Characterization of human deoxyribonuclease I gene
(DNASE1) promoters reveals the utilization of two
transcription-starting exons and the involvement of Sp1
in its transcriptional regulation
Yoshihiko Kominato
1
, Misuzu Ueki
2
, Reiko Iida
3
, Yasuyuki Kawai
4
, Tamiko Nakajima
1
, Chikako
Makita
1
, Masako Itoi
1
, Yutaka Tajima
1
, Koichiro Kishi
1
and Toshihiro Yasuda
2
1 Department of Legal Medicine and Medical Genetics, Gunma University, Japan
2 Division of Medical Genetics and Biochemistry, University of Fukui, Japan
3 Division of Legal Medicine, University of Fukui, Japan
4 Third Division of Internal Medicine, University of Fukui, Japan
Deoxyribonuclease I (DNase I, EC 3.1.21.1) is an
enzyme that preferentially attacks double-stranded

Fax: +81 776 61 8149
Tel: +81 776 61 8287
E-mail: [email protected]
Database
The nucleotide sequences reported here
have been submitted to the GenBank/
EMBL/DDBJ Data Bank with accession
numbers AB188151 and AB188152
(Received 22 March 2006, revised 8 May
2006, accepted 15 May 2006)
doi:10.1111/j.1742-4658.2006.05320.x
Levels of deoxyribonuclease I (DNase I) activity in vivo have been shown
to be altered by physiological and ⁄ or pathological processes. However, no
information is available on the regulation of DNase I gene (DNASE1)
expression in vivo or in vitro. We first mapped the transcription start sites
of DNASE1 in human pancreas and in the DNase I-producing human pan-
creatic cancer cell line QGP-1, and revealed a novel site  12 kb upstream
of exon 1, which was previously believed to be the single transcription-
starting exon. This initiation site marks an alternative starting exon,
designated 1a. Exons 1 and 1a were used simultaneously as transcription-
starting exons in pancreas and QGP-1 cells. Promoter assay, EMSA and
chromatin immunoprecipitation analysis with QGP-1 cells showed the pro-
moter region of exon 1a in which the Sp1 transcription factor is specifically
involved in promoter activity. This is the first to be identified as a tran-
scription factor responsible for gene expression of vertebrate DNase I
genes. Furthermore, RT-PCR analysis indicated alternative splicing of
human DNASE1 pre-mRNA in pancreas and QGP-1 cells. Only two tran-
scripts among eight alternative splicing products identified can be transla-
ted to produce intact DNase I protein. These results suggest that human
DNASE1 expression is regulated through the use of alternative promoter

PCI remain to be elucidated. Delineation of the
molecular basis for our observations is essential to
evaluate the elevated DNase I activity in the sera of
patients with AMI and to validate the use of serum
DNase I activity as a new diagnostic marker for the
early detection of AMI. To elucidate the molecular
basis of this phenomenon, it is important to under-
stand the regulatory mechanism of the human DNase I
gene (DNASE1) expression.
Previous molecular–genetic studies have shown that
the human DNase I gene consists of at least nine
exons spanning > 3.2 kb of genomic DNA at chromo-
some 16p13.3; exon 2 includes the translation initiation
codon (ATG) and the first exon is believed to include
only the 5¢-UTR of the mRNA [15,16]. However, com-
parison of the sequence of the 5¢-UTR of human pan-
creatic DNASE1 mRNA reported previously [15]
with the human genomic sequence showed that the
5¢-terminal 15 nucleotides of the 5¢-UTR are not found
in the human genomic sequence [16], whereas the 3¢
segment of 143 nucleotides in the 5¢-UTR matches the
genomic sequence. Thus, the transcription initiation
site of exon 1 has not yet been definitively identified.
To our knowledge, no information is available on the
regulation of vertebrate DNASE1 expression in vivo or
in vitro, including characterization of the promoter
region of the gene and the associated transcriptional
factors. Therefore, delineation of the transcriptional
regulation of human DNASE1 may provide clues to
the mechanisms underlying ischemia-induced elevation

Human DNase I activity is mainly demonstrable in
pancreas, alimentary tract and pituitary [3,18], and
most serum DNase I appears to be produced from
those tissues. Moreover, our survey of DNase I-produ-
cing cell lines showed a high level of DNase I activity
and its transcripts in QGP-1 cells, which were estab-
lished by Kaku et al. [19] from a human pancreatic
islet cell carcinoma (possibly D cells) as a carcinoem-
bryonic antigen-secreting cell line. A similar 5¢-RACE
was performed with cDNA synthesized from total
RNA of QGP-1 cells. The DNA sequences of the
5¢-RACE products were determined for five transform-
ant clones. Four clones contained a 417-bp 5¢-RACE
DNA product that appeared to be a hybrid between
exon 1 and the upstream genomic DNA of DNASE1:
the sequence of the 3¢ portion in those products was
identical to that of exons 1–3 from the 5¢-end of the
reverse primer DN+231 to a position +156 relative
Y. Kominato et al. Promoters of human deoxyribonuclease I gene
FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3095
A
B
Fig. 1. The nucleotide sequence of the
5¢-flanking region in the human DNase I
gene. (A) The sequence located between
positions )100 and +250 relative to the
5¢-end in DNASE1 exon 1. +1 above the
sequence indicates the 5¢-end of exon 1.
Open circles indicate locations of 5¢-ends
of the DNASE1 transcripts, determined by

lysis. The position and identity of mutations
at )11944 to )11941 are indicated in oligo-
nucleotide m91,50 and reporter construct
)197HmSp.
Promoters of human deoxyribonuclease I gene Y. Kominato et al.
3096 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS
to the beginning of exon 1, as shown in Fig. 1A.
Beyond that point, however, the sequence of the 5 ¢
portion showed 100% identity with that of genomic
DNA  12 kb upstream of the DNASE1 exon 1
(Fig. 1B). More interestingly, the products lacked the
sequence between positions +1 and +155 in exon 1.
This comparison with the upstream genomic sequence
of DNASE1 allowed us to demonstrate the presence of
an alternative exon, which we named exon 1a. The
donor splice site between exon 1a and the subsequent
intron had GT, whereas the acceptor site between the
subsequent intron and the 5¢-end at position +156 in
exon 1 had AG. Therefore, the splice sites seem to be
compatible with a splicing junction.
Confirmation of utilization of exon 1a as
a transcription initiation exon and occurrence
of alternative splicing
To examine whether exon 1a is used as a transcription
starting exon in QGP-1 cells and pancreas, and to con-
firm the splicing junction between exon 1 and the
upstream DNA, RT-PCR was carried out using a pri-
mer specific for exon 1a and a reverse primer comple-
mentary to the sequence in exon 8. DNA fragments of
different sizes were amplified from the RNA of QGP-1

DN+385 DN+721
DN-105
DN-9
+1
A
B
Fig. 2. RT-PCR analysis to detect the transcription-starting exon in
DNASE1 of QGP-1 cells and pancreas. (A) RT-PCR analysis. Total
RNA prepared from QGP-1 cells or pancreas was reverse-tran-
scribed with random primer, and the resulting single-strand cDNA
was used as a template for PCR analysis. The DNASE1 amplifica-
tion was performed using either distinct starting exon-specific pri-
mer DN)110 (left) or DN)144 (right) and a common reverse primer
DN+785 complementary to exon 8 of the DNASE1. PCR products
were electrophoresed through a 1.5% agarose gel and stained with
ethidium bromide. The amplified fragments were named A–J. A
1 kb Plus DNA Ladder was used as a molecular size marker. (B)
Splicing patterns of the amplified fragments A–J. Nucleotide
sequences of these fragments were determined and then com-
pared. Schematically represented DNASE1 was aligned with the
RT-PCR products amplified, using a set of each starting exon-speci-
fic primer (DN)110 or DN)144) and the DN+785 primer, which are
represented by arrows. Open boxes represent the DNASE1 exons,
and a vertical broken line indicates the splicing junction between
exon 1a and a portion of exon 1. The thick straight lines represent
the intron sequence. +1 indicates the position of the transcription
start site of exon 1. Dashed v-shaped lines in RT-PCR amplified
fragments A–J indicate regions that are removed by splicing,
whereas a dashed line in exon 7 of fragment C represents a dele-
tion of 39 bp. The thick lines indicate intron 6 of 75 bp in fragments

transcripts A and H and second, transcripts B–G, I
and J that all lack exon 3. Only two DNASE1 tran-
scripts, those corresponding to the amplified products
A and H, can be translated to produce intact DNase I
protein. By contrast, 3¢-RACE using total RNA from
QGP-1 cells and pancreas as a template gave a single
band with a sequence identical to that reported previ-
ously [16], in addition to the observation of a specific
cleavage ⁄ polyadenylation site located in the 3¢-flanking
region of the gene at position 142 downstream of the
stop codon. Thus, DNASE1 splicing variants appear
to share a common 3¢-UTR.
Quantitative real-time RT-PCR was performed
using each distinct starting exon-specific primer and
a common reverse primer complementary to exon 2
to determine the relative abundance by comparing
the copy number of transcripts containing exon 1a
with the number starting from exon 1. The abun-
dance of transcripts starting from exon 1 was 10-fold
higher than that of the transcripts starting from
exon 1a in pancreas, whereas it was half in QGP-1
cells.
To examine whether transcription starting from
both exon 1a and exon 1 results in the production of
DNase I enzyme, we transfected the expression plasmids
ex-pDN1a and ex-pDN1, containing the sequences cor-
responding to the DNASE1 cDNAs starting from either
exon 1a or exon 1, respectively, into COS-7 cells and
then determined the levels of DNase I activity secreted
into the medium of the cells transfected with each plas-

expression vector ex-pDN, the Kozak sequence just upstream from the coding region of DNASE1 is contained and indicated by a closed
box. In the expression vector ex-pDN1, the gray box represents the whole sequence of exon 1. In the expression vector ex-pDN1a, the
sequence between +4 and +102 relative to the transcription start site of exon 1a, indicated by the open box, is ligated with the part of
exon 1 between positions +156 and +243. The resulting DNase I activity in the medium secreted from each transfected cells was normal-
ized by coexpressed b-galactosidase activity, and is shown in the right panel. The mean values and standard deviations were calculated from
five independent experiments. The activity of the expression plasmid ex-pDN was assigned an arbitrary value of 1.0. The DNase I activities
of the cells transfected with ex-pDN1 were statistically significantly lower than those of cells transfected with ex-pDN or ex-pDN1a
(P<0.05).
Promoters of human deoxyribonuclease I gene Y. Kominato et al.
3098 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS
findings indicate that transcripts starting from either
exon 1a or exon 1 are translated to produce intact
DNase I protein.
Characterization of the promoter region of
exons 1a and 1 in the human DNase I gene
Because 5¢-RACE analysis identified two transcription-
starting exons used in DNASE1, we characterized the
promoters that regulate transcription of the DNASE1
messages containing exons 1 or 1a. To examine pro-
moter activity in the 5¢-flanking region of exon 1 in
DNASE1, we first obtained the )1386M construct by
introducing the )1386 to +268 sequence of DNASE1
into the promoterless pGL3–basic vector upstream of
the luciferase coding sequence. The reporter plasmid
was transfected into QGP-1 cells, followed by assay of
luciferase activities (Fig. 4A). pGL3–promoter vector
containing the SV40 promoter and pGL3–basic vector
without the promoter sequence were used as positive
and negative controls, respectively. The relative lucif-
erase activity of the )1386M construct was at least

indicating that negative regulatory elements are present
in the )11965 to )11944 region. Furthermore, deletion
of the upstream end from position )11944 to )11931
resulted in a fivefold decrease in luciferase activity,
suggesting that elements important for distal promoter
function are contained within the deleted region.
Inspection of the sequence between )73 and )60
upstream of the transcription start site of exon 1a
revealed a putative binding site for Sp1 transcription
factor and related proteins, as shown in Fig. 1B. To
evaluate whether the Sp1-binding site in the DNASE1
distal promoter is crucial for expression, a mutated
binding site was introduced into the )197H construct,
resulting in the loss of 80% of luciferase activity
(Fig. 4B). The data show that the Sp1 site is import-
ant for the DNASE1 distal promoter function
involved in transcription from exon 1a. To demon-
strate whether the sequence between )11944 and
)11931 bound Sp1 transcription factor, EMSA was
carried out using nuclear extracts prepared from
QGP-1 cells (Fig. 5). The oligonucleotide 90,51 probe
produced a major up-shifted band when the probe
was incubated with the nuclear extracts (lanes 1 and
7). Formation of the up-shifted complex, indicated by
the arrow, was decreased by the addition of compet-
ing unlabeled self oligonucleotide or Sp1 oligonucleo-
tide (lanes 2 and 4), but not by addition of
oligonucleotide m90,51 containing the same mutation
of the Sp1 site in )197HmSp construct as well as
oligonucleotide mSp1 with a mutated Sp1-binding site

to bind weakly to the promoter, although EMSA
failed to show a supershifted band with anti-Sp3 IgG
(data not shown). These results provide direct evi-
dence for Sp1 binding to the DNASE1 distal promo-
ter. The data demonstrate that Sp1 is involved in the
DNASE1 distal promoter function in transcription
from exon 1a.
Discussion
In previous studies, we were the first to determine the
genomic structure of the mammalian DNase I gene;
the human gene is located on chromosome 16p13.3, is
 3 kb long and contains nine exons interrupted by
eight introns [16]. Subsequently, the mouse [22], rat
[23] and bovine [24] genes have been shown to be sim-
ilar to the human gene. In this study, we investigated
the upstream region of the human gene. 5¢-RACE ana-
lysis of QGP-1 cells and pancreas revealed the presence
of a novel exon, named exon 1a, 12 kb upstream of
the original exon 1 in human DNASE1. Furthermore,
because the full-length 5¢-UTR of the transcript from
exon 1 was determined, the 5¢ boundary of exon 1
could be confirmed and was not in agreement with ear-
lier studies [15]. RT-PCR analysis and promoter assay
of the 5¢-upstream regions of both exons 1 and 1a clar-
ified that human DNASE1 utilizes two transcription-
starting exons simultaneously for expression of the
gene. Accordingly, the gene organization of human
DNASE1 should be corrected to 10 exons interrupted
by nine introns spanning  15 kb of genomic DNA.
Although the DNA region corresponding to exon 1a

−54M
−33M
−28M
−17M
+1M
luc
−116M
luc
−1386M
−138
A
6
B
−13952
−13866
−12068
−13202
−12395
luc
−1.0−2.0kb
Relative luciferase activities
in QGPI cells
0 1
luc
luc
luc
luc
luc
luc
luc

3100 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS
the regulation of gene expression [25]. The level of
transcription initiation can vary between alternative
promoters, the turnover or translation efficiency of
mRNA isoforms with different leader exons can differ,
and alternative promoter usage can lead to the genera-
tion of protein isoforms differing in amino acid
sequence. Human DNASE1 pre-mRNA is transcribed
from different transcription start sites, exons 1 and 1a,
resulting in generation of two kinds of gene transcript.
However, only the sequences of the 5¢-UTR are differ-
ent between them, because these transcripts share all
the other exons, exons 2–9, which contain the entire
coding region. Thus, use of alternative promoters in
DNASE1 results in no generation of protein isoforms.
The 5¢-UTR of eukaryotic mRNA influences the initi-
ation step of protein synthesis and thereby in part
determines the translational efficiency of the transcript
[26]. In fact, as shown in Fig. 3, the translational effi-
ciency of the transcript from exon 1 was about half
of that from exon 1a. We used genetyx software
(GENETYX Corp., Tokyo, Japan) to search for poss-
ible secondary structure in the nucleotide sequence of
the 5¢-UTR in the transcripts starting from exons 1
and 1a, and found that the 5¢-UTR of the transcript
from exon 1 has a higher content of stem-loop struc-
ture than does that from exon 1a. Because stable
stem–loop structures are known to cause significant
suppression of translation, the distinctive secondary
structures of the 5¢-UTRs in these transcripts could

cells. DNA–protein interaction was investigated using radiolabeled
probe 90, 51 (lanes 1–5, 7–8) or m 90, 51 (lane 6) in the presence
or absence of a 200-fold molar excess of competing unlabeled
oligonucleotides or antibodies as indicated. The major shifted com-
plex is indicated by the arrow. Oligonucleotides Sp1 and mSp1 con-
tained the wild and mutant types of Sp1 site, respectively (lanes 4
and 5). The nuclear extract was preincubated with anti-PDX-1 (lane
8) or anti-Sp1 IgG (lane 9). A supershifted band with antibody to
Sp1 is indicated by the arrowhead.
Fig. 6. ChIP assays of the Sp1-binding status at the endogenous
DNASE1 distal promoter in QGP-1 cells with anti-Sp1, -Sp2, -Sp3,
and -Sp4 IgG. The amplified DNASE1 distal promoter sequences in
the input and bound fractions are shown. The PCR products of
97 bp were electrophoresed through a 2% agarose gel and stained
with ethidium bromide.
Y. Kominato et al. Promoters of human deoxyribonuclease I gene
FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS 3101
the human DNASE1 gene was performed with QGP-1
cells; using promoter deletion analysis we could survey
the upstream regions responsible for transcription from
exons 1 and 1a (Fig. 4), in the latter of which a poten-
tial Sp1 site in the promoter contributes to its basal
activity. EMSA experiments (Fig. 5), ChIP assay
(Fig. 6) and the introduction of mutations in the
reporter gene construct revealed that binding of Sp1 to
the upstream region of DNASE1 exon 1a controlled
the basal distal promoter activity. This is the first to
be identified as a transcription factor responsible for
gene expression of vertebrate DNase I genes. Sp1 is
the founding member of a growing family of transcrip-

streptomycin.
5¢- and 3¢-RACE analysis
5¢-RACE was performed using the 5¢-GeneRacer kit (Invi-
trogen Corp.) according to the manufacturer’s instruction.
Total RNAs isolated from QGP-1 cells using the acid
guanidine thiocyanate ⁄ acid phenol method [29] and human
pancreas (BD Biosciences Clontech, Palo Alto, CA) were
employed for the RACEs. Five micrograms of each total
RNA was treated with bovine intestinal phosphatase, fol-
lowed by incubation with tobacco acid pyrophosphatase
and ligation with the GeneRacer RNA oligo. cDNA
was synthesized by using Superscript III and the DNASE1-
specific primer DN+254, the sequence of which was 5¢-
TAGGTGTCTGGTGCATCCTG-3¢.5¢-Ends were PCR
amplified from these cDNA templates with a primer to the
GeneRacer RNA oligo and the DNASE1-specific primer
DN+232, the sequence of which was 5¢-TGAGGTTGTC
CAGCAGCTTC-3¢.3¢-RACE was performed using the
3¢-RACE system (Invitrogen Corp.) according to the manu-
facturer’s instruction. Five micrograms of each total RNA
was subjected to 3¢-RACE under the same conditions
described previously [30]. The sequences of the DNASE1-
specific forward primers used were 5¢-GACACCTTCAA
CCGAGAGCC-3¢ (DN+385) and 5¢-ATGCTGCTCCG
AGGCGCCGT-3¢ (DN+721). Conditions for these amplif-
ications were 95 °C for 9 min, 40 cycles of 94 °C for 1 min,
55 °C for 1 min, 72 °C for 2 min, followed by incubation
at 72 °C for 10 min. PCR amplifications were performed in
a50lL reaction mixture containing 10 pmol of each pri-
mer, 1.25 units of AmpliTaq Gold (Applied Biosystems,

GCTTGG-3¢ (DN)110), corresponding to the sequence in
exons 1 and 1a of the DNASE1, respectively. Another start-
ing exon-specific amplification of the DNASE1 message was
performed using each distinct starting exon-specific primer
Promoters of human deoxyribonuclease I gene Y. Kominato et al.
3102 FEBS Journal 273 (2006) 3094–3105 ª 2006 The Authors Journal compilation ª 2006 FEBS
and the reverse primer DN+89, of which the sequence was
5¢-ATGTTGAAGGCTGCGATCTTCAG-3¢, complement-
ary to exon 2 of the DNASE1. Conditions for these amplifi-
cations were 95 °C for 9 min, 30 cycles of 94 °C for 1 min,
60 °C for 1 min, and 72 °C for 2 min, followed by incuba-
tion at 72 °C for 10 min. Determination of the nucleotide
sequences of the amplified fragments were described above.
Plasmids
The different length of 5¢-upstream sequence of exons 1 and
1a in the DNASE1 were PCR-amplified using sequence-spe-
cific primers corresponding to the sequence deposited in the
GenBank with Accession no. AC006111 and subcloned into
a firefly luciferase reporter vector, the pGL3–basic vector
(Promega, Madison, WI). Nomenclature used for the var-
ious reporter constructs is based on the nature of the inser-
ted fragments. Letter symbol M reflects the restriction
enzyme cleavage site of MlnI at +268 relative to the tran-
scription start site of exon 1 in the DNSAE1, letter symbol
H reflects the restriction enzyme cleavage site of HindIII at
+90 relative to the transcription start site of exon 1a,
whereas numerals indicate the endpoints of the primers used
for PCR. For example, )197H construct contains the frag-
ment bordered with PCR primer sequence starting at )197
relative to the transcription start site at one end and HindIII

DN+89, respectively, into a pCR2.1 plasmid vector.
For all the constructs, sequencing was performed over
the entire region of the inserted sequences. Plasmid DNA
was purified by using HiSpeed Plasmid Kit (QIAGEN
GmbH, Hilden, Germany).
Transfection and luciferase assay
Transient transfection experiments into QGP-1 cells were
performed with Lipofectamine Plus reagent (Invitrogen
Corp.); 1 lg of firefly luciferase reporter and 0.01 lgof
pRL-SV40 Renilla luciferase reporter (Promega) were used
for each analysis. QGP-1 cells were split, 18–24 h prior to
transfection, into a six-well tissue culture plate (Becton
Dickinson Labware, Franklin Lakes, NJ) at 1 · 10
5
ÆmL
)1
.
At the time of transfection, cells were washed once with
Opti-MEM I-reduced serum medium (Invitrogen Corp.)
containing neither fetal bovine serum nor l-glutamine.
Plasmid DNA was suspended in 100 lL of Opti-
MEM I-reduced serum medium, followed by mixture with
6 lL of Lipofectamine Plus reagent at room temperature
for 10 min. Four microliters of Lipofectamine Plus reagent
were diluted in 100 lL of Opti-MEM I-reduced serum med-
ium. The two solutions were combined at room tempera-
ture for 15 min, followed by the addition of 0.8 mL of
Opti-MEM I-reduced serum medium. The mixture was then
overlaid onto the cells. The cells were incubated for 3 h
prior to addition of 1 mL of Opti-MEM I-reduced serum

Santa Cruz Biotechology (Santa Cruz, CA). A 200-fold
molar excess of unlabeled competitors over the radiolabe-
led probe was used for competition analyses. For super-
shift experiments, 2 lL of polyclonal rabbit anti-PDX-1
IgG or anti-Sp1 IgG (Santa Cruz Biotechnology) was
added to the nuclear extract, and preincubated on ice for
15 min prior to the addition of radiolabeled probe.
ChIP analysis
ChIP assay was performed using chromatin immunoprecipi-
tation assay kit (Upstate, Lake Placid, NY) according to the
manufacturer’s instruction. Anti-Sp1 IgG was purchased
from Upstate, whereas anti-Sp2, anti-Sp3 and anti-Sp4 IgGs
were obtained from Santa Cruz Biotechnology. PCR was
performed to amplify the region between ) 105 and )9 relat-
ive to the transcription start site of exon 1a in DNASE1
using primers DN)105 and DN)9, the sequences of which
were 5¢-CCAGCCTGGCTGGTTATCAGTCC-3¢ and 5¢-
GAGCTCTTCCACACCAGACGCA-3¢, respectively. Con-
ditions for these amplifications were 95 °C for 9 min, 37
cycles of 94 °C for 1 min, 65 °C for 1 min, and 72 °C for
2 min, followed by incubation at 72 °C for 10 min. The PCR
products were electrophoresed through a 2% agarose gel and
were stained with ethidium bromide. The sequences of the
amplified fragments were determined as described above.
Enzyme assay
Enzyme activity of DNase I was determined by the single
radial enzyme diffusion (SRED) method as described previ-
ously [7]. The human specific DNase I activity in the med-
ium secreted from QGP-1 cells was calculated by
subtraction of bovine DNase I activity in the fresh medium

260-nm. For each assay, a standard curve was prepared
using serial dilutions of template plasmid DNA with known
copy numbers in log steps from 2 · 10
7
copies to 2 · 10
2
copies in a 2-lL volume. All samples to be compared were
run in the same assay. After completion of the PCR ampli-
fication, the data were analysed with the lightcycler
ver.3.5 (Roche). The threshold cycle was calculated using
the sequence detection software as the cycle number at
which the fluorescence of the reporter dye crossed the
threshold in log-linear range of PCR. The copy numbers of
the respective DNASE1 cDNA were quantified by interpo-
lating the results from the threshold cycles.
Acknowledgements
This work was supported in part by Grant-in-Aid for
Scientific Research from the Ministry of Education, Sci-
ence, Sports and Culture, Japan (15209023, 17659196 to
TY, 17659161 to MU and 16209023 to KK).
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