by a Novel Three-Dimensional Gel Method - Molecular and Cellular ...

Vol. 14, No. 2

MOLECULAR AND CELLULAR BIOLOGY, Feb. 1994, p. 1520-1529

0270-7306/94/$04.00+0

Analysis of an Origin of DNA Amplification in Sciara coprophila by a Novel Three-Dimensional Gel Method CHUN LIANGt AND SUSAN A. GERBI* Division of Biology and Medicine, Brown University, Providence, Rhode Island 02912 Received 5 August 1993/Returned for modification 18 October 1993/Accepted 19 November 1993

The replication origin region for DNA amplification in Sciara coprophila DNA puff IV/9A was analyzed with a novel three-dimensional (3D) gel method. Our 3D gel method involves running a neutral/neutral 2D gel and then cutting out vertical gel slices from the area containing replication intermediates, rotating these slices 90° to form the third dimension, and running an alkaline gel for each of the slices. Therefore, replication intermediates are separated into forks and bubbles and then are resolved into parental and nascent strands. We used this technique to determine the size of forks and bubbles and to confirm the location of the major initiation region previously mapped by 2D gels to a 1-kb region. Furthermore, our 3D gel analyses suggest that only one initiation event in the origin region occurs on a single DNA molecule and that the fork arc in the composite fork-plus-bubble pattern in neutral/neutral 2D gels does not result from broken bubbles.

Studies on the mechanism of eukaryotic DNA replication have been largely limited to viral systems in which specific origins of replication are well defined. This reflects the difficulty in identifying and elucidating chromosomal origins of replication. A search for cellular replication origins has been prompted by the development of two-dimensional (2D) gel techniques (5, 21). Neutral/neutral 2D gels developed by Brewer and Fangman (5) separate replication intermediates (forks, bubbles, etc.) derived from a given restriction fragment from each other and from nonreplicating linear DNA. This is achieved by running the first dimension of a low-percentage gel at a low voltage, which resolves DNA primarily by mass, and the second dimension of a higher-percentage gel at a higher voltage so as to separate DNA by shape as well as by mass. Sequential hybridization of the 2D gel blots with probes from different restriction fragments will indicate whether a given fragment contains a replication origin (a bubble arc is seen) or is replicated by a passing fork (a fork arc is seen). A recent modification of this 2D gel method using digestion by a second restriction enzyme before running the second dimension of the gel allows determination of the direction of fork movement (13).

The first dimension of neutral/alkaline 2D gels developed by Huberman et al. (21) is the same as that of neutral/neutral 2D gels, but the second dimension is an alkaline gel which separates replication intermediates into nascent and parental strands and resolves them by size. By hybridizing the 2D gel blots with short probes from different areas within a restriction fragment, the direction of fork movement can be deduced from the size distribution of nascent strands detected by each probe. A probe at the end where forks enter the fragment will detect nascent strands of all sizes, while a probe at the other end will detect only very large nascent strands. The map position where fork movement changes from one direction to another is presumed to be the origin of bidirectional replication. The presence of replication bubbles can also be directly demonstrated by this approach. A short * Corresponding author. t Present address: Cold Spring Harbor Laboratory, Cold Spring

Harbor, NY 11724.

probe complementary to an origin located within the restriction fragment should detect small (as well as large) nascent strands from newly initiated bubbles. 2D gel methods were first developed by using the yeast 2,um plasmid as a model system. These studies showed that replication of the plasmid initiates at or near the single ARS (autonomously replicating sequence) element in the plasmid (5, 21). Subsequent 2D gel analyses also suggested that single replication origins colocalize with ARS elements on chromosomes of the yeast Saccharomyces cerevisiae (8, 14, 24, 29, 38), although some ARS elements appear to be silent and do not function as chromosomal replication origins (12, 33). In contrast to mapping origins to discrete sites in plasmids and S. cerevisiae, 2D gels suggest that chromosomes of metazoans (7, 10, 11, 20, 23, 27, 32, 34) and the yeast Schizosaccharomyces pombe (37) have multiple initiation sites in a broad zone. The findings of a zone of initiation raise a question that cannot be solved by 2D gels: is there more than one initiation event on a single DNA molecule in the origin region, or is it the case that there is just one initiation on a molecule but different molecules initiate replication at different sites within the origin zone? Data to be presented in this report suggest that the latter situation exists. Previously, electron microscopy studies revealed clusters of microbubbles and large stretches of single-stranded DNA in cells that are active in replication (1, 3, 4, 16, 19, 28), but seldom in nonreplicating DNA (28). These observations led to the strand separation hypothesis: the DNA region containing microbubbles melts into a large single-stranded region (3). In this model, initiation does not utilize replication bubbles; instead, DNA synthesis could start at many sites on both DNA strands within the melted region and be followed by ligation of all nascent DNA. Another model proposes that replication may initiate as microbubbles which fuse into larger bubbles that progress outward (26). Neither of these models is readily testable by 2D gels. Data to be presented in this report do not support either the strand separation or the microbubble model for Sciara DNA puff amplification. 2D gels suggest that the ARS element in the yeast 2,um plasmid is the only initiation site (5). There is no fork signal overlapping the bubble arc in a given restriction fragment (5). This is also true for ARS elements on chromosomes of 1520

VOL. 14, 1994

3D GEL ANALYSIS OF A DNA AMPLIFICATION ORIGIN

the yeast S. cerevisiae (Y' ARS [14] and ARS 306 [38]) as well as in bovine papillomavirus (36) and one locus in the slime mold Physarum polycephalum (2). However, when genomic DNA from metazoans (7, 10, 11, 20, 23, 27, 32, 34) and in some cases from the yeast S. cerevisiae (6, 14, 17, 25) or S. pombe (37) is analyzed, a complete fork arc in addition to a bubble arc is observed in the same fragment. These fork-plus-bubble patterns have been interpreted to mean that bubbles are unstable and break into fork-like structures during handling and/or that the fragment is sometimes replicated by forks that are from other replication origins located either outside and/or inside the fragment being tested. The possibility of some bubbles breaking into forks cannot be directly tested by 2D gel methods. The data to be presented here suggest that the fork arc in the composite fork-plusbubble pattern does not result from broken bubbles. We have designed a novel 3D gel method to investigate these and other questions about replication origins. Our model system is the developmentally regulated DNA amplification in DNA puff II/9A in salivary gland chromosomes of the fungus fly Sciara coprophila. DNA puffs are specific loci not only of active transcription but also of up to almost 20-fold DNA amplification in polytene chromosomes of late-larval salivary glands (35). Puff II/9A contains two genes (II/9-1 and II/9-2) which are about 3 kb apart and have 85% sequence similarity with each other (9). We have shown previously by neutral/neutral 2D gels that initiation of DNA amplication at this locus is confined to an area of no larger than 6 kb just upstream of gene II/9-1. Furthermore, our data obtained by using neutral/alkaline 2D gels indicated that there is a -1-kb major initiation region within the 6-kb area (23). Replication forks were seen to move outward from the -1-kb origin region in a bidirectional manner. Our results from 3D gels presented in this report confirm the location of the 1-kb major initiation region and give new insights into interpretations of the previous 2D gels. (This research was done in partial fulfillment of requirements for a Ph.D. degree to Chun Liang from Brown University.) MATERIALS AND METHODS DNA preparation. Salivary gland dissection (from -1,500 DNA puff stage female larvae), genomic DNA isolation via a CsCl gradient, restriction digestion, and benzoylated naphthoylated DEAE-cellulose (BND-cellulose) chromatography were done as described elsewhere (23). About 150 ,ug of DNA was obtained, and all DNA from the caffeine wash (-7 ,ug), which is enriched for replication intermediates, was loaded onto the first dimension of the 3D gels. Electrophoresis. The first two dimensions of the 3D gel were carried out by the neutral/neutral 2D gel method of Brewer and Fangman (5), with adjustments to the gel conditions as follows. The first dimension is a 0.35% gel run at 0.5 V/cm for 66 h, and the second dimension is a 0.75% gel run at 3 V/cm for 21 h. Pulsed-field gel grade agarose (Boehringer Mannheim) was used in both dimensions. After the first two dimensions were run, the gel was soaked in H2O (to get rid of excess ethidium bromide), and gel slices for the third dimension were cut out with the help of a mold made of a piece of unexposed, developed X-ray film. The mold has lanes cut out at desired positions according to the first-dimension size markers and the position of the bulk DNA in the 2D gel, which is visualized briefly under long-wavelength UV light. Each gel slice was turned 900 and placed in the trough of a premade alkaline gel (1.2%). The

1521

troughs were sealed with melted agarose, and the gels were soaked and then run at 1 V/cm for 19 h in an alkaline buffer which is the same as that used in neutral/alkaline 2D gels (21). Blank gel slices to replace the gel slices that were cut out for the third dimension were inserted into the remnants of the 2D gel to stabilize it for blotting. Saran Wrap was kept under the remnants of the 2D gel for added support during handling before blotting. Blotting and hybridization. Blotting of 3D gels and the remnants of the 2D gel onto Biotran (+) membrane (ICN Biochemicals) was carried out as described previously (23). Hybridization was the same as the second method used by Liang et al. (23). The filters were exposed to Kodak X-Omat AR films for 1 to 7 days with Cronex Lightning Plus intensifying screens (Du Pont). Stripping of blots was done by pouring on a boiling solution of 0.1% sodium dodecyl sulfate-0.lx SSC (lx SSC is 0.15 M NaCl plus 0.015 M sodium citrate) and then shaking at room temperature for -5 min; this was repeated twice. Exposure of the stripped filters showed that probes had been removed by this procedure. RESULTS Strategy of the 3D gel method. Our 3D gel method is a combination of a neutral/neutral 2D gel with an alkaline agarose gel as the third dimension. Restricted DNA is first run out on a neutral/neutral 2D gel which separates replication intermediates from each other and from nonreplicating linear DNA. Narrow gel slices perpendicular to the first dimension are then cut out such that each gel slice carries bulk DNA, forks, and bubbles in three different places along the slice (see the diagram in Fig. 1A). Gel slices are cut out from known positions at an interval (e.g., every kilobase) according to the size markers in the first dimension, and each gel slice is named in this report by the first-dimensional size (e.g., the 9-kb slice). Each of these gel slices is rotated 900 and put into a preformed trough in an alkaline agarose gel (see the diagram in Fig. 1B). After the troughs are sealed with melted agarose, the gels are soaked and then run in an alkaline buffer to resolve the denatured DNA by size. Therefore, both forks and bubbles that have been separated from each other by the 2D gel are further resolved into parental and nascent strands (Fig. 1B). Sequential hybridization of the blots for a given restriction fragment with the entire fragment as probe and with small probes across the fragment will give useful information about the replication origin in this fragment, as will be described in the following sections. The remnants of the 2D gel after the gel slices had been cut out to run the third dimension can also be blotted and hybridized so that one can see where the gel slices have been taken from relative to the fork and bubble arcs in the 2D gel. The distances from the bulk DNA to the fork arc and to the bubble arc in each gel slice can be used to confirm the fork and bubble signals in 3D gels. If an origin zone is too large to be included in one restriction fragment, other fragments can also be analyzed by reprobing the same set of blots, provided that the cut-out gel slices cover the size range for all fragments of interest. The sizes of replication forks and bubbles can be measured more accurately in 3D gels than in neutral/neutral 2D gels. To localize a replication origin by the neutral/neutral 2D gel technique, one needs to estimate the sizes of replication intermediates according to size markers in the first dimension, assuming an equal rate of migration for replication intermediates and linear molecules of the same mass. With this assumption, the point of bubble-to-fork transition has

1522

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conditions. 3D gels can solve this problem by separating forks and bubbles into single strands and resolving them solely by size in the third dimension. When EcoRI-digested Sciara DNA from puff stage larval salivary glands is run out on a neutral/neutral 2D gel, only the 5.5-kb EcoRI fragment which contains the origin region in the DNA puff II/9A locus gives a fork-plus-bubble pattern while all other EcoRI fragments tested do not produce bubble arcs (reference 23, and see the map in Fig. 1 of this report). Therefore, we have focused our 3D gel analysis on this 5.5-kb EcoRI fragment which contains the initiation

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FIG. 1. Strategy (A and B) and actual data (C and D) of the 3D gel method. (A) The first two dimensions of the 3D gel method are a neutral/neutral 2D gel. A 2D pattern with both fork and bubble arcs is depicted. Six vertical bars indicate the gel slices that are cut out to run the third dimension. The horizontal bar at the top represents the loading well for the second dimension, and the square at its left is the well for the first dimension. (B) The third dimension of one of the gel slices (* in panel A) is depicted. The third dimension is run in an alkaline buffer to resolve the single-stranded DNA by size. After hybridization, four spots are expected: fork parental and nascent strands and bubble parental and nascent strands. See the text for more detail. (C) The remnants of the neutral/neutral 2D gel (after six gel slices were cut out for the third dimension) were hybridized with the entire 5.5-kb EcoRI restriction fragment from puff II/9A. Arrows and numbers (in kilobases) indicate the positions and sizes of the gel slices according to the first-dimension markers. The signal in the 9-kb slice on the fork arc, which is seen in a lighter exposure of Fig. 1C as a gap (data not shown), is due to overexposure of the neighboring signals of the fork arc. (D) The third dimension of the 9-kb slice hybridized with the 5.5-kb EcoRI fragment. Four spots are seen: two are 5.5 kb and derived from parental strands of fork and bubble; the fork nascent strand is -2.8 kb and the bubble nascent strand is -3.0 kb, measured at the centers of the spots by comparison with a 1-kb ladder size marker. (Bottom) An EcoRI restriction map of the DNA puff II/9A locus. II/9-1 and II/9-2 are two transcription units at this puff. Arrows indicate the direction of transcription. The stippled bar (below) represents the origin region, and the oval shows the major initiation region.

been used to calculate the location of an origin of replication (5). However, migration in the first dimension is likely to be influenced by molecular shape; replication intermediates might not run at the same rate as linear molecules of the same mass even under low-percentage-gel and low-voltage

EcoRI-digested DNA was enriched for replication intermediates by BND-cellulose chromatography, and the enriched fraction was run out on a neutral/neutral 2D gel. Six vertical gel slices were then cut out, at 6-, 7-, 8-, 9-, 10.5-, and 12-kb positions according to the size markers in the first dimension. Each gel slice was turned 90° to run in a gel of the third dimension in an alkaline buffer. These 3D gels as well as the remnants of the 2D gel were blotted onto filters and were first hybridized with the entire 5.5-kb EcoRI fragment as probe. Figure 1C shows the autoradiograph of the residual 2D gel. A fork-plus-bubble pattern interrupted by six vertical lanes where the gel slices were taken is observed as expected. The absence of a crossover arc is due to BNDcellulose chromatography selecting against homologous recombination intermediates since they lack sufficient singlestrandedness. The 6-kb slice is very close to the linear fragment (5.5 kb), and the 12-kb slice is just out of the area of forks and bubbles derived from the 5.5-kb EcoRI fragment. The 3D gels of these two slices did not yield useful information (data not shown) and will no longer be considered in this report. Figure 1D shows the 3D gel of the 9-kb slice. Four spots are seen as expected: two are 5.5 kb and derive from the parental strands of forks and bubbles; the other two are 2.8 and 3.0 kb (measured at the center of the signal spots) and derive from the nascent strands of the forks and bubbles, respectively. A fork with 2.8-kb nascent strands derived from a 5.5-kb fragment is about 50% replicated. Figure 1C shows that the 9-kb gel slice was cut out right through the apex of the fork arc. This agrees with the neutral/neutral 2D gel assignment that 50% replicated forks are at the peak of the fork arc (5). However, the forks and bubbles in the 9-kb slice with total sizes of 8.3 kb (5.5 kb parental plus 2.8 kb nascent) and 8.5 kb (5.5 kb parental plus 3.0 kb nascent), respectively, run at a 9-kb position in the first dimension under our conditions. This result and those from similar size measurements in the 3D gels of the 7-, 8-, and 10.5-kb slices (Table 1) show that replication intermediates run more slowly than linear mole-

1523


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% Replication FIG. 2. Molecular shapes affect DNA migration in the first dimension. Different symbols indicate data taken from different gel slices (see Table 1). Shape influence (percent) is defined as the difference between the length of linear DNA and replication intermediates migrating to the same position in the first dimension divided by the length of the linear DNA. For example, for the 7-kb slice, shape influence = (7.0 kb - 6.7 kb)/7.0 kb x 100% = 4.3%. The extent of replication (percent) is the size of the nascent strand divided by the size of the parental strand. The 0 and 100% replication points are hypothetical. Shape influence for forks increases with the extent of replication up to 50% replication and decreases afterwards, while the increase continues for bubbles throughout the entire extent of replication.

cules of the same sizes in the first dimension of a 0.35% gel run at 0.5 V/cm (these conditions were chosen to reduce shape influence; see reference 22). The shape influence would probably be even greater under the original conditions as first published (0.4%, 1 V/cm) for the first dimension of neutral/neutral 2D gels (5). How much does molecular shape influence DNA migration in the first dimension? We define shape influence (percent) as the difference between the length of linear DNA and replication intermediates migrating to the same position in the first dimension divided by the length of the linear DNA (Table 1). A plot of shape influence against the extent of replication (Fig. 2) shows that shape influence for forks increases with the extent of replication up to 50% replication and decreases afterwards, while the increase continues for bubbles throughout the entire extent of replication. Therefore, shape influence for replication intermediates in the first dimension is similar to, although less pronounced than, that in the second dimension. 3D gels confirm the location of the major initiation region mapped by 2D gels. 3D gels can be used to localize replication initiation sites by using short probes to detect bubble nascent strands in 3D gels. A slice taken from the 2D gel for the third dimension is like a snapshot of a moment in time of an expanding replication bubble. If a given probe fails to detect bubble nascent strands, this probe must be away from the closest initiation site of bidirectional replication by a distance at least one-half the size of the replication bubble. For example, if a bubble has expanded 2 kb at an equal rate for both directions, the nascent strands would extend out 1 kb on either side of the origin. A probe within 1 kb of the origin would detect the nascent strands in the 3D gel, whereas a probe further than 1 kb from the initiation site would not. By use of 2D gels, we have previously identified within the

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FIG. 3. The 3D gel of the 7-kb slice agrees with the location of the major initiation region mapped by 2D gels. The same blot of a 3D gel of the 7-kb slice was sequentially hybridized with the entire 5.5-kb EcoRI fragment (full length) and six shorter probes (A to F; open boxes) from the 5.5-kb EcoRI fragment (the two long vertical lines in the bottom part of Fig. 3 represent the EcoRI sites). The differences in the shapes of the signal spots in 3D gels of the 7-kb slice shown here and the 9-kb slice shown in Fig. 1D and 4 are due to different curvatures of the fork and bubble arcs at different positions of the 2D gel from which the slices for the third dimension were taken. Interpretive diagrams of the hybridization signals are shown below the corresponding actual data. The sizes of the nascent strands of bubbles and forks are -1.4 and -1.2 kb, respectively, measured at the centers of the signal spots on the 3D gel. The positions of the major initiation region (stippled oval), of gene II/9-1, a bubble initiated from the major initiation region, and of forks derived from minor initiation near the ends of the fragment are depicted in the lower part of the figure. The intensities of bubble nascent strand signals detected by probes A to F are consistent with the location of the major initiation region mapped previously by 2D gels (23).

5.5-kb EcoRI fragment a -1-kb major initiation region located about 2 to 3 kb upstream of Sciara DNA puff gene 11/9-1 (reference 23, and see the map in Fig. 3 of this report). As will now be described, 3D gels confirm the location of the -1-kb major initiation region. Hybridization of a 3D gel of the 7-kb slice with the entire 5.5-kb EcoRI fragment as probe shows that it contains 1.4-kb bubble and 1.2-kb fork nascent strands (Fig. 3, leftmost panel, full-length probe). When the same blot is sequentially hybridized with shorter probes across the 5.5-kb EcoRI fragment, probes at or near the major initiation region should produce primarily bubble nascent strand signals. Probes distant from the major initiation region would detect only nascent strands from minor initiation bubbles found elsewhere in the 6-kb origin region, thus giving weak bubble nascent strand signals. Probes near the ends of the 5.5-kb EcoRI fragment should detect fork nascent strands as well. These forks are derived from minor initiation events elsewhere in the 6-kb origin region. Our previous 2D gel study showed that there are no forks entering the 6-kb origin region from outside; analysis of the direction of fork movement indicated that all forks appear to move outward from the 6-kb region in a bidirectional manner

1524

LIANG AND GERBI

(23). As shown in Fig. 3, probe C, which maps within the left half of the major initiation region, gave the strongest bubble nascent strand signal of any of the probes used from this 5.5-kb fragment. Probe D, which maps just next to the right border of the major initiation region, detected a bubble nascent strand signal slightly weaker than that given by probe C, as the 1.4-kb bubble nascent strands initiated at the major initiation region extend into the probe D area. Probes C and D detected almost no fork nascent strand signals, since the 1.2-kb nascent strands of forks are at the ends of the 5.5-kb fragment and are therefore not complementary to probe C or D. Probes A, B, E, and F, which are all away from the major initiation region, produced weak or no bubble nascent strand signals. Probes A, B, and F, which are all within 1.2 kb of the ends of the 5.5-kb EcoRI fragment, detected fork nascent strands as expected, and probe E, which is farther than 1.2 kb from the right end of the fragment, cannot hybridize to fork nascent strands. These results agree with the location of the major initiation region we previously mapped by neutral/alkaline 2D gels (23). Moreover, the differential fork and bubble hybridization patterns revealed by the various probes further validate the interpretations of fork and bubble arcs in neutral/neutral 2D gels. Probes A, B, and F near the ends of the fragment detected strong fork nascent strand signals even though forks are from minor initiations near the ends of the fragment, compared with relatively weak bubble nascent strand signals detected by probes C and D at or near the major initiation region. This could be explained if bubbles are recovered less well than forks in the DNA preparation. In support of this assumption, it has been reported that different DNA preparation procedures yield different ratios of bubbles to forks; moreover, phenol extraction of DNA results in poor or no recovery of bubbles (references 11 and 33 and our unpublished data). Despite this difficulty, which we also encountered in our previous neutral/alkaline 2D gels at the same locus, the -1-kb major initiation region was able to be mapped by restriction of DNA at or near the major initiation region (23). That analysis allowed comparison of nascent strands between forks from the two ends of the restriction fragment, thus overcoming the inability to compare bubble nascent strand signals with those of forks. In this report, bubble nascent strands are separated from fork nascent strands in 3D gels; therefore, the relative amounts of bubble nascent strands from different areas within the origin region can be compared with one another without interference from fork signals. The 3D gels of other gel slices were also sequentially hybridized with the same set of probes as that used for the 7-kb slice. Figure 4 shows the results for the 9-kb slice, which contains larger bubbles with 3.0-kb nascent strands. Excluding the full-length 5.5-kb probe (Fig. 4, left-most panel, which is the same as Fig. 1D), the strongest bubble nascent strand signals were produced by Probes C and D followed by probe E and then probe B, while probes A and F gave almost no bubble nascent strand signals. These data would place the bubble with 3-kb nascent strands in the position shown in Fig. 4. Probes B and E detected weaker bubble nascent strand signals than probes C and D because only parts of probes B and E can hybridize to the bubble nascent strands. The deduced center of the bubble is about 2.3 kb upstream of gene II/9-1. This agrees with the results shown in Fig. 3 for the 7-kb slice and also with results from the 8- and 10.5-kb slices (data not shown). Therefore, these

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FIG. 4. The 3D gel of the 9-kb slice also agrees with the location of the major initiation region mapped by 2D gels. The same blot of a 3D gel of the 9-kb slice was sequentially hybridized with the set of probes used for Fig. 3. Pa, parental strands; Na, nascent strands. The leftmost panel (hybridized with the 5.5-kb EcoRI fragment) is the same as that in Fig. 1D. The sizes of the nascent strands of bubbles and forks are -3.0 and -2.8 kb, respectively, measured at the centers of the signal spots on the 3D gel. The weak smear beneath the nascent fork spot in blots A and B probably resulted from random nicking; it is not significant and does not interfere with the analysis. The signal intensities of bubble nascent strands relative to bubble parental strands detected by probes A to F can be best explained by placing the bubble as shown. See the text for further details.

3D gel data confirm the location of the major initiation region we previously mapped by neutral/alkaline 2D gels. Only one initiation event seems to occur on a single DNA molecule in the origin region. It has been shown by 2D gels that there is a zone of multiple initiation sites of DNA replication in metazoans (7, 10, 11, 20, 23, 27, 32, 34) and in the yeast S. pombe (38). We have used 3D gels to investigate the possibility that multiple initiation events may occur simultaneously on a single DNA molecule in the origin region at the DNA puff locus. Multiple initiation events occurring on a single molecule would result in smaller bubble nascent strands than those of single bubbles of the same total mass, so that hybridization signals would appear below the nascent strand spot of single bubbles. A hypothetical molecule with two bubbles each containing 1.5 kb of nascent DNA is depicted in Fig. 5A. If this molecule comigrated in the 2D gel with single bubbles containing 3.0 kb of nascent DNA, then it would release 1.5-kb nascent strands in the bubble lane in the 3D gel of the 9-kb slice (see the diagram "Multiple Bubble Nascent" in Fig. 5D). If molecules with multiple bubbles did not comigrate with single bubbles of the same mass or did not migrate along the bubble arc in the second dimension, then the third dimension would detect a heterogeneous population of molecules with multiple bubbles seen as a smear of signal below the 3.0-kb bubble nascent strand spot or below a 3.0-kb size in the area between the bulk DNA and bubble lanes. However, our 3D gels do not support the existence of multiple bubbles, as there is no hybridization signal in the bubble lane below the bubble nascent strand spot in the 3D gel of the 9-kb slice or in the area between the bulk DNA and


VOL. 14, 1994

1525

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C FIG. 5. Only one initiation event seems to occur on a single DNA molecule in the origin region. For DNA molecules, solid lines represent the parental strands and dotted lines represent the nascent strands. Numbers indicate the sizes of nascent strands (in kilobases). (A) Diagrams of molecules with two (Multiple Bubbles) and one (Single Bubble) initiation event. (B) A diagram of two initiation events on the same molecule, giving rise to a double fork as bubbles expand into the restriction sites bounding the fragment. (C) A molecule with clustered microbubbles (double or single stranded) is depicted. (D) Predicted hybridization patterns (hatched ovals) for the nascent strands of molecules with multiple bubbles ("Multiple bubble nascent"), double forks derived from multiple initiation events on the same molecule ("Double fork nascent"), and DNA with microbubbles ("Microbubble nascent"). None of these was observed (Fig. 1D), suggesting that multiple bubbles and microbubbles are unlikely; only one initiation event seems to occur on any one DNA molecule.

bubble lanes (Fig. 1D). This is also true for the 3D gels of other slices (see Fig. 3 for the 7-kb slice; additional data not shown). Furthermore, if there were two or more initiation bubbles on the same DNA molecule, a double fork would be generated as bubbles expand and reach restriction sites (see the diagram in Fig. 5B). The double fork depicted in Fig. 5B has a total of 2.8 kb (1 kb + 1.8 kb) of nascent plus 5.5 kb of parental DNA and might comigrate (again, a nonessential assumption) in the 2D gel with the single forks in the 9-kb slice. Nascent strands released from this double fork would appear in the fork lane below the 2.8-kb single fork nascent strand spot in 3D gels (see the diagram "Double Fork Nascent" in Fig. 5D; a smear would be expected for a collection of double forks of various sizes). This was not seen: there is no significant hybridization signal below the 2.8-kb fork nascent strand spot in the 3D gel of the 9-kb slice or below 2.8 kb in the area between the bulk DNA and bubble lanes (Fig. 1D). Similarly, no double-fork nascent strands could be detected in 3D gels of other slices (see Fig. 3 for the 7 kb slice; additional data not shown). Taken together, these results suggest that only one initiation event occurs on any one DNA molecule in the origin region of the DNA puff. Our 3D gels also suggest that molecules with many unligated nascent strands (predicted by the strand separation hypothesis) do not seem to occur in DNA puff amplification. Bubbles with unligated nascent strands would release nascent strands in 3D gels that are smaller than those from single bubbles to give a smear in the bubble lane or in the area between the bulk DNA and bubble lanes (the same prediction as for multiple bubbles), yet this was not seen, as discussed above.

It is unknown where microbubbles would migrate on a neutral/neutral 2D gel. Under appropriate conditions, including those in our studies, even very small bubbles that form the beginning portion of a bubble arc are separated from small forks as well as from linear fragments. For example, Fig. 1 in reference 23 shows bubble arcs arising from the 1 x spots of the linear fragments. In this report, bubbles with -1.4 kb of nascent DNA are well separated from forks (Fig. 3); a longer exposure of Fig. 1C indicates that the bubble arc also arises from the spot of the linear 5.5-kb fragment (data not shown). Microbubbles are observed by electron microscopy to be in the size range of about 200 to 500 bp (1, 4, 19, 28). A series of these microbubbles should endow DNA molecules with behavior in 2D gels of nonlinear molecules. For example, if molecules with microbubbles were found along the bubble arc, the DNA resolved into parental and nascent strands in the third dimension would release nascent strands from microbubbles that would migrate to a 0.2- to 0.5-kb area in the bubble lane below the nascent strand spot of single bubbles (see the diagrams in Fig. 5C and D). If instead molecules with microbubbles migrated in 2D gels along the fork arc or along the diagonal of linear bulk DNA, or anywhere between linear DNA and the bubble arc, then nascent strands released from microbubbles would migrate in the third dimension to a 0.2- to 0.5-kb area anywhere between the bulk DNA and bubble lanes. In fact, no signal of 0.2 to 0.5 kb was found between the bulk DNA and bubble lanes (Fig. 1D and 3; additional data not shown). A control experiment was carried out to show that small nascent strands from microbubbles could indeed have been detected if they occurred. A dilution series of a 1-kb DNA ladder (a size marker from Gibco-BRL) with fragments of sizes at every kilobase value from 1 to 12 kb plus fragments

1526

MOL. CELL. BIOL.

LIANG AND GERBI

(The 7 kb slice) I

-

..........

.~~~~........ . ;.*....

ForkkT

Bubble breaks

5.5 kb

.2kb

-'C

o

-

1.2kb

*

II/9-1

Probe C Probe D

I

I

3Major

-4

-2

initiation region

A

Bubble -*-Parental

-1

0

1.2

.: 'z .-

kbdf 4V1'4 kb

ubble "Broken bubble nascent Bnascent"

(The 9 kb slice) --,---

-

2.8kb

JBulk

= Parental

|Bubble breaks mik

~2.8 kb

c

FokBule

05.5 kb

-

Nascent -

2.8 kb

3-0 kb

.-_ -~"Broken,~1.7 kcb

-::2 - -

28kb 2

bubble -l parental" 81.0 kb 17k

c FIG. 6. The fork arc in the fork-plus-bubble pattern in neutral/neutral 2D gels does not result from broken bubbles. (A) A diagram of a bubble with 1.2-kb nascent strands. If this bubble breaks at one of the single-stranded gaps (arrowheads), it would give rise to fork-like structures with 1.2-kb nascent strands. If the fork arc in the 2D gel came from these fork-like molecules, their nascent strands would be expected to migrate in the fork lane of the 3D gel of the 7-kb slice as depicted in panel B ("Broken bubble nascent") and would be complementary to probes C and D of Fig. 3. This was not observed, as both probes detected nascent strands only in the bubble lane (Fig. 3C and D), indicating that the 1.2-kb nascent strand spot in the fork lane did not contain broken bubble nascent strands. (C) A diagram of a bubble with 2.8-kb nascent strands which, if it breaks at one of the single-stranded gaps (arrowheads), would give rise to fork-like structures. Each fork-like molecule would be resolved into five single-stranded pieces of four different sizes in the third dimension: two nascent strands (both are 2.8 kb), one intact parental strand (5.5 kb), and two pieces from the broken bubble (1.0 and 1.7 kb). The two pieces from the broken bubble parental strand would be expected to migrate in the fork lane in the 3D gel of the 9-kb slice as diagrammed in panel D ("Broken bubble parental"). This was not observed (Fig. 1D), suggesting that the 9-kb slice did not contain broken bubbles.

of 1.6 and 0.5 kb and several fragments from 200 to 450 bp alkaline gel under the same conditions as was the third dimension. The blot was hybridized with the same DNA marker as probe. The 500-bp fragment and larger fragments showed up as discrete bands; fragments between 200 and 450 bp are detected as diffuse bands in a 1.2% alkaline gel (data not shown). By comparing signal intensities in the control and 3D gel experiments, we conclude that any significant amount of nascent strands from microbubbles could have been detected had it been present. However, as stated above, no signal of 0.2 to 0.5 kb was found for amplifying DNA from Sciara DNA puff II/9A. These results suggest that microbubbles are probably not used in Sciara DNA puff amplification unless they are mostly single stranded, i.e., without nascent strands. The fork arc in the fork-plus-bubble pattern in neutral/ neutral 2D gels does not result from broken bubbles. When yeast 2,um plasmid was analyzed by neutral/neutral 2D gels, a fork arc or a bubble arc is observed in a given restriction fragment, indicating a single fixed origin of replication in the plasmid (5). However, when genomic DNA from metazoans

was run on an

(7, 10, 11, 20, 23, 27, 34) and in some cases from the yeast S. cerevisiae (6, 14, 17, 25) or S. pombe (37) is analyzed, both fork and bubble arcs are observed in the same fragment. One explanation for the presence of the fork arc is that bubbles are unstable and break into fork-like molecules during handling (14, 25, 37). We have tested this possibility directly with 3D gels. If a bubble breaks, the break would likely occur at one of the two single-stranded gaps found in trans (indicated by the arrowheads in Fig. 6A and C). As discussed in the previous section on the 3D gel of the 7-kb slice (Fig. 3), probes C and D detected bubble nascent strands but not fork nascent strands. The latter are detected only at the ends of the fragnent not covered by either probe C or D. However, if the fork arc in the 2D gel contained fork-like (branched) molecules from broken bubbles (see the diagram in Fig. 6A), nascent strands from the broken bubbles should be detected by probes C and D in the fork lane in the 3D gel (see the diagram "Broken Bubble Nascent" in Fig. 6B). This is not the case: probes C and D detected nascent strands in the

VOL. 14, 1994


bubble lane but not significantly in the fork lane (Fig. 3C and D). Similar analysis was also done for the 3D gel of the 8-kb slice (not shown), which contains 2.0- and 2.2-kb nascent strands from forks and bubbles, respectively (see Table 1). Probe D in Fig. 3 is 2.2 and 2.6 kb away from the right and left ends, respectively, of the 5.5-kb EcoRI fragment. Therefore, the 2.0-kb fork nascent strands in the 8-kb slice are not complementary to probe D and will not hybridize to it. Again, nascent strands were detected only in the bubble lane and not in the fork lane in the 3D gel (data not shown). This analysis cannot be done for the 9-kb (Fig. 4) or the 10.5-kb (not shown) slice because the real fork nascent strands are longer than one-half of the 5.5-kb fragment and will be hybridized by any probe, thereby masking any possible nascent strand signal from broken bubbles in the fork lane. The data presented above show that forks contained in the 7- or the 8-kb slices were bona fide replication forks probably from minor initiation events elsewhere in the 6-kb origin region and do not seem to come from broken bubbles. This is also supported by the following analysis for the 9-kb slice. A broken bubble would release single-stranded DNA of four different sizes in the alkaline third dimension: one intact parental strand, two nascent strands of the same size, and two different pieces from the broken parental strand (Fig. 6C). The sizes of the two broken pieces from the parental strand depend on the size of the bubble and the location of the initiation site. For example, the bubble depicted in Fig. 6C has replicated 2.8 kb of the 5.5-kb EcoRI fragment leaving a total of 2.7 kb of unreplicated DNA at the ends. Then, the parental strand broken pieces would be 1 and 1.7 kb and would migrate to an area in the fork lane below the 2.8-kb fork nascent strand spot in the 3D gel (see the diagram "Broken Bubble Parental" in Fig. 6D). As shown in Fig. 1D, there is no significant hybridization signal in the fork lane below the fork nascent strand spot in the 3D gel of the 9-kb slice, suggesting that the 9-kb slice contained bubbles and forks but no significant amount of broken bubbles. Taken together, these results suggest that the fork arc in the 5.5-kb EcoRI fragment is not a result of bubbles breaking into forks. DISCUSSION There has been a wave of efforts to identify and characterize eukaryotic DNA replication origins since the development of 2D gel replicon mapping techniques. 2D gels showed that ARS elements are indeed replication origins in the yeast plasmid (5, 21) and in some chromosomal locations of 2pm the yeast S. cerevisiae (8, 14, 24, 29, 38). 2D gels have also identified replication origins in Epstein-Barr virus (15), bovine papillomavirus (31, 36), and P. polycephalum (2). However, in metazoans multiple initiation sites have been found by 2D gels in a region (7, 10, 11, 20, 23, 27, 32, 34) of up to 55 kb (10) rather than in fixed locations. These observations raise questions about the mechanism of replication initiation in metazoans. Multiple initiation sites were also found in a region of 3 to 4 kb in the yeast S. pombe (37). We have investigated the origin region in Sciara DNA puff II/9A in more detail with 3D gels to gain further information than is possible with just 2D gels. Our 3D gel method can be used to determine the size of forks and bubbles by measuring their nascent strands and to localize replication origins by using short probes to detect bubble nascent strands which are separated from bubble parental strands as well as from fork parental and nascent strands. The data presented in this report confirm the location of the -1-kb major initiation

1527

region previously mapped by neutral/alkaline 2D gels and further validate fork and bubble patterns in neutral/neutral 2D gels. We have also used 3D gels to test the possibility of multiple initiation events occurring on a single DNA molecule in the 6-kb origin region in the DNA puff locus. Our data show that this is unlikely in Sciara DNA puff amplification, as no nascent strands smaller than those expected for single forks and single bubbles were observed in 3D gels. This seems to indicate that initiation at one site prevents initiation at neighboring sites on the same molecule. Our 3D gel analysis does not support the strand separation model for Sciara DNA puff amplification. When forks and bubbles are resolved as single strands in the alkaline third dimension, nascent strands of about 200 to 500 bases expected from microbubbles are not observed below the nascent strand spots of full-length bubbles or elsewhere in 3D gels. Furthermore, molecules without microbubbles but with many unligated nascent strands would release nascent strands in 3D gels smaller than those from single bubbles, yet this is not seen. It is possible that microbubbles and molecules with many unligated nascent strands are very unstable and easily lost in DNA preparation, or they may be transient in nature and fuse into large bubbles so rapidly that they are not represented in the sample. Notice that Okazaki fragments (ca. 200 bp) are not detected by the 3D gels either, which is attributable to rapid ligation of Okazaki fragments during DNA synthesis. However, electron microscopy studies reported that microbubbles as well as large singlestranded regions are more abundant than replication forks and bubbles. Therefore, our data suggest that it is unlikely that initiation of DNA amplification in Sciara DNA puff II/9A is via a strand separation mechanism. Similarly, electron microscopy of replicating Chinese hamster ovary DNA with the amplified dihydrofolate reductase gene failed to detect microbubbles (18). It should be pointed out that the neutral/alkaline 2D gel method (21) can be used in principle to test if molecules contain microbubbles and/or unligated nascent strands, since the second dimension of an alkaline gel would release nascent strands smaller than those predicted for single forks and bubbles. However, because forks and bubbles are not separated from the bulk DNA in the first dimension, background from nicked, nonreplicating DNA would render analysis difficult if not impossible. Neutral/neutral 2D gel studies have raised another question about replication origins when both a complete fork and a bubble arc are seen in the same restriction fragment of genomic DNA. Different interpretations have been given to the fork arc in different cases on the basis of additional available information. For example, rRNA genes in the yeast S. cerevisiae show a very strong fork arc in addition to a very weak bubble arc (6, 13, 24). The intrepretation of this fact has been that most of the rRNA genes are replicated by passing forks. This is supported by direction of fork movement analysis showing that most rDNA units are replicated by unidirectional forks that traverse the repeat units from one end to the other (24). This also agrees with an electron microscopy study (30). Several other ARS-containing chromosomal fragments also show fork-plus-bubble patterns in which the bubble arc signals are either stronger (ARS 307 and ARS 309 [17]), weaker (ARS 1 [14]), or of about the same intensity (ARS 305 [25] and ARS 501 [14]) as the fork arcs in the portions where the two arcs overlap. In all of these cases, analysis of the direction of fork movement in the flanking fragments show

1528

LIANG AND GERBI

that replication forks move away from the ARS-containing fragments, indicating that the ARS-containing fragments are not replicated by forks emanating from replication origins located outside the ARS-containing fragments. Therefore, in these cases the fork arcs are thought to come from bubbles which broke from mechanical shear and/or from nucleases. However, our present 3D gel study shows that the fork arc in the origin region at the Sciara DNA puff amplification locus does not result primarily from broken bubbles. Another possible explanation for the fork-plus-bubble pattern is that there is population polymorphism for the initiation site within the fragment being analyzed. Some molecules initiate replication at the central area of the fragment, thus giving rise to a bubble arc, while bubbles initiated on other molecules near either end of the fragment will expand into the restriction site and become forks which then traverse the fragment, thus resulting in an almost complete fork arc. In Sciara DNA puff II/9A, analysis of the direction of fork movement shows that replication forks move away from the origin region in a bidirectional manner, indicating that there are no forks entering the -6-kb origin region from replication origins that are outside the origin region (23). We have now shown by 3D gels that the fork arc in the 5.5-kb EcoRI fragment does not result from broken bubbles. Therefore, the presence of a complete fork arc in addition to a bubble arc in the 5.5-kb EcoRI fragment can be best explained as different DNA molecules initiating replication at different sites that are inside the origin region. In other words, a given molecule only has a single initiation event, but initiation sites can vary between different molecules. The majority of initiations occur within the -1-kb region (perhaps even at a single site) that we previously mapped by 2D gels (23) and confirmed here by 3D gels. Occasional initiations can occur elsewhere in the 6-kb origin region, giving rise to population polymorphism observed as the fork-plus-bubble pattern in 2D gels. ACKNOWLEDGMENTS We are grateful to Heidi S. Smith for maintenance of the flies and assistance with larval dissections. We appreciate helpful discussions with Joel Huberman and Arthur Landy. This work was supported by a grant from the National Institutes of Health (GM 35929) to S. A. Gerbi. REFERENCES 1. Balgari, C. T., F. Amaldi, and M. Buongiorno-Nardelli. 1978. Electron microscopic analysis of replicating DNA of sea urchin embryos. Cell 15:1095-1107. 2. Benard, M., and G. Pierron. 1992. Mapping of a Physarum chromosomal origin of replication tightly linked to a developmentally-regulated profilin gene. Nucleic Acids Res. 20:33093315. 3. Benbow, R. M., M. F. Gaudette, P. J. Hines, and M. Shioda. 1985. Initiation of DNA replication in eukaryotes, p. 449-483. In A. L. Boynton and H. L. Leffert (ed.), Control of animal cell proliferation, vol. I. Academic Press, Inc., New York. 4. Bozzoni, I., C. T. Baldari, F. Amaldi, and M. BuongiornoNardelli. 1981. Replication of ribosomal DNA in Xenopus laevis. Eur. J. Biochem. 118:585-590. 5. Brewer, B. J., and W. L. Fangman. 1987. The localization of replication origins on ARS plasmids in S. cerevisiae. Cell 51:463-471. 6. Brewer, B. J., and W. L. Fangman. 1991. Mapping replication origins in yeast chromosomes. Bioessays 13:317-322. 7. Delidakis, C., and F. C. Kafatos. 1989. Amplification enhancers and replication origins in the autosomal chorion gene cluster of Drosophila melanogaster. EMBO J. 8:891-901. 8. Deshpande, A. M., and C. S. Newlon. 1992. The ARS consensus

MOL. CELL. BIOL. sequence is required for chromosomal origin function in Saccharomyces cerevisiae. Mol. Cell. Biol. 12:4305-4313. 9. DiBartolomeis, S. M., and S. A. Gerbi. 1989. Molecular characterization of DNA puff II/9A genes in Sciara coprophila. J. Mol. Biol. 210:531-543. 10. Dikwel, P. A., and J. L. Hamlin. 1992. Initiation of DNA replication in the dihydrofolate reductase locus is confined to the early S period in CHO cells synchronized with the plant amino acid mimosine. Mol. Cell. Biol. 12:3715-3722. 11. Dijkwel, P. A., J. P. Vaughn, and J. L. Hamlin. 1991. Mapping of replication initiation sites in mammalian genomes by twodimensional gel analysis: stabilization and enrichment of replication intermediates by isolation on the nuclear matrix. Mol. Cell. Biol. 11:3850-3859. 12. Dubey, D. D., L. R. Davis, S. A. Greenfeder, L. Y. Ong, J. Zhu, J. R. Broach, C. S. Newton, and J. A. Huberman. 1991. Evidence suggesting that the ARS elements associated with silencers of the yeast mating-type locus HML do not function as chromosomal DNA replication origins. Mol. Cell. Biol. 11: 5346-5355. 13. Fangman, W. L., and B. J. Brewer. 1991. Activation of replication origin within yeast chromosomes. Annu. Rev. Cell Biol. 7:375-402. 14. Ferguson, B. M., B. J. Brewer, A. E. Reynolds, and W. L. Fangman. 1991. A yeast origin of replication is activated late in S phase. Cell 65:507-515. 15. Gahn, T. A., and C. L. Schildkraut. 1989. The Epstein-Barr virus origin of plasmid replication, oriP, contains both the initiation and termination sites of DNA replication. Cell 58:527535. 16. Gaudette, M. F., and R. M. Benbow. 1986. Replication forks are underrepresented in chromosomal DNA of Xenopus laevis embryos. Proc. Natl. Acad. Sci. USA 83:5953-5957. 17. Greenfeder, S. A., and C. S. Newlon. 1992. A replication map of a 61-kb circular derivative of Saccharomyces cerevisiae chromosome III. Mol. Biol. Cell 3:999-1013. 18. Hamlin, J. L., P. A. Dikwel, and J. P. Vaughn. 1992. Initiation of replication in the Chinese hamster dihydrofolate reductase domain. Chromosoma 102(Suppl.):s17-s23. 19. Hardman, N., and D. A. F. Gillespie. 1980. DNA replication in Physarum polycephalum. Eur. J. Biochem. 106:161-167. 20. Heck, M. M. S., and A. C. Spradling. 1990. Multiple replication origins are used during Drosophila chorion gene amplification. J. Cell Biol. 110:903-914. 21. Huberman, J. A., L. D. Spotila, K. A. Nawotka, S. M. ElAssouli, and R. D. Leslie. 1987. The in vivo replication origin of the yeast 2,um plasmid. Cell 51:473-481. 22. Krysan, P. J., and M. P. Calos. 1991. Replication initiates at multiple locations on an autonomously replicating plasmid in human cells. Mol. Cell. Biol. 11:1464-1472. 23. Liang, C., J. D. Spitzer, H. S. Smith, and S. A. Gerbi. 1993. Replication initiates at a confined region during DNA amplification in Sciara DNA puff II/9A. Genes Dev. 7:1072-1084. 24. Linskens, M. H. K., and J. A. Huberman. 1988. Organization of replication of ribosomal DNA in Saccharomyces cerevisiae. Mol. Cell. Biol. 8:4927-4935. 25. Linskens, M. H. K., and J. A. Huberman. 1990. Ambiguities in results obtained with 2D gel replicon mapping techniques. Nucleic Acids Res. 18:647-652. 26. Linskens, M. H. K., and J. A. Huberman. 1990. The two faces of higher eukaryotic DNA replication origins. Cell 62:845-847. 27. Little, R. D., T. H. K. Platt, and C. L. Schildkraut. 1993. Initiation and termination of DNA replication in human rRNA genes. Mol. Cell. Biol. 13:6600-6613. 28. Micheli, G., C. T. Baldari, M. T. Cam, G. D. Cello, and M. Buongiorno-Nardelli. 1982. An electron microscope study of chromosomal DNA replication in different eukaryotic systems. Exp. Cell Res. 137:127-140. 29. Rivier, D. H., and J. Rine. 1992. An origin of DNA replication and a transcription silencer require a common element. Science 256:659-663. 30. Saffer, L. D., and 0. L. J. Miller. 1986. Electron microscopic study of DNA replication in rDNA of Saccharomyces cerevi-

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siae. Mol. Cell. Biol. 6:1148-1157. 31. Schvartzman, J. B., S. Adolph, L. Martin-Parras, and C. L. Schildkraut. 1990. Evidence that replication initiates at only some of the potential origins in each oligomeric form of bovine papillomavirus type 1 DNA. Mol. Cell. Biol. 10:3078-3086. 32. Shinomiya, T., and I. Sawako. 1991. Analysis of chromosomal replicons in early embryos of Drosophila melanogaster by two-dimensional gel electrophoresis. Nucleic Acids Res. 19: 3935-3941. 33. Umek, R. M., H. K. LiAskens, D. Kowalski, and J. A. Huberman. 1989. New beginning in studies of eukaryotic DNA replication origins. Biochim. Biophys. Acta 1007:1-14. 34. Vaughn, J. P., P. A. DUkwel, and J. L. Hamlin. 1990. Replication initiates in a broad zone in the amplified CHO dihydrofolate reductase domain. Cell 61:1075-1087.

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35. Wu, N., C. Liang, S. M. DiBartolomeis, H. S. Smith, and S. A. Gerbi. 1993. Developmental progression of DNA puffs in Sciara coprophila: amplification and transcription. Dev. Biol. 160:7384. 36. Yang, L., and M. Botchan. 1990. Replication of bovine papillomavirus type 1 DNA initiates within an E2-responsive enhancer element. J. Virol. 64:5903-5911. 37. Zhu, J., C. Brun, H. Kurooka, M. Yanagida, and J. A. Huberman. 1992. Identification and characterization of a complex chromosomal replication origin in Schizosaccharomyces pombe. Chromosoma 102(Suppl.):s7-s16. 38. Zhu, J., C. S. Newlon, and J. A. Huberman. 1992. Localization of a DNA replication origin and termination zone on chromosome III of Saccharomyces cerevisiae. Mol. Cell. Biol. 12:47334741.