Download a new function for the nucleolus

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
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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
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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
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