Hexamethylenebisacetamide-mediated induction of murine erythroleukemia cells: relationship between expression of beta major and beta minor globin genes, globin mRNA synthesis and commitment to erythroid differentiation

A switch in beta globin gene expression is operated in murine Friend erythroleukemia cells due to the inducing agent used. The competence of Friend cells to express beta major globin genes is operated within 8 hours exposure to hexamethylenebisacetamide. This early feature of induced differentiation is expressed in the absence of beta globin mRNA synthesis and is not suppressed by the corticosteroid hormone dexamethasone, which by contrast inhibits later stages of induced-mediated commitment to erythroid differentiation such as globin mRNA accumulation and heme synthesis.     

Analysis of murine S-glutathionyl hemoglobins and beta globin haplotype by dynamic capillary isoelectric focusing

Genetic variation in the number and reactivity of beta globin sulfhydryl groups causes variation in erythrocyte redox status in mouse populations. These experiments demonstrate the use of capillary isoelectric focusing for measuring endogenous S-glutathionyl hemoglobin and identifying mouse beta globin (Hbb) haplotype in inbred and outbred mouse strains with mono-cysteinyl or di-cysteinyl beta globins. Hbb haplotype can be readily determined in all strains based on characteristic differences in peak profiles or on peak mobility shift induced by thiol exchange with glutathione disulfide in vitro. This method could prove useful for in vivo study of factors that influence thiol protein modification.     

Hypersensitive site 4 of the human beta globin locus control region.

The Locus Control Region (LCR) of the human beta globin gene domain is defined by four erythroid-specific DNasel hypersensitive sites (HSS) located upstream of this multigene cluster. The LCR confers copy number dependent high levels of erythroid specific expression to a linked transgene, independent of the site of integration. To assess the role of the individual hypersensitive sites of the LCR, we have localized HSS4 to a 280bp fragment that is functional both in murine erythroleukaemia (MEL) cells and in transgenic mice. This fragment coincides with the major area of hypersensitivity 'in vivo' and contains a number of DNasel footprints. Bandshift analysis shows that these footprints correspond to binding sites for the erythroid specific proteins GATA1 and NF-E2 and a number of ubiquitous proteins, including jun/fos, Sp1 and TEF2.     

Transcriptional and translational control of the biphasic increase in ornithine decarboxylase activity in liver

The requirements for protein and RNA synthesis for each of the two increases in liver ornithine decarboxylase activity after the injection of unoperated rats with a solution containing glucagon and after 70% hepatectomy were studied with cycloheximide and actinomycin D. Protein synthesis is required for both increases whereas RNA formation is essential for the first elevation only. The second increase appears to be dependent upon RNA that is made during the period of the first rise in activity.The two rises in the decarboxylase activity may be caused by different stimuli. After the injection of the mixture with glucagon, the first elevation is accompanied by increases in hepatic tyrosine aminotransferase activity and in the rate of transport into liver cells of the model amino acid, α-aminoisobutyrate. Neither an increase in the aminotransferase activity nor in amino acid uptake occurs, however, during the period of the second elevation in the decarboxylase activity.     

Rapid induction of thymosin beta 4 in concanavalin A-stimulated thymocytes by translational control

The expression of thymosin beta 4, an ubiquitous peptide of high cellular content, was studied in concanavalin A-stimulated rat thymocytes within the first 3 h after activation of the cells. An early 6.3-fold increase of the peptide occurred after 1 h of stimulation amounting to 0.4% of the total cellular protein. This increase coincided with that of thymosin beta 4 biosynthesis measured by [35S]methionine incorporation. The share of thymosin beta 4 synthesis in total protein synthesis 1 h after addition of concanavalin A amounts to 1% but no elevation of the corresponding mRNA was observed. These data suggest that a translational control mechanism is involved in this rapid induction. Consequently, actinomycin D did not inhibit thymosin beta 4 induction in contrast to cycloheximide. The peaks of maximal thymosin beta 4 levels and biosynthesis were followed by rapid decreases of these parameters suggesting a function of thymosin beta 4 in the early phase of T cell activation.     

Differential expression of mouse beta/goat beta c, mouse beta/goat beta F, and mouse beta/goat epsilon II hybrid globin genes in murine erythroleukemia cells.

We assembled three hybrid beta-globin genes by fusing the mouse beta-major promoter and initial transcribed region to one of three goat beta-like globin gene bodies: beta c (preadult), beta F (fetal), or epsilon II (embryonic). Thymidine kinase (tk)-deficient murine erythroleukemia (MEL) cells were cotransformed with one of these constructs and a separate plasmid bearing the tk gene. Half of the 24 cell lines containing either the mouse beta/goat beta c or mouse beta/goat beta F genes expressed the transferred genes at significant levels; in many cases the hybrid genes were, like the endogenous beta-globin genes, inducible with dimethyl sulfoxide. We obtained 13 cell lines containing the mouse beta/goat epsilon II hybrid gene, 6 of which were cotransfected with a mouse beta/human beta fusion gene known to function in MEL cells. In contrast to the results with the other fusion genes, the mouse beta/goat epsilon II hybrid was very poorly expressed: in two separate experiments, 0 of 13 and 2 of 13 lines showed significant mouse beta/goat epsilon II RNA levels after induction. In all these lines the endogenous mouse beta and cotransfected mouse beta/human beta genes were expressed. As an initial test of possible reasons for the inactivity of the mouse beta/goat epsilon II hybrid, we recloned this fusion gene into a tk-bearing plasmid, adjacent to the tk gene. Of 12 cell lines transformed with this plasmid, 11 produced mouse beta/goat epsilon II RNA; in 6 cases the expression was both strong and dimethyl sulfoxide inducible.     

Rabbit globin pseudogene psi beta 2 is a hybrid of delta- and beta-globin gene sequences

The evolutionary history of the rabbit globin pseudogene psi beta 2 was studied by completing its nucleotide sequence and aligning the sequence with that of the rabbit adult globin gene beta 1 and the human minor adult globin gene delta. The 5' flanking region and exon 1 of psi beta 2 were most similar to rabbit beta 1, but the large intervening sequence and the 3' untranslated region were most similar to human delta. Intron 1 and exon 2 were equally similar to both delta and beta 1. This pattern indicates that psi beta 2 was originally a delta-like gene that acquired the 5' portion of gene beta 1 by intrachromosomal gene conversion. The presence of a delta-globin gene sequence in both rabbits and humans shows that it is an ancient gene, predating the mammalian radiation that occurred over 85 Myr ago. Delta has shown a pronounced tendency to be altered in its 5' end during the course of mammalian evolution. Quantitative divergence analysis shows that the ancestor to rabbit psi beta 2 was active until 20-30 Myr ago, during which time the lagomorph beta-globin gene family apparently functioned without a pseudogene.      

Protein synthesis and the cell cycle: centrosome reproduction in sea urchin eggs is not under translational control

The reproduction, or duplication, of the centrosome is an important event in a cell's preparation for mitosis. We sought to determine if centrosome reproduction is regulated by the synthesis and accumulation of cyclin proteins and/or the synthesis of centrosome-specific proteins at each cell cycle. We continuously treat sea urchin eggs, starting before fertilization, with a combination of emetine and anisomycin, drugs that have separate targets in the protein synthetic pathway. These drugs inhibit the postfertilization incorporation of [35S]methionine into precipitable material by 97.3-100%. Autoradiography of SDS-PAGE gels of drug-treated zygotes reveals that [35S]methionine incorporates exclusively into material that does not enter the gel and material that runs at the dye front; no other labeled bands are detected. Fertilization events and syngamy are normal in drug-treated zygotes, but the cell cycle arrests before first mitosis. The sperm aster doubles once in all zygotes to yield two asters. In a variable but significant percentage of zygotes, the asters continue to double. This continued doubling is slower than normal, asynchronous between zygotes, and sometimes asynchronous within individual zygotes. High voltage electron microscopy of serial semithick sections from drug-treated zygotes reveals that 90% of the daughter centrosomes contain two centrioles of normal appearance. From these results, we conclude that centrosome reproduction in sea urchin zygotes is not controlled by the accumulation of cyclin proteins or the synthesis of centrosome-specific proteins at each cell cycle. New centrosomes are assembled from preexisting pools of ready-to-use subunits. Furthermore, our results indicate that centrosomal and nuclear events are regulated by separate pathways.     

Stability of globin mRNA in terminally differentiating murine erythroleukemia cells

The stability of globin mRNA in terminally differentiating MEL cells has been reevaluated. Previously, it had been reported that globin mRNA has a half-life of approximately 17 hr in terminally differentiating MEL cells. We show that the previous measurements of this parameter were confounded by physical instability of differentiating MEL cells. By using culture conditions that physically stabilize end-stage cells we show that the stability of globin mRNA in terminally differentiating MEL cells is equal to the value observed for ribosomal RNA, a half-life greater than 60 hr.