Behavior of indivdual maternal pA+ RNAs during embryogenesis of Xenopus laevis

DEVELOPMENTAL

BIOLOGY

94,

‘79-86

(1982)

E3ehavior of Individual Maternal pA+ RNAs (during Embryogenesis of Xenopus laevis HILDUR Department

V. COLOT AND MICHAEL

of Biology, Brandeis University,

Waltham,

ROSBASH Massachusetts

02254

Receiued May 10, 1982; accepted June 18, 1982 Of the 10 Xenopus oocyte cDNA clones previously examined in this laboratory (L. Golden, U. Schafer, and M. Rosbash, 1980, Cell 22,835-844), 5 are complementary to RNAs which decrease in abundance during early development. We have further examined the behavior during embryogenesis of these 5 sets of clone-complementary RNAs. The results indicate that for 3 of these 5 sets of RNAs there is an increase in the per embryo levels of RNA. Thus, 8 of the 10 clones originally examined are complementary to RNAs which increase in amount during early embryogenesis. One of the remaining two clones is complementary to (at least) 4 RNAs which vary somewhat in their levels during embryogenesis. The last clone (XOC 2-7) is complementary to an RNA species which is largely destroyed at late blastula or early gastrula. This, RNA is therefore the only maternal sequence, of the ten clones examined, which unambiguously decreases in amount during embryogenesis. The data also show that XOC 2-7 RNA is largely adenylated at oocyte maturation and then deadenylated during subsequent embryogenesis while another clone, XOC l-2, is largely deadenylated at oocyte maturation. The results also suggest that a large fraction of oocyte RNAs are present in early embryos (and in liver) and are largely the same size as in oocytes.

Recombinant DNA technology makes it possible to examine many of these events in finer detail. First, the use of clones permits the examination of events not detectable by in vitro protein synthesis. Moreover, it permits the examination of mRNAs too rare to be easily detectable by irz vitro protein synthesis. Our laboratory has utilized Northern gel analysis to examine changes in the abundance of a number of RNAs in the pA+ RNA populations during Xenopus oogenesis. Since the total pA+ RNA content remains constant during much of oogenesis, changes in the abundance of individual pA+ RNAs reflect changes in the per oocyte levels of these sequences (Golden et al., 1980). These same clones were also used in a preliminary comparison of the RNA populations of Xenopus tadpoles, liver, and oocytes (Golden et al., 1980) (Table 1). In agreement with a more extensive study using colony hybridization (Dworkin and Dawid, 1980), no general rules could be drawn from the relative abundance of these mRNAs in tadpole as compared to oocytes; some mRNAs increased in abundance and others decreased in abundance during the first 3 days of embryogenesis. The interpretation of abundance changes during early development in Xenopus, however, is not straightforward. In contrast to the relatively static situation which occurs during oocyte vitellogenesis (Golden et al., 1980) the mRNA content (and pA+ RNA content) of Xenopus embryos increases approximately 6- to lo-fold between fertilization and the stage 41 tadpole (Sagata et al., 1980; Weiss et al., 1981). Consequently, a change in the relative abundance of an mRNA sequence in these

INTRODUCTION

During the first 3 days of embryonic development in the South African clawed. toad, Xenopus, a large number of changes take place which serve to transform the large unicellular unfertilized egg into a fully formed, swimming tadpole. This process is almost certainly the result of developmental information of both maternal and zygotic origin. In particular, informational RNA of maternal (maternal mRNA)l and zygotic origin code for the spectrum of proteins synthesized during this period. The mRNA population of Xenopus oocytes and the changes that this population undergoes during embryogenesis have been examined by a number of investigators. Both quantitative changes in the amount of total messenger RNA present at various stages of embryogenesis (Sagata et al., 1980; Weiss et al., 1981) as well as qualitative changes which take place in this mRNA population (Perlman and Rosbash, 1978; Brock and Reeves, 1978; Ballantine et al., 1979) have been described. Changes have also been reported in the amount, length, and subcellular location of poly(A) during both oocyte maturation and elmbryogenesis (Darnbrough and Ford, 1976; Sagata et al., 1980). It may also be the case that mRNAs are differentially expressed (translational control) during early embryogenesis to account for some of the changes which take place in in uivo protein synthesis (Ballantine et al., 1979; Sturgess et al., 1980). All of these studies serve to underscore the complexity of the molecular events which occur during early embryogenesis. 79

0012-1606/82/110079-08$02.00/O Copyright All rights

0 1982 by Academic Press. Inc. of reproduction in any form reserved.

80

DEVELOPMENTALBIOLOGY TABLE

1

ANALYSISOF~~ OOCYTE(XOC)CDNA xoc clones”

Northern oocyte signal*

CLONES

Liver pA+ abundance’

Tadpole pA+ abundanced

Tadpole pA+ per embryo”

Down l-2 2-4 2-6 2-l 6-35

2 1 2 4 3

Down Down Down Down Down

Down Down Down Down Down

UP ? HP Down UP

UP l-6 l-7 1-31

3 3 2

UP UP Same

HP HP Same

UP UP

2-9

1

UP

UP

UP UP

6-34

2

HP

UP

UP

’ As analyzed in Golden et al. (1980). The first set of five are down clones in that the abundance of the complementary RNA decreases by the tadpole stage. The second set of five are up clones in that the abundance and therefore the levels per embryo of the complementary RNA increase by the tadpole stage. * Data from Golden et al. (1980). 5 = Strongest signal, 1 = weakest signal on a Northern blot to oocyte RNA. c,dData from Golden et al. (1980). Abundance, relative to oocyte pA+ RNA, of liver and tadpole pA’ RNA, respectively. e Behavior of clone-complementary RNAs on a per embryo basis.

two pA+ RNA populations does not necessarily give a meaningful indication of the change during development in the per embryo level of that sequence. This is especially relevant to the five oocyte clones examined which are complementary to RNAs which decrease in abundance during embryogenesis (Golden et al., 1980). The per embryo levels of some of these RNAs might remain relatively constant or even increase somewhat during embryogenesis while the abundance decrease is due to a greater increase (6- to lo-fold) in total pA+ RNA per embryo. Therefore, we examined these five clones in more detail. By comparison to our previous study, we utilized more RNA and examined fixed numbers of oocytes and embryos so that comparisons on a per embryo basis could be made. These changes provide unambiguous evidence for one mRNA which is destroyed during early embryogenesis. They also suggest a number of additional sequence-specific phenomena, chiefly at the level of polyadenylation, which take place during oocyte maturation and early embryogenesis. MATERIALS

AND

METHODS

Oocytes and embryos. Mature stage 6 Xenopus oocytes and embryos were prepared as previously described (Rosbash, 1981; Golden et al., 1980). RNA preparations. RNA and pA+ RNA was prepared as in Rosbash (1980). Fixed numbers of oocytes, eggs, and embryos were used in parallel experiments. Prior to application to oligo(dT)-cellulose the same amount

VOLUME94. 1982

of yeast RNA was added to each sample of embryo RNA, usually 10 pg yeast RNA for 200 embryos (N l-2 mg RNA). Gels of developmental stages were hybridized to Xenopus probes and subsequently to the yeast plasmid pYll-10 (Woolford and Rosbash, 1979), to verify that the recovery of pA+ RNA was uniform for the different RNA samples. Only lane 3 of Fig. 4 was visibly deficient in the recovery of yeast pA+ RNA. Northern blots. Formaldehyde gel electrophoresis was performed by a minor modification of the method of Rave et al. (1979). Electrophoresis was as in Bruskin et al. (1981) except that 1.5% agarose gels were utilized. The gel was rinsed for 30 min in 20 X SSC (1 X SSC = 0.15 h4 NaCl, 0.015 M Nas citrate, pH 7.4) and blotted to nitrocellulose (Thomas, 1980) in 20 X SSC. The filter was rinsed for 1 min in 4 X SSC and baked for 2 hr at 80°C in a vacuum oven. In our hands, the formaldehyde gel procedure affords better resolution, somewhat better signals, and much lower noise levels. Consequently long exposures are more informative, especially for faint bands and for smears. One experiment (Fig. 2) employs our previous CH3Hg-DBM protocol (Golden et al., 1980). Radioactive DNAs. Clones were nick-translated as described (Golden et al., 1980) using 0.2 pg cloned DNA and 50 PC1 32P dCTP (New England Nuclear-supplied in Tricine) in a final volume of 20 ~1. Specific activities of l-5 X lo8 cpm/pg were routinely achieved. Reactions were terminated by the addition of 10 ~1 stop buffer (Golden et al., 1980), and passed through a Pasteur pipet containing approximately 1.0 ml Bio-Gel P-60 (Bio-Rad Laboratories) in TE (0.01 A4 Tris, 0.001 M EDTA, pH 8.3). The excluded volume was collected by using blue dextran and bromocresol red as color indicators of the excluded and included volume, respectively. (The use of P-60 and the dyes was suggested by E. Myerowitz). Hybridization. The nitrocellulose was moistened in 2 X SSC. Then 10 ml of prehybridization mix (5 X Denhardt’s solution (Denhardt, 1966), 50 m&f Na phosphate, pH 6.5,5 X SSC, 50% formamide, 0.2 mg/ml sonicated denatured salmon sperm DNA) was added and incubated for 1 hr at 42’C. The solution was removed and replaced by 10 ml of hybridization mix (1 X Denhardt’s solution, 20 mM Na phosphate, pH 6.5,5 X SSC, 50% formamide, 10% dextran sulfate, 0.1 mg/ml sonicated denatured salmon sperm DNA, 5-10 X lo6 cpm denatured probe). Incubation was usually 16-18 hr at 42°C. The nitrocellulose was washed 3 X 10 min in 2 X SSC, 0.1% SDS at room temperature, 2 X 30 min in 0.1 X SSC, 0.1% SDS at 50-55°C. The paper was then exposed to Kodak XAR-5 film at -70°C with intensifying screens.

COLOT

AND

ROSBASH

Maternal

RNase H. Incubation of total RNA with RNase H (Vournakis et al., 1975) was as indicated by the supplier (BRL). RNA (10 pg) was incubated with 0.5 pg oligo(dT) in 10 ~1 for 3 min at 50°C and then for 30 min at 37°C. Approximately 0.25 units of enzyme was added and the incubation continued for an additional 30 min at 37°C. The volume was increased to 300 ~1 with 0.2 M NaOAc and the RNA was extracted with phenol and precipitated with ethanol. Half (5 pug) of each sample was analyzed on Northern gels. Poly(U)-Sepharose. As described by Palatnik et al. (1979), 1.8 mg total RNA. from mature oocytes and from eggs was analyzed by thermal elution from poly(U)-Sepharose (Pharmacia). Fractions (1%ml) were collected at 2.5”C increments from a 0.5-ml column of p(U)-Sepharose. Control experiments indicated that this volume was sufficient to collect most of the RNA that would elute at any temperature. tRNA (10 pg) carrier was added to each fraction, and the RNA was precipitated with ethanol and resuspended in 10 ~1 H,O. Of each fraction, 4 ~1 was analyzed on formaldehyde gels as shown in Fig. 7.

mRNAs

81

in X. laevis Embryos

1234

I234

A FIG. 2. Hybridization of cDNA clones to pA+ RNA from staged embryos. RNA was isolated from 200 mature oocytes, and from sets of 200 embryos at the gastrula (stage g/10), neurula (stage 18/20), and the feeding tadpole stage (stage 41). pA+ RNA from each of these RNAs was isolated, electrophoresed in CH,Hg, blotted to DBM paper, and hybridized with nick-translated clone DNA. (A) Probe = XOC l2. (B) Probe = XOC 2-6. Lane 1, oocyte pA+ RNA; lane 2, gastrula pA+ RNA; lane 3, neurula pA+ RNA; lane 4, tadpole pA+ RNA.

RESULTS

As an initial step toward reanalyzing these five clones, they were nick-translated and hybridized to different amounts of oocyte and liver RNA. An example of two of these is shown in Fig. 1 in which clones XOC l-2 and XOC 2-6 were hybridized to oocyte RNA and liver RNA

FIG. 1. Hybridization of cDNA clones to oocyte and liver pA+ RNA. RNAs were prepared, electrophoresed in formaldehyde, blotted to nitrocellulose, and hybridized tto nick-translated clone DNA as under Materials and Methods. (A) F’robe = XOC 1-2. (B) Probe = XOC 26. Lane 1,0.5 pg oocyte pA+ R.NA; lane 2,5 pg oocyte pA+ RNA; lane 3, 0.5 pg liver pA+ RNA; lane 4, 5 pg liver pA+ RNA. These bands have not been precisely sized., but are smaller than 18 S rRNA. In addition XOC 2-6 RNAs are smaller than XOC 1-2 RNAs.

(Fig. 1). Previously we were unable to detect liver pA+ RNA complementary to clone XOC l-2 (Golden et al., 1980). However, a band of the same molecular weight is clearly visible in lane 4 (Fig. 1A) which contains 5 pg of liver pA+ RNA. This is also the case for clone XOC 2-4 (data not shown), the other oocyte clone previously reported as having no detectable complementary RNA in liver pA+ RNA. Therefore, all 10 oocyte clones examined have detectable complementary pA+ RNAs in liver and tadpoles. The only qualitative exception which we have found is shown in Fig. 1B. Clone XOC 2-6 hybridizes to an RNA doublet in oocyte RNA, but in liver RNA the ratio of the two bands is substantially altered such that a band of molecular weight similar to the oocyte lower band is the predominant RNA, as previously indicated (Golden et al., 1980). All other clones visualize a similar if not identical band pattern in all three RNA sources: oocytes, tadpoles, and liver. Although present in both liver and tadpoles, these five sets of oocyte RNAs are of higher abundance in oocyte pA+ RNA than in tadpole pA+ RNA or in liver pA+ RNA (Golden et al., 1980). In order to examine changes during embryogenesis in the per embryo levels of these RNAs, the five clones were hybridized to Northern gels in which RNA isolated from equal numbers of embryos was applied to adjacent lanes. Figure 2 shows an analysis of this kind for XOC l-2 (Fig. 2A) and XOC 2-6 (Fig. 2B). In both cases, the RNA levels remain approximately constant during early embryogenesis and

82

DEVELOPMENTAL BIOLOGY

12345678

-

-

FIG. 3. Hybridization of clone XOC 2-4 to pA+ RNA from 8 developmental stages. pA+ RNA from 100 embryos (or 100 mature oocytes) was electrophoresed in formaldehyde, blotted to nitrocellulose, and hybridized to nick-translated XOC 2-4. Lane 1, mature oocyte; lane 2, 2-8 cell; lane 3, stage 6/7; lane 4, stage 9/10; lane 5, stage 18/20; lane 6, stage 28/30; lane 7, stage 37/39; lane 8, stage 41. Arrows indicate approximate positions of 28 S and 18 S rRNAs.

then increase between the neurula (lane 3) and tadpole stage (lane 4). Hence, for these two clones and for XOC 6-35 (data not shown) “down” in abundance is really “up” on a per embryo basis. It must be the case that the total pA+ RNA content per embryo rises somewhat faster than the levels (per embryo) of these three clonecomplementary RNAs. The data for the two remaining clones are more complex. XOC 2-4 hybridizes to four RNA bands in oocyte RNA (Fig. 3). All four of these bands are faintly detectable in liver RNA (data not shown) and throughout embryogenesis (Fig. 3). The changes in the embryonic pattern are complex and not interpretable in any simple way. The three upper bands might represent intron-containing precursor RNAs, stored in the oocyte and processed during embryogenesis to yield a mature mRNA, the lowest molecular weight form. The changes which take place during embryogenesis, however, do not obviously suggest such a relationship. It is equally likely that each RNA is an individual mRNA which shares sequence homology with the other three RNAs. In addition, preliminary data indicate that at least some of the changes in the levels of the individual XOC 2-4 RNAs may reflect adenylation changes in the individual RNA species which take place during early embryogenesis (data not shown). A different pattern is observed for the remaining clone, XOC 2-7. The RNA complementary to this clone decreases on a per embryo basis as well as on an abundance basis (Fig. 4). This occurs between stages 7 and 10. This level remains relatively constant throughout the balance of embryogenesis. It is interesting that there

VOLUME 94, 1982

is also a visible increase in the size of the band in unfertilized egg RNA as well as a subsequent decrease in RNA size which occurs during early embryogenesis. It is possible that the decrease of XOC 2-7 pA+ RNA during early embryogenesis is due to deadenylation and not due to actual RNA degradation. To test such an hypothesis, XOC 2-7 was hybridized to a similar gel which contained alternate lanes of total and pAP RNA, isolated from embryos of different stages of development (Fig. 5). The data demonstrate that the same decrease observed in pA+ RNA is also visible in total RNA. Consequently, the decrease is almost certainly due to the degradation of most XOC 2-7 RNA during early development. Furthermore, there is relatively little detectable signal in pA- RNA, indicating that almost all of this mRNA is adenylated in oocytes (lanes 1, 2) and that most is adenylated throughout embryogenesis. Consistent with the data in Fig. 4, there is a marked difference in the mobility of the band from oocyte RNA and from egg RNA which is probably due to further adenylation (increase in the size of the already present pA tail) of XOC 2-7 RNA which occurs during maturation of Xenopus eggs (see below). There is also a subsequent decrease in the size and width of the band during cleavage and blastula (Figs. 4 and 5). This decrease occurs in parallel with an increase in the amount of detectable pA- XOC 2-7 RNA. It would appear, therefore, that the degradation of the XOC 2-7 sequence occurs concomitant with a shortening of the pA tail and an increase in the absolute amount of pAP XOC 2-7 RNA. Subsequent to gastrulation, the amount of XOC 2-7 RNA remains relatively constant, the band size increases and

I

2345678

FIG. 4. Hybridization of clone XOC 2-7 to pA+ RNA from 8 developmental stages. pA+ RNA from 25 embryos (or 25 oocytes or 25 unfertilized eggs) was electrophoresed in formaldehyde, blotted to nitrocellulose, and hybridized to nick-translated XOC 2-7. Recovery of pA+ RNA, as assayed by hybridization to the yeast RNA (see Materials and Methods), was relatively poor in lane 3. Lane 1, mature oocyte; lane 2, unfertilized egg; lane 3, 2-8 cell; lane 4, stage 617; lane 5, stage 9/10; lane 6, stage 11/13; lane ‘I’, stage 18/20; lane 8, stage 28/30.

COLOT AND ROSBASH

Maternal

becomes wider, and the amount of pA- RNA decreases to an undetectable level (lanes 11-16). In order to verify that the difference in the mobility of XOC 2-7 RNA from oocytes and eggs is due to a difference in the pA tail length, two different kinds of assays were performed. First, oocyte RNA and egg RNA were treated with RNase H in the presence of oligo(dT) prior to Northern gel analysis (Vournakis et al., 1975). The difference in the mlobilities of the two RNAs was essentially eliminated as treatment with RNAse H produced a relatively narrow band of approximately the same size from both soumces of RNA (Fig. 6). Second, RNA from oocytes and unfertilized eggs was fractionated by thermal elution from poly(U)-Sepharose. This technique has been shown to fractionate RNAs on the basis of pA tail length (Palatnik et al., 1979). The data (Figs. 7C, D) show that XOC 2-7 RNA from oocytes elutes from poly(U)-Sepharose at a lower temperature than XOC 2-7 RNA from eggs, consistent with the (proposed) smaller size pA tail of XOC 2-7 RNA from oocytes than from eggs. It may also be the case that the postgastrula increase in the size and the width of XOC 2-7 RNA (Fig. 5, lanes g-16) is due to a larger and more heterogenous pA tail, but this has not been directly established. In contrast to XOC 2-7 RNA, the size of XOC l-2 RNA decreases at oocyte maturation (Figs. 8 and 2A, lanes 1 and 2). Experiments using thermal elution from poly(U)-Sepharose indicate that, as expected, this size decrease is due to substantial deadenylation of the XOC l-2 sequence (Figs. 7A, B). These two examples, XOC 2-7 and XOC 1-2, suggest that at least some of the adenylation and deadenylation which occur at oocyte maturation affect different maternal sequences in different ways.

1

2 3

4

5

6 7 8

910111213141516

FIG. 5. Hybridization of clone XOC 2-7 to total RNA and pA- RNA from developmental stages. 5 pg total RNA and 5 pg pA- RNA, isolated from the indicated stages, were electrophoresed in adjacent lanes and blotted to nick-translated XOC 2-7. Lanes 1, 2, oocyte total and pARNA; lanes 3, 4, unfertilized egg total and pA- RNA; lanes 5, 6, 2-8 cell total and pA- RNA; lanes 7, 8, stage 6/7 total and pA- RNA; lanes 9, 10, stage 9/10 total and pA- RNA; lanes 11, 12, stage 11/13 total and pA- RNA; lanes 13, 14, stage 18/20 total and pA- RNA; lanes 15, 16, stage 28/30 total and pA- RNA.

mRNAs

in X. laevis Embryos

83

FIG. 6. Treatment of oocyte and egg RNA with RNAse H. 10 pg total RNA from oocytes and from eggs was treated with RNAse H in the presence of oligo(dT). The RNA was phenol extracted, ethanol precipitated, and 5 rg was assayed (on formaldehyde-nitrocellulose Northern gels) with nick-translated XOC 2-7. Lane 1, mock-digested (no enzyme) oocyte RNA; lane 2, digested oocyte RNA; lane 3, mockdigested unfertilized egg RNA; lane 4, digested unfertilized egg RNA.

DISCUSSION

Of the 10 oocyte cDNA clones originally examined, 4 were complementary to RNAs which increased in abundance during embryogenesis and one was complementary to RNA, the abundance level of which was about the same in oocytes and tadpoles. These 5 sequences, therefore, must increase dramatically in their per embryo levels during embryogenesis. The data presented in this report show that of the other 5 sequences which decrease in abundance during embryogenesis, 3 actually increase in their per embryo levels. It is therefore likely that at least 8 of the 10 maternal sequences examined are resynthesized during embryogenesis. It is also possible that one or both of the other two sequences are also resynthesized since a decrease in amount does not necessarily exclude the possibility that the sequence is also transcribed during embryogenesis. It has been shown by two other laboratories that there is a dramatic change in the amount and length of pA tails during oocyte maturation (Darnbrough and Ford, 1976; Sagata et al., 1980). These studies indicate that the absolute amount of pA per oocyte decreases by approximately a factor of 2, the absolute amount of pA+ RNA also decreases by approximately a factor of 2, and that the average size of pA increases somewhat. The data presented in this report suggest that different pA+ RNAs are affected differently during oocyte maturation, i.e., that polyadenylation and deadenylation occur to different extents on individual pA+ RNA sequences. The pA+ RNA complementary to XOC 2-7 undergoes a substantial increase in the length of its pA tail during oocyte maturation. We assume that this change is detectable by agarose gel electrophoresis because the RNA is sufficiently short that the pA sequence represents a substantial fraction of its length. This increase can also be

84

DEVELOPMENTALBIOLOGY

I

2

3

4

5

6

7

6

9

IO

II

FIG. 7. Thermal elution from poly(U)-Sepharose. 1.8 mg of mature oocyte and 1.8 mg of unfertilized egg RNA was fractionated by thermal elution from poly(U)-Sepharose. Fractions were collected, ethanol precipitated, and analyzed by formaldehyde nitrocellulose Northern blotting. One 30-slot gel was run which contained the fractions from both RNA samples. The nitrocellulose was hybridized first with XOC 1-2, the radioactivity removed, and the filter then hybridized with XOC 2-7. (A) Oocyte fractions, XOC 1-2 hybridization. (B) Egg fractions, XOC 1-2 hybridization. (C) Oocyte fractions, XOC 2-7 hybridization. (D) Egg fractions, XOC 2-7 hybridization. Lane 1,25”C eluate; lane 2, 35OC eluate; lane 3, 37.5’C eluate; lane 4, 4O’C eluate; lane 5, 43°C eluate; lane 6, 45°C eluate; lane 7, 475°C eluate; lane 8, 50°C eluate; lane 9, 525°C eluate; lane 10, 55°C eluate; lane 11, 55’C eluate with 90% formamide.

VOL~JME 94. 1982

detected during in vitro maturation of Xenopus oocytes (data not shown), as expected from the experiments cited above which concern themselves with the total pA+ RNA population (Darnbrough and Ford, 1976; Sagata et al., 1980). There is no detectable decrease in the level of XOC 2-7 RNA in eggs as compared to oocytes and no large amount of XOC 2-7 pA- RNA (nonoligo(dT) binding) in either RNA population. Thus, the data argue that both the size and the amount of pA associated with XOC 2-7 RNA increase during oocyte maturation, in contrast to the total population of pA. While the data suggest that the major effect on XOC 2-7 RNA is an increase in pA size, there is a hint that a small fraction of XOC 2-7 RNA is deadenylated at oocyte maturation. There is consistently a small amount of XOC 2-7 RNA in egg pAP RNA, more than is visible in oocyte pA- RNA. Furthermore, there is a detectable signal in the 25°C eluate of the poly(U)-Sepharose column for egg RNA and none for oocyte RNA (Figs. 7C, D; lane 1). The data suggest that while most XOC 2-7 RNA molecules are lengthened, some may be shortened. We have also been able to detect on gels the size change that XOC l-2 undergoes at maturation (Fig. 8). This is presumably because, like XOC 2-7 RNA, the RNA is sufficiently small that the changes in pA length make a detectable difference in gel mobility. The data support the notion that different sequences respond differently to maturation. It should be noted that XOC l-2 and XOC 2-7 are the only two clones that we have examined from this point of view. It should also be noted that pA size changes of even large RNA molecules should be detectable by using thermal elution from poly(U)-Sepharose and Northern blotting. Unfortunately, almost all of the RNAs analyzed in this study (other than XOC 2-7) are present at levels too low to detect easily in total or pAP RNA by Northern blotting. Therefore we do not know whether any of these other sequences are present as pA- RNA in oocytes or embryos. This is an important consideration because the large amount of both adenylation and deadenylation which takes place during oocyte maturation and early embryogenesis might influence significantly the interpretation of measurements which are made uniquely on pA+ RNA. Indeed, there are reports of large amounts of pAP mRNA in Xenopus embryos (Miller, 1978). This possibility affects our conclusions as well, in that some of the increases in RNA levels which occur during embryogenesis might be due to adenylation of previously unadenylated sequences. In the case of XOC 2-7 RNA, however, we have documented that the decrease occurs in total RNA as well as pA+ RNA and therefore can conclude that the sequence decrease is not due to the conversion of pA+ to pAP RNA. Clearly, the data do not

COLOT AND ROSBASH

Maternal

FIG. 8. Hybridization of clone XOC l-2 to oocyte and egg RNA. pA+ RNA from 25 oocytes (lane 1) and 25 eggs (lane 2) was electrophoresed in formaldehyde, blotted to mtrocellulose, and hybridized with nicktranslated XOC l-2. The lower band in both lanes is “left-over” from a previous hybridization with XOC 2-7.

exclude the possibility that other maternal sequences are also degraded durin.g embryogenesis and that this degradation is obscured by zygotic transcription which takes place subsequent to the midblastula transition (Newport and Kirschner, manuscript in preparation). The only remaining caveat applies to all uncharacterized cDNA clones. It is formally possible that only a part of the oocyte XOC 2-7 RNA sequence disappears during embryogenesis, e.g., the 3’-noncoding portion of the mRNA. It is interesting to note that there has been reported a small but detectable decrease in the total amount of pA+ RNA which takes Iplace at late blastula-early gastrula (Sagata et al., 1980). This decrease takes place without any detectable change in the average size of total poly(A), indicating that there is some deadenylation of some pA+ RNA and/or some degradation of some pA+ RNA sequences (Sagata et al., 1980). It may be the case that XOC 2-7 RNA belongs to a class of RNA sequences which are largely degraded during early embryogenesis. This possibility is currently under investigation. The continual improvement of Northern gel technology (see Materials and Methods) is providing more information about clone-complementary RNAs. Indeed, a reanalysis of the same clones is providing more detail about the behavior of individual RNAs during embryogenesis as well as new smears or bands not previously detected. In the case of XOC 2-7 RNA there is a relatively low-molecular-wieight smear which is present in oocyte pA+ RNA and #absent or present in greatly reduced amount in egg RNA (Fig. 4, arrow). It seems unlikely that this smear is due to degraded RNA since there is a relatively clear gap in the autoradiograms between the major XOC 2-7 RNA and this smear, unlike

mRNAs

in X. laeuis Embryos

85

the continuous smear directly below an RNA band which is indicative of degradation. A similar lower molecular weight smear has been reported for chicken (Yactin mRNA (Ordahl et al., 1980). We do not know the significance of this XOC 2-7 oocyte smear nor the significance of its relative absence in eggs and embryos. The oocyte clones were originally selected as giving a range of abundance levels on colony hybridization with oocyte cDNA and as being positive when hybridized to oocyte RNA on Northern gels. No clones which hybridized positively with oocyte RNA were eliminated from the analysis. However, we cannot exclude the possibility that some selection, either during or subsequent to the cloning of these ovary pA+ sequences, was inadvertently introduced. This general caveat notwithstanding, the analysis indicates that much of the oocyte sequence complexity is present in somatic cells and is in a form similar to that found in the oocyte. Moreover, the data suggest that at least 8 of these 10 oocyte cDNA clone-complementary RNAs are resynthesized in embryos. This tentative conclusion is in general agreement with the results of RNA-cDNA hybridization analysis (Perlman and Rosbash, 1978; Rosbash, 1981). Clearly more information, with more clones, will be required to establish this generalization definitively. We wish to thank Allan Jacobson for helpful suggestions with the thermal elution from poly(U)-Sepharose, Linda Hyman and Lynna Hereford for helpful discussions, Mary Ann Osley for a careful reading of the manuscript, and Tobie Tishman for preparing the manuscript. This work was supported by a grant (HD 08887) from the NIH to M.R. REFERENCES BALLANTINE, J. E. M., WOODLAND, H. R., and STURGESS, E. A. (1979). Changes in protein synthesis during the development of Xenopzu laeuis. J. Embryol. Exp. Morphol. 51, 137-153. BROCK, H. W., and REEVES, R. (1978). An investigation of de nouo protein synthesis in the South African clawed frog, Xenopus laeuis. Deuelop. Biol. 66, 128-141. BRUSKIN, A. M., TYNER, A. L., WELLS, D. E., SHOWMAN, R. M., and KLEIN, W. H. (1981). Accumulation in embryogenesis of five mRNAs enriched in the ectoderm of the sea urchin pluteus. Deuelop. Biol. 87, 308-318. DARNBROUGH, C., and FORD, P. J. (1976). Cell-free translation of messenger RNA from oocytes of Xenopus laeuis. Deuelop. Biol. 50, 2855301. DENHARDT, D. T. (1966). A membrane-filter technique for the detection of complementary DNA. Biochem. Biophys. Res. Commun. 23, 641-646. DWORKIN, M. B., and DAWID, I. B. (1980). Use of a cloned library for the study of abundant poly(A)+ RNA during Xenopus laeuis development. Develop. Biol. 76, 449-464. GOLDEN, L., SCHAFER, U., and ROSBASH, M. (1980). Accumulation of individual pA+ RNAs during oogenesis of Xenopus lawis. Cell 22, 835-844. HEREFORD, L., and ROSBASH, M. (1977). Number and distribution of polyadenylated RNA sequences in yeast. Cell 10, 453-462.

86

DEVELOPMENTAL BIOLOGY

MILLER, L. (1978). Relative amounts of newly synthesized poly(A)+ and poly(A) messenger RNA during development of Xenopus Levis. Develop. Biol. 64, 118-124. NEWPORT, J., and KIRSCHNER, M., manuscript in preparation. ORDAHL, C. P., TILGHMAN, S. M., OVITT, C., FORNWALD, J., and LARGEN, M. T. (1980). Structure and developmental expression of the chick cy-actin gene. Nucl. Acids Res. 8, 4989-5005. PALATNIK, C. M., STORTI, R. V., and JACOBSON, A. (1979). Fractionation and functional analysis of newly synthesized and decaying messenger RNAs from vegetative cells of Dictyostelium discoideum. J. Mol. Biol. 128, 371-395. PERLMAN, S., and ROSBASH, M. (1978). Analysis of Xenopus laevis ovary and somatic cell polyadenylated RNA by molecular hybridization. Develop. Biol. 63, 197-212. RAVE, N., CRKVENJAKOV, R., and BOEDTKER, H. (1979). Identification of procollagen mRNAs transferred to diazobenzyloxymethyl paper from formaldehyde agarose gels. Nucl. Acids Res. 6, 3559-3567. ROSBASH, M. (1981). A comparison of Xenopus laeuis oocyte and embryo mRNA. Develop. Biol. 87, 319-329. SAGATA, N., SHIOKAWA, K., and YAMANA, K. (1980). A study on the

VOLUME 94, 1982

steady-state population of poly(A)+ RNA during early development of Xenopus laevis. Develop. Biol. 77, 431-448. SCHAFER, U., GOLDEN, L., HYMAN, L. E., COLOT, H. V., and ROSBASH, M. (1982). Some somatic sequences are absent or exceedingly rare in Xenopus oocyte RNA. Develop. Biol. 94, 87-92. STURGESS, E. A., BALLANTINE, J. E. M., WOODLAND, H. R., MOHUN, P. R., LANCE, C. D., and DIMITRIADIS, G. J. (1980). Actin synthesis during the early development of Xenopus laevis. J. Embryol. Exp. Morphol. 58, 303-339. THOMAS, P. (1980). Hybridization of denatured RNA and small DNA fragments transferred to nitrocellulose. Proc. Nat. Acad. Sci. USA 77, 52015205. VOURNAKIS, J. N., EFSTRATIADIS, A., and KAFATOS, F. C. (1975). Electrophoretic patterns of deadenylated chorion and globin mRNAs. Proc. Nat. Acad. Sci. USA 72, 2959-2963. WEISS, Y. C., VASLET, C. A., and ROSBASH, M. (1981). Ribosomal protein mRNAs increase dramatically during Xenopus development. Develop. Biol. 87, 330-339. WOOLFORD, J. L., and ROSBASH, M. (1979). The use of R-looping for structural gene identification and mRNA purification. Nucl. Acids Res. 6, 2483-2497.