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Assessment of the naked DNA content in oligonucleosome preparations
NAKE:I)
I)SA
('OS'1'EN'I'
3X5
IN OI,I(:ONI'('I,E:OSOM~S
APPENDIX Assessment of the Naked DNA
2Europea~~,
Content
Molrcdar
D 79 Hridd6ury.
F~dwnl
in Oligonucleosome
Hioloyy
Preparations
-/-
Laboratory
R~pu61ic of &wn~ny
In ord~~r to analyze for contarrlitlation by frets I)NA in oligonuclrosomt~ prepa.rations a series of elrct,ron microsropic photographs has IWTI evaluated. From defined mixtures of I)NA with oligonucleosomes the sensitivit? of t,his Assam method u as checked. It could 1~ &duced that our nucleosom~ preparations usc~l for transcription experiments n-rre frclcaof naked DNA
1. Introduction Early investigations on the in vitro transcription of chromatin could not rigorousl,v exclude the possibility that the template activity of chromatin was mainly due t)o et rrl. the presence and transcription of deproteinized DNA regions. Varshavsky (1974,197(i) observed t,hat shearing exposed long st,retches of naked DNA. No11 et (xl. (1!$75) also proposed that shear seems to damage the nucleosome st)ructure. Both for mononucleosomes and core part,icles (Lilley et al., 1979: Cotton & Hamkalo. 1981) and for reconstitutes on circular DNA (Wasylyk et al.. 1979) a contaminat,ion of these preparations with naked DNA has been report’ed, in both cases the contamination being high enough to account for the transcription observed. Lille) et nl. (1979) indeed argued that transcription in core particle preparations was mainly due to naked core DNA, which presumably derived from an artifactual loss of histones. which has been observed previously (Simon et nl., 1978). For our transcription studies with oligonucleosomes (Pflugfelder & Sonnenbichler, main t>ext) it was, t,herefore, necessary t)o measure if and to what extent oligonwleosorne preparations contained free DNA molecules. W:e describe here experiments in which we determined the sensit)ivitp of an electron microscopic assay by which we analyzed the content of naked DNA molecules in our oligonucleosome preparations.
2. Results and Discussion The method of Dubochet DNA and DX&protein t Ikdi~~atd
uf ~1. (1971) allows t)he simultaneous visualization complexes in electron microscxopy. Limiting
to tlw rrrernory of Micharl
(~n~~rrrc~.
ot for
:Giti
c:. 0. I’rI,I’(:PE:I,I)I’K
87’ AL
quant)itativc evaluat,ion was t)hat it was not known if deprot,einized l)S=\ and nueleosomes were retained with equal effic+nc*y on t,he posit,ively charged grid. In order to elwidatc this problem we used a series of defined mixtures of free oligonucleosomes and corresponding DNA fragments in order to compare the theoretical mixing ratios with those found by electron microscopic determinations. For a quantitative treatment it will be assumed that a given oligonuc,lrosornc~ preparation n’(i)t contains a crrt,ain amount of free rm4 D’(i): n’(i)
If free DNA contaminating n(i):
=
n(i)+
I)‘(i)
arises from the dissociation of nuclrosomes the concentration of DNA D’(i) would be proportional to t)hr nwleosome concentration I)‘(i)
= k x n(i).
with k as an arbitrary proportionality constant. For artificial mixtures x is t)he input ratio of I)(i)/n’(
i) :
V(i)
” = n(i) x (1 +l) The ratio
of DNA
fragments
D(i)+
D’(i) t 0 real oligonucleosomes
n(i) is y :
D(i) + k x n(i) .Y =
n(i)
Numbers of real nucleosomes n(i) and DNA f?agments U(i) in a given sample can be obtained by counting molecules ret,ainrd on the grids. To allow for differential retent’ion ofn(i) and D(i) on the grids we introduced the proportionality constants 6 and v which correlate concentrat,ions to specimen numbers. Thus : GID(i)+kxn(i)/ ,Y = ~ v x n(i) and 6
6
y=-xk+-x(l+k)xr. ” ” In order to compare the input ratios J with the observable ratios !/ a constant amount, of oligonucleosomes (n(6) and n(7)) which had been fixed with glutaraldehyde was mixed with varying amounts of deproteinized DNA of the corresponding lengths. The samples were prepared for electron microscopy as described in t,he legend to Figure A 1. Then in a series of photographs nueleosomes and DK’X fragments were counted separately. From the evaluation of 100 electron micrographs (for an example see Fig. Al ) with 16,000 objects on 30 grids from nine
PII:. Al. Electron microscopy ofoligonucleosome/DNA mixtures. Nucleosomes were fixed at a concentration of35 pg/ml with @5% glutaraldehyde for 20 min at WC’. Hrptanucleosomes were in 5 mm-triethanolamine (pH 7.9). 10 mM-ammonium chloride, 0.2 mm-EDTA; hexanucleosomes were in 5 mM-Tris (pH 7.9). 20 mM-ammonium acetate. After fixing the samples were diluted to l/20 of the DSA concentration a.nd l/5 of the buffer concentration. 1)s.~ was added t,o give thv drsired mixing ratios. Copper grids ~vcre exposed to a glow discsharye in prnt~lamine atmosphere (Dubochet P/ ol.. 1971 ). .4 drop of sample n as added fol :$OS. the grids were wash4 twictb \I ith water for 10 s each. treat4 twice for 30 s each u ith lo,, uranyl acetate in 80”,, ethanol and ww H ash4 again v ith u ate), a$ tlohcril& nl~vv. H~~t\\-cvn the straps tht aqueous phase was rt~~o~ed 1)~ tiltcr Papa ‘I’hv &ctron rrricn)graphs WWP take11 nt H magnification of’41~.000 x u itI1 i+ l’hilil,s :101 clrc*tron mi~.ro
Inputratiosx = E FII:. AP. Comparison of the input (I) and observed (y) ratios. The wncentrations of nuclcosomes DSA fragments were determined fluorimetrically as described in the main text,. Roth were mixed to desired input ratios and prepared for electron microscopy. From the observed numbers of DNA oligonuclwsomr molecules the true D(i)/n(;) ratios were calculated. A linear regression line was fitted the points. (0) Hexanucleosomrs (n(6). D(6)); (m) hept anucleosomrs (n(7). I)(7)): (A) theoretical for IO”, DNA contamination (k = 0.1).
and the and to line
artificial mixtures these ratios were derived. Starting concentrations n’(i) and D(i) were determined fluorimetrically (Setaro & Morley. 1976). Figure A2 compares the experimental data with a curve for which a IO?, contamination with DNA (II = O-1) has been assumed. The experimental curve fitted by regression analysis (correlation coefficient, 0.99) yielded an intercept with t,he y axis at 0.01. The slope was 0.81. The line within t,he experimental error thus passes through the origin. With (S/V) x k equal to zero, k has to be zero, since S/v as judged from the electron microscopic data cannot deviate much from unity. With k equal to zero S/v corresponds to the slope. Nurleosomes seem to possess a slightly higher affinity for the grids under the preparation condition used. Our data demonstrate t,hat our oligonucleosome preparations before transcription did not contain naked DNA. This finding is in agreement with the transcription data presented in the main text. Assuming that tjranscription of oligonucleosomes was solely due to naked DNA contaminat,ion (Lilley it al.. 1979) we should have found more than 30yo DNA (k = 0.3), because 30”/, was the lowest relative template activity observed in our experiments. This is clearly not compatible with the findings reported here. Furthermore, crosslinking of the t’emplates with glutaraldehyde and formaldehyde completely abolishes the transcription of nucleosomes, but has no effect on the transcription of DNA (unpublished observations). Hexanucleosomes under our preparation conditions have a sedimentation value has constant of 30 S (Kittlaus, 1980). E‘ree DNA wit,h a similar sedimentat’ion a length of about 40.000 base-pairs (Crothers & Zimm, 1965). Lye csould not’ detect’
NAKEI)
1)S.A COSTE:X’I’
IN 01,I~:ONI’~l,~OSOhlES
:ci!)
any DNA molecules of this size or those which were longer than one would expert from published compaction ratios (Elgin 8: Weintraub. 1975). Prolonged incubation of the nucleosomes (2 weeks at 4”(‘), or repeated freezing and thawing, does not lead to the appearance of free DNA. Also the kineticbehavior of such preparations was unchanged by the treat’ment.
(‘ottou. Ii. LV. & Hamkalo. H. A. (1981). .Vucl. dcids 8e.s. 9. 4-458. (‘rothrrs. I). bl. & Zimm, H. H. (1965). ./. Mol. Riol. 12. 525%536. Dultochc~t. ,J.. 1)wommun. 11.. Zollingrr. 11. & Krllrnbrrger. E. (I!)‘71 ). .1. TTtrcl.sfruct. f&s. 35. 147 167.
Elgin. S. (‘. it. & N’eintraul). H. (1975). A4,ruz(. Kw. Bio&n/.
44, 725~~77-1. Kittlaus. W. (1980). Thesis, Ludwig Jlaximilians rniversitbit,. Miinchen. Lilley, I). 31. ,I.. .Jacob. M. F. &, Houghton, bl. (1979). S~.cl. dcida H&. 7. 377~:X1!). Nell. II.. Thomas. J. 0. R: Kornbrrg, K. I). (1075). Scie,,cr. 187. 1%X--1206 Srtaro. F. & Morley, C. (:. 1). (1976). Awl. Riochw~. 71. 313-317. Simon. R. H.. C’atnrrini-Otero. R. I). & Felsenf~4d. (:. (1978). .VUC/. .-Icids Rcs. 5. 48OkCWi. Varshavsky. A. J.. Ilgin. Y. V. & (:rorgier. (:. I’. (J9i-c). Snfuw (Lordo~). 250. 602 606. Varshavsky. .4. J.. Hakayw. V. V.. Ilyin. I-. V.. Hayrv. A. A. .Jr k &orgit~v. (:. 1’. (1978). K//r. .J. Rioch~nr. 66. 21 lL223. Il’asylyk. IS., Thevenin. (i.. Oudct. P. & Chatnlton. I’. (1979). -1. Nol. Riol. 128. 41 1 440.
Ed&d
by I’. Chamhon
I)SA
('OS'1'EN'I'
3X5
IN OI,I(:ONI'('I,E:OSOM~S
APPENDIX Assessment of the Naked DNA
2Europea~~,
Content
Molrcdar
D 79 Hridd6ury.
F~dwnl
in Oligonucleosome
Hioloyy
Preparations
-/-
Laboratory
R~pu61ic of &wn~ny
In ord~~r to analyze for contarrlitlation by frets I)NA in oligonuclrosomt~ prepa.rations a series of elrct,ron microsropic photographs has IWTI evaluated. From defined mixtures of I)NA with oligonucleosomes the sensitivit? of t,his Assam method u as checked. It could 1~ &duced that our nucleosom~ preparations usc~l for transcription experiments n-rre frclcaof naked DNA
1. Introduction Early investigations on the in vitro transcription of chromatin could not rigorousl,v exclude the possibility that the template activity of chromatin was mainly due t)o et rrl. the presence and transcription of deproteinized DNA regions. Varshavsky (1974,197(i) observed t,hat shearing exposed long st,retches of naked DNA. No11 et (xl. (1!$75) also proposed that shear seems to damage the nucleosome st)ructure. Both for mononucleosomes and core part,icles (Lilley et al., 1979: Cotton & Hamkalo. 1981) and for reconstitutes on circular DNA (Wasylyk et al.. 1979) a contaminat,ion of these preparations with naked DNA has been report’ed, in both cases the contamination being high enough to account for the transcription observed. Lille) et nl. (1979) indeed argued that transcription in core particle preparations was mainly due to naked core DNA, which presumably derived from an artifactual loss of histones. which has been observed previously (Simon et nl., 1978). For our transcription studies with oligonucleosomes (Pflugfelder & Sonnenbichler, main t>ext) it was, t,herefore, necessary t)o measure if and to what extent oligonwleosorne preparations contained free DNA molecules. W:e describe here experiments in which we determined the sensit)ivitp of an electron microscopic assay by which we analyzed the content of naked DNA molecules in our oligonucleosome preparations.
2. Results and Discussion The method of Dubochet DNA and DX&protein t Ikdi~~atd
uf ~1. (1971) allows t)he simultaneous visualization complexes in electron microscxopy. Limiting
to tlw rrrernory of Micharl
(~n~~rrrc~.
ot for
:Giti
c:. 0. I’rI,I’(:PE:I,I)I’K
87’ AL
quant)itativc evaluat,ion was t)hat it was not known if deprot,einized l)S=\ and nueleosomes were retained with equal effic+nc*y on t,he posit,ively charged grid. In order to elwidatc this problem we used a series of defined mixtures of free oligonucleosomes and corresponding DNA fragments in order to compare the theoretical mixing ratios with those found by electron microscopic determinations. For a quantitative treatment it will be assumed that a given oligonuc,lrosornc~ preparation n’(i)t contains a crrt,ain amount of free rm4 D’(i): n’(i)
If free DNA contaminating n(i):
=
n(i)+
I)‘(i)
arises from the dissociation of nuclrosomes the concentration of DNA D’(i) would be proportional to t)hr nwleosome concentration I)‘(i)
= k x n(i).
with k as an arbitrary proportionality constant. For artificial mixtures x is t)he input ratio of I)(i)/n’(
i) :
V(i)
” = n(i) x (1 +l) The ratio
of DNA
fragments
D(i)+
D’(i) t 0 real oligonucleosomes
n(i) is y :
D(i) + k x n(i) .Y =
n(i)
Numbers of real nucleosomes n(i) and DNA f?agments U(i) in a given sample can be obtained by counting molecules ret,ainrd on the grids. To allow for differential retent’ion ofn(i) and D(i) on the grids we introduced the proportionality constants 6 and v which correlate concentrat,ions to specimen numbers. Thus : GID(i)+kxn(i)/ ,Y = ~ v x n(i) and 6
6
y=-xk+-x(l+k)xr. ” ” In order to compare the input ratios J with the observable ratios !/ a constant amount, of oligonucleosomes (n(6) and n(7)) which had been fixed with glutaraldehyde was mixed with varying amounts of deproteinized DNA of the corresponding lengths. The samples were prepared for electron microscopy as described in t,he legend to Figure A 1. Then in a series of photographs nueleosomes and DK’X fragments were counted separately. From the evaluation of 100 electron micrographs (for an example see Fig. Al ) with 16,000 objects on 30 grids from nine
PII:. Al. Electron microscopy ofoligonucleosome/DNA mixtures. Nucleosomes were fixed at a concentration of35 pg/ml with @5% glutaraldehyde for 20 min at WC’. Hrptanucleosomes were in 5 mm-triethanolamine (pH 7.9). 10 mM-ammonium chloride, 0.2 mm-EDTA; hexanucleosomes were in 5 mM-Tris (pH 7.9). 20 mM-ammonium acetate. After fixing the samples were diluted to l/20 of the DSA concentration a.nd l/5 of the buffer concentration. 1)s.~ was added t,o give thv drsired mixing ratios. Copper grids ~vcre exposed to a glow discsharye in prnt~lamine atmosphere (Dubochet P/ ol.. 1971 ). .4 drop of sample n as added fol :$OS. the grids were wash4 twictb \I ith water for 10 s each. treat4 twice for 30 s each u ith lo,, uranyl acetate in 80”,, ethanol and ww H ash4 again v ith u ate), a$ tlohcril& nl~vv. H~~t\\-cvn the straps tht aqueous phase was rt~~o~ed 1)~ tiltcr Papa ‘I’hv &ctron rrricn)graphs WWP take11 nt H magnification of’41~.000 x u itI1 i+ l’hilil,s :101 clrc*tron mi~.ro
Inputratiosx = E FII:. AP. Comparison of the input (I) and observed (y) ratios. The wncentrations of nuclcosomes DSA fragments were determined fluorimetrically as described in the main text,. Roth were mixed to desired input ratios and prepared for electron microscopy. From the observed numbers of DNA oligonuclwsomr molecules the true D(i)/n(;) ratios were calculated. A linear regression line was fitted the points. (0) Hexanucleosomrs (n(6). D(6)); (m) hept anucleosomrs (n(7). I)(7)): (A) theoretical for IO”, DNA contamination (k = 0.1).
and the and to line
artificial mixtures these ratios were derived. Starting concentrations n’(i) and D(i) were determined fluorimetrically (Setaro & Morley. 1976). Figure A2 compares the experimental data with a curve for which a IO?, contamination with DNA (II = O-1) has been assumed. The experimental curve fitted by regression analysis (correlation coefficient, 0.99) yielded an intercept with t,he y axis at 0.01. The slope was 0.81. The line within t,he experimental error thus passes through the origin. With (S/V) x k equal to zero, k has to be zero, since S/v as judged from the electron microscopic data cannot deviate much from unity. With k equal to zero S/v corresponds to the slope. Nurleosomes seem to possess a slightly higher affinity for the grids under the preparation condition used. Our data demonstrate t,hat our oligonucleosome preparations before transcription did not contain naked DNA. This finding is in agreement with the transcription data presented in the main text. Assuming that tjranscription of oligonucleosomes was solely due to naked DNA contaminat,ion (Lilley it al.. 1979) we should have found more than 30yo DNA (k = 0.3), because 30”/, was the lowest relative template activity observed in our experiments. This is clearly not compatible with the findings reported here. Furthermore, crosslinking of the t’emplates with glutaraldehyde and formaldehyde completely abolishes the transcription of nucleosomes, but has no effect on the transcription of DNA (unpublished observations). Hexanucleosomes under our preparation conditions have a sedimentation value has constant of 30 S (Kittlaus, 1980). E‘ree DNA wit,h a similar sedimentat’ion a length of about 40.000 base-pairs (Crothers & Zimm, 1965). Lye csould not’ detect’
NAKEI)
1)S.A COSTE:X’I’
IN 01,I~:ONI’~l,~OSOhlES
:ci!)
any DNA molecules of this size or those which were longer than one would expert from published compaction ratios (Elgin 8: Weintraub. 1975). Prolonged incubation of the nucleosomes (2 weeks at 4”(‘), or repeated freezing and thawing, does not lead to the appearance of free DNA. Also the kineticbehavior of such preparations was unchanged by the treat’ment.
(‘ottou. Ii. LV. & Hamkalo. H. A. (1981). .Vucl. dcids 8e.s. 9. 4-458. (‘rothrrs. I). bl. & Zimm, H. H. (1965). ./. Mol. Riol. 12. 525%536. Dultochc~t. ,J.. 1)wommun. 11.. Zollingrr. 11. & Krllrnbrrger. E. (I!)‘71 ). .1. TTtrcl.sfruct. f&s. 35. 147 167.
Elgin. S. (‘. it. & N’eintraul). H. (1975). A4,ruz(. Kw. Bio&n/.
44, 725~~77-1. Kittlaus. W. (1980). Thesis, Ludwig Jlaximilians rniversitbit,. Miinchen. Lilley, I). 31. ,I.. .Jacob. M. F. &, Houghton, bl. (1979). S~.cl. dcida H&. 7. 377~:X1!). Nell. II.. Thomas. J. 0. R: Kornbrrg, K. I). (1075). Scie,,cr. 187. 1%X--1206 Srtaro. F. & Morley, C. (:. 1). (1976). Awl. Riochw~. 71. 313-317. Simon. R. H.. C’atnrrini-Otero. R. I). & Felsenf~4d. (:. (1978). .VUC/. .-Icids Rcs. 5. 48OkCWi. Varshavsky. A. J.. Ilgin. Y. V. & (:rorgier. (:. I’. (J9i-c). Snfuw (Lordo~). 250. 602 606. Varshavsky. .4. J.. Hakayw. V. V.. Ilyin. I-. V.. Hayrv. A. A. .Jr k &orgit~v. (:. 1’. (1978). K//r. .J. Rioch~nr. 66. 21 lL223. Il’asylyk. IS., Thevenin. (i.. Oudct. P. & Chatnlton. I’. (1979). -1. Nol. Riol. 128. 41 1 440.
Ed&d
by I’. Chamhon