J. Mol. Biol. (1976) 107, 385-390
Location and Magnification o f 5 S R N A Genes in Saccharomyces
cerevisiae We have compared the amount of DNA complementary to the 5 S, 5"8 S and 4 S RNAs in a strain of Saccharomyces cerevisiae monosomie for chromosome I and a related diploid. The monosomic strain contains approximately 25% less DNA complementary to both 5 S and 5-8 S RNA than the diploid. The amount of DNA complementary to 4 S RNA is the same in both strains. These results indicate that the same relative proportions of the 5 S RNA genes, and the 5-8 S genes as well as the 18 S and 26 S RNA genes are present on chromosome I. Moreover, we have observed that the monosomic strain reverted to normal levels of DNA complementary to 5 S, 5"8 S, 18 S and 26 S RNA when continuously cultured for several months even though the strain remained monosomic for chromosome I by genetic criteria. This co-ordinate recovery in gene number is consistent with the close association of the 5 S, 5.8 S, 18 S and 26 S ribosomal RNA genes in yeast. Yeast ribosomes contain four species of RNA. The large ribosomal subunit contains one molecule each of 26 S, 5-8 S and 5 S R N A while the small subunit contains a single molecule of 18 S RNA. The 18 S, 26 S and 5.8 S R N A are derived from the same high molecular weight precursor, while the 5 S R N A is thought to be synthesized independently (Udem & Warner, 1972). The haploid genome of the yeast Saccharomyces cerevisiae contains approximately 140 genes each for the 5 S, 5.8 S, 18 S and 26 S RNAs (Schweizer et al., 1969; Rubin & Sulston, 1973). Approximately 60% of the 18 S and 26 S r R N A genes are located on chromosome I based on the observation that strains monosomic ( 2 n - - l ) for chromosome I contain about 30% less genes coding for r R N A than related diploid strains as determined b y saturation D N A r R N A hybridization (Oyen, 1973; Finkelstein et al., 1972; K a b a c k et al., 1973) and by quantitation of the rDNAt-containing satellite (Kaback et al., 1973). Furthermore, in experiments in which intact chromosomal D N A was sedimented in sucrose gradients, rDNA was shown to be associated with two size-classes of D N A molecules, one of which was associated with chromosome I (Finkelstein et al., 1972). Unlike other eucaryotes studied, in which the genes coding for 5 S R N A are not linked to the r R N A genes (Wimber & Steffenson, 1971 ; Aloni et al., 1971), yeast 5 S R N A genes are interspersed in some manner with the D N A coding for 18 S and 26 S r R N A (Rubin & Sulston, 1973). Rubin & Sulston (1973) have suggested that 5 S R N A genes m a y be arranged in an alternating fashion with 18 S and 26 S r R N A genes. However, a strict relationship between a single 5 S R N A gene and a single r R N A gene has not been shown. I f 5S R N A genes alternate with the r R N A genes we would expect the same proportion of 5 S R N A and r R N A genes to map on chromosome I. To test whether or not the same proportion of 5 S R N A genes map on chromosome I as 18 S and 26 S r R N A genes we adopted the following strategy. Strains aneuploid in a chromosome containing the gene for a specific R N A should contain different levels of DlqA complementary to t h a t R N A when compared to D N A from euploid strains. Thus, D N A isolated from a strain monosomic for a chromosome carrying 5 S R N A genes will hybridize less 5 S ~fAbbreviation used: rDNA, genes coding for rRNA. 385
386
D. B. KABACK, H. O. H A L V O R S O N AND G. M. R U B I N
RI~A than D N A from a diploid strain. The difference would be one-half the amount mapping on t h a t chromosome. Therefore, if 50 to 60% of the genes for 5 S are on chromosome I, a strain monosomic for chromosome I should contain 25 to 30% less D N A complementary to 5 S R N A t h a n a diploid. Purified 5 S R N A was hybridized to the D N A extracted from X1221a-7c, a strain monosomic for chromosome I t (Bruem] & Mortimer, 1970), and to the D N A extracted from DK8, a diploid strain made by crossing two haploids derived from X1221a-7c. The results (Fig. l(a)) show t h a t D N A derived from the diploid saturates at 0.042% while the D N A derived from the monosome for chromosome I saturates at approximately 0.032%, 24% less t h a n the diploid. Thus, approximately 48% of the 5 S R N A genes are located on chromosome I. The 5.8 S R N A was also hybridized to the same D N A preparations as used in the 5 S R N A experiments. The 5-8 S R N A is derived from the same precursor molecule as the 18 S and 26 S rRNAs (Udem & Warner, 1972) and should show the same percentage difference between the monosome and diploid strains in saturation hybridization levels as has been observed for 18 S and 26 S r R N A (Oyen, 1973; Finkelstein et al., 1972; K a b a c k et al., 1973). The results shown in Figure l(b) show an average 25% difference in the amount of D N A complementary to 5.8 S R N A between the monosome (0-056%) and the diploid (0-076%). Thus, there are approxim a t e l y equal numbers of 5 S R N A genes and 5-8 S R N A genes on chromosome I. Similar differences in hybridization levels to the D N A of these strains were reported with 18 S and 26 S rRNA. The monosome saturated at 1.60~ while the diploid saturated at 2-25%, a difference of 29% less rDNA in the monosomic strain. The respective differences between 24, 25 and 29 ~/o for each of the R N A species hybridized are insignificant. Since there is an inherent error of • in determining the specific activity of a given [SH]DNA preparation, a n y comparison between two strains in the percentage of DNA complementary to a given R N A can be =t=10% in error. As a control to check t h a t DNA from both strains was hybridizing with the same efficiency and the observed differences in hybridization of 5 S and 5-8 S R N A were not due to some artifact caused by a large error in the specific activity determinations of the individual D N A preparations, saturation hybridizations were carried out with 4 S R N A (tRNA). I n yeast external super-suppressors of m a n y alleles have been found t h a t m a p all over the yeast genome. M a n y of these suppressors are thought to be loci for t R N A genes (Hawthorne & Leupold, 1974). Assuming 4 S R N A genes are distributed over the whole yeast genome, the strain monosomic for chromosome I should contain approximately the same number of 4 S R N A genes as the diploid. Hybridization of 4 S R N A to D N A isolated from the monosome and the diploid show {Fig. l(c)) t h a t the levels of D N A complementary to 4 S R N A in these two strains are approximately the same. Non-saturation in this experiment is probably due to contaminating radioactive R N A which is not completely competed out by the nonradioactive r R N A added (Rubin & Sulston, 1973). Non-saturation does not affect our results since we are only concerned with the relative amounts of hybridization between the two strains, which in this case appear to be the same. t Strains used were the same as described previously (Kaback et al., 1973). Genetic analysis was done as described by Mortimer & Hawthorne (1969). In every D N A preparation we tested X 122 l a7c for monosomy by dissecting a few tetrads, which invariably showed first division 2 : 2 segregation of survivors]non-survivors, due to assortment of the haploid chromosome I into only 2 of the 4 spores. We further showed that the adel gene was hemizygous in the monosomic strain (D. B. Kaback, unpublished results).
LETTERS
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Fzc. 1. S a t u r a t i o n h y b r i d i z a t i o n of a2P-labeled 5 S, 5-8 S a n d 4 S R N A to [ a H ] D N A isolated f r o m t h e diploid D K 8 a n d t h e m o n o s o m e for c h r o m o s o m e I, X1221a-7c. D N A w a s labeled to a p p r o x i m a t e l y 103 c t s / m i n p e r ~g b y g r o w i n g cells to e a r l y s t a t i o n a r y p h a s e in 1.0% (w/v) y e a s t e x t r a c t , 2 % (w/v) p e p t o n e , 2 % (w/v) d e x t r o s e ( Y E P D ) w i t h 1 ~Ci [ G - 3 H ] a d e n i n e / m l (New E n g l a n d Nuclear). [ a H ] D N A w a s isolated b y a m o d i f i c a t i o n o f a p r o c e d u r e u s e d b y K f i c n z i et al. (1973). T h e p r o c e d u r e w a s identical e x c e p t before t h e CsCI c e n t r i f u g a t i o n t h e ]ysate w a s e x t r a c t e d twice w i t h c h l o r o f o r m / i s o a m y l alcohol, ( 2 4 : 1 , v / v ) . T h e p h a s e s were s e p a r a t e d b y c e n t r i f u g a t i o n a t 8000 r e v s / m i n for 20 m i n in a Sorvall H B - 4 rotor. T w o vol. ice-cold 9 5 % e t h a n o l were a d d e d to t h e a q u e o u s p h a s e a n d t h e D N A spooled o n a glass rod. T h i s D N A w a s d i s s o l v e d in 0-1 x SSC (SSC is 0.015 M-NaCI, 0.0015 ~ - N a citrate, p H 7.0) a n d b a n d e d in CsCI as p r e v i o u s l y d e s c r i b e d (Kiienzi, et al. 1973). F r a c t i o u s c o n t a i n i n g D N A were pooled, d i a l y z e d a g a i n s t 1 x SSC to r e m o v e CsC[ a n d p r e c i p i t a t e d w i t h 2 vol. 9 5 % e t h a n o l . T h e p r e c i p i t a t e d D N A w a s d i s s o l v e d in 0-1 x SSC for use. 32p-labeled R N A s were p r e p a r e d as d e s c r i b e d b y R u b i n (1975). H y b r i d i z a t i o n s were p e r f o r m e d b y a m o d i f i c a t i o n of t h e p r o c e d u r e of Gillespie & S p i e g e l m a n (1965). A p p r o x . 260 ~g n a t i v e D N A in 3.5 m l 0.1 x SSC w a s h e a t e d a t 100~ for 10 rain. T h e h e a t e d D N A s o l u t i o n w a s t h e n q u i c k l y p o u r e d into 17.4 m l 2 X SSC + 3.4 m l 2 N - N a O H t h a t h a d b e e n prechilled to 0~ T h e N a O H w a s to p r e v e n t r e p e t i t i v e s e q u e n c e s f r o m r c a n n e a l i n g . T h e D N A s o l u t i o n w a s b r o u g h t to p H 7-0-t-0"2 w i t h 1 N-HCI. T h e n 3 m l (22 ~ g D N A ) of t h e n e u t r a l i z e d s o l u t i o n w a s loaded o n t o 1 Schleicher a n d Schuell 27-ram B6 nitrocellulose filter a n d a l l o w e d to p a s s t h r o u g h w i t h o u t a n y suction. T h e filters were w a s h e d twice w i t h 5 m l 2 • SSC w i t h o u t s u c t i o n , twice w i t h 5 m l 2 x SSC w i t h s l i g h t s u c t i o n , dried face u p for 5 rain to 1 h on W h a t m a n no. 1 filter p a p e r a n d t h e n b a k e d i n v a c u o for 4 h a t 80 to 85~ T h e filters were t h e n c u t into 4 to 6 sections. H y b r i d i z a t i o n s were done in 10-ml d i s p o s a b l e s c i n t i l l a t i o n vials c o n t a i n i n g a g i v e n c o n c e n t r a t i o n of R N A d i s s o l v e d in 5 m l 2 x SSC -[- 0 - 2 % s o d i u m d o d e c y l sulfate. F o r t h e 4 S R N A h y b r i d i z a t i o n , in a d d i t i o n to a=P-labeled 4 S R N A , 40 ~ g n o n - r a d i o a c t i v e 18 S a n d 26 S R N A / m l w a s a d d e d to e a c h r e a c t i o n m i x t u r e . F i l t e r s were a d d e d a n d i n c u b a t e d for 16 h a t 65~ w i t h g e n t l e s h a k i n g . All d e t e r m i n a t i o n s were b a s e d o n t h e a v e r a g e of 3 filters c o n t a i n i n g t h e D N A o f e a c h s t r a i n w h i c h were all i n c u b a t e d in t h e s a m e vial for a g i v e n R N A c o n c e n t r a t i o n . T h e filters were t h e n w a s h e d for 1 h b y g e n t l e swirling t h r o u g h 5 s u c c e s s i v e c h a n g e s o f 25 m[ 2 x SSC. F i l t e r s were t h e n i n c u b a t e d for 1 h a t 30~ in a s o l u t i o n o f 50 Izg p a n c r e a t i c R N A a s e / m l (Calbiochem) a n d 10 u n i t s Tz R N A a s e / m l (Calbiochem) in 2 X SSC. T h e R N A a s e s o l u t i o n h a d b e e n p r e v i o u s l y h e a t e d for 10 rain a t 95~ to d e s t r o y D N A a s o a c t i v i t y . F i l t e r s were t h e n w a s h e d w i t h 5 c h a n g e s o f 2 x SSC, p l a c e d face u p o n W h a t m a n no. 1 filter p a p e r to r e m o v e m o s t of t h e s a l t a n d t h e n d r i e d u n d e r a h e a t l a m p . F i l t e r s were c o u n t e d in a B e c k m a n LS-255 liquid scintillat i o n c o u n t e r u s i n g a t o l u e n e - b a s e d s c i n t i l l a t i o n fluid. - - O - - O - - , DK8; --O--O--, X1221a-7e. (a) 5 S R N A , (b) 5-8 S R N A , (c) 4 S R N A .
388
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H . O. H A L V O R S O N
A N D G. M, R U B I N
During the course of this work we have observed what may amount to complete reversion to the wild-type level of DNA complementary to 5 S, 5.8 S, 18 S and 26 S RNA in the strain monosomie for chromosome I. An adenine prototroph of X1221a-7c which was previously shown to be deficient in rDNA (Kaback et al., 1973) was continually transferred on Y E P D (see the legend to Fig. 1) agar plates for five months (approx. 300 generations). The amount of DNA complementary to 5 S and 5.8 S RNA had increased to a level that was within 10% of the level in the diploid DK8 (Fig. 2(a) and (b)). Moreover, hybridization of 18 S and 26 S rRNA to DNA isolated from the monosome t h a t had been cultured showed the same hybridization level as to DNA isolated from the diploid (Table 1). The 4 S RNA hybridizations again gave the same level in both strains (Fig. 2(c)). Although it appears t h a t the level of 5 S, 18 S and 26 S RNA genes increases to a point slightly greater than the diploid, while the 5.8 S RNA gene level increases to slightly below the diploid level, these differences cannot be considered significant for the reasons described earlier, since they are both within 10% of the control diploid, DK8. We conclude that a strain initially deficient in rDNA had increased the number of these genes to approximately the wild-type level. The revertant strain remained monosomic for chromosome I as judged by tetrad analysis (Mortimer & Hawthorne, 1969) which invariably gave 2:2, first division segregation of survivors/non-survivors. As further evidence for chromosome I monosomy we showed that the adel gene was hemizygous (D. Kaback, unpublished data). This phenomenon may be analogous to the magnification of rDNA reported in Drosophila melanogaster bb mutants which are deficient in these genes (Ritossa, 1968 ; (a)
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Fro. 2. Saturation hybridization of a2P-labeled 5 S, 5.8 S and 4 S R N A to [3H]DNA from the diploid, DK8 and from the strain monosomic for chromosome I, X1221a-7c ade + which h a d been cultured for 5 months on Y E P D (see the legend to Fig. 1) plates. All procedures were the same as described in the legend to Fig. 1. DK8 ( - - 9 X1221a-70 ade + ( - - 0 - - 0 - - ) . (a) 5 S RNA, (b) 5-8 S RNA, (c) 4 S RNA.
L E T T E R S TO THE E D I T O R
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TABLE 1
Percentage of D N A complementary to 18 S a~ut 26 S R N A in the diploid, DK8, and the strain monosomic for chromosome I that had been cultured for five montl~s 18 S and 26 S RNA concentration (2 pg/ml) DK8 (cultured) DK8 (uncultured) X1221a-7c ade +
2.16 2.21 2.17
(3 pg]ml) 2-58 2-55 2-72
Hybridization of 32P-labeled 18 S and 26 S RNA to 3H-labeled DNA was carried out at 2 RNA concentrations, below saturation and at saturation. Hybridizations were done as described in the legend to Fig. l. Atwood, 1969). The observation that in yeast the 5 S RNA gene number reverts concurrently with that of the genes for other rRNAs is consistent with their physical linkage. The observed magnification of rDNA in yeast raises the question of the accuracy of our quantitation of the amount of rDNA mapping on chromosome I. I t is not possible to determine if any increase in the amount of rDNA has already occurred in the original monosomic strain. Thus, the estimates given above for the amount of 5 S and 5.8 S R N A genes as well as previous estimates for the amount of 18 S and 26 S R N A genes on chromosome I (Oyen, 1973; Finkelstein et al., 1972; Kabaek et al., 1973) m a y be inaccurate. The distribution of rDNA in sucrose gradients of chromosome-sized molecules in an unrelated haploid strain suggest that about 70~/o of the rDNA is associated with chromosome I (Finkelstein et al., 1972). These results imply that a monosome for chromosome I should contain 35% less r D N A than the diploid. This difference is within 10% of all the differences we measured between the original monosome t h a t had not been cultured and the diploid and is considered insignificant. I t is, therefore, unlikely that much if any r D N A magnification has occurred in the original monosomie strains. Whether or not partial rDNA magnification has occurred does not affect our finding that the relative proportions of all genes found on chromosome I are approximately the same in all cases examined. These results are a strong indication of the close association of these genes in yeast and arc consistent with an alternating arrangement of the 5 S R N A genes with the genes for the 5.8 S, 18 S and 26 S rRNAs. We thank James Haber and John Sulston for advice and Pieter Wensink for critically reading the manuscript. This work was supported by a grant from the National Institutes of Health (AI 10610). One of us (D. B. K.) was supported by a U. S. Public Health Service training grant. Another author (G. M. R.) was supported by a National Science Foundation predoetoral fellowship. Rosenstiel Basic Medical Sciences Research Center Department of Biology, Brandeis University Waltham, Mass. 02154, U.S.A.
DAVID B. KABACK HARLYN O. HALVORSON
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, CB2 2QH, England
GERALD M. RUBINt
Received 22 August 1975, and in revised form 16 April 1976 ~fPresent address: Department of Biochemistry, Stanford University, School of Medicine, Stanford, Calif. 94305, U.S.A.
390
D.B.
K A B A C K , H. O. H A L V O R S O N A N D G. M. R U B I N
REFERENCES Aloni, Y., Halten, L. E. & A t t a r d i , G. (1971). J. Mol. Biol. 56, 555-563. Atwood, K. C. (1969). Genetics, (Suppl. 1) 61, 319-327. Bruenn, J. & Mortimer, R. K. (1970}. J. Bacteriol. 102, 548-551. Finkelstein, D. B., Blamire, J. & Marmur, J. (1972). Nature New Biol. 240, 279-281. Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829-842. Hawthorne, D. C. & Leupold, U. (1974). Current Topics Microbiol. Immunobiol. 64, 1-47. K a b a c k , D. B., Bhargava, M. M. & Halvorson, H. O. (1973}. J. Mol. Biol. 79, 735-739. K~enzi, M. T., Tingle, M. A. & Halvorson, H. O. (1973). J. Bacteriol. 117, 80-88. Mortimer, R. K. & Hawthorne, D. C. (1969). I n The Yeasts (Rose, A. H. & Harrison, J. S., eds}, pp. 385-460, Academic Press, New York. Oyen, T. B. (1973). F E B S Letters, 30, 53-56. Ritossa, F. (1968). Proc. Nat. Aead. Sci., U.S.A. 66, 509-516. Rubin, G. M. (1975}. I n Methods in Cell Biology (Prescott, D., ed.), vol. 12, pp. 45-64, Academic Press, New York. Rubin, G. M. & Sulston, J. E. (1973). J. Mol. Biol. 79, 521-530. Schweizer, E., MacKechnie, C. & Halvorson, H. O. (1969). J. Mol. Biol. 40, 261-277. Udem, S. A. & Warner, J. R. (1972). J. Mol. Biol. 65, 227-242. Wimber, D. E. & Steffenson, D. M. (1971). Science, 170, 639-641.
Location and Magnification o f 5 S R N A Genes in Saccharomyces
cerevisiae We have compared the amount of DNA complementary to the 5 S, 5"8 S and 4 S RNAs in a strain of Saccharomyces cerevisiae monosomie for chromosome I and a related diploid. The monosomic strain contains approximately 25% less DNA complementary to both 5 S and 5-8 S RNA than the diploid. The amount of DNA complementary to 4 S RNA is the same in both strains. These results indicate that the same relative proportions of the 5 S RNA genes, and the 5-8 S genes as well as the 18 S and 26 S RNA genes are present on chromosome I. Moreover, we have observed that the monosomic strain reverted to normal levels of DNA complementary to 5 S, 5"8 S, 18 S and 26 S RNA when continuously cultured for several months even though the strain remained monosomic for chromosome I by genetic criteria. This co-ordinate recovery in gene number is consistent with the close association of the 5 S, 5.8 S, 18 S and 26 S ribosomal RNA genes in yeast. Yeast ribosomes contain four species of RNA. The large ribosomal subunit contains one molecule each of 26 S, 5-8 S and 5 S R N A while the small subunit contains a single molecule of 18 S RNA. The 18 S, 26 S and 5.8 S R N A are derived from the same high molecular weight precursor, while the 5 S R N A is thought to be synthesized independently (Udem & Warner, 1972). The haploid genome of the yeast Saccharomyces cerevisiae contains approximately 140 genes each for the 5 S, 5.8 S, 18 S and 26 S RNAs (Schweizer et al., 1969; Rubin & Sulston, 1973). Approximately 60% of the 18 S and 26 S r R N A genes are located on chromosome I based on the observation that strains monosomic ( 2 n - - l ) for chromosome I contain about 30% less genes coding for r R N A than related diploid strains as determined b y saturation D N A r R N A hybridization (Oyen, 1973; Finkelstein et al., 1972; K a b a c k et al., 1973) and by quantitation of the rDNAt-containing satellite (Kaback et al., 1973). Furthermore, in experiments in which intact chromosomal D N A was sedimented in sucrose gradients, rDNA was shown to be associated with two size-classes of D N A molecules, one of which was associated with chromosome I (Finkelstein et al., 1972). Unlike other eucaryotes studied, in which the genes coding for 5 S R N A are not linked to the r R N A genes (Wimber & Steffenson, 1971 ; Aloni et al., 1971), yeast 5 S R N A genes are interspersed in some manner with the D N A coding for 18 S and 26 S r R N A (Rubin & Sulston, 1973). Rubin & Sulston (1973) have suggested that 5 S R N A genes m a y be arranged in an alternating fashion with 18 S and 26 S r R N A genes. However, a strict relationship between a single 5 S R N A gene and a single r R N A gene has not been shown. I f 5S R N A genes alternate with the r R N A genes we would expect the same proportion of 5 S R N A and r R N A genes to map on chromosome I. To test whether or not the same proportion of 5 S R N A genes map on chromosome I as 18 S and 26 S r R N A genes we adopted the following strategy. Strains aneuploid in a chromosome containing the gene for a specific R N A should contain different levels of DlqA complementary to t h a t R N A when compared to D N A from euploid strains. Thus, D N A isolated from a strain monosomic for a chromosome carrying 5 S R N A genes will hybridize less 5 S ~fAbbreviation used: rDNA, genes coding for rRNA. 385
386
D. B. KABACK, H. O. H A L V O R S O N AND G. M. R U B I N
RI~A than D N A from a diploid strain. The difference would be one-half the amount mapping on t h a t chromosome. Therefore, if 50 to 60% of the genes for 5 S are on chromosome I, a strain monosomic for chromosome I should contain 25 to 30% less D N A complementary to 5 S R N A t h a n a diploid. Purified 5 S R N A was hybridized to the D N A extracted from X1221a-7c, a strain monosomic for chromosome I t (Bruem] & Mortimer, 1970), and to the D N A extracted from DK8, a diploid strain made by crossing two haploids derived from X1221a-7c. The results (Fig. l(a)) show t h a t D N A derived from the diploid saturates at 0.042% while the D N A derived from the monosome for chromosome I saturates at approximately 0.032%, 24% less t h a n the diploid. Thus, approximately 48% of the 5 S R N A genes are located on chromosome I. The 5.8 S R N A was also hybridized to the same D N A preparations as used in the 5 S R N A experiments. The 5-8 S R N A is derived from the same precursor molecule as the 18 S and 26 S rRNAs (Udem & Warner, 1972) and should show the same percentage difference between the monosome and diploid strains in saturation hybridization levels as has been observed for 18 S and 26 S r R N A (Oyen, 1973; Finkelstein et al., 1972; K a b a c k et al., 1973). The results shown in Figure l(b) show an average 25% difference in the amount of D N A complementary to 5.8 S R N A between the monosome (0-056%) and the diploid (0-076%). Thus, there are approxim a t e l y equal numbers of 5 S R N A genes and 5-8 S R N A genes on chromosome I. Similar differences in hybridization levels to the D N A of these strains were reported with 18 S and 26 S rRNA. The monosome saturated at 1.60~ while the diploid saturated at 2-25%, a difference of 29% less rDNA in the monosomic strain. The respective differences between 24, 25 and 29 ~/o for each of the R N A species hybridized are insignificant. Since there is an inherent error of • in determining the specific activity of a given [SH]DNA preparation, a n y comparison between two strains in the percentage of DNA complementary to a given R N A can be =t=10% in error. As a control to check t h a t DNA from both strains was hybridizing with the same efficiency and the observed differences in hybridization of 5 S and 5-8 S R N A were not due to some artifact caused by a large error in the specific activity determinations of the individual D N A preparations, saturation hybridizations were carried out with 4 S R N A (tRNA). I n yeast external super-suppressors of m a n y alleles have been found t h a t m a p all over the yeast genome. M a n y of these suppressors are thought to be loci for t R N A genes (Hawthorne & Leupold, 1974). Assuming 4 S R N A genes are distributed over the whole yeast genome, the strain monosomic for chromosome I should contain approximately the same number of 4 S R N A genes as the diploid. Hybridization of 4 S R N A to D N A isolated from the monosome and the diploid show {Fig. l(c)) t h a t the levels of D N A complementary to 4 S R N A in these two strains are approximately the same. Non-saturation in this experiment is probably due to contaminating radioactive R N A which is not completely competed out by the nonradioactive r R N A added (Rubin & Sulston, 1973). Non-saturation does not affect our results since we are only concerned with the relative amounts of hybridization between the two strains, which in this case appear to be the same. t Strains used were the same as described previously (Kaback et al., 1973). Genetic analysis was done as described by Mortimer & Hawthorne (1969). In every D N A preparation we tested X 122 l a7c for monosomy by dissecting a few tetrads, which invariably showed first division 2 : 2 segregation of survivors]non-survivors, due to assortment of the haploid chromosome I into only 2 of the 4 spores. We further showed that the adel gene was hemizygous in the monosomic strain (D. B. Kaback, unpublished results).
LETTERS
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Fzc. 1. S a t u r a t i o n h y b r i d i z a t i o n of a2P-labeled 5 S, 5-8 S a n d 4 S R N A to [ a H ] D N A isolated f r o m t h e diploid D K 8 a n d t h e m o n o s o m e for c h r o m o s o m e I, X1221a-7c. D N A w a s labeled to a p p r o x i m a t e l y 103 c t s / m i n p e r ~g b y g r o w i n g cells to e a r l y s t a t i o n a r y p h a s e in 1.0% (w/v) y e a s t e x t r a c t , 2 % (w/v) p e p t o n e , 2 % (w/v) d e x t r o s e ( Y E P D ) w i t h 1 ~Ci [ G - 3 H ] a d e n i n e / m l (New E n g l a n d Nuclear). [ a H ] D N A w a s isolated b y a m o d i f i c a t i o n o f a p r o c e d u r e u s e d b y K f i c n z i et al. (1973). T h e p r o c e d u r e w a s identical e x c e p t before t h e CsCI c e n t r i f u g a t i o n t h e ]ysate w a s e x t r a c t e d twice w i t h c h l o r o f o r m / i s o a m y l alcohol, ( 2 4 : 1 , v / v ) . T h e p h a s e s were s e p a r a t e d b y c e n t r i f u g a t i o n a t 8000 r e v s / m i n for 20 m i n in a Sorvall H B - 4 rotor. T w o vol. ice-cold 9 5 % e t h a n o l were a d d e d to t h e a q u e o u s p h a s e a n d t h e D N A spooled o n a glass rod. T h i s D N A w a s d i s s o l v e d in 0-1 x SSC (SSC is 0.015 M-NaCI, 0.0015 ~ - N a citrate, p H 7.0) a n d b a n d e d in CsCI as p r e v i o u s l y d e s c r i b e d (Kiienzi, et al. 1973). F r a c t i o u s c o n t a i n i n g D N A were pooled, d i a l y z e d a g a i n s t 1 x SSC to r e m o v e CsC[ a n d p r e c i p i t a t e d w i t h 2 vol. 9 5 % e t h a n o l . T h e p r e c i p i t a t e d D N A w a s d i s s o l v e d in 0-1 x SSC for use. 32p-labeled R N A s were p r e p a r e d as d e s c r i b e d b y R u b i n (1975). H y b r i d i z a t i o n s were p e r f o r m e d b y a m o d i f i c a t i o n of t h e p r o c e d u r e of Gillespie & S p i e g e l m a n (1965). A p p r o x . 260 ~g n a t i v e D N A in 3.5 m l 0.1 x SSC w a s h e a t e d a t 100~ for 10 rain. T h e h e a t e d D N A s o l u t i o n w a s t h e n q u i c k l y p o u r e d into 17.4 m l 2 X SSC + 3.4 m l 2 N - N a O H t h a t h a d b e e n prechilled to 0~ T h e N a O H w a s to p r e v e n t r e p e t i t i v e s e q u e n c e s f r o m r c a n n e a l i n g . T h e D N A s o l u t i o n w a s b r o u g h t to p H 7-0-t-0"2 w i t h 1 N-HCI. T h e n 3 m l (22 ~ g D N A ) of t h e n e u t r a l i z e d s o l u t i o n w a s loaded o n t o 1 Schleicher a n d Schuell 27-ram B6 nitrocellulose filter a n d a l l o w e d to p a s s t h r o u g h w i t h o u t a n y suction. T h e filters were w a s h e d twice w i t h 5 m l 2 • SSC w i t h o u t s u c t i o n , twice w i t h 5 m l 2 x SSC w i t h s l i g h t s u c t i o n , dried face u p for 5 rain to 1 h on W h a t m a n no. 1 filter p a p e r a n d t h e n b a k e d i n v a c u o for 4 h a t 80 to 85~ T h e filters were t h e n c u t into 4 to 6 sections. H y b r i d i z a t i o n s were done in 10-ml d i s p o s a b l e s c i n t i l l a t i o n vials c o n t a i n i n g a g i v e n c o n c e n t r a t i o n of R N A d i s s o l v e d in 5 m l 2 x SSC -[- 0 - 2 % s o d i u m d o d e c y l sulfate. F o r t h e 4 S R N A h y b r i d i z a t i o n , in a d d i t i o n to a=P-labeled 4 S R N A , 40 ~ g n o n - r a d i o a c t i v e 18 S a n d 26 S R N A / m l w a s a d d e d to e a c h r e a c t i o n m i x t u r e . F i l t e r s were a d d e d a n d i n c u b a t e d for 16 h a t 65~ w i t h g e n t l e s h a k i n g . All d e t e r m i n a t i o n s were b a s e d o n t h e a v e r a g e of 3 filters c o n t a i n i n g t h e D N A o f e a c h s t r a i n w h i c h were all i n c u b a t e d in t h e s a m e vial for a g i v e n R N A c o n c e n t r a t i o n . T h e filters were t h e n w a s h e d for 1 h b y g e n t l e swirling t h r o u g h 5 s u c c e s s i v e c h a n g e s o f 25 m[ 2 x SSC. F i l t e r s were t h e n i n c u b a t e d for 1 h a t 30~ in a s o l u t i o n o f 50 Izg p a n c r e a t i c R N A a s e / m l (Calbiochem) a n d 10 u n i t s Tz R N A a s e / m l (Calbiochem) in 2 X SSC. T h e R N A a s e s o l u t i o n h a d b e e n p r e v i o u s l y h e a t e d for 10 rain a t 95~ to d e s t r o y D N A a s o a c t i v i t y . F i l t e r s were t h e n w a s h e d w i t h 5 c h a n g e s o f 2 x SSC, p l a c e d face u p o n W h a t m a n no. 1 filter p a p e r to r e m o v e m o s t of t h e s a l t a n d t h e n d r i e d u n d e r a h e a t l a m p . F i l t e r s were c o u n t e d in a B e c k m a n LS-255 liquid scintillat i o n c o u n t e r u s i n g a t o l u e n e - b a s e d s c i n t i l l a t i o n fluid. - - O - - O - - , DK8; --O--O--, X1221a-7e. (a) 5 S R N A , (b) 5-8 S R N A , (c) 4 S R N A .
388
D.B.
KABACK,
H . O. H A L V O R S O N
A N D G. M, R U B I N
During the course of this work we have observed what may amount to complete reversion to the wild-type level of DNA complementary to 5 S, 5.8 S, 18 S and 26 S RNA in the strain monosomie for chromosome I. An adenine prototroph of X1221a-7c which was previously shown to be deficient in rDNA (Kaback et al., 1973) was continually transferred on Y E P D (see the legend to Fig. 1) agar plates for five months (approx. 300 generations). The amount of DNA complementary to 5 S and 5.8 S RNA had increased to a level that was within 10% of the level in the diploid DK8 (Fig. 2(a) and (b)). Moreover, hybridization of 18 S and 26 S rRNA to DNA isolated from the monosome t h a t had been cultured showed the same hybridization level as to DNA isolated from the diploid (Table 1). The 4 S RNA hybridizations again gave the same level in both strains (Fig. 2(c)). Although it appears t h a t the level of 5 S, 18 S and 26 S RNA genes increases to a point slightly greater than the diploid, while the 5.8 S RNA gene level increases to slightly below the diploid level, these differences cannot be considered significant for the reasons described earlier, since they are both within 10% of the control diploid, DK8. We conclude that a strain initially deficient in rDNA had increased the number of these genes to approximately the wild-type level. The revertant strain remained monosomic for chromosome I as judged by tetrad analysis (Mortimer & Hawthorne, 1969) which invariably gave 2:2, first division segregation of survivors/non-survivors. As further evidence for chromosome I monosomy we showed that the adel gene was hemizygous (D. Kaback, unpublished data). This phenomenon may be analogous to the magnification of rDNA reported in Drosophila melanogaster bb mutants which are deficient in these genes (Ritossa, 1968 ; (a)
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Fro. 2. Saturation hybridization of a2P-labeled 5 S, 5.8 S and 4 S R N A to [3H]DNA from the diploid, DK8 and from the strain monosomic for chromosome I, X1221a-7c ade + which h a d been cultured for 5 months on Y E P D (see the legend to Fig. 1) plates. All procedures were the same as described in the legend to Fig. 1. DK8 ( - - 9 X1221a-70 ade + ( - - 0 - - 0 - - ) . (a) 5 S RNA, (b) 5-8 S RNA, (c) 4 S RNA.
L E T T E R S TO THE E D I T O R
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TABLE 1
Percentage of D N A complementary to 18 S a~ut 26 S R N A in the diploid, DK8, and the strain monosomic for chromosome I that had been cultured for five montl~s 18 S and 26 S RNA concentration (2 pg/ml) DK8 (cultured) DK8 (uncultured) X1221a-7c ade +
2.16 2.21 2.17
(3 pg]ml) 2-58 2-55 2-72
Hybridization of 32P-labeled 18 S and 26 S RNA to 3H-labeled DNA was carried out at 2 RNA concentrations, below saturation and at saturation. Hybridizations were done as described in the legend to Fig. l. Atwood, 1969). The observation that in yeast the 5 S RNA gene number reverts concurrently with that of the genes for other rRNAs is consistent with their physical linkage. The observed magnification of rDNA in yeast raises the question of the accuracy of our quantitation of the amount of rDNA mapping on chromosome I. I t is not possible to determine if any increase in the amount of rDNA has already occurred in the original monosomic strain. Thus, the estimates given above for the amount of 5 S and 5.8 S R N A genes as well as previous estimates for the amount of 18 S and 26 S R N A genes on chromosome I (Oyen, 1973; Finkelstein et al., 1972; Kabaek et al., 1973) m a y be inaccurate. The distribution of rDNA in sucrose gradients of chromosome-sized molecules in an unrelated haploid strain suggest that about 70~/o of the rDNA is associated with chromosome I (Finkelstein et al., 1972). These results imply that a monosome for chromosome I should contain 35% less r D N A than the diploid. This difference is within 10% of all the differences we measured between the original monosome t h a t had not been cultured and the diploid and is considered insignificant. I t is, therefore, unlikely that much if any r D N A magnification has occurred in the original monosomie strains. Whether or not partial rDNA magnification has occurred does not affect our finding that the relative proportions of all genes found on chromosome I are approximately the same in all cases examined. These results are a strong indication of the close association of these genes in yeast and arc consistent with an alternating arrangement of the 5 S R N A genes with the genes for the 5.8 S, 18 S and 26 S rRNAs. We thank James Haber and John Sulston for advice and Pieter Wensink for critically reading the manuscript. This work was supported by a grant from the National Institutes of Health (AI 10610). One of us (D. B. K.) was supported by a U. S. Public Health Service training grant. Another author (G. M. R.) was supported by a National Science Foundation predoetoral fellowship. Rosenstiel Basic Medical Sciences Research Center Department of Biology, Brandeis University Waltham, Mass. 02154, U.S.A.
DAVID B. KABACK HARLYN O. HALVORSON
Medical Research Council Laboratory of Molecular Biology Hills Road, Cambridge, CB2 2QH, England
GERALD M. RUBINt
Received 22 August 1975, and in revised form 16 April 1976 ~fPresent address: Department of Biochemistry, Stanford University, School of Medicine, Stanford, Calif. 94305, U.S.A.
390
D.B.
K A B A C K , H. O. H A L V O R S O N A N D G. M. R U B I N
REFERENCES Aloni, Y., Halten, L. E. & A t t a r d i , G. (1971). J. Mol. Biol. 56, 555-563. Atwood, K. C. (1969). Genetics, (Suppl. 1) 61, 319-327. Bruenn, J. & Mortimer, R. K. (1970}. J. Bacteriol. 102, 548-551. Finkelstein, D. B., Blamire, J. & Marmur, J. (1972). Nature New Biol. 240, 279-281. Gillespie, D. & Spiegelman, S. (1965). J. Mol. Biol. 12, 829-842. Hawthorne, D. C. & Leupold, U. (1974). Current Topics Microbiol. Immunobiol. 64, 1-47. K a b a c k , D. B., Bhargava, M. M. & Halvorson, H. O. (1973}. J. Mol. Biol. 79, 735-739. K~enzi, M. T., Tingle, M. A. & Halvorson, H. O. (1973). J. Bacteriol. 117, 80-88. Mortimer, R. K. & Hawthorne, D. C. (1969). I n The Yeasts (Rose, A. H. & Harrison, J. S., eds}, pp. 385-460, Academic Press, New York. Oyen, T. B. (1973). F E B S Letters, 30, 53-56. Ritossa, F. (1968). Proc. Nat. Aead. Sci., U.S.A. 66, 509-516. Rubin, G. M. (1975}. I n Methods in Cell Biology (Prescott, D., ed.), vol. 12, pp. 45-64, Academic Press, New York. Rubin, G. M. & Sulston, J. E. (1973). J. Mol. Biol. 79, 521-530. Schweizer, E., MacKechnie, C. & Halvorson, H. O. (1969). J. Mol. Biol. 40, 261-277. Udem, S. A. & Warner, J. R. (1972). J. Mol. Biol. 65, 227-242. Wimber, D. E. & Steffenson, D. M. (1971). Science, 170, 639-641.
