Absence of nucleolar dominance in mouse-human heterokaryons

Autoradiographic analysis of [³H]uridine incorporation 48 h after polyethylene glycol-mediated cell fusion indicates that nucleolar RNA synthesis persists in both human and mouse nuclei in interspecific heterokaryons. The absence of nucleolar dominance in heterokaryons has been confirmed by zinc-dithizone nucleolus-specific staining, and is true even when there are considerably more nuclei of one species than of the other in the heterokaryon. Studies of actinomycin D-induced nucleolar segregation indicate that the zinc-binding proteins responsible for zinc-dithizone staining are located in a different nucleolar component than the protein responsible for silver staining.     

Intracellular antigen migration in interspecific myoblast heterokaryons

Intracellular migration of species-specific nuclear antigens was studied in chick-rat heterokaryons. These cells were produced by virus-induced or spontaneous fusion of different chick cells with rat myoblasts or myotubes. Chick erythrocyte nuclei introduced into rat myogenic cells increased in volume and were reactivated to synthesize RNA. As the chick erythrocyte nuclei enlarged, they rapidly accumulated rat nuclear antigens. Rat nucleolar and nucleoplasmic antigens assumed a distribution in the chick nuclei corresponding to that in rat nuclei. In hybrid myotubes formed by the spontaneous fusion of chick myoblasts and rat myoblasts antigen exchange was at a much lower level. Some exchange of both rat and chick nuclear antigens could, however, be detected also in this system. Thus chick nuclear envelope and nucleolar antigens migrated into the rat myoblast nuclei and assumed an intranuclear localization analogous to that in chick nuclei. On the basis of these results it appears that antigenic nuclear macromolecules are constantly exchanged between the rat and chick nuclear compartments and the cytoplasm of the heterokaryon. During the rapid nuclear swelling which occurs when chick erythrocyte nuclei are activated in rat myoblast heterokaryons, the inward migration of rat nuclear antigens into the chick erythrocyte nucleus is more impressive than the migration of chick antigens into the rat nuclei.     

Heterokaryons in the analysis of genes and gene regulation.

Cytological and chemical analysis of heterokaryons, the immediate product of cell fusion, offer new possibilities for studying the factors responsible for genetic regulation in eukaryotic cells. In comparison with proliferating cell hybrids the heterokaryon state offers the important advantage that a heterokaryon contains two complete genomes since chromosome loss does not occur, but since segregation and recombination are absent, heterokaryons cannot be used for gene mapping in the same way as proliferating cell hybrids. However, if two cell types carrying different genetic defects are fused the analysis can be used for studies of gene complementation. The biological information obtained with heterokaryons has emphasized the role of the cytoplasm in the control of nuclear activity. When a G1 nucleus is brought into contact with the cytoplasm of an S phase cell the G1 nucleus is stimulated to synthesize DNA. If the nucleus is brought into a mitotic cell, the chromatin of the G1 nucleus is forced to condense into prematurely condensed chromosomes. Inactive nuclei such as the dormant chick erythrocyte nucleus will be stimulated to initiate RNA and DNA synthesis when brought into contact with an active cytoplasm by cell fusion. Specific nuclear proteins have been shown to be responsible for this process of reactivation. Other inactive nuclei such as the nuclei of macrophages and spermatozoa have likewise been shown to be reactivated by fusion with active cells. The degree of activation in all of these cases appears to be determined by the state of the active cell. Inactive nuclei are activated to the same level as the active nucleus but seldom beyond this level. If differentiated cells are fused with undifferentiated cells, usually the differentiated character is lost rapidly after fusion. This observation is in agreement with several studies on proliferating cell hybrids indicating some type of negative control of differentiated properties. In heterokaryons obtained by fusion of cells of a similar type of histotypic differentiation usually coexpression of the differentiated markers is observed.     

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.     

Evidence for endogenous polypeptide-mediated inhibition of cell-cycle transit in human diploid cells

Previous studies have shown that the senescent phenotype is dominant with respect to DNA synthesis in fusions between late passage and actively replicating human diploid fibroblasts. Brief postfusion treatments with the protein synthesis inhibitor cycloheximide (CHX) or puromycin have been found to significantly delay (by 24-48 h) the inhibition of entry into DNA synthesis of young nuclei in heterokaryons after fusion with senescent cells. A significant fraction of the senescent nuclei incorporated tritiated thymidine in CHX-treated heterokaryons. The optimal duration of exposure to CHX was 1-3 h immediately after fusion, although treatments beginning as late as 9 h after fusion elevated the heterokaryon labeling index. Prefusion treatments with CHX were without a significant effect. These results are consistent with the interpretation that regulatory cell cycle inhibitor(s) which are dependent upon protein synthesis may be present in heterokaryons between senescent and actively replicating cells.     

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.     

Cell cycle re-entry of quiescent mammalian nuclei following heterokaryon formation

When 3T3 mouse fibroblasts are made quiescent by serum deprivation and are then fused with tsAF8 hamster fibroblasts synchronized by a combination of high temperature block and hydroxyurea, the nuclei of binucleated heterokaryons which are formed enter S phase asynchronously in media containing low levels of serum. The tsAF8 nuclei of these biphasic heterokaryons enter S phase shortly after fusion, as do the tsAFS nuclei of homokaryons in the same culture. In contrast, the nuclei of the biphasic heterokaryons which have been contributed by quiescent 3T3 enter S phase only after a lag following fusion. This suggests that the quiescent nucleus within the heterokaryon is stimulated by factor(s) from the more advanced cell to re-enter the cell cycle in the absence of serum. In contrast to factors which induce the immediate synthesis of DNA, these factors may be those responsible for the transition of a cell from a non-proliferating to a proliferating state.     

Sexual diploids of Aspergillus nidulans do not form by random fusion of nuclei in the heterokaryon

The sexual stage of Aspergillus (Emericella) nidulans consists of cleistothecia containing asci, each with eight ascospores. The fungus completes the sexual cycle in a homokaryotic or a heterokaryotic mycelium, respectively. The common assumption for the last 50 years was that different nuclear types are not distinguishable when sexual development is initiated. When cultured on a medium limited for glucose supplemented with 2% sorbitol, sexual development of A. nidulans is slowed and intact tetrads can be isolated. Through tetrad analysis we found that unlike haploid nuclei fuse preferentially to the prezygotic diploid nucleus. When heterokaryons are formed between nuclei of different genetic backgrounds, then recombinant asci derived from opposite nuclei are formed exclusively. Strains in the same heterokaryon compatibility group with moderate differences in their genetic backgrounds can discriminate between the nuclei of a heterokaryon and preferentially form a hybrid diploid nucleus, resulting in 85% recombinant tetrads. A. nidulans strains that differ at only a single genetic marker fuse the haploid nuclei at random for formation of diploid nuclei during meiosis. These results argue for a genetically determined "relative heterothallism" of nuclear recognition within a heterokaryon and a specific recruitment of different nuclei for karyogamy when available.     

Internuclear transfer of genetic information in kar1-1/KAR1 heterokaryons in Saccharomyces cerevisiae.

Heterokaryons of Saccharomyces cerevisiae have been constructed utilizing the kar1-1 mutation, which prevents nuclear fusion during conjugation (J. Conde and G. Fink, Proc. Natl. Acad. Sci. U.S.A. 73:3651-3655, 1976). Each heterokaryon contained two haploid nuclei that were marked on several chromosomes. They segregated haploid progeny (cytoductants), most of which have the nuclear genotype of one or the other of the heterokaryon parents, but they occasionally segregated progeny having a recombinant genotype (exceptional cytoductants). Exceptional cytoductants receive the majority of their genome from one parent (the recipient) and a minority from the other (the donor). Transfer of two markers from the donor nucleus to the recipient is rarely coincident for markers located on different chromosomes but is nearly always coincident for those markers located on the same chromosome, suggesting that whole chromosomes are transferred from the donor nucleus to the recipient. In crosses of kar1-1 X KAR1 parents, either nucleus may act as a recipient or donor with equal probability. Recipient nuclei acquired 9 of the 10 chromosomes examined, with frequencies which were inversely correlated with the size of the chromosome. When a chromosome is acquired by the recipient nucleus, it either replaces its homolog or exists in a disomic condition. Haploid progeny emanating from kar1 X KAR1 crosses are frequently inviable. I tested whether this inviability might be the result of chromosome loss by donor nuclei. Viability of progeny from kar1 X KAR1 heterokaryons was improved when the parental nuclei were diploid to an extent consistent with the hypothesis, and diploid progeny which had become monosomic were recovered from these heterokaryons. The following sequence of events accounts for chromosome transfer in kar1 X KAR1 heterokaryons. After cell fusion, each nucleus in the heterokaryon has a probability of about 0.38 of losing one or more chromosomes. A nucleus sustaining such a loss can become a donor in a chromosome transfer event. If the other nucleus does not sustain a mortal chromosome loss, it can become a recipient in a transfer event. The chance of acquiring a chromosome lost by the donor is greater for smaller chromosomes than for larger ones and is about 0.05 for the average chromosome.     

Complementation and noncomplementation in heterokaryons of three unlinked pigment mutants of the fowl.

This study shows that melanocyte heterokaryons formed between cells of the blue and recessive white genotypes complement one another to produce normal pigmentation, while heterokaryons of the blue and pinkeye genotypes fail to complement. The simplest interpretation of these findings is that the blue and recessive white mutations affect different aspects of pigment synthesis so that when both kinds of nuclei exist in the same cytoplasm, they can correct (complement) each other's defect. On the other hand, the blue and pinkeye mutations, although unlinked, apparently affect the same aspect of pigment synthesis so that when both kinds of nuclei are in a common cytoplasm, they cannot correct each other's defect. This suggests that one of these two loci exerts some kind of control, or "regulation," over the other. It has previously been shown that recessive white--pinkeye heterokaryons can complement. Thus, only two heterokaryon complementation groups are evident within the three mutants examined.     

Test for temporal or spatial restrictions in gene product function during the cell division cycle.

The ability of a functional gene to complement a nonfunctional gene may depend upon the intracellular relationship of the two genes. If so, the function of the gene product in question must be limited in time or in space. CDC (cell division cycle) gene products of Saccharomyces cerevisiae control discrete steps in cell division; therefore, they constitute reasonable candidates for genes that function with temporal or spatial restrictions. In an attempt to reveal such restrictions, we compared the ability of a CDC gene to complement a temperature-sensitive cdc gene in diploids where the genes are located within the same nucleus to complementation in heterokaryons where the genes are located in different nuclei. In CDC X cdc matings, complementation was monitored in rare heterokaryons by assaying the production of cdc haploid progeny (cytoductants) at the restrictive temperature. The production of cdc cytoductants indicates that the cdc nucleus was able to complete cell division at the restrictive temperature and implies that the CDC gene product was provided by the other nucleus or by cytoplasm in the heterokaryon. Cytoductants from cdc28 or cdc37 crosses were not efficiently produced, suggesting that these two genes are restricted spatially or temporally in their function. We found that of the cdc mutants tested 33 were complemented; cdc cytoductants were recovered at least as frequently as CDC cytoductants. A particularly interesting example was provided by the CDC4 gene. Mutations in CDC4 were found previously to produce a defect in both cell division and karyogamy. Surprisingly, the cell division defect of cdc4 nuclei is complemented by CDC4 nuclei in a heterokaryon, whereas the karyogamy defect is not.     

Trikaryon formation and nuclear selection in pairings between heterokaryons and homokaryons of the root rot pathogen Heterobasidion parviporum

Pairings between heterokaryons and homokaryons of Agaricomycete fungi (he-ho pairings) can lead to either heterokaryotization of the homokaryon or displacement of the homokaryotic nucleus through migration of nuclei from the heterokaryon into the homokaryon. In species of Agaricomycetes with multinucleate cells (>2 nuclei per cell), he-ho pairings could result in the stable or transient formation of a hypha with three genetically different nuclei (trikaryons). In this study, he-ho pairings were conducted using the multinucleate Agaricomycete Heterobasidion parviporum to determine whether trikaryons can be formed in the laboratory and whether nuclear genotype affects migration and heterokaryon formation. Nuclei were tracked by genotyping the heterokaryotic mycelium using nucleus-specific microsatellite markers. The data indicated that certain nuclear combinations were favored, and that nuclei from some strains had a higher rate of migration. A high percentage of trikaryons (19 %) displaying three microsatellite alleles per locus were identified among subcultures of the he-ho pairings. Using hyphal tip and conidial isolation, we verified that nuclei of three different mating types can inhabit the same mycelium, and one of the trikaryotic strains was judged to be semi-stable over multiple sub-culturing steps, with some hyphal tips that retained three alleles and others that reduced to two alleles per locus. These results demonstrate that nuclear competition and selection are possible outcomes of heterokaryon-homokaryon interactions in H. parviporum and confirm that ratios of component nuclei in heterokaryons are not strictly 1:1. The high rate of trikaryon formation in this study suggests that fungi with multinucleate cells may have the potential for greater genetic diversity and recombination relative to dikaryotic fungi.     

Positive and negative regulation of replication in hybrid cells]

DNA replication blockage in various differentiated cells was investigated on the model of heterokaryons. Two distinct types of DNA synthesis regulation in heterokaryons "differentiated cell + proliferating cell" were revealed: I. Neutrophils and nucleated erythrocytes efficiently prevented the entry of non-malignant proliferating cells nuclei into the S-period but usually failed to substantially inhibit the replication in malignant cells nuclei. Both "mortal" and immortalized proliferating cells activated the DNA synthesis in neutrophil and chicken erythrocyte nuclei. II. Macrophages did not influence the DNA synthesis in the nuclei of non-malignant cells in heterokaryons but drastically inhibited that in the nuclei of malignant cells. Only immortalized cells reactivated DNA synthesis in the nuclei of macrophages. These data show that the mechanisms maintaining differentiated cells in non-proliferating state are not uniform. Nucleated erythrocytes were shown to suppress the duplication of centrioles in partner cells. The possibility of the blockage of DNA replication upon the fusion of two proliferating cells (fibroblast + leukemia cell) was demonstrated for the first time in the present work. The influence of various oncogenes upon the regulation of DNA synthesis in heterokaryons was investigated in detail. New modifications of the methods of cell fusion, enucleation and heterokaryon identification were proposed.     

Tad, a Line-like Transposable Element of Neurospora, Can Transpose between Nuclei in Heterokaryons

The Tad transposon of Neurospora crassa appears to be a LINE-like element with very restricted distribution within the genus Neurospora. When forced heterokaryons were constructed between strains which did and did not contain Tad, the nuclei of the naive nuclear type rapidly acquired Tad elements. The elements acquired by naive nuclei are active, since they can pass Tad to other naive nuclei in subsequent heterokaryons. When heterokaryons are passaged by serial transfer, the load of acquired Tad elements appears to increase, indicating that transposition is continuing in these heterokaryons, even after all of the naive nuclei have acquired Tad. In normal heterokaryons of Neurospora, nuclei do not fuse. An experiment to test for the possibility that Tad promotes nuclear fusion gave negative results. Thus Tad appears to have a cytoplasmic intermediate in its transposition. When heterokaryon incompatible strains were cocultured, there was no indication that Tad elements could be transferred to the naive strain, suggesting that Tad is not a virus. These data are consistent with the transposition of Tad via RNA and cDNA intermediates, as has been postulated to occur with LINE-like elements.     

Reactivation of rat peritoneal leukocyte and mast cell nuclei in heterokaryons with actively growing mouse cells

Leukocytes and mast cells of rat peritoneal exudate (PE) were fused in vitro with actively growing mouse cells. Segmented ring-shaped nuclei of granulocytes undergo drastic changes which result in dispersion of tightly condensed chromatin and gradual disappearance of the opening in the centre of the nucleus. These changes are paralleled by a resumption of RNA and DNA synthesis, as shown by autoradiography with [3H]uridine and [3H]thymidine. Solid inactive nuclei of mast cells, lymphocytes, monocytes and macrophages also resume DNA replication and high level of RNA synthesis. Fusion of thymidine kinase-deficient 3T3-4E cells with PE cells results in the incorporation of [3H]thymidine into the nuclei of heterokaryons. This may be considered evidence of the phenotypic expression of rat thymidine kinase gene in heterokaryons. A similar way in which segmented and non-segmented dormant nuclei undergo reactivation suggests that the reversibility of nuclear inactivation is a common feature of differentiated somatic cells.     

Factors affecting the frequency of concealed heterokaryosis in Saccharomyces cerevisiae

In this work, studies on the phenomenon of concealed heterokaryosis that we previously detected in the saccharomycetes yeast strains were continued. New approaches to high effectiveness of isolation of cytoductants carrying the concealed nucleus were implemented, and the composition of individual concealed heterokaryons, zygotic clones, and the first zygotic buds was analyzed by a micromanipulator. The relationship between a delay in the growth of the parental strain (a potential donor of the concealed nucleus) and a decline in the frequency of the appearance of concealed heterokaryons (HKC) was observed. It is assumed that different replication rates of two nuclei of the heterokaryon probably underlie the appearance of HKC. A drastically decreased level of replication of one of the parental nuclei may be connected with the fact that binuclear buds appear extremely rarely and give rise to the rapidly "purified" progeny consisting of cells carrying the second nucleus with normal replication. A lack of the phenotype allows rare binuclear cells to persist as concealed heterokaryons. HKC may be detected only when cells of either parental type are isolated on the corresponding selective media.     

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.