FIFTEENTH CIBA MEDAL LECTURE
Molecular mechanisms in the control of gene expression during
development
J. B. GURDON
MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K.
Nature of the problem, and background information
The problem which I will discuss in this lecture is how a
single-celled fertilized egg develops into a highly organized
functional organism. In all animals the egg is an exceptionally
large single cell, containing superficially homogeneous yolky
cytoplasm. Within a few days the egg undergoes many rounds
of cell division, to become a free-swimming larva containing
thousands of cells, of which many are fully specialized adult
cell-types, such as erythrocytes. In the case of the African frog,
Xenopus laeois, which I shall refer to extensively in this talk, the
main events of early development are summarized in Fig. 1. A
generalization which applies to all animals except for mammals
(in which the connection between the embryo and the maternal
placenta alters normal events), is that no increase in dry weight
takes place between the egg and hatched larval stages. Indeed
the environment makes no known material or other contribution
to early development. All of the materials needed to form a fully
functional larva are already present in an egg in some form,
largely yolk. Early development consists of the conversion of the
components of an egg into those of a tadpole. During this
process, extreme specialization in gene expression takes place in
some cells; for example, an erythrocyte contains about 10’
molecules of globin, whereas other cells contain few if any
globin molecules. This must result from intense expression of the
single-copy globin genes in some cells, and their relatively
complete non-expression in other cells.
In trying to explain early development in molecular terms,
there is one fundamental concept which nearly all embryologists subscribe to. This is that eggs or early embryos contain
determinants which are unequally distributed in the cytoplasm
and which are partitioned out during cleavage, so that different
cells of a blastula acquire different concentrations of these
determinants (see Fig. 2).
Determinants may be thought of as substances which activate
or repress genes. No-one believes that there is, in egg cytoplasm,
a determinant for each gene which is subject to developmental
The Fifteenth CIBA Medal Lecture
Delivered at a Meeting of the Biochemical Society
on 24 July 1980 at The University of Sheffield,
Sheffield, U.K.
DR. J. B. GURDON
Blastula
Tail-bud tadpole
Swimming tadpolc
Time
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months-1
7-1
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1-
13
day-1
3-
davs-
BIOCHEMICAL SOCIETY TRANSACTIONS
14
control. Rather it is thought that a limited number of different
determinants cause initial differences in gene activity. Once the
first differences have been established it is not difficult to
understand how further differences may arise as a result of
interactions between cells, such as ones which occur during
embryonic induction processes associated with the folding of cell
layers.
I propose to summarize what can be said about some aspects
of gene control in development. I shall emphasize work of my
colleagues and myself involving the manipulations of amphibian
material, since this is the area with which I am most familiar. It
does not seem to me to be useful to discuss or investigate
mechanisms by which determinants come to be localized where
they are within cells, until some of these determinants
themselves have been identified.
Levels of gene control
There are many steps between the initial transcription of a
gene and the formation of its eventual protein product. In
principle, gene activity could be regulated at any one, or all, of
these steps. There are good reasons now for excluding the two
extreme ends of the range of possibilities. One is that the genes
themselves become irreversibly altered so that they are
incapable of activity in cells where they are not expressed. The
other extreme view is that all genes are always transcribed into
mRNA in all cells, but that the mRNA is translated into protein
only in those cell-types in which expression occurs.
The first clear evidence against irreversible gene changes
during cell differentiation came from a nuclear-transplantation
experiment of the kind illustrated in Fig. 3. In the original
experiment (Gurdon, 1962) nuclei from larval intestine cells
were transplanted to enucleated eggs, some of which then
developed into adult frogs. Subsequently, nuclei from
keratinized skin cells of adult frog foot-web tissue were
transplanted to eggs, some of which formed swimming larvae
with fully functional muscle, nerve, blood and other cell-types.
In both these experiments genetic markers were used, either a
nucleolar marker resulting from a deletion of ribosomal genes
(Elsdale et al., 1958) or in subsequent experiments an albino
mutant (Hoperskaya, 1975). Since blood cells (and globin gene
expression) are wholly unrelated, embryologically and biochemically, to intestine cells or to skin cells, these experiments
showed that genes which are developmentally 'switched off' are
not only present, but also can be reactivated by exposure to
components of eggs or early embryos. It is worth mentioning
that there is no known way of converting an intestine or skin cell
into a blood cell, or of inducing globin synthesis in intestine or
skin cells. Recently, other kinds of experiments involving
restriction enzyme analyses of genomic DNA (i.e. Potter &
Thomas, 1977) have supported the concept of a constant
genome in development, except for the case of antibodyproducing cells.
The other extreme view mentioned above predicts that
mRNA characteristic of one kind of specialized cell (such as
globin mRNA of an erythrocyte) would not be translated if
introduced into other quite different cells, such as muscle or
nerve. After the surprising discovery (Gurdon et al., 1971; Lane
et al., 1971) that purified globin mRNA is efficiently translated
when injected directly into living oocytes, it soon became
technically possible to test the translation of mRNA species in
specialized cells. It would be technically difficult and timeconsuming to inject mRNA directly into muscle or nerve cells;
therefore we injected rabbit globin mRNA into eggs (actually
ones which had started but not completed their first cleavage)
and then grew these eggs until they had reached the tadpole
stage. Then a piece of axial tissue composed almost entirely of
muscle and nerve cells was dissected away from the rest of the
tadpole, and both pieces of tadpole were incubated in labelled
amino acids. Subsequent analysis showed that the muscle and
nerve fragment synthesized rabbit globin just as well as the rest
of the tadpole (Fig. 4), and hence that there is no evident
inability of one kind of cell to translate messages normally
present only in other entirely unrelated cells (Gurdon et al.,
1974; Woodland et al., 1974). This conclusion is in agreement
with subsequent work involving the direct injection of globin
mRNA into cells (Stacey & Allfrey, 1976). and with the results
of using various cell-free systems (Lodish, 1976).
The purpose of referring to these mRNA-injection experiments was not only to establish the point that translational
control does not appear to be the level at which the major gene
activity differences between cells are established. The other
interest in these experiments is that they were probably the first
to establish injected living oocytes as a generally useful
biochemical reagent. An oocyte is a growing egg-cell which is
readily obtained from the ovary of an adult female (Fig. 5). The
efficiency of mRNA translation is enormously high in living
oocytes, compared with cell-free systems, in terms of the
number of proteins eventually formed from each mRNA
molecule. This is largely because an injected oocyte will
continue to translate injected messages for as long as it can be
-
Keratiniaed
I
Adult frog
First nuclear transfers
r&
skin cells
in culture
Blastula
(partial)
Fertilized egg
2-cell
4-cell
Fig. 2. Diagram to show presumed partitioning of determinants
in early development
The symbols represent determinants for an endodermal tissue,
such as intestine (A), or for an ectodermal tissue, such as
nervous system (.).
The determinants are shown to be
randomly arranged in the fertilized egg, but to become
progressively localized towards the two poles of the egg so as to
be separated at the four-cell stage. This is the stage when the
ancestral cells of the intestine and nervous system become
separate (lower and upper pairs of cells respectively).
Serial nuclear transfers
w
Tadpole
Fig. 3. Summarji of a nuclear transplantation experiment in
which the nucleus of a keratinired skin cell is serially
transplanted into u.v.-enucleated eggs
See Gurdon et al. (1975) for details. UV, u.v.-enucleated egg.
1981
15
FIFTEENTH CIBA MEDAL LECTURE
mRNA
Inoubation in
labelled amino acids
I
Globin chains
Fig. 4. Diagram ofan experiment in which puriJed rabbit globin
mRNA was introduced into muscle and nerve cells
mRNA was injected into two-cell eggs, which were grown into
tadpoles. The tadpoles were dissected into one fragment, nearly
all of which consisted of muscle or nerve cells. This fragment, as
well as the other fragment, was shown to synthesize rabbit
globin (from the persistent injected mRNA) when appropriately
labelled and analysed. IFrom Woodland et al. (1974).1
(b)
oocyte
with yolk
but no
injection
pipette
-
maintained in culture (up to 3 weeks) whereas cell-free systems
are rarely very productive after 1-2 h. The efficiency of mRNA
translation in injected oocytes has attracted biochemists to their
use whenever very small amounts of mRNA are available. Up
until this time oocytes. which had first been used as ‘living
test-tubes’ many years before in connection with studies on the
control of DNA synthesis (Gurdon, 1967), had been regarded
as useful only to embryologists having a special interest in
oogenesis.
Analysis of gene activity by the use of cloned single genes
The conclusion so far is that the major differences between
cells depend on the control of gene activity at the general level of
follicle
cell layer
yolk
accumulation
B
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SV40 DNA
Fig. 5 . Xenopus oocytes
Photograph with a microinjection pipette (a),
and a drawing ( b ) to indicate the main features
of oocytes and ovarian tissue. [From Gurdon
(1977).1
Fig. 6. PuriJed DNA injected into occvtes is
expressed as proteins
Water (A) or SV40 circular DNA (B) was
injected into the nuclei of Xenopus oocytes.
which were incubated for 3 days, after which
labelled amino acids were added to the incubation medium. Twelve hours later proteins were
extracted and analysed by two-dimensional
gel electrophoresis, and fluorographed. For
further details see Gurdon et al. (1978).
BIOCHEMICAL SOCIETY TRANSACTIONS
16
transcription, including post-transcriptional processing etc. It
seemed clear that there would be an enormous advantage in
being able to follow the behaviour of single genes if it were
possible to inject numerous copies of one gene rather than to
transplant whole nuclei, in which some loo00 genes may be
undergoing changes in activity. Three specific examples of
information that can be gained by injecting single genes will be
given below, but first it may be useful to summarize the reasons
for believing that meaningful results can be obtained by injecting
purified genes as naked D N A into oocytes.
The first success came from experiments in which SV40 DNA
was injected into oocytes. By virtue of experience gained from a
very extensive analysis of sectioned oocytes (Gurdon, 1976), it
was possible to deposit injected material more often than not in
the nucleus of an oocyte, even though yolk and pigment make it
impossible to see the nucleus during injection. In these early
experiments (Mertz & Gurdon, 1977) it immediately became
clear that injected D N A was transcribed only if it was deposited
in the oocyte nucleus. Analysis of DNA-injected oocytes which
had been labelled with amino acids showed that some
SV40-coded proteins were synthesized (Fig. 6,and De Robertis
& Mertz, 1977). This last result proved that some meaningful
I NJ ECTE D
CONTROL
r 10
30
transcripts were made from injected DNA, but did not exclude
the possibility that transcription from the injected DNA was
more or less random, and that a few of the random transcripts
were able to be translated into the proteins which they coded for.
The fidelity or accuracy of transcription was first established by
using purified 5 s DNA, which codes for 5 s ribosomal RNA
(Brown & Gurdon, 1977). In these experiments all of the RNA
transcribed by injected D N A was exactly the correct size ( 5 S),
and was transcribed by the correct (coding) strand of the DNA.
The transcription from the injected DNA was so extensive that
in some cases it amounted to well over half of all labelled
transcripts made by an injected oocyte (Fig. 7).
In the first experiments, genomic DNA enriched for 5 S genes
was used. It was next established that segments of chromosomal
DNA, containing just one 5 S gene, and cloned in bacteria, also
produced extensive and accurate transcripts after injection into
oocytes (Brown & Gurdon, 1978). It was established by the
work of Wyllie et al. (1978)that much of the D N A injected as
double-stranded circles was rapidly converted into a nucleoprotein complex. We therefore decided to try to demonstrate by
the most decisive way possible, using the nuclear spreading
technique of Miller & Beatty (1969), that injected genes are
correctly transcribed. Nuclei were manually isolated from
DNA-injected oocytes, and subjected to the Miller spreading
technique. The results of using a recombinant bacterial plasmid
which contained a single ribosomal gene are described by
Trendelenburg & Gurdon (1978). Most of the small circular
molecules recovered consisted of D N A complexed in the normal
way with nucleosomes, and evidently untranscribed. On the
other hand a substantial number of transcription complexes
were seen in which a typical dense cluster of ribosomal
transcripts was visible. The correct length of the transcribed
region of DNA, and of the increasingly long lateral transcripts,
as well as the characteristically nucleosome-deficient spacer
DNA, all established the remarkable fidelity with which the
injected ribosomal genes were transcribed. Together these
results showed that correct transcription can take place from
injected DNA molecules containing single eukaryotic genes.
Evidently these injected DNA molecules are converted into
mini-chromosomes, which as far as the quality of transcription
is concerned behave much like the same genes in a normal
chromosome. It should be mentioned that linear molecules of
DNA are subject to exonuclease degradation (Wyllie et al.,
1978), and molecules of the length that can be cloned should be
injected as circles. Three examples are now given of the uses to
which gene injection into oocytes has been, or is being, put.
Transcriptional intermediates
Fig. 7. Purified SS DNA is accurately transcribed after injection
into Xenopus oocyte nuclei
DNA containing 5 S genes was injected into oocytes together
with a labelled nucleotide triphosphate. The next day RNA was
extracted, analysed by gel electrophoresis, and the gel radioautographed. The black band at the top of each gel track represents
mainly ribosomal RNA. The black bands 9cm down the gel are
5 s RNA, which is absent in the controls (no DNA injected).
IFrom Gurdon & Brown (1978).]
For a long time it has been believed, and more recently
established, that some genes are transcribed initially into a
precursor RNA which is subsequently cut up to yield a shorter
stable mRNA, the final gene product. Unless one is dealing with
multiple copy genes, which are synthesizing a significant
proportion of the total cellular RNA, as in the case with
ribosomal genes, it is very hard to identify these precursors or
early transcriptional intermediates. This is especially so if they
are short-lived. It is clearly of much importance to know the
initial and subsequent transcripts of a gene, since it could well be
that the expression of a gene is regulated by the selective
processing of intermediates as well as by transcription itself.
Gene injection into oocytes is proving to be an invaluable
technique by which to identify the initial transcripts as well as
processing intermediates. Enormous numbers of copies of a gene
can be injected into a single oocyte nucleus; typically about lo*
copies of an average-sized (5000 base pairs) length of DNA are
injected. This has two consequences. First a high proportion,
sometimes over 5096, of the total labelled transcripts of an
injected oocyte are products of that single gene, whereas the
transcripts of a single kind of gene usually constitute much less
than 0.1%)of all new RNA. Secondly, the enzymic machinery of
1981
FlFTEENTH ClBA MEDAL LECTURE
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5’ Leader
1
PPP
108
104
92
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\
Int. Seq.
17
3’ Traller
Fig. 8. Diagram showing the processing of transcripts synthesized from oocyte-injected
DNA, which includes a yeast tyrosine tRNA gene
The numbers on the left represent the length in bases of the tRNA gene transcripts, as seen
by gel electrophoresis. The right-hand part of the diagram shows the composition of the
early transcripts. [From Melton et al. (1980).1
the oocyte is sometimes unable to process the intermediates as
fast as usual because of their large abundance. Consequently
injected oocytes may accumulate large numbers of the normally
short-lived intermediates and initial transcripts. This approach
has been used particularly successfully by De Robertis & Olson
(1979) to identify a previously unknown initial transcript and
subsequent intermediates for a yeast transfer RNA gene.
Advantage has been taken by Melton & Cortese (1979) and by
Melton ef al. (1980) of the ability to isolate oocyte nuclei to
establish which of the many processing events take place in the
nucleus or in the cytoplasm (Fig. 8).
It is remarkable that amphibian oocytes can correctly process
the transcripts of foreign genes, such as the transfer RNA genes
of yeast and nematodes; this is also true of the chicken
ovalbumin gene, which is transcribed by polymerase I1
(Wickens er al., 1980). Indeed it is beginning to look like a valid
generalization that the processing of transcripts (as also the
secondary modification of proteins, see review by Lane &
Knowland, 1974) are not species-specific, and, in so far as
oocytes can carry out processing and modifications typical of
specialized cells, the enzymes needed for these events also seem
not to be cell-type specific.
Inverse genetics
In trying to investigate gene control, an immediate objective is
to map sequences of DNA, in and around a gene, which are
needed for accurate transcription; these include the polymerase
attachment site and sequences needed for the correct initiation
and termination of transcription. Ideally this type of investigation would be carried out by genetic analysis, but generally in
higher organisms, and certainly in the vertebrates, the life cycle
is sufficiently long as to make conventional genetic analysis
totally unrealistic. But once it is possible to determine the
biological activity of cloned D N A species, the dependence of
genetic analysis on successive generations, and hence on the
length of the life cycle, can be eliminated. For example a
eukaryotic gene can be cloned in a plasmid or in A bacteriophage, and then mutated. Mutants can at this stage be
described in detail, and if necessary sequenced, to determine the
exact site and nature of the mutation. The various mutant D N A
Vol. 9
species can then be injected into oocytes to test the biological
effect of the mutation (inverse genetics):
(1) Clone chromosomal DNA.
(2) Mutagenize and reclone.
(3) Sequence; identify mutations.
(4) Inject DNA molecules into oocytes-test
of mutants.
biological effects
This type of experiment has been carried out very successfully by Brown and colleagues, with 5 s genes transcribed in
isolated oocyte nuclei which work as satisfactorily as injected
oocytes for transcribing polymerase 111 genes (Birkenmeier et
al., 1978). Sakonju et al. (1980) showed that the promoter for
5 S genes lies in the middle of the gene. Using injected oocytes,
Grosschedl & Birnstiel (1980) have mapped the promoter for
histone genes to the 5’ side of the structural gene.
There is no obvious reason why this procedure should not be
used extensively to map DNA sites involved in the transcription
of genes in general. It now seems that genes transcribed by
polymerase I1 may be more difficult to test biologically because
1 phage and most commonly used plasmid DNA species have
promoters in them which are recognized by the oocyte
polymerase 11, and which therefore confuse any attempt to
analyse promoters within a cloned eukaryotic gene. This
difficulty seems to be overcome by re-isolating and circularizing
the cloned insert DNA, as has been done for histone genes
(Grosschedl & Birnstiel, 1980).
Within the last few years another assay procedure for cloned
DNA species has come into wide use. This is the transformation of cultured cells by precipitated DNA. However, the
transformed cells have to be propagated extensively and there is
a real possibility that the initially cloned D N A may be
subsequently cut down in size, or fused to other D N A species
during this period. In contrast, DNA molecules injected into
oocytes are not propagated and sufficient transcripts for
analysis have been made after incubation for only a few hours.
Re-isolation of DNA-protein complexes
A third quite different application of gene-injection experiments is to use oocytes to ‘fish out’ gene control molecules. The
BIOCHEMICAL SOCIETY TRANSACTIONS
18
600
800
-
600
500
200
I
0
0
10
20
30
40
Fraction no.
-
0
5
10
15
20
25
Fraction no.
Fig. 9. Re-isolation of DNA injected into oocytes as DNA-protein complexes
DNA of SV40 or of a 5 S gene-containing plasmid had been labelled during growth, and
was injected into oocyte nuclei. Two days later the oocytes were homogenized and the
homogenate was centrifuged on a sucrose gradient (a).The labelled DNA sedimented as a
peak much further down the gradient than would pure DNA, which would have remained
near the top of the gradient (fractions 35-40). The peak of labelled material was fixed in
formaldehyde and banded on a CsCl gradient (b). The injected DNA now bands in the
position expected of a DNA-protein complex containing about equal weights of DNA
and protein. [From Gurdon & Brown (1978) and Wyllie et al. (1978).1
idea is to inject multiple copies of a gene as purified DNA, and
then to re-extract the injected genes as nucleoprotein complexes.
In this way it might be possible to identify proteins which
interact with these genes, or DNA sequences to which oocyte
molecules might bind. The so-called ‘foot-printing’ procedure
could be used to identify protected regions of the injected, and
re-isolated DNA molecules. The injection of lo* genes into each
of about 50 oocytes should yield enough of a protein which
binds to each gene to describe the protein, perhaps after
iodinating it, after its removal from the DNA-protein
complexes.
Partial success towards this eventual objective has been
achieved by extracting oocytes injected with 3H-labelled DNA.
The extract is run down a sucrose gradient, in which a peak of
label is found in the region expected for DNA converted into a
nucleosomed structure (i.e. with an equal weight of protein)
(Fig. 9a). This complex has been further purified by fixing the
sucrose gradient peak of )H radioactivity counts in formaldehyde and centrifuging to equilibrium in a CsCl gradient (Fig.
9b). Further purification should be possible by use of the HMG
affinity column of Weisbrod et al. (1981). This column binds
transcriptionally active, but not inactive, chromatin by virtue of
the affinity of active chromatin for the ‘high mobility group’
non-histone chromosomal proteins 14 and 17.
If this experimental approach eventually proves useful, this
will be an area in which injected oocytes are likely to have a
distinct advantage over other possible experimental systems,
since their enormous size makes it possible to introduce and
recover substantial amounts of D N A and protein.
Nuclei injected into oocytcs
Although the injection of purified D N A molecules into
oocytes, and the introduction of D N A into cultured cells by
transformation, is giving much useful information, it is by no
means certain that either of these procedures will be able to
answer the ultimately most important question of how the
activity of genes is regulated. It is quite conceivable that the use
of cloned genes will permit the identification of DNA sequences
involved in the process of transcription, but not of the sites or
molecules concerned with determining whether or not transcrip
tion takes place. There are several reasons for worrying about
this possibility. When eukaryotic genes are cloned in bacteria,
they are unmethylated or incorrectly methylated; there is some
evidence that gene repression involves methylation. The length
of D N A that can be cloned in vectors currently in use is limited
to a maximum of about 50000 base pairs, and with many
systems only much shorter lengths (up to 20000 base pairs) can
be cloned. There is a real possibility that D N A of more than
50000 base pairs is needed to enable it to take up some special
conformation necessary for regulation. It is also possible that
higher ‘organisms could have regulatory genes located some
distance from the structural genes which they control, as has
been found for some bacterial genes. In the latter case,
regulatory genes might well not be included in the same cloned
1981
FIFTEENTH CIBA MEDAL LECTURE
19
length of DNA as their structural genes. Apart from these
theoretical considerations, it is true, as far as I know, that no
cloned gene has yet been shown to be subject to correct
regulation when introduced into living cells. In view of these
arguments, we have tried to find out whether it is possible to
detect the activity of genes when introduced into oocytes as a
suspension of whole nuclei, rather than as purified DNA. If this
were possible, nuclei could be taken from cells in which it is
known that certain genes are in a repressed inactive state. If
these genes were to remain repressed, it would be possible to
remove molecules from the nuclei until the genes are reactivated, and hence identify the molecules responsible for the
inactive state. If the inactive genes are activated after injection
into oocytes, it might be possible to find out how the genes have
been changed as they are transformed from an inactive into an
active state.
Pleurodeles
Xenopus
To detect the activity of genes in nuclei injected into oocytes
is much more demanding technically than in the case of nuclei
transplanted to eggs. In eggs, a single nucleus divides numerous
times to yield an embryo with about 50000 cells, and hence with
50000 copies of a unique gene. In contrast, nuclei injected into
oocytes do not divide; even though one can inject some 200
nuclei into each oocyte, it is very hard to see the products of
only 200 copies of a gene. However, the precision and sensitivity
of biochemical techniques improve continually and are now
sufficiently good to justify attempting the analysis of nucleusinjected oocytes.
In our initial investigation of this area, we injected cultured
adult kidney cell nuclei into oocytes, which were labelled with
amino acids overnight 3 days after injection, and the results
were assessed by two-dimensional protein-gel analysis (De
Robertis & Gurdon, 1977). The aim of these experiments was to
demonstrate the activation of genes characteristically active in
oocytes but inactive in kidney cells. In order to see such an effect
it was necessary to inject nuclei of Xenopus laevis into oocytes
of a different species (Pleurodeles waltlii), whose oocytesynthesized proteins were clearly distinguishable by 2D gel
analysis. Because oocytes synthesize their characteristic proteins
at a substantial rate from a large store of accumulated mRNA, it
would be very hard to demonstrate convincingly the activation
of oocyte-active genes in oocytes of the same species. The design
of these experiments as well as a diagram of the results is
shown in Fig. 10. It is evident that genes making proteins
characteristic of oocytes have been activated. We know
nothing more about these oocyte-active genes, except that they
are transcribed by polymerase 11, and we have not identified
.their initial transcripts.
In order to try to extend this type of analysis, we have turned
our attention to genes which synthesize 5 s ribosomal RNA.
These are especially suitable for our purpose because there are
two main classes of 5 s genes (Wegnez et al., 1972; Ford &
Southern, 1973; Brown, 1979). One class, the 5 s somatic genes,
of which there are about 400 copies per haploid chromosome
set, behave as if they are always active in all cells. The other
class, the 5 s oocyte genes, are present in about 20000 copies
per chromosome set, are fully active in oocytes and at least lo00
times less active per gene than the 5 s somatic genes in somatic
cells (see Fig. 11). But to investigate the regulation of the 5 s
genes in oocytes it is necessary to distinguish the 5 s RNA
products of the two kinds of genes. Until now this has had to be
done by the separation of oligonucleotides, a procedure which is
very time-consuming and which would severely limit the number
of samples which could be analysed. But the difficulties of this
analysis have been totally eliminated by L. J. Korn’s non-
I
1
1
Pleurodeles
oocytes
1
Pleurodeles
wcytes
with Xenopus
kidney nuclei
I
1
1
Xenopus
kidney cells
Xenopus
wcytes
Fig. 10. Summary of an experiment in which developmentally
‘switched o r genes are reactivated in oocytes
Cultured Xenopus kidney cell nuclei were injected into oocytes
of Pleurodeles, and the labelled proteins of the injected oocytes
analysed by two-dimensional gel electrophoresis. I Details from
De Robertis & Gurdon (1977).1
Tadpole
oocytes
5 s-vm
(20000 genesl
II
Blastula
Frog
Egg
’
+
+
+
++i
Fig. 1 1. Developmental regulation of 5 S genes
In adult cells, each 5 Soocficgene is lo00 times less active than each 5 Ssomatlcgene.
See the text for details, and references to original work.
VOl.
9
BIOCHEMICAL SOCIETY TRANSACTIONS
20
denaturing gel-electrophoresis system recently developed in this
laboratory. By this procedure, which separates RNA species on
the basis of their secondary structure, R N A species of identical
length and differing in only 1-2% of their bases can be fully
separated.
Using this procedure, we have analysed the 5 S RNA made by
oocytes injected with somatic nuclei. The first very encouraging
result is that substantial amounts of 5 s DNA are synthesized by
oocytes injected with nuclei, compared with the very low level of
5 s RNA synthesis by control uninjected oocytes by use of
their endogenous 5 S genes. Regarding the state of the 5 S oocyte
genes which were transcriptionally inactive in the donor cells, we
find that they have often been fully reactivated in the injected
oocytes. In some experiments, the 5 s oocyte genes have
remained repressed, but in these cases the treatment of the nuclei
with salt and detergent (a procedure which removes much of the
non-histone chromosomal protein) causes reactivation. These
results, which are described together with the non-denaturing
gel-electrophoresis procedure by L. J. Korn & J. B. Gurdon
(1981), need to be pursued further before any conclusions
can be drawn about the nature of the controls which regulate
5 s gene activity. There is hope that the material which is
removed from nuclei to reactivate these can eventually be
identified, possibly by the progressive removal of chromosomal
components in small steps. It is, however, clear at this stage that
the developmental switch-off of S S oocyte genes depends on
some rather easily reversible event. The ease with which the 5 S
RNA synthesized by relatively few nuclei can be seen suggests
that it may be possible to apply this same approach to single
copy genes, such as globin.
The oocyte as a universal gene-reactivating cell
There are several hints that oocytes may possess conditions
which activate all genes. Their own chromosomes are very
extensively dispersed into a lampbrush condition, which is
associated with intense RNA synthesis (Callan, 1963). There is
also a report that oocytes contain abundant globin transcripts
(Perlman el al., 1977). Somatic nuclei injected into oocytes
enlarge and increase their rate of RNA synthesis by 50-100
times (Gurdon, 1976). We have seen that some polymerase I1
genes as well as 5 s oocyte genes can be activated in nuclei
injected into oocytes.
Should it turn out in future experiments that oocytes d o
themselves actively transcribe nearly all genes, and that they can
activate nearly all transcriptionally inactive genes injected into
them as nuclei, this would raise the interesting possibility of
using oocytes to investigate non-expressed genes of somatic
cells. The prenatal analysis of cells collected by amniocentesis is
at present restricted to a few genes which are normally expressed
and to a few others whose existence can be verified by use of
appropriate cDNA probes. It might greatly extend the
usefulness of amniocentesis if a wide range of inactive genes
could be made to be expressed by injecting nuclei into oocytes.
The expression of single-copy genes in oocytes would still
require the use of cDNA probes for recognition of specific
transcripts, but the formation of transcripts would test the ability
of genes to transcribe, as well as making possible a test of the
normality of the transcripts. Neither of these kinds of information can come from the use of cDNA probes hybridized to
unexpressed genes in the DNA of cells collected by
amniocentesis.
Oocytes and eggs as living test-tubes
Although the early microinjection experiments with amphibian eggs required considerable technical expertise, especially
the transplantation of single nuclei to eggs, the microinjection of
solutions into oocytes is not technically demanding, and has
come to be used as a general biochemical procedure. Oocytes
were first used as living test-tubes to provide a negative control
for the induction of DNA synthesis by eggs injected with
purified D N A (Gurdon, 1967). The more general interest in
oocytes stemmed from the finding that purified mRNA is very
efficiently translated into protein when injected into them, as
indicated above. Success with oocytes as a message translation
system encouraged attempts to use them for transcription
experiments. In fact the synthesis of correct transcripts from
purified D N A was first achieved by the use of injected oocytes
(Mertz & Gurdon, 1977;Brown & Gurdon, 1977). Subsequently
a crude cell-free system has been developed by using an
isolated oocyte nucleus (Birkenmeier et al., 1978). As in the case
of message translation, a major advantage of injected oocytes is
that they continue to operate with a very high activity for many
days or even weeks. It is of interest that injected oocytes will
transcribe SV40 D N A by polymerase 11, its correct enzyme, but
the same D N A is transcribed by polymerase 111 in the cell-free
isolated-nucleus system. Another difference between injected
oocytes and systems at present in use in uitro is that initial
transcripts are usually processed to completion in living cells,
and consequently oocytes can be used to obtain complete
expression of a protein from purified DNA. The most recent use
of oocytes as living gene-expression systems, that is the
transcription of genes in whole nuclei as described above,
promises to be of considerable value. It is remarkable that
nuclei injected into oocytes have their 5 S genes transcribed los
times more efficiently than the same nuclei incubated in uitro.
Clearly it is desirable eventually for cell-free systems to be
developed which make use of only purified components. Injected
oocytes serve as a guide for the development of cell-free
systems; but no cell-free system seems likely to have the ability
to continue transcription or translation as long as a living cell.
The time taken to inject an oocyte is minimal; an average of
over 100 per hour is easy to achieve. The large size of an oocyte
means that substantial amounts of label can be introduced. For
labelled precursors that are efficiently incorporated, such as
[ 35S]methionine, about lo6 c.p.m. can be incorporated into the
proteins of a single oocyte during an overnight incubation. For
these various reasons, I believe that injected oocytes will
continue to be of considerable use as general biochemical
reagents, quite apart, I hope, from contributing directly to the
eventual understanding of gene control in development.
It is evident from the list of references that I am greatly indebted to
many colleagues who have worked with me in a scientific or technical
capacity; I have benefited immeasurably from their advice and help.
But I would like particularly to thank two whose names do not appear
in publications, but whose help has been of enormous benefit to myself
and my colleagues: Mrs. Barbara Rodbard, who has been responsible
for secretarial and other more administrative matters, and Janet Davey,
who has been responsible for the care of our Xenopus colony. Both
have been giving us invaluable help for many years.
References
Birkenmeier, E. H., Brown, D. D. & Jordan, E. (1978) Cell I S ,
1077- 1086
Brown, D. D. (1979) in Mechanisms of Cell Change (Ebut, J. D. &
Okada, T. S., eds.), pp. 65-70, Wiley, New York
Brown, D. D. & Gurdon, J. B. (1977) Proc. Natl. Acad. Sci. U S A .
74,2064-2068
Brown, D. D. & Gurdon, J. B. (1978) Proc. Natl. Acad. Sci. U.S.A. 75,
2849-2853
Callan, H. G. (1963) Int. Rev. Cytol. 15, 1-34
De Robertis, E. M. & Gurdon, J. B. (1977) Proc. Nafl. Acad. Sci.
U S A . 74,2470-2474
De Robertis, E. M. & Mertz, J. (1977) Cell 12, 175-182
De Robertis, E. M. & Olson, M. V. (1979) Nature (London) 278,
137-143
Elsdale, T. R.3 Fischberg. M. 8~Smith, S . (1958) Exp. Cell Res. 14,
642-643
Ford, P.J. & Southern, E. M. (1973) Nature (London) New Biol. 241,
7-11
Grosschedl, R. & Birnstiel, M. L. (1980) Proc. Natl. Acad. Sci. U.S.A.
77. 1432-1436
1981
FIFTEENTH CIBA MEDAL LECTURE
Gurdon, J. B. (1962)J. Embryol. Exp. Morphol. 10,622-640
Gurdon, J. B. (1967)Proc. Natl. Acad. Sci. U.SA. 58,545-552
Gurdon, J. B. (1977)Proc. R . Soc. London Ser. B. 198,211-247
Gurdon, J. B. (1976)J. Embryol. Exp. Morphol. 36,523-540
Gurdon, J. B. & Brown, D. D. (1978)D w . Biol. 67,346-356
Gurdon, J. B., Lane, C. D., Woodland, H. R. & Marbaix, G. (1971)
Nature (London) 233, 177-182
Gurdon, J. B., Woodland, H. R. & Lingrel, J. B. (1974)D w . Biol. 39,
125- I33
Gurdon, J. B., Laskey, R. A. & Reeves, 0. R. (1975)J. Embryol. Exp.
Morphol. 34,93-I 12.
Gurdon,J. B., Wyllie, A. H. & De Robertis, E. M. (1978)Philos. Trans.
R. Soc. London Ser. B 283,367-372
Hoperskaya, 0.A. (1975)J. Embryol. Exp. Morphol. 34,253-264
Korn, L. J. & Gurdon, J. B. (1981)Nature (London)in the press
Lane, C. D. & Knowland, J. S. (1974)in The Biochemistry of Animal
Development (Weber, R. A., ed.), vol. IV, Academic Press, New
York, London
Lane, C. D., Marbaix, G. & Gurdon, J. B. (1971)J. Mol. Biol. 61,
73-91
Lodish, H.F. (1976)Annu. Rev. Biochem. 45,39-72
Melton, D. A. & Cortese, R. (1979)Cell 18, 1165-1 172
Vol. 9
21
Melton, D. A., De Robertis, E. M. & Cortese, R. (1980)Nature
(London) 284, 143-148
Mertz, J. & Gurdon, J. B. (1977)Proc. Natl. Acad. Sci. U S A . 74,
1502- 1506
Miller, 0. L. & Beatty, B. R. (1969)Genetics Suppl. 61, 133-000
Perlman, S. M., Ford, P. J. & Rosbash, M. M. (1977)Proc. Natl. Acad.
Sci. U S A . 74,3835-3839
Potter, S. S . & Thomas, C. C. (1977) Cold Spring Harbor Symp.
Quant. B i d . 42, 1023- 1031
Sakonju, S., Bogenhagen, D. F. & Brown, D. D. (1980)Cell 19,13-25
Stacey, D. W. & Allfrey, V. G. (1976)Cell 9,725-732
Trendelenburg, M. F. & Gurdon, J. B. (1978)Nature (London) 276,
292-294
Wegnez, M., Monier, R. & Denis, H. (1972)FEBS Lett. 25, 13-18
Weisbrod, S.,Groudine, M. & Weintraub, H. (1981) Cell in press
Wickens, M. P., Woo, S., O'Malley, B. W. & Gurdon, J. B. (1980)
Nature (London) 285,628-634
Woodland, H. R., Gurdon, J. B. & Lingrel, J. B. (1974)D e n Biol. 39,
134-140
Wyllie, A. H.,Laskey, R. A., Finch, J. & Gurdon, J. B. (1978)Deo.
Biol. 64, 178-188