Proc. Nati. Acad. Sci. USA Vol. 88, pp. 7026-7030, August 1991 Biochemistry
An approach for isolation of mutants defective in 35S ribosomal RNA synthesis in Saccharomyces cerevisiae (mi mutation/rpal90 mutation/rpal3S mutation/GAL7-35S rDNA fusion/plasmid dependence) YASUHISA NOGI*, LOAN VU, AND MASAYASU NOMURAt Department of Biological Chemistry, University of California at Irvine, Irvine, CA 92717
Contributed by Masayasu Nomura, May 22, 1991
We have developed a method to isolate muABSTRACT tants of Saccharomyces cereviswae that are primarily defective in the transcription of 35S ribosomal RNA (rRNA) genes by RNA polymerase I. The method uses a system in which the 35S rRNA gene is fused to the GAL7 promoter and is transcribed by RNA polymerase II under control of the GAL regulatory system. Chromosomal mutations affecting components specifically involved in synthesis of 35S rRNA by RNA polymerase I can be suppressed by this hybrid gene in the presence of inducer (galactose) but not ii its absence. We looked for mutants the growth of which depended on the presence of plasmid expressing the hybrid gene. For this purpose, we used a red/whitecolony color assay as the initial screen followed by a test for galactose-dependent growth. We have thus isolated many mutants and identified at least nine genes (RRN1-RRN9) involved in 35S rRNA synthesis, two of which correspond to known RNA polymerase I subunit genes RPA190 and RPA135. In eukaryotic cells ribosomal RNA (rRNA) genes are transcribed by a unique RNA polymerase, RNA polymerase I (pol I), and this reaction, together with subsequent processing and assembly into ribosomal subunits, occurs in the nucleolus. In addition to pol I, other components are expected to participate in rRNA gene transcription either directly or indirectly. Studies on transcription of cloned rRNA genes by using cell-free extracts from a variety of organisms have revealed at least two trans-acting protein factors involved in initiating transcription (for reviews, see ref. 1-3). One of the factors, termed upstream binding factor (UBF), was recently purified to homogeneity from human cells (4), and its cDNA has been cloned (5). Nevertheless, in vitro studies alone with the available cell-free systems obviously are inadequate for understanding the mechanism and regulation of rRNA synthesis in vivo. For example, some features of nucleolus structure, which are absent in the cell-free systems, might play important roles for transcription and/or its regulation. Alternatively, transcription factors might be unstable or lost during extract preparation. Furthermore, characterization of functional and regulatory roles of each pol I subunit may not be easy by using only the in vitro systems now available. An obvious approach complementary to the in vitro approach is a genetic one, and we have initiated this approach using the yeast Saccharomyces cerevisiae as an experimental organism. In this organism pol I consists of "43 different polypeptide subunits (for review, see ref. 6). Three of these subunits are known to be shared by all three nuclear RNA polymerases (pol l, pol II, and pol III); two others are shared by pol I and pol III, whereas the remaining ones, including the largest (A190) and the second largest (A135) subunits, are unique to pol I. The genes for large rRNAs (35S rRNA genes
or 35S rRNA-encoding DNA) are tandemly repeated on chromosome XII, and the initial transcripts, 35S rRNA transcripts, undergo a series of posttranscriptional processing steps, leading to the production of 18S, 5.8S, and 25S rRNAs (for reviews, see refs. 7 and 8). In our genetic studies, we first carried out in vitro mutagenesis of the RPAJ90 gene encoding the A190 subunit (9) and isolated and characterized several temperature-sensitive mutants that have alterations in this subunit (10). We then isolated and studied extragenic suppressors of these temperature-sensitive mutants (11); one of them was identified as the gene (RPAJ35) for the A135 subunit (12), and another was identified as the gene for a non-pol I protein that is apparently required for structural integrity of the nucleolus (R. Yano, M. Oakes, and M.N., unpublished work). This type of analysis is one way to isolate mutants with alterations in subunit polypeptides of pol I and other components interacting with pol I. Another way to isolate mutants is to mutagenize yeast cells and screen for the desired phenotype-i.e., defects in the synthesis of rRNA in this case. However, simple screening of temperaturesensitive mutants by assaying RNA and protein accumulation at nonpermissive temperatures, which was done in the past (13), did not yield any desired mutants; most mutants originally so isolated were defective in splicing ribosomal protein mRNA, thus indirectly affecting rRNA accumulation (ref. 14; for a review, see ref. 15). In this paper, we describe a method to screen for mutants primarily defective in rRNA synthesis. The method uses a system in which the 35S rRNA gene is fused to the GAL7 promoter (called GAL7-35S rDNA) and is transcribed by pol H under control of the GAL regulatory system (16). This hybrid gene, when present on a multicopy plasmid and induced by galactose, was shown to suppress the growth defects of a temperature-sensitive rpal9O mutant and those of a mutant in which the RPAJ35 gene was deleted (16). Thus, -lethal mutations that can be suppressed by this hybrid gene in the presence of inducer (galactose), but not in its absence, most likely affect components specifically involved in 35S rRNA synthesis. Mutants carrying such lethal mutations must maintain the plasmid carrying the GAL7-35S rDNA hybrid gene for growth and can be detected by using a red/white-colony sectoring assay (17-19) as the initial screen, followed by a test for galactose-dependent growth. Usingthis approach, we have isolated many mutants and identified at least seven additional genes involved in rRNA
synthesis.
MATERIALS AND METHODS Strains, Media, and Genetic Methods. Yeast strains used are listed in Table 1. Construction and structure of plasmid Abbreviations: pol I, 1I, and III, RNA polymerase I, II, and III, respectively; rRNA, ribosomal RNA. *Present address: Laboratory of Molecular Genetics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7026
Biochemistry: Nogi et al. Table 1. Yeast Designation Strain CH1305 NOY418 NOY396 NOY421
Proc. Natl. Acad. Sci. USA 88 (1991)
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strains and plasmids Description
MATa ade2 ade3 Ieu2 ura3 lys2 can1* Strain CH1305 carrying pNOY103 MATa ade2-1 ura3-1 his3-11 trpl-) leu2-3,112 can) MATa ade2 ade3 leu2 ura3 trpl his can) (constructed by crossing strain CH1305 with NOY396) MATa rpal90-3 ade2 leu2 ura3 trpl his can) MATa rpa)35::LEU2 ade2-1 ura3-1 his3-11 trpl-) leu2-3,112 can) pNOY102t
NOY422 NOY446 Plasmid pNOY102 pNOY103
High-copy-number plasmid carrying GAL7-35S rDNA, URA3, 2Mu ampt High-copy-number plasmid carrying GAL7-35S rDNA, ADE3, URA3, 2,u, amp (see Fig. 1)
*Ref 19.
tFormerly called NOY408-la, see ref. 16.
tRef. 16.
pNOY103 are described in Fig. 1. YEP-glucose medium is 1% Bacto yeast extract (Difco)/2% Bacto peptone (Difco)/2% glucose. YEP-galactose medium is the same, except 2% galactose is substituted for glucose. Synthetic galactose medium is 2% galactose/0.67% Bacto-yeast nitrogen-base (Difco), which, when indicated, was supplemented with amino acids or bases as described by Sherman et al. (21). The concentration of Casamino acids used as a supplement was 0.5%. Synthetic glucose medium is the same as synthetic galactose medium, except that 2% glucose is substituted for galactose. For making solid medium, 2% agar was added. Isolation of Mutants Defective in 35S rRNA Synthesis. To use a colony-sectoring assay, we used NOY418 as a starting parental strain; this strain is identical to strain CH1305 (19), which is ade2 ade3, but carries plasmid pNOY103. It is known that ade2 yeast cells accumulate a red pigment but ade2 ade3 cells do not. Because pNOY103 carries ADE3, strain NOY418 forms red colonies as long as the strain maintains the plasmid by selection. Without selection, strain NOY418 tends to lose the plasmid and forms colonies with white sectors or nearly completely white colonies. Strain NOY418 was grown overnight in synthetic glucose medium (supplemented with Casamino acids and adenine but 2 t ---amp a
URA3
XI/S
GALU7-T E
pNOY1 03 B
E3
ADE
25S
GAL7-P
5.8S
1 8S
FIG. 1. Structure of plasmid pNOY103. pNOY103 was constructed from pNOY102 (16) by first converting the unique Sma I site of pNOY102 to a Xho I (X) site by using a Xho I linker (CCTCGAGG) and then inserting the 5-kb BamHI (B)-Sal I (S) fragment carrying ADE3 (derived from pDK255; see ref. 19) between the BamHI and Xho I sites of the resultant pNOY102 derivative. The two fusion sites (B and X/S) created in this step are indicated. The GAL7-35S rDNA hybrid gene consists of 35S rDNA (from +1 to +6922 with respect to transcription start site; this includes 18S, 5.8S, and 25S rRNAcoding regions and the enhancer element (20) (E, shown as black boxes) and the GAL7 promoter and terminator (GAL7-P and GAL7-T; shown as grey boxes). DNA derived from pBR322 carrying the B-lactamase gene (amp) is shown as a line. Other yeast elements, including the 2-Am plasmid origin of replication (2A), are also shown.
without any uracil addition) to a cell density (OD6N) of U5 (=1 x 108 cells per ml). Cells were centrifuged, washed with 0.1 M sodium phosphate buffer, pH 7.0, and resuspended in the same buffer to give a cell density (A6N) of -13. Fifty microliters of ethylmethane sulfonate was added to 1.7 ml of the cell suspension and incubated at 30'C for 1 hr with shaking. Ten milliliters of 5% thiosulfate was then added to 0.1 ml of the mutagenized cell suspension, and the mixture was left for -40 min at room temperature. Cells were plated on YEP-galactose to give -200 colonies per plate. (The ethylmethane sulfonate treatment under the conditions described gave 40%o survivors.) Plates were first incubated overnight at room temperature and then at 360C for an additional 10-12 days. Potential nonsectoring red colonies on YEP-galactose were picked, restreaked on YEP-galactose plates, and incubated at 360C for 4-5 days. Colonies showing the same nonsectoring red-color phenotype were then streaked on two plates, YEP-galactose and YEP-glucose plates, and incubated at 360C for 6-7 days. Mutants that failed to grow on YEP-glucose but formed nonsectoring red colonies on YEP-galactose were kept, after single colony purification, as candidate mutants defective in 35S rRNA synthesis. These mutants were once more streaked on YEPgalactose and YEP-glucose plates, and their growth on these plates was examined at 360C and 250C to find whether these mutations that require the plasmid-encoded GAL7-35S rDNA hybrid gene for growth are or are not temperaturesensitive mutations. Dominance-Recessive Test and Complementation Test. Mutants isolated from strain NOY418 were initially crossed with "wild-type" strain NOY421 and two pol I mutant strains, one (NOY422) carrying the temperature-sensitive mutation rpa)90-3 and the other (NOY446) carrying the deletion mutation rpa)35::LEU2 (complemented by GAL7-35S rDNA on a plasmid). Mating was done in YEP-galactose liquid medium at 30'C (or at 250C when a temperature-sensitive mutant, such as NOY422, was involved). Diploids were then selected on supplemented minimal galactose plates with appropriate selectable markers (lys2 and trp) in this case), and their growth on glucose plates was examined at 360C. Because the temperature-sensitive rpaJ90-3 allele is somewhat leaky at 36WC, complementation tests with the strain carrying rpaJ90-3 were usually done at 380C. Mutants that complemented both rpaJ90-3 and rpa135::LEU2 were further analyzed to define new complementation groups. For this purpose, the opposite mating type (MA Ta) and suitable genetic markers (trp) L YS2) were introduced into appropriate mutants by crossing them with NOY421, and these a-mating-type mutant strains were crossed with original mutant strains. The ability of diploids to grow on glucose medium at 360C was then examined to find
7028
Biochemistry: Nogi et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
whether two mutations in question belong to the same complementation group. Analysis of RNA Labeled in Vivo. Cells were grown in synthetic galactose medium supplemented with Casamino acids, tryptophan, and adenine at 36°C to a cell density (OD6w) of =0.2. Each culture was divided into two parts (usually 8 ml for each). Glucose was added to one culture to a final concentration of 2%, and the other culture served as control. After culture incubation at 36°C for 1 hr, [3H]uridine (174 mCi/mg, 100 ,uCi/ml; 1 Ci = 37 GBq) was added, and after 30-min incubation the labeling was stopped by immersing the culture flasks into a dry ice/ethanol bath. RNA was isolated and analyzed by electrophoresis on a 2% polyacrylamide/0.5% agarose composite gel as described (16, 22, 23).
independent mutants that require plasmid pNOY103 as well as inducer galactose for growth at 36°C. As described below, these mutants are defective in rRNA synthesis in glucose and require rRNA synthesis from the GAL7-35S rDNA hybrid We call these mutants rrn (rRNA-synthesis defective). As described, growth on glucose was tested both at 36°C and 25°C. Several mutants grew on glucose at 250C but not at 36°C, indicating that they carry temperaturesensitive mutations that affect 35S rRNA synthesis by pol I at 36°C but not at 250C. Complementation Tests of rrn Mutants. Each of the 24 rrn mutants isolated from strain NOY418 was first crossed with wild-type strain NOY421. In every case, diploids grew on glucose at 36°C, indicating that the mutations are recessive. Mutants were then classified into complementation groups. For this purpose, we used two a-mating-type strains, NOY422 and NOY446, each carrying known pol I-specific mutations, rpal9O-3 and rpal35::LEU2, respectively, to find whether any of the new mutations are in these two genes, which encode pol I subunits. In addition, strains with the opposite mating type (a) carrying rrn mutations and other markers convenient for selection were constructed. Mutants were crossed with these a-mating-type strains, and resultant diploids were examined for their ability to grow on glucose medium at 36°C. Results of these complementation tests are summarized in Table 2. Altogether, nine complementation groups were clearly demonstrated; one of them corresponds to rpal90, and another corresponds to rpal35. The other complementation groups, RRN3-RRN9, are defined by rrn mutations carried by seven mutants, 74, 225, 242, 195, 801, 784, and 1100. It should be noted that these mutants were all crossed with wild-type NOY421, and the RRN:rrn pattern of segregation was 2:2, indicating that each mutant bears a mutation in a single gene. [In these tetrad analyses, spore germination was done at 25°C for temperature-sensitive rrn mutations, and the recovery of two rrn segregants was very high. However, the recovery of rrn haploid segregants was not high for other mutants, presumably because of nonuniform distribution of pNOY103 and requirement of the plasmid in high-copy number for growth of rrn mutants (see ref. 16). Nevertheless, results of the tetrad analysis clearly supported the conclusion of a single mutation for these mutants. We also note that some mutants (944 and 837) appear to gene for growth.
RESULTS A System for Isolation of Mutants Defective in rRNA Synthesis. As described, both rpal90 and rpal35 mutations, which cause specific defects in transcription of 35S rRNA genes, can be suppressed by the GAL7-35S rDNA hybrid gene on a multicopy plasmid in the presence of galactose. Thus, starting with a strain carrying such a plasmid, a screen for mutants specifically defective in 35S rRNA synthesis can be devised to identify those cells able to grow on galactose medium but not on glucose medium. Such mutants must maintain the plasmid carrying the GAL7-35S rDNA hybrid gene for growth. We, therefore, used a red/white-colony color-sectoring assay (17-19) as the initial screen followed by a test for galactose-dependent growth. As a starting strain, we used NOY418, which is ade2 ade3 and carries ADE3 in addition to the GAL7-35S rDNA gene on a 2I.m-based multicopy plasmid. It is known that yeast strains carrying ade2 form red colonies from accumulation of red pigment, but ade2 ade3 strains form white colonies. Thus, NOY418 forms red colonies when grown on medium that selects for the plasmid (synthetic plates without uracil) but forms red/ white-sectored or almost totally white colonies on YEPgalactose plates because the 2,um-based plasmid, pNOY103, is unstable without selection. After ethylmethane sulfonate mutagenesis of NOY418, we looked for nonsectoring red colonies on YEP-galactose plates and then tested growth of these colonies on glucose medium. We thus isolated 24 Table 2. Classification of rrn mutants by complementation tests
Independent mutants
Representative Gene RRNI (RPA190) RRN2 (RPA13S) RRN3 RRN4 RRNS RRN6 RRN7 RRN8 RRN9
Othert
mutant
395 193
Strains used for complementation test to define RRN genes* NOY422 NOY446 (rpal90) (rpaI35) 74cr 225a 242cr 195ac 801a 784a 1100ar + + + + + + + + + + + + + + + + -
74
+
+
225(ts)
+ +
+ +
+
+
+ + + +
+ + + +
242 195
801(ts) 784(ts) 1i00 944(ts)
+
-
+ + + + + +
+ + + + + +
+
+ +
+ + + + +
+ + +
+ + + +
+ + + +
+ + +
+ + + + +
+
+
+ + + + + +
+
isolated, no. ts
0 3
Non-ts 1 2
Total 1 5
1
3
4
2
0
2
0 0
3 2
3
1 2
0 1
1 3
0
1
1
2 0 (837, 944)
2
2
ts, temperature-sensitive. *74a carries the original rrn3 mutation in mutant 74 but carries MA Ta trpl LYS2 and was obtained as a haploid segregant after crossing 74 with NOY421. Strains 225cr, 242a, 195a, 801a, 784a, and 1100a were constructed in a similar way, carry MATa, and were used for complementation analyses. tStrains 944 and 837 did not show clean 2:2 (RRN:rrn) segregation in tetrad analysis in the cross with strain NOY421 (RRN) and, therefore, may carry more than one mutation. However, all mutants in other complementation groups (RRNl-RRN9) tested showed complementation with 944, indicating the mutation(s) in 944 contributing to the rrn phenotype is unique and, thus, defining at least one complementation group. Complementation tests with 837 showed some ambiguous results, and characterization of mutation(s) in 837 needs further studies.
Biochemistry: Nogi et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
contain more than one mutation and were left unclassified (see the legend for Table 2)]. Isolation of mutants defective in each of the two known pol I subunit genes was expected and justifies our strategy for isolating mutants specifically defective in 35S rRNA synthesis. Direct Demonstration of Defects in 35S rRNA Synthesis in rrn Mutants. The phenotype of rrn mutants described above indicates that these mutants are defective in synthesis of 35S rRNA from the chromosomal rRNA genes, and their growth depends on transcription of the plasmid-encoded GAL7-35S rDNA hybrid gene by pol II. By [3H]uridine pulse-labeling experiments, we confirmed this conclusion directly. Mutant strains representing each of seven new RRN complementation groups (RRN3-RRN9) were grown in synthetic galactose medium, and [3H]uridine pulse-labeling experiments were done with and without repression of the GAL7 promoter by glucose (24, 25), as was done in our previous study (16). As shown in Fig. 2, except for mutant 784 (rrn8), synthesis of large rRNA (18S, 25S, and 5.8S rRNAs and other precursor rRNAs) was strongly inhibited by glucose, relative to synthesis of 5S and tRNAs for every mutant analyzed. Thus, the mutations in each of these additional genes (except RRN8) preferentially affect synthesis of 35S rRNA. For mutant 784, total incorporation of [3H]uridine into RNA in the presence of glucose was 3- to 4-fold less than that in the control without glucose, and the amounts of radioactive large rRNAs accumulated relative to 5S RNA and tRNA were similar to those seen without glucose (Fig. 2, lanes 13 and 14). Therefore, the addition of glucose apparently inhibits not only the synthesis of large rRNA, but also that of SS RNA and tRNA. Although we cannot explain the unexpected results for this mutant 1 2 3 4 5 6 25S rRNA18S rRNA-
im-- i; 0!.. _
WW
7029
without further experiments, we retain this and other mutants that belong to the same complementation group as rrn8 mutants.
DISCUSSION AND CONCLUSION The method described here has proven very effective for isolating mutants defective in transcription of 35S rRNA genes by pol I and, thus, identifying components involved in this process. Of 24 independent mutants studied, 22 mutants were unambiguously classified into nine complementation groups, two of which correspond to known pol I subunit genes, RPA190 and RPA135, respectively. The remaining seven complementation groups define additional genes and, except for RRN8, their essential role for 35S rRNA transcription was confirmed directly by [3H]uridine-pulse labeling experiments. The two unclassified mutants (837, 944) appeared to have more than one mutation. At least one of the mutation(s) carried by 944 appears distinct from the above nine complementation groups (see the legend for Table 2). Thus, the number of RRN genes found may be 10 or more. In addition, from the distribution of mutants into nine complementation groups, the mutagenesis of RRN genes clearly has not been saturated. We note in this connection that mutations detected by the current method include completely lethal mutations as well as conditionally lethal mutations in RRN genes and, hence, one could hope to approach identification of all possible RRN genes. Among 24 independent mutants isolated, five had rpal35 mutations, whereas only one mutant had a rpal90 mutation, even though the RPA190 gene is larger than RPA135. Why 7 8 9 10 11 12
am
. W _ "
13 14 15 16
I'
6
'~~~~~~~~
~~~'
5.8S rRNA 5SRNAtRNA-
lU b
S
_
_
FIG. 2. Polyacrylamide/agarose gel electrophoresis of RNA synthesized in various rrn mutants and the parent strain growing in galactose medium with (even-numbered lanes) and without (odd-numbered lanes) further glucose addition. Parent strain NOY418 (lanes 1 and 2) and rrn mutants 416 (rrn3; lanes 11 and 12), 225 (rrn4; lanes 9 and 10), 242 (rrn5; lanes 7 and 8), 793 (rrn6; lanes 5 and 6), 801 (rrn7; lanes 3 and 4), 784 (rrn8; lanes 13 and 14), and 1100 (rrn9; lanes 15 and 16) were grown at 36°C (or at 30°C for 416 and 242) in galactose medium. At a cell density (A6w) of -0.2, cultures were divided into two parts, and glucose (final concentration 2%) was added to one part; the other served as control. At 1 hr after glucose addition, cells were pulse-labeled with [3H]uridine for 30 min (or 60 min for 784). RNA was isolated from each culture, and samples containing -7 X 104 cpm of [3H]RNA were analyzed by electrophoresis on a polyacrylamide/agarose composite gel. Autoradiograms of the dried gels are shown. Results from three separate experiments appear in lanes 1-6, lanes 7-12, and lanes 13-16, respectively. We note that autoradiograms obtained after longer gel exposures indicated that 5.8S rRNA synthesis in all rrn mutants (except 784) was inhibited by glucose, as was that of 18S and 25S rRNAs.
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Biochemistry: Nogi et al.
many more mutations were detected in the second largest subunit, the A135 subunit, is unclear. Both RPAJ90 and RPA135 are essential genes, but RPA135 may have more essential sites sensitive to mutagenesis; our initial color screening during mutant isolation tends to discard leaky mutants that could grow slowly without the GAL7-35S rDNA hybrid gene, and many point mutations induced by ethylmethane sulfonate in the RPAJ90 gene might be this type of mutation in contrast to those induced in the RPA135 gene. As mentioned earlier, S. cerevisiae pol I appears to consist of -13 polypeptide subunits, but five of them are shared by the other RNA polymerase(s), and, therefore, we do not formally expect to detect mutations in these five subunits by using the present mutant isolation method (for qualification of this statement, see below). In addition, some polypeptides detected in purified pol I preparations have not been rigorously proven as genuine components and are possibly partially or completely dispensable for 35S rRNA synthesis. Because we have found at least 9 RRN genes (including RPA190 and RPA135), the newly identified genes probably include those coding for proteins different from known pol I subunits. Although this mutant-isolation method is designed to isolate mutants with defects in components specifically involved in transcription of 35S rRNA genes, the proteins encoded by some of these RRN genes just identified may participate not only in transcription of 35S rRNA genes by pol I but also in other cellular functions, including pol II and pol III functions; the mutations may abolish the pol I function completely but may affect the other functions only partially without causing lethality; thus, the mutants can be rescued by transcription of the GAL7-35S rDNA gene by pol II in the presence of galactose. In preliminary experiments, we observed that some rrn mutants isolated (e.g., mutant 784) grow in galactose medium more slowly than mutants carrying rpal90 or rpaJ35 deletions (e.g., strain NOY446), suggesting that the possibility considered here may, in fact, be the case. In any event, cloning and analysis of the RRN genes defined in this work as well as in vitro transcription studies of 35S rRNA genes (see refs. 26-28) with extracts from these rrn mutants should make a significant contribution to studies of the mechanism of rRNA synthesis and its regulation in this model
eukaryotic organism. We thank Drs. C. Holmes and L. H. Hartwell for supplying yeast strains and plasmids and Drs. L. E. McAlister-Henn, J. Keener, and T. Menees for comments on the manuscript. This work was sup-
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An approach for isolation of mutants defective in 35S ribosomal RNA synthesis in Saccharomyces cerevisiae (mi mutation/rpal90 mutation/rpal3S mutation/GAL7-35S rDNA fusion/plasmid dependence) YASUHISA NOGI*, LOAN VU, AND MASAYASU NOMURAt Department of Biological Chemistry, University of California at Irvine, Irvine, CA 92717
Contributed by Masayasu Nomura, May 22, 1991
We have developed a method to isolate muABSTRACT tants of Saccharomyces cereviswae that are primarily defective in the transcription of 35S ribosomal RNA (rRNA) genes by RNA polymerase I. The method uses a system in which the 35S rRNA gene is fused to the GAL7 promoter and is transcribed by RNA polymerase II under control of the GAL regulatory system. Chromosomal mutations affecting components specifically involved in synthesis of 35S rRNA by RNA polymerase I can be suppressed by this hybrid gene in the presence of inducer (galactose) but not ii its absence. We looked for mutants the growth of which depended on the presence of plasmid expressing the hybrid gene. For this purpose, we used a red/whitecolony color assay as the initial screen followed by a test for galactose-dependent growth. We have thus isolated many mutants and identified at least nine genes (RRN1-RRN9) involved in 35S rRNA synthesis, two of which correspond to known RNA polymerase I subunit genes RPA190 and RPA135. In eukaryotic cells ribosomal RNA (rRNA) genes are transcribed by a unique RNA polymerase, RNA polymerase I (pol I), and this reaction, together with subsequent processing and assembly into ribosomal subunits, occurs in the nucleolus. In addition to pol I, other components are expected to participate in rRNA gene transcription either directly or indirectly. Studies on transcription of cloned rRNA genes by using cell-free extracts from a variety of organisms have revealed at least two trans-acting protein factors involved in initiating transcription (for reviews, see ref. 1-3). One of the factors, termed upstream binding factor (UBF), was recently purified to homogeneity from human cells (4), and its cDNA has been cloned (5). Nevertheless, in vitro studies alone with the available cell-free systems obviously are inadequate for understanding the mechanism and regulation of rRNA synthesis in vivo. For example, some features of nucleolus structure, which are absent in the cell-free systems, might play important roles for transcription and/or its regulation. Alternatively, transcription factors might be unstable or lost during extract preparation. Furthermore, characterization of functional and regulatory roles of each pol I subunit may not be easy by using only the in vitro systems now available. An obvious approach complementary to the in vitro approach is a genetic one, and we have initiated this approach using the yeast Saccharomyces cerevisiae as an experimental organism. In this organism pol I consists of "43 different polypeptide subunits (for review, see ref. 6). Three of these subunits are known to be shared by all three nuclear RNA polymerases (pol l, pol II, and pol III); two others are shared by pol I and pol III, whereas the remaining ones, including the largest (A190) and the second largest (A135) subunits, are unique to pol I. The genes for large rRNAs (35S rRNA genes
or 35S rRNA-encoding DNA) are tandemly repeated on chromosome XII, and the initial transcripts, 35S rRNA transcripts, undergo a series of posttranscriptional processing steps, leading to the production of 18S, 5.8S, and 25S rRNAs (for reviews, see refs. 7 and 8). In our genetic studies, we first carried out in vitro mutagenesis of the RPAJ90 gene encoding the A190 subunit (9) and isolated and characterized several temperature-sensitive mutants that have alterations in this subunit (10). We then isolated and studied extragenic suppressors of these temperature-sensitive mutants (11); one of them was identified as the gene (RPAJ35) for the A135 subunit (12), and another was identified as the gene for a non-pol I protein that is apparently required for structural integrity of the nucleolus (R. Yano, M. Oakes, and M.N., unpublished work). This type of analysis is one way to isolate mutants with alterations in subunit polypeptides of pol I and other components interacting with pol I. Another way to isolate mutants is to mutagenize yeast cells and screen for the desired phenotype-i.e., defects in the synthesis of rRNA in this case. However, simple screening of temperaturesensitive mutants by assaying RNA and protein accumulation at nonpermissive temperatures, which was done in the past (13), did not yield any desired mutants; most mutants originally so isolated were defective in splicing ribosomal protein mRNA, thus indirectly affecting rRNA accumulation (ref. 14; for a review, see ref. 15). In this paper, we describe a method to screen for mutants primarily defective in rRNA synthesis. The method uses a system in which the 35S rRNA gene is fused to the GAL7 promoter (called GAL7-35S rDNA) and is transcribed by pol H under control of the GAL regulatory system (16). This hybrid gene, when present on a multicopy plasmid and induced by galactose, was shown to suppress the growth defects of a temperature-sensitive rpal9O mutant and those of a mutant in which the RPAJ35 gene was deleted (16). Thus, -lethal mutations that can be suppressed by this hybrid gene in the presence of inducer (galactose), but not in its absence, most likely affect components specifically involved in 35S rRNA synthesis. Mutants carrying such lethal mutations must maintain the plasmid carrying the GAL7-35S rDNA hybrid gene for growth and can be detected by using a red/white-colony sectoring assay (17-19) as the initial screen, followed by a test for galactose-dependent growth. Usingthis approach, we have isolated many mutants and identified at least seven additional genes involved in rRNA
synthesis.
MATERIALS AND METHODS Strains, Media, and Genetic Methods. Yeast strains used are listed in Table 1. Construction and structure of plasmid Abbreviations: pol I, 1I, and III, RNA polymerase I, II, and III, respectively; rRNA, ribosomal RNA. *Present address: Laboratory of Molecular Genetics, Keio University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160, Japan. tTo whom reprint requests should be addressed.
The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. 7026
Biochemistry: Nogi et al. Table 1. Yeast Designation Strain CH1305 NOY418 NOY396 NOY421
Proc. Natl. Acad. Sci. USA 88 (1991)
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strains and plasmids Description
MATa ade2 ade3 Ieu2 ura3 lys2 can1* Strain CH1305 carrying pNOY103 MATa ade2-1 ura3-1 his3-11 trpl-) leu2-3,112 can) MATa ade2 ade3 leu2 ura3 trpl his can) (constructed by crossing strain CH1305 with NOY396) MATa rpal90-3 ade2 leu2 ura3 trpl his can) MATa rpa)35::LEU2 ade2-1 ura3-1 his3-11 trpl-) leu2-3,112 can) pNOY102t
NOY422 NOY446 Plasmid pNOY102 pNOY103
High-copy-number plasmid carrying GAL7-35S rDNA, URA3, 2Mu ampt High-copy-number plasmid carrying GAL7-35S rDNA, ADE3, URA3, 2,u, amp (see Fig. 1)
*Ref 19.
tFormerly called NOY408-la, see ref. 16.
tRef. 16.
pNOY103 are described in Fig. 1. YEP-glucose medium is 1% Bacto yeast extract (Difco)/2% Bacto peptone (Difco)/2% glucose. YEP-galactose medium is the same, except 2% galactose is substituted for glucose. Synthetic galactose medium is 2% galactose/0.67% Bacto-yeast nitrogen-base (Difco), which, when indicated, was supplemented with amino acids or bases as described by Sherman et al. (21). The concentration of Casamino acids used as a supplement was 0.5%. Synthetic glucose medium is the same as synthetic galactose medium, except that 2% glucose is substituted for galactose. For making solid medium, 2% agar was added. Isolation of Mutants Defective in 35S rRNA Synthesis. To use a colony-sectoring assay, we used NOY418 as a starting parental strain; this strain is identical to strain CH1305 (19), which is ade2 ade3, but carries plasmid pNOY103. It is known that ade2 yeast cells accumulate a red pigment but ade2 ade3 cells do not. Because pNOY103 carries ADE3, strain NOY418 forms red colonies as long as the strain maintains the plasmid by selection. Without selection, strain NOY418 tends to lose the plasmid and forms colonies with white sectors or nearly completely white colonies. Strain NOY418 was grown overnight in synthetic glucose medium (supplemented with Casamino acids and adenine but 2 t ---amp a
URA3
XI/S
GALU7-T E
pNOY1 03 B
E3
ADE
25S
GAL7-P
5.8S
1 8S
FIG. 1. Structure of plasmid pNOY103. pNOY103 was constructed from pNOY102 (16) by first converting the unique Sma I site of pNOY102 to a Xho I (X) site by using a Xho I linker (CCTCGAGG) and then inserting the 5-kb BamHI (B)-Sal I (S) fragment carrying ADE3 (derived from pDK255; see ref. 19) between the BamHI and Xho I sites of the resultant pNOY102 derivative. The two fusion sites (B and X/S) created in this step are indicated. The GAL7-35S rDNA hybrid gene consists of 35S rDNA (from +1 to +6922 with respect to transcription start site; this includes 18S, 5.8S, and 25S rRNAcoding regions and the enhancer element (20) (E, shown as black boxes) and the GAL7 promoter and terminator (GAL7-P and GAL7-T; shown as grey boxes). DNA derived from pBR322 carrying the B-lactamase gene (amp) is shown as a line. Other yeast elements, including the 2-Am plasmid origin of replication (2A), are also shown.
without any uracil addition) to a cell density (OD6N) of U5 (=1 x 108 cells per ml). Cells were centrifuged, washed with 0.1 M sodium phosphate buffer, pH 7.0, and resuspended in the same buffer to give a cell density (A6N) of -13. Fifty microliters of ethylmethane sulfonate was added to 1.7 ml of the cell suspension and incubated at 30'C for 1 hr with shaking. Ten milliliters of 5% thiosulfate was then added to 0.1 ml of the mutagenized cell suspension, and the mixture was left for -40 min at room temperature. Cells were plated on YEP-galactose to give -200 colonies per plate. (The ethylmethane sulfonate treatment under the conditions described gave 40%o survivors.) Plates were first incubated overnight at room temperature and then at 360C for an additional 10-12 days. Potential nonsectoring red colonies on YEP-galactose were picked, restreaked on YEP-galactose plates, and incubated at 360C for 4-5 days. Colonies showing the same nonsectoring red-color phenotype were then streaked on two plates, YEP-galactose and YEP-glucose plates, and incubated at 360C for 6-7 days. Mutants that failed to grow on YEP-glucose but formed nonsectoring red colonies on YEP-galactose were kept, after single colony purification, as candidate mutants defective in 35S rRNA synthesis. These mutants were once more streaked on YEPgalactose and YEP-glucose plates, and their growth on these plates was examined at 360C and 250C to find whether these mutations that require the plasmid-encoded GAL7-35S rDNA hybrid gene for growth are or are not temperaturesensitive mutations. Dominance-Recessive Test and Complementation Test. Mutants isolated from strain NOY418 were initially crossed with "wild-type" strain NOY421 and two pol I mutant strains, one (NOY422) carrying the temperature-sensitive mutation rpa)90-3 and the other (NOY446) carrying the deletion mutation rpa)35::LEU2 (complemented by GAL7-35S rDNA on a plasmid). Mating was done in YEP-galactose liquid medium at 30'C (or at 250C when a temperature-sensitive mutant, such as NOY422, was involved). Diploids were then selected on supplemented minimal galactose plates with appropriate selectable markers (lys2 and trp) in this case), and their growth on glucose plates was examined at 360C. Because the temperature-sensitive rpaJ90-3 allele is somewhat leaky at 36WC, complementation tests with the strain carrying rpaJ90-3 were usually done at 380C. Mutants that complemented both rpaJ90-3 and rpa135::LEU2 were further analyzed to define new complementation groups. For this purpose, the opposite mating type (MA Ta) and suitable genetic markers (trp) L YS2) were introduced into appropriate mutants by crossing them with NOY421, and these a-mating-type mutant strains were crossed with original mutant strains. The ability of diploids to grow on glucose medium at 360C was then examined to find
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Proc. Natl. Acad. Sci. USA 88 (1991)
whether two mutations in question belong to the same complementation group. Analysis of RNA Labeled in Vivo. Cells were grown in synthetic galactose medium supplemented with Casamino acids, tryptophan, and adenine at 36°C to a cell density (OD6w) of =0.2. Each culture was divided into two parts (usually 8 ml for each). Glucose was added to one culture to a final concentration of 2%, and the other culture served as control. After culture incubation at 36°C for 1 hr, [3H]uridine (174 mCi/mg, 100 ,uCi/ml; 1 Ci = 37 GBq) was added, and after 30-min incubation the labeling was stopped by immersing the culture flasks into a dry ice/ethanol bath. RNA was isolated and analyzed by electrophoresis on a 2% polyacrylamide/0.5% agarose composite gel as described (16, 22, 23).
independent mutants that require plasmid pNOY103 as well as inducer galactose for growth at 36°C. As described below, these mutants are defective in rRNA synthesis in glucose and require rRNA synthesis from the GAL7-35S rDNA hybrid We call these mutants rrn (rRNA-synthesis defective). As described, growth on glucose was tested both at 36°C and 25°C. Several mutants grew on glucose at 250C but not at 36°C, indicating that they carry temperaturesensitive mutations that affect 35S rRNA synthesis by pol I at 36°C but not at 250C. Complementation Tests of rrn Mutants. Each of the 24 rrn mutants isolated from strain NOY418 was first crossed with wild-type strain NOY421. In every case, diploids grew on glucose at 36°C, indicating that the mutations are recessive. Mutants were then classified into complementation groups. For this purpose, we used two a-mating-type strains, NOY422 and NOY446, each carrying known pol I-specific mutations, rpal9O-3 and rpal35::LEU2, respectively, to find whether any of the new mutations are in these two genes, which encode pol I subunits. In addition, strains with the opposite mating type (a) carrying rrn mutations and other markers convenient for selection were constructed. Mutants were crossed with these a-mating-type strains, and resultant diploids were examined for their ability to grow on glucose medium at 36°C. Results of these complementation tests are summarized in Table 2. Altogether, nine complementation groups were clearly demonstrated; one of them corresponds to rpal90, and another corresponds to rpal35. The other complementation groups, RRN3-RRN9, are defined by rrn mutations carried by seven mutants, 74, 225, 242, 195, 801, 784, and 1100. It should be noted that these mutants were all crossed with wild-type NOY421, and the RRN:rrn pattern of segregation was 2:2, indicating that each mutant bears a mutation in a single gene. [In these tetrad analyses, spore germination was done at 25°C for temperature-sensitive rrn mutations, and the recovery of two rrn segregants was very high. However, the recovery of rrn haploid segregants was not high for other mutants, presumably because of nonuniform distribution of pNOY103 and requirement of the plasmid in high-copy number for growth of rrn mutants (see ref. 16). Nevertheless, results of the tetrad analysis clearly supported the conclusion of a single mutation for these mutants. We also note that some mutants (944 and 837) appear to gene for growth.
RESULTS A System for Isolation of Mutants Defective in rRNA Synthesis. As described, both rpal90 and rpal35 mutations, which cause specific defects in transcription of 35S rRNA genes, can be suppressed by the GAL7-35S rDNA hybrid gene on a multicopy plasmid in the presence of galactose. Thus, starting with a strain carrying such a plasmid, a screen for mutants specifically defective in 35S rRNA synthesis can be devised to identify those cells able to grow on galactose medium but not on glucose medium. Such mutants must maintain the plasmid carrying the GAL7-35S rDNA hybrid gene for growth. We, therefore, used a red/white-colony color-sectoring assay (17-19) as the initial screen followed by a test for galactose-dependent growth. As a starting strain, we used NOY418, which is ade2 ade3 and carries ADE3 in addition to the GAL7-35S rDNA gene on a 2I.m-based multicopy plasmid. It is known that yeast strains carrying ade2 form red colonies from accumulation of red pigment, but ade2 ade3 strains form white colonies. Thus, NOY418 forms red colonies when grown on medium that selects for the plasmid (synthetic plates without uracil) but forms red/ white-sectored or almost totally white colonies on YEPgalactose plates because the 2,um-based plasmid, pNOY103, is unstable without selection. After ethylmethane sulfonate mutagenesis of NOY418, we looked for nonsectoring red colonies on YEP-galactose plates and then tested growth of these colonies on glucose medium. We thus isolated 24 Table 2. Classification of rrn mutants by complementation tests
Independent mutants
Representative Gene RRNI (RPA190) RRN2 (RPA13S) RRN3 RRN4 RRNS RRN6 RRN7 RRN8 RRN9
Othert
mutant
395 193
Strains used for complementation test to define RRN genes* NOY422 NOY446 (rpal90) (rpaI35) 74cr 225a 242cr 195ac 801a 784a 1100ar + + + + + + + + + + + + + + + + -
74
+
+
225(ts)
+ +
+ +
+
+
+ + + +
+ + + +
242 195
801(ts) 784(ts) 1i00 944(ts)
+
-
+ + + + + +
+ + + + + +
+
+ +
+ + + + +
+ + +
+ + + +
+ + + +
+ + +
+ + + + +
+
+
+ + + + + +
+
isolated, no. ts
0 3
Non-ts 1 2
Total 1 5
1
3
4
2
0
2
0 0
3 2
3
1 2
0 1
1 3
0
1
1
2 0 (837, 944)
2
2
ts, temperature-sensitive. *74a carries the original rrn3 mutation in mutant 74 but carries MA Ta trpl LYS2 and was obtained as a haploid segregant after crossing 74 with NOY421. Strains 225cr, 242a, 195a, 801a, 784a, and 1100a were constructed in a similar way, carry MATa, and were used for complementation analyses. tStrains 944 and 837 did not show clean 2:2 (RRN:rrn) segregation in tetrad analysis in the cross with strain NOY421 (RRN) and, therefore, may carry more than one mutation. However, all mutants in other complementation groups (RRNl-RRN9) tested showed complementation with 944, indicating the mutation(s) in 944 contributing to the rrn phenotype is unique and, thus, defining at least one complementation group. Complementation tests with 837 showed some ambiguous results, and characterization of mutation(s) in 837 needs further studies.
Biochemistry: Nogi et al.
Proc. Natl. Acad. Sci. USA 88 (1991)
contain more than one mutation and were left unclassified (see the legend for Table 2)]. Isolation of mutants defective in each of the two known pol I subunit genes was expected and justifies our strategy for isolating mutants specifically defective in 35S rRNA synthesis. Direct Demonstration of Defects in 35S rRNA Synthesis in rrn Mutants. The phenotype of rrn mutants described above indicates that these mutants are defective in synthesis of 35S rRNA from the chromosomal rRNA genes, and their growth depends on transcription of the plasmid-encoded GAL7-35S rDNA hybrid gene by pol II. By [3H]uridine pulse-labeling experiments, we confirmed this conclusion directly. Mutant strains representing each of seven new RRN complementation groups (RRN3-RRN9) were grown in synthetic galactose medium, and [3H]uridine pulse-labeling experiments were done with and without repression of the GAL7 promoter by glucose (24, 25), as was done in our previous study (16). As shown in Fig. 2, except for mutant 784 (rrn8), synthesis of large rRNA (18S, 25S, and 5.8S rRNAs and other precursor rRNAs) was strongly inhibited by glucose, relative to synthesis of 5S and tRNAs for every mutant analyzed. Thus, the mutations in each of these additional genes (except RRN8) preferentially affect synthesis of 35S rRNA. For mutant 784, total incorporation of [3H]uridine into RNA in the presence of glucose was 3- to 4-fold less than that in the control without glucose, and the amounts of radioactive large rRNAs accumulated relative to 5S RNA and tRNA were similar to those seen without glucose (Fig. 2, lanes 13 and 14). Therefore, the addition of glucose apparently inhibits not only the synthesis of large rRNA, but also that of SS RNA and tRNA. Although we cannot explain the unexpected results for this mutant 1 2 3 4 5 6 25S rRNA18S rRNA-
im-- i; 0!.. _
WW
7029
without further experiments, we retain this and other mutants that belong to the same complementation group as rrn8 mutants.
DISCUSSION AND CONCLUSION The method described here has proven very effective for isolating mutants defective in transcription of 35S rRNA genes by pol I and, thus, identifying components involved in this process. Of 24 independent mutants studied, 22 mutants were unambiguously classified into nine complementation groups, two of which correspond to known pol I subunit genes, RPA190 and RPA135, respectively. The remaining seven complementation groups define additional genes and, except for RRN8, their essential role for 35S rRNA transcription was confirmed directly by [3H]uridine-pulse labeling experiments. The two unclassified mutants (837, 944) appeared to have more than one mutation. At least one of the mutation(s) carried by 944 appears distinct from the above nine complementation groups (see the legend for Table 2). Thus, the number of RRN genes found may be 10 or more. In addition, from the distribution of mutants into nine complementation groups, the mutagenesis of RRN genes clearly has not been saturated. We note in this connection that mutations detected by the current method include completely lethal mutations as well as conditionally lethal mutations in RRN genes and, hence, one could hope to approach identification of all possible RRN genes. Among 24 independent mutants isolated, five had rpal35 mutations, whereas only one mutant had a rpal90 mutation, even though the RPA190 gene is larger than RPA135. Why 7 8 9 10 11 12
am
. W _ "
13 14 15 16
I'
6
'~~~~~~~~
~~~'
5.8S rRNA 5SRNAtRNA-
lU b
S
_
_
FIG. 2. Polyacrylamide/agarose gel electrophoresis of RNA synthesized in various rrn mutants and the parent strain growing in galactose medium with (even-numbered lanes) and without (odd-numbered lanes) further glucose addition. Parent strain NOY418 (lanes 1 and 2) and rrn mutants 416 (rrn3; lanes 11 and 12), 225 (rrn4; lanes 9 and 10), 242 (rrn5; lanes 7 and 8), 793 (rrn6; lanes 5 and 6), 801 (rrn7; lanes 3 and 4), 784 (rrn8; lanes 13 and 14), and 1100 (rrn9; lanes 15 and 16) were grown at 36°C (or at 30°C for 416 and 242) in galactose medium. At a cell density (A6w) of -0.2, cultures were divided into two parts, and glucose (final concentration 2%) was added to one part; the other served as control. At 1 hr after glucose addition, cells were pulse-labeled with [3H]uridine for 30 min (or 60 min for 784). RNA was isolated from each culture, and samples containing -7 X 104 cpm of [3H]RNA were analyzed by electrophoresis on a polyacrylamide/agarose composite gel. Autoradiograms of the dried gels are shown. Results from three separate experiments appear in lanes 1-6, lanes 7-12, and lanes 13-16, respectively. We note that autoradiograms obtained after longer gel exposures indicated that 5.8S rRNA synthesis in all rrn mutants (except 784) was inhibited by glucose, as was that of 18S and 25S rRNAs.
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Biochemistry: Nogi et al.
many more mutations were detected in the second largest subunit, the A135 subunit, is unclear. Both RPAJ90 and RPA135 are essential genes, but RPA135 may have more essential sites sensitive to mutagenesis; our initial color screening during mutant isolation tends to discard leaky mutants that could grow slowly without the GAL7-35S rDNA hybrid gene, and many point mutations induced by ethylmethane sulfonate in the RPAJ90 gene might be this type of mutation in contrast to those induced in the RPA135 gene. As mentioned earlier, S. cerevisiae pol I appears to consist of -13 polypeptide subunits, but five of them are shared by the other RNA polymerase(s), and, therefore, we do not formally expect to detect mutations in these five subunits by using the present mutant isolation method (for qualification of this statement, see below). In addition, some polypeptides detected in purified pol I preparations have not been rigorously proven as genuine components and are possibly partially or completely dispensable for 35S rRNA synthesis. Because we have found at least 9 RRN genes (including RPA190 and RPA135), the newly identified genes probably include those coding for proteins different from known pol I subunits. Although this mutant-isolation method is designed to isolate mutants with defects in components specifically involved in transcription of 35S rRNA genes, the proteins encoded by some of these RRN genes just identified may participate not only in transcription of 35S rRNA genes by pol I but also in other cellular functions, including pol II and pol III functions; the mutations may abolish the pol I function completely but may affect the other functions only partially without causing lethality; thus, the mutants can be rescued by transcription of the GAL7-35S rDNA gene by pol II in the presence of galactose. In preliminary experiments, we observed that some rrn mutants isolated (e.g., mutant 784) grow in galactose medium more slowly than mutants carrying rpal90 or rpaJ35 deletions (e.g., strain NOY446), suggesting that the possibility considered here may, in fact, be the case. In any event, cloning and analysis of the RRN genes defined in this work as well as in vitro transcription studies of 35S rRNA genes (see refs. 26-28) with extracts from these rrn mutants should make a significant contribution to studies of the mechanism of rRNA synthesis and its regulation in this model
eukaryotic organism. We thank Drs. C. Holmes and L. H. Hartwell for supplying yeast strains and plasmids and Drs. L. E. McAlister-Henn, J. Keener, and T. Menees for comments on the manuscript. This work was sup-
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