Molecular Cloning of the Murine Adenosine Deaminase Gene from aGenetically Enriched Source: Identification and Characterization of
the Promoter RegionDIANE E. INGOLIA,' MUAYYAD R. AL-UBAIDI,' CHO-YAU YEUNG,'t HELENE A. BIGO,2 DAVID A.
WRIGHT,2 AND RODNEY E. KELLEMSl*The Verna and Marrs McLean Department of Biochemistry, Baylor College of Medicine,1 and Department of Genetics,
The University of Texas M. D. Anderson Hospital and Tumor Research Institute,2 Houston, Texas 77030
Received 23 June 1986/Accepted 2 September 1986
A genomic library was prepared with DNA from a genetically enriched mouse cell line in which amplifiedcopies of the adenosine deaminase (ADA) gene account for over 5% of the genome. Overlapping cosmid clonesencompassing the entire ADA structural gene were isolated from this genomic library and used for subsequentstructural and functional analyses. Nuclease protection and primer extension analyses served to identify thelocation of multiple transcription initiation sites at the 5' end of the structural gene. Promoter activity wasfound by functional analyses to reside within a 240-base-pair fragment which contains the transcriptioninitiation sites. Sequences upstream of the transcription initiation sites are very G+C rich (77%) and includea 22-nucleotide stretch of deoxyguanylate residues and two potential Spl transcription factor-binding sites.Comparison of the mouse and human ADA gene promoters revealed the presence of several regions that arehighly conserved with regard to both sequence content and location and may represent genetic elements whichare involved in ADA gene expression.
Adenosine deaminase (ADA; EC 3.5.4.4) is an enzyme ofpurine metabolism that is present in virtually all mammaliantissues. The level of enzyme activity varies substantially in atissue-specific manner and in certain tissues is subject todevelopmental regulation. The highest activities are found incells of the T-lymphocyte lineage in which ADA levels aredevelopmentally controlled. Enzyme activity peaks at thecortical thymocyte stage of T-cell development and declinesas T-cell maturation proceeds (1, 22, 26). Pharmacologicallyinduced ADA deficiency in rats blocks the proliferation anddifferentiation of subcapsular cortical thymocytes which arethe precursors of the cortical thymocytes (2). In humans thegenetic deficiency of ADA is associated with a form ofs-vere combined immunodeficiency disease which is char-acterized by a lack of functional B and T lymphocytes (17,41). Together these data provide evidence for the essentialrole that ADA plays in T-lymphocyte development. ADAlevels are also developmentally regulated in gastrointestinaltissues. The level of enzyme activity in the stomach andintestine of mice is very low at birth, increases dramaticallywithin the first 2 weeks of life, and achieves relatively highlevels in the adult animal (30). Within the brain certainhypothalamic neurons have elevated levels of ADA activity(40). These neurons are also enriched in adenosine uptakesites and may play a role in adenosinergic neurotransmissionpathways. In most other tissues ADA levels are quite low,with the lowest levels occurring in liver, lung, and fetalplacental tissues (7, 30, 49). An elevated level of the enzymein erythrocytes is a dominantly inherited condition which isassociated with a form of hemolytic anemia in humans (39,47). Thus, the pattern of ADA expression has the followinginteresting characteristics. (i) The enzyme is expressed invirtually all types of cells; (ii) the extent of expression varies
* Corresponding authort Present address: Center for Genetics, The University of Illinois
at Chicago, Chicago, IL 60612.
over 1,000-fold in a tissue-specific manner; and (iii) the levelof expression is developmentally regulated in more than onetype of cell or tissue.The pattern ofADA gene expression is unlike that of most
other genes characterized to date, the majority of which fallinto two classes. One class includes the globin, ovalbumin,and immunoglobulin genes which are expressed at highlevels in a restricted set of terminally differentiated celltypes. The other class of genes consists of those such asdihydrofolate reductase and thymidine kinase, which areexpressed at relatively low levels in essentially all cell types.ADA falls into a third category because it is expressed in alltissues but shows a level of expression that varies substan-tially among different cell types. Thus, the parameters con-trolling ADA expression may be quite different from thosecontrolling the expression of most genes currently underinvestigation.To address a number of questions regarding ADA gene
structure, expression, and developmental regulation, it isnecessary to have molecular clones of the structural gene.As a first step toward achieving this objective, we isolatedmammalian cells with amplified copies of the ADA gene (52).In the most highly drug-resistant cell lines, ADA levels areelevated approximately 11,000-fold relative to those in theparental cells, and the enzyme accounts for over 75% of thesoluble protein (23). These cell lines have enabled us topurify large amounts of the enzyme to homogeneity, preparemonospecific antisera, and obtain full-length and functionalcopies of the ADA cDNA (23, 53). Here we report themolecular cloning of the ADA structural gene from a genet-ically enriched source and the structural and functionalcharacterization of the promoter region.
MATERIALS AND METHODSMaterials. Alanosine was obtained from the Drug Synthe-
sis and Chemistry Branch, and 2'-deoxycoformycin wasobtained from the Natural Products Branch, Division of
4458
MURINE ADENOSINE DEAMINASE GENE 4459
Cancer Treatment, National Cancer Institute. Restrictionendonucleases, Klenow fragment, and calf intestinal alkalinephosphatase were from Boehringer Mannheim Biochemic-als, Indianapolis, Ind. T4 polynucleotide kinase was fromNew England BioLabs, Inc., Beverly, Mass. Avian myelo-blastosis virus reverse transcriptase was from Life Sciences,Inc., St. Petersburg, Fla. SP6 polymerase, RNasin, and theplasmid vector pSP64 were from Promega Biotech, Madi-son, Wis. The cosmid vector pCV108 was a gift of Y.-F. Lauand Y. W. Kan (29). The plasmids pSVOCAT and pSV2CATare described by Gorman et al. (19). The MOLT-4 cell line(GM2219B) was obtained from the Human Genetic MutantCell Repository.
Cel lines and culture conditions. B-1 cells were derivedfrom LMTK- Cl-iD mouse cells and selected for resistanceto 1.1 mM adenosine-0.05 mM alanosine-1 mM uridine and2'-deoxycoformycin as previously described (51). The num-ber following the B-1 designation indicates the level ofresistance to 2'-deoxycoformycin in micromolar concentra-tion. The B-1/50 and B-1/100 cells overproduce ADA 4,300-and 7,900-fold, respectively, relative to the parental Cl-iDcell line (23). Maintenance and harvest of these mouse celllines were as described previously (52). MOLT-4 cells are ahuman T-lymphoblastoid line (38). MOLT-4 cells weregrown in RPMI 1640 medium containing 10% horse serum.JEG-3, a human choriocarcinoma cell line (27), and VA-2, ahuman fibrosarcoma cell line (42), were maintained inDulbecco modified Eagle medium supplemented with 10%horse serum.
Construction of cosmid library and isolation of genomicclones. High-molecular-weight DNA was extracted fromB-1/100 cells and partially digested with MboI by the proto-col of Dillela and Woo (12). The partially digested DNA wasphosphatase treated and inserted into the BamHI site of thecosmid vector pCV108, using a modification of the proce-dure of Ish-Horowicz and Burke (24). The modification liesin the preparation of the cosmid cloning arms which wasdone by using ClaI-BamHI and SalI-BamHI digestions ofthe vector. The cosmid library was plated and colonyhybridization was performed as described previously (12).The probe used to screen the library was a mixture ofuniformly labeled, single-stranded RNA probes generated bythe in vitro transcription of the 310-base-pair (bp) 5' PstI andthe 1-kilobase (kb) 3' PstI fragments of the mouse ADAcDNA pADA5-29 (53) inserted into the PstI site of SP64.The plasmids were linearized with EcoRI before transcrip-tion which was done by the protocol supplied by PromegaBiotech. The cosmid clones were subcloned by isolating thedesired restriction fragments on low-melting-temperatureagarose gels and ligating them into appropriate vectors (10).DNA blot analysis. DNA isolated from the B-1/100 cell line
or purified cosmid DNA was digested with EcoRI andfractionated on 0.8% agarose gels. The gel was transferred tonylon membranes in 0.4 M NaOH by the method of Reedand Mann (43). The filter was probed with labeled plasmidDNA prepared by the procedure of Feinberg and Vogelstein(16). Prehybridization, hybridization, and washing of thefilter were performed at 68°C as described by Reed andMann (43).
Isolation of RNA. Total cytoplasmic RNA was preparedfrom Cl-iD and B-1/50 cells as previously described (52).RNA from the MOLT-4 cells was prepared by the aboveprotocol with the following modification. The cell pellet wassuspended in 7 volumes of 0.01 M Tris hydrochloride (pH7.4)-0.01 M NaCl-1.5 mM MgCl2 and was homogenized byfour strokes in a loose-fitting Dounce homogenizer.
Poly(A)+ RNA was prepared by oligo(dT)-cellulose chroma-tography (6), with the modification that KCI was usedinstead of NaCl to precipitate the poly(A)+ RNA. TotalRNA was extracted from Xenopus laevis oocytes by themethod of Gurdon and Wickens (21).
Si nuclease analysis. Si nuclease assays were performedessentially as described previously (4). A 240-bp EcoRI-NcoI fragment was 5' end labeled by labeling the Ncol sitesof the subcloned 2.7-kb NcoI fragment with T4 polynucleo-tide kinase. The labeled 2.7-kb NcoI fragment was thendigested with EcoRI, and the 240-bp EcoRI-NcoI fragmentwas isolated from a 5% polyacrylamide gel. Total cytoplas-mic RNA (40 to 160 ,ug) from either Cl-iD or B-1/50 cells washybridized to 0.25 to 0.35 ,ug of end-labeled DNA. Hybrid-izations were carried out in 80% formamide under conditionsofDNA excess at 58°C for 8 to 12 h. After digestion with 800to 1,000 U of Si nuclease per ml, the protected fragmentswere electrophoresed on 8% polyacrylamide-urea gels.DNA sequence analysis. The 240-bp EcoRI-NcoI fragment
was sequenced by the method of Maxam and Gilbert (34).Both strands of the fragment were sequenced.Primer extension. Synthetic oligonucleotide primers were
labeled with T4 polynucleotide kinase. The primer was thenhybridized to RNA for 1 h at 30°C in a final volume of 6.5 ,ulof 150 mM Tris hydrochloride (pH 8.3)-150 mM KCl-30 mMMgCl2-20 U of RNasin. After hybridization the followingwere added: 2 ,ul of each of the four deoxynucleosidetriphosphates (10 mM), 2 ,ul of actinomycin D (400 ,ug/ml), 12to 16 U of reverse transcriptase, and H20 to bring the finalvolume to 20 ,ul. The mixture was incubated at 46°C for 30min to 1 h. The DNA was ethanol precipitated and electro-phoresed on 8% polyacrylamide-urea gels.
Construction of plasmids pN2.7CAT, pXN.80CAT, andpEN.24CAT. The 2.7-kb NcoI genomic fragment was in-serted into the HindIlI site of pSVOCAT by blunt-endligation to generate the plasmid pN2.7CAT. pXN.80CATwas derived from pN2.7CAT by complete digestion ofpN2.7CAT with XbaI. The desired 5.3-kb fragment wasisolated on a low-melting-temperature agarose gel and circu-larized. Plasmid pEN.24CAT was derived from pXN.80CATby partial digestion of pXN.80CAT with EcoRI followed bycomplete digestion with NdeI. After the use of the Klenowfragment to fill in the EcoRI and NdeI ends, the desired4.7-kb fragment was isolated on a low-melting-temperatureagarose gel and then circularized to yield plasmidpEN.24CAT. This plasmid contains a 60-bp deletion ofpBR322 sequences located between the NdeI and HindIIIsites of pSVOCAT.Gene transfer. For transient expression experiments 20 ,ug
of each plasmid was introduced into JEG-3 and VA-2 cellsby the calcium phosphate precipitation technique (20) withthe following modifications. Transfections were carried outon monolayers that were 30 to 45% confluent, and the cellswere exposed to the DNA-CaPO4 precipitate for 12 to 18 h.For stable expression experiments, 18 ,ug of each plasmidwas cotransfected into the LMTK- Cl-iD cell lines with 2 ,ugof the construct pHC79-2cos/tk (31), which contains theherpesvirus thymidine kinase gene, by using calcium phos-phate precipitation as above. Stable transformants wereselected for by their ability to grow in medium containinghypoxanthine, aminopterin, and thymidine (32). The result-ant stable transformants were pooled in groups of 11 to 20independent colonies and grown in mass culture beforeharvest.CAT assays. Transfected cell lines were harvested and
assayed for chloramphenicol acetyltransferase (CAT) activ-
VOL. 6, 1986
4460 INGOLIA ET AL.
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FIG. 1. Visualization of amplified sequences in B-1/100 cells andDNA blot analysis of ADA cosmid clones isolated from a B-1/100library. (A) Genomic DNA from the B-1/100 (lanes 1 and 3) and theCl-1D (lanes 2 and 4) cell lines was digested to completion withEcoRI and fractionated on a 0.8% agarose gel. Lanes 1 and 2 contain5 ,ug of DNA; lanes 3 and 4 contain 10 ,ug of DNA. The gel wasstained with ethidium bromide and photographed with UV illumina-tion. (B) DNA blot analysis of the ADA gene in the B-1/100 cell lineand two ADA cosmid clones. Genomic DNA from B-1/100 cells andpurified cosmid DNA was digested to completion with EcoRI,fractionated on a 0.8% agarose gel, and transferred to a nylonmembrane. The blot was probed with radiolabeled pADA5-29, afull-length ADA cDNA. Lanes: 1, 1.1 ,ug of cGAM4.5 DNA; 2, 0.6,ug of cGAM2.4 DNA; 3, 7 jig ofDNA from the B-1/100 cells. Lane3 was exposed for twice the length of time as lanes 1 and 2. DNAconcentration was determined by the diphenylamine reactionmethod (45).
ity as described previously (19) with the exception that 0.5,uCi of [14C]chloramphenicol was used per assay. Assayswere performed with amounts of cell extract that gaveresults within the linear range of the reaction.Thymidine kinase assays. Cell extracts from transfected
Cl-1D cells were prepared as described above for CATassays and were assayed for thymidine kinase activity asdescribed previously (5).
MicroinJection of X. laevis oocytes. Oocytes were injectedas described previously (21) with the modification that thefollicles were removed from the oocytes before injection.After a 24-h incubation at 18°C, the injected oocytes weremechanically disrupted with a sterile pipette tip. The lysatewas cleared by centrifugation for 10 min at 4°C in a micro-centrifuge.
RESULTS
Isolation of the ADA gene from a genetically enrichedsource. A cosmid library was constructed with DNA fromthe B-1/100 cell line which contains amplified copies of theADA gene. This cell line contains enough amplified DNA toallow restriction enzyme fragments to be visualized asdistinct bands over the background smear of digested,nonamplified DNA (Fig. 1A). These repeated restrictionenzyme fragments are not seen in the parental Cl-1D cellline. The moderately repetitive, interspersed, 1.3-kb EcoRImouse repeat (8) is visible in both the parental and amplifiedcell lines. A rough estimate of 121 kb is obtained as the
FIG. 2. Restriction map of the mouse ADA gene. (A) Thelocations of the EcoRI sites (E) and two of the NcoI sites (N) areshown. Two overlapping cosmid clones which span the entirestructural gene are shown under the map. The location of the 0.6-kbEcoRI fragment marked by the asterisk has not been preciselydetermined owing to the large fragment sizes present at the 3' end ofthe ADA gene. The exon-containing EcoRI fragments are shown bythe bold lines. The thin lines designate fragments containing noncod-ing sequences. (B) Fine map of the 2.7-kb NcoI fragment. Thisfragment was subcloned into the NcoI site of pADA5-29 and into theHindIlI site of pSVOCAT. The translation start codon (ATG) isindicated.
minimum size of the amplification unit in the B-1/100 cell lineby summing the sizes of the bands seen upon digestion of theDNA with EcoRI.The cosmid library was screened with the extreme 5' and
3' PstI fragments of our full-length ADA cDNA, pADA5-29(53). Forty-three clones containing ADA gene sequenceswere isolated. The entire structural gene is contained on twooverlapping cosmid clones, cGAM2.4 and cGAM4.5. Asexpected, these two clones contain between them all three ofthe exon-containing EcoRI fragments seen when EcoRI-digested B-1/100 DNA is probed with the entire cDNA (Fig.1B, lane 3). Cosmid cGAM4.5 contains the 8.3- and 1.5-kbEcoRI fragments of the ADA gene (Fig. 1B, lane 1). The5.1-kb band seen in cGAM4.5 was shown to be a fragment ofthe 11-kb band by probing a similar blot with a portion of thecDNA specific for the 11-kb band (data not shown). CosmidcGAM2.4 contains the 11- and 8.3-kb EcoRI fragments of theADA gene (Fig. 1B, lane 2). The 8.3-kb band seen in bothcosmid clones appears disproportionately intense relative tothe 8.3-kb band seen in the B-1/100 DNA. This is due to thesignal contributed by hybridization of pBR322 sequences inthe probe to the linearized cosmid vector which is approxi-mately 8.1 kb in size and therefore migrates close to the8.3-kb EcoRI fragment of the ADA gene. When similar blotsare hybridized with probes devoid of pBR322 sequences,this increased intensity of the 8.3-kb band in EcoRI-digestedcosmid clones is not seen (data not shown). A restrictionmap of the ADA gene and the relative positions of thecosmids cGAM2.4 and cGAM4.5 are shown in Fig. 2A. Ourcollection of cosmid clones contains sequences that flank theADA structural gene by approximately 20 kb on either side(data not shown).ADA transcripts have multiple start sites which map within
a 240-bp EcoRI-NcoI fragment. To map the 5' boundary ofthe ADA gene, the 2.7-kb NcoI fragment immediately up-stream of the ATG start codon was subcloned. The restric-tion map of this NcoI fragment is shown in Fig. 2B. This
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FIG. 3. Identification of the transcription initiation sites of theADA gene. (A) SI nuclease analysis was performed with the 240-bpEcoRI-NcoI fragment which was 5' end labeled at the NcoI site. Theprobe was hybridized to increasing amounts of total cytoplasmicRNA from B-1/50 cells (lanes 2 to 4), treated with Si nuclease, andelectrophoresed as described in the text. Lanes 1 and 5 contain totalcytoplasmic RNA from Cl-iD cells and tRNA, respectively, hybrid-ized and Si treated and serving as negative controls. The amount ofRNA in micrograms used in each reaction is shown at the top ofeach lane. The Si-resistant hybrids are run alongside a Maxam-Gilbert sequencing ladder of the labeled probe. Arrows indicate thefive major transcription initiation sites. The number in parenthesesindicates the location of the cap sites along the sequence of the240-bp fragment. (B) Primer extension analysis was performed withthe 25-nucleotide primer described in the text. The primer waslabeled at the 5' end and hybridized to 20 ,ug of poly(A)+ RNA fromthe Cl-iD cells (lane 1) and 1 ,ug of poly(A)+ RNA from the B-1/50cells (lane 2). Primer extension was carried out, and the productswere electrophoresed as described in the text. Arrows designate thecap sites as described in panel A. The size markers are 5'-end-labeled 4X HaeIII fragments. M.W., Molecular weight.
fragment was 5' end labeled at the NcoI site and thencleaved with EcoRI to generate a 240-bp probe for Sinuclease protection experiments. This probe was hybridizedto total cytoplasmic RNA isolated from both the B-1/50 cellline and the Cl-iD cell line. The B-1/50 cell line containsamplified ADA genes and serves as an enriched source ofADA mRNA sequences. Along with tRNA, the nonamplifiedCl-iD cell line serves as a negative control in this experimentsince the abundance of ADA mRNA is very low in this cellline as determined by RNA blotting analysis (51). Afterhybridization and treatment with Si nuclease, the protectedfragments were run on a denaturing polyacrylamide gel witha sequencing ladder of the probe as the size standards. Thisprobe protected five relatively abundant fragments in anRNA-dependent manner (Fig. 3A, lanes 2 to 4).
Primer extension experiments were also performed tolocate the 5' termini of the ADA transcripts. A 25-nucleotideprimer complementary to nucleotide positions -53 to -77upstream of the ATG start codon (see Fig. 4) was 5' endlabeled and hybridized to poly(A)+ RNA isolated from both
B-1/50 and Cl-iD cells. The hybrids were extended, and theproducts were run on a denaturing polyacrylamide gel.Multiple primer extension products were seen, and theyshow a pattern very similar to that of the Si-resistantproducts (Fig. 3B). The primer extension products thatcorrespond to the two fastest-migrating Si-resistant prod-ucts run very close to the primer and are better visualized inFig. 6B, lane 5. Primer extension experiments were alsocarried out after the denaturation of the RNA template withmethyl mercury hydroxide (33) to minimize the effects ofsecondary structure in the RNA which may prevent full-length extension of the primer. No difference was seen inprimer extension experiments performed with RNA tem-plates treated with or without methyl mercury hydroxide(data not shown).The location of the 5' termini of the ADA messages
identified by both primer extension and Si mapping agrees towithin 3 nucleotides. The positions of the 5' termini of theADA mRNAs were assigned relative to the position that theSl-resistant fragments migrated along the sequencing ladder.It has been shown that Si analysis of the mouse P-globinmRNA produces Sl-resistant fragments that extend 4 to 5nucleotides upstream of the cap nucleotide (50). This pro-tection is postulated to be due to steric hindrance of Sidigestion of the probe by the cap structure. Therefore, theactual cap sites of the ADA mRNAs may be located severalnucleotides downstream of the 5' termini of the Sl-resistantfragments.The EcoRI-NcoI fragment containing the multiple tran-
scription start sites was sequenced. The sequence and thepositions of the transcription start sites are shown in Fig. 4.The sequences located around the multiple start sites arevery G+C rich, with a 71% G+C content for the entire240-bp EcoRI-NcoI fragment. The region upstream of thetranscription initiation sites contains no obvious CAAT orTATA boxes, which are found in the promoters of a numberof eucaryotic genes (3, 9). This EcoRI-NcoI fragment con-tains two decanucleotide sequences which match the con-sensus sequence for the transcription factor Spl-bindingsite,
G GGCTGGGCGGAAT
(25). This putative promoter fragment does not contain anydirect repeat elements greater than 9 bp in length. Twoimperfect inverted repeats are found; they are mutuallyexclusive. The most stable cruciform structure that could beformed by these inverted repeats is found between nucleo-tides -104 to -94 and nucleotides -71 to -60 (Fig. 4). Thiswould result in a hairpin structure with a i9-bp loop and an11-bp stem containing one mismatched base pair and a i-bploop.The 240-bp EcoRI-NcoI fragment has promoter activity. To
locate the sequences necessary for promoter activity, vari-ous amounts of 5'-flanking sequences of the ADA gene werefused to the bacterial CAT gene and tested for expression invivo. These constructs contain either 2.56 kb, 660 bp, or 100bp of sequences upstream of the major transcription initia-tion sites (Fig. 5A). In these constructs the CAT gene isunder the transcriptional control of the inserted ADA se-quences since the starting vector, pSVOCAT, lacks a pro-moter. The vector pSV2CAT, in which the CAT gene isunder the control of the simian virus 40 enhancer andpromoter, was used as a positive control in the transfectionexperiments. All three ADA-CAT constructs functioned
FIG. 4. Nucleotide sequence of the 240-bp EcoRI-NcoI fragment. The arrows pointing downward indicate the positions of the multipletranscription initiation sites. The size of the arrow indicates the relative abundance of each cap site. The boxes contain sequences fitting theSpl transcription factor-binding site consensus sequence. The dashed arrows indicate the imperfect inverted repeat described in the text. Thelocation of the 25-mer utilized in primer extension experiments is shown by the solid line. The nucleotides are numbered with the A of theATG codon as + 1. Negative numbers refer to upstream sequences.
equally well when transfected into a variety of human celllines under transient assay conditions (Fig. SB; Table 1). Thelargest construct, pN2.7CAT, was also introduced into themouse Cl-iD cell line with a thymidine kinase construct, andstable transformants were assayed. pN2.7CAT functionedapproximately 10-fold better than pSV2CAT in mouse cellsunder stable expression conditions (Table 1). In addition tostudies in cultured mammalian cell lines, all three ADA-CATconstructs were microinjected into X. laevis oocytes withsimilar results (Table 1; Fig. 6A). These various expressionstudies show that as little as 100 bp upstream of the majortranscription initiation sites are sufficient for promoter activ-ity. We did not make 3' deletions into the 240-bp EcoRI-NcoI fragment; therefore, we cannot rule out the contribu-tion of sequences downstream of the multiple start sites topromoter activity.To determine whether transcription initiated at appropri-
ate sites within the ADA promoter region of the ADA-CATconstructs, primer extension experiments were performedon total RNA extracted from injected Xenopus oocytes.Oocytes were utilized in preference to cultured mammaliancells for these studies because oocytes display a much higherlevel of transcriptional activity from exogenously addedtemplates, a feature which makes the transcription productseasier to detect. Furthermore, as shown above, the expres-sion of the ADA-CAT constructs in oocytes is qualitativelysimilar to that obtained after introduction of these constructsinto cultured mammalian cells. The five major transcription
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initiation sites utilized in the B-1/50 cells were also used inthe injected oocytes (Fig. 6B). In addition, there were twoupstream sites (located above the 118-bp marker in lanes 3and 4) utilized in injected oocytes that were not seen inprimer-extended B-1/50 mRNA. The frequency of utilizationof the various start sites is different in the injected oocytes ascompared with the B-1/50 cell line. Most notable are the startsites at positions -92 and -93, which are relatively minorsites in the B-1/50 cells, whereas they are the major startsites utilized in the injected oocytes. We can conclude fromthis experiment that these two constructs containing either2.56 kb or 100 bp of upstream sequences, when injected intoXenopus oocytes, produce similar amounts of mRNA whichinitiate, for the most part, at the same sites utilized incultured mouse cells, albeit with a preference for the -92and -93 initiation sites.Mouse and human ADA promoter fragments share regions
of homology. The sequence of the mouse ADA gene pro-moter located within the 240-bp EcoRI-NcoI fragment wascompared with an analogous 230-bp EcoRI-NcoI fragmentfrom the human ADA gene promoter (48) to search forconserved sequences which may constitute essential pro-moter elements. Alignment of the promoter regions fromthese two species revealed four areas of homology which areat least 10 bp in length and show at least 90% homology.These regions are shown boxed in Fig. 7. The spacingbetween these regions of homology was also conserved towithin 2 nucleotides.
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FIG. 5. Functional analysis of ADA gene sequences required for promoter activity. (A) ADA-CAT constructions. The 2.7-kb NcoIfragment was inserted into the HindIlI site of pSVOCAT to create pN2.7CAT. Successive deletions of this construct were made to generatepXN.80CAT and pEN.24CAT as described in the text. Abbreviations: E, EcoRI; N, NcoI; X, XbaI. (B) Assay of CAT activity in JEG-3 cellstransfected with the ADA-CAT constructions. A 20-,ug sample of each plasmid was transfected into JEG-3 cells. After 48 h cell extracts wereprepared and assayed for CAT activity. Lanes 1 to 5 show CAT activity in JEG-3 cells transfected with the plasmids pSV2CAT, pSVOCAT,pN2.7CAT, pXN.80CAT, and pEN.24CAT, respectively. The location of unacetylated chloramphenicol (CM) and the two monoacetylatedproducts, i-acetate chloramphenicol (1) and 3-acetate chloramphenicol (3), are shown.
MOL. CELL. BIOL.
,MURINE ADENOSINE DEAMINASE GENE 4463
TABLE 1. Expression of ADA-CAT constructions
CAT activity' in transient expression assays Stable expression in Cl-1D cellsPlasmid JEG-3b VAb2b Xenopus CAT TK CAT activity/
a Percent conversion of [14Clchloramphenicol per minute per milligram at 37'C.b Values given are the mean with the range in parentheses. Each point given represents a minimum of three separate transfection experiments.c For each plasmid a minimum of 10 injected oocytes were homogenized and assayed.d Percent conversion of [14C]TdR to [14C]d-TMP per minute per milligram at 37'C.' ND, Not determined.
Si mapping and primer extension analysis of the mouseADA gene showed that the two regions of homology nearestthe ATG codon (positions -22 to -32 and -92 to -114, Fig.7) are included on the majority of the mouse ADA mRNAs.The reported start site for the human ADA gene, located atnucleotide -95 (11, 48), excludes from human ADA mRNAthe region sharing 21 of 23 nucleotides of homology with themouse gene. It was surprising to us that this highly con-served region (-92 to -114 in the mouse gene and -98 to-120 in the human gene, Fig. 7) would be included in mouse
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FIG. 6. Expression of ADA-CAT constructions in Xenopusoocytes. (A) Assay of CAT activity in microinjected oocytes.Oocytes were injected with 3.5 ng of plasmid DNA. After a 24-hincubation period, extracts were prepared and assayed for CATactivity. Lanes 1 to 4 show CAT activity in extracts from oocytesinjected with pSVOCAT, pN2.7CAT, pXN.80CAT, andpEN.24CAT, respectively. (B) Primer extension analysis of tran-scripts from ADA-CAT constructions microinjected into oocytes.Total oocyte RNA (10 jig) from uninjected oocytes (lane 1) or
oocytes injected with pSVOCAT (lane 2), pN2.7CAT (lane 3), or
pEN.24CAT (lane 4) was subjected to primer extension analysis.Lane 5 contains the extension products from 1 jig of poly(A)+ RNAfrom B-1/50 cells. The arrows indicate the locations of the five majortranscription initiation sites seen in B-1/50 cells as described in thelegend to Fig. 3. The locations of the 118- and 72-bp 4X HaeIIIfragments are indicated. 3, 1, and cm are defined in the Legend toFig. 5.
ADA mRNA but be excluded from human ADA mRNA.However, we confirmed the location of the reported startsite for the human ADA gene by Si nuclease protectionanalysis. Total cytoplasmic RNA from the human MOLT-4cell line was hybridized to the 230-bp EcoRI-NcoI fragmentlocated immediately upstream of the ATG codon in thehuman ADA gene. A single protected fragment was seenwhose 5' terminus is located at nucleotide -95 (data notshown). Therefore, we conclude that the highly conservedregion located at nucleotides -92 to -114 in the mouse geneand -98 to -120 in the human gene (Fig. 7) is indeedexcluded from human ADA mRNA.
DISCUSSION
We isolated the mouse ADA structural gene, which spansapproximately 27 kb, and characterized the promoter regionboth structurally and functionally. The gene was isolatedfrom a cosmid library made with DNA extracted from a cellline, B-1/100, which overproduces the ADA enzyme 7,900-fold relative to the parental Cl-iD cell line. Assuming thatthe amount of overproduction of enzyme corresponds to acomparable increase in the copy number of the ADA gene aswe have shown earlier for the B-1/25 cell line (51), one cancalculate that ADA gene sequences account for approxi-mately 5% of the genome in the B-1/100 cell line (thecalculation is as follows: [7,900 (copy number) x 27 kb (sizeof ADA gene)]/[3 x 106 kb (size of mammalian haploidgenome) + 7,900 (copy number) x 121 kb (minimum size ofamplified unit)] = 5.4%). We directly determined the per-centage of the B-1/100 genome composed of amplified ADAgenes by comparing the amount of ADA gene sequences inB-1/100 DNA with that present in cosmid clones whichcontain known amounts of ADA structural gene sequences.Such comparisons (e.g., Fig. 1B) indicate that the amplifiedADA genes account for approximately 10% of the genome ofthe B-1/100 cells. This value is in reasonably good agreementwith the calculated value of 5% discussed above.The cosmid library was constructed from DNA derived
from a cell line that was selected for the presence ofamplified ADA gene sequences. Although we had previouslyshown that the enzyme derived from the amplified cell linesis indistinguishable from enzyme derived from the parentalCl-iD cell line by a number of biochemical criteria (23), wewere nevertheless concerned that the ADA gene sequencespresent in this cell line might have undergone some alter-ation(s) during the amplification process. However, we haveevidence that the ADA genes present in the B-1/100 ampli-fication mutants are indistinguishable from the ADA genepresent in nonamplified mouse cell lines. The 5'-most 16 kb
FIG. 7. Sequence of the mouse and human ADA gene promoter fragments. The mouse ADA gene sequences are shown above the humanADA gene sequences. Regions of sequence homology of at least 10 bp in length sharing .90% homology are shown in boxes. Asterisksindicate nonhomologous nucleotides within the boxed region of homology. The arrows pointing downward indicate the cap sites of the threemost abundant mouse ADA gene transcripts. The arrow pointing upward indicates the cap site of the human ADA gene transcripts. Thesequences are numbered with the A of the ATG codon as + 1. Dots are placed every 10 nucleotides.
of the mouse ADA gene have also been isolated in ourlaboratory (M. R. Al-Ubaidi, unpublished observations)from a library made with DNA derived from mouse spleen.Comparison of the restriction maps ofADA genomic clonesisolated from the two different DNA libraries revealed nodifference over that portion of the gene shared between theseclones. The 240-bp EcoRI-NcoI fragment was subclonedfrom genomic clones derived from the mouse spleen celllibrary. DNA sequence analysis of this fragment showed it tobe identical to the 240-bp EcoRI-NcoI fragment described inthis paper. From such comparisons we conclude that theB-1/100 cell line serves as an enriched source of structurallyunaltered ADA gene sequences.The 5' boundary of the mouse ADA gene was determined
by both Si nuclease and primer extension analysis. Multipletranscription start sites were found that map within the240-bp EcoRI-NcoI fragment. Promoter activity was shownto reside in this EcoRI-NcoI fragment by functional analysis.The addition of up to 2.5 kb of 5'-flanking sequences did notincrease promoter activity in the cell lines used for thefunctional assays.
Structural analysis of the mouse ADA promoter regionshowed it to be extremely G+C rich and to give rise tomultiple transcription initiation sites. Sequences upstream ofthe multiple start sites lack the TATA or CAAT box con-sensus sequences which are found approximately 20 and 80bp, respectively, in front of the cap site of a number of genes(3, 9). Many genes which lack the TATA consensus se-quence have multiple start sites in agreement with the notionthat the TATA box is responsible for positioning of the startsite (9, 37, 44, 46). The mouse ADA promoter fragmentcontains two 10-bp sequences which fit the decanucleotideconsensus sequence
G GGCTGGGCGGAAT,
which shows binding with the transcription factor Spl (25).Spl has now been shown to bind to and activate transcrip-tion from several viral and cellular promoters which containGC box elements (for a review, see reference 25). The geneswhose promoters contain proven or potential Spl-bindingsites represent a diverse group that does not show a commonpattern of expression or regulation. Included in this groupare genes such as dihydrofolate reductase, which is ex-pressed at low levels in all cell types, and genes such asADA, which is expressed in cell types but shows a 1,000-folddifference in expression between cell types.A run of 22 deoxyguanylate residues which overlaps at its
3' end with-a potential Spl transcription factor-binding site islocated upstream of the transcription initiation sites of the
mouse ADA gene (nucleotides -211 to -185; Fig. 4).Homopurine-homopyrimidine stretches are located in the5'-flanking region of a number of eucaryotic genes (for areview, see reference 15) in which they have been shown tobe hypersensitive to S1 nuclease. Sensitivity to S1 nucleasehas been interpreted as a reflection of an altered DNAsecondary structure. S1-hypersensitive sites have beenfound in the 5'-flanking region of actively transcribed genes(28, 36), lending support to the notion that the 5' ends oftranscriptionally active genes are characterized by regions ofaccessible DNA secondary structure. A deoxyguanylatetract found in the 5'-flanking region of the chicken P-globingene (13, 18) closely resembles the deoxyguanylate tractfound in the 5'-flanking region of the mouse ADA gene inthat each deoxyguanylate tract overlaps at its 3' end with apotential Spl-binding site. In the chicken 3-globin gene thisdeoxyguanylate tract has been shown to be S1 sensitive, andthere is evidence to suggest that this region is free ofnucleosomes in cells that are expressing 3-globin (35). Re-cently, it has been shown that this deoxyguanylate tractbinds a protein that is present in chicken erythrocyte nucleibut absent in oviduct nuclei (14). It will be interesting to seewhether the deoxyguanylate tract located in the 5'-flankingregion of the mouse ADA gene confers any altered DNAsecondary structure to this region as has been shown forhomopurine-homopyrimidine tracts located in the 5'-flankingregion of a number of genes and whether any biologicallyrelevant proteins bind to this region of the ADA gene.A comparison of the mouse and human ADA gene pro-
moter regions revealed several areas of extensive sequencehomology. It is interesting to note that not only are regionsof sequence homology observed, but the spacing betweenthese regions is also conserved. The two most upstreamregions of homology contain potential Spl-binding sites. Theregion of sequence homology closest to the ATG codon ispresent on the mRNAs of the mouse and human ADA genesand may serve a posttranscriptional role in ADA geneexpression. We were surprised to find that one region ofhomology (-92 to -114 in the mouse gene and -98 to -120in the human gene; Fig. 7) is present on the majority of themouse ADA mRNAs but is excluded from the human ADAmRNAs. Another unexpected difference between the mouseand human ADA genes is the presence of multiple transcrip-tion initiation sites in the mouse gene but a single initiationsite in the human gene as neither the mouse nor human genepromoter regions contain a canonical TATA box.
Studies are under way to delineate which sequence ele-ments are essential for ADA promoter activity. Becausestrong sequence homology is frequently the result of evolu-tionary conservation of functional sequence information, the
MOL. CELL. BIOL.
MURINE ADENOSINE DEAMINASE GENE 4465
highly conserved nucleotide sequence elements of the mouseand human ADA promoter regions may constitute geneticelements essential for the functioning of these promoters. Inaddition, the availability of the structural gene sequenceswill allow us to address questions concerning the tissue-specific and developmentally stage-specific regulation of themouse ADA gene.
ACKNOWLEDGMENTS
We are grateful to Grant MacGregor for the gift of human ADAgene sequences and to Marian Jackson, Connie Clancey, and UrsulaWeiss for critical comments on the manuscript.
This research was supported by Public Health Service grantAI20402 from the National Institutes of Health, American CancerSociety grant CD-268, Robert A. Welch Foundation grant Q-893,and March of Dimes grant 6-393 to R.E.K., Public Health Servicegrant RR05425 from the National Institutes of Health to C.-Y.Y.,and Public Health Service grant CA16672 from the National CancerInstitute to D.A.W. Alanosine was kindly provided by the DrugSynthesis and Chemistry Branch, and 2'-deoxycoformycin by theNatural Products Branch, of the Developmental Therapeutics Pro-grams, Division of Cancer Treatment, National Cancer Institute.D.E.I. and M.R.A. are predoctoral fellows of the Robert A. Welchfoundation, and R.E.K. is a recipient of Research Career Develop-ment Award CA00828 from the National Cancer Institute.
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