The investigation of the ribosomal RNA sites in yeast DNA by the hybridization technique

416

BIOCHIMICA ET BIOPHYSICAACTA

BBA 96038

T H E INVESTIGATION OF T H E RIBOSOMAL RNA SITES IN YEAST DNA BY T H E H Y B R I D I Z A T I O N T E C H N I Q U E

J. RETIkL AND R. J. PLANTA Biochemisch Laboratorium, Vrije Universiteit, Amsterdam (The Netherlands) (Received July 4th, I968)

SUMMARY

By the hybridization technique of GILLESPIE AND SPIEGELMAN,the fraction of Saccharomyces carlsbergensis DNA complementary to homologous ribosomal RNA (rRNA) was found to be 1. 9 % corresponding to a relatively high number of rRNA cistrons on yeast DNA. Possible contributions to this high hybridization percentage by contaminating messenger RNA (mRNA) or by contaminating mitochondrial nucleic acids could be excluded. The formed yeast rRNA-DNA hybrid proved to have a high thermal stability and a narrow melting range, demonstrating its great homogeneity. The two rRNA components (17 S and 26 S), purified by disc-gel electrophoresis, showed considerable cross-hybridization, indicating that the base sequences of the two RNA components are very similar.

INTRODUCTION B y means of hybrid formation between DNA and radioactive-labeled homologous RNA, it has been shown that about 0.3--0. 4 % of the total genome in bacteria is complementary to ribosomal RNA (rRNA). This indicates more than one site for each rRNA component in bacterial D N A I - ! About the same fraction of the DNA from plants s and insects 6,7 forms specific hybrids with homologous rRNA, whereas, the hybridization percentage for r~RNA in mammalian cells varies from 0.03 to 0.005% (refs. 8-1o). These values for higher organisms correspond to a much larger number of rRNA cistrons than in bacteria, as the molecular weight of the DNA in more complex organisms shows a progressive increase. Yeast has a relatively low content of DNA per cell with a molecular weight approximately ten times higher than that in bacteria. Yeast, however, is characterized by a high rate of rRNA synthesis proceeding b y a mechanism resembling that of higher organisms n, and b y a high content of cytoplasmic ribosomes. These data might point to a relatively high proportion or rRNA cistrons in yeast. We have investigated the number of rRNA sites in yeast D N A by the technique Abbreviations: rRI~A, ribosomal RNA; mRNA, messenger RNA; SSC buffer, o. 15 M NaClo.o15 M sodium citrate. Biochim. Biophys. Acta, 169 (I968) 416-429

RIBOSOMALRNA SITES IN YEAST DNA

417

of hybridization as described by GILLESPIE AI~D SPIEGELMANTM. The experimental data presented in this paper, show that about 2 % of the yeast DNA is complementary to rRNA, corresponding to a number of rRNA cistrons in yeast comparable to that of higher organisms. In addition it appears from competitive hybridization experiments, that there is a great structural similarity between the sites for each of the two rRNA components.

MATERIALS AND METHODS

Strains The strains of Saccharomyces carlsbergensis used in these studies were N'o. 74 from the British National Collection of Yeast Cultures and Sce/I/317, a methioninerequiring mutant. Bacillus licheni]ormis S 244 (wild type) was kindly provided by Dr. A. H. STOUTHAMER (Botanical Laboratory, Vrije Universiteit, Amsterdam, The Netherlands). Petite mutants of S. carlsbergensis No. 74 were prepared by means of acridine orange according to a method essentially the same as described by BOLDEIO3. Petite mutants thus obtained had the following characteristics: they form small colonies on agar plates; do not consume oxygen; lack cytochrome a and b; and are not able to grow in a medium with glycerol as the only carbon source. All yeast cultures were grown in the media as previously described 1~, unless otherwise indicated. B. licheni]ormis was grown in brain-heart infusion b~oth at 37 °.

Reagents EMe-3H]Methionine (3.0 C/retool) was obtained from Schwarz BioResearch Inc. (Orangeburg, N.Y., U.S.A.); EaFI]uridine (2.73 C/retool) from the Radiochemical Centre (Amersham, England); E3~p]phosphate from N.V. Philips-Duphar (Petten, The Iqetherlands). Bovine pancreatic ribonuclease (EC 2.7.7.16) (Type Ilia), micrococcal deoxyribonuclease (EC 3.1.4.5) (Grade II, ribonuclease-free)and calfthymus DNA (Type I) were purchased from Sigma Chemical Company (St. Louis, Mo., .U.S.A.); Tl-ribonuclease (EC 3.1.4.8) (B grade, prepared by the Sankyo Co. from Takadiastase) and pronase (B grade) from Calbiochem. AG (Luzern, Switzerland). Acrylamide and N,N'-methylenebisacrylamide were purchased from Fluka AG (Buchs, Switzerland); macaloid (Langer and Co, Ritterhude, W.-Germany) was prepared for use as previously reported 11.

Isolation o! DNA Yeast protoplasts, prepared from a yeast culture as described previously11, were carefully lyzed in 3 × SSC buffer, o.oi M EDTA, (pH 7.5) (SSC buffer is o.15 M NaCl-o.oI5 M sodium citrate) containing per 12oo g sucrose and 2o g sodium dodecyl sulphate, by incubating for 3 h at room temperature, with occasional shaking. From these lysates the nuclear DNA was isolated according to a modification of the method of MA~MUR14; the purification procedure included an extraction with phenol at 5o °, precipitation with 2 vol. of 96 ~o ethanol at --2o ° and removal of RIgA by a combined treatment with pancreatic ribonuclease (5oo/~g/ml, free of deoxyribonuclease) Biochim. Biophys. Acta, I69 (1968) 416-429

418

j . RETEL, R. J. PLANTA

and T1 ribonuclease (250 units/ml, free of deoxyribonuclease) in o.I × SSC, I mM EDTA, (pH 7.5), followed by pronase digestion and extractions (3-5 times) with phenol at room temperature. DNA was further purified by repeated precipitation with 0.54 vol. of isopropylalcohol and winding on a glass rod. The DNA thus obtained, still contained about ~o % RNA. This contamination was completely removed by hydrolysis at pt{ 13 of 20 h at room temperature. Subsequent dialysis proceeded for 12 h against o.I ×SSC. Isopycnic CsCI centrifugation at 25 ° according to FLA~M, BOND AND BURR~5 showed, that the pure DNA bands as a broad peak at a buoyant density of 1.7o2 g/ml (Fig. i) in close agreement with the value reported by lgouNo-

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Fig. I. Density-gradient centrifugation of yeast DNA. 95/~g y e a s t DNA was added to 3.5 ml of a CsC1 solution in o.i × SSC with a density of 1.7Ol g/ml. Centrifugation was performed in the SW 39 rotor of tile Spinco model L ultracentrifuge a t 33 ooo rev./min for 6o k at 25 °.

LOU, JAKOB AND SLONIMSKI~6 for nuclear yeast DNA, but different from the value (1.692) found by TEWARI, V6TSCH AND MAHLER17. DNA from B. licheni[ormis was prepared, after disruption of the cells b y grinding them with alumina, in essentially the same way as described above. Calf-thymus DNA was purified by treatments with ribonuclease and pronase, phenolextractions, followed by precipitation with isopropylalcohol as described above.

Isolation o/rRNA Protoplasts were lyzed at o ° in o.oi M Tris-HC1 (pH 7.7), o.oi M NaC1, o.oi M MgC12, 0.5 % Triton X-Ioo containing per 1:2 ml of pure fl-mercaptoethanol (Sigma) and 3 ml of macaloid suspension (15 mg/ml). After centrifugation at 3° ooo × g at 4 ° for 30 rain, the ribosomes were sedimented from the supernatant at 200 ooo × g for 60 rain at 4 ° in the SW 50 rotor of the Spinco model L-2 ultracentrifuge. The rRNA components were extracted from the pellet by p h e n o l - 1 % sodium dodecyl sulphate treatment at o ° and purified by centrifugation through a 5-2o % sucrose gradient n. In some cases further purification was carried out by polyacrylamide gel electrophoresis according to the method of LOENIN~is. Complete separation of the two rRNA components was achieved by electrophoresis on 2.6 % polyacrylamide gels (with o.13 % methylene bisacrylamide as a cross-linker). The separated rRNA's were reBiochim. Biophys. Acla, I69 (1968) 416-429

RIBOSOMALRNA SITES IN YEAST DNA

419

covered from the gels by closing the lower end of the gel cylinders with a dialysis membrane and allowing the fastest moving component to run off the gel; the electrophoresis was then interrupted and the I~NA component, present in only a small volume (o.2 ml) of electrophoresis buffer, could be collected. The other ribosomal RNA component was next isolated in tile same manner. 32P-labeled rRNA was obtained from yeast and from B. licheni]ormis by growing the organism in the presence of [~2Plphosphate (o.7 ffC/ml of medium). [3Hlmethyl-labeled yeast rRNA was prepared from the methionine-requiring mutant, which has been starved of methionine for 3 h; the methionine-deprived cells were converted into protoplasts, which were then preincubated at 29 ° in the methionine-free culture medium for 45 min (ref. II), and subsequently labeled with [Me-3H]methionine (4 #C/ml; 3.o C/mmole) for 2 h.

Sedimentation characteristics o/ the yeast rRNA components I m g unlabeled yeast rRNA and appropriate small amounts of a~P-labeled rat liver rRNA and [~H]uridine-labeled B. licheni/ormis RNA in I ml of o.I M ~aC1, o.oi M disodium-EDTA (pH 5.o), were layered on the top of a linear 5-zo % sucrose gradient made up in o.i ~ NaC1, o.oi M disodium-EDTA (pH 5.0). Sedimentation coefficients and molecular weights were determined by centrifugation at 22 500 rev./ min in the SW 25-1 rotor of the Spinco model L ultracentrifuge at 4 ° according to the method described by CLICK AND TI~T~9. The sedimentation pattern after centrifugation for 18 h is presented in Fig. 2. Using B. licheni/ormis rRNA (16.5 and 23.5 S)

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F i g . 2. D e t e r m i n a t i o n o f t h e s e d i m e n t a t i o n c o e f f i c i e n t o f y e a s t r R N A ' s b y s u c r o s e g r a d i e n t a n a l y s i s . F o r d e t a i l s see t h e t e x t . - - - - - - , u n l a b e l e d y e a s t r i b o s o m a l R N A ; - - , 82P-labeled rat liver r R N A ; . . . , [ a H ] u r i d i n e l a b e l e d B. licheni/o~mis r R N A .

Biochim. Biophys. Acta, 1 6 9 ( 1 9 6 8 ) 4 1 6 - 4 2 9

420

j. RETEL, R . J . PLANTA

as a marker, the sedimentation coefficients for yeast rRNA were determined to be 17 and 26.5 S. For rat liver rRNA, used as a control in the same run, the values were 18. 7 and 31.7 S in agreement with the results of CLICK aND TINT19. The molecular weights of the two yeast rRNA components were calculated to be 1.24" lO6, resp. o.6o.lo 6 daltons, in close agreement with the values determined by ]3RUENING AND BOCKz° by means of analytical ultracentrifugation.

Hybridization The hybridization technique used was the nitrocellulose-membrane procedure of GILLESPIE AND SPIEGELMAN12 with some modifications. Alkaline-denatured DNA at a concentration of 25/,g/ml was immobilized on nitrocellulose filters (Membranfilter MF 30, 24 mm diameter) in the presence of 3 × SSC (pH 6.9). Usually 80-90 % of the DNA was retained on the filter. Filters, charged with 60/~g DNA were incubated with radioactive-labeled RNA in 2 ml of 3 × SSC (pH 6.9) at 63 ° for 16 h in a stoppered scintillation vial. After hybridization the filters were washed extensively with 3 ×SSC (pH 6.9) and treated with ; o # g pancreatic ribonuclease (flee of deoxyribonuclease) and 50 units T 1 ribonuclease (flee of deoxyribonuclease) in 2 ml of 2 × SSC (pH 7.6) at 3 °0 for I h and again washed. Prior to counting in a Nuclear Chicago (model 725) liquid scintillation counter, DNA and RNA were digested with deoxyribonuclease (6/~g/m]) and ribonuclease (IO/~g/ml) in I × SSC, o.I mM MgC12 (pH 7.3) in order to enhance the counting efficiency in Bray's liquid. To correct for unspecific adsorption filters charged with calf thymus or B. licheni/ormis DNA were also incubated. This background represented about 4-5 % of the radioactivity, found cn the filters with homologous DNA.

RESULTS

Characteristics o~ the hybridization reaction In studies of DNA-RN'A hybrid formation, a careful evaluation of the experimental conditions is required, as several factors may influence the hybridization result. It is well known that the D~A employed must be completely denatured, and that the DNA and RNA used must be free of (basic) protein and especially of ribonuclease 12. Moreover, the hybridization efficiency and the properties of the hybrid depend on the reaction conditions employed during the hybrid formation. This raises the question whether the hybridization reactions are completely locusspecific. It was suggested 1~ that treatment with pancreatic ribonuclease is necessary for removal of unpaired RNA regions in the hybrid complex and for reduction of the non-specific adsorption of RNA. On the other hand, it was stated by other authors ~1 that treatment with ribonuclease causes also a reduction in the percentage hybridization of true homologous reactions. The influence of ribonuclease treatments in our hybridization system is shown in Table I. Since the saturation plateau for rRNA in bacterial systems had already been carefully examined ~,lg, it was adopted as a useful control system. As was to be expected from previous work (e.g. ref. 12) treatment with pancreatic ribonuclease reduced considerably the level of labeled RNA complexed with DNA. However, Biochim. Biophys. Acta, 169 (1968) 416-429

RIBOSOMAL R N A SITES IN YEAST D N A

421

TABLE I EFFECT

OF TREATMENT

WITH

RIBONUCLEASE

ON THE PLATEAU

VALUE

2o/~g a2P-labeled rlRI~A isolated f r o m B. licheni]ormis, resp. S. carlsbergensis w a s hybridized with 6 o # g h o m o l o g o u s D N A immobilized Oil a MF 3 ° filter in 2 ml of 3 × SSC for 16 h at 63 °. Some of t h e filters were t h e n t r e a t e d w i t h pancreatic ribonuclea~e (IO/~g/ml, i h), o t h e r s w i t h pancreatic ribonuclease ( i o # g / m l ) and T t ribonuclease (25 u n i t s / m l ) for i h. The filters were assayed for szp c o u n t s as described in MATERIALS AND METHODS.

Organism

Ribonuclease treatment

% r R N A to D N A in hybrid

B. licheni/ormis

None Pancreatic Pancreatic Pancreatic Pancreatic

1.4 o. 7 o.4 2.8 1.9

S. carlsbergensis

ribonuclease ribonuclease + T t ribonuclease ribonuclease ribonuclease + T 1 ribonuclease

under our conditions this level could be reduced still further for both systems investigated by treatment with T x ribonuclease. The hybridization percentage of 0.4 %, then obtained for the B. licheni[ormis system, is in close agreement with the values reported for other members of the genus Bacillus s,19. Therefore,°,we considered the combined treatment with pancreatic ribonuclease+T 1 ribonuclease as essential, and in subsequent experiments the combined ribonuclease treatment was included as a routine step. It should be noted that a reiteration of the ribonuclease treatment did not change the hybridization percentage further, indicating that the label persistently retained represents a stable kind of hybrid. When increasing amounts of szP-labeled yeast rt~NA were annealed with a fixed amount of homologous DNA, a saturation plateau of the hybrid RNA counts on the filters was reached at a RNA/DNA input ratio of about o.I (Fig. 3). Varying between 1.8 and 2.1%, with an average of 1. 9 %, the percentage of yeast DNA

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Fig. 3. D e t e r m i n a t i o n of the p l a t e a u value for t o t a l r R N A (26 S + 17 S). 60 # g S. carlsbergensis D N A immobilized on a MF 3 ° filter w a s hybridized w i t h v a r y i n g a m o u n t s of h o m o l o g o u s s2p_ labeled r R N A . F o r f u r t h e r details see MATERIALS AND METHODS. Fig. 4. T h e r m a l dissociation curve of t h e y e a s t r R N A - D N A hybrids. H y b r i d i z a t i o n w a s perf o r m e d as described in MATERIALS AND METHODS. After t h e c o m b i n e d t r e a t m e n t w i t h pancreatic ribonuclease and T 1 ribonuclease, the filters were h e a t e d for io mitt in 2 ml of 2 × SSC (pH 6.9) in a s t o p p e r e d scintillation vial to v a r i o u s t e m p e r a t u r e s as indicated in t h e figure. The filters were t h e n t r e a t e d agairt w i t h p a n c r e a t i c ribormclease ( i o / t g / m l ) and Tx ribonuclease (25 u n i t s / m l ) , w a s h e d and a s s a y e d for 8~p counts.

Biochim. Biophys. Acta, 169 (1968) 416-429

422

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RETI~L, R. J. P L A N T A

apparently coding of rRNA is rather high as compared with the values reported for other organisms. Now McCARTHY~ has drawn attention to the possibility that for higher organisms the maximal hybridization percentage will provide an overestimate of the proportion of DNA sites actually responsible for the synthesis of the RNA concerned. Because of the existence of related base sequences in the DNA of higher organisms, the hybridization reaction is not only an association between a gene and its RNA product as in bacterial systems, but also one between an RNA molecule and a DNA segment similar but not identical to the DNA site, which produced that RNA molecule. So DNA-RIgA hybrids of various degrees of mismatching may be formed. The heterogeneity and the stability of the total hybrid complex in mammalian systems was shown to be dependent on the hybridization conditions ~2. Therefore, we have investigated a possible influence of the hybridization conditions upon the saturation plateau for yeast rRNA. As shown in Table II, variation within the limits TABLE

1I

I N F L U E N C E OF HYBRIDIZATION CONDITIONS U P O N T H E P L A T E A U V A L U E I~F 30 filtersare charged with 6o ~ g D I ~ A from S. carlsbeygensis and incubated with homologous a2P-labeled r R N A under different hybridization conditions as indicated in the Table. For further details see MATERIALS A N D METHODS.

Hybridization conditions

% r R N A to DNA in hybrid

Salt concn.

Temp.

Time

i × SSC 3 × SSC 6 × SSC

63 ° 63 ° 63 °

16 16 16

1.8 2.0 2.0

3 x SSC 3 >< SSC 3 x SSC

60 ° 65 ° 7 °0

16 16 16

2.o 2.0 1.9

3 x SSC 3 × SSC 3 × SSC

63 ° 63 ° 63 °

16 24 4°

1.9 1.8 1.8

(h)

applied of the conditions such as salt concentration, temperature and time of hybridization, had no significant effect on the plateau value. In addition, the properties of the DNA-RNA hybrid formed under standard conditions (3 x SSC, 63 °, 16 h) was assessed b y means of its thermal stability (Fig. 4). The thermal dissociation of the yeast r~RNA-DNA complex in 2 × SSC occurred over a small temperature range, indicating a considerable homogeneity of the hybrids in guanine plus cytosine (G + C) content. The high thermostability of the hybrid, characterized b y a Tm (or midpoint of the dissociation curve) of approx. 89 °, indicates too that the association between yeast ribosomal RNA and homologous DNA is rather specific. The Tm found is close to that, which might be predicted from the base composition of yeast ribosomal RNA n. So it seems reasonable to assume that the saturation value found is a true estimate of the fraction of the yeast genuine actively synthesizing rRNA. However, we considered the possibility that the high hybridization percentage might be due Biochim. Biophys. Acta, 169 (1968) 4 1 6 - 4 2 9

RIBOSOMAL RNA SITES I~ YEAST DNA

423

to contamination by messenger R N A - D N A hybrids or by hybrids of mitochondrial nucleic acids.

Possible contamination by m R N A mRNA represents only a small percentage of total cellular RNA, but it is complementary to a large proportion of the genome. Consequently, the hybridization efficiency for mRNA is much higher than for rRNA. At a sufficiently low RNA/DNA input, e.g. in the range used in the saturation experiments with rRNA (Fig. 3), all mRNA present will be bound. So, only a slight contamination of the rRNA preparation by mRNA would strongly increase the hybridization value. However, if the r~RNA preparation contains mRNA, no definite saturation plateau as in Fig. 3 would be achieved. Since the opposite was found, a strong contamination of the rRNA b y mRNA is not very likely. This conclusion is supported b y the results of the thermal dissociation studies (Fig. 4), unless contaminating mRNA-DNA hybrids have the same G + C content as the r~RNA-DNA hybrids. In order to distinguish any possible influence of mlZNA contamination on the hybridization results, different approaches have been applied. First we tried to remove from the yeast ribosomes possibly adherent mRNA by a short treatment with pancreatic ribonuclease or b y dialysis of the ribosome suspension against an EDTAcontaining buffer. However, the rRNA isolated from these treated ribosomes turned out to be broken down extensively. Next we tried to remove possible mRNA contamination from our rRNA preparation b y other means. 32P-labeled rRNA, partially purified b y centrifugation through a 5-20 % sucrose gradient, was subjected to disc electrophoresis on 2.6 °/o polyacrylamide gels. In this way the two ribosomal RNA components were completely separated from each other and, more important, from possible RNA contaminants having a different chain length. Both rRNA components were collected separately from the gels as described in MATERIALS AND METHODS and used for competition experiments (described later), or combined for the determination of the saturation plateau. This improved purification did not have any effect on the saturation-plateau value found before. Lastly we repeated the saturation experiments with r~RNA labeled in vivo with EMe-3Hlmethionine. It is believed that rRNA and transfer RNA are methylated in vivo, while mRNA is not 23. Consequently, annealing DNA with a E3H;methyllabeled rRNA preparation will yield a plateau value, which does not contain a contribution of the mlZNA possibly present. Since we found in this way exactly the same plateau value as with 82P-labeled rRNA, the conclusion that contamination b y mRNA does not contribute at all to the high plateau value seems to be justified.

Possible contamination by mitochondrial nucleic acids Both DNA and rRNA used for the hybridization experiments, might be contaminated b y the corresponding mitochondrial nucleic acids. Assuming that mitochondrial DNA codes for mitochondrial rRNA, it can be calculated that a single set of rRNA cistrons would represent at least IO % of the mitochondrial genome ~. Therefore, already a slight contamination of our DNA preparation b y mitochondrial DNA might make a considerable contribution to the hybridization perBiochim. Bioph.vs. Acta, 169 (1968) 416-429

424

j . R E T E L , R. J . P L A N T A

centage under investigation, if our r R N A preparation also contained mitochondrial rRNA. DNA prepared as described under MATERIALSAND METHODS from yeast grown under standard conditions 11 gave an asymmetrical band in preparative equilibrium density-gradient centrifugation in CsC1 at 1.7o2 g/ml (Fig. i). Essentially the same pattern was obtained b y analytical CsC1 equilibrium density-gradient centrifugation in the Spinco Model E analytical ultracentrifuge equipped with ultraviolet optics. Although no distinct band was observed at 1.687 g/ml, the density of yeast mitochondrial DNA ~, there was some tailing in this region of the gradient, which might be derived from a small amount of contaminating mitochondrial DNA. Since the absence of mitochondrial DNA in our DN'A preparations cannot be established with certainty b y CsCl-gradient centrifugation, we looked at the problem from the RNA side. We therefore investigated the hybridization percentage for r:RNA isolated from yeast cells grown under various conditions and from cells with a different content of mitochondria. The results are summarized in Table I I I . The same hybridization TABLE III rRNA OBTAINED FROM DIFFERENT YEAST CULTURES 82P-labeled RNA was prepared from: (I) vigorously aerated yeast cultures in media n containing 1% glucose; (2) yeast cells, grown under strict anaerobic conditions in the same media but with 5 % glucose; (3) petite mutants. Di%A was isolated from a yeast (S 74) culture as described in THE PLATEAU VALUE FOR

MATERIALS AND METHODS.

Growth conditions

Aerobiosis --glucose repression Anaerobiosis +glucose repression Petite mutants

% rRNA to DNA in hybrid

1.9 1.9 2.0

plateau value was found for rRNA isolated from cells grown under strictly aerobic conditions in a glucose-poor medium and hence with a relatively high content of mitochondria, and for r R N A from cells in which the biogenesis of mitochondria was strongly suppressed b y anaerobiosis in a glucose-rich medium, and for rRNA from petite mutants which have no functioning mitochondria at all. From these findings we conclude that the high hybridization plateau value found for yeast rRNA does not contain any contribution from mitochondrial components. Evidently, the nuclear DNA from yeast contains segments complementary to rRNA, representing about 1.94-0.1% of the total nuclear genome. Thus assuming a molecular weight of 13 • lO 9 daltons for yeast nuclear DNA ~5, and a molecular weight of 1.2 and 0.6.10 s daltons, respectively, for the two rRNA species (see MATERIALS AND METHODS), the yeast genome m a y be calculated to possess about 140 cistrons each coding for a single set of rRNA. This number is comparable to that ot higher organisms.

Competition in hybridization between the two r R N A species

In order to investigate the hybridization characteristics of the two separate Biochim. Biophys. Acta, 169 (1968) 416-429

RIBOSOMAL R N A SITES IN YEAST D N A

425

rRNA species, 26-and I7-S rRNA, were highly purified and completely separated from each other by means of polyacrylamide-gel electrophoresis on 2.6 % gels as stated before. The proportion of ribonuclease resistant hybrid formed by annealing a fixed amount of yeast DNA with increasing amounts of each rRNA component are shown in Fig. 5. The saturation plateaus for the two rRNA species are nearly alike; they are, identical to the plateau value found for both components together (c/. Fig. 3). These findings suggest that, under the conditions employed, both rRNA A

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Fig. 5. Determination of the plateau value for each of the two rRBIA components purified by polyacrylamide gel electrophoresis. For details see MATERIALS AND METHODS (¢t" also Fig. 3). I - O , 26-S RNA; C)-- -O, I7-S RNA. Fig. 6. Competition in hybridization of I7-S and 26-S rRNA. Hybridization of mixtures of (A) zzP-labeled 26-S r R N A and unlabeled I7-S rRNA, or (B) 3*P-labeled I7-S r R N A and unlabeled 26-S rRNA, was carried out at a labeled RNA/DNA input of o. 3 (cf. Fig. 3). The hybridization value in the presence of cold R2CA as percentage of the control (without cold RNA) is plotted against the percentage of cold RNA present in the RNA mixture, - - , theoretical curve for complete competition; • - - - O, experimental curve.

species can form hybrids with the same DNA segments. If this assumption is correct, the two £RNA components will compete with each other in hybrid formation. The results of competitive hybridization experiments with nonradioactive I7-S rRNA and radioactive 26-S rRNA, and vice versa, are presented in Fig. 6. Using sufficiently high labeled-RNA/DNA inputs to attain saturation, the relative hybridization value in the presence of a given amount of unlabeled competitor RNA will be linearly related to the fraction of unlabeled RNA in the mixture (labeled plus unlabeled ~RNA) according to the equation H L H ~ - - L-FU -- I

U L+U

where FI and I-£mare the hybridization values in the presence and in the absence of unlabeled competitor RNA, and L and U are the amounts of labeled and unlabeled RlffA, respectively. If the labeled and unlabeled RNA compete completely for the same sites on DNA, a plot of H/Hm versus U / L + U will intersect the ordinate and the abscissa at IOO (%). If the two R1VA components give only partial cross-hybridization, the linear plot will still intersect the ordinate at IOO,but the slope will be less than in the case of complete competition. The slope is then a measure for the degree of similarity in base sequences of the two R1VA components. As can be seen, highly purified unlabeled I7-S rRNA competes almost completely with highly purified, labeled Biochim. Biophys. Aeta, 169 (1968) 416-429

426

j. RETEL, R.J. PLANTA

26-S rRN'A (Fig. 6A), and vice versa (Fig. 6B). Tiffs indicates that the base sequences of 26-S rRNA and I7-S rRNA from yeast are very similar. The fact that I7-S rRNA can almost completely supersede 26-S rRNA in the hybridization reaction, even when the mixture comprises more than 50 % of I7-S rRNA, suggests that the 26-S rRN'A consists of two regions, both very similar in base sequence with I7-S rRNA. The similarity of base composition for both rRNA components 11 is consistent with this assumption. On the other hand, evidence was obtained that the base sequences of 26-S and I7-S yeast rRNA are not completely identical; disc electrophoresis on polyacrylamide gels of T 1 ribonuclease digests of purified 26-S and I7-S RNA revealed the existence of some differences26. DISCUSSION

The formation of stable hybrids of RNA with denatured strands of homologous DNA provides a valuable method for estimating the proportion of the genome specific for a class of RNA. However, as already stated by 1VfcCARTHY22, the interpretation of studies of DNA-RNA hybrid formation requires some caution, especially in systems of higher organisms. The hybridization reactions between RNA and homologous DNA from higher organisms are not completely locus-specific, because of the existence of families of related base sequences on the genome of higher organisms. The predominant reaction product formed in this case is one between the RNA in question and DNA stretches which are not completely complementary to that RNA. Since the members of such families of related genes possess various degrees of similarity of base sequence, hybrids of various degrees of mismatching may be formed. The properties of this hybrid complex depend on the reaction conditions employed during its formation. Under less stringent conditions, such as temperatures insufficiently high, the range of unspecific hybrids formed is rather extensive. Now, the degree of specificity of the hybrid complex formed may be assessed by means of its thermal stability. The temperature at which a hybrid dissociates at a given salt concentration is a function of its base composition and the degree of base pairing which exists; and the slope of the thermal transition is a measure for the degree of heterogeneity of the hybrids present. This kind of thermal dissociation study led McCARTHY2~ to assume, that the many rRN'A cistrons in mammalian DNA are similar, but not identical, in sequence, so that hybridization occurs between rRNA having one base sequence and a DNA gene representing another rRNA species. Our studies on the hybridization of yeast rRNA with homologous DNA indicate that a relatively high proportion of the yeast DNA forms hybrids with rRNA. We have obtained conclusive evidence that this high level of hybridization is neither due to contamination of our r~RNA preparation by mRNA nor to contamination by mitochondrial nucleic acids (Table III). It was shown that variation of the hybridization conditions within reasonable limits did not influence the hybridization result (Table II). Moreover, it appeared from thermal dissociation studies that the hybrids formed under our hybridization conditions are very stable and homogeneous (Fig. 4) with a Tm of 89 ° in 2 × SSC, which is close to the one expected from the base composition of yeast rRNA n. We therefore conclude that the saturation value found may be considered as a correct estimate of the proportion of DNA homologous with r~RNA. Bio~him. Biophys. Acta, 169 (1968) 416-429

RIBOSOMALRNA SITES II~ YEAST DNA

427

Recently FUKtmARA~ found a hybridization percentage of 2.5 % for the rRNA from the very related S. cerevisiae. The difference may be due to the difference in organism, and also to the different hybridization conditions used by this author. The rather low incubation temperature of 45 °, employed by F U K U H A R A , may give rise to the formation of less specific hybrids as argued before. Furthermore, FUKUHARAdid not find a definite saturation plateau, which might be an indication of the presence of mRNA in his rRNA preparation. Moreover, it should be noted that a supplementary treatment with T1 ribonuclease of the hybridization products was found to be necessary to remove thoroughly unpaired RNA stretches present on the filters (Table I). Treatment with pancreatic ribonuclease alone, as FUKUHARAdid, appeared to be insufficient in this respect, at least under our hybridization conditions. That the ribonuclease treatments removed only mispaired RNA and did not affect specific hybrids may be derived from Fig. 4. This shows that after annealing below 7o°, repeated ribonuclease treatment does not change the hybridization value. The levels of annealing obtained indicate that there is a large multiplicity of D1VA stretches apparently complementary to yeast rRNA. From the experimental data it can be calculated that about 14o cistrons for total rRNA are present on the yeast genome. Although it remains to be settled whether all these sites are really active in rRNA synthesis, the high proportion of rRNA cistrons may explain the high content of cytoplasmic ribosomes and the high rate of rRNA synthesis in this organism11. A comparison of yeast with other organisms, both eukaryotes and prokaryotes, shows that yeast, ill many respects resembling the prokaryotes and having a DNA content only about ten times higher than bacteria, has a number of rRNA cistrons comparable to that of higher organisms, which have a DNA content per haploid genome some fifty times higher than in yeast (Table IV). The biosynthesis of rRNA in yeast 11 follows a similar pattern as that in higher organisms. So yeast, in spite of its much lower DNA content, resembles the other eukaryotes, both in the relatively high number of rRNA cistrons and in the mechanism of rRNA synthesis. During

TABLE

IV

NUMBER OF r R N A ClSTRONS ON VARIOUS GENOM]~S Values in parenthesis indicate reference numbers.

Organism

E. coli

B. subtilis S. carlsbergensis Pisum sativum Drosophila melanogaster Xenopus laevis Chicken HeLa cells Human

Molecular weight

% r R N A to DNA in hybrid

Number of r R N A cistrons (per haploid genome) 5-9 3 5 14o IO0 135 5o0-9oo IOO-2OO 200-300 13o-2oo

DNA

Total r R N A

2.8. lO 9 (27) 1. 3 - lO 9 (28) 2.0" 109 (29) 13" lO 9 (25) 6" IO 10 (3 o) 12- lO 1° (6) 1.8. lO TM (31) 1.3" lO TM (32) 1.2. lO TM (8) 7" lO12 (9)

1.66. lO 6 (33) 1.66. i o e (19)

0.27-0.56 0.38

1.84" lO 6 1.86" 106 ( I 9 ) 2.4 " 106 (34) 2.23 "Ioe (35) 1 . 8 9 " 1 o 6 (36) 2.66" IO e (19) 2.66" lO 6 (19)

1.9 0. 3 (5) 0.27 (6) o.o6-O, l l (31,38) o . o 1 5 - o . o 2 6 (IO) 0 . 0 0 5 - o . 0 0 8 (8,4) o . o 0 5 - o . 0 o 8 (8,4)

(lO,4) (37)

Biochim. Biophys. Acta, 169 ( i 9 6 8 ) 4 1 6 - 4 2 9

428

j. RETEL, R.J. PLANTA

the evolution of life, a progressive increase in the DNA content per haploid cell and changes in DNA base sequence have accompanied the development of more complex organisms. It is most likely that gene duplication followed by translocation is responsible for the increase of DNA content. The data, summarized in Table IV, permit the speculation that gene duplication in the course of the evolution has started with sequences coding for 1RNA species with a general function (rRNA, transfer RNA), then followed b y a multiplication of messenger cistrons correlated with the increasing structural and frunctional complexity of the organisms. The evolution of base sequences takes place at different rates at different sites in the genome. From different comparative studies it is known that certain sequences are remained relatively conserved, rRNA and transfer RNA cistrons are examples of such conservative regions. The narrow melting range of the yeast rRNA-DNA hybrid indeed suggests, that the divergency in base sequences among the rRNA cistrons in yeast is very small, as distinct from mammalian cells. The two rRNA components from yeast show considerable cross-hybridization indicating a great similarity in the base sequence of the 26-S and I7-S rRNA species (Fig. 6). Since I7-S rRNA is able to compete for much more than 50 % with 26-S rRNA it may be assumed that 26-S rRNA consists of two regions similar in base sequence to I7-S rRNA. The two '17 S' regions in 26-S rRNA and the I7-S rRNA itself could then be related to each other in a manner analogous to the genes coding for the a- and fl-chain of hemoglobin, and myoglobin. Cross-hybridization of the two rRNA components was found before for Escherichia coli by ATTARDI, I-[UANG AND KABAT4. On the other hand, no cross-hybridization of the rRNA components was observed for Bacilli 8,a9 and for Drosophila melanogaster ~4. The opposite results for E. coli and for B. subtilis were confirmed very recently by 3IANGIAROTTIet al. 4°, using the same hybridization procedure. Apparently, it depends only on the organism used, whether cross-hybridization is found or not. Although the failure to detect differences between two RNA species with the hybridization technique does not establish identity, it is inferred from different data that the two rRNA components from yeast have a very similar base sequence. The competititve interaction in hybridization, the complete similarity in base composition n and the same methylation degree (J. RET~L, T. J. STOOF, R. C. VAN DEN BOS AND R. J. ]~LANTA, unpublished results), all point to a great structural similarity. There are, however, also some differences as judged by the electrophoretic analysis of T 1 ribonuclease digests of purified 27-S and I6-S rRNA; differences, which are too small to be detected with the hybridization technique.

ACKNOWLEDGEMENTS

The present investigations have been partly sponsored b y the Netherlands Foundation for Chemical Research (S.O.N.) and the Netherlands Organization for the Advancement of pure Research (Z.W.O.). The authors are grateful to Dr. P. BORST and Dr. A. M. KROON for stimulating discussions; to Mr. G. PAUW for his collaboration in some of these experiments; to Mr. J. L. 1V[OLENAAR,~¢~issV. C. t{. F. DE REGT, Mr. H. BREMAN and Mr. H. SNIPPE for skillful technical assistance. Biochim. Biophys. Acta, 169 (z968) 416-429

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Biochim. Biophys. Acta, 169 (1968) 416-429