Pattern of chick gene activation in chick erythrocyte heterokaryons

The reactivation of chicken erythrocyte nuclei in chick-mammalian heterokaryons resulted in the activation of chick globin gene expression. However, the level of chick globin synthesis was dependent on the mammalian parental cell type. The level of globin synthesis was high in chick erythrocyte-rat L6 myoblast heterokaryons but was 10-fold lower in chick erythrocyte-mouse A9 cell heterokaryons. Heterokaryons between chick erythrocytes and a hybrid cell line between L6 and A9 expressed chick globin at a level similar to that of A9 heterokaryons. Erythrocyte nuclei reactivated in murine NA neuroblastoma, 3T3, BHK and NRK cells, or in chicken fibroblasts expressed less than 5% chick globin compared with the chick erythrocyte-L6 myoblast heterokaryons. The amount of globin expressed in heterokaryons correlated with globin mRNA levels. Hemin increased beta globin synthesis two- to threefold in chick erythrocyte-NA neuroblastoma heterokaryons; however, total globin synthesis was still less than 10% that of L6 heterokaryons. Distinct from the variability in globin expression, chick erythrocyte heterokaryons synthesized chick constitutive polypeptides in similar amounts independent of the mammalian parental cell type. Approximately 40 constitutive chick polypeptides were detected in heterokaryons after immunopurification and two-dimensional gel electrophoresis. The pattern of synthesis of these polypeptides was similar in heterokaryons formed by fusing chicken erythrocytes with rat L6 myoblasts, hamster BHK cells, or mouse neuroblastoma cells. Three polypeptides synthesized by non-erythroid chicken cells but less so by embryonic erythrocytes were conspicuous in heterokaryons. Two abundant erythrocyte polypeptides were insignificant in non-erythroid chicken cells and in heterokaryons.     

DNA repair synthesis in human heterokaryons. III. The rapid and slow complementing varieties of xeroderma pigmentosum.

Patients with Xeroderma pigmentosum and defective DNA excision repair can be distinguished as a rapid (r-XP) and slow (s-XP) complementing variety. When fused with normal cells, fibroblasts from the r-XP are complemented rapidly and in the absence of protein synthesis while those from the s-XP are complemented slowly by a process partly, but not entirely, dependent on protein synthesis. Heterokaryons with different ratios of r-XP to s-XP nuclei (i.e. 1:1-5 and 1-5:1) and control heterokaryons containing one normal and 1-5 r- or s-XP nuclei show that if cell fusion and incubation is conducted in medium preventing protein synthesis, the rXP cells do not complement the s-XP partner at all and, conversely, that the latter is not as effective as normal cells at complementing the rXP partner. On the contrary, if protein synthesis is permitted, the 2 types of XP cells complement each other in a gene dose-dependent manner and to an extent similar to that observed in the control heterokaryons. These findings indicate that the r- and s-XP varieties are caused by mutations at different loci and suggest that the products of these loci interact to produce a functional unit which is present in normal control cells but absent in the XP strains. The relationship between the complementation groups described here and those already reported in the literature being investigated.     

Preparation and characterization of chick erythrocyte nuclei from heterokaryons

A method for the isolation of reactivated chick erythrocyte nuclei from heterokaryons was developed. The heterokaryons were produced by fusing chick erythrocytes with HeLa or L cells in the presence of inactivated Sendai virus. At various time intervals after fusion nuclei were isolated directly from the monolayer by treatment with an acidic detergent solution. Chick erythrocyte nuclei were then separated from other nuclei (HeLa or L cell) by centrifugation on sucrose gradients. The purified preparation of reactivated chick erythrocyte nuclei was shown to be free from other nuclei and cytoplasmic contamination. By using L cells which had been labelled with ³H-leucine before fusion or heterokaryons labelled after fusion it was demonstrated that labelled mouse proteins migrate from the cytoplasm of the heterokaryons into the reactivating chick erythrocyte nuclei. ³H-uridine labelling of heterokaryons made by fusing UV-irradiated chick erythrocytes with L cells failed to reveal any significant migration of mouse RNA into the chick erythrocyte nuclei.     

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 tissuespecific 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.     

Aphidicolin inhibits repair of DNA in UV-irradiated human fibroblasts

Aphidicolin, a specific inhibitor of DNA polymerase α, is shown to inhibit DNA repair in human diploid fibroblasts. Although aphidicolin has no apparent effect on the DNA of unirradiated cells, it causes a large number of strand breaks to accumulate in UV-irradiated cellular DNA. The number of breaks is the same as the number observed following a similar dose of ultraviolet light when cells are treated with arabinofuranosyl cytosine (araC) and hydroxyurea (HU), known inhibitors of repair. Moreover, two-dimensional paper chromatography shows that aphidicolin completely blocks removal of pyrimidine dimers. These observations are discussed in light of the proposed roles of DNA polymerases α β in DNA replication and repair and the action of aphidicolin on polymerase α.     

DNA repair within nucleosome cores of UV-irradiated human cells

We have compared the distributions of repair synthesis and pyrimidine dimers (PD) in nucleosome core DNA during the early (fast) repair phase and the late (slow) repair phase of UV-irradiated human fibroblasts. As shown previously [Lan, S. Y., & Smerdon, M. J. (1985) Biochemistry 24, 7771-7783], repair synthesis is nonuniform in nucleosome core particles during the fast repair phase, and the distribution curve can be approximated by a model where repair synthesis occurs preferentially in the 5' and 3' end regions. In this report, we show that, during the slow repair phase, [3H]dThd-labeled repair patches are much more uniformly distributed in core DNA, although they appear to be preferentially located in sequences degraded slowly by exonuclease III. This change in distribution cannot be explained by an increase in patch size during slow repair, since the size of these patches actually decreases to about half the size measured during the fast repair phase. Furthermore, PD mapping within core DNA at the single-nucleotide level demonstrated that, at least within the 30-130-base region from the 5' end, there is little (or no) selective removal of PD during the fast repair phase. However, the nonuniform distribution of repair synthesis obtained during fast repair throughout most of the core DNA region (approximately 40-146 bases) is accounted for by the nonuniform distribution of PD in core DNA. The near-uniform distribution of repair synthesis observed during slow repair may result from more extensive nucleosome rearrangement and/or nucleosome modification during this phase.     

Reactivation of chick erythrocyte nuclei in heterokaryons with rat hepatoma cells

The concentration of adenosine 3′,5′-cyclic monophosphate (cAMP) was measured in the parotid gland of the mouse after intraperitoneal injection of a beta-adrenergic drug, isoproterenol. A marked elevation of cAMP was observed 10 min after the administration, followed by a return to the control level within 40 min. A small peak was found around 14 h, onset of the stimulated DNA synthesis being observed at 20 h. A close relationship was found between the cAMP level at 10 min and the rate of DNA synthesis at 24 h in animals given different doses of the drug. However, DNA synthesis could not be induced by adrenalin in spite of a significant increase in the cAMP concentration. Furthermore, X-irradiation or certain metabolic inhibitors (actinomycin D and cycloheximide), administered prior to isoproterenol, completely inhibited the stimulated DNA synthesis without affecting the cAMP elevation at 10 min. It is concluded that the critical step in the initiation of stimulated DNA synthesis may be located at a period later than the initial cAMP elevation.     

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.     

Interferon activates DNA repair genes in UV-irradiated human cells]

Unscheduled DNA synthesis in human fibroblasts pretreated with natural and recombinant interferons was studied by scintillated radiometry. UDS was increased in fibroblasts pretreated 7 days before UV-irradiation. Newly synthesized proteins induced in cells after interferon treatment were compared with heat shock proteins.     

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: -1-tosylamide-2-leucyl chloromethyl ketone (TLCK), N-α-tosyl- -arginine methyl ester (TAME) and N-benzoyl- -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.     

Nucleo-cytoplasmic protein migration during the activation of chick erythrocyte nuclei in heterokaryons

The reactivation of the chick erythrocyte nucleus was studied after erythrocytes were induced to fuse with rat epithelial cells in the presence of Sendai virus. The chick nucleus swells, shows an increase in dry mass and protein content and resumes RNA synthesis. Nucleoplasmic antigens characteristic of the rat cell are found to migrate into the erythrocyte nucleus. The rate of uptake of these molecules, which are believed to be proteins, appears to be directly related to increases in nuclear size, ³H-uridine incorporation and RNA polymerase activity. The polymerase activity which increases during the first days after cell fusion is sensitive to α-amanitin but relatively resistant to actinomycin D. At later time points there is an increase in α-amanitin resistant polymerase activity which probably reflects the appearance of ribosomal RNA synthesis.When heterokaryons containing different proportions of rat: chick nuclei are compared, reactivation is found to proceed most rapidly in those containing a high rat: chick nuclear ratio. As the number of erythrocyte nuclei in heterokaryons increases, the rate of reactivation in the individual nuclei is progressively reduced suggesting that the erythrocyte nuclei compete with each other for macromolecules of specific importance for the activation process.     

Mechanisms of impairment of DNA repair in human cells. Interferons stimulated DNA repair in xeroderma pigmentosum cells

DNA repair synthesis and strand break DNA repair induced by 4-nitroquinoline-1-oxide and UV-irradiation in Xeroderma pigmentosum lymphocytes and fibroblasts pretreated by leucocyte interferons were studied. Stimulation of DNA repair synthesis in interferon-pretreated Xeroderma pigmentosum cells, defective in incision, was detected. No such effect was noted for strand break DNA repair. Hence, antimutagenic activity of interferons in human cells is connected with their modificating effect on DNA repair.