Highly specific transcription of globin sequences in isolated reticulocyte nuclei

Vol. 77, No. 4, 1977

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

H I G H L Y S P E C I F I C T R A N S C R I P T I O N OF G L O B I N S E Q U E N C E S

IN

ISOLATED RETICULOCYTE NUCLEI Eric J. B. Fodor and Paul Doty Department of Biochemistry and Molecular Biology Harvard University Cambridge, Massachusetts 02138 Received July 6,1977 Summary. Chicken reticulocyte nuclei have been isolated under conditions where the endogenous RNA polymerase activity is retained and catalyzes the incorporation of ribonucleotide triphosphates into RNA linearly for at least 5 hours at 25°C after an initial burst of activity. Transcription has also been carried out utilizing 5-mercurated uridine triphosphate. Hybridization of the mercurated RNA, isolated from the endogenous RNA by affinity chromatography on sulfhydryl Sepharose with globin cDNA reveals that 0.24 percent of the transcript is globin sequence: this is about 100-fold higher than that reported for chromatin with E. coli RNA polymerase. Although transcription of chromatin offers many opportunities

for observing template availability,

from several drawbacks. that many otherwise

MOst importantly are:

"endogenous"

it suffers

the possibility

factors required for specificity

or selectivity may be removed during purification;

and,

the

wide-spread use of E. coli polymerase as a template probe may lead to artifactual information

(i).

Isolated nuclei from specific tissues have recently been shown by several investigators to support the synthesis of RNA

(3,4,5,6).

As a consequence it has become possible to

study in vitro transcription under minimally disruptive conditions.

Abbreviations: SDS, sodium dodecyl sulfate; EDTA, ethylene diamine tetra-acetic acid; cDNA, DNA complementary in sequence to globin RNA; HEPES, N-2 hydroxyethylpiperazine-Ni-2-ethane sulfonic acid

Copyright © 1977 by Academic Press, Inc. All rights o/ reproduction in any/orm reserved.

1478 1SSN 0006-291X

Vol. 77, No. 4,1977

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The introduction by Dale and Ward

(2) of a novel technique

for fractionating oligonucleotides by substitution with mercury and subsequent isolation by their affinity to Sepharose-SH has opened the possibility to study RNA transcripts produced under controlled conditions and separate the de novo transcripts an otherwise contaminating pool of homologous products.

from

This

technique has recently been utilized to demonstrate the synthesis of specific sequences in isolated nuclei (3,7,8).

The work presented

(3,4,5) and ehromatin

(9) here analyzes the transcriptional

specificity of isolated chicken reticulocyte nuclei utilizing the Hg-UTP technique with respect to the production of globin messenger RNA. Materials and Methods. 5-mercury uridine triphosphate (Hg-UTP) was synthesized from UTP (2), and purified and characterized as previously described ( 8 ) . ~-Amanatin was obtained from Sigma Chemical Co. [3H]UTP, [3H]GTP and ~-[32p]UTP were from New England Nuclear Corp. Anemia was produced in white leghorn roosters by intramuscular injection of acetylphenylhydrazine in a 50% ethanol solution on a schedule of 70, 40, 20 and I0 mg/day. On the 5th day blood was collected directly into 50 ml of isotonic saline containing 300 units of heparin (Sigma grade I). Cells were washed twice by centrifugation in isotonic saline and the buffy coat removed. The wash was repeated once more and the cells suspended in 30 ml lysis buffer: 0.3 M sucrose, 5 mM Mg (OAc)2 , i0 mM tris, pH 7.5, 0.5 mM dithiothreitol. Triton X-100 was then added to a concentration of 0.2~ and the cells lysed by 60 strokes of a tight fitting Dounce homogenizer. All extraction steps were performed at 10°C. The homogenate was mixed with an equal volume of the same buffer in 2 M sucrose, layered over a i0 ml cushion of the same buffer in 1.5 M sucrose and centrifuged for 30 minutes at 8,000 rpm in the Sorvall HB4 rotor. This procedure was repeated if lysis was incomplete. The resulting grayish-white pellet was then suspended in 25~ glycerol, 5 mM Mg(OAc)2 , i0 mM tris, p H 7.5, 5 mM dithiothreitol, and 0.2 mM EDTA, at roughly 108 nuclei/ml, and stored under liquid N 2. Microscopic examination reveals that the nuclei are at least 98~ free of cytoplasmic tags; however, some membrane contamination was evident.

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Vol. 77, No. 4, 1977

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

For RNA synthesis an equal volume of freshly [hawed nuclei was added at 25°C to a reaction mixture of; 50 mM HEPES, pH 8.1, 5 mM Mg(OAc)2 , 1 mM MnCl 2, 12 mM B-mercapto-ethanol, 150 mM KCl, I0~ glycerol, 0oi mM ATP, CTP, GTP, i0 ~M UTP, or 50 ~M Hg UTP and 2 x 106 cpm [3H]UTP or [32p]UTP. The mixture was gently agitated every 30 minutes, and at various times, 50 ~i aliquots were spotted directly onto Whatman DEAE filter paper, prespotted with 0.5 M EDTA, pH 8. The filter disks were washed three times with 0.3 M HCOONH4, pH 8, and three times with 0.25 M NH4HC03, and twice with 95% ethanol at i0 ml/sample, and dried under a heat lamp. The filters were counted in 5 ml of Scintiverse (New England Nuclear). Alternatively, the samples were precipitated with bovine serum albumin and i0~ trichloroacetic acid and processed as previously described (8). After the desired time, the reaction mixture was made 2~ in SDS, heated briefly to 45°C and shaken. Nucleic acid was extracted by adding an equal volume of chloroform-isoamyl alcohol-phenol (47:3:50), heating to 40°C for 8 minutes with shaking and then centrifuging at 7,000 rpm for 15 minutes to separate the aqueous phase. The aqueous phase was reextracted and finaliy precipitated with 3 volumes of ice cold ethanol overnight at -20°C. Purification of the Hg-RNA was essentially as outlined before "(8), except, after ethanol precipitation, the RNA + DNA was dissolved in column buffer (0.01 M tris, pH 7.5) without SDS and digested for approximately 15 minutes with i0 ~g/ml RNAse free DNAse (Worthington, Biochemical Corp.), made 0.2~ in SDS, heated several minutes at 40°C, and developed over a preequilibrated Sepharose-SH column (8). Hybridization of the isolated Hg-RNA tO mixed s-and ~-globin cDNA was identical to that reported previously (8). The fraction of transcript that was globin was calculated as before, or using a Rot of 6.5 x 10 -4 (12) for pure globin sequence. Results.

The incorporation of [3H]GTP into RNA as a function of

time under several conditions is shown in Figure 1.

With the

normal complement of four trinucleotides the synthesis is essentially linear, most curve).

after an initial burst,

for 5 hours

(upper-

The next lower curve shows that when Hg-UTP

replaces UTP the rate is diminished by about 20~ as others have observed.

Kinetic analysis,

ing a compound analysis

although probably represent-

(more than one polymerase)

the K for the Hg-UTP is 22 ~M m

(3)

(data not shown),

1480

reveals that

roughly 3 fold

Vol. 77, No. 4, 1977

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

I

I

{

1600

(3.

1200

BOO

400

L

L

I00

200 TIME(minutes)

L

500

Figure I. Transcription of l08 nuclei in 2 ml of reaction mixture was carried out according to methods with the following exceptions. [3H]GTP was used with a specific activity 7 x 104 cpm/n mole--~ ~ . [ 3 transcription with 15 ~M GTP, 20 p/4 UTP, 0.i mM CTP and ATP. --~ ~ w ~ - - transcription with 15 p~M GTP, 50 p~4 Hg-UTP 0.i mM CTP and ATP. ---O O ~ as with Hg-UTP, plus 6 ~g/ml ~-Amanatin.

higher than that for UTP while the V The lowest curve shows ~-Amanatin.

the

that RNA polymerase

period of linear synthesis

of about 1200o

addition apply,

incorporated

Furthermore,

7-[32p]ATP

of mercurated

and

is occurring.

[3H]UTP are the

a plausible mean chain length

the net synthesis

RNA by 108 nuclei in 5 hours corresponds

Yields

the long

into RNA and that in 200 minutes

indicating

140 globin mRNA molecules

(i0).

suggests that reinitiation

This is supported by finding that

molar ratio is 1:300,

II and/or III

for the bulk of the synthesis

If normal rates of nucleotide

simultaneously

is essentially unchanged.

marked suppression produced by 6 ~g/ml

This indicates

have been responsible

max

of about 2 ~g of

to the synthesis

of

per nucleus. RNA were usually 80% of that expected

1481

Voi. 77, No. 4,1977

,

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BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

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Figure II. RNA was synthesized as per methods from 2 x l08 nuclei with 4 x 104 epm/n mole. [3H]UTP and i0 ~LM UTP--O---C~ O - or 50 ~M H g - U T P - ~ [] [] extracted at 250 minutes, and developed over a 1.5 x 3 cm column of sulfhydryl sepharose (Sulfhydryl content was 1.2 ~ Mole of SH/ml resin). The wash included the 50% dimethyl sulfoxide wash as previously described (7). RNA is eluted with 0.i M ~ mercaptoethanol in the column buffer. FlOw rate was 25 ml/hour, at 2 ml/fraction. Figure IIi. Hybridization of Hg-RNA to globin cDNA. After elution from the sulfhydryl column and precipitation, the mercurated RNA was lyophilized and 1 ~g brought up in 70 ~i containing 0.2 M Na phosphate, pH 6.8, 0.5% SDS, 5 mM EDTA and 0.036 ng/~l globin cDNA. The solution was then overlayed with mineral oil, heated to 97 ° for 5 minutes, and incubated at 68 ° . The fraction hybridized (double strand) was determined with S-I nuclease as before (7):__ 0 O O RNA isolated from a single column run, _ _ ~ O RNA was isolated as above, but, reprecipitated, brought up in column buffer, heated to 98°C for 5 minutes, cooled quickly and rechromatographed over the sulfhydryl column.

from the time course after extraction and precipitation, completely labile to 5 ~g/ml of pancreatic ribonuclease.

and The

affinity chromatography of the de novo synthesized RNA on a column of sulfhydryl sepharose is displayed in Figure 2. Superimposed on the same graph is the result of a similar experiment except that only UTP was present. eluted in this case.

1482

2 o radioactivity was

Vol. 77, No. 4,1977

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

The h y b r i d i z a t i o n of isolated Figure

of g l o b i n

RNA is shown

in Figure

3 of 0.32 ~ 0.05,

calculated

a value

for the c o n t e n t described

be c a l c u l a t e d

from a double

3.

In a g r e e m e n t

yields

a value

transcript

m R N A by

is globin.

were

the result

with

RNA-RNA

SDS,

ethanol heated

then eluted

roughly

9 0 % of the applied

sequence 3 that run

over

the amount

fractional

noted by Z a s l o f f

a pre-equilibrated

the r e i s o l a t e d content

and F e l s e n f e l d

and S w e r d l o w

(6) have

from Friend

cell n u c l e i

bound

from this

shown

that

on the second globin seen

does

(i) is low.

affinity

not show a d e c r e a s e production

In agreement,

the de novo g l o b i n

1483

RNA

from Figure

second

antistrand

do not c o n s i s t

heating

determinations.

Hg-RNA

of globin,

column

quenched

After

it can be

from p r e v i o u s

messenger,

Sepharose-SH

as before.

of the RNA

globin

up in column b u f f e r immediately

radioactivity

in

of the

the affinity

Furthermore,

is i n d i s t i n g u i s h a b l e

in the

brought

shown

treatment

containing

anti-sense

the h y b r i d i z a t i o n

Since

strand),

also

or not the

of m e r c u r a t e d

is negligible.

Discussion:

this

contaminating

(one strand

and h y b r i d i z e d

mRNA may

fraction

TO check w h e t h e r of trapping

is

of the data

for the

in

g l o b i n mRNA.

of g l o b i n

analysis,

for 5 minutes,

column,

hence

the rate

precipitated,

and d e v e l o p e d

pass:

synthesized

fraction

anti-sense

in ice,

value

of 0.2 ~ 0.03 p e r c e n t

duplexes

to 98°C

concentrations

From the Rotl/2

r e c i p r o c a l plot

with

the m e r c u r a t e d

eluant was

3.

of 0.24 + 0.03 p e r c e n t

forming

the other

(8) the

which

transcripts

at various

of in vitro

As p r e v i o u s l y

Figure

cDNA

of R N A - R N A

as Orkin

transcripts

duplexes.

Vol. 77, No. 4, 1977

Hence,

BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS

the a r t i f a c t u a l

(i) does not appear Despite with

that

difficulties

data

per nuclei.

measured

by the H g - U T P

time

that RNA p o l y m e r a s e

reaction

condition

reaction

to p r o c e e d

Under

these

equivalent sion that

conditions

yields

This

more

globin

in the c h r o m a t i n

of the g l o b i n random

gene

initiated

Acknowledgments: of his m a n u s c r i p t

a 100-fold

remains

is c a l c u l a t e d

the tentative

product

per u n i t

being

transcription

In the nuclei,

We w i s h to thank Dr. before publication.

the same h i g h

(13)

is the randomly of the

transcription value but

a 100-fold

Stuart Thanks

conclu-

w i t h E. coli

of g l o b i n mRNAs

is supressed

1484

the

for comparison.

of the genome

at roughly

transcription

by assuming

chrematin

faster

case.

If the

globin m R N A per genome

the p r o d u c t i o n

along w i t h

for

(37 ° ) and

in c h r o m a t i n

one can draw

w i t h most

results

in both cases.

of 5/2.5

than i00 times more

is c o n s i s t e n t

is 140

at 25 ° , then the c h r o m a t i n

by a factor

transcribed gene

slower

From

at 25°C

the c h r o m a t i n m u s t be m a d e

in excess

of r e t i c u l o c y t e

whereas

striking.

in 5 hours

are a c c o m m o d a t e d

From this,

of r e t i c u l o c y t e

and t e m p e r a t u r e

RNA p r o d u c t i o n

transcription

DNA than nuclei,

with

~g DNA and 90 m o l e c u l e s

of DNA.

polymerase

was

times

should be reduced

to be i00 ~g/100

same.

2.5

that

allowance

(i hr)

differences

are

of this w o r k

Globin mRNA production

technique

assuming

results

transcription

For c o m p a r i s o n

in r e a c t i o n

the results

it can be shown ~g DNA.

transcription

transcription.

and one similarity

on nuclei

molecules

in c h r o m a t i n

in nuclear

RNA p o l y m e r a s e

is 0.75 ~g RNA/100

difference

found

in c o m p a r i n g

one d i f f e r e n c e

the above there

to exist

of E. coli

chromatin,

behavior

or more.

Orkin for a copy also to Dr. J.

Vol. 77, No. 4,1977

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Beard for the A.M.V. reverse transcriptase. We also wish to thank Dr. Su-Yun Chung-Wooley for her advice and critical reading of this manuscript. This work was supported by NIH Grant HI) 01229. References 1 2 3 4 5 6 7 8. 9. i0. ii. 12. 13.

Zasloff, M., and Felsenfeld, G., (1977) Biochem. Biophys. Res. Comm., 7_~5, 598-603. Dale, R. M., and Ward, D. C., (1975) Biochemistry i_~4, 2458-2469. Smith, M. M., and Huang, R. C., (1976) Proc. Nat. Acad. Sci. USA 7_~3, 775-779. Beebee, T. J. C., and Butterworth, P. H. W., (1976) Eur. J. Biochem., 66, 543-550. Ernest, M. J., Schutz, G., and Fergelson, P., (1976) Biochemistry, i__55, 824-829. Orkin, S. H., and Swerdlow, P. S., (1977) Proc. Nat. Acad. Sci. USA, in press, June. Towle, H. C., Tsai, M., Tsai, S. Y., and O'Malley, W., (1977) J. Biol. Chem., 252, 2396-2404. Crouse, G. F., Fodor, E. J. B., and Doty, P., (1976) Proc. Nat. Acad. Sci. USA 7_~3, 1564-1567. Fodor, E. J. B. and Doty, P., (1977) Fed. Proc., 3_~6, 2885. Longacre, S. S., and Rutter, W., (1977) J. Biol. Chem. 252, 273-283. Longacre, S. S., and Rutter, W., (1977) J. Biol. Chem. 252, 2742-2752. Axel, R., Cedar, H., and Felsenfeld, G., (1973) Proc. Nat. Acad. Sci. USA 7__O0, 2023-2032° Crouse, G. F., and Doty, P., (1977) manuscript submitted.

1485