/ . Embryol. exp. Morph. Vol. 49, pp. 139-152, 1979
Printed in Great Britain © Company of Biologists Limited 1979
J39
Adenylation and ADP-ribosylation in the mouse
1-cell embryo
By R. J. YOUNG 1 AND K. SWEENEY1
From the Reproductive Biology Unit,
Department of Obstetrics and Gynecology,
Cornell University Medical College
SUMMARY
The incorporation of [ H]adenosine into cold trichloroacetic acid (TCA) insoluble material
by the mouse 1-cell embryo has been studied. Incorporation of label was high immediately
after fertilization, then decreased over the next 7 h with the sharpest decline occurring 3-5 h
after fertilization. A small maximum was observed at the time of pronuclear DNA synthesis.
Actinomycin D at a concentration which inhibited the cleavage of 1-cell embryos by 50 %
had little effect on this incorporation, which in the period 1-6 h post-fertilization was shown
by autoradiography to be confined to the ooplasm of the newly fertilized ovum. [3H]Adenosine and poly ([3H]A) were released from embryo RNA labelled 1-3 h after fertilization with
[3H]adenosine by digestion with a mixture of ribonucleases A and Tx. The poly ([3H]A)
segments were hydrolysed by alkali to 3'-[3H]AMP and [3H]adenosine ([3H]AMP/[3H]adenosine = 5/1), and by snake venom phosphodiesterase to 5'-[3H]AMP but very little [3H]adenosine. These results suggest that adenylation of RNA occurs soon after fertilization, that this
is a cytoplasmic event, and that most of the newly synthesized poly ([3H]A) segments are
joined to pre-existing poly (A) tracts. The unusual polynucleotide, poly (ADP-ribose), identified by its resistance to alkali and the release of 2'-(5"-phosphoribosyl)-5'[3H]AMP on incubation with snake venom phosphodiesterase, was also found in the ribonuclease digest.
3
INTRODUCTION
Although techniques for the culture in vitro of mammalian ova and embryos
have been developed, relatively few biochemical studies of the preimplantation
development of mammalian embryos have been made. Such investigations have
been confined to rabbit and mouse embryos and the difficulty in obtaining large
numbers of these embryos is most probably the reason for the small number of
studies carried out. An important aspect in embryonic development is when does
transcription of the embryonic genome commence, and if RNA synthesis is
required through preimplantation development. Inhibitors of RNA synthesis
can prevent cleavage of rabbit embryos as early as the 2-cell stage and even the
first cleavage of mouse embryos (Manes, 1973; Golbus, Calarco & Epstein,
1973) suggesting that transcription of the embryonic genome is necessary at all
1
Authors' address: Reproductive Biology Unit, Department of Obstetrics and Gynecology,
Cornell University Medical College, 1300 York Avenue, New York, New York 10021, U.S.A.
140
R. J. YOUNG AND K. SWEENEY
stages of preimplantation development. Itis possible, however, that the inhibition
of embryonic cleavage is unrelated to the effects that inhibitors actinomycin D
and a-Amanitan have on RNA synthesis since the former can cause changes in
cellular metabolism unrelated to RNA synthesis and the latter affects nucleolar
integrity (Manes, 1969; Golbus et al. 1973; Epstein, 1975).
The synthesis of heterogeneous RNA and tRNA has been reported to occur
in the rabbit embryo as early as the 2-cell and 4-cell stages (Manes, 1969, 1971,
1973) but a recent study casts doubt on the validity of the early results and
suggests that the rabbit embryonic genome is not active prior to the 16-cell
stage (Manes, 1977).
There is no doubt that the mouse embryonic genome is active before the
16-cell stage. The identification of paternally specific /?-glucuronidase by the
4- to 8-cell stage shows that mRNA synthesis occurs at least by the 4-cell stage
(Wudl & Chapman, 1976). Synthesis of RNA has been demonstrated in the
2-cell embryo and although the RNAs transcribed have not been biochemically
characterized, gel electrophoresis suggests that rRNA is a transcript (Knowland
& Graham, 1972). A recent histochemical study also suggests that the rRNA
genes are active at the 2-cell stage (Engel, Zenzes & Schmid, 1977). RNA
polymerase, however, is inactive in the late 1 -cell embryo (Warner & Hearn,
1977), but within 3 h after fertilization a very low level of RNA synthesis is
detectable, and [3H]guanosine is incorporated into RNA, predominately as the
7-methyl derivative at the 5'-terminus of pre-existing mRNA (Young, 1977;
Young, Sweeney & Bedford, 1978). Nevertheless, autoradiography has not
demonstrated incorporation of nucleosides into cytoplasmic macromolecules or
consistent labelling of pronuclei, but it has shown RNA polymerase activity in
a polar body (Mintz, 1964; Moore, 1975; Young et al. 1978), suggesting that the
embryonic genome is not active and that maternal RNA is utilized for development of the 1-cell embryo. The very low level of [3H]uridine or [3H]guanosine
incorporation by the 1-cell embryo has prevented biochemical characterization
of the RNA even when large numbers of embryos were used (Knowland &
Graham, 1972; Young et al. 1978). However, higher levels of [3H]adenosine are
incorporated into TCA insoluble material (Young, 1976) and this has permitted
limited biochemical characterization of the product. This communication reports experiments which suggest that soon after fertilization adenosine is incorporated predominately into the 3'-end of pre-existing RNA, and a polymer
of ADP-ribose is synthesized.
MATERIALS AND METHODS
Collection and culture of embryos
Virgin random bred Swiss female mice 7-12 weeks old were superovulated by
sequential injection of pregnant mare's serum and human chorionic gonadotropin (HCG) as previously described (Young et al. 1978). Mated females were
Adenylation and ADP-ribosylation in mouse embryos
141
sacrificed at 2 h intervals commencing approximately 0-5 h after fertilization
(15-16 h post-HCG), 1-cell embryos collected and incubated at 37 °C in Whitten's
medium containing 10mg/m.l bovine serum albumin overlayered with silicon
oil in an atmosphere of 5 % CO2, 5 % O2, 90 % N 2 as previously described
(Younger/. 1978). [2,8-3H]Adenosine (30-50 Ci/mmole, New England Nuclear)
was present in the medium at a concentration of 100-500 ^Ci/ml and actinomycin D (Calbiochem) was used at a concentration of 0-01 jug/m). Embryos
collected from ten females and divided into two groups were used for each time
point; one group was incubated in medium only and the other group in medium
containing actinomycin D. After incubation for 2 h, embryos were washed
four to six times with medium containing adenosine (0-1 mg/ml) and transferred in batches of 20-40 to lysing buffer (Young et al. 1978). The percentage
of fertilized ova that were pronuclear was determined at the start and end of the
2 h incubation period and 2-6 h later in a separate control culture in order to
monitor the development of the embryos. Labelling of large numbers of embryos
(up to 600/100/d) was carried out with gentle agitation on a reciprocating
shaker.
The ability of silicon oil to extract actinomycin D from the incubation medium
was checked by either gentle agitation for 24 h at 37 °C of a solution of actinomycin D covered with silicon oil or by vigorous shaking at 37 °C for 16 h of a
mixture of silicon oil and an aqueous solution of actinomycin D. Measurement
of the absorbance at 436 nm of the aqueous solution showed that > 98 % of the
actinomycin D remained in the aqueous phase.
Measurement of [sH]adenosine incorporation
After lysis of embryos by repeated freeze-thawing, uptake and incorporation
of label were determined by either placing the lysate onto paper discs and measuring the radioactivity before and after washing with cold TCA, or by precipitation with cold TCA and measuring the radioactivity in the precipitate and
supernatant (Young, et al. 1978). Background for the two methods was determined by adding an aliquant of the final wash medium to lysing buffer and
carrying this mixture through the precipitation or paper disc procedure. The
background was 15-25 cpm, and samples were counted to an error of 3-5 %.
Three measurements, each using 20-40 embryos, were averaged to give the
value for each time point in each experiment. Experiments were repeated five
times. Labelling times are the time post-HCG when embryos were placed into
culture for a 2 h incubation.
Isolation of^H-adenosine-labelledRNA and poly (A) tracts
Washed labelled embryos were transferred to 0-2 ml of 0-1 M Tris-HCl, pH
7-4, containing 15/tg yeast tRNA (Sigma) and 1 % sodium dodecyl sulphate
and lysed by repeated freeze-thawing. Labelled material was precipitated with
two volumes of ethanol in the presence of 2 % sodium acetate, pH 5, at - 2 0 °C
IO
EMB 49
142
R. J. YOUNG AND K. SWEENEY
for 16-20 h. The precipitate was collected by cenirifugation at 10000#, redissolved in 1 % sodium dodecyl sulphate, 0-1 M Tris-HCl, pH 8, containing
20 jug ATP and reprecipitated with ethanol at — 20 °C in the presence of 2 %
sodium acetate, pH 5. RNA was isolated from the precipitate as described by
Perry, La Torre, Kelly & Greenberg (1972) or Brawerman (1973) and recovered
from the aqueous layer by ethanol precipitation as above, and washed with cold
70 % aqueous ethanol. Some [3H]ATP was retained with the RNA even after
three precipitations.
Poly (A) tracts were released from [3H]adenosine-labelled RNA by digestion
of the RNA with a mixture of ribonucleases A (Worthington) and Tx (Calbiochem) essentially as described (Rodriguez-Pousada & Hayes, 1976) except that
sodium dodecyl sulphate was omitted and poly (A) (Miles) 3 A260 units and
tRNA (lO^g/ml) was added. The digest was examined by paper chromatography in solvent A (see below) followed by paper electrophoresis in the reverse
direction. Control experiments with poly ([3H]A) showed that the higher salt
conditions of Darnell, Wall & Tushinski (1971) did not alter the result and a
lower salt concentration (0-2 M) was generally used since this did not affect the
subsequent chromatographic separation. However, incubation of labelled embryo RNA for long periods with ribonuclease A in the absence of NaCl and
carrier poly (A) and tRNA did result in breakdown of the poly (A) tracts.
Chemical and enzyme digestion
Alkaline hydrolysis was carried out in 0-3 M KOH at 37 °C for 16-20 h. The
hydrolysate was desalted with perchloric acid in the cold before either chromatography or electrophoresis. Snake venom phosphodiesterase (phosphatasefree, Worthington) digestion was carried out in 001 M-MgCl2, 0-1 M Tris-HCl,
pH 7-5 or pH 8-8. Pronase (nuclease-free, Calbiochem) was incubated at 10
mg/ml in 005 M Tris-HCl, pH 7-5, for 1 h before use. All enzyme digestions
were carried out at 37 °C.
Electrophoresis and chromatography
Paper electrophoresis was performed on Whatman no. 1 paper as previously
described (Young & Fraenkel-Conrat, 1971). Buffers used were 0-05 M citrate,
pH 5, or 0 0 5 M triethylammonium acetate, 0 0 0 1 M EDTA, pH 5, when a
volatile buffer was required. Paper chromatography was carried out with
Whatman no. 1 paper; if electrophoresis was to follow chromatography, development was stopped when the solvent front had moved 10 cm beyond the
origin, the paper dried and electrophoresis carried out in the reverse direction.
Polyethyleneimine cellulose sheets (Baker) were used for thin layer chromatography. Solvent systems were: A, isopropyl alcohol:7-2 N ammonium hydroxide: water; 7:1:2 (v/v); B, 1 M acetic acid until the front had moved 5-6 cm
then development continued with 0-9 M acetic acid - 0-3 M-LiCl. The dried
Adenylation and ADP-ribosylation in mouse embryos
143
chromatograms or electrophorograms were cut into 0-5 cm strips, and the
radioactivity eluted from paper with 1 M ammonium hydroxide or from thin
layer sheets with 0-05 M acetic acid - 0-3 M-LiCl in 7 M urea before counting in a
toluene-based scintillation fluid (4 g Omnifluor/1) (New England Nuclear) containing the emulsifier BBS3 (Beckman).
Polyacrylamide gel electrophoresis was carried out as described (Fowlkes &
Young, 1970). Radioactivity in polyacrylamide gels was measured by solubilizing 1 mm slices at 60 °C with a mixture of nine parts NCS (Amerham-Searle)
and one part water, cooling at —15 °C for 3 h and counting in a Beckman
model LS 350 liquid scintillation spectrophotometer after addition of scintillation fluid without BBS3. Poly ([3H]A) was obtained from Schwartz/Mann,
poly A of average length 16, (A)-6 (range 10-23), was purchased from Miles and
adenosine phosphates (A)3_5 were obtained from PL Biochemicals. Poly (ADPribose) was a gift from L. Burzio; 2'-(5"-phosphoribosyl)-5'-AMP was obtained
by digestion of this polymer with snake venom phosphodiesterase.
A utoradiography
Autoradiography was carried out as described (Luthardt & Donahue, 1973).
The first slide was developed after 3 days, and sample slides developed every
2 days until exposure was satisfactory. Kodak NTB2 emulsion was used. The
slides were stored at 4 °C.
RESULTS
Incorporation of [^Hjadenosine
The time course of [3H]adenosine uptake and incorporation into TCA insoluble material by mouse 1-cell embryos was studied to determine the period
during development when adenosine incorporation was maximal. [3H]adenosine
was taken up and incorporated into cold TCA insoluble material throughout the
period 17-27 h post-HCG (ca. 1—11 h after fertilization). Uptake and incorporation of [3H]adenosine was highest soon after fertilization but these levels decreased within 2 h to about 50 % of this value and then slowly declined by a
further 40 % over the next 8 h (Fig. 1). In other experiments the level of [3H]adenosine incorporation fell to less than 20 % of the initial value by 28 h postHCG. Similar incorporation curves were obtained with different concentrations
of [3H]adenosine (100-500 /tCi/ml) and a levelling in incorporation occurred at
a [3H]adenosine concentration of about 300 /tCi/ml, suggesting that the internal
pool could be saturated and the decline in incorporation represented a decrease
in the rate of synthesis. A small peak was observed at approximately 23-25 h
post-HCG when pronuclear DNA synthesis is at its maximum (Luthardt &
Donahue 1973; Siracusa, Coletta & Monesi, 1975; Young et al. 1978). This peak
was found in each of five experiments occurring as early as 22-24 h and as late
as 24-26 h post-HCG. Actinomycin D at a concentration (0-01 ^g/ml) which
inhibited cleavage of 1-cell embryos by 50 % did not inhibit [3H]adenosine
144
R. J. YOUNG AND K. SWEENEY
17
21
Time post HCG (h)
25
Fig. 1. Time course of [3H]adenosine uptake and incorporation by 1-cell embryos
in the presence and absence of actinomycin D. • , TCA insoluble, no actinomycin D;
O, TCA insoluble, + actinomycin D; A, TCA soluble, no actinomycin D; A, TCA
insoluble, + actinomycin D. Each point is the time post-HCG when embryos were
placed in culture medium for 2 h. The figure shows an experiment in which the radioactivity was measured by the filter disc method. Each experimental point is the average
value from two to three batches of 20-40 embryos. [3H]Adenosine concentration
was200/*Ci/ml.
incorporation (Fig. 1), suggesting that [3H]adenosine was not incorporated
into newly made RNA.
Autoradiographic examination of one-cell embryos incubated with [3H]adenosine earlier than 21 h post-HCG showed that the cytoplasm but not pronuclei
was uniformly and heavily labelled in agreement with earlier studies (Moore,
1973; Young et al 1978). Thus [3H]adenosine incorporation appears to be a
cytoplasmic event.
Adenylation and ADP-ribosylation in mouse embryos
145
210
60
65
70
Gel slice number
Fig. 2. Polyacrylamide gel electrophoresis profile of 3H-adenosine-labelled material
from 1-cell embryos. Embryos incubated 1-3 h post-fertilization with [3H]adenosine
at 500/*Ci/ml were lysed by freeze-thawing and the labelled RNA isolated by
extraction with chloroform-phenol.
The nature of the zH-adenosine-labelled material
Since [3H]adenosine incorporation by 1-cell embryos was highest soon after
fertilization, only material labelled with [3H]adenosine 1-3 h after fertilization
was characterized. This material, isolated from embryos by phenol extraction
or ethanol precipitation, was insensitive to DNase but most (60-75 %) of the
radioactivity became TCA soluble after incubation with RNase A. Alkali
hydrolysed the labelled material to 3'-[3H]AMP and [3H]adenosine with the
ratio of the former to the latter varying from three to eight in different experiments. [3H]adenosine was therefore present in internal positions of RNA as well
as at the 3'-terminus.
When embryo lysates were extracted by procedures which liberate RNA
containing poly A (Perry et al. 1972; Brawerman, 1973) and the labelled RNA
examined by electrophoresis in 5 % polyacrylamide gels, peaks of radioactivity
were found near the top of the gel and near 4S RNA (Fig. 2). If the lysate was
extracted with 80 % aqueous phenol only, a method which does not efficiently
release RNA containing poly A, reduced levels of radioactivity were found in
the gel, and only one peak was present in the 4S region. However, if embryo
lysates were digested with Pronase before phenol extraction, more radioactivity
was present in the gel and the gel profile was similar to that shown in Fig. 2.
These results indicate that 1-3 h after fertilization [3H]adenosine is incorporated
into large and small RNA and some of the RNA may contain poly A tracts.
146
R. J. YOUNG AND K. SWEENEY
240
200
160
— 120
5
Distance (cm)
10
15
20
Fig. 3. Electrophorogram of ribonucleases A and Tx digest of 3H-adenosinelabelled embryo RNA. Labelled RNA was isolated by the method of Brawerman
(1973) from 1-cell embryos incubated with [3H]adenosine at 500/tCi/ml 1-3 h
post-fertilization, and digested with ribonucleases A and Tx. The digest was analysed
by paper chromatography in solvent A and paper electrophoresis at pH 5 as described in Materials and Methods. The dried electrophorogram was cut into 1 cm
strips, placed in scintillation fluid (4 g Omnifluor/1) and the radioactivity measured.
Labelled material was eluted from the paper strips with 1. M - N H 4 0 H . The origin is
0; direction of chromatography is to the left and electrophoresis to the right.
Because of self-absorption only 10-15 % of the radioactivity on paper is measured,
but the method does allow the location of radioactive peaks.
The poly (A) segments
Alkaline hydrolysis suggested that if segments of contiguous [3H]adenosine
residues were present in labelled embryo RNA then some would be short, less
than 9 nucleotides long, and thus may not be efficiently retained by immobilized
poly (U) (Nudel et al. 1976). This consideration and the difficulty in obtaining material suggested that attempts to isolate the poly([3H]A) segments
released from the RNA by digestion with ribonucleases A and Ty by binding to
Adenylation and ADP-ribosylation in mouse embryos
147
immobilized poly (U) or to characterize them by gel electrophoresis would be
unfruitful. Because lower members of a homologous series can be separated
by paper electrophoresis at acid pH (Smith, 1967), the use of this technique was
investigated. Paper electrophoresis of (A)i5 (range 10-23) at pH 5 did not resolve
the individual polymers; most remained at the origin but bands with mobility
0-24-0-65 with respect to 3'-AMP were present in the electrophorogram showing
that polymers longer than (A)10 do move from the origin. The poly ([3H]A)
segments released from labelled embryo RNA by digestion with ribonucleases
A and Tj were therefore analysed by paper chromatography in solvent A followed
by paper electrophoresis at pH 5. Instead of the expected radioactive peaks with
mobility near 3'-AMP and markers (A)2-(A)5, the digest was resolved by this
method into three large sharp radioactive peaks, one with the same mobility as
marker ATP, a second at the origin of the electrophorogram, and the third
behind the origin (Fig. 3). The poly ([3H]A) segments therefore appear to be
longer than the nine nucleotides indicated by alkaline hydrolysis.
The 3H-adenosine-labelled material in the peak with the same electrophoretic
mobility as ATP was completely hydrolysed to 5'-AMP by snake venom phosphodiesterase, co-migrated with marker ATP as a single radioactive band on
thin layer chromatography in l-6M-LiQ, and was not hydrolysed by 0-3 M
KOH. This peak therefore is [3H]ATP. However, [3H]ATP was not a product
of enzymic digestion since it was also found when the undigested 3H-adenosinelabelled RNA was examined by paper electrophoresis. Repeated precipitation
of the RNA with ethanol did not remove the contaminating [3H]ATP, but the
amount retained was greatly reduced by brief (5-10 sec) shaking of the RNA
pellet with cold 10 % TCA on a vortex mixer. The contaminating [3H]ATP
could be removed by repeated extractions with cold TCA; however, this was
avoided since hydrolysis of the RNA or isomerization of the internucleotide bond
could occur and ATP was clearly separated by paper electrophoresis from the
products of enzymic digestion.
The radioactive peak behind the origin co-migrated with added adenosine
marker on both paper chromatography and paper electrophoresis and was
identified as [3H]adenosine. In three experiments 33-42 % of the radioactivity
was released as [3H]adenosine from the labelled RNA following its digestion
with ribonucleases A and Tv Thus a large proportion of the [3H]adenosin.e
incorporated into RNA by the 1 -cell embryo is present at the 3'-terminus
adjacent to cytidine, uridine or guanosine.
The 3H-adenosine-labelled material in the peak at the origin amounted to
35-48 % of the total radioactivity in the RNA, and was completely hydrolysed
by both snake venom phosphodiesterase and 0-3 M KOH, showing it to be
poly ([3H]A). The [3H]AMP/[3H]adenosine ratio in the alkaline hydrolysate was
5/1 (449 cpm/90 cpm) but very little [3H]adenosine (48 cpm; [3H]AMP, 1242
cpm) was found in the snake venom phosphodiesterase digest.
The small broad peak with mobility between (A)3 and (A)5 was found in every
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R. J. YOUNG AND K. SWEENEY
experiment. Very little (< 20%) of this labelled material was hydrolysed to
[3H]AMP or [3H]adenosine by 0-3 M KOH and the electrophoretic mobility of
the remainder was unchanged after incubation with alkali. This result suggested
that an alkaline resistant 3H-adenosine-labelled compound with a relatively high
electrophoretic mobility, possibly a compound with several phosphate residues
as well as oligo ([3H]A) segments were present in the small peak. However,
snake venom phosphodiesterase completely hydrolysed the 3H-adenosinelabelled material in the peak giving two 3H-adenosine-labelled compounds which
were identified as [3H]AMP and 2'-(5"-phosphoribosyl)-5'-[3H]AMP (phosphoribosyl-AMP) by thin layer chromatography with added markers in solvent B
(Chambon et al 1966; Lehmann, Kirk-Bell, Shall & Whish, 1974; Young &
Sweeney, 1978). A small amount of radioactivity also co-migrated with adenosine, but no radioactivity was found with marker adenosine-3',5'-diphosphate.
Since adenosine polyphosphates (e.g. ppAp) which have high electrophoretic
mobility are hydrolysed to adenosine-3',5'-diphosphate by snake venom phosphodiesterase, this result shows that the alkaline resistant 3H-adenosine-labelled
compound is not an adenosine polyphosphate. On the other hand, poly (ADPribose) is resistant to alkali but is hydrolysed by snake venom phosphodiesterase to phosphoribosyl-AMP, a reaction which is characteristic of such polymers
and is used as a method for their detection (Chambon et al. 1966; Hasegawa,
Fujimara, Shimizu & Sugimura, 1967; Nishizuka, Ueda, Nakazawa & Hayaishi,
1967; Reeder et al. 1967; Hayaishi & Ueda, 1977). Identification of phosphoribosyl-AMP in the snake venom phosphodiesterase digest therefore shows that
the alkali resistant 3H-adenosine-labelled compound is a polymer of ADPribose. The ratio of phosphoribosyl-AMP to AMP found in two experiments was
less than one. This value is probably low because of the presence of some oligo
([3H]A) segments in the small peak; nevertheless the result does suggest that the
polymer is a dimer of ADP-ribose since the polymer chain length is given by the
ratio phosphoribosyl-AMP/AMP.
DISCUSSION
Previous autoradiographic studies of nucleoside or nucleotide incorporation
by the mouse 1-cell embryo have failed to demonstrate RNA polymerase activity
in pronudei or cytoplasm of the embryo (Mintz, 1964; Moore, 1974; Young
et al. 1978). Biochemical techniques also have not detected extensive incorporation of [3H]uridine or [3H]guanosine into RNA (Woodland & Graham, 1969;
Knowland & Graham, 1972; Young, 1977; Young et al. 1978), results which
suggest that development of the 1-cell embryo is dependent on maternal RNA.
The present studies are in agreement with this conclusion since they also show
that the pronuclei of the newly fertilized ovum are inactive, but in contrast
with earlier studies, they show that the ooplasm of the fertilized ovum actively
incorporates [3H]adenosine into macromolecules. Most of the [3H]adenosine
label was found, after digestion of the [3H]adenosine-labelled RNA with
Adenylation and ADP-ribosylation in mouse embryos
3
3
149
ribonucleases A and Tl5 as [ H]adenosine and poly ([ H]A) segments which
remain at the origin on pH 5 paper electrophoresis. Liberation of [3H]adenosine
from labelled embryo RNA by these enzymes means that this nucleoside is at
the 3'-terminus of the RNA and the penultimate nucleoside is either guanosine,
undine or cytosine. The 1 -cell embryo is active in protein synthesis (Epstein &
Smith, 1974; Van Blerkom & Brockway, 1975), and turnover of the adenosine
in the -CCA terminus of tRNA may explain the presence of [3H]adenosine in
the digest and the appearance of labelled RNA in the 4S region of the gel
(Fig. 2).
Alkali releases nucleosides only from the 3'-terminus of a polynucleotide;
therefore the presence of [3H]adenosine in the alkaline hydrolysate of the poly
([3H]A) segments at the origin of the electrophorogram shows that these segments are at the 3'-terminus of the RNA and that their average length
([3H]AMP/[3H]adenosine = 5/1) was six nucleotides long. Because poly (A)
tracts longer than ten nucleotides long move from the origin on pH 5 electrophoresis, the poly ([3H]A) segments at the origin are most probably joined to
unlabelled poly (A) tracts, a conclusion supported by snake venom phosphodiesterase digestion of the poly ([3H]A) segments. Snake venom phosphodiesterase liberates nucleosides only from the 5'-terminus of a polynucleotide chain
and since little [3H]adenosine was present in the enzyme digest of the poly
([3H]A) segments, few of these segments terminate in a 5'-[3H]adenosine residue,
i.e. most are linked to unlabelled poly (A) tracts. It is likely then that RNA containing poly (A) is present in the unfertilized ovum. The recent demonstration of
RNA containing poly (A) in the unfertilized mouse ovum (G. Stull, personal
communication) and that adenylation of RNA occurs in the unfertilized ovum,
(Young & Sweeney, 1978) agrees with this conclusion. Whether the the cytoplasmic adenylation observed in the 1-cell embryo is turnover of poly (A), or
lengthening of pre-existing poly (A) tracts is unknown. The observation that the
amount of poly (A) in the ovum of the mouse (G. Stull, personal communication) and rabbit (Schultz, 1975) does not increase after fertilization may mean
that the cytoplasmic adenylation is turnover of the poly (A) tracts in embryo
RNA rather than their net lengthening. Further, the decrease in [3H]adenosine
incorporation occurring immediately after fertilization (Fig. 1) suggests that
poly (A) synthesis largely ceases soon after fertilization. This behaviour is in
marked contrast to that of the sea urchin embryo in which extensive and dynamic
turnover of poly (A) occurs and results in a net increase in their length (Wilt,
1977) and contributes to the doubling of the amount of poly (A) after activation
(Slater, Slater & Gillespie, 1972; Wilt, 1973).
Poly (ADP-ribose) is present in a variety of tissues covalently linked to proteins by bonds which differ in their lability at neutral or alkaline pH values
(Nishizuka, Veda, Honjo & Hayaishi, 1968; Wong, Poirier & Dixon, 1977) and
a recent study has demonstrated the presence of a monomer and pentamer in
unfertilized mouse ova (Young & Sweeney, 1978). The dimer found in the 1-cell
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R. J. YOUNG AND K. SWEENEY
embryo may have arisen by modification of the pentamer or monomer after
fertilization. However, since neither the pentamer or monomer was released
from protein and isolated with RNA (Young & Sweeney, 1978), it is likely that
the dimer was synthesized after fertilization and if it were linked to protein this
bond is more labile than those joining the pentamer and monomer to protein.
Covalent modification of cellular proteins by poly (ADP-ribose) has been
associated with a number of regulatory activities such as chromatin function and
protein and nucleic acid metabolism (see Hilz & Stone, 1976; Hayaishi & Ueda,
1977). Soon after fertilization chromatin of both the ovum and the fertilizing
sperm changes from a highly condensed to a diffused state, DNA is replicated,
and a change occurs in the pattern of proteins synthesized. Modification of
proteins in the newly fertilized ovum by ADP-ribosylation reactions may play
a role in these changes.
The gift of poly (ADP-ribose) from Dr L. Burzio is gratefully acknowledged. This investigation was supported by The Rockefeller Foundation and National Institutes of Health
Grant HD 10230.
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{Received 4 July 1978, revised 4 October 1978)