Somatic cell hybrids were derived by fusion of mouse erythroleukemia cells with fractionated human marrow enriched in erythroblasts, or with chinese hamster fetal liver erythroid cells. Such interspecific hybrid cells, when isolated in suspension culture, had retained nearly all the mouse chromosomes and had lost most of the human or chinese hamster chromosomes. However, two such hybrids (one human, the other hamster) studied 4-6 weeks after fusion, were found to contain several non-mouse chromosomes. RNA extracted from the human marrow X erythroleukemia hybrid annealed equally to both human and mouse globin complementary DNA, indicating that coexpression of the globin genes of each species had occurred in the hybrid cells. Mouse Hybrids between somatic cells provide an experimental system in which the coexpression of specific structural genes of two parent cell types can be examined (1-3). To study the regulation of the globin genes, we have used cell hybrids formed with a murine erythroleukemia line. Induction of mouse hemoglobin synthesis by dimethylsulfoxide (Me2SO) has been observed in a number of these erythroleukemia cell lines (4-6), but fusion of erythroleukemia cells with fibroblasts of mouse or human origin produces hybrid cells in which extinction of globin mRNA synthesis has occurred at the level of transcription or mRNA processing (7-9). We have previously described a hybrid cell line, formed by fusion of human bone marrow to erythroleukemia cells (7), which had a low level of constitutive hemoglobin synthesis that was markedly amplified on exposure of the hybrid cells to Me2SO. This line, when studied 10 weeks after fusion, retained an average of 1 to 3 human chromosomes per metaAbbreviations: cDNA, complementary DNA; CM-cellulose, carboxymethyl cellulose; Me2SO, dimethylsulfoxide; HAT medium, F-14 medium supplemented with aminopterin, hypoxanthine, and thymidine as formulated by Littlefield (10); G-6-P dehydrogenase, glucose-6-phosphate dehydrogenase; 6-P-GA dehydrogenase, 6-phosphogluconate dehydrogenase; 1S = one diploid set of chromosomes; 2S = two diploid sets of chromosomes; CH, chinese hamster. *This is paper II of a series. Paper I is ref. 7. phase, and only mouse globin was produced. In this paper, we report further studies of interspecific somatic cell hybrids derived from fusion of the Friend mouse erythroleukemia cell with hematopoietic cells of human and chinese hamster origin. Two hybrid lines containing several nonmouse chromosomes exhibited co-expression of the mouse and hamster, or mouse and human globin genes on exposure of hybrid cells to Me2SO. METHODSCell Fusions. A 6-thioguanine-resistant clone of the Friend mouse erythroleukemia cell line (clone 745) was used. This line had a spontaneous reversion frequency of less than 10-8 (7), even under fusion conditions. Cells from human marrow aspirates were enriched in erythroid cells by centrifugation through a discontinuous albumin gradient (15-30%) to reduce the number of nonnucleated cells and then fractionated by gra...
Hemoglobin switching, which occurs in all classes of vertebrates as well as in certain invertebrates, is due to developmental regulation of different globin genes which are typically arranged in clustered families. By fusing erythroid cells of different developmental programs, trans-acting factors that regulate this switch in gene expression have been detected [Ramseyer et al. (1989): Dev Biol 133:262-271]. Adult erythroid cells of one anuran species, Xenopus laevis, were fused with tadpole erythroid cells of another frog, Rana catesbeiana, creating developmental erythroid heterokaryons that synthesize adult Rana globin mRNA and hemoglobins. The results show that factors from adult Xenopus erythroid cells are capable of inducing adult Rana globin gene expression in the Rana tadpole erythroid cell nucleus. We have used the cross-induction of adult Rana hemoglobin synthesis in these adult Xenopus/Rana tadpole erythroid heterokaryons to address two practical questions, answers to which may be helpful in isolating developmental stage-specific globin gene regulatory proteins: 1) Are erythroblasts which are actively expressing globin mRNAs and hemoglobins richer in specific globin-inducing activities than other stages of erythroid cellular differentiation? 2) Do mature, circulating erythrocytes still have the activities necessary to mediate the cross-induction of Hb synthesis? The results reported here show that the answers to both questions are affirmative and show that quiescent, fully differentiated adult erythroid cells are still capable of expressing the trans-activator(s). These findings show that factors which mediate the metamorphic hemoglobin switch are conserved between these two genera of frogs.
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