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Chloroplast ribosomal RNA genes in the chloroplast DNA of Euglena gracilis
J. Mol. Biol. (1973) 77, 125-132
hloroplast Ribosomal RNA Genes in the Chloroplast DNA of Euglena gracilis JAMES R. Y. RAWSON AND ROBERT EASELKORN
Department of Biophysics, University of Chicago Chicago, Ill., U.S.A. (Received 25 July 1972, nd in revised form 22 January 1973) Euglena ehloroplast DNA has a buoyant density in C&I of 1.686. Shearing this UN-4 produces a satellite band at density 1.700. The satellite, easily lost during preparative C&l gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 x lo6 daltons of DNA.
1. Introduction Chloroplasts of the green alga Euglena gracilis contain ribosomes that differ in several respects from the organism’s cytoplasmic ribosomes. Chloroplast ribosomai RNAs have lower molecular weights than their cytoplasmic counterparts, and different average nucleotide composition (Rawson & Stutz, 1969). Chloroplast ribosomal RNAs are synthesized at extremely low rates (if at all) in cells grown in the dark; when cells grown in the dark are exposed to light, these RNAs are synthesized preferentially (Brown & Haselkorn, 1971). Based on the observation that chloroplast ribosomal RNA anneals to chloroplast DNA (Scott & Smillie, 1967; Stutz & Rawson, t970), it has been ooncluded that the genes for these RNAs are contained in the chloroplast DNA. The molecular weight of Euglena chloroplast DNA has been measured in several ways. Electron microscopic determinations of the contour length of intact circles gave values corresponding to a molecular weight of 92 x lo6 (Manning & Richards, 1972u;). The kinetics of renaturation of Euglena chloroplast DNS imply a unique genome size consistent with this value (Stutz, 1970). The chloroplast ribosomal RNAs have molecular weights of 1.1 x IO6 and OG%X lo6 (Rawson & Stutz, 1969), therefore, each imegral complement of chloroplast ribosomal RNA genes should comprise 3*So/0 of the chloroplast DNA, or, in terms of DNA capable of annealing with ribosomal RNA, 1.9%. Published values for the fraction of chloroplast DNA ca,pable of annealing with ehloropfast ribosomal RNA are somewhat lower than 1*9%, both for Euglewx and for higher plants (Scott & Smillie, 1967; Tewari $ Wildman, 1970; Ingle et al., 1970). Such lower numbers can be int,erpreted to mean that either the molecular weight of chloroplast DNA is higher than 92 x lo”, or that some chloroplast “ chromosomes ” lack genes for ribosomal RNA. In this report we show that, depending on the molecular weight of the ehloropla& DNA, traditional methods for the preparation of chloroplast DNA tend to lose, selectively, the genes for ribosomal RNA. With a method for avoiding such losses, 125
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we find that 1.9% of Euglena chloroplast DNA anneals with chloroplast ribosomal RNA, implying that each “chromosome ” of chloroplast DNA contains exactly one gene for each chloroplast ribosomal RNA.
2. Materials and Methods (a) Cell growth Axenic cultures of Euglena gracilis var. Z were grown to stationary phase in an autotrophic medium (Rawson & Stutz, 1969), harvested in a Sharples centrifuge and stored at -60°C. (b) Isolation of chloroplast DNA Chloroplast DNA devoid of nuclear or mitochondrial DNA was isolated from chloroplasts purified by flotation on gradients of Renografm (Brown & Haselkorn, 1972). Residual protein and oligoribonucleotides from RNase digestion were removed by banding the DNA in 20-ml preparative CsCl equilibrium density gradients in the Spin00 type 60 rotor at 35,000 revs/mm for 60 h. CsCl gradients were fractionated so as to assure that all DNA, regardless of its G + C content, was recovered. The resulting DNA was dialyzed against 0.1 x SSC (SSC is 0.15 &f-NaCl, 0.015 M-sodium citrate), 10m4 M-EDTA (pH 80), concentrated in vacua, and further dialyzed against the same buffer. DNA was stored at -20°C at concentrations of about 200 pg/ml. (c) Isolation of ribosomal RNAs Chloroplast ribosomal RNAs were isolated from purified ribosomes prepared as previously described (Rawson & Stutz, 1969). The ribosome suspensions were deproteinized with 2% sodium dodecyl sulfate and immediately layered onto 35-ml convex exponential gradients in O-1 M-Nacl and 0.01 M-Tris.HCl (pH 7.9) of the type C = 1*4- 1.1 exp-Y’2s, where C is the molarity of the sucrose being delivered into the gradient tube at the volume equal to V. Centrifugation was carried out in a Spinco SW27 rotor at 27,000 revs/min for 26 h. The large and small ribosomal RNAs were fractionated, dialyzed against SSC, concentrated iti vacua, and dialyzed against the same buffer. RNA samples were stored at -20°C at concentrations of 100 to 200 pg/ml. Pure cytoplasmic ribosomal RNA can be prepared routinely from cytoplasmic ribosomes, but chloroplast ribosomal RNA is always subject to contamination by cytoplasmic ribosomal RNA. For this reason, before the preparative isolation of .the RNA, the RNA from the chloroplast ribosomes used for these experiments was resolved on polyaorylamide gels to determine both the purity and the ratio of 23 S to 16 S RNA. The gels were run long enough to resolve chloroplast RNA from cytoplasmic RNA completely. As seen by ultraviolet scans of the gels, cytoplasmic ribosomal RNA contaminated this ohloroplast ribosomal RNA by less than 5%, and the molar ratio of 23 S to 16 S RNA was 1: 1. (d) Labeling of ribosomal RNA Chloroplast ribosomal RNA was labeled in. vitro with [3H]dimethylsulfate (Smith et al., 1967). The high molecular weight chloroplast ribosomal RNAs were fractionated from sncrose gradients, dialyzed against SSC, re-adjusted to 2% sodium dodecyl sulfate, and extracted twice with phenol-10% cresol and 8-hydroxyquinoline saturated with SSC, 1O-4 M-EDTA. The deproteinized aqueous phase was dialyzed overnight against 2 1 of 6 x SSC to remove residual phenol and the salt concentration was again lowered to 0.1 x SSC by dialysis. The RNA was concentrated in dialysis bags and redialyzed against 0-l x SSC, yielding an RNA concentration of 200 pg/ml. The sample was adjusted to O-5 M-sodium acetate and precipitated with 2 vol. of ethanol. The precipitate was collected acetate. Five millicuries of L3H]dimethylby centrifugation and redissolved in O-5M-SOdiUm sulfate (spec. act. 385 mCi/mmol, New England Nuclear) was extracted from its shipping vial with a total of 2.0 ml of anhydrous ethyl ether and added to the aqueous RNA solution. The dimethyl-sulfate was forced into the aqueous phase by evaporating the ether with filtered Nz. The reaction mixture was shaken for 18 h at room temperature.
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The reaction was stopped by adding 2 vol. of ethanol and collecting the precipitate bg centrifuge&ion. The RNA was re-precipitated as described above until all radioactivity remaining in the solution was trichloroacetio acid-precipitable (4 precipitations). The RNA was then dissolved in water and stored at -20°C. The resulting 3H-labeled met,hylated RNA had a specific activity of 4860 cts/min/pg. When examined in a sucrose gradient with B’~cherichicc co& 23 S and 16 S ribosomal RNA it had sedimentation coefficients in $he range of 6 to 10 S. (e) DNA-RNA hybridization Hybridization was carried out according to Gillespie & Spiegelman (1965) with some modifications. DNA samples were denatured with 0.1 vol. of 1 m-Nash, cooled at O”@, neutralized with 0.1 vol. of 2 M-NaH,PO, and adjusted to 4 x SSC with cold 20 x SSC. Denatured DNA was adsorbed onto nictrocellulose filters (Schleicher and Schuell, E6), washed with 50 ml of 4 x SSC and dried first at room temperature for 1 h and then in a vacuum oven at 80°C for 2 h. The hybridization reaction was carried out at 70°C for 16 h in glass scintillation vials containing, unless otherwise specified, 2.0 ml of the RNA mixture. The reaction mixture contained the appropriate RNA, 4 x SSC a,nd 0.5% sodium dodecyl sulfate. The dodecyl sulfate lowered the non-specific binding of RNA to blank filters to less than 0.01 y0 of the input and did not alter the hybridization efficiency. All RNAs (labeled or unlabeled) used for hybridization were altered 3 times through nitrocellulose filters to remove residual basic proteins and any possible RNA containing poly(A) sequences. Filters were washed in batches of up to ‘75 filters/2 1 flask. Filters were first rinsed in 2 x SSC, then washed 3 times by shaking in 2 x SSC (20 ml/filter) for 10 min. Unhybridized RNA was eliminated by digestion with pancreatic RNase (25 fig/ml? 5 ml/filter) for 1 h at room temperature. The filters were again washed 3 times with 2 x SSC (20 rnli filter), dried and counted. All hybridization data were corrected for the binding of RNA to blank filters. (f ) Equilibriwm density gradient centr$%gation Analytical CsCl gradients were run in a Spinco model E ultracentrifuge at 44,000 revs,/ min for 20 h at 25°C. Buoyant densities were determined relative to the phage SPOl DNA (p = 1.742 g/cm2). Preparative CsCl gradients were run in a Spinco SW65 rotor using polyallomer tubes, Gradients of 4.0 ml were centrifuged for 42 h at 40,000 revs/mm ak 25°C. (g) Molecular weight determination of DNA The molecular weights of the various DNA preparations were determined by band sedimentation in 3 M-CsCl (p = I.38 g/cm2). so qrO, was calculated as described by Bruner & Vinograd (1965) and the so20,w values determined using a correction factor of 1.6. Freifeider’s (1970) equation was used to convert so20,w to molecular weight. (h) Preparatiort of DNAs of various molecular weights Chlosoplast DNA, isolated as described above, was sheared in high salt (77 M-CsCl) by passing the DNA through a 26-gauge needle 15 times. Sonicated chloroplast DNA was also prepared in 7.7 M-CsCl by subjecting the DNA solution to 4 bursts of 15 s each of the Bronson sonicator at position no. 7. The DNA was kept on an ice bath during both modes of shearing.
3. Results The average density
in CsCl of Euglena nuclear DNA is 1.707; of chloroplast DNA, composit,ion of the chloroplast ribosomal RNAs require a G f C content in DNA of 51o/o, corresponding to a density of 1.710 in CsCl. Thus, if a preparation of Euglena chloroplast DNA is partially contaminat,ed with nuclear DNA, and subjected t#o preparative centrifugation in a CsCl gradient in order to remove the contaminant, careful removal of material at density l-686 could result
1.686. The nucleotide
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in the preferential loss of ribosomal RNA genes at density greater than 1.686. But nuclear DNA must be removed, because chloroplast ribosomal RNA will anneal, albeit with reduced efficiency, with the nuclear genes for cytoplasmic ribosomal RNA (p = 1.722, G + C = 62%) (J. Rawson, unpublished observations). The development of a technique for the isolation of chloroplasts, and hence chloroplast DNA, uncontaminated by nuclear DNA, makes the requisite experiments feasible (Brown & Haselkorn, 1972). The distribution of Euglena chloroplast DNA, prepared from nuclear DNA-free chloroplasts, in analytical CsCl density gradients is shown in Figure 1. The gradient in Figure l(a) contained DNA as it is prepared from the chloroplasts; the average double strand molecular weight, based on zone centrifugation, was 1.25 x 10’. The material in (b) was sheared to a molecular weight of 3.3 x 10s ; in (c), the sample was sonicated to yield material with an average molecular weight of 6.9 x 105. The point of the demonstration is that lowering the average molecular weight of the chloroplast DNA permits the separation of a discrete class of DNA molecules, of density 1.700, from the bulk of the chloroplast DNA.
I.700
1.686
Buoyant density
FIG. 1. Densitometer tracings of analytical CsCl equilibrium density gradients of chloroplast DNA of varying molecular weights. DNA was prepared from Renografk-purified chloroplasts as described in the text. The DNA was sheared (b) or sonicated (c) and centrifuged to equilibrium. 1.5 ,~g of phage SPOl DNA (p = 1.742 g/ cmz) was used as the density reference. Patterns (a), (b) and (c) represent 2.5 pg chloroplast DNA of molecular weight 12.5 x 106, 3.3 x 10s and 6-9 x 105, respectively.
The same DNA samples were centrifuged in preparative CsCl gradients, and individual fractions were annealed with 3H-labeled chloroplast ribosomal RNA. In the high molecular weight sample, DNA sequences complementary to chloroplast ribosomal RNA are found in that part of the gradient containing most of the ohloroplast DNA (Fig. 2(a), fractions 13 to 15), as well as regions containing DNA of higher density, fractions 8 to 11. In the samples of DNB that had been sheared or sonicated
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(Fig 2(b) and (c)), all of the DNA complementary to chloroplast ribosomal RNA is in a region of the gradient of density greater than that of the bulk of chloroplast DNA. We interpret these results to mean that some, and perhaps all, of the genes for chloroplast ribosomal RNA are located in the class of DNA molecules of density la700 released from the bulk of chloroplast DNA by shearing.
Y”
E
E
’
0
(b)
2 0
0 Fi Y
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- 150
B 2 p ,r
::1, ::
5
-
IO
15
100
-
Fraction no
FIQ. 2. tive CsCl legend to collected, with 5.0 --a--@--,
Hybridization of chloroplast ribosomal RNA to chloroplast DNA fractions from preparedensity gradients. Chloroplast DNA (15 pg/gradient) of varying molecular weights (see Fig. 1) was banded in 4.0 ml CsCl density equilibrium gradients. Fractions of 0.15 ml were denatured and fixed to nitrocellulose filters. The filters were annealed in batches of e pg [3H]CH,/ml chloroplast ribosomal RNA at 70°C for 16 h. ( L -4nso; 3H (cts/min).
The results shown in E‘igures 1 and 2 illustrate the point made in the Introdu&ion : if chloroplast DNA is purified by preparative CsCl centrifugation in order to isolate DNA of density l-686 for the determination of the number of ribosomal RNA genes, the determination will be low to the extent that ribosomal RNA genes are sheared from the bulk of chloroplast DNA. Since the method for preparation of chloroplast+z used in this work provides material free of nuclear DNA, we include the preparative &Cl gradient in the purification of chloroplast DNA only to remove protein and oligosibonucleotides. When filters containing chloroplast DNA were annealed with 9
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increasing amounts of 3H-labeled chloroplast ribosomal RNA, the amount of RNA annealed, at saturating levels of RNA, corresponded to 1.9% of the DNA (Fig. 3). Assuming equal specific activities of the two ribosomal RNA species, equimolar ratios of the two RNAs (see Materials and Methods), and lOOo/oefliciency of hybridization at saturation, the value of 1.9% corresponds to one gene for each ribosomal RNA in each ‘ichromosome” of molecular weight 92 x 106.
0
02
04
06
’ IO
’ 20
’ 30
RNA/DNA OS IO ’ ’ 4.0 50 [RNA] pg/mt
II2
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’ 60
7&i
16
FIG. 3. Saturation of chloroplast DNA with chloroplast ribosomal RNA. Chloroplast DNA (5.0 pg/filter) was annealed with varying amounts of [3H]CH, chloroplast ribosomal RNA (4860 cts/ min). Each point represents the average of duplicate determinations. 1.9% DNA saturation corresponds to 460 cts/min/filter.
4. Discussion The average number of cistrons for ribosomal RNA in each complement of chloroplast DNA can be determined by quantitative hybridization of ribosomal RNA with chloroplast DNA. The chloroplast DNA used for hybridization must be free of nuclear DNA, and must contain all the components of chloroplast DNA. Earlier estimates of the fraction of Euglena chloroplast DNA complementary to ribosomal RNA of 1.0 to 1.2% (Scott & Smillie, 1967; Stutz & Rawson, 1970) were low because the chloroplast DNA, taken from preparative CM3 gradients, lacked a portion of the chloroplast DNA rich in cistrons for ribosomal RNA. In a more recent report Stntz & Vandrey (1971) found that 6% of Euglena chloroplast DNA, prepared by another procedure that preserves (and possibly enriches) for the ribosomal RNA cistrons, hybridizes with chloroplast ribosomal RNA. We find it difficult to evaluate this report. Stutz & Vandrey prepare Euglena chloroplasts by flotation on a gradient of Ludox, and then chromatograph the chloroplast DNA on a column of methylated albumin. Approximately 20% of this DNA is the satellite at p = l-701. Nevertheless, their value of 6% hybridization with chloroplast ribosomal RNA is more than three times higher than we have ever observed for any chloroplast DNA preparation. Stutz & Vandrey (1971) carry out DNA-RNA hybridization in 2 x SSC at 65”C, while our conditions are somewhat more stringent, 4 x SSG at 70°C. This is not likely to be a significant difference, since we found the extent of hybrid formation
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between EugEena cytoplasmic ribosomal RNA and total Euglena DNA to be independent of temperature between 66 and 74°C in 4 x SEX (J. Rawson & R. Brown, ~np~bhshed observations). ~~~e~ chloroplasts contain DNA molecules that can be isolated as covaIent1~ closed, supercoiled circles (Manning & Richards, 1972a). Up to one-third ofthe chloroplast DNA is found as circles or as linear molecules of length equal to that of the circles; the remainder are linear, distributed about means corresponding to haK or quarter length molecules. Half or quarter length circles are not observed. Since a reasonable proportion of big circles can be preserved, the absence of circles from the populations of shorter molecules is interpreted to mean that all of the shorter moleaules are derived by breakage from full length circles. How many classes of full length circles are there? The molecular weight of a single cireIe is 92x 1~3. The oomplexity of Euglenu chloroplast DNA was originally determined by renaturation kinet,ics to be 180x 10” (Stutz, 1970), but this result did not take into account the very low G + C content of that DNA. It has been suggested (Manning et al,, 1971) that a, correction for the G + C content would bring the complexity down to 92 But we know very little about the distribution of G-C pairs in the chloroplast and, in the absence of such information, it would seem that any “correct~ion” is risky. If we assume that the kinetic complexity is actually 92 x 106, then there can be only one class of DNA molecules, and each molecule must contain one cistron for each cbloroplast ribosomal RNA species. Suppose the complexity is 180x lOa, then there are two classes of DNA molecules ; each could contain one cistron for each ribosomal RNA, or one DNA molecule could contain two cistrons for each RNA. In the latter case, the ribosomal RNA cistrons cannot be repeated in tandem, for the following reason: from the base composition of chloroplast ribosomal RNA, DNA coding for chloroplast ribosomal RNA must have a buoyant density of 1.710. When chloroplast DNA is sheared to an average molecular weight of 3-3 x 106, approximately the size of one set of ribosomal RNA cistrons, the DNA has an average buoyant density of 1.700 (Fig. 2(b)). Therefore the ribosomal RNA cistrons must be linked at both. ends to DNA of very low G + C content (p = l-690), and they cannot be joined in tandem. Manning & Richards (19723) report that the proportion of Euglenu chloroplast NA found in the satellite at p = I.700 is higher by 50% in cells growing exponentially than in cells grown into stationary phase. In general, one expects the two situa tions to differ by virtue of the fact that DNA molecules are replicating in cells growing exponentially, but are completed in stationary phase cells (Yosbikawa & Sueoka, 1963)” If the 25% of the completed ohloroplast DNA molecule found in the p L- P-700 satelhte is closest to the origin of DNA replication, then Manning & Richards’ (197%) observation is easily understood: the satellite will comprise, on the average, 40% of the DNA in growing DNA molecules. It is not necessary to postulate independent replication of the satellite. The assumption that the satellite is that part 01 the DN.L4 closest to the origin of replication is not unreasonable; in Bacillus subtilis the genes for ribosomal RNA are very close to the chromosome origin (Qishi & Sueoka, 1965). While the p = L-700 satellite contains the genes for ribosomal RNA, those genes are onIy a small part (
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The total number of genes per cell for chloroplast ribosomal RNA is quite high. Exponentially growing Euglem contain approximately 350 molecules of chloroplast DNA (0. Richards, personal communication), each of which contains one cistron for ribosomal RNA. This is nearly as many as the nuclear genes for cytoplasmic ribosomal RNA, which we estimate to be 450 (J. Rawson & R. Haselkorn, unpublished observations). The chloroplast genes for ribosomal RNA are subject to regulation; they are rarely transcribed in the dark (Brown & Haselkorn, 1971); in the light, chloroplast ribosomal RNA synthesis accounts for 10% of the cell’s ribosomal RNA. Presumably initiation is less frequent on the chloroplast ribosomal RNA cistrons than on their nuclear counterparts. The number and distribution of ribosomal RNA genes on ehloroplast DNA from other organisms is similar to what we find for Euglena. In each case the major uncertainty is the size of the chloroplast “chromosome ” : measurements of the kinetic complexity of chloroplast DNA from Chlamydomonas (Bastia et al., 1971), tobacco (Tewari & Wildman, 1970) and lettuce (Wells & Birnstiel, 1969) all give values around 200x 106. However, as is true for Euglena, the unknown contribution of nucleotide sequence distribution to the renaturation kinetics introduces an uncertainty of perhaps a factor of two in the “chromosome” size. In each case chloroplast ribosomal RNA
hybridizes with around 1% of the chloroplast DNA, leading to the conclusion that each “chromosome” contains one to three cistrons for ribosomal RNA. This work was supported by grant GB17514 from the National Science Foundation. One of us (J. R. Y. R.) was a postdoctoral fellow of the National Institutes of Health. REFERENCES Bastia, D., Chiang, K.-S., Swift, H. & Siersma, P. (1971). Proc. Nat. Acad. Sci., U.X.A. 68, 1157.
Brown, R. D. & Haselkorn, R. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 2536. Brown, R. D. & Haselkorn, R. (1972). &o&m. Biophys. Acta, 259, 1, Bruner, J. & Vinograd, J. (1965). Biochim. Biophys. Acta, 108, 18. Freifelder, D. (1970). J. Mol. Biol. 54, 567. Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829. Ingle, J., Possingham, J. V., Wells, R., Leaver, C. J. & Loening, V. E. (1970). Xymp. Sot. Exptl. Biol. 24, 303. Manning, J. E. & Richards, 0. C. (1972a). Biochim. Biophys. Acta, 259, 285. Manning, J. E. & Richards, 0. C. (19726). Biochemistry, 11, 2036. Manning, J. E., Wolstenholme, D. R., Ryan, R. S., Hunter, J. A. & Richards, 0. C. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 1169. Oishi, M. & Sueoka, N. (1965). Proc. Nat. Acad. Sci., U.S.A. 54, 483. Rawson, J. R. & Stutz, E. (1969). Biochhim. Biophys. Acta, 190, 368. Scott, N. S. & Smillie, R. N. (1967). Biochem. Biophys. Res. Commua. 28, 598. Smith, K. D., Armstrong, J. L. & McCarthy, B. J. (1967). Biochim. Biophys. Acta, 142, 323.
Stutz, E. (1970). E%BS Letters, 8, 25. Stutz, E. & Rawson, J. R. (1970). Biochim. Biophys. Acta, 209, 16. Stutz, E. & Vandrey, J. P. (1971). FEBS Letters, 17, 277. Tewari, K. K. & Wildman, S. G. (1970). Symp. Sot. Exptl. Biol. 24, 147. Wells, R. & Birnstiel, M. (1969). Biochem. J. 112, 777. Yoshikawa, H. & Sueoka, N. (1963). Proc. Nut. Acad. Sci., U.S.A. 49, 559.
hloroplast Ribosomal RNA Genes in the Chloroplast DNA of Euglena gracilis JAMES R. Y. RAWSON AND ROBERT EASELKORN
Department of Biophysics, University of Chicago Chicago, Ill., U.S.A. (Received 25 July 1972, nd in revised form 22 January 1973) Euglena ehloroplast DNA has a buoyant density in C&I of 1.686. Shearing this UN-4 produces a satellite band at density 1.700. The satellite, easily lost during preparative C&l gradient centrifugation of chloroplast DNA, contains the genes for chloroplast ribosomal RNA. Pure Euglena chloroplast DNA is shown to contain one set of ribosomal RNA genes for each 90 x lo6 daltons of DNA.
1. Introduction Chloroplasts of the green alga Euglena gracilis contain ribosomes that differ in several respects from the organism’s cytoplasmic ribosomes. Chloroplast ribosomai RNAs have lower molecular weights than their cytoplasmic counterparts, and different average nucleotide composition (Rawson & Stutz, 1969). Chloroplast ribosomal RNAs are synthesized at extremely low rates (if at all) in cells grown in the dark; when cells grown in the dark are exposed to light, these RNAs are synthesized preferentially (Brown & Haselkorn, 1971). Based on the observation that chloroplast ribosomal RNA anneals to chloroplast DNA (Scott & Smillie, 1967; Stutz & Rawson, t970), it has been ooncluded that the genes for these RNAs are contained in the chloroplast DNA. The molecular weight of Euglena chloroplast DNA has been measured in several ways. Electron microscopic determinations of the contour length of intact circles gave values corresponding to a molecular weight of 92 x lo6 (Manning & Richards, 1972u;). The kinetics of renaturation of Euglena chloroplast DNS imply a unique genome size consistent with this value (Stutz, 1970). The chloroplast ribosomal RNAs have molecular weights of 1.1 x IO6 and OG%X lo6 (Rawson & Stutz, 1969), therefore, each imegral complement of chloroplast ribosomal RNA genes should comprise 3*So/0 of the chloroplast DNA, or, in terms of DNA capable of annealing with ribosomal RNA, 1.9%. Published values for the fraction of chloroplast DNA ca,pable of annealing with ehloropfast ribosomal RNA are somewhat lower than 1*9%, both for Euglewx and for higher plants (Scott & Smillie, 1967; Tewari $ Wildman, 1970; Ingle et al., 1970). Such lower numbers can be int,erpreted to mean that either the molecular weight of chloroplast DNA is higher than 92 x lo”, or that some chloroplast “ chromosomes ” lack genes for ribosomal RNA. In this report we show that, depending on the molecular weight of the ehloropla& DNA, traditional methods for the preparation of chloroplast DNA tend to lose, selectively, the genes for ribosomal RNA. With a method for avoiding such losses, 125
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we find that 1.9% of Euglena chloroplast DNA anneals with chloroplast ribosomal RNA, implying that each “chromosome ” of chloroplast DNA contains exactly one gene for each chloroplast ribosomal RNA.
2. Materials and Methods (a) Cell growth Axenic cultures of Euglena gracilis var. Z were grown to stationary phase in an autotrophic medium (Rawson & Stutz, 1969), harvested in a Sharples centrifuge and stored at -60°C. (b) Isolation of chloroplast DNA Chloroplast DNA devoid of nuclear or mitochondrial DNA was isolated from chloroplasts purified by flotation on gradients of Renografm (Brown & Haselkorn, 1972). Residual protein and oligoribonucleotides from RNase digestion were removed by banding the DNA in 20-ml preparative CsCl equilibrium density gradients in the Spin00 type 60 rotor at 35,000 revs/mm for 60 h. CsCl gradients were fractionated so as to assure that all DNA, regardless of its G + C content, was recovered. The resulting DNA was dialyzed against 0.1 x SSC (SSC is 0.15 &f-NaCl, 0.015 M-sodium citrate), 10m4 M-EDTA (pH 80), concentrated in vacua, and further dialyzed against the same buffer. DNA was stored at -20°C at concentrations of about 200 pg/ml. (c) Isolation of ribosomal RNAs Chloroplast ribosomal RNAs were isolated from purified ribosomes prepared as previously described (Rawson & Stutz, 1969). The ribosome suspensions were deproteinized with 2% sodium dodecyl sulfate and immediately layered onto 35-ml convex exponential gradients in O-1 M-Nacl and 0.01 M-Tris.HCl (pH 7.9) of the type C = 1*4- 1.1 exp-Y’2s, where C is the molarity of the sucrose being delivered into the gradient tube at the volume equal to V. Centrifugation was carried out in a Spinco SW27 rotor at 27,000 revs/min for 26 h. The large and small ribosomal RNAs were fractionated, dialyzed against SSC, concentrated iti vacua, and dialyzed against the same buffer. RNA samples were stored at -20°C at concentrations of 100 to 200 pg/ml. Pure cytoplasmic ribosomal RNA can be prepared routinely from cytoplasmic ribosomes, but chloroplast ribosomal RNA is always subject to contamination by cytoplasmic ribosomal RNA. For this reason, before the preparative isolation of .the RNA, the RNA from the chloroplast ribosomes used for these experiments was resolved on polyaorylamide gels to determine both the purity and the ratio of 23 S to 16 S RNA. The gels were run long enough to resolve chloroplast RNA from cytoplasmic RNA completely. As seen by ultraviolet scans of the gels, cytoplasmic ribosomal RNA contaminated this ohloroplast ribosomal RNA by less than 5%, and the molar ratio of 23 S to 16 S RNA was 1: 1. (d) Labeling of ribosomal RNA Chloroplast ribosomal RNA was labeled in. vitro with [3H]dimethylsulfate (Smith et al., 1967). The high molecular weight chloroplast ribosomal RNAs were fractionated from sncrose gradients, dialyzed against SSC, re-adjusted to 2% sodium dodecyl sulfate, and extracted twice with phenol-10% cresol and 8-hydroxyquinoline saturated with SSC, 1O-4 M-EDTA. The deproteinized aqueous phase was dialyzed overnight against 2 1 of 6 x SSC to remove residual phenol and the salt concentration was again lowered to 0.1 x SSC by dialysis. The RNA was concentrated in dialysis bags and redialyzed against 0-l x SSC, yielding an RNA concentration of 200 pg/ml. The sample was adjusted to O-5 M-sodium acetate and precipitated with 2 vol. of ethanol. The precipitate was collected acetate. Five millicuries of L3H]dimethylby centrifugation and redissolved in O-5M-SOdiUm sulfate (spec. act. 385 mCi/mmol, New England Nuclear) was extracted from its shipping vial with a total of 2.0 ml of anhydrous ethyl ether and added to the aqueous RNA solution. The dimethyl-sulfate was forced into the aqueous phase by evaporating the ether with filtered Nz. The reaction mixture was shaken for 18 h at room temperature.
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The reaction was stopped by adding 2 vol. of ethanol and collecting the precipitate bg centrifuge&ion. The RNA was re-precipitated as described above until all radioactivity remaining in the solution was trichloroacetio acid-precipitable (4 precipitations). The RNA was then dissolved in water and stored at -20°C. The resulting 3H-labeled met,hylated RNA had a specific activity of 4860 cts/min/pg. When examined in a sucrose gradient with B’~cherichicc co& 23 S and 16 S ribosomal RNA it had sedimentation coefficients in $he range of 6 to 10 S. (e) DNA-RNA hybridization Hybridization was carried out according to Gillespie & Spiegelman (1965) with some modifications. DNA samples were denatured with 0.1 vol. of 1 m-Nash, cooled at O”@, neutralized with 0.1 vol. of 2 M-NaH,PO, and adjusted to 4 x SSC with cold 20 x SSC. Denatured DNA was adsorbed onto nictrocellulose filters (Schleicher and Schuell, E6), washed with 50 ml of 4 x SSC and dried first at room temperature for 1 h and then in a vacuum oven at 80°C for 2 h. The hybridization reaction was carried out at 70°C for 16 h in glass scintillation vials containing, unless otherwise specified, 2.0 ml of the RNA mixture. The reaction mixture contained the appropriate RNA, 4 x SSC a,nd 0.5% sodium dodecyl sulfate. The dodecyl sulfate lowered the non-specific binding of RNA to blank filters to less than 0.01 y0 of the input and did not alter the hybridization efficiency. All RNAs (labeled or unlabeled) used for hybridization were altered 3 times through nitrocellulose filters to remove residual basic proteins and any possible RNA containing poly(A) sequences. Filters were washed in batches of up to ‘75 filters/2 1 flask. Filters were first rinsed in 2 x SSC, then washed 3 times by shaking in 2 x SSC (20 ml/filter) for 10 min. Unhybridized RNA was eliminated by digestion with pancreatic RNase (25 fig/ml? 5 ml/filter) for 1 h at room temperature. The filters were again washed 3 times with 2 x SSC (20 rnli filter), dried and counted. All hybridization data were corrected for the binding of RNA to blank filters. (f ) Equilibriwm density gradient centr$%gation Analytical CsCl gradients were run in a Spinco model E ultracentrifuge at 44,000 revs,/ min for 20 h at 25°C. Buoyant densities were determined relative to the phage SPOl DNA (p = 1.742 g/cm2). Preparative CsCl gradients were run in a Spinco SW65 rotor using polyallomer tubes, Gradients of 4.0 ml were centrifuged for 42 h at 40,000 revs/mm ak 25°C. (g) Molecular weight determination of DNA The molecular weights of the various DNA preparations were determined by band sedimentation in 3 M-CsCl (p = I.38 g/cm2). so qrO, was calculated as described by Bruner & Vinograd (1965) and the so20,w values determined using a correction factor of 1.6. Freifeider’s (1970) equation was used to convert so20,w to molecular weight. (h) Preparatiort of DNAs of various molecular weights Chlosoplast DNA, isolated as described above, was sheared in high salt (77 M-CsCl) by passing the DNA through a 26-gauge needle 15 times. Sonicated chloroplast DNA was also prepared in 7.7 M-CsCl by subjecting the DNA solution to 4 bursts of 15 s each of the Bronson sonicator at position no. 7. The DNA was kept on an ice bath during both modes of shearing.
3. Results The average density
in CsCl of Euglena nuclear DNA is 1.707; of chloroplast DNA, composit,ion of the chloroplast ribosomal RNAs require a G f C content in DNA of 51o/o, corresponding to a density of 1.710 in CsCl. Thus, if a preparation of Euglena chloroplast DNA is partially contaminat,ed with nuclear DNA, and subjected t#o preparative centrifugation in a CsCl gradient in order to remove the contaminant, careful removal of material at density l-686 could result
1.686. The nucleotide
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in the preferential loss of ribosomal RNA genes at density greater than 1.686. But nuclear DNA must be removed, because chloroplast ribosomal RNA will anneal, albeit with reduced efficiency, with the nuclear genes for cytoplasmic ribosomal RNA (p = 1.722, G + C = 62%) (J. Rawson, unpublished observations). The development of a technique for the isolation of chloroplasts, and hence chloroplast DNA, uncontaminated by nuclear DNA, makes the requisite experiments feasible (Brown & Haselkorn, 1972). The distribution of Euglena chloroplast DNA, prepared from nuclear DNA-free chloroplasts, in analytical CsCl density gradients is shown in Figure 1. The gradient in Figure l(a) contained DNA as it is prepared from the chloroplasts; the average double strand molecular weight, based on zone centrifugation, was 1.25 x 10’. The material in (b) was sheared to a molecular weight of 3.3 x 10s ; in (c), the sample was sonicated to yield material with an average molecular weight of 6.9 x 105. The point of the demonstration is that lowering the average molecular weight of the chloroplast DNA permits the separation of a discrete class of DNA molecules, of density 1.700, from the bulk of the chloroplast DNA.
I.700
1.686
Buoyant density
FIG. 1. Densitometer tracings of analytical CsCl equilibrium density gradients of chloroplast DNA of varying molecular weights. DNA was prepared from Renografk-purified chloroplasts as described in the text. The DNA was sheared (b) or sonicated (c) and centrifuged to equilibrium. 1.5 ,~g of phage SPOl DNA (p = 1.742 g/ cmz) was used as the density reference. Patterns (a), (b) and (c) represent 2.5 pg chloroplast DNA of molecular weight 12.5 x 106, 3.3 x 10s and 6-9 x 105, respectively.
The same DNA samples were centrifuged in preparative CsCl gradients, and individual fractions were annealed with 3H-labeled chloroplast ribosomal RNA. In the high molecular weight sample, DNA sequences complementary to chloroplast ribosomal RNA are found in that part of the gradient containing most of the ohloroplast DNA (Fig. 2(a), fractions 13 to 15), as well as regions containing DNA of higher density, fractions 8 to 11. In the samples of DNB that had been sheared or sonicated
CHLOROPLAST
rRNA
GENES
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223
(Fig 2(b) and (c)), all of the DNA complementary to chloroplast ribosomal RNA is in a region of the gradient of density greater than that of the bulk of chloroplast DNA. We interpret these results to mean that some, and perhaps all, of the genes for chloroplast ribosomal RNA are located in the class of DNA molecules of density la700 released from the bulk of chloroplast DNA by shearing.
Y”
E
E
’
0
(b)
2 0
0 Fi Y
(c) 05-
- 150
B 2 p ,r
::1, ::
5
-
IO
15
100
-
Fraction no
FIQ. 2. tive CsCl legend to collected, with 5.0 --a--@--,
Hybridization of chloroplast ribosomal RNA to chloroplast DNA fractions from preparedensity gradients. Chloroplast DNA (15 pg/gradient) of varying molecular weights (see Fig. 1) was banded in 4.0 ml CsCl density equilibrium gradients. Fractions of 0.15 ml were denatured and fixed to nitrocellulose filters. The filters were annealed in batches of e pg [3H]CH,/ml chloroplast ribosomal RNA at 70°C for 16 h. ( L -4nso; 3H (cts/min).
The results shown in E‘igures 1 and 2 illustrate the point made in the Introdu&ion : if chloroplast DNA is purified by preparative CsCl centrifugation in order to isolate DNA of density l-686 for the determination of the number of ribosomal RNA genes, the determination will be low to the extent that ribosomal RNA genes are sheared from the bulk of chloroplast DNA. Since the method for preparation of chloroplast+z used in this work provides material free of nuclear DNA, we include the preparative &Cl gradient in the purification of chloroplast DNA only to remove protein and oligosibonucleotides. When filters containing chloroplast DNA were annealed with 9
130
J. R. Y. RAWSON
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increasing amounts of 3H-labeled chloroplast ribosomal RNA, the amount of RNA annealed, at saturating levels of RNA, corresponded to 1.9% of the DNA (Fig. 3). Assuming equal specific activities of the two ribosomal RNA species, equimolar ratios of the two RNAs (see Materials and Methods), and lOOo/oefliciency of hybridization at saturation, the value of 1.9% corresponds to one gene for each ribosomal RNA in each ‘ichromosome” of molecular weight 92 x 106.
0
02
04
06
’ IO
’ 20
’ 30
RNA/DNA OS IO ’ ’ 4.0 50 [RNA] pg/mt
II2
14
’ 60
7&i
16
FIG. 3. Saturation of chloroplast DNA with chloroplast ribosomal RNA. Chloroplast DNA (5.0 pg/filter) was annealed with varying amounts of [3H]CH, chloroplast ribosomal RNA (4860 cts/ min). Each point represents the average of duplicate determinations. 1.9% DNA saturation corresponds to 460 cts/min/filter.
4. Discussion The average number of cistrons for ribosomal RNA in each complement of chloroplast DNA can be determined by quantitative hybridization of ribosomal RNA with chloroplast DNA. The chloroplast DNA used for hybridization must be free of nuclear DNA, and must contain all the components of chloroplast DNA. Earlier estimates of the fraction of Euglena chloroplast DNA complementary to ribosomal RNA of 1.0 to 1.2% (Scott & Smillie, 1967; Stutz & Rawson, 1970) were low because the chloroplast DNA, taken from preparative CM3 gradients, lacked a portion of the chloroplast DNA rich in cistrons for ribosomal RNA. In a more recent report Stntz & Vandrey (1971) found that 6% of Euglena chloroplast DNA, prepared by another procedure that preserves (and possibly enriches) for the ribosomal RNA cistrons, hybridizes with chloroplast ribosomal RNA. We find it difficult to evaluate this report. Stutz & Vandrey prepare Euglena chloroplasts by flotation on a gradient of Ludox, and then chromatograph the chloroplast DNA on a column of methylated albumin. Approximately 20% of this DNA is the satellite at p = l-701. Nevertheless, their value of 6% hybridization with chloroplast ribosomal RNA is more than three times higher than we have ever observed for any chloroplast DNA preparation. Stutz & Vandrey (1971) carry out DNA-RNA hybridization in 2 x SSC at 65”C, while our conditions are somewhat more stringent, 4 x SSG at 70°C. This is not likely to be a significant difference, since we found the extent of hybrid formation
CHLOROPLAST
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GENES
OF E, CRACIEIX
131
between EugEena cytoplasmic ribosomal RNA and total Euglena DNA to be independent of temperature between 66 and 74°C in 4 x SEX (J. Rawson & R. Brown, ~np~bhshed observations). ~~~e~ chloroplasts contain DNA molecules that can be isolated as covaIent1~ closed, supercoiled circles (Manning & Richards, 1972a). Up to one-third ofthe chloroplast DNA is found as circles or as linear molecules of length equal to that of the circles; the remainder are linear, distributed about means corresponding to haK or quarter length molecules. Half or quarter length circles are not observed. Since a reasonable proportion of big circles can be preserved, the absence of circles from the populations of shorter molecules is interpreted to mean that all of the shorter moleaules are derived by breakage from full length circles. How many classes of full length circles are there? The molecular weight of a single cireIe is 92x 1~3. The oomplexity of Euglenu chloroplast DNA was originally determined by renaturation kinet,ics to be 180x 10” (Stutz, 1970), but this result did not take into account the very low G + C content of that DNA. It has been suggested (Manning et al,, 1971) that a, correction for the G + C content would bring the complexity down to 92 But we know very little about the distribution of G-C pairs in the chloroplast and, in the absence of such information, it would seem that any “correct~ion” is risky. If we assume that the kinetic complexity is actually 92 x 106, then there can be only one class of DNA molecules, and each molecule must contain one cistron for each cbloroplast ribosomal RNA species. Suppose the complexity is 180x lOa, then there are two classes of DNA molecules ; each could contain one cistron for each ribosomal RNA, or one DNA molecule could contain two cistrons for each RNA. In the latter case, the ribosomal RNA cistrons cannot be repeated in tandem, for the following reason: from the base composition of chloroplast ribosomal RNA, DNA coding for chloroplast ribosomal RNA must have a buoyant density of 1.710. When chloroplast DNA is sheared to an average molecular weight of 3-3 x 106, approximately the size of one set of ribosomal RNA cistrons, the DNA has an average buoyant density of 1.700 (Fig. 2(b)). Therefore the ribosomal RNA cistrons must be linked at both. ends to DNA of very low G + C content (p = l-690), and they cannot be joined in tandem. Manning & Richards (19723) report that the proportion of Euglenu chloroplast NA found in the satellite at p = I.700 is higher by 50% in cells growing exponentially than in cells grown into stationary phase. In general, one expects the two situa tions to differ by virtue of the fact that DNA molecules are replicating in cells growing exponentially, but are completed in stationary phase cells (Yosbikawa & Sueoka, 1963)” If the 25% of the completed ohloroplast DNA molecule found in the p L- P-700 satelhte is closest to the origin of DNA replication, then Manning & Richards’ (197%) observation is easily understood: the satellite will comprise, on the average, 40% of the DNA in growing DNA molecules. It is not necessary to postulate independent replication of the satellite. The assumption that the satellite is that part 01 the DN.L4 closest to the origin of replication is not unreasonable; in Bacillus subtilis the genes for ribosomal RNA are very close to the chromosome origin (Qishi & Sueoka, 1965). While the p = L-700 satellite contains the genes for ribosomal RNA, those genes are onIy a small part (
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The total number of genes per cell for chloroplast ribosomal RNA is quite high. Exponentially growing Euglem contain approximately 350 molecules of chloroplast DNA (0. Richards, personal communication), each of which contains one cistron for ribosomal RNA. This is nearly as many as the nuclear genes for cytoplasmic ribosomal RNA, which we estimate to be 450 (J. Rawson & R. Haselkorn, unpublished observations). The chloroplast genes for ribosomal RNA are subject to regulation; they are rarely transcribed in the dark (Brown & Haselkorn, 1971); in the light, chloroplast ribosomal RNA synthesis accounts for 10% of the cell’s ribosomal RNA. Presumably initiation is less frequent on the chloroplast ribosomal RNA cistrons than on their nuclear counterparts. The number and distribution of ribosomal RNA genes on ehloroplast DNA from other organisms is similar to what we find for Euglena. In each case the major uncertainty is the size of the chloroplast “chromosome ” : measurements of the kinetic complexity of chloroplast DNA from Chlamydomonas (Bastia et al., 1971), tobacco (Tewari & Wildman, 1970) and lettuce (Wells & Birnstiel, 1969) all give values around 200x 106. However, as is true for Euglena, the unknown contribution of nucleotide sequence distribution to the renaturation kinetics introduces an uncertainty of perhaps a factor of two in the “chromosome” size. In each case chloroplast ribosomal RNA
hybridizes with around 1% of the chloroplast DNA, leading to the conclusion that each “chromosome” contains one to three cistrons for ribosomal RNA. This work was supported by grant GB17514 from the National Science Foundation. One of us (J. R. Y. R.) was a postdoctoral fellow of the National Institutes of Health. REFERENCES Bastia, D., Chiang, K.-S., Swift, H. & Siersma, P. (1971). Proc. Nat. Acad. Sci., U.X.A. 68, 1157.
Brown, R. D. & Haselkorn, R. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 2536. Brown, R. D. & Haselkorn, R. (1972). &o&m. Biophys. Acta, 259, 1, Bruner, J. & Vinograd, J. (1965). Biochim. Biophys. Acta, 108, 18. Freifelder, D. (1970). J. Mol. Biol. 54, 567. Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829. Ingle, J., Possingham, J. V., Wells, R., Leaver, C. J. & Loening, V. E. (1970). Xymp. Sot. Exptl. Biol. 24, 303. Manning, J. E. & Richards, 0. C. (1972a). Biochim. Biophys. Acta, 259, 285. Manning, J. E. & Richards, 0. C. (19726). Biochemistry, 11, 2036. Manning, J. E., Wolstenholme, D. R., Ryan, R. S., Hunter, J. A. & Richards, 0. C. (1971). Proc. Nat. Acad. Sci., U.S.A. 68, 1169. Oishi, M. & Sueoka, N. (1965). Proc. Nat. Acad. Sci., U.S.A. 54, 483. Rawson, J. R. & Stutz, E. (1969). Biochhim. Biophys. Acta, 190, 368. Scott, N. S. & Smillie, R. N. (1967). Biochem. Biophys. Res. Commua. 28, 598. Smith, K. D., Armstrong, J. L. & McCarthy, B. J. (1967). Biochim. Biophys. Acta, 142, 323.
Stutz, E. (1970). E%BS Letters, 8, 25. Stutz, E. & Rawson, J. R. (1970). Biochim. Biophys. Acta, 209, 16. Stutz, E. & Vandrey, J. P. (1971). FEBS Letters, 17, 277. Tewari, K. K. & Wildman, S. G. (1970). Symp. Sot. Exptl. Biol. 24, 147. Wells, R. & Birnstiel, M. (1969). Biochem. J. 112, 777. Yoshikawa, H. & Sueoka, N. (1963). Proc. Nut. Acad. Sci., U.S.A. 49, 559.