a protein similar to the 67 kda laminin binding protein ...of projects focused on cytoskeletal...

245

Journal of Cell Science 108, 245-256 (1995)Printed in Great Britain © The Company of Biologists Limited 1995

A protein similar to the 67 kDa laminin binding protein and p40 is probably a

component of the translational machinery in Urechis caupo oocytes and

embryos

Eric T. Rosenthal* and Linda Wordeman†

University of Hawaii, Pacific Biomedical Research Center, 41 Ahui Street, Honolulu, HI 96813, USA

*Author for correspondence†Present address: Department of Physiology and Biophysics, School of Medicine, SJ-40, University of Washington, Seattle, WA 98195, USA

Oocytes of the echiuroid, Urechis caupo, contain anabundant maternal mRNA that encodes a protein verysimilar to LBP/p40, originally identified as a non-integrin67 kDa laminin binding protein. We have sequenced theUrechis caupo mRNA for LBP/p40, and a similar mRNAfrom the Hawaiian sea urchin, Tripneustes gratilla. Both ofthe encoded proteins, as well as LBP/p40 proteins fromother sources, share significant homology in the amino 2/3of the proteins, but diverge extensively at the carboxylends. LBP/p40 protein is present in growing and in full-grown U. caupo oocytes. The protein concentrationremains constant for the first 48 hours of embryogenesisand then begins to decline. In sucrose gradients run with

homogenates from coelomocytes, oocytes, and earlyembryos, all of the LBP/p40 protein appears to be associ-ated with either polysomes or free 40 S ribosomal subunits.In later embryos, an increasing proportion of the proteinis found in the soluble fraction. Immunohistochemistryindicates that LBP/p40 is uniformly distributed in early U.caupo embryos, with no localization at the cell surface. Inlater embryos LBP/p40 is localized in specific parts of theembryo which may correspond to neural tissue.

Key words: Urechis caupo, Tripneustes gratilla, maternal mRNA,laminin binding protein, p40, polysome, ribosome

SUMMARY

INTRODUCTION

Animal oocytes contain a large pool of messenger RNAmolecules, referred to as maternal mRNA. Many of thesematernal mRNAs are translationally inactive until after theoocyte is activated by fertilization or hormonal stimulation, butothers are associated with the translational machinery ofgrowing and full-grown oocytes. Presumably, these lattermRNAs are used to synthesize proteins that are required foroogenesis, for maintenance of the oocyte in a viable state, orfor use during the early stages of embryogenesis. As part of aproject aimed at identifying the features that regulate the trans-lation of different maternal mRNAs in oocytes and embryos,over a dozen translationally controlled maternal mRNAs fromthe echiuroid, Urechis caupo, have been sequenced. Virtuallyall of these mRNAs encode proteins that can be identified byvirtue of their similarity to amino acid sequences present in theGenBank and EMBL databases (Rosenthal, 1993).

One of the U. caupo maternal mRNAs sequenced encodesa product very similar to a 67 kDa laminin binding protein (67kDa LBP), originally isolated from extracts of mammaliancells by affinity chromatography on laminin-Sepharosecolumns (Wewer et al., 1986). This protein binds laminin withhigher affinity than the integrins, is found in a wide variety ofmammalian cells, and is particularly abundant in tumor cell

lines (Mecham, 1991). A partial cDNA clone for the human 67kDa LBP was originally selected from an expression library byscreening with a monoclonal antibody raised against proteinpurified from human tissue (Wewer et al., 1986). Subsequently,full-length cDNA clones were obtained, from a variety ofmammals, by investigators specifically interested in lamininbinding proteins (Rao et al., 1989; Grosso et al., 1991), as wellas groups studying gene expression in transformed cells (Yowet al., 1988; Satoh et al., 1992a,b; Kondoh et al., 1992), trans-lational control in mouse cells (Chitpatima et al., 1988), devel-opment of the embryonic eye (Rabacchi et al., 1990), andmitotic phosphoproteins (Westendorf et al., 1990). MessengerRNAs encoding similar proteins have also been cloned as partof projects focused on cytoskeletal reorganization in hydra(Keppel and Schaller, 1991), growth factor receptors in flies(Melnick et al., 1993), and DNA binding proteins in yeast(Ellis et al., 1994). The amino acid sequence of the 67 kDaLBP from different eukaryotic sources is highly conserved.There are also prokaryotic proteins from a variety of sourceswhich show significant sequence similarities to the 67 kDaLBP (An et al., 1981; Kromer and Arndt, 1991).

Laminin, a major component of the extracellular matrix,functions in cell adhesion, migration, proliferation and differ-entiation. Cells interact with laminin by employing cell surfacereceptors, including integrins, and non-integrin proteins like

246

E. T. Rosenthal and L. Wordeman

the 67 kDa LBP (Mecham, 1991; Tryggvason, 1993). We wereinitially excited to discover that U. caupo oocytes contained amaternal store of mRNA for the 67 kDa LBP, since thissuggested that it might mediate one or more early develop-mental processes, such as the morphogenic movements respon-sible for gastrulation, or the initial differentiation of embryonictissues. Therefore, we decided to investigate the function of the67 kDa LBP during U. caupo embryogenesis.

After we began these studies, however, questions arose con-cerning the relationship between the 67 kDa LBP and themRNA thought to encode the protein (Rao et al., 1989;McCaffery et al., 1990; Groso et al., 1991; Mecham, 1991;Keppel and Schaller, 1991; Auth and Brawerman, 1992). Thehuman mRNA, and the similar mRNAs that have been clonedfrom a wide range of organisms, all encode proteins withmolecular masses of approximately 34 kDa. None of theseproteins have signal sequences or obvious membrane spanningdomains, and most laboratories have found their location to beintracellular. Furthermore, two groups have demonstrated thatthe protein encoded by the cloned mammalian mRNA is asso-ciated with ribosomes (McCaffery et al., 1990; Auth andBrawerman, 1992). One of these laboratories, which calls theprotein ‘p40’, has also demonstrated that, in most mouse cells,the mRNA is found in untranslated messenger ribonucleopro-tein particles (Chitpatima et al., 1988).

This paper presents results from our investigations into theexpression and function of this protein, which we will hence-forth refer to as LBP/p40, during U. caupo development. Wehave determined the temporal pattern of expression for themRNA and the protein, and produced antibodies to use inlocalizing the protein during development. Our experiments donot support the contention that LBP/p40 functions as a lamininbinding protein in U. caupo embryos. Instead, we have foundthat maternal, embryonic and adult coelomocyte U. caupoLBP/p40 protein is associated with polysomal ribosomes andprobably with 40 S ribosomal subunits. Changes in the natureof this association during U. caupo embryogenesis suggest thatLBP/p40 plays a novel role in protein synthesis. We have alsoidentified, and sequenced, an mRNA for LBP/p40 from theHawaiian sea urchin, Tripneustes gratilla.

MATERIALS AND METHODS

Culture of oocytes and embryos from U. caupoAdult U. caupo were collected at Bodega Bay, California and main-tained in refrigerated aquaria. The methods for the isolation ofimmature oocytes, collection of mature gametes, fertilization, andculture of embryos have all been described previously (Rosenthal andWilt, 1986).

cDNA cloning, sequencing and analysisThe original cDNA clone to U. caupo LBP/p40 was isolated from thecDNA library described by Rosenthal and Wilt (1986). AdditionalcDNA clones were isolated from a library constructed in the λEXloxvector system by Novogen, Inc. (Madison, WI). The cDNA used toconstruct this library was synthesized using poly(A)+ RNA from U.caupo immature oocytes, mature oocytes and embryos 2 hours afterfertilization (Rosenthal, 1993). cDNA synthesis was primed with botholigo(dT) and random oligonucleotides.

cDNA clones to the Tripneustes gratilla mRNA were isolated froma λGT10 cDNA library generously provided by Drs Ian and Barbara

Gibbons. The poly(A)+ RNA used for this library was extracted fromdeciliated T. gratilla blastula embryos. Multiple, overlapping cloneswere isolated to obtain the entire protein sequence.

cDNA inserts were sequenced with the dideoxy chain-terminationmethod of Sanger et al. (1977) using a Sequenase sequencing kit (USBiochemicals, Cleveland, OH). Multiple restriction fragments fromthe cDNA inserts were subcloned into M13mp18 or M13mp19 inorder to obtain overlapping sequences from both strands of the DNA.In order to determine the complete 5′ sequence of the U. caupoLBP/p40 mRNA, an oligonucleotide complementary to a regionspanning the initiation codon was synthesized and used for RNAsequencing (McSwiggin, 1990). Sequencing project management andsequence analysis were performed with a software package fromIntelligenetics (Palo Alto, CA), accessed through Bionet or theGenbank On-line Service.

Databases were searched with either the FASTA program (Pearsonand Lipman, 1988) using the GenBank On-line Service E-mail serversor the BLAST program (Altschul et al., 1990) using the NCBInetwork service. The alignments of the sequences shown in Figs 2and 3 were performed with the CLUSTAL V software (Higgins et al.,1991).

Analysis of mRNAThe procedures used for northern blotting, RNA extraction, fraction-ation of mRNA into poly(A)+ and poly(A)− fractions, and determina-tion of mRNA association with polysomes have all been described byRosenthal et al. (1983). Rabbit reticulocyte lysate in vitro translationreactions and electrophoresis of proteins on SDS-polyacrylamide gelswere conducted as described by Rosenthal and Wilt (1986).

In vitro transcription‘Wild-type’ mRNA was produced from plasmid 1340, which containsthe entire U. caupo LBP/p40 mRNA sequence inserted into the EcoRIsite of pGEM2. For in vitro transcription, this plasmid was linearizedwith XbaI. In vitro transcription reactions were performed using aMEGAscript SP6 kit from Ambion, Inc. (Austin, TX).

Antibody productionIn order to produce antibody against the U. caupo LBP/p40 proteinwe first expressed it in bacteria with the pET vector system describedby Rosenberg et al. (1987). Antibody RP1340 was generated usingcDNA clone 1340, which contains the entire LBP/p40 codingsequence. The 1340 plasmid was cut with NheI and EcoRI, producinga fragment with the entire 3′ end of the mRNA, but missing 160 basesfrom the 5′ end. This fragment was ligated into NheI-EcoRI-cutplasmid pET5A, a generous gift from F. W. Studier. The recombinantprotein consists of a methionine, encoded by the vector sequence,followed by a truncated p40 protein sequence, missing only the first30 amino acids. Proteins from IPTG-induced transformed bacteriawere subjected to electrophoresis on SDS-polyacrylamide gels(Laemmli, 1970) and the band containing the recombinant LBP/p40protein was excised. This gel slice was ground up to a fine mush bypressure between two plastic Petri dish lids, lyophilized, dissolved inphosphate-buffered saline, and injected into rabbits to raise polyclonalantisera.

A second fusion protein was produced to facilitate affinity purifi-cation of the rabbit antibodies. This protein was produced using theQIAexpress system distributed by Qiagen (Chatsworth, CA). A BclI-BamHI fragment from plasmid 1340 was ligated into BamHI-cut, calfintestinal phosphatase-treated plasmid pQE31. Recombinant plasmidswith the LBP/p40 fragment in the correct orientation were trans-formed into E. coli M15[pREP4] for expression of the fusion protein,which consisted of the entire U. caupo LBP/p40 protein, plus 5 aminoacids encoded by the last 15 bases of the mRNAs 5′ untranslatedregion, and 10 amino acids encoded by the pQE31 vector. The 10vector-encoded amino acids include the 6 histidines used for purifi-cation of the fusion protein on nickel ion columns. Induction of the

247LBP/p40 in

U. caupo development

fusion protein with IPTG, and its purification on Ni-NTA resin weredone according to the Qiagen instruction manual (Crowe and Henco,1992). Purified protein was coupled to Bio-Rad (Richmond, CA)Affigel-15 according to manufacturer’s instructions. The Affigel-LBP/p40 was used to affinity purify rabbit antibodies from theRP1340 serum by the protocol described by Harlow and Lane (1988).Antibody RP1340ap consists of the antibodies eluted from theAffigel-LBP/p40 with 100 mM glycine, pH 2.5.

One experiment utilized a 1:1:1 mixture of monoclonal antibodiesto neurofilament proteins 68 (Sigma #N5139), 160 (Sigma #N5264),and 200 (Sigma #N5389). This mixture was diluted 1:30 for staining.

Immunoprecipitations and western blottingImmunoprecipitations and western blot analysis of proteins werecarried out according to the protocols described by Harlow and Lane(1988). For the western blots, all incubations were carried out in 1×Tris-buffered saline (TBS), 0.2% Tween-20, 5% non-fat instant milk.Secondary antibodies, obtained from Promega (Madison, WI), weregoat anti-rabbit conjugated to horseradish peroxidase. Detection wascarried out with the ECL reagents manufactured by Amersham, Inc.(Arlington Hts, IL). Quantification of some western blot results wascarried out with an Ambis (San Diego, CA) optical imaging system.

ImmunohistochemistryFor immunohistochemistry, pelleted U. caupo oocytes and embryoswere fixed for 30 minutes in 5% paraformaldehyde in 50 mMpotassium phosphate, 10 mM magnesium chloride, 5 mM EGTA, pH9.0. After fixation the cells were resuspended and stored in 100%methanol at −20°C. Embryos from fertilization to gastrula (18 hours)are surrounded by a fertilization membrane which was removed by amodification of the procedure developed by Mitchison and Sedat(1983). Embryos in methanol were resuspended inheptane:methanol:EDTA (0.5 M), 10:9:1, and shaken vigorously.Devitellinated embryos fall to the bottom of the tube, instead of col-lecting at the methanol:heptane interface.

For immunostaining, the cells in methanol were rehydrated with aseries of cold methanol:Tris-buffered saline (TBS) solutions, withincreasing ratios of TBS. This was followed by a 2 hour incubationin 2% glycine in TBS, and a 2 hour incubation in 5% normal goatserum in TBS. Cells were labelled for 4 hours, or overnight, at 4°C,with preimmune or immune serum diluted 1:500 in TBS, pH 7.4, with0.5% BSA, 0.5% ovalbumin, and 0.1% Triton X-100 (TBTO). Thelabelled cells were then washed with TBTO, incubated at 4°C for 2to 4 hours with rhodamine-conjugated goat anti-rabbit IgG secondaryantibodies in TBTO, and finally washed in TBTO. For whole mountobservations, the cells were mounted in 90% glycerol in TBS. Forsections, the labelled cells were dehydrated in methanol andembedded in Spurr’s low viscosity resin (Spurr, 1969). 4 µM sectionswere mounted in Fluormount (Gallard Schlesinger, Carle Place, NY).All photographs were taken with a Zeiss (Thornwood, NY) Axiophotfluorescence photomicroscope using Kodak (Rochester, NY) SO-115technical pan film.

Sucrose gradientsIn order to determine if LBP/p40 was associated with ribosomes, U.caupo oocyte, embryo, and coelomocyte homogenates were cen-trifuged through sucrose gradients. Some homogenates wereincubated before fractionation with either 25 mM EDTA at 4°C, orRNAse A at 37°C, for 10 minutes. Homogenates and gradients wereprepared as described previously (Rosenthal et al., 1983). SW41 cen-trifuge tubes were filled with 9 ml linear 15% to 40% sucrosegradients and underlayed with 3 ml of 50% sucrose. Centrifugationwas for 3 hours at 38,000 rpm in a SW41 rotor. After centrifugation,each gradient was collected in 9 or 14 fractions, which were precipi-tated with 2.5 volumes of ethanol. The precipitated proteins werecollected by centrifugation, washed once with ethanol, and driedunder vacuum. Each fraction was dissolved in an equal volume of

protein gel buffer and equal volumes were loaded on gels for westernblotting.

RESULTS

The U. caupo and T. gratilla LBP/p40 amino acidsequences are very similar to sequences from otherorganisms, although the carboxyl segments of theproteins diverge extensivelyA cDNA clone for LBP/p40 was originally selected from acDNA library constructed from poly(A)+ mRNA isolated fromU. caupo oocytes and 2-cell embryos (Rosenthal et al., 1993).In order to obtain the complete sequence of this mRNA addi-tional cDNA clones were identified by re-screening the librarywith the original, partial cloned sequence, and the full sequencewas obtained by Sanger sequencing of overlapping subcloneson both strands of the DNA. We confirmed that we had theentire 5′ sequence of the mRNA, right up to the 7mG ‘cap’ byusing reverse transcriptase to sequence the mRNA directly,using a primer complementary to bases 71 to 98 of the mRNA(underlined in Fig. 1). We also obtained multiple cDNA clonesspanning the complete coding region of a similar mRNA fromthe Hawaiian sea urchin, T. gratilla (GenBank accessionnumber U02371). The sea urchin amino acid sequence ispresented here for inclusion in a comparison of the proteinsequences of LBP/p40 from different sources. Detailedanalysis of LBP/p40 expression during sea urchin developmentwill be presented elsewhere (M. Hung et al., unpublished data).

The U. caupo mRNA contains a single start codon upstreamof the correct site for initiation (Fig. 1). This upstream startcodon is followed by an in-frame termination codon 2 basesbefore the correct initiation codon. The presence of thisupstream ATG codon does not appear to inhibit translation ofthe mRNA, since in vitro transcribed versions of the wild-typemRNA translate very well (Fig. 5, lane H). As expected, apolyadenylation signal hexanucleotide, AATAAA, is locatedclose to the 3′ end of the mRNA.

Fig. 2 shows an alignment of LBP/p40 sequences fromdifferent eukaryotic organisms. The U. caupo and T. gratillasequences were determined by us. All of the other sequenceswere obtained from Genbank with the NCBI Retrieve MailServer. Three additional mammalian sequences from theGenbank database, for mouse, bovine and hamster proteins,were not included in the alignment because they are virtuallyidentical to the human sequence (data not shown). Thealignment shows that all of the LBP/p40 proteins are verysimilar in the amino terminal 2/3 of the protein, although theydiverge extensively toward the carboxyl terminus. Althoughthe alignment in Fig. 2 gives the impression that there is littlerelationship between the carboxyl portions of the differentproteins, aligning sequences two at a time demonstrates thatthere are similar segments in this region. An example of thisis shown in Fig. 3, where the divergent carboxyl portion of theU. caupo protein is aligned with individual sequences from theproteins included in the multiple alignment. The proteins fromall the organisms share some characteristics in this region. Theunusually high frequency of tryptophan has already beenpointed out by Keppel and Schaller (1991). In both the U.caupo and T. gratilla proteins, 5 of the 9 tryptophans are found

248 E. T. Rosenthal and L. Wordeman

Fig. 1. Sequence of U. caupo LBP/p40 mRNA. The amino acidsequence of the protein encoded by the mRNA is shown above thenucleic acid sequence, using the single letter code. Asterisks mark anATG codon upstream of the correct start codon. An oligonucleotidecomplementary to the underlined bases was used for directsequencing of the mRNA. Potential sites for N-glycosylation aremarked with a ‘ngl’ above the amino acid, potential sites for caseinkinase II phosphorylation are marked with a ‘ck2’, potential sites forprotein kinase C are marked with a ‘pkc’, and potential sites fortyrosine phosphorylation are marked with a ‘tyk’. The carboxylregion alanine-proline rich segment is marked by a bar above theamino acids. The AATAAA polyadenylation hexanucleotide isshown in lower case letters.

in the carboxyl 1/3 of the proteins, where they are oftenpreceded by aspartic acid. The U. caupo and T. gratillaproteins both have a long stretch close to the carboxyl terminusthat is composed almost entirely of alanine and proline. Thisfeature is not found in any of the other LBP/p40 proteins,although in A. thaliana there are some shorter stretches ofalanine and proline at the carboxyl terminus.

There are also bacterial, mitochondrial and chloroplastsequences which show significant sequence similarity to theLBP/p40 proteins. Of these, the archaebacterium, H. maris-

morti, gene matches best with the eukaryotic LBP/p40 proteins(data not shown). Its function has not been identified, but it isencoded within an operon containing, among other things,genes for 3 ribosomal proteins (Kromer and Arndt, 1991). Theeubacterium, E. coli, protein has been identified as ribosomalprotein S2 (An et al., 1981). Homologues to the S2 ribosomalprotein have also been identified in the mitochondria of yeastand the chloroplasts of plants (Davis et al., 1992). All of thenon-eukaryotic sequences were left out of the alignment in Fig.2 because they are substantially more divergent than theeukaryotic sequences, but examples of such alignments can beseen elsewhere (Kromer and Arndt, 1991; Davis et al., 1992;Melnick et al., 1993; Tohgo et al., 1994). No other eukaryoticribosomal proteins have been identified as homologues toprokaryotic and plastid S2.

LBP/p40 mRNA accumulates during U. caupooogenesis, is degraded during early embryogenesis,and re-synthesized during later developmentIt has been shown previously that LBP/p40 mRNA (identifiedas ‘518’ in earlier work) accumulates to a steady-state level bystage 2 of U. caupo oogenesis (mid-sized oocytes, 40-77 µMin diameter), and declines slightly in full-grown oocytes(Rosenthal and Wilt, 1986). Following fertilization, the amountof LBP/p40 mRNA per embryo declines steadily for 6 hours,until there is very little left in the 56-cell embryo (Fig. 4). Atthe 92-cell stage, 10 hours after fertilization, the amount of thismRNA per embryo begins to increase, presumably due to newtranscription. The mRNA in 10-hour and later embryos appearsto be slightly larger than the mRNA from earlier stages (Fig.4). This is because the newly transcribed mRNA is polyadeny-lated, and the maternal LBP/p40 mRNAs have very shortpoly(A) tails (data not shown). Fig. 4 also shows that two adulttissues, gut and body wall, contain LBP/p40 mRNA.

These results are identical to those obtained with other U.caupo maternal mRNAs from the class that is translated inoocytes, and not in early embryos (Rosenthal et al., 1993;Rosenthal, 1993; E. T. Rosenthal, unpublished data). Theassignment of the LBP/p40 mRNA, previously named ‘518’mRNA, to this class was demonstrated by Rosenthal et al.(1993) with northern blots of RNA extracted from polysomegradients. This method has also been used to show that thenewly transcribed LBP/p40 mRNA present in 24-hourembryos is associated with polysomes (data not shown).

Three lines of evidence indicate that LBP/p40 mRNAs fromall stages of development have the same sequence: (1) Thesequence of the 5′ untranslated region of the RNA fromoocytes, embryos, coelomocytes, gut and body wall isidentical. This was established by direct sequencing of mRNAsfrom these sources with an oligonucleotide complementary toa 5′ sequence at the start of the coding sequence (data notshown). (2) Sequences of LBP/p40 clones from a coelomocytecDNA library match with the sequence determined from theoocyte cDNA clone (data not shown). (3) A single band isdetected on Southern blots of EcoRI-digested genomic DNA(data not shown), suggesting that there is only one geneencoding this protein.

LBP/p40 protein accumulates during U. caupooogenesis and persists after fertilizationWe produced polyclonal antibody RP1340, which reacts with

249LBP/p40 in U. caupo development

Fig. 2. CLUSTAL V alignment of eukaryotic LBP/p40 proteins.Protein sequences were aligned with the CLUSTAL V software(Higgins et al., 1991). Asterisks mark positions where all 7 proteinshave an identical amino acid, and full stops mark positions where atleast one of the proteins has a conservative substitution. The GenBankaccession numbers for the sequences included in this figure are: T.

gratilla (sea urchin), U02371; U. caupo (Urechis), U02370; H. sapiens (human), J03799; C. viridissima (hydra), X63849; D. melanogaster(fly), M90422; A. thaliana (plant), X69056; S. cerevisiae (yeast), M88277; U02370.

Fig. 3. CLUSTAL V alignment of LBP/p40 protein carboxyl termini. In order to show the unusual relationships of the carboxyl segments ofLBP/p40 proteins from diverse sources, the U. caupo protein’s carboxyl region was aligned individually with the homologous region from eachof the proteins included in Fig. 2. A ‘>’ in Fig. 2 indicates the start of the area defined as the carboxyl terminal in this figure. This correspondsto amino acid number 212 in the U. caupo protein and in the proteins from all of the other sources except A. thaliana, where it is amino acidnumber 216. The abbreviations for the different sources of the proteins are the same as in Fig. 2.

the U. caupo LBP/p40, by immunizing rabbits with a bacteri-ally expressed fusion protein. RP1340 was used to immuno-precipitate LBP/p40 from homogenates of [35S]methionine-labelled U. caupo oocytes and embryos, and from an in vitrotranslation of poly(A)+ RNA from Urechis oocytes (Fig. 5).Fig. 5 also includes the in vitro translation products of mRNAtranscribed in vitro from plasmid 1340, which contains the fullcoding sequence of the protein (lane H). U. caupo LBP/p40does not undergo any detectable post-translational modifica-tions in vivo, since the bands obtained from the in vitro trans-

lations (lanes F and H) comigrate with the bands immunopre-cipitated from in vivo material (lanes A and C). Although theamino acid sequence predicts a molecular mass of 34 kDa, theU. caupo protein has an apparent molecular mass of 54 kDaon SDS-polyacrylamide gels. Similar results have beenobtained with LBP/p40 proteins from other sources.

It is possible to immunoprecipitate radioactively labelledLBP/p40 from U. caupo oocytes (lane A) and 24-hour embryos(lane C). Although there is a great deal of background in theimmunoprecipitations performed with 24-hour embryos there


Fig. 4. Northern blot analysis of LBP/p40 mRNA concentrationsduring early U. caupo embryogenesis and in selected adult tissues.Total RNA from embryos at different times after fertilization, inhours, was electrophoresed in a 1% agarose gel containingformaldehyde. The gels were blotted to nitrocellulose filters, whichwere then hybridized to radioactively labelled full-length probe tothe U. caupo LBP/p40 mRNA. Each lane contains RNA from anequal number of embryos, amounting to approximately 1 µg perlane. Lane G contains 1 µg of RNA from the adult animal’s gut, andlane BW contains 1 µg of RNA from the adult body wall.

Fig. 5. Immunoprecipitation of LBP/p40. Rabbit polyclonalantiserum raised against bacterially expressed U. caupo LBP/p40was used to immunoprecipitate proteins from [35S]methionine-labelled oocytes and embryos, and from in vitro translation reactions.Lanes are: A, oocyte homogenate precipitated with rabbit immuneserum; B, 2-cell homogenate precipitated with immune serum; C, 24-hour embryo homogenate precipitated with immune serum; D, 24-hour embryo homogenate precipitated with pre-immune serum; E, invitro translation with no added mRNA precipitated with immuneserum; F, in vitro translation with oocyte poly(A)+ RNA precipitatedwith immune serum; G, in vitro translation with oocyte poly(A)+

RNA precipitated with pre-immune serum. Lanes H, I and J containin vitro translation products from: H, in vitro transcribed LBP/p40mRNA; I, oocyte poly(A)+ RNA; J, no added mRNA.

is only one band present in the material precipitated by theimmune serum (lane C) that is not present in the material pre-cipitated by the preimmune serum (lane D). No labelledLBP/p40 is immunoprecipitated from 2-cell embryos, indicat-ing that the protein is not being synthesized at that stage. Theseresults are in perfect agreement with the previously cited datafrom the polysome gradients, which showed that the mRNA istranslated in oocytes and 24-hour embryos, but not in 2-cellembryos.

Accumulation of LBP/p40 protein during U. caupo devel-opment was also investigated using western blots and antibodyRP1340ap, which consists of affinity-purified antibodies fromthe RP1340 serum. RP1340ap gives an extremely clean andintense signal from a single band on western blots. Fig. 6shows blots done with oocytes at different stages of growth(Fig. 6A), and embryos at different times, in hours (Fig. 6B)or days (Fig. 6C), after fertilization. Each lane in Fig. 6A wasloaded with an equal amount of protein, so the relativelyconstant levels of LBP/p40 detected at each stage actuallyrepresent an increase in the amount of the protein per oocyte.This follows from the fact that the oocytes are getting muchlarger as oogenesis proceeds, ie. the mid-size oocytes in laneF3 have an average volume close to 7 times the averagevolume of the previtellogenic oocytes in lane F5. Thepolysome data, which shows that LBP/p40 is being synthe-sized during oogenesis, predicts this result.

During the first 48 hours of embryogenesis there is nochange in the amount of LBP/p40 per embryo (Fig. 6B and C).Maternal LBP/p40 protein must be stable during the first 10hours of embryogenesis, since the maternal LBP/p40 mRNAis not being translated during this period, and is eventuallydegraded. In later embryos, LBP/p40 must be turning over,since its concentration remains constant while levels of themRNA increase, and this new mRNA is being translated. Inembryos 3 to 5 days after fertilization the concentration ofLBP/p40 declines (Fig. 6C). The rabbit antibodies detect onlya single band at all these stages.

LBP/p40 is associated with polysomes and possibly40S ribosomal subunits in U. caupo oocytes andembryosTwo groups have reported that LBP/p40 is associated withpolysomes and possibly with ribosomes in mouse cells(McCaffery et al., 1990; Auth and Brawerman, 1992). Todetermine if this was also the case in U. caupo, we centrifugedpost-mitochondrial supernatants from oocytes, embryos atvarious stages, and coelomocytes, on sucrose gradients. TheLBP/p40 content in different fractions was assayed by westernblotting, and the position of the ribosomal subunits was deter-mined by measuring the OD260 (Figs 7, 8 and 9). Fig. 7


Fig. 6. Accumulation of LBP/p40 protein during U. caupo oogenesisand embryogenesis. Western blots were probed with affinity-purifiedrabbit polyclonal antibodies raised against U. caupo LBP/p40(RP1340ap). Horseradish peroxidase-conjugated anti-rabbitantibodies were used in conjunction with a chemiluminescentdetection protocol to visualize the antibodies. Each lane in Acontains equal amounts of protein from oocytes at different stages ofgrowth. Lane F5 contains protein from previtellogenic oocytes (10-39 µM), lane F3 contains mid-size oocytes (40-77 µM), lane F1contains large oocytes (77-125 µM), and lane FG contains full-grown oocytes (125 µM). The lanes in B and C contain protein fromequal numbers of oocytes or embryos at different times, in hours (B) or days (C) after fertilization. The lanes on the edges in Bcontain bacterial fusion protein encoded by the pQE31 vectorconstruct described in Materials and Methods.

Fig. 7. Association of LBP/p40 with ribosomes before and afterfertilization in U. caupo. Gradients were run as described inMaterials and Methods with homogenates from U. caupo oocytes (O panels) or 2-cell embryos (E panels). Western blots of thegradient fractions were probed with antibody RP1340ap and detectedwith chemiluminescence. Fraction 1 is from the bottom of thegradient, and fraction 9 is from the top. Panels marked ‘+edta’ showgradients run with homogenates treated with 25 mM EDTA, whichshifts all of the LBP/p40 to the fractions containing soluble protein.

Fig. 8. Association of LBP/p40 with the 40 S ribosomal subunit.This gradient was run with homogenate from 2-cell U. caupoembryos. Conditions were as described in Materials and Methods,except that the centrifugation time was doubled to 6 hours, and onlythose fractions containing ribosomal subunits were assayed. Westernblots of the gradient fractions were probed with antibody RP1340ap,detected with chemiluminescence and quantitated with an Ambisoptical image analyzer. The upper graph shows an A260 trace of therelevant portion of the gradient, aligned with a bar graph showing theamount of LBP/p40 protein in each corresponding gradient fraction.

compares the sedimentation of LBP/p40 protein in U. caupooocytes and 2-cell embryos. In these gradients, most of the 40S and 60 S ribosomal subunits are found in fractions 7 and 6,respectively. There are no 80 S ribosomes present due to thehigh salt concentration (0.5 M KCl) used in the gradients.Polysomes are found in fractions 1 through 4. LBP/p40 proteinis found entirely in gradient fractions corresponding to thelocation of polysomes or 40 S and 60 S ribosomal subunits.More of the protein is found in the polysomal fractions of theembryo gradient than in the oocyte gradient, probably becausethere are more polysomes in embryos. EDTA treatment of thepost-mitochondrial supernatants prior to centrifugation shiftsall the LBP/p40 into the top of the gradients. This is consistentwith results from the mouse system, where it was demonstratedthat EDTA treatment causes the LBP/p40 to move into thesoluble protein fraction (McCaffery et al., 1990; Auth andBrawerman, 1992).

In order to identify more specifically which of the ribosomalsubunits co-sediments with LBP/p40, another gradient using 2-cell embryo homogenate was split into 28 fractions. Compar-ison of the LBP/p40 concentration in each fraction, usingwestern blots, with an A260 trace of the gradients, indicates thatLBP/p40 co-sediments with the 40 S ribosomes (Fig. 8).

We also looked at the distribution of LBP/p40 in later stage


Fig. 9. Association of LBP/p40 with ribosomes in U. caupocoelomocytes and embryos. Gradients were run as described inMaterials and Methods with homogenates from U. caupocoelomocytes or embryos at different stages. Western blots of thegradient fractions were probed with antibody RP1340ap and detectedwith chemiluminescence. Fraction 1 is from the bottom of thegradient, and fraction 14 is from the top. The gradients were loadedwith coelomocyte homogenate (A), 8-cell embryo homogenate (B),5.5-hour embryo homogenate (C), 16-hour embryo homogenate (D),16-hour embryo homogenate after incubation with RNAse (E), and16-hour embryo homogenate after incubation with EDTA (F).

embryos, where there are many more polysomes present. Thegradients shown in Fig. 9 were collected in 14 fractions, andthe X-ray films used in the chemiluminescent detectionprocedure were exposed for very short periods in order not toobscure data from the tops of the gradients. Consequently,these exposures are too light to show the presence of LBP/p40in the polysome fractions, except in the case of the 16-hourembryos. Longer exposures do show the presence of LBP/p40

in the polysome containing fractions from coelomocytes, 9-cellembryos and 5.5-hour embryos (data not shown). In thesegradients, the 40 S ribosomal subunit peak is between fractions9 and 10, and the 60 S subunit peak is between fractions 8 and9. Since these gradients were run in high salt, there are no 80S ribosomes, except in the RNAse-treated sample (Fig. 9D),where they are located primarily in fractions 7 and 8. Com-parison of Fig. 9D and E demonstrates that RNAse treatmentshifts LBP/p40 from the polysome region of the gradient to the80 S region. This is consistent with LBP/p40 being associatedwith ribosomes that are actively engaged in translatingmRNAs. Fig. 9F once again shows that EDTA shifts all theLBP/p40 protein into the soluble fraction.

The most intriguing result illustrated in Fig. 9 is the obser-vation that a decreasing percentage of the LBP/p40 co-sediments with the 40 S ribosomes as embryogenesis pro-gresses. In oocytes and 2-cell embryos (Fig. 7), and in 8-cellembryos (Fig. 9B), virtually all of the LBP/p40 is found in thesame fractions as the 40 S ribosomal subunits. In 5.5-hour, 48-cell embryos, a substantial percentage of the protein is in thesoluble fraction (Fig. 9C), and in 16-hour, 148-cell embryos,almost all of the LBP/p40 not present in the polysomes issoluble (Fig. 9D). Coelomocytes resemble oocytes and earlyembryos, with virtually all of the LBP/p40 appearing to beassociated with 40 S ribosomal subunits (Fig. 9A). It is worthpointing out that significant amounts of new LBP/p40 are notsynthesized for at least 10 hours after fertilization, so thesoluble LBP/p40 seen in 5.5-hour embryos must be derivedfrom the same maternal store of the protein that sedimentedwith the 40 S subunits at earlier stages.

Localization of LBP/p40 protein during U. caupodevelopmentWe stained U. caupo oocytes and embryos with the anti-LBP/p40 antiserum in order to determine whether or not theprotein was selectively localized within cells, or in differentcells of the embryo. The data shown here were obtained usingthe RP1340 antisera from rabbits - we have obtained identicalresults with polyclonal antibodies from mice (data not shown).These rabbit and mouse antisera stain single bands on westernblots of oocyte and embryo protein (Fig. 6, and other data notshown). Observations were made on both sectioned (Fig. 10)and whole-mounted (Fig. 11) material.

In oocytes and in embryos immediately after fertilization(Fig. 10b and c) the protein is evenly distributed in thecytoplasm. The full-grown oocytes in these figures show thelarge ‘dent’ that is characteristic of U. caupo oocytes beforefertilization. This indentation disappears after fertilization.LBP/p40 does not display any localization in 17-hour embryos(Fig. 10d), which are just beginning gastrulation, and remainsevenly distributed until the trochophore larvae is formedapproximately 2 days after fertilization.

Since relatively few changes occur in larval structure duringthe first few days of the trochophore (Newby, 1940) we havechosen 5-day larvae to illustrate the localization of LBP/p40 inadvanced U. caupo embryos. In sectioned material (Fig. 10eand f) the LBP/p40 is restricted to the outer epidermis, and thecells lining the oesophageal crop (arrowhead). Label isexcluded from cells lining the larval stomach and intestines(arrows). When whole mount embryos are viewed (Fig. 11),LBP/p40 labelling is particularly strong between the epidermis


Fig. 10. Indirect immunofluorescence microscopy of U. caupooocytes and embryos labelled with anti-LBP/p40 antiserum.(a) Unfertilized oocyte labeled with preimmune serum, 4 µMsection; (b) unfertilized oocyte and 1-hour embryo (arrow)labeled with RP1340, 4 µM section; (c) growing oocytes atvarious stages labeled with RP1340, whole mounts; (d) 17-hourembryo (midgastrula) labeled with RP1340, 4 µM section;(e) sagittal section through a 5-day trochophore embryo labeledwith RP1340 (arrowhead points to labelled cells of oesophagealcrop and arrows point to unlabelled cells of the stomach andintestine); (f) phase image of same field as shown in e. Bar in b,50 µM.

and endoderm (arrowheads in Figs 11a and 11d). We also con-sistently see labelling of filamentous structures in the embryos(Fig. 11c). Since these are suggestive of neurons we stained 5-day embryos with a mixture of commercially obtained mono-clonal antibodies against 68 kDa, 160 kDa, and 200 kDa neu-rofilament proteins. These antibodies stained the samefilamentous structures that stain with RP1340 (data not shown).

DISCUSSION

LBP/p40 mRNA is a member of a large pool of U. caupomaternal mRNAs which are translated in growing and in full-grown oocytes (Rosenthal et al., 1993). After fertilization,these maternal mRNAs are no longer associated withpolysomes. By 24 hours after fertilization the translationallyinactive maternal mRNA has been degraded, and transcriptionin the embryo has produced high levels of new LBP/p40mRNA which is used for protein synthesis. A similar patternis seen with many other U. caupo maternal mRNAs, includingthose encoding 2 ribosomal proteins, elongation factor 1A, andα-tubulin (Rosenthal, 1993; E. T. Rosenthal, unpublishedresults). Other U. caupo maternal mRNAs, encoding cyclins,the small subunit of ribonucleotide reductase, and a plakoglo-bin homologue, behave in the opposite manner. These mRNAsare inactive in oocytes at all stages, and are only translated afterfertilization (Rosenthal, 1993; Rosenthal et al., 1993).

The identification of LBP/p40 as a high-affinity laminin

binding protein was first made by Wewer et al. (1986), whoisolated a human cDNA clone for a 67 kDa protein that hadbeen purified on laminin affinity columns. Although there isgood evidence substantiating a role for the 67 kDa protein inlaminin binding, the identification of the LBP/p40 mRNA asthe source of the protein is very controversial (for review seeMecham, 1991). It is beyond the scope of this report to repeatall of the arguments surrounding this issue, which has beenaddressed in depth elsewhere (Rao et al., 1989; McCaffery etal., 1990; Mecham, 1991; Groso et al., 1991; Keppel andSchaller, 1991; Auth and Brawerman, 1992; Tohgo, et al.,1994). Like other LBP/p40 proteins, the U. caupo and T.gratilla versions lack any signal sequence or obviousmembrane spanning domain, and the U. caupo LBP/p40mRNA does not yield a processed product when translated inthe presence of dog pancreatic microsomes (data not shown).So far, we have been unsuccessful in attempts to bind the invitro translation product of U. caupo LBP/p40 mRNA tomouse laminin-Sepharose.

LBP/p40 genes have been identified in many diverseorganisms, including S. cerevisiae and A. thaliana. Yeasts andplants do not contain laminin or interact with laminin-contain-ing extracellular matrixes, so it is very unlikely that LBP/p40functions as a laminin binding protein in these organisms. Thepresence of LBP/p40 in U. caupo coelomocytes, which floatfreely in the coelomic cavity, is also inconsistent with a role inlaminin binding.


Fig. 11. Indirect immunofluorescencemicroscopy of 5-day U. caupo whole mountembryos labelled with anti-LBP/p40 serum.(a and c) RP1340 serum staining of wholemount 5-day embryos at two different levelsof focus. Note accumulation of label betweenepidermis and endoderm (arrowheads); (b) preimmune serum staining of wholemount 5-day embryos; (d) another 5-dayembryo labeled with RP1340 (arrowheads asin a); (e) phase image of same embryosshown in a and c. Bar in b, 50 µM.

Four lines of evidence indicate that LBP/p40 is associatedwith the 40 S ribosomal subunit, and with polyribosomes: (1)there is a weak, but significant sequence similarity betweenLBP/p40 and the S2 ribosomal proteins from prokaryotes,mitochondria and chloroplasts (An et al., 1981; Kromer andArndt, 1991; Davis et al., 1992); (2) the phenotype ofDrosophila LBP/p40 mutants is consistent with it functioningas a ribosomal protein (Melnick et al., 1993); (3) carefulimmunofluorescence observations on tissue culture cells showLBP/p40 co-localizing with ribosomes (McCaffery et al.,1990); and (4) LBP/p40 associates with polysomes, andpossibly 40 S ribosomal subunits, in cells (McCaffery et al.,1990; Auth and Brawerman, 1992; Tohgo et al., 1994; thisreport).

We used western blots to analyze the sedimentation ofLBP/p40 in sucrose gradients run with extracts of U. caupooocytes, embryos and coelomocytes. Our results are similar tothose obtained by Auth and Brawerman (1992) in finding thata portion of the LBP/p40 sediments with polysomes. RNAsetreatment of the cell extracts shifts this LBP/p40 to the 80 Sribosome peak, indicating that the protein’s association withpolysomes is via the ribosome. In oocytes and in very earlyembryos, where there are very few polysomes, all of theLBP/p40 not associated with polysomes sediments as thoughit were associated with the 40 S ribosomal subunit. The sameresult is obtained with coelomocytes, which also contain veryfew polysomes. In later embryos, where the translational

machinery is becoming more active, an increasing proportionof the protein is found as soluble material. These variations inthe distribution of LBP/p40 strongly imply that LBP/p40 is notan integral ribosome protein, despite its similarity to prokary-otic and plastid ribosomal protein S2.

Our immunohistochemistry does not show any LBP/p40localization at the cell surface in oocytes or embryos, and untilthe trochophore larvae there is no localization to any particu-lar cell type. Initially, we had hoped to implicate this proteinin morphogenic processes during early embryogenesis, but ourresults with immunostained gastrulae indicate that it is veryunlikely that this protein plays any role in mediating cell inter-actions with the extracellular matrix during gastrulation. Theimmunostaining pattern we observed in 5-day trochophorelarvae is intriguing, but since there is very little backgroundinformation concerning embryogenesis in U. caupo, we arelimited in the conclusions we can draw from our results. Theaccumulation of LBP/p40 at the interface between theendoderm and epidermis might be expected of an extracellularmatrix receptor, and the staining of structures which also stainwith neurofilament antibodies is interesting, since laminin par-ticipates in the process of neurite guidance during developmentof the nervous system (Kleinman et al., 1990; Nurcombe,1992).

Another possibility is that the distribution of LBP/p40 in 5-day larvae reflects the mitotic activity of cells in differentregions of the embryo. We do not yet know enough about U.


caupo embryogenesis to identify which cells are dividing inthe 5-day embryo, but in sea urchins, LBP/p40 is mostabundant in the more mitotically active regions of the pluteuslarvae (M. Hung et al., unpublished data).

All of the LBP/p40 sequences currently available are verysimilar in the amino terminal 2/3 of the proteins, with sub-stantial divergence occurring in the carboxyl third of theproteins. In contrast to the alignments displayed in Figs 2 and3, an alignment of the four available mammalian LBP/p40protein sequences shows that they are virtually identical fortheir entire lengths (data not shown). This suggests that therole(s) played by the protein in mammalian cells places a con-straint on the carboxyl region’s structure that is not present inthe non-mammalian sequences, and raises the possibility thatLBP/p40 may not have exactly the same function in differentorganisms. Unfortunately, there are no non-mammalian, ver-tebrate sequences available to indicate whether or not all ver-tebrate LBP/p40 proteins share a high level of similarity intheir carboxyl regions.

We cannot completely rule out the possibility that thisprotein, or perhaps a protein sharing significant sequenceidentity with it, functions as a laminin binding protein.However, we feel that the preponderance of the evidence fromour work in U. caupo, and work in other laboratories, supportsa role for LBP/p40 in protein synthesis. Many of the charac-teristics displayed by LBP/p40, such as the changes we havedocumented in the sedimentation of the protein during U.caupo development, the highly conserved amino acidsequences of proteins identified in a wide variety of organisms,and the elevated levels of the protein in tumor cells, illustratethe importance of further investigations into its exact function.

We thank Kristie Okazaki, Michele Bahr and Cheryl Phillipson fortechnical assistance, and Ian and Barbara Gibbons for the use of theirT. gratilla cDNA library. We are grateful to F. W. Studier for helpwith expressing protein in the pET vectors. Steve Benson, GordonVansant, Billie Swalla and Bill Jeffrey provided useful assistance andadvice with immunological techniques, and Francis Lefcort gener-ously provided the monoclonal antibodies used to stain neurofilamentproteins. We also thank Ian Gibbons for his detailed contributions tothe writing of this manuscript. This work was supported by NIH grantHD23130 to E.T.R. and a Helen Hay Witney Foundation fellowshipto L.W.

REFERENCES

Altschul, S. F., Gish, W., Miller, W., Myers, E. W. and Lipman, D. J.(1990). Basic local alignment search tool. J. Mol. Biol. 215, 403-410.

An, G., Bendiak, D. S., Mamelak, L. A. and Friesen, J. D. (1981).Organization and nucleotide sequence of a new ribosomal operon inEscherichia coli containing the genes for ribosomal protein S2 andelongation factor Ts. Nucl. Acids Res. 9, 4163-4172.

Auth, D. and Brawerman, G. (1992). A 33-kD polypeptide with homology tothe laminin receptor: component of translation machinery. Proc. Nat. Acad.Sci. USA 89, 4368-4372.

Chitpatima, S. T., Makrides, S., Bandyopadhyay, R. and Brawerman, G.(1988). Nucleotide sequence of a major messenger RNA for a 21 kilodaltonpolypeptide that is under translational control in mouse tumors. Nucl. AcidsRes. 16, 2350.

Crowe, J. and Henco, K. (1992). The QIAexpressionist Qiagen, Chatsworth,CA.

Davis, S. C., Tzagoloff, A. and Ellis, S. R. (1992). Characterization of a yeastmitochondrial ribosomal protein structurally related to the mammalian 68-kDa high affinity laminin receptor. J. Biol. Chem. 267, 5508-5513.

Ellis, S., Miles, J. and Formosa, T. G. (1994). Characterization of NAB1, a

yeast gene coding for a protein homologous to the mammalian 67 kDalaminin receptor. FASEB J. 8, A1312.

Grosso, L. E., Park, P. W. and Mecham, R. P. (1991). Characterization of aputative clone for the 67-kilodalton elastin/laminin receptor suggests that itencodes a cytoplasmic rather than a cell surface receptor. Biochemistry 30,3346-3350.

Harlow, E. and Lane, D. (1988). Antibodies: A Laboratory Manual, 1st edn.Cold Spring Harbor Press, Cold Spring Harbor.

Higgins, D. G., Bleasby, A. J. and Fuchs, R. (1991). CLUSTAL V: improvedsoftware for multiple sequence alignment. Comput. Appl. Biosci. 8, 189-91.

Keppel, E. and Schaller, H. C. (1991). A 33 kD protein with sequencehomology to the ‘laminin binding protein’ is associated with the cytoskeletonin hydra and in mammalian cells. J. Cell Sci. 100, 789-797.

Kleinman, H. K., Sephel, G. C., Tashiro, K., Weeks, B. S., Burrous, B. A.,Adler, S. H., Yamada, Y. and Martin, G. R. (1990). Laminin in neuronaldevelopment. Ann. NY Acad. Sci. 580, 302-310.

Kondoh, N., Schweinfest, C. W., Henderson, K. W. and Papas, T. S. (1992).Differential expression of S19 ribosomal protein, laminin-binding proteinand human lymphocyte antigen class I mRNAs associated with coloncarcinoma progression and differentiation. Cancer Res. 52, 791-796.

Kromer, W. J. and Arndt, E. (1991). Halobacterial S9 operon. Threeribosomal protein genes are cotranscribed with genes encoding a tRNA(leu),the enolase, and a putative membrane protein in the archaebacteriumHaloarcula (halobacterium) marismortui. J. Biol. Chem. 266, 24573-24579.

Laemmli, E. K. (1970). Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4. Nature 227, 680-685.

McCaffery, P., Neve, R. L. and Drager, U. C. (1990). A dorso-ventralasymmetry in the embryonic retina defined by protein conformation. Proc.Nat. Acad. Sci. 87, 8570-8574.

McSwiggin, J. A. (1990). RNA sequencing. Comments (USB) 17, 1-8.Mecham, R. P. (1991). Receptors for laminin on mammalian cells. FASEB J. 5,

2538-2546.Melnick, M. B., Noll, E. and Perrimon, N. (1993). The Drosophila stubarista

phenotype is associated with a dosage effect of the putative ribosome-associated protein D-p40 on spineless. Genetics 135, 553-564.

Mitchison, T. J. and Sedat, J. (1983). Localization of antigenic determinantsin whole Drosophila embryos. Dev. Biol. 99, 261-264.

Newby, W. W. (1940). The embryology of the echiuroid worm, Urechis caupo.Memoirs, American Philosophical Society, Philadelphia, PA.

Nurcombe, V. (1992). Laminin in neural development. Pharm. Ther. 56, 247-264.

Pearson, W. R. and Lipman, D. J. (1988). Improved tools for biologicalsequence comparison. Proc. Nat. Acad. Sci. USA 85, 2444-2448.

Rabacchi, S. A., Neve, R. L. and Drager, U. C. (1990). A positional markerfor the dorsal embryonic retina is homologous to the high-affinity lamininreceptor. Development 109, 521-531.

Rao, C. N., Castronovo, V., Schmitt, M. C., Wewer, U. M., Claysmith, A. P.,Liotta, L. A. and Sobel, M. E. (1989). Evidence for a precursor of the high-affinity metastasis-associated murine laminin receptor. Biochemistry 28,7474-7486.

Rosenberg, A. H., Lade, B. N., Chui, D., Lin, S., Dunn, J. J. and Studier, F.W. (1987). Vectors for selective expression of cloned DNAs by T7 RNApolymerase. Gene 56, 125-135.

Rosenthal, E. T., Hunt, T. and Ruderman, J. V. (1983). Selective translationof mRNA controls the pattern of protein synthesis of during earlydevelopment of the surf clam, Spisula solidissima. Cell 20, 487-494.

Rosenthal, E. T. and Wilt, F. H. (1986). Patterns of mRNA accumulation andadenylation during oogenesis in Urechis caupo. Dev. Biol. 117, 55-63.

Rosenthal, E. T. (1993). Sequence analysis of translationally controlledmaternal mRNAs from Urechis caupo. Dev. Genetics 14, 485-491.

Rosenthal, E. T., Bahr, M. and Wilt, F. H. (1993). Regulation of maternalmessenger RNA translation during oogenesis and embryogenesis in Urechiscaupo. Dev. Biol. 155, 297-306.

Sanger, F., Nicklen, S. and Coulson, A. R. (1977). Sequencing with chain-terminating inhibitors. Proc. Nat. Acad. Sci. USA 74, 5463-5467.

Satoh, K., Narumi, K., Isemura, M., Sakai, T., Abe, T., Matsushima, K.,Okuda, K. and Motomiya, M. (1992a). Increased expression of the 67 kDa-laminin receptor gene in human small cell lung cancer. Biochem. Biophys.Res. Commun. 182, 746-752.

Satoh K., Narumi, K., Sakai, T., Abe, T. and Motomiya, M. (1992b).Cloning of 67-kDa laminin receptor cDNA and gene expression in normaland malignant cell lines of the human lung. Cancer Let. 62, 199-203.

Spurr, A. R. (1969). A low-viscosity epoxy resin embedding medium forelectron microscopy. J. Ultrastruc. Res. 26, 31-43.


Tohgo, A., Takasawa, S., Munakaka, H., Yonekura, H., Hayashi, N. andOkamoto, H. (1994). Structural determination and characterization of a 40kDa protein isolated from rat 40S ribosomal subunit. FEBS Let. 340, 133-138.

Tryggavason, K. (1993). The laminin family. Curr. Opin. Cell. Biol. 5, 877-882.

Westendorf, J. M., Rao, P. N. and Gerace, L. (1990). Molecular cloning ofmitotic phosphoproteins. J. Cell Biol. 111, 93A.

Wewer, U. M., Liotta, L. A., Jaye, M., Ricca, G. A., Drohan, W. N.,Claysmith, A. P., Rao, C. N., Wirth, P., Coligan, J. E., Albrechtsen, R.,

Mudry, M. and Sobel, M. E. (1986). Altered level of laminin receptormRNA in various human carcinoma cells that have different abilities to bindlaminin. Proc. Nat. Acad. Sci. USA 83, 7137-7141.

Yow, H., Wong, J. M., Chen, H. S., Lee, C., Davis, S., Steele, G. D. andChen, L. B. (1988). Increased mRNA expression of a laminin-bindingprotein in human colon carcinoma: complete sequence of a full-length cDNAencoding the protein. Proc. Nat. Acad. Sci. USA 85, 6394-6398.

(Received 3 April 1994 - Accepted 19 September 1994)

a protein similar to the 67 kda laminin binding protein ...of projects focused on cytoskeletal...

Documents