Abundant species of poly(A)-containing RNA from Saccharomyces cerevisiae

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Biochimica et Biophysica Acta, 4 1 4 ( 1 9 7 5 ) 2 6 3 - - 2 7 2 © Elsevier Scientific P u b l i s h i n g C o m p a n y , A m s t e r d a m - - P r i n t e d in T h e N e t h e r l a n d s

BBA 98472

ABUNDANT SPECIES O F POLY(A)-CONTAINING R N A F R O M SACCHAROMYCES CEREVISIAE *

CHANNA SHALITIN and ISHAK FISCHER

Department of Biology, University of California, San Diego, La Jolla, Calif. (U.S.A.) and Department of Biology, Technion-Israel Institute of Technology, Haifa, (Israel) ** (Received August 6th, 1975)

Summary Hybridization experiments using uniformly labeled poly(A) R N A derived from Saccharomyces cerevisiae strains carrying the "killer character" showed that: (1) these molecules appear to be transcribed from repetitive DNA sequences. (2) there are approximately 35 DNA template sequences that are transcribed into poly(A) RNA. It is concluded that under the R N A extraction procedure used, most of the poly(A) R N A represents killer-RNA as judged by the dependence of the kinetic complexity of poly(A) R N A on the genomic complexity of killer-RNA.

Introduction It n o w seems well established that yeast nuclear poly(A)-containing R N A is in fact m R N A and is post-transcriptionally processed. After transcription these molecules are transported from the nucleus into the cytoplasm and enter the polysomes [1,2,3]: However, the origin of this poly(A) R N A is still unclear. The Saccharomyces cerevisiae strains used in the first t w o of these studies were found to contain a killer factor [4] which is composed of t w o species of double-stranded RNA, one of mol.wt 2.5 • 106 and a second 1.4 • 106 [5,6]. In addition, Vodkin et al. suggested that the double-stranded R N A of killer strains contains stretches of poly(A) [6]. These poly(A) sequences were found to be covalently linked to m R N A molecules [1]. In this study an a t t e m p t was made to determine whether some of the poly(A) m R N A is a specific transcript of the killer factor. On the basis of molecular hybridization experiments of uniformly-labeled poly(A) RNA, it is concluded that the R N A which survives the extraction procedure has a sequence complexity of 4.8--9.6 • 106 daltons • Thi~ investigation received financial support from the World Health Organization. • * Present address.

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which is related to the estimated molecular weight of the killer factor [5,6]. It seems that t h e genome contains in the neighborhood of 35 cistrons which code for this kind of m R N A . Materials and Methods

Strains The strains of Saccharomyces cerevisiae used in this study were kindly supplied by L. Hartwell, A364A (a, adel, ade2, ural, his7, lys2, gall, K*) * was converted into a petite strain by W. Oertel. The killer determinant is retained in this p- strain [7]. X2180-1B (a, wild type K-) and X2180A (a, wild type K-) were from the collection of T.R. Manney. The diploid strain, homozygous for the temperature sensitive m u t a t i o n cdc4 (314-D5), has been previously described [8] and was found in our laboratory to contain a killer factor by replica plating onto a lawn of 106 sensitive X2180-1Ba cells on buffered methylene blue medium [4]. After 48 h incubation at room temperature, killer colonies were identified as those that produce a zone of clearing of the sensitive cells.

Chemicals [U-14C] Adenine [specific activity 287 m C i / m m o l ] , and [5,6-3H] uracil [specific activity 53 Ci/mmol] were purchased from the Radiochemical Center, Amersham. Sarkosyl NL 30 was the gift of the Geigy Chemical Corporation.

Preparation of 14C.labele d DNA and fixation to filters A364Ap- cells were grown in synthetic medium [9] supplemented with 0.1 pCi/ml [U- ~4 C] adenine to a concentration of 2 • 107 cells/ml. Spheroplasts were prepared from these cells as described by Hutchison and Hartwell [10]. 14 C-labeled nuclear DNA that is free of mitochondrial DNA was prepared by lysing spheroplasts with 4% sodium dodecyl sulfate, incubating for 3 h at 63°C with 1 mg/ml pronase (Calbiochem, preheated 10 min 85 ° C) and then subjecting the lysate to two CsCl equilibrium centrifugation steps. 14 C-labeled DNA (800 cpm/pg) in 0.1 X SSC was denatured by treatment with 0.3 M NaOH for 20 min at 30 ° C. 1 pg of denatured 14 C-labeled DNA was fixed to each 24 mm filter (Schleicher & Schuell Bac-T-Flex type B-6).

Preparation of 3H-uniformly labeled R N A Cells were grown to a concentration of 5 • 106 cells/ml in synthetic medium [9] supplemented either with 0.2 pg/ml or with 2 pg/ml of non-radioactive uracil for the growth of strain A364A or 314-D5, respectively. The cells were labeled by adding 10--50 pCi/ml [5,6-3H]uracil for 3 h at 25°C (>1 generation). The incorporation was stopped by chilling. The cells were collected by centrifugation, washed in cold water and resuspended at a concentration of 108 cells/ml in 0.1 M sodium acetate buffer, pH 5.2, containing 100 * K, the p h e n o t y p e s o f strains w i t h regaxd to t h e i r killing ability. K + d e n o t e s the p r e s e n c e o f t h e " k i l l e r s u b s t a n c e " w h i c h kills sensitive cells, a n d K - d e n o t e s the n o n k i U e r sensitive p h e n o t y p e . A b b r e v i a t i o n : SSC, standard saline-citrate s o l u t i o n ( 0 . 1 5 M s o d i u m c h l o r i d e / 0 . 0 1 5 M s o d i u m citrate, p H 7.2).

265 pg/ml polyvinyl sulfate (Sigma) and 2% sodium dodecyl sulfate. Subsequently the cells were frozen in an Eaton pressure cell and passed through the cell under pressure of 16 000 lb/inch 2 . Following pronase digestion (1 mg/ml, 3 h at 63°C), the R N A was extracted at 68°C by water-saturated phenol. Finally, the R N A was dialyzed vs 6 X SSC and traces of phenol were removed by cold ether extractions. R N A concentrations were calculated from the absorbance at 260 nm, with an extinction coefficient E ~ m , of 250. Preparation of nonlabeled R N A followed a similar procedure. Isolation o f p o l y ( A ) R N A To prepare poly(A) RNA, 100--500 #g 3H-labeled R N A in 1 X SSC containing 0.5% sarkosyl was passed through a Sepharose 4B column (0.7 ml packed vol) to which poly(U) (Miles) had been covalently attached (12.5 mg poly(U) per 100 ml of packed Sepharose) by the technique of Wagner, Bugianese and Shen (1971) [11] or by using Pharmacia CNBr-activated Sepharose 4B. The poly(A) containing RNA was eluted at room temperature with either water or with 40% formamide. No further radioactive material was eluted by washing the column with 90% formamide. The concentrations of poly(A) R N A were calculated from the specific activity of uniformly labeled RNA. Prepal"ation o f 3H-labeled rRNA X2180-1B ~ cells were grown and labeled as described above. Purified ribosomes and ribosomal subunits were prepared as described by Warner [12]. Isolated ribosomal subunits were ethanol precipitated in 1 M NaCl and resuspended in 0.1 M sodium acetate buffer, pH 5.2, containing 100 pg/ml polyvinyl sulfate. The rRNA was extracted at 68°C by water-saturated phenol. Finally, the 3H-labeled r R N A was dialyzed vs 6 × SSC and was further purified by passing it through a column of poly(U)-Sepharose to remove any poly(A)-containing mRNA. DNA . R N A hybridization procedure For hybridization each 24 mm [14C]DNA filter paper was cut into 4 circles of 6 mm diameter. Each 6 mm filter contained approximately 0.125 pg of DNA. Filters containing no DNA served as control. Hybridization was carried o u t at 50°C in 2 ml Lancer Analyzer cups (flat b o t t o m with wings, Sherwood Medical Indust. Inc., St. Louis, Mo.) in a vol. of 0.4 ml of 4 X SSC containing 1% Sarkosyl and 25% Formamide (v/v; Merck). Before hybridization, the 3H-labeled R N A was preincubated in the same solution for 20--24 h at 50°C to allow self-annealing of RNA before the hybridization reaction with the [~4C] DNA filters. The a m o u n t of RNA self-annealed was determined by its RNAase resistance and did not exceed 14% of the total R N A (Shalitin, C. and Fisher, I., unpublished). At the end of the incubation period, filters were blotted in tissue paper to eliminate any excess solution containing labeled RNA and soaked twice in 200 ml of 2 X SSC for 15 min at room temperature without stirring. Digestion of any non-hybrid R N A which survived the washing procedure was accomplished by treatment with RNAase (2 #g/ml in 2 X SSC for 30 min at 30°C). The filters were washed again with 200 ml 2 X SSC and with 50 ml of H 2 0 before drying and counting in 4 ml toluene scintillator in

266

mini-vials in a Packard Tri-Carb scintillation counter. For counting, the channels were set for simultaneous counting of 3H and ’ 4C, with correction for spillover between them. The approximate counting efficiencies were 44% for 3Hand 14C. Gel electrophoresis Samples of RNA to be analyzed by gel electrophoresis were first treated with 2 M LiCl to precipitate ribosomal RNA [ 131. Analysis by gel electrophoresis was done in 2.8% acrylamide/0.14% methylene bis acrylamide/0.5% agarose composite gels [14]. The electrophoresis buffer was 0.04 M Tris/acetate (pH 7.6) containing 2 mM ethylenediaminetetraacetic acid (EDTA) and 0.02 M sodium acetate. The gels were run first without samples at 5 mA/gel for 45 min. The samples were then added and subjected to electrophoresis for 4 h at 5 mA/gel. After removal from the running tubes, the gels were rinsed in 0.4 M citrate buffer, pH 4.5, for 15 min, stained in 0.1% toluidine blue 0 in the same buffer for 25 min and destained in distilled water. Results The following experiments were performed to determine the optimal conditions for poly(A) RNA * DNA hybridization. The Gillespie and Spiegelman procedure (1965) [ 151 was used with the low temperature formamide technique of McConaughy et al. [16].

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Fig. 1. Extent of hybridization of 314-D5 poly(A) RNA as function of temperature. Filters contained approximately 0.1 pg [14C] DNA. The jH-labeled poly(A) RNA concentration was 1.4 pg (sp. activ. 6 . lo4 cpm//& well in excess of the complementary DNA. The hybridization procedure was as described in Materials and Methods. The time of incubation was 24 h at 5O’C. Filters were counted for 100 min each. Background radioactivity bound to DNA-free filters (2-3 cpm) was subtracted. Fig. 2. Extent of hybridization of 314-D5 poly(A) RNA as function of formamide concentration. The 3H-labeled poly(A) RNA concentration was 0.4 I.rg(sp. activ. 4 * lo4 cpm/j&. The hybridization mixture contained approximately 0.1 fig [ 14C] DNA and was incubated for 20 h at 50°C as described in Materials and Methods. All data were corrected for binding to blank filters without DNA (l-3 cpm).

267

(a) Effect o f temperature on the extent o f poly(A) R N A . DNA hybridization. An experiment was done to determine the optimal temperature for maximal extent of the reaction in 4 × SSC 27% formamide, 1% Sarkosyl. The results are shown in Fig. 1. The counts hybridized were taken as a measure of the extent of the reaction. As seen in Fig. 1, the temperature at which the reaction extent is maximal (50°C) is rather high for poly(A) RNA, possibly due to increased stability of RNA • DNA hybrids under the experimental conditions [17]. The optimal temperature found for rRNA hybridization, was 55°C, close to that presented for poly(A) RNA (data not shown). (b) Effect o f formamide concentration on extent o f poly(A) hybridization. Aqueous formamide was previously found [18] to increase the retention of DNA by membrane filters and to decrease nonspecific adsorption of RNA to such filters. Therefore, the optimal formamide concentration for maximal extent of the reaction at 50°C in 4 × SSC, 1% Sarkosyl was determined. As shown in Fig. 2, the reaction extent is maximal in 25% formamide. Saturation hybridization experiments. Once the optimal conditions for poly(A) RNA • DNA hybridization were known, it was possible to determine by this technique the saturation value and the sequence complexity of the annealed poly(A) RNA [17]. Fig. 3 presents a hybridization saturation curve of yeast poly(A) RNA with nuclear DNA under these conditions. Saturation is reached at concentrations of poly(A) RNA above 0.2 pg per reaction tube. At saturation, 2.5% of the nuclear DNA hybridizes with poly(A) RNA. Gel electrophoresis o f killer RNA. To estimate the genomic complexity of killer RNA, ribosomal RNA was removed by LiC1 precipitation [13] and the LiCl-soluble fraction was analyzed by gel electrophoresis. The results are shown in Fig. 4. Molecular weight estimates are based on comparisons of the migration of killer RNA with that of reovirus double-stranded RNA [19]. Two species of

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[3.] ~ y (A) R.A (~g) Fig. 3. S a t u r a t i o n h y b r i d i z a t i o n c u r v e o f n u c l e a z D N A w i t h p o l y ( A ) R N A i s o l a t e d f r o m s w a i n 3 1 4 - D 5 . S p e c i f i c a c t i v i t y o f the p o l y ( A ) R N A w a s 1 . 0 • 105 c p m / ~ Increasing a m o u n t s o f p o l y ( A ) R N A were h y b r i d i z e d t o n i t r o c e l l u l o s e filters c o n t a i n i n g a p p r o x i m a t e l y 0.1 ~ g o f [ 1 4 C ] l a b e l e d D N A f o r 2 4 h a t 50°C as described i n M a t e r i a l s and Methods. Duplicate annealing e x p e r i m e n t s g a v e d a t a t h a t agreed w i t h i n 10%.

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Fig. 4. C o m p a r i s o n of d o u b l e - s t r a n d e d R N A g e n o m e s e g m e n t s e x t r a c t e d f r o m K + a n d K - y e a s t strains. A p p r o x . 3 0 0 - - 4 0 0 Mg n o n - l a b e l e d R N A s a m p l e s p r e p a r e d as d e s c r i b e d in Materials a n d M e t h o d s w e r e s u b j e c t e d t o e l e e t r o p h o r e s i s on 2.8% p o l y a c r y l a r n i d e gels, s t a i n e d in 0.1% t o l u i d i n e b l u e O a n d d e s t a i n e d in distilled w a t e r . D o u b l e - s t r a n d e d r e o v i r u s R N A has b e e n a n a l y z e d as (a) c o n t r o l ; (b) X 2 1 8 0 - 1 B ~ {K"] ; (c) X 2 1 8 0 - 1 A a [ K - ] ; (d) A 3 6 4 A p - [ K + ] . M i g r a t i o n is f r o m t o p t o b o t t o m f o r 4 h.

double-stranded RNA are observed in K * strains (2.5 • 106 and 1.4 • 106 daltons). However in K- strains used, the smaller species of double-stranded RNA (1.4 • 106 daltons) is missing, and the a m o u n t of the larger piece of double-stranded RNA (2.5 • 106 daltons) is greatly elevated. This finding is in accord with previous observations [6]. In view of the results reported here, we have tested Vodkin's suggestion [6] concerning the presence of poly(A) sequences attached to the doublestranded RNA fractions. Uniformly labeled 3H- or 14 C-RNA preparations were subjected to analysis by gel electrophoresis. The double-stranded RNA fractions were eluted for 16 h at 50°C from the stained gels into 88C containing 1% sodium tri-isopropylnaphthalene-sulphonate. Following poly(U)-Sepharose chromatography of the double-stranded RNA fractions, we have been able to show t h a t about 50% of the molecules were bound to the poly(U)-Sepharose column. It is therefore concluded that at least 50% of the double-stranded RNA molecules contain poly(A) sequences, or else all of them contain rather short poly(A) sequences (less than 30 nucleotides) which bind to the poly(U)Sepharose column with 50% efficiency [20]. T i m e course o f h y b r i d i z a t i o n . The time course of the hybridization of poly(A) RNA to DNA immobilized on filters was followed under conditions of RNA excess at the optimal temperature. The percent of RNA hybridized at each time is expressed as a double reciprocal plot (Fig. 5, upper part) [17,21]. T h e t l l 2 of the reaction may then be determined, i.e. the time taken to

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F i g . 5. L e f t part: D o u b l e r e c i p r o c a l p l o t o f t h e a p p r o a c h to saturation o f p o l y ( A ) R N A isolated f r o m strain 3 1 4 - D 5 . A t 1/% saturation = 0 . 0 2 , 5 0 % o f t h e s a t u r a t i o n value has b e e n r e a c h e d , R i g h t part: T h e t i m e c o u r s e o f h y b r i d i z a t i o n . 0 . 4 7 # g o f p o l y ( A ) R N A (sp. activ. 1 . 0 • 1 0 5 c p m / # g ) w a s h y b r i d i z e d to 0.1 # g 1 4 C - l a b e l e d D N A as d e s c r i b e d in Materials and M e t h o d s . 1 0 0 % s a t u r a t i o n = 2 . 5 ng p o l y ( A ) R N A bound per 100 ng DNA.

reach 50% of the saturation value, and the reaction expressed as Crtl/2 where Cr is the molar concentration of the ribonucleotides in solution. For a homogenous RNA species present in excess, the constant Crt 1/2 is directly proportional to the base sequence complexity of the RNA, and within certain limits, is independent of both the amount of complementary DNA and the degree of fragmentation of the RNA [17]. By comparing the Crtl/2 of the RNA in question to that of an RNA of known complexity, it is possible to determine whether the gene transcripts form one, two or more families of base sequences. Thus, when the concentration of RNA is in vast excess to that of complementary DNA (by a factor of 200), it is possible to measure the complexity of RNA sequences that contain covalently linked poly(A) sequences. These sequences represent 2.5% of the DNA (Fig. 3). As shown in Fig. 5 the tl / s of the reaction (the time taken to reach 50% of the saturation value) is 2.5 h. Since the molar concentration* of ribonucleotides in solution was 2.85 • 10 -6, we find that 314-D5K ÷ poly(A) RNA has a Crtl/2 of 24.0 +- 1.6" 10 -3 mol × s/1. For comparison, the Crtl/2 value of yeast rRNA ( 1 . 3 . 1 0 6 and 0 . 7 . 1 0 6 daltons mol. wt [22] was tested under our annealing conditions. It was found to be 5.0 -+ 0.6 • 10 -3 mol × s/l**, and was used to calculate the sequence complexity of poly(A) RNA. Using this method, we arrive at a sequence complexity of 9.6 + 0.6 • 106 daltons for 314-D5 K÷ poly(A) RNA (Table I). This is about twice the genomic complexity of killer double-stranded RNA [ 5 , 6 ] . * V a l u e c o r r e c t e d for c o m p l e m e n t a r y p o l y ( A ) R N A ( S h a l i t i n , C. and Fischer, I., u n p u b l i s h e d ) w h i c h

c a n n o t b e c o n s i d e r e d as freely r e a c t i v e R N A in s o l u t i o n . C~1/2 value o f y e a s t r R N A c a l c u l a t e d f r o m F i g . 1 in F i n k e l s t e i n , Blamire & M a r m u r [ 2 3 ] , is s o m e w h a t h i g h e r t h a n ours ( 1 0 -2 tool × s / l ) d u e t o the d i f f e r e n t a n n e a l i n g c o n d i t i o n s u s e d b y t h e s e a u t h o r s ( 5 0 % f o r m a m i d e , 2 × S S C , 3 3 ° C and 0 . 1 % s o d i u m d o d e c y l s u l f a t e ) w h i c h m a y h a v e s l o w e d the a n n e a l i n g rate.

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270 TABLEI D E P E N D E N C E O F Crt ½ O F P O L Y ( A ) R N A ON G E N O M I C C O M P L E X I T Y O F K I L L E R R N A (a) Rou~,h e s t i m a t e b a s e d on Fig. 4 a n d ref. 5. (b) C a l c u l a t e d f r o m Fig. 5 a n d f r o m parallel e x p e r i m e n t s p e r f o r m e d w i t h strains A 3 6 4 A p - ( K + ) , X 2 1 8 0 - 1 B ~ ( K - ) a n d X 2 1 8 0 - 1 A a ( I C ' ) . (c) Crt ½ values are u s e d to c a l c u l a t e t h e s e q u e n c e c o m p l e x i t y o f p o l y ( A ) R N A b y c o m p a r i s o n o f its Crt ½ value t o t h e Crt ½ value o f r R N A 45.0 +- 0.6 • 1 0 - 3 tool X s/1 ( s e e t e x t ) ; tool. w t . 1.3 • 106 a n d 0.7 • 106 [ 2 2 ] ). T h r e e a n n e a l i n g e x p e r i m e n t s for p o l y ( A ) R N A using d i f f e r e n t p r e p a r a t i o n s of [ 1 4 C ] n u c l e a r D N A a n d p o l y ( A ) R N A gave Crt ½ values t h a t agreed w i t h i n 10%.

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Calculated complexity

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Furthermore, t h e data of Table I indicate that the kinetic complexity of poly(A) R N A is related to the genomic complexity of killer R N A (see Fig. 4). Thus it is plausible that poly(A)-containing m R N A sequences thus extracted represent mainly killer R N A molecules. On the basis of this assumption it becomes possible to calculate the redundancy of D N A complementary to these sequences. As shown in Table II, the percent of D N A saturated is directly proportional to the genomic complexity of the killer RNA. In haploid S. cerevisiae the redundancy of these cistrons which specify the killer R N A is about 35. Moreover, in a K- strain which is missing the small double-stranded RNA, the redundancy of the large double-stranded RNA is similar to that of the doublestranded R N A in a K ÷, strain which contains both small and large species of double-stranded RNA. This finding presented here (Table II) is of interest in view of the assumption that the two species of double-stranded R N A are informationally distinct [6] as judged by the lower saturation level that is attained when only the large double-stranded R N A is present.

T A B L E II E S T I M A T I O N O F K I L L E R C I S T R O N S I N N U C L E A R D N A O F S. C E R E V I S I A E (a) D a t a f r o m Fig. 3 a n d 5. (b) D a t a f r o m a p~rallel e x p e r i m e n t p e r f o r m e d w i t h strain X 2 1 8 0 - 1 B a ( K - ) . (c) Based o n t h e a s s u m p t i o n o f 1 . 2 5 • 1 0 1 0 d a i t o n s f o r S. cerevisiae h a p l o i d n u c l e a r D N A [ 2 6 ] . (d) Calcul a t e d c o m p l e x i t y f r o m T a b l e I. K +

D N A h y b r i d i z e d (%) D N A h y b r i d i z e d ( d a l t o n s ) (c) M o l e c u l a r w e i g h t o f R N A (d) Number of cistrons

2.5 (a) 6 . 2 • 108 9.6 • 106 32

K-

1.69 (b) 4.2 • 108 5.6 • 106 37

271 Discussion The experiments described in this report lead to t w o main conclusions regarding the nature of the sequences present in poly(A) m R N A isolated from whole S. cerevisiae cells. First, some of the m R N A molecules appear to be transcribed from repetitive DNA sequences. The second conclusion is that the redundancy of the DNA template [24] transcribed into abundant poly(A) R N A is a b o u t 35 sequences (Table II). These conclusions are based mainly on saturation-hybridization experiments (Figs 3 and 5) and on measurement of the rate of poly(A) m R N A • D N A hybrid formation (Fig. 5). The fraction of m R N A that is not retained by poly(U)-Sepharose has generally been thought to result from breakages in m R N A during isolation; since there is only one stretch of poly(A) in each mRNA, a single breakage would release a molecule altogether lacking poly(A). Thus, it seems that the abundant species of poly(A) RNA retained by poly(U)-Sepharose following our R N A extraction procedure, selectively resist breakage of the poly(A) stretch. It is plausible that most of the poly(A) m R N A retained by poly(U~Sepharose following our isolation procedure represents a precursor for killer-RNA as judged by the dependence of the kinetic complexity of poly(A) RNA on the genomic complexity of killer-RNA (Table I). It therefore seems probable that approximately 80% of the m R N A (mol. wt 9.6 + 0.6 • 106 ) is degraded before mature killer-RNA appears in the form of double-stranded R N A (mol. wt 2.5 • 106 and 1.4 • 106 ). That such post-transcriptional processing of yeast prem R N A might occur has been previously suggested [2]. DNA renaturation experiments have shown that there is little redundant DNA in yeast cells, and that most of the DNA has a kinetic complexity of 0.92 1010 daltons [25]. While the location of the DNA coding for killer factor R N A is still unknown, it seems to be non-mitochondrial [7]. Our results, using poly(A)-containing R N A suggest that approximately 2.5% of the DNA with the density of nuclear DNA encodes for killer-RNA and that there are about 35 cistrons for this R N A in the haploid genome of S cerevisiae. However, the possibility exists that some classes of poly(A) R N A may be indetectable by the experimental technique used here because they are present at a lower concentration than the least concentrated sequences observed to react and hence fail to react significantly with the complementary DNA sequences. If the class of poly(A) R N A described here, belongs to a longer-lived class of mRNA, the abundancy of this class is due to degradation of the other classes. On the assumption that most of the poly(A) RNA prepared by our isolation procedure represents killer-RNA, the data can also be used to calculate the number of copies there are per cell of killer precursor mRNA. Based on the fact that 108 haploid cells contain a b o u t 55/~g of RNA (Hartwell, personal communication) of which a b o u t 3% is poly(A)-containing m R N A (Shalitin, C. and Fischer, I., unpublished), the minimal no. of abundant RNA copies ( ~ 9 • 106 daltons) per haploid cell equals approx. 1000. The number of copies of R N A killer formed in the yeast cell under different growth conditions, is n o w under investigation. Moreover, present studies are directed at establishing the identity of abundant poly(A) R N A with RNA isolated from whole virus-like particles.

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Acknowledgements Part of this work was performed while the senior author was on sabbatical leave at the Department of Biology, University of California, San Diego, La Jolla, California. The authors wish to thank E.P. Geiduschek in whose laboratory this study was initiated, for his support and useful discussions. Thanks are due to Mike Philip and Rivka Pusin for capable technical assistance. The authors acknowledge with thanks the support of the Technion Research Fund. We also t h a n k Aaron J. Shatkin for his generous gift of reovirus RNA sample.

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Note added in proof (Received October 22nd, 1975) Recently, Whitney and Hall (personal communication) have found S. cerevisiae nuclear DNA to contain about 5.8 • 109 daltons of DNA in a haploid nucleus instead of 1.25 • 1010 daltons previously described [26]. Taking 5.8 • 109 daltons as the DNA content of a haploid nucleus, our results suggest a b o u t 16 template sequences transcribed into a b u n d a n t poly(A) RNA.