Gene, 4 (1978) 37--49 37 © Elsevier/North-Holland Biomedical Press, Amsterdam --Printed in The Netherlands
ISOLATION AND ANALYSIS OF RECOMBINANT DNA MOLECULES CONTAINING YEAST DNA (pMB9 plasmid; E. coli; yeast nuclear DNA; genes for tRNA and rRNA; abundant m R N A ) THOMAS D. PETES ''s , JAMES R. BROACH 2, PIETER C. WENSINK 3, LYNNA M. HEREFCRD 3, GERALD R. FINK 4 and DAVID BOTSTEIN'
'Department of Biology, Massachusetts Institute of Technology, Cambridge, MA; zCold Spring Harbor Laboratory, Cold Spring Harbor, N Y 11724; 3Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02154; 4Department of Genetics, Development and Physiology, Cornell University, Ithaca, NY; and SDepartment of Microbiology, University of Chicago, 920 E. 58th Street, Chicago, IL 60637 (U.S.A.)
(Received December 21~t, 1977) (Accepted May 5th, i 9 7 8 ) SUMMARY
2500 recombinant plasmids containing insertions of yeast nuclear DNA have been cloned in Eseherichia coli. It can be calculated that about 85% of the yeast genome is represented in this collection. The clones have been characterized by hybridization to purified RNA species. Of the 2000 clones examined, 75 contain insertions of yeast ribosomal DNA, 201 contain insertions of yeast tRNA genes, and 26 contain DNA sequences that are complementary to abundant mRNA species.
INTRODUCTION
The yeast Saccharomyces cerevisiae has a genome that is much smaller than higher eukaryotes. Although estimates vary, several independent methods yield a nuclear genome size of about 9 • 109 daltons (Hartwell, 1974). Despite the small size of the genome, many of the details of cell structure, chromosome structure and replication, and macromolecular synthesis in yeast are very similar to those processes in higher eukaryotic cells. Since the yeast genome is small, one method of analysis of yeast chromosome structure and function is construction of a large number of recombinant molecules containing insertions of yeast DNA. If a sufficient number of such plasmids can be constructed, most of the sequences in the yeast genome should be represented. We have isolated 2500 molecules containing insertions of yeast nuclear DNA; b y calculation, 85% of the yeast genome should be present in this collection. As apreliminary characterization of the inserted sequences, 2000 of the clones have been examined for sequences homologous to yeast tRNA, ribosomal RNA, and messenger RNA.
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Since the collection of recombinant plasmids has been used by many laboratories,we feel that it is useful to present, in a preliminary way, the general characteristicsof this collection and the detailsof its construction. Several other workers (Kramer et al.,1976; Ratzkin and Carbon, 1977; Beckmann et al.,1977) have also constructed large collectionsof recombinant bacteriophages or plasmids containing yeast D N A . The collection of recombinant clones described in this paper has several advantages over those cons t r u ~ using restrictionenzymes. The size of the D N A inserted into the recombinant plasmids was larger (10 kb) than that reported for the other collections. Since the recombinant plasmids in our collection were constructed from randomly sheared fragments of yeast nuclear D N A (Lobban and Kaiser, 1973), recombinant plasmids with overlapping D N A sequences were present. In addition, when recombinant molecules are constructed with restrictionenzymes, only those genome fragments with a cohesive end (generated by the restrictionenzyme) on both ends of the fragment can be cloned. This requirement specificallyexcludes the yeast chromosome ends. Finally, the recombinant clone collection described in this paper is the only one in which the total collection has been hybridized, in separate experiments, to ribosomal RNA, messenger RNA and transfer RNA. Since the recombinant clm~es were screened in the same ordered array for each experiment, relationships among the hybridization patterns were established (for example, what proportion of the plasmids which hybridize to messenger RNA also contained tRNA genes). MATEV.IALSAND METHODS
Strains The yeast stre.inused as a source of D N A for cloning was the diploid strain + D 4 (provided by L.H. Hartwell). The genotype of the strain is: a/a gall/gall adel/adel ural/ural his7/+ +/his5 lys2/+ +/lysl tyrl/+ +/leu2 ade2/+. Ribosomal R N A was isolated from the haploid yeast strain A364a (a gall adel ade2 ural tyrl his7 lys2),one of the haploid parents of +D4. Messenger RNA and transfer RNA were isolated from strain R95-4A, a GAL ÷ derivative of strain $288C. The plasmid vector used was the small plasmid pMB9 (Rodriguez et al., 1977) which confers resistance to tetracycline upon bacteria which contain it. The host bacterial strain for the transformation experiments was HB101 which is a derivative of E. coil K-12 and has the genotype: hsm-, hsr-, recAgal- pro- str ~ (Boyer and Roulland-Dussoix, 1969). Plasmid D N A Plasmid DNA was isolated by a minor modification of t h e cleared-lysate method of Clewell and Helinski (1969), Triton X-100 wasused to lyse the cells after the lysozyme treatment; a preliminary centrifugation at 17 000 rev./min in the Sorvall SS34 rotor for 30 min prior to equilibrium banding in
39
CsCloethidium bromide was performed. The former modification gives slightly higher yields; the latter removes insoluble material which otherwise sometimes obscured the DNA bands. Sizing of plasmids was performed first by measurement in the electron microscope (Davis et al., 1971). Aqueous spreads of circular plasmid DNA relaxed by treatment with DNase were measured relative to linear pMB9 molecules produced by digestion with the site-specific endonuclease EcoRI. A second method was to use supercoiled plasmids of known length to calibrate 0.9% agarose gels containing 0.5% ethidium bromide (Sharp et al., 1973); most length measurements were done with this simple method.
Isolation of high molecular weight yeast nuclear DNA Yeast cells contain at least three types of DNA: nuclear DNA, mitochondrial DNA, and a class of extranuclear plasmid molecules (Hartwell, 1974). Since the mitochondrial and plasmid DNA molecules are usually isolated as fragments which are considerably smaller than nuclear DNA (Petes et al., 1973), in vitro recombinants constructed from unfractionated yeast DNA would overrepresent these species. Consequently, the procedure described below was designed to isolate selectively the largest DNA molecules of the yeast cell. Cells from the yeast strain +D4 were grown in 2 liters of SD minimal medium (Mortimer and Hawthorne, 1969) supplemented with 2% glucose, 20 ~g/ml adenine, 20/~g/ml uracil, and 50/~g/ml tyrosine. Labeled ['4C] uracil (New England Nuclear; 0.5 mCi/mg) was added at a concentration of 15 ~Ci/liter to allow monitoring of DNA purity and recovery. Cultures were grown with aeration at 30°C to an absorbance (660 nm) of 1.2. The cells were harvested by centrifugation and washed in 1/10 vol. of 0.05 M EDTA (pH 8). The protocol for formation of spheroplasts was similar to that used by Cryer et al. (1975). The cell pellet was resuspended in 25 ml of 0.05 M Tris, 0.03 M ~-mercaptoethanol (pH 9.5). The cells were incubated at room temperature for 15 min and then centrifuged and resuspended in 1 M sorbitol, 0.001 M EDTA. The cells were converted to spheroplasts by addition of 2 ml of an aqueous (1 mg/ml) solution of zymolyase (Kirin Brewing Co.) to the suspension, followed by incubation at 37°C. Efficacy of spheroplast formation was monitored by observation i n t h e light microscope after treatment of aliquots with 2% sodium dodecyl sarcosinate. After 80% of the cells had been converted to spheroplasts, the mixture was centrifuged in the Sorvall SS-34 rotor at 2000 rev./min for 2 min. The pellet was gently resuspended in a small volume of 0.1 M Tris, 0.1 M EDTA, 0.15 M NaCI, 0.3 M ~-mercaptoethanol, pH 9.5. An additional 20 ml of the same buffer containing 4% sodium lauryl sarcosinate was then added and the mixture was incubated at 45°C. After 15 min, 25 ml of a buffer containing 0.1 M Tris, 0.1 M EDTA, 0.15 M NaCI, 0.3 M ~-mercaptoethanol, and 4% sodium lauryl sarcosinate was added and the mixture was shifted to 70°C for 15 min. The lysate was centrifuged at 5000 rev./min and the supernatant decanted. The supernatant was layered (10 ml/gradient) onto sucrose gradients prepared in
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B e c ~ a n SW27 tubes as follows: 4 ml of a high-density mixture (20% sucrose in 80% sodium ioth~lsmate [Angio-Conray, Mallinckrodt Pharmaceuticals] ) was put in the bottom and overlaid with a 24 ml gradient of 5% to 20% sucrose in 0.02 M Tris, 0.02 MEDTA, 0.2 M NaCI, 0.1% sodium lauryl sarcosinate, pH 8, The sample was added t o t h e gradients t h r o u ~ a large,bOre plastic pipette. ~ The gradients were centrifuged a t 1 3 500 rev./min for 17 h at 21°C. Fractions were slowly collected through a hole made by an 18-gauge needle. Fractions were assayed for D N A by determining the number of alkali-stable acid-precipitable counts in each fraction (Petes and Fangman, 1972). The fractions containing the largest DNA were pooled and dialyzed exhaustively against 0.01 M Tris, 0.005 M EDTA, 0.15 M NaCI. The dialyzed fractions Were pooled and the DNA banded to density equilibrium in CsCI gradients (Petes and Fangman, 1972). No peak of DNA was observed banding at the position of yeast mitochondrial DNA. Fractions containing the yeast nuclear DNA were pooled and dialyzed against 0.01 M Tris, 0.001 M EDTA, pH 8. Recovery of the DNA varied in three preparations from 25% t o 60%. The DNA recovered was larger than intact T4 DNA; i.e. :>200 kilobases (kb) in length.
Shear of yeast nuclear DNA Yeast DNA (20 mg/ml) in 0.01 M Tris, 0.001 M EDTA, and 2.5 M NaCI (pH 8) was sheared by two passages through a syringe fitted with a 25-gauge needle (Pyertiz et al., 1972). All manipulations were done at 5°C. Examination of the sheared DNA with the electron microscope indicated that most of the molecules were between 10 and 25 kb. Recombination in vitro The recombinant molecules were constructed by the hompolymer taft method described by Wensink et al. (1974). The only change was the use of the terminal transferase reaction conditions of Roychoudbury et al. (1975), in which Co 2÷ was the only divalent cation. Oligo-dT tails of average length 280 nucleotides were added to the EcoRI-cleaved pMB9 DNA; oligo-dA tails of average length 100 nucleotides were added to the sheared yeast nuclear DNA. Transformation and selection o f recombinant clones A modification of the CaCl2method described by Mandel and Higa (1970) was used. HB101 (Boyer and Roulland-Dussoix, 1969) cells growing exponentially in LB medium (Miller, 1972) at a density of 5 • 10 s cells/ml were collected by centrifugation and resuspended in 1/2 vol. of ice-cold 0.05 M CaCI2., After ~15 m re.at . . . . 0 oC t h.e bacteria . . c e n ~.g. .e.d. were agam a n d resu~ pended in 0.05 M CaCI2 at 1/1 this suspension wereadded to~ ed 1001~Iof a buffer (0.01 M Tris, 0.01 M CaCl2, 0,01 M:MgCI~, pH 7) a n d 1 , 20
41
/~1 of DNA. After the addition of cells, the mixture was kept on ice for 25 rain. The tubes were then shifted to 37°C for 2 rain and put at room temperature for 10 min. I ml of LB medium was added to each tube and the tubes were shaken at 37°C for 30 rain. 2.5 ml soft agar (5 g NaC1, 8 g Bacto nutrient broth, 6.5 g agar in I liter of water) was added to each tube and the mixture was poured onto LB plates containing 10 ~g/ml tetracycline. Transformants were picked after the plates had been incubated 2--3 days at 37°C. i
Storage and handling of the set of recombinant clones Recombinant clones rapidly lose viability when stored in agar stab tubes or on agar plates. For permanent storage, we add an equal volume of a freezing media to a saturated culture in LB media. The freezing media contains twice the concentration of components in the media used by Hogness and Simmons (1964). To aid in storage, subcloning and colony hybridization, we have adopted the general procedure for handling large numbers of clones which has been developed by Brenner et al. (1975); Weiss and Milcarek (1974); and Stern and Wensink (unpublished). The colonies are grown and stored in 144 well polystyrene microtiter plates (Linbro Scientific, Inc.) which are sterilized by UV light and filled with media using a 144 tube multipipette. Paper or microtiter plate replicas are made with a 144 prong replicator. The plates are covered with a plastic adhesive film (M. and C. Specialties) and stored either in liquid nitrogen vapor or at-70°C.
Colony hybridization The colony hybridization procedures used are a modification of the technique developed by Grunstein and Hogness (1975). The modification used for colonies grown on nitrocellulose paper was developed by J. Lis and L. Prestige (unpublished) and that used for colonies grown on Whatmann 3MM paper was developed by R. Stern and P. Wensink (unpublished). Colonies from each microtiter plate were replicated onto sterile sheets of Whatmann 3MM paper or nitrocellulose paper which were lying on large trays containing LB agar and tetracycline. The replicas were made with the replicator previously mentioned and were grown for 24 h at 37°C. Whatmann filters containing the colonies were placed in 1-1iter baths of the following solutions: (1) 0.5 N NaOH for 7 min, (2) two baths of I M Tris • HCI (pH 7.4) for 2 min per bath, and (3) 1.5 M NaCl, 0.5 M Tris • HCI (pH 7.4) for 4 min. The filters were rinsed in 95% ethanol. Nitrocellulose sheets were subjected to an identical lysing and washing regime. However, instead of being immersed in solutions, the filters were laid on blotting paper saturated with the appropriate solutions. The nitrocellulose filters were then sucked dry and rinsed in two washes of 95% ethanol. The filters were sucked dry and baked under vacuum at 80°C for 2 h. (a) Hybridization of ribosomal RNA. Total yeast RNA was isolated from the strain A364a (Hereford and Rosbash, 1977). The 18S and 24S RNA molecules were purified by centrifugation (15 h at 27 000 rev./min in the
42
SW27 rotor) through 15- 30% sucrose gradients (0.01 M Tris, 0.001 M EDTA, 0.1 M NaCI, 0.5% sodium dodecyl sulfate, pH 7.4). Those fractions containing 18S and 24S RNA were pooled and precipitated with 2 vol. of ethanol. The RNA was resuspended in 0.2 M sodium acetate (pH 5.5) and reprecipitated with 9. vol. of ethanol. This procedure was repeated once. The R N A was then washed twice with 70% ethanol, resuspended at a concentration of 2 mg/ml, and stored at-20°C. The RNA was cleaved to 100- 200 nucleotides by incubation at 90°C for 60 min in 0.005 M glycine, 0.01 mM EDTA (pH 9.15). The RNA was labeled by the kinase labeling procedure of Maxam and Gilbert (19"/7). The specific activity of the labeled RNA was 1- 2-107 cpm//~g. The hybridization buffer contained 50% formamide, 4.25 × SSC (0.64 M NaCI, 0.064 M trisodium citrate), and 0.1 M phosphate buffer (0.05 M of both K2HPO4 and KH2PO4). The volume of hybridization buffer per filter was 5 ml, and the amount of radioactivity per filter was l 0 s cpm. Hybridization was done in plastic bags at 37°C for 18 h. Washing of the filters and autoradiography were done as described by Grunstein and Hogness (1975). (b) Hybridization with messenger DNA. Labeled messenger RNA was prepared by a modification of the in vivo labeling procedure described by Rubin (1974). Strain R95-4A was grown at 30°C in low phosphate YEPA medium (1% yeast extract, 2% peptone, 2% potassium acetate treated with magnesium sulfate and ammonium hydroxide as described; Rubin, 1974) to a density of 107 ceUs/ml. Carrier-free [32p] orthophosphate was added to a concentration of 100 ~Ci/ml, and growth was continued. After I h, cycloheximide was added to a concentration of 100 pg/ml and the cells were poured over crushed ice. After harvesting by centrifugation, the cells were washed with and resuspended in 1/100 vol. polysome buffer (25 mM Tris.HCl, pH 7.5, 25 mM NaCl, 5 mM MgCl2, I mg/ml heparin). Cells were then broken by two 1-min treatments in a Braum homogenizer. After the cell debris was removed by centrifugation at 30 000 × g for 5 min, the supernatant was made 1% in SDS and extracted with phenol/chloroform (1/1). The RNA was then precipitated with 2 vol. of ethanol. The precipitate was resuspended in 0.1 M NaCI and reprecipitated with 2 vol. of ethanol. The precipitate was then collected and dried. Poly(A)-containing RNA was purified by poly(U) sepharose chromatography and aclTlamide gel electrophoresis. The dried RNA precipitate was resuspended in 2 ml buffer A (0.5 M NaCl, 0.01 M Tris.HCl, pH 7.5), incubated at 95°C for I min, and then quick-chilled on ice. This solution was then applied to a 1-ml poly(U) sepharose column (Pharmacia equilibrated with buffer A). The column was washed with approx. 20 ml buffer A, and the poly(A)-containing RNA was eluted with distiUed, deionized water. The poly(A)-containing RNA was precipitated with 1/10 vol. 1 M NaCI and 2 vol. ethanol, collected by centrifugation, and dried. The RNA was then electrophoresed on a 3.5% acrylamide 98% formamide cylindrical gel, as described by Duesberg and Vogt (1973). After electrophoresis the labeled RNA was located by auto-
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radiography of the gel, and the region containing the bulk of the labeled RNA was cut out. This section of the gel was combined with an equal volume of 10 X SSC and dispersed by several strokes of a dounce homogenizer. The gel suspension was then sonicated until no turbidity was apparent in the solution. This solution was used without further treatment as messenger RNA probe. Hybridization was performed at 42°C for 16 h in 50% formamide, 5 × SSC, 200 mg/ml polyadenylic acid, and 106 to 107 cpm labeled RNA/filter, in a volume of either 2 ml (for nitrocellulose filter hybridization) or 4 ml (for Whatman filter hybridization). After hybridization, the filters were incubated at 42°C in 100 ml 50% formamide, 5 × SSC/fflter for 16 h. The filters were then washed and autoradiographed as described (Grunstein and Hogness, 1975}. (c) Hybridization of transfer RNA. Labeled transfer RNA was purified from total in vivo labeled RNA by DEAE cellulose chromatography and polyacrylamide electrophoresis. The follow-through fraction from poly(U) sepharose chromatography in vivo labeled RNA was diluted tenfold with 0.01 M Tris. HCI, pH 7.5, and applied to a 2-ml DEAE cellulose column equilibrated with 50 mM NaCl, 10 mM Tris.HC1, pH 7.5. The column was washed with approx. 20 ml equilibration buffer and then with several 1-ml portions of 1.0 M NaCl, 10 mM Tris.HC1, pH 7.5. The RNA eluted by the 1.0 M NaCI wash was precipitated with 2 vol. ethanol, collected, and dried. The tRNA was further purified by polyacrylamide gel electrophoresis. For preparation of crude tRNA, the post-DEAE cellulose RNA sample was electrophoresed on a 10% polyacrylamide 98% formamide cylindrical gel (Duesberg and Vogt, 1973}. After electrophoresis, the 4S to 4.5S region was located by autoradiography, cut out, and prepared as described for isolation of messenger RNA probe. For isolation of purified tRNA probes, the post~DEAE cellulose fraction was subjected to two-dimensional polyacrylamide gel electrophoresis as described by Piper et al. (1977). After electrophoresis, the pattern of tRNA spots was visualized by autoradiography and the regions corresponding to spots previously identified as the UCG-decoding serine tRNA and the leucine II tRNA {Piper et al., 1977) were cut out of the gel. The tRNA species were eluted from the excised gel pieces by incubation overnight at 4°C in 0.1 M NaCI, 10 mM Tris-HC1, pH 7.5, 1 mM EDTA, after the gel was homogenized with several strokes in a dounce homogenizer. After removal of polyacrylamide gel fragments by centrifugation through glass wool, the solutions were used directly for hybridization. Hybridization of crude or purified tRNA probes was performed as described for that of messenger RNA except that polyadenylic acid was omitted from the hybridization mixture and a posthybridization incubation on 50% formamide, 5 × SSC was not performed.
Clone nomenclature The recombinant plasmids are labeled pY1 (indicating that the clone is a yeast DNA-containing piasmid from the first preparation of yeast DNA)
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followed by the miczotiter dish number (1-,20)and the position on the microtiter dish. The rows in the microtiter d ~ e s are labeled A through L and the columns labeled 1 through 12. A complete description of a single clone, therefore, might be p Y 1 (2A-3), ~ i s clone Would b e located on microtiter dish No, ~2in r o w A, column 3. RESULTS
Construction of the recombinant plasmids The recombinant molecules were constructed by the technique developed by Lobban and Kaiser (1973) and used by Wensink et al. (1974) to clone Drosophila DNA. A homopolymer "taft" of dAMP residues is added to the ends of sheared yeast nuclear DNA. This technique has the advantage of a positive selection for insertion of foreign DNA into the plasmid carrying the tetracycline-resistance genes since the cleaved plasmid, after addition of the homopolymers, can no longer transform cells to tetracycline resistance by itself. In our experiments, the transformation efficiency of yeast DNA and plasmid DNA annealed together was about 30-fold higher than either species alone. The small number of transform~nts that arise with pMB9 alone are probably the result of plasmid DNA. which was not cleaved with EcoRI, since a small number of uncleared molecules were observed with the electron microscope. Evidence for presence of yeast DNA in the plasmids in the transformants was obtained by isolating plasmid DNA from several of the transformants and analyzing this DNA on agarose gels. Recombinant plasmids had a slower mobility, indicating that they were larger in size than the original pMB9 pla~ mid. One of the twenty plasmids contained no detectable insertion of yeast DNA; the remainder contained an insertion whose average length was 10 kb. The recombinant plasmids when digested with EcoRI and HindIII restriction enzymes showed patterns on agarose gels which were different from that of pMB9. The recombinant plasmids also differed from pMB9 DNA in contour length as measured by electron microscopy and in buoyant density as measured in CsC1 density gradients. Hybridization of clones to yeast RNA probes 2000 of the 2500 clones were hybridized in separate experiments to either purified 18S and 24S yeast ribosomal RNA, yeast messenger RNA sequences, or yeast tRNA. The technique of colony hybridization was used for this analysis. Approx. 15% (298/2000 clones) of the strains containing recombinant plasmids hybridized to one or more of the labeled yeast RNA species. Almost all (295/298) of these clones showed one of five patterns of hybridization shown in Table I. Each of these patterns will be discussed individually. The largest classof clones, type 11,hybridized only to yeast t R N A . This result suggests that the yeast t R N A Cistrons are widely spaced throughout the
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TABLE I HYBRIDIZATION PATTERNS OF CLONES TO PURIFIED YEAST rRNA, mRNA, AND tRNA I n all t h e s e e x p e r i m e n t s , t w o t h o u s a n d c l o n e s w e r e s c r e e n e d b y c o l o n y h y b r i d i z a t i o n to 32P-labeled p u r i f i e d y e a s t r R N A , m R N A or t R N A .
Hybridization p a t t e r n
I II HI IV V Other
Number of clones with pattern
H y b r i d i z a t i o n to p r o b e s rRNA
mRNA
tRNA
194 18 57 19 7 3
+ + -
+ + + +
+ + +
yeast genome and not highly clustered. In addition to hybridizing the clones with total yeast tRNA, we have used UCG-decoding serine tRNA and leucine II tRNA as hybridization probes. Three clones containing sequences complementary to the former probe, and six clones containing sequences complementary to the latter probe, have been identified. These results are consistent with the idea that many different tRNA genes are present in the type I clones. Type II clones represent plasmids which hybridize both to the 18S and 24S ribosomal RNA and to the messenger RNA hybridization probe. Since mRNA isolated on poly(U) columns is known to be contaminated by ribosomal RNA, all clones containing yeast ribosomal DNA would hybridize to both probes. Independent evidence that type II clones contain only yeast ribosomal DNA has been provided by restriction analysis of these clones (Petes, Hereford and Skryabin, J. Bacteriol., in press). Type III clones hybridize to all RNA probes. Since the clones hybridize to purified ribosomal RNA, .hey must contain insertions of yeast ribosomal DNA. The hybridization to the mRNA probe, as described previously, is the result of contamination of the probe with ribosomal RNA. The hybridization of these clones to purified tKNA has two possible interpretations. Either some tRNA genes are interspersed with ribosomal DNA cistrons, or the tRNA hybridization probe is contaminated with yeast ribosomal RNA fragments. The fourth class of hybridization pattern is shown by those clones that hybridize only to the mRNA probe and therefore represent DNA sequences whose transcripts are sufficiently abundant to be detected in these experiments. ~ e V clones hybridize both to mRNA and to tRNA. These clones presumably contain both genes which are transcribed into abundant messenger RNA sequences and genes which code for tRNA.
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DISCUSSION
The 2500 clones containing insertions of yeast DNA should contain about 85% of the yeast nuclear genome. This calculation was made according to the PoP,son equation: • .... P = (1 - [ T / G ] )N
where P equals the probability of missing a particular yeast DNA sequence, T equals the average size of the inserted DNA (10 kb), G equals the yeast genome size (13.5 • 103 kb), and N equals the total number of recombinant plasmids. An observation which suggests that most of the yeast genome is represented in this collection is that ura3, a yeast gene present in a single copy per haploid genome, has been found in two different recombinant clones of two thousand examined (M. Bach and D. Botstein, personal communication). In these experiments, the recombinant DNA molecules containing ura3 were identified by complementation of a p y r F mutant of E. coll. In addition, a recombinant clone from this collection has been tentatively shown to contain yeast histone genes (L.M. Hereford and G. Lauer, personal communication). Of the 2000 clones that were analyzed, 75 hybridized to ribosomal RNA. This frequency (3.7%) is close to the predicted proportion of ribosomal DNAcontaining clones, since Schweizer et al. (1969) showed that 2.4% of yeast nuclear DNA hybridized to ribosomal RNA and, therefore, 4.8% of the yeast DNA should represent ribosomal DNA genes. This result indicates that ribosomal DNA sequences were not preferentially retained or lost during the cloning procedure. One surprising result of these experiments was the small number of recombinant plasmids (26/2000 clones) which hybridized specifically to mRNA. This observation does not, of course, imply that only a small proportion of the yeast genome is transcribed. Hereford and Rosbash (1977) have shown that yeast messenger RNA species are present in the cell in at least three classes of abundance. About 20 mRNA species are present at a frequency of 100 copies per cell, 400 species are present at a frequency of 10 copies per cell, and about 2500 species are present as a single copy in the cell. It is likely that in our experiments only the most abundant transcribed species of RNA are being detected. This conclusion is confirmed by the observation that in mENA hybridization experiments in which tenfold greater amounts of probe are used, approximately 40% of the clones show hybridization of probe (Broach, unpublished observations; Hereford, unpublished observations). This is approximately the proportion of clones one would expect to contain genes corresponding to the very abundant and moderately abundant messenger RNA classes.
The largest class of clones that hybridized to a n y o f the RNA probes consisted of Ciones hybridizing t o tRNA r(201/2000 ciones).~About 0.064- ~).08% of nuclear DNA hybridizes to tRNA (Schweizer et ~al., 1969). The proportion
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of clones expected tohybridize to tRNA depends on how the tRNA genes are arranged in the genome. If all the tRNA genes were clustered, only a few clones would be expected to hybridize to the tRNA probe (about 0.0007 × 2000 = 1.4 clones). If the tRNA genes are unlinked, the expectation is quite different. The number of tRNA genes can be estimated from the data of Schweizer et al. (1969) to be about 300. Since the genome size is 13 500 kb, there is l tRNA gene per 45 kb. Since the average size of the inserted sequences is 1 0 kb, about 20% of the clones would be expected to hybridize to tRNA if the tRNA genes were completely unlinked. The experimental observation, therefore, that 10% of the clones hybridize to tRNA suggests that the tRNA genes are scattered throughout the genome and not extensively clustered. The conclusion is supported by the data of Hawthorne and Leupold (1974), which showed that the 8 tyrosine-inserting ochre suppressors (Gilmore et al., 1971) mapped at different positions in the yeast genome. Beckmann et al. (1977) have shown that the yeast tRNA genes are generally unlinked in experiments similar to those reported here. In conclusion, our preliminary characterization of this set of 2500 yeast/ pMB9 hybrid plasmids suggests that it is a fair representation of the yeast nuclear genome, even though it is certainly incomplete. These results seem to us to encourage further use of this collection in studies of the yeast genome. ACKNOWLEDGEMENTS
T.D. Petes was supported by an N.I.H. postdoctoral fellowship. J.R. Broach was supported by an N.I.H. postdoctoral grant 5T32-0917602. P.C. Wensink was supported by N.I.H. grant GM21626. L.M. Hereford was supported by an N.I.H. grant (GM23549) to M. Rosbash. G.R. Fink was supported by N.I.H. grant GM15-408. The work was also supported by N.I.H. grants (GM18973 and GM21253) to D. Botstein and a Research Career Development award to D. B, REFERENCES Beckmann, J.S., Johnson, P.F. and Abelson, J., Cloning of yeast transfer RNA genes in Escherichia coli, Science, 196 (1977) 205--208. Boyer, H.W. and Roulland-Dussoix, D., A complementation analysis of the restriction and modification of DNA inEscherichia coli, J. Mol. Biol., 41 (1969) 459--472. Brenner, M., Tisdale, D. and Loomis, W., Techniques for rapid biochemical screening of large numbers of cell clones, Exp. Cell Res., 90 (1975) 249--252. Clewcll, D. and Helinski, D., Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event, Biochemistry, 9 (1970) 4428--4440. Cryer, D.R., Eccleshall, R. and Marmur, J., Isolation of yeast DNA, in Prescott (Ed.), Methods in Ceil Biology, Vol. 12, Academic Press, New York, 1975, pp. 39--44. Davis, R.W,, Simon, M. and Davidson, N., Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, in Grossman and Moldave (Eds.), Methods in Enzymology, Vol. 21, Academic Press, New York, 1971, pp. 413--428.
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Duesberg, P.H, andVogt,. P.K,; Gel electrophoresls of avian leukosis and mrcoma viral RNA in formamide: comparison with other viral U d cellular RNAspecies, J. Virol., 12 (1973) 594--599,~ . ~ - :~ ..... Gilmore, R.~ stewart, 3. and Sherman, F-, Amino acid rep_~cements ~sult'_mg from super" of iso-l,c y t o e ~ o m e C from yeast, J. MoL m o L, -.. Hartwell,~L,H.Saccharomyces cerevisiae eel[cycle; Baeteri0LRev~; 38 (19.74) 164--198. Hawthorne, D.C... .and . . . .LeUpolct, . U,, Suppressor mutations~ in. Jyeast, . . . in . .Arber~et~ ~ aL(Eds.),Current Topics m Mierobiologyand I m m u n o l ~ , VoL 64, Springer-Ver!~, Berlin, 1974, pp. 1--47. • ~ Hereford, L.M. and Rosbash, M., Nu_mberand~ tribution of polyadenylated RNA sequences in yeast, Ceil, I0(1977) 453-'462. ....... ...... Hogness, D.$. and Simmons, J;R,•Breakage of ~dg DNA: chemie~ and genetic characterization of each isolated half-molecule, J. MoL Biol,,9 (1964) 411--438. Kramer, R,A., Cameron, J.R. and Davis, R,W., Isolation of becteriophage X containing yeast ribosomal RNA genes: screening by in situ RNA hybridization~ • ~ to plaques,• Cell, 8 (1976) 227--232. Lobban, P.E. and Kaiser, A.D. Enzymatic end, to-end joining of DNA molecules, J. Mol. Biol., 78 (1973)453--471. Mandel, M. and Higa, A., Caleium.dependentbacteriophage DNA infection, J. Mol. Biol., 53 (1970) 159--162. Maxam, A.M' andGflbert~ W., New method for sequencing DNA, Proc. Natl. Acad. 8ci. usA, 74 (1977) 560-564, Miller, J.H., Experiments in molecular genetics, Cold Spring Harbor Laboratory, New York, 1974, p. 433. Mortimer, R.K. and Hawthorne, D.C., Yeast genetics, in Rose and Harrison (Eds.), The Yeasts, Vol. 1, Academic Press, New York, 1969, pp. 385--460. Petes, T.D. and Fangman, W.L., Sedimentation properties of yeast chromosomal DNA, Proc. Natl. Acad, Sci. USA, 69 ('1972) 1188-~-1191. Petes, T.D., Byers, B. and Fangman, W.L., Size and structure of yeast chromosomal DNA, Proc. Natl, Acad. SeL USA, 70 (1972) 3072--3076, Piper, P.W. and Wasserstein, M., Separation of Saccharomyces cerevisiae tRNA's on two dimensional polyacrylamide gels as applied to investigations of mutational alterations of tRNA's that produce nonsense suppressors, Eur. J. Biochem., 80 (1977) 103--109. Pyeritz, R.E., Schlegel, R.A. and Thomas, C.A., Hydrodynamic shear breakage of DNA may produce single-chained terminals, Biochim. Biophys. Aeta, 272 (1972) 504-509. Ratzkin, B. and Carbon, J., Functional expression of cloned yeast DNA in Escherichia coli, Proc. Natl. Acad. Sci. USA, 74 (1977) 487--491. Rodriguez, R.L., Bolivar, R., Goodman, H.M., Boyer, H.W. and Betlach, M.C., Construction and characterization of cloning vehicles, in Neitlich, Rutter and Fox (Eds.), Proceedings of the ICN-UCLA Symposia on Molecular and Cellular Biology, Academic Press, San Francisco, in press. Roychoudbury, R., Jay, E. and Wu, R., Terminal labeling and addition of homopolymer tracts to duplex DNAfragments by terminal deoxynucle0tidyl transferme, Nucleic. Acids Res., 3 (1976) 101--116. Rubin, G.M., Three forms of the 5.88 ribosomal RNA species in Saccharomyces cerevisiae, Eur. J. Biochem,, 41(1974) 197,202. ~. . . . . Schweizer, E., MacKechnie, C. and Halvorson, H.O., The redundancy of ribosomal and transfer RNA genes in 8accharomyces cerevisiue, J.~MoL Biol., 40 (1.969) 261--277.
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Weiss, B. and Milcarek, C., Mass screening for mutants with altered DNases by microassay techniques, in Grossman and Moldave (Eds.), Methods in Enzymology, Vol. 29, Academic Press, New York, 1974, pp. 180--193. Wensink, P.C., Finnegan, D.J., Donelson, J.E. and Hogness, D.S., A system for mapping DNA sequences in the chromosomes of Drosophila melanogaster, Cell, 3 (1974) 315--325. Communicated by F.E. Young.
ISOLATION AND ANALYSIS OF RECOMBINANT DNA MOLECULES CONTAINING YEAST DNA (pMB9 plasmid; E. coli; yeast nuclear DNA; genes for tRNA and rRNA; abundant m R N A ) THOMAS D. PETES ''s , JAMES R. BROACH 2, PIETER C. WENSINK 3, LYNNA M. HEREFCRD 3, GERALD R. FINK 4 and DAVID BOTSTEIN'
'Department of Biology, Massachusetts Institute of Technology, Cambridge, MA; zCold Spring Harbor Laboratory, Cold Spring Harbor, N Y 11724; 3Rosenstiel Basic Medical Sciences Research Center, Brandeis University, Waltham, MA 02154; 4Department of Genetics, Development and Physiology, Cornell University, Ithaca, NY; and SDepartment of Microbiology, University of Chicago, 920 E. 58th Street, Chicago, IL 60637 (U.S.A.)
(Received December 21~t, 1977) (Accepted May 5th, i 9 7 8 ) SUMMARY
2500 recombinant plasmids containing insertions of yeast nuclear DNA have been cloned in Eseherichia coli. It can be calculated that about 85% of the yeast genome is represented in this collection. The clones have been characterized by hybridization to purified RNA species. Of the 2000 clones examined, 75 contain insertions of yeast ribosomal DNA, 201 contain insertions of yeast tRNA genes, and 26 contain DNA sequences that are complementary to abundant mRNA species.
INTRODUCTION
The yeast Saccharomyces cerevisiae has a genome that is much smaller than higher eukaryotes. Although estimates vary, several independent methods yield a nuclear genome size of about 9 • 109 daltons (Hartwell, 1974). Despite the small size of the genome, many of the details of cell structure, chromosome structure and replication, and macromolecular synthesis in yeast are very similar to those processes in higher eukaryotic cells. Since the yeast genome is small, one method of analysis of yeast chromosome structure and function is construction of a large number of recombinant molecules containing insertions of yeast DNA. If a sufficient number of such plasmids can be constructed, most of the sequences in the yeast genome should be represented. We have isolated 2500 molecules containing insertions of yeast nuclear DNA; b y calculation, 85% of the yeast genome should be present in this collection. As apreliminary characterization of the inserted sequences, 2000 of the clones have been examined for sequences homologous to yeast tRNA, ribosomal RNA, and messenger RNA.
38
Since the collection of recombinant plasmids has been used by many laboratories,we feel that it is useful to present, in a preliminary way, the general characteristicsof this collection and the detailsof its construction. Several other workers (Kramer et al.,1976; Ratzkin and Carbon, 1977; Beckmann et al.,1977) have also constructed large collectionsof recombinant bacteriophages or plasmids containing yeast D N A . The collection of recombinant clones described in this paper has several advantages over those cons t r u ~ using restrictionenzymes. The size of the D N A inserted into the recombinant plasmids was larger (10 kb) than that reported for the other collections. Since the recombinant plasmids in our collection were constructed from randomly sheared fragments of yeast nuclear D N A (Lobban and Kaiser, 1973), recombinant plasmids with overlapping D N A sequences were present. In addition, when recombinant molecules are constructed with restrictionenzymes, only those genome fragments with a cohesive end (generated by the restrictionenzyme) on both ends of the fragment can be cloned. This requirement specificallyexcludes the yeast chromosome ends. Finally, the recombinant clone collection described in this paper is the only one in which the total collection has been hybridized, in separate experiments, to ribosomal RNA, messenger RNA and transfer RNA. Since the recombinant clm~es were screened in the same ordered array for each experiment, relationships among the hybridization patterns were established (for example, what proportion of the plasmids which hybridize to messenger RNA also contained tRNA genes). MATEV.IALSAND METHODS
Strains The yeast stre.inused as a source of D N A for cloning was the diploid strain + D 4 (provided by L.H. Hartwell). The genotype of the strain is: a/a gall/gall adel/adel ural/ural his7/+ +/his5 lys2/+ +/lysl tyrl/+ +/leu2 ade2/+. Ribosomal R N A was isolated from the haploid yeast strain A364a (a gall adel ade2 ural tyrl his7 lys2),one of the haploid parents of +D4. Messenger RNA and transfer RNA were isolated from strain R95-4A, a GAL ÷ derivative of strain $288C. The plasmid vector used was the small plasmid pMB9 (Rodriguez et al., 1977) which confers resistance to tetracycline upon bacteria which contain it. The host bacterial strain for the transformation experiments was HB101 which is a derivative of E. coil K-12 and has the genotype: hsm-, hsr-, recAgal- pro- str ~ (Boyer and Roulland-Dussoix, 1969). Plasmid D N A Plasmid DNA was isolated by a minor modification of t h e cleared-lysate method of Clewell and Helinski (1969), Triton X-100 wasused to lyse the cells after the lysozyme treatment; a preliminary centrifugation at 17 000 rev./min in the Sorvall SS34 rotor for 30 min prior to equilibrium banding in
39
CsCloethidium bromide was performed. The former modification gives slightly higher yields; the latter removes insoluble material which otherwise sometimes obscured the DNA bands. Sizing of plasmids was performed first by measurement in the electron microscope (Davis et al., 1971). Aqueous spreads of circular plasmid DNA relaxed by treatment with DNase were measured relative to linear pMB9 molecules produced by digestion with the site-specific endonuclease EcoRI. A second method was to use supercoiled plasmids of known length to calibrate 0.9% agarose gels containing 0.5% ethidium bromide (Sharp et al., 1973); most length measurements were done with this simple method.
Isolation of high molecular weight yeast nuclear DNA Yeast cells contain at least three types of DNA: nuclear DNA, mitochondrial DNA, and a class of extranuclear plasmid molecules (Hartwell, 1974). Since the mitochondrial and plasmid DNA molecules are usually isolated as fragments which are considerably smaller than nuclear DNA (Petes et al., 1973), in vitro recombinants constructed from unfractionated yeast DNA would overrepresent these species. Consequently, the procedure described below was designed to isolate selectively the largest DNA molecules of the yeast cell. Cells from the yeast strain +D4 were grown in 2 liters of SD minimal medium (Mortimer and Hawthorne, 1969) supplemented with 2% glucose, 20 ~g/ml adenine, 20/~g/ml uracil, and 50/~g/ml tyrosine. Labeled ['4C] uracil (New England Nuclear; 0.5 mCi/mg) was added at a concentration of 15 ~Ci/liter to allow monitoring of DNA purity and recovery. Cultures were grown with aeration at 30°C to an absorbance (660 nm) of 1.2. The cells were harvested by centrifugation and washed in 1/10 vol. of 0.05 M EDTA (pH 8). The protocol for formation of spheroplasts was similar to that used by Cryer et al. (1975). The cell pellet was resuspended in 25 ml of 0.05 M Tris, 0.03 M ~-mercaptoethanol (pH 9.5). The cells were incubated at room temperature for 15 min and then centrifuged and resuspended in 1 M sorbitol, 0.001 M EDTA. The cells were converted to spheroplasts by addition of 2 ml of an aqueous (1 mg/ml) solution of zymolyase (Kirin Brewing Co.) to the suspension, followed by incubation at 37°C. Efficacy of spheroplast formation was monitored by observation i n t h e light microscope after treatment of aliquots with 2% sodium dodecyl sarcosinate. After 80% of the cells had been converted to spheroplasts, the mixture was centrifuged in the Sorvall SS-34 rotor at 2000 rev./min for 2 min. The pellet was gently resuspended in a small volume of 0.1 M Tris, 0.1 M EDTA, 0.15 M NaCI, 0.3 M ~-mercaptoethanol, pH 9.5. An additional 20 ml of the same buffer containing 4% sodium lauryl sarcosinate was then added and the mixture was incubated at 45°C. After 15 min, 25 ml of a buffer containing 0.1 M Tris, 0.1 M EDTA, 0.15 M NaCI, 0.3 M ~-mercaptoethanol, and 4% sodium lauryl sarcosinate was added and the mixture was shifted to 70°C for 15 min. The lysate was centrifuged at 5000 rev./min and the supernatant decanted. The supernatant was layered (10 ml/gradient) onto sucrose gradients prepared in
40
B e c ~ a n SW27 tubes as follows: 4 ml of a high-density mixture (20% sucrose in 80% sodium ioth~lsmate [Angio-Conray, Mallinckrodt Pharmaceuticals] ) was put in the bottom and overlaid with a 24 ml gradient of 5% to 20% sucrose in 0.02 M Tris, 0.02 MEDTA, 0.2 M NaCI, 0.1% sodium lauryl sarcosinate, pH 8, The sample was added t o t h e gradients t h r o u ~ a large,bOre plastic pipette. ~ The gradients were centrifuged a t 1 3 500 rev./min for 17 h at 21°C. Fractions were slowly collected through a hole made by an 18-gauge needle. Fractions were assayed for D N A by determining the number of alkali-stable acid-precipitable counts in each fraction (Petes and Fangman, 1972). The fractions containing the largest DNA were pooled and dialyzed exhaustively against 0.01 M Tris, 0.005 M EDTA, 0.15 M NaCI. The dialyzed fractions Were pooled and the DNA banded to density equilibrium in CsCI gradients (Petes and Fangman, 1972). No peak of DNA was observed banding at the position of yeast mitochondrial DNA. Fractions containing the yeast nuclear DNA were pooled and dialyzed against 0.01 M Tris, 0.001 M EDTA, pH 8. Recovery of the DNA varied in three preparations from 25% t o 60%. The DNA recovered was larger than intact T4 DNA; i.e. :>200 kilobases (kb) in length.
Shear of yeast nuclear DNA Yeast DNA (20 mg/ml) in 0.01 M Tris, 0.001 M EDTA, and 2.5 M NaCI (pH 8) was sheared by two passages through a syringe fitted with a 25-gauge needle (Pyertiz et al., 1972). All manipulations were done at 5°C. Examination of the sheared DNA with the electron microscope indicated that most of the molecules were between 10 and 25 kb. Recombination in vitro The recombinant molecules were constructed by the hompolymer taft method described by Wensink et al. (1974). The only change was the use of the terminal transferase reaction conditions of Roychoudbury et al. (1975), in which Co 2÷ was the only divalent cation. Oligo-dT tails of average length 280 nucleotides were added to the EcoRI-cleaved pMB9 DNA; oligo-dA tails of average length 100 nucleotides were added to the sheared yeast nuclear DNA. Transformation and selection o f recombinant clones A modification of the CaCl2method described by Mandel and Higa (1970) was used. HB101 (Boyer and Roulland-Dussoix, 1969) cells growing exponentially in LB medium (Miller, 1972) at a density of 5 • 10 s cells/ml were collected by centrifugation and resuspended in 1/2 vol. of ice-cold 0.05 M CaCI2., After ~15 m re.at . . . . 0 oC t h.e bacteria . . c e n ~.g. .e.d. were agam a n d resu~ pended in 0.05 M CaCI2 at 1/1 this suspension wereadded to~ ed 1001~Iof a buffer (0.01 M Tris, 0.01 M CaCl2, 0,01 M:MgCI~, pH 7) a n d 1 , 20
41
/~1 of DNA. After the addition of cells, the mixture was kept on ice for 25 rain. The tubes were then shifted to 37°C for 2 rain and put at room temperature for 10 min. I ml of LB medium was added to each tube and the tubes were shaken at 37°C for 30 rain. 2.5 ml soft agar (5 g NaC1, 8 g Bacto nutrient broth, 6.5 g agar in I liter of water) was added to each tube and the mixture was poured onto LB plates containing 10 ~g/ml tetracycline. Transformants were picked after the plates had been incubated 2--3 days at 37°C. i
Storage and handling of the set of recombinant clones Recombinant clones rapidly lose viability when stored in agar stab tubes or on agar plates. For permanent storage, we add an equal volume of a freezing media to a saturated culture in LB media. The freezing media contains twice the concentration of components in the media used by Hogness and Simmons (1964). To aid in storage, subcloning and colony hybridization, we have adopted the general procedure for handling large numbers of clones which has been developed by Brenner et al. (1975); Weiss and Milcarek (1974); and Stern and Wensink (unpublished). The colonies are grown and stored in 144 well polystyrene microtiter plates (Linbro Scientific, Inc.) which are sterilized by UV light and filled with media using a 144 tube multipipette. Paper or microtiter plate replicas are made with a 144 prong replicator. The plates are covered with a plastic adhesive film (M. and C. Specialties) and stored either in liquid nitrogen vapor or at-70°C.
Colony hybridization The colony hybridization procedures used are a modification of the technique developed by Grunstein and Hogness (1975). The modification used for colonies grown on nitrocellulose paper was developed by J. Lis and L. Prestige (unpublished) and that used for colonies grown on Whatmann 3MM paper was developed by R. Stern and P. Wensink (unpublished). Colonies from each microtiter plate were replicated onto sterile sheets of Whatmann 3MM paper or nitrocellulose paper which were lying on large trays containing LB agar and tetracycline. The replicas were made with the replicator previously mentioned and were grown for 24 h at 37°C. Whatmann filters containing the colonies were placed in 1-1iter baths of the following solutions: (1) 0.5 N NaOH for 7 min, (2) two baths of I M Tris • HCI (pH 7.4) for 2 min per bath, and (3) 1.5 M NaCl, 0.5 M Tris • HCI (pH 7.4) for 4 min. The filters were rinsed in 95% ethanol. Nitrocellulose sheets were subjected to an identical lysing and washing regime. However, instead of being immersed in solutions, the filters were laid on blotting paper saturated with the appropriate solutions. The nitrocellulose filters were then sucked dry and rinsed in two washes of 95% ethanol. The filters were sucked dry and baked under vacuum at 80°C for 2 h. (a) Hybridization of ribosomal RNA. Total yeast RNA was isolated from the strain A364a (Hereford and Rosbash, 1977). The 18S and 24S RNA molecules were purified by centrifugation (15 h at 27 000 rev./min in the
42
SW27 rotor) through 15- 30% sucrose gradients (0.01 M Tris, 0.001 M EDTA, 0.1 M NaCI, 0.5% sodium dodecyl sulfate, pH 7.4). Those fractions containing 18S and 24S RNA were pooled and precipitated with 2 vol. of ethanol. The RNA was resuspended in 0.2 M sodium acetate (pH 5.5) and reprecipitated with 9. vol. of ethanol. This procedure was repeated once. The R N A was then washed twice with 70% ethanol, resuspended at a concentration of 2 mg/ml, and stored at-20°C. The RNA was cleaved to 100- 200 nucleotides by incubation at 90°C for 60 min in 0.005 M glycine, 0.01 mM EDTA (pH 9.15). The RNA was labeled by the kinase labeling procedure of Maxam and Gilbert (19"/7). The specific activity of the labeled RNA was 1- 2-107 cpm//~g. The hybridization buffer contained 50% formamide, 4.25 × SSC (0.64 M NaCI, 0.064 M trisodium citrate), and 0.1 M phosphate buffer (0.05 M of both K2HPO4 and KH2PO4). The volume of hybridization buffer per filter was 5 ml, and the amount of radioactivity per filter was l 0 s cpm. Hybridization was done in plastic bags at 37°C for 18 h. Washing of the filters and autoradiography were done as described by Grunstein and Hogness (1975). (b) Hybridization with messenger DNA. Labeled messenger RNA was prepared by a modification of the in vivo labeling procedure described by Rubin (1974). Strain R95-4A was grown at 30°C in low phosphate YEPA medium (1% yeast extract, 2% peptone, 2% potassium acetate treated with magnesium sulfate and ammonium hydroxide as described; Rubin, 1974) to a density of 107 ceUs/ml. Carrier-free [32p] orthophosphate was added to a concentration of 100 ~Ci/ml, and growth was continued. After I h, cycloheximide was added to a concentration of 100 pg/ml and the cells were poured over crushed ice. After harvesting by centrifugation, the cells were washed with and resuspended in 1/100 vol. polysome buffer (25 mM Tris.HCl, pH 7.5, 25 mM NaCl, 5 mM MgCl2, I mg/ml heparin). Cells were then broken by two 1-min treatments in a Braum homogenizer. After the cell debris was removed by centrifugation at 30 000 × g for 5 min, the supernatant was made 1% in SDS and extracted with phenol/chloroform (1/1). The RNA was then precipitated with 2 vol. of ethanol. The precipitate was resuspended in 0.1 M NaCI and reprecipitated with 2 vol. of ethanol. The precipitate was then collected and dried. Poly(A)-containing RNA was purified by poly(U) sepharose chromatography and aclTlamide gel electrophoresis. The dried RNA precipitate was resuspended in 2 ml buffer A (0.5 M NaCl, 0.01 M Tris.HCl, pH 7.5), incubated at 95°C for I min, and then quick-chilled on ice. This solution was then applied to a 1-ml poly(U) sepharose column (Pharmacia equilibrated with buffer A). The column was washed with approx. 20 ml buffer A, and the poly(A)-containing RNA was eluted with distiUed, deionized water. The poly(A)-containing RNA was precipitated with 1/10 vol. 1 M NaCI and 2 vol. ethanol, collected by centrifugation, and dried. The RNA was then electrophoresed on a 3.5% acrylamide 98% formamide cylindrical gel, as described by Duesberg and Vogt (1973). After electrophoresis the labeled RNA was located by auto-
43
radiography of the gel, and the region containing the bulk of the labeled RNA was cut out. This section of the gel was combined with an equal volume of 10 X SSC and dispersed by several strokes of a dounce homogenizer. The gel suspension was then sonicated until no turbidity was apparent in the solution. This solution was used without further treatment as messenger RNA probe. Hybridization was performed at 42°C for 16 h in 50% formamide, 5 × SSC, 200 mg/ml polyadenylic acid, and 106 to 107 cpm labeled RNA/filter, in a volume of either 2 ml (for nitrocellulose filter hybridization) or 4 ml (for Whatman filter hybridization). After hybridization, the filters were incubated at 42°C in 100 ml 50% formamide, 5 × SSC/fflter for 16 h. The filters were then washed and autoradiographed as described (Grunstein and Hogness, 1975}. (c) Hybridization of transfer RNA. Labeled transfer RNA was purified from total in vivo labeled RNA by DEAE cellulose chromatography and polyacrylamide electrophoresis. The follow-through fraction from poly(U) sepharose chromatography in vivo labeled RNA was diluted tenfold with 0.01 M Tris. HCI, pH 7.5, and applied to a 2-ml DEAE cellulose column equilibrated with 50 mM NaCl, 10 mM Tris.HC1, pH 7.5. The column was washed with approx. 20 ml equilibration buffer and then with several 1-ml portions of 1.0 M NaCl, 10 mM Tris.HC1, pH 7.5. The RNA eluted by the 1.0 M NaCI wash was precipitated with 2 vol. ethanol, collected, and dried. The tRNA was further purified by polyacrylamide gel electrophoresis. For preparation of crude tRNA, the post-DEAE cellulose RNA sample was electrophoresed on a 10% polyacrylamide 98% formamide cylindrical gel (Duesberg and Vogt, 1973}. After electrophoresis, the 4S to 4.5S region was located by autoradiography, cut out, and prepared as described for isolation of messenger RNA probe. For isolation of purified tRNA probes, the post~DEAE cellulose fraction was subjected to two-dimensional polyacrylamide gel electrophoresis as described by Piper et al. (1977). After electrophoresis, the pattern of tRNA spots was visualized by autoradiography and the regions corresponding to spots previously identified as the UCG-decoding serine tRNA and the leucine II tRNA {Piper et al., 1977) were cut out of the gel. The tRNA species were eluted from the excised gel pieces by incubation overnight at 4°C in 0.1 M NaCI, 10 mM Tris-HC1, pH 7.5, 1 mM EDTA, after the gel was homogenized with several strokes in a dounce homogenizer. After removal of polyacrylamide gel fragments by centrifugation through glass wool, the solutions were used directly for hybridization. Hybridization of crude or purified tRNA probes was performed as described for that of messenger RNA except that polyadenylic acid was omitted from the hybridization mixture and a posthybridization incubation on 50% formamide, 5 × SSC was not performed.
Clone nomenclature The recombinant plasmids are labeled pY1 (indicating that the clone is a yeast DNA-containing piasmid from the first preparation of yeast DNA)
44
followed by the miczotiter dish number (1-,20)and the position on the microtiter dish. The rows in the microtiter d ~ e s are labeled A through L and the columns labeled 1 through 12. A complete description of a single clone, therefore, might be p Y 1 (2A-3), ~ i s clone Would b e located on microtiter dish No, ~2in r o w A, column 3. RESULTS
Construction of the recombinant plasmids The recombinant molecules were constructed by the technique developed by Lobban and Kaiser (1973) and used by Wensink et al. (1974) to clone Drosophila DNA. A homopolymer "taft" of dAMP residues is added to the ends of sheared yeast nuclear DNA. This technique has the advantage of a positive selection for insertion of foreign DNA into the plasmid carrying the tetracycline-resistance genes since the cleaved plasmid, after addition of the homopolymers, can no longer transform cells to tetracycline resistance by itself. In our experiments, the transformation efficiency of yeast DNA and plasmid DNA annealed together was about 30-fold higher than either species alone. The small number of transform~nts that arise with pMB9 alone are probably the result of plasmid DNA. which was not cleaved with EcoRI, since a small number of uncleared molecules were observed with the electron microscope. Evidence for presence of yeast DNA in the plasmids in the transformants was obtained by isolating plasmid DNA from several of the transformants and analyzing this DNA on agarose gels. Recombinant plasmids had a slower mobility, indicating that they were larger in size than the original pMB9 pla~ mid. One of the twenty plasmids contained no detectable insertion of yeast DNA; the remainder contained an insertion whose average length was 10 kb. The recombinant plasmids when digested with EcoRI and HindIII restriction enzymes showed patterns on agarose gels which were different from that of pMB9. The recombinant plasmids also differed from pMB9 DNA in contour length as measured by electron microscopy and in buoyant density as measured in CsC1 density gradients. Hybridization of clones to yeast RNA probes 2000 of the 2500 clones were hybridized in separate experiments to either purified 18S and 24S yeast ribosomal RNA, yeast messenger RNA sequences, or yeast tRNA. The technique of colony hybridization was used for this analysis. Approx. 15% (298/2000 clones) of the strains containing recombinant plasmids hybridized to one or more of the labeled yeast RNA species. Almost all (295/298) of these clones showed one of five patterns of hybridization shown in Table I. Each of these patterns will be discussed individually. The largest classof clones, type 11,hybridized only to yeast t R N A . This result suggests that the yeast t R N A Cistrons are widely spaced throughout the
45
TABLE I HYBRIDIZATION PATTERNS OF CLONES TO PURIFIED YEAST rRNA, mRNA, AND tRNA I n all t h e s e e x p e r i m e n t s , t w o t h o u s a n d c l o n e s w e r e s c r e e n e d b y c o l o n y h y b r i d i z a t i o n to 32P-labeled p u r i f i e d y e a s t r R N A , m R N A or t R N A .
Hybridization p a t t e r n
I II HI IV V Other
Number of clones with pattern
H y b r i d i z a t i o n to p r o b e s rRNA
mRNA
tRNA
194 18 57 19 7 3
+ + -
+ + + +
+ + +
yeast genome and not highly clustered. In addition to hybridizing the clones with total yeast tRNA, we have used UCG-decoding serine tRNA and leucine II tRNA as hybridization probes. Three clones containing sequences complementary to the former probe, and six clones containing sequences complementary to the latter probe, have been identified. These results are consistent with the idea that many different tRNA genes are present in the type I clones. Type II clones represent plasmids which hybridize both to the 18S and 24S ribosomal RNA and to the messenger RNA hybridization probe. Since mRNA isolated on poly(U) columns is known to be contaminated by ribosomal RNA, all clones containing yeast ribosomal DNA would hybridize to both probes. Independent evidence that type II clones contain only yeast ribosomal DNA has been provided by restriction analysis of these clones (Petes, Hereford and Skryabin, J. Bacteriol., in press). Type III clones hybridize to all RNA probes. Since the clones hybridize to purified ribosomal RNA, .hey must contain insertions of yeast ribosomal DNA. The hybridization to the mRNA probe, as described previously, is the result of contamination of the probe with ribosomal RNA. The hybridization of these clones to purified tKNA has two possible interpretations. Either some tRNA genes are interspersed with ribosomal DNA cistrons, or the tRNA hybridization probe is contaminated with yeast ribosomal RNA fragments. The fourth class of hybridization pattern is shown by those clones that hybridize only to the mRNA probe and therefore represent DNA sequences whose transcripts are sufficiently abundant to be detected in these experiments. ~ e V clones hybridize both to mRNA and to tRNA. These clones presumably contain both genes which are transcribed into abundant messenger RNA sequences and genes which code for tRNA.
46
DISCUSSION
The 2500 clones containing insertions of yeast DNA should contain about 85% of the yeast nuclear genome. This calculation was made according to the PoP,son equation: • .... P = (1 - [ T / G ] )N
where P equals the probability of missing a particular yeast DNA sequence, T equals the average size of the inserted DNA (10 kb), G equals the yeast genome size (13.5 • 103 kb), and N equals the total number of recombinant plasmids. An observation which suggests that most of the yeast genome is represented in this collection is that ura3, a yeast gene present in a single copy per haploid genome, has been found in two different recombinant clones of two thousand examined (M. Bach and D. Botstein, personal communication). In these experiments, the recombinant DNA molecules containing ura3 were identified by complementation of a p y r F mutant of E. coll. In addition, a recombinant clone from this collection has been tentatively shown to contain yeast histone genes (L.M. Hereford and G. Lauer, personal communication). Of the 2000 clones that were analyzed, 75 hybridized to ribosomal RNA. This frequency (3.7%) is close to the predicted proportion of ribosomal DNAcontaining clones, since Schweizer et al. (1969) showed that 2.4% of yeast nuclear DNA hybridized to ribosomal RNA and, therefore, 4.8% of the yeast DNA should represent ribosomal DNA genes. This result indicates that ribosomal DNA sequences were not preferentially retained or lost during the cloning procedure. One surprising result of these experiments was the small number of recombinant plasmids (26/2000 clones) which hybridized specifically to mRNA. This observation does not, of course, imply that only a small proportion of the yeast genome is transcribed. Hereford and Rosbash (1977) have shown that yeast messenger RNA species are present in the cell in at least three classes of abundance. About 20 mRNA species are present at a frequency of 100 copies per cell, 400 species are present at a frequency of 10 copies per cell, and about 2500 species are present as a single copy in the cell. It is likely that in our experiments only the most abundant transcribed species of RNA are being detected. This conclusion is confirmed by the observation that in mENA hybridization experiments in which tenfold greater amounts of probe are used, approximately 40% of the clones show hybridization of probe (Broach, unpublished observations; Hereford, unpublished observations). This is approximately the proportion of clones one would expect to contain genes corresponding to the very abundant and moderately abundant messenger RNA classes.
The largest class of clones that hybridized to a n y o f the RNA probes consisted of Ciones hybridizing t o tRNA r(201/2000 ciones).~About 0.064- ~).08% of nuclear DNA hybridizes to tRNA (Schweizer et ~al., 1969). The proportion
47
of clones expected tohybridize to tRNA depends on how the tRNA genes are arranged in the genome. If all the tRNA genes were clustered, only a few clones would be expected to hybridize to the tRNA probe (about 0.0007 × 2000 = 1.4 clones). If the tRNA genes are unlinked, the expectation is quite different. The number of tRNA genes can be estimated from the data of Schweizer et al. (1969) to be about 300. Since the genome size is 13 500 kb, there is l tRNA gene per 45 kb. Since the average size of the inserted sequences is 1 0 kb, about 20% of the clones would be expected to hybridize to tRNA if the tRNA genes were completely unlinked. The experimental observation, therefore, that 10% of the clones hybridize to tRNA suggests that the tRNA genes are scattered throughout the genome and not extensively clustered. The conclusion is supported by the data of Hawthorne and Leupold (1974), which showed that the 8 tyrosine-inserting ochre suppressors (Gilmore et al., 1971) mapped at different positions in the yeast genome. Beckmann et al. (1977) have shown that the yeast tRNA genes are generally unlinked in experiments similar to those reported here. In conclusion, our preliminary characterization of this set of 2500 yeast/ pMB9 hybrid plasmids suggests that it is a fair representation of the yeast nuclear genome, even though it is certainly incomplete. These results seem to us to encourage further use of this collection in studies of the yeast genome. ACKNOWLEDGEMENTS
T.D. Petes was supported by an N.I.H. postdoctoral fellowship. J.R. Broach was supported by an N.I.H. postdoctoral grant 5T32-0917602. P.C. Wensink was supported by N.I.H. grant GM21626. L.M. Hereford was supported by an N.I.H. grant (GM23549) to M. Rosbash. G.R. Fink was supported by N.I.H. grant GM15-408. The work was also supported by N.I.H. grants (GM18973 and GM21253) to D. Botstein and a Research Career Development award to D. B, REFERENCES Beckmann, J.S., Johnson, P.F. and Abelson, J., Cloning of yeast transfer RNA genes in Escherichia coli, Science, 196 (1977) 205--208. Boyer, H.W. and Roulland-Dussoix, D., A complementation analysis of the restriction and modification of DNA inEscherichia coli, J. Mol. Biol., 41 (1969) 459--472. Brenner, M., Tisdale, D. and Loomis, W., Techniques for rapid biochemical screening of large numbers of cell clones, Exp. Cell Res., 90 (1975) 249--252. Clewcll, D. and Helinski, D., Properties of a supercoiled deoxyribonucleic acid-protein relaxation complex and strand specificity of the relaxation event, Biochemistry, 9 (1970) 4428--4440. Cryer, D.R., Eccleshall, R. and Marmur, J., Isolation of yeast DNA, in Prescott (Ed.), Methods in Ceil Biology, Vol. 12, Academic Press, New York, 1975, pp. 39--44. Davis, R.W,, Simon, M. and Davidson, N., Electron microscope heteroduplex methods for mapping regions of base sequence homology in nucleic acids, in Grossman and Moldave (Eds.), Methods in Enzymology, Vol. 21, Academic Press, New York, 1971, pp. 413--428.
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