Demonstration of Promoter Activity and Alternative Splicing in the Region 5' to Exon 1 of the AFC Gene1

Home , Promoter activity

[CANCERRESEARCH54,2991â€”2995,June1, 1994] Demonstration of Promoter Activity and Alternative Splicing in the Region 5' to Exon 1 of the AFC Gene1

Andrew Thliveris,2 Wade Samowitz, Non Matsunami, Joanna Groden, and Ray White

School of Medicine [A. T.J, Department ofPathology [W. S.J, and Department of Human Genetics and Howard Hughes Medical Institute [N. M., R. W.J, University of Utah, Salt Lake City, Utah 84112, and Department ofMolecular Genetics, Biochemistry, and Microbiology, University ofCi,winnati Cincinnat4 Ohio 45267 [J. G./

ABSTRACT MATERIALS AND METhODS

Screening of fetal brain and fetal retina complementary DNA (cDNA) Isolation of Exons 0.1, 0.2, and 0.3, 5' to Exon 1. Fetal brain and fetal libraries and exon-connection experiments using brain cDNA have iden retina cDNA libraries (Stratagene, San Diego, CA) were plated and tified three exons 5' to exon 1 ofthe adenomatous polyposis coli gene. The screened with a 5 â€˜-probe derived from APC, according to standard meth exons are termed (from 5'-3') 0.3, 0.1, and 0.2; exoas 0.1 and 0.2 are ods. This probe was radiolabeled and amplified by the PCR using primers contiguous genomically. Library screening revealed alternatively spliced in exons 1 (V38LIS) and 3 (V28.RP) (Table 1), with a 5'-APC cDNA clone cDNAs containing the following combinations of 5'-exons: 0.3 + 1 + 2, (11) as template, as follows: 200 ng template; 8 @l[2.5m@tdTTP, dGTP, 0.3 + 2, 0.1 + 0.2 + 1 + 2, and 0.1 + 1 + 2. Exon-connection experiments and dATPJ; 30 @l[32PJdCTP(3000 Ci/mmol); 5 @llox buffer (Perkin also identified these four forms in mRNAs from tissues and cultured cell Elmer); 1 p.1each primer (20 p@M);and1 unit Taq polymerase, in double lines, along with two additional forms, 0.1 + 0.2 + 2 and 0.1 + 2. The distilled H20 to 50 @lunder PCR conditions (94Â°C for 2 mm, 52Â°C for 1 multiple splice forms may lead to proteins of differing activity; for exam mm, 72Â°Cfor 1 mm, for 30 cycles). pie, products derived from cDNAs without exon 1 will lack most of a Relative Ordering ofExons 0.1, 0.2, and 0.3. Exons 0.1, 0.2, and 0.3 were heptad-repeat domain that supports formation ofhomodimers. No mRNA ordered using a AgtlOlibrary constructed from YAC 310D8, which spans the species combining 0.3 with either 0.1 or 02 were identified The existence polyposis locus (11). Individual phage containing a human insert were spotted of two apparently separate 5'-ends ofAPC suggests the possibility of two onto bacterial lawns in numeric order and transferred to a nitrocellulose independent promoters. The genomic sequence adjacent to exon 0.3 con membrane by standard techniques. These filters were hybridized with several fers promotor activity when cloned in a chlorampheaicol acetyltransferase probes prepared in the following way: (a) radiolabeled PCR fragments from expression vector and transfected into a colon cancer cell line. exons 1 (primers V38LIS and V38.RP) and 0.1 (primers V52.UP and RP) were prepared as described above with YAC 310D8 as template. Primer sequences are given in Table 1; (b) an XbaI restriction fragment obtained from A clone INTRODUCTION 168 (see â€œResultsâ€•)wasisolated with GeneClean (BiolOl, La Jolla, CA) and radiolabeled using a random primer method (Stratagene). APC3 is an autosomal dominant inherited disorder caused by ab Confirmation of an exon sequence in phage clones was accomplished by errant alleles of a gene (APC) on chromosome Sq (1, 2). The numer PCR amplificationusing phage DNA as templateand exon 1 (V38L1S and ous colonic polyps typical of this disease place patients at high risk of V38.RP), exon 0.1 (V52.UP and RP), exon 0.2 (V59.UP and RP), and exon 0.3 (V64.UP and V55.RP) as primers. colon carcinoma. One model for development of colorectal cancer, in Isolation of Genomic Sequence Flanking Exons 0.1, 0.2, and 0.3. An sporadic as well as inherited cases, invokes tumor progression through chored and inverse PCR were used to isolate genomic sequences flanking each accumulation of genetic events in a clonal lineage of colonic epithelial exon. Inverse PCR (1) utilized opposing primers in each of the three exons: for cells; mutation in APC appears to occur at an early stage of the exon 0.1, V54.RP and V53.UP; for exon 0.2, V71.UP and RP; for exon 0.3, tumorigenic process. Mutations ofAPC also have been implicated in V72.UP and RP or V73.UP and RP. Template DNA was prepared by digesting gastric cancer (3), and allelic loss of APC is often observed in YAC 310D8 with HpaI, NlaI, and AluI restrictionenzymes. DNAS were esophageal (4), pancreatic (5), and hepatocellular carcinomas (6). ligated to maximize circularization, and flanking sequences were amplified by PCR and sequencedas describedby Grodenet a!. (1). Following initial localization of the unknown gene responsible for The genomic sequence 5â€˜toexon 0.1was isolated using anchored PCR (12) APC to the long arm of chromosome S by cytogenetic observations with one anchored primer (â€œAncâ€•inTable 1) and either V70.RP or V71.RP as (7) and linkage analyses (8, 9), APC was identified through isolation the opposing primer. The anchored template was prepared by digesting phage of several germ line mutations in the form of stop codons and clone 108 DNA with Sau3A and ligating an anchored linker (â€œTLBSâ€•inTable frameshift mutations (1, 2). Thus far, 15 coding exons have been 1). Resulting fragments were sequenced by dideoxy termination (13) with reported; a consensus Kozak sequence occurs in exon 1. fluorescently tagged M13 primers on an Applied Biosystems (Foster City, CA) In the work reported here, three exons â€˜toexon 1 were revealed model 373A DNA sequencer. by probing cDNA libraries. Expression of these exons in various Exon Connection. RNAwas extractedfromtissuesandvariouscarcinoma tissues was analyzed. Using different methodology, others have cell lines as described by Chomczynski and Sacchi (14). cDNA was made detected the same three exons and one additional exon (10). Here through reverse transcription of 1 @goftotal RNA; synthesis was primed by we describe novel splice forms and correlate our findings with random hexamers (Perkin-Elmer Corp., Norwalk, CF). PCR then was per formed on the cDNA using oligonucleotide primers from different exons. The theirs. In addition, we have isolated genomic sequences surround following exon connections were performed (primers in parentheses, see Table ing the 5'-exons and have identified promoter activity in the region 1): 0.3 to 1 (V58.RP and V38.RP); 0.3 to 2 (V58.RP and V24.RP); 0.3 to 3 upstream of exon 0.3. (V58.RP and V32.UP); 0.1 to 1 (V52.RP and V38.RP); 0.1 to 3 (V52.RP and V32. UP); and 0.2 to 3 (V59.UP and V32.UP). Received 12/3/93; accepted 4/5/94. Construction of CAT Expression Plasmids and Ascertainment of CAT The costs of publication of this article were defrayed in part by the payment of page Activity. pCAT basic (Promega) was digested with SphI and XbaI, and charges. This article must therefore be hereby marked advertisement in accordance with vector backbone was purified on a 1% medium electroendosmosis agarose 18 U.S.C. Section 1734 solely to indicate this fact. I A. T. was supported by a grant from the Howard Hughes Medical Institute. R. W. is gel (15). Inserts for plasmids pAT175, 176, and 177 were synthesized using an Investigator of the Howard Hughes Medical Institute. PCR (94Â°Cfor2 mm, 52Â°Cfor1 mm, 72Â°Cfor1 mm, for 30 cycles) with 2 To whom requests for reprints should be addressed, at University of Utah, Eccles primer pairs V100.Xba/V101.Sph, V97.Xba/V96.Sph, V94.SphIV98.Xba, Institute of Human Genetics, Bldg. 533, Salt Lake City, UT 84112. respectively (Table 1), and YAC 310D8 as template. Products were di 3 The abbreviations used are: APC, adenomatous polyposis coli; cDNA, complemen tary DNA; PCR, polymerase chain reaction; OTF, octamer transcription factor; CAT, gested with SphI and XbaI and purified on a 1% medium electroendosmosis chloramphenicol acetyltransferase. agarose gel as described previously. Ligations of insert to vector and 2991

PROMOTER AND 5' APC EXONS

TablesequencesÂ°Universal 1 PCR primers used for amplification ofAPC intronic and exonic probe contained a novel sequence 5' to exon 1. This sequence was bottom.Primerprimer (UP) and reverse primer (RP) sequences are shown at the isolated and designated exon 0.1. SequenceV24.RP Exon-connection experiments were performed originally on cDNA RP-CAATCrGTCCAGAAGAAGCCAV28.RP prepared from brain RNA, since the strong PCR signal in brain cDNA RP-CTCrCITFCrCAAGUCVFCfAV32.UP UP-CGGCrFCCATAAGAACGGAGV38.RP RP-GTAAGATGATrGGAAUATmCl@AV38LIS @ UP-AGGTCCAA000TAGCCAAGGV52.UP - x@â€”w- UP-GGAACAGCATCGAGCCAACCV52.RP RP-CACGAGGCCGCCGGAAGCV53.UP UP-GGUGGCFCGATGCTGUCCV54.RP RP-GCCrGAGCCCGCAGGTCCV55.RP RP-GCAGCACCTCCATfCI'OTCfCV58.RP # cDNA isolates RP-GAGACAGAATGGAGGTGCFGCV59.UP UP-TGAAGCAC1'CAGlTGCmCV59.RP RP-CCAGTGGCCAGAAGTACGAGV64.UP UP-ATGCGGACCAGGGCGCrCV70.RP -a-3 1 k@1 5 RP-GAGAACTGAGOGTGGTACAGV71.UP UP-GCGCCCCCFATGTACGCCFV71.RP RP-ACFGAGTGCflCACCFTCCfCV72.UP UP-CGACATGTGGCFGTA1TGGTGV72.RP RP-CFGGTCCGCATCCAGCGGAV73.UP UP-GT000CGCACGTGACCGAV73.RP RP-AGCGCCCFGGTCCGCATCV94.Sph 3 ATI'GCATGCGGCFGAGGCAGGAGAATCG V96.Sph AUOCATGCGA1TFGTAGTAGCTAACAGCAC [email protected] A@[email protected] iT@@@i2@1 0 ATETCFAGATfGGA1TVFGTCCITCAACCI'CV101.Sph A1TGCATOCCCGCATCCAGCGGATTACAC Ancâ€• UP-TGTAAAACGACGGCCAGTACAG 1 FR .1@5bGATCCFGTGATUP i@I 1 r@;1 TGTAAAACGACGGCCAGTRP CAGGAAACAGCfATGACC

a Tm of 52Â°C for all primers, with the following PCR conditions unless otherwise 0 stated: 200 ng template; 8 pi [2.5 msi d1TP, dCl'P, dGTP, dAli']; 5 @dlOX buffer Fig. 1. Alternative splice forms identified by exon connection with brain cDNA as (Perkin-Etmer); 1 p.1each primer (20 psi); and 1 unit Taq polymerase, in double-distilled template (shown as boxes on left) or by screening of fetal brain or fetal retina (FR) cDNA H2O to 50 ,.Llunder PCR conditions 94Â°Cfor 2 mm, 52Â°Cfor 1 mm, 72Â°Cfor 1 mm, for libraries. The number of cDNA isolates is shown at right for each form. Exon 0.3 did not 30 cycles. connect with exons 0.1 or 0.2. b Primers for anchored PCR.

transformation into Escherichia coli selecting for ampicillin resistance @o@oiÃ§oico were performed according to standard protocol. Recombinant plasmids @ â€˜S were sequenced using a dideoxy termination method (13), with fluores cently tagged M13 primers on an Applied Biosystems model 373A DNA sequencer. Twenty-four h before transfection, 5 x 10-i human colon carcinoma cells @ (cell line HCT116) were seeded onto 60-mm dishes in McCoy 5A medium I@__ (GIBCO)supplementedwith 10%fetal calf serum(Hyclone). At the time of transfection, cells were washed three times with serum-free medium. Then, 20 @.dofpremixed Transfectam (Promega),preparedaccordingto the instructions

of the manufacturer, and 15 @tgofDNA were added to the cells in 5 ml of -@ serum-free medium. Twenty-four h after the addition ofthe DNAlFransfectam @- mixture, the medium was replaced with 5 ml of medium containing 10% fetal calf serum. Thirty-six h later, the cells were washed three times with ice-cold phosphate-buffered saline and harvested by scraping into 0.5 ml of 0.25 M Tris-HC1(jH 8.0). Transfections were done in triplicate for each reporter gene construct. Cells were lysed by three cycles of freeze-thawing and then heated to 60Â°Cfor 10 min to inactivate endogenous acetylases. Aliquots of cell lysate (50 @d)wereused for each CAT assay. Protein content for each assay was measured by densitometry and found to be consistent. A liquid scintillation counting assay (16) with reaction time of 3 h and total reaction volume of 125 1246 @lwasused to determine CAT activity. 357

RESULTS 8

Isolation of 5'-Exons of the APC Gene. Forty-four cDNA clones 9 were isolated by screening a fetal brain library with a probe harboring Fig.2. Identificationofmultiple5'-spliceformsfromexon-connectionexperiments. PCR with primers in exons 0.3 and 1, 0.3 and 3, 0.1 and 1, and 0.1 and 3 was performed sequence from exons 1â€”3.Several splice forms were identified (Fig. on cDNAmade from brain mRNA.Numbersbelowlanes correspondto the exonic 1): five cDNA isolates had an exon arrangement of 0.3 + 1 + 2, one compositionofeachPCRband(determinedbysequencing)asfollows:1, 0.3 + 1; 2, 0.3+ 1 + 2 + 3;3,0.3+ 2 + 3;4,0.1+ 0.2+ 1;5,0.1+ 1;6,0.1+ 0.2+ 1 + @ had an arrangementof0.3 + 2, and three contained 0.2 + 1 + 2. One 2 + 7,0.1 + 0.2 + 2 + 3; 8,0.1 + 1 + 2 + 3; 9,0.1 + 2 + 3. Themarkerlaneis cDNA clone isolated from the fetal retina library using an exon 1-to-3 a 100-basepairladder(Gibco-BRL). 2992

ciâ€˜-:N,-x exon 0. 1 were isolated. Exon 0. 1 sequence was immediately contig uous and 5' to exon 0.2. The sequences of exons 0.1, 0.2, and 0.3, and a,xx wwx corresponding genomic sequences, are shown in Fig. 4. Promoter Activity Associated with DNA Sequence 5' to Exon @92 + 0.3. The genomic sequence 5' to exons 0.1 and 0.3 was analyzed with @.l68 ++ + a â€œfindâ€•algorithm(17) using the transcription factor database (18) in 175-base pair segments with 50-base pair overlaps. The 100 base pairs A108 + + immediately 5' to exon 0.3 contained a GC-rich region (75%) with a single putative SP1 site. Within 624 base pairs 5' to exon 0.3 were a )@_q9 + single putative OTF-binding site (19, 20) and two atypical TATA boxes (at positions 237 and 619; see Fig. 4). Three plasmids were Fig. 3. Overlapping human genomic phage clones. 5'-Exons (cx.) are shown in their positions relative to the phage contiguous map. +, the appropriate PCR product was seen constructed using a CAT expression vector and different amounts of in experiments using various exon primers, with phage DNAS as templates. Dotted line, sequence 5' to exon 0.3 to allow separation of these promoter dc 3'-end of clone 99, indicating the uncertain extent of its overlap with clone 108. ments. Plasmid pAT177 contained both the OTF and SP1 sites in genomic orientation, and pAT175 contained both sites in the opposite allowed identification of multiple splice forms (Fig. 2). PCR failed to orientation. Plasmid pAT176 contained the SP1 site in genomic on reveal a connection between exon 0.3 and either 0.1 or 0.2. PCR entation (Fig. 5). The plasmid with both sites in genomic orientation between exons 0.3 and 1 revealed a single band of expected size. PCR gave a 15-fold increase in CAT activity when transformed into colon between exons 0.3 and 3 revealed two bands, one of expected size and cancer cell line HCT1 16, while CAT activities of the other constructs one corresponding to a message missing exon 1; this was verified by were near background. The genomic sequence 5 â€˜to exon 0. 1 was sequencing. PCR between exons 0.1 and 1 revealed two bands, a unremarkable except for a single SP1 site 11 base pairs 5' from the larger species consistent with an exonic sequence of 0.1 + 0.2 + 1 ending of the cDNA clone identifying exon 0.1 (Fig. 4). and a band consistent with a message in which exon 0.2 is spliced out. Sequencing of four bands resulting from PCR between exons 0.1 and DISCUSSION 3 revealed splice forms 0.1 + 0.2 + 1 + 2, 0.1 + 0.2 + 2, 0.1 + 1 + 2,and 0.1+ 2. We have identified three exons 5â€˜to the APC gene and have Tissue-specific Expression of the New APC Exons. Exon-con discovered six alternative splice forms involving exons 0.1, 0.2, 0.3, nection experiments performed with RNA from cultured cell lines 1, and 2 of APC. Four of the splice forms (0.3 + 1 + 2, 0.3 + 2, (Epstein-Barr virus-transformed lymphocytes; lung, colon, and breast 0.2 + 1 + 2, 0.1 + 1 + 2) were identified in cDNA libraries. carcinomas; and normal dermal fibroblasts) and in fresh tissue (colon Exon-connection experiments confirmed these four and identified two and heart), from exon 0.3 to exon 2, revealed the same two splice additional splice forms (0. 1 + 0.2 + 2, 0. 1 + 2). No splice forms forms seen in brain. The smaller band without exon 1 was decreased connecting exon 0.3 with either 0.1 or 0.2 were found. The apparently in intensity, but its presence was confirmed by hybridization with a ubiquitous expression of all of these 5'-exons followed the pattern probe to exon 1 (data not shown). Exon connection between 0. 1 and previously noted in experiments with exons downstream of exon 2(1). 3 on cDNAs from these tissues and cell lines revealed two prominent Moreover, data presented here identify no obvious tissue specificity, forms consistent with the presence or absence of exon 0.2 in most although in many cell lines, splice forms containing exon 1 appeared tissues, although all four forms were seen in heart (0.1 + 0.2 + 1 + to be more prominent than those without this exon. 2, 0.1 + 0.2 + 2, 0.1 + 1 + 2, and 0.1 + 2). Exon connection between Several putative promoter elements exist 5' to exon 0.3: an OTF at exons 0.2 and 3 revealed splice forms with and without exon 1, base pairs 323â€”333,an SP1 at base pair 927, and a GC-rich region although the splice form without exon 1 was decreased in intensity (75%) at base pair 857â€”957(Fig. 4). When this region was cloned into (data not shown). a CAT expression vector and transfected it into colon cancer cell line Relative Order of 5' Exons. Exons 0.1, 0.2 and 0.3 were ordered HCT116, we observed a 15-fold increase in CAT activity. If one using a AgtlO human genomic library constructed from YAC 310D8. separates the OTF and SP1 sites, CAT expression falls to background; When 248 phage isolates were spotted onto a nitrocellulose mem this result is consistent with the notion that the region containing the brane, radiolabeled exon 1 identified A clones 92 and 168. A radio putative OTF site plays a role in expression of APC. More detailed labeled 80-base pair fragment from exon 0.1 identified phage clones analysis of this region will be required to identify elements which may 168 and 108. Primers from exons 0.1 and 0.2 amplified correctly sized contribute to such specificity and to determine starting sites for products when these phage clones were used as templates. To extend transcription. However, further study using expression vectors and the phage-contiguous map, a radiolabeled restriction fragment from directed mutagenesis will help clarify the significance of the various phage 168 was used to identify phage 99; primers from exon 0.3 elements upstream of APC. amplified a correctly sized product with phage 99 as template. Exons 0.1, 0.2, and 0.3 also have been identified by investigators In an attempt to order these exons further, we designed exon using a different methodology (10), but several differences are evident connection experiments using primers in 0.1, 0.2 and 0.3 in both between their data and ours: (a) the authors failed to describe splice orientations with genomic DNA as template. Primers with 3'-orien forms missing exon 1. The removal of exon 1 is of potential impor tation in exon 0.1 connected with primers in 5'-orientation in exon tance because this exon encodes part of a heptad repeat (1 1) involved 02. Primers in exon 0.3 failed to connect with primers in exons 0.1 or in homodimerization (21, 22) and possibly in interactions with other 0.2. The phage-contiguous map and exon-connection studies are sum proteins. The removal of this exon in some splice forms would be marized graphically in Fig. 3. expected to alter dimerization, and indeed, its absence has been shown Isolationof GenomicSequence5' to Exons 0.1, 0.2, and 0.3. to alter homodimerization in vitro (21, 22). The presence of splice Genomic sequences 5 â€˜toeach of the new exons were sought by forms lacking exon 1 in all of the tissue types examined, although inverse PCR using YAC 310D8 DNA as template to identify the apparently in lower quantities than other forms, suggests that APC promoter region of APC. More than 1.1 kilobases of genomic se may have multifunctionality within a given cell; (b) the other quence 5' to exon 0.3 and 0.24 kilobases of genomic sequence 5' to authors described a brain-specific exon that connected with exon 2993

*xona .1 and .2

27 54 297 324 ATC TAT TAA CTG 0CC TGC AGC ACA M@r CAG AGA AGO CCA GTA AGT GCT GCA ACT CCT CAT AAA ATT GGG AAT TTA AGC ATC ACC TOG TTC OAT TTA AAT OCA ATG TAG

81 108 OTT 351 378 GAG ACT COG CTG CCT A@ CAG CAA TOG CTC A@ GGA CAG AAC ARC GAA GCA OTO AS? TTG CAT TAA AAT ACT ACA TFA AAG CCT CAG ATT TOT AGT AGC TAA CAG CAC

135 162 405 432 ccc ccc AAGCOGA@GCAGCACCCAT@GCGCCT0CCCATAACAGOCTCTAGTCT 1@rC TAT GTA TOT GTC AGO GAG TGC TOT AAA TAC TTC ATA TAT ATT AAC TCC TOT

189 216 459 486 cco roc TOT GGGas@Gcca GCAACA @TCTCA@ CATGCGCATTOT AGTCTT CCC M@ CTG TAC TTC TOT TOG COT TrT ATA GAG CAG GAA ATT GAA ACA CTG AGA GOT Pro Gly Cys Gly Lys Pro Ala Thr Pro Leu Thr His Ala His Cys Ser L.eu Pro

243 270 513 540 ACC TCC CAC AAG ATG GCG GAG GGc AAG TAG CAA GGG GGc GGG GiG TOG CCC CCC TAA GTA ACT AAA G@1@ACA GAG CTA GAG TGA GAG GAG TAA AGC TIC AAC TCA CCC Thr Ser His Lys MET Ala Glu Gly I.ys - Gln Gly Gly Gly Val Tro Pro Pro Gly Arg Arg

297 324 567 594 GAA CCC TAG CCC CTG CTS GGG COG GAC CTG COG OCT CAG CCC COG GAG CTG COG AAC CCA GAC TTC CAG ACT TOT CAT crc GAG TAC TAA OCT GOT AGC ATA OCT TTr - GltI Ala Pro Leu Leu Glv Glv Asp Leu Am Ala Gin Ala Sin Glu L.eu Am Lys Pro Ser Az-g Cys Ser Gly Gly Thr Cys Gly Lu Arg Pro Gly Ser Cys Gly

Exon 1 351 Exon .2 378 621 648 ACC GAG G@i-rGGC TOG ATG CTG TTC CCA QOT ACT G?r OTT GGC TOT TOG TGA GGA CTG OTA ACT A@ TTT AAT TCA AAT ATA ATT CGA GTC ATC TAT CTA ACA ACT CAT . Thr Glu Val Glv Sam MET Leu Pha Pro Glv Thr Val Val Giv Cvs Tm Glv Pro Arg Leu Ala Arg Cys Cya Sam Gin

405 432 675 702 AGO TGA AGC ACT CAG TTG CCT TCT COG GCC TCG GCG CCC CCT ATG TAC GCC TCC CAC TOT GAC AAC TCA GTG ACT TOT AAT GTA AAA TTA TTC ATT GTA A1@ CAT TTA . Ama Ser Thm Gin Leu Pro Ser Ama Ala Ser Ala Pro Pro MET Tvr Ala Ser

459 486 729 756 cr0 GGC TOG GGT CCC GTC GCC CCT TTG ccc OCT TOT GTA CCA CCC TCA OTT CTC ATA TM 3-PG TTT CTC TOT OCT OCA AAA ATC ATA OCA ATC GAG ATG TAA â€˜ITT ACT Leu Glv Ser Glv Pro Val Ala Pro Leu Pro Ala Ser Val Pro Pro 5cr Val Leu . Phe lie

513 540 783 810 GGG TCC TOG AGC ACC GGC GGC AGC AOG AGC TGC GTC CCC CAG GAG ACC AAG AGC ACT CTC CCT CCC ACC TCC CCC ATC TiC TGC TAA TCC TTC TGC CTG COG ACC TCC Glv Scm Tm 5cr Thr Glv Glv Sam Ama See Cvs Val Ama Gin Glu The Lvs 5cr Thr Leu Pro Pro Thr Scm Gly lie Leu Cys . 5cr Phe Cys Leu Arg Thr 5cr

567 594 837 864 CCC 0CC 0CC OCT cur ACT TOT GGc CAC TOG GCG AGC GTC TOG CAG GTG AGT GAG CCC GAC TOT TTA CTA TGC OrG TCA ACT CCC ATC AAC TTC C@CTGCT TCC TOG GGA Pro Glv Giv Ala Ama Thr 5cr Glv His Tm Ala Sam Val Tm Gln Pro Asp 5cr Leu Leu Cys Val 5cr Thr Ala lie ken Phe L.eu Ala Cys Trp Gly

621 648 891 918 OCT GCA CCC ATT GAC GTC TCC TCC COG CAA A@ TTC CTC GGC TTT 0CC CCG CCC CTG GGG CCG CGA GGG CAT ACC CCC GAG CCC TAC GGG OCT AGO GCT AGG CAG GCT I@eu Giy Pro Arg Gly His Thr Pro Glu Gly Tyr Gly Ala Arg Ala Arg Gin Ala

675 702 spi. 945 972 CTG CTC GGC ACC CTA COG TGC TCG CCC CGA CTC TOT GGC TCT C@CF CTC TCC ATC GTG COG 1@rG mc ono CCC CTG TOC CCC ACT CCC GAG TOG GGG TOG GGA AGC OGA Val Arg Leu Gly Gly Ala Leu Cys Pro Thr Ala Glu @ys Glv Sam Glv Scm Giv

729 756 999 1026 TCT CAC CCT CTC CCC TCC CCC CAC TCC CCA TTC AGC CCT CCA GTT GGC CCC TOG GAG AGA AGC AGC TOT GTA ATC CCC TOG ATG CCC ACC AGG GCG CTC CCC ATT CCC Glu Ama Ser Scm Cvs Val lie Am Tm MET Ama Thr Ama Ala Let Pro lie Pro

783 1053 1080 CTT TGC AGO TCC TNC APT CrC ACC CAG GGA NrC GGG GTT GTC 0CC AGC CCC CCC ACT GGC TOG GIG TOG CCC CAC CCC ACC GAC ATG TOG CTG Val Glv 5cr Pro Pro lie Glv Tm Val Tro Ala His Val Thr Aso MET Tmo@Leu

1107 1134 TAT TOG TGC AGC CCC CCA GGG TOT CAC TOG AGA CAG AAT GGA OCT OCT 0CC GGA Tvr Tro Cvs Scm Pro Pro Glv Cvs His Tm Ama Gin Asn Glv Glv Ala Ala Glv 5*0* .3

1161 1188 27 54 CTC GGA AAT 0CC GTA OCT OCT GGA CCC ACC ATG 0CC AGG CTT OCT GCG COG GGA GCA CCT CCC TOT NAG TOG CAG TTC ACT TOG HAG OCT GAG GCA GGA GAA TOG CTT Leu Giv A@snGlv

1215 1242 81 108 GGG 000 AAG GTG OTT â€˜PlC CCT CCC ACT GTC TTA AAC CGA TOG COT TTC CTT 0CC GAA CCC GGG AGO TOG AGO TTG CAG TGA GCG GAG ATT GCA CCA CTG CAC TOG CCT

1269 1296 135 162 ACA COG TCC ACT GCA GCA TOG CAA ACC AGG AGG CAG COG CGT COT CCC CCC CCC GOT GAC AGA GCA AGA CTC TAT CTC AAA AAA CAA ACA AAG MA ACT TGA AGT ATA

1323 1350 189 216 CCC CAC TGC AGC ACT GGA GAT GGA TTT COT GTA CCT CGG ATC CAG OCT TTT TGA GTA TCC @I-rr TAA ATF TTA AAT AGA TAA TAG AAA CTG OTT TCC CCC CAT 1@A AAC

1377 1404 243 270 CAG AAG AGO AAG AAG CON GAG 000 TAG P.50 TOT TAA COG GAG TOT OCT GAG AAA CAG AAT ?@A AGT @rAACT CTA TAT ATT CTr GAC AGT CTG CAT TTT GTC CTT CAA Fig. 4. Genomic sequence corresponding to exons 0.1 and 0.2 and to exon 0.3. Numbering begins arbitrarily at one end of the most 5' genomic clone. Exonic sequences defined by cDNA clones, with their corresponding amino acids, are underlined. In the first underlined sequence, roman type indicates amino acid sequences in the frame consistent with splicing of exon2 to 0.2 + 0.1;italicsindicatetheframeconsistentwithsplicingofexon2 to exon0.1.Notethesplicejunctionanddonorsitebetweenexons0.1and0.2at basepair352, depicted by bold type. Putative promoter elements are also indicated in bold type.

0.3; we did not observe this splice form with PCR amplification exon 0.3 sequence 5' to the reported equivalent â€œexon1Aâ€•;(d) our using primers in exons 1 and 0.3 and ethidium bromide staining; sequence shows a one-base pair deletion in exon 0.2 when com (c) several cDNA clones have been isolated here that extend the pared to its reported equivalent, â€œexonlB.â€•The sequence of exon 2994

Plasmid CATactivity Burt, R., Hughes, J., Warrington, J., McPherson, J., Wasmuth, J., La Paslier, D., Abderrahim, H., Cohen, D., Leppert, M., and White, R. Identification and character ization of the familial adenomatous polyposis coli gene. Cell, 66: 589â€”600, 1991. pCATbasic i 25901 +/-125 2.Kinzler,K.,Nilbert,M.,Su,L-K.,Vogelstein,B.,Bryan,T.,Levy,D.,Smith,K., SV4O Preisinger, A., Hedge, P., McKechnie, D., Finniear, R., Markham, A., Groffen, J., Boguski, M., Altschul, S., Horii, A., Ando, H., Miyoshi, Y., Miki, Y., Nishisho, I., @ pAT175 Sl@1 O'rF 420 +/- 75 and Nakamura, Y. Identification of FM' locus genes from chromosome 5q21. Science (Washington DC), 253: 661â€”665, 1991. 3. Horii, A., Nakatsuru, S., Miyoshi, Y., Ichii, S., Nagase, H., Kato, Y., Yanagisawa, A., pAT176 217 +/-7 and Nakamura, Y. The APC gene, responsible for familial adenomatous polyposis, is mutated in human gastric cancer. Cancer Res., 52: 3231â€”3233, 1992. 4. Boynton, R. F., Blount, P. L., Yin, J., Brown, V. L., Huang, Y., Tong, Y., McDaniel, pAT177 I 6337 +/- 375 T., Newkirk, C., Resau, J. H., Raskind, W. H., Haggitt, R. C., Reid, B. J., and Meltzer, Off SP1 S. I. Loss of heterozygosity involving the APC and MCC loci occurs in the majority of human esophageal cancers. Proc. Nat]. Acad. Sci. USA, 89: 3385-3388, 1992. (â€”) 178 +1-27 5. Neuman, W. L, Wasylyshyn, M. L, Jacoby, R., Erroi, F., Angriman, I., Montag, A., Fig. 5. APC-CAT constructs with orientation (arrows) and OTF or SP1 promoter Brasitus, T., Michelassi, F., and Westbrook, C. A. Evidence for a common molecular element(s) indicated. CAT activity is expressed in cpm. (â€”),vector-only control. Bound pathogenesis in colorectal, gastric, and pancreatic cancer. Genes Chromosomes aries of genomic fragments used to construct the CAT plasmids are as follows (numbers Cancer,3:468â€”473,1991. correspond to numbering of nucleotides of exon 0.3 in Fig. 4): pAT175, nucleotides 6. Ding, S.F.,Habib,N. A., Dooley,J.,Wood,C., Bowles,L., andDeihanty,J.D. Loss 253â€”1005;pAT176,nucleotides 378â€”987;pAT177,nucleotides 51â€”1157.SV4O,simian of constitutional heterozygosity on chromosome 5q in hepatocellular carcinoma virus40. without cirrhosis. Br. J. Cancer, 64: 1083â€”1087,1991. 7. Herrera, L, Kakati, S., Gibas, L, Pietrzak, E., and Sandberg, A. Brief clinical report: Gardner syndrome in a man with an interstitial deletion of 5q. Am. J. Med. Genet., 0.2 was confirmed with independent cDNA clones, as well as 25:473â€”476,1986. genomic clones. As a result, in-frame termination codons are 8. Bodmer, W., Bailey, C., Bodmer, J., Bussey, H., Ellis, A., Gorman, P., Lucibello, F., Murday, V., Rider, S., Scambler, P., Sheer, D., Solomon, E., and Spurr, N. Local generated in the splice form 0.1 + 0.2 + 2. If this splice form is ization of the gene for familial adenomatous polyposis on chromosome 5. Nature expressed, perhaps a downstream start codon is invoked. (Lond.), 328: 614â€”616, 1987. Differential expression could conceivably lead to variation in APC 9. Leppert, M., Dobbs, M., Scambler, P., O'Connell, P., Nakamura, Y., Stauffer, D., Woodward, W., Burt, R., Hughes, J., Gardner, E., Lathrop, M., Wasmuth, J., Lalouel, phenotype- or tissue-specific manifestations of the disease. In tissues J-M., and White, R. The gene for familial polyposis coli maps to the long arm of expressing splice forms which remove exon 1, a new start sequence chromosome 5. Science (Washington DC), 238: 141 1â€”1413,1987. is required. Potential in-frame starts with atypical Kozak sequences 10. Hori, A., Nakatsuru, S., Ichii, S., Nagase, H., and Nakamura, Y. Multiple forms of the were found in exon 0.3 at base pairs 1000 and 1072 and in exon 0.1 APC gene transcripts and their tissue-specific expression. Hum. Mol. Genet., 2: 283â€”287,1993. at base pair 197 (Fig. 4). Alternatively, translation may start at an 11. Joslyn, J., Carlson, M., Thliveris, A., Albertsen, H., Gelbert, L., Samowitz, W., ATG downstream from exon 1. The splice form containing exons Groden, J., Stevens, J., Spirio, L, Robertson, M., Sargeant, L., Krapcho, K., Wolff, 0.1 + 0.2 + 2 has in-frame stop codons in 0.1 and 0.2, suggesting that E., Burt, R., Hughes, J. P., Warringion, J., McPherson, J., Wasmuth, J., I.e Paslier, D., Abderrahim, H., Cohen, D., Leppert, M., and White, R. Identification of deletion translation starts at a downstream ATG. mutations and three new genes at the familial polyposis locus. Cell, 66: 601â€”613, The mapping data reported here suggest that 0.3 may be the most 1991. 5'-exon of APC, making the upstream genomic sequence the most 12. Roux, K. H., and Dhanarajan, P. A strategy for single site PCR amplification of probable site of promoter elements. Since exon-connection experi dsDNA: priming digested cloned or genomic DNA from an anchor-modified restric tion site and a short internal sequence. Biotechniques, 8: 48â€”57,1990. ments failed to connect exon 0.3 with exons 0.1 or 0.2, two possibil 13. Sanger, F., Nicklen, S., and Coulson, A. DNA sequencing with chain-terminating ities can be entertained. One is that other exons exist between 0.3 and inhibitors. Proc. NatI. Acad. Sci. USA, 74: 5463â€”5467,1977. 0.2, making the distance between these exons too large for PCR 14. Chomczynski, P., and Sacchi, N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal. Biochem., 162: 156â€” amplification. [Two fetal brain clones containing exon 0.1 diverge at 159, 1987. either base pair 262 or 325 of the genomic sequence (see Fig. 4), 15. Maniatis, T., Fritsch, E., and Sambrook, J. Molecular Cloning: A Laboratory Manual, suggesting that unidentified exons may exist upstream from 0.1. pp. 150â€”169. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press, 1982. 16. Seed, B., and Sheen, J. Y. A simple phase-extraction assay for chloramphenicol However, the sequence surrounding the divergence point at base pair acetyltransferase activity. Gene, 67: 271â€”277,1988. 262 is not supportive of a splice junction.] Another possibility is that 17. Devereux, J., Haeberli, P., and Smithies, 0. A comprehensive set of S analysis a second promoter region and exon may originate 5' of exon 0.3. With programs for the VAX. Nucleic Acids Res., 12: 381â€”395,1984. respect to this possibility, two promoters are involved in specific 18. Ghosh, D. A relational database of transcription factors. Nucleic Acids Res., 18: 1749â€”1756,1990. regulation of the porphobilinogen deaminase gene (23), and four 19. Kemler, I., and Schaffner, W. Octamer transcription factors and the cell-type speci alternative dystrophin transcripts in brain are regulated by different ficity of immunoglobulin gene expression. FASEB J., 4: 1444â€”1449,1990. promoters (24). 20. Sturm, R. A., Das, G., and Herr, W. The ubiquitous octamer-binding protein Oct-l contains a POU domain with a homeobox subdomain. Genes Dev., 2: 1582â€”1599, 1988. ACKNOWLEDGMENTS 21. Su, L-K., Johnson, K. A., Smith, K. J., Hill, D. E., Vogelstein, B., and Kinzler, K. Association between wild-type and mutant APC gene products. Cancer Res., 53: The authors thank Steve Gerken for help with scanning the databases, Ed 2728â€”2731,1993. Meenen for preparationof oligonucleotides, and Ruth Foltz for editing the 22. Joslyn, G., Richardson, D., White, R., and Alber, T. Dimer formation by the manuscript. N-terminus of the APC protein. Proc. NatI. Acad. Sd. USA, 90: 11109â€”11113, 1993. 23. Beaumont, C., Porcher, C., Picat, C., Nordmann, Y., and Grandchamp, B. The mouse porphobilinogen deaminase gene structural organization, sequence, and transcrip REFERENCES tional analysis. J. Biol. Chem., 264: 14829â€”14834, 1989. 24. Gorecki, D. C., Monaco, A. P., Derry, M. J., Walker, A. P., Barnard, E. A., and 1. Groden, J., Thliveris, A., Samowitz, W., Carlson, M., Gelbert, L, Albersen, H., Barnard, P. J. Expression of four alternative dystrophin transcripts in brain regions Joslyn, 0., Stevens, J., Spirio, L., Roberston, M., Sargeant, L, Krapcho, K., Wolff, E., regulated by different promoters. Hum. Mol. Genet., I: 505â€”510,1992.

2995

Downloaded from cancerres.aacrjournals.org on September 30, 2021. © 1994 American Association for Cancer Research. Demonstration of Promoter Activity and Alternative Splicing in the Region 5 ′ to Exon 1 of the APC Gene

Andrew Thliveris, Wade Samowitz, Nori Matsunami, et al.

Cancer Res 1994;54:2991-2995.

Updated version Access the most recent version of this article at: http://cancerres.aacrjournals.org/content/54/11/2991

E-mail alerts Sign up to receive free email-alerts related to this article or journal.

Reprints and To order reprints of this article or to subscribe to the journal, contact the AACR Publications Subscriptions Department at [email protected].

Permissions To request permission to re-use all or part of this article, use this link http://cancerres.aacrjournals.org/content/54/11/2991. Click on "Request Permissions" which will take you to the Copyright Clearance Center's (CCC) Rightslink site.