Nucleic Acids Research Volume 144Nme Number 5 1986 96NcecAisRsac

Vlum

Methidiumpropyl-EDTA-ironalI) cleavage of ribosomal DNA chromatin from Dictyostelgum

discoideum

Roger W.Parish, Elizabeth Banz and Penelope J.Ness1

Plant Biology Institute, University of Zurich, Zollikerstr. 107, CH-8008 Zurich and 'Cell Biology Institute, ETHZ, Honggerberg, CH-8093 Zurich, Switzerland Received 18 October 1985; Revised and Accepted 6 December 1985

ABSTRACT We have used methidiumpropyl-EDTA-iron(II) LMPE-Fe(II)] in parallel with micrococcal nuclease to investigate the chromatin structure of the extrachromosomal palindrome ribosomal RNA genes of Dic.tvos.telium. Confirming our earlier results with micrococcal nuclease (1,2), MPE*Fe(II) digested the coding region of rapidly transcribing rRNA genes as a smear, indicating the absence or severe disruption of nucleosomes, whereas in slowly transcribing rRNA genes, a nucleosomal ladder was produced. In the central non-transcribed spacer region of the palindrome, MPE*Fe(II) digestion resulted in a normal nucleosomal repeat, whereas micrococcal nuclease gave a complex banding pattern. The difference is attributed to the lower sequence specificity of MPE-Fe(II) compared to micrococcal nuclease. In the terminal region of the palindrome, however, both substances gave a complex chromatin digestion pattern. In this region the DNA appears to be packaged in structures strongly positioned with respect to the underlying DNA sequence.

INTRODUCTION The changes that occur in chromatin structure during transcription are still uncertain. There is disagreement about the fate of the nucleosome, some authors believing it remains unchanged while others suggest conformational changes or even histone release from the DNA are necessary prerequisites for transcription (reviewed in Reeves, 1984; see

Discussion). We are studying the chromatin structure of ribosomal RNA (rRNA) genes in Dictyostelium discoideum (1-4). The rRNA genes are located on extrachromosomal palindromic dimers, about 88 x 103 base pairs long (5,6) which are repeated about 90 times per haploid genome (7). Each dimer encodes a 36S primary transcript, which is processed to yield mature 26S, 17S and 5.8S rRNAs. Two distal regions of the palindrome code for 5S RNA (8). After limited digestion with micrococcal nuclease, the rRNA coding regions of exponentially growing vegetative cells reveal no periodic

C I R L Press Umited, Oxford, England.

2089

Nucleic Acids Research structure or DNA fragments of nucleosomal size (1,2). The coding but not the non-coding regions of the palindrome are accessible to restriction enzymes (3). Electron microscopy of lysed nucleoli (2,3,9) or of denatured DNA which has been psoralen-closslinked in nucleoli (4) indicates the nontranscribed spacer is packaged into nucleosome-like structures. However, micrococcal nuclease digestion of the spacer region gives irregular patterns of DNA fragments, which may derive from several nucleosome repeats and "half-nucleosomes" (2,10). Micrococcal nuclease has a marked sequence preference, introducing cleavages into purified DNA at specific positions (11,12). Since cutting at the same sites has been observed in chromatin (12,13), interpretation of data can be problematic. Micrococcal nuclease can also cleave nucleosome core particles internally (at roughly 10 bp intervals). Hence, any preferred cutting sequence at these regions will give an erroneous impression of nucleosome phasing when restriction enzyme cleavage of core particle DNA is used to test for phasing (14). Methidiumpropyl-EDTA (MPE) cleaves DNA efficiently and with low sequence specificity in the presence of Fe(II), 02 and reducing agents (15,16). MPE.Fe(II) footprinting has been used to determine the preferred binding sites and binding site sizes of drugs on DNA (16-18). It cleaves chromatin into oligonucleosomes, apparently due to a high preferential cleavage in linker DNA, and has been used in nucleosomal positioning studies (19-22). Here we report the use of MPE.Fe(II) to study the chromatin structure of the ribosomal DNA of D. discoideum. The results are compared to those obtained with micrococcal nuclease. Data from these comparative studies clarify our earlier data on the structure of the central spacer region, and indicate that nucleoprotein complexes at the ends of the palindrome are strongly positioned with respect to DNA sequence.

MATERIALS AND METHODS Preparation of nucleoli and nuclei. D. discoideum cells, axenic strain Ax2, were grown in shaking liquid culture with HL-5 medium (23). Nucleoli and nuclei were isolated from vegetative cells as previously described (2, 10, 24) with the following modifications: lysis with Triton X-100 was with cold (0-4 C) solutions, but was carried out at room temperature; the nucleolar and 2090

Nucleic Acids Research nuclear pellets were washed in 20 mM Tris-HCl (pH 7.8), 2 mM CaCl2, and resuspended in 20 mM Tris-HCl (pH 7.8). Slugs were obtained by washing vegetative cells with K/PO4 buffer (20 mM potassium phosphate, pH 6.2) and about 4.5 x 109 cells were resuspended in 25 ml of the same buffer. The 25 ml cell suspension was spread evenly on 2% agar in K/PO4 in 28 x 48 cm aluminium trays (each containing 400 ml agar; final cell concentration 3.5 x 106 cells/cm2). When almost dry, two trays were placed face to face and incubated in the dark at 230C for about 15 h. The slugs were washed off the trays with cold K/PO4 onto nylon filters (25 pm "Nybolt", Swiss Bolting Cloth MFG Co. Ltd). Non-aggregated cells wash through the filter. The slugs were scraped into K/PO4 and disaggregated by squeezing them through a syringe (0.8 x 80 mm). The cells were passed through the 25 pm filter and nuclei isolated as described above.

klicrococcal nuclease digestion. Washed nucleoli or nuclei from vegetative cells were resuspended in 20 mM Tris (pH 7.8), 0.5 mM CaCl2, and incubated at 37 0C with 75 units of micrococcal nuclease/ml (Worthington) (10). An enzyme concentration of 15 units/ml was used for slug nuclei. Incubation times varied from 15 sec to 10 min. The "zero" digestion was incubated without enzyme at 370C for 2 min. The reaction was stopped by adding EDTA to 20 mM and sodium dodecyl sulphate to 0.5%. After treating with 200 pg/ml proteinase K (10) the samples were extracted twice with phenol/chloroform/isoamylalcohol (25/24/1, by vol.) and precipitated with ethanol. The precipitates were washed with 70% ethanol, resuspended in RNase A (2 pg/ml) and incubated at 370C for 30 min before applying to gels (26). Pure DNA was cut with micrococcal nuclease at 00C; 20 pg of DNA in 200 p1 of 20 mM Tris (pH 7.8), 0.5 mM CaC12, was incubated with 10 units of enzyme for 1-15 min. MPE* Fe (II) digestin Washed nucleoli or nuclei were resuspended in 20 mM Tris-HCl (pH 7.5). The complex was prepared separately by mixing equimolar quantities of MPE (stored as a 1 mM stock solution in water at -20°C) and freshly prepared ferrous ammonium sulphate and diluting with buffer to the required concentration. The mixture was preincubated with nucleoli, nuclei or DNA for 1 min at 25°C at a concentration of 50 pM MPE-Fe(II), 50 pM ferrous ammonium sulphate. 10 pM of each was used for digestion of purified DNA. The reaction, carried out at 250 was initiated by 2091

Nucleic Acids Research adding freshly dissolved dithiothreitol to a final concentration of 2 mM (nuclei or nucleoli) or 1 mM (DNA). Aliquots of reaction mixture were withdrawn at times ranging from 2-64 min and mixed with 5 mM bathophenanthroline disulphonate (4,7-diphenyl-1,10-phenanthroline disulphonate, Sigma) to stop the cleavage reaction. The "zero" control was treated only with bathophenanthroline disulphonate. A further control involved incubation for 1 min with ferrous ammoniumsulphate and dithiothreitol only before adding bathophenanthroline disulphonate. Sodium dodecyl sulphate, EDTA and proteinase K were added to 0.5%, 20 mM and 200 pg/ml respectively, and DNA was extracted and purified as described for micrococcal nuclease digestion. DNA Purification. DNA was prepared from nucleoli or nuclei using caesium chloride/ethidium bromide gradients (5). The DNA band was extracted with isopentyl alcohol, dialysed extensively against 10 mM Tris-HCl, 1 mM EDTA (pH 8), adjusted with 0.3 M sodium acetate to pH 4.5 and precipitated with ethanol. Gel electrophoresis. Micrococcal nuclease and MPE Fe(II) fragments were analyzed on horizontal gels 25 cm x 20 cm x 0.3 cm. Gels contained 2.3% Sigma type II agarose. The electrophoresis buffer contained 36 mM Tris, 30 mM NaH2PO4, 1 mM EDTA. Gels were run overnight at 4 0C, 40 V. Size markers were HpaII digested pBR322, a double digest of X)DNA with EcoRl and HindIII, and HincII digested g X174. Transfer of DNA onto DBM p)aper. DBM paper was prepared as previously described (2, 26). Gels were stained with ethidium bromide and photographed. They were soaked for 30 min at room temperature in 0.5 M NaOH, for 30 min in 1 M sodium acetate buffer (pH 4.5) and for 15 to 30 min in cold transfer buffer (1 M sodium acetate, pH 4.5). The DNA was transferred overnight at 40C to DBM paper (27). Plasmids, Plasmids used were: pEcoIV from the non-coding region of the rDNA palindrome (2), pEcoVII from the coding region (2) and pCTl (or pEcoX) which contains sequences from about 1 kb of the terminus of the rDNA (28).

2092

Nucleic Acids Research

b

a

1980 1590 1370 940 830 560

MONOMER 2

3

4

5

6

Fig.

1.

(a) MPE Fe(II) digestion of

Dictgostelium nucleoli. Nucleoli were incubated with 50 pM MPE-Fe(II) for 2, 4, 8, 16, 32, 64 min (lanes 3-8).

C

Controls: incubated for 1 min in buffer (lane 1) or in reaction mix minus MPE-Fe(II) (lane 2). (b) M icrococcal nuclease digestion of nucleoli. Nucleoli were incubated with 75 units of micrococcal nuclease for 15, 30, 60, 120, 240, 360 sec (lanes 2-7) or without enzyme for 120 sec (lane 1). (c) MPE-Fe(II) digest of nucleolar DNA. Pure DNA war digested with 50 pM MPE*Fe(II) at 25 C for 1, 2, 4, 8, 16 min (lanes 2-6). Control: incubation for 1 min, dithiothreitol omitted (lane 1). M, marker (bp).

I M

-I

2

3

4

5

6

2093

Nucleic Acids Research Hybridization. DNA on filters was prehybridized and hybridized with translated plasmids as previously described (2).

32p

nick-

RESULIM

Micrococcal nuclease

and MPE F e(II) digestion of nucleoli from vegeta-

tie c*lMicrococcal nuclease and MPE-Fe(II) cut preferentially the linker region between nucleosomes (29,30 and 19-22). D. discoideum nucleoli isolated from vegetative cells were treated with either micrococcal nuclease or MPE*Fe(II) for different times and the resulting DNA fragments separated on a 2.3% agarose gel (Fig. 1). Micrococcal nuclease digestion of nucleoli (Fig. lb) generates a series of bands that differ from those of nuclei (2,10). Although the typical double band of monomer DNA (about 146 and 170 bp) was present (31,32), the oligonucleosomes consisted of two or more bands. Moreover, bands were present between the repeats. We originally suggested that several different nucleosomal repeats and "half-nucleosomes" were superimposed to form this pattern (10). MPE*Fe(II) cleavage resulted in a more "typical" nucleosomal repeat (Fig. la) The sharp monomer bands of micrococcal nuclease digested material may result from trimming of the linker regions. The exonuclease activity of micrococcal nuclease has been reported (11). The monomer produced by MPE * Fe(II) was both broader (spreading from 146-200 bp) and a little larger than the monomer resulting from micrococcal nuclease action. Cartwright et al. (19) digested Dxpr aQsw 1 nuclei with MPEFe(II) and found the nucleosome bands migrated slightly slower than the corresponding oligomers produced by micrococcal nuclease. This was attributed to the single-strand nicking activity of the MPE-Fe(II) complex (15). They showed that incubation of purified DNA with Si nuclease resulted in greatly increased digestion and also in a shift in the MPEFe(II) fragment pattern on gels to resemble that obtained with micrococcal nuclease. However, in our experiments we found that S1 nuclease digestion did not significantly change the patterns obtained with MPE-Fe(II) except occasionally where much of the DNA remained near the top of the gel following MPE'Fe(II) digestion. In such cases, S1 nuclease digestion then generated the normal MPE Fe(II) pattern (not shown). 2094

Nucleic Acids Research Palindrome centre

Polindrome

end

V

V

EcoRI

j

= xob

o

17 5-8 26S kb

0

10

20

5S

30

40

Fig. 2. EcoRl restriction map of the rDNA of Dictyostelium discoideum. A number of high molecular weight DNA fragments were shared by both digests (Fig. 1). The relationship of these bands to the nucleoso-

mal repeat is unclear. Digestion of nucleolar DNA with micrococcal nuclease (2) or with MPE Fe(II) (Fig. lc) gave a smear on ethidium bromide stained gels.

a

h

1980

1590 1370

-mm

940

4_ 830 560

1

3

4

5

6

7

8

M

1

2

3

4

5

6

7

M

Fig. 3. Hybridization of DNA fragments with pEcoVII, the coding region, after digestion of nucleoli from vegetative cells by MPE-Fe(II) or micrococcal nuclease. DNA fragments were separated on 2.3% agarose gels, blotted onto DBM paper and hybridized. (a) MPE.Fe(II) digestion for 2, 4, 8, 16, 32, 64 min (lanes 3-8). Controls: as in Fig. 1 (lanes 1 and 2). (b) Micrococcal nuclease digestion for 15, 30, 60 120, 240, 360 sec (lanes 2-7) or 120 sec without enzyme (lane 1). 2095

,~ ~ ~

Nucleic Acids Research

...

....

Fig. 4. Hybridization of DNA fragments with pEcoVII, the coding region, after digestion of nuclei from vegetative cells by MPE-Fe(II) or micrococcal nuclease. (a) Micrococcal nuclease digestion for 15, 30, 60, 120, 240 sec (lanes 2-6) or 120 sec without enzyme (lane 1). (b) MPE Fe(II) digestion for 2, 4, 8, 16, 32 min (lanes 3-7). Controls: as in Fig. 1 (lanes 1 and 2). 1-4).

Digestio ptterns in the transcribed region of the rRNA gene The plasmid pEcoVII contains the sequence complementary to 5.8 S rRNA as well as parts of those complementary to 17 S and 26 S rRNA (Fig. 2). The entire sequence is present in the 8500 base rRNA precursor (33). When the plasmid was used as a hyridization probe, following digestion of vegetative nucleoli with micrococcal nuclease, a continuous distribution of DNA fragments was observed (Fig. 3b; ref. 2). At longer digestion times, a weak nucleosomal repeat was seen with an average repeat length of 160 bp. Identical results was obtained with MPE Fe(II) (Fig. 3a). In order to ensure that nucleolar isolation techniques had not disrupted the chromatin structure, the same experiment was performed on nuclei from vegetative cells. Once again, both digestions gave a smear 2096

Nucleic Acids Research

b

a

,i.

622

622 527

527_ 404

309

404 _

M

309

2

3

4

5

6

2

3

5

6

7

M

Fig. 5. Hybridization of DNA fragments with pEcoVII, the coding region, after digestion of nuclei from slug cells with MPE*Fe(II) or micrococcal nuclease. (a) Micrococcal nuclease digestion for 0, 15, 30, 60, 120, 300 sec (lanes 1-6). (b) MPE*Fe(II) digestion for 0, 1, 2, 4, 8, 16, 32 min (lanes 1-7).

(Fig. 4a and b), indicating the coding region in the majority of the genes is not packaged into regularly repeating units. Pure DNA cut with MPE Fe(II) (result not shown) or with micrococcal nuclease (2) and hybridized with pEcoVII gave a smear. When Dictyostelium amoebae are plated onto nutrient-free agar a programme of differentiation is initiated. About 15 h after plating, at the so-called slug stage, rRNA synthesis is up to 80 % lower than in vegetative cells (34,35). Since nucleoli from this stage are smaller and difficult to isolate, nuclei were used in the digestion experiments. When the DNA fragments from micrococcal nuclease digestion were hybridized with pEcoVII, the bulk of the DNA showed a characteristic nucleosomal repeat (Fig. 5a; ref. 2). A similar banding pattern was obtained with MPE *Fe(II), although the high molecular weight regions were more smeared (Fig. 5b). Nevertheless, the change from vegetative nuclei (see 2097

Nucleic Acids Research a

52 ,% 4 2 .,!

Fig. 6. Hybridization of DNA fragments with pEcoIV, the spacer region, after digestion of nucleoli or pure DNA of vegetative cells with MPE-Fe(II) or micrococcal nuclease. (a) Micrococcal nuclease digestion of nucleoli for 0, 15, 30, 60, 120, 240 sec (lanes 1-6) or (b) MPE Fe (II) digestion of nucleoli for 2, 4, 8, 16, 32 min (lanes 3-7). Controls: as in Fig. 1 (lanes 1 and 2). (c) MPE -Fe (II) digestion of pure nucleolar DNA for 2, 4, 8, 16 min (lanes 1-4).

2098

Nucleic Acids Research Fig. 4b) is readily apparent. The nucleosomal repeat obtained with MPE-Fe(II) was about 185 bp, whereas micrococcal nuclease gave a repeat of 170 bp. Digestion pattern of the central spacer region The EcoRl fragment IV derives from the central spacer region of the palindrome (Fig. 2) which is not transcribed (7). Micrococcal nuclease digestion of vegetative nucleoli produced a distinctive pattern of DNA fragments hybridizing with pEcoIV which did not fit into a simple repeat (Fig 6a, Ref 2). With MPE-Fe(II) digestion a normal nucleosomal ladder was generated (Fig. 6b) in which the the double bands in the dimer and trimer region of Fig. 6a were replaced by broad single bands. The strongest micrococcal nuclease product (marked with an arrow in Fig. 6a) is not over-represented after MPE.Fe(II) digestion. The MPE-Fe(II) digestions resulted in a repeat of 160-165 bp. The pattern generated by micrococcal nuclease digestion probably arose partly from sequence specific cutting. When pure DNA was cut with micrococcal nuclease and hybridized with pEcoIV a smear was seen with some faint bands, attributed to sequence-specific cutting (ref. 2). Pure DNA cut with MPE-Fe(II) and hybridized with pECOIV gave a smear (Fig. 6c). Digestion pattern of the terminus of the palindrome The plasmid pCTl carries the EcoRl fragment from the terminal 1 kb of the palindrome (Fig. 2) (28). A characteristic pattern of DNA fragments was obtained when pure DNA was cut with micrococcal nuclease and hybridized with pCTl (Fig. 7b). Hence, sequence specific cutting is quite dramatic (see Discussion). MPE*Fe(II) digestion also produced a number of blurred bands (Fig. 7a). These bands were similar in size to those obtained with micrococcal nuclease, however MPE-Fe(II) generated a lot more background smearing, indicating cleavage was less specific than with the nuclease. One end of many of the digestion products will be the end of the palindrome, hence sequence-specific cuts in the DNA will show up more clearly when pCTl is used as a hybridization probe, particularly at low extents of digestion. Micrococcal nuclease digestion of nucleoli and hybridization of DNA fragments with pCTl resulted in a pattern of sharp bands (Fig. 7d). MPE-Fe(II) digestion produced an identical pattern (Fig. 7c) except that longer micrococcal nuclease digestions produced additional fragments in the 250 - 450 bp region (Fig. 7d and e). Extended MPE *Fe(II) digestion

2099

Nucleic Acids Research did not result in an appearance of new bands but simply a fading of the pattern (Fig. 7c). The monomer DNA generated by micrococcal nuclease was about 190-210 bp and was reduced to the core size of 146 bp by longer

cli

iM.I N T ' EMF.

5: -

;., .:

f

_

'

;

''t

, /O

r.M

t;

.:i:

4i ..

2100

,,1

7'

e

.:

Nucleic Acids Research

-

....

Ati', B:

w.....

*^L

:

_ _. _. .-

..

*:'''.7".i i:x$:

.', ... ',

.::

.::

.:

D

1

2

3

4

5

6

7

8

M

Fig. 7. Hybridization of DNA fragments with pCTl, the end of the palindrome, after digestion of nucleoli or pure nucleolar DNA with MPE*Fe(II) or micrococcal nuclease. (a) MPE*Fe(II) digestion of pure nucleolar DNA and 1, 2, 4, 8 and 16 min (lanes 2-6) or for 2 min without dithiothreitol (lane 1). (b) Micrococcal nuclease digestion of pure nucleolar DNA for 0, 1, 2, 5, 10 and 15 min (lanes 1-6). N, nucleoli digested with nuclease. (c) MPE-Fe(II) digestion of nucleoli for 2, 4, 8, 16, 32, 64 min (lanes 2-7). Control: MPE omitted (lane 1). (d) Micrococcal nuclease digestion of nucleoli for 0, 15, 30, 60, 120, 240 and 360 sec (lanes 1-7). (e) Micrococcal nuclease digestion of nucleoli for 15, 30, 60, 90, 180, 300 and 600 sec (lanes 2-8) or without enzyme for 300 sec (lane 1). Lane D contains pure nucleolar DNA digested with micrococcal nuclease.

digestion (Fig. 7e). We interpret the higher molecular weight products as nucleosome multimers, as indicated on Fig. 7c. For shorter extents of micrococcal nuclease digestion a repeat of about 190 bp was found up to the hexamer, although the trimer was a little shorter than predicted (about 180 bp). The tetramer and hexamer were relatively strong bands. The extra bands resulting from longer nuclease treatment seemed to be products of trimer and dimer digestion (Fig. 7d, e). However, the sizes indicate they did not derive from simply shortening of the terminal 2101

Nucleic Acids Research linkers and imply cutting is also occurring within the nucleosomal DNA. This may be related to the sequence specificity of the enzyme (see Discussion). Comparison of the band sizes obtained following micrococcal nuclease digestion of naked DNA or nucleoli (Fig. 7b, lanes 6 and N; Fig. 7e, lanes D and 2) shows that they do not coincide. This indicates that the sequences preferentially cleaved in naked DNA are protected in chromatin.

DISCUSSION Previous experiments involving the digestion of D. discoideum with micrococcal nuclease indicated that the transcribed and spacer regions of the rDNA palindrome have different chromatin structures (2). Digestion of the active region resulted in a smear of DNA fragments on gels, ie. fragments of a wide range of sizes. However, in stages of differentiation where rRNA synthesis was greatly reduced, the coding region was condensed into nucleosomes. The DNA banding patterns of the spacer regions were more complex than the nucleosome repeats of the coding region. Ethidium bromide stained gels of DNA from nucleoli digested with micrococcal nuclease showed a complex pattern of bands and we proposed that several different repeats and "half-nucleosomes" may be superimposed (10). Micrococcal nuclease has a strong preference for specific DNA sequences (11 - 13). Sequence specific cutting by micrococcal nuclease has been found, for example, in the flanking regions of progophila heat-shock genes (36, 37) and the spacer region of Physarnm rRNA genes (38). Strong sequence-specific cutting in the coding regions were not observed. Although comparison with micrococcal nuclease digestion of protein-free DNA was made in our original experiments, we wished to extend the results by using MPE'Fe(II), which has a much less marked sequence preference (15, 19). MPE Fe(II) digestion of D. discoideum nucleoli did not result in the sharp double bands associated with monomer, dimer and trimer nucleosomes following micrococcal nuclease treatment. The bands between the repeats were also no longer present (see later). The broad oligomer bands may reflect the single-strand cutting activity of MPE-Fe(II), however S1 nuclease treatment did not sharpen the bands. The monomer obtained with micrococcal nuclease consists of two DNA bands, the larger (166-180 bp) includes the chromatosome (166 bp). A lysine-rich protein 2102

Nucleic Acids Research brings the two DNA strands together at their exit point from the nucleosome, protecting about 20 bp from digestion (32) The second band is the 146 bp DNA of the nucleosome core particle. MPE-Fe(II) treatment gave a broad band in the region 146-200 bp. Chromatin structure of the coding region Digestion of chromatin from the transcribed region of the rRNA genes of vegetative cells with MPE-Fe(II) produced a continuous distribution of DNA fragments. After rather extensive digestion, a faint repeat was observed which we propose originates from gene copies that are not being transcribed. The coding region in slug cells, where the rate of rRNA synthesis is reduced, showed a typical nucleosomal repeat pattern. These results are identical to those obtained with micrococcal nuclease (2). They indicate that the nucleosomal structure changes when transcription occurs, although whether this leads to the loss of some or all of the histones is not known. The lack of periodicity could also result from an undefined linker length between the nucleosomes. However, electron microscopy of DNA molecules cross-linked in nucleoli with psoralen show no sign of protection by nucleosomes in the coding region (4). Biochemical evidence is accumulating from studies on yeast (39) TetIa4 men (40, 41), DrQoophila (42), D. discoideum (1, 2, 4) and mouse (43) that active rRNA genes are particularly sensitive to micrococcal nuclease and no longer possess typical nucleosomal packaging. Active rRNA genes in XenoQuiw are protected from micrococcal nuclease digestion, but also appear to have a non-nucleosomal structure (44). However, Prior et al. (45) propose that core histones are bound to the transcribed DNA of Physarum rRNA genes. As the D. discoideum rRNA genes become less active, in slugs, nucleosomes form on the coding region (this paper, and ref. 2). There does not appear to be a sub-population of nucleosome-free, transcribing gene copies in slugs, but rather a structural change of all copies (4). The notion that nucleosomes and transcription complexes co-exist on slowly transcribed genes may explain the contradictions in the literature. We currently favour a model in which the transcription complex, having gained access to the DNA, carries the machinery capable of opening the nucleosome structure and facilitating transcription. Wasylyk and Chambon (46) found that nucleosomes inhibit transcription in a reconstituted system where polymerase I alone was added, so additional 2103

Nucleic Acids Research factors must be responsible. we are currently analysing the characteristics of purified transcription complexes from Ditvosatelimw nucleoli. A similar model can probably be applied to polymerase II and III genes, where results of nuclease digestion experiments are also conflicting (reviewed in 47). Using the technique of "protein-image" hybridization, which involves cross-linking of histones to DNA, Karpov et al. (48) have proposed that the coding region of the DosoPilA hsp 70 gene loses all histones when the transcription rate is very high and retains them when it is low. Chromatin structure of the non-coding region The irregular DNA-banding pattern obtained when the central spacer region of the palindrome (EcoIV fragment) is cut by micrococcal nuclease can be attributed in part to sequence-specific cutting, since MPE-Fe(II) gives a normal pattern. Electron microscopy of DNA cross-linked in nucleoli with psoralen supports the idea that there is a regular array of nucleosomes in this part on the gene (4). We are currently investigating whether the nucleosomes in this region are also phased with respect to DNA sequence, as they appear to be in Tetrnthvmena (49). We have located putative DNA topoisomerase activity, in nuclei, within EcoRl fragment IV (50). The activity is at two fixed positions, which may in turn exert a positioning effect on neighbouring nucleosomes. Hybridization of digests with pCTl, which carries the terminal 1 kb of the palindrome, gave intriguing results. The rDNA termini of fl. digggideUM are heterogeneous in length and possess an irregular, simple sequence repeat with the general formula [CnT]m; n varies from 1 to 8 and m from 18 to 34 (28). (In pCTl, m is 17 and the simple terminal repeat is 100 bp long.) Four nearly perfect repeats of a 29 bp sequence are present immediately proximal to the CnT satellite. The majority of the rDNA molecules in our cell line, however, appear to have a rather similar length. Band X after EcoRl digestion is slightly blurred but certainly does not spread from 1000 to 1300 (see, eg., ref. 3). Micrococcal nuclease showed strong preference for some of the DNA sequences in this region, digestion of naked DNA producing a family of discrete sizes. MPE'Fe(II) cleaved the DNA with some specificity, the bands being superimposed on a smear. However, the bands were weaker and more diffuse than the micrococcal nuclease bands. Nucleoli digestion with micrococcal nuclease produced a band pattern entirely different from that obtained with DNA. Moreover, MPE-Fe(II) and micrococcal 2104

Nucleic Acids Research nuclease digestion patterns of nucleoli were identical. Analysis of fragment sizes indicated a repeat of around 190-200 bp may occur. The relative amounts of the oligomers varied considerably, the tetramer and hexamer being present in higher concentrations than the trimer and pentamer. This may reflect higher order structure. Indirect end-labelling experiments in the central part of the terminal spacer (51) have suggested dinucleosomes with a repeat of 400-500 bp are released preferentially by micrococcal nuclease. The sequences specifically cut by micrococcal nuclease in naked DNA were protected in chromatin, probably by nucleosomes. Extensive micrococcal nuclease but not MPE Fe(II) digestion reduced the sizes of dimers and trimers, we speculate that this is due to cutting within the nucleosome. This effect is presumably related to the presence of preferred cutting sequences, and may contribute to the "half-nucleosomes" seen on ethidium bromide stained gels (10). In the coding region, where no strong sequence specific cutting by micrococcal nuclease was detected, longer digestions did not lead to the reduction in dimer and trimer

sizes. The results suggest that nucleo-protein structures are precisely positioned in the terminal region of the palindrome, the micrococcal nuclease sensitive sites being preferentially associated with the core of the protective complex. Evidence for nucleosome positioning has been found in the spacer region between the Hl and H3 genes of pros. i15 and in parts of the yeast centromere sequences (13, 19, 52). Gottschling and Cech (20) found that the molecular ends of Oxytricha macronuclear DNA possess a terminal complex involving about 100 bp with nucleosomes phased inward from this complex. Edwards and Firtel (51) have used used indirect end-labelling to map micrococcal nuclease and DNase I sites in the terminal 10 kb of Dictvostelium rDNA. They found that the sites in naked DNA attacked most readily by both nucleases were the most protected sites in chromatin and proposed that the phasing signal and the nuclease-sensitive site may be centred within the nucleosome and may be identical. This agrees with our results, which also suggest the chromatin in this region is highly ordered with respect to DNA sequence. ACKNOWLEDGEMENT We thank A. Weiner and P.B. Dervan for generously providing us with pCTl and MPE respectively. This work was supported by a grant from the 2105

Nucleic Acids Research Schweizerische Nationalfonds zur Forderung der wissenschaftlichen Forschung. REFERENCES

1. Labhart, P., Ness, P., Banz, E., Parish, R.W. and Koller, Th. (1983) Cold Spring Harbor Symp. Quant. Biol. Vol. XLVII, 557-564. 2. Ness, P.J., Labhart, P., Banz, E., Koller, Th. and Parish, R.W. (1983) J. Mol. Biol. 166, 361-381. Erratum 169, 639-640. 3. Labhart, P., Banz, E., Ness, P.J., Parish, R.W. and Koller, Th. (1984) Chromosoma 89, 111-120. 4. Sogo, J., Ness, P.J., Widmer, R., Parish, R.W. and Koller, Th. (1984) J.Mol. Biol. 178, 897-920. 5. Maizels, N. (1976) Cell 9, 431-438. 6. Cockburn, A.F., Taylor, W.C. and Firtel, R.A. (1978) Chromosoma 70, 19-29. 7. Firtel, R.A., Cockburn, A., Frankel, G. and Herschfield, V. (1976) J. Mol. Biol. 102, 831-852. 8. Frankel, G., Cockburn, A.F., Kindle, K.L. and Firtel, R.A. (1977) J. Mol. Biol. 109, 539-558. 9. Grainger, R.M. and Maizels, N. (1980) Cell 20, 619-623. 10. Widmer, R., Fuhrer, S. and Parish, R.W. (1979) FEBS Lett. 106, 363-369. 11. Horz, W. and Altenburger, W. (1981) Nucl. Acids Res. 9, 2643-2658. 12. Dingwall, C., Lomonossoff, P. and Laskey, R.A. (1981) Nucl. Acids Res. 9, 2659-2672. 13. Bloom, K.S. and Carbon, J. (1982) Cell 29, 305-317. 14. McGhee, J.D. and Felsenfeld, G. (1983) Cell 32, 1205-1215. 15. Hertzberg, R.P. and Dervan, P.B. (1982) J. Am. Chem. Soc. 104, 313-315. 16. Van Dyke, M.W., Hertzberg, R.P. and Dervan, P.B. (1982) Proc. Natl. Acad. Sci. USA 79, 5470-5474. 17. Van Dyke, M.W. and Dervan, P.B. (1983) Nucl. Acids Res. 11, 5555-5567. 18. Van Dyke, M.W. and Dervan, P.B. (1984) Science 225, 1122-1127. 19. Cartwright, I.L., Herzberg, R.P., Dervan, P.B. and Elgin, S.C.R. (1983) Proc. Natl. Acad. Sci. USA 80, 3213-3217. 20. Gottschling, D.E. and Cech, T.R. (1984) Cell 38, 501-510. 21. Cartwright, I.L. and Elgin, S.C.R. (1984) EMB0 J. 3, 3101-3108. 22. Eissenberg, J.C., Kimbrell, D.A., Fristrom, J.W. and Elgin, S.C.R. (1984) Nucl. Acids Res. 12, 9025-9038. 23. Cocucci, S.M. and Sussman, M. (1970) J. Cell Biol. 45, 399-407. 24. Charlesworth, M.C. and Parish, R.W. (1977) Eur. J. Biochem. 75, 241-250. 25. Parish, R.W., Stalder, J. and Schmidlin, S. (1977) FEBS Lett. 84, 63-66. 26. Alwine, J.C., Kemp, D.J., Parker, B.A., Reiser, J., Renart, J., Stark, G.R. and Wahl, G.M. (1979) Methods Enzymol. 68, 220-242. 27. Southern, E.M. (1975) J. Mol. Biol. 98, 503-517. 28. Emery, H.S. and Weiner, A.M. (1981) Cell 26, 411-419. 29. Igo-Kemenes, T.,Horz, W. and Zachau, H.G. (1982) Annu. Rev. Biochem. 51, 89-121. 30. Cartwright, I.L., Abmayr, S.M., Fleischman, G., Lowenhaupt, K., Elgin, S.C.R., Keene, M.A. and Howard, G.C. (1982) CRC Crit. Rev. Biochem. 13, 1-86. 2106

Nucleic Acids Research 31. Parish, R.W., Schmidlin, S., Fuhrer, S. and Widmer, R. (1980) FEBS Lett. 110, 236-240. 32. Parish, R.W. and Schmidlin, S. (1985) Nucl. Acids Res. 13, 15-30. 33. Batts-Young, B., Maizels, N. and Lodish, H.F. (1977) J. Biol. Chem. 252, 3952-3960. 34. White, G.J. and Sussman, M. (1961) Biochim. Biophys. Acta 53, 285-293. 35. Soll, D.R. and Sussman, M. (1973) Biochim. Biophys. Acta 319, 312-322. 36. Keene, M.A. and Elgin, S.C.R. (1981) Cell 27, 57-64. 37. Keene, M.A. and Elgin, S.C.R. (1984) Cell 36, 121-129. 38. Pauli, V.H., Seebeck, T. and Braun, R. (1982) Nucl. Acids Res. 10, 4121-4133. 39. Lohr, D. (1983) Biochemistry 22, 4527-4534. 40. Borchsenius, S., Bonven, B., Leer, J.C. and Westergaard, 0. (1981) Eur J. Biochem. 117, 245-250. 41. Palen, T.E. and Cech, T.R. (1983) Nucl. Acids Res. 11, 2077-2091. 42. Udvardy, A., Louis, C., Han, S. and Schedl, P. (1984) J. Mol. Biol. 175, 113-130. 43. Davis, A.H., Reudelhuber, T.L. and Garrard, W.T. (1983) J. Mol Biol. 167, 133-155. 44. Spadafora, C. and Crippa, M. (1984) Nucl. Acids Res. 12, 2691-2704. 45. Prior, C.P., Cantor, C.R., Johnson, E.M., Littau, V.C. and Allfrey, V.G. (1983) Cell 34, 1033-1042. 46. Wasylyk, B. and Chambon, P. (1979) Eur. J. Biochem. 98, 316-328. 47. Reeves, R. (1984) Biochim. Biophys. Acta 782, 343-393. 48. Karpov, V.L., Preobrazhenskaya, O.V. and Mirzabekov, A.D. (1984) Cell 36, 423-431. 49. Palen, T.E. and Cech, T.R. (1984) Cell 36, 933-942. 50. Ness, P.J., Parish, R.W. and Koller, Th. (1986) J. Mol. Biol. In press.

51. Edwards, C. and Firtel, R.A. (1984) J. Mol. Biol. 180, 73-90. 52. Worcel, A., Gargiulo, G., Jessee, B., Udvardy, A., Louis, C. and Schedl, P. (1983) Nucl. Acids Res. 11, 421-429.

2107