Molec. gen. Genet. 151,229-244 (1977) © by Springer-Verlag 1977

Construction and Restriction Endonuclease Mapping of Hybrid Plasmids Containing Saccharomyces cerevisiae Ribosomal DNA Jane Harris Cramer, Frances W. Farrelly, Joy T. Barnitz, and Robert H. Rownd Laboratory of Molecular Biology and Department of Biochemistry, University of Wisconsin, 1525 Linden Drive, Madison, Wisconsin 53706, USA

Summary. Fragments produced by partial digestion of Saccharomyces cerevisiae ribosomal D N A (rDNA) with the restriction endonuclease EcoRI were ligated in vitro to the bacterial plasmid RSF2124. The resulting hybrid plasmids were cloned in Escherichia coli. Three hybrid plasmids which contain at least one intact repetitive unit of the multiple, tandem sequences of the yeast r D N A genes have been further characterized. These plasmids have been used to construct a map of the EcoRI, SmaI, HindII and HindIII restriction sites in the individual repetitive units of yeast rDNA.

Introduction The ribosomal R N A (rRNA) genes in the yeast Saccharomyces cerevisiae provide a useful system for the study of gene expression and control in eucaryotes. There are approximately 140 copies of the r R N A genes per haploid genome (Schweizer, MacKechnie, and Halvorson, 1969). In CsC1 density gradients r D N A (previously called ~ DNA) has a density of 1.705 g/cc, whereas the density of the remainder of the nuclear DNA, called c~ DNA, is 1.699 g/cc (Cramer, Bhargava, and Halvorson, 1972). Hybridization studies have shown that in addition to the genes for the 18S and 25S rRNAs the 7 D N A contains the genes for 5S R N A and possibly some t R N A species (Cramer et al., 1972; Aarstad and Oyen, 1975). The 18S, 25S and 5.8S rRNAs are transcribed together as part of a 35S precursor molecule which has a molecular weight of 2.5 x 106 (Udem and Warner, 1972). Approximately 20% of this precursor is removed and degraded during subsequent processing (Udem and Warner, 1972). The genes for the 35S r R N A precursor are interspersed with those for the 5S RNA, probably in an alternating arrangement

(Rubin and Sulston, 1973), but the 5S R N A is synthesized independently of the other r R N A species (Udem and Warner, 1972; McLaughlin, 1974). Restriction endonuclease and denaturation mapping experiments (Cramer, Farrelly, and Rownd, 1976; Cramer, unpublished results) have shown that the r D N A consists of a tandem head-to-tail array of homogeneous genetic units, each of which has a molecular weight of 5.6 x 106. Each unit presumably contains genes for the 35S precursor and 5S rRNAs and a small amount of nontranscribed spacer DNA. These units are arranged in the genome in clusters which are surrounded by ~ D N A (Cramer et al., 1972). Approximately 70% of the r R N A genes are located on chromosome I, whereas the chromosomal location of the remainder is unknown (Kaback, Bhargava, and Halvorson, 1973; Finkelstein, Blamire, and Marmur, 1972). Using the restriction endonuclease EcoRI, we have constructed hybrid plasmids between purified yeast r D N A and the bacterial plasmid RSF2124, a ColEI derivative which carries resistance to ampicillin (So, Gill, and Falkow, 1975). RSF2124 has several features which make it an especially useful cloning vehicle. It has a single EcoRI site which is in a gene affecting colicin biosynthesis; when foreign D N A is inserted at this site colicin is no longer produced. Therefore, the properties of ampicillin resistance and failure to produce colicin can be used to screen the bacterial transformants for hybrid plasmids. EcoRI cleaves each r D N A repetitive unit at seven sites (Cramer et al., 1976). Therefore we have cloned partial digestion fragments of this D N A in order to obtain hybrid plasmids that contain at least one complete r D N A repetitive unit. We have isolated a number of hybrid plasmids which contain yeast rDNA. Three of t h e s e - p J H C l 1, pJHC23, and p J H C 3 5 - c o n t a i n yeast D N A segments which are larger than one r D N A repetitive unit. These

230 plasmids have been characterized in greater detail and they have been used to prepare a map of the EcoRI, S i n a i , H i n d I I , a n d H i n d I I I r e s t r i c t i o n s i t e s in t h e repetitive units of yeast rDNA.

Materials and Methods Strains. S. cerevisiae Y55 (Schweizer et al., 1969) a wild type diploid was obtained from H.O. Halvorson, E. coli Cla, a prototrophic C strain (Sasaki and Bertani, 1965) was obtained from B. Weisblum and E. coli HB101, r B m B- p r o - gal recA- streptomycin R (Boyer and Roulland-Dussoix, 1969), was obtained from P. Wensink. Yeast rDNA Preparation. S. cerevisiae cells were grown to stationary phase in YEP (Cramer et al., 1972), disrupted with a French press and the D N A was isolated from whole cells by the method of Bhargava, Cramer, and Halvorson (1972). Yeast rDNA was purified by Hg ++/CszSO4 density gradient centrifugation as described in Cramer et al. (1972). The amount of c~D N A contamination in the rDNA preparations was determined by analytical CsC1 density gradient centrifugation. Plasmid D N A Preparation. E. coli cells containing plasmids were grown to mid-log phase in Penassay broth (Difco) and chloramphenicol was added to a final concentration of 170 lag/ml to increase the number of copies of the plasmid (Clewell, 1972). The cells were incubated for an additional 12 15 h, harvested, and plasmid D N A was isolated by the cleared lysate procedure as detailed in Guerry, LeBlanc, and Falkow (1973). The D N A in the cleared lysate supernatant was precipitated by adding polyethylene glycol (Carbowax, Union Carbide) to a final concentration of 10% (w/v), NaC1 to a final concentration of 0.5 M and subsequently incubating this solution at 4 ° C for at least 2 h. The precipitate was collected by centrifugation for 5 rain at 3000 rpm in a Sorvall SS34 rotor, dissolved in 1/10 x SSC (SSC is 0.15 M NaCI, 0.015 M Na citrate, pH 8.0), and the closed circular plasmid D N A was further purified by ethidium bromide-CsC1 density gradient centrifugation. Ethidium bromide was added to the D N A to a final concentration of 250 lag/ml. The solution was adjusted to a density of 1.598 g/cc with solid CsC1, and the gradients were centrifuged for 60 h at 35,000 rpm or 36 h at 45,000 rpm in a Beckman Ti50 rotor. The band containing the closed circular D N A was visualized under illumination with ultraviolet light and was extracted from the gradient with a syringe through the side of the centrifuge tube. The ethidium bromide was removed by extracting 3 times with an equal volume of isoamyl alcohol, and the D N A solution was dialyzed exhaustively against 1/10 SSC. Restriction Endonuclease Preparation. The restriction endonuclease EcoRI was prepared from E. coli RY13 as previously described (Tanaka and Weisblum, 1975). Sinai was purified from Serratia marcescens strain SB using a modification of the procedure of Tanaka and Weisblum (1975). Late log phase cells were disrupted with a French press, the nucleic acids were removed by streptomycin sulfate precipitation, and the proteins were fractiouated by ammonium sulfate (Grand Island Biochemical ComlSany, enzyme research grade) precipitation as previously described (Tanaka and Weisblum, 1975). Proteins which precipitated between 50 and 80% saturation with ammonium sulfate were resuspended in ExBT (Extraction Buffer plus Triton: 0.01 M phosphate buffer, pH 7.0, 0.001 M EDTA, and 0.007M fl-mercaptoethanol, containing 0.015% Triton X-1.00), dialyzed against this same buffer, and applied to a DEAE cellulose column. The column was washed

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast rDNA with 200 ml ExBT (until the 0D280 of the effluent reached background levels) and the endonuclease was eluted with a 0 M to 0.6 M KC1 concentration gradient in ExBT. Active fractions, which were eluted between 0.25 M and 0.35 M KC1, were pooled, dialyzed against ExBT and applied to a phosphocellulose column (Whatman, P-11). This column also was washed with 200 ml ExBT prior to eluting the Sinai with a concentration gradient of 0.2 M to 0.8 M KC1 in ExBT. Washing the columns prior to starting the elution gradient removes nuclease activity which masks the presence of SmaI in the column fractions. Active fractions from the phosphocellulose column, emerging around 0.6 M KC1, were pooled, concentrated by dialysis against ExB (extraction buffer with no Triton X-100) containing 0.3 M NaC1 and 50% glycerol, and were stored at - 2 0 ° C. HindII and HindIII were purchased from P-L Biochemicals, Inc. and New England BioLabs. Restriction Endonuclease Digestion. Small quantities of D N A (1-2 lag for agarose, 5 10 gg for gradient acrylamide) for analytical gels were brought to a volume of 50 lal in 1/10 SSC, and 5 lal of enzyme plus 5 gl of a 10-fold concentrated solution of the appropriate buffer were added. The buffers for the enzyme digestions were: 0.09 M Tris HC1 and 0.01 M Mg SO4, pH 7.4 for EcoRI; 0.015 M Tris HC1, 0.001 M MgClz, 0.15 M KC1, pH 9.0 for SmaI; 0.01 M Tris HC1, 0.0067 M MgC12, 0.06 M NaC1, pH %4 for HindII and HindIII. The digestion mixture was incubated at 37°C 1-3 h for EcoRI and Sinai, or 8-12 h for HindII and HindIII. When D N A was digested with two different enzymes, the mixture was dialyzed vs 1/10 SSC after the initial digestion, then the second enzyme and the appropriate buffer were added and the incubation continued. If the sample was to be electrophoresed, 20 lal of BJ solution (60% sucrose, 0.04 M EDTA, 0.005% Bromphenol blue) was added when digestion was complete. If the D N A was to be used for electron microscopy, BJ was omitted and the sample was heated to 65 ° C for lOmin to stop the reaction. When larger amounts of D N A were digested for preparative purposes, the same procedures were followed with all components increased in proportion to the amount of D N A used. Electrophoresis. (i) Agarose gels. Cylindrical analytical agarose gels (11 x 0.6 cm) were 0.6% or 1% in E buffer (Electrophoresis buffer: 0.04 M Tris HC1, 0.02 M Na acetate, 0.001 M NazEDTA, pH 7.9, with 0.5 lag/ml ethidium bromide). D N A samples containing BJ (see above) were loaded onto the gels and electrophoresed at 50 or 70 V from 3-5 h depending on the size of the fragments to be examined. Large scale 1.0% agarose gels (Tanaka et al., 1976) were used to prepare restriction fragments for mapping studies. (ii) Gradient acrylamide gels. Gradient acrylamide gels were prepared using a modification of the method of Jeppeson (1974) in an apparatus made by Hoeffer Scientific Instruments (Model SE500). These slab gels ( 9 x 1 6 x 0 . 3 cm) were composed of two parts, a 3% to 8% gradient acrylamide "running gel" which was 7 cm high, topped by a 2-cm-high 3% "stacking" gel made up in 1/5 the electrolyte concentration of the running gel. Acrylamide, N,N'-methylenebisacrylamide (bisacrylamide), and N,N,N',N'-tetramethylethylenediamine (TEMED) were from Eastman Kodak and ammonium persulfate was from Mallinckrodt, E buffer was the same as that used for agarose gels. A plug gel was formed at the bottom of the gel mold from 2 ml of a solution containing 8.0% acrylamide, 0.2% bisacrylamide, 0.2% TEIVIED and 0.04% ammonium persulfate. 5 ml of "layering solution" containing 0.2% TEMED and 0.01% ammonium persulfate were layered over the plug gel. The gradient acrylamide gel was then formed by displacement from the bottom of the gel mold by feeding the solution through 1 mm diameter polyethylene tubing inserted along the side of the gel mold and immersed in the layering solution. The gradient maker contained 30 ml of a degassed solution with 3.0% acrylamide~

J.H. Cramer et aI. : Hybrid Plasmids Containing Yeast rDNA 0.15% bisacrylamide and 10% sucrose in E buffer in the mixing chamber and 20 ml of a degassed solution with 8.0% acrylamide, 0.2% bisacrylamide and 25% sucrose in E buffer in the other chamber. The sucrose was included to stabilize the acrylamide gradient and Bromphenol blue was added to the 8.0% acrylamide solution to act as a visible indication of the linearity of the gradient. Immediately prior to pouring the gradient, TEMED and ammonium persulfate were added to the 3.0% acrylamide solution to final concentrations of 2.0% and 0.01%, respectively, and ammonium persulfate was added to the 8% acrylamide solution to a final concentration of 0.006%. The concentrations of TEMED and ammonium persulfate were carefully adjusted to avoid convection during polymerization of the gel and ensure smooth formation of the acrylamide gradient. The 3% acrylamide solution was allowed to flow into the gel mold until the levels of solution in the two chambers were equal, then the valve between the two chambers was opened and the remainder of the gradient formed. The tubing was removed and the gel allowed to polymerize for 4 h. With a template in place at the top of the gel mold, the layering solution was removed and the stacking gel solution (3.0% acrylamide and 0.15% bisacrylamide in 1/5 E buffer plus 0.083% TEMED and 0.083% ammonium persulfate) was added until the level reached the top of the gel mold. The stacking gel was allowed to polymerize for 4 h, after which E buffer was added to the upper chamber of the gel apparatus and the template was carefully removed. The template formed wells which were 4 mm wide and I0 to 15 mm deep. DNA samples containing BJ (see above) were layered into the wells and run in E buffer at 50 volts for 12 14 h. The gel was removed from the electrophoresis apparatus, stained for 1 h in a solution of 2 gg/ml ethidium bromide, and photographed. No destaining was necessary. The DNA bands in both agarose and acrylamide gels were visualized and photographed as previously described (Tanaka et al., 1976).

Extraction of DNA from Agarose Gels. Bands containing DNA were visualized by illumination with ultraviolet light and cut from the gel with a razor blade. The gel slices were crushed by forcing tSem through a 25 gauge hypodermic needle, mixed with an approximately equal volume of 1/10 SSC and frozen and thawed three times. The agarose was removed by centrifugation at 12,000 rpm for 30 min in a Sorvall SS34 rotor, and the supernatant was concentrated by one of two methods. In the first, the supernatant was reduced to a volume of approximately 100 gl by dialysis vs 35% polyethylene glycol (Carbowax, Union Carbide), and dialyzed exhaustively against 2 x SSC, then against 1/10 SSC. In the second, the NaC1 concentration of the supernatant was adjusted to 0,3 M, and the DNA was precipitated by the addition of two volumes 95% ethanol. After remaining at - 4 ° C for at least 12 h, the precipitate was collected by centrifugation at 10,000 rpm for 30 min in a Sorvall HB4 rotor, and resuspended in 100 ~tl of 1/10 x SSC.

Construction of Hybrid Plasmids: Cloning in E. coll. RSF2124 was digested with EcoRI to convert the molecules to linear form. Purified yeast rDNA ( < 5% e~DNA) was also digested with EcoRI under conditions designed to yield partial digestion fragments large enough to include at least one complete rDNA repetitive unit. Equal aliquots of rDNA were digested with serial dilutions (1/2, 1/4,. .... 1/64) of EcoRI for 15 min and the extent of the digestion reaction in each sample was analyzed by electrophoresis on 1% agarose gels. The digestion reaction (in this case, the one with a 1/32 dilution of EcoRI) which contained large partiaI digestion fragments and no detectable complete digestion fragments was used in the ligation reaction. The restriction endonuclease reactions for both RSF2124 and rDNA were terminated by heating the DNA to 65 ° C for 5 rain

231 and the two DNAs were mixed and incubated with DNA ligase as previously described (Tanaka and Weisblum, 1975). The ligated DNA sample was used to transform E. coil Cla cells according to the method of Cohen, Chang, and Hsu, 1972. The transformed cells were diluted 30-fold into enriched medium (containing per 1 of H 2 0 : 5 g peptone, 5 g yeast extract, 1 g K2HPO4 and 2 g glucose), divided into five different growth tubes, grown 2 h at 37 ° C and the cells in each tube were plated on enriched medium containing 100 ~tg/ml ampicillin. Ampicillin resistant colonies were then assayed for colicin production (Clowes, 1968), and plasmid DNA was prepared by the chloramphenicol amplification-cleared lysate technique (see above) from colonies which failed to produce colicin. A portion (20-30 lal) of the DNA in the cleared lysates was mixed with 20 p.1 BJ plus 0.5-1 ~g purified RSF2124 DNA to act as a molecular weight marker, and electrophoresed on 0.6% agarose gels. The remainder of the cleared lysate was heated to 65 ° C for 15 rain, extracted once with chloroform-isoamyl alcohol (24:1 mixture), dialyzed exhaustively against 1/10 SSC, digested with EcoRI, and electrophoresed on 1.0% agarose gels to determine which rDNA EcoRI fragments were present.

Electron Microscopy. Intact hybrid plasmid DNA and restriction fragments were mounted for electron microscopy by the method of Inman and Schn6s (1970) and rotary shadowed with platinum. Either d~XRF DNA, which has a molecular weight of 3 . 4 x 10 6 (Davis, Simon and Davidson, 1971) or PM-2 DNA, which has a molecular weight of 6.39 × 106 (Kriegstein and Hogness, 1974), was included with the DNA to be examined as an internal molecular weight standard. Grids were examined and photographed with a Phillips 300 electron microscope. Length measurements were made on a digitizer (Numonics Corp.) interfaced to a programmable calculator and plotter (Hewtett-Packard).

Results Construction o f H y b r i d Plasmids between Yeast r D N A and R S F 2 t 2 4 Bacterial plasmid RSF2124 DNA and yeast rDNA were digested separately with EcoRI to generate cohesive ends (Hedgpeth, Goodman and Boyer, 1972), a n n e a l e d t o o n e a n o t h e r a t l o w t e m p e r a t u r e s , a n d c o v a l e n t l y l i n k e d w i t h D N A l i g a s e as d e t a i l e d in Experimental Procedures. The yeast rDNA used in t h e c l o n i n g e x p e r i m e n t s w a s j u d g e d t o b e a t l e a s t 9 5 % p u r e b a s e d o n its p r o f i l e in a n a l y t i c a l CsC1 d e n sity g r a d i e n t s . R S F 2 1 2 4 h a s o n l y o n e E c o R I site a n d w a s d i g e s t e d t o c o m p l e t i o n w i t h E c o R I t o c o n v e r t it t o linear form. Yeast rDNA was partially digested with E c o R I as d e t a i l e d in E x p e r i m e n t a l P r o c e d u r e s . T h e rDNA digestion mixture which contained the largest partial digestion fragments and no detectable complete digestion fragments was used in the ligation reaction. The yeast partial digestion fragments were n o t s e p a r a t e d i n t o size c l a s s e s p r i o r t o l i g a t i o n b u t i n s t e a d t h e e n t i r e d i g e s t i o n r e a c t i o n w a s u s e d . E. coli was transformed with the ligated DNA and transformants were selected first for ampicillin resistance and t h e n f o r loss o f c o l i c i n p r o d u c t i o n . D N A f r o m c l o n e s containing prospective hybrid plasmids was then

232

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast rDNA Table 1. Molecular weights of EcoRI, EcoRI + SmaI, and EcoRI + HindIII restriction fragments of pJHC11 Fragment source

Molecular weights ( x 10 6) EcoRI

a

b

c

Fig. 1a-c. Gradient acrylamide gel electrophoresis of hybrid plasmid DNA. DNA from hybrid plasmids was purified, digested with EcoRI, and electrophoresed on gradient acrylamide gels as described in Experimental Procedures. a pJHC11, b pJHC23, e JHC35

screned for p l a s m i d size a n d for the presence of yeast r D N A E c o R I fragments. A n u m b e r of plasmids which c o n t a i n some or all of the yeast r D N A E c o R I fragments have been isolated. W e have n o t observed a n y plasmids which c o n t a i n yeast D N A E c o R I fragments other t h a n the seven E c o R I fragments o b t a i n e d by digestion of purified r D N A . Three plasmids which c o n t a i n all seven r D N A E c o R I f r a g m e n t s - p J H C l l , p J H C 2 3 a n d p J H C 3 5 - w e r e selected for further study. Figure 1 shows the E c o R I digestion p a t t e r n of these three plasmids o n a gradient acrylamide gel. Eight fragments are visible for each plasmid. The largest f r a g m e n t is linear R S F 2 1 2 4 D N A , which has a m o l e c u l a r weight of 7.4 x 106 (B. W e i s b l u m , personal c o m m u n i cation). The r e m a i n i n g seven fragments are from the yeast D N A p o r t i o n of the p l a s m i d a n d have the same

RSF2124

7.4a

Yeast rDNA A B C D E F G

1.79b 1.46 1.19 0.41 0.34 0.22 0.17

EcoRI + SmaI

EcoRI + HindIII

6.7" 0.7 a A B-1 C D E F G B 2

1.79 1.31c 1.19 0.41 0.34 0.22 0.17 0.15c

7.4" A B-1 C-1 D E F G C-2 B-2

1.79 1.37a 1.07° 0.41 0.34 0.22 0.17 0.12~ 0.09d

Molecular weight values obtained from B. Weisblum b The molecular weight of this and all other fragments, unless otherwise indicated, was taken from Cramer et al., 1976 c This pair of fragments arose by cleavage of EcoRI fragment B. The molecular weight of EcoRI-SmaI B-1 was determined by measuring its contour length in the electron microscope. The molecular weight of EcoRI-SmaI B- 2 was calculated by subtracting the molecular weight of B 1 from that of the original EcoRI B fragment d This pair of fragments arose by cleavage of EcoRI fragment B, The molecular weight of EcoRI-HindIII B- 1 was determined by coelectrophoresis with DNA markers of known molecular weight, the EcoRI fragments of the R plasmid N R ! (Tanaka et al., 1976). The molecular weight of B 2 was calculated by subtracting the molecular weight of B-1 from that of the original EcoRI B fragment e This pair of fragments arose by cleavage of EcoRI fragment C. The molecular weight of EcoRI-HindIII C 1 was determined by coelectrophoresis with DNA markers of known molecular weight (Tanaka et al., 1976). The molecular weight of C-2 was calculated by subtracting the molecular weight of C- 1 from that of the original EcoRI C fragment

m o l e c u l a r weights as the E c o R I fragments from purified r D N A ( C r a m e r et al., 1976). They have been designated A t h r o u g h G to be consistent with the n o m e n c l a t u r e of the yeast r D N A E c o R I fragments. The m o l e c u l a r weights of the p J H C 11 E c o R I fragments are given in Table 1. It is clear f r o m a n e x a m i n a t i o n of the gel p h o t o g r a p h s in Figure 1 that some o f the D N A b a n d s fluoresce more brightly t h a n w o u l d be expected if all b a n d s were present in stoichiometric a m o u n t s . F o r example, in the gel p a t t e r n for p J H C 3 5 , r D N A fragments E c o R I E a n d E c o R I F are present in increased p r o p o r t i o n s relative to the other r D N A fragments. T h u s the h y b r i d plasmids a p p a r e n t l y cont a i n m o r e t h a n one copy of some of the r D N A E c o R I fragments a n d therefore include a segment of yeast D N A which is larger t h a n a single r D N A repetitive u n i t of 5.6 x l 0 6 daltons.

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast r D N A Table 2. Molecular weights of SmaI and HindIII restriction fragments of pJHC11, pJHC23, and pJHC35 DNA source

Restriction fragment

Molecular weights ( x 106) Undigested

SmaI

HindIII

Yeast rDNA

A B

5.60"

4.0" 1.6 a

pJHC11

A B C D E

8.2 c 5.6 ~ 2.7 °

9.5 c 4.0 ~ 1.60 c'a

Sum of fragments Plasmid molecular weight A B C Sum of fragments Plasmid molecuIar weight

16.6 b

pJHC23

A B Sum of fragments Plasmid molecular weight

16.5 16.5

8.0 u 6.2 u

15.1 16.7

14.5 b

14.2 14.2

8.6 b 4.0 b 1.65 b 14.25 14.3

14.0 b

11.5 c 2.7 ° 14.2 14.2

11.9 b 1.66 b 13.56 13.6

pJHC35

Sinai + HindIII

6.8 ~ 4.0 ~ 2.27 c 1.17 °'d 0.43 c, d 14.67 16.3

a Molecular weights from Cramer et al., 1976 b Molecular weights determined by measuring contour lengths in the electron microscope as described in Experimental Procedures c Molecular weights determined by coelectrophoresis with D N A fragments of known molecular weight, the EcoRI fragments of the R plasmid NR1 (Tanaka, et al., 1976) d These fragments are present in two copies per plasmid and are counted twice in calculating total plasmid molecular weights

The molecular weights of the three plasmids as determined by measuring their contour lengths in the electron microscope are shown in Table 2. Since the RSF2124 portion of the plasmid has a molecular weight of 7.4 x 106, the yeast DNA portion must account for approximately 9.2 x 106, 7.1 x 106 and 6.6x 106 daltons in p J H C l l , pJHC23 and pJHC35, respectively. These results are consistent with our observation that the plasmids contain more than one copy of the some of the rDNA EcoRI fragments. The fact that the three plasmids have all seven yeast rDNA EcoRI fragments does not necessarily mean that they contain an intact monomer unit as it exists in yeast rDNA. For example, separate EcoRI partial digestion fragments might have associated with one another in the ligation mixture before being linked to RSF2124. Therefore, before proceeding with our mapping studies, we digested the plasmids with two other restriction enzymes, SmaI and HindIII, to determine whether the order of rDNA EcoRI frag-

233

ments in the plasmids is the same as in yeast rDNA. If the order of EcoRI fragments were altered, the positions of the SmaI and HindIII restriction sites would be changed and the sizes of the plasmid fragments would be different from those obtained by digestion of purified yeast rDNA alone. In order to understand the SmaI and HindIII digestion results with the different plasmids, it may be helpful to refer to their restriction maps shown in Figures 3 and 8. Digestion of pure yeast rDNA with SmaI yields one fragment with a molecular weight of 5.6 x 106 (Cramer et al., 1976). The SmaI cleavage site is in EcoRI fragment B (Cramer, unpublished results). Therefore in order to obtain an intact rDNA SmaI fragment from a plasmid, it would have to contain two copies of rDNA EcoRI fragment B. HindIII digestion of rDNA yields two fragments, rDNA HindIII A with a molecular weight of 4.0 × 106 and rDNA HindIII B with a molecular weight of 1.6x 106 (Cramer et al., 1976). Since HindIII has two cleavage sites per rDNA repetitive unit, we should observe at least one of these two fragments in each plasmid if all of them actually do include one complete repetitive unit. The locations of the two HindIII cleavage sites are in EcoRI fragments B and C (see below). Therefore, the presence of both rDNA HindIII fragments would require that two copies of either rDNA EcoRI fragment B or EcoRI fragment C be present in the rDNA portion of the plasmid. In addition to generating SmaI and HindIII fragments which contain only rDNA, digestion of the plasmids with these two enzymes yields restriction fragments which contain segments of both rDNA and RSF2124 DNA. For this reason, plasmid fragments generated by digestion with restriction enzymes other than EcoRI are labeled with capital letters in order of decreasing size with no effort to designate whether the source of the DNA was RSF2124 or rDNA. The SmaI and HindIlI restriction fragments which include both rDNA and RSF2124 DNA are discussed with the mapping experiments in the following sections since analyses of their molecular weights and their restriction patterns after subsequent digestion with EcoRI have been useful in determining the restriction fragment maps of the plasmids. Figure 2 shows the SmaI and HindIII digests of p J H C l l , pJHC23, and pJHC35 compared to those of yeast rDNA after electrophoresis on 0.6% agarose gels. The molecular weights of these restriction fragments are given in Table 2. For each plasmid the molecular weight totals of the SmaI fragments are close to those obtained for the intact circular DNA molecule. The SmaI digest of pJHC11 contains three fragments. Since RSF2124 has only one SmaI site, the other two cleavage sites are apparently in the

234

Sma I

a bcd

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast rDNA

Hind 111

e f g h

Fig. 2a-h. Agarose gel electrophoresis of SmaI and HindIII digests of hybrid plasmids and yeast rDNA. Hybrid plasmid D N A and yeast rDNA were purified, digested with SmaI (gels a through d) or HindIII (gels e through h) and electrophoresed on 0.6% agarose gels as described in Experimental Procedures. a, e yeast rDNA; b, f p J H C l l ; e, g pJHC23; d, h pJHC35

yeast rDNA portion of the molecule. The plasmid SmaI fragment B has the same molecular weight as the yeast monomer rDNA unit and coelectrophoreses with the rDNA SmaI fragment when the two different digests are electrophoresed together as described by Tanaka et al. (1976) in a 0.6% agarose gel (data not shown). The simplest explanation of these results is that a single rDNA fragment containing two SmaI sites, i.e. a repetition of rDNA EcoRI fragment B, has been incorporated into pJHC11. It seems unlikely that a fragment of molecular weight 5.6 × 106 would arise fortuitously by the annealing of two different rDNA EcoRI fragments. However, we cannot rule

out the possibility that the rDNA in excess of the 5.6 × 106 dalton Sinai fragment has come from a separate rDNA EcoRI fragment, pJHC23 and pJHC35 have only two Sinai fragments, apparently arising from the single cleavage site in RSF2124 and one site in their rDNA segment. Digestion of either pJHCll or pJHC23 with HindIII yields three fragments. HindIII fragments B and C from each of these plasmids have the same molecular weights as the rDNA HindIII fragments and coelectrophorese with them in 0.6% agarose gels (data not shown). We would except to obtain at least one copy of each of the rDNA HindIII fragments from pJHC11 since the results of the Sinai digestion have shown that this plasmid includes a repetition of EcoRI fragment B. The sum of the molecular weights of the three pJHCll HindIII fragments is 15.1 × 106, less than expected from molecular weight determinations of the intact plasmid and of its Sinai fragments. This discrepancy is explained by the fact that the plasmid contains two copies of the 1.6 x 106-dalton HindIII fragment (see below). Thus the rDNA segment in pJHCll must include two copies of rDNA EcoRI fragment C in addition to two copies of rDNA EcoRI fragment B (see Fig. 3). In the case of pJHC23, we know from the SmaI digestions that the plasmid does not contain a repetition of rDNA EcoRI fragment B. Therefore in order to yield both rDNA HindIII fragments it must contain a repetition of rDNA EcoRI fragment C (see Fig. 8). HindIII digestion of pJHC35 yields only two HindIII fragments, the smaller of which is equivalent to the smaller rDNA HindIII fragment. Therefore pJHC35 apparently has only one copy of both rDNA EcoRI fragment B and fragment C (see Fig. 8). These results are consistent with what we would expect from the molecular weight of the rDNA portion of pJHC35. It contains only about 1.0 x 10 6 daltons of yeast DNA in excess of that required for a complete rDNA unit and this is not enough to account for an additional copy of either rDNA EcoRI fragment B (1.46 × 10 6 daltons) or EcoRI fragment C (1.19 × l06 daltons) (see Table 1). Although the digestion results cited above do not provide unequivocal evidence that the order of all the yeast rDNA EcoRI fragments in the plasmids is the same as in rDNA purified directly from yeast, they strongly suggest that this is the case, especially in pJHCll. This conclusion was supported by the mapping experiments discussed below.

Restriction Endonuclease Mapping of pJHCl l We chose to concentrate our initial mapping efforts on pJHCll since it contains the largest segment of

J.H. Crameret al. : HybridPlasmidsContainingYeast rDNA yeast rDNA and since it yields an intact rDNA Sinai fragment, free of plasmid DNA, upon digestion with that enzyme. Our mapping strategy included the following approaches. We determined the molecular weights and the stoichiometry of the fragments of p J H C l l generated by digestion with SmaI and HindIII. Then the orientations of these fragments with respect to one another and the locations of the EcoRI cleavage sites were determined by digesting the plasmid with combinations of SmaI+HindIII, SmaI+ EcoRI, and HindIII+EcoRI, and by redigesting purified SmaI-HindIII restriction fragments with EcoRI. Ill addition, the location of the HindII sites on pJHC11 was determined by the same approachan analysis of the digestion products from HindII alone and in combination with other enzymes. An ambiguity in the order of HindII fragments was resolved by calculating the molecular weights of HindII incomplete digestion fragments and thereby determining which final fragments were adjacent to one another. In the mapping studies, fragment molecular weights are used as the basis for establishing the identities or relationships of different fragments. Because of the errors involved in our molecular weight measurements (in most cases less than_+3%), the values are not always identical or strictly additive; however, they are within the expected margin of error and therefore substantiate our conclusions. As shown in Figure 2b and Table 2, the SmaI digest of pJHCll has three fragments. Two of the three SmaI sites must be in the yeast rDNA portion of the plasmid and cleavage at these sites yields pJHC11 SmaI fragment B which is equivalent to the SmaI fragment from yeast rDNA. The RSF2124 portion of pJHCll has a molecular weight of 7.4 x 106 and includes one SmaI site which is located 0.7 x 106 daltons from the single RSF2124 EcoRI cleavage site (Tanaka and Weisblum, 1975). Therefore SmaI fragments A and C of pJHCll must contain portions of both rDNA and RSF2124 DNA. From a consideration of the sizes of these fragments, we can deduce that fragment A (8.2 x 106 daltons) must contain the large portion of RSF2124 (6.7 x 106 daltons) plus 1.5 x 106 daltons of rDNA, and fragment C (2.7 x 106 daltons) must contain the small portion of RSF2124 (0.7 x 106 daltons) and 2.0 x 106 daltons of rDNA. Thus, the amount of rDNA which is present in excess of the rDNA unit size is 3.5 x 106 daltons. Digestion of pJHCll with HindIII also yields three fragments (Fig. 2f, Table 2). HindIII fragments B (4.0x 106 daltons) and C (1.6 x 106 daltons) are equivalent to the two fragments obtained by digestion of purified yeast rDNA with HindIII and their combined molecular weight equals that of the individual rDNA units. RSF2124 has no cleavage sites for

235 HindIII (Farrelly, unpublished results). Thus, pJHC 11 HindIH fragment A (9.5 x 106 daltons) must contain all of RSF2124 and the remainder of the rDNA. As shown below, pJHCll includes two copies of the 1.6 x 106 dalton HindlII fragment (pJHC11 HindIII fragment C) per plasmid. Therefore, the molecular weight values calculated for pJHCll and for its rDNA segment from the sums of the SmaI and HindIII restriction fragments agree very well with one another and with the values determined for the intact plasmid. Digestion ofpJHC11 with both SmaI and HindIII yields five fragments, which have the molecular weights shown in Table 2. HindIII fragment A (9.5 x 106 daltons) is apparently cleaved by SmaI to give two fragments with molecular weights of 6.8 x 106 and 2.27 x 106, HindIII fragment B is not cleaved, and HindlII fragment C (1.6 x 106 daltons) is cleaved by SmaI to give two fragments with molecular weights of 1.17x 106 and 0.43x106. These changes in the restriction pattern account for only two SmaI cleavage sites. Since there are three SmaI sites in the plasmid and we know that two of them are in the yeast rDNA portion of the molecule, the simplest explanation of these data is that HindIII fragment C is present in two copies. This agrees with the prediction made on the basis of the discrepancy in total molecular weights of the Sinai and HindIII fragments. Figure 3 shows the positions of the SmaI and HindIII cleavage sites on the restriction map of pJHCll as deduced from these results. The locations of the SmaI and HindIII cleavage sites with respect to the pJHCll EcoRI fragments were then determined by digesting with each of these enzymes in combination with EcoRI. Figure4b shows the results of sequential digestion of pJHC11 with EcoRI and then SmaI. The RSF2124 portion of the plasmid is split into two fragments with molecular weights of 6.7x 106 and 0.7x 106 daltons as expected, and rDNA EcoRI fragment B is cleaved to yield two fragments, EcoRI-SmaI B- 1 and EcoRISmaI B-2 with molecular weights of 1.31 x 106 and 0.15x 106, respectively. When examining the fragments which result from cleavage of rDNA EcoRI fragments by a second enzyme, we will label them with the two enzymes (e.g. EcoRI-SmaI), the letter of the original EcoRI fragment with a superscript minus sign (e.g. B ), and then number them in order of decreasing size. The results of digestion of pJHCll with EcoRI plus HindIII are shown in Figure 4d. The migration rates of the RSF2124 fragment and of rDNA EcoRI fragments A, and D through G are unaffected, while rDNA EcoRI fragments B and C both migrate slightly faster through the gel, indicating that they

236

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast rDNA Hind "iT

H i n drf

Hindll HindlI

a b

Fig. 3. Restriction fragment map of hybrid plasmid pJHC11 DNA. The orientation of the restriction fragments was determined as described in the text. In the outermost circle the RSF2124 portion of the plasmid is represented by a solid line ( - - ) and the yeast rDNA portion is represented by a sawtooth line (~w). The SmaI, HindII and HindIII cleavage sites are labeled and are indicated by the long divider lines. The EcoRI cleavage sites are indicated by the short divider lines and the yeast rDNA EcoRI fragments are labeled with their appropriate letters. In the inner circles the entire plasmid is represented by a solid line ( - - ) . The middle circle shows the pJHC 11 SmaI-HindIII fragments with the cleavage sites for these two enzymes designated by the short divider lines. The center circle shows the pJHCll HindII+III fragments on the outside and the pJHC11 HindTI fragments on the inside. The HindII and the HindIII cleavage sites are indicated

Hind rr

c

are smaller t h a n the c o r r e s p o n d i n g E c o R I fragments. These two f r a g m e n t s will be referred to as E c o R I H i n d I I I B - 1 a n d C - 1 . T h e two smaller segments cleaved f r o m the E c o R I f r a g m e n t s will be referred to as E c o R I - H i n d I I I f r a g m e n t B - 2 a n d f r a g m e n t C - 2 . T h e m o l e c u l a r weights o f the p J H C l l E c o R I , E c o R I + S m a I , a n d E c o R I + H i n d I I I f r a g m e n t s are s h o w n in T a b l e 1. F r o m these results, r D N A E c o R I f r a g m e n t s B a n d C can be m a p p e d with respect to

d

Fig. 4a-d. Gradient acrylamide gel electrophoresis of EcoI + SmaI, and EcoRI + HindIII digestion of pJHC 11. Purified pJHC11 was digested in sequence with EcoRI and then either SmaI, or HindIII, and electrophoresed on gradient acrylamide gels. A sample of pJHC11 digested with EcoRI alone was included as an electrophoretic marker, pJHC11 digested with EcoRI alone a and e; EcoRI + SmaI b; and EcoRI +HindIII d the S m a I a n d H i n d I I I cleavage sites as s h o w n in Figure 3. By subtraction, we calculated t h a t a p p r o x i m a t e l y 0.16 x 106 daltons o f D N A b e t w e e n E c o R I f r a g m e n t s B and C were u n a c c o u n t e d for. This value is very close to the size o f r D N A E c o R I f r a g m e n t G ( 0 . 1 7 x 106 daltons), and we speculated that this f r a g m e n t m i g h t be l o c a t e d b e t w e e n E c o R I f r a g m e n t s B and C on the map. T o o b t a i n f u r t h e r i n f o r m a t i o n a b o u t the E c o R I

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast rDNA

a

bcd

e

237

f

map of the plasmid pJHC11, the following experiment was performed. Approximately 70 gg of pJHC11 was digested sequentially with HindIII and SmaI. Figure 5a shows a portion of the pJHC11 SmaI-HindIII digest on an analytical 1% agarose gel. The digestion products were separated on a 1% preparative agarose gel, and the DNA was extracted as described in Experimental Procedures. Each of the five SmaI-HindIII fragments was then digested with EcoRI and electrophoresed on the gradient acrylamide gel shown in Figure 5 c f . An EcoRI digest of pJHC11 has been included on the gel for comparison (Fig. 5b). Two EcoRI fragments are visible in the digest of pJHC11 SmaI-HindIII fragment A (6.8 x 106 daltons) (Fig. 5c). The larger one (6.7x 106 daltons) is the RSF2124 portion of the fragment and the smaller one (0.34 x 106 daltons) is rDNA fragment E. A third fragment between the HindIII site and the rDNA EcoRI E fragment junction, EcoRI-HindIII B 2, would be expected in this digest, but is too small to be seen under these electrophoresis conditions. Therefore EcoRI fragment E is adjacent to RSF2124 on one end of the yeast rDNA segment in pJHC11 and E must be adjacent to EcoRI fragment B in

Fig. 5a-f. EcoRI digestion of purified SmaI-HindIII fragments of pJHC 11. Purified pJHCI 1 was digested sequentially with SmaI and HindIII. The pattern of these fragments on a 1% analytical agarose gel is shown in a. The SmaI-HindIII fragments were separated on a preparative 1% agarose gel, the D N A was extracted and the purified fragments were digested with EcoRI as described in Experimental Procedures. The EcoRI restriction patterns of intact pJHC11 b; SmaI-HindlII fragment A c; SmaI-HindIII fragment B d; SmaI-HindIII fragment C e; and SmaI-HindIII fragment D f, on a gradient acrytamide gel are shown. The positions of the EcoRI fragments are shown to the left of gel b and the positions of the EcoRI fragments altered by cleavage with HindIII or Sinai are shown to the right of gel f

the rDNA since fragment B's position with respect to the HindIII and SmaI sites has already been determined. SmaI-HindlII fragment C (2.27x 106 daltons) yields three fragments after EcoRI digestion (Fig. 5 e ) - t h e 0.7x106 dalton RSF2124 segment, EcoRI fragment D (0.41 x 106 daltons) and EcoRIHindIII C-1 (1.07 x 106 daltons). It must therefore contain the other RSF2124-rDNA junction, and rDNA EcoRI fragment D must be between the 0.7 x 106 RSF2124 segment and the rDNA EcoRIHindIII C 1 fragment. SmaI-HindIII fragment B includes rDNA EcoRI fragments A,D,E,F and EcoRI-HindIII C- 1 (Fig. 5d). It should also include EcoRI-HindIII B-2; however, this fragment is too small to be seen under these particular electrophoresis conditions. We already know that rDNA EcoRI fragment E is adjacent to rDNA EcoRI fragment B so EcoRI E must be next to EcoRI-HindlII B-2 on one end of SmaI-HindIII fragment B, and rDNA EcoRI fragment C, which contains the HindIII site, must be on the other end. As mentioned above, EcoRI fragment D must be adjacent to rDNA EcoRI fragment C. From the data

238

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast rDNA

RSF2124 Hindl]

RSF2124 Hind'IT

pdHCll Hind lI+lrr

pJHCII HindK

PJHCil Hindf[

Fig. 6a-e. Agarose gel electrophoresis

a

b

c

shown to this point, the only ambiguity in the order of the rDNA EcoRI fragments contained in SmaIHindlI fragment B is the relative order of EcoRI fragments A and F. Experiments with pJHC35 (shown below) have demonstrated that F is next t o E and therefore the order of these rDNA EcoRI fragments is -E-F-A-D-C-. SmaI-HindIII fragment D is composed of the portion of rDNA EcoRI fragment B (designated B- in Fig. 50 which remains after SmaI and HindIII cleavage. We have been unable to cleave SmaI-HindIII fragment E with EcoRI (data not shown). The reason is not clear since this fragment must contain at least one EcoRI restriction site, i.e. the junction between rDNA EcoRI fragments B and C. In addition we can deduce that it contains rDNA EcoRI fragment G, both on the basis of the absence of rDNA EcoRI fragment G from all the other SmaI-HindIII fragments and on the basis of the SmaI-HindIII fragment E molecular weight. SmaI-HindIII fragment E (0.43x106 daltons) includes EcoRI-HindIII C-2 (0.12 x 106 daltons) and EcoRI-SmaI B-2 (0.15 x 106 daltons). However, 0.16x106 daltons of DNA, a value very close to the size of EcoRI fragment G (0.17 x 106 daltons), is unaccounted for. The location of rDNA EcoRI fragment G between fragments B

d

e

of RSF2124 and pJHC11 HindII digests. Purified RSF2124 or pJHC11 DNA was digested with HindII alone or HindII in combination with a second restriction endonuclease as indicated and the digests were electrophoresed on ! % agarose gels. a RSF2124 digested with HindII; b RSF2124 digested with HindII+EcoRI; c pJHC11 digested with HindII+III; d pJHC11 digested with HindII; e pJHC11 digested with HindII + EcoRI

and C was confirmed by digestion of the 1.6 x 106 dalton HindIII fragment from pJHC23 with EcoRI (see below). The location of the pJHC11 EcoRI fragments is shown in Figure 3. Before attempting to map the HindII cleavage sites in the rDNA of pJHCll, we examined the HindII and the HindII+EcoRI cleavage pattern of RSF2124 alone. Figure 6a and b shows the gel electrophoresis pattern of these fragments, and their molecular weights are given in Table 3. HindII cleaves RSF2124 into two fragments (Fig. 6a) and a subsequent digestion with EcoRI splits the larger of these (Fig. 6b). Since the yeast rDNA in the hybrid plasmids has been inserted at the EcoRI site, RSF2124 HindII-EcoRI fragments A and B will both be associated with rDNA segments in a HindH digest of pJHCll. RSF2124 HindII fragment B (or HindIIEcoRI fragment C) will be unchanged in a HindII digest of pJHC11. Previous results have given us some information about the location of the HindlI cleavage sites in yeast rDNA (Cramer et al., 1976). A mixture of HindlI +III cleavages yeast rDNA into six fragments with the molecular weights shown in Table 3. The two largest fragments have the same molecular weight and migrate together in agarose or acrylamide gels. The

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast r D N A

Table 3. Molecular weights of HindII, H i n d I I + I I I , and H i n d I I + EcoRI restriction fragments D N A source

Restriction fragment

Molecular weights ( x 106) HindII + III

RSF2124

A B C

Yeast r D N A

A B C D F, F

1.60 b 1.60 b 1.03 b 0.90 b 0.42 b 0.08 ~

pJHCll

A B C D E F G H I

5.08 a 2.50 a 1.73 a 1.60 d 1.60 a 1.03 d 0.90 a 0.42 a 0.08 °

HindII

HindII ÷ EcoRI

5.48 a 1.70"

3 -48a 1.98" 1-73a

6.60 e 3.14 ~ 2.58 ~ 1.74 e 1.03 ~ 0.99 ° 0.42 e

3-50f 2.10f 1.79 ~ 1.27 f 1.19 f 1.03 f 0.43 0.34 f'g

a Molecular weights determined by coelectrophoresis with D N A fragments of known molecular weight, the EcoRI fragments of file R plasmid N R 1 (Tanaka et al., 1976) b Molecular weights taken from Cramer et al., 1976 c The molecular weight for this fragment is only approximate and has been estimated from the difference between p J H C l l H i n d I I + I I I G and pJHC11 HindII F, and the difference between pJHC 11 HindII + I I I B and pJHC11 HindII C Molecular weights assumed to be the same as for the HindlI + IlI fragments of yeast r D N A e Molecular weights and fragment identities established by coelectrophoresis with a pJHC11 HindII + III digest f Molecular weights arid fragment identities established by coelectrophoresis with a pJHC11 EcoRI digest g Molecular weights for p J H C l l HindII-EcoRI fragments smaller than fragment H were not calculated

smallest fragment, F, is too small to be observed under our electrophoresis conditions and has been demonstrated only indirectly (see below). Only two fragments are obtained by digestion of yeast rDNA with HindIII (see Table 2). rDNA HindIII fragment A includes rDNA HindII +III fragments A,C,D, and E; rDNA HindIII fragment B includes rDNA HindII+III fragments B (Cramer et al., 1976) and F (see below). The orientation of the HindII restriction sites in the rDNA HindIII fragments was not determined. Figure 6 shows a H i n d I I + I I I digest of pJHCll compared with a HindII and a HindI[ + EcoRI digest. The molecular weights of the restriction fragments in these digests are given in Table 3. The identities of the fragments in the different digests have been

239

determined by comparing their molecular weights and by coelectrophoresing combinations of the different digests. A H i n d I I + I I I digest of pJHC11 yields nine fragments. Seven bands are visible on the gel (Fig. 6c) since fragments D and E migrate together and the smallest fragment, I, is not visible on the gel under these conditions. Fragments D through I of pJHC11 DNA are presumably the same as the rDNA H i n d l I + I I I fragments A through F, respectively. pJHCll HindII+III fragment C is equivalent to RSF2124 HindII fragment B. The other two pJHC11 HindII+III fragments, A and B, are composed of the rest of the RSF2124 DNA and portions of rDNA (see Fig. 3). Fragment A (5.08 x 106 daltons) must contain the 3.5x 106 dalton portion of RSF2124 (RSF2124 HindII-EcoRI fragment A), since fragment B is not large enough, and therefore also includes an rDNA segment of approximately 1.6 x 106 daltons which has no HindII or HindIII sites. We can conclude from the size of this rDNA segment that it must contain almost all of rDNA HindII+III fragment A or B and that it lies on the left-hand side of the map as shown in Figure 3, since there is a HindIII site 0.43 x 106 daltons into the rDNA portion of the plasmid on the right-hand side of the map. From experimental results on the location of pJHC11 HindIII fragments discussed earlier we can determine that rDNA HindII +III fragment B (pJHC11 HindlI +III fragment E), which is included in pJHCll HindIII fragment C, cannot be located next to RSF2124. Therefore, rDNA HindII + I I I fragment A must be in this position, pJHCll HindII+III fragment B (2.5 x 106 daltons) contains the segment of RSF2124 which is 1.98x106 daltons (RSF2124 HindII-EcoRI fragment B) plus a segment of rDNA which is approximately 0.5x 106 daltons; this then lies on the right-hand side of the map. Comparison of the pJHCll HindII+III digest with the pJHCll HindII digest (Fig. 6d) provides further information about the HindII restriction map of this plasmid (see Fig. 3). pJHC11 HindII + I I I fragments F and H are equivalent to pJHCll HindlI fragments E and G (rDNA H i n d t I + I I I fragments C and E), respectively. Since these two fragments are generated by HindII digestion alone, they must be the two interior fragments in the 4.0 x 106 dalton HindIII fragment, pJHCll HindII fragment D and HindII + III fragment C are the same and are equivalent to RSF2124 HindII fragment B. Two pieces of information from pJHC11 digests indicate that rDNA HindII+III fragments A and B are adjacent to one another. First, pJHC11 H i n d I I + I t t fragments D and E (which are equivalent to rDNA H i n d I I + I I I fragments A and B) are missing from the HindII digest,

240

but a new fragment, pJHCll HindII B, with a size approximately equal to their combined molecular weight appears. Second, pJHCll HindII+III A, which contains part of RSF2124 plus almost all of rDNA HindII+III A, is not present in the HindlI digest but is replaced by pJHCll HindII A which is larger by 1.5 x 106 daltons, approximately the size of pJHCll HindII+III fragment E (rDNA HindlI +III B). Two other observations support the conclusion that pJHC11 HindII + I I I fragment D (rDNA HindlI +III fragment A) is adjacent to pJHC 11 HindlI +III fragment E (rDNA HindII+III fragment B) and that pJHCll HindII÷III fragment I (rDNA HindlI + I I I fragment F) is on the other side of pJHC11 HindII + I I I fragment E (rDNA fragment B). pJHC11 HindII+III fragment G (rDNA HindII+III fragment D) is missing from the pJHCll HindII digest and a fragment which is slightly larger, pJHCll HindII F, appears. In addition pJHC11 HindII + I I I B disappears and is replaced by pJHC11 HindII C. In both cases the size difference has been demonstrated by coelectrophoresis of the two digests and the HindII fragments are larger by only about 0.08 x 106 daltons. This is, therefore, approximately the size of the proposed pJHC11 HindII +III fragment I (rDNA HindlI +III fragment F) which we have not yet observed directly. These results place pJHCll HindII+III fragment I (rDNA HindII+III fragment F) between pJHCll HindII+III fragments E and G (rDNA HindII + I I I fragments B and D). The only ambiguity remaining in the HindII map is the relative position of pJHCll HindII+III fragments F and H (rDNA HindII +III fragments C and E). A double digest of pJHC11 with HindII + EcoRI confirms the positions of the HindII ÷III fragments as assigned above, pJHC11 HindII-EcoRI fragments A,B, and C are equivalent to RSF2124 HindIIEcoRI fragments A,B, and C. pJHCll HindlIEcoRI fragment C (RSF2124 HindII-EcoRI fragment C) is approximately the same size as rDNA EcoRI fragment A. We know, however, from the position of the HindII cleavage sites that EcoRI fragment A is not present in the pJHCll HindII-EcoRI digest. pJHCll HindII-EcoRI fragments F and G, which are equivalent to pJHCll H i n d l I ÷ I I I fragments F and H (rDNA HindII+III fragments C and E) are present. There is no fragment in the pJHC11 HindII ÷EcoRI digest which is equivalent to rDNA EcoRI fragment B. However, p J H C l l HindIIEcoRI fragment D is the size expected for rDNA EcoRI fragment B after cleavage at its HindII site. p J H C l l HindII-EcoRI fragment E is equivalent to rDNA EcoRI fragment C, and pJHCll HindIIEcoRI fragments G and H are the same as rDNA EcoRI fragments, D and E. pJHCll HindII-EcoRI fragments smaller than I were not analyzed.

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast r D N A Table 4. Molecular weights of HindII partial digestion fragments

Partial fragment

1 2 3 4 5

Experimentally determined molecular weights

Calculated molecular weights m a p order -G-H-F-D

m a p order -G-F-H-D

3.07 2:55 2.33 1.46 1.23

HFD FD GHF HF GH

FHD GFH HD GF FH

3.05 2.63 2.35 1.45 1.32

3.05 2.35 2.02 1.93 1.45

The positions of pJHCll HindII+III fragments F and H (rDNA HindII+III fragments C and E) remain undetermined since they do not include cleavage sites for any of the other restriction enzymes we have used. Therefore, a different approach was used to map these two fragments. The 4.0x 106 dalton HindIII fragment (which contains pJHCll HindII + I I I fragments D, F, G and H) was purified from a HindIII digest of pJHC11 and was partially digested with HindII. The proposed molecular weights of the partial digestion fragments for the two possible orders of the pJHC11 H i n d l I + I I I fragments in the HindIII fragment, - G-H-F-D - or - G-F-H-D - , are shown in Table 4. The molecular weights of the partial fragments were experimentally determined by coelectrophoresis with DNA markers of known molecular weight and are also given in Table 4. From a comparison of these values, we can conclude that the order of the pJHCll HindII+III fragments is - G-H-F-D - . Therefore the order of the rDNA HindII+III fragments is - D-E-C-A - . The positions of the HindII cleavage sites are shown in Fig. 3.

Restriction Mapping of pJHC23 and pJHC35 We noted in the previous section that there were some ambiguities in the EcoR! map of pJHCll. In an attempt to resolve these ambiguities and to determine whether the order of rDNA EcoRI fragments in all three of the plasmids studied was the same, we did two additional experiments. In the first, pJHC23 was digested with HindIII, the resulting three fragments (see Fig. 2f) were purified, and each fragment was digested to completion with EcoRI. Figure 7a d shows the EcoRI patterns of pJHC23 HindIII fragments A, B, and C compared to an EcoRI digest of the intact plasmid after electrophoresis on gradient acrylamide gels. The largest HindIII fragment contains RSF2124, EcoRI-HindIII fragment C-1 and a very small fragment which must be either EcoRIHindIII fragment B-2 or C-2. Therefore rDNA

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast rDNA

a b c d

e

f

241

g h

Fig. 7a-h. Gradient acrylamide gel electrophoresis of EcoRI digests of restriction fragments from pJHC23 and pJHC35. Purified pJHC23 was digested with HindlII, and pJHC35 was digested with SmaI. The individual restriction fragments were isolated and each was digested to completion with EcoRI as described in Experimental Procedures. The EcoRI digests of a intact pJHC23 DNA; b pJHC23 HindIII fragment A; e pJHC23 HindIII fragment B; d pJHC23 HindIII fragment C; e intact pJHC35 DNA; f pJHC35 SmaI fragment A; g intact pJHC35 DNA and h pJHC35 SmaI fragment B are shown after electrophoresis on gradient acrylamide gels. The positions of the EcoRI fragments are indicated on the left of each gel set, and the positions of the EcoRI fragments altered by cleavage with SmaI or HindIII are shown on the right

EcoRI fragment C is adjacent to one end of RSF2124 in pJHC23. Since, as we discussed earlier, this plasmid has two copies of rDNA EcoRI fragment C but only one copy of fragment B, the small fragment we observe must be from EcoRI fragment C and the second copy of EcoRI fragment C must be adjacent to the other end of RSF2124 (see Fig. 8). Digestion of pJHC23 HindIII fragment B yields rDNA EcoRI fragments A, D, F, E, EcoRI-HindIII C-1 and another very small fragment, apparently EcoRI-HindII B-2. The location o f these EcoRI fragments in pJHC23 HindIII B is consistent with the EcoRI map obtained for pJHC11 and we have assumed they are in the same order. The smallest pJHC23 HindIII fragment yields rDNA EcoRI-HindIII fragment B - l , plus rDNA EcoRI fragment G and rDNA EcoRIHindIII fragment C-2. These results confirm the location of rDNA EcoRI fragment G between EcoRI fragments B and C. From these data we have constructed the restriction map of pJHC23 shown in Figure 8. In the second experiment we digested pJHC35

with SmaI, purified the fragments and subsequently digested them with EcoRI. Figure 7e h shows the EcoRI patterns of pJHC35 SmaI fragments A and B compared with an EcoRI digest of the intact plasmid. The larger of the Sinai fragments contains the 6.7×106 dalton SmaI-EcoRI RSF2124 fragment, rDNA EcoRI fragments A, C, D, E, F, G, and EcoRI-SmaI fragment B-2. The results with pJHC35 are also consistent with the rDNA EcoRI fragment order in pJHCll. Thus, if we assume the EcoRI restriction map of the two plasmids is the same, we can locate EcoRI fragment E next to RSF2124 in this portion of the plasmid. Digestion of the smaller pJHC35 SmaI fragment yields the remaining segment of RSF2124, plus rDNA EcoRI fragments E, F, and EcoRI-SmaI B-1. From these results we can locate EcoRI fragment F between fragments A and E. It cannot be on the other side of A next to D. The restriction map of pJHC35 is shown in Figure 8. Thus, analysis of the three hybrid plasmids yields an internally consistent order for the fragments

242

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast rDNA

pJHC 23

pdHC 35

Fig. 8. Restriction fragment maps of hybrid plasmids pJHC23 and pJHC35. The orientation of the restriction fragments was determined as described in the text. The RSF2124 portion of the plasmids is represented by a solid line ( - - ) and the yeast rDNA portion is represented by a sawtooth line (N~). The SmaI and HindIII cleavage sites are labeled, the EcoRI cleavage sites are represented by the short divider lines, and the yeast r D N A EcoRI fragments are labeled with the appropriate letter

t A "t~.J,.L

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ized in this paper. Some of the other hybrid plasmids which we have screened included several, but not all, of the seven rDNA EcoRI fragments. These plasmids have not been further characterized to date; however, the combinations of rDNA EcoRI fragments which they contain are consistent with the order of rDNA EcoRI fragments which has been determined for pJHCll. None of the plasmids we have examined have contained yeast DNA EcoRI fragments other than those observed in a digest of purified rDNA. Therefore, it appears that we have not cloned any non-ribosomal DNA fragments or any specific "saddle" fragments which span the interface between rDNA and non-ribosomal DNA. Two of the hybrid plasmids, pJHCll and pJHC35, have proved to be unstable in the E. coli Cla host and appear to delete a part of the yeast DNA region of the molecule to yield plasmids containing less rDNA. We have not explored the mechanisms of this process and do not know whether it

produced from rDNA by digestion with four different restriction endonucleases. Discussion

We have constructed a number of hybrid plasmids composed of segments of yeast rDNA attached to the bacterial plasmid vector RSF2124. Because the yeast rDNA contains multiple EcoRI cleavage sites per individual repetitive unit, we used a yeast rDNA digestion mixture that contained almost exclusively large partial digestion fragments in the reannealing reaction. We used the entire digestion mixture in the cloning experiment, rather than purifying fragments of a certain size, in order to avoid manipulating the DNA and exposing it to degradation. A number of hybrid plasmids containing different amounts of yeast rDNA have been obtained. Three of these plasmids which include yeast segments larger than an individual rDNA unit have been character-

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Fig. 9. Restriction map of yeast rDNA. Each horizontal line represents two complete repeat lengths of yeast r D N A beginning at the Sinai cleavage site. Each repeat has a molecular weight of 5.6 x 106. Short unmarked divider lines intersecting the top line represent EcoRI cleavage sites; cleavage sites of SmaI, HindlI and HindIII are labeled. The letters under each line mark the restriction fragments produced by digestion with the enzyme or enzymes indicated to the left. Those restriction fragments which are cleaved by SmaI are shown only in part at the far left and far right ends of the map

J.H. Cramer et al. : Hybrid Plasmids Containing Yeast rDNA is c o n f i n e d to t h e s e two p l a s m i d s o r w h e t h e r it m i g h t also e v e n t u a l l y o c c u r in the o t h e r h y b r i d plasmids. N o r have we s t u d i e d the structure o f the resulting p l a s m i d s except to n o t e t h a t they do n o t include all seven r D N A E c o R I f r a g m e n t s a n d t h a t no new E c o R I f r a g m e n t s a p p e a r to be generated. R e c e n t l y we t r a n s f o r m e d a rec A - host, E. coli HB101, with purified p J H C I 1 D N A , a n d have n o t o b s e r v e d a n y instability o f the p l a s m i d . H o w e v e r , o t h e r s (A. C o h e n , p e r s o n a l c o m m u n i c a t i o n ) have o b s e r v e d instability o f yeast r D N A - c o n t a i n i n g p l a s m i d s in this E. coli strain. Three hybrid plasmids, pJHCll, pJHC23, and p J H C 3 5 , have been used to d e t e r m i n e the m a p o f the E c o R I , Sinai, H i n d I I a n d H i n d I I I restriction sites in yeast r D N A which is s h o w n in F i g u r e 9. Since there is o n l y one S m a I site p e r r D N A repetitive unit, we have used this site to o r i e n t the restriction m a p . H o w e v e r , we d o n o t k n o w the o r i e n t a t i o n o f the r D N A S i n a i site with respect to the ends o f the r D N A repetitive units as they exist in the intact yeast c h r o m o s o m e s . M a p p i n g i n f o r m a t i o n f r o m the three plasraids indicates t h a t e a c h p l a s m i d c o n t a i n s a different p e r m u t a t i o n o f the r D N A E c o R I f r a g m e n t order, with different r D N A E c o R I f r a g m e n t s being p r e s e n t in duplicate. In a d d i t i o n , the yeast r D N A segments are inserted into R S F 2 1 2 4 in b o t h d i r e c t i o n s with the o r i e n t a t i o n s in p J H C 2 3 a n d p J H C 3 5 being o p p o site to t h a t in p J H C l l . H o w e v e r , the m a p o r d e r o f the E c o R I f r a g m e n t s a p p e a r s to be identical in all three p l a s m i d s . These results s u p p o r t o u r earlier conclusions t h a t the i n d i v i d u a l r D N A repetitive units are h o m o g e n e o u s in size a n d c o m p o s i t i o n ( C r a m e r et al., 1976). A d e t a i l e d r e s t r i c t i o n m a p o f the y e a s t r D N A s h o u l d be h i g h l y useful in l o c a t i n g R N A c o d i n g regions, s p a c e r sequences, R N A p o l y m e r a s e b i n d i n g sites a n d o t h e r i m p o r t a n t f u n c t i o n a l regions. S o m e i n f o r m a t i o n is a v a i l a b l e r e g a r d i n g the l o c a t i o n o f the r R N A c o d i n g sites in the E c o R I m a p . The 18S r R N A h y b r i d i z e s to r D N A E c o R I f r a g m e n t C, 26S R N A h y b r i d i z e s to r D N A E c o R I f r a g m e n t s A a n d E, a n d 5S R N A h y b r i d i z e s to r D N A E c o R I f r a g m e n t B ( K r a m e r , C a m e r o n a n d Davis, 1976; A.P. Bollon, p e r s o n a l c o m m u n i c a t i o n ) . N o i n f o r m a t i o n was given on r D N A E c o R I f r a g m e n t D. These results are consistent with the m a p o r d e r we have r e p o r t e d f o r the r D N A E c o R I fragments. U s i n g the restriction sites f o r the o t h e r enzymes we have used, it s h o u l d be possible to locate the p o s i t i o n s o f the R N A c o d i n g regions m o r e precisely. In a d d i t i o n to their usefulness in m a p p i n g the restriction f r a g m e n t s o f y e a s t r D N A , the h y b r i d plasmids s h o u l d be v a l u a b l e in studies o f the t r a n s c r i p t i o n a n d r e p l i c a t i o n o f yeast r D N A b o t h in vivo a n d in vitro.

243 Acknowledgements. We would like to thank Bernard Weisblum

for advice regarding the design of the cloning experiment and for performing the ligase reaction. We also acknowledge his assistance in the preparation of restriction endonucleases. This research was supported by National Science Foundation grant GB41551 to J.H.C. and R.H.R. and U.S. Public Health Service research grant GM14398 to R.H.R.F.W.F. and J.T.B. are predoctoral trainees supported by a U.S. Public Health Service research training grant.

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244 Kriegstein, H.J., Hogness, D.S. : Mechanism of DNA replication in Drosophila chromosomes: Structure of replication forks and evidence for bidirectionality. Proc. nat. Acad. Sci. (Wash.) 71, 135-139 (1974) McLaughlin, C.S. : Yeast ribosomes : Genetics. In: Ribosomes (Nomura, M., Tissi6res, A. and Lengyel, P. eds.), pp. 815-827. New York: Cold Spring Harbor Press 1974 Rubin, G.M., Sulston, J.E.: Physical linkage of the 5S cistrons to the 18S and 28S ribosomal RNA cistrons in Saccharomyces cerevisiae. J. molec. Biol. 79, 521-530 (1973) Sasaki, I., Bertani, G. : Growth abnormalities in Hfr derivatives of Escherichia coli strain C. J. gen. Micro. 40, 365-376 (1965) Schweizer, E., MacKechnie, C., Halvorson, H.O. : The redundancy of ribosomal and transfer RNA genes in Saccharomyces cerevisiae, J. molec. Biol. 40, 261-277 (1969)

J.H. Cramer etal. : Hybrid Plasmids Containing Yeast rDNA So, M., Gill, R., Falkow, S.F.: The generation of a ColE1-Ap r cloning vehicle which allows detection of inserted DNA. Molec. gen. Genet. 142, 23%249 (1975) Tanaka, N., Cramer, J.H., Rownd, R.H. : EcoRI restriction endonuclease map of the composite R plasmid NR1. J. Bact. 127, 619-636 (1976) Tanaka, T., Weisblum, B. : Construction of a colicin E1-R factor composite plasmid in vitro : Means for amplification of deoxyribonucleic acid. J. Bact. 121, 354-362 (1975) Udem, S.A., Warner, J.R. : Ribosomal RNA synthesis in Saccharomyces verevisiae. J. molec. Biol. 65, 227-242 (1972) Communicated

b y F. K a u d e w i t z

Received November 16, 1976