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Isolation of cloned ribosomal protein genes from the yeast Saccharomyces carlsbergensis
Gene. 14 (1981) 279-287 Elsevier/North-HollandBiomedicalPress
279
Isolation of cloned ribosomal protein genes from the yeast Saccbaromyces carlsbergensis (Recombinant DNA; small-size mRNA; R-looping; immunological screening; two-dimensional gels; colony bank)
Godfried H_P.M. BoHen, Louis H. Cohen, Willem H. Mager, Antonius W. Klaassen and Rudi J. Phnta
Bioehemiseh Laboratorium, Vri/e Universiteit, 1081 HV Amsterdam, (TheNetherlands) (Received February 12th, 1981) (Accepted March 31st, 1981)
SUMMARY A colony bank of yeast DNA obtained by cloning Hindlll.generated fragments of total yeast nuclear DNA in Escherichia coli K-12 with the vector pBR322, was screened with a radioactive RNA probe enriched for a subset of ribosomal protein ,,nRNAs. The selected recombinant DNA molecules were hybridized with poly(A)-containing mRNA under R-loop conditions. From the DNA-RNA hybrids the respective mRNAs were melted off and translated in vitro in a rabbit reticulocyte cell-free system. The translational products were analyzed by immunoprecipitation with antibodies raised against ribosomal proteins. The identity of the ribosomal protein geneproducts was further established by electrophoresis on two-dimen~ional gels. At least 15 recombinant DNA molecules were shown to contain ribosomal protein genes. Four of them, i.e. Y65, Y89, Y113 and Y138, have been characterized preliminarily.
INTRODUCTION Ribosome biosynthesis involves the simultaneous expression of a large number of ribosomal protein genes. In bacteria the genes cooing for ribosomal proteins are clustered and arranged in ~ number of common transcriptional units (Jaskunas et al., 1974). Evidence has been obtained that in bacteria regulation of the coordinate synthesis of the ribosomal proteins takes place not only on transcriptional level but also on translational level (Denn,s and Nomurz, 1975;
"Abbreviations:bp, base pairs; Md; megadalton;SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI, 0.015 M Na- citrate, pH 7.8; TEA, trichloro acetic acid; U, unit.
Fallon et al., 1979; Yates et al., 1980; Fill et al., 1980). In eukaryotes little is known about the genetic organization of the ribosomal protein genes nor about the way their expression is regulated coordinately. Recently, several authors reported the isolation of cloned eukaryotic DNA fragments containing a ribosomal protein gene as a first step towards a better understanding of the regulated expression of these genes (Woolford et al., 1979; Vaslet et al., 1980; Meyuhas and Perry, 1980). For the same purpose we had fractionated from the yeast Saccharomyces cadsbergensis a small-size mRNA fraction which is relatively enriched for a subset of ribosomal protein mRNAs (as ,tescribed by Bollen et al., 1980)and, thus, could be employed to detect ribosomal protein
0378-1119/81/0000-0000/$02.50 0 1981 Elsevier/North-HollandBiomedicalPress
280 genes in a yeast DNA bank. In this paper we describe the isolation and partial characterization of ribosomal protein genes from thi~ yeast strain.
MATERIALS AND METHODS
(a) Isolation of nucleic acids High Mr nuclear DNA was isolated essentially as described previously (Ret61 and Planta, 1972). Smallsize mIL'4A used as a probe in colony hybridization experiments was isolated from poly(A)-containing RNA by preparative sucrose gradient centrifugation (Boilen et al., 1980). (b) Cloning of HindlH.generated fragments of yeast DNA in E. coli The 31asmid pBR322 .~as linearized with Hindlll i:~ 30 mM Tris- HCI (pH 7.9), 12 mM NaCI and 10 mM MgCI2 by incubation during 2 h at 37°C. Subsequently~ the DNA was dephosphorylated with bacterial a~kaline phosphatase during 1 h at 37°C after the addition of 0.1 volume of 1 M Tris. HCI (pH 8.0) (Maxam and Gilbert, 1977). The reaction was termi. nated by extraction with phenol and chloroform and precipitation with ethanol. Yeast nuclear DNA was digested with HindIll as described above for pBR322, then heated to 65°C during 5 min and extracted with phenol and chloro. form. The DNA was precipitated with ethanol and dissolved in sterile redistilled water. After addition of "ligase buffer" the mixture was combined with the linearized, dephosphorylated pBR322 (in a 1 : 2 yeast DNA/pBR322 weight ratio) in a final volume of 50/d containing 66 mM Tris" HCI (pH 7.6), 6.6 mM MgC12, 0.1 mM ATP, 0.66 mM dithiotreitol and 1 mgfinl bovine serum albumin. An excess of T4 DNA ligase (Miles Lab.) was then added and the mixture was incubated at 4°C during 18 h (Meyerink, 1979). E. coli K-12 type 5K was transformed with the whole ligation mixture (Cohen and Chang, 1973). Transformants were selected for ampicillin resistance. As a control linearized and dephosphorylated pBR322 was ligated and transformed in E. coli 5K.
(c) Colony hybridization Both ribosomat RNAs and mRNAs to be used as hybridization probes were labelled in vitro by the following procedure. RNA was precipitated with ethanol and resuspended in freshly prepared 0.05 M Na-carbonate to a concentration of 300/g/ml and incubated at 50°C for 80 min. Under these conditions RNA was fragmented to pieces of about 200 nucleotides (Goldbach, 1978). The sample was neutralized by the addition of I vol. of 80 mM Tris-HCI (pH 7.6) and I vol. of 0.05 M HCI. After the addition of I voL of 20 mM MgCI2, 20 mM 2-mercaptoethanol the 5' ends of the RNA fragments were labelled with [~'-32P]ATP by 1"4 polynucleotide kinase (Boehringer). The mixture was incubated at 37°C for 30 rain. The reaction was stopped by the addition of SDS to a final concentration of 0.5% (w/v). The labelled RNA was purified by Sephadex G-50 gel filtration. The specific activity of the labelled RNA was approx. 2 - 7 X 10s cpm//zg RNA. The labelled RNAs were used for screening a colony bank containing Hindlll-generated fragments of yeast DNA. The whole bank was f'~.ed on Millipore HA filter grids (0A5 /an pores) each containing 103 colonies. The filters were prepared for hybridization essentially according to the method of Gmnstein and Hogness (1975). Each f'dter (diameter 11 cm) was moistened with 5 X SSC, 50% (v/v) formamide, containing about 10-20X 106 cpm (approx. 3 #g RNA)in a total volume of ~ ml. The solution was covered with mineral oil and the filters were incubated during 18 h at 42°C to allow hybridization. Then the filters were washed in 2XSSC, treated with pancreatic ribonuclease (20/zg/ml) and washed again. After drying, the filters were exposed dum~g 20-36 h to Kodak XRI fdm. (d) Isolation of recombinant DNAs The E. coli clones were grown overnight at 37°C in 200 ml Brain Heart Infusion medium (Oxoid), conraining 50 / ~ m l ampicillin. Cells were harvested by centrifugation for I0 rain at 3 000 X g at 4°C and washed with 25% (w/v) sucrose, 30 mM Tris. HCI (pH 8.0) at 4°C. They were resuspended in 1.3 ml of the same buffer. Then 0.5 ml c~ a lysozyme solution (Sigma; 8 mg/ml) was added and the suspension was incubated for 5 min at room temperature. The cells
281 were lysed by a second incubation after the addition of 0_3 ml 0.5 M EDTA (pH 8.0) and 3 ml "Brij-mix" (1% w/v Brij-58, 10 mM sodium deoxycholate, 60 mM EDTA, 50 mM Tris- HCI, pH 8.0) for 15 min at 20°C. Cell debris was removed by centdfugation for 30 min at 85 000 Xg at 4°C in a 50.2 Ti rotor (Beckman), 4.5 g of cesium chloride was added to the supematant as well as ethidium bromide up to a final concentration of 0.1 mg/ml. The volume of the final solution was adjusted to 6 ml with a solution containing 30 mM "Iris. HCI (pH 8.0), 0.5 mM EDTA and 50 mM NaCI (TES). Cesium chloride equilibrium centrifugation was performed during 20 h at 42 000 rev./ min at 4°C in a 50 Ti rotor (Beckman). The plasmid DNA, visible under UV light as a single band, was collected by means of a hypodermic needle and syringe from the side of the tube. Ethidium bromide was removed by extracting the solution at least three times with an equal volume of isoamyl alcohol, and finally the DNA solution was dialysed extensively against TES.
(e) Digestion of plasmid DNA with BamHl Purified plasmid DNA was linearized by digestion with the restriction endonuclease BamH! (Bethesda Research Laboratories Inc.). Incubation was performed for 2 h at 37°C in the presence of BamHl (1.5 U//zg DNA), 6 mM MgCI~, I mM dithiotreitol, 150 mM NaCI and 6 mM Tris. HCI (pH 7.9). The enzyme was removed by phenol extraction and the linearized DNA was collected by ethanol precipitation.
(f) R-loop hybridization and hybrid purification A pool of 3 or 4 different plasmids, 3/ag each, was hybridized to 15-18/ag of yeast poly(A)-containing RNA under R-loop conditions, and the DNA/RNA hybrids were purified from the unhybridized RNA by gel f'dtration on a Biogel A150m column (Biorad i~aboratories) as described by Woolford and Rosbash (1979). The hybrid-containing fraction (excluded volume; 3 ml) was ethanol-precipitated after the addition of 13 /ag of Bacillus Iicheniformis ribosomal RNA. The precipitated nucleic acids were washed with 70% (v/v) ethanol and dissolved in 6/,d sterile redistilled water.
(g) In vitro translation of hybridized mRNA mRNA, hybridized to the plasmid DNA, was released from the hybrid by incubating the 6/1! hybrid solution for 15 s at 100°C. The final translation mixture was obtained by adding 24/d rabbit reticulocyte lysate (Amersham) and 3/.d of L-[3SS]methionine (Amersham, 5/zCi//zl, spec. act.. >1000 Ci/mmol). Translation took place during 2 h at 30°C. (h) Gel electrophoresis of proteins synthesized in
vitro Analysis of the translation products was performed by electrophoresis in 10-15% polyacrylamide gradient gels in the presence of SDS as previously described (Bollen et al., 1980). Ribosomal proteins were identified by two-dimensional gel electrophoresis as described by Kaltschmidt and Wittmann (1970) with the following modification. Proteins synthesized in vitro (about 10/d) were precipitated with 100 pJ 20% (w/v) TCA and subsequently washed with 5% (w/v) TCA, acetone and ether (Howard et al., "1975). The precipitate was redissolved in 6 M urea, 1 mM dithiotreitol and 25% (w/v) sucrose. The protein sample was neutralized with 1 M unbuffered Tris and applied on top of the first dimension gel along with 0A mg unlabelled ribosomal proteins. The time of electrophoresis (23 h at 180 V) in the first dimension was doubled (see Kruiswijk and Planta, 1974). (i) Staining and autoradiography Two-dimensional gels were stained in a solution containing 0.2% (w/v) Coomassie Brilliant Blue R250, 45% (v/v) methanol and 9% (v/v) acetic acid during 2.5 h, destained in 7% (v/v) acetic acid and prepared for fluorography according to the method of Laskey and Mills (1975). SDS.contalning polyacrylamide gels were not stained, but f'Lxedovernight in a solution containing 25% (v/v) methanol and 9% (v/v) acetic acid.
(j) Preparation of antisera against yeast ribosomal proteins Ribosomal proteins, isolated and purified as described by Kruiswijk and Planta (1974) were fractionated by chromatography on a phosphocellulose
282 column. About 25 fractions were obtained, each contair=ing 2 - 6 ribosomal protein species. All fractions were injecte~ into rabbits separately. This p.-ocedure of p~eparing yeast n'bosomal protein fractiens as well as the characterization of antisera raised against them wflI be descn'bed in detail elsewhere (Bollen, G.H.P.M., Mager, W.H. and Planta, R.I., manuscript in preparation).
(k) lmmunoprecipitation of ribosomal proteins synthesized in vitro lmmunoprecipitation of the in vitro translation products was performed according to a modification of the procedure described by Kessler (1976). 5 gl of the transla~on mixture was preincubated in the presence of 100 gl of a 5% (v/v) Staphylococcus aureus suspension in immunoprecipitation buffer (1% (v/v) Triton X-100, 05% (w/v) sodium-deoxycholate, 0.1% SDS, 150 mM NaCI and 20 mM Na-phosphate pH 7.2). After incubation for 30 rain the sample was centrifuged for 1 rain at 12 000 × g. The supernatant was mixed with 5 gl of a mixture of 13 sera raised against different ribosomal protein fractions as described ~bove, and incubated for 18 h at 4°C. Subsequently 50/,d of a 10% (v/v) Staphylococcus aureus suspension in immunopre¢ipitation buffer was added and incul:ation was continued for 30 min. The Staphylococcus aureus immune complex was col. lected by eentrifugation for 1 rain at 12000Xg, resuspended in immunoprecipitation buffer and centrifuged through a layer of 30% (w/v) sucrose in the same buffer for 2 min at 12 000 Xg. The pellet was washed twice with immunoprecipitation buffer, once vAth this buffer without detergents and then resusp~nded in 25 bd of SD$ sample b:fffer (Bollen et al., 1980). The immune complex was dissociated by hea!ing for 3 rain at 100°C and Staphylococcus aureus was removed by centrifugation "for 10 rain at 12 000 X ~ at 200C. The labelled proteins in the supernatant were analysed by electrophoresis on SDS polyacrylami~e slab gels as described above.
RESULTS AND DISCUSSION (a) Isolation a~d screening of recombinant DNA molecules containing yeast DNA In order to obtain a yeast DNA bank total yeast nuclear DNA, digested with Hindlll, was cloned in E. coli K-12 5K after recombination with the plasmid vector pBR322 (see MATERIALS AND METHODS). We isolated about 10000 ampicillin. resistant transformants. Assuming that the haploid yeast genome contains 101° daltons of DNA and that the Hindlll-generated DNA fragments have an average size of about 2.5 Md it can be calculated that the probability that this bank contains all sequences present in the yeast genome is about 80% (Petes et al., 1978). Colony hybridization experiments were performed using a small-size poly(A)~eontaining mRNA fraction isolated from S. carlsbergensis as a probe (Bollen et al., 1980). Recently, we have demonstrated that this mRNA fraction is enriched in mRNAs coding for at least 30 ribosomal proteins; the concentration of the individual ribosomal protein mRNAs was estimated to be about 1.5% of the mRNA sequences present in this probe (Bollen et al., 1980). This extent of enrichment might be sufficient to give a reproducible signal in the colony hybridization test according to Gmn. stein and Hogness (1975). Since the small.size mRNA fraction always is contaminated with ribosomal RNA we first hybridized the colonies to purified ribosomal RNA labelled in vitro as described in MATERIALS AND METHODS. In 5 000 clones examined we found 160 recombinants (=3.2%)hybridizing with 17S rRNA and another 190 hybridizing with 5S rRNA. These groups of clones contain recombinant DNAs carrying the 4.1 X 106 and 1.7 X l0 s fragment of the ribosomal repeat, respectively (Meyerink et al., 1976). This frequency is close to the predicted proportion of ribosomal DNA-containing clones. By hybridization with the small-size mRNA fraction 350 clones (i.e. 7%) out of 5 000 clones could be selected. The hybridization response could be divided into two classes: for 47 clones the hybridization was stronger than for the remaining 300 clones. The yield of 7% of clones containing DNA complementary to yeast mRNA is in agreement with the findings of Woolford et al. (1979) and suggests, as also stated by Petes et al. (1978), that only abundant or moderately abun-
283 dant mRNAs hybridize under the conditions used. This probably also explains why two distinguishable hybridization signals were observed in the colony hybridization experiments.
(b) Characterization of the selected recombinants In order to characterize the recombinant DNAs present in the clones selected as described above, we isolated the plasmid DNAs and hybridized them under R-loop conditions with total poly(A)-containing mRNA according to the procedure designed by Woolford and Rosbash (1979) (see MATERIALS AND METHODS). Subsequently, the products encoded by the recombinant DNAs were analyzed by translating the complementary mRNAs in vitro. First, the recombinant DNA molecules were linearized with the restriction endonuclease BamHl which cuts pBR322 DNA once. Most of the plasmids investigated appeared to have no additional BamHl-site. Those plasmids which were digested into fragments smaller than 3 Md were omitted. In a first test a pool of 3 or 4 linearized recombinant DNA molecules, 3/.tg each, were hybridized to 15-18 gg poly(A)-containing RNA. The R.loop containing molecules (and the double.stranded DNAs) were separated from unhybridized mRNA by gel filtration on a Biogel Al50m column. From the excluded volume DNA was precipitated with ethanol. The precipitate was dissolved in 6 ~d sterile redistilled water and then the RNA was melted from the hybrid by incubation for 15 s at 100°C. The complementary mRNAs were translated in a rabbit reticulocyte cellfree system in the presence of [3SS]methionine. The proteins synthesized in vitro were analyzed by three different methods, viz. (i) SDS-polyacrylamide gel electrophoresis, (ii) immunoprecipitation and (iii) 2D.gel dectrophoresis. (i) SDS-polyacrylamide gel electrophoresis. The aim of this type of electrophoretie analysis was to prove both the selectivity of the procedure used and the persistence of proper translational capacity of the complementary mRNAs during the procedure. A typical gel is shown in Fig. IA. Obviously, the use of different combinations of reccxnbinant DNAs gives rise to the synthesis in vitro ot sets of distinct polypeptides. The Mr of most of the proteins synthesized in vitro is < 50 000, as could be expected since the clones were selected upon hybridization with small-
size mRNA. Under the conditions used nearly all mRNAs are hybridized to their complementary DNA sequences. Still it is difficult to draw any conclusions as to the abundancy of the isolated mRNAs from the different intensities of the protein bands observed in the autoradiogram, since both the content of methionine in these polypeptides and the translational capacity in vitro of the individual mRNAs are unknown. fii) Immunoprecipitation with anti-ribosomal protein serum. Subsequently we looked for the presence of ribosomal proteins among the translation products. In a first approach we made use of antisera raised against a set of 13 different ribosomal protein fractions isolated by chromatography on phosphocellulose columns of total ribosomal protein. We incubateC the translation products in ~¢ presence of the combined sera, then ,he antigen-antibody complexes were recovered by absorption to fLxed Staphylococcus aureus znd subsequentJy analyzed on the same type of SDS-containing polyacrylamide gradient gels as described above. A typicaJ result in presented in Fig. 1B. The lanes indicated in Fig. 1B correspond to those, indicated in Fig. 1A. Lane 12 shows the ribosomal proteins that are precipitated by the pooled antisera from the translation products when the cell-free system is programmed with total poly(A)-containing mRNA. From the protein bands visible in the other lanes of Fig. 1B it can be concluded that apparently several ribosomal proteins are encoded by at least one of the recombinant DNAs present in 5 out of 11 of the plasmid mixtures. In the example presented in this figure at least six ribosomal protein recombinants are present among the 43 plasmid DNAs investigated. In the same way additional sets of recombinant DNA molecules appeared to contain at least another 9 plasmids carrying genes coding for a ribosomal protein. The frequency of 15 ribosomal protein gene-contaiaing plasmids out of about 140 candidate-recombinants investigated so far most likely is too low because the antisera used are raised against only a subset of ribosomal proteins. Moreover, only methionine-containing proteins could be detected. The hybridization procedure was repeated for a number of individual recombinant DNA molecules. Analysis of the immunoprecipitated proteins synthesized in vitro on mRNAs derived from individual RNA/DNA duplexes again was performed by electro-
284
Fig. 1. E[ectrop~oresls on SDS-polyacrylamkle gradient gels of proteins synthesized in vitro on mRNAs complementary to several recombinant DI~'As. (A) Combinations of 3-5 recombinant DNAs were hybridized with poly(A)-containing mRNA under R-loop conditions (see MATERIALS AND METHODS). After melting off the complementary mRNAs, trarslation in vitro was performed in a reticelocyte cell-free system in the presence of [3SSlmethionine. The proteins synthesized in vitro were analyzed on 10-15% polyacrylamide gradient gels containing SDS and the labelled bands were made visible by autoradiography. Lanes 1-11 show the translation products encoded by the various r~ombinant DNA molecules. Lane 12 shows the pro~lucts obtained by translatien in vitro of total pc~ly(A)-containing mRNA. The bands which are in common in each lane represent products synthesized by endogenous mRNA. The gel was exposed for autoradiography during 2 days. (B) Proteins were synthesized in vitro and precipitated with "antisera raised tgainst r~bosomal proteins. Antisera raised against a number of ribosomal protein fractions were obtained as described in MATERIALS AND METHODS. The same translation products as analyzed in panel A were incubated in the presence of the combined ar~tisera (see MATERIALS AND METHODS). The immunoprecipitated proteinswere analyzed by electrophoresis on the same type of gels as shown in panel A. The following mixtures of recombinant DNAs gave rise to immunoprecipitated polypeptides: Y110, Yl 11, Y112, Yl 13 (lane 3), Y118, Y119, YI20, YI21 (lane 5), Y133, Y135, Y137 (lane 8), Y148, Y149, YIS0 (lane 9) and Y155, YI56, Y157 (lane l l). Lane 12 shows the analysis of immunoprecipitated ribosomal proteins synthesized in vitro o ~ total poiy(A)-containing mRNA. The gel was exposed for autoradiography during 14 days.
phoresis in SDS gels. The result is shov, n in Fig. 2. in lanes 2 , 3 , 4 and 5 the bands are v~lble correspondinf~ to the immunoprecipitated translation products o / the m R N A s hybridized to the plasmids isolated from the clones Y65, Y138, Y89 and Y113, respectively. The recombinant DNA from clone Y65 codes for at least one and possibly two ribosomal proteins. The major band corresponds to a ribosomal protein with an Mr o f 15 000; the minor with a polypeptide with a n M r o f 12 000. Clones Y138, Y89 and Y113 each code for
Fig. 2. Electrophoresis on SDS-polyacrylamide gradient gels of immunoprecipitated proteins encoded by individual recombinant DNAs. The gone products of clone Y65.. Y 138, Y89 and Y113 were analyzed individually in a similar way as described in the legend to Fig. lB. Lane 1: immunoprecipitated n'bosomal proteins synthesized in vitro on total poly(A)-containing mRNA. Lane 2, 3, 4 and 5: immunoprecipitated proteins encoded by recombinant DNAs Y65, Y138, Y89 and YII3, respectively. The gel was exposed for autoradiography during 14 days.
285 one ribosomal protein with Mrs o f 17000, 2£~000 and 30 000, respectively. fill) 2D-gel electrophoresis. Finally, the identity of the ribosomal proteins synthesized in vitro on the mRNAs hybridized to the selected recombinants was established on two-dimensional gels according to Kaltschrnidt and Wittmann (1970). In comparison
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with the m e t h o d published earlier (Kruiswijk and Hanta, 1974) the time of electrophoresis in the first dimension was doubled. As a consequence of this modification nearly all basic ribosomal proteins are resolved as can be seen in Fig. 3B (ef. the schematic representation in Fig. 3A). Figs. 3C, D, E and F show the two-dimension~l
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Fig. 3. Analysis of proteins encoded by individual recombinant DNAs on two-dimensional gels. Electrophoresi~ was performed as described previously (KmiswUk and Planta, 1974), except that the time of electrophoresis in the first dimension was doubled. (A) Schematic representation of the marker prof'fle; (B) analysis of total 80S ribosomal proteins, stained with Coomassie Brilliant Blue; C, D, E and F: TCA-precipitated proteins synthesized in vitro on the complementary mRNAs of recombinant DNAs, Y65, Y89, Y113 and Y138, respectively. On each gel 0.4 mg ribosomal proteins were added as markers. After staining the gels were exposed for autoradiography during two weeks.
2~ electropherograr3s of the proteins encoded by the cloned DNAs Y65, Y89, Y I I 3 and Y138. respecti~ely, Y65 appears to code for at least one [3sS]n~ethionine4abe~led protein, tha~ comigrates with the n~bosomal marker protein $31 in the same gel (Fig. 3C). The minor protein spot in Fig. 3C may represent $33 although its identity is not established with certainty since it does not comigrate exactly with $33. A possible explanation for this phenom~on is that the in vitro translation system is unable ~o c a m / o u t posttranslation~ modification steps which may occur in vivo. Another possibility is that the "minor" polypeptide is a pr~nature termination product of the synthesis in vitlo of the "major" ribosomal protein. Experiments are under investigation to discriminate between these two possibilities. Gone Y89 contains sequences that code for ribosomal protein L16 (Fig. 3D)whereas the cloned DNA fragment YI13 apparently codes for ribosomal pro°tein L6/SIO (Fi~. 3E). The two-dimensional electropherogram of the proteins encoded by clone Y138 reveals two spots: one major, comigra~ing with ribosomal protein L25 and one minor which probably is identical to protein L27/$21. By electrophoresis on SDS gels of the immunoprecipb ated proteins encoded by clone Y138 only one, rather weak, band was observed (Fig. 2). However, proteins L25 and $21 have almost equal Mrs (Kruiswijk ~t al., 1978). lr~ addition -o the sequences coding for ribosomal proteins some of the recombinant DNAs identified solar probably carry other, non-ribosomal protein genes, as suggested by analysis on SDS gels similar to that presented m Fig. 1A (not sho~wn). The length of the inserted Hindlll-generated fragments in the plasmids Y65, Y89, Y113 and Y138 as analyzed by electrophoresis on 1% agarose gels is 5090, 5870, 5080 and 3020 bp:, respectively (result not shown). This relatively smal~ size of the inserted yeast DNA sequences suggests that the genes are rather proximate to each other. Physical and genetic maps are currently in preparation. R-loop analysis under the electron microscope revealed that most genes identified so far on cloned yeast DNA fragments are intact (BoUen, G.H.P.M., Molenaar, C.M.T~,, Cohen, L.H., Mager, W.H. and Planta, R.J., manuscript in preparation).
ACKNOWLEDGEMENTS This work was supported in part by the Netherlands Foundation for Chemical Research (SON)with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO). We thank Mrs. M.L. Goei-Soen, Mrs. M.M.C. Duin and Mr. R.D. Fontijn for skilful technical assistance during part of this work. In addition the authors are indebted to Dr. J. Biewenga who enabled us to prepare antisera in the Histologisch Laboratorium, Vrije Universiteit, Amsterdam.
REFERENCES Bollen, G.H.P.M., Mager, W.H., Jenneskens, L.W.and Plant~, R.J.: Small-size mRNAs oode for ribosomal proteins in yeast. Cur. J. Biochem. 105 (1980) 75-80. Cohen, S.N. and Chang, A.C.Y.: Recirc~larization and autonomous replication of a sheared R-factor DNA segment in Escherichia coli transformants. Proc. Natl. Acad. Sci. USA 70 (1973) 1293-1297. Dennis, P.P. and Nomura, M.: Regulation of the expression of ribosomal protein genes in E. coll. J. Mol. Biol. 99 (1975) 61-76". Fallon, A.H., Jinks, C.S., Strycharz, G.D. and Nomura, M.: Regulation of ribosomal protein synthesis in Escherichla coli by selective mRNA inactivation. Proc. Natl. Acad. Sci. USA 76 (1979) 3411-3415. Fill, N.P., Friesen, J.D., Downing, W.L. and Dennis, P.P.: Post-transcriptional regulatory mutants in a ribosomal protein-RNA polymense operon of E. coll. Cell 19 (1980) 837-844. Goldbach, R.W.: Structure and repfication of Tetrahymena pyriformis mitochondrial DNA. Ph.D. Thesis,Universiteit van Amsterdam, Amsterdam, 1978. Grunstein, H. and Hogness, D.S.: Colony hybridization: A method for the isolation of cloned DNAs that contain a specific gene. Proc. Nail. A~td. Sci. USA 72 (1975) 3961-3965. Howard, G.A., Smith, R.L. and Gordon, J.: Ribosomal proteins: simplification of the methods to prepare them for gel electrophoresis. Anal. Biochem. 67 (1975) 110-114. Jaskunas, S.R., Lindahl, L. and Davies, J., in Nomura, M., Tissi~es, A. and Lengyel, P. (Eds.), Ribosomes. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, 1974, pp. 333-368. Kessler, S.W;: Rapid isolation of antigens from cells with a staphyloccal protein A-antibody absorbent: parametersof the interactions of antibody antigen complexes'with protein A. J. Immunol. 117 (1976) 136-I42. Kaltschmidt, E. and Wittmann, H.G.: R~osomal proteins,
287 VII. Two-dimensional polyacrylamide gel eleetrophoresis for fingerprinting of ribosomal proteins. Anal. Biochem. 36 (1970) 401-412. Kmisw~k, T. and Pianta, R.J.: Analysis of the protein composition of yeast ribosomal subunits by two-dimensional polyacrylamide gel electrophoresis. Mol. Biol. Rep. 1 (1974) 409-415. Kruisw~k, T., Planta, R.J. and Mager, W.H.: Quantitative analysis of the protein composition of yeast r~osomes. Eur. J. Biochem. 83 (1978) 245-252. Laskey, R.A. and Mills, A.D.: Quantitative film detection of 3H and 14C in polyacwlamide gels by fluorography. Eur. J. Biochem. 56 (1975) 335-341. Maxam, A.M. and Gilbert, W.: A new method for sequencing DNA. Proc. Natl. Acad. Sci. USA 74 (1977) 560-564. Meyerink, J.H., Ret61, J., Planta, R.J. and He~dekamp, F.: Topographical analysis of yeast ribosomal DNA by cleavage with restriction endonucleases. Mol. Biol. Rep. 2 (1976) 393-400. Meyerink, J.H.: Orgat~ization of yeast ribosomal DNA. Ph.D. Thesis, Vrije Universiteit, Amsterdam, 1979. Meyuhas, O. and Perry, R.P.: Construction and identification of cDNA clones for mouse ribosomal proteins: applica-
tion for the study of r-protein gene expression. Gene 10
(1980) 113-129. Petes, T.D., Broach, J.R., Wensink, P.C., Hereford, L.M., Fink, G.R. and Botstein, D.: Isolation and analysis of recombinant DNA molecules containing yeast DNA. Gene 4 (1978) 37-49. Ret~l, J. and Planta, R.J.: Nuclear satellite DNAs of yeast. Biochim. Biophys. Acta 281 (1972) 299-309. Vaslet, Ch.A., O'ConneIl, P., Izguierdo, M. and Rosbash, M.: Isolation and mapping of a cloned ribosomal protein gene of Drosophila melanogaster. Nature 285 (1980) 674-676. Woolford, J.L. and Rosbash, M.: The use of R-looping for structural gene identification and mRNA purificat:.ou. Nucl. Acids Res. 6 (1979) 2483-2497. Woolford, J.L., Hereford, L.H. and Rosbash, M~: Isolation of cloned DNA sequences containing ribosomal protein genes from Saecharomyces cere~isiae. CeU 18 (1979) 1247-1259. Yates, J.L., Arfsten, A.E. and Nomura, M.: In vitro exp~eqsion of E. coli ribosomal protein genes: autogenous inhibition of translation. Proc. Natl. Acad. Sci. USA 77 (1980) 1837-1841. Communicated by A. van der Eb.
279
Isolation of cloned ribosomal protein genes from the yeast Saccbaromyces carlsbergensis (Recombinant DNA; small-size mRNA; R-looping; immunological screening; two-dimensional gels; colony bank)
Godfried H_P.M. BoHen, Louis H. Cohen, Willem H. Mager, Antonius W. Klaassen and Rudi J. Phnta
Bioehemiseh Laboratorium, Vri/e Universiteit, 1081 HV Amsterdam, (TheNetherlands) (Received February 12th, 1981) (Accepted March 31st, 1981)
SUMMARY A colony bank of yeast DNA obtained by cloning Hindlll.generated fragments of total yeast nuclear DNA in Escherichia coli K-12 with the vector pBR322, was screened with a radioactive RNA probe enriched for a subset of ribosomal protein ,,nRNAs. The selected recombinant DNA molecules were hybridized with poly(A)-containing mRNA under R-loop conditions. From the DNA-RNA hybrids the respective mRNAs were melted off and translated in vitro in a rabbit reticulocyte cell-free system. The translational products were analyzed by immunoprecipitation with antibodies raised against ribosomal proteins. The identity of the ribosomal protein geneproducts was further established by electrophoresis on two-dimen~ional gels. At least 15 recombinant DNA molecules were shown to contain ribosomal protein genes. Four of them, i.e. Y65, Y89, Y113 and Y138, have been characterized preliminarily.
INTRODUCTION Ribosome biosynthesis involves the simultaneous expression of a large number of ribosomal protein genes. In bacteria the genes cooing for ribosomal proteins are clustered and arranged in ~ number of common transcriptional units (Jaskunas et al., 1974). Evidence has been obtained that in bacteria regulation of the coordinate synthesis of the ribosomal proteins takes place not only on transcriptional level but also on translational level (Denn,s and Nomurz, 1975;
"Abbreviations:bp, base pairs; Md; megadalton;SDS, sodium dodecyl sulfate; SSC, 0.15 M NaCI, 0.015 M Na- citrate, pH 7.8; TEA, trichloro acetic acid; U, unit.
Fallon et al., 1979; Yates et al., 1980; Fill et al., 1980). In eukaryotes little is known about the genetic organization of the ribosomal protein genes nor about the way their expression is regulated coordinately. Recently, several authors reported the isolation of cloned eukaryotic DNA fragments containing a ribosomal protein gene as a first step towards a better understanding of the regulated expression of these genes (Woolford et al., 1979; Vaslet et al., 1980; Meyuhas and Perry, 1980). For the same purpose we had fractionated from the yeast Saccharomyces cadsbergensis a small-size mRNA fraction which is relatively enriched for a subset of ribosomal protein mRNAs (as ,tescribed by Bollen et al., 1980)and, thus, could be employed to detect ribosomal protein
0378-1119/81/0000-0000/$02.50 0 1981 Elsevier/North-HollandBiomedicalPress
280 genes in a yeast DNA bank. In this paper we describe the isolation and partial characterization of ribosomal protein genes from thi~ yeast strain.
MATERIALS AND METHODS
(a) Isolation of nucleic acids High Mr nuclear DNA was isolated essentially as described previously (Ret61 and Planta, 1972). Smallsize mIL'4A used as a probe in colony hybridization experiments was isolated from poly(A)-containing RNA by preparative sucrose gradient centrifugation (Boilen et al., 1980). (b) Cloning of HindlH.generated fragments of yeast DNA in E. coli The 31asmid pBR322 .~as linearized with Hindlll i:~ 30 mM Tris- HCI (pH 7.9), 12 mM NaCI and 10 mM MgCI2 by incubation during 2 h at 37°C. Subsequently~ the DNA was dephosphorylated with bacterial a~kaline phosphatase during 1 h at 37°C after the addition of 0.1 volume of 1 M Tris. HCI (pH 8.0) (Maxam and Gilbert, 1977). The reaction was termi. nated by extraction with phenol and chloroform and precipitation with ethanol. Yeast nuclear DNA was digested with HindIll as described above for pBR322, then heated to 65°C during 5 min and extracted with phenol and chloro. form. The DNA was precipitated with ethanol and dissolved in sterile redistilled water. After addition of "ligase buffer" the mixture was combined with the linearized, dephosphorylated pBR322 (in a 1 : 2 yeast DNA/pBR322 weight ratio) in a final volume of 50/d containing 66 mM Tris" HCI (pH 7.6), 6.6 mM MgC12, 0.1 mM ATP, 0.66 mM dithiotreitol and 1 mgfinl bovine serum albumin. An excess of T4 DNA ligase (Miles Lab.) was then added and the mixture was incubated at 4°C during 18 h (Meyerink, 1979). E. coli K-12 type 5K was transformed with the whole ligation mixture (Cohen and Chang, 1973). Transformants were selected for ampicillin resistance. As a control linearized and dephosphorylated pBR322 was ligated and transformed in E. coli 5K.
(c) Colony hybridization Both ribosomat RNAs and mRNAs to be used as hybridization probes were labelled in vitro by the following procedure. RNA was precipitated with ethanol and resuspended in freshly prepared 0.05 M Na-carbonate to a concentration of 300/g/ml and incubated at 50°C for 80 min. Under these conditions RNA was fragmented to pieces of about 200 nucleotides (Goldbach, 1978). The sample was neutralized by the addition of I vol. of 80 mM Tris-HCI (pH 7.6) and I vol. of 0.05 M HCI. After the addition of I voL of 20 mM MgCI2, 20 mM 2-mercaptoethanol the 5' ends of the RNA fragments were labelled with [~'-32P]ATP by 1"4 polynucleotide kinase (Boehringer). The mixture was incubated at 37°C for 30 rain. The reaction was stopped by the addition of SDS to a final concentration of 0.5% (w/v). The labelled RNA was purified by Sephadex G-50 gel filtration. The specific activity of the labelled RNA was approx. 2 - 7 X 10s cpm//zg RNA. The labelled RNAs were used for screening a colony bank containing Hindlll-generated fragments of yeast DNA. The whole bank was f'~.ed on Millipore HA filter grids (0A5 /an pores) each containing 103 colonies. The filters were prepared for hybridization essentially according to the method of Gmnstein and Hogness (1975). Each f'dter (diameter 11 cm) was moistened with 5 X SSC, 50% (v/v) formamide, containing about 10-20X 106 cpm (approx. 3 #g RNA)in a total volume of ~ ml. The solution was covered with mineral oil and the filters were incubated during 18 h at 42°C to allow hybridization. Then the filters were washed in 2XSSC, treated with pancreatic ribonuclease (20/zg/ml) and washed again. After drying, the filters were exposed dum~g 20-36 h to Kodak XRI fdm. (d) Isolation of recombinant DNAs The E. coli clones were grown overnight at 37°C in 200 ml Brain Heart Infusion medium (Oxoid), conraining 50 / ~ m l ampicillin. Cells were harvested by centrifugation for I0 rain at 3 000 X g at 4°C and washed with 25% (w/v) sucrose, 30 mM Tris. HCI (pH 8.0) at 4°C. They were resuspended in 1.3 ml of the same buffer. Then 0.5 ml c~ a lysozyme solution (Sigma; 8 mg/ml) was added and the suspension was incubated for 5 min at room temperature. The cells
281 were lysed by a second incubation after the addition of 0_3 ml 0.5 M EDTA (pH 8.0) and 3 ml "Brij-mix" (1% w/v Brij-58, 10 mM sodium deoxycholate, 60 mM EDTA, 50 mM Tris- HCI, pH 8.0) for 15 min at 20°C. Cell debris was removed by centdfugation for 30 min at 85 000 Xg at 4°C in a 50.2 Ti rotor (Beckman), 4.5 g of cesium chloride was added to the supematant as well as ethidium bromide up to a final concentration of 0.1 mg/ml. The volume of the final solution was adjusted to 6 ml with a solution containing 30 mM "Iris. HCI (pH 8.0), 0.5 mM EDTA and 50 mM NaCI (TES). Cesium chloride equilibrium centrifugation was performed during 20 h at 42 000 rev./ min at 4°C in a 50 Ti rotor (Beckman). The plasmid DNA, visible under UV light as a single band, was collected by means of a hypodermic needle and syringe from the side of the tube. Ethidium bromide was removed by extracting the solution at least three times with an equal volume of isoamyl alcohol, and finally the DNA solution was dialysed extensively against TES.
(e) Digestion of plasmid DNA with BamHl Purified plasmid DNA was linearized by digestion with the restriction endonuclease BamH! (Bethesda Research Laboratories Inc.). Incubation was performed for 2 h at 37°C in the presence of BamHl (1.5 U//zg DNA), 6 mM MgCI~, I mM dithiotreitol, 150 mM NaCI and 6 mM Tris. HCI (pH 7.9). The enzyme was removed by phenol extraction and the linearized DNA was collected by ethanol precipitation.
(f) R-loop hybridization and hybrid purification A pool of 3 or 4 different plasmids, 3/ag each, was hybridized to 15-18/ag of yeast poly(A)-containing RNA under R-loop conditions, and the DNA/RNA hybrids were purified from the unhybridized RNA by gel f'dtration on a Biogel A150m column (Biorad i~aboratories) as described by Woolford and Rosbash (1979). The hybrid-containing fraction (excluded volume; 3 ml) was ethanol-precipitated after the addition of 13 /ag of Bacillus Iicheniformis ribosomal RNA. The precipitated nucleic acids were washed with 70% (v/v) ethanol and dissolved in 6/,d sterile redistilled water.
(g) In vitro translation of hybridized mRNA mRNA, hybridized to the plasmid DNA, was released from the hybrid by incubating the 6/1! hybrid solution for 15 s at 100°C. The final translation mixture was obtained by adding 24/d rabbit reticulocyte lysate (Amersham) and 3/.d of L-[3SS]methionine (Amersham, 5/zCi//zl, spec. act.. >1000 Ci/mmol). Translation took place during 2 h at 30°C. (h) Gel electrophoresis of proteins synthesized in
vitro Analysis of the translation products was performed by electrophoresis in 10-15% polyacrylamide gradient gels in the presence of SDS as previously described (Bollen et al., 1980). Ribosomal proteins were identified by two-dimensional gel electrophoresis as described by Kaltschmidt and Wittmann (1970) with the following modification. Proteins synthesized in vitro (about 10/d) were precipitated with 100 pJ 20% (w/v) TCA and subsequently washed with 5% (w/v) TCA, acetone and ether (Howard et al., "1975). The precipitate was redissolved in 6 M urea, 1 mM dithiotreitol and 25% (w/v) sucrose. The protein sample was neutralized with 1 M unbuffered Tris and applied on top of the first dimension gel along with 0A mg unlabelled ribosomal proteins. The time of electrophoresis (23 h at 180 V) in the first dimension was doubled (see Kruiswijk and Planta, 1974). (i) Staining and autoradiography Two-dimensional gels were stained in a solution containing 0.2% (w/v) Coomassie Brilliant Blue R250, 45% (v/v) methanol and 9% (v/v) acetic acid during 2.5 h, destained in 7% (v/v) acetic acid and prepared for fluorography according to the method of Laskey and Mills (1975). SDS.contalning polyacrylamide gels were not stained, but f'Lxedovernight in a solution containing 25% (v/v) methanol and 9% (v/v) acetic acid.
(j) Preparation of antisera against yeast ribosomal proteins Ribosomal proteins, isolated and purified as described by Kruiswijk and Planta (1974) were fractionated by chromatography on a phosphocellulose
282 column. About 25 fractions were obtained, each contair=ing 2 - 6 ribosomal protein species. All fractions were injecte~ into rabbits separately. This p.-ocedure of p~eparing yeast n'bosomal protein fractiens as well as the characterization of antisera raised against them wflI be descn'bed in detail elsewhere (Bollen, G.H.P.M., Mager, W.H. and Planta, R.I., manuscript in preparation).
(k) lmmunoprecipitation of ribosomal proteins synthesized in vitro lmmunoprecipitation of the in vitro translation products was performed according to a modification of the procedure described by Kessler (1976). 5 gl of the transla~on mixture was preincubated in the presence of 100 gl of a 5% (v/v) Staphylococcus aureus suspension in immunoprecipitation buffer (1% (v/v) Triton X-100, 05% (w/v) sodium-deoxycholate, 0.1% SDS, 150 mM NaCI and 20 mM Na-phosphate pH 7.2). After incubation for 30 rain the sample was centrifuged for 1 rain at 12 000 × g. The supernatant was mixed with 5 gl of a mixture of 13 sera raised against different ribosomal protein fractions as described ~bove, and incubated for 18 h at 4°C. Subsequently 50/,d of a 10% (v/v) Staphylococcus aureus suspension in immunopre¢ipitation buffer was added and incul:ation was continued for 30 min. The Staphylococcus aureus immune complex was col. lected by eentrifugation for 1 rain at 12000Xg, resuspended in immunoprecipitation buffer and centrifuged through a layer of 30% (w/v) sucrose in the same buffer for 2 min at 12 000 Xg. The pellet was washed twice with immunoprecipitation buffer, once vAth this buffer without detergents and then resusp~nded in 25 bd of SD$ sample b:fffer (Bollen et al., 1980). The immune complex was dissociated by hea!ing for 3 rain at 100°C and Staphylococcus aureus was removed by centrifugation "for 10 rain at 12 000 X ~ at 200C. The labelled proteins in the supernatant were analysed by electrophoresis on SDS polyacrylami~e slab gels as described above.
RESULTS AND DISCUSSION (a) Isolation a~d screening of recombinant DNA molecules containing yeast DNA In order to obtain a yeast DNA bank total yeast nuclear DNA, digested with Hindlll, was cloned in E. coli K-12 5K after recombination with the plasmid vector pBR322 (see MATERIALS AND METHODS). We isolated about 10000 ampicillin. resistant transformants. Assuming that the haploid yeast genome contains 101° daltons of DNA and that the Hindlll-generated DNA fragments have an average size of about 2.5 Md it can be calculated that the probability that this bank contains all sequences present in the yeast genome is about 80% (Petes et al., 1978). Colony hybridization experiments were performed using a small-size poly(A)~eontaining mRNA fraction isolated from S. carlsbergensis as a probe (Bollen et al., 1980). Recently, we have demonstrated that this mRNA fraction is enriched in mRNAs coding for at least 30 ribosomal proteins; the concentration of the individual ribosomal protein mRNAs was estimated to be about 1.5% of the mRNA sequences present in this probe (Bollen et al., 1980). This extent of enrichment might be sufficient to give a reproducible signal in the colony hybridization test according to Gmn. stein and Hogness (1975). Since the small.size mRNA fraction always is contaminated with ribosomal RNA we first hybridized the colonies to purified ribosomal RNA labelled in vitro as described in MATERIALS AND METHODS. In 5 000 clones examined we found 160 recombinants (=3.2%)hybridizing with 17S rRNA and another 190 hybridizing with 5S rRNA. These groups of clones contain recombinant DNAs carrying the 4.1 X 106 and 1.7 X l0 s fragment of the ribosomal repeat, respectively (Meyerink et al., 1976). This frequency is close to the predicted proportion of ribosomal DNA-containing clones. By hybridization with the small-size mRNA fraction 350 clones (i.e. 7%) out of 5 000 clones could be selected. The hybridization response could be divided into two classes: for 47 clones the hybridization was stronger than for the remaining 300 clones. The yield of 7% of clones containing DNA complementary to yeast mRNA is in agreement with the findings of Woolford et al. (1979) and suggests, as also stated by Petes et al. (1978), that only abundant or moderately abun-
283 dant mRNAs hybridize under the conditions used. This probably also explains why two distinguishable hybridization signals were observed in the colony hybridization experiments.
(b) Characterization of the selected recombinants In order to characterize the recombinant DNAs present in the clones selected as described above, we isolated the plasmid DNAs and hybridized them under R-loop conditions with total poly(A)-containing mRNA according to the procedure designed by Woolford and Rosbash (1979) (see MATERIALS AND METHODS). Subsequently, the products encoded by the recombinant DNAs were analyzed by translating the complementary mRNAs in vitro. First, the recombinant DNA molecules were linearized with the restriction endonuclease BamHl which cuts pBR322 DNA once. Most of the plasmids investigated appeared to have no additional BamHl-site. Those plasmids which were digested into fragments smaller than 3 Md were omitted. In a first test a pool of 3 or 4 linearized recombinant DNA molecules, 3/.tg each, were hybridized to 15-18 gg poly(A)-containing RNA. The R.loop containing molecules (and the double.stranded DNAs) were separated from unhybridized mRNA by gel filtration on a Biogel Al50m column. From the excluded volume DNA was precipitated with ethanol. The precipitate was dissolved in 6 ~d sterile redistilled water and then the RNA was melted from the hybrid by incubation for 15 s at 100°C. The complementary mRNAs were translated in a rabbit reticulocyte cellfree system in the presence of [3SS]methionine. The proteins synthesized in vitro were analyzed by three different methods, viz. (i) SDS-polyacrylamide gel electrophoresis, (ii) immunoprecipitation and (iii) 2D.gel dectrophoresis. (i) SDS-polyacrylamide gel electrophoresis. The aim of this type of electrophoretie analysis was to prove both the selectivity of the procedure used and the persistence of proper translational capacity of the complementary mRNAs during the procedure. A typical gel is shown in Fig. IA. Obviously, the use of different combinations of reccxnbinant DNAs gives rise to the synthesis in vitro ot sets of distinct polypeptides. The Mr of most of the proteins synthesized in vitro is < 50 000, as could be expected since the clones were selected upon hybridization with small-
size mRNA. Under the conditions used nearly all mRNAs are hybridized to their complementary DNA sequences. Still it is difficult to draw any conclusions as to the abundancy of the isolated mRNAs from the different intensities of the protein bands observed in the autoradiogram, since both the content of methionine in these polypeptides and the translational capacity in vitro of the individual mRNAs are unknown. fii) Immunoprecipitation with anti-ribosomal protein serum. Subsequently we looked for the presence of ribosomal proteins among the translation products. In a first approach we made use of antisera raised against a set of 13 different ribosomal protein fractions isolated by chromatography on phosphocellulose columns of total ribosomal protein. We incubateC the translation products in ~¢ presence of the combined sera, then ,he antigen-antibody complexes were recovered by absorption to fLxed Staphylococcus aureus znd subsequentJy analyzed on the same type of SDS-containing polyacrylamide gradient gels as described above. A typicaJ result in presented in Fig. 1B. The lanes indicated in Fig. 1B correspond to those, indicated in Fig. 1A. Lane 12 shows the ribosomal proteins that are precipitated by the pooled antisera from the translation products when the cell-free system is programmed with total poly(A)-containing mRNA. From the protein bands visible in the other lanes of Fig. 1B it can be concluded that apparently several ribosomal proteins are encoded by at least one of the recombinant DNAs present in 5 out of 11 of the plasmid mixtures. In the example presented in this figure at least six ribosomal protein recombinants are present among the 43 plasmid DNAs investigated. In the same way additional sets of recombinant DNA molecules appeared to contain at least another 9 plasmids carrying genes coding for a ribosomal protein. The frequency of 15 ribosomal protein gene-contaiaing plasmids out of about 140 candidate-recombinants investigated so far most likely is too low because the antisera used are raised against only a subset of ribosomal proteins. Moreover, only methionine-containing proteins could be detected. The hybridization procedure was repeated for a number of individual recombinant DNA molecules. Analysis of the immunoprecipitated proteins synthesized in vitro on mRNAs derived from individual RNA/DNA duplexes again was performed by electro-
284
Fig. 1. E[ectrop~oresls on SDS-polyacrylamkle gradient gels of proteins synthesized in vitro on mRNAs complementary to several recombinant DI~'As. (A) Combinations of 3-5 recombinant DNAs were hybridized with poly(A)-containing mRNA under R-loop conditions (see MATERIALS AND METHODS). After melting off the complementary mRNAs, trarslation in vitro was performed in a reticelocyte cell-free system in the presence of [3SSlmethionine. The proteins synthesized in vitro were analyzed on 10-15% polyacrylamide gradient gels containing SDS and the labelled bands were made visible by autoradiography. Lanes 1-11 show the translation products encoded by the various r~ombinant DNA molecules. Lane 12 shows the pro~lucts obtained by translatien in vitro of total pc~ly(A)-containing mRNA. The bands which are in common in each lane represent products synthesized by endogenous mRNA. The gel was exposed for autoradiography during 2 days. (B) Proteins were synthesized in vitro and precipitated with "antisera raised tgainst r~bosomal proteins. Antisera raised against a number of ribosomal protein fractions were obtained as described in MATERIALS AND METHODS. The same translation products as analyzed in panel A were incubated in the presence of the combined ar~tisera (see MATERIALS AND METHODS). The immunoprecipitated proteinswere analyzed by electrophoresis on the same type of gels as shown in panel A. The following mixtures of recombinant DNAs gave rise to immunoprecipitated polypeptides: Y110, Yl 11, Y112, Yl 13 (lane 3), Y118, Y119, YI20, YI21 (lane 5), Y133, Y135, Y137 (lane 8), Y148, Y149, YIS0 (lane 9) and Y155, YI56, Y157 (lane l l). Lane 12 shows the analysis of immunoprecipitated ribosomal proteins synthesized in vitro o ~ total poiy(A)-containing mRNA. The gel was exposed for autoradiography during 14 days.
phoresis in SDS gels. The result is shov, n in Fig. 2. in lanes 2 , 3 , 4 and 5 the bands are v~lble correspondinf~ to the immunoprecipitated translation products o / the m R N A s hybridized to the plasmids isolated from the clones Y65, Y138, Y89 and Y113, respectively. The recombinant DNA from clone Y65 codes for at least one and possibly two ribosomal proteins. The major band corresponds to a ribosomal protein with an Mr o f 15 000; the minor with a polypeptide with a n M r o f 12 000. Clones Y138, Y89 and Y113 each code for
Fig. 2. Electrophoresis on SDS-polyacrylamide gradient gels of immunoprecipitated proteins encoded by individual recombinant DNAs. The gone products of clone Y65.. Y 138, Y89 and Y113 were analyzed individually in a similar way as described in the legend to Fig. lB. Lane 1: immunoprecipitated n'bosomal proteins synthesized in vitro on total poly(A)-containing mRNA. Lane 2, 3, 4 and 5: immunoprecipitated proteins encoded by recombinant DNAs Y65, Y138, Y89 and YII3, respectively. The gel was exposed for autoradiography during 14 days.
285 one ribosomal protein with Mrs o f 17000, 2£~000 and 30 000, respectively. fill) 2D-gel electrophoresis. Finally, the identity of the ribosomal proteins synthesized in vitro on the mRNAs hybridized to the selected recombinants was established on two-dimensional gels according to Kaltschrnidt and Wittmann (1970). In comparison
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with the m e t h o d published earlier (Kruiswijk and Hanta, 1974) the time of electrophoresis in the first dimension was doubled. As a consequence of this modification nearly all basic ribosomal proteins are resolved as can be seen in Fig. 3B (ef. the schematic representation in Fig. 3A). Figs. 3C, D, E and F show the two-dimension~l
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Fig. 3. Analysis of proteins encoded by individual recombinant DNAs on two-dimensional gels. Electrophoresi~ was performed as described previously (KmiswUk and Planta, 1974), except that the time of electrophoresis in the first dimension was doubled. (A) Schematic representation of the marker prof'fle; (B) analysis of total 80S ribosomal proteins, stained with Coomassie Brilliant Blue; C, D, E and F: TCA-precipitated proteins synthesized in vitro on the complementary mRNAs of recombinant DNAs, Y65, Y89, Y113 and Y138, respectively. On each gel 0.4 mg ribosomal proteins were added as markers. After staining the gels were exposed for autoradiography during two weeks.
2~ electropherograr3s of the proteins encoded by the cloned DNAs Y65, Y89, Y I I 3 and Y138. respecti~ely, Y65 appears to code for at least one [3sS]n~ethionine4abe~led protein, tha~ comigrates with the n~bosomal marker protein $31 in the same gel (Fig. 3C). The minor protein spot in Fig. 3C may represent $33 although its identity is not established with certainty since it does not comigrate exactly with $33. A possible explanation for this phenom~on is that the in vitro translation system is unable ~o c a m / o u t posttranslation~ modification steps which may occur in vivo. Another possibility is that the "minor" polypeptide is a pr~nature termination product of the synthesis in vitlo of the "major" ribosomal protein. Experiments are under investigation to discriminate between these two possibilities. Gone Y89 contains sequences that code for ribosomal protein L16 (Fig. 3D)whereas the cloned DNA fragment YI13 apparently codes for ribosomal pro°tein L6/SIO (Fi~. 3E). The two-dimensional electropherogram of the proteins encoded by clone Y138 reveals two spots: one major, comigra~ing with ribosomal protein L25 and one minor which probably is identical to protein L27/$21. By electrophoresis on SDS gels of the immunoprecipb ated proteins encoded by clone Y138 only one, rather weak, band was observed (Fig. 2). However, proteins L25 and $21 have almost equal Mrs (Kruiswijk ~t al., 1978). lr~ addition -o the sequences coding for ribosomal proteins some of the recombinant DNAs identified solar probably carry other, non-ribosomal protein genes, as suggested by analysis on SDS gels similar to that presented m Fig. 1A (not sho~wn). The length of the inserted Hindlll-generated fragments in the plasmids Y65, Y89, Y113 and Y138 as analyzed by electrophoresis on 1% agarose gels is 5090, 5870, 5080 and 3020 bp:, respectively (result not shown). This relatively smal~ size of the inserted yeast DNA sequences suggests that the genes are rather proximate to each other. Physical and genetic maps are currently in preparation. R-loop analysis under the electron microscope revealed that most genes identified so far on cloned yeast DNA fragments are intact (BoUen, G.H.P.M., Molenaar, C.M.T~,, Cohen, L.H., Mager, W.H. and Planta, R.J., manuscript in preparation).
ACKNOWLEDGEMENTS This work was supported in part by the Netherlands Foundation for Chemical Research (SON)with financial aid from the Netherlands Organization for the Advancement of Pure Research (ZWO). We thank Mrs. M.L. Goei-Soen, Mrs. M.M.C. Duin and Mr. R.D. Fontijn for skilful technical assistance during part of this work. In addition the authors are indebted to Dr. J. Biewenga who enabled us to prepare antisera in the Histologisch Laboratorium, Vrije Universiteit, Amsterdam.
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