restriction enzymes have limited access to dna sequences in

The EMBO Journal vol.9 no.8 pp.2603 - 2609, 1990

Restriction enzymes have limited access to DNAsequences in Drosophila chromosomes

R.S.Jack and H.Eggert

Institut fur Genetik der Universitat zu Koln, Zulpicher Strasse 47,D 5000 Koln 1, FRG

Communicated by K.Rajewsky

Sequence specific DNA binding proteins in eukaryoticcells must efficiently locate their binding sites inchromosomes. Restriction enzymes provide a simplemodel system with which to investigate the factors whichinfluence this process. We have used P element mediatedtransformation to introduce a DNA fragment contain-ing a set of characterized restriction sites into theDrosophila germline. Embryonic nuclei prepared fromthese transgenic animals were treated with restrictionenzymes to probe the accessibility of the target restric-tion sites. The results show that the insert is within anaccessible region of the chromosome and that restrictionsites within the inserted sequence can be cut. However,the rate of cutting is biphasic. At each restriction site,a fraction of the chromosomes is cut rapidly after whichthe remainder is refractory. Similar levels of incompletecutting are obtained when the same P element constructis examined at a different chromosomal location, whendifferent sequence elements are introduced into the Pelement vector or when the experiment is carried out onnuclei from different embryonic stages. These results arediscussed in terms of how sequence specific DNA bindingproteins may locate their genomic targets in vivo.Key words: Drosophila/chromosome/restriction enzyme

IntroductionNumerous studies have demonstrated the important contribu-tion of sequence specific DNA binding proteins in the con-trol of gene expression in eukaryotes. It is also now clearthat strategies of gene regulation which are successful in pro-karyotes are also exploited in higher cells (Quian et al.,1989). However, eukaryotic genomes are structurally morecomplex than those of prokaryotes. The DNA is assembledinto chromatin which in turn is organized into large struc-tural domains (Mirkovitch et al., 1986). Wrapping sequencesaround the nucleosome need not preclude binding of proteinsto the DNA, but it must frequently hinder it substantially(Chao et al., 1980; Richmond et al., 1984). How then doesa sequence specific DNA binding protein efficiently locateits target sequence(s) in chromatin?One possibility would be that the sequences to which the

protein binds are kept permanently accessible in thechromosome. This might be achieved by accurately defin-ing the placement of nucleosomes in the surrounding areausing DNA sequences which are either preferentially in-cluded in (Shrader and Crothers, 1989) or excluded from

(Prunell, 1982; Satchwell et al., 1986) the nucleosome. Theresult would be that potential protein binding sites wouldform constitutive hypersensitive sites and would be associatedwith precisely positioned nucleosomes. Though situationslike this do occur (Simpson, 1990), constitutive nucleasehypersensitive sites-other than those associated with boundproteins-are not generally found in controlling regions ofgenes. Indeed, the capacity of specific DNA sequences todirect nucleosome placement in vivo is unclear. Directbinding studies have shown that many isolated DNAfragments can position nucleosomes formed over them invitro (Ramsay, 1986; Pina et al., 1990). However, bindingcompetition experiments indicate that there is only a marginalfree energy advantage in forming a nucleosome over sucha positioning sequence rather than over bulk DNA. Mostsuch positioning sequences are thought to be unlikely to havean autonomous capacity to position nucleosomes in vivo(Shrader and Crothers, 1989).An alternative means of ensuring efficient protein-DNA

interaction in eukaryotes would be if the proteins bounddirectly after replication, when the genome is availablebriefly as DNA (Brown, 1984). In the case of the Xenopus5S RNA gene transcription factor TFIIA, it has beendemonstrated that the protein can locate its target sequencein a chromatin template in the absence of DNA replication,both in activated egg extracts in vitro (Wolffe and Brown,1987) and by micro-injection of TFIIA mRNA in vivo(Andrews and Brown, 1987). At least in this case, replica-tion is not a necessary precondition for protein binding.The use of a specific transcription factor to determine how

a sequence specific DNA binding protein gains access to thechromosome limits the analysis to a small number of siteswithin the genome. On the other hand, non-specificnucleases, which do permit a global analysis, suffer fromthe drawback that they are able to digest the genome com-pletely. For this reason, it is not possible to carry out limitdigests with them. As a consequence it is hard to determinewhether a moderate degree of nuclease sensitivity resultsfrom moderate accessibility in all chromosomes or whetherit represents averaged accessibility within a mixedpopulation.To avoid this problem, we have exploited the special pro-

perties of restriction enzymes. These sequence specific DNAbinding proteins leave a quantitative record of their successin binding by introducing a double-stranded break into theDNA. Unlike nonspecific nucleases, restriction enzymes canbe permitted to digest chromatin to the limit of their ability.In a limit digest, the fraction of all chromosomes in whicha particular site is accessible is given simply by the fractionof this site which was cut. We find that restriction sites in-troduced into the Drosophila germline on P element vectorsare cut rapidly in embryonic nuclei and hence must be readilyaccessible. However, any given site is cut in only a fractionof all chromosomes. This phenomenon is not restricted to

2603© Oxford University Press


a particular site of P element insertion, is evident using dif-ferent but related P element vectors and is not restricted toa particular stage of embryogenesis.

ResultsExperimental designP element transformation was used to insert a DNA frag-ment containing a set of characterized restriction sites intothe Drosophila genome. Within this model substrate are twocopies of a hypersensitive site modified in vitro so as to con-tain restriction sites at the edge of the hypersensitive region.The site we used spans the heat shock promoter of theDrosophila hsp7O gene. It generates a broad area ofhypersensitivity centred around the transcription start (Wu,1980). About 100 bp of the 5' end of the site can be removedwithout affecting either the function of the promoter (Dudlerand Travers, 1984; Simon et al., 1985) or the ability of thetruncated site to generate a full sized hypersensitive regionwhen re-introduced into the chromosome (Costlow, et al.,1985). We inserted a fragment of polylinker within thisdispensible region of the hypersensitive site with the expecta-tion that the polylinker restriction sites would be renderedaccessible within the chromosome. The vector used totransform Drosophila is shown in Figure lb. Two copiesof the modified hypersensitive site are separated by 1.4 kbof spacer DNA taken from the transcribed region of the wild-type rosy locus (Keith et al., 1987).

Accessibility of the Hind!i! sitesWe first determined whether the restriction sites introducedat the edge of the hypersensitive sites are accessible in thechromosome. Embryonic nuclei were digested with HindIHfor 2 or 5 min. DNA was prepared from the digested nucleiand cut to completion with XAoI. In the absence of cuttingby HindHII, two fragments of 12 and 9 kb are recovered(Figure 1, lane 2). The 12 kb XhoI fragment extends acrossthe insert to the next XhoI site in the chromosome. The 9 kbfragment is derived from the endogenous rosy locus. IfHindm sites 2 and 3 are readily available, brief digestionwith HindIm should yield fragments of 3.0 and 4.4 kb (seemap in Figure lb). Both of these fragments were recovered(Figure la, lane 4), so the restriction enzyme does indeedhave access to its recognition sequence at the edge of themodified hypersensitive site.Much longer incubation of nuclei with HindIH prior to

preparation of the DNA and digestion with XhoI gives theresult shown in Figure 2. In addition to the two fragmentsH2 and H3 seen previously, three further fragments (H4, H5and R) are also present. The identities of these bands wereestablished from their molecular weights and from thepatterns of hybridization with pUC-rosy (Figure 2a), theisolated rosy fragment (Figure 2b) and pUC (Figure 2c).Two of them (H4 and H5) result from cutting at additionalHindmII sites within the recombinant insert (see map inFigure 2). Since site 5 is cut about as well as sites 2 or 3,the entire region must be in an accessible chromatin con-formation. The fragment labelled R is produced by cuttingat the Hindm site which lies at the 3' end of the wild-typerosy locus. This fragment is relatively over-represented onthe gels because each diploid genome contains two copiesof the rosy locus but only one copy of the recombinant insert.From the amount of remaining 12 kb XhoI fragment, we

...r._.n

Fig. 1 Restriction digestion at the modified hypersensitive sites in thechromosome in transgenic line R2A. a. Southern blot analysis. Lane 1displays size markers. Lanes 2-4 show complete XhoI digests ofDNA prepared from nuclei which had been treated for 0 minutes(lane 2), 2 minutes (lane 3), or 5 minutes (lane 4) with HindIll.Positions of the 3.0 kb H2 and the 4.4 kb H3 fragments discussed inthe text are shown. b. Map of the transformation vector. Cross-hatched boxes at the ends of the construct represent the P elementends. Black boxes show the positions of the modified hsp7Ohypersensitive sites. The heat shock promoters within them arerepresented by the arrows which indicate the direction of transcription.The fragment between these sites is an EcoRI-PstI fragment from therosy transcription unit. The five HindHI sites within the vector arelabelled H1-H5. We refer to the fragment generated fromchromosomes cut at H2 as the H2 fragment, that generated by cuttingat H3 as the H3 fragment, etc. The black bar above the map shows theextent of sequences shared with the pUC-rosy hybridization probe. TheH2 and H3 fragments are shown by the lines under the map.H = HindIm, X = XhoI.

estimate that in 50% of the chromosomes none of the HindIHsites 2-5 was cut. Furthermore, despite the long incuba-tion time, very little of the 1.4 kb fragment lying betweenHindIII sites 2 and 3 is recovered (Figure 2a, lane 3, seealso Figure 5a). In these embryonic nuclei, the HindHIclearly only achieved incomplete digestion. The basis forthis partial digestion pattern is considered in more detail.

Kinetics of cuttingWe have measured the rates of restriction digestion withinthe nuclei at HindUI sites 2 and 5. The time courses areshown in Figure 3. To our surprise, cutting at each of thesesites follows saturation kinetics and reaches a plateau within15 min. When cutting at site 2 ceases, only 15% of therecombinant chromosomes had been cut at this position.These results provides us with the means to interpret thepartial digest shown in Figure 2.Approximately 15% of the chromosomes are accessible

at site 2 and are cut rapidly. The remaining 85% are in-accessible at this site and are not cut. Similarly, 10% ofall chromosomes are accessible at site 5 but not at sites 2,3 or 4. They are cut rapidly at site 5 only and give rise tothe H5 fragment. In the same way, chromosomes accessibleat site 4 but not at sites 3 or 2 will give rise to the H4 frag-

2604

Access of restriction enzymes to chromosomes

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Fig. 2. HindIII digestion of embryonic nuclei from the transformant R2A. a. lane 1, markers. DNA from line R2A was digested to completion withXhoI (lane 2) or with HindIll and XhoI (lane 4). Lane 3, nuclei were digested with HindHI for 60 min, after which the DNA was prepared and cut

to completion with XhoI. The fragments H2-H5 which hybridize to the pUC-rosy probe and are discussed in the text are labelled. Fragment R isderived from the wild-type rosy locus. The filter was stripped and reprobed with the rosy insert fragment (b) and a duplicate filter was probed withpUC12 (c). The map (d) shows the positions of the five HindIll sites and the origin of the fragments H2-H5. The bars above the map show thesequences which are complementary to the pUC-rosy probe (a), the rosy insert fragment (b) and the pUC12 probe (c). Other conventions in the map

are as in Figure 1.

ment. Finally chromosomes accessible at site 3 but not atsite 2 will generate the H3 fragment. Each of these restric-tion sites is being presented on two distinct populations ofchromosomes. The accessible population is rapidly and com-pletely cut. The inaccessible population is not restricted atany detectable rate. This, rather than incomplete cutting ofa homogenous population of chromosomes, explains thepartial digestion pattern shown in Figure 2.

No lack of enzymeThe plateau in the rate of restriction after 15 min of diges-tion is not due to lack of enzyme. Nuclei were prepared anddivided into three aliquots. Aliquot A was incubated for60 min with 200 U/ml of HindlIl. Aliquot B was incubatedin digestion buffer alone for 30 min, after which 200 U/mlHindIll was added for a further 30 min. Aliquot C was

incubated for 30 min with 200 U/ml of HindU, after whichan equal amount of enzyme was added and the digestionallowed to proceed for a further 30 min. DNA was preparedfrom all the samples, and cut to completion with XhoI, andthe yield of the H2 fragment was determined by micro-densitometry. The result is shown in Figure 4. Since theyield of the 3 kb fragment from aliquot B is not significantlydifferent from that from aliquot A, a 30 min incubation indigestion buffer does not cause a change in the nuclei suchthat the enzyme can no longer enter. The yield of the 3 kbfragment from aliquot C is clearly not double the yield fromaliquot A or B. Thus, adding fresh enzyme after the plateauhas been reached does not increase the extent of digestioneven though the enzyme can certainly gain access to thenuclei. Our inability to cut these HindfII sites to completionis therefore not due to lack of enzyme.

No chromosomal position effectsTo rule out a unique chomosomal position effect, a secondindependent transgenic line, R3A, was used. Analogousresults were obtained from this insert which is on

chromosome 3 (Figure 5A). In this recombinant, the XhoI

fragment which covers the insert is shorter than that in R2Aand runs under the 9 kb XhoI fragment derived from the

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10 20 30 40 50 60 70 80

INCUBATION TIME (MINUTES)

Fig. 3. Quantification of restriction digestion rates at HindIll sites 2and 5. Nuclei prepared from fly line R2A were incubated with Hindulfor the indicated times after which genomic DNA was prepared andcut to completion with XhoI. Autoradiograms were analysed bymicrodensitometry. The fraction of chromosomes cut was estimatedfrom the total amount of genomic DNA loaded and from the amountof each fragment determined by reference to the quantificationstandards run on the same gel. Percentage of cut chromosomes isplotted against the digestion time. Cutting at site 2, 0. Cutting at site5, A.

wild-type rosy locus (Figure SA, lane 2). Chromosomes cutat HindHII site 2 yield an XhoI-HindIll fragment which co-

migrates with the H5 fragment. Apart from this the resultis similar to that obtained with line R2A.Figure Sb shows the result obtained using a transgenic line

containing a construct in which a fragment of different sizeand sequence has been placed between the two modifiedhypersensitive sites. The insert in this case is on the Xchromosome. Once again, very analogous results are

obtained. The fragments recovered indicate that each cutchromosome acquired on average only one cut in the region

2605

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Fig. 4. Incomplete digestion is not due to lack of enzyme. Column A.Nuclei prepared from R2A were incubated with HindIH for 60 min,after which DNA was prepared from them and cut to completion withXhoI. Microdensitometry was used to measure the signal intensity ofthe 3.0 kb H2 fragment. Column B. Nuclei were incubated for 30 minin digestion buffer alone prior to the addition of HindIII. Digestionwas for a further 30 min. Subsequent analysis was as for column A.Colunm C. Nuclei were incubated with HindIII for 30 min afterwhich fresh HindIII was added and the digestion continued for afurther 30 min. Subsequent analysis was as for A and B. The relevanttracks from the autoradiogram are inserted into the columns. Theseconfirn that the overall pattern of digestion is the same in all cases.

covered by the probe and that about half of the recombinantchromosomes were not cut at all. The phenomenon of rapidbut incomplete cutting therefore applies to P elements in-serted on the X chromosome (Figure 5b) as well as on bothmajor autosomes (Figures 2 and 5a). It is not restricted toa particular insert sequence nor is it dependent on the precisesize of the fragment between the modified hypersensitivesites.

A site cut almost to completionIn contrast to the incomplete restriction digestion seen at theseHindIH sites, the XAoI site is cut much more completely.This is seen in Figure 6 in which nuclei from the transgenicline R2A were incubated with XhoI. DNA was then preparedand cut to completion with Hindm. The HindIlI fragmentextending from sites 1 to 2 is 4.6 kb in length (see map inFigure lb). The extent of cutting at the XAoI site within thenuclei can therefore be judged by the fraction of the 4.6 kbHindm fragment which is reduced in size to 3.0 kb. Densit-ometry of the autoradiogram indicates that - 75% of thesite was cut in the chromosome. This excludes the possibilitythat the HindIfl results are due to a non-specific interactionduring the preparation of the nuclei in which any exposedpiece of DNA is covered with 85% efficiency. Were thisthe case then the XhoI site should be no more than 15%digestible.The endogenous rosy locus yields a HinduI fragment of

7.2 kb. Cutting at the unique XhoI site within the locus wouldreduce this band to 4.2 kb. Densitometry shows that < 5 %of this XAoI site was cut within the nuclei. Thus it is nota general peculiarity of chromosomal XhoI sites that theyare always efficiently cut.

Fig. 5. HindIH digestion of nuclei from other transgenic lines. Nucleiwere prepared and treated exactly as for the experiment shown inFigure 2. a. The pattern of digestion with R3A which carries a singlecopy of the same vector as is present in R2A. b. The digestion patternobtained with an X chromosomal insert of a vector in which the whitepromoter region was placed between HindlIl sites 2 and 3. The bandabove the H5 fragment in lane 3 is a HindIII-XhoI fragment from thewild-type white locus. Conventions on the figure are as in Figures 1and 2.

*4-_

4*4m

Fig. 6. Cutting the unique XhoI site. Nuclei from line R3A. Lane 1,DNA cut to completion with HindIll. Lanes 2-4, nuclei cut withXhoI for 5, 10 or 20 min respectively prior to preparation of thegenomic DNA which was then cut to completion with HindfIl. The7.2 kb HindIII fragment spans the wild-type rosy locus. Cutting theunique XhoI site reduces this band to 4.2 kb. The 4.6 kb HinduIfragment spans the insert from HindIH sites 1 to 2 (see map in Figure2). Cutting at the XhoI site reduces this fragment to 3.0 kb. The 14 kbHindHI fragment is the insert between HindIII sites 2 and 3.Hybridization probe was pUC-rosy.

Restriction digestions at the Xhol and Hindill sites areindependentIncubation of nuclei from the transgenic line R3A with bothHindm and XhoI gives the result shown in Figure 7. Wesee the same spectrum of bands H2 - H5 as is found whenthe HindIII digestion is carried out in the nuclei followedby a complete XhoI digest of the DNA. Thus within a singlechromosome, cutting at the XIzoI site does not interfere withcutting at the HindlIl sites and vice versa. This allows usto rule out the possibility that these sites are coupled by in-clusion within a conformationally stressed domain.

Different embryonic stages yield the same resultThe experiments reported so far were all carried out usingembryos collected over an 18 h period. To determine

2606

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Fig. 7. Restriction digestion at two neighbouring sites in onechromosome. Lane 1, markers. Lane 2, nuclei from R3A embryoswere incubated for 30 min with both HindIHl and XhoI. Genomic DNAwas prepared and analysed without further digestion. Hybridizationprobe was p

NI 1 2 3 4kb-

4.6- -H43.9-

3.0 - --H2

2.4- _

1.66b

1.4- *^

Fig. 8. Accessibility of HindIll sites 2-5 in nuclei from differentembryonic stages. Lane M, markers. Lane 1, XhoI digest of DNAfrom line R3A. Nuclei from 0-18 h embryos (lane 2), 12-18 hembryos (lane 3) and 0-6 hour embryos (lane 4) were incubated withHindIII after which the DNA was prepared and cut to completion withXhoI.

whether the chromosomes which were cut at each of thedifferent HindlIl sites originated from different cell pop-ulations, we prepared nuclei from 0-6 h embryos and from12-18 h embryos. The majority of embryonic cell divisionstake place during the first 6 h of development (Campos-Ortega and Hartenstein, 1985). Conversely, the 12-18 hembryos contain a diverse mixture of end differentiated cells.The result of an experiment on these two populations ofnuclei is shown in Figure 8. The patterns of restrictiondigestion within the nuclei are not significantly different innuclei from these different stages.

DiscussionRapid but incomplete cutting at each Hindil siteIn these experiments we use restriction enzymes as modelsequence specific DNA binding proteins. Each of the Hindmsites H2-H5 is cut rapidly but incompletely in the

embryonic nuclei. Hindm sites 2 and 3 are on polylinkerfragments. The BamHI and PstI sites which lie next to themare cut in embryonic nuclei in the same way and to the sameextent as the Hindm sites (data not shown). The XhoI sitein the P element is likewise cut rapidly but incompletelythough, for reasons which are not yet clear, it is cut to agreater extent than any of the Hindm sites.Although the curious pattern of restriction digestion that

we obtain is not limited to a particular position in thegenome, this is not the same as saying that it will be en-countered at all chromosomal locations. P elements-onwhich our vectors are based-frequently insert into the 5'upstream regions of genes. A high resolution in situ analysisof the Notch locus indicates that the 5' region, within whicha large number of P element insertions have been recovered,is in the form of open chromatin. The DNA packing ratioof this open region is compatible with it being in the formof a 10 nm chromatin fibre. In contrast, the transcriptionunit itself is more compacted and may be in the form of a30 nm fibre (Rykowski et al., 1988). By using P elementvectors we may therefore be restricted to examining whathappens in open chromatin. This notion is in line with theready accessibility of all four Hindm sites 2-5 comparedwith the considerably less accessible XhoI site in theendogenous rosy gene.

Many different populations of the recombinantchromosomesFor each of the HindmI sites 2-5, two distinct populationsof chromosomes are present prior to the addition of enzyme.In one the site is readily accessible and is rapidly cut, inthe other the site is cryptic and is cut very slowly if at all(Figure 3). The existence of two distinct chromosomepopulations for each of four independently cut Hindmll sitesrequires a total of 16 separate populations of the recombinantchromosome. How can these different populations begenerated? One way would be if in different cell types orlineages the P element insert within the chromosome wasfolded in different ways so that in each cell type only oneof the HindHI sites 2-5 was accessible. If in cell type Aonly site 2 could be cut, only site 3 in cell type B, etc., then16 lineages would be sufficient to yield the 16 structuralisomers necessary to explain our results. While we cannotformally exclude this possibility, the experiments shown inFigures 2, 5 and 8 place severe constraints on it. Neitherthe site into which the P element inserts (compare Figures 2and 5a) nor the sequence of the DNA fragment lying be-tween HindI sites 2 and 3 (compare Figure 5a and b) affectsthe pattern of cutting we obtain. Thus the generation ofstructural isomers of the P element insertion within thechromosome would have to be sequence independent. Inaddition the experiment in Figure 8 indicates that if the dif-ferent fragments H2- H5 are derived from different celltypes or lineages, then each of these lineages must contributeroughly the same fraction of cells to the embryo during thefirst 6 h of development as they do during the final hoursof embryogenesis. Indeed, simply on the basis of thenumbers of different populations required, it would seemmuch more likely that the phenomenon has a purelystochastic basis.

2607


Randomly distributed nucleosomesThe rapid but very incomplete cutting which we see mightbe simply explained if the region of the insert betweenthe HindIlI sites 2 and 5 is associated with randomlydistributed nucleosomes which block access to each site with85% probability. The fact that chromosomes which havebeen cut at HindHI site 5 are not subsequently cut at sites4, 3 or 2 tells us that in these restricted chromosomes thenucleosomes are essentially immobile. Is this a reflectionof the situation in vivo?Were nucleosomes in open regions of the chromosome

static in vivo, as they are in vitro, then our results suggestthat a DNA binding protein may be able to locate its recogni-tion sequence in only 15% of all chromosomes. A recogni-tion sequence on the X chromosome in males wouldtherefore be accessible to a trans-acting factor in only everysixth cell. For many essential functions this would beunacceptably inefficient. However, were the nucleosomesin such open regions of chromatin to engage in randomjostling motion then, at each instant, a different 15% of theDNA sequence would be accessible and the factor could bindefficiently. The surprisingly small sequence dependentdifferences in nucleosome binding free energies (Shrader andCrothers 1990) suggest that this may well be possible. Thestructure of chromatin is known to be very sensitive to smallchanges of ionic strength and buffer composition (Widom,1986). It is therefore conceivable that regions of structurallydynamic chromatin within the chromosome are frozen intoimmobility during the course of the experiment.The genomic targets of sequence specific DNA binding

proteins may thus be located in open regions of thechromosome within which efficient access to the DNA isensured by random nucleosome movement. This wouldobviate a requirement for special mechanisms which per-manently maintain these target sequences as nucleosome freestructures.

Materials and methodsConstruction of the modified hypersensitive siteA recombinant (pHTA5'-108) in which a HindHI linker had been inserted108 bp upstream of the transcription start of a Drosophila hsp7O gene(Pelham, 1982) was used as a source of the hypersensitive site. pHT 5'-108was cut with Bgll, flush ended with T4 polymerase, and cut with HindIII;the 500 bp fragment containing the hypersensitive site and the heat shockpromoter was gel purified. This fragment was inserted into pUC12 whichhad been cut with XbaI, flush ended with Klenow and recut with HindIII.The resulting clone (pUC-H) was identified by double-stranded dideoxysequencing. The 98 bp AluI fragment from the lac UVS promoter was gelpurified and ligated into SmaI cut pUC12 to yield the clone pUC-O. Theorientation of the operator was determined by sequencing. The clonepUC-OH was generated by cutting pUC-H with SacI, flush ending withT4 polymerase, adding HindHI linkers and excising the hypersensitive sitecontaining fragment with HindIll. This fragment was gel purified and ligatedinto HindIll cut pUC-O. The identity and orientation of the resulting clonewas determined by sequencing. The modified hypersensitive site thusgenerated contains, within the hypersensitive region, a copy of the lacoperator as well as HindIII, PstI, XbaI and BamHI restriction sites.

Construction of the transformation vectorsThe Drosophila transformation vector pUChsneo (Steller and Pirotta, 1985)was cut at the unique BamHI site and flush ended with Klenow. pUC-OHwas cut with EcoRI, flush ended and cut with SniaI; the modified hyper-sensitive site containing fragment was gel purified. This fragment was ligatedinto the BamHI cut transformation vector. The clone neoHO was recoveredand the orientation of the insert determined by sequencing. neoHO was cutwith SmaI and a flush ended 1. 1 kb EcoRI-PstI fragment from within thecoding region of the rosy gene (co-ordinates + 1 to + 1105, Keith et al.,

1987) was ligated into it. A second clone was constructed in the same way;a fragment covering the promoter region of the white gene (co-ordinates+3230 to +4828, O'Hare et al., 1984) was inserted into neoHO. Eachof these clones was then cut with EcoRI and flush ended with Klenow; asecond copy of the modified HSS was inserted to yield the final transforma-tion vectors which were identified by sequencing.

Fly transformationFly transformation was carried out by micro-injection of the clones intopreblastoderm embryos (Rubin and Spradling, 1982) along with the helperclone p7r 25.7wc (Karess and Rubin, 1984). GO flies were crossed to wild-type and recombinant progeny were selected on G418 containing medium.Standard balancer crosses were used to establish homozygous or balancedlethal stocks. Two fly lines which contain a single copy of the rosy constructwere used in these experiments. In the line R2A the insertion is on the secondchromosome and is homozygous lethal. In the line R3A the insertion ison the third chromosome and is homozygous viable. In addition a third linewas used in which the white construct was inserted on the X chromosome.

Preparation of nucleiEmbryos were collected from population cages over an 18 h period. Theywere washed with water and 5 ml settled volume of embryos was thendechorionated by suspension for 2 min in 50 ml of ice-cold 2% sodiumhypochlorite. Dechorionated embryos were collected on a Nitex filter washedwith cold water and then suspended in 200 ml of Buffer A (10 mM TrispH 7.5, 50 mM KCI, 0.5 mM EDTA, 0.5 mM EGTA, 0.5 mM DTT,0.75 mM spermidine). The embryos were disrupted in an all glasshomogeniser (Wheaton) by two strokes of the B pestle, filtered throughNitex and the crude nuclei peileted at 1930 x g at 40C. Nuclei were washedfour times by gentle resuspension in Buffer A followed by three washesin Buffer A supplemented with 0.3% NP40. Digestions (1 ml) were carriedout in digestion buffer (50 mM Tris, pH 7.5, 50 mM NaCI, 4 mM MgCl2)at 37°C using 150-200 U/mi of the appropriate restriction enzyme. Prepara-tions which contained a significant fraction of lysed nuclei, as evidencedby aggregation during preparation or digestion, were discarded as theirchromatin structure may be severely disorganised (Noll et al., 1975).

Analysis of digestion productsRestriction enzyme digests of nuclei were terminated by the addition of SDSto 0.5%, EDTA to 25 mM and Proteinase K to 1 mg/mi. After incubationovernight at 37°C they were extracted twice with phenol and twice withchloroform, and then ethanol precipitated. An aliquot of each sample wasthen cut to completion with appropriate restriction enzymes to permit mappingof the sites cut within the nuclei. Digests were analysed on 0.9% agarosegels. DNA was transferred to nylon membrane (Hybond, Amersham) byalkaline transfer (Rigaud et al., 1987) and hybridized with probes labelledusing a random primed labelling kit (Boehringer).

MicrodensitometryAutoradiograms were quantitated using a Joyce - Loebl Chromoscan 3densitometer. Each gel was loaded with a set of two-fold serial dilutionsof linearized pUC-rosy and of this plasmid with the insert excised. The signalintensities derived from the dilution series were used to determine the linearresponse range of the film and to provide quantification standards for relatingsignal intensity to the percentage of chromosomes cut.

AcknowlednementsWe thank H.R.B.Pelham and G.M.Rubin for DNA clones, Benno Muller-Hill and Klaus Rajewsky for lively discussion, Anna Starzinski-Powitz andWalter Vielmetter for critical readings of the manuscript and theBundesministerium fur Forschung und Technologie for support.

References

Andrews,M.T. and Brown,D.D. (1987) Cell, 51, 445-453.Brown,D.D. (1984) Cell, 37, 359-365.Campos-Ortega,J.A. and Hartenstein,V. (1985) 7he Embryonic Develop-

ment of Drosophila melanogaster. Springer-Verlag.Chao,M.V., Gralla,J.D. and Martinson,H.G. (1980) Biochemistry, 19,

3254-3260.Costlow,N.A., Simon,J.A. and Lis,J.T. (1985) Nature, 313, 147-149.Dudler,R. and Travers,A.A. (1984) Cell, 38, 391-398.Karess,R.E. and Rubin,G.M. (1984) Cell, 38, 135-146.

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Keith,T P., Riley,M.A., Kreitman,M., Lewontin,R.C., Curtis,D. andChambers,G. (1987) Genetics, 116, 67-73.

Mirkovitch,J., Spierer,P. and Laemmli,U.K. (1986) J. Mol. Biol., 190,255-258.

Noll,M., Thomas,J.O. and Kornberg,R.D. (1975) Science, 187,1203-1206.

O'Hare,K., Murphy,C., Levis,R. and Rubin,G.M. (1984) J. Mol. Biol.,180, 437-455.

Pelham,H.R.B. (1982) Cell, 30, 517-528.Pina,B., Briggemeier,U. and Beato,M. (1990) Cell, 60, 719-731.Prunell,A. (1982) EMBO J., 1, 173-179.Quian,Y.Q., Billeter,M., Otting,G., Muiller,M., Gehring,W.J. and

Wuthrich,K. (1989) Cell, 59, 573-580.Ramsay,N. (1986) J. Mol. Biol., 189, 179-188.Richmond,T.J., Finch,J.T., Rushton,B., Rhodes,D. and Klug,A. (1984)

Nature, 311, 532-537.Rigaud,G., Grange,T. and Pictet,R. (1987) Nucleic Acids Res., 15, 857.Rubin,G.M. and Spradling,A.C. (1982) Science, 218, 348-353.Rykowski,M.C., Parmlee,S.J., Agard,D.A. and Sedat,J.W. (1988) Cell,

54, 461-472.Satchwell,S.C., Drew,H.R. and Travers,A.A. (1986) J. Mol. Biol., 191,659-675.

Shrader,T.E. and Crothers,D.M. (1989) Proc. Natl. Acad. Sci. USA, 86,7418-7422.

Simon,J.A., Sutton,C.A., Lobell,R.B., Glaser,R.L. and Lis,J.T. (1985)Cell, 40, 805-817.

Simpson,R.T. (1990) Nature, 343, 387-389.Steller,H. and Pirotta,V. (1985) EMBO J., 4, 167-171.Widom,J. (1986) J. Mol. Biol., 190, 411-424.Wolffe,A.P. and Brown,D.D. (1987) Cell, 51, 733-740.Wu,C. (1980) Nature, 286, 854-860.

Received on April 2, 1990

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restriction enzymes have limited access to dna sequences in

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