ABSTRACT
Deoxycytidylate deaminase is an allosteric enzyme whose
impairment can lead to deoxynucleotide imbalances that affect the
fidelity of DNA synthesis. A DNA fragment encompassing the gene for
deoxycytidylate deaminase has been isolated from a human lung
fibroblast genomic library and sequenced in both directions through
26,764 base pairs. The previously isolated cDNA, which was used to
establish the amino acid sequence for this enzyme (Weiner, K. X. B.,
Weiner, R. S., Maley, F., and Maley, G. F.(1993) J. Biol. Chem. 268, 12983-12989) was instrumental in isolating this gene.
The gene consists of five exons of about 100 base pairs each, separated
by four introns. The most striking feature of the genomic structure is
that the second and third exons are separated by an intron of about 20
kilobases. The chromosomal location of the deaminase gene was
determined by fluorescence in situ hybridization as 4q35,
which is the extreme end of this chromosome. The position of this gene
on chromosome 4, in addition to the role of its product in limiting
potentially detrimental mutations, suggests that the normal operation
of both the gene and its product is important to the well being of the
organism.
Deoxycytidylate deaminase (EC 3.5.4.12) catalyzes the
deamination of dCMP to dUMP, a reaction that provides the nucleotide
substrate for thymidylate synthase (EC 2.1.1.45). The activity of the
enzyme is allosterically regulated by the ratio of dCTP to dTTP not
only in eukaryotic cells but also in T-even phage-infected Escherichia coli, with dCTP acting as an activator and dTTP an
inhibitor (Maley and Maley, 1972; Maley and Maley, 1990). Since the
deaminase and the synthase are elevated in rapidly dividing cells, such
as those associated with tumors, embryos (Maley and Maley, 1959), and
regenerating liver (Maley and Maley, 1960), a role for these enzymes in
DNA replication is clearly implied. Evidence supporting this thesis has
been obtained in studies with cells that are deficient in the deaminase
(Bianchi et al., 1987; Robert de Saint Vincent et
al., 1980), which results in altered intracellular dCTP and dTTP
pools and an increase in mutation rate (Sargent and Mathews, 1987;
Meuth, 1989; Kohalmi et al., 1991). Because of the potential
significance of this enzyme to the fidelity of replication and its role
in providing substrates for DNA synthesis, dCMP deaminase might serve
as a useful target for inhibitors that complement the inhibition of
thymidylate synthase.
We have recently cloned the human dCMP
deaminase cDNA and expressed the functional protein in E. coli (Weiner et al., 1993) to about 30% of its cellular
protein. In this report we have used this cDNA to isolate the deaminase
gene from a human lung fibroblast genomic library. This information
should provide further insight into the regulation of this important
enzyme at the genomic and perhaps translational levels of expression,
where in the latter case preliminary data have been obtained indicating
that the deaminase may autoregulate its own synthesis (
)similar to that described for thymidylate synthase (Chu et al., 1991).
EXPERIMENTAL PROCEDURES
Materials
The human lung fibroblast (WI38)
genomic library, which was a partial Sau3A1 digest inserted at
the XhoI site of the
FIX II vector, was purchased from
Stratagene (Palo Alto, CA). Although the XhoI restriction
site was not reformed by the insertion, inserts could, however, be
removed with NotI. Hybond-N nylon membranes
([
-
P]dCTP and
[
-
P]dATP) were from Amersham Corp.
Oligonucleotide primers were synthesized at the molecular genetics core
facility of the Wadsworth Center using a Millipore model 8750 DNA
synthesizer.
Isolation of Human dCMP Deaminase Gene
Clones
Screening of the human lung fibroblast genomic library (1
10
recombinants) was performed according to
standard procedures (Benton and Davis, 1977; Sambrook et al.,
1989). Phage plaques were lifted in duplicate onto nylon membranes,
denatured in 0.5 N NaOH, 1.5 M NaCl, and neutralized
in 1.5 M NaCl, 1.0 M Tris-HCl, pH 7.5. The membranes
were baked for 2 h at 80 °C and prehybridized for 2 h at 42 °C
in 6
SSC (0.15 M NaCl, 0.015 M sodium
citrate, pH 7.0) containing 5
Denhardt's solution
(Sambrook et al., 1989), 1% SDS, 50% formamide, and 10%
dextran sulfate. Hybridization was continued for 20 h in the same
buffer with 0.2 mg/ml salmon sperm DNA and
P-labeled
pBluescript (Stratagene) (1
10
cpm/filter)
containing the protein coding region of dCMP deaminase (designated
pCD12 in Weiner et al.(1993)). Positive clones were
plaque-purified, and phage DNA was isolated as described previously
(Maniatis et al., 1978; Yamamoto et al., 1970) and
analyzed by Southern blotting and DNA sequence analysis.
DNA Isolation and Southern Blotting Analysis
Human
genomic DNA was isolated essentially as described by Blin and
Stafford(1976). DNA was digested to completion with various restriction
enzymes (10 units/µg of DNA) for 2 h at 37 °C. Reaction
mixtures were concentrated in a Speed-Vac concentrator (Savant). The
restricted DNA was then electrophoresed through a 1% agarose gel using
the CHEF II pulse field apparatus (Bio-Rad) in 0.5
TBE (45
mM Tris borate, 45 mM boric acid, 1 mM EDTA)
gel running buffer for 22 h with a 1-6-s ramped pulse time. DNA
was transferred to Hybond-N nylon membranes essentially as described by
Southern(1975) and probed with
P-labeled pCD12 cDNA.
Hybridization conditions were as described previously (Weiner et
al., 1990). Autoradiograms were exposed to Kodak X-OMat film with
intensifying screens for 8 days at -70 °C.
DNA Sequence Analysis
The dCMP deaminase genomic
clones
CD7,
CD21, and
CD23 were digested with either SacI or XbaI to generate a series of overlapping
fragments and subcloned into the pBluescript vector. The nucleotide
sequence of these fragments was determined by the Sanger dideoxy chain
termination method (Sanger et al., 1977) using synthetic
18-21-mer oligonucleotides (Sequenase, U. S. Biochemical Corp).
The various DNA fragments were linked as described in the DNASIS manual
(Hitachi America, Ltd.).
Localization of the Deoxycytidylate Deaminase Gene by
Fluorescence in Situ Hybridization
The chromosomal location of
this gene was performed by Bios Laboratories Inc., New Haven, CT. A
genomic clone (
CD21) containing three of the exons of dCMP
deaminase (Fig.1) was labeled with digoxigenin dUTP by nick
translation. The labeled probe was combined with 0.33 µg/µl
sheared human DNA and hybridized to normal human metaphase cells in a
solution containing 50% formamide, 2
SSC, and 10% dextran
sulfate. Normal human metaphase cells were prepared from
phytohemagglutinin-stimulated and bromodeoxyuridine-synchronized
peripheral blood lymphocytes. Hybridization signals were detected with
anti-digoxigenin fluorescein isothiocyanate; chromosomes were then
counterstained with propidium iodide and analyzed. This experiment
resulted in specific labeling of the most terminal portion of the long
arm of a B-group chromosome. In order to distinguish whether this gene
is located on chromosome 4 or 5, a probe was prepared that cohybridized
specifically with the centromere of chromosome 4 and the genomic clone.
A total of 90 metaphase cells were examined, 59 of which showed
specific labeling of chromosome 4. This experiment clearly demonstrated
that the centromere of chromosome 4 was labeled and that the gene which
encodes dCMP deaminase is localized to band 4q35.
Figure 1:
Organization of introns and exons in
the deoxycytidylate deaminase gene. The location and size of the
CD DNA fragments used to obtain the sequence of the gene are
shown. For further details see ``Experimental
Procedures.'' The numberedbars indicate the
location of the CD exons, while the letteredregions present the intron regions. See Table1for the number of
base pairs in each.
RESULTS AND DISCUSSION
Restriction Fragment Analysis of the Human Fibroblast
Genomic Library
The genomic library from human lung fibroblasts
was restricted with BamHI, XbaI, EcoRI, and HindIII/BglII, and following pulse field
electrophoresis in 1.0% agar it was subjected to a Southern analysis
(Southern, 1975) using
P-labeled human deaminase cDNA
(pCD12) as the probe (Weiner et al., 1990). Each digest
yielded two radioactive bands, the first about 10-11 kb (
)and the second varying from 8.1 for BamHI, 5.7
for XbaI, 3.0 for EcoRI, and 1.6 kb for HindIII/BglII. From what is known now about the DNA
sequence of the CD gene and its restriction map, the two bands result
from exon clustering at the 5`-end and 3`-ends of the gene,
respectively, with a large intron interspersed (Fig.1).
Isolation of dCMP Deaminase Gene Clones
About 1
10
recombinants from the human lung fibroblast
genomic library were screened with pCD12 (Weiner et al.,
1993), which yielded three positive clones,
CD2,
CD7, and
CD21. The phage DNA was isolated from each clone, digested with NotI to release the insert, and analyzed by pulse field gel
electrophoresis (data not shown). The sizes of the released inserts
were 15 kb for
CD2, 17 kb for
CD7, and 17 kb for
CD21,
respectively.Southern blot analysis was used to determine whether
these three clones overlapped. The phage DNA from all three clones was
digested with NotI, and the resulting restriction fragments
were separated by pulse field electrophoresis on a 1% agarose gel and
probed with various oligonucleotides to span the entire length of the
deaminase cDNA. The results obtained revealed that
CD7 encompassed
exons associated with the 5`-portion of the deaminase gene, and
CD21 contained exons associated with the 3`-end of the deaminase
gene.
CD2 appeared to be included entirely within
CD21.
Although
CD7 and
CD21 did not overlap, further probing of the
genomic library yielded another clone,
CD23, which enabled the
complete sequence to be obtained since it overlapped
CD7 and
CD21 (Fig.1). The size of
CD23 was 17 kb, but Fig. 1only depicts the region sequenced.
Structural Organization of the dCMP Deaminase
Gene
Structural analysis of the three genomic clones revealed
the deaminase gene to encompass about 26 kb of DNA. As shown in Table1and Fig. 1the gene contains five exons and four
introns, with the introns located within the protein-coding region of
the deaminase. Interestingly, intron 2 is quite large, containing 20 kb
of DNA, which separates exon 2 from exon 3. All of the exons are quite
small, averaging about 100 base pairs each, and are separated by
introns containing their characteristic GT-AG signatures (Table1).An analysis of the potential transcription factor
binding sites within the 5`-flanking region of the deaminase gene as
shown in Table2revealed potential TATA and CAATT box binding
motifs, as well as several potential AP-1 (Fos-Jun protein complex),
SP-1 (GC box binding protein), and CTF/NF-1 DNA-binding sites (Faisst
and Meyer, 1992). There are also three potential cAMP response elements
as well as four half-sites for interaction with the estrogen receptor.
Whether these DNA binding motifs function in vivo as
cis-acting elements remains to be determined.
Chromosomal Location of the Deaminase Gene
A dCMP
deaminase probe (pCD21) was used to define the chromosomal locus of the
deaminase gene, which was located at 4q35, the very end of this
chromosome (Fig.2). Deletions in this region may therefore have
grave consequences for the organism. However, until such time as
``gene knockout'' experiments are performed, it will not be
possible to know the extent to which deletions in this gene affect the
organism. These studies are currently in progress.
Figure 2:
Chromosomal location of the
deoxycytidylate deaminase gene as determined by fluorescence in
situ hybridization.