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AJEBAK 50 (Pt. 7) 827-832 (1972) A NEW FUNCTION FOR THE NUCLEOLUS by HENRY HARRIS (From the Sir William Dunn School of Pathology, South Parks Road, Oxford, England.) I wish to describe how the technique of cell fusion has been used to reveal a new function for that traditionally mysterious organelle, the nucleolus. It is now well known that cells from diflFerent animal species can be fused together by means of inactivated viruses to produce hybrid cells that combine in various ways the genetic complements of the parent cells (Harris and Watkins, 1965). These hybrid cells, both in their initial multinucleate and subsequent mononucleate state (fusion of nuclei occurs at mitosis), have during the last few years been used for a wide variety of genetical and physiological investigations (Harris, 1970). The experiments I propose to discuss were done with a special kind of hybrid cell that differs in one important respect from all others that have so far been studied. When two somatic cells are fused together, the fused cell normally contains both the nuclei and the cytoplasms of the two parent cells, but this is uot, in general, the case when one of the parent cells is a nucleated erythrocyte. (The erythrocytes of birds, reptiles, fish and several other animal orders retain their nuclei in the fully differentiated state, even though these nuclei are totally inert.) The inactivated virus now generally used to produce cell fusion, the Sendai virus, is a haemolytic virus and, at the concentrations used to produce fusion, causes haemolysis of nucleated erythrocytes. Fusion then occurs between the membranes of the empty erythrocyte "ghosts" and those of the other cell type in the combination. The erythrocyte nucleus is thus incorporated into the cytoplasm of the other cell type, but the resulting heterokaryon receives little or no contribution of cytoplasm from the erythrocyte. What is achieved in this situation is thus essentially a nuclear transfer. When the inert nucleus of a chick erythrocyte is introduced into the cytoplasm of a tissue culture cell from the same or a different animal species, it undergoes reactivation (Harris, 1965; 1967). The mechanism of this reactivation has been intensively studied (Bolund, Ringertz and Harris, 1969; Bolund, Darzynkiewicz and Ringertz, 1969; Ringertz, Carlsson, Ege and Bolund, 1971), and it is clear that the chick erythrocyte nucleus not only resumes the synthesis of RNA and DNA but also eventually determines the synthesis of chick-specific proteins in the initially completely foreign cytoplasm (Harris, Sidebottom, Grace and Bramwell, 1969; Harris and Cook, 1969; Cook, 1970). It was during an 828 HENRY HARRIS examination of the ability of reactivated chick erythrocyte nuclei to determine the synthesis of chick-specific proteins iri the cytoplasm of mouse tissue culture cells that my attention was drawn to the nucleolus. It happened in the following way. The first proteins investigated were surface antigens. Antigens characteristic of each animal species are present on the surface of somatic cells in culture and can be detected with great specificity and sensitivity by immunological methods (Watkins and Grace, 1967). Although a small amount of chick-specific antigen was introduced into the chick-mouse heterokaryon when the membrane of the chick erythrocyte "ghost" fused with the membrane of the mouse cell, it seemed reasonable to suppose that when the chick erythrocyte nucleus was reactivated, and began to synthesize RNA, chick-specific antigens would begin to be synthesized and the concentration of these antigens on the surface of the cell would increase. In fact, the opposite occurred. No synthesis of chick-specific antigens could be detected despite the reactivation of the chick erythrocyte nucleus, and the adventitious antigens introduced into the surface of the heterokaryon by the process of fusion itself were eliminated. By the fourth or fifth day after fusion no chick-specific surface antigens could be detected in the cultures. I noticed during the course of these experiments that the chick erythrocyte nucleus, although it underwent great enlargement on reactivation, did not, for at least three or four days after cell fusion, develop any structure that corresponded to the prominent nucleolus normally seen in the nuclei of cells in culture. By the fourth day after cell fusion almost all heterokaryons undergo mitosis during which, in a variety of ways, the erythrocyte nuclei disappear as discrete entities. Since the erythrocytes came from normal birds, there was no reason to suspect a genetic defect aflFecting nucleolar development, and it was therefore of interest to see what would happen if mitosis of the heterokaryons was inhibited. This was done by appropriate doses of X irradiation, and it was then found that the erythrocyte nuclei persisted in the heterokaryons as separate recognizable structures for much longer periods. Under these conditions nucleoli began to develop within the erythrocyte nuclei from the fifth day onwards, and, as the nucleoli made their appearance, chick-specific antigens reappeared on the surface of the heterokaryons and progressively accumulated (Harris et al., 1969). It was found that nucleoli developed in chick embryo erythrocytes more rapidly than in adult hen erythrocytes, and more rapidly in the erythrocytes of younger embryos than in those of older ones. In all cases, there was a clear correlation between the time at which nucleoli appeared in the reactivated erythrocyte nuclei and the time at which chick-specific antigens reappeared on the surface of the cell. This suggested that the ability of the reactivated chick erythrocyte nucleus to determine the synthesis of chick-specific surface antigens was in some way linked to the development of the nucleolus. Similar experiments were done with a soluble enzyme. The A9 cell is a mutant mouse cell line that lacks the enzyme inosinic acid pyrophosphorylase, which converts hypoxanthine to inosinic acid (Littlefield, 1964). Cells that lack A NEW FUNCTION FOR THE NUCLEOLUS 829 this enzyme cannot incorporate hypoxanthine into nucleic acids. The enzyme can therefore be assayed directly in cell homogenates, and indirectly in individual cells, by autoradiographic methods that measure the ability of the cells to incorporate labelled hypoxanthine into nucleic acid. When chick erythrocyte nuclei are introduced into A9 cells they undergo enlargement and reactivation in the usual way; but this reactivation does not result in the appearance of inosinic acid pyrophosphorylase activity in the heterokaryons until nucleoli appear in the erythrocyte nuclei. When this occurs the enzyme makes its appearance and the heterokaryons acquire the ability to incorporate labelled hypoxanthine into nucleic acid (Harris and Cook, 1969). Electrophoretic examination of the inosinic acid pyrophosphorylase synthesized under these conditions reveals that it is chick, not mouse, enzyme (Cook, 1970). Again, over a range of adult and embryonic erythrocytes, there was a close correlation between the appearance of the nucleoli in the reactivated erythrocyte nuclei and the appearance of the enzyme. Other enzyme markers behave in the same way. When the erythrocyte nuclei were introduced into mutant cells which were triply defective, lacking inosinic acid pyrophosphorylase, adenylic acid pyrophosphorylase and nucleoside permease, the missing enzymes again made their appearance within the one cell when nucleoli appeared in the erythrocyte nuclei (Clements, personal communication). Diphtheria toxin provides another species-specific marker. Mouse cells are at least a hundred thousand times more resistant to the toxin than chick cells, so that, at an appropriate concentration, susceptibility to the toxin may be used as a species-specific marker (Dendy and Harris, 1972). Mouse cells into which chick erythrocyte nuclei were introduced remained insensitive to the destructive action of the toxin until nucleoli developed in the erythrocyte nuclei, but after this point the heterokaryons became progressively more susceptible (Deak, Sidebottom and Harris, 1972). It was thus clear that a correlation existed between the time at which the reactivated erythrocyte nuclei developed nucleoli and the time at which chickspecific proteins began to be made in the hybrid cell. Several explanations for this correlation were considered. For a variety of reasons, it seemed worthwhile to explore the possibility that the nucleolus might be the centre of some general regulatory mechanism governing the flow from nucleus to cytoplasm of the RNA that carries the instructions for protein synthesis. This idea found support in some experiments in which parts of cells such as nuclei or nucleoli were inactivated by a microbeam of ultraviolet light. It was found that prior to the development of nucleoli there was no detectable transfer of RNA from the erythrocyte nuclei to the cytoplasm of the heterokaryon; and the flow of RNA from nucleus to cytoplasm in normal mononucleate cells also ceased when the nucleolus was selectively inactivated (Sidebottom and Harris, 1969). Two alternative interpretations of this series of observations were, however, advanced. One proposed that there were species-specific restrictions on the translation of RNA, in particular that the RNA made on chick genes could not be translated by mouse cytoplasmic components. On this model, RNA bearing instructions for the synthesis of chick proteins did pass to the cytoplasm of the 830 HENRY HARRIS cell before development of the nucleolus in the chick erythrocyte nucleus, but this RNA could not be translated until chick ribosomes were produced, an event which would be expected to coincide with the development of the nucleolus. (It was assumed that the amount of RNA involved would be too small to be detected by radioactive labelling.) The whole corpus of experiments on interspecific hybrid cells argues against species-specific restrictions on tbe translation of RNA. For example, man-mouse bybrid cells can be constructed in which the great majority of the human chromosomes are rapidly eliminated, and eventually cell lines can be derived in which a single human chromosome is retained in an otherwise entirely mouse chromosome set (Weiss and Green, 1967). Genes carried on this residual human chromosome are expressed in the mouse cell, and genes on a number of diflEerent single human chromosomes have been shown to be expressed under these conditions. But these man-mouse hybrid cells do not synthesize any detectable amount of human 28S ribosomal RNA (Eliceiri and Green, 1969; Bramwell and Handmaker, 1971). Ghick-mouse hybrid cells can be made which contain only fragments of chick genetic material, too small to be detected in conventional chromosome preparations; but genes located in these fragments can determine the synthesis of chick-specific proteins in tbe cytoplasm of these otherwise completely mouse cells (Schwartz, Cook and Harris, 1971). Again, the chick-mouse hybrids do not synthesize any detectable amount of cbick 28S ribosomal RNA. It therefore seems very unlikely that mouse eytoplasmic components are unable to translate RNA made on chick genes, a conclusion that, in any case, finds strong support in experiments with cell-free and other systems which also show absence of species-specificity in the translation of RNA (Lockard and Lingrel, 1969; Housman, Pemberton and Taber, 1971; Rhoads, McKnight and Schimke, 1971; Gurdon, Lane, Woodland and Marbaix, 1971). The second alternative interpretation that has been proposed for the experiments on tbe reactivation of the erytbrocyte nucleus is that tbe association between the appearance of the nucleolus and the onset of chick-speeific protein synthesis is essentially fortuitous. It could be argued that these two processes happen to occur simultaneously, but that they are not functionally related. Experiments have now been done which eliminate tbis argument also (Deak et at, 1972). If tbe association between the development of the nucleolus in the reactivated erytbrocyte nucleus and the onset of synthesis of chick-specific proteins were fortuitous, then one would expect that inactivation of the nucleolus after the syntbesis of chick-specific proteins had been established would be without effect on this syntbesis. Experiments were therefore done with all the chickspecific markers that I have described to see whether the synthesis of these markers, once established, was affected by inactivation of the nucleolus in the chick erythrocyte nucleus. The following groups of heterokaryons, each containing one reactivated chick erythrocyte nucleus and one mouse nucleus, were compared: unirradiated cells; cells in which an extranucleolar region of the erythrocyte nucleus was inactivated; cells in which one of two nucleoli in the erythrocyte nucleus was inactivated; cells in which a single nucleolus in the A NEW FUNCTION FOR THE NUCLEOLUS 831 erythrocyte nucleus was inactivated; and cells in which the whole erythrocyte nucleus was inactivated. Inactivation of extranucleolar regions of the erythrocyte nucleus, or of one of two nucleoli in the erythrocyte nucleus, was without effect on the synthesis of chick-specific markers: heterokaryons in which the erythrocyte nuclei were treated in this way were indistinguishable from unirradiated cells in their ability to synthesize inosinic acid pyrophosphorylase, chick-speciflc surface antigens and the receptors for diphtheria toxin. But in heterokaryons in which a single nucleolus in the erythrocyte nucleus was inactivated the synthesis of all these markers decayed. For inosinic acid pyrophosphorylase the rate of decay after inactivation of the erythrocyte nucleolus was measured and found to be comparable to that resulting from inactivation of the whole of the erythrocyte nucleus. It thus appears, that for a range of different and unrelated chick-specific markers, synthesis fails to occur before the development of the nucleolus in the erythrocyte nucleus and ceases when the nucleolus is inactivated. It might be argued that the genes for these markers might by chance be situated at the nucleolar site and might therefore be inactivated directly by the microbeam. The fact that the synthesis of the chick-specific markers is unaffected when only one of two nuceoli in the erythrocyte nucleus is inactivated makes this unlikely; for it would then be necessary to propose that a structural gene for each marker is present at both nucleolar sites and that the gene in the unirradiated nucleolar region compensates for the loss of its partner. For the chick-specific surface antigens this proposal is especially difficult to accept, for it has been shown that the genes determining the synthesis of species-specific surface antigens are widely distributed throughout the chromosome set (Weiss and Green, 1967). It might still be argued, although there are grounds for considering the idea improbable, that all active structural genes are located at, or in close association with, the nucleolus; but a model of this kind implies some function for the nucleolus in the expression of structural genes, and is thus a version of our general proposition and not an argument against it. It is, in any case, clear that the association between the development of the nucleolus in the erythrocyte nucleus and the onset of synthesis of chick-specific markers is not fortuitous: some function located at, or close to, the nucleolus is required for the full expression of the structural genes. The problem that now faces us is how, in biochemical terms, the nucleolar region exercises this control over gene expression. The evidence that we have so far been able to gather appears to indicate that some mechanism closely associated with the nucleolus controls the flow to the cytoplasm, not only of the RNA made at the nucleolar site, but also of high molecular weight RNA made elsewhere in the nucleus; impairment of nucleolar function appears to stem the flow of all high molecular weight RNA from nucleus to cytoplasm. But measurements of RNA flow are subject to serious technical uncertainties, and a good deal of rather dull, but unavoidable, biochemical work will need to be done before a satisfactory picture emerges of how, in molecular terms, the nucleolus achieves this overriding regulatory role in the expression of structural genes. HENRY HARRIS 832 REFERENCES. BoLUND, L., DARZYNKIEWICZ, Z., and RIN- GERTZ, N. R. (1969): 'Growth of hen erythrocyte nuclei undergoing reactivation in heterokaryons.' Expl Cell Res., 56, 406. BoLUND, L., RiNGERTZ, N. R., and HARRIS, H. 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