Transcription and reisolation of the simian virus 40 nucleoprotein complex

BIOCHEMICAL

Vol. 76, No. 3, 1977

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TRANSCRIPTION AND REISOLATION OF THE SIMIAN VIRUS 40 NUCLEOPROTEIN COMPLEX Mark R. Hall Department Received

March

of Cell and Molecular Georgia, Augusta,

Biology, Georgia

Medical 30902

College

of

30,1977

WY: Nucleoprotein complexes, containing cellular histones associated man Virus 40 DNA, were isolated from virus infected cells. These complexes supported RNA synthesis when incubated with E. coli DNAdependent RNA polymerase. Under the conditions of assay, transcripts synthesized by E. coli polymerase were shown to be highly specific for viral DNA by hy6rimion assay. Of particular importance is the demonstration that the viral nucleoprotein complexes isolated from the transcription mixture have not been physically altered. INTRODlJCTION Viral

nucleoprotein

complexes @PC), isolated

(SV40) or polyoma virus have been reported

virions

to contain

from Simian Virus 40

(1,2) and from virus four cellular

infected

histones

cells

(7,8).

(3-6),

These histones,

H2A, H2B, H3, and H4, are also found in the nucleosomes characteristic cellular viral

chromatin NPCs contain

to cellular

microscopic

have shown that

between viral

on transcription

NPC and cellular

chromatin

provides

(12,13).

models for studying of a defined

the effects

genome.

The present

evidence that the viral

model chromosomes (mini-chromosomes) of their

in structure

suggests the

Indeed,

have shown that polyoma and SV40 NPCs can be transcribed

and in addition

the

(10,ll).

and endogenous polymerases

ation

studies

20 nucleosomes and are similar

that NPCs might be ideal

chromosomal proteins studies

Electron

approximately

chromatin

The similarity possibility

(9).

of

report

recent

by exogenous

supports these studies

NPCs can be utilized

in a transcription

of

assay without

as alter-

structure. MATERIALSANDMETHODS

African green monkey kidney cells ((X-1) were infected with strain WI911 SV40 virus at a multiplicity of lo-20 pfu/cell. At 40 h postinfection, cultures were pulsed with [3H]TdR (15 Ci/r&i) for 120 min at 14 uCi/ml, Copyright 0 1977 by Academic Press, Inc. All rights of reproduction in any form reserved.

698

ISSN

0006-291X

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AND BlOPHYSlCAL

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Nucleoprotein complexes containing SV40 DNAwere extracted 5.0 ml/culture. as previously described (6). The crude lysates were cleared of nuclear aggregates and large cellular debris by centrifugation at 800 x g for 10 min at 4”. The resulting NPC containing supernatant was divided into three fractions for transcription studies. One fraction was deproteinized and closed circular supercoiled viral DNAisolated from propidium diiodide-CsCl gradients. The second fraction was concentrated by pressure dialysis in order to produce a “crude NPC fraction” containing viral DNA at a concentration of 2.0 ug/ml. Finally, the third fraction was fractionated by a combination of hydroxyapatite (HA) chromatography and velocity centrifugation previously described by Meinke et al. (8) to produce “purified NPCs”. Velocity centrifugation of complexes in linear 5 to 20% (w/w) sucrose gradients, and buoyant density centrifugation of glutaraldehyde fixed complexes as well as collection, processing and scintillation counting of centrifuged samples have all been previously described (5,6). In vitro transcription of SV40 NPCsand viral DNAwas carried out by a Ed-tion of Astrins (14) assay system. Except where indicated, RNAwas synthesized from NPCtemplates or purified viral DNAat 1.0 pg of DNAper assay. Each reaction also contained, in a total volume of 1.0 ml, 20 units of 5. coli DNA-dependent RNApolymerase (Miles Laboratories), 15 I&I KCl, 40 mMTRIS-HCl (pH 7.9), 0.1 n&l dithiothreitol, 5 I&I MgC12, 0.05% Triton X-100? 40 mMNaCl, 2 mMEDTA, and 1 I&I each, ATP, GTP, CI’P, and UTP ([3H]UTP 10 uC1). The reaction was terminated after 15 min incubation at 37”C, by addition of 5.0 ml of cold 10%TCA. The acid-precipitable material was collected on glass fiber filters (WhatmanGF/C), dried and counted in a liquid scintillation counter. To prepare RNAfor filter hybridization, the preceeding reaction mixtures were incubated for 3 hr at 37’C. The reaction was terminated by addition of SDSto a final concentration of 1.0%. RNAwas extracted with phenol saturated with acetate buffer pH 5.2 (13) and collected by ethanol precipitation. For DNA-RNAhybridization, SV40 DNAI, purified through propidium diiodideCsCl gradients, was heat denatured and immobilized onto 25 mmnitrocellulose filters (Schleicher 8 Schuell Co., type B6) by the methods of Aloni et al. (15). DNA-RNAhybrid analyses were performed in scintillation vials containing one blank filter and one filter containing either 2.5 or 5.0 ug of viral DNA. Incubations were for 24 hrs at 65’C with viral RNAin 2.0 ml 2 x SSC (SSC is 0.15 M NaCl, 0.015 M sodium citrate). The filters were then chilled, washed in 2 x SSCand exposed to 20 pg/ml pancreatic ribonuclease in 2 x SSC for one hr at room temperature. Filters were then rinsed in 2 x SSC, dried and counted in a scintillation counter. RESULTS In Vitro

Transcription

of SV40 DNA, Crude NPCs, and Purified

Prior to use in the transcription

NPCs

assay, aliquots of the purified

NPCswere analyzed to assure that they maintained the sedimentation velocity (61s) and density purified

(1.437 g/cm3) previously

NPCpreparation

coefficients 1.445 g/c+.

utilized

reported for SV40 NPCs (6).

The

in these studies had sedimentation

of between 58 and 61s and buoyant densities between 1.435 and

Vol. 76, No. 3, 1977

Table 1.

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AND BIOPHYSICAL RESEARCH COMMUNICATIONS

In Vitro Transcription of SV40 DNA and Nucleoprotein 5 E. coli DNA-Dependent RNA Polymerase.

RNA Synthesis *(cpm[3H]DI'P Incorporated)

Template crude

+180

NPC

Purified

Complexes

NPC

970

SV40 DNfl

4100

* RNA was synthesized from NPC templates containing 1.0 vg of DI\EAper assay or from 1.0 pg of purified Form I SV40 DNA as described in Materials and Methods. These values are corrected for approximately 85 cpm of incorporation in the absence of template. ' Corrected activity.

Purified utilized

for approximately

SV40 DNA and NPCs as well as crude NPCs were prepared and

as templates

and Methods. supported

120 cpm of endogenous polymerase

for -.E. -coli

The results

incorporation

presented

RNA catalyzed

However, it is evident

poor template

which supports

approximately

20% as much [3H]UTP as purified

that purified

viral

purified

as described

in Table 1 show that all

of [3H]UTP into

dependent RNA polymerase. is a relatively

RNA polymerase

DNA was at least

The best evidence that is accurate,

was obtained

sized from either

complex.

DNA immobilized Unfortunately, mixtures

viral

filters

we could not recover

to allow filter

hybridization

RNA synthesized

from purified

the same extent

as RNA synthesized

It is also apparent a template

of NPCs by 5 &RNA experiments.

DNA or purified

on nitrocellulose

of only

from purified 700

RNA syntheto viral

and Methods).

RNA from crude NPC reaction

experiments.

NPCs hybridized

polymerase

NPCs was hybridized

(see Materials

sufficient

as

as crude complex.

by DNA-RNA hybridization

purified

DNA

that the crude complex

4 times as efficient

transcription

three templates

by -E. coli

incorporation

complex and 20 to 22 times as effective

in Materials

As shown in Table 2,

to viral viral

DNA to approximately DNA. This suggests

BIOCHEMICAL

Vol. 76, No. 3, 1977

Hybridization of [3H]-Labeled RNASynthesized 7In Vitro from Purified SV40 Nucleoprotein Complexes or Purified Supercoiled SV40 DNA.

Table 2.

[ 3H]RNA Input wwl

Template for RNASynthesis Purified

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

NPC

sv40 DNA

[ 3H]RNAHybridized (CPM) 2.5 ug DNA 5.0 1-g DNA (% of Input)

3,200

2,685

2,750

84.9

11,000

9,690

9,800

88.6

RNAwas synthesized from each template by the procedure described in Materials and Methods except that the incubation time was increased from 15 min to 3 hr. At the end of the incubation period, RNAwas extracted and purified and DNA-RNAhybridization performed as described in Materials and Methods. All values have been corrected for background counts (l-2% of input) of [3H]RNA bound to blank filters.

that viral

DNAsequences in NPCsare transcriptionally

transcribed

Recovery of Nucleoprotein

Since one of the main objectives

physical

Complex Templates

of these studies is to determine the

of NPCsfor use as model chromosomes,it was essential that the integrity

Therefore,

of the complex be retained following

purified

[3EI]-thymidine

characterize

complexes (PI-NPCs) and compare them with pretranscripNPCs. As shown in Figure 1, the sedimentation velocity

labelled F’I’-NPCsis identical

labelled pretranscriptional

to assist in determination

NPCs. A [“Cl-thymidine

sedimentation coefficients

to viral

viral

(arrow over fraction

of their

as 60 S. fixed SV40 NPCsis

Density analyses of glutaraldehyde

701

35)

for the complexes.

DNAallows calculation

The average density reported for glutaraldehyde 1.437 + .008 g/cm” (6).

labelled purified

of sedimentation coefficients

of the NPCsrelative

of

to that of [14C]-thymidine

DNAmarker (21 S) was also present in this gradient

The position

transcription.

several experiments were performed to physically

posttranscriptional tional

and are

faithfully.

Posttranscriptional

suitability

active

fixed

Vol. 76, No. 3, 1977

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

IO@

9

0

IO 20 30 40 FRACTION NUM BE R

IO 20 30 FRACTION NUMBER

0

Figure 1. Velocity sedimentation, in a linear sucrose gradient, of SV40 nucleoprotein complexes. After 15 min incubation in the transcription assay mixture (see Materials and Methods), an aliquot of [3H]-thymidine labeled NPC (0.15 ml) was removed from the assay mixture mixed with 0.05 ml of [14C]-thymidine labeled marker SV40 NPC and [j4C]-thymidine labeled viral DNA (10 lambda), and centrifuged as described (5,6). An arrow marks the position of [14C]-thymidine labeled, purified viral DNA included as a marker. Figure 2. Equilibrium centrifugation of glutaraldehyde-fixed SV40 nucleoprotein complexes in linear CsCl gradients. After 15 min incubation in the transcription assay mixture (see Materials and Methods), aliquots (0.25 ml) of t3H]-thymidine labeled NPC were removed and centrifuged on 5% to 20% sucrose gradients. Peak fractions from each gradient were pooled, mixed with ['4C]-thymidinelabeled marker NPCs, and treated with glutaraldehyde (5,6). Aliquots (0.2 ml) of the glutaraldehyde fixed complexes were layered on preformed linear CsCl gradients and centrifuged to equilibrium as previously described (5,6).

pretranscriptional linear

and posttranscriptional

CsCl gradients

as previously

that these two WC preparations their

density

Although

not shown, standard

of 1.444 is within

of the DNA extracted

NPCs exhibit

similar

described

had identical

apparent

gradients

NPCs were conducted on preformed

patterns

cells

buoyant densities.

diiodide-CsCl

from pretranscriptional of banding of viral supercoiled

the same ratios

cellular

2)

In addition,

and posttranscriptional DNA with approximately

histones

90%

form.

bound to viral

as are found in eukaryotic 702

(Figure

equilibrium

DISCUSSION complexes isolated from polyoma and SV40 virus

have been shown to contain

approximately

It is evident

the normal range for SV40 NPCs.

propidium

of the DNA being in the closed circular Nucleoprotein

(5,6).

chromatin

infected DNA in (11).

The

BIOCHEMICAL

Vol. 76, No. 3, 1977

similarity

of viral

be ideal

although

has led to the proposal

the effects

presented

here indicate

report

that

was deproteinized

viral

a template

DNA. Similarly,

crude NPCs were almost totally It was suggested that,

in addition

in Table 1, indicate crude extracts

that

as templates

to restriction

or ribonucleases similar

of SV40 NPCs.

RNA synthesis

inhibitory

polymerase

of transcription

(13).

as

also contained Cur results,

free of soluble

of transformed

cells

RNA polymerase.

by histones cellular presented

substances might be present

Recent work has shown that both SV40 subviral

by --E. coli

the SV40 viral

for RNA synthesis.

There is an approximately

when NFCs are purified

sequences in chromatin

that

in a study of polyoma WCs (13),

invactive

of RNA synthesis

DNA. This is in

for -E. coli

in the polyoma NPCs, the crude preparations

accurately

on gene

SV40 NPCs are transcriptionally

(12) which demonstrated

core complex was only 30% as effective

inhibitors

that NPCs might

of chromosomal proteins

at a reduced rate as compared to viral

agreement with a previous

present

RESEARCH COMMUNICATIONS

(16).

The results active

NPCs and chromatin

models for studying

expression

AND BIOPHYSICAL

five fold increase host cell

in in

proteins.

cores (12) and viral

DNA

(SV3T3) (14) can be transcribed

Significantly,

the sequences transcribed

--in vitro from SV3T3 chromatin were the same as those present in transformed cells (14). Our present work supports these studies and indicates (Table 2) that E. coli

polymerase

whether these transcripts Most important,

represent

relative

complexes for studies

intact

template

marked contrast

to recent reports

bound proteins,

(17,18).

rather

However,

true messages remains to be determined. of these viral

of gene expression,

DNA-histone

is the evidence that

from the transcription

evidence that transcription

the DNA-protein

the assay mixture

copy SV40 NFCs -__ in vitro.

to the utility

on control

complexes may be isolated is strong indirect

accurately

can

solutions,

is actually

than some deproteinized

occurring

except the polymerase,

that sarcosyl in these assays.

703

on

form and is in

in which sarcosyl was incorporated

It is likely

This

removed all

into DNA

The recovery of

Vol. 76, No. 3, 1977

unaltered transcribing

BIOCHEMICAL

NPCs after unaltered

template-complex

--in vitro

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

transcription

NPCs, reisolating

(DNA or Protein),

offers

the possibility

the NPC template,

transcribing

of

modifying

the

it again and comparing

transcripts. ACKNOWLEDGEMENTS:The author thanks Ms. Brenda Kozyra and Mr. Warren Hall for skillful technical assistance. This investigation was supported by Grant Number CA 17167, awarded by the National Cancer Institute and by Grant Number NP-189 from the American Cancer Society. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18.

hang, E-S., M.K. Estes, and J.S. Pagano. (1972) J. Virol. 9: 923-929. McMillen, J., and R.A. Consigli. (1974) J. Virol. 14: 1326-T536. Green, M.H., H.J. Miller, and S. Hendler. (1971) SC. Nat. Acad. Sci., U.S.A. 68: 1032-1036. White, K, and R. Eason. (1971) J. Virol. 8: 363-371. Goldstein, D., M.R. Hall, and W. Meinke. (1973) J. Virol. 12: 887-900. Hall, M.R., W. Meinke, and D.A. Goldstein. (1973) J. Virol.2: 901-908. Pett, D.M., M.K. Estes, and J.S. Pagano. (1975) J. Virol. 157379-385. Meinke, W., M.R. Hall, and D.A. Goldstein. (1975) J. VirolTl5: 439-448. Kornberg, R.D. (1974) Science 184: 868-871. Griffith, J.D. (1975) Science m: 1202-1203. Cremisi, C., P.F. Pignatti, Oxroissant, and M. Yaniv. (1976) J. Virol. 17: 204-211. iiing, E-S., M. Nonoyama, and J.S. Pagano. (1972) J. Virol. 9: 930-937. Shmookler, R.J., J. Buss, and M.H. Green. (1974) Virology 577122-127. Astrin, S.M. (1973) Proc. Nat. Acad, Sci., U.S.A. 70: 23047308. Aloni, Y., E. Winocour, and L. Sacks. (1968) J. Mar Biol. 31: 415-429. Germond, J-E., B. Hirt, P. Oudet, M. Gross-Bellard, and P. -bon. (1975) Proc. Nat. Acad. Sci., U.S.A. 72: 1843-1847. Gariglio, P., and S. Mousset. (1975) EBS Letters 56: 149-155. Green, M.H., and T.L. Brooks. (1976) Virology 72: X0-120. -

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