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Molec. gen. Genet. 147, 153-168 (1976)

© by Springer-Verlag 1976

Characterization of Yeast Ribosomal DNA Fragments Generated by EcoR1 Restriction Endonuclease Kamalendu Nath and Arthur P. Bollon BiochemistryDepartment, The Universityof Texas, Health ScienceCenter, Dallas, Texas 75235, USA

Summary. The action of Escherichia coli restriction endonuclease R1 (EcoR1) on DNA isolated from Saccharornyces cerevisiae (strain MAR-33) generates three predominent homogenously sized DNA fragments (species of 1.8, 2.2 and 2.5 kilo nucleotide base pairs (KB). Many DNA species of molecular weight greater than 2 million daltons can be recognized upon incomplete EcoR1 digestion of yeast DNA. Four additional DNA species ranging from 0.34).9 KB can be identified as the second major "class of EcoRl-yeast DNA products. Hybridization with radioactive ribosomal RNA (rRNA) and competition with nonradioactive rRNA show that of the three predominent EcoRl-yeast DNA species, the 2.5 KB species hybridizes only with the 25S rRNA while the lighter 1.8 KB species hybridizes with the 18S rRNA. The intermediate DNA species of 2.2 KB hybridizes to a small extent with the 25S rRNA and could be a result of the presence of the 2.5 KB DNA species. The mass proportions and hybridization values of these 3 DNA species account for about 60% of the total ribosomal DNA (rDNA). The 5 EcoRl-yeast DNA species of less than 0.9 KB (4 major and 1 minor species) hybridize to varying degrees with the 2 rRNA and can be grouped in two classes. In one class there are 3 DNA species that hybridize exclusively with the 18S rRNA. In the second class there are 2 DNA species that besides hybridizing predominently with the 25S rRNA also hybridize with the 18S rRNA. The 7 EcoRl-yeast DNA species (excluding the 2.2 KB DNA species) that hybridize with the two rRNA account for nearly a 5 million dalton DNA segment, which is very close to the anticipated gene size of rRNA precursor molecule. If the 2.2 KB DNA species is a part of the rDNA that is not transcribed or 5 sRNA then the cistron encoding the rRNA in S. cerevisiae has at least 8 EcoR1 recognition sites resulting in 8 DNA frag-

ments upon digestion with the EcoR1. Consideration is given to the relationship of the rRNA species generated by EcoR1 digestion and the chromosomes containing ribosomal cistrons.

Introduction Our interest in the studies of gene regulation, mainly of the isoleucine-valine (ilv) biosynthetic pathway in Saccharomyces cerevisiae (Bollon and Magee, 1971; Bollon, 1974; Bollon, 1975), have led us to attempt the amplification and isolation of the yeast ilv and in Escherichia coli. The procedure is based on the joining of a DNA fragment to a bacterial plasmid followed by transformation in E. coli and selection for the transformants that carry the desired DNA (Cohen et al., 1972; Morrow et al., 1974; Hershfield et al., 1974; Tanaka and Weisblum, 1975 ; Kedes et al., 1975; Glover et al., 1975). In early experiments the E. coli restriction enzyme endonuclease R1 (EcoR1) that generates a cohesive end of four nucleotides long (Sgarmella, 1972; Mertz and Davis, 1972; Hedgpeth et al., 1972) and a plasmid, PSC101, that confers resistance to tetracycline (Cohen and Chang, 1973) have been used with some success in attempts at eucaryotic gene amplification in E. coli (Cohen and Chang, 1974; Morrow et al., 1974; Kedes et al., 1975; Glover et al., 1975), Our approach for the isolation of the yeast iIv genes is based on the assumption that the isoleucinevaline biosynthetic pathway in S. cerevisae and E. coli are similar (Bollon, 1974; Umbarger, 1971). In addition, the transformation of a particular yeast ilv gene in a strain of E. coli that carries a deletion in the corresponding bacterial ilv gene, under auxotrophic conditions, might enable the E. coli strain to grow and thus amplify the particular yeast ilv gene.

154

K, Nath and A.P. Bollon: EcoR1 Endonuclease Generated Yeast r D N A

While the existence of a transcriptional eucaroytic gene product has been established in the case of Xenopus laevis ribosomal DNA (rDNA) (Morrow et al., 1974), no evidence for a translated eucaryotic gene product, functional or otherwise, has yet been reported to occur in the procaryote E. coli 1. Hence as part of a systematic approach, our initial attempts have been focused on the generation of discrete species of S. cerevisiae DNA followed by their identification and amplification. The satellite DNA in S. cerevisiae, most of which is reiterated rDNA (Schweitzer et al., 1969; Cramer et al., 1972; Kabach et al., 1973) comprises more than 10% of the total nuclear DNA (Schweitzer et al., 1969; Cramer etal., 1972). We have previously observed that when yeast DNA is treated with the restriction enzyme EcoR1 various discrete DNA species appear which are identified as sharp bands upon electrophoresis on agarose gels (Nath and Bollon, 1975). Since these discrete EcoR1 generated yeast DNA species probably arose from reiterated genes it was not surprising that of the three major DNA species tested, two species hybridized with ribosomal RNA (rRNA). In this report we show that of the 9 discrete DNA species identified by electrophoresis on agarose gels, at least 6 and possibly 7 DNA species hybridize with rRNA, indicating the presence of several EcoR1 sites in the yeast ribosomal gene cluster; furthermore, several species may contain ribosomal spacer DNA which may be involved in the processing ofrRNA. The relationship of the 18S and 25S ribosomal genes to each other and to chromosome 1 which contains 70% of the ribosomal genes (Finkelstein et al., 1972) of Saccharomyces cerevisiae is discussed. Materials and Methods Materials. All enzymes were obtained from commercial sources. RNase-free DNaseI (DPFF), RNase T1, micrococcal nuclease and lysozyme were obtained from Worthington. Pancreatic RNase (Gr. III) was obtained from Miles-Seravac, the snail enzyme Glusulase was obtained from Endo laboratories, chromatographically purified Proteinase K was obtained from E M Laboratories, 2x crystallized g-amylase was obtained from Seikagaku Fine Biochemicals (Tokyo, Japan) and the E. coli restriction endonuclease R1 (EcoR1) was obtained from Miles Laboratories. The R N a s e solutions used were heat treated as follows: Pancreatic RNase at a concentration of 10 m g / m l in 5 m M acetate buffer, p H 5.0, was incubated in a boiling water bath for 20 min and then adjusted to 20 m M Tris.HC1, pH 8.1. RNase T1 was dissolved in 20 m M Tris.HC1, pH 8.1, at concentrations of 10,000 unit/ml (20 gg/ml) and incubated for 10 min in a boiling water bath. [3H]-Adenme used was either [2-3H] which was obtained from Schwartz-Mann, or [8-3H] which was obtained from New England

1 Successful transformation of a yeast histidine gene in E. coh has just been reported by K. Struhl, J.R. Cameron, and R.W. Davis in Proc. nat. Acad. Sci. (Wash.) 13, 1471 1475 (1976)

Nuclear. Both [aH]-adenine preparations had specific activities of .greater than 20 Ci/mmole. [8-14C]-Adenine of specific activity 55 mCi/mmole was obtained from Schwartz-Mann. Agarose and ethidium bromide were obtained from Sigma, yeast nitrogen base and other reagents for culture media were obtained foom Difco, nuclease free sucrose was obtained from Schwartz-Mann, optical grade caesium chloride (CsC1) was obtained from Harshaw Chemical, linear polyacrylamide (mol. wt. > 5 x 106) was obtained from Gallard-Schlesinger Chemical (BDH) and nitrocellulose m e m b r a n e filters (25 mm/0.45 g) were obtained from Schleicher and Schuell. The photographic negatives were scanned in an instrument called Quick Scan (Helena Lab, P.O. Box 752, Beaumont, Texas 77704). Isolation of Nonradioactive DNA. S. cerevisiae strain MAR-33 (Betz et al., 1971) was grown in 10 liters of a medium containing 1% yeast extract, 1% bacto peptone (Y media) and 3% glucose at 30 ° C with aeration. The cells were harvested at an absorbance of 0.52 at 600 nm. Nearly 40 g of wet cells were collected and washed once with 4 vol (4 ml/gm) of deionized distilled water and then resuspended in 2 vol (80 ml) of a solution containing 45 m M E D T A z, p H 9.0 and 320 m M 2-mercaptoethanol. After incubation at room temperature for 45 min, the cells were collected and resuspended in 2 vol of a solution containing 100 m M EDTA, p H 9.5 and 1 M sorbitol. The cells were next treated with 0.1 ml of a glusulase solution per g of cells at 30 ° C for 45 min and conversion into spheroplasts was monitored under a phase contrast microscope. The spheroblasts (nearly 20 g) were next collected by centrifugation in the cold, and resuspended in 4 vol of a solution containing 100 m M E D T A , p H 8.0, and 150 m M NaC1. This culture was incubated with 4.3 units (2.3 gg) of e-amylase and 4 m g of pancreatic R N a s e at 38 ° C for 60 min. The complete lysis of spherplasts were attained by the addition of 1% SDS. This suspension was supplemented with 6.5 m g of proteinase K and incubated first at 38 ° C for 3 h and then at 63 ° C for 30 min. The crude lysate was mixed with an equal volume of a solution containing chloroform and isoamyl alcohol at ratios of 24 to 1. The phases were separated by centrifugation. Nucleic acids from the upper aqueous phase was "spooled" on a glass rod after the addition of 2 v o l of 95% ethanol, and dissolved in 8 ml of 0.1 x SSC. This solution was incubated with 3 m g of pancreatic RNase' and 43 units of e-amylase for 2 h at 38 ° C. After the addition of 1 m g of RNase, the solution was dialysed at room temperature (7 times) against 250 ml of SSC containing a few drops of chloroform. Nucleic acid from the contents inside the dialysis tubing (15 ml) was again spooled after the addition of 2 vol of 95% ethanol and redissolved in 8 ml of 0.1 x SSC. This solution was incubated with 2 m g of pancreatic RNase at 3 8 ° C for 60 min followed by the addition of 4 m g of fresh RNase. The solution was then dialyzed as before with 6 changes of SSC. The contents of the dialysis tubing were further incubated with 2 m g of RNase for 2 h at 38 ° C. This solution was made to 0.3 M with sodium acetate and the D N A from this solution was spooled after dropwise addition of 0.6 vol of isopropranol. The D N A was dissolved in 0.1 x SSC. A total of 6.3 m g D N A was obtained and stored at a concentration of 1 m g / m l in SSC. Isolation of Radioactive DNA. S. cerevisiae strain MAR-33 was grown in 2 liters of yeast nitrogen base m e d i u m supplemented with 2% glucose and 0.5 mCi of [14C]-adenine at 30 ° C in a rotary z Abbreviation used. EDTA, ethylenedinitrilo tetraacetic acid; SDS, sodium dodecyl sulphate; SSC, a solution containing 150 m M NaC1 and 15 m M sodium citrate at p H 7.0; CsC1, Cesium chloride; r R N A , ribosomal R N A ; 18S, the r R N A from the 40S ribosomal subunit; 25S, the r R N A from the 60S ribosomal subunit previously referred to as 26S; r D N A , ribosomal D N A and EcoR1, Escherichia call restriction endonuclease R1

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solution was divided into 3 x 1 ml samples and each fraction was mixed with 6 ml of a saturated CsC1 solution (at 24 ° C), 0.3 ml of SSC and 0.7 ml water. This mixture was overlaid with 1.5 ml of light paraffin oil and centrifuged in a Beckman rotor 40 at 32,000 rpm and at 19° C for 74 h. Ten drop fractions were collected from the b o t t o m of the tubes using a peristaltic pump. The upper one third of the gradient containing the total D N A was adjusted to a density of 1.70 g/cm 3 with CsC1 and recentrifuged for 82 h. The bulk of the D N A in this second gradient appeared at a density of about 1.70 g/cm 3 with no trace of contaminating R N A material (Fig. 1 A). The D N A from the gradient was spooled in the presence of 2 vol of cold absolute alcohol, washed repeatedly with alcohol and taken in 1.1 ml of water. The D N A solution was first dialysed against 0.1 x SSC (2 changes) and then against a solution containing 10 m M Tris.HC1, 10 m M NaCI and 2 m M MgCI2, p H 7.5 (3 changes). A total of 560 pg D N A with a specific activity of 4,500 cpm/lag was obtained.

Isolation of Radioactive rRNA. A 25 ml culture of S. cerevisiae

I-g] m Fig. 1A and B. Preparative and analytical ultracentrifuge profile of [14C]-DNA from S. cerevisiae strain MAR-33: A One of the two preparative CsC1 gradient profile of [I~C]-DNA obtained after centrifugation of 400 pg of D N A in 8 ml of a CsC1 solution at a density of 1.70 g/cm 3 in a Beckman rotor 40 at 32,000 rpm and at 19 ° C for 82 h. 12 drop fractions ( ~ 0 . 2 6 1331)were collected from the b o t t o m of each tube using a p u m p and 1 gl portions were dissolved in 0.5 ml of water for radioactivity determination. The fractions included by the arrows were pooled and is represented as the yeast D N A preparation Y-2 in Table 1. B The D N A preparation Y-2 at 8 pg/ml and at a density of 1.71 g/cm 3 in CsC1 was centrifuged in an analytical ultracentrifuge at 40,000 rpm and at 20 ° C for 21 h. The equilibrium profile obtained represents m as the mltochondrial, c~ as the nuclear and ? as the satellite D N A of densities 1.683, 1.699 and 1.705 g/cm 3 respectively. The dashed line at the b o t t o m is a profile prior to the start of the centrifugation

shaker. The culture was harvested at an absorbance of 0.63 at 650 nm. The 4 g of cells thus collected were washed and incubated with E D T A and mercaptoethanol for 13 h at 30 ° C as mentioned above. The spheroplasts formed with glusulase by incubation for 60 min at 30 ° C were incubated with 2.1 units of e-amylase, 1 m g of pancreatic R N a s e and 47 units of R N a s e T1 at 38 ° C for 30 min. SDS was added to 2% followed by the addition of 1 m g of proteinase K. The incubation was continued for 3 h at 3 8 ° C followed by 30 min at 60 ° C. Nucleic acid was separated from the crude extract by extraction with chloroform-isoamyl alcohol and spooled in the presence of ethanol. The nucleic acid was taken in 2 ml of 0.2 x SSC and incubated with 2.1 units of e-amylase, 0.5 m g of pnacreatic RNase and 19 units T1 at 38 ° C for 30 min. After a second addition of both R N a s e the mixture was dialyzed 2 x against 1 liter of SSC. The contents (2.5 m g nucleic acid) were further incubated with the two R N a s e and a-amylase followed by proteinase K and then extracted with chloroform-isoamyl alcohol. The nucleic acid (about 2 mg) was spooled in ethanol and dissolved in 3 ml of 0.07 x SSC. This

MAR-33 grown in yeast nitrogen base mediun containing 2.5% glucose was supplemented with 1.5 ml of a [3HI-adenine solution (1 mCi/ml) and the culture was harvested at an absorbancy of 0.40 at 600 rim. Approximately 0.2 g of cells were obtained and contained 35% of the input radioactivity in an acid insoluble form. Spheroplasts were formed as above and they were washed with a 2 ml solution contaning 1 M sorbitol and 0.1 M EDTA, p H 7.5. The spheroplasts were suspended in 1 ml of buffer A which contained 1 0 m M Tris.HC1, p H 7.7, 1 0 0 m M NaC1 and 3 0 r a M MgC12. They were next treated on ice with 0.5% deoxycholate for 10 rain foilowed by 0.75% Brij-58 for 5 r a i n with occasional mixing by a vortex mixer. The crude lysate was centrifuged at 15,000 rpm in Sorvall rotor SS-34 for 15 min at 4 ° C and the pellet was reextracted with deoxychoiate and Brij-58 as described above. The ribosomes in the total supernatant (2 ml) were next pelleted by centrifugation at 4 ° C for 2 h in a Beckman rotor 40 at 40,000 rpm through a 1 ml layer of a 15% sucrose solution prepared in bzffer A. The ribosomes were dissociated by resuspension in 0.5 ml of buffer B that contained 10 m M Tris.HCl, pH 7.7, 100 m M NaC1 and 0.01 m M MgC12. The suspension was overlaid on 17 ml of a 15 to 38% linear sucrose gradient prepared in buffer B. The ribosomal subunits were separated by centrifugation at 4 ° C for 16 h at 21,000 rpm in a Beckman rotor SW27. 33 x 13 drop fractions were coIlected from the bottom with a peristaltic pump. The fractions containing the 60S and 40S subunits were pooled separately, adjusted to 0.5 M LiC1, mixed with 3 vol of absolute alcoIhol, stored overnight at - 2 0 ° C and then centrifuged at 4 ° C for 30 min in a Beckman rotor 30 at 30,000 rpm. The two pellets were suspended in 0.5 ml of buffer C which contained 10 m M Tris .HC1, pH 7.7, 10 m M EDTA, p H 8.0, 100 m M LiC1 and 0.5% SDS. This suspension was overlaid on 16 ml of a 15 to 30% linear sucrose gradient prepared in buffer C over 1 ml of 60% sucrose. The gradient was centrifuged in a rotor SW27 at 26,000 rpm and at 20 ° C for 21 h. As shown in Figure 2 the gradient containing the 18S r R N A showed the presence of a contaminating peak corresponding to the 25S r R N A peak in the second gradient. The major fractions from the 18S and 25S r R N A peaks were collected. The R N A was precipiated with ethanol in the presence of 0.68 M LiC1 and resuspended in 0.25 ml of b~ffer D which contained 2 x S S C and 0.1% SDS. The r R N A fractions thus separated were extracted 2 x with 0.25 ml of neutralized phenol, equilibrated against buffer D. The phenol phase was reextracted 2 x with 0.1 ml of buffer D and the combined aqueous phase was extracted 2 x with 2 ml of diethyl ether. The dissolved ether was removed and the aqueous phase was concentrated at 37 ° C to a volume of 0.10 to 0.15 ml by aeration. The r R N A solutions were dialysed 2 x against 125 ml of

156

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Fig. 2. Isolation and sedimentation patterns of [3H]-18S and [3H]25S r R N A : 115 gg of 40S and 160 gg Of 60S ribosomal subunits were dissociated with SDS and centrifuged in a 15 to 30% sucrose gradient in a Beckman rotor SW27 at 26,000 rpm and at 2 0 ° C for 21 h as described in Experimental Procedure. 20 drop fractions ( ~ 0 . 4 4 ml) were collected and the absorbance at 260 n m as well as the a m o u n t of radioactivity of 1 ~tl portions were determined. The fractions within the horizontal lines were pooled as 18S or 25S [3H]-rRNA

buffer D and finally passed through a 13 m m m e m b r a n e filter. The preparation outlined above gave 20 gg of 18S and 90 gg of 25S r R N A . Both R N A species had a specific radioactivity of about 300,000 cpm/gg. Other preparations yielded r R N A of half this specific radioactivity. Isolation of Nonradioactive rRNA. The [3H]-t8S r R N A in Figure 2 was contamined with 25S r R N A (see also Figs. 12 and 13). To avoid such cross contamination, an attempt was made to purify the two ribosomal subunits through repeated centrifugation in sucrose gradients. But this resulted not only in lower yields, but also in degraded R N A , presumably as a result of ribosome b o u n d nuclease action. Large quantities of noncontaminated r R N A species were then obtained by selective pooling of fractions as shown in Figure 3. Ribosomes were obtained essentially by the method described above from 0.9 g of the yeast cells grown in 50 ml of Y media supplemented with 2% glucose (w/v). The ribosomal subunits were

Fig. 3. Isolation and sedimentation patterns of nonradioactive 18S and 25S r R N A : Yeast ribosomes equivalent to 25 A260 units were dissociated into subunits and centrifuged i n 15 to 30% sucrose gradients at low Mg ++ (10 gM). The 4 ( u ) 15 drop fractions of 60S and 2 of 40S from 6 gradients were combined separately and dissociated with SDS. Eight A260 units of 18S and 13 A26 o units of 25S r R N A thus obtained were recentrifuged through a sucrose gradient containing SDS. Five ( u ) 30 drop fractions from 18S and 25S r R N A were collected. The absorbance at 260 n m was determined in a Beckman spectrophotometer DU25 where an absorbance of greater than 2,8 is represented by open circles

separated by centrifngation at 4 ° C through 6 gradients containing 17 ml of 15 to 30% sucrose in buffer B at 26,000 rpm for 16 h. Fractions from the opposite portions of the subunit fraction were collected and the two r R N A were obtained by centrifugation at 19 ° C for 19.5 h through a 15 to 30% sucrose gradient in buffer C at 26,500 rpm (Fig. 3). Even after rejection of most of the r R N A in the two sucrose gradients nearly 300 gg of each of the R N A species were obtained.

RNA-DNA Hybridization. This procedure is a modification of a published method (Finkelstein et al., 1972). The D N A solution was diluted to 1 ml with water and was incubated at 100 ° C for 3 rain followed by 10 min in ice water. The solution was further treated with 0.1 N N a O H for 60 min at room temperature. This solution was cooled in ice, neutralized with HCI and made to 6 x SSC. This solution was diluted with 10 ml of 6 x SSC and passed slowly through a Schleicher and Schuell m e m b r a n e filter (Gillespie and Spiegelman, 1965) presoaked in cold 6 x SSC for more than 60 min. The filter was washed twice with 15 ml of 6 x SSC and dried first at r o o m temperature for 60 min and then at 80 ° C in vacuo for 60 min. 6.5 m m filter pieces were then punched out of the 24 m m filter and used for hybridization. The reverse side of filter containing the denatured D N A was numbered with a pencil.

157

K. Nath and A.P. Bollon: EcoR1 Endonuclease Generated Yeast r D N A

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Fig. 4, Electrophoretic patterns of yeast D N A treated with EcoR1 for various time intervals: A 240 pl solution contained 90 gg of yeast D N A (Y-l) and the reaction buffer consisting of 50 m M Trizma, 50 m M NaC1 and 10 m M MgCI 2 adjusted to p H 7.5 with HC1. After withdrawing a 15 gl portion (0 rain) the reaction was initiated with a 5 pl preparation of EcoR1 (12 unit/gl). At designated intervals, 30 pl portions were withdrawn and the digestion was halted by addition of 1 gl of 0.25 M E D T A followed by incubation at 65 ° C for 5 min. 15 gl samples containing 5.6 pg of D N A was made to a final vol of 40 pl which included 5 gl of glycerol and 5 gl of 0.1% Bromophenol Blue and applied on 1% agarose tube gels. 12 such gels were electrophoresed at 5 m A m p for 23,5 h, Both the gel and the electrophoresis buffer contained 0.5 gg/ml of ethidium bromide. The gels were photographed using 2 mineral lamps under long wave length and exposed for 3 min. Bormophenol Blue indicates the position of the migration of the dye in the gels

The hybridization reaction was carried out in 8 m m wide tubes (9.6 cm long). The reaction solution (0.2 ml) contained 50% formamide, 2 x S S C , 0.1% SDS, the radioactive R N A and usually 5 D N A containing filters each separated by a G F / A glass fiber filter (Whatman). The incubation was carried out at 33 ° C (Bonner et al., 1967) usually for 17 to 22 h with mild shaking. To determine the a m o u n t of hybrid D N A - R N A molcules formed, the filters were washed together m the following m a n n e r : with 120 ml of cold 2 x SSC for 10 rain; with 60 ml of 2 x S S C containing pancreatic R N a s e at 20 gg/ml and R N a s e T1 at 0.8 unit/ml shaken at 33 ° C for 30 min; twice with 120 rnl of 2 x SSC for 15 min each; and finally rinsed with 100 ml of deionized distilled water. The filters were dried at 80 ° C in vacuo for 60 min, dissolved in 0.5 ml of ethyl acetate and the radioactivity was determined in 5 ml of a scintillation fluid containing Triton-toluene-PPO and POPOP at concentrations of 600 m l - 1,000 m l - 3.9 g and 1 g respectively. M e m b r a n e filters containing no D N A generally retained 0.01 to 0.05% of the input R N A radioactivity.

Digestion of DNA with EcoR1. In a typical reaction 30 p,1 of reaction mixture contained 50 m M Tris .HC1, p H 7.5, 50 m M NaC1, 10 m M MgClz, 5 to 15 p,g of yeast D N A and 1 to 2 ~tl of EcoR1 solution (1 Colicin E1 or 2 SV 40 units/pl). Although digestion of yeast D N A by EcoR1 is complete in less than 10 rain at 37 ° C (Fig. 4) the D N A was usually digested for 60 rain. The reaction was stopped by the addition of 2 pl of 0.25 M EDTA, p H 9.6, followed by incubation at 65 ° C for 5 rain and then quick chilling in ice.

Gel Electrophoresis. Agarose was refluxed at I00 ° C in the electrophoresis buffer composed of either 40 m M Tris.HC1, 20 m M sodium acetate and 5 m M EDTA, p H 8.0, used for tube gels, or 36 m M T r i z m a base, 30 m M N a 2 H P O 4 and 10 m M E D T A , p H 7.8 used for slab gels. The agarose solution was brought to 5 8 ° C and if desired ethidium bromide (0.5 pg/ml) was added. The agarose was cast into gels in 6 m m x 14 cm glass tubes or 15 × 16 cm slab plates (3 m m thick). Prior to using the tube gels a dialysis tubing was secured with

158

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Fig. 5. EcoRl: D N A profile of 2 different yeast D N A preparations, ). and calf thymus D N A : The first 6 samples were treated by electrophoresis in a 1.4% agarose slab gel utilizing a Tris-phosphate-EDTA buffer, pH 7.8. Electrophoresis was carried out on the 7th sample in a 1% agarose gel utilizing a Tris-acetate-EDTA buffer, pH 8.0. The 1.4% and 1% agarose gels were run in the absence of ethidium bromide. The first 4 samples represent 10 gl of yeast D N A preparations Y-1 and Y-2. The 3rd and the 4th D N A samples were treated with 1 gl of EcoR1 for 45 rain. The 5th sample consists of 2.5 gg of 2 D N A digested with 1 gl of EcoR1 for 45 rain. The 6th sample consists of 10 ~tg of calf thymus D N A digested first with 2 ~1 of EcoR1 for 1 h followed by another 1 gl of EcoR1 for an additional 1 h. The last sample consists of a mixture of 2.5 gg o f EcoRl-yeast D N A and 2.5 gg of EcoR1-2 DNA. The slab gel was electrophoresed for 30 min at 15 m A m p followed by 5 h at 60 mAmp, while the tube gel was electrophoresed at 0.6 m A m p for 16.5 h. The slab and the tube gels were stained with 2 gg/ml and 0.5 lag/ml of ethidium bromide respectively for 30 rain followed by destaining in the electrophoresis buffer for 30 rain

a rubber ring on the open end and the glass tube was inverted so as to slide the gel against the perforated dialysis tubing. The sample containing 30 gl of D N A sample; 5 I11 of glycerol; and 5 ~1 of a solution containing 0.1% Bromophenol Blue (BB) was put on the top of the gel. The sample was then overlaid with electrophoresis buffer containing 0.5 gg/ml ethidium bromide when ethidium bromide was included in the gel. Electrophoresis was generally carried out at 4 mAmp per gel for 2.5 h at room temperature at which time the dye generally moves down 60 to 70% of the gel. The 4% polyacrylamide slab gel consisted of a bisacrylamide to acrylamide ratio of 1:20. The polymerization was carried out by the addition of 1.2 ml of a 10% solution of ammonium persulphate and 0.12 ml of T E M E D (N,N,N',N'-tetramethylethylene diamine) per 100 ml of solution. The 10 mm slots of the slab agarose and acrylamide gels (10 slots per gel) were loaded with 70 gl of a solution containing 30 ~1 of D N A sample, 30 gl of a 2% linear polyacrylamide solution

in the electrophoresis buffer, 5 gl each of glycerol and 0.1% BB. The sample was laid under the electrophoresis buffer and the electrophoresis was carried out initially at 15 or 20 m A m p for 30 rain followed by 60 or 75 mAmp for 5 h at 24 ° C. If the electrophoresis was performed in the absence of ethidium bromide, the gels were stained with electrophoresis buffer containing ethidium bromide (2 gg/ml) for 30 min. The gels were then washed with plain electrophoresis buffer for 10 to 30 rain. The gels were photographed with either short or long ultraviolet light using 2 mineral lamps and a 35 mm SLR camera equipped with a 55 M microlens, ektachrome X film and an orange Kodak filter Wratten number 12. The film was exposed at an f stop of 5.6 for 5, 10 or 20 rain.

Elution of DNA from Agarose Gels. The segments of the agarose gel containing the discrete D N A species (appearing as bands) were cut with a razor blade and the D N A was elnted as follows. The

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0.37

50

159 and the solution was concentrated to i to 3 ml by aeration. W h e n 3 tube gels, electrphorised with 10 gg of EcoRl-yeast D N A were separated into 4 segments (Fig. 10), and the D N A eluted from them, a total of 23 gg of D N A was eluted by the above procedure, giving a recovery value of 77%. Generally higher molecular weight D N A such as untreated yeast D N A or EcoRl-yeast D N A from Segment I gave poorer yields ( ~ 5 0 % ) .

Analytical Ultracentrifugation. The average sedimentation and molecular weight values of yeast D N A were obtained essentially as described by Studier (1965). A solution containing 10 to 25 lag of D N A / m l , 1 M NaC1 and 50 m M sodium citrate, pH 7.0 was centrifuged at 2 0 ° C in a 12 m m centerpiece 4 ° C sector A N H t i rotor at 36,000 rpm. Pictures were taken at 8 rain intervals with ultraviolet optics. The equihbrium density profile of yeast D N A was obtained (Fig. 1 B) by centrifugation of 1 to 25 gg D N A in CsC1 adjusted to a density (at 24 ° C) of 1.705_+0.005 g/cm 3 (0.8 ml saturated CsC1 solution per 1.05 ml final volume) at 40,000 rpm for 21 h at 20 ° C. The photographic plate was exposed for 15 s and the negatives were scanned. The proportion of mitochondrial D N A was estimated by weighing the paper encompassing the nuclear and mitochondrial D N A peaks.

2O Z 0

Results

gb

5.0

Action o f Restriction Endonuclease EcoR1 on Yeast DNA

_3

2.0 ZE GO

%

0.5 C) LU _J 0

0.2 0.1

I I 8181~ 0,2

0,4

0,6

0 8

l IO

RELATIVE MOBILITY

Fig. 6. Determination of the molecular weights of the Eco R 1-generated yeast D N A f r a g m e n t s : T h e relative mobility of the D N A bands is the ratio of the distance migrated to that of the total gel distances and was calculated from the E c o R 1 - D N A profiles of Figure 5. The molecular weight of the 5 EcoRl-calf thymus D N A is based on the reported values of 10,000; 2,100; 1,550; 1,300 and 970 base pairs for the 5 bands respectively in Figure 5. One nucleotide base pair is assumed to correspond with a molecular weight of 660 daltons. The molecular weights of the 9 EcoRl-yeast D N A fragments agrees well with that determined previously utilizing the 6 EcoRI-2 D N A fragments as standards on 1% agarose gel (Nath and Bollon, 1975)

gel pieces were homogenized in a 13 x 100 m m tissue grinder (Pyrex brand, C o m i n g 7725) in the presence of buffer E which contained 10 m M Tris.HC1, p H 7.7 and 5 m M E D T A , pH 8.0. This slurry was centrifuged in a Sorvall rotor SS-34 at 15,000 rpm for 15 min at 4 ° C. The bulk of the D N A from the gel could be recovered in the supernatant. The agarose pellet was vortexed with neutralized phenol and after the addition of buffer E it was incubated at 60 ° C for 15 rain. The aqueous phase was combined with the supernatant fraction described above. The combined sample was reextracted with phenol followed by an ether extraction. The ether was removed

The primary result of the endonuclease EcoR1 cleavage of yeast D N A is a reduction in the average D N A size. In Table 1 it can be seen that EcoR1 treatment of a yeast D N A preparation Y-1 of an average molecular weight of 7 million daltons is reduced approximately fourfold. In addition, when the EcoR1 treated D N A is analyzed by electrophoresis in 1.0% or 1.4% agarose gels, 3 predominent D N A species appear (Figs. 4 and 5). As can be seen in Figure 4, during the initial period of EcoR1 treatment, large DNA species are visible. The 3 dominent EcoR1 species ap-

Table 1

DNA preparations

Properties

Y-I

Nonradioactive D N A +EcoR1 treatment

21

7

5

12

1.5

-

CsC1 purified [14C]-DNA

33

Y-2

Average size a S20,w

Daltons ( x 106)

27

Per cent mitochondrial DNA b

7

" The average size values were determined by the method of Studier (1965) b Proportion of mitochondrial D N A was estimated from the density equilibrium profile of the isolated D N A as depicted in Figure 1 B

K. Nath and A.P. Bollon: EcoR1 Endonuclease Generated Yeast rDNA

160

TOP'

,+

Y- 2 I

TOP

+

I

23

4

5

67

89

Fig. 7. Complete EcoR1 profile of [14C]-yeast DNA Y-2: The profile at the top of the figure is the scan Of the untreated yeast DNA Y-1 and Y-2 in Figure 5. The EcoR1 profile of Y-1 has been reported previously (Nath and Bollon, 1975). The appearance of additional bands can be seen in the lower EcoR1 profile of Y-2 DNA between bands 1 and 2 and some minor peaks between 4 and 5. Band 10 is absent in this preparation. The gel above is a 1% agarose gel containing 0.5 gg/ml ethidium bromide through which 10gg of Y-2 DNA digested with EcoR1 for 60 rain has been electrophoresed at 4 mAmp for 2.5 h

pear at 3 min. The last traces of intermediate D N A species are observed at 6 min whereas at 8 rain the stable E c o R l - y e a s t D N A pattern is established. The appearance of intermediate sized D N A species in Figure 4 can also be found when limiting concentrations of EcoR1 are used for digestion (Nath and Bollon, 1975). The stable yeast D N A pattern generated by EcoR 1 treatment is reproducibly obtained using different preparations of yeast D N A and using different agarose gel concentrations (0.6, 1.0, 1.4 and 2.0%). EcoR1 treatment of a radioactive yeast D N A preparation Y-2 which is approximately 4 times larger in size than Y-1 (Table 1) results in the generation of the same 3 dominant yeast D N A species (Fig. 5). As shown in Figure 6 these 3 species numbered previously as bands 2, 3, and 4 represent D N A species of 2.50, 2.17 and 1.79 (KB) when compared with EcoR1 fragments of bacteriophage lambda (Allet

et al., 1973; Thomas and Davis, 1975) and calf thymus D N A (Botchan, 1974; Philippsen et al., 1975). These average molecular weight values are the same when analyzed on 1% (Tube) or 1.4% (slab) agarose gels utilizing Tris-acetate buffer or Tris-phosphate buffer respectively (Fig. 5). Besides the 3 dominant EcoR1 generated yeast D N A species seen in Figures 4 and 5, numerous other enriched D N A species are generated by EcoR1. As shown in Figure 7, in the optical scan of a 1% agarose gel containing EcoR1 treated yeast D N A , 2 or possibly 3 more E c o R l - y e a s t D N A bands larger than 2.50 KB are recognized. Previously we had identified only one band in this region of about 4 million daltons (band 1 in Fig. 6, N a t h and Bollon, 1975) utilizing D N A preparation Y-1. The prominence of the 2 additional bands (between bands 1 and 2 in Fig. 7) have resulted from the use of the D N A preparation Y-2. Thus it appears that EcoR1 cleavage of larger

K. Nath and A.P. Bollon: EcoR1 Endonuclease Generated Yeast rDNA

16l

T( )P

+

IO-

40

A

B

C

D

E

F

G H

I

J

Fig. 8. Effects of nucleases on the EcoRl-yeast DNA profile: In all samples above I0 gg of DNA was used. Sample E contains DNA preparation Y-2 while all of the other samples contain DNA preparation Y-1. The 1% agarose tube gels (A D) containing 0.5 gg/ml of ethidium bromide were electrophoresed at 4 mAmp/gel for 15 h. Samples A and C are whole yeast DNA and samples B and D are EcoR1 treated yeast DNA. Samples C and D were treated with 0.5 gg of pancreatic DNase I for 30rain at 37° C. The 4% acrylamide slab gel (E-J) was electrophoresed for 30 min at 20 mAmp followed by 3 h at 60 mAmp and 2 h at 75 mAmp. It includes EcoR1-Y-2 DNA (sample E) ; EcoRI-Y-I DNA (sample F) ; and sample F treated with 10 gg of pancreatic RNase and 9.4 unit of RNase T1 at 37° C (sample G) or at 65° C for 30 min (sample H); sample F treated with 5 I~g of pancreatic DNase I (sample I): and sample I treated with the two RNase (sample J). Following the electrophoresis the acrylamide gel was stained with 2 gg/ml of ethidium bromide

DNA such as Y-2 which is also less heterogenous in size range than Y-1 (compare the gel scans of Y-1 and Y-2 in Fig. 7) yields higher molecular weight DNA species than cleavage of smaller sized DNA such as Y-1. In addition to the high molecular weight EcoR1 generated DNA species, 4 major DNA species are evident in Figure 7 (bands 6 through 9). These DNA species correspond to about 700, 640, 450 and 370 base pairs (Fig. 6) when compared with EcoR1calf thymus DNA fragments or SV40 DNA fragments generated by Hind III treatment (not shown) utilizing either 1.4% agarose or 4% acrylamide gel electrophoresis. There are other minor bands between bands 4 and 6 in Figure 6. One such band which is marked 5 was identified previously utilizing DNA preparation Y-1. Although DNA preparation Y-1 contained a very

prominent lower molecular weight band which appeared independent of EcoR1 treatment (band 10 in Fig. 8), the yeast DNA preparation Y-2 which was purified through a CsC1 gradient was devoid of this species (Fig. 7). It can be seen either in the 1% agarose or the 4% acrylamide gels shown in Figure 8, that digestion of the Y-2 DNA preparation with pancreatic DNase results in the disappearance of all DNA species except that corresponding to band 10 (C, D, I). The combined actions of pancreatic RNase and RNase T1 at 37°C or at 65°C did not affect the appearance of band 10 or any of the other bands (G, H, J). Band 10 would appear to result from the presence of small oligonucleotides in the DNA preparation. EcoR1 treatment thus generates 7 major DNA species, three of which are most prominent in 1% agarose gels (bands 2 to 4) and four are prominent in 4% acrylamide gels (bands 6 to 9).

K. Nath and A.P. Bollon: EcoRl Endonuclease Generated Yeast rDNA

162 5

./'

z r',2

z 0

o

0 o

1

S:

N

f-

m >.-i-

•

I

Fig. 9. Hybridization of total yeast D N A with total yeast rRNA (18S+25S): The hybridization mixture containing 50% formamide, 2% SSC, 0.1% SDS, and [3H]-rRNA consisting of a mixture of 18S+25S (2:1) and the 6.5ram membrane filters containing the denatured-immobilized [14C]-DNA were incubated at 33°C for 17 h and treated as described in the Experimental Procedure. • total yeast D N A ; o total yeast DNA eluted from a 1% agarose gel. In all cases the 24 mm membrane filters were loaded with 5 gg of denatured DNA. At the end of the hybridization reaction, the 6.5 mm filters retained between 300 to 350 ng of D N A representing a D N A retention value of 80 to 95%. Under the conditions of radioactivity measurement the [3H]-rRNA was 100 or 300 cpm/ ng for the two separate r R N A preparations used while the [14C]D N A was 3 to 4 cpm/10 ng after all necessary corrections were made

!

10 20 TIME (HRS)

0

I

I

2

4

RIBOSOMAL

TOP

I

RNA (/zgIml) +

I

I

I

6 SEGMENT

132

d~

•

~

0

& 0~ "

z4 13

5 N

£n~2 nn >"1-

g,. 0

I

2 4 RIBOSOMAL RNA (/~g/ml)

6

Fig. 10. Localization of the r D N A in the EcoRl-yeast D N A profile obtained by electrophoresis on a 1% agarose gel: Three 1% agarose gels were electrophoresed with 10 gg each of EcoRl-yeast D N A and were divided into 4 segments as indicated in the figure. The [14C]-DNA eluted from the segments were hybridized as described in the legend to Figure 9. The 24 mm filters were loaded with 2.17, 1.45, 0.96 and 0.57gg of D N A obtained from segments I, II, III and IV respectively. At the end of the experiment the 6.5mm filters retained an average of 170 and l l 5 n g of D N A in the case of segments I and II (100% retention) and 53 and 30 ng of D N A in the case of segments III and IV (70% retention)

163

K. Nath and A.P. Bollon: EcoR1 Endonuclease Generated Yeast rDNA BANDS

Identification of EcoR1 Generated rDNA Fragments When [14C]-labeled yeast D N A Y-2 was subjected to analytical ultracentrifugation thre e primary classes of D N A were recognized (Fig. 1 B): mitochondrial D N A (m) which has a density of 1.683 g/cm3; bulk nuclear D N A (e) which has a density of 1.699 g/cm 3 ; and heavy satellite D N A (7) which has a density of 1.705 g/cm a. The profile of yeast D N A shown in Figure 1 B is in agreement with work done by other investigators (Cramer et al., 1972; Goldberg et al., 1972); except for the enhancement of the heavy satellite D N A (7) shoulder. The presence of this heavy satellite D N A (7) raised the possibility that some of the discrete EcoRl-generated yeast D N A species could be rDNA. The identification of r D N A among the EcoR1 generated D N A species involved the elution of the [14C]-DNA from 1% agarose gels and hybridization of the eluted D N A with [3H]-rRNA (100-300 cpm/ ng). In Figure 9 it can be seen that total untreated [t ~C]-DNA eluted from agarose gels hybridized similar to control D N A which was not applied to agarose gels. The saturation value of nearly 2.4% D N A hybridizing with r R N A is close to the reported values for total yeast nuclear D N A (Schweizer et al., 1969). We did not correct our hybridization values for the presence of mitochondrial D N A since we did not undertake a complete recovery of nuclear DNA, mainly from the first CsC1 gradient. In addition, the 7 band in Figure 1 B as mentioned was greater than reported by others. The hybridization reaction in Figure 9 (insert) was complete by 14 h and in all the experiments described the reaction was carried out for nearly 20 h. EcoR1 treated yeast D N A which was analyzed by electrophoresis in 1% agarose gels was next divided into 4 segments as shown in Figure 10 and eluted from the gel. The discrete EcoR1-DNA species in the 4 segments included band 1 in segment I, bands 2 to 4 in segment II, bands 5 to 7 in segment III and bands 8 and 9 in segment IV. As shown in Figure 10, 1% of segment I which included nearly 40 to 60% of the EcoRl-yeast D N A maximally hybridized with a mixture of 25S and 18S r R N A used at a ratio of 1:2 respectively. Segment II consisting of nearly 30% of the EcoR1 yeast D N A accounted for the bulk of the hybridization with r R N A (about 60%). In addition to segment II, segments III and IV gave the maximal hybridization value of nearly 5% with the mixture of 25S and 18S r R N A indicating that each segment contains some rDNA. EcoRl-yeast D N A bands 2, 3 and 4 were next eluted individually from several 1% agarose gels and hybridized with a mixture of 25S + 18S r R N A at D N A concentrations proportional to the total D N A (each

8~

/f/--°

,/

2 °

o m

4

Z O N

f

r'n >"1-

't

2I; /

Am

I

3

I

4 6 RIBOSOMAl_RNA (Fg/ml) Fig. 11. Hybridizationof the 3 major EcoRl-yeast DNA fragments obtained from segment II with the total rRNA: [14C]-DNA from the 3 major bands (2, 3 and 4 each of which comprise about one third the segment II) were eluted from the gel and were hybridized for 20 h with total rRNA as described in the legend to Figure 9. In all the cases the 24 mm filters were loaded with 500 ng of DNA and the retention of DNA at the end of the experimentwas 100% 0

2

band included nearly 10% of the total DNA). It can be seen in Figure 11 that D N A eluted from bands 2 and 4 but not form band 3 accounted for the bulk r R N A hybridization obtained with segment II described above. The D N A obtained from bands 2 and 4 hybridized with the r R N A mixture to an extent of 8% and 6% respectively while D N A from band 3 hybridized only to an extent of 1%. Thus of three major EcoR1 generated D N A species at least two (bands 2 and 4) contain r D N A accounting for 60% of the total rRNA.

Characterization of Bands 2 through 4 D N A eluted from bands 2, 3 and 4 were next hybridized with [3H]-25S and 18S r R N A separately (Fig. 12). At R N A concentrations of about 2 gg/ml, 8% of D N A eluted from band 2 gave a maximal hybridization with 25S rRNA. At a similar R N A concentration 6% of D N A eluted from band 4 gave a maximal hybridization with 18S rRNA. These maximal hybridization values are the same as those obtained with total r R N A (25S+ 18S) shown in Fi-

164

K. Nath and A.P. Bollon: EcoRl Endonuclease Generated Yeast rDNA

8

BANDS

al

e-

BANDS

2

~6

o

//°/"

Z a

o

4

Z

24 N E] r'r rr~ "-r-

2 3

, i

oN 4

o° ~ 0

I

I

I

2

4

6

0

2

25S

6

4

18S

Fig. 12. Hybridization of the 3 major EcoR1-DNA fragments with the 18S or 25S rRNA: The hybridization details are given in the legends to Figure 11 except that in these experiments the [3H]-rRNA species used were either 18S or 25S

Br-j.--

--.BANOS 2 2.3/zg/ml

6

[3H] 2 5 S

~ o

rRNA

-oBANDS 4

I

Z

4:

2.5 /~g/ml [:31"t]IBS r R N A

~

k

,_

TOTAL D N A

° ........

, ...............................

"

•

•

•

I

0

I

--o

t',,,\. 3

,4

20 40 60 [HI 1BS/PH] 25 S RIBOSOMAL RNA

-

' l ~~ , ~

TOT,,

.......

I

0

I

* ~

ONA

I

20 40 60 [H] 25 S/[31-t] 18S RIBOSOMAL RNA

Fig. 13. Competition hybridization of the 3 major EcoR1-DNA fragments with 18S or 25S rRNA: The details of hybridization are given in the legend to Figure 12 except that 27 gg/ml or 134 pg/ml of nonradioactive 18S rRNA was added to the solution of 2.3 ~tg/ml of [3H]-25S rRNA for the competition experiment. In the case of the [3H]-18S rRNA (2.5 pg/ml) 27 pg/ml or 133 gg/ml of nonradioactive 25S rRNA was present. For the total [14C]-DNA hybridization experiments 5 ~tg of denatured DNA was used on each 24 mm membrane filter with a final DNA retention of 100%

K. Nath and A.P. Bollon: EcoR1 Endonuclease Generated Yeast rDNA Table 2 DNA preparation from

% DNA hybridized with [all]-18S rRNA

[3H]-25S rRNA

-

+25S"

-

+18Sa

Band 2 Band 3 Band 4

4.92 0.63 5.99

0.13 0.17 5.70

7.80 0.70 1.37

7.82 0.79 0.41

Total DNA

1.64

0.84

1.88

1.66

a Prehybridized with nonradioactive 25S or 18S as indicated above The 6.5 mm filters containing the denatured immobilized DNA were prehybridized with nonradioactive rRNA at concentrations of 9.0 lag/ml of 18S or 7.6 gg/ml of 25S for 22 h at 33° C and then washed 2x with 100ml of 6xSSC for 15min each. The excess salt was soaked on a paper and the filters were subsequently dried first at room temperature and then at 80° C in vacuo. The competition was carried out with two concentrations of [3HJrRNA; 3.8 and 7,6 lag/ml of 25S rRNA or 2.5 and 5.0 Ixg/ml of 18S rRNA. The hybridization values were very close at both [3HI rRNA concentrations; the average value of which is presented in this Table. Approximately 0.5 lag of DNA isolated from the bands 2, 3, and 4, and 5 lag of total DNA were denatured and loaded on 24 mm S & S filters, The 6.5 mrn filter at the end of the processing contained 40 to 45 ng of DNA from bands 2, 3, and 4 (100% retention) and 250 to 300 ng of total DNA (80%) retention)

gure 11. Hence, the additional hybridization of D N A from band 2 with 18S r R N A would indicate that the 18S r R N A preparation is contaminated with 25S r R N A fragments. This is evident from the hybridization kinetics of D N A from band 2 where no saturation value is attained even at a concentration of 18S r R N A of 5 gg/ml. A direct answer to the question of 25S r R N A contamination was obtained with the competition experiments in Figure 13 and the pre-hybridization experiments in Table 2. In the presence of 60 fold excess of nonradioactive 18S r R N A , D N A from bands 2 and 3 maximally hybridize about 8% and 1% with [3H]-25S r R N A . The D N A from band 4 shows almost no hybridization with [3H]-25S r R N A in the presence of nonradioactive 18S r R N A and the total D N A hybridizes to about 1.6% with 25S rRNA. Conversely, using more than 50-fold excess of nonradioactive 25S r R N A , 6% of the D N A eluted from band 4 maximally hybridizes with [3H]-18S rRNA. Nearly all of the [3H]-18S r R N A hybridization of D N A eluted from bands 2 and 3 is competed out. A b o u t 0.8% of the total D N A hybridizes with 185' r R N A giving the ratio of 25S to 18S as 2 to 1. Similar results were obtained when denatured [14C]-DNA immobilized on the filter was first hybridized with nonradioactive r R N A (Table 2). Pre-

165

hybridization with 25S r R N A results in nearly 6% and 0.8% of the D N A from band 4 and the total D N A to hybridize with [3H]-18S rRNA. Hybridization with D N A from bands 2 and 3 were less than 0.2%. Conversely, D N A from bands 2, 3 and 4 as well as total D N A , when first hybridized with nonradioactive 18S r R N A give a subsequent hybridization value of about 7.8%, 0.8%, 0.4% and 1.7% respectively with [3H]-25S. It is concluded that 25S r D N A is present in band 2 and nominally in band 3 and that 18S r D N A is present in band 4. Due to slight overlapping of bands 2 and 3 (seen on gel scan in Fig. 7) the nominal 25S r D N A in band 3 could represent contamination of D N A species representing band 2.

Characterization of Bands 5 through 9 D N A in bands 5 through 9 are not only small in size but also constitute a very small proportion of the total EcoRl-yeast D N A (less than 10%). In addition, after heat and alkali denaturation about 50% of the D N A could be retained on the filter. Hence the amount of D N A from these 5 bands used for hybridization was larger than its proportion in the whole EcoRl-yeast D N A . As shown in Table 3, D N A from bands 8 and 7 gave the highest hybridization value with total rRNA. The values being nearly 6 and 5% respectively. This is largely due to the hybridization with the [3H]-25S r R N A corresponding to a value of about 4%. Nearly 2 and 1% of the D N A from bands 8 and 7, in addition, hybridized with [3H]-I 8S r R N A in the presence of an excess of nonradioactive 25S rRNA. The hybridization of D N A eluted from band 7 with 18S r R N A may result from the contamination of D N A from band 6 (see again gel scan in Fig, 7). D N A from band 6 like that of band 9 preferentially hybridizes with 18S r R N A to an extent of about 3%. Unlike the bands 6 and 7, the bands 8 and 9 appear to be electrophoretically well separated and hence band 8 may truly represent a dual hybridization with both 18S and 25S r R N A but at a ratio of about 2 to 1. Band 5 is a minor peak and D N A from this band showed a preference for hybridization with 18S. Thus 18S r D N A is also present in bands 6, 8 and 9 and possibly in band 5 while 25S r D N A is present in bands 7 and 8.

Discussion With few exceptions both the 18S and 25S r R N A species in all organisms arise from a single r R N A

166

K. N a t h and A.P. Bollon: EcoR1 Endonuclease Generated Yeast r D N A

precursor molecule. Although various values have been assigned for such a precursor rDNA molecule in yeast, namely, 42S (Retel and Planta, 1970), 38S (Tabor and Vincent, 1969) and 35S (Udem and Warner, 1972), the latter value may be the most reasonable estimate. Udem and Warner has shown that the process of rRNA maturation involves the cleavage of the 35S RNA into 27S and 20S RNA which in turn are processed into 25S+ 5.8S and the 18S rRNA species respectively (Udem and Warner, 1972; Udem and Warner, 1973). The 5.8S RNA stays hydrogen bonded with the 25S rRNA (Udem and Warner, 1972; Helser and McLaughlin, 1975). During the processing into the 3 final rRNA species nearly 20% of the precursor rRNA is lost. The DNA segment coding for the 35S rRNA precursor is about 5 million daltons comprising 2.6, 1.4 and 0.12 million daltons of 25S, 18S and 5.8S rDNA respectively. If the total S. cerevisiae nuclear genome is assumed to be 1.25 x 10 ~° daltons, then the satellite DNA constitutes a genome of at least 1.25 x 109 daltons. Since the 5S RNA has been shown to be adjacent to the rRNA cistrons (Rubin and Sulston, 1973), the satellite DNA then may contain at least 4 defined species of DNA comprising a total molecular weight of about 5 million daltons. The 25S (and 5.8S), 18S and 5S RNA are p~esent in about 140 to 150 copies (Schweizer etal., 1969; Rubin and Sulston, 1973) mostly as clusters of 10 to 30 (Kaback et al., 1973). Thus if all of the satellite DNA codes for only these 4 RNA species, then the segment of DNA representing each copy would be about 8 to 9 million daltons. This could mean that about 40% of the DNA is not transcribed either as 35S or 5S RNA. Portion or all of this DNA could be involved in the processing or regulation of transcription of the rRNA genes. The 3 EcoR1-DNA species in bands 2, 7 and 8 (Fig. 6) comprise a total of 2.4 million daltons which is nearly 90% of the size of the 25S rDNA. The total size of the EcoR1-DNA species from bands 4, 6 and 9 is about 1.9 million daltons which is about 140% of the size of the 18S rDNA. Consequently, the EcoR1-DNA species from bands 4, 6 and 9 must contain DNA portions other than 18S rDNA. Although the hybridization of the DNA species from band 7 with 18S rRNA may represent a contamination of the DNA from band 6 (Fig. 7), the DNA species from band 8 is well separated from band 9 and may truly represent a DNA segment that contains portions of both 18S and 25S rDNA. These 6 bands (2, 7, 8 and 4, 6, 9) together would represent a single DNA species of about 4.3 million daltons ; including band 5 it becomes about 4.8 million daltons, very close to that estimated for the precursor DNA encoding the 35S rRNA.

Identification of the DNA species in band 3 is uncertain. While the hybridization with the 25S rRNA is low, yet it could result from a contamination of the DNA species from band 2. On the other band the amount of DNA in band 3 as judged by the relative intensities and the molecular weight of the DNA species from this region is comparable to that from bands 2 and 4. While segment II in Figure 10 comprising bands 2, 3 and 4 amount to about 30% of the total EcoR1-DNA each of the 3 bands constitute about one third of the segment II or 10% of the total DNA. The peaks of the bands 2, 3 and 4 in the scan of Figure 7 is about 20% of each of the bands or about 2% of the total EcoR1-DNA. Moreover, the generation of these 3 DNA species seem to occur simultaneously, probably from similar EcoR1precursor DNA segments (Fig. 4). If the DNA species from band 3 was then included along with the DNA species from 7 other bands (2 and 4 through 9) a DNA segment of about 6 million daltons is obtained. This value is about 20% higher than the DNA representing the 35S rRNA but about 30% lower than a copy of about 150 pieces of satellite DNA. Our preliminary attempts in hybridizing the DNA species from bands 2 through 9, mainly from band 3, with [3H]-methyl 35S rRNA precursor molecule was without success probably due to the low quantity and the low specific activity of the precursor RNA used. Even if the DNA species from band 3 is not a part of the ribosomal cistron it may be a part of the satellite DNA or include 5 sRNA due to its reiterated nature. sites on yeast satellite DNA and at least 7 on total rDNA. In our discussion above we have assumed a We may have recognized at least 8 EcoR1 cleavage value of 5 x 10 6 daltons for the rRNA precursor molecule (35S) mainly because this value seems to be the size species reproducibly obtained from S. cerevisae (results not presented; and Udem and Warner, 1973). On the other hand the rRNA precursor molecule from S. carlsbergensis has been reported to be as large as 42S which is 6.2 x 106 daltons (Retel and Planta, 1970; Retel and Von Keuben, 1975). If such a 42S precursor rRNA species does exist in S. cerevisiae then the 8 EcoR1-DNA fragments can be accounted for. It should be pointed out though that obvious differences may exist between these two yeast strains. Thus whereas the 18S and the 25S rRNA in S. cerevisiae show no sequence homology (Fig. 13, Table 3) these two rRNA species in S. carlsbergensis show in excess of 85% sequence homology (Retel and Planta, 1970), It will be interesting to compare the EcoR1 generated pattern of total yeast DNA with the pattern obtained from isolated pure yeast satellite DNA. The relationship of the EcoR1 generated rDNA

K. Nath and A.P. Bollon: EcoRl Endonuclease Generated Yeast rDNA Table 3

DNA preparation from band

5 6 7 8 9

DNA hybridized with Total [aH]-rRNA

[3H]-18S +[H]-25S

[3H]-25S +[H]-18S

2.2% 3.9% 4.9% 5.9% 3.1%

1.5% 2.6% 1.1% 1.9% 3.4%

0.6% 0.6% 4.0% 3.5% 0.3%

The 250 pl hybridization mixture contained 4.8 gg/ml of [3H]18S+25S (2: 1) rRNA or 5.3 pg/ml [3H]-18S+57 gg/ml 25S rRNA or 7.2 gg/ml [3H]-25S+67 ~tg/ml 18S rRNA. The 24 mm filter was loaded with 400 ng of denatured DNA of each species. The 6.5 mm filters contained nearly 50% of the DNA for bands 5, 8 and 9, and 80 to 90% for bands 6 and 7 at the end of experiments

fragments to the ribosomal spacer D N A and to chromosome 1 which codes for 70% of the ribosomal cistrons should be considered. Due to similarity in size of band 3 with bands 2 and 4 and the quantity of band 3 (2% of the total EcoR1-DNA) it can be speculated that band 3 contains ribosomal spacer D N A or some other reiterated D N A species such as 5 sRNA. In addition, some of the lower bands (6, 7, 8, 9) may also contain ribosomal spacer D N A or some other reiterated D N A species. The low degree of hybridization of bands 6, 7, 8 and 9 with r R N A may then be due to the presence of only small amounts of 18S or 25S D N A associated with the D N A of each band where the remaining non 25S or 18S D N A is ribosomal spacer DNA. Alternatively the small sizes of the lower bands may result in a low efficiency of hybridization or that the lower bands 6, 7, 8 and 9 contain a class of reiterated D N A in addition to segments of the ribosomal genes. It remains to be established if the ribosomal gene cluster on chromosome 1 differs with non chromosome 1 ribosomal gene organization; if the ribosomal gene organization differs it must then be established whether any of the EcoR1 generated r D N A fragments are specific for only chromosome 1. Acknowledgment. We thank Dr. J. LoSpalluto for operation of the analytical ultracentrifuge, Mr. Richard Todd for the slab gels, and Mr. Terry Webb and Mr. Bob Parker for the photography of the gels and Dr D. Finkelstein for helpful discussions. K. Nath was supported by a Moss Heart Fellowship and the American Cancer Society. This investigation was supported by grants from the United States National Science Foundation, the American Cancer Society and The Robert A. Welch Foundation.

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Communicated by G. O'Donovan Received April 2, 1976