Initiation of transcription of rDNA in rice

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Vol. 160, No. 3, 1989

Pages 1117-1123

May 15, 1989

INITIATION OF TRANSCRIPTION

OF rDNA IN RICE

K.Nandabalan and J.D.Padayatty

C e n t e r for Genetic E n g i n e e r i n g and D e p a r t m e n t of B i o c h e m i s t r y I n d i a n I n s t i t u t e of S c i e n c e B a n g a l o r e - 5 6 0 012, I n d i a Received March 17, 1989

Three direct repeats of 320, 340 and 238 n u c l e o t i d e s were detected u p s t r e a m to t h e 5' end of t h e 185 rRNA gene of an rDNA u n i t p r e s e n t on a 9.8 k b EcoRl f r a g m e n t of the r i c e D N A . The p r i m e r extension a n a l y s i s showed that the s i t e of i n i t i a t i o n of t r a n s c r i p t i o n i s i n t h e 1st r e p e a t at an A, t h e 623rd n u c l e o t i d e u p s t r e a m to t h e 5' end of t h e 18S rRNA gene. Different stretches of the i n t e r g e n i c s p a c e r DNA l i n k e d to t h e C h l o r a m p h e n i c o l a c e t y l t r a n s f e r a s e gene were t r a n s c r i b e d i n t h e i n t a c t n u c l e i of r i c e ~ e m b r y o s . The 51 n u c l e a s e p r o t e c t i o n z a n a l y s i s of t h e transcripts u s i n g [v P ] - l a b e l l e d Chloramphenicol acetyl t r a n s f e r a s e gene as t h e p r o b e s h o w e d the p r e s e n c e of m u l t i p l e p r o m o t e r s for F D N A transcription. ~ 1 9 8 9 A c a d e m i c Press, Inc. SUMMARY:

The all

other

rDNA i s to

genes

the

at even

for

the

eukaryotes, transcribed

185,

are

as

origin

between

transcription

of

are

start

the

identification

point

and

the rice v a r i e t y

related

located

the

promoters

the

of of

the

the

site

in

long

with

the

precrusor repeats for

of

rDNAs

species

within

long

25S

rRNAs

+20

to

rRNA in

initiation

the

plants,

repeats

like (1).

in The

initiation occurring upstream (IGS). show

(2).

in

tandem

intergenic spacer

transcription

closely

and

arranged

a single unit

18S rRNA gene i n

the

5.8S

The

Nucleotide

very

sequences

little

homology

promoters

for

the

rDNA

-150

nucleotides,

the

+1

being

(3).

In

IGS

of

of

this an

transcription

study, rDNA of

we

report

unit,

spacer

the

rDNA

in

IR20.

MATERIALS AND METHODS

S1 n u c l e a s e p r o t e c t i o n a s s a y This was done a c c o r d i n g to a m o d i f i c a t i o n (4) of t h e of H a r r i n g t o n and C h i k a r a i s h i ( 5 ) . Primer extension analysis Primer extension anaylsis was done according to the of T u r n b o u g ~ 9 et a l . ( 6 ) . N u c l e a r RNA (10 pg) (4) was m i x e d ng of 5 ' - [ ~" ~ - - 1 a-b-elled 15- met universal primer and 50 ng ABBREVIATIONS

: CAT-Chloramphenicol

acetyl

transferase;

method

method with 3 of the

IGS-Intergenic

spacer. 0006-291X/89 $1.50 1117

Copyright © 1989 by Academic Press, Inc. All rights of reproduction in any form reserved.

Vol. t60, No. 3, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

recombinant single stranded DNA bearing the insert, hybridized in 25 pl of 20 mM Tris-HCl, pH 8.0, 0.I M NaCI, 0.i mM EDTA at 60°C for 4 hr. Then, it was cooled to room temperature, 25 pl of the buffer containing 0.i M Tris-HCl, pH 8.0, I mM each of dATP, dCTP, dGTP, dTTP and 10 units of reverse transcriptase were added and incubated at 37°C for i hr. The extended products were precipitated with ethanol, dissolved in 80% formamide containing i0 mM NaOH, 1 mM EDTA, 0.025% Xylene cyanol and 0.025% Bromophenol blue, heated at 90°C for 3 min, chilled in ice and analysed on a sequencing gel, where the same insert DNA was sequenced. rDNA-Chloramphenicol acetyl transferase (CAT) gene constructs The RF DNA from the Ml3mpl8 or Ml3mpl9 clones bearing the 3.3 kb EcoRI-BamHI, the 0.35 kb BamHI or the 0.6 kb .B.amHI-SacI fragment covering the IGS of the rDNA "unit was prepared and digested with EcoRI and HindIII to release the fragments bearing the inserts. The 35SCATpUCI3 plasmid (7) (supplied by Nam-Hai Chua, Rockefeller University, New York) was restricted with BamHI and HindIII, the 0.8 kb fragment was separated by agarose gel electrophoresis and eluted. The HindIII-BamHI 0.8 kb fragment contained only the structural gene for CAT. The DNA fragments from the IGS were ligated to the 0.8 kb HindIII-BamHI fragment bearing the CAT gene and cloned at the EcoRI-BamHI sites in pBR322. Transcription in intact nuclei Transcription in intact nuclei was carried out by a modification of the method of Jost et al. (8). Nuclei (i0 O.D. units at 260 nm) isolated from 24 hr germinated rice embryos (4) were suspended in i00 pl of i0 mM Hepes, pH 7.9, 75 mM KCI, 0.5 mM dithiothreitol, i0 mM creatine phosphate and 20% glycerol. It was frozen in liquid nitrogen, thawed in ice, plasmid DNA (i pg), 600 pM each of ATP, CTP, GTP and UTP and i0 units of RNasin were added and incubated at 26°C for i hr with or without ~-Amanitin (0.5 ~g). The nuclei were then lysed with 0.5% SDS, deproteinized, and the nucleic acids were precipitated with ethanol. The DNA was then removed by digestion w i t h DNaseI (10 pg) a t 37°C for 2 h r and t h e RNA was d e p r o t e i n i z e d and p r e c i p i t a t e d w i t h e t h a n o l .

RESULTS AND DISCUSSION

Sequence of t h e IGS u p s t r e a m to the i8S rRNA gene By

screening

with

[32p]

unit

was

protection 3.3

and

the

and/or (9).

of

of

gene

sizes

by

and

the is

320,

1482

read

obtained

kb

at

from the

published

genes

the

sequence

340

nucleotides

rDNA

by

$1

nuclease

(data

not

shown).

analysis

BamHI-SacI

cloned

dideoxy

chain

termination

in

the

IGS

(Fig.2).

the

18S The of

starting

upstream The

rRNA IGS three from

the

rRNAs,

were

presence

1118

phage

fragments

(10).

by

238

the

of

[32p]

4 the

( F i g . 1)

0.35 k b BamHI, 0 . 6 k b

ladders

characterized and

with

Charon

containing

mapped

sequence

the

end

)t

insert

were

HindIII,

by

DNA i n

kb

fragments

nucleotides

5'

rice 9.8

restriction

sequenced

of

was

a

nucleotide

1.9

SacI-BamHl

sequence

gene

that

rRNA

kb

rRNA

restriction

and

of

bearing

The

EcoRI-BamHI,

0.7

library

clone

the

analyses

kb

nucleotides to

of

M13mp19 A

rRNA

EcoRI

a

obtained.

hybridization

The

an

rRNAs,

in

to

was

upstream long

of

18S 86

identical

to

the

direct

526th

method the

sequence

gene

the

M13mp18

18S

repeats

nucleotide

Vol. 160, No. 3, 1989

EcoRl

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Hindl[l

Hindlll

BamHI Sacl

C[al

BamHl

BgIll BamHI EcoRI

J.

185

0 255

6

5.85

~

q

(

I

255

9.8 kb

v//1//i~ v/zz////J

i

i

CAT

v//////zA

FiR.l: Organization of rRNA genes, s e q u e n c i n g s t r a t e g y and t h e CAT c o n s t r u c t s . The r e s t r i c t i o n fragment was cloned in M13mp18 or M13mp19, transfected into E.coli MvIIg0, grown, the recombinant phage was prepared, single stranded DNA was i s o l a t e d and s e q u e n c e d a c c o r d i n g to the dideoxy chain termination method. The d i r e c t i o n and e x t e n t of t h e n u c l e o t i d e s e q u e n c e o b t a i n e d a r e i n d i c a t e d . The rDNA-CAT c o n s t r u c t s were made as described under Materials and Methods. The hatched a r e a r e p r e s e n t s t h e CAT gene. The i n i t i a t i o n of t r a n s c r i p t i o n i s m a r k e d b y an a r r o w ( F i g . 2 ) .

upstream

to

and

3rd,

76,

83

and

repeats the

5'

end

of

2rid

and

3rd

120

the

Within

near each

nucleotides

10096

rRNA are

However, homology,

repeat long,

i8S

repeats

6496 r e s p e c t i v e l y .

display

ends.

about

the and

there which

8ene.

the while

is by

The

ist

homologous

a

to

central

duplication

2nd,

of

occur

conserved could

1st

extent

region

variations highly

and the

of

these towards

block have

of

given

CGTGTAGGATCAGATG•ACAATACGGAAACAGACGGAAATTAGTTGCCCATTTGACAAAGCGCGATGTATTTCGCCTTGTTTTTGGCCTGGAATGCTTCG

-- 760

GATCTCTTGATCGTATA~CGCTCCTCAACGATGCCGATATAGCCGGCCAACAGCTTAATTG~TTTGCGTCCCGGTGTGGTG~TCCTTGCGGGGTTCGAGA

-- 660

GAAGGGAAATGTTGCTAGTACGGT~GAAGCTTGAGTGGAGGCACCGATGTGATGAAATTGCTACGCGAACGGGACGGTGGTGCATCTAACCTTACACGG $ 3 •GGAAAGGATTGAAAGGTTGGTA•GGGA••GTTT•AAGGAAAGTGCACCTGACAAAGCGCGGTGTTTTTCGC•TTGTTTT•GACCTGGAATGCTTCGGAT

--560

CCCTTAAACGCATACCGCACCTAAATGATGCCGGTATAGCCGGCCAACAGCTTAATTGCTTTGCGTACCCGGTGTAGCTCGCTTCG~GGGGTCTCCGAGA

-- 360

GAAGGGTAGATCTTGGTCAGTCAGTACGGTC~GAGCTCTGAGGGTGGAGGCTCACTGAGTGGTGGACTGG~ACTGAAAGTAAGCCTGGCCTACGCCCCTT

-- 260

-- 460

GAAATGGTTCAGGCGCCCAACA•ATGATCCAACAACCTGGCGCGTGGCAAGGATTGAGAGGTTGGTACGAGGCCCGTTTAAGGAAAGTGGCCG•CCTCTG --160 2 Ba,mHl ACGAAGGCGCGGTGTTGTTCGCCTTGTTTTCAACTCTGAATGCTTGGGATCCCTTGATCGTATAACGCCCTCAACGATGCCGATATAGC•GGCCAACAGC -- 60 TTAATTG~TTTGCGTCCCGGTGTG~TCCTTGGGGGGTTCGAGAGAAGGGAAATGTTGGTAGTACGGTC~GAGCTTGAGTGGAGGCACTGATGTGATGAAA +

40

GTAACTGGC•ACGC•C•TTGAAATGGTTCATGTG•CCATCACTT•GATTCCAACAACGCTTACAGTGGACCTCGATCATCGTCTTATAGCAGACCCACTT #140 I CGTAAGCAAGGTGGGATGTTTGAACGGCTTTGGTCGTTGTGGTG•GTGAGAAGCTATGGACATTGATTGCTCCGGAGGCCCCCCGTTGTTGAGCGGCCGA + 240 CTCTCGGGACTGTCATTTCAATCAAGAAATA•CGGTTTTGTACTTGTTCCCCCTCAGGAAGTGCTTGCGTTCTTCTACCCGTTATGCG•GAAGTTGATGC + 340

TTGCA•AGATTGCCTTTGTCGC•TACACCCTTGGCGTGTC•TGTCGCCACATA•CATGATAGTGTGCCTGTGTGTCGGCTAAACCGGCATCACCCAGGAC

5~cl

-I-440

ACATGTCTCTTGCCATTGGTCTCGGATGACGCTTGTATGCGAG•TCGTGGTCCTTTCGACGTGCCTCACAAAAGTTCCGTCGGAATTAATTGACG•TTGG -]-540 CTCGGCTGCCTTGAATGTTTTAGAGCATGGCCTCTGAGCTACGATTGGTAGCCTGCTGCCAACTTGTCAATGAGGAC•TTGCTACCTGGTTGATC•TGC• + 6 4 0 AGTAGTCATATGCTTGTCTCAAAGATTAAGCCATGCATTGTGCAAGTATGAACTAATTGAACTGTGAA+

708

Fi~.2. N u c l e o t i d e s e q u e n c e of the IGS. The 5' end of t h e 185 rRNA gene and t h e IGS u p s t r e a m to i t w e r e s e q u e n c e d ( F i g . i ) , and a s e q u e n c e of 1568 n u c l e o t i d e s was r e a d from t h e l a d d e r s . The 185 rRNA gene i s m a r k e d b y a wavy l i n e , the repeats are underlined, the highly c o n s e r v e d r e g i o n s in t h e r e p e a t s are o v e r l i n e d , t h e p u t a t i v e p r o m o t e r and t h e p r o m o t e r l i k e s e q u e n c e s a r e b o x e d and t h e t r a n s c r i p t i o n i n i t i a t i o n s i t e i s m a r k e d b y an a r r o w , w h i c h i s t h e +1 n u c l e o t i d e . 1119

Vol. 160, No. 3, 1989

rise

to

in

the

the

longer

region

repeats

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In

downstream

to

upstream

to

present

in the

repeats.

rDNA r e p e a t

length

rice

and

the

wheat

25S

the

rRNA

18S

(11)

the

gene

rRNA

repeats

(4)

differ

gene.

i n r i c e may be a t t r i b u t e d

The

present from

the

heterogeneity

to e i t h e r

the

differing

n u m b e r of r e p e a t s or to the p r e s e n c e of d i f f e r e n t k i n d s of r e p e a t s .

I n i t i a t i o n of rDNA t r a n s c r i p t i o n The

0.6

hybridized nuclear and a

size

3'end

to

the

18S

1st

repeat

gene.

kb

an

A,

(12) within

and

initiation There

between

(16),

(5),

an

starts in

A,

(Fig.2) A

+32

to

G.

and

In

of

dyad

of

the

-168

as

(unpublished

stem

and

the

all

maize,

of

the and

structure by

the

The

of

In

of

symmetry

the

and the of

rDNA (15) the

mouse

melanogaster

(5)

initiates where

it

rDNA t r a n s c r i p t i o n

+9 to -35

nucleotides

transcription

200

of

(15),

always

nucleotides

in

rice.

the

dominant

nucleotides

spanning

of

is

Zuker

transcription could

of

radish

transcription

rat

for

the

0.35

crustacean

start

radish

and

insert

The

BamHI-SacI

Drosophila

rDNA the

initiation

dyad

(11), or

programme

the

kb

region

(17)

140

'fold'

0.6

the

18S PRNA

transcription

promoter

for

about

with

transcription

wheat,

the

method.

before

(18)

obtained upstream

within

to

(14),

the

human

with

of

is

the

promoter

symmetry,

region

of

radish

the

data).

the maize

wheat

cases,

the

the

analyzing

was

initiates

product.

in

(14),

case

secondary

(20)

in

homology

comparison

predicted

also,

maize

the

constitute

(13) while

Xenopus l a e v i s

almost in

extended

transcription

much

(17),

laevis,

may

feature

in

a X.

region

rice

except

with

mouse,

not

to

by

nucleotide

upstream

was

and

transcriptase

product

624th

extension

upstream

fly

long

hybridizing

primer

M13mpl8

primer

determined

the

nucleotides

primer

rDNA

at

in

reverse

transcription

the

repeat

for

human

However,

with

1st

was

initiation

any

was

molecules

by

Tse-tse

the site

623

immediately

yield

T

the

precursor detected

fragment not

is

using

nucleotide

the

cloned universal

by

product

A 179

which

be

initiates

(19).

gene.

rRNA

Artemia

rat

(Fig.3).

Thus,

did

repeats.

extended

rRNA

fragment

the

gel

(Fig.l) 15-mer

extended

with

not

BamHl

the

was

corresponded

at

could

primer

of

Shorter

DNA

fragment

5'-[32p]-labelled

The

sequencing

whose

BamHI-SacI

the

RNA.

the

on

kb

to

and

occurs

possibly

play

Steigler in a

the role

i n t h e i n i t i a t i o n of t r a n s c r i p t i o n .

M u l t i p l e p r o m o t e r s for rDNA t r a n s c r i p t i o n The

3.3

rDNA-CAT of

the

kb

EcoRI-BamHI,

constructs,

358 p r o m o t e r ,

0.35

kb

BamHI and

(Fig.l),

the

and

CAT gene i n pBR322

the

CAT gene

1120

in

the

pUC13 were

0.6

kb

under used

BamHI-SacI

the

influence

as

templates

Vol. 160, No. 3, 1989

A

C

G

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

T

1

1

2

3

4

5

kb

Q b

Q

Q

Fig.3. The site of initiation of rDNA transcription. by the primer extension analysis as described Methods. Lane l-the primer extended product.

This under

was determined Materials and

Fig.4. The SI nuclease protection ~analysis of the transcripts from the rDNA-CAT constructs. The 3 ' [ ~Zp ]_labelled 0.8 kb HindIII-BamHI fragment bearing the CAT gene was hybridized to the transcripts of the rDNA-CAT constructs, digested with S1 nuclease and the protected fragments were analysed by a 8 M urea-5~ PAGE using HaeIII fragments of ~ x174 RF DNA as size markers. Protected DNA fragments from : Lane i - CAT gone with the 35S promoter in pUCI3; 2-CAT gene in pBR322 ; 3-3.3 kb EcoRI-BamHI-CAT construct ; 4-0.35 kb BamHI-CAT c o n s t r u c t , and 5-0.6 k b BamHI-SacI-CAT c o n s t r u c t .

for

transcription

to

the

the

358CATpUCI3

3

' -

[ 32p ]

in end DNA,

intact

nuclei.

labelled bearing

0.8 the

CAT 1121

The

transcripts

kb

HindIII-BamHI gene,

digested

were

hybridized

fragment, with

$I

from nuclease

Vol. 160, No. 3, 1989

and

the

the

2)

protected

rDNA-CAT

0.8

kb

fragments

constructs

fragment

showed

the

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

that

the

which

protected

the

produced

transcripts

the

CAT

gene

indicating

the

that

there

of

the

the

rDNA

control

were

rDNA

of

in

present

further

of

that

the

dyad

been

presence the

this

in

spacer

the

decreased may

to

be the

serve

the

repeats

be

around

as

of

in

The

rice

(23)

efficiency a

are

when

sink

of for

and

of

mouse

sequences may

also

promoters mouse

have

(24).

The

hypothesis

in

through

be

initiation

sequences

genes

eukaryotes enhancement'

( 25 ).

In

X.

laevis

termination

of

the

tandem

transcription the

under

faithful

the

s u p p o r t s the

'read

IGS

melanogaster and

which

for like

recycled

premature

gene

like

those

rRNA

the

control

Spacer

rDNA

repeated

factors

and

promoter

X.laevis

in

D.

promoter

data).

the

the

responsible

the

shown) showed

CAT

of

protected

not

Transient

under

constructs

results

transcription

spacer

(Fig.2)

also

(data

the

while,

transcripts

which

These

(21).

a m e c h a n i s m known as

true

not

cells

(lane

transcribed,

rDNA-CAT

rDNA

(22).

tandemly

transcription

proved

promoters

of

did

All the

band

produced

the

rRNAs.

sequences

case

promoters

not

any

~ -Amanitin

rDNA

(unpublished

through

the

was

repeats

regions

transcription

which

in

may

is

PAGE.

protected

of

plasmids carrying

Drosophila

3rd

which

All

of

for

reported

symmetry

may b e e n h a n c e d in

promoters

and

The

1).

of

upstream

M urea-5~

35S p r o m o t e r

plasmid

been

2nd

pBR322

35S-CAT

cultured

8

absence

transcription

heterologous

detected of

the

has

transcription.

exhibit also

the

the

presence

promoter

in

The

(lane

Recombinant

the

promoters

present

the

multiple

of

in

gene

in

while

transcribed

expression

CAT

unit.

3-5).

gene

by

transcripts

i n f l u e n c e of the

specific

are

lanes

CAT the

analysed

produced

(Fig.4,

CAT gene u n d e r

were

transcription

(25).

The

factors

spacer and

thus

e n h a n c e t h e t r a n s c r i p t i o n of rDNA. ACKNOWLEDGMENTS

This work was supported by the grant No.SP/SO/D83/86 from the D e p a r t m e n t of Science and T e c h n o l o g y , G o v e r n m e n t of I n d i a . The C e n t e r for Genetic E n g i n e e r i n g was e s t a b l i s h e d by the D e p a r t m e n t of B i o t e c h n o l o g y , G o v e r n m e n t of I n d i a . REFERENCES

1. A p p e l s , R . and H o n e y c u t t , R . L . (1986). In DNA s y s t e m a t i c s ( S . K . D u t t a , E d . ) , V o l . I I , p p . 8 1 - 1 3 5 . CRC P r e s s , F l o r i d a . 2. Kohorn, B.D. and Rae, P.M.M. (1982) Proc. Natl. Acad. Sci. USA, 79, 1501-1505. 3. S o l l n e r - W e b b , B. and Tower, J. (1986) A n n . R e v . B i o c h e m . 55, 801-830. 4. N a n d a b a l a n , K. and Padayatty, J.D. (1988) Biochem. Biophys. Res. Comm. 157, 68-74. 5. H a r r i n g t o n , C.A. and C h i k a r a i s h i , D.M. (1983) Nucleic Acids Res. 11, 3317-3332. 1122

Vol. 160, No. 3, 1989

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

6. T u r n b o u g h , C . L . , Kerr,J.K.H., Funderburg,W.R., Donahere,J.P. and Powell,F. (1987) J . B i o l . Chem. 262, 10239-10245. 7. O d e l l , J . T . , Nagy,F. and Chua,N-H. (1985) Nature, 313, 810-812. 8. J o s t , J . P . , Geisser,M. and Seldran,M. (1985) Proc.Natl.Acad.Sci. USA, 82, 988-991. 9. S a n g e r , F . , Nicklen,S. and Coulson,A.R. (1977) Proc.Natl.Acad.Sci. USA, 74, 5463-5467. 10. T a k a i w a , F . , Oono,K. and Sugiura,M. (1984) Nucleic Acids Res. 12, 5441-5448. 11. B a r k e r , R . F . , Harberb,N.P., Jarvis,M.G. and Flavell,R.B. (1988) J . M o l . B i o l . 201, 1-17. 12. G i l , I . , Gallego,M.E., Renart,J. and C r u c e s , J . (1987) Nucleic Acids Res. 15, 6007-6016. 13. Cross,N. and Dover,G. (1987) Nucleic Acids Res. 15, 15-30. 14. McMullen,M.D., Hunter,B., Phillips,R.L. and Rubenstein,I. (1986) Nucleic Acids Res. 14, 4953-4968. 15. Delcasso-Tremousaygue,D., Grellet,F., Panabieres,F., Ananiev,E.D. and Delseny,M. (1988) E u r . J . B i o c h e m . 172, 767-776. 16. Grummt,I. (1982) Nucleic Acids Res. 9, 6093-6102. 17. L e a r n e d , R . M . , S m a t e , S . T . , Haltiner,M.M. and Tjian,R. (1982) P r o c . N a t l . Acad.Sci. USA, 80, 3558-3562. 18. B a k k e n , A . , Morgan,G., Sollner-Webb,B., Roan,J., Busby,S. and Reeder,R. (1982) P r o c . N a t l . Acad.Sci. USA, 79, 56-60. 19. Kohorn,B.D. and Rae,P.M.M. (1982) Nucleic Acids Res. 10, 6879-6886. 20. Zuker,M. and S t i e g l e r , P . (1981) Nucleic Acids Res. 9, 133-148. 21. Grimaldi,G. and N o c e r a , P . P . D . (1986) Nucleic Acids Res. 14, 6417-6432. 22. Grummt,I. and Skinner,J.A. (1985) Proc. Natl.Acad.Sci. USA, 82, 722-726. 23. DeWinter,R.F.J. and Moss,T. (1986) Nucleic Acids Res. 14, 6041-6051. 24. Grummt,I., Kuhn,A., Bartch,I. and Rosenbauer,H. (1986) Cell, 47, 901-911. 25. Mitchelson,K. and Moss,T (1987) Nucleic Acids Res. 15, 9557-9596.

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