Cell, Vol. 18. 1247-i

259, December

1979.

Copyright

0 1979

by MIT

Isolation of Cloned DNA Sequences Ribosomal Protein Genes from Saccharomyces cerevisiae John L. Woolford, Jr.,* Lynna M. Hereford and Michael Rosbash Rosenstiel Basic Medical Sciences and Department of Biology Brandeis University Waltham, Massachusetts 02254

Research

Center

Summary Yeast mRNA enriched for ribosomal protein mRNA was obtained by isolating poly(A)+ small mRNA from small polysomes. A comparison of cell-free translation of this small mRNA and total mRNA, and electrophoresis of the products on two-dimensional gels which resolve most yeast ribosomal proteins, demonstrated that a 5-10 fold enrichment for ribssomal protein mRNA was obtained. One hundred different recombinant DNA molecules possibly containing ribosomal protein genes were selected by differential colony hybridization of this enriched mRNA and unfractionated mRNA to a bank of yeast pMRg hybrid plasmids. After screening twenty-five of these candidates, five different clones were found which contain yeast ribosomal protein gene sequences. The yeast mRNAs complementary to these five plasmids code for %-methionine-labeled polypeptldes which co-mlgrate on two-dimensional gels with yeast ribosomal proteins. Consistent with previous studies on ribosomal protein mRNAs, the amounts of mRNA complementary to three of these cloned genes are controlled by the RNA2 locus. Although two of the flve clones contain more than one yeast gene, none contain more than one identifiable ribosomal protein gene. Thus there is no evidence for “tight” linkage of yeast ribosomal protein genes. Two of the cloned ribosomal protein genes are single-copy genes, whereas two other cloned sequences contain two different copies of the same ribosomal proteln gene. The fifth plasmid contains sequences which are repeated in the yeast genome, but it is not known whether any or all of the ribosomal protein gene on this clone contains repetitive DNA. Introduction Many complex processes occurring during cellular proliferation or differentiation of eucaryotes involve the coordinate expression of a set or sets of genes. Ribosome biosynthesis is an excellent example of such a process, in that it involves the expression of a number of genes and the assembly of the products of these genes into a functional unit essential for cell l Present address: Department of Biological Sciences, tute of Science, Carnegie Mellon University, 4400 Pittsburgh, Pennsylvania 15213.

Mellon InstiFifth Avenue,

Containing

growth. Procaryotic ribosome structure and biogenesis have been studied in great detail in E. coli (for review see Nomura, Tissieres and Lengyel, 1974). Almost all of the 53 different proteins and three different ribosomal RNA molecules are present in equimolar quantities in E. coli ribosomes (Hardy, 1975). Many if not all of these components are synthesized coordinately and stoichiometrically under a variety of conditions of cellular growth rate and metabolism (Kjeldgaard and Gausing, 1974; Dennis and Nomura, 1975). The coordinate expression of at least some of the E. coli ribosomal protein genes is regulated at the level of transcription, and perhaps also at the level of translation (Dennis and Nomura, 1975; Fallon et al., 1979). By genetic, physiological and biochemical methods, it has been demonstrated that the expression of the ribosomal protein (rp) genes of the baker’s yeast, Saccharomyces cerevisiae, is also coordinately regulated. Perhaps the most compelling evidence in favor of the coordinate control of these genes is the effect of any of ten complementary conditional lethal mutations, -RNA2-RNA11 (Hartwell, McLaughlin and Warner, 1970). These mutations, originally selected on the basis of their ability to prevent the accumulation of ribosomal RNA under nonpermissive conditions, have a dramatic and specific effect on the synthesis of almost all ribosomal proteins. After a shift-up to the nonpermissive temperature, there is a rapid decrease in the synthesis of ribosomal proteins (43 out of 48 examined) (Gorenstein and Warner, 1978). There is a parallel decrease in the concentration of these ribosomal proteins’ mRNAs as assayed by in vitro translation, while the concentration of nonribosomal mRNA or total mRNA is unaffected (Warner and Gorenstein, 1977). The kinetics with which this decrease takes place suggest that the coordinate decrease of ribosomal protein mRNA is mediated at the level of transcription, or shortly thereafter. Further progress in understanding the nature of this coordinate control has been hindered by the paucity of bona fide ribosomal protein mutations in yeast as well as other eucaryotes. (We define bona fide as exhibiting an electrophoretic or primary sequence difference from a previously identified ribosomal protein.) Progress has also been hindered by the lack of nucleic acid probes for ribosomal protein mRNA sequences, such as can be provided by recombinant DNA technology. Such cloned ribosomal protein genes would also provide insight into the chromosomal organization of these genes, which in turn might provide further insight into the ways in which these genes are coordinately controlled. In this paper, we report the isolation and preliminary characterization of recombinant DNA molecules containing yeast ribosomal protein genes as a first step towards these goals. These recombinant DNA mole-

Cell 1240

cules were identified and isolated from a colony bank of yeast-pMB9 plasmids (Petes et al., 1978) by a two step procedure. First, candidate colonies were identified by their ability to hybridize well with a radioactive probe enriched for a subset of ribosomal protein mRNAs. Second, the mRNAs coded for by these candidate plasmids were assayed by a sensitive method previously developed by us for this purpose (Woolford and Rosbash, 1979). Of these candidates, five were shown to code for bona fide ribosomal proteins. Our preliminary characterization of these cloned sequences reveals interesting features and suggests that they will be of considerable use in further studies on the organization and expression of ribosomal protein genes.

A

SDS Sucrorc

Gradient

1

Results Isolation of mRNA Enriched for Ribosomal Protein mRNA and Differential Colony Hybridization To design a rational method for identifying ribosomal protein mRNA complementary sequences from among a collection of yeast recombinant DNA molecules, it is necessary to consider the intracellular concentration of ribosomal protein mRNAs. Although definitive experiments are lacking, the available data from this and from other laboratories (see Hereford and Rosbash, 1977b) suggest that each ribosomal protein mRNA constitutes approximately O.l -0.2% of the total mRNA, values which are rather low for obtaining reproducible colony hybridization signals (see Discussion). We therefore decided to isolate mRNA enriched for ribosomal protein mRNAs in order to prepare a probe for colony hybridization. Our scheme for purification of an mRNA fraction enriched for rp mRNA took advantage of the fact that a large fraction of ribosomal proteins are of relatively low molecular weight (Warner and Gorenstein, 1979; Otaka and Kobata, 1978) and are encoded by small mRNA (Hackett, Egberts and Traub, 1978) which is found predominantly on small polysomes (Mager and Planta, 1976). We therefore isolated the small mRNA from small polysomes of yeast. A gradient profile of polysomes from yeast is shown in Figure IA; the polysomes were centrifuged to increase the resolution between small polysomes and larger ones. RNA extracted from the small polysomes included some large RNA, perhaps due to understaffing of these mRNAs with ribosomes, and was therefore centrifuged on denaturing sucrose gradients and pooled as shown in Figure 1B to obtain a fraction of RNA substantially enriched for small RNA. Since Warner and Gorenstein (1977) had previously demonstrated that most of the mRNA coding for ribosomal proteins contains poly(A), a final mode of enrichment for rp mRNAs was to chromotograph the small RNA from small polysomes on oligo(dT)-cellulose. This fractionated poly(A)+ RNA

B

Figure

1. Isolation

of Small mRNA From

Small Polysomes

(A) Polysomes were prepared from exponentially growing yeast cells and were centrifuged for 5 hr at 27,000 rpm through a linear IO40% sucrose gradient, so that smaller polysomes were resolved from larger ones. A profile of the optical density at 260 nm is shown. RNA was extracted from the smaller polysomes. indicated by the hatched area. (B) Aliquots of the small polysomal RNA were centrifuged for 16 hr at 39,000 rpm on 15-30% sucrose gradients to resolve small RNA from larger RNA. The profile of poly(A>containing RNA, assayed by ‘H poly(U) hybridization, is shown. The smaller RNA was pooled from the indicated fractions and chromatographed over oligo(dTt cellulose to purify the poly(A&containing RNA. (C) Denaturing gel electrophoresis of the purified RNA samples. (1) Poly(A)+ small polysomal small RNA: (2) poly(A)small polysomal small RNA: (3. 4) unfractionated yeast RNA: (5. 6) unfractionated yeast poly(A)+ RNA.

is indeed small, as the majority of it has an electrophoretic mobility similar to QS globin RNA (Figure 1 C). To verify that this fractionated mRNA is intact small mRNA and that it, is indeed enriched for rp mRNA, the fractionated RNA and total mRNA were translated in a wheat germ cell-free system. Equal amounts of the 35S-methionine-labeled polypeptide products were electrophoresed on SDS-polyacrylamide gels and on

Yeast 1249

Ribosomal

Protein

Genes

two-dimensional gels which resolve most of the yeast ribosomal proteins (Gorenstein and Warner, 1977). The SDS gel profiles indicate that the small RNA codes almost entirely for small proteins less than 25,000 daltons in molecular weight (data not shown). A comparison of the fluorograph from the two-dimensional gels indicates that the small RNA is significantly enriched for mRNA translatable into the identifiable yeast ribosomal proteins (Figure 2). In addition, the enrichment is only for small ribosomal proteins less than 25,000 daltons in molecular weight, as indicated by the darkened spots in Figure 2D. There was no visible enrichment for some of the small ribosomal proteins. It is probable that some of these ribosomal proteins are relatively poor in methionine and therefore not readily visible by in vitro translation with 35Smethionine (see Warner and Gorenstein, 1977). From a qualitative examination of the two gels, we estimated

that the small mRNA was enriched approximately 510 fold for the indicated ribosomal proteins, raising their concentration in the small mRNA to approximately 0.5-2%. Since the small mRNA contained at least 25 ribosomal protein mRNAs at an increased concentration of 0.5-2.0%, it was radioactively labeled in vitro with 32P using polynucleotide kinase and hybridized to the PMBS/yeast DNA-containing colonies in parallel with kinased total poly(A)+ mRNA. Approximately 125 colonies were reproducibly positive with this enriched probe; approximately 100 of these colonies were at least as positive-and in most cases considerably more positivewhen hybridized to the enriched probe than to the unfractionated probe (data not shown). These 100 colonies were considered as candidates for containing cloned ribosomal protein genes and were examined further, as described below.

0

Acid

Urea

__c

SDS

1

J Figure RNA

2. Two-Dimensional

Gel Electrophoresis

of Cell-Free

Translation

Products

of Total Poly(A)+

mRNA

and Poly(A)’

Small Polysomal

Small

Total Yeast poly(A)+ mRNA and poly(A)+ small polysomal small mRNA were each translated in a wheat germ cell-free translation system containing %-methionine. and 5 X 1 O5 cpm each of the labeled products were electrophoresed on two-dimensional gels which resolve most of the yeast ribosomal proteins. (A. 6) Autoradiogram of gel of polypeptides translated from (A) total poly(A)+ RNA and (B) poly(A)+ small polysomal small RNA; (C) Coomassie brilliant blue-stained gel of total yeast protein; (D) schematic diagram of yeast ribosomal proteins resolved by the gel. Solid spots correspond to those 35S-methionine-labeled polypeptides whose mRNAs are enriched in the poly(A)+ small polysomal small RNA compared with total poly(A)+ RNA.

Cell 1250

Screening Candidates to Identify Clones Containing Ribosomal Protein Genes Having identified 100 candidates for ribosomal protein gene-containing clones, we selected 40 of these at random for further examination. Recombinant DNA from these 40 clones was isolated and was linearized by digestion with a restriction enzyme that had only one site in the plasmid. These DNAs were then divided into ten pools of four plasmids each, and their complementary RNAs were purified and translated as described in Experimental Procedures. The protein products of the mRNA complementary to each pool of cloned DNA were analyzed by four different types of acrylamide gel electrophoresis systems. First they were electrophoresed on SDS-polyacrylamide slab gels (Laemmli, 1970) to determine the number and molecular weight of the different proteins encoded by each plasmid pool. Each pool of DNAs encoded several proteins, and all but one of the visible proteins was smaller than 25,000 daltons in molecular weight (Woolford and Rosbash, 1979; our unpublished observations). Since most ribosomal proteins are basic as well as small, the protein products were also electrophoresed on two types of one-dimensional gels designed to resolve basic proteins: on 4% acrylamide at acid pH in the presence of urea (Mets and Bogorad, 1974); and under similar conditions, but with the addition of the nonionic detergent Triton X-l 00 (Alfageme et al., 1974). By these criteria, each pool of plasmid DNAs was found to hybridize to mRNA which coded for at least one basic protein that migrated similarly to a protein present in purified ribosomes (data not shown). Finally, the polypeptide products from each pool were electrophoresed on the ribosomal protein two-dimensional gel described above (Gorenstein and Warner, 1976). By this criterion, each of the ten recombinant DNA pools coded for at least one 35S-methionine-labeled protein that co-migrated with an unlabeled protein present on purified yeast ribosomes. Since each pool of four plasmids was positive for the presence of ribosomal protein genes, the recombinant DNAs were rescreened individually and assayed by the same acrylamide gel systems. Of the first 25 plasmids screened, five plasmids were found to hybridize to mRNA which coded for a protein that co-migrated exactly in two-dimensional electrophoresis with a bona fide ribosomal protein. Two of the recombinant DNA molecules, pY lo-78 and pY 1 l-40, code for a protein that co-migrates with ribosomal protein 39. Plasmid pY 9-90 codes for a protein which co-migrates with ribosomal protein 63 (Figure 3A), pY 13-86 codes for a protein which comigrates with ribosomal protein 52 (Figure 3B) and plasmid pY 1 l-l 38 codes for ribosomal protein 51. Some of these plasmids are complementary to additional mRNAs in that more than one protein band is visible on at least one of these four gel systems (Table

1). The presence of more than one yeast structural gene on some of these plasmids is not surprising considering the size of the yeast DNA inserts in these plasmids and the general organization of the yeast genome previously described (Hereford and Rosbash, 1977a; Kaback, Angerer and Davidson, 1979). Synthesis of mRNAs Complementary to the Cloned Ribosomal Protein Genes Is Controlled by the RNA2 Locus as Predicted The synthesis of three of the four ribosomal proteins identified in the previous section (numbers 51, 52 and 63) has been shown to be under the control of the RNA2 locus (Gorenstein and Warner, 1976); at the nonpermissive temperature, the intracellular concentration of these mRNAs rapidly decreases (Warner and Gorenstein, 1977). In contrast, the synthesis of ribosomal protein 39 is insensitive to the RNA2 locus and is therefore an example of the five identified exceptional ribosomal proteins. To further verify that these four proteins, identified above on the basis of co-electrophoresis in two dimensions, are indeed ribosomal proteins, we examined the effect of the RNA2 locus on the levels of these mRNAs. Poly(A)+ RNA isolated from an ma2 strain after 1 hr at the nonpermissive temperature and identical amounts of control poly(A)+ RNA from wild-type yeast were hybridized in parallel incubations to the five plasmid DNAs described above. Since the R loop incubations are in plasmid DNA excess and go to kinetic completion (Woolford and Rosbash, 1979) the relative amount of a radioactive protein is a function of the relative concentration of its mRNA in the cells from which the RNA was isolated. Densitometer tracings of the autoradiograph shown in Figure 4 confirm that the major mRNA complementary to pY 9-90, identified as coding for rp 63 on the basis of coelectrophoresis, and the major mRNA complementary to pY 1 l-l 38, identified as coding for rp 51, are decreased 3-5 fold in the ma2 mutant cell after 1 hr at the nonpermissive temperature. In contrast, the major mRNA complementary to pY 10-78, identified as coding for rp 39, is present at an approximately equal concentration in the two RNA populations. Similarly, the amount of mRNA complementary to pY 1 l40, also coding for rp 39, was equivalent in mutant and wild-type RNA, whereas the level of mRNA complementary to pY 13-86, coding for rp 52, was depleted in the mutant compared with the wild-type mRNA (data not shown). We therefore conclude that these four proteins are indeed correctly assigned and that these five plasmids are correctly identified as containing yeast ribosomal protein gene sequences. Mapping of Genes within the Recombinant Plasmid DNAs Several of our five ribosomal protein clones code for more than one protein. To examine the arrangement

Yeast 1251

Ribosomal

Protein

Genes

A-

* Figure

3. Two-Dimensional

Gel Electrophoresis

of Translation

Products

of mRNA

Hybridized

to Individual

Ribosomal

35S-methionine-labeled translation products of mRNA complementary to (A) pY 9-90 and (8) pY 13-66. (C) Stained electrophoresis of yeast ribosomal proteins. The arrows, from left to right, indicate the ribosomal proteins encoded (rp 39). pY 1 l-l 36 (rp 51) and pY 13-66 (rp 52).

of these structural genes, we determined the location of most or all of the genes on each plasmid. Physical maps of each plasmid were constructed by cleavage with thirteen different restriction endonucleases (Figure 5). Measurements of the sizes of the restriction fragments derived from the plasmids indicated that the yeast DNA sequences were quite small, from 15 8.0 kb. The genes in these plasmids were then mapped by electron microscopy. As expected, one small R loop was observed in each molecule of pY 1 O78 and pY 1 l-40 (Figure 6B), having a length appropriate for the size of rp 39 (-450 bp). Consistent with the fact that pY S-SO codes for one major small protein and a second minor protein was the observation of

Protein

Clones

pattern of two-dimensional gel by pY 9-90 (rp 631, pY 1 O-76

one small R loop in most molecules and a second slightly larger R loop in about 38% of the molecules (Figure 6A). One small R loop was observed in all pY 13-86 molecules. pY 1 l-l 38 had a more complex population of R loops; almost every molecule contained one small R loop near the “left end” of the yeast insert. Frequently, this R loop was present on the same molecule with one of two small R loops on the “right end” of the molecule (Figure 60; less frequently, all three R loops were present. In no cases were any structures observed that suggested the presence of introns in any of these genes, although structures smaller than 100 bp may not have been detected. These data are summarized in Table 2.

Cell 1252

Genomic Organization and Copy Number of Cloned rp Gene Sequences To verify that the gene organization in the cloned sequences is a faithful representation of that in the yeast genome and to examine the frequency with which these sequences are present in the yeast genome, each of the plasmid DNAs was radioactively labeled in vitro and hybridized to yeast genomic DNA cleaved with restriction endonucleases, separated by agarose gel electrophoresis and transferred to nitrocellulose paper (that is, Southern blots). Since the recombinant plasmids consist of randomly sheared yeast DNA inserted into pMB9 DNA, a recombinant Table

1. Characterization

of Ribosomal

Protein

Clones

Colony Hybridization Signal

Size of insert (kb)

Total mRNA

Small mRNA

Proteins’

Basic Protein@

Ribosomal Proteins”

S-90

2.5

++

+++

2

2

#63

1 O-78

5.1

++

++

1

1

#39

11-40

1.6

++

++

1

1

#39

11-136

5.1

+

+++

3

3

#51

13-66

6.2

+

++

1

1

#52

Plasmid

’ The number of total proteins was determined by electrophoresis on 17% acrylamide SDS gels. b The number of basic proteins is defined as the number of bands separable on acid urea Triton gels (see Experimental Procedures). ’ Ribosomal proteins are defined by co-electrophoresis on two-dimensional gels with purified ribosomal proteins, according to the nomenclature of J. Warner and C. Gorenstein (personal communication).

plasmid that contains only single-copy DNA should hybridize to the same yeast genomic restriction fragments as are present in the cloned DNA, plus two others derived from the ends of the inserted sheared yeast DNA. As shown in Figure 7, this is the case for pY 11-l 38 and pY 13-86. On the other hand, pY 990 hybridizes to at least 25-30 different Hind ill fragments, and therefore must contain a sequence or sequences that are repeated in the yeast genome. The same results were obtained for all three of these clones hybridized to yeast DNA digested with Barn HI and Eco RI. Since pY 1 O-78 and pY 11-40 code for the same protein, it was of interest to determine whether each clone contains the same or overlapping sequences from the yeast genome or whether the cloned sequences contain two independent copies of the same gene. These possibilities were directly tested by hybridization of the two plasmids to genomic fragments and to each other. As shown in Figure 7, each plasmid hybridized to the appropriate genomic restriction fragments plus only one additional fragment. In each case, this additional fragment contained the rp 39 gene sequence from the opposite clone. Identical results were obtained using DNA digested with Barn HI, Eco RI, Xba I, Pst I and Sal I, indicating that there are not one but two copies of sequence information which includes at least a significant fraction of the information for rp 39. The lack of common restriction enzyme sites around the pY 1 O-78 and pY 1 l-40 genes (none of the thirteen restriction enzymes used cleave within the rp 39 structural gene) and the inability of radioactive pY 1 O-78 to hybridize to two additional bands when hybridized to Xba I- or Hind Ill-digested genomic

@

39, encoded by pY 1O-76; (51) ribosomal protein 51, translation products of: (1) wild-type mRNA hybridized type mRNA hybridized to pY 1 O-78 and pY 1 l-l 0; (4) 138 and pY 1 l-l 0; (6) -ma2 mRNA hybridized to pY -DhIA

.

Figure 4. SDS-Polyacrylamide Gel Electrophoresis of Translation Products of ma2 and Wild-Type RNA Hybridized to Cloned Ribosomal Protein Genes

Poly(A)’ mRNA isolated from ma2 yeast (fs368) grown at the restrictive temperature (37°C) and wild-type yeast (A364A) grown at 23°C was hybridized to a mixture containing 3 pg of each plasmid DNA containing riboSOmat protein genes and 3 kg of plasmid pY 11-l 0 DNA containing a gene coding for an abundant yeast mRNA (Woolford and Rosbash, 1979) whose synthesis is not affected by the ma2 mutation. The purified mRNAs were translated in a wheat germ lysate containing “S-methionine, and equal cpm of radioactive polypeptide products were electrophoresed in parallel lanes of a gel containing a 1 O-l 5% gradient of polyactylamide. The gel was fluorographed and exposed to XR5 X-ray film for 20 hr at -70°C. (C) polypeptide encoded by pY 11-l 0; (39) ribosomal protein encoded by pY 1 l-l 36; (63) ribosomal protein 63, encoded by pY s-90. Each lane contains to pY 9-90 and pY 1 l-l 0; (2) ma2 mRNA hybridized to pY 9-90 and pY 1 l-10; (3) wildma2 mRNA hybridized to pY 1 O-78 and pY 1 l-l 0; (5) wild-type mRNA hybridized to pY 1 t 1 l-l 38 and pY 1 l-1 0; (7) wild-type poly(A)+ mRNA: (8) ma2 poly(A)+ mRNA; (9) no added

Yeast 1253

Ribosomal

Protein

Genes

pY 13-86 I 0 Figure

I

I

I

I

I

I

2

3

4

5

5. Restriction

Maps

of Plasmids

Containing

Ribosomal

I

I

I

I

I

I

I

6 7 Kilobases

8

9

IO

II

12

13

I

Protein

Genes

Linear maps of the recombinant plasmids containing pMBB DNA (open bar) linked to randomly sheared yeast DNA (thin solid line) by poly(dA). poly(dT) connectors are shown, beginning at the PMBB Eco RI site where the yeast DNA was inserted. Locations of restriction enzyme sites were derived by single and combined enzyme digests and by electrophoresis of the resulting fragments on 1% agarose gels and 4% polyacrylamide gels as described in Experimental Procedures. Bacteriophage X DNA digested with Eco RI, pMB9 DNA digested with Eco RI or Hae Ill, adenovirus DNA cut with Barn HI, a gift from 6. Roberts and pPWl28 DNA, a recombinant plasmid containing Drosophila melanogaster DNA cut with Eco RI and Hind Ill were used as molecular weight standards. The locations and size of yeast DNA sequences complementary to yeast poly(A)+ RNA are indicated by thick solid bars and were determined by hybridization of radioactively labeled mRNA or cDNA to plasmid DNA restriction fragments or by electron microscopic observation of R loops formed between poly(A)+ RNA and the plasmid DNA, as described in Results and Experimental Procedures.

DNA suggest that only a fraction of the yeast DNA in pY 1 l-40 shares sequence homology with the yeast DNA in pY 1 O-78. Hybridization of radioactive pY ll40 DNA to restriction fragments of pY 1 O-78 revealed that none of the sequences in pY 1 O-78 to the right of the Barn HI site adjacent to the rp 39 gene are present in pY 1 l-40 (data not shown). The Bgl-Eco RI and Bgl-Barn HI fragments containing the pY 1 O-78 gene do hybridize to pY 11-40 DNA as expected if the two structural gene sequences cross-hybridize. Since there were no restriction sites discovered to the left of the pY 1 O-78 gene, it is not known whether pY 1 l-40 contains any sequences in common with this region.

In any case, the data argue convincingly that there are two independent copies of at least some of the structural information for rp 39. Discussion This paper reports the isolation and preliminary characterization of five cloned ribosomal protein genes from the budding yeast, Saccharomyces cerevisiae. The identification of recombinant DNA genes containing ribosomal protein genes posed a significant problem. Most DNA sequences that have been isolated by recombinant DNA technology have been identified

Cdl 1254

Table 2. Electron Protein Clones

B

Figure 6. Electron Micrographs PolyfA)+ RNA and Recombinant Protein Genes

of R Loops Formed between Yeast Plasmid DNAs Containing Ribosomal

Yeast poly(A)+ RNA was hybridized in slight RNA excess to linearized plasmid DNA to form R loops, incubated with 5.7% glyoxal and spread

Microscopic

Plasmid

Molecules

Mapping

Gene 1

of R Loops

Gene 2

in Ribosomai

Gene 3

Average Size of Each R LOOP (bp)

pY1 O-70

28

28

-

-

432

pY1 l-40

27

27

-

-

270

pYQ-90

29

29

10

-

311.672

pYll-138

33

29

20

11

387,470.352

pY13-86

37

37

-

-

380

using abundant RNAs as hybridization probes (for examples, see Dawid and Wahli, 1979). Certain genes have been isolated using complementation assay8 with bacterial aUXOtrOph8-for example, the HIS-3 or URAS genes of yeast (Struhl, Cameron and Davis, 1976; Bach, Lacroute and Botstein, 1979). and Other8 using immunological procedures (Broome and Gilbert, 1978). Since none of these approaches were applicable with certainty, we chose to identify ribosomal protein gene8 by colony hybridization with radioactive mRNA. A sequence which represent8 0.51% of the total RNA appears to be near the lower limit with which one can reproducibly identify colonie8 by Grunstein-Hogness colony hybridization (P. Gergen et al., unpublished results). To increase the effective concentration of some of the ribosomal protein mRNAs from their usual concentration of approximately 0.1% to this abundant level of approximately l%, we chose to fractionate mRNA on the basis of size and to utilize as a hybridization probe the small mRNA fraction which codes for Small proteins. When used as a probe on our colony bank, this RNA fraction reproducibly reacts with approximately 100 colonies. If we make the reasonable assumption that this small mRNA is enriched for approximately 25% of yeast mRNA, and if this 25% is organized in a manner similar to the entire complement of moderately abundant mRNAs, there would be approximately 400 colonies reproducibly positive with all moderately abundant mRNAs. Since this value is similar to the number of moderately abundant gene8 previously identified by an analysis of RNA-cDNA hybridization kinetics (Hereford and Rosbash, 1977a), these data also suggest that most of these genes are not tightly clustered in the yeast genome. Were there strong clustering of these genes-that is, were most moderately abundant genes directly adjacent to each other-we should have obtained a smaller number of positive colonies. in formamide for microscopy as described in Experimental Procedures. (A) A pY 9-90 DNA molecule, cut at the single Barn HI site in pMB9, containing two R loops: (B) a pY lo-78 DNA molecule, cut at the single Hind Ill site in pMB9. containing one R loop: CC) a pY 1 l138 DNA molecule, cut at the pMB9 Barn HI site, containing two R loops corresponding to the “left-most” and “middle” genes.

Yeast 1255

Ribosomal

Protein

Genes

5

Figure 7. Yeast Genomic Hind III Restriction Fragments Complementary to Recombinant Plasmid DNA Containing Ribosomal Protein Genes Plasmid DNAs and Saccharomyces cerevisiae A364A DNA were digested with Hind Ill. electrophoresed on a 1% agarose gel. transferred to nitrocellulose and hybridized with 10’ cpm of each plasmid DNA radioactively labeled in vitro by nick translation. The left lane in each pair is yeast genomic DNA and the right lane is the plasmid DNA. The labeled probes were (I 1 pY Q-90, (2) pY 1 O-78, (3) pY ll40. (4) pY 1 l-1 36 and (5) pY 13-88.

To examine these candidate clones and to determine which of them contain bona fide ribosomal protein genes, we exploited a method that we had previously designed for this purpose (Woolford and Rosbash, 1979). Of the first 25 candidates analyzed, five ribosomal protein genes were identified. It is of some interest to consider the degree of success achieved by such a screen. 20% (5/25) of the plasmids examined contained ribosomal protein genes. Also, each pool of four plasmids examined (10 x 4 = 40 plasmids examined) contained at least one protein visible on a two-dimensional ribosomal protein gel. These data suggest that there may exist approximately 15 ribosomal protein clone8 in the remaining 75 candidate clones not yet examined individually. Since our original mRNA fractionation enriched for approximately 25 ribosomal protein mRNAs of lower molecular weight, the data suggest that many of these genes would be uncovered in a subsequent analysis of the remaining 75 candidate clones. They also suggest that these gene8 are sufficiently far away from each other (not clustered) so that few of the clones contain more than a single ribosomal protein gene. The small size of the eucaryotic inserts in this library and the

small number of clones examined, however, render this conclusion somewhat uncertain. To verify that each of these recombinant plasmids contain8 significant homology with the identified mRNA8, R loops formed between the plasmid DNAs and mRNA were examined in the electron microscope. Under the conditions of annealing used in these experiments, all abundant and moderately abundant mRNAs should form R loops; even rare mRNAs should form R loops at a perhaps low but measurable frequency (Kaback et al., 1979). The examination of these structures, in combination with the hybridization of radioactive mRNA and cDNA to restriction fragments of the plasmids (data not shown), ha8 provided a fairly accurate map of the structural genes for each of these plasmids (Figure 5 and Table 2). For all five plasmids, the size of the R loops was consistent with the observation that all but one of these genes code for proteins smaller than 25,000 daltons. This degree of homology (300-500 bp), in conjunction with the data from the genome blots discussed below, suggests that at least two of the ribosomal protein gene8 identified in this report are bona fide single-copy genes. The data supporting such a conclusion are presented in Figure 7. Two of the five clones hybridize largely, if not exclusively, to unique restriction fragments. These genome blots also suggest that neither of these cloned yeast segments ha8 undergone sequence rearrangement during cloning or propagation. The data, in sum, suggest that the R loops on these two plasmids are unique and due to single-copy mRNAs which are also responsible for the production of the individual proteins. The other three plasmids are somewhat different. First, clone pY9-90 must contain repetitive DNA in that a large number of genomic restriction fragments are complementary to this clone. The specificity of it8 protein spot (631, however-and its control by RNA2-suggest that the ribosomal protein gene will prove to be single-copy and that the repetitive DNA lies outside the structural gene. This hypothesis will be tested by the analysis of SUbClOD of this plasmid. The other exception is provided by plasmids pY1 l-40 and pYlO-78, both of which code for ribosomal protein 39, a protein previously identified to be on the large ribosomal subunit. Since both plasmids appear to form R loops with the same or very similar mRNAs, it was reasonable to expect that these plasmids would share sequency homology. A hint that this sequence homology might be restricted to a limited fraction of the eucaryotic DNA in these plasmids comes from the restriction maps. The Hind Ill site to the right of the structural gene in pY1 I-40 is absent from either side of the structural gene in pYlO-78. While this result is tempered somewhat by the fact that there is relatively little cloned yeast DNA to the left of the structural gene in pY1 O-78, it was consistent with the view that there are two copies of at least a significant fraction of the structural gene information

Cell 1256

for ribosomal protein 39. This hypothesis is consistent with the genome blots presented in Figure 7 and described above. Results indicating that the rp39 gene is also duplicated in a second strain of yeast, Y55, have recently been obtained (data not shown). These data are interesting in light of the fact that ribosomal protein 39 is one of the few ribosomal proteins not controlled by the RNA2 locus. While no information exists to indicate that both gene copies are complete and/or are transcriptionally active, perhaps these copies differ in their ability to respond to the temperature-sensitive mutation; one copy may be sensitive and the other insensitive to the general effects of RNA2 on ribosomal protein mRNA metabolism. It isresting that this putative “two gene” situation is similar to the description of yeast histone genes H2A and H2B. For this pair of genes, there are two copies of each gene. Only the structural genes are duplicated: the flanking sequence information is unique for both pairs of genes (Hereford et al., 1979). While these data unambiguously identify five genes containing sequence information for ribosomal proteins, they do not answer the question of whether ribosomal protein genes are clustered in yeast. For each of these plasmids there is only one unambiguous ribosomal protein spot on the two-dimensional ribosomal protein gels. Although we do not yet know which R loop or structural gene corresponds to the identified ribosomal protein gene, the single-copy nature of almost all of this sequence information (four of five clones save the duplicated region of plasmids pY 1 l-40 and pY 1 O-78) suggests strongly that one R loop corresponds to the identified ribosomal protein gene and the others correspond to the additional proteins coded for by these plasmids. It is probable that for each plasmid the most frequent R loop corresponds to the identified ribosomal protein mRNA. Therefore many if not all of the additional proteins coded for by these DNAs are probably not ribosomal proteins. The fact that all of the ribosomal proteins are not resolved on these gels, and the fact that all of the proteins coded for by these plasmids have not been unambiguously identified by two-dimensional electrophoresis, however, make this conclusion uncertain. Furthermore, there would appear to be some clustering or organization of small and basic proteins, in that eleven of the proteins coded for by these plasmidsand identified by these procedures-are both small and basic. A more careful examination of these proteins and their assignment to individual subclones is in progress. The general question of the linkage or clustering of ribosomal protein genes in yeast-and in eucaryotes in generalis unanswered. The few antibiotic-resistant mutants that have been mapped are not linked (Skogerson, McLaughlin and Wakamata, 1973, Schindler, Grant and Davies, 1974, Grant, Schindler and Davies, 1976). On the other hand, none of these

have been unambiguously identified as ribosomal protein mutants. Although the DNAs described in this communication give no indication of linked ribosomal protein genes, it should be emphasized that the way in which these clones were identified-through the use of a small mRNA probe-might be responsible for identifying a highly unrepresentative subfraction of the ribosomal protein genes. A region of the yeast genome which contains adjacent ribosomal protein genes, coding for both high molecular weight and low molecular weight ribosomal proteins, might well have been ignored in our screen, since an enrichment for small mRNA would cause a simultaneous loss of large mRNA. This putative region of the genome might then be less positive with a small mRNA probe than with total mRNA, and consequently not be chosen for further analysis. Correspondingly, a region of the genome in which several small mRNAs are tightly clustered would be, and probably was, selected for by the use of a small mRNA probe. To explore this situation more fully, recombinant DNA molecules containing ribosomal protein genes within larger inserts of yeast DNA-and ribosomal protein genes identified, on the basis of criteria other than their complementarity to small mRNA-should be examined. Experiments of this nature are currently in progress. To our knowledge, this is the first report of the isolation of recombinant DNA containing ribosomal protein genes from any eucaryotic organism. Much has been learned about the organization of these genes in E. coli; this organization has proved increasingly important to an understanding of the ways in which these genes are coordinately controlled during ribosome biosynthesis. It is our intention to use these five plasmid DNAs to study the organization of these genes in yeast. We expect that this organization will also prove important for an understanding of the ways in which these eucaryotic genes are coordinately controlled. Experimental

Procedures

Strains and Media Saccharomyces cerevisiae strain A364A (Hartwell et al., 1970) was grown at 23OC in YM-1 liquid media (Hartwell. 1967) supplemented with 25 pg/ml adenine and uracil and 2% glucose. E. coli K12 strain HE101 , containing recombinant DNA molecules, was grown in L media, liquid or solid, containing 10 fig/ml tetracycline. Preparation of Nucleic Acids Poly(A)+ RNA was prepared from logarithmically grown wild-type (A364A) and ma2 (ts368) yeast cells as described previously (Hereford and Rosbash, 1977b). PolyfA)+ RkA enriched for ribosomal protein mRNA was isolated as follows. Polysomes were prepared as described in Hereford and Rosbash (1977a). except that the supernatant from the lysed cells was centrifuged for 5 hr at 27.000 rpm at 4OC in a Beckman SW27 rotor in order to better resolve smaller polysomes from larger polysomes. Fractions containing each were pooled, SDS and EDTA @H 7.9) were added to final concentrations of 0.5% and 50 mM. respectively and the polysomal material was precipitated with 2.5 vol of -2O’C ethanol. RNA was extracted from the precipitate by the method of Kirby (1965). resuspended in 0.1 ml

Yeast Ribosomal 1257

Protein

Genes

of a solution containing 99% DMSO and 0.1 mM Tris (pH 7.5) incubated at 37°C for 5 min, mixed with 0.4 ml SDS buffer [5 mM Tris-HCI (pH 7.5). 1 mM EDTA (pH 7.5). 0.1 M NaCI. 0.5% SDS], heated at 88°C for 1 min, layered on 15-30% (w/v) sucrose gradients in SDS buffer and centrifuged for 18 hr at 39,000 rpm and 22’C in a Beckman SW40 rotor. Fractions were assayed for total RNA by measuring optical density at 280 nm. and for poly(A)+ RNA by hybridization with radioactive poly(U) (Rosbash and Ford, 1974). Fractions containing -5-11 S RNA were pooled, and the RNA was precipitated by the addition of 2.5 vol of -20°C ethanol and NaCl to 0.2 M. The polyfA)+ RNA from this preparation of small RNA from small polysomes was isolated by three cycles of column chromatography on oligddT)-cellulose (T2) (Collaborative Research). The various preparations of RNA were analyzed on 2.5-5.0% acrylamide gels (Spradling. Pardue and Penman, 1977). Yeast DNA was isolated from spheroplasts as described by Hereford and Rosbash (1977a). Recombinant DNA molecules containing the plasmid vector pMB9 with insertions of yeast DNA, constructed by Petes et al. (1978). were purified from Brijdeoxycholate-treated bacterial spheroplasts by CsCI-ethidium bromide density gradient centrlfugation. as described by Petes et al. (1978). Rabbit globin mRNA was purified from reticulocytes by the method of Lockard and RajBhandary (1978). Celf-Free Tranefetion RNA samples were translated in a wheat germ extract (Roberts and Patterson, 1973) containing 35S-methionine (New England Nuclear, -1000 Ci/mmole) or ‘H-lysine (-70 Ci/mmole) and 3H-Ieucine (-70 Ci/mmole) (New England Nuclear). Preparation of Riboeomal Protefne Purified ribosomes and rlbosomal subunits were purified from 800 ml of exponentially growing yeast cells by a modification (Pearson and Haber. 1977) of the procedure of van der Zeijst, Kool and Boemeis (1972). The purified subunits were pooled and ribosomal proteins were prepared from them as described by Warner and Gorenstein (1979). These purified ribosomal proteins, as well as total yeast protein extracted as detailed by Warner and Gorenstein (1977). were used as nonradioactive standards for gels. Gel Electrophoreelo of Proteins Unlabeled protein, and radioactive proteins synthesized in vitro, were analyzed by a number of different gel systems. SDS slab gels containing either 15 or 17% polyacrylamide or a linear 7.5-l 5% or 1 O15% gradient of polyacrylamide stabilized by a 5-25% (w/v) sucrose gradient were run according to the method of Laemmlf (1970). X-ray film exposed to gelscontaining radioactive polypeptides was scanned using a Joyce Loebel microdensitometer. Acid urea Triton gels were run as described by Alfageme et al. (1974). Ribosomal proteins were analyzed on two-dimensional gels run as described by Gorenstein and Warner (1978). Gels containing radioactively labeled proteins were subjected to fluorography at -80°C (Banner and Laskey, 1974) using Kodak XR5 X-ray film. Colony Hybrldfzation Messenger RNA preparations to be used as hybridization probes were radioactively labeled in vitro at 5’ OH termini as described by Maizels (1978). RNA molecules were hydrolyzed for 45 min at 90°C in 0.05 M glycine (pH 9.5) to an average size of 80-100 nucleotides. Y-~*P-ATP synthesized from “P-phosphate (New England Nuclear) and ATP as described by Maxam and Gilbert (1977) was transferred to the 5’ OH termini of the nicked RNA by polynucleotide kinase. a gift from W. McClure. The labeled RNA was ethanol-precipitated twice and chromatographed over oligo(dT)-cellulose to remove the labeled 3’ termini fragment containing poly(A). The flow-through fractions were pooled, ethanol-precipitated and resuspended in buffer for hybridization. By this method, RNA was labeled to specific activities of 5 x font02 x lO’cpm/pg. The colony bank containing yeast DNA was screened with these probes by the colony hybridization protocol of Grunstein and Hogness

(1975). with the modiftcations of J. Lis. L. Prestidge and D. S. Hogness @ersonal communication). The yeast bank contains approximately 2000 colonies (Petes et al., 1978) fixed on sixteen different nitrocellulose filters, to which a total of 1.8-3.2 x IO’ cpm of radioactive RNA was hybridized at 37’C for 18 hr in sealed plastic bags. After hybridization and washing, dried filters were exposed to Kodak XR5 film at -8O’C using DuPont Cronex Lightning Plus intensifier screens. Those colonies containing plasmids containing rDNA sequences were identified by hybridization with purified rRNA (Petes et al., 1978) and were removed from the collection. Reetrictfon Endonucleeee Dlgeetion and Electrophorwle of DNA Restriction endonucleases were purchased from either New England Biolabs or Bethesda Research Laboratories and were used as directed. All digestions were performed at 37°C for l-4 hr at DNA concentrations of 100-400 pgs/ml in 20-100 ~1. When DNA was digested with two different enzymes, the enzyme requiring the lower monovalent cation concentration was used first. The resultant DNA restriction fragments were electrophoresed at 4 V/cm for 18 hr on 0.7-1.0% agarose gels run in 50 mM Trisacetate (pH 8.05). 20 mM NaAc, 2 mM Na*EDTA, 18 mM NaCl (Sharp, Sugden and Sambrook. 1973). Small fragments of DNA (1000 bp to 50 bp in length) were electrophoresed on 4.5% acrylamide gels using 90 mM Tris-borate (pH 8.3). -2.5 mM EDTA (Maniatis, Jeffrey and Van de Sande, 1975). Gels were stained for 30 min with 0.5 f&ml ethidium bromide, destained and photographed with polaroid film using ultraviolet illumination. Radioectlvefy Labeled DNA Plasmid DNA was radioactively labeled in vitro to specific activities of lo’-1 Or’ cpm/Fg using the “nick translation” activity of DNA polymerase I (Rigby et al., 1977) according to the procedure of Maniatis et al. (1975). with modifications as described by Hereford and Rosbash (1977a). Radioactive cDNA complementary to poly(A) RNA was synthesized using a-32P-dCTP (New England Nuclear) and AMV reverse transcriptase, a gifl from J. Beard (Life Sciences Inc.), and was purified as previously described (Hereford and Rosbash, 1977a). Filter Hybridlzation DNA was transferred from agarose or acrylamide gels according to the method of Southern, (1975). except that 20 x SSC was used in the transfer rather than 8 X SSC. When DNAs labeled in vitro by nick translation were hybridized to the DNA transferred to filters, the filters were presoaked for 2 hr at 80°C in Denhardt’s solution (Denhardt, 1988) plus 8 x SSC. The radioactive DNA was denatured by heating in H,O at 100°C for 2 min before hybridizing in Denhardt’s solution, 8 X SSC. 0.5% SDS and 100 Fg/ml denatured salmon sperm DNA for 18 hr at 80°C in a sealed plastic bag. The filters were subsequently washed in the hybridization buffer at 50°C for 45 min, followed by four successive 30 min washes in 2 x SSC at 8OOC. When cDNA was hybridized to the filter-bound DNA, filters were presoaked in 5x Denhardt’s solution, 8 X SSC and 0.1% SDS for 1 hr at 23’C. then in the same solution for 1 hr at 80°C followed by 5x Denhardt’s solution, 8 x SSC, 0.2% SDS and 50 &ml denatured salmon sperm DNA for 3 hr at 80°C. Hybridization was performed in the above buffer for 18 hr at 80°C. The filters were washed twice in the hybridization buffer for 30 min at 80°C. then 4 times in 2 X SSC at 80°C and finally twice in 0.2 x SSC at 8O’C. Filters were exposed to X-ray film, as described for colony hybridization filters. Formation of R Loops and Electron Microscopy For identification of the proteins encoded by particular recombinant DNA molecules by purification of the complementary RNAs and subsequent cell-free translation (Woolford and Rosbash, 1979). the R loops were formed between plasmid DNA hybridized in excess and the complementary yeast mRNAs and were purified away from unhybridized noncomplementary RNA by gel filtration chromatography in high salt buffer. Generally, 3 pg of each DNA, linearized by digestion with a restriction enzyme and phenol-extracted, were hy-

Cell 1258

bridized to 3 ~g of yeast poly(A)+ RNA in 100 fil of buffer containing 0.1 M PIPES (pt-i 6.8). 0.001 M EDTA, 0.4 M NaCl and 70% (v/v) deionized and recrystallized formamide. Hybridizations were performed for 4 hr at linearly decreasing temperatures of 55’-45°C. Subsequent chromatographic isolation of the molecules containing R loops was as described by Woolford and Rosbash (1979). For electron microscopic observations, R loops were formed as described above and in Woolford and Rosbash (1979) except that incubations were carried out for 24 hr at 52°C with 0.1 Fg of restriction enzyme-linearized, phenol-extracted plasmid DNA and 20 Fg of poly(A)+ yeast RNA. By using an equivalent or excess amount of complementary RNA to drive the reaction, and by incubating for longer periods of time, a Rot should be reached sufficient to form R loops between a middle abundant mRNA (-0.1% of total mRNA) and most of the DNA molecules. Following hybridization, the R loops were stabilized by reaction with 0.14 M glyoxal at 12’C for 2 hr as described by Kaback et al. (1979). Subsequently the mixture was chromatographed on 2 ml of Agarose Al 50m equilibrated with 0.6 M NaCI. 0.01 M Tris (pH 7.5) 0.001 M EDTA to purify the R loops away from the majority of the unhybridized RNA. The R loops were then spread in 50% formamide hyperphase solutions onto a 15% formamide hypophase as described by Davis, Simon and Davidson (1971). The grids were stained with 1O-’ M uranyl acetate in ethanol, rotaryshadowed with platinum-palladium (80.20) and viewed with a Phillips 301 electron microscope using circular double-stranded pMB9 DNA (5.4 kb) as a length standard. Biohazard Consld~atlona All plasmids containing yeast or Drosophila DNA segments were propagated under PPEKl containment, in compliance with the NIH Guidelines for Recombinant DNA Research.

We gratefully acknowledge our colleagues L. Golden. C. Vaslet. T. Barnett, P. Gergen, J. Loewenberg. P. Wensink. B. Roberts and N. Pearson for helpful discussions and materials. J.L.W. was supported by Postdoctoral Fellowship and MR. by a Research Career Development Award from the National Institutes of Health. L.M.H. was supported by a New England Medical Foundation Fellowship. This study was supported by two grants from the NIH. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 16 USC. Section 1734 solely to indicate this fact. September

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