Reactivation Of Chick Erythrocyte Nuclei Research Articles

DNA replication and H5 histone exchange during reactivation of chick erythrocyte nuclei in heterokaryons.

Fusion of terminally differentiated chick erythrocytes (CE) with replicating quail myoblasts or established L6J1 rat myoblasts results in reactivation of DNA synthesis in the dormant CE nuclei and in suppression of DNA synthesis in the myoblast nuclei. The nuclei of primary quail myoblasts are more effectively inhibited than the nuclei of established rat myoblasts. Inhibition of DNA replication occurs not only by preventing G1 nuclei from entering S-phase but also by blocking nuclei in S-phase and by delaying nuclei in G2 from undergoing mitosis and starting a new DNA replication cycle. No inhibition of DNA synthesis could be observed when mouse erythrocytes, i.e., erythrocytes lacking nuclei, were fused with rat myoblasts to generate mouse-globin-containing L6J1 cybrids.--Reactivation of CE nuclei is associated with a loss of the tissue-specific H5 histone variant. Complete elimination of H5 histone, however, does not seem to be a necessary prerequisite for the initiation or completion of DNA replication in CE nuclei since H5 antigens are found on reactivated G1, S, and G2 nuclei.

Changes in nuclear antigens during reactivation of chick erythrocyte nuclei in heterokaryons.

Reactivation of dormant chick erythrocyte (CE) nuclei was studied by fusing chick red cells with rat myoblasts, HeLa cells, and chick fibroblasts. Heterokaryons representing different stages of nuclear reactivation were fixed and examined for nuclear antigens using polyclonal patient autoantisera reacting with mammalian (human, mouse, and rat) nuclear envelope, nucleoplasm, and chromatin (DNA) antigens. Reactivation of CE nuclei was associated with marked changes in nuclear antigenicity. In rat myoblast and HeLa heterokaryons the CE nuclei acquired mammalian nucleoplasmic and nuclear envelope antigens in the corresponding nuclear subcompartments. Drastic changes in nuclear antigenicity were noted also in heterokaryons stained with DNA antisera. The compact chromatin of nuclei in mature chick erythrocytes showed little binding of DNA antibodies. Isolated nuclei on the other hand gave a strong immunofluorescence. CE nuclei in heterokaryons were strongly positive during the early stages of nuclear reactivation but then exhibited decreased reactivity. An unexpected finding was a marked reduction in the capacity of mammalian nuclei in heterokaryons to bind DNA-antibodies. This observation is discussed in relation to the previous finding that in CE-heterokaryons these nuclei often show reduced transcription and replication. The present results indicate that in heterokaryons both types of nuclei exchange macromolecules of regulatory importance via the common cytoplasm.

Interaction of human and chick DNA repair functions in UV-irradiated xeroderma pigmentosum-chick erythrocyte heterokaryons

Fusion of chick erythrocytes with human primary fibroblasts results in the formation of heterokaryons in which the inactive chick nuclei become reactivated. The expression of chick DNA repair functions was investigated by the analysis of the DNA repair capacity after exposure to ultraviolet (UV) irradiation of such heterokaryons obtained after fusion of chick erythrocytes with normal human or xeroderma pigmentosum (XP) cells of complementation groups A, B, C and D. Unscheduled DNA synthesis (UDS) in normal human nuclei in these heterokaryons is suppressed during the first 2–4 days after fusion. The extent and duration of this suppression is positively correlated with the number of chick nuclei in the heterokaryons. Suppression is absent in heterokaryons obtained after fusion of chicken embryonic fibroblasts with XP cells (complementation group A and C). Restoration of DNA repair synthesis is found after fusion in XP nuclei of all complementation groups studied. It occurs rapidly in XP group A nuclei, starting one day after fusion and reaching near normal human levels after 5–8 days. In nuclei of the B, C and D group increased levels of UDS are found 5 days after fusion. At 8 days after fusion the UDS level is about 50% of that found in normal human nuclei. The pattern of UDS observed in the chick nuclei parallels that of the human counterpart in the fusion. A fast complementation pattern is also observed in chick fibroblast-XP group A heterokaryons resulting within 24 h in a UDS level comparable with that in chick fibroblast-normal human heterokaryons. In heterokaryons obtained after fusion of chick fibroblasts with XP group C cells UDS remains at the level of chick cells. These data suggest that reactivation of chick erythrocyte nuclei results in expression of repair functions which are able to complement the defects in the XP complementation groups A, B, C and D.

Cell cycle dependence of the reactivation of chick erythrocyte nuclei after transplantation into mouse L929 cell cytoplasts.

AbstractMonolayer cultures of cytoplasts were formed from synchronized mouse L929 cells in the G1, S and G2 phases of the cell cycle by cytochalasin‐induced enucleation. Chick embryo erythrocytes were then fused to the cytoplasts using Sendai virus. Extensive reactivation of the dormant erythrocyte nuclei‐determined by nuclear swelling, nucleoli formation and incorporation of 3H‐uridine into RNA‐was observed upon fusion to the three types of cytoplasts. By all of these criteria, reactivation was greatest when nuclei were introduced into G1 cytoplasts.Similar experiments in which DNA synthesis was measured showed that both G1 and G2 cytoplasts almost completely lacked the ability to induce DNA synthesis in the chick nuclei. But at least 26% of the erythrocyte nuclei within S cytoplasts exhibited significant levels of DNA synthesis. Cytoplasts prepared from cells three hours after entry into the S phase, which is six to seven hours in length in L929 cells, possessed the maximum capacity to induce DNA replication in chick nuclei. As many as 60% of the nuclei introduced into 3‐hour S phase cytoplasts synthesized significant quantities of DNA.The effect of cycloheximide, an inhibitor of protein synthesis, on erythrocyte nuclear reactivation within G1 phase cytoplasts was also investigated. Protein synthesis was necessary to maximize the reactivation of erythrocyte nuclei. However, it appeared that protein synthesis occurring prior to enucleation was more important in the reactivation process than synthesis occurring following enucleation.

Reactivation of chick erythrocyte nuclei in young and senescent WI38 cells

The chromatin of the dormant chick nucleus is dispersed in the heterokaryons made by Sendai virus fusion of phase II WI38 cells with chick erythrocyte nuclei. The erythrocyte nucleus resumes RNA synthesis and enters into DNA synthesis with the host nucleus. In the heterokaryons of phase III WI38 cells and chick erythrocytes, the nuclear chromatin is not dispersed and RNA synthesis occurs at a reduced rate. The differences in the physiological state of the young and senescent cells measured by [ 3H]uridine incorporation into nuclear RNA is reflected in the extent of reactivation of the chick erythrocyte nuclei in the cytoplasm of these cells. The reactivation of the chick nucleus in enucleated fibroblasts parallels the nucleated cells. The results of these studies are interpreted as evidence that there is a specific loss of nuclear function in the senescent cells.

Ultrastructure of chick erythrocyte nuclei undergoing reactivation in heterokaryons and enucleated cells

The reactivation of chick erythrocyte nuclei after Sendai virus induced fusion of chick erythrocytes with intact or anucleate rat myoblasts or rat epithelial cells was studied by electron microscopy. Both in heterokaryons and in reconstituted cells formed by the fusion of chick red cells with anucleate rat L6 myoblasts the amount of highly condensed chromatin in the chick nuclei decreased with time after fusion at the same time as the proportion of dispersed chromatin increased. Nuclear organelles, typical of active nuclei but absent in the nuclei of unfused erythrocytes, appeared during reactivation. The percentage of chick nuclei containing a nucleolus was low 24 h after fusion but increased so that almost all nuclei contained one or more nucleoli 120 h after fusion. In reconstituted cells the frequency of nucleoli was much lower than in heterokaryons. In other respects, the erythrocyte nuclei introduced into anucleate rat cells underwent a normal reactivation and appeared to be well integrated with the cytoplasm. Thus, the nuclear envelope consisted of two normal leaflets in direct contact with the cytoplasm. Nuclear pores were observed in front of interchromatin channels. A normal cytoplasmic geometry appeared to be re-established since the Golgi apparatus occupied a position close to the poles of the chick nucleus.

Reactivation of chick erythrocyte nuclei in heterokaryons with temperature-sensitive Chinese hamster cells.

Chinese hamster cell line K12 is temperature-sensitive for the initiation of DNA synthesis. K12 cells synchronized by serum deprivation were collected in early G1(G0). Heterokaryons were formed by fusing chick erythrocytes with serum-starved K12 cells through the use of UV-irradiated Sendai virus. At the permissive temperature (36.5 degrees C), erythrocyte nuclei in heterokaryons enlarged, the chromatin dispersed, and erythrocyte nuclei synthesized DNA at about the same time as the K12 nuclei. At the restrictive temperature (41 degrees C), erythrocyte nuclei enlarged, but neither erythrocyte nor K12 nuclei initiated DNA synthesis. When erythrocyte nuclei were fused with Wg-1A cells, the wild-type parent for ts K12 cells, both kinds of nuclei synthesized DNA at 36.5 degrees C and 41 degrees C. Activation of erythrocyte nuclei was inefficient in heterokaryons incubated in low-serum medium. The results indicate that serum factors and a cellular function defined by the K12 mutation are required for activation of chick erythrocyte nuclear DNA synthesis.

Reactivation of chick erythrocyte nuclei in BHK derived cells with multiple biochemical lesions

Fusion experiments employing chick erythrocytes and multiply biochemically marked BHK-derived cell lines have given clear evidence that reacquisition of enzyme activity in all markers followed the same time course and correlated with the appearance of a microscopically detectable nucleolar structure in the reactivating erythrocyte nucleus. The studies additionally provide evidence of passive transfer at the time of fusion of a considerable amount of APRT activity and a lesser amount of HGPRT activity but not of the other enzyme activities followed.

Reactivation of chick erythrocyte nuclei after fusion with enucleated cells

Inactivated Sendai virus was used to fuse nucleated chick erythrocytes with mouse L and A9 cells which had been enucleated by centrifugation in the presence of cytochalasinB. The enucleation step removed the nuclei from more than 99% of the cells. During the fusion step, chick erythrocyte nuclei were introduced into 20% of the enucleated mouse cytoplasms. This resulted in the formation of a large number of "reconstituted cells" where practically all the cytoplasm originated from the mouse cell while the nucleus was of chick origin. The chick erythrocyte nuclei appeared to become well integrated into the mouse cytoplasms since they increased dramatically in size and dry mass, formed nucleolus-like bodies, and resumed RNA synthesis. This, however, did not prevent a gradual decrease in the rate of protein synthesis in the cytoplasm after the removal of the mouse nucleus. Protein synthesis decayed at a similar rate in both reconstituted and enucleated cells. The majority of these "cells" died within 48 h and all of them within 5 days after enucleation/fusion. By contrast, the small number of L cells which failed to become enucleated multiplied rapidly. The results obtained suggest that the reactivation of the chick erythrocyte nuclei is not fast enough to rescue the enucleated mouse cytoplasms.

Chapter 5 Chemical and Structural Changes Within Chick Erythrocyte Nuclei Introduced Into Mammalian Cells by Cell Fusion

This chapter discusses the recent studies on chick erythrocyte nucleus, as it undergoes the reactivation reaction. The chapter describes the macromolecular and structural changes within the erythrocyte nucleus. The chick erythrocyte is a differentiated cell and its nucleus is very low in activity related to macromolecular synthesis. The inactivation occurs gradually during erythropoiesis and is paralleled by the changes in nuclear composition and condensation of the chromatin. When a chick erythrocyte nucleus is introduced, by cell fusion into the cytoplasm of a mammalian cell, the first event is redistribution of the cellular p301 of mammalian nuclear protein. The chapter discusses the fate of chick nuclear material and the changes in protein composition within the reactivating chick nuclei. In addition, the changes in nucleic acid metabolism (RNA and DNA synthesis) are also discussed in this chapter. The cause-effect relationships suggest that one event during the reactivation may depend on the other independent events, thus leading to the reactivation of the chick erythrocyte genome. The general problems in analyzing the experiments are the time sequence of events, competition for rate-limiting factors, and uncoupling reactions. After the reactivation of chick erythrocyte nuclei in mammalian tissue culture cells, the chick nucleus acquires a broad spectrum of the mammalian proteins, because at the early stages of reactivation the mammalian nucleus is the only one specifying the synthesis of nuclear proteins in the heterokaryon. Among the mammalian nucleospecific proteins that accumulate, some proteins should be considered as signals of direct regulatory importance. Further analysis of the reactivation reaction should lead to an identification of such signals.

Suppressive effect of protease inhibitors on heterokaryons containing chick erythrocyte nuclei

Nuclei of active cells (HeLa, mouse fibroblasts) partnered with chick erythrocyte nuclei in heterokaryons are suppressed, as judged by a decreased rate of 3H-uridine incorporation and diminished nuclear binding of 3H-actinomycin D. The extent to which active partner nuclei are suppressed, the extent to which erythrocyte nuclei are reactivated, and the degree of sensitivity of heterokaryons towards certain inhibitors of proteolytic enzymes, all correlate strongly with the ratios of erythrocyte nuclei to active nuclei. Thus, reactivation of individual erythrocyte nuclei is reduced progressively and active nuclei are suppressed progressively as the ratio of erythrocyte nuclei per active nucleus in heterokaryons increases. This erythrocyte nuclear-dose dependent suppression is markedly amplified when heterokaryons are grown in the presence of protease inhibitors. The protease inhibitors found to affect heterokaryons are low molecular weight (<400) inhibitors of trypsin-like enzymes: l-1-tosylamide-2-leucyl chloromethyl ketone (TLCK), N-α-tosyl- l-arginine methyl ester (TAME) and N-benzoyl- l-arginine amide (BAA). They affect heterokaryons at concentrations comparable to the minimal concentrations at which they inhibit trypsin. Nonfused HeLa cells, mouse fibroblasts, or their homokaryons are refractory to protease inhibitors at these concentrations. Reactivation of chick erythrocyte nuclei in a heterokaryon may involve release of suppressors ordinarily confined to the erythrocyte nucleus, with subsequent redistribution of suppressor among all the nuclei of the heterokaryon. Under these circumstances the state of nuclear activity will depend on the quantity of suppressor per individual nucleus; within the erythrocyte nucleus the suppressors will decrease its rate of reactivation, when they migrate into an active nucleus they will suppress it. These suppressors, either in transit between the nuclei, or within the nuclei, may be hydrolysed by intracellular proteases.

Chick-erythrocyte nucleus reactivation in heterokaryons: suppression by inhibitors of proteolytic enzymes.

Reactivation of chick-erythrocyte nuclei in heterokaryons (obtained by Sendai virus-induced fusion of chick erythrocytes with HeLa cells) is suppressed by specific inhibitors of trypsin and trypsin-like enzymes. N-alpha-tosyl-L-lysyl-chloromethane and N-alpha-tosyl-L-arginine methylester inhibit erythrocyte nuclear enlargement and suppress RNA and DNA synthesis in nuclei of erythrocytes and HeLa cells in heterokaryons at concentrations that only minimally influence individual HeLa cells or HeLa homokaryons. Although other unknown mechanisms of action cannot be formally excluded, the data are interpreted as fitting best with an intracellular site of action of the protease inhibitors studied, and as suggesting a role for cellular proteases in reactivation of chick-erythrocyte nuclei in heterokaryons.

Reactivation of chick erythrocyte nuclei in heterokaryons with rat hepatoma cells

Chick erythrocyte nuclei were fused with rat hepatoma cells which express a number of liver functions; among other proteins, they secrete serum albumin. Like other cells (A9, HeLa), these rat hepatoma cells induce the reactivation of the erythrocyte nuclei, which increase in size, resume the synthesis of RNA and acquire nucleoli. In hepatoma cells deficient in the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT) the reactivated chick erythrocyte nuclei will induce the synthesis of this enzyme. In populations of fused cells containing 15 to 30% of chick nuclei, the amount of induced HGPRT is more than 1% of the level found in HGPRT + rat hepatoma cells. On the other hand no secretion of chick albumin can be detected. Given the sensitivity of the method, one can conclude that if any chick albumin is secreted, it is at a rate equal to less than 0.1% of the rate of rat albumin secretion. This result shows either that chick albumin is synthesized but not secreted by the heterokaryons; or that the reactivation of the erythrocyte nucleus is selective (only some chick genes are expressed) and that the rat hepatoma cells cannot induce these reactivated nuclei to acquire the same pattern of gene activity as themselves.