©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Chromosomal Location and Structural Organization of the Human Deoxycytidylate Deaminase Gene (*)

(Received for publication, May 10, 1995; and in revised form, June 15, 1995)

Karen X. B. Weiner (§) Joanna Ciesla (¶) Anita B. Jaffe Roy Ketring Frank Maley Gladys F. Maley (**)

From theWadsworth Center for Laboratories and Research, New York State Department of Health, Albany, New York 12201-0509

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

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.


INTRODUCTION

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 (^1)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 ([alpha-P]dCTP and [alpha-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 10^6 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 10^7 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 (^2)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 10^6 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.




FOOTNOTES

*
This work was supported in part by Grant CA44355 from the National Cancer Institute (to F. M.) and Grant MCB-9316321 from the National Science Foundation (to G. F. M.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBank®/EMBL Data Bank with accession number(s) L39874[GenBank].

§
Current address: SUNY Health Science Center, Dept. of Biochemistry and Molecular Biology, Syracuse, NY 13210.

Current address: Nencki Institute of Experimental Biology, Dept. of Cellular Biochemistry, 3 Pasteur St. 02-093, Warsaw, Poland.

**
To whom correspondence should be addressed: Wadsworth Center for Laboratories and Research, New York State Department of Health, P. O. Box 509, Albany, NY 12201-0509. Tel.: 518-474-9623; Fax: 518-473-2900.

^1
F. Maley and E. Chu, unpublished data.

^2
The abbreviation used is: kb, kilobase(s).


ACKNOWLEDGEMENTS

We thank Judith Valentino for excellent assistance in preparing this manuscript.


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©1995 by The American Society for Biochemistry and Molecular Biology, Inc.