the mechanism of denucleation and the regulation of globin gene and oncogene expression during erythroid terminal differentiation. As (?) erythroid denucleation occurred only in mammalian erythrocytes but not in other vertebrate animals, we have postulated the hypothesis of existence of an erythroid differentiation factor which functions to suppress nuclear activity and lead to the denucleation in terminal differentiation, and must be genarated evaluationally during phylogenesis in mammalian erythropoiesis. Through a series of repeated hybridization experiments by using reticulocytes fusion with murine and human mylomal cell lines, we have provided evidence for the existence of a differentiation factor which regulate malignant mitosis and gene expression of mylomal cells/erythroleukemia cells. This factor which is named erythroid differentiation—denucleation factor(EDDF) could alter the ultrastructure of nuclear matrix-intermediate filament(MN-IF) system and suppress the gene expression of vimentin and c-myc genes, but activate the globin gene expression. The above results provide a solid basis for our ongoing projects to purify EDDF from erythropoietic tissues.As the spleen is one of the hematopoietic organs in erythropoiesis, in the present study, the splenic erythroblasts obtained from Balb/c mice infected with FVA by Koury's techniques which cell numbers increase more then tenfold were used to produce sufficient materials for the purification of EDDF. Bioassays, sequence analysis and m(?)lecular cloning etc. were also carried out. The results were as follows:...