somes, DNA fiber autoradiography and electron microscopy could not provide this information sincc the identity of the individual molecules under observation could not be determined. What was needed was an origin mapping technique that physically separated rcplication intermediates from non-replicating moleCllle5 and unambiguously identified specific segments of a chromosome. Agarose gel electrophoresis and Southern hybridization seemed ideal for this p u r w

Summary The replicon hypothesis, first proposed in 1963 by Jacob and Brenner''), states that DNA replication is controlled at sites called origins. Replication origins have been well studied in prokaryotes. However, the study of eukaryotic chromosomal origins has lagged behind, because until recently there has been no method for reliably determining the identity and location of origins from eukaryotic chromosomes. Here, we review a technique we developed with the yeast Saccharomyces cerevisiae that allows both the mapping of replication origins and an assessment of their activity. Two-dimensional agarose gel electrophoresis and Southern hybridization with total genomic DNA are used to determine whether a particular restriction fragment acquires the branched structure diagnostic of replication initiation. The technique has been used to localize origins in yeast chromosomes and assess their initiation efficiency. In some cases, origin activation is dependent upon the surrounding context. The technique is also being applied to a variety of eukaryotic organisms. Introduction Eukaryotic chromosomes are replicated by the activation of multiple origins within them. Early work with DNA fiber autoradiography and electron microscopy revealed that origins are spaced at intervals that range from about SO to 300kb and that this spacing is conserved among bpecies as distantly related as ycast, mammals, and higher plants (reviewed in Ref. 2). The appealing notion that origins are located at specific sites on chromosomes was strongly wpported when Stinchcomb et aZ.(3),using yeast, introduced a plasmid test for origins of replication. They found that some. but not all, restriction fragrncnts derived from yeast chromosomal DNA could confer on recombinant plasmids the ability to be maintained as autonomous mini-chromosomes in yeast. These scquences are defined as Autonomously Replicating Sequence elements or ARS elements. While ARS elements have the functional property expected for origins of replication, physical proof was needed that replication was indeed initiating from these sites on the plasmids, and furthermore, that replication was initiating from these sites in their normal locations within yeast chromo~

Origin Mapping by 2-Dimensional Agarose Gel Electrophoresis In 1981, Bell and Byers(4) reported the use of a 2-dimensional gel system to separate and characterizc branched recombination intcrmediatcs and illustrated their technique with restriction fragments from the yeast 2 micron plasmid. Since replication intermediates are also branched, a logical extension of their work was to use 2-D gels to analvze restriction fr agments containing replication forks('.('). The two dimensions of electrophoresis differ in several respects. The first dimension is run at low voltage in low gel concentration to optimize the invei-se relationship between the mass of a DNA fragment and its mobility. The second dimension is run at high voltage in a gel of higher agarose concentration with the direction of electrophoresis at 90" to the original direction of electrophoresis. Ethidium bromide is included in the second dimcnsion and electrophoresis is carried out at 4°C. This combination of conditions, since it exaggerates the contribution of the shape of a molecule to its mobility, causes restriction fragments that contain one or more branches to be reduced in their mobility in the second dimension relative to linear fragments of equivalent mass. We wished to explorc the utility of the Bell and Byers 2-D gels system for analyzing replication intermediates and chose as a starting point the multiple copy native 2 micron plasmid!'). This plasmid has a single ARS element and replicating intermediates have a thetashape consistent with the existence of a single replication origin('). We reasoned that if there were a single, specific origin of replication located at the plasmid ARS then cutting the 6 kb plasmid in half, with one cut at the ARS and the other 180" opposite, would reducc the theta-shaped replication intermediates to the simplest of branched forms: two 3 kb restriction fragments each with a single fork that resembles a simple Y structure (Fig. 1A). When these plasmids are derived from an asynchronously growing cell pop"lalion they would vary in the extent of their replication - from just initiated to nearly completed - and the simple Ys would therefore range in mass from 3 kb to 6 kb and correspond to fragments with incrcasing lengths of daughter duplexes. Replication intermediates would therefore not occupy a single spot on a 2-D gel but would produce a continuum ranging between 3 and 6 kb in mass. I f the degree of retardation in the

A

C

B

t

D

Fig. 1. Generation of branched forms from a replicating circular DNA molecule. Through the appropriate choice of restriction enzyme it is possible to gciicratc thrce basic replicating structures from replicatine circular molecules simple Ys (A arid D). bubbles (R and D), anddouble Ys (C). ~

A

B

C

Simple Y

Bubble

D Double Y

Bubble-Y

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.-f3-

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0

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3 kb

6 kb

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6kb

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Fig. 2. 2-D gel patterns genzrated by the three basic forms of replication intermediates. The hypothetical fragment examined in each panel is 3 kb. See the description in the text.

sccond dimension is proportional to the degree of branching, then the displacement from the arc of linears would be greatest when the length of a branch extending lrom an otherwise linear molecule is greatest (Fig. 2A). For simple Y molecules this maximum would occur when the molecule IS 50% replicated. With further replication. t h e degree of branching actually decreases since thc molcculcs can adopt a conformation in which two replicated arm\ bccomc aligned during electrophoresis, with the unreplicated portion of thc moleculc producing a centrally located side branch. As the molecule nears 100 % replication, the linear portion of the inoleciile approaches a 6 k b linear with a very short, unreplicated side branch (SCC Fig. 2A). The expccted distribution of simple Ys in a 2-D gel is illustrated in Fig. 2A. The 2 micron plasmid halves, generated by cleaving at the ARS fcqucnce and 180" opposite the ARS and detected from the total

complement of yeast nucleic acids by Southern hybridization: give this pattern (Fig. 3A). To confirm that a 2-D gel pattern with this shape is indeed generated by replication intermediates containing a single fork: Elissa Sena examined replicating mitochondria1 DNA that was first separated from other yeast nucleic acids by its unique low buoyant density('). When cleaved and examined on 2-1)gels it gave the pattern shown in Fig. 2A. More importantly, when DNA was recovered from various portions of the arc of replication intermediates and examined in the electron microscope, it had precisely the predicted structures. These results unequivocally establish the electrophoretic pattern of simple Y restriction fragments. Two other simple replication structures can be gencratcd from circular theta intermediates by restriction cleavage. If plasinids are cleaved at a single site 180" from the origin of replication, a series 01fragments containing internal cyc or bubble forms would result (Fig. 1B). If plasmids are cleaved at a single site that lies near the origin of replication, a series oC molecules with forks approaching each other from both ends would result (Fig. IC). Neither the bubbles nor the double Ys approach a linear shape as they near completion ol' replication, and therefore, neither of these structures are expected to return to the arc of linears as their mass approaches 12 kb. For example, as the forks meet in thc double Y replication intermediates, the highly branched restriction fragments fall apart into 6 kb linear molecules. The 2-D gel patterns for bubbles and double Ys are shown in Figs 2B and 2C. The 2-D gel patterns produced by bubble molecules and by double Y molecules were determined, not directly by electron microscopy, but indirectly by demonstrating that diffcrcnt restriction digests alter the gel patterns in predictable ways!5). We reasoned that the electrophoretic pattern of bubble molecules could be deduced if thc plasmids are cleaved asymmctrically so that the origin of replication does not lie at the exact ccntcr of the fragment (Fig. 1D). As the two forks proceed bidirectionally outward from the origin, one fork will reach its nearest restriction site before the other fork reaches the other restriction site. In this situation, cleavage of the initial, 'young' replication intermediates would produce restriction fragments with internal bubbles, but 'old' replication intermediates would contain only a single fork and finish their replication as simple Ys. The pattern of replication intermediates on a 2-D gel would consist of two types of branched structures - 'young' bubbles and 'old' simple Ys - with a discontinuity between the two partial patterns (Fig. 2D). Since the migration behavior of simple Ys was established by electron microscopy, the shape of the 'young' arc from discontinuous patterns in which the molecules complete their replication as simplc Ys must define the arc of bubbles. A n example is shown in Fig. 3B. Joel Huberman concurrently and independently derived a different 2-dimensional agarose gel procedure

B

A

2 p m Xbal

D

C

ARS1 Ncol

2 pm EcoRI

rDNA Nhel

Fig. 3. 2-D gel patterns obtained from yeast DNA sequences. A . 2 micron DNA cleaved into two nearly equal sized fragments with XhaI (as illustrated in Fig. 1A) is examined by hybridization tu a probe specific to one of the fragments. B. 2 micron DNA clcaved with EcoRl (as illustrated in Fig. 1D) is examined by hybridization to a prohe specific for the fragments containing the ARS. Because the plasmid is present in two isomeric forms. the two EcoRT fragments containing the ARS are diffcrent in size. In cach isomer thr AKS lies asymmetrically within the EcoRl fragment. Consequently the gel pattern reveals two sets of bubble-to-simple Y arc transitions as illustrated in Fig. 2D. C . The chromosome IV copy o f ARSl was cleaved at flanking NcoI sites so that thc ARS is asymmctrically locatcd within the fragment. Hybridization to the cloned NcoI fragment detects the expected bubble-to-simple Y transition. In addition. a faint signal corresponding to a complete simple Y arc appears to be present. A complete simple Y arc implies that the ARSl fragment is sometimcs replicatcd by a replication fork passing through it: rather than by the activation of forks at ARSl. However, quantitative estimates from this and other gels indicates that ARSl is used as an origin in more than YO % of S phascs. D. Thc portion of the rDNA repcating unit that contains the ARS elemcnr was clcaved from the tandem cluster on chromosome XI1 by NheT digestion and detected by a probe from the non transcribed spacer. The ARS is in the center of this fragmcnt. Thr strong signal for the complete simple Y arc and fainter signal for the bubble arc indicate that most repeats are replicated by a fork passing through them. Estimates from this and other gels indicates that only 12 to 17% of t h e repeats contain an activc origin. The proinincnt spot of hybridization on the arc of simple Ys at a size where they are nearly completed in their replication results from a replication fork barrier (RFB) (Brewer and Fangman, 1988; Linskens and Huberman. 1988).

for the analysis of replication intermediates. In the Hubcrman procedure, restriction fragments are also separated by mass in the first dimension of electrophoresis. However, the second dimension is an alkaline gel that separates the denatured nascent DNA strands from the full length parental strands. Sequences are detected by using a series of probes along the fragment of interest. Replication fork direction and origin location are deduced from the observed lengths of nascent single strands when probes from different regions of the fragment are used. (For a discussion of the neutral/ alkaline 2 D gel technique see Refs. 8,s) When is an ARS an Origin? The results we obtained with the 2 micron plasmid and a recombinant plasmid that contains the chromosomal sequence ARSl confirmed that replication was initiating within or near the sequences that function as ARSs('). Tt was important next to determine whether this conclusion would hold for ARSs in their normal chromosomal contexts. The principles of origin mapping with 2-D gels are also amenable to the analysis of restriction fragments from the large DNA molecules of chromosomes. Total DNA is simply cleaved with an appropriate restriction enzyme and the fragment of interest detected by hybridization. Depending upon the sites of cleavage relative to a chromosomal origin of

replication, any of the pattcrns illustrated in Fig. 2 will be produced. The first chromosomal origin to be ed with this technique was from chromosome and was shown to coincide with a previously identificd ARS element("). The chromosomal copy of ARSl was also examined ('la) and found to function as an origin (Fig. 3C). The limited analysis so far indicates that chromosomal origins of replication map within (or near) sequences that provide ARS function on recombinant plasmids. In other words, no chromosomal sequence that has bccn found to act as an origin on the chromosome has failed to function as an ARS on a recombinant plasmid. Surprisingly, the converse is not true. A number of sequences identified as ARSs on plasmids apparently do not function: or function very inefficiently, as origins of replication in their native chromosomal locations. While all origins may be ARS clcments, not all ARS elements are necessarily chromosomal origins. The inactive state of ARS elements as origins on chromosomes has been observed in several different contexts: (1) A single copy ARS adjacent to the centromere (CEN) of chromosome I11 is not a chromosomal origin ( S . Greenfedder and C. Newlon, personal communication). (2) Based on the kinetics of replication of restriction fragments near the left telomere of chromosome 111, it was suggested that several ARS clcments there are inactive as chromo-

E:86

soma1 origins(l2).This inactivity has bccn confii-med by 2-D gel analysis (J. Huberman and C. Newlon, personal communications). (3) An ARS is present in thc nontranscribed spacer of each repeat of the ribosomal KNA gene cluster, vet origins are found in only a sub-set of those repeatsfl’,13). All of these findings reveal that the origin activity of some ARS clcments is influenced by chromosomal context. It will be o f grcat interest to determine what features of the flanking DNA arc responsible for the silencing of potential origins and whether these ARS elements are cryptic origins that are activated under some growth conditions. These silent origins may be relevant to the change in origin density that has been observed in different developmental or culture condition^"^) in higher eukaryotes. Contextual control may involve specific silencer sequences that act in cis or may involve broader features of chromosome structure, such as proximity to telomercs, centroineres. or transcriptionally active sequences. Tandemly Repeated Origins in the rRNA Gene Cluster The 200 copies of yeast ribosomal RNA genes arc found on chromosome XI1 in a tandem array of 9 kb repeating units, each of which contains two transcription units, one for the 35 s rKNA that gives rise to the 28, 5.8, and 18 S rRNAs and one for the 5 S rRNA. The ARS element is located in the non-transcribed spacer between the 5‘ ends of these two divergent transcription units(18) (see Fig. 4). Based on the size of t h e nascent single strands from rDNA replicons, Walmsley er al. (19) suggested that active origins were spaced roughly five repeats apart. Infrequent origin use was also indicated by an electron microscopic analysis(2”’ and by 2 D gel analyses(’”~’”).Since all repeats in the rDNA cluster probably have a functional ARS, features other than the primary sequence of the repeat must influence origin use. Among the possible controlling factors might be variability in chromatin organization among different repeats, a higher order chromosome folding in the nucleolus, or some (unknown) regulatory event that activates or silences the transcription of particular repeats. Transcri tion across an ARS can interfere with origin function(’ ) and transcription may influence or

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1 8 , 5 8 , 28s

f

t

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5s

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R

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Fig. 1. 4 xhemdtic diagram of the rDNA gene clustcr The 1s 5 8 and 28 S ribosoinal RNAs are tianscribed in a single 6 6 kb transcript bv RNA polymerdw T The 5 Y t r a n w i p t of 120b i \ copied from the opposite strand bv RNA polymeiase I11 Between the 5’ end5 of these t w o trdnscript\ lies an ARS element and between thc 3’ ends of the ti anscripts lies a replication fork barrier (RTB) that halts the progre\\ of leftward, hut not rightwaid. moving folks (Biewer and Fangman, 1988. Linshens and Huberman. 1988) Evidence for the RFB and the oiigiii are gircii in Fig 31,

impede the movement of replication lorks once initiation has so it seems reasonable to speculate that transcription may also influence origin selection. For example, the positive and negative domains of supercoiling that flank an actively transcribed gene(*?)may result in a local environment that is either hospitable or hostile for rcplication initiation. Origin Mapping in Other Organisms With its small genome size and relatively small amounts of repeated DNA, rapid progress is being made in the mapping of replication origins in yeast chromosomes using 2-D gels. This technique is also being successfully used with a variety of higher eukaryotes and their viruses to explore the nature of their origins. The replication initiation sites for several animal viruses have been localized using 2-D els (bovine papilloma Epstein Barr virus(’$. In general, the sites previously defined by functional tests appear to coincide with the actual sites of initiation of replication. However, in the case of bovine papilloma virus the actual site o f initiation was shown by 2-D gel analysis to map near but outside of the region shown to be required for replication by cloning studies(”). The results with origins in higher eukaryotic chromosomes have been even more intriguing. An origin of replication used for the amplification of chorion genes in Drosoplzila follicle cells has bcen localized to the region that contains the Amplification Control Element (ACE). Instead of finding a unique single origin, it appears that initiation may occur over a range of sites flanking the A aimilar rebult has been found in the dih drofolate reductase (DHFR) amplicon of hamster cellsJ9). In this case the amplification process itself was not examined; instcad the replication of the tandemly repeated DHFR cluster was analyzed in stable cell lines. While origin activity was localized to a 30 kb region, the specific site of initiation appeared to vary at random within this broad initiation zone. Most recently, a crippled Epstein Barr virus plasmid whose maintenance in human cells is driven in part by a human genomic insert was shown to initiate replication at five or more different sites, possibly including one in vector sequences(”). In conjunction with the observations on bovine papilloma virus, these results may mean that if specific sequences in the mammalian genome are required for origin function they may cause initiation to occur at multiple sites distributed over a broad region. Within this region the sites may be chosen at random. Application of the 2-D gel mapping technique to single copy origins in higher eukaryotic chromosomes is limited in part by the amount of DNA that can be run on a gel and additionally by the level of detection by hybridization. The sensitivity of probing continues to improve, but the most important breakthroughs have come in the methods of enriching for replicating intermediates. Natural or induced synchrony helps. However, the enrichments afforded by using BND-

cellulose to recover molecules with partial singlestranded character(’) and/or by isolating the nuclearmatrix-bound fraction of DNA(”) is making the technique feasible for single-copy mammalian sequences.

Other Origin Mapping Techniques Other origin mapping procedures currently being used complement our technique. The laboratory of Joel Huberman, using their neutral/alkaline technique, have arrived at the same conclusions as those reached with our neutral/neutral technique concerning the localization of the 2 micron plasmid origin@), the infrequent use of origins in the yeast rDNA cluster(13), and the identification of both active and inactive ARSs on yeast chromosome 111 (Ref. 10, and J. Huberman and C. Newlon, unpublished). In addition, Vaughn et al. (29), using the neutral/alkaline gels to analyze the DHFR origin region, obtained results confirming that initiation occurred at random places within the origin region. Both 2 D gel techniques have technical limitations. To obtain mapping information with the alkaline technique, the membrane filters must be repeatedly reprobed with multiple small probes. To be certain an origin hasn’t been missed with our gel system, overlapping restriction fragments must be analyzed. Neither technique can currently map an origin with nucleotide resolution. Both techniques rely, to different extents, on maintaining the parcntal strands in an intact or un-nicked form. In the alkaline second dimension of the Huberman technique, the hybridization signal from nascent strands runs below the arc of parental strands and is therefore subject to contaminating signal produced by any random single strand breaks that have occurred in the parental strands. A potential problem with our 2-D gcl systcm is that if a break were to occur in a parental strand at a replication fork it would lead to underestimation of branched structures, including the bubble forms that are diagnostic of origin activity. Within the past few years additional origin mapping techniques that rely on the switching of strand polasity of Okazaki fragments(”) or on the segregation of nucleosomes in the presence of protein synthesis inhibitors(33)have been used to examine the amplified D H F R region. The results obtained with these techniques do not agree completely with the results obtained by Vaughn et ~ l . ( ’ ~ )For . a discussion of these conflicting data the reader is directed to a mini-review by Linskens and Huberman(34). Because of the assumptions upon which these techniques are based, only bidirectional origins can be detected: unidirectional origins would be missed. A third technique, dependent on the ability to amplify specific fragments by PCR from size-separated, purified nascent strands, has given promising results with SV40 and a mammalian cellular gene r e g i ~ n ( ~ ’ ” ~ ) .

Prospects The importance of DNA replication is reflected in the organization and precision with which cells carry out the duplication of their genomes. In eukaryotes, a particularly intriguing aspect o f the regulation o f replication is the observation that each chromosomal DNA molecule is replicated exactly once every cell cycle. While we as yet have no specific clues as to how cells impose this strict control, the general mechanism of control of chromosome duplication appears to act by regulating the initiation of new rounds of synthesis lrom the origins. To begin to understand the ‘oncc and only once’ control over DNA replication i t may be essential to identify the origins. The 2-D gel technique for identifying and mapping origins of replication that we developed with yeast, and the other new techniques, help open up this important control for analysis. From initial studies. origins in yeast chromosomes appear to be simple in that initiation appears to take place at a localized position at or near an ARS. The picture of higher eukaryotic origins that is emerging from the 2-D gel analyses of the DHFR and chorion gene regions and human D N A plasmids is more complicated. A n ‘origin‘ may actually consist of multiple alternative initiation sites distributed over a broad region. Whether these cases arc typical of animal origins remains to be seen. Other replicatioll origins need to be examined. The finding that yeast origins can be silenced by context makes it likely that activc origins exist in chromosomes that are not active on plasmids. Such a ‘reverse’ context effect might explain the failure to establish a reliable ARS test in mammalian cells. The inability of all yeast ARS elements to act as functional origins within their native chromosome context is somewhat surprising. This observation indicates that while the ARS element may be necessary for initiation, it is not sufficient. Other features of the chromosomc must somehow act in cis to limit the potential for origin activity. In the rDNA cluster, it appears that each cell cycle a sub-set of rcpcats is chosen to havc its origins activated. The ability to easily change the chromosomal context of sequences in yeast chromosomes will prove invaluable in dissecting the contextual rules that regulate activation of both single copy and repeated origins. Acknowledgements Our work on chromosome replication is supported by Public Health Service grant NIGMS 18926. We thank Betsy Ferguson. Daniel Lockshon and M. K. Raghuraman for useful comments on the manuscript, and Re thank our colleagues in the field for allowing us to cite their unpublished work. References 1 JACUU. 1 . A N D BRENNER. S. (196?). Sur la regulation dc la qnthese? du DNA chen les bacteria: I’hgpothese du replicon. C. R. Acud. 5 i , Paris 256.

2‘18-300.

2 E D ~ N H ~11. K CJ .I .AND I I U H L K M AJ~. . A . (lY75). Eukaryotic chromosome replication. Annu. liev. Genet. . 9. 245-284. 3 STINCHCOMB, D. T., SIRUHI., K. AND DAVIS, R . w. (1979). Isolation and charactenLaLion of a y e a t chrumoaomal replicator. Natirw 282. 39-43 4 BELL.L. AUD BYERS,B. (1Y79). Occurrence of crossed strand-exchange forms in yeast DNA during meiosis. Proc. ?VarlAcad. Sci. USA 76, 344-3449. 5 BREWER. B. J. A N D F ~ U ~ C ~W. M L. A N(1987). , The localization of replication origin5 on ARS plasmids in S. cerevisiae. Cell 51. 463-471. 6 BREWER, B. I.. SENA.E. P. A N D FANGMAN. W. L. (1988). Analyris of replication intermediatcs by two-diinensiorial agarose gel electrophoresis. Cnncer C'elh 6, 229-234. 7 NELVLON,C. S . . ULVEVISH,R . J.. SKI, P. A. AND RL I~ F IS C.. (1981). Keplication origins used in vivo in yeast. lC',V- OCLA Symp. Moi. Celi. Diol. 22. 501-515. 8 HUBERMAN, J. A , , SPoTIL.4, L. D., KALVOTKA, K.A , . EL-AssouLI. S. M. A N D Dams, L. R. (1987). Thc in vir-o replication origin of the yeast 2 p i plasmid. cell 51, 473-481. 9 N ~ ~ , O T K AK., A. . ~ K D HGBLRWAF, J. A. (1988). Two-dimenaional gel electrophoretic method of mapping DNA replicons. Mol. Cel!. Bio!. 8. 1408-1413. 10 HUBERMAN. J. A , . ZHU.J . , DAVIS.L. R . AND NEWLON, C. S. (1988). Close association of a DNA replication origin and an ARS element on chromosome I11 of thc ycast. Sacrhnromyces cerevisiae. A'trcl. Acids Res. 16, 6373-6384. 11 PALZKILL. T. G.. OLIVER. S. G. A P ~ DNEWLON. C. S. (1986). DNA sequence analysis of ARS elements from chromosome 111 of Saccharorrryces cerebisiae: identification of a new conserved sequence. iVtfcl. Acids Res. 14, 6247-6264. 11a FERGUSON, B. M.. BREWER, B. J., REYNOLDS, A . E. AKD FAXGMAX, W. L. (1991). A yeast origin of replication is activatcd laic in S phase. Cell 65, 507-515, 12 REYUOLDS, A . E.. MCCARROLL. R. M., NFWI 06,C. s. AND F.SKCMAr\, h'. L. (1989). Time uf replication of AKS elements along yeast chromoaome 111. Molec. Cell. Biol. 9 . 4488-4494. 13 LIUSKEKS, hl. H. K. AND HUBERMAK. J. A. (1988). Organization of rcplication of ribosomal DNA in Sacclzarorriyces cerebipiae. Molec. Cell. B i d . 8. 492774915. 14 BRERER, B. J. AND F~KGMAN, W. L. (1988). A replicalion fork barrier at the 3' er.d oE yedsl ribosomal KNA genes. C'dl55, 637-643. 15 BLL-MFNTIIAL. f\. B.. KRIEGSTLIN. 11. J. AND HOGNESS. U . (lY74). The unit of DNA replication in Drosophila melanognster chromosomes. Cold Spr-ing HarD. S y n p . Quant. B i d . 38, 205-223. 16 CAI.TAN. H. G. (1974). DNA replication i n thc chromo~omesofeukaryotes. Cold S j m n g Harh. S p p . Quart/. R i d . 38. 195-203. 17 TAYLOR, J. H. A N D HOZIER.J. C. (1976). Evidence lor a four micron 57, 341-350. replication unit in CHO cells. Cl~rwr~usunzu 18 KOUPRIKA, N. Y . AND LARIONOV, V. L. (1983). The study of a rDNA replicator in Sncchn~oniyces.Cwr. Genet. 7, 433 438. 19 WALMSLBY,K. $1.. JOHNSION, L. H . , WILLIAMSON. D. H. AND OLIVLR. S . G. (1984).Replicon aizc of yeast ribosomal DNA. Mol. Gen. Genet. 195,260-266. 20 SAFFER, L. D. AXUDMILLER,0 . L.. JR(1986). Electron microscopic study of Sacchai.oinyces cerevisiae rDNA chromatin replication. Molec. Cell. Biol. 6. 1148-1157.

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Bonita J. Brewer and Walton L. Fangrnan are at the Department of Genetics, SK-50, University of Washington, Seattle, WA 98195, USA.