Lorsque ce travail a été amorcé, GARIGLIO et MOUSSET venaient de mettre en évidence que la transcription virale tardive des cellules per missives infectées par le virus SV 40 se faisait à partir d'un complexe de transcription non intégré dans le génome cellulaire (GARIGLIO et MOUSSET,
1975). Le but de notre travail a consisté à déterminer si le complexe de
transcription tardive du virus SV40 se présente ou non sous forme de mini chromosome, c'est-à-dire si les nucléosomes sont présents dans la chro matine virale transcrite "in vivo".
I. "Isolation and charaaterization of an active miniahromosome Llopis, R. and Gariglio, P. ( 1976). Arch Internatl .
Physiologie et Biochimie, 1087.
II. "The template of the isolated native SV40 transariptional
complexes is a minichvomosome".
Gariglio, P., Llopis, R., Oudet, P. and Chambon, P. (1976) Accepted for publication to J. Mol. Biol.
III. "Levels of SV40 transcriptional complex in productively
infected cells".
SOCIÉTÉ BELGE DE BIOCHIMIE BELGISCHE VEREMGING VOOR BIOCHEMIE
98® réunion, Bruxelles 23 octobre 1976
R. Llopis et P. Gariglio (Laboratoire de Génétique, Département de Biologie moléculaire. Université Libre de Bruxelles).
Isolement et caractérisation d’un minichroinosomc actif pour la transcription.
Au cours de ces cinq dernières années, des progrès considérables ont été faits dans l’éclaircissernent de la structure intime de la chromatine des eucaryotes (Kornberg, 1974). A riieure actuelle, on sait que le matériel génétique des eucaryotes a une structure définie, constituée par la répétition d’une unité discrète — les nucléosomes —, reliés par de petits ponts de vingt paires de bases de DNA. Chaque nucléosome est formé par un octamère — deux molécules de chacune des histones H2a. H2b. H3 et H4 — entouré par plus ou moins deux cents paires de bases de DNA.
Green et al. (1971) et Wiiite & Eason (1971) ont isolé des complexes nucléo-protéiques à partir de cellules infectées par le virus du polyome et S\N0 respectivement. Ces complexes contiennent du DNA viral et quatre histones cellulaires (Golds tein et al., 1973).
Griffith (1974) a montré que les nucléoprotéincs de SV40 isolées par Wiiite & Ea.sün (1971) possédaient une structure identique à celle de la chromatine des cellules eucaryotes. Ce complexe yiral a été nommé « minichromosome ». Cependant, un
important composant de la chromatine. l’acti\ité de la polymérase endogène du RNA est absent de ce minichromosome.
Gariguo & Mousskt (1975) ont extrait à l'aide du détergent Sarkosyl, à partir de cellules permissives infectées par SV40. un complexe nucléoprotéique viral, ayant perdu la plupart des pro téines basiques, mais conservant l'activité de transcription endogène.
Green & Broüks (1976) en employant des méthodes d'extrac tion plus douces, solubilisent une fraction des nucléoprotéines virales ne manifestant pas d'activité transcriptionnelle. Celle-ci peut cependant être activée en présence de Sarkos_\l.
Notre travail se situe dans ce contexte. Nous avons utilisé des méthodes d’extraction qui ne détruisent ni la structure intime des minichromosomes, ni leur potentialité de transcription.
En combinant les etfcts du détergent Triton X-IOO et du sulfate d’ammonium il est possible de solubiliser environ 40 % du matériel extrait par le Sarkosyl. Ce matériel présente la densité de la chro matine, est potentiellement actif en transcription et peut être aisé ment purifié.
Nous avons également abordé l’étude du rôle joué par les pro téines basiques dans l'inhibition de la transcription. Nous avons essayé, par différentes méthodes, d'activer le complexe, en impo sant comme seule condition que ne soit pas détruite l'association DNA-histones. Il a été possible, à des concentrations 0.2 m en SLiIfiite d'ammonium, d'atteindre des niveaux de transcription aussi élevés que ceux atteints en présence du Sarkosyl. Ceci suggère que, du moins dans les conditions que nous avons utilisées, la présence des historiés n’empêche pas la transcription du DN.A par la polymérase endogène.
Nous pensons que ce système, tout en étant très simple, peu., servir de modèle pour l’étude de la structure de la chromatine ainsi que de la régulation de l’expression génétique chez les eucaryotes.
REFERENCES
G\rk;lio. P. & Moi'S,si.r, S. (1975) i'füiS Lcn. 56, 149-155.
GoiDsrtiN. T., IIm.l, M. R. l'i Miinkf. W. (197.') ,/. i'iioUis-y 12. .S87-900, Guiin. m. h. it Hkdoks, T. (I97(i) sous l'rcssc.
CiKhEN, M. H.. Mm.u n, H. I. & Hlndiir. S. (1971) Proc. ,\oi. Aciul. Sci. U.S..i. 6S, lO.tMO.U).
Grifiitii, J. D. (1974) Science i IVn.'ilniiytonJ 187. 1202-120.'. ■KoRNRLRCi, R. D. (1974) Science ( iVashinton) IS4. 8(>S-S71. Wmiit. m. X: E.vson. R. (1971) J. i'iroUn;y 8, .V'.'-.171.
THE TEMPLATE OF THE ISOLATED NATIVE SV40 TRANSCRI PTlONAL COMPLEXES IS A MINICHROMOSOME."
Patricio Gariglio, Rafael Llopis, Pierre Oudet and Pierre Chambon.””
Laboratoire de Génétique Moléculaire des Eucaryotes du CNRS, U.44 de 1'INSERM, Institut de Chimie Biologique, Faculté de Médecine, 67085 STRASBOURG - France, (phone : 88 - 36 56 70)
Running title : Native SV40 transcribing mi ni chromosome.
A preliminary account of this work has been presented at the 12th FEBS Meeting (Dresden, DDR, July 1978).
SUMMARY
Native SV40 transcriptional complexes containing active DNA-dependent RNA polymerase B enzymes that hâve
initiated synthesis in vivo were extracted by sait from
nuclei of infected cells late in infection. These complexes can synthesize RNA chains longer than the viral genome
under conditions that do not irreversibly alter the struc
ture of the viral chromatin. The in vitro synthesized RNA
allowed the séparation of most of the viral transcriptional complexes from transcriptionally inactive SV40 mi ni chromoso mes. Density studies indicate that about the same amount of protein is associated v/ith both the bulk of the viral mi ni chromosomes and the transcriptional complexes. Parallel shifts in the sédimentation of bulk mi ni chromosomes and transcribing complexes at increasing ionic strength and after nucléosome "exchange" strongly suggest that the actively transcribed viral genome has a structure similar to that of the bulk of viral chromatin. In agreement with the biochemical results, électron microscopy studies strongly suggest that the template for late viral transcription has a minichromos orne-1ike structure.
INTRODUCTION
The démonstration that the bulk of eukaryotic chromatin is organized in a repeating subunit structure, the nucléosome (for references, see Kornberg, 1977, and Chambon, 1977).raised the questi as to whether ail genes are packaged in a similar manner since any mode! of chromatin structure should ultimately also account, at the molecular level, for the sélective transcription of only a part of the genome. Micrococcal nuclease digestion studies (for references, see Bellard et al., 1977; Chambon, 1977), and light and électron microscopie immunological studies (Elgin et al,, 1977; Jamrich et al., 1977; McKnight et al., 1977) hâve suggested that histones are présent on actively transcribed ribosomal and non-ribosoma1 genes, but that the compact nucleosomal structure is altered in some way. Using the method of Miller (Miller and Beatty, 1969; Miller and Bakken, 1972) various investigators hâve shown that compact nucléo somes are absent on actively transcribed ribosomal genes (for refe rences, see Chambon, 1977, and Scheer, 1978). Whether compact
nucléosomes are présent or not in non-ribosomal transcription units is still debatable, mainly because undefined transcribing genes
hâve been examined. Indeed, a détermination of the DNA packing
ratio is required to demonstrate that parti cl es, wi th nucleosomal size and staining properties, which could be seen on a transcriptional unit are compact nucléosomes. This direct détermination is ob-
viously impossible when the examined transcriptional unit corresponc to an unknov/n gene. In any case, it has been observed using
the électron microscopy method of Miller that the chromatin
segments associated with nascent non-ribosomal ribonucleoprotein fibers exhibit a reduced bead periodicity v/hen compared to inactive chromatin, and that no nucleosome-like beads are visible on the very active transcriptional units of lampbrush chromosomes (for
references, see Chambon, 1977, and Scheer, 1978).
Simian Virus 40 offers a model System to study whether nucléo somes are présent on a well defined non-ribosomal transcriptional unit. Late during the infection cycle the SV40 genome is présent in nuclei of infected cells in the form of a circular chromatin-1ike structure consisting of about 20 nucléosomes, the mi ni chromosome, which can be extracted free of cellular chromât in (Griffith, 1975; Bellard et al., 1976; for other references, see Chambon, 1977). Green and Brooks,( 1976 ) hâve previously reported that at least a fraction of the SV40 complex responsible for "late" transcription ("late" SV40 transcriptional complex) can be extracted with the bulk of the mi ni chromosome. Since the SV40 genome is v/ell defined, this obser- bation offered a possibility to investigate whether the SV40 "late" transcriptional complex is in the form of a mi ni chromosome,
that is, whether nucléosomes are présent on transcriptionally active chromatin. We report here biochemical and électron microscopy stu- dies which indicate that the viral transcriptional complexes which can be extracted from nuclei late in infection consist of mini chromosomes transcribed by RNA polymerase B molécules.
MATERIALS AND METHODS
Chemiaals and Enzymes.
14
Enzymes and Chemicals were as follows : [ C]-thymidine
3
(53.6 mCi/mmol) and [ H]-thymidine (25 Ci/mmol) from CEA (France), [^H]-UTP (13.6 Ci/mmol) and [a-^^P]-CTP (>300 Ci/mmol) triethyl- ammonium sait from The Radiochemica1 Centre (England), ribonucleic triphosphates from P.L. Biochemicals (USA), Sarkosy1 NL-30 solutio from Ciba-Geigy (Switzerland) , sucrose (RNase free, density
gra-!
dient grade) from Schwartz/Mann (USA), pheny1 methy1 su 1fony1 fluoride (PMSF), sodium dodecyl sulfate (SOS), dithiothreitoi
(DTT) and RNase A (type IIA) from Sigma (USA), diethyIpyrocarbonati from Serva (Germany). E.coli tRNA (Boehringer, Germany) was further purified by treatment with protéinase K in the presence of SDS follov/ed by phénol extraction (Bellard et al., 1973). Pancreatic DNase I (Sigma, USA) was made RNase-free as described elsewhere (Bn'son and Chambon, 1976 ). Protéinase K (Merck) was preincubated for 30
min at 37°C before use. a-amanitin and 0-[ Hl-methyldemethyl-y- amanitin was a gift of Drs. T. Wieland and H. Faulstich (Heidel berg). Actinomycin D was a gift of Dr. Jolies (Rhône-Poulenc).
Dialysis bags were purchased from Sartorius GmbH (Germany). Filter^ GF/C, Whatman 3 MM and DE/81 (DEAE cellulose paper) were from
Whatman. Other Chemicals were analytical grade. SV40 DNA Form I was prepared as described previously (Mandel and Chambon, 1974b).
Ail glassware was freed of ribonucléase contamination either by heating for 12 hours at 180°C or treating with 0.2% diethyl- pyrocarbonate for 1 hour and thoroughly washing with stérile water. Most solutions were autoclaved before use.
Cells and Virus.
Africain green monkey kidney cells (CVl) were grov/n as pre- viously described (Mandel and Chambon, 1974b) in Pétri dishes with Dulbecco's modified Eagle medium, supplemented by 10% foetal calf sérum. Virus stocks of SV40 (Strain 777) were passaged at low multiplicity. For SV40 nucleoprotein complex préparation the cells were adsorbed with partially purified SV40 (0.5 ml) at a multiplicity of about 10 PFU/cell for 60 min at 37°C. After infec tion the cells were incubated in the above medium supplemented by 2% foetal calf sérum.
Isolation of SV40 ohromatin aontaining transcriptional oomvlexss.
fi \
CVl cells (1 X 10 /plate , 3 days before infection) were grown and infected as described above. DNA was routinely labelled with
14
[ C]-thymidine (0.025 yCi/ml) from 20 to about 40 hours after
in-vJ 5 X 10^ cells/plate)/
fection. At 40-42 hours post-infection, the mono IayerWwere wàsned twice with buffer A (25 m.M Tris-HCl pH 7.9, 140 mM NaCl, 5 mM
KCl, 1 mM Na2HP04) and SV40 chromatin was prepared according to
the following procedure modified from Bellard et al. (1976). To each plate, 1 ml of buffer A was added and cells were scraped off the plate with a rubber policeman and pooled in a centrifuge tube. Ail subséquent operations were carried out at 2-4°C. The cells were pelleted (5 min at 1,500 g), then suspended by gentle pipett-
ing in 0.25 ml per plate of hypotonie buffer B (25 mM Tris-HCl pH 7.9, 2 mM EDTA, 1 mM DTT, 0.2 mM PMSF) and layered in a centri
fuge tube containing from the bottom to the top : 4 ml of 0.8 M
sucrose in buffer C (20 mM Tris-HCl pH 7.5, 150 mM NaCl, 0.2 mM EDTA, 1 mM DTT, 0.2 mMPMSF), 2 ml of 0.5 M sucrose in buffer C
contaim'ng 0.25% Triton X-100, and 2 ml of 0.3 M sucrose in buffer C. After centrifugation (15 min at 5,000 g) the purified nuclei were resuspended (0.25 ml per plate) in 0.25% Triton X-100, 10 mH
Tris-HCl pH 7.9, 1 mM DTT, 0.2 mM PMSF. NaCl was added in
order to obtain a final concentration of 0.4 fi and the solution was incubated for 30 min and centrifuged for 10 min at 17,000 g. The supernatant containing the viral chromatin (10-20 pg SV40 DNA per plate) was carefully removed from the cell chromatin pellet. The spécifie activity of the []- 1abe11ed complex was about 1,000 cpm/yg DNA when counted on DE/81 (see below) or Whatman 3 MM fil- ters washed with 3% trichloroacetic acid (TCA) and éthanol, and about 2,000 cpm/yg DNA if spotted directly on GF/C filters, dried and counted in 10 ml of a toluene based scintillation mixture.
Assay for RNA polymerase aotivity.
!
The standard reaction mixture (125 yl) contained 4-8 yg of
viral chromatin DNA, 88 mM Tris-HCl pH 7.9, 288 mM NaCl, 0.18%
Triton X-100, 0.18 mM PMSF, 150 mM (NH4)2S04, 2.2 mM MnCl2, 1-7 mM
DTT, 1 mM ATP, CTP and GTP each, and 90 yM [^H]-UTP (spécifie
activity usually between 200-900 cpm/pmol, unless stated otherwise) The reaction was carried out at 32°C for the time indicated in the legend to figures and synthesis stopped in ice water by
chilling and adding EDTA to a final concentration of 10 mM. RNA synthesis was determined by measuring the incorporation of
[^H]-UMP into material retained on DE/81 filters after 5 to 6 washings with a solution containing 10% (w/v) Na2HP0^-12H20 , 0.5%
Na^P20^, 0.1% sodium dodecyl sulfate followed by two washings with
H2O and éthanol. The filters were dried and treated at room tem
10 ml of a toluene based scintillation mixture. Blank values obtained by adding EDTA at 0 time of incubation were subtracted.
Extraction and sédimentation analysis of REA synthesized in vitro.
3 volumes of 0.13% SDS (in H2O) contai ning 65 yg/ml protéinase
K were added to the RNA synthesizing reaction mixture, which was further incubated for 15 min at 37°C. Purified tRNA (10 ug/ml) was added and the samples were extracted at room température with one volume of phénol saturated with 0.05 M acetate buffer pH 5.1, and then with ch 1 oroform: i soamy 1 alcohol (24:1). After éthanol préci pitation (16 hours at -20°C in the presence of 0.1 il NaCl), the RNA was collected by centrifugation (60 min at 17,000 g). The précipi tâtes were resuspended in 0.1 ml 96% formamide, 20 mM PIPES
(Piperazine-N ,N‘-bis [2-ethane sulfonic acid]) pH 7.8, 2 mM EDTA, 'were/
«heated at 47°C for 15 min,rapidly chilied to 0°C and diluted with 0.8 ml of cold stérile water.
The formamide-treated RNA sample (0.3 ml) was layered on to a 10-30% (w/v) sucrose gradient contai ning 50 mM Tris-HCl pH 7.9,
100 mM NaCl , 1 mM EDTA and 0.1% SDS and centrifuged for 110 min at 56,000 rpm (20°C)in an SW60 rotor. Fractions were collected on Whatman 3 MM filters from the bottom of the tube. The filters
^washed-were washed 10 times with 3% TCA,'/twice with éthanol, dried and
14
counted in a toluene-based scintillation mixture. [ C]-uridine- labelled (14,000 cpm/yg) 4S, 18S and 28S rRNAs (a gift of Dr.
B. Wasylyk ) were phénol-extracted at 25°C from HeLa cells and puri fied through a 15-30% sucrose gradient.
Sédimentation analysis of the viral chromatin.
Before or after incubation for RNA synthesis, samples of viral chromatin (0.3 ml) were layered on to 3.7 ml linear 5-20% (w/v)
sucrose gradients in 10 mM Tris-HCI pH 7.9, 0.2 mM EDTA, 0.4 M NaCl ,
1 mM DTT and 0.25 mM PMSF and centrifuged for 55 min at 57,000 rpm
in a Spinco SW60 rotor at 2°C, unless stated otherwise. Fractions
were collected from the bottom of the tubes directly onto Whatman 3 MM filter squares and acid-precipitable counts were determined as described above. If the material contained a high level of [^H]-
UTP, the top five fractions were usuaily not collected. Kilham rat
virus (a gift of Dr. M. Gunther, Villejuif) was used as a 122S
sédimentation marker (Salzman and Loi, 1970).
Eleotvon miorosaopy.
Viral chromatin was isolated and incubated 60 min for RNA synthesis as described above. The incubation mixture (0.25 ml) was sedimented as in Fig. 4. Fractions (115 yl ) were collected, 10 i^l aliquots v/ere spotted onto 3 MM Whatman filters and the acid précipi table counts were determined (see above). Fractions were pooled as
described in Fig. 14 and dialyzed for 30 min at 4°C
against 100 ml of 8 mM Triethanolamine (TEA) pH 7.9. For visuaiization of nucleoprotein complexes the samples were diluted to about 0.3ygDNA/ ml in 8 mM TEA pH 7.9, adsorbed on charged grids and processed for Visualization as previously described (Oudet et al., 1975) (Method A).
the
In some cases, in order to partially extend^ribonucleoprotein (RNP) chains, samples were dialyzed for 20 min against 0.2 mM EDTA pH 7.5, and then processed as described above (Method B). Alternately the RNA chains were extended and coated with bactériophage T4 gene 32
protein (Method C). Fractions from the sucrose gradient containing transcriptional complexes were dialyzed 30 min against 80 mM TEA-HCl pH 7.5. The fractions were then treated with bactériophage T4 gene 32
protein according to Delius et al. (1973) and Wasylyk et al. (1979) except that 210 yg/ml of gene 32 protein was used. The excess of T4 gene 32 protein was removed by chromatography on a sepharose 4B column equilibrated with 80 mM TEA-HCl pH 7.5. Samples were then processed as described above. Samples were also spread in parallel according to a modification of the method of Davis et al. (1971) (Method D). Aliquots of the
pooled fractions described above werefirst dialyzed for 10 min in 10 mM Tris-HCl pH 7.5 containing 100 mM NaCl, then diluted and spread. 50 yl of the spreading solution containing 2.5 yg
cytochrome (type VI, Sigma) and 0.015 yg of DNA in 30% deionized formamide, 10 mM EDTA and 100 mM Tris-HCl pH 8.5 were spread onto bidistilled stérile water. In some cases the samples were treated with 1 yg/ml RNase (DNase free) for 10 min at room température before adsorption on grids or spreading.
RESULTS
I. Charaaterization of the RNA polymerase activity présent in SV40
chromatin extracted by sait from nualei of infeoted oells.
Viral DNA was labelled with [^^C]-thymidine from 20 to 40 hours after infection, and viral mi ni chromosomes were extracted at about 40 hours from purified nuclei as described under mate-
rialsand Methods..About 60 to 70* of the DNA which could be
extracted by the Hirt method (1967) was released under our extrac tion conditions (10-20 yg of SV40 DNA per plate dépending on the experiment). In agreement vn’th our previous report (Bellard et al 1976), électron microscopy examination of the viral chromatin préparation extracted under these conditions showed that it was free of cellular chromatin and consisted of SV40 mi ni chromosomes containing about 20 nucléosomes.
Under optimal incubation conditions (see below) the amount 3
of [ HJ-UMP incorporated by this extract increased linearly with the amount of SV40 chromatin présent in the incubation mixture
O
(Table la). Dépending on the experiment, 2-4 pmoles of [ H]-UMP were incorporated per yg of SV40 chromatin (expressed as DNA) in
30 min at 32°C. This incorporation corresponds to about 90% of the incorporation which can be obtained with transcriptional complexes solubilized with Sarkosyl (Gariglio and Mousset, 1977 ) and incubated for RNA synthesis under identical conditions (not
3
shown). The [ Hj-UMP incorporation was not stimulated by the addi tion of DNA, but, as shown in Table Ib, the activity was strongly inhibited by actinomycin D and a low concentration of a-amanitin (0.3 yg/ml). The reaction product was RNA as indicated by the requirement for ail four nucleoside triphosphates (not shown)
and by its complété digestion with pancreatic RNase (Table le). These results indicate that the viral chromatin préparation con- tains a DNA -dépendent RNA polymerase activity belonging to class B (Chambon, 1975), presumably in the form of a viral transcrip- tiona1 comp1 ex.
In order to establish the optimum time period for isolating the viral transcriptional complex, we hâve determined the RNA polymerase activity of the nuclear extract at various times after
infection. In addition, the amount of viral DNA and its rate of synthesis were followed throughout the same time period. As
3
shov;n in Fig. 1, [ H]-thymi di ne incorporation i nto viral DNA began at about 20 hours after infection and reached a maximum rate by about 40 hours. The total amount of viral DNA and the RNA
polymera-, • T
se activity in the extracted viral chromatin increased in parallel from about 20 to 40 hours post-infection, and reached a plateau level by 40-45 hours. These results suggest very strongly that new SV40 transcriptiona1 complexes were formed in proportion to the increase in the amount of viral DMA during the 20 to 40 hour period after infection.
II. Sait aotivati-on and ki-netias of Rl^A synthesis by the SV40
tvan-scriptional complex.
As shown in Fig. 2, a and b, RNA synthesis is markedly
stimulated by the presence of ammonium sulfate in the incubation mixture, both in the absence and in the presence of a saturating concentration (0,.1%) of Sarkosyl , which is known to dissociate
most proteins from chromatin (Green et al., 1975) and to inactivate RNA polymerase molécules not engaged in a transcriptional complex