469

Biochimica et Biophysica Acta, 454 ( 1 9 7 6 ) 4 6 9 - - 4 7 9 © El sev ie r /Nor th -Hol l and B i o m e d i c a l Press

BBA 98767

CHARACTERIZATION OF THE RNA TRANSCRIBED IN VITRO FROM NATIVE MAMMALIAN DNA BY ESCHERICHIA COLI RNA POLYMERASE

A N G E L A L O N S O *, G.D. BIRNIE, L. K L E I M A N **, A.J. M A C G I L L I V R A Y and J O H N P A U L

Beatson Institute for Cancer Research, 132 Hill Street, Glasgow, G3 6UD (U.K.)

(Received April 20th , 1976)

Summary

High-molecular-weight native mouse DNA was transcribed with Escherichia coli RNA polymerase under low salt conditions, and the nature of the DNA se- quences transcribed determined by molecular hybridization. The results indi- cated that E. coil RNA polymerase does not transcribe the sequences in native mouse DNA randomly under these conditions. First, hybridization with a large excess of mouse DNA showed that no more than 5% of the RNA synthesized had been transcribed from repeated sequences in the DNA. Second, hybridiza- tion with tracer amounts of labelled non-repeated mouse DNA indicated that the bulk of the RNAhad been transcribed from less than 1% of the non-repeated sequences and only about 10% had been transcribed from a further 25% of these sequences; the remaining non-repeated sequences in the DNA, amounting to 50% of the genome, were not represented in the RNA synthesized in vitro to any detectable extent. Third, the proportion (40%) of complementary DNA transcribed from mouse-liver nuclear polyadenylated RNA which hybridized with the RNA synthesized in vitro was significantly greater than would have been expected if transcription had been random.

The data have also been interpreted as indicating the presence of two types of initiation site for E. coli RNA polymerase in the non-repeated sequences in mouse DNA. The frequencies of their occurrence have been calculated to be one per 150 000 base-pairs and one per 5000 base-pairs, respectively.

* Present address: Institut fiir experimentel le Pathologie, Deutsches Krebsforschungszentrum, 69 Heidelberg (G.F.R.).

** Present address: Lady Davis Institute for Medical Research, Jewish General Hospital, Montreal, Quebec H3T 1E2 (Canada).

470

Introduct ion

Bacterial RNA polymerases, in particular Escherichia coli RNA polymerase, have been widely used to transcribe chromatin in vitro, in experiments designed to determine the way in which chromatin proteins impose specific limitations on the sequences transcribed in the DNA to which they are bound. The tran- scription of native DNA in vitro by E. coli RNA polymerase has been studied extensively (see ref. 1), but most of the information available has been ob- tained using bacteriophage DNAs as templates. Despite the extensive use of this enzyme for the transcription of chromatin in vitro, there have been few studies of the actual sequences in the RNA transcribed by E. coli RNA polymerase from native mammalian DNAs. Although Cedar and Felsenfeld [2] showed that there are a large number of sites (about one per 1200 base-pairs) in native calf- thymus DNA on which initiation of transcription by E. coli polymerase occurs, it was not possible to tell from their experiments whether the initiation sites are clustered or are distributed evenly throughout both repeated and non-re- peated sequences in the DNA. However, data obtained from DNA • RNA hy- bridization experiments by Bishop and his collaborators [3,4] have indicated that the initiation sites recognised by E. coli RNA polymerase are not equally distributed in mammalian DNA. These experiments showed that, although a large proport ion of the repeated DNA sequences were transcribed by the en- zyme, only a small proport ion of the RNA synthesized had been transcribed from these sequences, and a much larger proport ion comprised transcripts from non-repeated sequences.

As part of our studies on the transcription of chromatin in vitro, we under- took a detailed examination of the sequences in the RNA transcribed in vitro by E. coli RNA polymerase from native mouse DNA, using D N A . RNA hybridi- zation techniques which have recently been developed to determine the relative abundances of the RNAs transcribed from different DNA sequences. The data in this report show that, in the transcribed RNA, (i) the repeated DNA sequen- ces are under-represented; (ii), at least 75% of the non-repeated sequences (that is, half of the genome) is represented to a vanishingly small extent, if at all; and (iii), a very small proport ion (less than 1%) of the non-repeated sequences are grossly over-represented.

Materials and Methods

All possible precautions were taken to eliminate ribonucleases and heavy met- al ions [5]. All glassware used in the synthesis of RNA, and in the isolation and hybridization of RNA and DNA (including capillaries), had been siliconized ( 'Repelcote ' ; Hopkins and Williams Ltd., Chadwell Heath, Essex, U.K.), then sterilized by rinsing with 0.1% aqueous diethylpyrocarbonate and drying at 100°C. Solutions in which hybridization reactions were done had been passed through Chelex-100 resin (Bio-Rad Laboratories, Richmond, Calif., U.S.A.) to remove heavy metal ions, then sterilized by treatment with diethylpyrocarbo- nate; excess die thylpyrocarbonate was destroyed by heating at 60~C for 18 h. Suspensions of Sephadex (Pharmacia, Uppsala, Sweden) were sterilized by shaking them with die thylpyrocarbonate (0.1%), then heating at 60 ° C for 18 h.

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High-molecular-weight DNA. Nuclei were prepared from livers of young adult mice by the sucrose/citric acid procedure [6] as described previously [5]. Na- tive DNA was extracted from these nuclei by the method of Gross-Bellard et al. [7]. The molecular weights of the DNA molecules were calculated from their contour lengths, measured in the electron microscope by Dr. Lesley Coggins using the aqueous technique of Davis et al. [8]. A total of 80 molecules, se- lected at random, were measured. The internal standard was SV40 DNA, kindly given by Dr. R. Eason. The DNA was stored at --20°C in 0.1 M NaCl.

RNA polymerase. The holoenzyme was extracted from E. coli (M.R.E. 600) as described by Burgess [9], omitting the phosphocellulose chromatography step. The enzyme was then absorbed to, and eluted from, a column of DNA- cellulose as described by Bautz and Dunn [10] except that high-molecular- weight calf-thymus DNA (prepared as described for mouse-liver DNA) was used in place of T4 DNA. This procedure successfully removed ribonuclease from the RNA polymerase, as judged by the failure of the enzyme preparation to de- grade E. coli ribosomal RNA.

Transcription of DNA. The incubation mixture contained 40 mM Tris • HC1, pH 7.9, 10 mM NaC1, 1 mM dithiothreitol, 0.1 mM EDTA, 12 mM MgC12, 1 mM MnC12, 0.4 mM ATP, 0.4 mM GTP, 0.4 mM CTP, 0.4 mM UTP and, in each 1 ml, 100 pg of mouse-liver DNA and 160 units [9] of E. coli RNA poly- merase. When labelled RNA was required, the UTP was replaced by 40 pM [ 3 H]- UTP (300 pCi/ml; Radiochemical Centre, Amersham, U.K.). After incubation at 37°C for 30 min, RNA was isolated from the mixture by the procedure de- scribed previously for the isolation of RNA from nuclei [5]. The RNA eluted in the void volume of the Sephadex G-100 column was precipitated by the ad- dition of NaC1 to 0.2 M and 2 vol. of ethanol; after storage overnight at --20°C, the precipitated RNA was recovered by centrifugation (10 000 × g for 15 min), washed with ethanol and dried under a stream of nitrogen. The RNA was de- salted by passage through a column of Sephadex G-50 and a 5 mm pad of Chelex-100 equilibrated with sterile distilled water, and lyophilized. The molec- ular weight of the RNA was measured by electrophoresis in 3.8% polyacryl- amide gels in 100% formamide, essentially as described by Staynov et al. [11].

Hybridization o f mouse non-repeated DNA with RNA. Highly labelled non- repeated ("unique") DNA was isolated from mouse Friend cells (clone M2) grown at 37°C in a 250 ml spinner culture [12]. When the cells were in mid-log phase of growth (8 • l 0 s cells/ml), 2.5 ml of a mixture of aminopterin (2 • 10 -s M), deoxyadenosine (7 • 10 -3 M) and glycine (3 • 10 -2 M) was added, followed 15 min later by 2.5 mCi of [Me-3H]thymidine (40.3 Ci/mmol; Radiochemical Centre, Amersham, U.K.). The cells were collected by centrifugation 23 h later, and washed with phosphate-buffered saline. DNA was isolated as described by Hell et al. [13], sheared by ultrasonication, desalted on Sephadex G-50 (with a pad of Chelex-100) in sterile distilled water, and lyophilized. The DNA was re- dissolved in 0.5 M NaC1, 25 mM HEPES, 0.5 mM EDTA, 50% formamide, pH 6.8, at 5 mg/ml, sealed in capillaries, heated at 70°C for 5 min and incubated at 43°C for 4.25 h (Cot -'- 250 mol • s • litre-'). Single- and double-stranded DNA were separated by hydroxyapat i te column chromatography [14]; the single stranded DNA was isolated and re-cycled through the hybridization and frac- t ionation procedure. The unhybridized fraction of the DNA was finally desalted

472

on Sephadex G-25 and lyophilized. This procedure yielded non-repeated mouse DNA of mean single-stranded molecular weight 5 • 104 daltons (as determined by alkaline sucrose-gradient centrifugation [13] ), and specific activity 8.7 • 105 counts/min/pg. When annealed with a 2000-fold excess of total, sheared mouse DNA, 85--90% of the labelled non-repeated DNA formed duplexes.

Appropriate volumes of RNA and labelled non-repeated DNA in sterile, dis- tilled water were mixed, lyophilized and redissolved in 0.24 M phosphate buffer (equimolar Na2HPO4 and NaH2PO4; pH 6.8) containing 0.1% sodium lauryl sulphate. Portions of the solution were sealed in glass capillaries, denatured by heating at 100°C for 5 min then incubated at 60°C. The capillaries were flushed out with 1 ml of 0.03 M phosphate buffer, pH 6.8, and single- and double- stranded molecules were separated by chromatography on hydroxyapat i te at 60°C [14]; the radioactivity in each fraction was measured by liquid scintilla- tion spectrometry in Instagel (Packard Instrument Ltd., Caversham, Berks., U.K.).

Hybridization of [3H]RNA with whole mouse DNA. DNA was isolated from whole mouse embryos as described by Hell et al. [13], sheared by ultrasonica- tion and desalted by chromatography on Sephadex G-50 (with a pad of Chelex- 100) in sterile distilled water. The mean single-stranded molecular weight of the DNA was 1 . 5 . 1 0 s. Appropriate volumes of [3H]RNA and sheared DNA in sterile distilled water were mixed, lyophilized and redissolved in 0.12 M phos- phate buffer (equimolar NaH2PO4 and Na2HPO4; pH 6.8) containing 0.1% so- dium lauryl sulphate. Portions of the solution were sealed in glass capillaries, denatured by heating at 100°C for 5 min then incubated at 65°C. The capil- laries were flushed out with 1 ml of 0.24 M phosphate buffer, and the amount of [3H]RNA which had formed stable hybrids was determined by measuring the proport ion which was not degraded by incubation with ribonuclease A (200 pg/ml) at 37°C for 20 min [4].

Hybridization of cDNA with RNA. Mouse-liver nuclei were prepared by the sucrose-citric acid method [5,6] and total RNA was isolated essentially as de- scribed previously [5] except that the nuclei were first lysed in sodium lauryl sarcinosate and incubated for 45 min with 500 pg/ml proteinase K [15] (British Drug Houses, Poole, Dorset, U.K.). Polyadenylated [poly(A) ÷ ] RNA was isolated following affinity chromatography on oligo(dT)-cellulose (Collaborative Re- search Inc., Waltham, Mass., U.S.A.) as described previously for polysomal poly(A) ÷ RNA [16]. The RNA was transcribed, and the cDNA (spec. act., 5000 cpm/ng) fractionated and isolated, as described previously [5]. The mean mo- lecular weight of the cDNA was 1 • 10 s daltons. The hybridization reactions were done at 43°C in 0.5 M NaC1, 25 mM HEPES, 0.5 mM EDTA, 50% forma- mide, pH 6.8 [5,16], and the proport ion of cDNA in hybrid was determined [ 5] by measuring the proport ion of radioactivity rendered acid soluble by incu- bation with ~S, nuclease (Sigma Chemical Co. Ltd., London, U.K.).

Results

Transcription of DNA The DNA used was prepared by the method of Gross-Bellard et al. [7] in or-

der that the template should be of high molecular-weight and as free as possible

473

of single-strand nicks. As calculated from contour lengths in electron micro- graphs, 90% of the DNA was in molecules greater than 6.5 • 106 daltons while the average molecular weight of the DNA was 13.2 • 106; estimation of the sin- gle-strand molecular weight by rate-zonal sedimentation in alkaline sucrose gra- dients indicated there was 1--2 scissions per DNA strand on average. The E. coli RNA polymerase was prepared as described by Burgess [9], omitting the phos- phocellulose column step in order to retain sigma factor. The enzyme was de- void of ribonuclease activity and was template-dependent; transcription of the DNA in vitro yielded 40--45 pg of RNA per 100/~g of DNA. Electrophoresis on denaturing formamide-polyacrylamide gels showed that most (80%) of the RNA synthesized consisted of molecules around 4 • l 0 s daltons; the remainder had a molecular weight of about 6 • 104 (Fig. 1).

Hybridization of RNA to non-repeated DNA To determine what proport ion of the non-repeated sequences in the high-

molecular-weight DNA was represented in the in vitro transcripts, a large (10 000-fold) excess of the ENA was hybridized with highly radioactive non- repeated mouse DNA. The reaction appeared to be complete when 12--13% of the non-repeated DNA had hybridized with the RNA (Fig. 2), indicating that, if transcription was asymmetric, about 25% of the non-repeated sequences in the genome had been transcribed by E. coli polymerase. The possibility that a higher proport ion of the non-repeated sequences had been transcribed cannot be ruled out, but it is clear that, if they were, the abundance of their transcripts was extremely low (less than 1% of the total RNA).

About 70% of the sequences in mouse DNA are non-repeated sequences [17].

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Fig. 1. Prof i le of R N A t r ansc r ibed in v i t ro f r o m na t ive m o u s e D N A af ter e l ec trophores i s in a po lyac ry l - a m i d e gel in 1 0 0 % f o r m a m i d e as desc r ibed in Mater ia ls and Methods .

Fig. 2. T i m e - c o u r s e of h y b r i d i z a t i o n of R N A transcribed in v i t ro f r o m na t ive m o u s e D N A w i t h non-re- pea ted m o u s e [ 3 H ] D N A . The c o n c e n t r a t i o n of R N A was 2 to 20 m g / m l , and the r a t io o f R N A : D N A ,

5 0 0 0 to 10 0 0 0 : 1. Se l f -annea l ing of the non-repea ted D N A was m e a s u r e d in h y b r i d i z a t i o n react ions d o n e in paral le l w i th E. c o U R N A at the s a m e c o n c e n t r a t i o n s ; the a m o u n t o f n o n - r e p e a t e d D N A which r e a n n e a l e d (1 - -2%) has been s u b t r a c t e d f r o m each point .

474

The base-sequence c o m p l e x i t y o f the hap lo id m o u s e g e n o m e is 1.8 • 10 ~ 2 (ref. 18) , so t h a t o f the n o n - r e p e a t e d sequences is 0.7 × 1.8 • 1012 da l tons , t ha t is, 1 .26 • 1012 da l tons . Since 12 - -13% of the n o n - r e p e a t e d D N A sequences hybr id - ized wi th the R N A syn thes ized in vi t ro , the to ta l base-sequence c o m p l e x i t y o f the R N A is 0 .125 × 1.26 • 1012 da l tons , t h a t is, 1.6 • 1011 da l tons . The ra te o f an R N A . D N A h y b r i d i z a t i o n reac t ion , as measu red b y the p a r a m e t e r Rot112 (R0 = init ial c o n c e n t r a t i o n of R N A in moles o f nuc leo t ide / l i t r e and t1/2 = t i m e in s fo r the reac t ion to be 50% c o m p l e t e d ) , is d i rec t ly p r o p o r t i o n a l to the base- sequence c o m p l e x i t y o f the R N A (see refs. 19,20) . Thus , since the Rotll2 of the r eac t ion (done u n d e r exac t l y the same cond i t ions of t e m p e r a t u r e , and salt and R N A c o n c e n t r a t i o n ) b e t w e e n globin m R N A and the c D N A t ransc r ibed f r o m it, was 9 • 10 -4 mo l • s • litre - t , and the base-sequence c o m p l e x i t y of globin m R N A is 4 • 10 s da l tons [21 ] , i t can be ca lcu la ted t h a t the Rot112 o f a h y b r i d i z a t i o n reac t ion wi th R N A of base-sequence c o m p l e x i t y 1 . 6 . 1 0 1 1 dal- tons shou ld be

1.6 • 1011

4" l 0 s × 9 • 10 -4 m o l • s • l i tre -1, i.e. 3.6 • 102 mol • s • l i tre -1.

However , the obse rved Rot1/2 of this r eac t ion was 3.5 • 103 m o l • s -1 ' litre -1 (Fig. 2) and, the re fo re , the p r o p o r t i o n o f the R N A which was driving the hy- b r id iza t ion r eac t ion was

3.6 • 102 i.e. 0.10.

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This means tha t , whereas a b o u t 12% of the n o n - r e p e a t e d D N A sequences had been t ranscr ibed , the t ranscr ip t s f r o m these sequences c o n s t i t u t e d on ly 10% o f the to ta l R N A syn thes ized .

Hybridization of RNA in DNA excess The p r o p o r t i o n o f the R N A h o m o l o g o u s to r epea t ed D N A sequences was de-

t e r m i n e d b y measur ing the ra te o f f o r m a t i o n o f r ibonuc lease- res i s tan t hybr ids in a r eac t i on b e t w e e n [ 3 H ] R N A t ransc r ibed f r o m h igh-molecu la r -weigh t D N A and a large (5500- fo ld ) excess of f r a g m e n t e d , to ta l m o u s e D N A (Fig. 3). A b o u t 5% of the [3 H] R N A b e c a m e r ibonuc lease- res i s tan t a t Co t values be low 102 mol • s • litre-1; since 2- -3% of the [ 3 H ] R N A b e c a m e r ibonuclease- res i s tan t w h e n i n c u b a t e d wi th E. coli DNA, this indicates t h a t on ly a small p r o p o r t i o n (less t han 5%) o f the R N A had been t ransc r ibed f r o m the r epea t ed sequences in the DNA. The r e m a i n d e r o f the [3 H] R N A which hybr id ized did so at an e x t r e m e l y s low rate . The C0t a t which the R N A - D N A reac t ion was ha l f - comple t e was 5 • 103 mol • s • litre -1, whereas the Cotl/2 of the reanneal ing reac t ion b e t w e e n the n o n - r e p e a t e d D N A strands was 1--2 • 103 m o l • s • l i tre -1. I t has been s h o w n t h a t the ra te of D N A • R N A hyb r i d i za t i on is s lower than t h a t o f D N A reanneal ing , a f ac to r o f 4 having been d e t e r m i n e d in reac t ions a t 37°C in 0 .45 M NaCl, 50% f o r m a m i d e [22] . I f a s imilar re la t ionsh ip holds in 0 .12 M phos- p h a t e a t 60 ° C, Fig. 3 shows tha t the bu lk o f the R N A which hyb r id i zed did so to n o n - r e p e a t e d D N A sequences .

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Fig . 3. T i m e - c o u r s e o f h yb r id i z a t ion o f [ 3 H ] R N A transcribed in v i tro f r o m nat ive m o u s e D N A w i t h sheared tota l m o u s e D N A . The c o n c e n t r a t i o n o f D N A w a s 5 t o 2 0 m g / m l , and the rat io o f D N A : [ 3 H I R N A w a s 5 5 0 0 : 1; the p r o p o r t i o n o f [ 3H] R N A w h i c h was r ibonuclease-res i s tant a f ter i n c u b a t i o n w i t h E. coli D N A ( 2 - - 3 % ) has n o t b e e n subtrac ted .

However, even at completion of the reaction, no more than 40% of the input RNA had formed ribonuclease-resistant hybrids. While some degradation of the RNA undoubtedly occurred during the course of the hybridization reaction, this was not severe enough to cause a detectable increase in the proportion of acid-soluble RNA fragments. Thus, although degradation may have caused some reduction in the rate of the hybridization reaction, this was insufficient to account for the observation that less than half of the input RNA formed hybrids. It is unlikely that this result was due to the synthesis of a significant proportion of non,complementary polynucleotide material in the transcription reaction since the reaction was template-dependent and, moreover, the DNA was transcribed under conditions known to suppress such reactions [ 1]. Similar results have been obtained by Melli et al. [4], who found that no more than 50--55% of the RNA synthesized in vitro on rat DNA formed duplexes when hybridized with a vast excess (10 s fold) of DNA. The failure of a large propor- tion of the [3H]RNA to hybridize in a DNA-driven reaction of this kind indi- cates that there is an excess of some of the sequences in the RNA over their complementary sequences in the DNA. It can be calculated that, in this type of reaction, 50% of the RNA would form hybrids at a ratio of DNA to RNA of 4000, even if the RNA sequences represented only 0.4% of the sequences in the DNA. Since the 10% of the RNA which had been transcribed from the non- repeated DNA sequences (Fig. 2) will have hybridized completely at this ratio of DNA to RNA, the remainder of the ttNA must have been transcribed from a very small proportion of the non-repeated s e q u e n c e s - certainly from less than 1% of these sequences and perhaps from as little as 0.1% of the DNA.

Hybridization of RNA to cDNA Single-stranded complementary DNA (cDNA) transcribed from mouse-liver

nuclear poly(A) ÷ RNA represents those RNA sequences which are transcribed and polyadenylated in vivo in the liver and which are, in part at least, precur-

476

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Fig. 4 . 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 o f c D N A t r a n s c r i b e d f r o m m o u s e - l i v e r n u c l e a r p o l y ( A ) * R N A w i t h

(a) u n f r a c t i o n a t e d m o u s e - l i v e r n u c l e a r R N A ( I l ) ; (b) m o u s e - l i v e r n u c l e a r p o l y ( A ) * R N A ( o - - o ) ;

a n d (c ) R N A t r a n s c r i b e d in v i t r o f r o m n a t i v e m o u s e D N A ( A - - A). T h e c o n c e n t r a t i o n o f R N A w a s 1

t o 1 0 m g / m l a n d t h e r a t i o o f R N A : c D N A , 3 0 0 t o 9 0 0 0 : 1. S e l f - a n n e a l i n g o f t h e c D N A ( . . . . . . ) w a s m e a s u r e d i n h y b r i d i z a t i o n r e a c t i o n s d o n e in para l l e l w i t h E. coli R N A at t h e s a m e c o n c e n t r a t i o n s .

sors of polysomal mRNA sequences (see ref. 23). Thus, measurement of the ex- tent to which this cDNA forms hybrids with RNA transcribed from DNA in vitro gives an indication of the extent of homology between the sequences in in vitro RNA transcripts and nuclear poly(A) ÷ RNA. When the cDNA was annealed with its template RNA, 80% formed S~-nuclease-resistant hybrids; a similar pro- portion hybridized with total nuclear RNA. In contrast, 30% of the cDNA hy- bridized when annealed with a 9000-fold excess of the RNA synthesized in vitro (Fig. 4). After normalising to allow for the proportion of the cDNA which was non-hybridizable, the data in Fig. 4 indicate that at least 40% of the cDNA was complementary to the sequences transcribed in vitro from mouse DNA.

Fig. 4 also shows that the rate of the reaction between the cDNA and the in vitro RNA transcripts was markedly slower than that of the reaction between the cDNA and its template RNA. Since it is not known whether the relative abundances of the hybridizing RNA sequences in the nuclear poly(A) ÷ RNA were similar to those in the RNA synthesized in vitro, it is not possible to make a definitive assessment of the concentration in the latter of those sequences which hybridized to the cDNA. However, if the relative abundances were sim- ilar, by comparing the Rott/2 values of the two reactions it can be estimated that they constituted less than 10% of the total RNA transcribed from DNA in vitro.

Discussion

The picture which emerges from these analyses is one of marked non-ran- domness of transcription of high-molecular-weight native mouse DNA by E. coli RNA polymerase ho loenzyme. First, although 20% of the sequences in mouse D N A belong to the middle-repetitive class [17] , only a very small proportion (less than 5%) of the RNA had been transcribed from these sequences. This con-

477

clusion is not at variance with previous data which indicated that a high propor- tion of the middle-repetitive sequences of mammalian DNA are transcribed by bacterial polymerase [3,24]. In those experiments, saturation of middle-repeti- tive DNA sequences was obtained in DNA • RNA hybridization reactions only at high ratios of RNA to DNA; moreover, less than 5% of the input RNA hy- bridized under the conditions used [3]. Second, although almost all of the RNA had been transcribed from the non-repeated DNA sequences of the mouse ge- nome, no more than a quarter of these DNA sequences were represented in the RNA. Moreover, even those non-repeated DNA sequences which had been tran- scribed were not all equally represented in the RNA; the major part of the RNA was composed of transcripts from less than 1% (possibly less than 0.1%) of the non-repeated DNA sequences, while the transcripts from the remaining sequen- ces made up no more than 10% of the total RNA synthesized. However, these data should not be taken to imply the existence of completely sharp demarca- tions between the abundance classes in the RNA synthesized. A more likely pattern is depicted in Fig. 5, which suggests the presence of an overlapping se- ries of abundance classes which fall into 3 main groups.

These data show that some of the sequences in non-repeated native DNA are preferentially transcribed by E. coli RNA polymerase in vitro. While it is pos- sible that the enzyme is capable of transcribing some sequences in native DNA more rapidly or frequently than others, a likelier proposition is that initiation of transcription occurs more readily on some sequences than on others. If this is correct, the pattern of transcription implies that there are two types of initiation site on the DNA, which could be analogous to (but not necessarily the same as) the class A and class B sites detected on phage DNA [25]. I t is un- likely that either one or both of these sites are ends and single-strand scissions in the DNA template since the DNA was prepared by a method which pro- duced high-molecular-weight DNA; moreover, ends and single-strand nicks might be expected to be randomly distributed throughout both the repeated

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478

and non-repeated sequences in the DNA. In addition, there is evidence that single-strand nicks in native DNA are not effective initiation sites for the syn- thesis of RNA chains [26].

Although the experiments were no t specifically designed for the purpose, by making a number of assumptions an estimate of the number of initiation sites on native mouse DNA can be obtained from the data. The sites giving rise to the major port ion of the RNA initiated transcription from no more than 1% of the non-repeated DNA sequences. Assuming asymmetry of transcription, the base-sequence complexi ty of the transcribed sequences was thus, at most, 0.01 X 1.9 • 109 base-pairs, that is 1.9 • 107 base-pairs. The molecular weight of the bulk of the RNA synthesized in vitro was 4 • l 0 s ; assuming that there was no degradation of the RNA after transcription, the base-sequence complexi ty of each DNA sequence transcribed was, therefore, 1.2 • 103 base-pairs. If there was only one initiation site per transcribed sequence then the number of initiation sites of this type was

1.9 • 107 i.e. about 1.6 • 104 per haploid genome. 1.2 103'

Making the same assumptions, the number of sites of the second type, which initiated transcription from 25% of the non-repeated DNA, can similarly be calculated to be about 4 • l 0 s per haploid genome. These data therefore sug- gest there is about one initiation site per 5000 base-pairs of non-repeated native mouse DNA whereas a more direct method of estimating this number indicated that there is one initiation site for E. coli polymerase per 1200 base-pairs on calf-thymus DNA [2]. The 4-fold discrepancy could reflect an intrinsic differ- ence between mouse and calf-thymus DNA; alternatively, it could be due to in- accuracies in the estimate from the data in this paper due, for example, to some clustering of the initiation sites. However, it should be noted that the technique used by Cedar and Felsenfeld [2] would not have distinguished between the two classes of initiation site which these data suggest are present.

Although Meilhac and Chambon [27] concluded that calf-thymus B and E. coli holoenzyme RNA polymerases initiate transcription at different sites on calf-thymus DNA, it is interesting to speculate on whether the sequences in na- tive mouse DNA which are transcribed in vitro by E. coli polymerase bear any relationship to the sequences which are transcribed in vivo. One possible clue is given by the exper iment in which cDNA homologous to nuclear poly(A) ÷ RNA was annealed with the RNA synthesized in vitro. If in vitro transcription were purely random with respect to those sequences represented in the cDNA, at most 25% of the cDNA would have been complementary to the sequences tran- scribed from DNA in vitro whereas, in point of fact, 40% of the cDNA formed hybrids. This difference is small, but significant. Moreover, the cDNA represents only the 3'-ends of the nuclear poly(A) ÷ RNA whereas RNA is transcribed in vivo from its 5'-end [28]. Thus, if transcription in vitro were initiated close to the same points as transcription in vivo, the ex ten t to which the cDNA hybrid- ized with RNA synthesized in vitro will have underest imated the degree of cor- respondence between the DNA sequences transcribed in vivo and those tran- scribed in vitro by E. coli RNA polymerase.

479

Acknowledgements

The Beatson Institute is supported by grants from M.R.C. and C.R.C.A.A. was on leave of absence from Deutscheskrebsforschungzentrum, Heidelberg, W. Germany, and was supported by an EMBO Fellowship; L.K. was an Ameri- can Leukemia Society Fellow. We are grateful to Dr. B.D. Young for invaluable advice and criticism, to Dr. Lesley Coggins for DNA molecular-weight deter- minations by electron microscopy, and to Dr. Anna Hell for a gift of high spe- cific activity mouse DNA.

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