Masuo Obinata

Probability that the commitment of murine erythroleukemia cell differentiation is determined by the c-myc level

During the commitment of mouse erythroleukemia cell differentiation, c-myc mRNA levels change dramatically. To examine the involvement of c-myc in the commitment of these cells, we have introduced the rat c-myc gene driven by inducible, heterologous (human metallothionein IIA) gene promoter into murine erythroleukemia cells and we have examined the ability of the transformed cells to undergo commitment to terminal differentiation. The induction of the exogenous c-myc gene expression inhibited the commitment of these cells. Time-dependent inhibition of the commitment was observed with the addition of zinc at an appropriate time after the induction with dimethyl sulfoxide. The result clearly indicated that late decline, not early decline, is required for the commitment. By examining the transformants expressing the exogenous c-myc mRNA at different levels, and the induction of the exogenous c-myc mRNA by varying the concentration of zinc, we demonstrated that the commitment may be determined by a stoichiometric amount of c-myc in the defined period. The data also suggest that the probability value for the commitment process occurring in a stochastic manner is well-correlated with the amount of c-myc mRNA.

The ‘pricking’ method: A new efficient technique for mechanically introducing foreign DNA into the nuclei of culture cells☆

Using the thymidine kinase gene-containing plasmid pTK-1 and the mouse fibroblast thymidine kinase-deficient LTK- cell line, we obtained results indicating that the plasmid molecules suspended in the external medium were introduced into the nuclei by pricking cells on the nuclei with a microneedle. Approx. 25% of the cells pricked in the presence of the plasmid pTK-1 showed TK gene expression, and 2% of the cells became stable TK+ transformants. This pricking method is applicable for introducing DNA into cell nuclei and is more efficient than the conventional calcium phosphate transformation method.

Differential expression of two abundant messenger RNAs during development of Sarcophaga peregrina.

Expression of two abundant mRNAs in the fat body of third instar larvae of Sarcophaga peregrina was studied using genomic clones of storage protein and 25K protein as probes. It was found that these two genes were expressed sequentially in the third instar with a 20-hr interval between their expressions. No amplification of genes was detected during larval development, suggesting that the high level of mRNA was due to efficient transcription. The contents of the two mRNAs in third instar larvae were about the same, although the synthesis of storage protein was much more than that of 25K protein.

Multi-gene structure of the storage protein genes of Sarcophaga peregrina.

Three recombinant phages containing chromosomal segments of Sarcophaga peregrina encoding storage protein were isolated. These clones were found to contain sequences hybridized with the messenger RNA of 75,000 Mr protein, a monomer of storage protein. From restriction analysis it was found that message-homologous regions are not identical, and thus are different genes with similar sequences. From the analysis of these clones it was concluded that the storage protein gene belongs to a multi-gene family.

Effect of c-myc gene expression on early inducible reactions required for erythroid differentiation in vitro.

By employing cell fusion between two genetically marked mouse erythroleukemia (MEL) cells in which an artificially introduced c-myc gene had been placed under the control of human metallothionein promoter, we investigated the mechanism of the suppressive action of c-myc gene expression in erythroid differentiation. The results indicated that the expression of the c-myc gene blocked the induction of dimethyl sulfoxide-inducible activity, one of the two early activities required for triggering the differentiation.

Induction of commitment of murine erythroleukemia cells (TSA8) to CFU-E with DMSO

The commitment of novel mouse erythroleukemic (MEL) cells (TSA8) to colony-forming units of erythroid (CFU-E) by dimethylsulfoxide (DMSO) was investigated. After exposure to the inducer in liquid culture, the cells were transferred to a semi-solid culture to examine their ability to form erythroid colonies which were dependent on erythropoietin. Exposure to DMSO for 2 days is optimum for CFU-E type colony formation and colonies induced in this manner are equivalent to CFU-E. The induction occurred in a synchronous manner. Partly stained colonies appeared prior to CFU-E formation and are thought to be a result of asymmetric cell division. Appearance of these partly stained colonies suggested that the number of erythropoietin receptors is important in the complete responsiveness to erythropoietin. TSA8 cells constitute a suitable model system in which to analyse the mechanism of commitment in early erythropoiesis.

Activation of the secretion of specific proteins from fat body following injury to the body wall of Sarcophaga peregrina larvae

Abstract When the body wall of Sarcophaga peregrina larvae was injured, the fat body was transiently activated to synthesize and secrete 70k and 27k proteins. It was found that genes for these proteins were activated at the time of the injury. The amount of resulting mRNA was too small to detect a translation product in vitro, using the reticulocyte lysate system, but the mRNA seemed to be translated quite efficiently in vivo, suggesting translational control of these mRNA.

Transformation of L cells with virus thymidine kinase genes introduced by red cell-mediated microinjection

Fusion of red cell ghosts containing foreign materials with cells results in the introduction of the materials into the cells (red cell-mediated microinjection). Until now, two-step dialysis has mainly been used for trapping proteins in the ghosts. Large-sized materials such as DNA, however, are rarely trapped in the ghosts, since the holes in the red cell membrane caused by osmotic shock are too small for such materials to pass through. In this study, we improved the trapping technique. Some of the Hind III fragments of lambda phage DNA as well as proteins could be trapped in the ghosts when the mixture of these materials and red cells were frozen at -80 degrees C for a short period followed by quick thawing. Red cell-mediated microinjection using ghosts containing plasmid pBR322 linked with a Herpes simplex viral thymidine kinase (tk) gene brought about transformation of tk-defective L cells, the efficiency of transformation was 1 out of 20 000-60 000 cells fused with the ghosts.

Unique pattern of gene expression in the erythroid precursor cells

Erythroid cells were fractionated by preformed Percoll density gradient from livers of 12.5 day old mouse fetuses. With combination of lysing of mature erythroid cells, the CFU‐E (colony forming unit of erythroid) was enriched as high as 30% pure. The mRNA levels of the rt‐genes previously cloned as genes expressed in the reticulocytes are estimated in the fractionated erythroid cells. These rt‐genes show a drastic change in expression during erythroid differentiation; Their expression was not detectable at the CFU‐E cell stage. But it reached to maximum at the polychromatic erythroblast (stage I) and then decreases with maturation. The result suggests that mRNA synthesis of these rt‐genes may be induced after the stimulation of erythropoietin.

Translation of Human Macrophage Activating Factor (For Glucose Consumption) Mrna in Xenopus Laevis Oocytes

Total messenger RNA was extracted from a human T cell hybridoma, clone H-E4-9, which strongly produced macrophage activating factor for glucose consumption (MAF-G). This messenger RNA gave rise to functional MAF-G when translated in Xenopus laevis oocytes. Isoelectric focusing of culture supernatants of the mRNA-microinjected oocytes and the H-E4-9 cells revealed that the former contained MAF-Gs with isoelectric points of pH 5.0 and 3.0 while the latter contained MAF-Gs with isoelectric points of pH 5.0 and pH 3.3. Sucrose density gradient centrifugation analysis showed that MAF-G mRNA prepared from H-E4-9 cells sedimented at about 11.5 S.