Isolation and Characterization of the Mouse Ornithine Decarboxylase Gene*

Mouseornithine decarboxylase (ODC) genomic clones were isolated from a bacteriophage X genomic library representing mouse myeloma 653-1 cells which overproduce ODC due to amplification of an active ODC gene. Sequence analysis of the amplified ODC gene revealed that ODC mRNA is encoded by 12 exons, 10 of which (exons 3 to 12) code for the ODC protein. Exon 12 also corresponds to the 3‘ noncoding region of the two species of ODC mRNA which are formed by alternative utilization of two polyadenylation signals separated from each other by 422 nucleotides. The transcription initiation site was mapped by S1 nuclease protection and by primer extension analysis. The 5’ flanking region is extremely rich in G+C and contains typical promoter motifs such as the TATA box and SP1 transcription factor binding sites. Joining the 5’ flank- ing region to the Escherichia coli chloramphenicol acetyltransferase structural gene and its introduction into mouse cells resulted in the expression of a high level of chloramphenicol acetyltransferase activity. Compar-ing the sequence of the ODC gene to our previously published sequence of ODC cDNA revealed a disagreement between the sequences located 5’ to the AuaI site and demonstrated that this region of our previously reported cDNA represents a cloning artifact. The portion of the correct 5’ noncoding region encoded by exon 1 is extremely rich in G+C and includes potential secondary structures which may be involved in transla- tional regulation of ODC mRNA. Ornithine


Isolation and Characterization of the Mouse Ornithine Decarboxylase Gene*
(Received for publication, October 22,1987)

Arieh Katz and Chaim Kahana
From the Department of Virology, The Weizmann Institute of Science, Rehovot 76100, Israel Mouseornithine decarboxylase (ODC) genomic clones were isolated from a bacteriophage X genomic library representing mouse myeloma 653-1 cells which overproduce ODC due to amplification of an active ODC gene. Sequence analysis of the amplified ODC gene revealed that ODC mRNA is encoded by 12 exons, 10 of which (exons 3 to 12) code for the ODC protein. Exon 12 also corresponds to the 3' noncoding region of the two species of ODC mRNA which are formed by alternative utilization of two polyadenylation signals separated from each other by 422 nucleotides. The transcription initiation site was mapped by S1 nuclease protection and by primer extension analysis. The 5' flanking region is extremely rich in G+C and contains typical promoter motifs such as the TATA box and SP1 transcription factor binding sites. Joining the 5' flanking region to the Escherichia coli chloramphenicol acetyltransferase structural gene and its introduction into mouse cells resulted in the expression of a high level of chloramphenicol acetyltransferase activity. Comparing the sequence of the ODC gene to our previously published sequence of ODC cDNA revealed a disagreement between the sequences located 5' to the AuaI site and demonstrated that this region of our previously reported cDNA represents a cloning artifact. The portion of the correct 5' noncoding region encoded by exon 1 is extremely rich in G+C and includes potential secondary structures which may be involved in translational regulation of ODC mRNA.
Ornithine decarboxylase (ODC;' EC 4.1.1.17) is the first and a key enzyme in the biosynthesis of polyamines in mammalian cells, since it provides the only route for de nouo synthesis of putrescine. Accumulating evidence suggests that ODC may be regulated at several control levels: production of mRNA (1-5), changes in the translatability of the mRNA (6, 7), changes in protein stability (8), and by post-translational interactions and modifications (6,7, [9][10][11][12]. High ODC activity and elevated levels of polyamines are characteristic of rapidly * This work was supported in part by Grant 85-00093/1 from the United States-Israel Binational Foundation, Jerusalem, Israel, by a grant from the Israel Cancer Research Fund, by a Fund for Basic Research administrated by the Israeli Academy of Sciences and Humanities, and by a grant from the Leo and Julia Forchheimer Center for Molecular Genetics at the Weizmann Institute of Science. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "adoertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. The nucleotide sequence(s) reported in thispaper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) 503 733. The abbreviations used are: ODC, ornithine decarboxylase; CAT, chloramphenicol acetyltransferase; kb, kilobase pairs. proliferating cells. Pharmacological and genetic studies demonstrated that growth-related cellular functions require sufficient intracellular concentration of polyamines; in fact, deficiency results in cessation of growth (13)(14)(15)(16)(17)(18). We have recently demonstrated that mitogenic induction of quiescent BALB/c 3T3 mouse fibroblasts results in transcriptional activation of an ODC gene and consequently in accumulation of ODC mRNA ( 5 ) . To further explore the mechanisms which regulate ODC at the transcriptional level, we have isolated ODC genes from a X phage genomic library representing mouse myeloma 653-1 cells (2) which massively overproduce ODC due to amplification of an ODC gene. The gene analyzed in the present study corresponds to the gene amplified in 653-1 cells. This ODC gene consists of 12 exons and 11 introns, the boundaries of which obey the gt-ag splice rule (19). Its sequence demonstrated that 10 of the exons represent coding sequences. The transcription initiation site was determined by S1 nuclease protection analysis and primer extension analysis. The sequence upstream to the AuaI site in exon 1 showed disagreement with the parallel region of our previously reported ODC cDNA (6). This together with the failure of that region of the cDNA to hybridize to ODC mRNA demonstrated that it represents a cloning artifact. The portion of the 5"noncoding region encoded by exon 1, is extremely rich in G+C, and computer analysis demonstrated potential formation of secondary structure which may be involved in regulating the translation of ODC mRNA. The region upstream to the transcription initiation site contains typical promoter motifs such as a TATA box and CCGCCC SP1 transcription factor binding sites (20,21). Fusing of this upstream region in front of the bacterial chloramphenicol acetyltransferase coding region and its introduction into mouse cells directed the synthesis of a chloramphenicol acetyltransferase protein, demonstrating its function as an active promoter. Exon 12 which contains the translation termination TAG triplet, contains also two AATAAA polyadenylation signals separated from each other by 422 nucleotides, whose alternative usage accounts for the two species of ODC mRNA observed in mouse cells (1-4, 22). Moreover, since both species of mRNA are overproduced in 653-1 cells and only one ODC gene was amplified in these cells, we conclude that both species are encoded by a single ODC gene.

EXPERIMENTAL PROCEDURES
Cloning of the ODC Gene-High molecular weight DNA from 653-1 cells was prepared (33) and partially digested with EcoRI to yield fragments of about 20 kb which were then dephosphorylated and ligated into the EcoRI site of the X-vector EMBL 4 (34). After packaging, recombinant phages were plated on the restrictive strain NM539 and screened with ODC cDNA as hybridization probe.
Analysis of DNA-Cellular DNA and DNA of the isolated clones were digested with restriction endonucleases, fractionated by electrophoresis in 1% agarose, transferred to nitrocellulose, and hybridized to radioactive probes as before (2, 11).
DNA Sequence Analysis-The insert of the X clone mODC-gl was split into three primary subclones representing the 6.7-kb and 14-kb EcoRI fragments and a 1.1-kb BamHI fragment overlapping the two EcoRI fragments. The two EcoRI subclones were further dissected to generate smaller overlapping subclones. The subclones were in the Bluescript plasmid (Stratagene) which enabled the isolation of singlestranded plasmid DNA upon infection of plasmid containing cells with bacteriophage M13. Each subclone was linearized at either end and subjected to a limited digestion with Ex0111 nuclease followed by S1 nuclease to generate a non-random series of deletions (23). Singlestranded DNA of the resulting deletions was hybridized to a 17-base pair sequencing primer and sequenced by the dideoxy chain termination method (24)(25)(26). Each nucleotide was sequenced a t least twice.
SI Nuclease Analysis-5-pg portions of total cellular RNA isolated from 653-1 cells by the guanidinium thiocyanate extraction (35) were hybridized with 5' end-labeled or uniformly labeled probes in 15 pl of buffer containing 80% formamide, 0.4 M NaCI, 40 mM 1,4-piperazinediethanesulfonic acid (pH 6.4), and 1 mM EDTA. After 12 h of hybridization at 60 "C the sample was diluted with 300 pl of ice-cold buffer containing 40 mM potassium acetate (pH 4.5), 2.5 mM ZnCln, 300 mM NaCl, 20% glycerol, and digested with 250 units of S1 nuclease (Pharmacia) for 30 min a t 30 "C. Protected fragments were resolved by electrophoresis in polyacrylamide-urea denaturing gel and visualized by autoradiography. End-labeling was performed by kinasing dephosphorylated genomic fragment with [-pR2P]ATP (5000 Ci/mmol) and T4 polynucleotide kinase. Uniformly labeled fragments were prepared by synthesizing the complementary strand using single-stranded template plasmid, sequencing primer, and Klenow enzyme in the presence of [a-"PIdCTP (600 Ci/mmol).
Primer Extension Analysis-A synthetic oligonucleotide complementary to nucleotides 113 to 130 of exon 1, corresponding to the 5' noncoding region of ODC mRNA, was end-labeled with [y"P]ATP and T4 polynucleotide kinase. 100 pg of total cellular RNA from 653-1 cells were hybridized with 0.2 pmol of the "P-labeled oligonucleotide in 60 pl of solution containing 250 mM NaCI, 25 mM Tris-HC1 (pH 7.5) for 1 h at 55 "C. The hybridization mix was diluted with 2 milliliters of buffer containing 500 mM NaC1, 10 mM Tris-HCI (pH 7.5), 0.1% sodium dodecyl sulfate and chromatographed on oligo(dT)cellulose to select polyadenylated RNA and remove excess of nonannealed primer. Extension of the annealed primer was with reverse transcriptase (Bethesda Research Laboratories) in the presence of dNTPs or dNTPs plus dideoxy-NTPs when sequencing of the RNA was required.
Computer Adysis-Nucleotide sequences were compared to the sequences in the Los Alamos and European Molecular Biology Laboratory data banks using the algorithm of Wilbur and Lipman (36). Potential secondary structures in ODC mRNA were determined using the method of Zuker and Stiegler (37).

RESULTS AND DISCUSSION
Isolation and Sequencing of an Active ODC Gene-As reported previously, the mouse genome contains a family of ODC-related genes (2, 4, 11) of which only one can be positively identified as an active gene, since its amplification gave rise to a-difluoromethylornithine (a suicide inhibitor of ODC (14)) resistance in mouse plasmacytoma cells (2, 4). In order to isolate this active ODC gene, high molecular weight DNA from a-difluoromethylornithine-resistant 653-1 cells (2) was partially digested with EcoRI and ligated into bacteriophage X-EMBL 4. 500,000 plaques of the resulting library were screened with ODC cDNA as hybridization probe, yielding two types of hybridizing clones, both of which contain the 6.7-kb EcoRI fragment previously demonstrated as the amplified ODC DNA fragment in 653-1 cells (2, 11) (Fig. 1). In addition to the 6.7-kb fragment, the first type of clones contains a 14-kb EcoRI fragment also amplified in 653-1 cells (Fig. 1) and the second type contains a 9-kb EcoRI fragment equally represented in 653-1 cells and in the parental 653 cells (Fig. 1). Since the first group of clones corresponds to the ODC gene amplified in 653-1 cells, one of these clones designated mODC-gl was selected for sequence analysis. For this purpose, several overlapping subclones were prepared in the Bluescript plasmid (Stratagene), and each was subjected to shown in A were transferred to nitrocellulose and hybridized with a fragment of ODC cDNA spanning the region between AvaI and Sun which represents the first and second exons. C, total genomic DNA isolated from 653-1 cells ( l a n e 1 ) and from their parental cells ( l a n e 2) was digested with EcoRI, fractionated in 1% agarose gel, transferred to nitrocellulose, and hybridized with nick-translated DNA of phage mODC-gl (A, lane I ) . Marker molecular weights in kilobase pairs are indicated. limited unidirectional digestion with exonuclease I11 followed by S1 nuclease to generate a successive set of deletions (23). Single-stranded plasmid DNA was prepared from each deletion by infecting plasmid-containing cells with M13 bacteriophage and sequenced by the dideoxy chain termination method (24)(25)(26). Schematic representation of the ODC gene and its nucleotide sequence are presented in Fig. 2. As shown, the ODC gene consists of 12 exons and 11 introns which obey the gt-ag splice rule (19) (Table I). The first two exons and 16 nucleotides of the third represent the 5' noncoding region of ODC mRNA ( Fig. 2 and Table 11). The rest of exon 3, exons 4 through 11, and the first 142 nucleotides of exon 12 encode the ODC protein and are in full agreement with the nucleotide sequence of the coding region of ODC cDNA (11,27). The remaining part of exon 12 corresponds to the 3' noncoding region of the two ODC mRNAs detected in mouse cells (1-4) which as recently suggested (22) are formed by alternative utilization of two AATAAA polyadenylation signals separated from each other by 422 nucleotides (positions 6068 and 6490, Fig. 2). Since both ODC mRNAs are overproduced in 653-1 cells (not shown), we conclude that both are transcribed from the same ODC gene which is amplified in these cells. The nucleotide sequence of the ODC gene was compared to DNA sequences in the Los Alamos and EMBL data banks: no significant homology was detected except to previously published ODC cDNA sequences.

5'
End of the ODC Gene"S1 nuclease and primer extension analyses were used to map the transcription initiation site. Two DNA fragments were employed for S1 nuclease analysis. The first spans the region between the AvaI site in exon 1 (position 143) and a BamHI site located 2 kb upstream, the second extends from a BamHI site in the first intron (position 347) to the above-mentioned upstream BamHI site. The first fragment was uniformly and end-labeled, while the second was uniformly labeled only. When these fragments were hybridized with 653-1 RNA and treated with S1 nuclease, a 143- bp fragment was protected with the first probe (Fig. 3, lanes hybridized with 653-1 RNA, and the primer was extended 1 and 2) and a 196-bp fragment with the second probe (lane with reverse transcriptase in the presence of deoxy-and 3). To further establish that the 5' end of exon 1 represent dideoxynucleotides to obtain the sequence of the RNA, or in the transcription initiation site the 5' end of ODC mRNA the presence of deoxynucleotides only (Fig. 4, lanes 1 and 2, was mapped by primer extension analysis. A 32P-labeled oli-respectively), and the length of the extended products was gonucleotide complementary to nucleotides 113 to 130 was determined by fractionation in a denaturing gel. As shown in the figure, we were able to determine four transcription initiation sites clustered in a region of 13 nucleotides, the predominant site (accounting for 90% of the initiations) corresponds to a G residue and is numbered as position +1 in Fig.  2. The three other minor initiation sites are mapped to +5, -4, and -8 (Fig. 4). The region 5' to the transcription initiation site contains typical promoter motifs such as a TATA box at -26 and three CCGCCC transcription factor SP1 binding sites (28, 29) a t -108, -179, -328, and its inverted form GGGCGG at -207. The 5' flanking region is also rich in G+C, particularly between -85 and -335 (81%), and then its A+T content increases (Fig. 2). Interestingly, one CCGCCC spl binding site and two of its inverted forms GGGCGG are present also in exon 1 corresponding to the 5' noncoding region of ODC mRNA (positions 28, 98, and 111, respectively).

TABLE I Exon-intron junctions of the mouse ODC gene
The sequence presented was determined by sequencing the entire ODC gene (see Fig. 2). Exon and intron sequences are represented by capital and lower-case letters, respectively. IVS stands for intervening sequence. The numbers below each IVS refer to their length. Mapping of the transcription initiation site by S1 nuclease analysis. 5 -p g portions of total cellular RNA from 653-1 cells was annealed to the indicated probes, the RNA-DNA hybrids were digested with nuclease SI and fractionated by electrophoresis in denaturing polyacrylamide gel (see "Experimental Procedures"). The probes used were: I , 5' end-labeled AuaI-BamHI fragment; 2, uniformly labeled AuaI-BamHI fragment; 3, uniformly labeled BamHI-BamHI fragment (see diagram on the right). The molecular weights indicated on the left represent HpaII digest of pBR322 DNA. bp, base pairs. whether this upstream region displays promoter activity, a pstI fragment spanning the region between +9 and -1940 was joined to the coding region of the Escherichia coli chloramphenicol acetyltransferase (30) with position +9 adjacent to the 5' of the chloramphenicol acetyltransferase gene, and introduced into mouse NIH/3T3 cells by DNA-mediated gene transfection (31) together with the plasmid pCHllO (Pharmacia) which contain the LacZ coding region under the regulation of the SV40 early promoter. A simian virus 40 promoter-chloramphenicol acetyltransferase construct was used as promoter activity control plasmid. 48 hours post-transfection chloramphenicol acetyltransferase activity was determined in cellular extract after normalizing for transfection efficiency by monitoring P-galactosidase activity. As shown in Fig. 5, chloramphenicol acetyltransferase activity was monitored in the transfected cells. The availability of the ODC gene and its promoter region will serve as a major tool in Lane 3 displays the sequence of the DNA which was obtained using the synthetic oligonucleotide as sequencing primer. The nucleotide sequence of the region encompassing the initiation sites is presented on the right with the initiations marked by asterisks.  studies aiming at the identification and characterization of specific sequences and putative cellular factors which by interaction mediate the mitogenic activation of the ODC gene in mammalian cells. 5' Noncoding Region of ODC mRNA-Comparison between the sequence of exons 1, 2, and the beginning of 3 with that of the 5' noncoding region of our previously reported mouse ODC cDNA (11) revealed extensive disagreement in the region 5' to the AuaI site (position 138 in exon 1 and position -172 in the cDNA). Since in the present study the 5' end of ODC mRNA was carefully mapped, including by direct sequencing of the mRNA, and since a subclone of the cDNA representing the region 5' to the AuaI site failed to hybridize with ODC mRNA (not shown), we concluded that this region of the cDNA represents a cloning artifact. Computer analysis of the correct sequence of exon 1 predicted possible secondary structure in this region of the mRNA (Fig. 6). Of particular interest is the first hairpin (position 1 to 118) which displays high free stabilizing energy (-69.9 kcal/mol) and whose formation is preferred over interaction with any other region of the mRNA. In considering this observation together with previous studies which demonstrated that polyamines negatively affect ODC mRNA translation (6, 7, 32), it is tempting to speculate that polyamines exert their effect on the translatability of ODC mRNA by stabilizing such potential secondary structures. This possibility will be tested in cells and in uitro using an authentic full length ODC cDNA isolated during the course of the present study.