Proc. Natl. Acad. Sci. USA Vol. 84, pp. 1550-1554, March 1987 Botany Light-regulated changes in DNase I hypersensitive sites in the rRNA genes of Pisum sativum (peas/chromatin/photoregulation/DNA/nucleolar organizer) LON S. KAUFMAN*, JOHN C. WATSONt, AND WILLIAM F. THOMPSON: Carnegie Institution of Washington, 290 Panama Street, Stanford, CA 94305 Communicated by Winslow R. Briggs, November 10, 1986 (receivedfor review May 23, 1986)

ABSTRACT We have examined the rDNA chromatin of transcribed genes (8), including the ribosomal RNA genes of Pisum sativum plants grown with or without exposure to light Tetrahymena (9-11), Xenopus (12), and Drosophila (13). for the presence of DNase I hypersensitive sites and possible However, DNase I hypersensitive sites have not yet been developmental changes in their distribution. Isolated nuclei reported in plant chromatin, and developmental changes in from pea seedlings were incubated with various concentrations the pattern of DNase I hypersensitivity have not been of DNase I. To visualize the hypersensitive sites, DNA purified reported for rDNA chromatin in any system. from these nuclei was restricted and analyzed by gel blot In the work reported here, we have examined the rDNA hybridization. We find that several sites exist in both the coding chromatin from pea buds for the presence of DNase I and noncoding regions of rDNA repeating units. Several of the hypersensitive sites. Using conditions that minimize the sites in the nontranscribed spacer region are present in the light activity of endogenous and proteases, we find that but are absent in the dark. Conversely, the hypersensitive sites DNase I hypersensitive sites exist in rDNA chromatin of within the mature rRNA coding regions are present in the dark isolated pea nuclei. When similar experiments are performed but absent in the light. There are two maojor'length variants of with nuclei isolated from the buds of dark-grown or light- the rRNA genes in P. sativum var. Alaska. The sites in the grown seedlings, we find several sites in the rRNA coding nontranscribed spacer region that appear during the light regions that are present in nuclei from dark-grown plants but treatment occur only in the shorter ofthese two length variants not in nuclei isolated from light-grown material. Conversely, in this cultivar. several DNase I hypersensitive sites in the nontranscribed spacers are present in nuclei from light-treated plants but are The cellular content of cytoplasmic rRNA in several plant absent from those of dark-grown seedlings. species is regulated by light (1), and light-regulated changes in the rate of nuclear rRNA gene transcription have been MATERIALS AND METHODS demonstrated in pea (2), Lemna (3), and barley (4). The overall transcriptional organization of rRNA genes is similar Seeds of Pisum sativum L. cv. Alaska (W. Altee Burpee, in plants and animals, but the plant genes tend to be more Werminster, PA) were imbibed for 5 hr and grown on two highly repeated than their animal counterparts (5). Ribosomal layers ofwater-soaked Kimpac (Kimberly-Clarke, Rosewell, RNA gene copy numbers can also vary substantially even GA) at 270C, 85% relative humidity, in absolute darkness among closely related genotypes within a species, indicating (14). Dark-grown plants were kept in these conditions for 7 that the number of nuclear rRNA genes in plants often days. Light-grown or light-treated plants were kept in the exceeds that needed to supply rRNA to the cytoplasmic dark for 4 days and then transferred to white light (102 ribosome pool (6). The presence of a large excess of gene ILmolmM2 sec-1; cool white fluorescent) for an additional 3 copies suggests that a mechanism must exist for determining days. The buds and hooks of the 7-day-old plants were which copies are transcriptionally active and which are silent. harvested into ice-cold containers under dim green light Regulation of rRNA by light may therefore (dark-grown plants) or in fluorescent room light (light-grown involve a recruitment ofmore genes into the transcriptionally plants). active pool. Nuclei were isolated as described by Watson and Thomp- The activity of individual rDNA loci (nucleolar organizers) son (15) except that the extraction and gradient buffers has been shown to be genetically determined (6, 7). For contained 5 mM sodium butyrate, 10 mM o-phenanthroline, example, the nucleolar organizer of the rDNA Aegilops 0.2% methylmethanethiosulfonate, and 0.1 mM phenylmeth- umbellulata chromosome 1 is dominant over all four Triticum ylsulfonyl fluoride. The final nuclear pellet was resuspended loci in chromosome addition lines containing this chromo- in DNase I buffer (250 mM sucrose/10 mM NaCl/10 mM some in a Triticum aestivum background (6). Since the rDNA Pipes, pH 7.0/3 mM MgCl2/3 mM CaCl2/5 mM sodium loci in these hybrids have rDNA repeats of different lengths butyrate/0.2% methylmethanethiosulfonate/0.1 mM phenyl- and distinguishable restriction patterns, molecular tests can methylsulfonyl fluoride/10 mM o-phenanthroline/5 mM 2- be designed to examine features of chromatin structure (e.g., mercaptoethanol) at a concentration of =250 jxg per ml of sensitivity to exogenous DNase I) and cytosine methylation DNA. ofthe DNA of each locus. Nucleolar dominance is correlated DNase I (Sigma) was added to aliquots of the nuclear with a more DNase I sensitive chromatin conformation and suspension to give final concentrations of 8.0, 2.4, 0.8, and a hypomethylation ofthe Aegilops rDNA (ref. 6; W.F.T. and R. B. Flavell, unpublished data). Abbreviations: NTS, nontranscribed spacer; L and S variants, longer It is well established in animal systems that DNase I and shorter variants of rDNA. hypersensitive sites are frequently associated with actively *Present address: Department of Biological Sciences, University of Illinois at Chicago, Chicago, IL 60680. tPresent address: Department of Botany, University of Maryland, The publication costs of this article were defrayed in part by page charge College Park, MD 20742. payment. This article must therefore be hereby marked "advertisement" *Present address: Departments of Botany and , North in accordance with 18 U.S.C. §1734 solely to indicate this fact. Carolina State University, Raleigh, NC 27695. 1550 Botany: Kaufman et al. Proc. Natl. Acad. Sci. USA 84 (1987) 1551 0.2 units/ml. Incubation was for 10 min at 370C. Additional RESULTS aliquots ofnuclei were also incubated at 37TC with no enzyme (sham digest) and at 0C with no enzyme (control digest). In the garden pea, there are 3000-4000 copies of the rRNA After 10 min, the reaction was stopped by the addition of a genes (18). Most of these genes, together with spacer se- large volume of ice-cold DNase I buffer lacking both phen- quences between them, are present in large tandem arrays at ylmethylsulfonyl fluoride and methylmethanethiosulfonate. one of the two nucleolar organizer loci (19). The basic The nuclei were pelleted immediately and resuspended in a monomer repeating unit of -9 kilobases (kb) contains the small volume of the buffer. (Phenylmethylsulfonyl fluoride 18S, 5.8S, and 25S rRNA cistrons and a "nontranscribed" and methylmethanethiosulfonate were omitted at this stage to spacer (NTS) =3 kb long (Fig. 1). About one-third of each avoid interference with subsequent proteinase K treatment.) NTS is occupied by a tandem array of short (180 bp) repeated The resuspended nuclei were lysed by the addition of 1 vol of elements, called the subrepeat array, which occurs just 2x lysis buffer (200 mM Tris HCl, pH 8.0/50 mM EDTA/1 downstream from the 3' end of the 25S rRNA gene. M NaCl/2% Sarkosyl) and the lysate was treated with In many pea varieties, including cv. Alaska, two major proteinase K (0.5 mg/ml) at 550C for 1.5 hr. DNA was length classes of rDNA are observed (17, 20), and the purified by banding in cesium chloride/ethidium bromide different forms are thought to be located at different nucleolar gradients as described (15). Purified DNA was exhaustively organizers (20, 21). The number of elements in this subrepeat digested with either BamHI (Bethesda Research Laborato- array is variable both between cultivars and between loci ries) or EcoRI (Bethesda Research Laboratories) in the ionic within a cultivar. In P. sativum cv. Alaska, the longer (L) conditions recommended by the supplier. The DNA restric- variant contains nine elements per subrepeat array, while the tion fragments were size-fractionated on agarose gels, trans- shorter (S) variant subrepeat arrays contain seven elements ferred to either GeneScreen (New England Nuclear) or Nylon each. Although other variants exist (16), they are present in 66 (Schleicher & Schuell), and hybridized with the appropri- much lower copy numbers and do not contribute significantly ate DNA probe as described (15). to the data presented here. Restriction digests of genomic Bands in the hybridization profiles that occurred in either DNA indicate several differences in sequence between the L control or sham digests were discounted when mapping and S variants (17). For example, the L variant, but not the DNase I hypersensitive sites. These bands probably repre- S variant, contains two closely spaced EcoRI sites within the sent restriction fragments from minor length variants known subrepeat array. This difference in sequence and the differ- to be present at low copy number in the pea genome (16). ences in length between the two variants allow us to distin- Control experiments were conducted to be certain that the guish between them in gel blot hybridization studies. DNase I hypersensitive sites mapped reflect properties ofthe DNase I hypersensitive sites were mapped by using the chromatin complex rather than properties of naked DNA. method of indirect end-labeling (22, 23). Small hybridization Both dark-grown and light-grown seedling buds and hooks probes are used that hybridize only to one end ofthe genomic were used to prepare total cell DNA or DNA from isolated restriction fragment being mapped. The length of a fragment nuclei in the absence of DNase I digestion. Purified DNAs end-labeled in this way directly defines the distances between were then subject to digestion with a series of DNase I a known restriction site end and the site of DNase I cleavage. concentrations and analyzed as described for DNase-treated A map of DNase I sites covering the entire rDNA repeating samples. In no case were we able to observe any hypersen- unit was established by using four different indirect end- sitive sites in purified DNA. labeling probes. Examples ofsites detected by each probe are Fragments of pea rDNA used as indirect end-labeling indicated to the right of each blot in Figs. 2 and 3. Fig. 4 is probes were prepared from various subclones of our original based on a compilation of mapping data from several blots pea rDNA clone pHA1 (17) as described (16). The location of similar to those illustrated as well as from different autora- each fragment is shown in Fig. 1. Fragments XB380, BE360, diographic exposures. and EBg520 were isolated from subclone pBH3.1 (16), while Several methods were used to assign the DNase I sites fragment EC510 was from subclone pB2.6 (16). XB380 is a observed in the digestion profiles to the L and/or S variants. 380-base-pair (bp) Xba I/BamHI fragment; BE360 is a 360-bp EBg520 was used to map sites between the EcoRI site in the BamHI/EcoRI fragment; EBg520 is a 520-bp EcoRI/Bgl II 25S coding sequence and the next EcoRI site downstream. fragment; EC510 is a 510-bp EcoRI/Cla I fragment. Since two EcoRI sites occur in the middle of the subrepeat E C B E Bg Hd X B

BE360 -EJXB380 FIG. 1. Map of the pea rDNA repeat and EC510 EBg520O location of the DNA fragments used as indirect end-labeling probes. Each rDNA repeat en- codes an 18S, 5.8S, and 25S RNA as shown at the top of the figure. The coding regions are a B E EE X B separated by NTS. There are two major NTS length classes in the pea cultivar Alaska, which we refer to as the L and S variants. Maps of the LI NTS regions of these variants are shown in SUBREPEAT REGION expanded form in the lower part of the figure. Both NTS regions contain a region of 180-bp repeats (subrepeat region). The L variant has nine of these subrepeats, and the S variant has seven. Note the presence of EcoRI sites in two B E X B of the subrepeat sequences in the L variant. The fragments used as indirect end-labeling probes (XB380, BE360, EBg520, and EC510) are shown Si 25i± 1 1 1 1 1 18iS at their approximate location on the map. Ar- SUBREPEAT REGION rows indicate the direction of mapping from 4 3 2 1 0 each probe. E, EcoRI; B, BamHI; C, Cla I; Bg, L 1- k Bgl II; Hd, HindIII; X, Xba I. 1552 Botany: Kaufman et al. Proc. Natl. Acad. Sci. USA 84 (1987)

EBg520 PROBE EC510 PROBE BE360 PROBE XB380 PROBE

S 80 24 08 C20 S 80 24 08 C2 C 8.0 2.4 0.8 02 S C 8.0 2.4 0.8 0.2 S C

9.6 -

6.6 -- L S LS 9.6 - S _ L S 01) c 6.6 - 4.3 E- 0) .1 0 c (Du _ ) C CC) E-" - 2 " 4.3 ~~ +-~~B- CO) Bea -o- (0 .tBf a) U>0) I# o >1>I~~~~~ 212- 0) 2.2 - _ MI 0 2.1 - U) 1I1 U) U) E- C2 n _ ___ m ..*, = =~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~23}0 U-C) it Df

FIG. 2. DNase I hypersensitive sites in the rDNA chromatin of dark-grown pea buds. DNase I hypersensitive sites in the rDNA chromatin of the buds and subapical hooks of pea seedlings grown in darkness for 7 days were visualized by using the method of indirect end-labeling as described in the text. The DNA fragments used as indirect end-labeling probes are described in the text and their map positions are indicated in Fig. 1. Maps to the right of each autoradiogram indicate sites mapped from the illustrated exposure ofthat autoradiogram. The methods used to assign the various sites to the L and/or S variant are described in the text. EcoRI digests were used with EBg520 and EC510 probes, while BamHI digests were used with BE360 and XB380 probes. C, control digest; S, sham digest. Numbers above lanes refer to units of DNase I per ml. Numbers to left oflanes indicate the size (in kbp) ofHindIII-digested X DNA markers. Arrowheads in the EC510 panel denote bands resulting from DNase I cleavage of hypersensitive sites within the rRNA coding region (see uppermost map in Fig. 4), which are not easily detectable in chromatin from light-treated plant material (cf. Fig. 3).

region of the L variant (1.2 kb downstream from the 25S An alternative method was used to confirm these assign- EcoRI site), no L variant DNase I hypersensitive sites will be ments. This method relies solely on the results of hybridiza- detected beyond these EcoRI sites. However, since the S tions with XB380 and BE360 and takes advantage of the two variant does not have these EcoRI sites, the EBg520 probe extra 180-bp subrepeats present in the L variant. When can be used to map S variant DNase I hypersensitive sites BE360 is used as a probe on BamHI digests, cleavage by throughout the NTS and on into the 3' end of the 18S gene. DNase I at the same position in both length variants will yield Thus, hypersensitive sites visualized by EBgS20, occurring two hybridizing bands differing in length by 360 bp. Howev- between the middle of the subrepeat region and the 5' end of er, when XB380 is used as the probe, DNase I cleavages in the 18S coding sequence (i.e., fragments longer than 1.2 kb) the same region of the spacer will produce hybridizing can only be derived from the S variant and were assigned fragments of identical length from both variants. If separate accordingly. Sites mapped from fragments <1.2 kb long were maps of this region are deduced from hybridizations with assigned to both variants. XB380 and BE360 and then aligned at the right-hand (in the Sites occurring in the NTS of the L variant were deter- 25S) BamHI site, bands appearing at the same positions in mined from hybridizations with BE360 and XB380 (Figs. 2 both maps can be assigned to the L variant. Similarly, when and 3). First, sites known to occur in the S variant (as the maps are aligned at the left-hand (in the 18S) BamHI site, determined from EBgS20, Figs. 2 and 3) were assigned to the bands appearing in the same positions on both can be S variant. Any remaining sites, mapped by either XB380 or assigned to the S variant. BE360, were assigned to the L variant. As the transcribed Examples of autoradiograms resulting from experiments sequences of the two length variants are indistinguishable with DNase I-treated nuclei of dark-grown plants are shown with the EC510 probe, any sites mapped by EC510 were in Fig. 2. A map deduced from these and other blots (Fig. 4, assumed to occur in both variants. solid arrows) shows the approximate location of all the EBg520 PROBE EC510 PROBE BE360 PROBE XB380 PROBE

S 24 08 02 80 C S En 24 08 02 0 8.0 2.4 0.8 02 S C 8.0 2.4 0.8 0.2 S C L S 9.6 - L S L,S L S 6.6 --- D) E- S a 43- 64b#O _ S B- 0c :M ., =m E- 8 CD co n7n 2.2 - 0 2.1 - 46E T B_U)Xr.0 vn 0 HC<8 U) U/) LO) R. |N OL 1 - a-

FIG. 3. DNase I hypersensitive sites in pea rDNA chromatin from light-grown plants. DNase I hypersensitivity was assayed as described in Fig. 2 in the rDNA chromatin ofbuds and hooks from pea seedlings that had been grown in darkness for 4 days followed by 3 days in continuous white light. Arrowheads in the EBg520 panel indicate the positions of three hypersensitive sites found in S variant chromatin of light-treated pea seedlings but not in chromatin from dark-grown (cf. Fig. 2) seedlings. Botany: Kaufman et al. Proc. Natl. Acad. Sci. USA 84 (1987) 1553 LIGHT

f f ff DARK

B E x B

25 i fflf fi f ;---; 18S DARK

SUBREPEAT REGION

B E x B FIG. 4. Maps of DNase I hy- I LIGHT persensitive sites in pea rDNA t fitt t b t chromatin. The approximate loca- 'S 25S 185 tion ofthe DNase I hypersensitive sites in rDNA chromatin of dark- DARK grown (solid arrows) or light-treat- 4 3 2 1 0 ed (open arrows) pea seedlings. B, kb BamHI; E, EcoRI; X, Xba I. observed DNase I hypersensitive sites. First, we note the positions of the constitutive sites described for L variant ladder ofbands with a 180-bp periodicity found when blots of chromatin. In addition, S variant chromatin exhibits DNase BamHI digests are probed with XB380 or BE360, indicating I sites 1.9 and 2.0 kb upstream from the right-hand BamHI that DNase I cuts preferentially within each subrepeat. In site in both light- and dark-grown plants. So of the five addition, several hypersensitive sites occur between the 3' hypersensitive sites in the proximal halfofthe S variant NTS, end of the subrepeats and the 5' end ofthe 18S coding region. two (at '1.9 and -2.0 kb) are present in both light- and As described above, mapping the NTS region with XB380, dark-grown plants, while three (at 1.0, 1.2, and 1.55 kb) are BE360, and EBg520 allowed us to determine in which variant light induced. these sites occur. The L variant contains three of the five In nuclei isolated from light-treated plants, we detect sites, '4.55, '1.2, and 4.0 kb upstream from the right-hand neither the cluster of hypersensitive sites within the 25S gene BamHI site. None of these sites is observed in the S variant nor the sites at the start of the 18S and 25S coding sequences chromatin (determined from experiments with the EBgS20 (Fig. 3). All of these sites were reproducibly observed in probe; Fig. 2). However, the S variant has a hypersensitive nuclei from dark-grown plant material. region containing at least two sites 1.9-2.0 kb upstream from the right-hand BamHI site, which is not seen in L variant chromatin. DISCUSSION Hypersensitive sites were not confined to spacer regions. We have mapped 17 discrete DNase I hypersensitive sites In addition to major sites near the middle of the 25S rRNA within the pea rDNA chromatin. These sites are distributed coding sequence (detected by EC510; Fig. 2), we detect sites throughout both the coding and noncoding regions of the near the start of both the 18S (by EBg520; Fig. 2) and 25S (by rDNA repeat. Since the method of indirect end-labeling EC510; Fig. 2) rRNA coding sequences. allows only one hypersensitive site to be visualized for each To examine possible alterations in the pattern of DNase I copy of the gene, our maps are necessarily composite hypersensitivity during light-induced leaf development, 4- pictures derived from a population of genes; it is therefore day-old etiolated seedlings were transferred to continuous impossible to determine whether all copies contain all the white light before being harvested on day 7. Fig. 3 shows sites we have mapped or whether there are subpopulations examples of the autoradiograms from DNase I experiments with different DNase I hypersensitive sites. The detection of with nuclei from these light-treated plants. A map of the hypersensitive sites is thought to be dependent on approximate location of the DNase I hypersensitive sites, DNA supercoiling in some systems (see ref. 24). Thus, where deduced from these and other similar blots, is shown in Fig. many potential sites exist, as in the rRNA genes, alterations 4 (open arrows). When BamHI digests are probed with in superhelicity after cleavage at one site may reduce the XB380 and BE360, we detect approximately one hypersen- sensitivity of neighboring sites to subsequent DNase I cleav- sitive site per subrepeat element, as in the case ofdark-grown ages. plants. However, the bands are less sharply defined than they Specific DNase I hypersensitive sites have not previously are for the dark-grown plants. Hypersensitive sites between been demonstrated in plant chromatin, although actively the 3' end ofthe subrepeats and the start ofthe 18S gene occur transcribed genes in plants have been shown to have an at similar positions in the L variant rDNA of both light- increased general sensitivity to DNase I (25, 26). As men- treated and control plants. Easily detectable sites map 41.55, tioned in the Introduction, DNase I hypersensitive sites have '1.2, and 41.0 kb upstream from the right-hand BamHI site. been found in the rDNA of several animal species (9-13). In marked contrast to the L variant, S variant rDNA However, developmental regulation of DNase I hypersensi- chromatin from light-grown plants exhibits several differ- tive sites in rDNA chromatin has not been reported. Macleod ences in DNase I hypersensitive site distribution when and Bird (27) reported an increase in the general DNase I compared to that of dark-grown controls. In three indepen- sensitivity of rDNA chromatin in Xenopus laevis during dent experiments, we observe three sites in S variant chro- embryo development, although no hypersensitive sites were matin from light-treated plants that are not detectable in demonstrated. In a separate study, La Volpe et al. (12), using nuclei isolated from plants grown in the dark. These induced both X. laevis and X. borealis, observed no difference sites are located 1.55, 1.2, and 1.0 kb upstream from the between the population of hypersensitive sites in embryonic right-hand BamHI site, positions that correspond to the (transcribing, dividing) cells and blood (nontranscribing, 1554 Botany: Kaufman et al. Proc. Natl. Acad. Sci. USA 84 (1987) nondividing) cells. Similarly, there is no difference in the be ofgreat interest to determine whether the same population population of sites in the rDNA of Tetrahymena pyriformis of rRNA genes exhibits the decrease in cytosine methylation between logarithmic phase (dividing, transcribing) cells and as well as the appearance ofthe DNase I hypersensitive sites. starved (nondividing, nontranscribing) cells (9). The clusters of repetitive elements within the NTS of We thank C. Abdelhamid for excellent technical assistance. This Drosophila and Xenopus may well be analogous to the is Carnegie Institution of Washington-Department of Plant Biology subrepeat array found within the NTS of plant rDNA. publication 902. This research was funded in part by U.S. Depart- ment of Grant and 82-CRCR-1-1559 to sites occur in the of Dro- Agriculture 85-CRCR-1081 Hypersensitive subrepeat region W.F.T. and in part by a research agreement between Shell Agricul- sophila NTS (13). Similar arrays of DNase I hypersensitive tural Chemical Co. and J.C.W. and W.F.T. sites have also been found in the subrepeat region of wheat rDNA chromatin (W.F.T., unpublished data). In this case, 1. Tobin, E. M. & Silverthorne, J. (1985) Annu. Rev. Plant both the subrepeats themselves and the DNase I sites show Physiol. 36, 569-593. a periodicity of 130-140 bp rather than the 180-bp periodicity 2. Gallagher, T. F. & Ellis, R. J. (1982) EMBO J. 1, 1493-1498. observed in pea rDNA chromatin. Thus, the spacing of 3. Silverthorne, J. & Tobin, E. M. (1984) Proc. Nati. Acad. Sci. DNase hypersensitive sites in this region seems related to the USA 81, 1112-1116. subrepeat structure of the DNA rather than being a simple 4. Mosinger, E., Batshauer, A., Schafer, E. 4 Apel, K. (1985) reflection of nucleosome periodicity. Eur. J. Biochem. 147, 137-142. 5. Long, E. 0. & Dawid, I. B. (1980) Annu. Rev. Biochem. 74, In Drosophila, these subrepeats contain sequences with 727-764. strong sequence homology to the (28, 29), and can 6. Flavell, R. B., O'Dell, M., Smith, D. B. & Thompson, W. F. act both in vitro (30) and in vivo (31) as sites for initiation of (1985) in Molecular Form and Function of the Plant Genome, transcription. These subrepeats and similar regions of the eds. van Vloten-Doting, L., Groot, G. S. P. & Hall, T. C. NTS in Xenopus may also possess enhancer activity (32). A (Plenum, New York), pp. 1-14. strong positive correlation between nucleolar dominance and 7. Martini, G. & Flavell, R. B. (1982) Chromosoma 84, 687-700. the number of subrepeats in the rDNA at different nucleolar 8. Elgin, S. R. C. (1981) Cell 27, 413-417. organizer loci in hexaploid wheat suggests a similar function 9. Palen, T. E. & Cech, T. R. (1984) Cell 36, 933-942. for the subrepeats in this species (6). Low stringency hybrid- 10. Borchsenius, S., Bonven, B., Leer, J. C. & Westergaard, 0. (1981) Eur. J. Biochem. 117, 245-250. izations using probes from the pea rDNA promoter region 11. Bonven, B. & Westergaard, 0. (1982) Nucleic Acids Res. 10, (L.S.K. and J.C.W., unpublished observation) as well as 7593-7608. sequence data from wheat (6) indicate that there is sequence 12. La Volpe, A., Taggart, M. McStay, B. & Bird, A. (1983) homology between subrepeat sequences and the promoter Nucleic Acids Res. 11, 5361-5380. region in plants as well. 13. Udvardy, A., Louis, C., Han, S. & Schedl, P. (1984) J, Mol. The fact that developmental changes can be observed in Biol. 175, 113-130. the DNase I hypersensitive sites of pea rRNA gene chromatin 14. Kaufman, L. S., Briggs, W. R. & Thompson, W. F. (1985) may be related to the large number of rDNA genes in pea and Plant Physiol. 78, 388-393. in as 15. Watson, J. C. & Thompson, W. F. (1986) Methods Enzymol. plants general (103-104 copies per haploid genome) 118, 57-75. compared with animals (102_103 copies; see ref. 5). A likely 16. Watson, J. C., Kaufman, L. S. & Thompson, W. F. (1986) J. consequence of the high reiteration frequency in plants is that Mol. Biol., in press. only a small fraction of the rRNA genes will be active at any 17. Jorgensen, R. A., Cuellar, R. E. & Thompson, W. F. (1986) given time. A further possibility is that different populations Plant Mol. Biol. 8, 3-12. of rRNA genes are expressed at different stages of develop- 18. Ingle, J., Timmis, J. N. & Sinclair, J. (1975) Plant Physiol. 55, ment. To the extent that the presence of certain DNase I 496-501. hypersensitive sites is reflective of gene activity, then tran- 19. Lamm, R. (1981) Hereditas 94, 45-52. scription of the L variant, whose promoter-region hypersen- 20. Ellis, T. H. N,, Davies, D. R., Castleton, J. A. & Belford, sitive sites do not differ between light- and dark-grown I. D. (1984) Chromosoma 91, 74-81. 21. Polans, N. O., Weeden, N. F. & Thompson, W. F. (1986) plants, may be constitutive with respect to light. However, Theor. Appl. Genet. 72, 289-295. the changes in the hypersensitive sites of the S variant, in 22. Wu, C. (1980) Nature (London) 286, 854-860. particular those in the promoter region, may reflect a light- 23. Nedospasov, S. A. & Georgiev, G. P. (1980) Biochem. Bio- induced increase in the rate of transcription of these genes phys. Res. Commun. 92, 523-539. and/or an increase in the number of these genes in an active 24. Weintraub, H. (1985) Cell 42, 705-711. conformation within the nucleolus. Consistent with this 25. Spiker, S., Murray, M. G. & Thompson, W. F. (1983) Proc. notion is the observation of Gallagher and Ellis (2) that the Natl. Acad. Sci. USA 80, 815-819. rate of rRNA synthesis in nuclei isolated from pea seedlings 26. Murray, M. G. & Kennard, W. C. (1984) Biochemistry 23, grown in the light is -2-fold greater than in nuclei isolated 4225-4232. 27. Macleod, D. & Bird, A. (1982) Cell 29, 211-218. from dark-grown plants. 28. Coen, E. S. & Dover, G. A. (1982) Nucleic Acids Res. 10, The DNase I hypersensitive sites we have mapped in the 7017-7026. NTS of both the L and S variants correspond very closely to 29. Moss, T., Mitchelson, K. & De Winter, R. (1985) Oxford Surv. Hpa II/Msp I (CCGG) sites we have mapped in the NTS of Eucaryotic Genes 2, 207-250. the pea rDNA (16). Moreover, developmental changes in 30. Kohorn, B. D. & Rae, P. M. M. (1982) Nucleic Acids Res. 10, cytosine methylation status of Hpa II/Msp I sites within the 6879-6886. NTS occur after transfer to the light. As in the case of DNase 31. Murtif, V. L. & Rae, P. M. M. (1985) Nucleic Acids Res. 13, I hypersensitive sites, the developmental changes in meth- 3221-3239. ylation status are observed primarily in the S variant. It will 32. Reeder, R. H. (1984) Cell 38, 349-351.