Molec. gem Genet. 148, 233-241 (197.6) © by Springer-Verlag 1976
Restriction Endonuclease Analysis of Ribosomal DNA from Saccharomyces cerevisiae Jane Harris Cramer, Frances W. Farrelly, and Robert H. Rownd Laboratory of Molecular Biology,Universityof Wisconsin, 1525LindenDrive, Madison,Wisconsin53706, U.S,A.
Summary. The size and the degree of homogeneity of the repetitive units in purified ribosomal DNA (7 DNA) from Saccharomyces cerevisiae have been analyzed by restriction endonuclease digestion and heteroduplex mapping. Digestion of the 7 DNA with EcoRI yields seven fragments, digestion with Hind II + I I I yields five fragments, digestion with Hind III alone yields two fragments, and digestion with Sma I yields one fragment. The sum of the fragment molecular weights after digestion with each of the endonucleases is 5.5-5.6 x 10 6. When the DNA strands of the Sma I fragment are dissociated and reannealed, only homoduplexes are formed. We have concluded from these results that the repeating units in yeast ribosomal DNA are 5.6 x 106 daltons and are homogeneous in size and composition.
Introduction DNA isolated from whole cells of Saccharomyces cerevisiae consists of three species distinguishable by their different densities in CsC1 gradients: a major component, c~,which has a buoyant density of 1.699 g/ cm3; and two minor components, /3, with a density of 1.683 g/cm 3, and 7, with a density of 1.705 g/cm 3, (Cramer, Bhargava and Halvorson, 1972; Moustacchi and Williamson, 1966). The/~ DNA is of mitochondrial origin (Tewari, Jayaraman and Mahler, 1965; Corneo et al., 1966) whereas both ~ and 7 DNA are located in the nucleus (Cramer et al., 1972). The 7 DNA contains the 18S and 25S ribosomal RNA (rRNA) genes, the 5S RNA genes and perhaps some of the tRNA genes (Aarstad and Oyen, 1975; Cramer et al., 1972). S. cerevisiae contains approximately 140 copies of the 18S and of the 25S rRNA genes (Schweizer, MacKechnie and Halvorson, 1969). These two RNA
species as well as the 5.8S rRNA are synthesized as part of a large common precursor which has a molecular weight of 2.5 x 106 (Udem and Warner, 1972). The 5S RNA genes are present in the same number of copies as the 18S and 25S genes and are closely linked to them (Rubin and Sulston, 1973; Kaback, personal communication); however, they are apparently transcribed separately from the rRNA precursor genes (Udem and Warner, 1972; McLaughlin, 1974). Approximately 70% of the rRNA genes are located on chromosome I (Finkelstein, Blamire and Marmur, 1972; Kaback, Bhargava and Halvorson, 1973); the chromosomal location of the remainder is unknown. The rRNA genes on any one chromosome are not all contiguous but appear to be arranged in small clusters of tandem repeating units which are interspersed with regions of e DNA (Cramer et al., 1972). Because of this clustered arrangement, only one band, with the density of ~ DNA, is Visible in CsC1 analytical gradients of yeast DNA preparations with molecular weights greater than 80 x 106 (Bhargava, Cramer and Halvorson, 1972). When the size of this DNA is subsequently reduced by shearing, the ~ and 7 DNA segments are sheared away from one another, and in preparations with molecular weights less than 30 × 106 the 7 DNA can be seen as a separate band (Cramer et al., 1972). We have analyzed purified 7 DNA from yeast DNA preparations of 20 30 × 106 daltons by restriction endonuclease digestion and heteroduplex mapping. Four different restriction endonucleases were used: EcoRI from Escherichia coli (Hedgpeth, Goodman and Boyer, 1972), Sma I from Serratia marcescens (Mulder, unpublished results) and Hind II + Hind III from Hemophilus inlTuenzae (Old, Murray and Roizes, 1975). The results of our experiments indicate that the 7 DNA consists of a tandem head-to-tail array of homogeneous genetic units. Each
J.H. Cramer et al. : Restriction Endonuclease Analysis of Yeast rDNA
Fig. 1. Schematic representation of the arrangement of repetitive rDNA units in the yeast chromosome. The homogeneous monomer units (~--~) have a molecular weight of 5.6.106 dalton s and are arranged in small clusters interspersed with c~DNA (~,,~,,,~)
unit has a molecular weight of 5.6 x l 0 6 and presumably contains a copy of the gene coding for the r R N A precursor, a 5S R N A gene and possibly a small amount of nontranscribed spacer DNA. Figure 1 illustrates our hypothesis of the arrangement of the repetitive 7 D N A monomer units in the yeast chromosomes. At present we have no information about the amount or nature of the e D N A which separates the ribosomal D N A (rDNA) clusters.
Materials and Methods Strains S. cerevisiae Y55 (Schweizer et al., 1969) a wild type diploid was obtained from H.O. Halvorson and was used in all these studies.
DNA preparation Yeast cells were disrupted with a French Press and the DNA was isolated by the procedure detailed in Bhargava et al. (1972). Purified ~ and ¢~ DNA were prepared by Hg + +/CszSO4 density gradient centrifhgation as previously described (Cramer et al., 1972). DNA from the density gradients was dialyzed exhaustively against 2 x SSC (SSC is 0.15 NaC1, 0.015 M Na3 citrate, pH 7) • and then against 1/10 SSC prior to digesting with restriction endonucleases or mounting for electron microscopy.
Sma I, 1-2 h for Hind III and 6-8 h for Hind II + I I I to achieve complete digestion of the DNA. In some cases an additional 5 lal of enzyme was added halfway through the digestion period. The length of incubation and amount of enzyme required to digest the DNA completely differed slightly among preparations of the same enzyme, presumably due to minor variations in enzyme activity. When samples were to be analyzed by agarose-ethidium bromide gel electrophoresis, reactions were terminated by the addition of 20 gl of BJ solution, which contains 60% sucrose (w/v) and 0.005% Bromphenol blue in 2 x electrophoresis buffer (see below). Bromphenol blue was used as a tracking dye during agarose gel electrophoresis. The two smallest EcoRI fragments of 7 DNA comigrate with the tracking dye and are obscured by it; therefore Bromphenol blue was omitted from BJ solution added to EcoRI digests.
Agarose Gel Electrophoresis DNA samples were analyzed on agarose-ethidium bromide gels according to the method of Sharp, Sugden and Sambrook (1973). Depending on the experiment, 0.6%, 1.0% or 1.4% (w/v) agarose gels (Sigma, electrophoresis grade) made up in electrophoresis buffer (E buffer) were used. E buffer contains 0.04 M Tris, 0.02 M sodium acetate, 0.001 M disodium ethylenediaminetetraacetate (Na2 EDTA) and 0.5 btg/ml ethidium bromide; the pH was adjusted to 7.9 with glacial acetic acid before the ethidium bromide was added. DNA samples were applied to the gels (0.6 x 11 cm) and electrophoresed in E buffer at 2.5 mA/gel (constant current) for 4 h. Gels were removed from the glass tubes and the DNA was visualized by fluorescence of the bound ethidium bromide under illumination with a short wavelength mineral light (UVS-54, Ultraviolet Products, Inc., San Gabriel, California). Photographic records were made with Type 57 film (ASA 3000) in a Polaroid MP-3 Land camera equipped with Wratten K2 (yellow) and 25 (red) filters. For quantitation of the DNA fragments the gels were photographed with Kodak 4 x 5 inch cut sheet Tri-X film (ASA 320) with the filter system used above. The Tri-X was developed 5 rain in Kodak HC-110 developer diluted 1:7 and the negatives were traced using a Joyce Loebel microdensitometer. The areas under the bands in the microdensitometer tracings were quantitated by resolving the individual curves, cutting them out and weighing them.
Restriction Enclonuclease Preparation The methods for preparing the restriction endonucleases EcoRI from Escherichia coli and Sma I from Serratia marcescens have been described (Tanaka and Weisblum, 1975). A mixture of Hind II + I I I was obtained from Hardy Chart and Hsu I (from Hemophilus suis) was the gift of M. Nomura. Hsu I cleaves at the same restriction site as Hind III (Richard Roberts, personal communication) and will be referred to as Hind III in our experiments.
Restriction Endonuclease Digestion Restriction endonuclease digestions were carried out in a total volume of 50 gl which contained 0.75-1.5 gg of DNA and 5 pl of enzyme in the appropriate buffer. Buffers in the digestion reaction mixtures were composed of 90 mM tris (hydroxymethyl) aminomethanehydrochloride (Tris-HC1) pH 7.4, 10 mM MgC1z for EcoRI, 15 mM Tris-HC1, pH 9, 6 mM MgCI~ and 15 mM KC1 for Sma I, and 10 mM Tris-HCl, pH 7.9, 6.7 mM MgC12 and 60 mM NaC1 for Hind II + III or Hind III alone. Reaction mixtures were incubated at 37° for 30-60 min for EcoRI, 1-2 h for
Extraction of DNA from Agarose Gels Separated DNA fragments were cut from the gels with a razor blade and the gel slices were crushed by forcing them through a 25 gauge hypodermic needle. The DNA was extracted as follows : 1.0 ml of E buffer was added to the crushed agarose, the mixture was frozen and thawed three times and the agarose was removed by centrifugation at 12,500 rpm for 30 rain in a Sorvall SS-34 rotor, 4° C. The supernatant was removed, concentrated ten-fold by dialysis against 25% polyethylene glycol (Carbowax, Union Carbide) in 0.001 M NazEDTA, pH 7, and then dialyzed exhaustively against 1/10 × SSC.
Electron Microscopy i) Contour Length Determinations. DNA was suspended in a buffer containing 0.02 M Na2CO3, 0.003 M Na2EDTA, and 11% HCHO with the pH adjusted to 8.6, mounted for electron microscopy by the method of Inman and Schn6s (1970), and rotary shadowed with platinum. 2 DNA was included with the Sma I restriction
J.H. Cramer et al.: Restriction Endonuclease Analysis of Yeast rDNA
Fig. 2. Agarose-ethidium bromide gel electrophoresis of 7 DNA after digestion with restriction endonucleases. 7 DNA was digested by the indicated enzyme, electrophoresed on agarose gels for 4-5 h, and the gels were photographed as described in Materials and Methods. The ~ DNA on gels (a) through (f) was highly purified; that in gel (g) was contaminated with c~ DNA. a EcoRI digest on 1.4% agarose gel. b Hind II + I I I digest on 1.4% agarose gel. e Hind II + I I I digest followed by Sma I digest on 1.4% agarose gel. d Hind III digest on 1.0% agarose gel. e Sma I digest on 1.0% agarose gel. f Undigested 7 DNA on 1.0% agarose gel. g Sma I digest on 1.0% agarose gel
fragment and P4 DNA was included with the EcoRI restriction fragments as internal length markers. Grids were examined and photographed with a Phillips 300 electron microscope. Sma I fragments were projected on a paper screen, traced and measured with a KeuffeI and Esser map measurer. Length measurements for EcoRI fragments were made on a digitizer (Numonics Corp.) interfaced to a programmable calculator and plotter (Hewlett-Packard). Length and molecular weight determinations were made relative to the marker DNAs. 2 has a length of 17.06 ~ (Younghusband, Egan and Inman, 1975) and a molecular weight of 30 x 106 (Freifelder, 1970); P4 DNA has a length of 4.l g and a molecular weight of 7.35 x 106 (Younghusband et al., 1975).
ii) Heteroduplex Formation. The complementary strands of Sma I fragments were dissociated and reannealed by the formamide renaturation technique of Davis, Simon and Davidson (1971). The DNA was allowed to reanneal for 1 2 h at 22 ° C. Cytochrome C was added directly to this DNA solution to a final concentration of 1 lag/ml and the heteroduplexes were spread, shadowed and examined as described for contour length determinations. The lengths of the heteroduplexed molecules were determined on a digitizer interfaced to a programmable calculator and plotter as described above. Lengths were measured relative to a carbon grating replica
calibration grid with 54,800 lines/inch (Pelco Electron Microscopy Supplies).
E c o R I Digestion E c o R I d i g e s t i o n o f p u r i f i e d 7 D N A f r o m S. cerevisiae yields seven fragments (Fig. 2a) which have been labeled A through G in order of their increasing mobility (and decreasing molecular weight) on agarose gels. D i g e s t i o n o f p u r i f i e d ~ D N A p r o d u c e s f r a g m e n t s t o o n u m e r o u s t o r e s o l v e u n d e r o u r gel c o n d i t i o n s . The molecular weights of the individual 7 DNA EcoRI restriction fragments have been determined by measuring their contour lengths in the electron micros c o p e ( T a b l e 1) a n d b y c o e l e c t r o p h o r e s i n g t h e m w i t h D N A s t a n d a r d s o f k n o w n size, t h e E c o R I f r a g m e n t s
J.H. Cramer et al. : Restriction Endonuclease Analysis of Yeast r D N A
Table 1. Molecular weights of ~ D N A restriction fragments Restriction enzyme Molecular weights of restriction fragments ( x 106) A B C D E F G Total molecular weight
Hind II + I I I
4.00 +0.02 b 1.60 __+0.02 °
1.60__+0.026 1.60 + 0.02 c 1.03__+0.028 0.90__+0.02 b 0.42 + 0.01 b
1.79__+0.05 " 1.46 __+0.06 ~ 1.19+0.06 " 0.41 +__0.02a 0.34__+0.02a 0.22 __+0.02 a 0.17__+0.01 a
(X 106) a The molecular weights of these fragments were determined from their contour lengths as measured in the electron microscope. The molecular weights of the remaining fragments were determined by coelectrophoresis in the same gels with D N A markers of known molecular weights. At least 50 molecules were measured for electron microscopic determinations. Values from coelectrophoresis experiments are based on averages from five different gels b Hind III fragment A contains Hind II + I I I fragments A, C, D, and E Hind III fragment B is equivalent to Hind II + III f r a g m e n t B and contains the Sma I restriction site
of NRI R plasmid DNA (Tanaka, Cramer and Rownd, 1976). The values obtained by the two methods are in close agreement and the relationship between the logarithms of the fragment molecular weights and their mobilities in our agarose gel system is linear over the size range of the 7 DNA EcoRI fragments (data not shown; see also Tanaka et al., 1976). The total molecular weight of the seven ~ DNA EcoRI fragments is 5.58 x 106. To determine if the seven 7 DNA EcoRI fragments observed on gels were present in equimolar amounts, we traced negative photographs of agarose-ethidium bromide gels (Fig. 3 A) and quantitated the area of each band in the tracings as described in Materials and Methods. Figure 3B shows the relationship between the logarithm of the mass and the mobility of the y DNA EcoRI fragments on 1.4% agarose gels. Each point on the graph is the average of values from four different gels and the line is a least-squares fit of the data. There is some deviation from the line for the smaller fragments. However this variation is probably due to experimental error in quantitating the area of the small bands and we have concluded that each of the seven fragments is present in one copy per 7 DNA repeating unit. Restriction mapping experiments (Farrelly, unpublished results) confirm that each of the EcoRI fragments is present at a single location in the restriction map of a y DNA monomer unit. We have never seen any variation in the number or size of the EcoRI fragments in many different 7 DNA preparations. Other investigators (Nath and
Bollon, 1975 ; Kramer, personal communication) have seen some of the same EcoRI fragments in other yeast strains as highly repeated bands above the background of~ DNA fragments in gels of whole cell DNA EcoRI digests.
Hind H + III Digestion When 7 DNA is digested with a mixture of the two Hemophilus enzymes Hind II and Hind III, four fragments are resolved on agarose-ethidium bromide gels (Fig. 2b). The molecular weight total of these four fragments (only 4x 106), as well as the intensity of fluorescence of the most slowly migrating band (Fig. 3C), suggested that this band contained more than one class of fragments. Quantitation of the areas under the curves on gel tracings of the Hind 7 DNA digests shows that the most slowly moving fragment band is a doublet (Fig. 3D) and that digestion with Hind II + I I I yields five fragments. The two largest fragments are very similar in size and they cannot be resolved even after electrophoresis to the bottom of 0.6% agarose gels which are 10 cm long. The molecular weights of the Hind II + I I I fragments (Table 1) have been determined by coelectrophoresis with the 7 DNA EcoRI fragments and with the EcoRI fragments of the R plasmid NRl (Tanaka et al., 1976). Hind II + I I I fragments A and B can be distinguished from one another by doing a subsequent digestion with Sma I (see below), which cleaves one of them into two smaller fragments, B'I and B'2, of approxi-
J.H. Cramer et al. : Restriction Endonuclease Analysis of Yeast rDNA
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