Specificity of chromatin transcription in vitro

356

Biochimica et Biophysica Acta, 6 5 3 ( 1 9 8 1 ) 3 5 6 - - 3 6 7 E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

BBA 99857

SPECIFICITY OF C H R O M A T I N T R A N S C R I P T I O N IN VITRO ASYMMETRIC T R A N S C R I P T I O N O F THE GLOBIN GENE BY ESCHERICHIA COLI R N A POLYMERASE

E T I E N N E P A Y S * a n d R. S T E W A R T G I L M O U R

Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow, G61 1BD (U.K.) (Received December 24th, 1980)

Key words: Chromatin transcription; Globin gene; R N A polymerase, (Mouse fetal liver)

Summary The transcription of globin genes in mouse foetal liver chromatin and nuclei by exogenous Escherichia coli RNA polymerase is prone to artifacts due to RNA
© E l s e v i e r / N o r t h - H o l l a n d B i o m e d i c a l Press

357 separation of endogenous RNA; apparently as a result of RNA-dependent transcription by E. coli RNA polymerase the endogenous RNA accompanies the newly synthesized transcripts through the isolation procedure as a double stranded molecule, one strand of which is mercurated. Secondly, it is becoming clear that the nature of the divalent cation present in the transcription reaction essentially determined whether the transcription is predominantly RNAdependent [9,10]. In order to define these conditions further we have examined the transcription of the globin gene in foetal mouse liver chromatin and nuclei using specific probes for both globin and anti-globin RNA sequences and using different ionic conditions for RNA synthesis by the bacterial polymerase. Materials and Methods Purification of E. coli RNA polymerase and mouse foetal liver chromatin was carried out as described previously [9]. Preparation of foetal liver nuclei. Nuclei were prepared by a modification of the method of Marzluff [11]. Approx. 1 g 14
358 7.9)/0.2 M NaC1/0.1% sodium dodecyl sulphate) and the nucleic acid fraction precipitated with 2.5 vol. ethanol. The precipitate was dissolved in 10 mM TrisHC1 (pH 7.9) and heated for 5 min at 100°C. After cooling quickly and adjusting to 0.2 M NaC1 and 1% SDS the material was applied to a 3 ml column of thiol agarose (thiopropyl-Sepharose 6 B, Pharmacia) at 20°C. The column was washed with 20 vol. column buffer and then eluted with 5 ml column buffer containing 0.05 M ~-mercaptoethanol. The mercurated transcripts were precipitated with ethanol and dissolved in water. Hybridization analysis. The synthesis of globin [3H]cDNA and the hybridization analysis are described in a previous paper [9]. Results

Transcription of nuclei and chromatin in presence of 5 mM MgCl2. The levels of globin RNA sequences in the transcripts of foetal liver nuclei and chromatin are shown in Table I and Fig. 1. In the absence of E. coli RNA polymerase, no globin RNA sequences are generated from chromatin templates. Under identical conditions however nuclear transcripts are found and yield 0.17 ng globin RNA/mg DNA; this level can be reduced to 0.1 ng/mg DNA by the inclusion of 10 pg/ml a-amanitin in the incubation. This background level of hybridisable RNA is probably due to endogenous globin RNA which has been trapped nonspecifically on the thiol agarose column; it represents about 0.05% of the amount of endogenous globin mRNA in the nuclei (200 ng/mg DNA). In the presence of E. coli RNA polymerase, transcription of nuclei with or without a-amanitin leads to the appearance of 1.40 and 1.52 ng globin RNA/ TABLE I TITRATION

OF GLOBIN RNA IN NUCLEAR

AND CHROMATIN

TRANSCRIPTS

A l l f i g u r e s are d e r i v e d f r o m s a t u r a t i o n v a l u e s o f s e p a r a t e t i t r a t i o n a n a l y s e s , n . m . , n o t m e a s u r a b l e s p e c t r o photometrically. RNA

Nuclear transcripts without polymerase - - c~ a m a n i t i n + c~ a m a n i t i n ( 1 0 p g / m l ) with polymerase -- ~ amanitin + amanitin + a d a e d m R N A ** + actinomycin D (250 pg/ml) Chromatin transcripts without polymerase with polymerase + a d d e d m R N A ** + actinomycin (250 pg/ml)

Yield of transcription (#g RNA/mg DNA)

Globin RNA in t r a n s c r i p t s (ng/~g RNA)

Globin RNA (ng/mg DNA template)

n.m. n.m.

n.m. n.m.

0.17 0.10

1.31 1.31 1.14 0.30

1.16 1.07 1.94 1.07

1.52 1.40 2.21 0.32

n.m. 1.5 1.5 0.3

n.m. 0.27 0.36 0.31

n.m. 0.41 0.54 0.09

* I n t h e s e i n c u b a t i o n s c a r r i e r E. c o l i t R N A w a s a d d e d a n d t h e i s o l a t e d R N A a n a b s o l u t e m e a s u r e m e n t o f g l o b i n R N A in t h e p r e s e n c e o f carrier. ** A d d e d m R N A : 5 0 0 n g e x o g e n o u s g l o b i n m R N A / m g D N A t e m p l a t e .

titrated with cDNA

t o give

359 8C ~

70

/

/

I

-

-

-

-

'

-

4

60 ~ 5C ~_ 4 0 N

:8 3O C

/°'

/

!

z

i 11 /o ,,o - /

10

jlz o / •

"

F

RNA/¢ DNA( # g / n g ) Fig. 1. 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 of E. coli R N A p o l y m e r a s e t r a n s c r i p t s of f o e t a l liver nuclei a n d c h r o m a t i n m a d e in t h e p r e s e n c e of Mg 2+. V a r y i n g a m o u n t s of p u r i f i e d t r a n s c r i p t w e r e h y b r i d i z e d t o 0 . 0 5 ng globin c D N A a n d $1 n u c l e a s e r e s i s t a n c e of t h e c D N A d e t e r m i n e d as d e s c r i b e d in Materials a n d M e t h o d s . o c o m p l e t e n u c l e a r t r a n s c r i p t i o n ; o, n u c l e i + 10 # g / m i c~-amanitin; A n u c l e i + 10 p g / m l ~ - a m a n i t i n + 2 5 0 # g / m l a c t i n o m y c i n D; o . . . . . . o, n u c l e i w i t h o u t a d d e d p o l y m e r a s e a n d also 1 0 / l g / m l a-amanitin o ...... o. o, c o m p l e t e c h r o m a t i n t r a n s c r i p t i o n ; A c h r o m a t i n + 2 5 0 # g / m l a c t i n o m y c i n D; , , ehromatin without added polymerase.

mg DNA, respectively, while for chromatin the same estimation gives 0.41 ng globin R N A / m g DNA. It has been established that under the same transcription conditions transcripts of adult mouse liver chromatin do n o t contain detectable amounts of globin R N A (data not shown). It is clear that the appearance of globin m R N A in the transcription products depends on the presence of E. coli R N A polymerase and an active globin gene. These globin R N A sequences are synthesized on DNA. There have been a number of reports of anomalous RNA-dependent transcription (endogenous R N A primed) in chromatin systems which can give rise to very pronounced artifacts [6--9]. Here we present evidence that globin m R N A sequences present in nuclear and chromatin transcripts have arisen de novo and in an DNAdependent manner as judged b y a number of criteria. Later on we show the ionic conditions of transcription strongly influence template specificity. Different experiments were designed to check the involvement of DNA transcription in the appearance of giobin R N A in nuclear or chromatin transcripts. A c t i n o m y c i n D sensitivity. In the presence of 2 5 0 / l g / m l actinomycin D the level of globin sequences in nuclear transcripts is reduced b y 80--90% to a b o u t 0.3 ng/mg DNA. Under identical conditions chromatin transcription was reduced by 80% to a level of around 0.09 ng/mg DNA (Table I). Although very little R N A is synthesized under these conditions, the residual insensitive transcripts hybridise to cDNA in an identical fashion to whole transcripts (Fig. 1). R N A - d e p e n d e n t transcription. The addition of exogenous giobin m R N A to the incubation mixture leads to an enrichment of globin R N A in the transcripts (Table I). However, this enrichment (0.13 and 0.69 ng globin R N A / 5 0 0 ng exogenous globin m R N A , or 0.026% and 0.14% of added RNA, for

360 chromatin and nuclear incubations, respectively) is of the same order of magnitude as the background of nonspecific adsorption on thiol agarose (0.05%) found in control incubations of adult liver chromatin and globin mRNA. It will be argued later that these background levels are probably not due to residual transcription of the m R N A by E. coli RNA polymerase. Globin R N A sequences are synthesized de novo. If the hybridizable globin R N A in transcripts has arisen by de novo synthesis, these should be mercurated and furthermore it should be possible to isolate [3H]cDNA hybrids on thiol agarose. Experiments of this t y p e are described in a previous paper [9]. It was found that this procedure gave rise to poor recoveries of [3H]cDNA hybrids from thiol agarose, despite the fact that hybridization was essentially complete. It appears that extensive demercuration of transcript during the first purification on thiol agarose greatly reduces its capacity to bind a second time. We observed that this effect could be minimized by reducing the concentration of mercaptoethanol in the elution buffer to 50 mM and further b y immediately extracting mercatoethanol from column eluates by passing the drops through diethyl ether. Table II shows data obtained with this modified procedure. The binding of [3H]cDNA to thiol agarose was calibrated with globin m R N A which had been maximally mercurated in vitro and subsequently purified on thiol agarose. Quantitative binding of HgmRNA-cDNA hybrids is obtained with this RNA. As a control, nuclei from adult mouse liver were transcribed with HgUTP and non-mercurated globin m R N A added to the purified transcripts. The resulting hybrids show extremely low levels of binding. In addition to providing a background level for the procedure, these data also demonstrate that mercury groups are not transferable from mercurated to non-mercurated RNA. Transcripts from both nuclei and chromatin show an equal capacity for rataining cDNA hybrids to thiol agarose with an efficiency very close to that observed for chemically mercurated globin m R N A . We conclude from this data that virtually all the globin m R N A transcripts synthesized in vitro with E. coli polymerase arise de novo and are not due to endogenous RNA contamination. The synthesis o f globin R N A sequences is completely asymmetric. Scheme I describes the preparation of an anti-globin cDNA probe. This probe hybridizes to 'sense' globin cDNA (Fig. 2A) and to anti-globin RNA (transcribed on a

T A B L E II BINDING OF GLOBIN 3H cDNA/RNA

HYBRIDS TO THIOL AGAROSE

RNA

G l o b i n Hg m R N A Nuclei transcripts Chromatin transcripts Globin m R N A + adult liver Hg-transcriPts

cDNA binding

Hybridization

%

%

70.5 50.6 53.7 0.15

cpm

7403 5320 5651 24

76.1 61.0 65.3 80.5

Binding of hybridized cDNA

% max

93.6 74.2 80.5 100

%

% control

92.0 83.0 82.2 0.18

100 90.2 89.3 0.2

361

globin mRNA template as described in Ref. 9) (Fig. 2B), but is unable to hybridize to globin mRNA. Yield (%) Globin m R N A

100

Rever,~e transcriptase actinomycin D

Glohin eDNA

19.1

DNA polymerase A [3H]dCTP

Globin eDNA/[ 3 H]anti eDNA

5.6

nuclease Sl

Globin eDNA/[ 3 H]anti eDNA hybrids

3.5

t h e r m a l d e n a t u r a t i o n and hybridization to a 100-fold excess of globin mRNA

Globin e D N A / m R N A hybrids + [3 H]anti eDNA hydroxyapatite

[3 H]anti eDNA Jt

2.5

alkaline s u c r o s e gradient

9 S[3H]anti eDNA

0.2

Scheme I. Preparation of anti-globin eDNA (Ref. 9).

Using this anti-globin probe, no antisense RNA was found in either chromatin or nuclear transcripts provided that transcription was carried out in the presence of magnesium (Fig. 3). However, substantial amounts of antisense RNA were detected in manganese-promoted reactions (Fig. 3 and Table III). Transcriptional specificity depends on the presence of magnesium. The data above suggest that the DNA
A

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B

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.

.

.

cDNA(ng/ng

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[

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. . 200

~

300 cDNA

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a

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;

(rig /rig)

Fig. 2. H y b r i d i z a t i o n t i t r a t i o n c u r v e s o f 3 H - l a b e l l e d '9 S' a n t i - g l o b i n e D N A w i t h the 'sense' g l o b i n c D N A ( A ) , or a n t i - g l o b i n R N A (B). ( A ) 3 H - l a b e l l e d a n t i - e D N A w a s h y b r i d i z e d t o a 3 H - l a b e U e d e D N A prepaxat i o n o f t h e s a m e s p e c i f i c r a d i o a c t i v i t y ; t h e m a x i m u m h y b r i d i z a t i o n value ( 6 5 % ) w a s d e t e r m i n e d b y a n n e a l i n g the 3 H - l a b e l l e d a n t i - e D N A w i t h a lazge e x c e s s of u n l a b e l l e d 'sense' e D N A . (B) 3 H 4 a b e l l e d a n t i - c D N A w a s h y b r i d i z e d t o 'antisense' R N A ( o ) or g l o b i n m R N A ( o ) . T h e a n t i s e n s e R N A w a s s y n t h e s i z e d o n a g l o b i n m R N A t e m p l a t e by E. coli R N A p o l y m e r a s e in t h e p r e s e n c e o f I m M MnCI 2 and H g U T P . It w a s p u r i f i e d as t h e transcripts (see the M e t h o d s s e c t i o n ) .

362

60 50

jj~.~4 40 ~4

3o I

2O

10 0

10

20 30 RNA/ANTI e D N A ( # g / n g )

4~)

Fig. 3. H y b r i d i z a t i o n t i t r a t i o n c u r v e s on 3 H 4 a b e l l e d '9 S' a n t i - g l o b i n c D N A w i t h c h r o m a t i n or nuclei transcripts, o ©, n u c l e i + 1 m M MnCl 2; • -', c h r o m a t i n + 1 m M MnC12; ~, nuclei + 1 m M MnCl 2 + 0 . 1 5 M KCl; O, n u c l e i + 1 m M MnC12 + 0 . 1 5 M K C I + 5 m M MgC12; •, n u c l e i + 0 . 1 5 M KC1 + 5 m M MgC12 ; m c h r o m a t i n + 5 m M MgCl 2. N o t i c e t h e d i f f e r e n c e in t h e abcissa scale, c o m p a r e d to Fig. 1.

value of separate titration curves of the t y p e shown in Fig. 1, using cDNA and anti-globin cDNA probes. For chromatin transcribed in the presence of MnC12, yields of transcripts are high. However, both sense and antisense globin RNA is found in roughly equal amounts. The presence of salt has little influence on this result. The plateau values for the two probes, however, are different. We have presented evidence in a previous paper [9] that transcription under these conditions is essentially RNA
OF THE

CATIONS

1.63 2.94 1.50 1.31 3.51 4.25

Yield RNA (~g)

DIVALENT

Nuclei 1 m M MnC12 1 m M MnC12 ~ 0 . 1 5 M KC1 5 m M MgC12 5 m M MgC12; 0 . 1 5 M K C I 5 m M MgC12; 1 m M MnCl 2 5 m M MgC12 ; 1 m M MnC12 ; 0 . 1 5 M KCI

OF

0.15 M KCI 1 m M MnC12 1 m M MnCI 2 ~ 0 . 1 5 M KC1

0 . 1 5 M KC1

EFFECT

25 23 1.6 1.54 1.35 1.44

Chromatin 1 m M MnC12 1 m M MnCI2~ 5 m M MgCl 2 5 m M MgCl 2, 5 m M MgC12~ 5 m M MgC12 ;

Conditions

TRANSCRIPTS

SUMMARY

TABLE III LEVELS

OF

0 1.33 0.80 1.16 1.52 1.89

0.076 0.091 0.20 0.23 0.21 0.25

Sense

GLOBIN

0.25 0 0 0 0 0

0.1 0.12 0 0 0 0

Antisense

Globin R N A (ng//lg)

ON THE

0 3.91 1.20 1.52 5.33 8.02

1.9 2.1 0.32 0.35 0.28 0.36

Sense

0.41 0 0 0 0 0

2.5 2.76 0 0 0 0

AND CHROMATIN

0 54 72 76 73 70

46 48 75 76 70 72

Sense

40 0 0 0 0 0

63 65 0 0 0 0

Antisense

Maximal hybridization to c D N A

R N A IN N U C L E A R

Antisense

ANTIGLOBIN

Globin RNA (ng/mg DNA)

AND

~o O~ co

364 globin m R N A it contains. The presence of salt potentiates this effect considerably. As can be seen in Table III, magnesium~atalysed nuclear transcripts contain 0.08%--0.19% globin m R N A sequences, compared with the much lower levels for the corresponding chromatin transcripts. It is possible that during the isolation of chromatin from nuclei, mechanical stress renders non-transcribable DNA accessible to polymerase, thereby lowering the overall specificity of transcription. Discussion

In this study we have e m p l o y e d a n u m b e r of criteria to examine transcription of the globin gene in nuclear and chromatin templates, namely, the sensitivity to actinomycin D, the prevalence of RNA
4o

.~60

80 i

10 -3

•

10 . 2

10 -1

1

Cot

Fig. 4. H y b r i d i z a t i o n of fully m e r c u r a t e d • X D N A t r a n s c r i p t s t o ~ X D N A . T r a n s c r i p t i o n w a s c a r r i e d o u t in t h e p r e s e n c e o f 20 m M ~ - m e r c a p t o e t h a n o l as d e s c r i b e d by Z a s l o f f a n d F e l s e n f e l d [ 7 ] a n d t h e n p u r i f i e d o v e r S e p h a d e x G 5 0 ( e ) or o v e r t h i o l a g a r o s e (o). H y b r i d i z a t i o n r e a c t i o n s c o m p r i s e d 2 0 0 ng ~ X 1 7 4 D N A a n d 20 ng t r a n s c r i p t l a b e l l e d w l t h [ 3 H ] G T P a n d w e r e c a r r i e d o u t a t 4 3 ° C in 5 0 % f o r m a m i d e . T h e Cotl/2 f o r u n m e r c u r a t e d t r a n s c r i p t s u n d e r t h e s e c o n d i t i o n s is 1.3 • 1 0 - 2 M • s.

365

observed under the various ionic conditions must represent spurious endogenous R N A contamination. The Original observations with fully mercurated R N A were carried out with q)X 174 DNA transcripts which were made in vitro in the presence of mercurated UTP and mercaptoethanol w i t h o u t exposure to thiol agarose chromatography. However, the chromatin transcripts were isolated using this procedure which involves heating and exposure to high concentrations of mercaptoethanol. In view of our own findings and those of Broeckhoven and de Wachter [14] who showed that demercuration can take place rather rapidly even during R N A synthesis and certainly on exposure to thiol reagents, it is possible that the hybridization observed is due to demercuration of genuine transcripts rather than to endogenous contaminating RNA. We investigated this matter further b y synthesizing labelled, fully mercurated OX 174 transcripts according to Zasloff and Felsenfeld [7] and purifying them with or without exposure to thiol sepharose chromatography. Fig. 4 shows the back-hybridization of these transcripts to OX 174 DNA. Two points emerge. We find that full substitution with mercurated UTP does n o t completely inhibit hybridization. The reaction is slower than predicted; however, upon purification over thiol agarose this discrepancy disappears and the Cotlj2 value obtained is close to that for unmercurated QX 174 transcripts. These results agree with the findings of Brown and Balmain [18] which show that mercaptoethanol can act as a ligand for mercury groups and permits hybridization of fully substituted transcripts. Here the mercaproethanol in the transcription reaction partially fulfils this role. However, their results also suggest that thiol groups can be lost during purification in thiol-free media. This could explain why both Zasloff and Felsenfeld [7] and Beebee and Butterworth [19] found no hybridization with fully mercurated transcripts. The important conclusion from our data is that thiol agarose chromatography enhances the hybridizability of fully mercurated transcripts probably due to demercuration. Because of these arguments, in Zasloff and Felsenfeld's experiments with Mn 2÷ and Mg 2÷ transcripts it is difficult to distinguish RNA
366 this disappears in the presence of Mg 2÷ or mixtures of the two cations. The results obtained with nuclei suggest that the artefacts of Mn 2÷ synthesis can be partially reversed by the presence of salt. Although R N A copying is no longer apparent only incomplete DNA transcription is observed. This result is found consistently with our system. However, we cannot offer an explanation for it at present. The fact that a double round of RNA
367

Thus there is a growing body of evidence to show that when artefactual errors are avoided E. coli R N A polymerase can initiate transcription on accessible sequences in both nuclei and chromatin. It is probable that active sequences in nuclei are in a conformation which permits access by E. coli RNA polymerase as well as by DNAase I [ 1 9 , 2 0 ] . Moreover although the exogenous polymerase was used initially only as a probe, the transcript is asymmetric in the conditions used. This latter observation must lead one to consider the possibility that in nuclei the bacterial polymerase behaves in a manner similar to the eukaryotic polymerase. In this connection it is interesting to note that in the work of Lescure et al. [21] the suggestion has been made that the binding sites for E. coli polymerase and eukaryotic polymerase II on polyoma D N A are similar. It has however been shown [22] that in vitro initiation of transcription of eukaryotic genes by E. coli R N A polymerase is not accurate, using purified cloned D N A as template in the presence of a cytosol extract. Acknowledgements The Beatson Institute is supported by grants from M.R.C. and C.R.C. One of us (E.P.) received finance from E.M.B.O. and from the Fonds National de la Recherche Scientifique (Belgium). References 1 Axel, R., Cedar, H. and Felsenfeld, G. (1973) l>roc. Natl. Acad. Sci. U.S.A. 70, 2 0 2 9 - - 2 0 3 2 2 Gflmour, R.S. and Paul, J. (1973) Proc. Natl. Acad. Sci. U.S.A. 70, 3 4 4 0 - - 3 4 4 2 3 Steggles, A.W., Wilson, G.N., Kantor, J.A., Picciano, D.J., Falvey, A.K. and Anderson, F.W. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 1 2 1 9 - - 1 2 2 3 4 Barret, T., Maryanka, D., Hamlyn, P.H. and Gould, H.J. (1974) Proc. Natl. Acad. Sci. U.S.A. 71, 5057--5061 5 Chiu, J.F., Tsai, Y.H., Sakima, K. and Hnilica, L.S. (1975) J. Biol. Chem. 250, 9 4 3 1 - - 9 4 3 3 6 Zasloff, M. and Felsenfeld, G. (1977a) Biochem. Biophys. Res. C o m m u n . 7 5 , 5 9 8 - - 6 0 3 7 Zasloff, M. and Felsenfeld, G. (1977b) Biochemistry 16, 5135--5145 8 Giesecke, K., Sippel, A.E., Nguyen Huu, N.C., Croner, B., Hynes, N.E., Wurtz, T. and Shutz, G. (1977) Nucleic Acids Res. 3943--3958 9 Pays, E., Donaldson, D. and Gilmour, R.S. (1978) Biochim. Biophys. Acta 5 6 2 , 1 1 2 - - 1 3 0 10 Tsai, M.J., Tsai, S.Y., Chang, C.W. and O'Malley, B.W. (1978) Biochim. Biophys. Acta 5 2 1 , 6 8 9 - - 7 0 7 11 Maxzluff, W.F., Murphy, E.C. and Huang, R.C. (1973) BiochemistrY 12, 3 4 4 0 - - 3 4 4 6 12 Mehotra, B.D. and Khorana, H.G. (1965) J. Biol. Chem. 240, 1750--1758 13 F o x, C.F., Robinso n, W.S., Haselkorn, R. and Weiss, S.B. (1964) J. Biol. Chem. 2 3 9 , 1 8 6 - - 1 9 3 14 V a n B r o e c k h o v e n , C. and de Wachter, R. (1978) Nucleic Acids Res. 5, 2133--2151 15 Hguyen-Huu, M.C., Sippcl, A.A., Hynes, N.E., Croner, B. a nd Schutz, G. (1978) l~oc. Natl. Acad. Sci. U.S.A. 7 5 , 6 8 6 - - 6 9 0 16 Reff, M.E. and Davidson, R.L. (1979) Nucleic Acids Res. 6, 275--287 17 Crouse, C.F., Fo dor , E.J.B. and D o t y , P. (1979) Nucleic Acids Res. 6 , 3 7 1 - - 3 8 3 18 Brown, D. and Balmain, A. (1979) Nucleic Acids Res. 7, 2357--2368 19 Weint~aub, H. and Groudine, M. (1976) Science 193, 84 8--855 20 Paul, J., Zollner, E.J., Gilmour, R.S. and Birnie, G.D. (1978) Cold Spring Harbor Syrup. Quant. Biol. 42,597--603 21 Lescure, B., Dauget, C. and Yaniv, M. (1978) J. Mol. Biol. 124, 87---96 22 Wasylyk, B., Kedlnger, C., Corden, J., Brison, O.Z. and C h a m b o n , P. (1980) Nature 2 8 5 , 3 6 7 - - 3 7 3