An Initiation Zone of Chromosomal DNA Replication at the Chicken Lysozyme Gene Locus*

The chicken lysozyme gene domain is distinguished by a broad knowledge of how its expression is regulated. Here, we examined the in vivo replication of the lysozyme gene locus using polymerase chain reaction amplification and competitive polymerase chain reaction of size-fractionated, nascent DNA strands. We found that DNA replication initiates at multiple sites within a broad initiation zone spanning at least 20 kilobases, which includes most of the lysozyme gene domain. The 5′ border of this zone is probably located downstream of the lysozyme 5′ nuclear matrix attachment region. Preferred initiation occurs in a 3′-located subzone. The initiation zone at the lysozyme gene locus is also active in nonexpressing liver DU249 cells. Furthermore, examining the timing of DNA replication at the lysozyme gene locus revealed that the gene locus replicates early during S phase in both HD11 and DU249 cells, irrespective of its transcriptional activity.


The chicken lysozyme gene domain is distinguished by a broad knowledge of how its expression is regulated.
Here, we examined the in vivo replication of the lysozyme gene locus using polymerase chain reaction amplification and competitive polymerase chain reaction of size-fractionated, nascent DNA strands. We found that DNA replication initiates at multiple sites within a broad initiation zone spanning at least 20 kilobases, which includes most of the lysozyme gene domain. The 5 border of this zone is probably located downstream of the lysozyme 5 nuclear matrix attachment region. Preferred initiation occurs in a 3-located subzone. The initiation zone at the lysozyme gene locus is also active in nonexpressing liver DU249 cells. Furthermore, examining the timing of DNA replication at the lysozyme gene locus revealed that the gene locus replicates early during S phase in both HD11 and DU249 cells, irrespective of its transcriptional activity.
In prokaryotic cells, DNA replicates from a unique, genetically defined DNA sequence, called origin of replication (or ori) (1). By contrast, DNA in higher eukaryotic cells replicates as multiple, independent replication units (replicons) (2). In every replicon, the initiation of replication occurs at an origin, and the nascent DNA is elongated unidirectionally or bidirectionally (3). It has been reported that actively transcribed genes are often replicated in early S phase, whereas repressed genes and highly repetitive genomic sequences are replicated during late S phase (4 -6).
Identification of origins of DNA replication is essentially required for an understanding of the DNA replication process in eukaryotic cells. In comparison with prokaryotic cells, identification of replication origins in higher eukaryotic cells, particularly at unique genomic sequences, has encountered great difficulties because of the high complexity of their genome (7). It was hampered so far by the lack of sensitive techniques for mapping or functional analysis of putative origins. The twodimensional gel electrophoresis technique described first by Brewer and Fangman (8) has been used to map origins of DNA replication in genomes with low complexity and to localize origins in amplified genomic sequences. Recently, extremely sensitive methods for mapping origins of DNA replication of single copy genes have been developed. These techniques were used successfully to identify origins of DNA replication, which reside approximately 17 kb 1 downstream from the 3Ј-end of the Chinese hamster dihydrofolate reductase (DHFR) gene (9 -12); reside 1.5 kb upstream of exon 1 of the human c-myc gene (13); are located upstream of the human ␤-globin gene (14); comap with the transcriptional enhancer of the heavy chain immunoglobulin gene (15); or are embedded within the transcriptional unit of the CAD (carbamoyl-phosphate synthetase, aspartate carbamoyltransferase, and dihydroorotase) gene (16). Origins of DNA replication are not always restricted to specific sequences. It has been reported that DNA replication can initiate from a broad initiation zone (3,6,17). For example, DNA replication initiates in a zone of Ͼ4 kb near the Schizosaccharomyces pombe ura4 gene (18), in the nontranscribed spacer (31 kb) of the human ribosomal DNA (19), in a ϳ6-kb region at the amplified Drosophila chorion genes (20), in a 10-kb region downstream of the Drosophila DNA polymerase ␣ gene (21), and at multiple sites in the histone gene repeating unit of Drosophila melanogaster (22,23). However, using methods with higher resolution, often small highly preferred OBRs were detected in such initiation zones. For example, two-dimensional gel analyses combined with a genetic analysis revealed that initiation events were concentrated at three autonomous replication sequence elements near the S. pombe ura4 gene (24). Similarly, in the case of human rRNA gene repeats, results obtained by the nascent-strand abundance analysis indicated that replication initiates at high frequency a few kb upstream of the transcribed region, whereas most low frequency initiation sites were distributed throughout the ribosomal DNA repeat unit (25).
The chicken lysozyme gene is embedded into a 21-24-kb chromatin domain displaying an elevated nuclease sensitivity (26,27). The 5Ј and 3Ј borders of this domain coincide with nuclear matrix attachment regions (MARs) (28). All known sequence elements involved in developmentally specific and cell-specific regulation of lysozyme gene expression reside within the domain. In this study, we describe the identification of an initiation zone of DNA replication at the chicken lysozyme gene locus. DNA replication starts at multiple sites within a broad initiation zone covering at least 20 kb. Our results strongly suggested that preferred initiation occurs in a 3Јlocated subzone. Furthermore, the initiation zone is functional not only in chicken lysozyme-expressing myelomonocytic HD11 cells but also in hepatic DU249 cells. The timing of DNA replication at the lysozyme gene locus seems also to be independent of the transcriptional activity of the lysozyme gene.

MATERIALS AND METHODS
Cell Culture and Isolation of Nascent DNA-Myelomonocytic HD11 cells (29) and hepatic DU249 cells (30) were grown in Iscove's modified Dulbecco's medium, supplemented with 8% fetal calf serum, 2% chicken serum, 100 units/ml penicillin, and 100 g/ml streptomycin at 37°C and 5% CO 2 . For preparation of 5-bromodeoxyuridine (BrdUrd)-labeled DNA, 10 8 exponentially proliferating cells were labeled with 20 M BrdUrd for 15 min. After labeling, all subsequent steps were performed under minimal light to protect nascent BrdUrd-labeled DNA strands against damage.
The preparation of BrdUrd-labeled nascent DNA was performed according to the method described by Vassilev and Johnson (13) with some modifications. Briefly, high molecular weight DNA was isolated by digestion with proteinase K, extraction with phenol-chloroform, and spooling from 70% ethanol. For size fractionation, spooled genomic DNA was denatured in 0.2 N NaOH and layered onto 5-15% (w/v) linear sucrose gradients. Gradients were centrifuged in a Beckman SW 40 rotor at 35,000 rpm at 15°C for 18 h and subsequently collected in 12 fractions. Only six size fractions were chosen for preparation of BrdUrdlabeled nascent DNA strands. They were purified by two cycles of immunoprecipitation using 30 l (in each immunoreaction) of an anti-BrdUrd monoclonal antibody (25 g/ml, Becton-Dickinson) and then dissolved in 20 l of TE buffer containing 10 mM Tris, pH 8.0, and 1 mM EDTA or in 400 l when used for competitive PCR. The size fractionation was monitored by alkaline agarose gel electrophoresis and hybridization to 32 P-labeled genomic DNA.
Cell Synchronization-Cells were synchronized according to the method described by Heintz and Hamlin (31). Briefly, HD11 and DU249 cells were cultured in 8.5-cm dishes to 80 -90% confluence and incubated in Iscove's modified Dulbecco's medium without isoleucin for 36 -48 h. The cells were subsequently arrested at the G 1 /S boundary by incubation in complete Iscove's modified Dulbecco's medium containing 20 g/ml aphidicolin (Sigma, Deisenofen, Germany) for at least 12 h.
For labeling with BrdUrd, G 1 /S boundary-arrested cells were washed three times with Iscove's modified Dulbecco's medium to remove aphidicolin and immediately released into S phase by incubation at 37°C for 1, 3, 5, or 8 h including a 45-min labeling with BrdUrd at the end of each time period. Genomic DNA was then isolated and sonicated to an average size of about 1000 bp. BrdUrd-labeled DNA from 5 g of sonicated genomic DNA was purified by two cycles of immunoprecipitation as described above and dissolved in 40 l of TE.
To amplify specific DNA fragments (lys A through lys F), 30 cycles of PCR were carried out in 50 l of standard buffer (Pharmacia) containing 1 M primers, four deoxyribonucleotides (200 M each), 2-2.5 units of Taq polymerase, and 2 l of purified BrdUrd-labeled DNA as template by a protocol consisting of 1 min of denaturation at 95°C, 2 min of annealing at 53°C, and 1 min of extension reaction at 72°C.
Slot Blotting and Hybridization-10 l of each PCR and 3 g of salmon sperm carrier DNA were NaOH-denatured and blotted onto nylon membranes (Appligene, Heidelberg, Germany) using a Hybri-Slot manifold (Life Technologies, Eggenstein, Germany). After baking for 30 min at 80°C, the nylon membranes were hybridized with oligonucleotides, which were 32 P-labeled with T4 polynucleotide kinase and [␥-32 P]ATP (3000 Ci/mmol) using the method described by Vassilev and Johnson (34), except that the hybridization temperature was 50°C. To quantify the hybridization signals, autoradiograms were scanned with an imaging densitometer from Bio-Rad (Munich, Germany).
Competitive PCR-Competitors for lys A through lys I (Fig. 3) were constructed by PCR using two external primers (see "Synthetic Oligonucleotides and PCR") and two internal primers as described by Diviacco et al. (35). Each internal primer contained a 20-nucleotide sequence unrelated to chicken genomic DNA, either 5Ј-ACCTGCAGGGAT-CCGTCGAC-3Ј (tail 1) or 5Ј-GTCGACGGATCCCTGCAGGT-3Ј (tail 2) as follows: lys A, 5Ј-tail 1-GTTGACTAGAGATTTCATCT-3Ј, 5Ј-tail 2-C-CTTCGTTGCAATGCAGTTT-3Ј; lys B, 5Ј-tail 1-GCCAAAGAGTCTGC- The resulting amplified competitors have the same sequence as lys A through lys I but an additional unrelated 20 nucleotides in the middle. They were isolated from agarose gels and quantified by coamplification with a known amount of chicken genomic DNA.
Competitive PCRs were performed in a 50-l reaction mixture under standard conditions with 1ϫ GeneAmp buffer, 1.25 units AmpliTaq Gold polymerase (Perkin-Elmer, Hamburg, Germany), four deoxyribonucleotides (200 M each), 1 M primers, 5 l of purified size-fractionated DNA together with increasing amounts of specific competitor as template by 43 cycles (1 min at 94°C and 1 min at 60°C, each) in a Perkin-Elmer thermal cycler. The amplified DNA fragments were fractionated on 5% polyacrylamide gels at 150 V for 2 h, stained with ethidium bromide, and quantified by using an ethidium bromide gel documentation system from Bio-Rad.
Southern Analysis of Size-fractionated BrdUrd-labeled DNA-Size determination of BrdUrd-labeled nascent DNA was performed by electrophoresis of 1 ⁄10 of the size-fractionated immunoprecipitated DNA in an alkaline 1% agarose gel containing 30 mM NaOH and 1 mM EDTA and transfer onto nylon membrane according to Southern (36). The nylon membrane was then baked for 30 min at 80°C and hybridized to 32 P-labeled, nick-translated genomic DNA as described by Church and Gilbert (37). After washing, the membrane was dried and exposed to x-ray film.
RNA Analysis-Poly(A) ϩ RNA was isolated from HD11 or DU249 cells by an oligo(dT)-cellulose adsorption method (38). Lysozyme gene expression was determined by an RNase protection assay (39) of poly(A) ϩ RNA using a 32 P-labeled lysozyme-specific antisense RNA probe that was prepared as follows. A 252-bp TaqI-KpnI fragment of the lysozyme cDNA containing part of exon 2, exon 3, and part of exon 4 (40) was cloned into pBluescript II SK ϩ (Stratagene, Heidelberg, Germany) (41). The recombinant plasmid was linearized with XbaI and used as template to synthesize 32 P-labeled lysozyme-specific antisense RNA in an in vitro transcription reaction with T7 RNA polymerase (Stratagene kit). The 32 P-labeled RNA probe was hybridized to 4 g of poly(A) ϩ RNA and then digested with RNase T1 and RNase A (39). Protected RNAs were separated by electrophoresis in a 5% polyacrylamide-8 M urea gel containing 1ϫ TBE (89 mM Tris, pH 8.0, 89 mM boric acid, 2 mM EDTA). After drying, the gel was exposed to x-ray film.
Immunoblotting of Purified Size-fractionated DNA-One-tenth of the purified nascent DNA was blotted onto a nitrocellulose membrane using a Hybri-Slot manifold (Life Technologies). After the membrane was baked under vacuum for 2 h at 80°C, incorporated BrdUrd was visualized by incubation with an anti-BrdUrd antibody and use of a phosphatase detection system (Proto Blot ® II AP system; Promega, Heidelberg, Germany).

RESULTS
Purification of BrdUrd-labeled, Size-fractionated Nascent DNA-In this study, we employed the nascent strand PCR assay developed by Vassilev and Johnson (13) to identify and localize origins of DNA replication at the single copy lysozyme gene locus in chicken myelomonocytes (HD11 cells). This cell line, transformed by the v-myc-encoding retrovirus MC29, constitutively expresses the gene at a low level (Refs. 29 and 41; see also Fig. 6). To isolate newly replicated DNA, exponentially growing HD11 cells were labeled with BrdUrd for 15 min. Nascent DNA was then size-fractionated by sedimentation in alkaline sucrose gradients and purified by two rounds of immunoprecipitation using an anti-BrdUrd antibody. The size of the purified nascent strands from six selected gradient fractions was determined by electrophoresis in alkaline agarose gels and Southern blot hybridization with labeled chicken genomic DNA followed by densitometric scanning (Fig. 1A). Average nascent strand size increased from 0.6 kb at the top to 10.5 kb near the bottom of the gradient (see also Table I). The level of BrdUrd incorporation in each size fraction was monitored by immunoblotting using an anti-BrdUrd antibody (Fig.  1B). Densitometric scanning of Fig. 1B revealed that this level, as expected from a continuous traveling of the replication forks, increased proportionally with the DNA length, suggesting an approximately equal number of nascent molecules in each size fraction (Fig. 1C). This furthermore indicated that the integrity of the nascent DNA strands had sustained the purification procedure.
An OBR Is Located 1.5-2.0 kb Upstream of the Chicken c-myc Gene-Originally, the nascent strand length assay was first applied to the human c-myc gene by localizing an initiation zone of DNA replication ϳ1.5 kb upstream of the first exon (13). To verify that we successfully established the assay, we adapted the method to the chicken c-myc gene. Size-fractionated, nascent DNA from HD11 cells was used to determine the abundance of two segments by PCR and specific hybridization, one located within the first intron (myc A) and the other located at the boundary between intron 2 and exon 3 (myc B) (see map in Fig. 2B) (33). We note that the amplified DNA segments are not present in the genome of the transforming retrovirus MC29 and that they do not contain Alu repeats or other types of repetitive sequences (42). As shown in Fig. 2A, size fraction 4 (3.8 kb) and the larger size fractions, 5 and 6, contained segment myc A in great abundance, while segment myc B was present in solely the largest size fraction, 6 (10.5 kb). Unfortunately, further mapping experiments were hampered by the lack of sequence information. Nevertheless, our results are consistent with the conclusion that an OBR centers between 1.5 and 2.0 kb upstream of the first exon. This compares well with the previous localization of the human c-myc gene OBR (see Fig. 2B) (13).
Initiation of DNA Replication at the Chicken Lysozyme Gene Locus-To analyze the replication pattern of the chicken lysozyme gene locus, we selected six DNA segments (lys A through lys F), whose abundance in the purified nascent DNA size fractions described above was determined by PCR amplification and hybridization (Fig. 3). By spanning ϳ48 kb, these segments cover the complete lysozyme gene domain (21-24 kb) and flanking regions (26 -28). Repetitive sequences are excluded from the selected segments and their immediate neighborhood, with the exception of segment lys E, which is located close to a moderately repeated sequence.
A prerequisite for the nascent strand PCR mapping assay is that the BrdUrd-labeled DNA purified by two rounds of immunoprecipitation is not contaminated with any trace of unlabeled broken genomic DNA. This was monitored by a control experiment, in which one or two rounds of immunoprecipitation were performed with genomic DNA isolated from cells that were not labeled with BrdUrd. After amplification of one of the selected segments, hybridization did not detect any amplified products when using two prior rounds of immunoprecipitation (Fig. 4A), indicating that the hybridization signals obtained with Brd-Urd-labeled DNA were specific to newly replicated DNA. Furthermore, we routinely performed three control experiments along with each segment-specific PCR amplification and hybridization. First, the segments were amplified from unfractionated genomic DNA. As shown in Fig. 4B, lane c, all probes efficiently hybridized to the respective amplified segments. Then assays, which were run without any DNA (Fig. 4B, lane c) or with yeast tRNA as pseudotemplate (Fig. 4B, lane c), did not yield any hybridizable products.
Following these controls, the nascent DNA size fractions described above were amplified at the selected segments, and the reaction products were then hybridized to specific probes. Autoradiograms were selected, which show approximately equal intensities of the hybridization signals with unfractionated genomic DNA (Fig. 4B, lane c). A quantitative evaluation of the results is presented in Table I. While segment lys A was not present in any of the selected size fractions, segments lys B through lys F were contained in great abundance in the two largest size fractions, 5 and 6 (Ͼ5 kb). Furthermore, segments lys C through lys F were present in size fraction 4 (3.8 kb), and segment lys E was also found in size fraction 3 (2.2 kb). These results identify a zone or a cluster of replication origins at the lysozyme locus. Since segment lys A was lacking in any selected size fraction and, furthermore, segment lys B was detectable in nascent strands as long as 5 kb, we suggest that the 5Ј border of the initiation zone is located ϳ2.5 kb downstream of lys B, e.g. ϳ8 kb upstream of the lysozyme gene promoter. Segment lys E is probably part of the initiation zone, while segment lys F is too far away (ϳ31 kb) to suggest that the initiation events monitored at this site belong to the initiation zone at the lysozyme gene. Reproducibly, we observed that the level of segments lys C and lys E in size fractions 3-6 is significantly higher than that of segments lys B and lys D. This would be compatible with the interpretation that two OBRs occur at the locus, i.e. one between lys B and lys C and another one between lys D and lys E. Alternatively, a larger group of initiation sites may occur at the lysozyme gene locus, and the higher level of segment lys C relative to segment lys B may simply reflect an elevated strength of the initiation site near lys C.
To examine these possibilities, we included three additional genomic probes, lys G, lys H, and lys I, so that the spacing of the probe sites was reduced to 4 -5 kb (Fig. 3). Furthermore, we employed a variant of the nascent strand PCR assay, the competitive PCR technique, in order to facilitate quantitation of the results. For each selected segment, we constructed a competitive fragment containing the same sequence as the genomic DNA except for a 20-bp insertion in the middle to allow resolution of the genomic and the competitor amplification products by polyacrylamide gel electrophoresis. A fixed amount of nascent DNA was mixed with varying known amounts of competitor for each primer set and amplified by 43 cycles of PCR. The reaction products were resolved by gel electrophoresis, and band intensities were determined by densitometric scanning.  Fig. 4B were quantified by densitometric scanning. The signal intensities of the size fractions were normalized to those in lane c of Fig. 4B. Data shown were mean values from four experiments. S.D. values were less than 30%. The average DNA size of the fractions was derived from the experiment in Fig. 1

FIG. 2. Nascent strand analysis of the c-myc gene.
A, segments myc A and myc B of the c-myc gene were amplified from purified nascent DNA of size fractions 1-6 ( 1 ⁄10 of each). The PCR products were blotted onto a nylon membrane and hybridized to labeled probes from segments myc A and myc B. As a control, segments myc A and myc B were amplified from 1 g of chicken genomic DNA, and 1 ⁄100 of each PCR was blotted for hybridization. B, the horizontal lines above the map of the chicken c-myc gene indicate the size of the shortest nascent strands encompassing probes myc A and myc B (filled squares), respectively. The center of the strands is marked by circles. The chicken c-myc gene OBR centers between Ϫ1.5 and Ϫ2.0 kb (filled bar). For comparison, the uppermost bar depicts the location of the OBR upstream of the human c-myc gene centered at Ϫ1.5 kb, as determined by Vassilev and Johnson (13).
The results presented in Fig. 5 are selective by showing only a single competitive PCR with a fixed amount of competitor for each segment. From the ratio between the PCR products of the complete measurements and the known number of added competitor molecules, we calculated the number of segment molecules in each size fraction. These calculations are summarized in Table II. They reveal that the abundance of segment lys A in all size fractions was very low. On the contrary, the abundance of segments lys B through lys I reached very high values, supporting our earlier suggestion that a zone or a cluster of initiation sites occurs at the lysozyme gene locus. Surprisingly, the highest abundance in particularly short nascent DNA strands was observed for segment lys H, located within the second intron of the gene. In the smallest size fraction, the abundance of lys H is ϳ15 times higher than that of lys A outside of the initiation zone and 3-4 times higher than that of others (lys G, C, I, and E). This result is not compatible with the notion that solely two OBRs, located upstream and down-stream of the gene, are operative. Furthermore, the abundance of segment lys G was intermediate between those of lys B and lys C, and similarly, the abundance of segment lys I was intermediate between those of lys D and lys E. Thus, the relative frequency of initiation shows a broad maximum ranging from lys C to lys E. In summary, the results obtained by the quantitative PCR assay are more compatible with the conclusion that a cluster of initiation sites occurs at the lysozyme gene and that sites within a 3Ј-located subzone initiate at an elevated frequency.
An Initiation Zone of DNA Replication at the Lysozyme Gene Locus Is Also Present in Nonexpressing Liver Cells-We also considered the possibility that the presence of an initiation zone of DNA replication at the lysozyme gene locus is dependent on the transcriptional activity of the locus. We therefore extended our studies to the lysozyme-nonexpressing hepatocyte cell line DU249, which is also transformed by the v-mycencoding virus MC29 (30). The sensitive RNase protection assay shown in Fig. 6 did not detect lysozyme RNA in DU249 cells. On the contrary, lysozyme RNA was well detected in  (1) or to two rounds (2) of immunoprecipitation. Immunoprecipitated DNA samples ( 1 ⁄10) were amplified by PCR with a primer pair in segment lys B and hybridized. B, segments lys A through lys F of the chicken lysozyme gene locus were PCR-amplified from nascent DNA size fractions 1-6 from HD11 cells. The PCR products ( 1 ⁄5 of each) were slot-blotted onto nylon membrane and hybridized to corresponding probes. As controls, segments lys A through lys F were PCR-amplified from unfractionated genomic DNA (c), with yeast tRNA as template (b), or without DNA (a) and subjected to hybridization.

FIG. 5. Competitive PCR on size-fractionated, nascent DNA.
Fixed amounts of nascent DNA of each size fraction were coamplified with varying known amounts of specific competitor DNA. Amplified products were then electrophoretically fractionated on 5% polyacrylamide gels and stained with ethidium bromide. The figure selects the results from coamplification with a single amount of competitor for each segment. As controls, 1 ng of genomic DNA was coamplified with the indicated amount of the competitors (g). HD11 cells. Furthermore, immunofluorescence studies using an anti-lysozyme antiserum revealed that greater than 95% of the HD11 cell population used in our experiments expressed lysozyme (data not shown). Size-fractionated, nascent DNA was then isolated from DU249 cells; amplified at segments lys A, lys C, lys D, and lys E; and hybridized. As in Fig. 4B, autoradiograms were selected that show equal signal intensities after control hybridization with unfractionated genomic DNA as template (Fig. 7, lower panel). The upper panel of Fig.  7 shows that each size fraction was devoid of segment lys A, while the abundance of segments lys C, lys D, and lys E progressively increased from the smaller size fractions to the larger ones. These results with nonexpressing DU249 hepatocytes are qualitatively similar to those obtained with expressing HD11 cells. Thus, they indicate that the initiation zone of DNA replication at the lysozyme gene locus is also operative in DU249 cells.
The Lysozyme Gene Locus Replicates Early during S Phase in Synchronized HD11 and DU249 Cells-To determine the timing of DNA replication of the lysozyme gene locus in HD11 and DU249 cells, synchronized cells were released from a block at the beginning of S phase and incubated for various times (1, 3, 5, and 8 h) including a terminal 45-min period, in which cells were labeled with BrdUrd. In parallel control experiments, cells that remained arrested at the G 1 /S border were also incubated with BrdUrd for 45 min. Replication at the lysozyme gene locus was then determined by the nascent strand PCR assay. Replicated BrdUrd-labeled DNA was purified by two rounds of immunoprecipitation, and the abundance of two genomic markers (lys B and lys D) was measured by PCR followed by hybridization. As shown in Fig. 8A, cells arrested at the G 1 /S border did not replicate any DNA at the lysozyme locus, demonstrating efficient inhibition of DNA polymerase ␣ by aphidicolin (43). The abundance of replicated DNA at both genomic loci was high in HD11 cells pulse-labeled at the end of a 3-h incubation period but low in cells pulse-labeled at the end of a 1-or 5-h incubation period. Replicated DNA from cells pulse-labeled at the end of an 8-h period contained only marginal quantities of both lysozyme gene markers. Very similar results were obtained with DU249 hepatocytes. These results indicate that the lysozyme gene locus replicates early in S phase in expressing HD11 as well as nonexpressing DU249 cells.
To examine replication of bulk genomic DNA, a slot blot loaded with purified BrdUrd-labeled DNA was hybridized to radiolabeled genomic DNA. Fig. 8B shows that genomic DNA replicated at all selected time periods but most intensively at the end of the 3-and the 5-h periods, corresponding to the middle of S phase. The low but detectable hybridization signal in cells arrested at the G 1 /S boundary may be due to the TABLE II Number of molecules of segments lys A through lys I in size-fractionated, nascent DNA Coamplifications of nascent DNA and specific competitors were carried out as described in the legend to Fig. 5. The PCR products were resolved by polyacrylamide gel electrophoresis and stained with ethidium bromide, and the intensity of each band was determined by densitometric scanning. The number of molecules of lys A through lys I in each size fraction was determined from the ratio between the amplified products from the nascent genomic DNA and the added competitor. The size of the nascent DNA strands was determined by alkaline agarose gel electrophoresis. A repetition of this experiment gave qualitatively similar results.  replication of mitochondrial DNA by DNA polymerase ␥, which is not inhibited by aphidicolin (43). DISCUSSION In the present study, we analyzed the replication pattern at the chicken lysozyme gene locus, applying two versions of the nascent strand PCR assay: one measures the level of genomic markers in size-fractionated nascent DNA by PCR and hybridization (13), and another one uses a competitive PCR to measure the abundance of such markers in size-fractionated nascent DNA (12). Results obtained from both methods indicate that the lysozyme gene locus harbors an active initiation zone of DNA replication. Since segment lys A was not contained in any nascent DNA size fraction, and segment lys B was first detectable in size fraction 5 (5 kb), we suggest that the 5Ј border of the initiation zone is located ϳ2.5 kb downstream of lys B, i.e. ϳ8 kb upstream of the lysozyme promoter (Fig. 3). On the other hand, it is presently not possible to deduce the 3Ј border of the initiation zone, due to the lack of sequence information between lys E and lys F and downstream of lys F. Since segment lys E (located 14 kb downstream of the promoter) maps between fractions 3 and 4 (ϳ3-kb median strand length), the initiation zone encompasses at least ϳ20 kb. The results of the competitive PCR assay using eight genomic probes with a spacing of the probe sites of 4 -5 kb furthermore argue that the zone is composed of a cluster of initiation sites. Quantitative analysis of the abundance of the various segments in each size fraction detects a broad maximum of relatively frequent initiations in a subzone ranging from lys C to lys I with a peak near lys H. Thus, these results are consistent with the suggestion that initiation sites near lys H fire more frequently than other sites located further away from lys H. However, our competitive PCR analysis suffers from a significant drawback. The distance between the probes used is too large (between 4 and 5 kb) to rule out the existence of small highly preferred OBRs. They are detectable only by using more closely spaced probes and a small nascent DNA size fraction (ϳ1000 bp), but not in larger size fractions which have a high risk to be contaminated by randomly broken genomic DNA (12). In other studies, where competitive PCR has been applied using closely spaced probes, relatively small (0.5-3-kb), highly preferred OBRs with high initiation activity (Ͼ10-fold over background) such as the human lamin B2 origin (44,45), the human rRNA gene origin (25), and the hamster DHFR gene origin (12) were identified. Our results show that the activity at lys H was only 3-4 times higher than that at lys G, C, I, and E. Hence, it is possible that initiation sites with higher activity are present between lys C and lys D. The use of further probes that are distributed 0.5-1 kb apart in this area will help to address this possibility satisfactorily.
A specific aspect of our data seems to contradict the suggestion of a uniform initiation zone at the lysozyme gene locus. If initiation is uniformly distributed in this zone, one would expect a linear correlation between the size and the number of nascent molecules, because an approximately equal number of nascent molecules has been analyzed for each size fraction. However, this is not the case. When the numbers of nascent molecules in the 3.0-kb size fraction are compared with those in the 4.5-kb fraction, a 4 -8-fold increase in the number of nascent molecules is observed in contrast to a 1.5-fold increase in size. The simplest possible explanation for this discrepancy is the existence of possibly multiple initiation sites located 1.5-2.25 kb away from the probes. Alternatively, the discrepancy may be caused by the fact that larger size fractions (Ͼ3 kb) are more contaminated by randomly broken genomic DNA than the smaller ones (12). In this case, the number of nascent molecules in larger size fractions may be overestimated.
A careful comparison of the results obtained from the two assays used also reveals some differences. For instance, the nascent strand PCR hybridization assay seems to indicate a more frequent initiation at lys C and lys E than at lys D, while the competitive PCR assay suggests that the initiation frequency at lys D is slightly higher than that at lys C and lys E. Since hybridization signals obtained from the nascent strand PCR hybridization assay were normalized to those from control PCR with genomic DNA, we attribute this discrepancy to differences in the amplification efficiency of different DNA samples, e.g. size-fractionated, immunoprecipitated DNA versus genomic DNA.
The nascent strand PCR analysis furthermore provides evidence indicating that replication initiates at an elevated frequency in a subzone near the 3Ј-end of the gene. Thus, our results on the initiation of replication at the lysozyme gene locus closely resemble those obtained for the DHFR gene in Chinese hamster ovary cells (46) and the human rRNA gene repeats (25). Two preferred initiation regions (termed ori-␤ and ori-␥) were identified within a large zone of multiple potential initiation sites downstream of the DHFR gene. Similarly, most of the initiation events in human ribosomal DNA occur in a short region upstream of the rRNA gene promoter. In contrast, the results obtained by two-dimensional gel electrophoresis methods argue in favor of a broad (55-kb) initiation zone downstream of the DHFR gene (47)(48)(49). Large zones of initiation have been also found within the Syrian hamster CAD gene (5 kb) (16), in the Drosophila histone gene repeat (5 kb) (22,23), in the amplified Drosophila chorion gene (10 kb) (20), and downstream of the Drosophila DNA polymerase ␣ gene (10 kb) (21). It might be a drawback that we solely used methods based on PCR amplification of nascent DNA strands to characterize an origin of replication at the lysozyme gene locus. However, since the nascent strand PCR assay identified a fixed location of replication origins at several genetic loci (13,34,50), we have confidence that our finding of a broad initiation zone is not an artifact of the method applied.
Interestingly, it was found that the initiation zone at the lysozyme gene locus is operative not only in myelomonocytic HD11 cells, which express the gene, but also in nonexpressing DU249 hepatocytes. The functional status of the initiation zone is thus independent of the transcriptional activity of the lysozyme gene. The localization of a 5-kb initiation zone of replication within the Syrian hamster CAD transcriptional unit FIG. 8. The lysozyme gene locus is replicated early in S phase in HD11 and DU249 cells. A, HD11 and DU249 cells arrested at the G 1 /S border were allowed to progress into S phase for 1 h (S 1 ), 3 h (S 3 ), 5 h (S 5 ), and 8 h (S 8 ). During the last 45 min of these time periods, cells were pulse-labeled with BrdUrd. As a control, cells that remained arrested (G 1 /S) were incubated with BrdUrd for 45 min. Newly synthesized DNA was then isolated, and amplified by PCR at segments lys B and lys D. Amplified DNA was immobilized on nylon membranes and hybridized with the corresponding probes. B, BrdUrd-labeled DNA from synchronized HD11 cells was immobilized on a nylon membrane and hybridized with 32 P-labeled genomic DNA.
(16) is a precedent for the compatibility of origin function and transcriptional activity. Furthermore, the timing of DNA replication at the lysozyme gene locus during S phase also seems not to be influenced by the transcriptional activity of the gene, since the locus is replicated early in S phase in expressing HD11 as well as nonexpressing DU249 cells. Examples for replication of tissue-specifically nonexpressed genes early in S phase have been previously reported (e.g. ␣-globin genes in lymphocytes) (51). It may be important that the initiation zone at the lysozyme gene locus contains most of the elements essential for regulation of developmentally and cell type-specific expression of the gene. It has been previously observed that the locus control region regulating expression of the human ␤-globin gene cluster is necessary for initiation of DNA synthesis at the 5Ј-end of the ␤-globin gene (52).
The initiation zone localized to the lysozyme gene starts closely downstream of the lysozyme 5Ј MAR and encompasses the 3Ј MAR (see map in Fig. 3). This is reminiscent of the DHFR gene, where ori-␤ and ori-␥ are 22 kb apart and lie on either side of a prominent MAR (53). Various previous observations suggest that the origins of DNA replication are determined by chromatin and nuclear structure in addition to DNA sequence. Newly replicated DNA is preferentially bound to a nuclear subcomponent recovered in the nuclear matrix and is organized into a limited number of nuclear loci, named replication factories (54,55). Replication of DNA introduced into either Xenopus eggs or egg extract does not occur unless DNA is first assembled into chromatin and nuclei (56,57). Recently, Xenopus egg extracts have been used to determine the requirements that govern site-specific initiation of replication at the amplified DHFR gene region (58). Replication initiated at the origin utilized in intact hamster cells when undamaged nuclei were added to the extract but nonspecifically when pure DNA was added. Higher eukaryotic replicons have average sizes between 50 and 300 kb (59). Thus, the initiation zone of replication at the lysozyme gene locus and its close association with two MARs suggest that the lysozyme locus as a whole fulfills an important function within a large chromatin organization. Potentially chromatin units much larger than that of the lysozyme domain originate on both sides of the locus, and the interplay between this chromatin structure and specific sequences finally determines the origin of replication at the lysozyme locus.