Development 111, 623-633 (1991)Printed in Great Britain © The Company of Biologists Limited 1991

623

'Unmasking' of stored maternal mRNAs and the activation of protein

synthesis at fertilization in sea urchins

LESLIE C. KELSO-WINEMILLER and MATTHEW M. WINKLER

Center for Developmental Biology, Department of Zoology, University of Texas, Austin, Texas 78712-1064, USA

Summary

The isolation and in vitro assay of maternal mRNPs hasled to differing conclusions as to whether maternalmRNAs in sea urchin eggs are in a repressed or 'masked'form. To circumvent the problems involved with in vitroapproaches, we have used an in vivo assay to determine ifthe availability of mRNA and/or components of thetranslational machinery are limiting protein synthesis inthe unfertilized egg. This assay involves the use of aprotein synthesis elongation inhibitor to create asituation in the egg in which there is excess translationalmachinery available to bind mRNA. Eggs were fertilizedand the rate of entry into polysomes of individualmRNAs was measured in inhibitor-treated and controlembryos using 32P-labeled cDNA probes. The fraction ofribosomes in polysomes and the polysome size were alsodetermined. The results from this in vivo approach

provide strong evidence for the coactivation of bothmRNAs and components of the translational machineryfollowing fertilization. The average polysome sizeincreases from 7.5 ribosomes per message in 15minembryos to approximately 10.8 ribosomes in 2hembryos. This result gives additional support to the ideathat translational machinery, as well as mRNA, isactivated following fertilization. We also found thatindividual mRNAs are recruited into polysomes withdifferent kinetics, and that the fraction of an mRNA inpolysomes in the unfertilized egg correlates with the rateat which that mRNA is recruited into polysomesfollowing fertilization.

Key words: fertilization, protein synthesis, maternalmRNA, translational control, masked mRNA.

Introduction

Fertilization of the sea urchin egg results in a 20- to 40-fold increase in the rate of protein synthesis in the twohours following fertilization (Hultin, 1952; Epel, 1967;Regier and Kafatos, 1977). This increase in the rate ofprotein synthesis is largely mediated by the mobiliz-ation of stored maternal mRNAs into polysomes (Grossand Cousineau, 1963; Denny and Tyler, 1964; Humph-reys, 1971). The molecular mechanisms that have beenproposed to explain the activation of these storedmaternal mRNAs can be conveniently divided into twonon-mutually exclusive general categories. These are anincrease in the availability of either (1) mRNA or (2)components of the translational machinery other thanmRNA. (Availability is defined as being in a form orintracellular location where it can be utilized fortranslation). The discovery that mRNA exists in the cellas RNA-protein complexes (messenger ribonucleopro-tein particles or mRNPs) led to the suggestion thatassociated proteins might be repressors of mRNAactivity in the unfertilized egg (reviewed by Davidson,1986). This theory has become termed the 'maskedmessage' hypothesis (Spirin, 1966). Four previous

studies have utilized the isolation and in vitro assay ofmRNPs from unfertilized eggs to determine if proteinsynthesis is regulated at the level of repression ofmRNPs (Jenkins et al. 1978; Ilan and Ilan, 1978; Moonet al. 1982; Grainger and Winkler, 1987). The con-clusions from these studies oscillate between the ideathat mRNA is masked or not masked in the unfertilizedegg. Many of these discrepancies can be attributed totechnical problems associated with the isolation orassay of mRNP activity.

Our approach to this question has been to develop anin vivo assay for studying mRNP activity that does notrely on mRNP isolation. Thus, we avoid the problem ofartifactually altering the activity of the mRNP orcomponents of the translational machinery (i.e. ribo-somes, initiation factors) during the isolation or assayprocess. The results from our study clearly show'thatsome form of 'masking' of mRNAs limits proteinsynthesis in the unfertilized egg. In addition, we findthat protein synthesis is limited by the activity of somecomponent(s) of the translational machinery. Theseexperiments also reveal that the rate of mobilization ofan mRNA into polysomes correlates with that mRNA'stranslational efficiency in the egg.

624 L. C. Kelso-Winemiller and Matthew M. Winkler

Materials and methods

Preparation of sea urchin post-mitochondrialsupernatants and glycerol density gradientcentrifugationA 1 % suspension of Strongylocentrotus purpuratus eggs inJamarin U Artificial Sea Water (Osaka, Japan) was dividedinto two equal aliquots. Anisomycin (10 /IM) was added to onealiquot of eggs 5 min prior to fertilization. Both anisomycin-treated and untreated eggs were fertilized by adding 1/20000volume sperm. Eggs and embryos were incubated at 15°Cwith constant stirring. Post-mitochondrial supernatants(PMS) were prepared (Grainger and Winkler, 1987) and 20OD260 units were adjusted to 0.1 % Triton X-100 and layeredonto 15-50% glycerol gradients in Cold IV buffer (0.25 MNaCl, 5mM EGTA, 5mM MgCl2, 1 min DTT, 10mM Pipes,pH 6.8) or Cold IV+ EDTA (10 mM EDTA) with a 7 ml 80 %glycerol-EDTA pad (Kelso-Winemiller et al. 1989). Gradi-ents were centrifuged for 5h at 28 000 revs min"1 in aBeckman SW 28 rotor at 4°C. Ten fractions were collectedfrom each gradient by upwards displacement with 50%sucrose-30% glycerol (w/v). The optical density wasmonitored at 254 nm with an Isco recording spectropho-tometer. Baker's yeast tRNA (Sigma) was added as a carrierto each fraction at a concentration of 10/^gml"1. 2.5 volumesof 95 % ethanol were added to each gradient fraction andallowed to precipitate at —20°C overnight.

Isolation of RNAGradient fractions were centrifuged at3600gfor30min. RNApellets were resuspended in 0.5 ml STEN buffer (0.5 % SDS10 mM EDTA, 50mM NaCl, 10 mM Tris, pH8), 250 ̂ g ml"1

proteinase K (Boehringer-Mannheim Biochemicals), andincubated at 40°C for 2h. Gradient fractions were extractedtwice with equal volumes of phenol:chloroform (50 % phenol,46% chloroform, 4% isoamyl alcohol (v/v), 0.1%8-hydroxyquinoline (w/v), saturated with 1M Tris, pH8) andonce with an equal volume of chloroform. 2.5 volumes of95 % ethanol were added to each gradient fraction and theRNA allowed to precipitate at —20°C overnight. Gradientfractions were centrifuged at 10 000 revs min"1 in a microfugeand RNA pellets were resuspended in double-distilled H2Oand stored at -80°C.

Preparation of mRNA probes from a cDNA libraryProbes used in hybridizations were prepared from cDNAclones selected from a 2h (2-cell stage) Strongylocentrotuspurpuratus cDNA library made from polysomal poly (A)+

RNA using a modified version of the ribonuclease H method(Gubler and Hoffman, 1983). Double-stranded cDNA mol-ecules were ligated into lambda Zap (Stratagene). Recombi-nant Bluescript plasmids were excised from lambda Zapaccording to Stratagene protocols. cDNA clones werehybridized to equal amounts of unfertilized egg RNA andmitochondrial RNA (Wells et al. 1982) to determine if any ofthem coded mitochondrial sequences. The signal was strongerwith the unfertilized egg RNA in all cases. To determine ifthere was a large percentage of mitochondrial sequencescoded by the cDNA used to prepare the library, labeledcDNA was hybridized to equal amounts of unfertilized eggand mitochondrial RNA. The signal from the mitochondrialRNA was barely detectable indicating that the librarycontained very few mitochondrial sequences. These cDNAclones coded for bonafide mRNAs based on three criteria: (1)presence of 3' poly (A) tails, (2) mobilization into polysomes

after fertilization, and (3) release from polysomes with EDTAtreatment. cDNA clone 53 (encoded an 'A' type cyclin)hybridized to 4.9 kb and 6.6 kb transcripts. cDNA clone 12hybridized to 4.5 kb and 5.8 kb transcripts. Construction ofthe plasmid Sp64-/31 coding containing the cDNA cloneencoding /} tubulin mRNAs was described by Harlow andNemer (1987). Either T3, T7 or SP6 polymerase were used togenerate radiolabelled RNA transcripts from cDNA clones asdescribed by Stratagene protocols. 50//Ci of [a-32P] CTP(~600Cimmol~1: ICN) was added to a standard reactionvolume of 25^1. A typical reaction yielded between 30-60 ngof [32P] RNA with a specific activity of 8x 108 cts min"' ng~ .Random primed 32P-labeled probes as described by Amer-sham protocols.

Determination of fraction of mRNA in polysomesRNA isolated from each fraction was applied to Hybond N(Amersham) nylon membranes under vacuum with a slotblotter (Schleicher and Schuell) and baked one hour at 80°C(White and Bancroft, 1982). Slot blots were hybridized withradiolabeled probes as described by Hurley et al. (1989)except that hybridizations were incubated at 65 °C, and50 jig ml"1 poly (A) RNA was added to the hybridizationsolution. Following hybridization, membranes were washedtwice with 40mM Na3PO4 (pH7.2), IITIM EDTA, 1 % SDS, at65 °C. Membranes were wrapped in plastic wrap andautoradiographed with Kodak XAR-5 X-ray film. Autoradio-graphs were exposed for appropriate time intervals to remainwithin the linear response of the film as monitored by astandard curve of unfertilized egg RNA. Slot blot autoradio-graphs of gradient RNA fractions were scanned using adensitometer and the areas under the peaks integrated.Gradient fractions 1-6 contained nontranslated mRNA andfractions 7-11 contained polysomal RNA. Parallel gradientscontaining EDTA were used to determine the fraction of non-EDTA releasable mRNA in the polysome fraction of thegradient. The fraction of each mRNA in polysomes wascalculated by subtracting the amount of non-EDTA releasablemessenger RNA in polysome fractions from the polysomalmessenger RNA in those fractions (7-11). This value wasdivided by the total amount of that messenger RNA in thegradient to give the fraction of mRNA in polysomes.

Determination of fraction of ribosomes in polysomesThe fraction of ribosomes in polysomes was determined usinga technique described by Martin (1973). Post-mitochondrialsupernatants were prepared from control and inhibitor-treated Strongylocentrotus purpuratus embryos at 15 minintervals following fertilization. Supernatants were treatedwith 10/igml"1 ribonuclease A in high salt (0.7 M) homogeniz-ation buffer. Ribonuclease A hydrolyzes mRNA not pro-tected by a ribosome converting polysomes into short messagefragments bound to 80S ribosomes. These 80S ribosome-mRNA complexes are resistant to dissociation by high saltwhereas free 80S ribosomes dissociate into 40S and 60Ssubunits. The amounts of 40S and 60S subunits and 80Sribosomes were quantified by fractionating homogenates on15-40% sucrose gradients made up in high salt buffer (0.7 MKCl,5mMMgCl2, lmMEGTA, 10 mM Hepes). The gradientswere centrifuged in a SW 41 rotor at 41000 revs min"1, for4.3 h, at 4°C. Absorption peaks (260 nm) of the 40S and 60Ssubunits and 80S ribosomes were integrated and the fractionof ribosomes in polysomes calculated by dividing the amountof SOS ribosomes by the total sum of the 40S and 60S subunitsand 80S ribosomes.

Unmasking of stored maternal mRNAs 625

Determination of number of ribosomes bound permessenger RNAStrongylocentrotus purpuratus control and inhibitor-treatedpost-mitochondrial supematants were prepared and fraction-ated on 33 ml linear glycerol gradients in a Beckman SW 28rotor at 28000 revs min"1, 2 h, 4°C. RNA was isolated from 11fractions and transferred to nylon membranes with a slotblotter. Slot blots were probed with either a 32P-labeled RNAtranscript of cDNA 53 or 12. Autoradiographs were scannedusing a densitometer and the areas under the peaksintegrated. A parallel gradient of homogenate from either 10or 12 h embryos was run at the same time so that polysomesedimentation into the gradient could be visualized andmeasured. The fraction of the total RNA for each gradientfraction was multiplied by 'x' (where x=average polysomesize) for each fraction and totaled to give the 'x' for theparticular developmental period. Fraction 11 was not used todetermine 'x'. Fraction 11 included a wash of the gradienttube which likely contained aggregated material.

Results

Previous studies utilized the isolation and in vitro assayof mRNPs from unfertilized eggs to determine ifprotein synthesis is regulated at the level of repressionof mRNPs. Our approach has been to .develop an invivo assay that does not rely on mRNP isolation. Thus,we have avoided the problem of artifactually alteringthe activity of the mRNA (i.e. mRNPs) or componentsof the translational machinery (i.e. ribosomes, in-itiation factors) during the isolation or assay process.The principle of this assay is to create artificially asituation where there is additional active translationalmachinery in the egg. Thus, the ratio of activetranslational machinery to mRNA in the egg isincreased. This can be accomplished by fertilizing eggsin the presence of saturating levels of a peptideelongation inhibitor. Under these conditions, asmRNAs move into polysomes, only one ribosome canbind each mRNA. (Note, we use the term polysome todescribe one or more ribosomes bound to an mRNAmolecule.) Further migration of the ribosome along themRNA is blocked by the inhibitor (Fan and Penman,1970; Lodish, 1971). If mRNA availability is the rate-limiting factor for egg protein synthesis, then we wouldexpect that messenger RNAs would bind ribosomeswith the same kinetics in control and inhibitor-treatedembryos. However, if the activation of translationalmachinery is the rate limiting factor, then the mRNA ofthe inhibitor-treated embryos, which now have avail-able extra translational machinery, would enter poly-somes at a faster rate. If both mRNA and translationalmachinery are being activated simultaneously, wewould expect results similar to those obtained if onlymRNA is being unmasked. This assay is only validwhen there is an excess of unutilized mRNA andtranslational machinery. For this reason our con-clusions are only drawn from experimental measure-ments made 15-30 min following fertilization whenboth of these components are potentially available. Bytwo hours post-fertilization most of the maternalmRNA has already been mobilized into polysomes.

To illustrate this assay, consider a hypotheticalsituation where 10 mRNAs and 100 ribosomes are inpolysomes at time T. Assume each mRNA can bind 10ribosomes so that when fully loaded the averagepolysome size is 10. Also assume that mRNAs show alinear recruitment into polysomes. In the first scenario,mRNA is limiting translation in the unfertilized egg,and is activated at fertilization. In this case, at timeTx/5, there would be 2 messages in polysomes in bothcontrol embryos and inhibitor-treated embryos. Con-trol embryo polysomes would contain a total of 20ribosomes with 10 ribosomes per mRNA, whereasinhibitor-treated polysomes would bind only 1 ribo-some per mRNA with a total of 2 ribosomes inpolysomes.

In contrast, consider an alternative scenario wheretranslational machinery (i.e. ribosomes), and notmRNA, is limiting protein synthesis in the egg and isactivated at fertilization. Then at time Ti/5, there wouldstill be 2 messages in polysomes in the control embryoswith 10 ribosomes attached to each. The inhibitor-treated embryos, however, would have available 20active ribosomes, each of which could bind an mRNA.Thus, all 10 mRNAs would be in polysomes. In thissituation where mRNA is not limiting protein synthesis,the inhibitor-treated embryos, which contain an excessof translational machinery, can mobilize mRNA intopolysomes to a gTeater extent than control embryos. Ina situation where both translational machinery andmRNA limit protein synthesis and are activatedsimultaneously, we would expect results similar to thoseif just mRNA is activated. We have made measure-ments similar to these examples described here. Whileour results are more complex than in this hypotheticalsituation, they indicate that both the availability ofmRNA and translational machinery limit proteinsynthesis in the unfertilized egg.

Kinetics of messenger RNA entry into polysomesTo measure the mobilization of mRNAs into poly-somes, we used labeled probes prepared from cDNAclones which are complementary to individual abundantmRNAs. These clones were selected from a cDNAlibrary made from polysomal poly (A) RNA from 2h(2-cell) Strongylocentrotus purpuratus embryos. cDNAclones coded for bonafide mRNAs on the basis of thefollowing three criteria: (1) presence of 3' poly (A)tails, (2) mobilization into polysomes after fertilization,and (3) release from polysomes with EDTA treatment.

One experimental difficulty encountered whenmeasuring the kinetics of mRNA mobilization intopolysomes involves the isolation of polysomal andnonpolysomal fractions of mRNA. Most often this isaccomplished by density gradient centrifugation. How-ever, the centrifugation conditions required to separateeffectively these two populations of mRNAs pelletsmost of the polysomes. We developed a method thatcompresses the polysome region of the gradient into asmall area (Kelso-Winemiller et al. 1989). This com-pression is accomplished by fractionating post-mito-chondrial supematants of sea urchin egg and embryo

626 L. C. Kelso-Winemiller and Matthew M. Winkler

homogenates on glycerol gradients containing an 80 %glycerol-EDTApad. When polysomes, which disaggTe-gate in EDTA, reach this interface the ribosomes arereleased from the mRNA. Further migration into thegradient is greatly reduced and polysomes are notpelleted.

To be assured that individual mRNAs were behavinglike the bulk of the total maternal message population,we measured the mobilization of several differentmRNAs into polysomes in both control and inhibitor-treated embryos. 32P-labeled RNA transcripts wereprepared from cDNA clones and hybridized with RNAfrom gradient fractions. The fact that large untranslatedmRNPs sediment in the same region of the gradient aspolysomes presents a problem when determining theamount of polysomal mRNA. Since these mRNPs arestable in EDTA and polysomes are not, parallelgradients run in EDTA allowed the determination ofthe proportion of mRNA in the polysome region of thegradient that was untranslated mRNP (Fig. 2). Thedifference between the non-EDTA and EDTA poly-some fractions gave the amount of polysomal mRNA inthe faster sedimenting region of the gradient. Forindividual mRNAs, the amount of non-EDTA releas-able mRNPs ranged from 0% to 17%. In Fig. 1A,RNA isolated from two separate embryo batches wasprobed with 32P-labeled RNA transcripts to messengerRNA 53. We have subsequently identified this mRNAby sequence analysis as an 'A' type cyclin. This mRNAconstitutes 0.32% of the mRNA in the egg. Eachembryo batch consisted of eggs pooled from severalfemales to reduce the possibility of individual variation.The non-EDTA releasable untranslated mRNPs weresubtracted from the polysome region of the gradientand the values were averaged from the two separateexperiments using different preparations of RNA. At15min post-fertilization, approximately 49% of themRNA was in polysomes in the control embryos and64% in the inhibitor-treated embryos. By 30min, 68%and 74 % of the mRNA was in polysomes in the controland inhibitor-treated embryos, respectively. In Fig. IB,RNA isolated from one batch of embryos was probedwith 32P-labeled RNA transcripts to messenger RNA12. This RNA constitutes 0.97% of the translatablemRNA in the egg. Non-EDTA releasable, untranslated

Fig. 1. Kinetics of mobilization of individual messengerRNAs into polysomes following fertilization.Strongylocentrotus purpuratus post-mitochondrialsupernatants were fractionated on glycerol gradientscontaining a 80% glycerol-EDTA pad. RNA isolated fromequal fractions was transferred onto nylon membranes witha slot blotter and hybridized with 32P-labelled antisenseRNA transcripts prepared from cDNA clones. Slot blotautoradiographs were scanned with a densitometer and theareas under the peaks integrated. (A) Average fraction ofmessenger RNA 53 in polysomes for two different embryobatches, (B) fraction of messenger RNA 12 in polysomesfor 1 embryo batch, and (C) fraction of f3 tubulinmessenger RNA in polysomes for 1 embryo batch. Solidcircles denote inhibitor-treated embryos and closed circlesdenote control embryos.

mRNPs were subtracted from the polysome region ofthe gradient. Both the control and experimentalembryos had approximately 55 % of their mRNA inpolysomes at 15min. By 45min, almost 90% of themRNA had entered polysomes in both the control andexperimental embryos. 32P-labeled RNA transcriptscomplementary to fi tubulin mRNA were also used toprobe RNA isolated from one batch of embryos(Fig. 1C). Tubulin message shows a much slower rate ofmobilization into polysomes. For example, only 28%

(A) Fraction of mRNA 53 (Cyclin A) In Polysomes

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Unmasking of stored maternal mRNAs 627

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Fig. 2. Separation of polysomesfrom nontranslated mRNPs onglycerol gradients. Strongylocentrotuspurpuratus post-mitochondrialsupernatants prepared from controland anisomycin-treated embryoswere fractionated on glycerolgradients containing an 80%glycerol-EDTA pad. RNA isolatedfrom equal fractions was transferredonto nylon membranes with a slotblotter and hybridized with 32P-labelled antisense RNA transcriptsprepared from cDNA clone 53. Slotblot autoradiographs were scannedwith a densitometer and the areasunder the peaks integrated. Totalrelative amounts of RNA from eachgradient were normalized to theOmin control gradient. Solid linesindicate non-EDTA gradients anddashed lines denote gradients with10 mM EDTA.

and 32 % of ft tubulin mRNA was in polysomes incontrol and inhibitor-treated embryos at 30min post-fertilization. Similar rates of tubulin mRNA recruit-ment into polysomes occur in Lytechinus pictus(Alexandraki and Ruderman, 1985). Only a smallportion of both a and /3 tubulin mRNAs are mobilizedinto polysomes within 30min after fertilization. Themobilization kinetics of these three different messagesinto polysomes reveal that mRNAs have different ratesof entry into polysomes.

It is interesting to note that approximately 10% ofmRNAs 12 and 53 are in polysomes in the unfertilizedegg, while tubulin is almost completely excluded frompolysomes. In another study, we described the recruit-ment of two general classes of maternal mRNAs intopolysomes at fertilization (Kelso-Winemiller andWinkler, unpublished data). We found that one class ofmRNAs, which include 12 and 53, were recruited intopolysomes very rapidly following fertilization. Over50 % of each of these mRNAs were in polysomes 30 minafter fertilization. The other class of maternal mRNAs,which include tubulin, were mobilized into polysomes

much more slowly. It appears that mRNAs that arerecruited more rapidly into polysomes at fertilizationare translated to a greater extent in the unfertilized egg.These results suggest that mRNA activation at fertiliz-ation is regulated by factors that show specificity forindividual messages. It is not clear if these unknown'factors' are the same as the 'masking' factor or arecomponents of the translational machinery that interactwith the message following unmasking. The fact thatmRNAs in inhibitor-treated eggs show similar mobiliz-ation kinetics into polysomes as do controls suggeststhat the difference in activation of these two classes ofmRNA lies at the level of 'unmasking'.

To confirm that these individual mRNAs werebehaving like bulk of the messages, we also measuredthe rate of mobilization of the total mRNA populationusing labeled cDNA prepared from poly (A)+ polyso-mal RNA from 2h embryos. RNA isolated from twoseparate embryo batches was probed with 32P-labeledcDNA (Fig. 3). We found that 25% of the bulk ofmRNAs in control embryos and 30% of the bulk ofmRNAs in inhibitor-treated embryos were in poly-

628 L. C. Kelso-Winemiller and Matthew M. Winkler

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Fig. 3. Kinetics of mobilization of total mRNA intopolysomes following fertilization. Slot blots described inFig. 1 were hybridized with 32P-labelled cDNA preparedfrom polysomal poly (A)+ RNA from 2hStrongylocentrotus purpuratus embryos. Closed circlesdenote the fraction of mRNA in polysomes in inhibitor-treated embryos. Open circles indicate the fraction ofmRNA in polysomes in control embryos. Graph representsaverage of 2 experiments using two different embryobatches.

somes at 15min post-fertilization. At 30min 38% and43 % of the mRNAs were in polysomes in control andexperimental embryos, respectively (Fig. 3). The rateof entry of the total mRNA is intermediate between therate of mobilization of the two fast mobilizing mRNAs53 and 12, and the slower mobilizing tubulin mRNA.This suggests that the mRNAs we studied provide areasonable representation of the message population asa whole.

The in vivo approach that we used circumventsproblems associated with biochemical isolation andassay of mRNPs. Our results suggest that mRNAavailability limits protein synthesis in the unfertilizedegg. The in vivo assay does not distinguish betweenmRNA and both mRNA and translational machinerybecoming activated and it is likely that some com-ponents) of the translational machinery also limitsprotein synthesis in the unfertilized egg.- This reasoningis based upon experiments that tested mRNP activity incell-free translation systems (Winkler et al. 1985; Colinet al. 1987; Winkler, 1988). In contrast to previous invitro studies, these experiments involved minimalbiochemical manipulation of mRNPs. The results fromthese experiments clearly showed the involvement ofboth translational machinery and mRNA in theactivation of protein synthesis at fertilization.

Measurements of mRNA entry into polysomes frominhibitor-treated embryos consistently show a slightlyhigher rate of mobilization as compared to mRNAsfrom untreated embryos (Figs 1 and 3). We speculatethat at fertilization the activation of mRNA precedesslightly the activation of the translational machinery.Thus, mRNAs in inhibitor-treated eggs have excess

unbound translational machinery available and are ableto mobilize into polysomes slightly faster. Their rate ofmobilization probably reflects the actual rate ofunmasking. In contrast, in untreated embryos, wherepolysomes are fully loaded, there is a slight excess ofactive mRNAs over translational machinery. As newmRNAs become active, there is a lag time betweenmRNA activation and ribosome binding. This lag timedecreases as more ribosomes become active. By 45 minpost-fertilization, the control and inhibitor-treatedembryos contain the same number of mRNAs inpolysomes (Fig. IB). Thus unmasking does not occurall at once following fertilization but continues over aperiod of time during early development.

Kinetics of ribosome mobilization into polysomesTo substantiate further the conclusion that mRNAavailability limits protein synthesis, we used a secondapproach that involves measuring the rate of ribosomeentry into polysomes. If mRNA availability is rate-limiting, untreated embryos should contain moreribosomes in polysomes than inhibitor-treated embryosby a factor proportional to the difference in polysomesize between untreated and inhibitor-treated embryos.This is because mRNAs from inhibitor-treated embryosshould bind only one ribosome. Note that whatevercomponent of translational machinery is limiting willultimately affect ribosome binding, and thus whenconsidering ribosomes in this context we mean thelimiting translational component.

The lack of concentrated OD260 absorbing material inthe polysome region of the gradient makes it difficult todiscern polysome peaks during sea urchin earlydevelopment. In order to measure the amount ofribosomes in polysomes, we used a more sensitiveprocedure described by Martin (1973). Control andinhibitor-treated egg and embryo homogenates weretreated with ribonuclease A, which hydrolyzes mRNAnot protected by ribosomes, converting polysomes intoshort message fragments bound to 80S ribosomes. Todistinguish free 80S ribosomes from those bound tomRNA, homogenates were treated with 0.7 M KC1.High salt dissociates free 80S ribosomes into theirconstituent 40S and 60S subunits, but does not affectmessage-bound 80S ribosomes. 40S and 60S subunitsand 80S ribosomes were separated by centrifugation onhigh salt sucrose gradients.

Using this technique, the fraction of ribosomes inpolysomes for inhibitor-treated and untreated embryoswas determined for six different embryo cultures. Theaverages of the percentages for these six experiments isshown in Fig. 4. From 0 to 120 min following fertiliz-ation, the fraction of ribosomes in polysomes increasedgradually from 1.8 % to 16.5 % in the control embryos.This is consistent with results from other investigatorsusing different methods that show approximately 20 %of the ribosomes in polysomes by the first 2h afterfertilization (Humphreys, 1971; Goustin and Wilt,1981). In the same time period, the fraction ofribosomes in polysomes in the anisomycin-treatedembryos increased from 1.7% to 9.3%.

Unmasking of stored maternal mRNAs 629

Control EmbiyosAnisomyctn-treatsd Embryos

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Fig. 4. Kinetics of ribosome entry into polysomes followingfertilization. This figure shows the average fraction ofribosomes in polysomes for six embryo batches at 15 minintervals following fertilization. Error bars indicatestandard error for each time point. Points lacking errorbars had standard errors less than ±0.5. Solid circlesindicate inhibitor-treated embryos and open circles denotecontrol embryos.

On the average 2.0-fold more ribosomes were foundin polysomes in untreated embryos compared toinhibitor-treated embryos (Fig. 4). Thus, ribosomes aremobilizing into polysomes to a greater extent in controlembryos than in inhibitor-treated embryos, even withadditional active translational machinery available inthe inhibitor-treated embryos. We found that theaverage polysome size in untreated embryos is 7.5ribosomes and in inhibitor-treated embryos is 3.2ribosomes (Fig. 5) (The fact that polysomes frominhibitor-treated embryos bind more than a singleribosome will be discussed below). Thus, if mRNAavailability limits protein synthesis, then polysomes ofuntreated embryos should contain 7.5/3.2 or 2.3-foldmore ribosomes than inhibitor-treated embryo poly-somes. The experimental (2.0 fold) and predicted (2.3fold) values for the difference in polysome size are inclose agreement clearly demonstrating that mRNAavailability is a factor limiting protein synthesis in theunfertilized egg.

There are several possible explanations why mRNAsin inhibitor-treated embryos bind not the theoreticalsingle ribosome, but an average of 3.2 ribosomes. First,anisomycin may not completely inhibit protein syn-thesis and movement of ribosomes down the mRNA,thus allowing additional ribosomes to bind. Wemeasured both the rate of protein synthesis and theamount of labeled nascent chains in polysomes ininhibitor-treated embryos. Inhibitor-treated eggs failedto show any incorporation of the [35S] methionine intoproteins after fertilization and lacked labeled peptidechains in polysomes indicating that ribosomes are not'creeping' down the messenger RNA (Shettles, B. andWinkler, M. M., unpublished data). Simultaneous

treatment with saturating amounts of both emetine andanisomycin did not reduce the amount of ribosomes inpolysomes over that seen with just one inhibitor.Emetine and anisomycin both inhibit elongation, but bydifferent mechanisms (Vazquez, 1979). Anisomycinbinds to the 60S subunit and blocks peptide bondformation, while emetine acts on the 40S subunit andprevents EF-2-dependent translocation by ribosomes.Thus, their effects would be expected to complementeach other. These observations collectively suggest thatanisomycin is completely inhibiting protein synthesis intreated eggs.

A more likely explanation is that more than oneribosome is binding each mRNA, even with the AUGinitiator codon blocked by the first ribosome. Previousinvestigators have found that two ribosomes are able tobind to Tobacco Mosaic Virus and Turnip YellowMosaic Virus RNA leader sequences when they areincubated in a wheat germ extract with anotherelongation inhibitor, sparsomycin (Tyc et al. 1984;Filipowicz and Haenni, 1979). The first ribosome bindsto the AUG initiator codon and the second ribosomebinds upstream from the initiator codon in the 5'untranslated region (3=69 nucleotides) of the mRNA.Similar formation of disomes, or trisomes, is probablyoccurring on sea urchin mRNAs, when embryos areincubated in the presence of anisomycin. There areseveral examples of eukaryotic mRNAs in whichtranslation starts from an initiator AUG downstreamfrom other non-initiating AUG codons (Racaniello andBaltimore, 1981; Darlix et al. 1982; Mulligan and Berg,1981). Sequence analysis of an abundant cDNA clonefrom our 2 h S. purpuratus library revealed the presenceof two additional AUG (out of frame) codons upstreamfrom the AUG initiator codon (Yoon and Winkler,unpublished results). Thus, it is likely that binding ofmultiple ribosomes 5' of the initiator AUG occurs in thesea urchin system.

Another issue raised by this work concerns changesin average polysome size following fertilization. Wefound that polysome size gradually increases duringearly development from 7.5 ribosomes in 15 minembryos to 9.8 ribosomes in 1 h embryos and by 2 h to10.8 ribosomes (Fig. 5). A number of other investi-gators have also measured polysome size duringdevelopment. Rinaldi and Monroy (1969) clearlyshowed with polyribosome profiles that the size ofpolysomes increases from 2 to 10 min and continues toincrease up to the 2-cell stage (90min). Martin andMiller (1983) determined polysome size from electronmicrographs of Lytechinus pictus eggs and embryos.Their polysome size measurements show a decrease inpolysome size following fertilization from an average11.96 ribosomes per mRNA in eggs to an average 7.14ribosomes per mRNA in 1 h embryos. It is possible thatthe centrifugation process used to spread polysomes onelectron microscope grids might have artifactuallyselected for larger polysomes in the eggs. Humphreys(1969, 1971) concluded that polysome size does notchange appreciably during the first 6 h of development(up to the 16-cell stage). However, inspection of

630 L. C. Kelso-Winemilter and Matthew M. Winkler

(A) 15 minute Control Embryos (B) 15 Minute Anisomycln Embryos

100 •

5 6 7 B 9 10 11 6 7 8 9 10 11

(C) One Hour Control Embryos (D) Two Hour Control Embryos

I I I I I M i l l

3 4 5 6 7 8

Fraction Number

9 10 11

100

1 2 3 4 5 6 7 8 9 10 11

Fraction Number

Fig. 5. Determination of number of ribosomes bound per messenger RNA. Strongylocentrotus purpuratus control andanisomycin-treated post-mitochondrial supernatants were prepared and fractionated on linear glycerol gradients. RNA wasisolated from 11 fractions and transferred to nylon membranes with a slot blotter. Slot blots were probed with a 32P-labelled RNA transcript of cDNA 53. Autoradiographs were scanned using a densitometer and the areas under the peaksintegrated. A parallel gradient of either 10 or 12 h homogenate was run at the same time so that polysome sedimentationinto the gradient could be visualized and measured. The sedimentation of polysomes in the gradient is indicated by theslash marks on the x-axis (i.e. l=monosome, 5=polysome with 5 ribosomes bound, etc.). The fraction of the total RNAfor each gradient fraction was multiplied by 'x' (where x=average polysome size) for each fraction and totaled to give the'x' for the particular developmental period. Fraction 11 was not used to determine 'x'. Fraction 11 included a wash of thegradient tube which likely contained aggregated material. (A) 15min control embryos, (B) 15min anisomycin embryos, (C)lh control embryos, (D) 2h control embryos.

Humphreys polysome profiles does reveal a slight shiftto larger polysome size by lh following fertilization.This is consistent with our results which show thatpolysome size increases during the first 2h of develop-ment by approximately 3 ribosomes per message. Apossible explanation for this increase is that the ratio ofactive translational machinery to mRNA increasesduring this period. Earlier, we discussed that mRNAactivation slightly precedes that of the translationalmachinery. As more translational machinery is acti-vated during early development more ribosomes will beable to bind to messages. An alternative possibility isthat polysomes require a long time period to reach thefully loaded state. Nelson and Winkler (1987) foundthat full loading of mRNAs in a reticulocyte lysaterequired at least 10 ribosome transit times. In eithercase, the increase in polysome size provides anadditional piece of evidence consistent with the idea

that translational machinery is activated continuouslyfollowing fertilization.

Discussion

Our results support the hypothesis that both mRNAand some component or components of the trans-lational machinery are limiting protein synthesis in theunfertilized egg of the sea urchin, Strongylocentrotuspurpuratus. Following fertilization both of these aregradually made available for protein synthesis in theembryo. In the past, most hypotheses regarding thequestion as to what is limiting protein synthesis in theegg have fallen into two general categories. These arethe limited availability of either (1) mRNA or (2) acomponent (s) of the translational machinery. Thesetwo hypotheses have often been presented as alterna-

Unmasking of stored maternal mRNAs 631

tive possibilities. Humphreys (1971) postulated that iftranslational machinery was limiting in the egg andmade available for protein synthesis at fertilization thenpolysome size would change. Since sea urchin eggs andembryos contain equal size polysomes (Humphreys,1971), he concluded that mRNA and not translationalmachinery is made available for protein synthesis atfertilization. Other experimenters (Giudice, 1973;Danilchik and Hille, 1981) looking at the activity ofvarious components of the translational machinery invitro have come to the opposite conclusion thattranslational machinery rather than mRNA is limitingprotein synthesis in the unfertilized egg.

However, these two hypotheses are not mutuallyexclusive. Several investigators have suggested thatboth translational machinery and mRNA are ratelimiting for protein synthesis in the unfertilized egg(Winkler et al. 1985; Colin et al. 1987; Lopo et al. 1988;for a review see Clemens, 1987). mRNPs from bothLytechinus pictus and Strongylocentrotus purpuratuseggs do not bind efficiently to preinitiation complexes inreticulocyte lysate translation systems (Winkler et al.1985; Grainger and Winkler, 1987; Lopo et al. 1988).This suggests that mRNAs in the egg are masked orunavailable for translation in the unfertilized egg.However, other experiments clearly show the involve-ment of translational machinery in the activation ofprotein synthesis at fertilization. Highly active cell-freetranslation systems have been developed to analyze themechanisms limiting protein synthesis during earlyembryogenesis (Winkler and Steinhardt, 1981; Colin etal. 1987; Hansen et al. 1987; Lopo et al. 1989). Thesecell-free systems demonstrate rates of protein synthesisand regulatory characteristics approaching those ob-served in vivo. If protein synthesis in the unfertilizedegg is limited by the supply of active mRNA, then theaddition of exogenous message should stimulate proteinsynthesis. If protein synthesis in the unfertilized egg islimited by deficiencies in the cell's translationalmachinery then the exogenous message would betranslated at the expense of endogenous message andoverall protein synthesis would remain the same.Protein synthesis is not increased in unfertilized eggcell-free translation systems when exogenous mRNAsalone are added. However, protein synthesis can bestimulated by addition of eukaryotic initiation factors(elF) eIF-2, eIF-4F, the guanine nucleotide exchangefactor (GEF), or soluble components of post-ribosomalsupernantants from reticulocyte lysates (Winkler et al.1985; Colin et al. 1987; Huang et al. 1987; Lopo et al.1988). The addition of increasing amounts of exogenousmRNA with purified initiation factors or S100 fractionsfrom rabbit reticulocytes causes an even greaterstimulation of protein synthesis (Colin et al. 1987;Winkler and Grainger, 1987). Thus initiation factorsexpand the capacity of the translational machinerywhich can then be utilized by the exogenous mRNA.Despite the expanded capacity of the translationalmachinery, the endogenous mRNAs still remainuntranslated suggesting that they are in some repressedform unavailable to the translational machinery.

Other evidence for the involvement of initationfactors in the activation of protein synthesis comes fromthe identification of inhibitor of translation in the egg(Hansen et al. 1987). Addition of this inhibitor to cell-free translation systems from sea urchin embryos orrabbit reticulocytes prevents translation. However, theinhibition can be reversed by the addition of exogenouseIF-4F to sea urchin embryo or reticulocyte cell-freesystems (Huang et al. 1987). Experimental resultsindicate that phosphorylation of one subunit of eIF-4Fat fertilization reverses the inhibitory effects (asreferenced in Lopo et al. 1988). Using intact cells,rather than a cell-free system, Colin (Colin and Hille,1987) also came to the conclusion that components ofthe translational machinery are limiting protein syn-thesis in the unfertilized sea urchin egg. Exogenousglobin mRNA injected into unfertilized eggs of Strongy-locentrotus droebachiensis and L. pictus had little effecton the overall rate of protein synthesis.

The frog Xenopus laevis is another organism in whichthe mechanisms for regulating protein synthesis duringdevelopment have been intensely studied. At matu-ration, the frog oocyte undergoes a 2- to 4-fold increasein the rate of protein synthesis. The control of thisprotein synthesis increase has been studied principallyby microinjecting mRNA into Xenopus oocyte cyto-plasm. Like the sea urchin, the addition of exogenousmRNA does not stimulate overall protein synthesis.This suggests that a component of the translationalmachinery is limiting in the oocyte (Laskey et al. 1977;Richter and Smith, 1981). On the other hand, themicroinjection studies of Lingrel and Woodland (1974)argue against translational machinery as the regulatorof protein synthesis. Their experiments show thatinjection of exogenous message into oocytes does notresult in a change in polysome size. Like Humphreys,they interpret their results to mean that proteinsynthesis is not regulated at the level of translationalmachinery since such events would lead to a change inpolysome size. Recently, Audet et al. (1987) havereported that microinjection of initiation factor eIF-4Astimulates translation in Xenopus oocytes. However, nodirect tests have been conducted on costimulation ofprotein synthesis by mRNAs and translational machin-ery components. It seems likely, however, that inXenopus dual levels of control similar to those in the seaurchins are operating.

Recently, Calzone et al. (1988) have studied theregulation of protein synthesis in Tetrahymena. Grow-ing Tetrahymena cells and starved-deciliated Tetrahy-mena cells show an increased rate of protein synthesisover starved Tetrahymena cells (Calzone et al. 1983).Since the polysome size and the peptide elongation rateremained constant during changes in the rate of proteinsynthesis, they suggested that messages could not becompeting for a limiting initiation factor. They alsoargued against differential affinity of messages for alimiting initiation factor because essentially the sameset of message sequences are found in polysomes beforeand after the change in the rate of protein synthesis.Thus, they concluded that the major mechanism that

632 L. C. Kelso-Winemiller and Matthew M. Winkler

regulates the rate of protein synthesis in Tetrahymena isthe regulation of the number of messages madeavailable for translation and not a change in thetranslational efficiency (polypeptides produced permRNA per unit time) of the mRNAs. They concludedthat unmasking of an untranslatable form of mRNAcould explain the changes in protein synthesis seenbetween starved and growing Tetrahymena cells.

Our results show that the regulation of the increase inprotein synthesis at fertilization is a complex process,which is controlled by components of the translationalmachinery as well as availability of the mRNA. We alsofind that mRNAs are not all simultaneously recruitedinto polysomes but show differential rates of mobiliz-ation. Some mRNAs mobilize into polysomes veryrapidly at fertilization while others enter polysomesmuch more slowly. Those mRNAs that are recruitedrapidly are translated to a greater extent in the egg.These results are consistent with masking factor(s) withdifferent affinities for different mRNAs similar to theRNA-binding proteins which bind specific messagesequences in Xenopus (Crawford and Richter, 1987).mRNAs that mobilize into polysomes with a faster ratewould have a lower affinity for a masking factor thanmRNAs that enter polysomes more slowly. Anotherpossibility is that different mRNAs have differentaffinities for components of the translational machin-ery. For example, it is possible that in the egg there isonly a limited supply of an active initiation factor whichbinds to individual mRNAs. At fertilization more of theinitiation factor becomes available to bind to messages.A likely candidate for such an initiation factor could beeIF-4F, the 5' cap binding protein. As discussedpreviously, eLF-4F activity is suppressed by an inhibitorin the egg which is inactivated at fertilization. Ray et al.(1983) identified eIF-4F as the limiting initiation factorimplicated in discrimination of mRNAs for translation.Those messages with a higher affinity for the limitedinitiation factor would be preferentially translated inthe egg and would be recruited into polysomes faster inthe embryo, fi tubulin exemplifies this hypothesis. It isbarely translated in the egg and is recruited intopolysomes at a slower rate than the bulk of themessages at fertilization. It is possible that classicalinitiation factors could act as mRNA recruitmentfactors and give results that appear similar to thosecaused by masking factors. This is reminiscent ofmodels proposed by Lodish (1974) regarding compe-tition of mRNAs for initiation factors. This type ofmodel, however, requires a more complex set ofassumptions than do unmasking models. In summary,our results show that mRNAs are made available forentry into polysomes following fertilization. We alsoconclude that components of the translational machin-ery are activated at fertilization and that their activationmay be slightly preceded by that of the messengerRNA. It is the synergistic effect of these two processesthat is responsible for the regulation of proteinsynthesis in the early sea urchin embryo.

We acknowledge the collaboration of Tim Hunt on

preliminary versions of some of the experiments. We thankPatricia Harlow and Martin Nemer for the fi tubulin cDNA.We also thank Lizabeth Richter-Mann for development of thehomogenization buffer that results in a high yield ofundegraded polysomes. We appreciate the assistance that JimGrainger and Carol Cutler Linder provided during manyphases of the project. This work was supported by PublicHealth Service grant HD 17722-06 to M.W. from theNational Institutes of Health.

References

ALEXANDRAS, D. AND RUDERMAN, J. V. (1985). Expression ofalpha- and /S-tubulin genes during development of sea urchinembryos. Devi Biol. 109, 439-451.

AUDET, R. G . , GOODCHILD, J . AND RlCHTER, J . D . (1987).

Eukaryotic initiation factor 4A stimulates translation inmicroinjected Xenopus oocytes. Devi Biol. 121, 58-68.

CALZONE, F. J., ANGERER, R. C. AND GOROVSKY, M. A. (1983).Regulation of protein synthesis in Tetrahymena: Quantitativeestimates of the parameters determining the rates of proteinsynthesis in growing, starved, and starved-deciliated cells. J. biolChem. 258, 6887-6898.

CALZONE, F. J., CALLAHAN, R. AND GOROVSKY, M. A. (1988).Direct measurement of tubulin and bulk message distributionson polysomes of growing, starved, and deciliated Tetrahymenausing RNA gel blots of sucrose gradients containing acrylamide.Nucleic Acids Res. 16, 9597-9609.

CLEMENS, M. (1987). Translational control. Developments indevelopment. Nature 330, 699-700.

COLIN, A. M., BROWN, B. D., DHOLAKIA, J. N., WOODLEY, C. L.,WAHBA, A. J. AND HILLE, M. B. (1987). Evidence forsimultaneous derepression of messenger RNA and the guaninenucleotide exchange factor in fertilized sea urchin eggs. DeviBiol. 123, 354-363.

COLIN, A. M. AND HILLE, M. B. (1986). Injected mRNA does notincrease protein synthesis in unfertilized, fertilized, or ammonia-activated sea urchin eggs. Devi Biol. 115, 184-192.

CRAWFORD, D. R. AND RICHTER, J. D. (1987). An RNA-bindingprotein from Xenopus oocytes is associated with specific messagesequences. Development 101, 741-749.

DANILCHIK, M. V. AND HILLE, M. B. (1981). Sea urchin egg andembryo nbosomes: Differences in translational activity in a cell-free system. Devi Biol. 84, 291-298.

DARLIX, J. L., ZUKER, M. AND SPAHR, P. F. (1982). Structure-function relationship of Rous sarcoma virus leader RNA.Nucleic Acids Res. 10, 5183-5196.

DAVIDSON, E. H. (1986). Gene Activity in Early Development. 3rded. Academic Press, Orlando, FL.

DENNY, P. C. AND TYLER, A. (1964). Activation of proteinsynthesis in non-nucleate fragments of sea urchin eggs. Biochem.Biophys. Res. Commun. 14, 245-249.

EPEL, D. (1967). Protein synthesis in sea urchin eggs: A Mate'response to fertilization. Proc. natn. Acad. Sci. U.S.A. 82,7637-7640.

FAN, H. AND PENMAN, S. (1970). Regulation of protein synthesisin mammalian cells. II. Inhibition of protein synthesis at thelevel of initiation during mitosis. J. molec. Biol. 50, 655-670.

FILIPOWICZ, W. AND HAENNI, A. (1979). Binding of ribosomes to5' terminal leader sequences of eukaryotic messenger RNAs.Proc. natn. Acad. Sci. U.S.A. 76, 3111-3115.

GIUDICE, G. (1973). The Sea Urchin Embryo: A DevelopmentalBiological System. Springer-Verlag, New York, pp. 123-136.'

GOUSTIN, A. S. AND WILT, F. H. (1981). Protein synthesis,polyribosomes, and peptide elongation in early development ofStrongylocentrotus purpuratus. Devi Biol. 82, 32-40.

GRAINGER, J. L. AND WINKLER, M. M. (1987). Fertilization triggersunmasking of maternal mRNAs in sea urchin eggs. Molec. cellBiol. 7, 3947-3954.

GROSS, P. R. AND COUSINEAU, G. H. (1963). Effects ofactinomycin D on macromolecule synthesis and early

Unmasking of stored maternal mRNAs 633

development in sea urchin eggs. Biochem. biophys. Res.Commun. 10, 321-326.

GUBLER, U. AND HOFFMAN, B. J. (1983). A simple and veryefficient method for generating cDNA libraries. Gene 25,263-269.

HANSEN, L. J., HUANG, W.-I. AND JAGUS, R. (1987). Inhibitor oftranslarional initiation in sea urchin eggs prevents mRNAutilization. J. biol. Chem. 262, 6114-6120.

HARLOW, P. AND NEMER, M. (1987). Developmental and tissue-specific regulation of ^J-tubulin gene expression in the embryo ofthe sea urchin Strongyloccntrotus purpuratus. Genes and Dev. 1,147-160.

HUANG, W.-I., HANSEN, L. J., MERRICK, W. C. AND JAGUS, R.(1987). Inhibitor of eukaryotic initiation factor 4F activity inunfertilized sea urchin eggs. Proc. natn. Acad. Sci. U.S.A. 84,6359-6363.

HULTIN, T. (1952). Incorporation of 15N-labelled glycine andalancine into the proteins of developing sea urchin eggs. ExplCell Res. 3, 41-111.

HUMPHREYS, T. (1969). Efficiency of translation of messengerRNA before and after fertilization in sea urchins. Devi Biol. 20,435-458.

HUMPHREYS, T. (1971). Measurements of messenger RNA enteringpolysomes upon fertilization of sea urchin eggs. Devi Biol. 26,201-208.

HURLEY, D. L., ANGERER, L. M. AND ANGERER, R. C. (1989).Altered expression of spatially regulated embryonic genes in theprogeny of separated sea urchin blastomeres. Development 106,567-579.

ILAN, J. AND ILAN, J. (1978). Translation of maternal messengerribonucleoprotein particles from sea urchin in a cell-free systemfrom unfertilized eggs and product analysis. Devi Biol. 66,375-385.

JENKINS, N. A., KAUMEYER, J. F., YOUNG, E. M. AND RAFF, R. A.(1978). Messenger ribonucleoprotein particles in unfertilized seaurchin eggs. Devi Biol 63, 279-298.

KELSO-WINEMILLER, L., DRAWBRIDGE, J. AND WINKLER, M. M.(1989). A new ultracentrifugation technique for analysis andisolation of polysomes. Nucleic Acids Res. 17, 4896.

LASKEY, R. A., MILLS, A. P., GURDON, J. B. AND PARTINGTON, G. '

A. (1977). Protein synthesis in oocytes of Xenopus laevis is notregulated by the supply of messenger RNA. Cell 1, 345-351.

LINGREL, J. B. AND WOODLAND, H. R. (1974). Initiation does notlimit the rate of globin synthesis in message-injected Xenopusoocytes. Eur. J. Biochem. 47, 47-56.

LODISH, H. F. (1971). Alpha and beta globin messengerribonucleic acid. Different amounts and rates of initiation oftranslation. / . biol. Chem. 246, 7131-7138.

LODISH, H. F. (1974). Model for the regulation of mRNAtranslation applied to haemoglobin synthesis. Nature 251,385-388.

LOPO, A. C , LASHBROOK, C. C. AND HERSHEY, J. W. B. (1989).Characterization of translational systems in vitro from threedevelopmental stages of Srongylocentrotus purpuratus. Biochem.J. 258, 553-561.

LOPO, A. C , MACMILLAN, S. AND HERSHEY, J. W. B. (1988).Translational control in early sea urchin embryogenesis:Initiation factor eIF-4F stimulates protein synthesis in lysatesfrom unfertilized eggs of Strongylocentrotus purpuratus.Biochem. 27, 351-357.

MARTIN, K. A. AND MILLER, O. L. (1983). Polysome structure insea urchin eggs and embryos: An electron microscopic analysis.Devi Biol. 98, 338-348.

MARTIN, T. E. (1973). A simple general method to determine theproportion of active nbosomes in eukaryotic cells. Expl CellRes. 80, 496-498.

MOON, R. T., DANILCHIK, M. V. AND HILLE, M. B. (1982). An

assessment of the masked message hypothesis. Sea urchin eggmessenger ribonucleoprotein complexes are efficient templatesfor in vitro protein synthesis. Devi Biol. 93, 389-403.

MULLIGAN, R. C. AND BERG, P. (1981). Factors governing theexpression of a bacterial gene in mammalian cells. Molec. cell.Biol. 1, 449-459.

NELSON, E. M. AND WINKLER, M. M. (1987). Regulation ofmRNA entry into polysomes. J. biol. Chem. 24, 11501-11506.

RACANIELLO, V. R. AND BALTIMORE, D. (1981). Molecular cloningof poliovirus cDNA and determination of the completenucleotide sequence of the viral genome. Proc. natn Acad. Sci.U.S.A. 78, 4887-4891.

RAY, B. K., BRENDLER, T. G., ADIYA, S., DANIELS-MCQUEEN, S.,

MILLER, J. K., HERSHEY, J. W. B., GRIFO, J. A., MERRICK, W.

C. AND THACH, R. E. (1983). Role of mRNA competition inregulating translation: Further characterization of mRNAdiscriminatory initiation factors. Proc. natn. Acad. Sci. U.S.A.80, 663-667

REGIER, J. C. AND KAFATOS, F. C. (1977). Absolute rate of proteinsynthesis in sea urchins with specific activity measurements ofradioactive leucine and leucyl t-RNA. Devi Biol 57, 270-283.

RJCHTER, J. D. AND SMITH, L. D. (1981). Differential capacity fortranslation and lack of competition between mRNAs thatsegregate to free and membrane-bound polysomes. Cell 27,183-191.

RINALDI, A. M. AND MONROY, A. (1969). Polynbosome formationand RNA synthesis in the early post-fertilization stages of thesea urchin egg. Devi Biol. 19, 73-86.

SPIRIN, A. S. (1966). On 'masked' forms of messenger RNA inearly embryogenesis and in other differentiating systems. Curr.Top. Devi Biol. 1, 1-38.

TYC, K., KONARSKA, M., GROSS, H. J. AND FILIPOWICZ, W. (1984).

Multiple ribosome binding to the 5' terminal leader sequence oftobacco mosaic virus RNA. Eur. J. biochem. 140, 503-511.

VAZQUEZ, D. (1979). Inhibition of protein synthesis. In MolecularBiology, Biochemistry, and Biophysics. (Kleinseller, A.,Springer, G. F. and Wittman, H. G., eds.) Vol. 30, Ch. 3.Springer-Verlag, New York.

WELLS, D. E., BRUSKIN, A. M., O'BROCHTA, D. A. AND RAFF, R.

A. (1982). Prevalent RNA sequences of mitochondrial origin insea urchin embryos. Devi Biol. 92, 557-562.

WHITE, B. A. AND BANCROFT, F. C. (1982). Cvtoplasmic DotHybridization: Simple analysis of relative mRNA levels inmultiple small cell or tissue samples. J. biol. Chem. 257,8569-8572.

WINKLER, M. (1988). Translational regulation in sea urchin eggs:A complex interaction of biochemical and physiologicalregulatory mechanisms. BioEssays 8, 157-161.

WINKLER, M. M. AND GRAINGER, J. L. (1987). Regulation ofprotein synthesis during early development in sea urchin eggs.In Molecular Biology of Invertebrate Development (J. D.O'Conner, ed.), UCLA Symposia on Molecular and CellularBiology, Vol. 66, pp. 95-114. Alan R. Liss, Inc., New York.

WINKLER, M. M., NELSON, E. M., LASHBROOK, C. AND HERSHEY,

J. W. B. (1985). Multiple levels of regulation of proteinsynthesis at fertilization in sea urchin eggs. Devi Biol. 107,432-439.

WINKLER, M. M. AND STEINHARDT, R. A. (1981). Activation ofprotein synthesis in a sea urchin cell-free system. Devi Biol. 84,432-439.

{Accepted 29 October 1990)