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.
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INTRODUCTION |
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 two-dimensional 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 kb1 downstream from the
3'-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 >4 kb near the
Schizosaccharomyces pombe ura4 gene
(18), in the nontranscribed spacer (31 kb) of the human ribosomal DNA
(19), in a ~6-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 5' and 3'
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 3'-located 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.
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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 µg/ml
streptomycin at 37 °C and 5% CO2. For preparation of
5-bromodeoxyuridine (BrdUrd)-labeled DNA, 108 exponentially
proliferating cells were labeled with 20 µM 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 BrdUrd-labeled nascent DNA strands. They were purified
by two cycles of immunoprecipitation using 30 µl (in each
immunoreaction) of an anti-BrdUrd monoclonal antibody (25 µg/ml,
Becton-Dickinson) and then dissolved in 20 µl of TE buffer containing
10 mM Tris, pH 8.0, and 1 mM EDTA or in 400 µl when used for competitive PCR. The size fractionation was
monitored by alkaline agarose gel electrophoresis and hybridization to
32P-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
G1/S boundary by incubation in complete Iscove's modified
Dulbecco's medium containing 20 µg/ml aphidicolin (Sigma,
Deisenofen, Germany) for at least 12 h.
For labeling with BrdUrd, G1/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 µg of sonicated genomic DNA was purified by two cycles of
immunoprecipitation as described above and dissolved in 40 µl of
TE.
Synthetic Oligonucleotides and PCR--
Oligonucleotide primer
pairs and hybridization probes chosen from the sequences of the chicken
lysozyme gene locus (28, 32)2
and of the chicken c-myc gene (33) (see also Figs.
2B and 3) were chemically synthesized by Pharmacia
(Freiburg, Germany) or MWG-Biotech (Ebersberg, Germany) as
follows: lys A, 5'-CGGGTATCATTAGTGCCGAG-3', 5'-CTGCCAGTATATCCTGGCAAA-3', and probe, 5'-CTGCATTGCAACGAAGGGTTGAC-3'; lys B, 5'-AGAGCGATGCTCAGTAAGGC-3', 5'-ATGCAGCTTGCTTCCTATGC-3', and
probe, 5'-CAGATCCCAGGAAGTGTAGATCC-3'; lys C,
5'-TCTTCCATGTTGGTGACAGC-3', 5'-ATCAATCCATGCCAGTAGCC-3', and probe,
5'-CCAGCTGAGGTCAAGTTACGAAC-3'; lys D, 5'-CAGTCGTGGAGTTGTATGCG-3',
5'-ATACAAGCAGCAATCTGGCC-3', and probe, 5'-CACTGCAGTGTGTGACAACTGAC-3';
lys E, 5'-GTGCTCTCATTGGATAGCCC-3', 5'-CCATTTCTGAAACCACTGCC-3', and
probe, 5'-GAAGGAGCCTACTCCTTACACAGTG-3'; lys F,
5'-TTAGAAGTCGACGAGTGTGGC-3', 5'-TTCTTCAACCAGAAGCAACC-3', and probe,
5'-CTGTGTGGAACCCATTCATCAGC-3'; lys G, 5'-TGAGAGGGGGTTGGGTGTAT-3', 5'-CGCTCTACGCATTCTGAAACA-3'; lys H, 5'-CCACTAGTGAAGGGGAGGAGA-3', 5'-AGTGCAGCTGCCAGAATACC-3'; lys I, 5'-GCAACACTTGGCAAACCTCAC-3', 5'-ACTACACGGCCTTCAGCACAG-3'; myc A (first intron):
5'-CTCCGCTTTACCCATCACTC-3', 5'-CTGGGCAAACTTTGCCAA-3', and probe,
5'-GAGGGCAAGAAGCATTTGCTTCTCC-3'; myc B (second intron),
5'-CGAAGGAATGAGCTGAAGC-3', 5'-TCCAAGAGTTCCTATGCACG-3', and probe,
5'-CACAGACTGATCGCAGAGAAAGAGC-3'.
To amplify specific DNA fragments (lys A through lys F), 30 cycles of
PCR were carried out in 50 µl of standard buffer (Pharmacia) containing 1 µM primers, four deoxyribonucleotides (200 µM each), 2-2.5 units of Taq polymerase, and
2 µl 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 µl of each PCR and 3 µg 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 32P-labeled with T4
polynucleotide kinase and [
-32P]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
5'-ACCTGCAGGGATCCGTCGAC-3' (tail 1) or 5'-GTCGACGGATCCCTGCAGGT-3' (tail
2) as follows: lys A, 5'-tail 1-GTTGACTAGAGATTTCATCT-3', 5'-tail
2-CCTTCGTTGCAATGCAGTTT-3'; lys B, 5'-tail 1-GCCAAAGAGTCTGCTGAATG-3',
5'-tail 2-CTGCTGGAATCAGGAAACTG-3'; lys C, 5'-TAIL
1-GAGGTCAAGTTACGAACTCA-3', 5'-tail 2-AGCTGGGGTCAATAAGTAAC-3'; lys D,
5'-tail 1-ATTTCAAGGAGAATGGATCG-3', 5'-tail 2-AAACCAGTACATACCCATAG-3'; lys E, 5'-tail 1-AGCCTACTCCTTACACAGTG-3', 5'-tail
2-CCTTCAAGAAAGAGAGAACC-3'; lys G, 5'-tail 1-CGTGGCATAGTGCCAGCAGT-3',
5'-tail 2-GCCTGCTTGTGACTCTGAGA-3'; lys H, 5'-tail
1-CTTAATCTAACCAAGGGGGA-3', 5'-tail 2-GTGAAACAACACTCATGGTC-3'; lys I,
5'-tail 1-ATCACTGGGGTCAGGACAGT-3', 5'-tail
2-GACCGTGTCCCACCTAAAGC-3'.
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-µl reaction mixture under
standard conditions with 1× GeneAmp buffer, 1.25 units AmpliTaq Gold
polymerase (Perkin-Elmer, Hamburg, Germany), four deoxyribonucleotides (200 µM each), 1 µM primers, 5 µl 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
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 32P-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)+ 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)+ RNA using a 32P-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+ (Stratagene, Heidelberg,
Germany) (41). The recombinant plasmid was linearized with
XbaI and used as template to synthesize
32P-labeled lysozyme-specific antisense RNA in an in
vitro transcription reaction with T7 RNA polymerase (Stratagene
kit). The 32P-labeled RNA probe was hybridized to 4 µg of
poly(A)+ 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 1× 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).
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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.

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Fig. 1.
Size determination of size-fractionated
BrdUrd pulse-labeled DNA. A, exponentially
proliferating HD11 cells were pulse-labeled with BrdUrd. Extracted DNA
was size-fractionated in alkaline sucrose gradients, and six selected fractions were
immunoprecipitated twice using an anti-BrdUrd antibody. Purified
nascent DNA samples ( of each size fraction) were separated
on an alkaline 1% agarose gel, Southern-transferred onto a nylon
membrane and hybridized with 32P-labeled genomic DNA.
Marker fragments were HindIII-digested DNA and a 400-bp
DNA ladder. B, purified BrdUrd-labeled DNA of the six chosen
fractions was slot-blotted onto a nitrocellulose membrane and reacted
with an anti-BrdUrd antibody to determine the level of incorporated
BrdUrd. C, the levels of BrdUrd incorporation were plotted
against the average DNA length of the size fractions.
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Table I
Relative abundance of segments lys A through lys F in
size-fractionated, nascent DNA
The hybridization signals in 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.
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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 ~1.5 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).

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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 ( 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 µg of chicken genomic DNA, and 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 1.5 and 2.0 kb (filled bar). For
comparison, the uppermost bar depicts the location of the OBR upstream
of the human c-myc gene centered at 1.5 kb, as determined
by Vassilev and Johnson (13).
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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 ~48 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.

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Fig. 3.
Map of the chicken lysozyme gene locus.
The lysozyme gene locus with the coding sequence (filled
box) is flanked by the 5' and 3' MAR (open
boxes). The arrow indicates the primary
transcription product of the gene. Locations of the PCR-amplified
segments (lys A through lys I) are shown below the
sequence.
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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 BrdUrd-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.

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Fig. 4.
Abundance of six segments of the lysozyme
gene locus in size-fractionated, nascent DNA. A, 10 µg of genomic DNA from BrdUrd-labeled HD11 cells (a) or
from unlabeled cells (b) were subjected to one round
(1) or to two rounds (2) of immunoprecipitation.
Immunoprecipitated DNA samples ( ) 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 ( 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.
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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 (>5 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 5' border of the initiation zone is located ~2.5 kb
downstream of lys B, e.g. ~8 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 (~31 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. 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 ~15 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 downstream 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 3'-located subzone initiate at an elevated frequency.

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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).
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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.
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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-myc-encoding 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 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.

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Fig. 6.
Lysozyme gene expression in chicken cell
lines HD11 and DU249. Poly(A)+ RNA was isolated from
HD11 and DU249 cells and subjected to an RNase protection assay using a
32P-labeled lysozyme-specific RNA probe. Five hundred and
2000 cpm of the labeled probe were loaded left and
right on the gel, respectively.
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Fig. 7.
An initiation zone of DNA replication at the
lysozyme gene locus in nonexpressing DU249 hepatocytes. The level
of segments lys A, lys C, lys D, and lys E was determined in
size-fractionated, nascent DNA from pulse-labeled DU249 hepatocytes
using the same conditions as in Fig. 4B.
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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
G1/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
G1/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.

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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 G1/S border were allowed to progress
into S phase for 1 h (S1), 3 h (S3),
5 h (S5), and 8 h (S8). During the
last 45 min of these time periods, cells were pulse-labeled with
BrdUrd. As a control, cells that remained arrested (G1/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 32P-labeled genomic DNA.
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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 G1/S boundary
may be due to the replication of mitochondrial DNA by DNA polymerase
, which is not inhibited by aphidicolin (43).
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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 5' border of
the initiation zone is located ~2.5 kb downstream of lys B,
i.e. ~8 kb upstream of the lysozyme promoter (Fig. 3). On
the other hand, it is presently not possible to deduce the 3' 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 (~3-kb median strand length), the initiation zone encompasses at
least ~20 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 (~1000
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 (>10-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 (>3
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 3'-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
(47-49). 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 (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 5'-end of the
-globin gene
(52).
The initiation zone localized to the lysozyme gene starts closely
downstream of the lysozyme 5' MAR and encompasses the 3' 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.
We thank D. Wulf and K. Zimmermann for
skillful technical assistance. We are grateful to the referees for
valuable suggestions and comments on the manuscript.