Globin gene expression in cultured erythroleukemic cells

Abstract

A cultured, murine erythroleukemic cell line, which initially contains no detectable hemoglobin, can be induced to synthesize hemoglobin in quantities comparable to those found in normal red blood cells. In order to distinguish between several molecular mechanisms which might explain this induction, radioactive DNA complementary to mouse globin messenger RNA was used as a hybridization probe to measure globin genes and mRNA quantitatively during this form of differentiation. The results indicate that the number of globin genes does not change as these cells accumulate hemoglobin and that there are less than five copies of the globin genes per haploid genome. On the other hand, differentiated cells accumulate, on the average, 7000 to 8000 molecules of globin mRNA per cell, compared with less than ten in each undifferentiated cell. The accumulation of globin mRNA ceases in the presence of actinomycin D, suggesting that it is dependent on de novo RNA synthesis. It is also inhibited by cycloheximide and puromycin, suggesting a requirement for continued protein synthesis. Although a mechanism involving the post-transcriptional stabilization of newly synthesized globin mRNA can not be ruled out, these results are most simply explained on the basis of transcriptional activation of globin genes. Further data suggest that the globin mRNA synthesized in these erythroleukemic cells is relatively stable (chemical half-life more than 10 h) and is indistinguishable from authentic reticulocyte globin mRNA. The amount of globin mRNA, synthesized at the rate of approximately 20 nucleotides per second, has been measured and found comparable to the amount in mouse reticulocytes.