10 ChromatiN Assembly of DNA Templates Microinjected Into Xenopus Oocytes

8/13/2019 10 ChromatiN Assembly of DNA Templates Microinjected Into Xenopus Oocytes

1/9

10Chromat in Assembly of DNA TemplatesMic ro in jec ted In to Xenopus OocytesDanie le Roche, Genev ieve A lmouzn i , and Jean-P ier re Qu ivy

SummaryThe packaging of deoxyribonucleic acid (DNA) into chromatin within the eukaryoticnucleus can affect processes such as DNA replication, transcription, recombination, andrepair. Therefore, studies aimed at understanding at the molecular level how theseprocesses are operating have to take into account the chromatin context. We present amethod to assemble DNA into chromatin by nuclear m icroinjection intoXenopusoocytes.This method allows in vivo chromatin formation in a nuclear environm ent. We provide theexperimental procedures for oocyte preparation, DNA injection, and analysis of the

assembled chromatin.Key Words:Microinjection; oocytes; chromatin assembly; nucleosomes.1 . In t roduc t ion

In the nucleus of eukaryotic cells, DNA is packaged into chromatin, a nucleopro-tein complex consisting of a basic repeating unit known as the nucleosome. A singlenucleosome contains two turns of DNA wrapped around a core histone octamer comprised of the histones H2A, H2B, H3, and H4 1). Nucleosomes represent the firstlevel of compaction in chromatin, the dynamics of which will influence access toenzymes involved in DNA metabolism 2). In addition to these basic components,linker histones and a variety of nonhistone proteins are incorporated to achieve com plete genome organization w ithin a higher-order chrom atin structure 3).Thus, studies aimed at understanding mechanisms such as transcription, replication, repair, or recombination at the molecular level have to incorporate the nucleosomes and chromatin com ponents. Although chromatin can be reconstituted using purehistones 4,5), the nucleosomal templates generated in this way do not necessarilypossess some physiological characteristics of native chromatin, such as the spacing ofthe nucleosomes, the diversity of histone posttranslational m odifications, or the pres ence of nonhistone-associated proteins.

From: Method s in Molecu lar Biology vol. 322: Xenopus Protocols: Cell Biology and Signal TransductionEdited by: X. J. Liu Hum ana Press Inc., To tow a, NJ139


2/9

740 Roche Almouzni and Quivy

germinalvesicle

animalpolevegetalpole

nuclear injection:ssDNA + a-^2pcJCTP DNA purificationand analysis

ssDNA I

Fig.1.Simplified scheme oftheexperimental strategy.Above:single-stranded circular DNA(ssDNA) molecules with a-^^P-dCTP are injected into the nucleus (germinal vesicle, dashedcircle) ofastage VI oocyte. The animal and vegetal poles are indicated. After3h at 18C, theDNA is extracted and purified.Below:following its injection, the circular naked ssDNA undergoes a complementary strand synthesis (dashed arrow). During this synthesis, a-^-P-dCTP isincorporated in the synthesized DNA (^^-P), and nucleosomes (N in circles) are deposited concomitantly. After 3 h, the reaction is complete and yields a radioactively labeled double-stranded closed circular DNA molecule (dsDNA) on which nucleosomes are assembled(about1 nucleosome for 185 bp).

Efficient chromatin assembly can be reproduced in vitro in crude extracts derivedfrom Xenopus oocytes or eggs 6-9), Drosophila embryos 10-12), or human cells13-16). Ho wev er, these extracts are usually not simultaneo usly com petent for transcription, repair, and replication. The injection of DNA templatesin Xenopus oocytesas a living test tub e 17) provides a convenient and highly efficient means to

assemble in vivo the DN A into chromatin in a nuclear com partment (germinal vesicle)proficient for transcription and DNA synthesis andrepair 18-23). Th us, the impact ofthe chromatin structure on these processes can be studied directly, provided that theDNA template contains adeq uate features such as sequence con trol elements, reportergenes, and so on. In this chapter, we describe this powerful approach for chromatinassembly and discuss potential applications.The experimental strategy is summarized in Fig.1. Following injection of a circular single-stranded D NA (ssDN A; M l 3 d erivative) into the nucleus (germinal vesicle)ofastage VI oocyte, complem entary-strand DN A synthesis oc curs, leading to a closedcircular double-stranded DNA (dsDNA) 21). Coupled to this process, nucleosomesare efficiently assemb led onto the DNA tem plate. If radioactive deox ycitidine-5 '-triphosphate (a-^^P) (a-32P -dC TP ) is coinjected with DN A, it will be incorporated duringsynthesis, enabling the concomitant labeling of the DNA assembled into chromatin.

After3h, the efficiency of nucleoso me assembly can be assessed by purification ofinjected D NA . Tw o assays can be performed: (1) a superco iling assay and (2) a mic ro-


3/9

Chromatin Assembly of DMAsuperco i l ing assay

Ir

141MNase assay

o ^ 0^ \ ^fc. o ^ \ ^ b

IIIntermediatespartial duplexes)

l l oFig. 2. Analysis of chromatin assembly.Left:Autoradiography ofasupercoiling gel showing a time-course of chromatin assembly coupled to DNA synthesis. DNA was extracted andpurified after15min, 30 min,1 h, 2 h, and 4 h following injection. The positions ofthesuper-coiled form (form I) and the nicked and closed relaxed forms (forms II and Ir, respectively) areindicatedonthe right. The intermediates reflecting partial duplexes for w hich complete complementary DNA synthesis is not achieved are indicated.Right:Autoradiography of the agarose

gel showing the oligonucleosomal DNA fragments produced by micrococcal nuclease digestion at the end of an assembly reaction (3 h) in the oocyte. Time of digestion in minutes isindicated on the top, and the positions of the fragments corresponding to the m ono-, di-, tri-,and tetranucleosome are indicated.

coccal nuclease assay (M Nase assay), which are presented onFig.2. The supercoilingassay makes use of the topological properties of closed circular DNA molecules.During nucleosome assembly, the progressive deposition of nucleosomes in the presence of topoisomerase activity leads to conformational changes easily detectable onclosed circular DNA molecules. Indeed topoisomerase activity allows the absorptionof the constraints generated during the process 24). After deproteinization, topo-isomers with an increasing nu mber of negative supercoils correlate w ith the number ofnucleosomes assembled. Resolution and detection of topoisomers is achieved usinggel electrophoresis. The accumulation of the supercoiled form (form I) provides asemiquantitative estimation concerning the extent of assembly that can be followed asa function of time seeFig.2, left). The MNase assay for chromatin assembly makesuse of MNase, which cleaves the most accessible regions in a chromatinized DNA.Cleavage occu rs preferentially in the linker region between adjacent core nu cleoso mes ,generating digestion products with sizes that are multiples of the basic nucleosomalunit. The corresponding DNA fragments, when analyzed by gel electrophoresis, giverise to a characteristic profile or nucleosomal ladder. The regularity of the pattern andthe spacing between adjacent bands provides information on the quality of the finalproduct obtained in the assembly reaction seeFig.2, right).


4/9


5/9

Chromatin Assem bly of DM A 1439. 70% Ethanol stored at -2 0 C

10. TE , pH 8.0: 10m M Tris-HCl, pH 8.0, 1mM EDTA, pH 8.0.11. 5X Loading buffer: 0.5% brom opheno l blue, 5mM EDTA, 50% glycerol.12. Agarose (Ultrapure. Sigma, Saint Lou is, M O).13. SOX TAE stock solution buffer: 242 gT ris-b ase , 57.1 m L glacial acetic acid, and 100 mL0.5 MEDTA, pH 8.0 dissolved in deionized water to a final volume of 1L.14. Intensifying scre ens for X-ray films.15. Am ersham Hyperfi lm M P (Amersham Biosciences, Buckingham shire, UK ).

2 3 Analysis by Mictoccocal DigestionAssay1. Oocyte hom ogenization solution see Subheading 2.2., item 1).2. MN ase (Roche, Mannheim , Germa ny) solution in water at 15 U/(xL.3. 100 mM CaClo solution.4. Items 2 to 6as in Subheading 2.2.5. 3 M Sodium acetate, pH 5.2.6. 100% Ethanol stored at -2 0 C .7. TE , pH 8.0: 10 mM Tris-HCl, pH 8.0, 1 mM ED TA , pH 8.0.8. 5X MNase loading buffer: 0.3 % Orange G (Sigma, Saint Louis, MO ), 5 mM ED TA ,

50% glycerol see Note 2).9. Agarose (Ultrapure, Sigma, Saint Lou is, M O, US A).

10. lOX TBE stock solution buffer: 108 g Tris-base, 55 g boric acid, and 40 mL 0.5 M ED TA ,pH 8.0, dissolved in deionized water to a final volume of L.11 . See Subheading 2.2., items 14,15.

3 Methods3 1 Oocytes and Injection ofSingle StrandedDNA

1. One adult female frog is anesthe tized on ice (at least 30 min ) and sacrificed.2. Imm ediately perform a ventral cut 1 to 2 cm long with a razor blade, pull out the ovarieswith pincets, and transfer into a glass Petri dish containing OR2 medium. Quickly rinseseveral times to eliminate blood. Using two pairs of forceps, tear apart and open up theovary lobes and cut with scissors until pieces of ovaries hom ogen eous in size (about1 cm^)

are obtained. Rinse again with OR2 to eliminate blood and lysed or broken oocytes. Tra nsfer about 7.5 to 10 mL into a new 50-mL tube and add the same volume of collagenasesolution. Incubate at room temperature to dissociate follicular cells for about 2 h on arolling shaker. Check regularly to avoid overtreatment seeNote 3). Appearance of individual oocytes dissociated from follicules or follicular membranes indicates that thecollagenase treatment is achieved. Rinse thoroughly w ith OR2 and eliminate by sedim entation the youngest stages (ref.28 :small and nonpigmented, which sediment the slowest)and all debris. Wash again two times with IX MBSH seeNote 4).Oocytes can be storedfor several days at 18C in IX MB SH com plem ente d wi th 10 | ig /m L g enta m icinsee Note 5).

3. Sort manually under a dissecting microscope healthy stage VI oocytes according toref. 28see Note 6).4. M ake 10 |j,L of a solution of the ssDNA to be injected at 100 ng /|iL in the injection buffer,including 3 nL of a-^-P-dCTP see Note 7).5. This solution is aspirated in a capillary co ntaining m ineral oil mo unted on the injectionsystem under the microscope seeNote 8).


6/9

144 Roche Almouznl and Quivy6. Inject this radioactive DN A solution into the nucleus located one-third deep in the animalpart of the oocyte see Note 9): a volume of 20 to 30 nL corresponding to 2 to 3 ng ofDNA is injected.7. Follow ing injection, transfer the ooc ytes at 18C in fresh IX MB SH medium and incubate for chosen times (usually 3 h for a complete assembly).

3 2 Analysisby SupercoilingAssay1. Collect 10 healthy oocy tes into an Epp endo rf tube see Notes 6 and 10).2. Re mo ve as mu ch as pos sible the M BS H buffer and place on dry ice or liquid nitrogen tostop the reaction. This allows the oocytes to be kept for a short time before they are allprocessed for analysis when several conditions of injections, time-points, or treatmentsare performed.3. Hom ogenize the oocytes by crushing them in 50 |xL of oocyte hom ogenization bufferwith a Pipetman tip and then by pipeting up and down several times.4. Add the same volum e (50 |J.L) of 2X stop mix, 3 ^L ribonuc lease A; incuba te 30 minat 37C.5. Add 3 |a.L protein ase K and incu bate for at least 2 h at 37 C.6. Add 100 |J,L phenol/chloroform /isoamyl alcohol and vortex 10 s minimum each tube.Cen trifuge 10 min at 15.000 g at room tem pera ture, collect 100 |iL of a clear aqu eousupper phase, and transfer into a clean tube. Be careful not to take material from the interphase. A second phenol/chloroform/isoamyl alcohol extraction can be performed if theupper phase is not clear.7. Add 2 |a,L of glycog en and precip itate DNA w ith1vo l of amm onium acetate (100 |iL) and2 vol (400 \ih) of cold 100% ethanol.8. Centrifuge 30 to 45 min at 15,000 ^ at 4C to collect the DN A pellet, w ash with 800 |a,L ofcold 70% ethanol, centrifuge 5 to 10 min at 15,000g at 4C, and carefully remove thesupernatant, dry the pellet in the Speed Vac, and resuspend in 10 [xL TE.9. Add 2.5 |J.L of 5X loading buffer, load on a 1% agarose gel in IX T AE without ethidiumbromide, and run at 1.5 V/cm until the dye migrates to the bottom of the gel seeNotes 11-13).

10. Exp ose the dried gel aga inst an X-ray film for autoradiog raphy to visualize the migrationpattern of the radiolabeled DN A. Use two intensifying screens and leave at -8 0 C for atleast 4 h. Exposition time may vary according to the efficiency of DNA synthesis and thenumber of oocytes used. Phosphorlmaging system can also be used (Storm, AmershamPharmacia Biotech. Uppsala, Sweden)

3 3 Analysis by Microccocal DigestionAssay1. Hom ogenize 20 to 30 healthy injected o ocytes in 200 nL of homogenization buffer seeSubheading 3.2., item 2.and Notes 6and 10).2. Adjust to 3mM CaCl2 with the 100 mM solution before addition of 60 units of MNaseand digest at room temperature.3. Re m ove 50-|xL aliquo ts at 0.5 , t, 2, and 5 min and imm ediately transfer to a tube containing 50 |iL of stop mix to stop the digestion.4. Add 3 |iL of RNase A and incubate at 37C for 30 min.5. Proceed to DNA extraction as inSub heading 3.2., steps 4to 6.6. Precipitate the DN A by adding 2\iL glycogen, 10 |iL 3M sodium acetate. 330 |xL 100%ethanol. Vortex and store at -80C for 30 min.


7/9

Chromatin Assem bly of DNA 1457. Centrifuge 30 min at 15,000g at 4 C , wash the pellet with 800 |iL 70% etha nol. centrifuge10 min at 15,000g at 4C before rem oving the ethan ol. Repeat twice . Dry the DNA pelletin the Speed Vac.8. Resuspe nd in 10 p-L TE and add 2.5 |iL of 5X MN ase load ing buffer.9. Load on a 1.3% a garose gel in 0.5X TB E buffer and run at 5 V/cm until the Orange G dyemigrates through two-thirds of the gel seeNotes 2 an d 12).

10. 5ee Subheading 3.2, step 10.4 Notes

1. The single-stranded M 13 DNA should be of high quality and not contaminated by dsDN A.It is obtained after pha ge purification and further purification through a CsC l gradient 29).2. If an ethidium bro mid e picture is requ ired, it is im porta nt not to use a loading buffercontaining bromophenol blue as this dye will migrate at about the same position as themononucleosomal DNA and will interfere with the analysis of the migration pattern. Thus,as an alternative to bromophenol blue. Orange G is used.3. The collagena se treatm ent is a critical step for successful inje ctions. Ov ertrea tm ent willresult in very fragile oocytes that will not support storage and the injection injury.Undertreatment will result in oocytes difficult to pierce. Therefore, frequent monitoringof the oocytes during collagenase treatment is strongly advised. Different treatment timescan be performed and the corresponding defolliculated oocytes stored. The best time treatment can be quickly determined at the time of injection.4. The MBSH co ntains Ca-* ions that inhibit the action of collagenase , therefore preventing

overtreatment on storage.5. Do not keep oocytes too concen trated. They should not be in contact with each other.6. The quality of the oocy te is cruc ial. Avoid taking any oo cyte that does not look healthyand that is not stage VI 28). Stage VI oocytes should have a white band separating theupper brown an imal pole from the whitish lower veg etal pole seeschem e of the oocyte inFig.1). They should not be grayish or have white spots on the anim al pole.7. Follow safety rules concerning the handling of radioactivity. Plexiglas screens and protection devices should be impleme nted in the microinjection system. Check after use thatthe injector is free of contamination.8. We use a nanoject injector (Drum mon d) moun ted on a Leica microm anipulator, w hich

we found most convenient for these types of injections. The volume of injection can beadjusted within a range of 4.6 to 73.6 nL. Capillaries used are first beveled (with thebeveler model EG -40, Narishige) to ensure the best penetration into the oocyte as well asreproducible injection without clotting the tip of the capillary.9. An increase in size of the oocyte is visualized b y quick swe lling, which is the sign of asuccessful nuclear injection. W hen rem oving the need le, only limited leaka ge of the cytoplasmic material should occur.

10. In these assays, the DNA is radioactive and may be subjected to radiolysis. A nalysisshould thus be performed rapidly to avoid degradation of DN A, w hich could lead to lossof detectable supercoiled forms or to smeary MNase digestion patterns.11. Beca use intercalation of ethidium brom ide modifies the topology of closed doub le-stranded molecules, its presence must be avoided during the electrophoresis to ensuregood separation of the supercoiled form (form I) and the nicked and closed relaxed forms(forms II and Ir).12. We use gels that are 20 cm long (model SG E, VW R International) to obtain good resolution of topoisomers and oligonucleosomal DNA fragments. If an ethidium bromide pic-


8/9

746 Roche Almouzni and Quivyture is required. soal< tlie gel in a1(ig/mL ethidium brom ide so lution and rinse 30 min inwater at room temperature. Visualize the DNA by placing the gel on an ultraviolet tran-silluminator. Note that, to be visible, at least 100 ng of DNA should have been loadedon the gel.

13 . The absolute num ber of superhelical turns, correspond ing to each assembled nucleosom e,can be determined by visualization of the plasmid topoisomers on a two-dimensional(2D) agarose gel 30).Furthermore, closed circular relaxed (Ir) and nicked (II) forms ofthe plasmid can only be separated in a 2D gel. In some cases, it can be informative todetermine the amount of nicked plasmids because these may correspond to plasmidsassem bled into chromatin in which there are nicks. The 2D gels are set up as classical IDgels, but after migration in the first dimension, the gel is rotated by 90, and chloroquineis added at 10 |ig/mL to the electrophoresis buffer. The gel is then left to equilibrate for45 m in in the dark. The second dimen sion electrophoresis is then run in the dark under thesame conditions as for run. Under those conditions, the closed circular relaxed form (Ir),indicating that no nucleosomes are assembled, migrates faster than the nicked form (II).During the second run, it is important to recirculate the running buffer to maintain aneven distribution of chloroquine.

A c k n o w l e d g m e n t sThis work was suppor ted by l a L igue Nat iona le con t re l e Cancer (Equ ipe l abe l l i see

la L i g u e ) ; E u r a t o m ( F I G H - C T - 1 9 9 9 - 0 0 0 1 0 a n d F I G H - C T - 2 0 0 2 - 0 0 2 0 7 ) ; th e C o m m i s s a ri a t a I ' E n e r g i e A t o m i q u e ( L R C 2 6 ) ; E u r o p e a n C o n t r a c t s R T N ( H P R N - C T - 2 0 0 0 -0 0 0 7 8 a n d H P R N - C T - 2 0 0 2 - 0 0 2 3 8 ) ; a n d C o l l a b o r a t i v e P r o g r a m m e b e t w e e n t h e C u r i eIn s t it u t e an d th e Co m m i s s a r i a t a I ' E n e r g i e A t o m i q u e (P IC Pa ram e t r e s E p i g en e t i q u es ) .References

1. Lug er, K., Mader, A. W., Richmo nd, R. K., Sargent, D. F., and Richm ond, T. J. (1997)Crystal structure of the nucleosome core particle at 2.8 Angstrom resolution. Nature 389,251-260 .2. W olffe, A. P. (1997) Chromatin: Structure and Function. Academic Press, New York.3. Va quero , A., Loyo la, A., and Reinb erg, D. (2003) The constantly changing face of chro

matin. Available at: http://sageke.sciencemag.org/cgi/content/full/sageke;2003/14/re4.4. Carru thers, L. M., Tse, C , W alker, K. P., and Hanse n, J. C. (1999) Assem bly of definednucleosomal arrays from pure components. Methods Enzymol. 304, 19-35.5. Clapier, C . R., Langst, G., Coron a, D. F., Becker, P. B. , and Nightingale, K. P. (2001)Critical role for the histone H4 N terminus in nucleosome remodeling by ISWl.Mol. Cell.

Biol. 21 , 875-883 .6. Laskey , R. A., M ills, A. D., and Morris, N. R. (1977) Assembly of SV40 chrom atin in acell free system from Xenopus eggs. Cell10, 237 -243 .7. Glikin, G. C , R uberti, I., and W orcel, A. (1984) Chromatin assembly inXenopus oocytes:in vitro studies. Cell37 , 33-4 1.8. Alm ouzn i, G. and Mechali, M. (1988) Assembly of spaced chromatin involvem ent of ATPand DNA topoisomerase activity. EMBO J.7, 4355 -4365 .9. Alm ouzn i, G. and M echali, M . (1988) Assembly of spaced chromatin promoted by DNAsynthesis in extracts from Xenopus eggs. EMBO J. 7, 665-672.

10. Nelson, T., Hsieh, T.-S., and Brutlag, D. (1979) Extractsof Drosophila embryos mediatechromatin assembly in vitro. Proc. Natl.Acad Sci. USA76, 5510-5514 .
http://sageke.sciencemag.org/cgi/content/full/sageke;2003/14/re4http://sageke.sciencemag.org/cgi/content/full/sageke;2003/14/re4


9/9

Chroma tin Assemb ly of DNA 14711 . Becker, P. B. and Wu, C. (1992) Cell-free system for assembly of transcriptionallyrepressed chromatin from Drosophila embryos . Mol. Cell. Biol. 12,2241-224 9 .12. Kam akaka, R. T., Bulger, M., and Kadonaga, J. T. (1993) Potentiation of RNA polym erase

II transcription by Gal4-VP16 during but not after DNA replication and chromatin assembly.Genes Dev. 7, 1779-1795.13. Stillman, B. (1986) Chromatin assembly during SV40 DN A replication in vitro. Cell45,555-565.14. Banerjee, S. and Cantor, C. R. (1990) Nucleo som e assem bly of simian virus 40 DNA in amammalian cell extract. Mol. Cell. Biol. 10, 2863-2873.15. Gruss. C , G utierrez, C , B urhnans, W . C , DePa mphilis , M. L., Koller, T., and Sogo, J . M.(1990) Nucleosome assembly in mammalian cell extracts before and after DNA replication. EMBO J.9, 2911-2922 .16. Krude, T. and Knippers, R. (1991) Transfer of nucleosomes from parental to replicatedchromatin. Mol. Cell. Biol. 11, 6257-6267.17. Brown, D. D. and Gurdon, J. B. (1977) High fidelity transcription of 5S DNA injected into

Xenopus oocytes.Proc. Natl.Acad Sci. USA74, 2064-2068.18. McK night, S. L. and Kingsbury, R. (1982) Transcriptional control signals of a eucaryoticprotein coding gene.Science 217,316-325 .19. W yllie, A., H., Laskey, R., A., Finch, J., and Gurdon, J., B. (1978) Selective DNA conservation and chromatin assembly after injection of SV40 into Xenopus oocytes. Dev. Biol.

64, 178-188.20. Ryoji, M. and Worc el. A. (1984) Chrom atin assemb ly in Zenop.s oocytes: in vivo studies.

CeH 37, 21-32 .21. Almouzni, G. and Wolffe, A. P. (1993) Replication-coupled chromatin assembly isrequired for the repression of basal transcription in vivo. Genes Dev. 7, 2033-2047.22 . Gaillard, P.-H., Martini, E., Kaufman, P. D., Stilman, B., M oustacc hi, E., and Alm ouzni,G. (1996) Chrom atin assembly coupled to DNA repa ir: a new role for chromatin assemblyfactor I. Cell 86, 887-896.23. Belikov, S., Gelius B., Alm ouzni, G., and W range , O. (2000) Ho rmo ne activation inducesnucleosome positioning in vivo. EMBO J. 16 , 1023-1033.24. Germ ond, J. E., Rouv iere-Yaniv, J., Yaniv, M., and Brutlag, D. (1979) N icking-closingenzyme assembles nucleosome-like structures in vitro. Proc. Natl. Acad Sci. USA 76,

3779-3783.25. Ray-G allet, D., Quivy J.-P., Scam ps, C , M artini, E. M ., Lipinski, M ., and Almou zni, G.(2002). HIRA is critical for a nucleosome assembly pathway independent of DNA synthesis.Mo/. Cell9, 1091-100.

26. Ahm ad, K. andHenikoff S. (2002). The histone variant H3.3 marks active chromatin byreplication-independent nucleosome assembly. Mol. Cell 9, 1191-200.27. Tagam i, H., Ray-G allet, D., Alm ouzni, G., and Nakatani, Y. (2004). Histone H3.1 andH3.3 complexes mediate nucleosome assembly pathways dependent or independent ofDNA synthesis. Cell 116, 51-61.28. Dum ont. J. N. (1972) Oogen esis in Xenopus laevis (Daudin).J. Morphol. 136, 153-180.29. Sam brok. J., Fritsch, E. F., and Man iatis, T. (1989). Molecular Cloning A LaboratoryManual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.30. Peck, L. J. and Wang, J. C. (1985) Transcriptional block caused by a negative supercoilinginduced structural change in an alternating CG sequence. Cell 40, 129-137.

10 ChromatiN Assembly of DNA Templates Microinjected Into Xenopus Oocytes

Documents

Transcript of 10 ChromatiN Assembly of DNA Templates Microinjected Into Xenopus Oocytes