Identification of cDNAs corresponding to mosquito ribosomal protein genes

182

Biochimica et Biophysica Acta, 950 (1988) 182-192

Elsevier BBA 91823

Identification of cDNAs corresponding to mosquito ribosomal protein genes *

Joan E. Durbin, Mavis R. Swerdel and Ann Marie Fallon Department of Molecular Genetics and Microbiology University of Medicine and Dentistry of New Jersey, School of Osteopathic Medicine, Piscataway, NJ (U. S.A.)

(Received 12 October 1987) (Revised manuscript received 15 February 1988)

Key words: Ribosomal protein gene; cDNA; (A. albopictus); (Mosquito)

Sequences encoding mosquito (Aedes albopictus) ribosomal proteins L8, L14 and L31 were identified from a cDNA library made from size-selected polyadenylated mRNA. Candidate cDNAs corresponding to moderately abundant mRNAs were screened by translation of hybrid-selected transcripts in wheat-germ lysates. Translation products were extracted with acetic acid and analyzed by electrophoresis in two dimensions in the presence of unlabeled ribosomal proteins. The identity of translation products that coelectrophoresed with purified ribosomal protein standards was supported by peptide mapping. The cDNAs corresponding to L8 (pL8) and L31 (pL31) hybridized to cytoplasmic mRNAs of 1.4 and 0.9 kb, respectively. In Southern blots of genomic DNA digested with BamHI, HindIII or EcoRI, the cDNA inserts from both pL8 and pL31 gave simple hybridization patterns suggestive of a low copy number for mosquito ribosomal protein genes.

Introduction With the availability of cloned gene sequences, information on the regulation of ribosomal protein (r-protein) and r R N A gene expression in developing organisms such as the frog Xenopus laevis [1,2] and Drosophila [3-5] is beginning to be accumulated. Nevertheless, at this time, few generalizations can be made as to how the expression of these essential genes is coordinated with the metabolic demands of growth and differentiation. Similarly, little is known about the regulation of

* This paper is dedicated to the memory of Dr. John A. Holowczak. Abbreviations: DBM, diazobenzyloxymethyl; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid. Correspondence (present address): A.M. Fallon, Department of Entomology, 1980 Folwell Avenue, University of Minnesota, St. Paul, MN 55108, U.S.A.

genes that code for ribosomal components during hormonally induced stimulation of gene expression in differentiated tissues. In m a n y mosquito species, the blood meal initiates the female reproductive cycle, which involves massive synthesis of vitellogenin by fat body cells. During the synthetic phase of the reproductive cycle, the abundance of ribosomes in the mosquito fat body increases by about 3-fold [6-8]; later in the cycle, ribosome content returns to previtellogenic levels. Synthesis of vitellogenin has been shown to be under transcriptional regulation [6,9], and several hormones, including the egg d e v e l o p m e n t n e u r o s e c r e t o r y h o r m o n e ( E D N H ) , 20-hydroxyecdysone, and juvenile hormone appear to play key roles in controlling vitellogenin gene expression [10,11]. It is not clear, however, whether these same hormones play any role in regulating the cycle of ribosome synthesis and degradation that accompanies vitellogenesis, nor is it clear whether the accumulation of ribo-

0167-4781/88/$03.50 © 1988 Elsevier Science Publishers B.V. (Biomedical Division)

183 somes in female fat body during the synthetic phase of the vitellogenic cycle limits the amount of vitellogenin that can be produced. With the long-term goal of examining directly how expression of genes that code for ribosomal components may be coordinated with synthesis of vitellogenin in the mosquito, we have set out to construct a cDNA library from which sequences corresponding to mosquito ribosomal protein genes could be isolated. In this paper we describe the identification of sequences corresponding to Aedes albopictus ribosomal proteins L8, L14 and L31 and their use for initial analyses of r-protein genes in cultured mosquito cells.

mitochondria were pelleted by centrifugation at 12000Xg for 20 min at 4°C. The supernatant was then mixed with an equal volume of 0.2 M Tris-HC1 (pH 7.5)/25 mM EDTA/0.3 M NaC1/2% SDS. Proteinase K (Sigma) was added to a final concentration of 0.2 mg/ml, and the sample was incubated at 37 °C for 30 min. The RNA was extracted twice with p h e n o l / chloroform/isoamyl alcohol (25:25:1) and once with chloroform/isoamyl alcohol (25 : 1), and was precipitated with 2.5 vol. of 100% ethanol. Polyadenylated RNA was obtained by passing the total cytoplasmic RNA over oligo(dT)-cellulose, type 3 (Collaborative Research) according to the procedure described by Schlief and Wensink [18].

Materials and Methods

Polyadenylated mRNA (100 #g), twice chromatographed over oligo (dT)-cellulose, and 3H-labeled E. coli rRNA size markers were dissolved in 0.75 ml of 5 mM Tris-HC1 (pH 7.5)/33% DMSO. The RNA was denatured at 65 °C for 1 min, cooled quickly on ice, and layered onto a linear 15-30% sucrose gradient in 5 mM Tris-HC1 (pH 7.5)/0.1 M NaC1/1 mM EDTA/0.5% SDS. After centrifugation in an SW41 rotor at 180000 × g for 16 h at 20 ° C, fractions (0.6 ml) were collected and three size ranges (6-10 S, 11-12 S, and 13-15 S) were pooled and precipitated with ethanol in the presence of 0.2 M sodium acetate. cDNA synthesis. Size-fractionated polyadenylated mRNA from A. albopictus cells (0.5 #g each of 6-10 S, 11-12 S and 13-15 S) was precipitated with ethanol and lyophilized with 0.8 ~tg actinomycin D. Oligo(dT) (800 ng in 9.5/~1 water) was added, and the samples were heated to 56 °C for 2 min and then chilled on ice. For reverse transcription, 4/~1 of 5 x buffer (250 mM Tris-HC1 (pH 8.3)/40 mM MGC12/250 mM KC1), 500 #M of each of the four deoxyribonucleotide triphosphates (dNTPs), 10 mM dithiothreitol, 10 t~Ci [a-32p]dATP (3000 Ci/mmol; New England Nuclear), 8 units human placental RNAase inhibitor, and 17 units AMV reverse transcriptase were included in a final volume of 20 ~tl. The reaction was incubated at 42.5 °C for 60 rain and stopped by the addition of EDTA (pH 8) to 25 mM and SDS to 0.25%. First-strand synthesis was monitored using DE81 paper [19] and by electrophoresis on alkaline agarose gels.

Cells and Media. The C7-10 cells used in this study were derived from the Aedes albopictus line of Singh and are a subclone of the C-7 cells described by Sarver and Stollar [12]. The cell line was derived from minced neonate larvae, but the precise tissue of origin is unknown. C7-10 cells were maintained as monolayer cultures at 28 ° C, 5% CO 2 in Eagle's minimal medium supplemented with nonessential amino acids, glutamine and 5% heat-inactivated fetal bovine serum. Suspension cultures were maintained in Joklik's modified minimal essential medium [13] containing 200 ~tM nonessential amino acids and 5% fetal bovine serum. C7-10 cells are diploid, with a model chromosome number of 6; in a typical population, less than 15% of the nuclei are tetraploid [14]. Ribosomal subunits from C7-10 cells grown in suspension culture were isolated by sucrose density gradient centrifugation [15]. Pooled subunits were recovered from sucrose by precipitation with ethanol [8]. Electrophoresis in two dimensions was carried out according to Johnston and Fallon [16]. Isolation of polyadenylated RNA from cell lysates. C7-10 cells (3- 109) from suspension culture were collected by centrifugation, washed three times with phosphate-buffered saline [17] and 4°C, and lysed by incubation with 10 volumes of 50 mM Tris-HC1 (pH 8) containing 150 mM sodium acetate, 7 mM magnesium acetate, 0.5% Nonidet P40, 25 /xg poly(vinyl sulfate) per ml and 1 mM dithiothreitol on ice for 15 min. Nuclei and

Size fractionation of polyadenylated mRNA.

184 The R N A - D N A hybrids resulting from firststrand synthesis were extracted with p h e n o l / chloroform/isoamyl alcohol (25 : 25 : 1), precipitated twice from 2 M ammonium acetate with 2 vol. of 100% ethanol, and dried. The following components were added to each first-strand reaction for second-strand synthesis: 10 ixl 4 x E. coli PolI buffer (400 mM Hepes (pH 6.9)/40 m M MgC12/60 m M fl-mercaptoethanol/280 m M KC1), each of the 4 dNTPs to 100 /~M, 10 /~Ci [a-32p]dATP, 0.4 /~1 of 15 m M fl-NAD, 2 U E. coli ligase, 24 U E. coli polymerase I, and 1 U RNAase H, in a final reaction volume of 40 /d. The reaction was incubated at 12°C for 60 rain, then at room temperature for 60 rain. Aliquots (1 /~l) were taken before and after incubation with the enzymes and spotted on DE81 paper as described above. Second-strand synthesis was stopped by the addition of 4 #1 0.25 M E D T A containing 5% SDS and tRNA (125 ng/~tl). The double-stranded cDNAs were extracted with phen o l / c h l o r o f o r m / i s o a m y l alcohol ( 2 5 : 2 5 : 1 ) and precipitated twice in the presence of 2 M ammonium acetate and 2 vol. of 100% ethanol. S1 nuclease digestion. Double-stranded c D N A samples (0.1-0.2/~g) were treated with $1 nuclease for 30 rain at 25 ° C, producing fragments half the size of the untreated samples when run on alkaline agarose gels. Samples were extracted with p h e n o l / chloroform/isoamyl alcohol (25 : 25 : 1) and precipitated twice in the presence of 2 M ammonium acetate and ethanol.

Homopolymeric tailing of plasmid vector DNA with poly(dC). The pBR322-SV40 recombinant plasmid used as a cloning vector contains SV40 D N A corresponding to the map position 0.71 to 0.86, which replaces the 2036 bp fragment between the pBR322 PvuII and HindIII sites [20]. Vector D N A was digested with KpnI, extracted with p h e n o l / c h l o r o f o r m / i s o a m y l alcohol (25 : 25 : 1) and precipitated with ethanol. Linearized plasmid (20 /~g) was lyophilized together with [3H]dCTP (24/~Ci; 30 C i / m m o l ; ICN) and dissolved in 25 /~1 of terminal transferase buffer (30 mM Tris-HC1 (pH 6.8)/140 m M sodium cacodylate/0.1 mM dithiothreitol/50 /~M dCTP; the dCTP concentration was at 100-fold molar excess over the estimated number of ends). The reaction mixture was warmed to 37 ° C, and cobalt

chloride was added to a final concentration of 1 mM. An aliquot (0.5/~1) was removed and spotted onto DE81 paper. Finally, 25 units of the terminal transferase enzyme were included. After 10 min at 37°C, it was estimated that 5-10 Cs had been added to the ends of the vector DNA. An additional 25 units of enzyme was added, and after a 10 min incubation the D N A was extracted with p h e n o l / c h l o r o f o r m / i s o a m y l alcohol ( 2 5 : 2 5 : 1 ) , precipitated with ethanol, and stored at - 2 0 o C. The size of the poly(dC) tail was confirmed by agarose gel electrophoresis after digestion of the pBR322-SV40 recombinant plasmid at a unique PvuII site [20] adjacent to the KpnI site used for linearizing the plasmid.

Homopolymeric tailing of cDNAs with poly(dG). The Sl-nuclease-treated, double-stranded cDNA molecules were tailed as described above, using [a-32p]dGTP at 1000-fold molar excess over the estimated number of ends. The tailing reaction was incubated for 7 min at 37°C. The tailed cDNAs were fractionated on 5-20% sucrose gradients in 10 m M Tris-HC1 (pH 7.5)/0.1 M NaC1/1 mM EDTA. After centrifugation in an SW41 rotor at 26500 rpm for 17 h at 4°C, 0.5 ml fractions were collected, and 20/xl of each was run on a 1% neutral agarose gel. The lengths of the cDNAs contained in the various fractions were estimated by comparison with 32p-labeled HindIII fragments of lambda DNA. Transformation. Poly(dG)-tailed inserts ranging in size from 0.5 to 5 kbp were annealed with the poly(dC)-tailed vector and used to transform the E. co# strain K802 ( hsdR +, hsdM + gal-, met-, supE) according to the method of Hanahan [21]. Ampicillin-resistant bacterial colonies (1-4 mm diameter) containing mosquito D N A sequences were transferred to ultraviolet-sterilized Whatman 541 paper discs by incubation at 37 ° C for 2 h, and screened with an Aedes m R N A probe as described by Gergen et al. [22]. The specific activity of the R N A probe was approx. 1 • 108 cpm//~g. Binding of plasmid DNA to DBM paper. Plasmid D N A was linearized with HindIII and bound to diazobenzyloxymethyl (DBM) paper according to the method of Alwine [23]. The DBM papers (1 cm 2) were washed twice with water at 4 ° C and twice with 0.2 M sodium acetate (pH 4) at 4°C, and each was incubated overnight at 4 ° C with

185 6-12 /~g of denatured plasmid DNA in 25/~1 of 30 mM potassium phosphate (pH 6.0). The papers were washed with 50 mM potassium phosphate (pH 6.5) and then stored at - 7 0 ° C. Hybrid selection of mRNAs. DBM papers with bound DNA were incubated for 3 h at 37 °C in prehybridization buffer consisting of 50% formamide/5 × SSC/0.1% SDS/1 mM EDTA (5 ml per 1 cm2 paper filter). The filters were next incubated for 30 rain at 65 °C in 10 mM Tris-HC1 (pH 7.8)/99% formamide/0.1% SDS (2 ml per filter). The DBM papers were annealed with polyadenylated RNA (10/~g in 25 #1 of hybridization buffer containing 50% formamide/5 x SSC/1 mM EDTA/0.1% SDS per 1 cm2 filter). The hybridizations were carried out between two sheets of parafilm at 37°C for at least 20 h. After the papers had been incubated with the RNA, they were washed twice at room temperature in 2 × SSC/1 mM EDTA/0.1% SDS, using 5 ml per filter. The filters were then washed three times for 30 min at 37 ° C in 0.5 x S S C / 5 0 % formamide/0.1% SDS/1 mM EDTA (2 ml per filter). The RNA bound to each paper was eluted for 5 rain at 65 °C with 0.4 ml of 20 mM Tris-HC1 (pH 7.8)/99% formamide/0.1% SDS. After elution, p h e n o l / c h l o r o f o r m / i s o a m y l alcohol (25:25 (1) extracted wheat-germ tRNA (4/~g) was added to the mRNA; nucleic acid was precipitated from 0.2 M sodium acetate (pH 5.4) with 2.5 vol. of 100% ethanol. RNA pellets were washed twice with 70% ethanol and translated in the wheat-germ system (Bethesda Research Laboratories) in the presence of [35S]methionine (25 /xCi/reaction; 1200 Ci/mmol; Amersham). Peptide mapping. Bands or spots were carefully cut from one- or two-dimensional polyacrylamide gels that had been fixed, stained with Coomassie blue, incubated with Econofluor for autoradiography, and dried. The spots were incubated for approx. 1 h in water and then for 1 h in proteolysis buffer (125 mM Tris-HC1 (pH 6.8)/10% glycerol (w/w)/0.1% SDS) without added enzyme. Enzyme solutions were made fresh for each experiment by dissolving the SV8 proteinase from Staphylococcus aureus in proteolysis buffer without SDS. After the upper reservoir had been filled with the electrophoresis running buffer, the sample gel slices were inserted into individual wells.

Proteolysis buffer (5-10 /~1) with enzyme (0.1-1 mg/ml) was added to each well. Additional proteolysis buffer was layered over the enzyme solution to a final volume of 25/~1. The gel was run at 15 mA until the dye front had moved through the stacker into the separation gel, and the power was turned off for 30 min. Finally, the gel was run to completion at a constant current of 30 mA [24]. The gel was fixed for 2 h in 10% acetic acid/50% methanol, and then washed in 50% methanol. It was incUbated overnight in 50% methanol and stained with silver nitrate [25]. Southern blotting. C7-10 DNA (10/tg per lane) was restricted and electrophoresed on 1% agarose gels (15 x 20 cm) for 14 h at 40 V. The gel was stained with ethidium bromide for 30 rain, exposed briefly to ultraviolet light, and DNA was transferred to nitrocellulose as described by Maniatis et al. [19]. The filters were soaked for 5 min in 6 x SSC, excess liquid was removed and filters were baked for 2 h at 80 °C under vacuum. The baked nitrocellulose filter was soaked for 2 rain in 6 x SSC and prehybridized in 5 x SSC containing 0.5% SDS, 5 x Denhardt's solution and denatured calf thymus DNA (100/tg per ml) for 4 h at 42 ° C. Hybridization was carried out in 5 x SSC containing 5 X Denhardt's solution, 0.5% SDS, 10 mM EDTA, 20 mM sodium phosphate (pH 7.4), 50% formamide and 100 /~g denatured calf thymus DNA per ml. 50 /~1 of hybridization buffer were used for each cm2 of nitrocellulose filter. The probes were cDNA inserts purified from low-melting agarose, and labeled by nicktranslation to specific activities from 107 to 108 c p m / # g of DNA. An amount of probe equivalent to (1-5)- 107 cpm was used for each hybridization. The probe was denatured by boiling for 5 min, chilled on ice, and mixed with the hybridization buffer. Filters were hybridized with agitation at 42°C for 12-16 h. The filters were washed for 40 min at 65 °C in 2 x SSC, for 30 min at 60 °C in 2 x SSC/50% formamide, for 40 min at 65 °C in 2 x S S C , and for 40 min at 55°C in 0.2x SSC/0.1% SDS. Northern blotting. Cytoplasmic RNA from mosquito cells was glyoxalated, run on a 2% agarose gel (15 /xg/lane), transferred to nitrocellulose paper, and hybridized to nick-translated plasmid DNA probes (5 • 107 cpm//~g) essentially

186

as described by Thomas [26]. Size markers were globin mRNA and the rRNAs from E. coli.

and 13-15 S were collected, and a portion of each size fraction was translated in a wheat-germ lysate in the presence of [35S]methionine to confirm that the mRNAs in the fractions were intact and contained potential r-protein sequences (Fig. 1B). The results suggested that, collectively, these m R N A fractions coded for acid-soluble proteins with sizes ranging from less than 10000 up to 45000 Da, and were therefore likely to contain message corresponding to the mosquito r-proteins [16]. Portions of the m R N A fractions in these size ranges were copied into double-stranded cDNAs using reverse transcriptase and DNA polymerase I as described in the Materials and Methods. Poly(dG)-tailed cDNA was size-fractionated on 5-20% sucrose gradients, a n n e a l e d with poly(dC)-tailed vector DNA, and used to trans-

Results and Discussion

In eukaryotic cells, r-protein mRNAs are small, moderately abundant, and polyadenylated [27,28]. In the mouse, for example, the polyadenylated m R N A fraction that sedimented at 12 S or less has been shown to be enriched 2.5-fold for r-protein messages [28]. On the basis of these observations, polyadenylated m R N A from A. albopictus cells was purified by oligo(dT) cellulose chromatography and fractionated on a 15-30% sucrose gradient in the presence of 3H-labeled E. coli rRNA size markers (Fig. 1A). Gradient fractions containing m R N A at approx. 6-10 S, 11-12 S,

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Fraction number Fig. 1. Size fractionation of polyadenylated RNA from A. albopictus cells. Panel A shows the size distribution of mRNAs after fractionation on the linear 15-30% sucrose gradient. Arrows indicate the position of 3H-labeled E. coli rRNA size markers. In panel B, lanes 1, 2 and 3 represent an autoradiogram of [35S]methionine-labeled in vitro translation products from pooled mRNA fractions 8-10, 11-12, and 13-14, respectively. In vitro translation was carried out using wheat-germ extract (BRL) containing 100 mM potassium acetate and 2.5 mM magnesium acetate. After 2 h at 22 ° C, the reaction was stopped by the addition of magnesium acetate to a final concentration of 0.1 M and 2 voi. of glacial acetic acid. After 1 h on ice, the acidified lysate was clarified by centrifugation (15000× g for 10 min) and acid-soluble proteins were recovered from the supernatant by precipitation with acetone (5 vol) at - 2 0 ° C for 1 h. The pellet was collected by centrifugation, washed with 90% acetone, dried, dissolved in sample buffer [29] and electrophoresed on a 12.5% SDS polyacrylamide gel. Values on the left represent the positions of molecular weight standards.

187 form E. coli to ampicillin resistance. Ampicillinresistant colonies were screened by hybridization [22] to A. albopictus m R N A that had been endlabeled with polynucleotide kinase and [T32p]ATP. Of 146 colonies screened, 58 gave a moderate hybridization signal, and of these, 35 were chosen for further analysis. To identify c D N A clones containing r-protein gene sequences, D N A from each of the 35 isolates was fixed to DBM paper and used to hybrid-select the corresponding mRNA. The selected mRNAs were eluted from the DBM paper and translated in the wheat-germ system in the presence of [35S]methionine. The translation products were enriched for ribosomal proteins by extraction with acetic acid and screened by SDS polyacrylamide gel electrophoresis [29]. The predominant translation products from hybrid-selected mRNAs representing 3 of the 35 clones were found to be soluble in acetic acid and had sizes of approx. 18 000 for the pC12 done, 27000 for pF3, and 32000 for pC8 (Fig. 2). Additional radioactive bands with decreased intensity that were also produced by these lysates possibly represent p r e m a t u r e termination products or products from hybrid selection of more than one m R N A by the c D N A (see also Refs. 4, 28, 30, 31). Our interpretation of the results shown in Fig. 2 was supported by control experiments (not shown) in which c D N A corresponding to Sindbis virus was used to hybrid-select RNA coding for the viral capsid protein which like r-proteins is soluble in acetic acid and has a low molecular weight. Translation of the hybrid-selected viral R N A yielded a predominant band of the expected size (29 kDa) as well as other bands with lower molecular mass. The bands corresponding to capsid protein and the smaller translation products were similar in relative intensity to those shown in Fig. 2 for the putative r-proteins and the smaller unidentified products, respectively. In contrast, translation of m R N A selected by the vector alone yielded a population of products with uniformly low intensity. A portion of each of the translation mixtures shown in Fig. 2 was further analyzed by two-dimensional polyacrylamide gel electrophoresis using a system developed by Sanders [32] for the resolution of small, basic proteins. The large and small

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pC6 pF3 pC 12 Fig. 2. SDS-gel analysis of [35S]methionine-labeledtranslation products from hybrid selected mRNAs. After elution from DBM paper, mRNAs were translated in wheat-germlysates as described in the legend to Fig. 1. Lysates were extracted with acetic acid, and soluble proteins were analyzed on 12.5% gels in duplicate. Of 35 plasmids tested, three plasmids hybridselected mRNAs that coded for basic proteins. Values on the left represent migration of molecular weight standards. Plasraids pC8 and pC12 contain cDNA representingmRNA fractions 11-12; pF3 was derived from mRNA fractions 8-10. Duplicate lanes represent translation products from two independent reactions.

subunit proteins, as well as total proteins from the mosquito ribosome have previously been mapped using this system [16]. The [35S]methionine-labeled candidate r-proteins were coelectrophoresed in both dimensions with purified large and small ribosomal subunit proteins on two separate gels, and the resulting Coomassie blue-stained gels were compared with their autoradiographs (Fig. 3). In the case of all three clones, pC8, pF3 and pC12, the major labeled protein product comigrated with a large subunit r-protein; when the same material was coelectrophoresed with purified small subunit

188

proteins, the radioactive spot did not superimpose over a stained spot (data not shown). The protein product from pC12-selected transcript migrated with r-protein L31, that of pC8-selected transcript

with r-protein L8, and that of pF3-selected mRNA with L14. In the remainder of this text, we will therefore refer to the clones by their r-protein designation: pC12=pL31; pC8=pL8; p F 3 = 0

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31 B Fig. 3. Two-dimensional electrophoresis of [35S]methionine-labeled translation products. Translation products were mixed with unlabeled ribosomal proteins and run on two-dimensional gels as described in Materials and Methods. Panels A and B represent a schematic diagram of the large subunit (LSU) proteins and a Coomassie-blue stained gel, respectively (see also Ref. 16). In panel A, the open circles represent faintly staining proteins that are not easily seen in the photograph. Panels labeled pC8, pF3 and pC12 represent autoradiograms of the in vitro translation products from the corresponding hybrid selected mRNAs. The identity of the r-protein spot was established by superimposing the autoradiogram over the stained, dried gels containing purified large or small subunit ribosomal proteins. No coeleetrophoresis was observed with small subunit proteins.

189

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Fig. 4. Peptide-mapping of the [ 35S]methionine-labeled proteins produced by translation of hybrid-selected mRNAs. Lanes 1, 4 and 7 represent intact L8, L14 and L31 proteins, respectively (partial digestion of L8 and L14 presumably resulted from diffusion of enzyme during preparation of the gel; see the Materials and Methods). Lanes 2, 5 and 8 represent silver-stained bands after digestion of L8, L14, and L31 with SV8 proteinase (0.3 mg/ml), and lanes 3, 6 and 9 represent autoradiograms of lanes 2, 5 and 8, respectively. In lane 9, the symbols ( < ) indicate faint digestion products.

pL14. On each of the autoradiographs, the major spot was accompanied by faint spots of lower molecular mass (see also Fig. 2), which did not comigrate with any stained protein from either the large or the small ribosomal subunit. To further support the identification of the in vitro translation products, the spots (containing labeled protein from the lysate and non-radioactive marker proteins) were cut from the second-dimension gels and subjected to partial digestion with SV8 proteinase (see also Ref. 30). The peptide fragments were separated on an SDS slab gel. Correspondence between the radiolabeled bands (methionine-containing peptides) and silverstained peptides (which may or may not contain methionine) confirmed the identity of L14 (Fig.

4). For LS, the pattern of radioactive bands matched that of the stained bands, with the exception of the lowest labeled band in lane 3. Since r-proteins may vary widely in the intensity with

which they bind stain (see, for example, Fig. 3, panels A and B, and Ref. 16) it is possible that L8 contains a low-molecular-weight, methionine-containing peptide that is not detectable by silver staining. In several different experiments using different amounts of enzyme, both the standard L31 and the labeled material behaved similarly, in that neither was efficiently digested by SV8 proteinase. Nevertheless, two faint digestion products (indicated in Fig. 3 by the symbols) showed good correspondence with faintly stained bands. TABLE I Original Final r-protein Insert designation designation molecular mass " size pC8 pF3 pC12

pL8 pL14 pL31

" See Ref. 16.

L8; 34000 L14; 27000 L31; 16000

mRNA size

300 bp 1.4 kb 150 bp 600 bp 0.9 kb

190 The c D N A inserts were recovered from the vector by digestion with KpnI, and the sizes of the cloned fragments, determined by electrophoresis on agarose gels, ranged from 150 to 600 bp (Table I). Ribosomal protein cDNAs of similar size were described by Meyuhas and Perry [28], who used a similar cloning strategy. On Northern blots [26], plasmids pL8 and pL31 each hybridized to a single predominant m R N A of approx. 1.4 and 0.9 kb, respectively, consistent with the sizes of polyadenylated mRNAs that could code for L8 and L31 (Table I). For reasons that remain unclear, we have been unable to detect the m R N A corresponding to pL14; it is possible that this difficulty is related to the small size of the c D N A insert. One of the most striking observations that has emerged from recent studies on the molecular biology of eukaryotic r-protein genes is that the copy number of these genes varies considerably in the different organisms that have been examined. In yeast, 26 of the 32 r-protein genes that have been identified occur in two genomic copies, both of which are expressed [33-35]. Of four Drosophila r-protein genes, three are single copy [4,36], while one (which may correspond to $6) appears to be present in multiple copies [4]. R-protein gene copy numbers tend to be higher in the vertebrates. From two to five copies of each of six Xenopus laevis r-protein genes have been reported [30]. In all mammalian cells that have been looked at, there are many (7-20) homologous sequences corresponding to each r-protein gene, and several of these r-protein gene families, including those for L7 [37], L18 [38], L30 [39], L32 [40] and S16 [41], have been examined extensively by varying the stringency of hybridization and washing conditions. In each case, only a single expressed gene has been found; other members of the gene family represent processed pseudogenes. To investigate the copy number of mosquito r-protein genes, hybridizations to A. albopictus genomic D N A were carried out using as probes nick-translated c D N A inserts from pL8 and pL31 (Fig. 5). Each of these probes detected two bands of different size and shghtly varying intensity on Southern blots of mosquito genomic D N A digested with each of three different restriction enzymes that did not cut within the c D N A probe. In replicate experiments, nitrocellulose strips con-

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Fig. 5. Copy number of mosquito r-protein genes. Genomic DNA was digested to completion with HindllI (H), EcoRI (R), or BarnHI (B) and separated on agarose gels as described in Materials and Methods. Southern blots were probed with the cDNA insert from pL8 and pL31; sizes on the right represent migration of HindlII-digested lambda DNA. These results were duplicated in several independent experiments; bands that appeared reproducibly are indicated on the figure by the symbols ( > ).

taining the labeled probes hybridized to digested genomic DNAs were washed at varying temperatures, from 4 5 ° C to 6 0 ° C , for 30 min in 2 x SSC/50% formamide. No additional bands were detected under the less stringent conditions. These results suggest that to a first approximation, there are few copies of the genes for L8 and L31 in the A. albopictus genome.

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Comparisons between cloned r-protein gene sequences from a variety of different organisms have contributed to an understanding of their structure [39-41], regulation [42] and evolution [43,44]. This is the first analysis of r-protein genes from an insect other than Drosophila, and we anticipate that as r-protein sequences of functionally homologous proteins become available, it will be of interest to identify evolutionary changes that have occurred in r-protein genes from these two species. Our choice of the mosquito as an experimental organism has been based on the potential advantages of this insect for investigating ribosome metabolism in differentiated tissues using cloned r-protein probes. Schmidt et al. [45] have recently shown that in the non-mitotic paragonial glands of Drosophila, stimulation of production of secreted protein appears to involve translational regulation of r-protein gene expression. It will therefore be of interest to use rRNA (Park and Fallon, unpublished data) and r-protein gene probes to learn whether a similar regulatory mechanism occurs in the fat body of blood-fed female mosquitoes in association with vitellogenin synthesis.

Acknowledgements This work was supported by grant AI 20385 from the National Institutes of Health. We thank E. Kells and P. Vendula for typing the manuscript and Dr. Russell Durbin for technical advice and helpful discussions.

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