Differential expression of alpha- and beta-globin genes in erythroleukemic cell lines.

The IW32, NN10, and IW201 cell lines are erythroleukemic cell lines isolated from the spleens of mice infected with the Friend virus. IW32 and NN10 cells can be induced toward erythroid differentiation and hemoglobin synthesis by hemin or butyrate. Both cell lines contain some mature alpha- and beta-globin mRNA before induction, and addition of the inducers greatly increases the amount of globin message. Unlike IW32 and NN10 cells, IW201 cells are only partially inducible. Uninduced 201 cells contain a small amount of alpha-globin mRNA but no detectable beta-globin message. After induction, the cells contain markedly increased amounts of alpha-globin mRNA but still do not express the beta-globin gene. Southern blot analysis with 10 restriction enzymes shows that the restriction map of the beta-globin gene in IW201 cells is indistinguishable from that in IW32 and NN10 cells.     

Regulated expression of the c-myb and c-myc oncogenes during erythroid differentiation

We have investigated the expression of the genes c-myb, c-myc, and alpha globin in murine erythroid cells at different stages of development, in viral-induced erythroleukemias, as well as in two mouse erythroleukemia cell lines that can be induced to terminally differentiate when exposed to dimethylsulfoxide. We find that there is a reciprocal correlation between the cell's production of messenger RNA for c-myb and globin. c-myc message shows a similar but less dramatic decrease coincident with globin RNA production. Initially with the administration of an inducing agent, dimethylsulfoxide, there is a rapid decrease of myc and myb mRNA, which is followed by signs of differentiation in the induced culture. We conclude that these oncogenes function in early maturational stages of development of these cells. In the erythroleukemic state these genes are down-regulated by forced differentiation and may play a direct role in influencing the state of differentiation of these cells.     

The isolation of the beta A-, beta C-, and gamma-globin genes and a presumptive embryonic globin gene from a goat DNA recombinant library

As an approach to understand how the expression of globin genes are regulated during development, clones containing globin DNA sequences were selected from a recombinant library of goat genomic DNA. The type of globin gene present in each of the recombinants was determined by cross-hybridization to the DNA of mouse alpha- and beta-globin cDNA-containing plasmids. Of 11 clones isolated, eight hybridized specifically to the DNA of the mouse beta-globin plasmid, while one clone hybridized only to the DNA of the alpha globin plasmid. The location of each globin sequence within its DNA insert was determined by a combination of restriction enzyme mapping and Southern transfer-hybridizations. Selected fragments were sequenced; comparisons of the amino acids coded for by these regions with those of the goat globins identified clones carrying beta A-, beta C-, and gamma-globin genes. Another recombinant coded for amino acid sequences resembling, but not identical with, the known goat globins, and was identified tentatively as containing an embryonic or epsilon-gene. Detailed analysis of the clone containing the beta C gene and an overlapping clone revealed that three other beta-like sequences are located 6, 12, and 21 kilobases on the 5'-side of the beta C gene. The globin sequence of the locus nearest to the beta C gene has an altered translation termination codon and, if transcribed and translated, would give a globin chain seven amino acids longer than the normal goat beta C-globin. In addition, the sequence following this termination codon is very AT-rich, unlike that of other globin genes. The recombinants described contain extensive regions of DNA surrounding the globin genes, making them useful for identifying regulatory sequences as well as determining the sequence organization of the goat globin genes.     

Expression of chimeric human beta- and delta-globin genes during erythroid differentiation

To determine whether sequences contained within the small intervening sequence (IVS 1) or large intervening sequence (IVS 2) are involved in the regulated expression of the human beta-globin gene, chimeric genes containing portions of the human beta- and delta-globin genes were stably transfected into mouse erythroleukemia (MEL) cells. Since MEL cells can be induced to differentiate in culture, the expression of the chimeric genes was compared to the expression of beta and delta both before and after the induction of erythroid differentiation. The expression of beta delta 1, a beta-globin gene containing delta IVS 1 in place of beta IVS 1, was comparable to the expression of a beta-globin gene both before and after erythroid differentiation. However, the base-line expression of human beta-globin genes containing delta IVS 2 in place of beta IVS 2 was dramatically decreased. Furthermore, the substitution of delta IVS 2 for beta IVS 2 prevented the regulated increase in expression of the beta-globin gene upon induction. The results also indicate that sequences present in beta IVS 2 are not sufficient for this induced increase in expression since the substitution of beta IVS 2 for delta IVS 2 in a delta gene does not increase the regulated expression of delta during differentiation. These experiments suggest that either the presence of delta IVS 2 in a beta gene interrupts sequences required for the induced expression of beta-globin or that sequences in beta IVS 2 act in concert with other beta globin sequences not present in the delta-globin gene to permit optimal expression.     

Expression of alpha- and beta-globin genes occurs within different nuclear domains in haemopoietic cells

The alpha- and beta-globin gene clusters have been extensively studied. Regulation of these genes ensures that proteins derived from both loci are produced in balanced amounts, and that expression is tissue-restricted and specific to developmental stages. Here we compare the subnuclear location of the endogenous alpha- and beta-globin loci in primary human cells in which the genes are either actively expressed or silent. In erythroblasts, the alpha- and beta-globin genes are localized in areas of the nucleus that are discrete from alpha-satellite-rich constitutive heterochromatin. However, in cycling lymphocytes, which do not express globin genes, the distribution of alpha- and beta-globin genes was markedly different. beta-globin loci, in common with several inactive genes studied here (human c-fms and SOX-1) and previously (mouse lambda5, CD4, CD8alpha, RAGs, TdT and Sox-1), were associated with pericentric heterochromatin in a high proportion of cycling lymphocytes. In contrast, alpha-globin genes were not associated with centromeric heterochromatin in the nucleus of normal human lymphocytes, in lymphocytes from patients with alpha-thalassaemia lacking the regulatory HS-40 element or entire upstream region of the alpha-globin locus, or in mouse erythroblasts and lymphocytes derived from human alpha-globin transgenic mice. These data show that the normal regulated expression of alpha- and beta-globin gene clusters occurs in different nuclear environments in primary haemopoietic cells.     

Expression of the human beta-globin gene after retroviral transfer into murine erythroleukemia cells and human BFU-E cells.

Replication-defective amphotropic retrovirus vectors containing either the human beta-globin gene with introns or an intronless beta-globin minigene were constructed and used to study beta-globin expression following gene transfer into hematopoietic cells. The beta-globin genes were marked by introducing a 6-base-pair insertion into the region corresponding to the 5' untranslated region of the beta-globin mRNA to allow detection of RNA encoded by the new gene in human cells expressing normal human beta-globin RNA. Introduction of a virus containing the beta-globin gene with introns into murine erythroleukemia cells resulted in inducible expression of human beta-globin RNA and protein, while the viruses containing the minigene were inactive. The introduced human beta-globin gene was 6 to 110% as active as the endogenous mouse beta maj-globin genes in six randomly chosen cell clones. Introduction of the viruses into human BFU-E cells, followed by analysis of marked and unmarked globin RNAs in differentiated erythroid colonies, revealed that the introduced beta-globin gene was about 5% as active as the endogenous genes in these normal human erythroid cells and that again the minigene was inactive. These data are discussed in terms of the potential treatment of genetic disease by gene therapy.     

Expression of adult and tadpole specific globin genes from Xenopus laevis in transgenic mice.

Transgenic mice were generated which carried the adult alpha and beta-globin genes and the major tadpole specific beta-globin gene of Xenopus laevis. The adult specific alpha and beta genes were found to express in erythroid tissues in adult mice, while the major tadpole specific beta gene (beta T1) was expressed in blood from 12.5 day embryos. The pattern of expression of the beta T1 gene during mouse development was consistent with its being regulated as an embryonic globin gene in the mouse. This observation suggests that some of the factors mediating globin switching have been conserved during the evolution of modern amphibia and mammals and raises interesting questions concerning the evolution of vertebrate globin gene switching.     

Analysis of the ratio of alpha- to beta-globin and globin messenger ribonucleic acid content of fractionated rabbit erythroid bone-marrow cells.

The ratio of alpha- to beta-globin mRNA was measured by hybridization of a constant amount of highly purified alpha- or beta-globin cDNA (complementary DNA) with increasing amounts of RNA in the range up to 20% cDNA hybridization, where an essentially linear reaction is obtained. Statistical analysis indicates that the ratio of alpha- to beta-globin can be measured within a maximal error of +/- 0.3 and in most cases is better than +/- 0.15. Under these conditions there is no significant deviation from the ratio of 1.3 in the alpha- to beta-globin mRNA ratio of RNA isolated from erythroid cells rich in pronormoblasts through to reticulocytes. If the ratio of alpha- to beta-globin mRNA exceeded 1.7 or was less than 0.9 in pronormoblasts, it would be detected in these experiments. The overall globin mRNA content increases to a maximal value in the fractions rich in basophilic normoblasts of 30,000--50,000 molecules/cell. However, the accuracy of these determinations is not as great as for the ratio determinations, and no significant deviations were seen except in the cells rich in pronormoblasts, which contained less globin mRNA than the later stages.     

Expression of the human gamma-globin gene after retroviral transfer to transformed erythroid cells.

Regulation of the human fetal (gamma) globin gene and a series of mutant gamma-globin genes was studied after retroviral transfer into erythroid cells with fetal or adult patterns of endogenous globin gene expression. Steady-state RNA from a virally transferred A gamma-globin gene with a normal promoter increased after induction of erythroid maturation of murine erythroleukemia cells and comprised from 2% to 23% of the mouse beta maj-globin RNA level. RNA expression from the virally transferred A gamma-globin gene comprised 23% of the endogenous G gamma- + A gamma-globin expression in K 562 cells after treatment with hemin. Expression from a virally transferred gamma- or beta-globin gene exceeded endogenous gamma- or beta-globin expression by a factor of 6 or more in the human erythroleukemia line KMOE, in which the endogenous globin genes are weakly inducible. In these experiments, no difference in expression was observed between the gene with the normal promoter and an A gamma-globin gene with a point mutation in its promoter (-196 C-to-T) that has been associated with hereditary persistence of fetal hemoglobin (HPFH). To test for cis-acting determinants located within the introns of the gamma-globin gene, expression was measured from a set of gamma-globin genes configured with either intron alone or with neither intron. In contrast to an intronless beta-globin gene, which is not expressed in MEL cells, the intronless gamma-globin gene was expressed in MEL cells at 24% of the level of an intron-containing gene.(ABSTRACT TRUNCATED AT 250 WORDS)