J. Embryol. exp. Morph. 97 Supplement, 197-209 (1986)
197
Printed in Great Britain © The Company of Biologists Limited 1986
The stability and movement of mRNA in Xenopus
oocytes and embryos
ALAN COLMAN AND DOUGLAS DRUMMOND*
MRC Developmental Biology Group, Department of Biological Sciences, University
of Warwick, Coventry CV4 7AL, UK
INTRODUCTION
The Xenopus oocyte has a distinguished history as an in vivo host for the
deposition by microinjection of a variety of macromolecules including DNA
(Colman, 1984), RNA (Soreq, 1985), proteins (Dingwall, Sharnick & Laskey,
1982) and polysaccharides (Paine, Moore & Horowitz, 1975). In these cases, the
oocyte has simply either provided an efficient production system for a product
encoded by an injected nucleic acid (Soreq, 1985; Krieg etal. 1984) or has been
used to investigate mechanisms regulating RNA (De Robertis, Lienhard &
Parison, 1982) and protein (Gurdon, 1970; Davey, Dimmock & Colman, 1985)
targeting within a living cell. However, microinjection also provides a method for
perturbing or simulating processes that occur normally within the oocyte, in order
to understand their molecular nature and regulation. For example, Bienz &
Gurdon (1982) during their investigations of heat-shock translational control in
Xenopus oocytes, argued that the heat-shock genes encoding the 70xl0 3 M r heatshock protein (hsp 70) must be constitutively active in oocytes; this was in contrast
to the behaviour of injected Drosophila hsp 70 genes in unstressed oocytes. Bienz
(1984) was subsequently able to simulate the natural situation and confirm her
prediction by cloning and then injecting the Xenopus hsp 70 gene into oocytes.
A good example of the perturbation of the system comes from studies involving
competition between injected and endogenous mRNAs for the available translation machinery (Laskey, Mills, Gurdon & Partington, 1977; Asselbergs, Van
Venrooij & Bloemendal, 1979; Richter & Smith, 1981). Most of the studies have
used large stage VI oocytes (Dumont, 1972) and the current conclusions are that
the oocyte has no spare translational capacity and therefore the low percentage
(<2 %) of ribosomes present in polysomes (Woodland, 1974) is not simply due to a
shortage of available mRNA. Recently similar studies on stage IV oocytes have
shown that considerable spare translational capacity exists in these cells (Taylor,
Johnson & Smith, 1985).
* Present address: Department of Zoology, University of Texas, Austin, Texas, USA.
Key words: messenger RNA, Xenopus, oocyte, movement, stability, anti-sense RNA, poly (A)
RNA, capping, polyadenylation.
198
A . COLMAN AND D . DRUMMOND
Finally, in a different design of experiment, Richter & Smith (1981) investigated
the relative translational efficiencies of different exogenous mRNAs which were
either coinjected or sequentially injected. They observed no competition between
certain mRNA classes and concluded that the mRNAs for secretory and nonsecretory proteins entered different translational pools.
In the quantitative interpretation of all the competition data cited above, it is
implicitly assumed that injected mRNAs diffuse rapidly and at similar rates within
the oocyte to achieve a homogenous, equilibrium distribution. However, various
studies attest to the complex spatial arrangement of the endogenous macromolecules in the oocyte. For example several groups have demonstrated distinct
gradients of specific classes of RNA within oocyte and fertilized egg cytoplasm
(Capco & Jeffery, 1982; Carpenter & Klein, 1982). Also yolk and other proteins
show a pronounced concentration gradient along the animal-vegetal axis of these
cells (Nieuwkoop & Faber, 1967). Given this complexity, the behaviour of injected
RNAs and proteins cannot be assumed. Studies of mRNA movement have been
made in fertilized Xenopus embryos, however, it is inappropriate to extrapolate
the findings to oocytes since the cytoplasmic consistency and organization of
developing embryos is very different from that existing in oocytes.
If the above assumptions regarding RNA diffusion in oocytes are incorrect, the
results from the competition studies may need reinterpretation and in any event
clarification of the issue is required. Resolution of this diffusion problem also now
seems more urgent due to the emergence of the technique of anti-sense RNA
injection (Melton, 1984) and the discovery of localized RNAs in Xenopus oocytes
(Rebagliati, Weeks, Harvey & Melton, 1985). The ability of injected anti-sense
RNA to hybridize with endogenous RNA will obviously be affected by the ability
of the injected RNA to diffuse within the oocyte or embryo. Likewise studies
whose objective is to determine mechanisms of RNA localization and whose
experimental strategy involves relocalization of injected RNAs, will need to
consider the rate of diffusion of injected RNA. In this paper we discuss results that
directly address the movement of injected RNAs within oocytes and embryos.
RESULTS AND DISCUSSION
Stability of injected mRNA in oocytes
The stability of an mRNA in oocytes is an important factor in the design and
interpretation of experiments to measure the movement. Stability has also proved
an issue of some contention over the years with grossly different values being
assigned to the same mRNAs by different workers (e.g. Furiuchi, La Fiandra &
Shatkin, 1977; cf. McCrea & Woodland, 1981). We have conjectured that variation between oocyte batches might be responsible for reported differences in
mRNA stability (Drummond, McCrea & Colman, 1985) and such batch-dependent variation has recently been observed by Taylor etal. (1985). However, of
particular concern to our studies was the report of Richter & Smith (1981), which
indicated that stability was also related to the amount of mRNA injected (globin
Stability and movement ofmRNA in Xenopus
199
or zein mRNAs) even when the same batch of oocytes was used. Such dosedependent stability could complicate diffusion studies since the diluting effect of
diffusion might make RNA molecules in the diffusion 'front' less susceptible to
degradation than those remaining at the injection site.
We have used two different methods to examine mRNA stability in oocytes. In
the first, poly (A) + mRNAs extracted from a variety of tissues were injected into
oocytes and total RNA recovered from the oocytes at various times after injection.
This RNA was then electrophoresed on denaturing agarose gels, transferred to
nitrocellulose and then hybridized to 32P-radiolabelled complementary DNA
(cDNA) probes. After exposure to X-ray film, the RNA stability was quantified
either by microdensitometry or by band excision and scintillation counting. A
typical autoradiograph obtained after rabbit globin mRNA injection is shown in
Fig. 1A whilst the derived quantification is indicated in Table 1. Alternatively, the
final hybridization step can be omitted if the injected RNA is radiolabelled. Under
these circumstances the autoradiograph is obtained by exposure of the dried-down
gel. Measurements made using this method are more accurate.
Two methods for producing radiolabelled RNA were used. First 32P-reovirus
RNAs were synthesized in vitro by adding radioactive nucleotide triphosphates to
disrupted reovirus virions (Skehel & Joklik, 1969). The resultant transcripts,
which are poly (A)", were identical to those formed during normal viral infection
and we will refer to these RNAs as natural poly (A)~ mRNAs. The second method
involves the in vitro transcription of any cloned gene using the Salmonella
typhimurium phage SP6 polymerase/vector system (Melton etal. 1984). This
method allows the transcription of either sense (i.e. coding) or anti-sense RNA.
However, it should be emphasized that even the 'sense' RNAs so obtained will
always differ slightly from their natural equivalents at the 5' end and probably
differ at the 3' end. We will refer to all these RNAs as synthetic RNAs. When the
stability experiment was repeated with radioactive RNAs the results shown in
Fig. 1B,C and Table 1 were obtained.
The general conclusion from the results in Table 1 is that all the RNAs (natural
or synthetic) are similarly stable over the first 24 h with approximately 50%
of injected RNA surviving intact. Further experiments (not shown but see
Drummond et al. 1985a; Drummond, Armstrong & Colman, 1985b) indicated that
this stability is not affected by the exact site of injection, nor by the amount of
RNA injected (ranges of 5-100 ngoocyte" 1 tested) nor finally by the participation
of the transcript in translation/Indeed similar stabilities were found for anti-sense
(i.e. noncoding) RNAs (Drummond etal. 1985b). We have not observed the
striking batch-to-batch variations observed by others (e.g. Taylor etal. 1985).
Effect of post-transcriptional modifications on mRNA stability in oocytes
(1) Capping
All eukaryotic and many viral mRNAs have a cap structure consisting of a 7methyl-guanosine base joined by a triphosphate bridge to their 5' end. In addition
A. COLMAN AND D . DRUMMOND
200
the first (and sometimes the second) nucleoside of the RNA is methylated
(Banerjee, 1980). The cap structure increases the stability of natural mRNAs
(McCrea & Woodland, 1981) and SP6 transcripts (Green, Maniatis & Melton,
1983; Krieg & Melton, 1984) injected into Xenopus oocytes. In the experiments
m
1
Oh
24h48h
100
10
0 24h48h"0
m
1 101
24h48hM1
~l
m
Oh 24h 48h ' 10 100'
8
Stage
9 1 8
9
MDNA
HS~
-M
H4LYS-
B
JL
Expt
1
2
Fig. 1. Stability of RNAs in oocytes and embryos. Various RNAs were injected into
oocytes (A-C) or fertilized eggs (D) and RNA extracted and analysed at the times or
developmental stages indicated. (A) Globin mRNA; (B) reovirus RNAs, 12S (5), 18S
(M); (C) SP6-lysozyme RNAs, with (A+) or without (A~) added poly (A); (D) SP6
lysozyme RNA (LYS), Xenopus histone H4 RNA (H4), Xenopus heat shock, hsp 70
RNA (HS). Marker (m) tracks contain 1,10 or 100 ng per track. Further details can be
found in Drummond etal. (l%5a,b).
Stability and movement ofmRNA in Xenopus
201
Table 1. Stability of RNAs in oocytes and embryos
Cell type
RNA type
Amount of RNA after specified time
Oh
Oocytes
Embryos
rabbit globin
chick ovalbumin
reovirus 12S
reovirus 18S
SP6 lys, no cap
... lme, cap
... 2me, cap
SP6 lys, lme cap
SP6 lys, lme cap, poly (A) +
SP6 chym lme cap
SP6 chym lme cap, poly (A) +
SP6 lys, lme cap
SP6 lys, H4 and hsp 70,
all lme cap
100
100
100
100
100
100
100
100
100
100
100
6h
—
—
—
—
61
90
82
—
—
—
—
Stage 1
(Oh)
100
[100]
Stage 7
(4h)
28
—
24 h
—
—
37
47
10
49
45
62
47
70
90
48 h
=50
=50
39
51
—
—
—
33
54
38
71
Stage 8 Stage 9
(5h)
(7h)
—
—
[35]
[26]
Oocytes or fertilized eggs were injected with 5 or 50 ng per cell of the RNAs indicated above.
After the indicated times, the cells were frozen, extracted and the RNA analysed and stability
values obtained as described in the text except for the values in parentheses. In these instances
microdensitometry readings from the autoradiographs and acid insolubility estimates on total
extracted nucleic acid were used. Further details can be found in Drummond etal. (1985a,b).
Abbreviations: lys, chick lysozyme; chym, calf prochymosin; H4, Xenopus histone H4; hsp 70,
Xenopus 70xl0 3 M r heat-shock protein.
shown in Fig. 1, the synthetic transcripts all contained a methyl group only on the
first nucleotide. We have compared the stability of transcripts containing methyl
groups on the first (monomethyl cap), the first and second (dimethyl cap) or on
neither (uncapped). The results are shown in Table 1. In contrast to a previous
report where uncapped transcripts were undetectable 15 min after injection (Krieg
& Melton, 1984), we found 61 % of uncapped transcript present after 6h. However, the possession of some form of cap was essential for longer-term stability.
(2) Polyadenylation
The role of poly (A) in stabilizing mRNAs injected into oocytes is controversial
with claims for a significant stabilizing effect for some mRNAs (globin, Marbaix
et al. 1975; histone, Huez et al. 1978) being countered by claims for no effect at all
on the stability of other mRNAs (interferon, Sehgal, Soreq & Tamm, 1978; ar-2u
globulin, Deshpande, Chatterjee & Roy, 1979). We have directly tested the effect
of polyadenylation on mRNA stability by post-transcriptionally adding poly (A)
tails to synthetic RNAs, and then assessing the effect on stability in oocytes of this
modification. As shown in Fig. 1C and Table 1 polyadenylation had only a slight
effect on stability over the first 24 h. However, after 48 h, the polyadenylated
202
A. COLMAN AND D. DRUMMOND
transcripts were clearly more stable irrespective of RNA type. Further experiments demonstrated that this stability was unrelated to mRNA translation
(Drummond et al. 1985ft).
In summary, we have demonstrated that all the RNAs tested were at least 50 %
stable over the first 24 h so long as a 5-terminal cap structure was present. For the
synthetic transcripts, stability was increased in the long term by the possession of a
poly (A) tail.
Stability of RNA in embryos
We have tested the stability of synthetic transcripts in embryos over the first 4 h
of development (i.e. from the 1-cell to the early- or mid-blastula stage). Three
different transcripts, encoding the Xenopus hsp 70 heat-shock protein, Xenopus
histone H4 and chicken lysozyme, were coinjected into fertilized eggs, and the
RNA recovered from the blastulae and analysed. The results shown in Fig. ID and
Table 1 show that all the RNAs are far less stable in embryos than oocytes over
similar periods of incubation. Again, as in oocytes, degradation seems to be an allor-none process with no detectable shift in radioactivity to gradually smaller
molecular weights. This indicates that degradation is either a progressive or a
cooperative process involving one or more nucleases respectively. We have not yet
investigated the effect of polyadenylation on the stability of these RNAs.
Movement of injected RNAs in oocytes
We have used two methods to examine the movement of injected RNA in
oocytes. Common to both methods was the injection of RNA (natural or
synthetic) at the animal or vegetal poles. In one method the oocytes were frozen to
-70°C at various times after injection and bisected into animal and vegetal halves.
RNA was then extracted from pooled halves and analysed electrophoretically etc.,
as described above (see Fig. 1). In the second method oocytes were fixed,
embedded in wax, sectioned and then exposed to photographic emulsion (Fig. 3);
this latter procedure has so far been restricted to the analysis of radioactive RNA
movement.
The experiments shown in Fig. 2A,B indicate that the rates of movement of
natural poly (A) + and poly (A)~ (reo mRNAs) are slow and differ according to the
site of injection. We see that movement away from the animal halves appears to be
much slower than movement in the opposite direction. This same trend was found
after injection of synthetic RNAs (Drummond etal. 1985ft; also Fig. 2B) with
striking confirmation of the slow movement coming from the in situ autoradiography (Fig. 3). However, when the appropriate quantification was derived
from the gels it appeared that the synthetic RNAs moved more rapidly than the
natural mRNAs. This difference was confirmed by coinjecting radioactive natural
poly (A)~ mRNAs (reo) and SP6 transcripts (lysozyme) as shown in Fig. 2B.
These relative rates of movement of the injected templates were not influenced by
RNA concentration nor by size in the range of 500-1800 bases (Drummond et al.
1985a,ft).
203
Stability and movement ofmRNA in Xenopus
A inj
1
Oh
A
24h
V
A
V
A
V
Vinj
m
48h'
1
1
Oh
A
10
24h
V
A
V
48h'
A
m
r
V
,
1 10
fV inj
•~Oh
A
B
24h'
V
A
V
' Oh
A
r~oh
A V
24h '
V
A
V
24f? " O h
A
V A V
A
24h '
V
-M
•-S
-- r
— Lys
• -/-ys
Exposure 2
Exposure 1
V/ny
/A /ny
A
V
1cell
A
8cell
V
A
V
1 cell
A
V
8cell
Fig. 2. Movement of RNA in oocytes and embryos. RNAs were injected into the
animal or vegetal pole of oocytes (A,B) or fertilized eggs (C). At the times indicated
total RNA was extracted from animal or vegetal halves and analysed on denaturing
gels (A) Globin mRNA; (B) lysozyme (Lys) and reovirus 12S (5) and 18S (M) RNAs;
panel (C) SP6 lysozyme RNA (Lys) and SP6 Xenopus histone H4 RNA (H4).
Abbreviations: A, animal halves; V, vegetal halves; A inj, injection into animal pole;
Vinj injection into vegetal pole. Marker tracks (m) as in Fig. 1. Arrows point to the
positions of 18 and 28S ribosomal RNAs. For further details see Drummond etal.
(19S5a,b).
204
A . COLMAN AND D .
DRUMMOND
Fig. 3. In situ autoradiography of injected oocytes. 32P-Lys+ dimethyl cap RNA was
injected into oocytes at the vegetal (A,B>C,E and G) or the animal pole (D,F, and H)
and the oocytes fixed within lOmin (A-D), 6h (E,F) or 24h (G,H) and sectioned.
After mounting on slides the oocyte sections were autoradiographed. The autoradiographed sections were visualized by phase-contrast (A) or dark-field (B-H)
illumination. Panels A,B show the same oocyte section. The bright rim around the
oocytes in panels B-H is due to the light scattering by dense pigment granules (not
silver grains) distributed around the periphery of the oocytes. The nucleus of the
oocyte is indicated (gv, germinal vesicle).
Stability and movement ofmRNA in Xenopus
205
One explanation for the difference in mobility between the synthetic templates
and natural mRNAs included in Fig. 2 could be the absence in the former of a poly
(A) tail. Although this could not be the only factor since the reovirus mRNAs are
also poly (A)~, we compared the movement rates of synthetic templates with and
without added poly (A) tails (Drummond et al. 19856). Polyadenylation did reduce
the rate of diffusion, however, the data were not sufficiently accurate for any
unequivocal interpretation as to the quantitative contribution of polyadenylation
to the new, slower rate. Other contributing factors may be the binding of the 3' or
5' regions of the natural poly (A) + and poly (A)~ mRNAs with components of the
cytosol. Precedents for such interactions exist in observations of the binding of
mRNAs to poly (A) (Richter & Evers, 1984) or 'cap'-binding proteins.(Zumbe,
Stahli & Trachsel, 1982). The synthetic transcripts used in our diffusion study
lacked much of their normal 5' and 3' regions.
Two main conclusions emerge from the experiments outlined above. First,
diffusion is generally slow irrespective of the source of the RNA. This behaviour
contrasts with the more rapid equilibration shown by the other, much smaller
RNAs (e.g. transfer RNAs, 5S, 7S and small nuclear RNAs, De Robertis etal.
1982). We presume that the slow rate can be explained by the size and conformation of these RNA molecules combined with the increased diffusional
pathlength imposed by the crowded cytoplasm. The second conclusion is that
diffusion away from the animal half is much slower than that in the opposite
direction. This might reflect the volumes of 'accessible' cytoplasm in the two
halves (see Drummond et al. 1985a). We measured the relative volumes accessible
to tritiated fucose and found the ratio to be 60:40 (animal:vegetal). This is
probably not a sufficient difference to account for our observations, however,
regions accessible to a small sugar molecule might be unavailable to the high
molecular weight RNAs.
How do these observations affect the interpretation of the competition experiments cited earlier? Clearly with diffusion being so slow, the competition for
translation observed between injected and endogenous mRNAs can only reflect
events in those localized regions populated by both RNA types. This makes
attempts to compare relative translational efficiencies or to measure maternal
mRNA pool sizes (Richter & Smith, 1981) even more difficult to interpret.
However, our results do not alter the conclusions regarding spare translational
capacity or different translational pools (Laskey et al. 1911 \ Richter & Smith, 1981)
except that a small (^5 %) translational capacity in large oocytes might be
obscured if only a limited proportion of the oocyte's endogenous protein synthesis
is exposed to competition due to slow mRNA diffusion.
The slow diffusion observed has implications for the design of experiments
where the desired aim is to 'neutralize' endogenous mRNA sequences by
hybridization to injected anti-sense RNA (Melton, 1984; Harland & Weintraub,
1985). In order to ensure sufficient hybridization we would suggest (a) injection of
supersaturating concentrations of anti-sense RNA and (b) injection of small
(i.e. = 100 bases) anti-sense RNA molecules generated either by transcription of
206
A. COLMAN AND D. DRUMMOND
truncated genes or by alkali digestion (see Cox, Deleon, Angerer & Angerer,
1984). In both cases we would recommend delaying the assay until 24 h (at least)
after injection, although it is clear that satisfactory results have been obtained with
shorter incubation periods (Melton, 1984; Harland & Weintraub, 1985).
A second problem area highlighted by these results concerns studies investigating the localization of mRNAs in oocytes. Rebagliati et al. (1985) have recently
found four RNAs to be localized to either the animal or vegetal regions of the
Xenopus oocyte. It is conceivable that these RNAs contain sequence(s) that target
the RNAs in some way to the appropriate region. One test of this possibility would
be to inject oocytes with a synthetic version of the localized RNA and look for
relocalization. If relocalization occurred then the sequences responsible could be
identified using a systematic mutagenesis/RNA-injection strategy. Our results
would indicate that such injection experiments would require many days during
which time pathological changes may begin to occur in the oocytes. An alternative
strategy might be to inject the gene encoding the localized RNA into the germinal
vesicle of the oocyte. In this way the oocyte itself will produce the studied
transcript. We have reported tentative evidence that transcripts produced in this
way are more rapidly distributed within the cytoplasm (Drummond et al. 1985a).
Movement of injected RNAs in embryos
We have examined the movement of synthetic RNAs in fertilized embryos by
similar methods to those used in oocytes. Embryos were injected at the 1-cell stage
in either the animal or vegetal pole, with a mixture of Xenopus histone H4 and
chicken lysozyme RNAs. At the 8-cell stage, the embryos were frozen, bisected
and analysed as shown in Fig. 2C. The results are highly reminiscent of those
obtained in oocytes 48 h after injection in that there is very little detectable
movement animal to vegetal in contrast to considerable movement in the opposite
direction. However, it should be emphasized that the 8-cell stage is reached within
2?h. This increased rate of movement might reflect the greater fluidity of the
cytoplasm seen after maturation and fertilization as well as the bulk redistribution
of the yolk shortly after fertilization (Wilt & Phillips, 1984).
Extra information was obtained when in situ autoradiography experiments were
performed. We coinjected chicken lysozyme and Xenopus /3-globin transcripts and
fixed the embryos at stage 7. Sample results are shown in Fig. 4. We anticipated
that cleavage would pose an additional obstacle to RNA movement and this
turned out to be the case. Clearly the distribution is localized at stage 7 even after
injection at the 1-cell stage. Localization can occur to animal (Fig. 4C-F) and
vegetal (Fig. 4G,H) areas. We cannot reconcile these data with the observations
of Capco & Jeffery (1981) who found that RNA extracted from the vegetal pole of
eggs would relocalize to this area within 2h of injection, regardless of the site of
injection. Froehlich, Bowder & Schutz (1977) reported that rabbit globin mRNA
injected into fertilized embryos became sixfold more concentrated in the animal
regions by the gastrula stage. However, these results are consistent with our
Stability and movement ofmRNA in Xenopus
Fig. 4. In situ autoradiography of injected embryos. Fertilized eggs were injected at
the 1-cell stage with 32P-labelled SP6 globin RNA and fixed immediately (A,B) or at
the blastula stage (C-H) and processed as in Fig. 3. The sites of injection were vegetal
(A,B5G,H) or animal (C-F). The sections were visualized by phase-contrast
(B,D,F,H) or dark-field illumination (A,C,E>G). Standard negatives were used to
print A-F whilst G,H were prepared from colour transparencies.
207
208
A. COLMAN AND D. DRUMMOND
observations since their rabbit globin mRNA was injected into the animal
hemisphere.
In summary then, we find that movement of injected RNA in embryos appears
much faster than that in oocytes, however, the rapid divisions ensure segregation
of injected RNAs. This poses problems for experiments involving injections of
sense or anti-sense RNA since homogenous distribution throughout the embryo
may be a desired objective. However, small reductions in temperature (i.e. 5°C
fall) have dramatic effects on the cell division rate and this might not be
accompanied by comparable effects on RNA diffusion. The best compromise
would therefore appear to involve injections into the vegetal hemisphere of a
fertilized egg followed by incubation at 15 °C or below until the 8-cell stage when
segregation of animal and vegetal hemispheres becomes finally complete.
This work was supported by a grant to A.C. from the British Medical Research Council. We
thank Dr P. Krieg for comments on the manuscript.
REFERENCES
F. A. M., VAN VENROOIJ, W. J. & BLOEMENDAL, H. (1979). Messenger RNA
competition in living Xenopus oocytes. Eur. J. Biochem. 94, 249-254.
BANERJEE, A. K. (1980). 5'-terminal cap structure in eukaryotic messenger ribonucleic acids.
Microbiol. Rev. 44,175-205.
BIENZ, M. (1984). Xenopus hsp 70 genes are constitutively expressed in injected oocytes.
EMBOJ. 3, 2477-2483.
BIENZ, M. & GURDON, J. B. (1982). The heat shock response in Xenopus oocytes is controlled at
the translational level. Cell 29, 811-819.
BIENZ, M. & PELHAM, H. (1982). Expression of a Drosophila heat shock protein in Xenopus
oocytes: conserved and divergent regulatory signals. EMBO J. 1, 1583-1588.
CAPCO, D. G. & JEFFERY, W. R. (1982). Transient localisations of messenger RNA in Xenopus
laevis. DevlBiol. 89,1-12.
+
CARPENTER, C. D. & KLEIN, W. H. (1982). A gradient of poly (A) RNA sequences in Xenopus
laevis eggs and embryos. Devi Biol 91, 43-49.
COLMAN, A. (1984). Expression of exogenous DNA in Xenopus oocytes. In Transcription and
Translation - A Practical Approach (ed. D. Hames & S. Higgens), pp. 49-69. Oxford: IRL
Press.
Cox, K. H., DELEON, D., ANGERER, L. & ANGERER, R. (1984). Detection of mRNAs in sea urchin
embryos by in situ hybridisation using asymmetric RNA probes. Devi Biol. 101, 485-502.
DAVEY, J., DIMMOCK, N. J. & COLMAN, A. (1985). Identification of the sequence responsible for
the nuclear accumulation of the influenza virus nucleoprotein in Xenopus oocytes. Cell 40,
667-675.
DE ROBERTIS, E. M., LIENHARD, S. & PARISOT, R. F. (1982). Intracellular transport of
microinjected 5S and small nuclear RNAs. Nature, Lond. 295, 572-577.
DESHPANDE, A. K., CHATTERJEE, B. & ROY, A. K. (1979). Translation and stability of rat liver
messenger RNA for ar2u-globulin in Xenopus oocytes. /. biol. Chem. 254, 8937-8942.
DINGWALL, C , SHARNICK, S. V. & LASKEY, R. A. (1982). A polypeptide domain that specifies
migration into the nucleus. Cell 30, 449-458.
DRUMMOND, D. R., MCCREA, M. A. & COLMAN, A. (1985a). Stability and movement of mRNAs
and their encoded products in Xenopus oocytes. /. Cell Biol. 100,1148-1156.
DRUMMOND, D. R., ARMSTRONG, J. & COLMAN, A. (1985/?). The effect of capping and
polyadenylation on the stability, movement and translation of synthetic mRNAs in Xenopus
oocytes. Nucl. Acid Res. 13, 7375-7394.
DUMONT, J. N. (1972). Oogenesis in Xenopus laevis (Daudin). I. Stages of oocyte development in
laboratory maintained animals. J. Morph. 136, 153-180.
ASSELBERGS,
Stability and movement ofmRNA in Xenopus
209
FROEHLICH, J.
P., BROWDER, L. W. & SCHUTZ, G. A. (1977). Translation and distribution of rabbit
globin mRNA in separated cell types of Xenopus laevis gastrulae. Devi Biol. 56, 356-371.
FURIUCHI, Y. A., LA FIANDRA, A. & SHATKIN, A. J. (1977). 5'-terminal structure and mRNA
stability. Nature, Lond. 266, 235-239.
GREEN, M., MANIATIS, T. & MELTON, D. (1983). Human and globin pre mRNAs synthesised in
vitro is accurately spliced in Xenopus oocyte nuclei. Cell 32, 681-694.
GURDON, J. B. (1970). Nuclear transplantation and the control of gene activity in animal
development. Proc. R. Soc. B 176, 303-314.
HARLAND, R. & WEINTRAUB, H. (1985). Translation of mRNA injected into Xenopus oocytes is
specifically inhibited by anti-sense RNA. /. Cell Biol. 101,1094-1099.
HUEZ, G., MARBAIX, G., HUBERT, E., CLEUTER, Y., LECLERQ, M. & CHANTRENNE, H. (1975).
Readenylation of poly adenylate-free globin mRNA restores its stability in vivo. Eur. J.
Biochem. 59, 589-592.
KRIEG, P. A. & MELTON, D. A. (1984). Functional messenger RNAs are produced by SP6 in vitro
transcription of cloned cDNAs. Nucl. Acid Res. 12, 7057-7070.
KRIEG, P. A., STRACHAN, R., WALLIS, E., TABE, L. & COLMAN, A. (1984). Efficient expression of
cloned complementary DNAs for secretory proteins after injection into Xenopus oocytes.
/. molec. Biol. 180, 615-643.
LASKEY, R. A., MILLS, A. D., GURDON, J. B. & PARTINGTON, G. A. (1977). Protein synthesis in
oocytes of Xenopus laevis is not regulated by the supply of messenger RNA. Cell 11, 345-351.
MARBAIX, G., HUEZ, G., BURNY, A., CLEUTER, Y., HUBERT, E., LECLERQ, M., CHANTRENNE, H.,
SOREQ, H., NUDEL, U. & LITTAUER, U. (1975). Absence of polyadenylate segment in globin
messenger RNA accelerates its degradation in Xenopus oocytes. Proc. natn. Acad. Sci. U.S.A.
72, 3065-3067.
MCCREA, M. A. & WOODLAND, H. R. (1981). Stability of non polyadenylated viral mRNAs
injected into frog oocytes. Eur. J. Biochem. 116, 467-470.
MELTON, D. A. (1984). Injected anti-sense RNAs specifically block messenger RNA translation
in vivo. Proc. natn. Acad. Sci. U.S.A. 82, 144-148.
MELTON, D. A., KRIEG, P. A., REBAGLIATI, M. R., MANIATIS, T., ZINN, K. & GREEN, M. R. (1984).
Efficient in vitro synthesis of biologically active RNA and RNA hybridisation probes from
plasmid containing a bacteriophage SP6 promoter. Nucl. Acid Res. 12, 7035-7056.
NIEUWKOOP, P. D. & FABER, J. (1967). Normal Table of Xenopus laevis. Second edn.
Amsterdam: North Holland.
PAINE, P. L., MOORE, L. C. & HOROWITZ, S. B. (1975). Nuclear envelope permeability. Nature,
Lond. 254, 109-114.
REBAGLIATI, M. R., WEEKS, D. L., HARVEY, R. P. & MELTON, D. A. (1985). Identification and
cloning of localised maternal RNAs from Xenopus eggs. Cell 42, 769-777.
J. D. & EVERS, D. C. (1984). A monoclonal antibody to an oocyte-specific poly (A)
RNA-binding protein. J. biol. Chem. 259, 2190-2194.
RICHTER, J. D. & SMITH, L. D. (1981). Differential capacity for translation and lack of
competition between mRNAs that segregate to free and membrane-bound polysomes. Cell
27,183-191.
SEGHAL, P., SOREQ, H. & TAMM, H. (1978). Does 3-terminal poly (A) stabilise human fibroblast
interferon mRNA in oocytes of Xenopus laevis. Proc. natn. Acad. Sci. U.S.A. 75, 5030-5033.
SKEHEL, J. J. & JOKLIK, W. K. (1969). Studies on the in vitro transcription of reovirus RNA
catalysed by reovirus cores. Virology 39, 822-831.
SOREQ, H. (1985). The biosynthesis of biologically active proteins in mRNA-microinjected
Xenopus oocytes. CRC Critical Reviews in Biochem. 18,199-238.
TAYLOR, M. A., JOHNSON, A. D. & SMITH, L. P. (1985). Growing Xenopus oocytes have spare
translational capacity. Proc. natn. Acad. Sci. U.S.A. 82, 6586-6589.
WILT, F. H. & PHILLIPS, C. R. (1984). Localisation of poly (A) inXenopus embryos. In Molecular
Biology of Development, pp. 23-36. New York: Alan R. Liss Inc.
WOODLAND, H. R. (1974). Changes in the polysome content of developing Xenopus laevis
embryos. Devi Biol. 40, 90-101.
ZUMBE, A., STAHLI, C. & TRACHSEL, H. (1982). Association of a MT50,000 cap-binding protein
with the cytoskeleton in baby hamster kidney cells. Proc. natn. Acad. Sci. U.S.A. 79,
2927-2931.
RICHTER,