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Heterogeneity of Zea mays protein body messenger RNA
Plant Science Letters, 18 (1980) 133--141
133
© Elsevier/North-HollandScientificPubli~Qn Ltd.
HETEROGENEITY OF ZEA MA YS PROTEIN BODY MESSENGER R N A
ULRICHMELCHER Department of Biochemistry, Oklahoma State University, Stiilwater, OK 74074 (U.S.A.)
(ReceivedJune 28th, 1979) (Revhdonreceivedand acceptedJanuary 2rid, 1980) SUMMARY To investigate whether the heterogeneity of zein polypeptides found in protein bodies of corn endosperm is reflected in a ~ heterogeneity of the protein body mRNA, a complementary DNA copy of the polyadenylic acid containing (poly(A)+)RNA of protein bodies was prepared using reverse transeriptase. This radioactive DNA hybridized as two distinct kinetic components, differing 70-fold in complexity, to an excess of the poly(A)+ RNA. By comparison with the hybridization profiles of complementary DNA driven by total protein body RNA and by RNA lacking polyadenylic acid (poly(A)-) it was determined that the more slowly hybridizing component was present in both poly(A)+ and poly(A)-RNA, while the faster hybridizing component was almost exclusively restricted to the poly(A)+ RNA. INTRODUCTION Protein bodies in the starchy endosperm of cereal seeds are the intracellular site of accumulation of a special group of proteins [1] and are formed during endosperm development from the rough endoplasmic reticulum (ER) [2]. They have a protein-containing core, derived from the ER lumen, a surrounding membrane, and polyribosomes attached to the c y t ~ plesmic face of the membrane. The polyribosomes of these protein bodies are responsible for the synthesis of the proteins which occur inside of the protein bodies [3,4]. The proteins sequestered within these bodies are a collection of numerous similar polypeptides [5] and are soluble in aqueous alcohol, a property which identifies them as members of the prolamine fraction of cereal proteins [6]. The prolamine fraction of corn, zein, is heterotenous as demonstrafed by separations obtained by gel filtration [7], hydroxylapetite chromatography [8], dodecyl sulfate polyacrylamide gel electrophoresis [9], and isoeiectric focusing [5]. The fractions obtained differ structtmflly
134 from one another as shown by amino acid analysis [8,10], peptide mal~ ping [8], and chemical modification [11]. Despite these differences, the polypeptides of the zein fraction are very similar to one another. Besides a solubility in alcohol, these polypeptides share several other properties. Most of the polypeptides have a molecular weight between 19 000 and 23 000 [9]. They have very similar amino acid compositions when separated either by isoelectric focusing or by hydroxylapatite chromatography [8,10]. A c o m m o n set of peptides produced by digestion with proteolytic enzymes account for over half of the peptides produced from every subfraction of zein examined [8]. A similar situation is found in the wheat prolamines, gliadins, where separable polypeptides have nearly identical amino acid sequences [ 12]. Since the amino acid sequences~or these polypeptides are coded for by mRNA molecules, one would predict that the mRNA population responsible for the synthesis of zein is also composed of a series of similar but not identical molecules. One method of testing this prediction is to study the hybridization kinetics of mRNA from protein bodies with complementary DNA molecules obtained by reverse transcription using RNA dependent DNA polymerase. Only a preliminary report of such a study has been pre~ sented [13]. I wish to report the results of hybridization experiments which suggest that protein body mRNA consists of two fractions which differ in their complexity and distribution among poly(A)+ and p o l y ( A ) RNA fractions. MATERIALS AND METHODS The growth and harvesting of corn 20--25 days after silk emergence was previously described [14]. A protein body-enriched fraction was isolated from a homogenate of kernels according to Larkins et al. [3]. This fraction was used as the starting material for isolation of poly(A)+ RNA, p o l y ( A ) RNA and total RNA. For the isolation of poly(A)+RNA and poly(A)-RNA, the polyribosomes were dissociated from the protein bodies with Triton X-100 and pelleted by centrifugation through buffered 1.75 M sucrose [3]. The polyribosomal pellet was suspended and applied to oligo (dT) cellulose as previously described [14]. The u n b o u n d fraction was extracted twice with an equal volume of chloroform/isoamyl alcohol (24 : 1) for 15 min at r o o m temperature. RNA in the aqueous phase was precipitated with 3 vols. of ethanol and recovered by centrifugation as poly(A)-- RNA. The material eluting from the oligo(dT)cellulose with 10 mM Tris-HC1 (pH 7.5) was heated briefly to 65°C. The sample was adjusted to 0.5 M NaC1 and reapplied to the oligo(dT)cellulose column. The procedure was repeated for a total of three cycles of binding and elution. The final eluate was adjusted to 0.3 M NaC1 and the RNA precipitated with 2 vols. of ethanol. The RNA recovered after centrifugation is designated poly(A)+ RNA. For the isolation o f total RNA, the protein body enriched pellet was stmpended
135
in 1.0 ml/g of kernels of 0.1 M Tris--HCl, I mM Na2EDTA, 1% SDS, 0.1 M NaCI, (pH 8.0) and extracted for 30 rain at room temperattwe with an equal volume of a I : 1 mixture of phenol and chloroform. The sample was centrifuged at 10 000 g for 10 min and the resulting aqueous phase tranafe~ed to a tube containing an equal volume of chloroform/isoamyl alcohol (24 : 1). The organic phase and interphase were extracted with a half volume of 0.1 M Tris-HC1, 1 mM Na2EDTA, 1% SDS (pH 9.0) for 30 rain at r o o m temperature. After centrifugation to separate phases, the aqueous phase was removed and added to the first aqueous phase. These were mixed with chloroform : isoamyl alcohol for 15 rain at room temperature. To the final aqueous phase obtained after centrifugation 2 vols. of ethanol were added. After storage overnight at - 2 0 ° C the precipitated RNA was r ~ covered by centrifugation, dried and used in the hybridization experiments.
cDNA synthesis DNA complementary to poly(A)+ RNA was synthesized in 50 raM Tfis-HC1~(pH 8.3), 60 mM NaCI, 6 mM MgCI2, 20 mM dithiothreitol, 0.8 mM each of dTTP, dATP, dGTP, 2.6 #M [SM]dCTP. (22.9 Ci/nmol, New England Nuclear~ 0.1 mg/ml actin0mycin D, 40/~g/ml oligo(dTl0) (P-L Biochemicals), 40/~g/ml poly(A)+RNA by addition of 120 Units avian myeloblastosis vires reverse transcriptase (obtained through J.W. Beard, Life Sciences, Inc.) to a 25 #1 reaction mixture. After a 60 rain incubation at 37°C, 100/~1 of 10 mM Na2EDTA were added to stop the reaction. Then 0.3 ml 0.1 M Tris--HC1 (pH 9.0) and 50/~g yeast tRNA were added. The solution was made 0.5% in SDS and extracted with an equal volume of phenol for 15 rain. After centrifugation to separate phases, the aqueous phase was recovered and 3 vols. o f ethanol added. The precipitate that formed at - 2 0 ° C overnight was collected by centrifugation and redissolved in 1.0 ml 0.3 N NaOH. It was heated to 100°C for 15 min, chilled and neutmUzed with 30 #1 N HC1. The solution was made 10 mM MgC12, 10 mM Tris--HC1 (pH 7) and 10 ~g yeast tRNA were added. After ethanol precipitation the nucleic acid was taken up in 0.3 M sodium acetate and reprecipitated with ethanol. The final precipitate was dissolved in water and stored at - 70°C. The size of the resulting cDNA was estimated by electrophoreais of the cDNA in a 1.4% agarose gel containing 0.03 N NaOH, 1 mM Na=EDTA. Denatured HaeIII restriction fragments o f [SH]~X 174 DNA RF (Bethesda Research Laboratories) served as size standards. DNA fragments in the gel were transferred to a nitrocellulose sheet [ t 5 ] and the sheet dipped in 5% PPO in toluene prior to fluorography at - 7 0 ° C using flash-activated X-ray film [16]. The resulting fluorograph was scanned with a deusitometer to determine the distribution of cDNA molecules. The size ranged from 300 nucleotides to 1300 nucleotides with a weight average of 600 nucleotides. cDNA.RNA hybridization RNA-DNA hybridization was carried o u t in small plastic vials in 0.3 M
136 100
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NaC1, 20 mM Tric-HC1 (pH 7.4) 1 mM EDTA, 1.0% SDS covered with paraffin oil to minimize evaporation. RNA was present in at least a 1000fold excess over DNA. Incubation was at 68°C. At various times aliquots were removed and diluted into $1 nuclease b~Lffer (30 mM sodium acetate, 3 mM ZnC12, 0.3 M NaC1, and 10/~g/ml wheat DNA (extracted from wheat germ by the procedure of Bendich and McCarthy [17]). One-half of the diluted sample was set aside for analysis of total radioactivity while the other half was digested with 10 units of $1 nuclease (P-L Biochemicals). In early experiments (Fig. 1), radioactivity was analyzed by trichloracetic acid precipitation after the addition of carrier protein and collection of the 100
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precipitates on glass fiber filters. Later experiments (Figs. 2 and 3) were analyzed by the DE.81 filter disk method of Maxwell et aL [18]. Data obtained with the latter procedure had lower background levels of hybrkiization and Considerably less scatter in the data points and therefore fewer determinations were made. The data were fit to theoretical RNA driven RNA-DNA hybridization equations using the computer program of Pearson et aL [19]. Equivalent Cro values were calculated using a correction factor for salt concentration [20]. A [3H]cDNA copy of globin mRNA (Miles Laboratories) was made as described above and used in hybridization with globin mRNA to provide a kinetic standard. RESULTS
moly(A)+RNA The poI¥(A)+RNA isolated from polyribosomes released from protein bodies of ~eveloping corn endosperm, when used as a template for in vitro protein synthesis, coded for the synthesis of precursom to zein polypeptides [14]. As judged by gel electrophoresis [14] and ethanol extraction (Fraij and Melcher, unpublished) little or no non-zein polypeptides were synthesized. This RNA was used as a template for the synthesis of eDNA using reverse tmnscriptase. The radioactive cDNA was use~ unfmctionated in hybridization reactions using the template RNA to ~ v e hybridization at 68°C in 0.3 M NaCL A t t h e highest £Crot tried (Fig. 2) 85--90% hybridization was obtained. The hybridization values could be fitted best ammaning two components of hybridization. The first accounts for 82% of the total cDNA radioactivity while the second accounts for 49% of the total. The
138 TABLE I CALCULATED PARAMETERS FOR HYBRIDIZATION OF RNA TO [3H]cDNA RNA
Component
Fraction
poly(A)+
A B
0.324 0.489
total
A B
0.324 b 0.489 b
1.74 0.092
51.1 21.0
poly(A)--
B
0.489 b
0.070
27.6
globin c
0.82
K
Dilution a
88.7 1.93
143.2
---
--
aobtained by dividing K for the component in poly(A)+ RNA by K. b Values fixed for computation. C W i t h [ 3H]globin cDNA. first c o m p o n e n t hybridized with a kinetic complexity only 1.6 times that o f globin m R N A - - c D N A hybridization indicative o f the presence of only 3--5 sequences o f t h e size of zein m R N A . The second hybridized with a kinetic constant approx. 50 times smaller than that of t h e first c o m p o n e n t and must thus represent a substantial n u m b e r o f different sequences.
Total R N A Total R N A extracted from protein bodies using chloroform/phenol to minimize t h e loss o f poly(A)+ sequences was also subjected t o hybridization with t h e same cDNA. Figure 2 shows that approximately the same extent o f m a x i m u m hybridization was achieved. The t w o kinetic components are n o t as distinct as t h e y were in Fig. 1. Nevertheless the data were fit best b y a t w o c o m p o n e n t solution (goodness o f fit 0.0223 as opposed as to 0.0398 for one component) with the proportion of the cDNA responsible for each c o m p o n e n t fixed at the values deduced from the data in Fig. 2 (Table I). The acceleration in hybridization o f the slow relative to the fast c o m p o n e n t must be due to either the selective loss of the slow c o m p o n e n t during ollgo(dT)cellulose chromatography or the selective loss o f t h e rapidly hybridizing c o m p o n e n t during chloroform extraction. A calculation of t h e a m o u n t b y which each c o m p o n e n t o f Fig. 2 has been diluted b y other R N A molecules reveals that t h e rapidly h y b r i d i z ~ g comp o n e n t has been diluted approx. 50-fold while the slow c o m p o n e n t is only diluted approx. 20-fold (Table I). Since the major R N A that dilutes these samples is r R N A and since t h e r R N A accounts for 98--99% of the total R N A in polyribosomes, a dilution factor o f 50 is expected. This indicates that there was no selective loss o f t h e rapidly-hybridizing c o m p o n e n t during chloroform extraction and raises the possibility that the slowly hybridizing c o m p o n e n t is selectively lost during oligo(dT)cellulose chromatography.
139
PoZy(A )- PJVA Since p o l y ( A ) - molecules would be selectively lost during oligo dT cellulose chromatography, I sought to test whether p o l y ( A ) - molecules contained the sequences complementary to the cDNA w/aich hybridized with slow kinetics to the poly(A)+ RNA. The effluent from the application of RNA to the oligo dT cellulose column was recovered and extracted with chloroform to remove contaminating proteins. This RNA was allowed to hybridize with the cDNA probe. As can be seen in Fig. 3 the extent of maximum hybridization was less than seen with the POly(A)+RNA, indicating that some sequences are present in only the polyadenylated form. The hybridization points are best fit assuming two components. Assigning the first c o m p o n e n t to the fraction of cDNA hybridizing to total RNA with slow kinetics (0.489) resulted in a better fit (goodness of fit 0.0257) than when this fraction was assigned the value of 0.3j24 (goodness of fit 0.0345), that of the faster hybridizing c o m p o n e n t of Fig. 1. The result suggests then that POly(A)-RNA was enriched for the sequences corresponding to the slower hybridizing c o m p o n e n t of Fig. 1. The slower c o m p o n e n t in the p o l y ( A ) - R N A experiment must then be due to some of the sequences contained within the rapid c o m p o n e n t of poly(A)÷ RNA. Since complete hybridization of this component was n o t obtained even at high ECrotvalues, less than 1% of these RNA sequences in total RNA are present in p o l y ( A ) RNA. To confirm these results, poly(A)- molecules were prepared in a second way. Total RNA was extracted with phenol at pH 7, a procedure which results in the selective loss of poly(A)+~ontaining molecules [21]. When this RNA was hybridized to the cDNA probe, a maximum extent of hybridization of only 50--60% was routinely observed. The hybridization points could be fit better with two components than with a single-component. Assigning a value of 0.49 to the fraction of the cDNA hybridizing first results in a good fit to the data, consistent with the hybridization of p o l y ( A ) - R N A prepared by oligo(dT)cellulose chromatography. That the extraction was specific for the extraction of only p o l y ( A ) - molecules was shown by passing the extracted RNA over oligo(dT)cellulose and collecting the material that did n o t bind. This RNA hybridized to the cDNA probe with the same kinetics as the RNA before passing through oligo(dT)cellulose. DISCUSSION The observation of t w o distinguishable hybridization components in maize protein b o d y RNA, one of which also has sequences present in nonpolyadenylated RNA permit two different interpretations. First, the RNA present in the corn sample could be predominantly zein n~RNA, a fraction of which has been randomly fragmented. Zein m R N A could contain two regions of sequence, one region which is c o m m o n to the RNAs for all the zein IEF vza~mts and t h e other being variable from one mRNA to the other. Such a hypothesis is consistent with the current concept of zein
140 protein structure. If this assumption is true, one can make several calculations on the length and complexity of the two regions. From the relative dilutions of the two kinds o f RNA it can be calculated that 4(~o of the slow c o m p o n e n t sequences are present in the poly(A)+ fraction and 60% in the poly(A)-- fraction. Correcting for the resulting overrepresentation of the rapid c o m p o n e n t in t h e poly(A)+ fraction, and using the fraction of the cDNA nucleotides that are complementary to each component, it can be calculated that the rapid c o m p o n e n t would account for 21% of the length of t h e message while the slow c o m p o n e n t would account for the remainder. The expected ratio of complexity on the basis of sequence length alone would thus be 3.8. The actual difference in complexity measured with poly(A)+ RNA was 70-fold. Thus there would be approx. 18 different slow c o m p o n e n t sequences for each rapid c o m p o n e n t sequence. It is perhaps fortutious that this corresponds roughly to the number of zein IEF variants [15]. The second possible interpretation of the data is that the rapid RNA c o m p o n e n t consists of m R N A for the two major distinguishable size classes of zein polypeptide, zein A and zein B. The slowly hybridizing c o m p o n e n t would represent mRNAs for the synthesis of the minor zein polypeptides, for membrane proteins, and other proteins that may be made on the protein bodies. Although t h e synthesis of precursors to minor zein polypeptides can be detected when this RNA is translated in vitro, the synthesis of other non-zein polypeptides has n o t been detected. This may of course be due to the small a m o u n t of each polypeptide expected. Distinction between these two possible explanations awaits further experimentation. These results differ from the results of Pedersen et al. [13] who used a population of poly(A)+RNA molecules sedimenting at 138 in a sucrose gradient as a template for the synthesis of cDNA. They observed a single hybridizationcomponent. Possible minor mRNA molecules n o t related to the major zein polypeptides and possible fragments of the major m R N A species would n o t be present in their preparation. This difference can account for the discrepancies between the results of Pederson et al. [13] and the present ones. ACKNOWLEDGEMENTS Dr. Dale Weibel (Okla. St. Univ.) and Mr. R a y m o n d Peck (Panhandle St. Univ.) are thanked for the samples of com. The technical assistance of Mr. Charles R e d m o n d and Mrs. Elizabeth Hood was greatly appreciated. Financial support was provided by the National Science Foundation (Grant PCM 76-01699) and the Oklahoma Agricultural Experiment Station of which this is Journal Article No. J. 3662. REFERENCES 1 D.D. Chrlstianson, H.C. Nielsen, U. Khoo, M.J. Wolf and J.S. Wall, Cereal Chen~, 46 (1969) 372.
141
2 3 4 5 6 7 8 9 10 11 19. 13 14 15 16 17 18 19 20 21
U. Khoo and M.J. Wolf, Am. J. Bot., 57 (1970) 1049.. B.A~ Larkins, R.A. Jones and C.Y. Tsal, Biochemistry, 15 (1976) 5506. B. Burr and F.A. Burr, Proc. Natl. Acad. Sci. U.S.A., 73 (1976) 515~ E. Glanazza, P.G. Righetti, R. Pioli, E. Galante and C. Soave, Maydica, 21 (1976) 1. R.B. Osborne, The Vegetable Proteins, Longmans, Green and Co., NY, 199.4. J.W. Panlk and J.S. Wall, Biochim. Biophys. Acta, 251 (1971) 57. B. Fralj and U. Melche~, Plant Physiol. (Suppl) 61 (1978) 40. K.H. Lee, R.A. Jones, A. Dalby and C.Y. Tsai, Biochem. Genetics, 14 (1976) 641. E. Gianazza, V. Viglienghy, P.G. Righetti, F. Salamini and C. Soave, Phytochemistry, 16 (1977) 315. U. Melcher and B. Fraij, Plant Physiol. (SuppL) 63 (1979) 94. J.A. Bietz, F.R. Huebner, J.E. Sanderson and J.W. Wall, Cereal Chem., 54 (1977) 1970. K. Pedersen, D.V. Glover, C.Y. Tsai and B.A. Larkins, Fed. Proc., 37 (1978) 1401. U. Melcher, Plant Physiol., 63 (1979) 354. E.M. Southern, J. Mol. Biol., 98 (1975) 503. R.A. Laskey and A.D. Milk, Eur. J. Biochem., 56 (1975) 335. A.J. Bendich and B.J. McCarthy, Proc. Natl. Acad. Sci. U.S.A., 65 (1970) 349. I.H. Maxwell, J. VanNess and W.E. Hahn, Nucleic Acids Res., 5 (1978) 9-033. W.R. Pearson, E.H. Davidson and R.J. Britten, Nucleic Acids Res., 4 (1977) 179.7. R.J. Britten, D.E. Graham and B.R. Neufeld, Methods Enzymol., 29 (1974) 363. G. Brawerman, Methods Enzymol., 30 (1974) 605.
133
© Elsevier/North-HollandScientificPubli~Qn Ltd.
HETEROGENEITY OF ZEA MA YS PROTEIN BODY MESSENGER R N A
ULRICHMELCHER Department of Biochemistry, Oklahoma State University, Stiilwater, OK 74074 (U.S.A.)
(ReceivedJune 28th, 1979) (Revhdonreceivedand acceptedJanuary 2rid, 1980) SUMMARY To investigate whether the heterogeneity of zein polypeptides found in protein bodies of corn endosperm is reflected in a ~ heterogeneity of the protein body mRNA, a complementary DNA copy of the polyadenylic acid containing (poly(A)+)RNA of protein bodies was prepared using reverse transeriptase. This radioactive DNA hybridized as two distinct kinetic components, differing 70-fold in complexity, to an excess of the poly(A)+ RNA. By comparison with the hybridization profiles of complementary DNA driven by total protein body RNA and by RNA lacking polyadenylic acid (poly(A)-) it was determined that the more slowly hybridizing component was present in both poly(A)+ and poly(A)-RNA, while the faster hybridizing component was almost exclusively restricted to the poly(A)+ RNA. INTRODUCTION Protein bodies in the starchy endosperm of cereal seeds are the intracellular site of accumulation of a special group of proteins [1] and are formed during endosperm development from the rough endoplasmic reticulum (ER) [2]. They have a protein-containing core, derived from the ER lumen, a surrounding membrane, and polyribosomes attached to the c y t ~ plesmic face of the membrane. The polyribosomes of these protein bodies are responsible for the synthesis of the proteins which occur inside of the protein bodies [3,4]. The proteins sequestered within these bodies are a collection of numerous similar polypeptides [5] and are soluble in aqueous alcohol, a property which identifies them as members of the prolamine fraction of cereal proteins [6]. The prolamine fraction of corn, zein, is heterotenous as demonstrafed by separations obtained by gel filtration [7], hydroxylapetite chromatography [8], dodecyl sulfate polyacrylamide gel electrophoresis [9], and isoeiectric focusing [5]. The fractions obtained differ structtmflly
134 from one another as shown by amino acid analysis [8,10], peptide mal~ ping [8], and chemical modification [11]. Despite these differences, the polypeptides of the zein fraction are very similar to one another. Besides a solubility in alcohol, these polypeptides share several other properties. Most of the polypeptides have a molecular weight between 19 000 and 23 000 [9]. They have very similar amino acid compositions when separated either by isoelectric focusing or by hydroxylapatite chromatography [8,10]. A c o m m o n set of peptides produced by digestion with proteolytic enzymes account for over half of the peptides produced from every subfraction of zein examined [8]. A similar situation is found in the wheat prolamines, gliadins, where separable polypeptides have nearly identical amino acid sequences [ 12]. Since the amino acid sequences~or these polypeptides are coded for by mRNA molecules, one would predict that the mRNA population responsible for the synthesis of zein is also composed of a series of similar but not identical molecules. One method of testing this prediction is to study the hybridization kinetics of mRNA from protein bodies with complementary DNA molecules obtained by reverse transcription using RNA dependent DNA polymerase. Only a preliminary report of such a study has been pre~ sented [13]. I wish to report the results of hybridization experiments which suggest that protein body mRNA consists of two fractions which differ in their complexity and distribution among poly(A)+ and p o l y ( A ) RNA fractions. MATERIALS AND METHODS The growth and harvesting of corn 20--25 days after silk emergence was previously described [14]. A protein body-enriched fraction was isolated from a homogenate of kernels according to Larkins et al. [3]. This fraction was used as the starting material for isolation of poly(A)+ RNA, p o l y ( A ) RNA and total RNA. For the isolation of poly(A)+RNA and poly(A)-RNA, the polyribosomes were dissociated from the protein bodies with Triton X-100 and pelleted by centrifugation through buffered 1.75 M sucrose [3]. The polyribosomal pellet was suspended and applied to oligo (dT) cellulose as previously described [14]. The u n b o u n d fraction was extracted twice with an equal volume of chloroform/isoamyl alcohol (24 : 1) for 15 min at r o o m temperature. RNA in the aqueous phase was precipitated with 3 vols. of ethanol and recovered by centrifugation as poly(A)-- RNA. The material eluting from the oligo(dT)cellulose with 10 mM Tris-HC1 (pH 7.5) was heated briefly to 65°C. The sample was adjusted to 0.5 M NaC1 and reapplied to the oligo(dT)cellulose column. The procedure was repeated for a total of three cycles of binding and elution. The final eluate was adjusted to 0.3 M NaC1 and the RNA precipitated with 2 vols. of ethanol. The RNA recovered after centrifugation is designated poly(A)+ RNA. For the isolation o f total RNA, the protein body enriched pellet was stmpended
135
in 1.0 ml/g of kernels of 0.1 M Tris--HCl, I mM Na2EDTA, 1% SDS, 0.1 M NaCI, (pH 8.0) and extracted for 30 rain at room temperattwe with an equal volume of a I : 1 mixture of phenol and chloroform. The sample was centrifuged at 10 000 g for 10 min and the resulting aqueous phase tranafe~ed to a tube containing an equal volume of chloroform/isoamyl alcohol (24 : 1). The organic phase and interphase were extracted with a half volume of 0.1 M Tris-HC1, 1 mM Na2EDTA, 1% SDS (pH 9.0) for 30 rain at r o o m temperature. After centrifugation to separate phases, the aqueous phase was removed and added to the first aqueous phase. These were mixed with chloroform : isoamyl alcohol for 15 rain at room temperature. To the final aqueous phase obtained after centrifugation 2 vols. of ethanol were added. After storage overnight at - 2 0 ° C the precipitated RNA was r ~ covered by centrifugation, dried and used in the hybridization experiments.
cDNA synthesis DNA complementary to poly(A)+ RNA was synthesized in 50 raM Tfis-HC1~(pH 8.3), 60 mM NaCI, 6 mM MgCI2, 20 mM dithiothreitol, 0.8 mM each of dTTP, dATP, dGTP, 2.6 #M [SM]dCTP. (22.9 Ci/nmol, New England Nuclear~ 0.1 mg/ml actin0mycin D, 40/~g/ml oligo(dTl0) (P-L Biochemicals), 40/~g/ml poly(A)+RNA by addition of 120 Units avian myeloblastosis vires reverse transcriptase (obtained through J.W. Beard, Life Sciences, Inc.) to a 25 #1 reaction mixture. After a 60 rain incubation at 37°C, 100/~1 of 10 mM Na2EDTA were added to stop the reaction. Then 0.3 ml 0.1 M Tris--HC1 (pH 9.0) and 50/~g yeast tRNA were added. The solution was made 0.5% in SDS and extracted with an equal volume of phenol for 15 rain. After centrifugation to separate phases, the aqueous phase was recovered and 3 vols. o f ethanol added. The precipitate that formed at - 2 0 ° C overnight was collected by centrifugation and redissolved in 1.0 ml 0.3 N NaOH. It was heated to 100°C for 15 min, chilled and neutmUzed with 30 #1 N HC1. The solution was made 10 mM MgC12, 10 mM Tris--HC1 (pH 7) and 10 ~g yeast tRNA were added. After ethanol precipitation the nucleic acid was taken up in 0.3 M sodium acetate and reprecipitated with ethanol. The final precipitate was dissolved in water and stored at - 70°C. The size of the resulting cDNA was estimated by electrophoreais of the cDNA in a 1.4% agarose gel containing 0.03 N NaOH, 1 mM Na=EDTA. Denatured HaeIII restriction fragments o f [SH]~X 174 DNA RF (Bethesda Research Laboratories) served as size standards. DNA fragments in the gel were transferred to a nitrocellulose sheet [ t 5 ] and the sheet dipped in 5% PPO in toluene prior to fluorography at - 7 0 ° C using flash-activated X-ray film [16]. The resulting fluorograph was scanned with a deusitometer to determine the distribution of cDNA molecules. The size ranged from 300 nucleotides to 1300 nucleotides with a weight average of 600 nucleotides. cDNA.RNA hybridization RNA-DNA hybridization was carried o u t in small plastic vials in 0.3 M
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NaC1, 20 mM Tric-HC1 (pH 7.4) 1 mM EDTA, 1.0% SDS covered with paraffin oil to minimize evaporation. RNA was present in at least a 1000fold excess over DNA. Incubation was at 68°C. At various times aliquots were removed and diluted into $1 nuclease b~Lffer (30 mM sodium acetate, 3 mM ZnC12, 0.3 M NaC1, and 10/~g/ml wheat DNA (extracted from wheat germ by the procedure of Bendich and McCarthy [17]). One-half of the diluted sample was set aside for analysis of total radioactivity while the other half was digested with 10 units of $1 nuclease (P-L Biochemicals). In early experiments (Fig. 1), radioactivity was analyzed by trichloracetic acid precipitation after the addition of carrier protein and collection of the 100
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Fig. 2. Kinetics of hybridization of total protein body RNA with [3H]cDNA, Upper line represents the computed best fit to the data points. The lower lines represent the hybridization of each component. '
187 I~ ~0
o
o
'
I
o
o
I
I
o
g ~60
o o
4O
2C 0
\ I
i
0
I
\ ~
,
Z
i
3
Log Ecr0t Fig. 3. Kinetics of hybridization of protein body poly(A)-l~NA with [ =H]cDNA. Upper line represents the computed best fit to the data points. Lower line represents the hybridization of the component.
precipitates on glass fiber filters. Later experiments (Figs. 2 and 3) were analyzed by the DE.81 filter disk method of Maxwell et aL [18]. Data obtained with the latter procedure had lower background levels of hybrkiization and Considerably less scatter in the data points and therefore fewer determinations were made. The data were fit to theoretical RNA driven RNA-DNA hybridization equations using the computer program of Pearson et aL [19]. Equivalent Cro values were calculated using a correction factor for salt concentration [20]. A [3H]cDNA copy of globin mRNA (Miles Laboratories) was made as described above and used in hybridization with globin mRNA to provide a kinetic standard. RESULTS
moly(A)+RNA The poI¥(A)+RNA isolated from polyribosomes released from protein bodies of ~eveloping corn endosperm, when used as a template for in vitro protein synthesis, coded for the synthesis of precursom to zein polypeptides [14]. As judged by gel electrophoresis [14] and ethanol extraction (Fraij and Melcher, unpublished) little or no non-zein polypeptides were synthesized. This RNA was used as a template for the synthesis of eDNA using reverse tmnscriptase. The radioactive cDNA was use~ unfmctionated in hybridization reactions using the template RNA to ~ v e hybridization at 68°C in 0.3 M NaCL A t t h e highest £Crot tried (Fig. 2) 85--90% hybridization was obtained. The hybridization values could be fitted best ammaning two components of hybridization. The first accounts for 82% of the total cDNA radioactivity while the second accounts for 49% of the total. The
138 TABLE I CALCULATED PARAMETERS FOR HYBRIDIZATION OF RNA TO [3H]cDNA RNA
Component
Fraction
poly(A)+
A B
0.324 0.489
total
A B
0.324 b 0.489 b
1.74 0.092
51.1 21.0
poly(A)--
B
0.489 b
0.070
27.6
globin c
0.82
K
Dilution a
88.7 1.93
143.2
---
--
aobtained by dividing K for the component in poly(A)+ RNA by K. b Values fixed for computation. C W i t h [ 3H]globin cDNA. first c o m p o n e n t hybridized with a kinetic complexity only 1.6 times that o f globin m R N A - - c D N A hybridization indicative o f the presence of only 3--5 sequences o f t h e size of zein m R N A . The second hybridized with a kinetic constant approx. 50 times smaller than that of t h e first c o m p o n e n t and must thus represent a substantial n u m b e r o f different sequences.
Total R N A Total R N A extracted from protein bodies using chloroform/phenol to minimize t h e loss o f poly(A)+ sequences was also subjected t o hybridization with t h e same cDNA. Figure 2 shows that approximately the same extent o f m a x i m u m hybridization was achieved. The t w o kinetic components are n o t as distinct as t h e y were in Fig. 1. Nevertheless the data were fit best b y a t w o c o m p o n e n t solution (goodness o f fit 0.0223 as opposed as to 0.0398 for one component) with the proportion of the cDNA responsible for each c o m p o n e n t fixed at the values deduced from the data in Fig. 2 (Table I). The acceleration in hybridization o f the slow relative to the fast c o m p o n e n t must be due to either the selective loss of the slow c o m p o n e n t during ollgo(dT)cellulose chromatography or the selective loss o f t h e rapidly hybridizing c o m p o n e n t during chloroform extraction. A calculation of t h e a m o u n t b y which each c o m p o n e n t o f Fig. 2 has been diluted b y other R N A molecules reveals that t h e rapidly h y b r i d i z ~ g comp o n e n t has been diluted approx. 50-fold while the slow c o m p o n e n t is only diluted approx. 20-fold (Table I). Since the major R N A that dilutes these samples is r R N A and since t h e r R N A accounts for 98--99% of the total R N A in polyribosomes, a dilution factor o f 50 is expected. This indicates that there was no selective loss o f t h e rapidly-hybridizing c o m p o n e n t during chloroform extraction and raises the possibility that the slowly hybridizing c o m p o n e n t is selectively lost during oligo(dT)cellulose chromatography.
139
PoZy(A )- PJVA Since p o l y ( A ) - molecules would be selectively lost during oligo dT cellulose chromatography, I sought to test whether p o l y ( A ) - molecules contained the sequences complementary to the cDNA w/aich hybridized with slow kinetics to the poly(A)+ RNA. The effluent from the application of RNA to the oligo dT cellulose column was recovered and extracted with chloroform to remove contaminating proteins. This RNA was allowed to hybridize with the cDNA probe. As can be seen in Fig. 3 the extent of maximum hybridization was less than seen with the POly(A)+RNA, indicating that some sequences are present in only the polyadenylated form. The hybridization points are best fit assuming two components. Assigning the first c o m p o n e n t to the fraction of cDNA hybridizing to total RNA with slow kinetics (0.489) resulted in a better fit (goodness of fit 0.0257) than when this fraction was assigned the value of 0.3j24 (goodness of fit 0.0345), that of the faster hybridizing c o m p o n e n t of Fig. 1. The result suggests then that POly(A)-RNA was enriched for the sequences corresponding to the slower hybridizing c o m p o n e n t of Fig. 1. The slower c o m p o n e n t in the p o l y ( A ) - R N A experiment must then be due to some of the sequences contained within the rapid c o m p o n e n t of poly(A)÷ RNA. Since complete hybridization of this component was n o t obtained even at high ECrotvalues, less than 1% of these RNA sequences in total RNA are present in p o l y ( A ) RNA. To confirm these results, poly(A)- molecules were prepared in a second way. Total RNA was extracted with phenol at pH 7, a procedure which results in the selective loss of poly(A)+~ontaining molecules [21]. When this RNA was hybridized to the cDNA probe, a maximum extent of hybridization of only 50--60% was routinely observed. The hybridization points could be fit better with two components than with a single-component. Assigning a value of 0.49 to the fraction of the cDNA hybridizing first results in a good fit to the data, consistent with the hybridization of p o l y ( A ) - R N A prepared by oligo(dT)cellulose chromatography. That the extraction was specific for the extraction of only p o l y ( A ) - molecules was shown by passing the extracted RNA over oligo(dT)cellulose and collecting the material that did n o t bind. This RNA hybridized to the cDNA probe with the same kinetics as the RNA before passing through oligo(dT)cellulose. DISCUSSION The observation of t w o distinguishable hybridization components in maize protein b o d y RNA, one of which also has sequences present in nonpolyadenylated RNA permit two different interpretations. First, the RNA present in the corn sample could be predominantly zein n~RNA, a fraction of which has been randomly fragmented. Zein m R N A could contain two regions of sequence, one region which is c o m m o n to the RNAs for all the zein IEF vza~mts and t h e other being variable from one mRNA to the other. Such a hypothesis is consistent with the current concept of zein
140 protein structure. If this assumption is true, one can make several calculations on the length and complexity of the two regions. From the relative dilutions of the two kinds o f RNA it can be calculated that 4(~o of the slow c o m p o n e n t sequences are present in the poly(A)+ fraction and 60% in the poly(A)-- fraction. Correcting for the resulting overrepresentation of the rapid c o m p o n e n t in t h e poly(A)+ fraction, and using the fraction of the cDNA nucleotides that are complementary to each component, it can be calculated that the rapid c o m p o n e n t would account for 21% of the length of t h e message while the slow c o m p o n e n t would account for the remainder. The expected ratio of complexity on the basis of sequence length alone would thus be 3.8. The actual difference in complexity measured with poly(A)+ RNA was 70-fold. Thus there would be approx. 18 different slow c o m p o n e n t sequences for each rapid c o m p o n e n t sequence. It is perhaps fortutious that this corresponds roughly to the number of zein IEF variants [15]. The second possible interpretation of the data is that the rapid RNA c o m p o n e n t consists of m R N A for the two major distinguishable size classes of zein polypeptide, zein A and zein B. The slowly hybridizing c o m p o n e n t would represent mRNAs for the synthesis of the minor zein polypeptides, for membrane proteins, and other proteins that may be made on the protein bodies. Although t h e synthesis of precursors to minor zein polypeptides can be detected when this RNA is translated in vitro, the synthesis of other non-zein polypeptides has n o t been detected. This may of course be due to the small a m o u n t of each polypeptide expected. Distinction between these two possible explanations awaits further experimentation. These results differ from the results of Pedersen et al. [13] who used a population of poly(A)+RNA molecules sedimenting at 138 in a sucrose gradient as a template for the synthesis of cDNA. They observed a single hybridizationcomponent. Possible minor mRNA molecules n o t related to the major zein polypeptides and possible fragments of the major m R N A species would n o t be present in their preparation. This difference can account for the discrepancies between the results of Pederson et al. [13] and the present ones. ACKNOWLEDGEMENTS Dr. Dale Weibel (Okla. St. Univ.) and Mr. R a y m o n d Peck (Panhandle St. Univ.) are thanked for the samples of com. The technical assistance of Mr. Charles R e d m o n d and Mrs. Elizabeth Hood was greatly appreciated. Financial support was provided by the National Science Foundation (Grant PCM 76-01699) and the Oklahoma Agricultural Experiment Station of which this is Journal Article No. J. 3662. REFERENCES 1 D.D. Chrlstianson, H.C. Nielsen, U. Khoo, M.J. Wolf and J.S. Wall, Cereal Chen~, 46 (1969) 372.
141
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