ALU RECOMBINATION IN p.53

@@@ Q@fl@D 2D@Th@ @KIID @ U Fil I I @i1 1@1@1 rnriiii U flT@1 U U0I I I I 5 10 15 20kb Fig. 1. Schematic representation of the p13 gene. Exons 1—11ofp53(0) and the Mu elements found in the p.53gene (open arrows) are indicated. Note that Alu 4, 7, 10, 14, and 15 are rearranged clusters, and that Alu 2, 14, and 17 are in the reverse orientation.

3'), with a predicted length of 8826 bp. The 3' genomic part of p53 was starting at nucleotide position 12993. Previous reports have indicated analyzed with primers F15 (5'-GATGCTACITGACTFACGATGGAG-3') that Hs766T harbors a homozygous G to A point mutation at codon and R18 (5'-GTCCTGGGTGC1TCTGACGCAC-3'), with a predicted length 181 of exon 5, changing an arginine to histidine (13). However, of 3781 bp. For analysis of the intron 1 Mu element, p53 was amplified with bidirectional DNA sequence analysis of Hs766T exon 5 did not show primers F8 (5'-CTGTCTGTAGACCTGTGTCCCTC-3') and Ri I using the this mutation. conditions described above. A schematic representation of the p53 deletion/insertion event in Analysis of PCR Products. For DNA sequence analysis, the region span fling exons 2—5wasamplified with primers Fl 1 and 5AS (5'-CAACCAGC Hs766T is shown in Fig. 4. The 5' breakpoint is located 10 bp from CCTGTCGTCTCTC-3') using Taq polymerase (Perkin-Elmer Corp., Nor the end of Mu element 11 in the p53 sequence (at position 11347), walk, CT) for 35 cycles at 95°Cfor30 s, 56°Cfor30 s, and 72°CforI mm. whereas the 3' breakpoint occurs within the last nucleotides of intron The predicted length of this PCR fragment was 2249 bp. The PCR product 4 (at position 12993), approximately 100 nucleotides after Alu ele from Hs766T was purified with QlAquick PCR columns (Qiagen, Valencia, ment 12. CA) according to the manufacturer's instructions. DNA sequence analysis was Analysis of Mu Elements in and around the Hs766T p53 Dde done using an ABI Prism DNA sequencing kit (Perkin-Elmer Corp.) and lion. The newly identified Mu sequence that was inserted into p53 analyzed on an ABI fluorescent sequencer. For sequence analysis of the 5 ‘Mu was compared with all known Mu elements in the human genome by elements, the F8-Rll TaqPlus PCR product was purified using QlAquick PCR the BLAST Version 2 algorithm. Interestingly, this comparison led to columns and analyzed as described above. DNA sequence comparisons were a closest match with an Mu element within intron 1 of the p53 gene. performed with the Wisconsin Package Version 9.0 (Genetics Computer Group, Madison, WI). Genome-wide searches were performed at the National The matching Mu element had 173 of 175 nucleotides (98%) identical Library of Medicine using the BLAST Version 2 algorithm. to a part of Mu 8 in the published sequence of p53 (nucleotide positions 8703—8982), whereas the next nearest match had only 147 Results of 175 nucleotides (86%) identical with the inserted Mu fragment. All Mu elements involved in the p53 rearrangement are highly homolo The pancreas cancer cell line Hs766T contains a large deletion that gous: (a) the identity between Mu 8 and Mu 11 is 79% (225 of 284 was first reported as an aberrant hybridization pattern on a p53 DNA bp); (b) the identity between Mu 11 and Mu 12 is 75% (214 of 286 blot (13). Subsequent analyses provided evidence that this deletion bp); and (c) the identity between Alu 8 and Mu 12 is 76% (217 of spans exons 2—4,because these exons could not be PCR-amplified 285 bp). from Hs766T (14). To investigate the possible involvement of Mu To establish the origin of the inserted Alu fragment in the Hs766T elements in the inactivation of the p53 gene in human cell lines, we p53 deletion, we analyzed Alu 8 from p53 in Hs766T, Y79, and chose to further analyze the p53 alteration in Hs766T. human placenta DNA. The sequence of the 175-bp inserted Mu The p53 gene was analyzed by the amplification of three separate fragment was identical to Alu element 8 from the Hs766T p53 gene. long PCR fragments in pancreas cancer cell line Hs766T, retinoblas Compared to the published p53 sequence, two thymines (at positions toma cell line Y79, and human placenta DNA. The PCR fragment 8759 and 8797) are replaced by cytosines in Hs766T. The change to spanning p53 exons 2—9was of the expected 4 kb in Y79 and in the a cytosine at position 8797 was also found in Y79 and in placenta normal human DNA but showed a truncated amplification product of DNA, but these samples had a thymine at position 8759. approximately 2 kb in Hs766T (Fig. 2). No normal-sized amplifica tion product was found in Hs766T, indicating loss of the other allele Discussion of p53. A minor amplification product of about 2 kb was seen in the normal control DNA; this fragment was not present in subsequent Inactivation of the p53 tumor suppressor gene in human cancer is analyses and presumably represents a PCR artifact. The 5' PCR usually due to a single point mutation leading to an amino acid change fragment migrated at the expected length of about 8.5 kb. Similarly, in the p53 protein (1). Such mutations have a dominant negative effect the 3' part of p13, which harbored exon 10 and part of exon 11, yielded the expected PCR product of about 4 kb for Hs766T, Y79, and normal human DNA (data not shown). Molecular Analysis of Genomic p53 Spanning Exons 2—5.To further characterize the deletion event in Hs766T, the region corre sponding to p53 exons 2—5was amplified and subjected to DNA sequence analysis. This region would result in a 2.2-kb PCR fragment in wild-type p53. The DNA sequence analysis revealed that exons 2—4 had been deleted from the Hs766T p53 gene, and that several Mu elements were present in and around the breakpoint. The DNA se quence of this area is shown in Fig. 3. At the 5' end, the DNA sequence in Hs766T is identical to the published p53 sequence up to nucleotide position 11348 (GenBank accession number, X54 156). The DNA sequence then continues with a l75-bp fragment (p53 nucleotide position 8737—8911) of an Alu element that was appar Fig. 2. Long PCR of the p53 coding region in Hs766T, Y79, and placenta DNA. The normal-sized amplification product ofabout 4 kb is visible in Y79 and placenta DNA, but ently inserted into the p53 gene. After this inserted Alu fragment, the the PCRproductof Hs766Tis only about2.3 kb. LaneM, markerLambdacut with DNA sequence is again identical to the published p53 sequence HindlIl; Lane 1, Hs766T; Lane 2, Y79; Lane 3, placenta DNA. 5334

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11347

p53 intron 1 ACTGTGCTCCAGCCTAGGTG ACAGAGAGAG1 actccatctc aaaaaaaaaa aaaaatacag \8737 gggcttgggg gctcacgcct gtaatc9@@ CACTTTAGGA GGCCAAGGGG GGCGGATCAC

CTGAGGTCGG GAGTTCAAGA CCAGCCTGAC CAACATGGTG AAACCCCGTC TCTACTAGAA

Mu 8 fragment

GTACAAAATT AGCCAGGTGT GGTGGCACAT GCTTGTAGTC CTAGCTACTC AGCAGGCTGA

8911 GGCAGGAGAA TCATTTGAAT CCGGG@\12993 aggag gttgcagtaa gcggagatag tgccactgta p53 intron 4 gtttgtttct ttgctgccgt gttcc__AGTTG CTTTATCTGT TCACTTGTGC CCTGACTTTC

Fig. 3. DNA sequence of the deletion/insertion site in the p.5.3gene in Hs766T. The p53 DNA sequence of Hs766T is indicated in uppercase letters, whereas the flanking sequences not present in Hs766T are indicated with lowercase letters. The partial chi sequence CCAG is underlined. A l75-bp fragment from Alu 8 was inserted between the breakpoints in introns 1 and 4. All sequences involved in the intron I breakpoint correspond to Mu DNA. The intron 4 breakpoint involves Alu 8 and a sequence 100 bp downstream from Alu 12.

over the wild-type protein. In addition, some point mutations may lead numbers of Mu elements in the genome and because they share signif to the generation of STOP codons causing premature termination of icant homology. Mso, Mu elements have been implicated in germ-line protein translation or even occur in consensus splice sites, leading to rearrangements in human familial cancer syndromes (8). aberrant p53 mRNAs that do not encode functional protein. It has We analyzed a large deletion spanning exons 2—4ofp53in pancreas been reported that p53 microdeletions (those that involve 3 or more cell line Hs766T at the molecular level. Previous studies had shown that bp) at the sites of small repeats are more common in pancreas cancer this cell line had a deletion of approximately 2 kb in the p53 gene (13), than in other cancer types (14). and that it had lost exons 2, 3, and 4 (14). The 5' breakpoint of the From a mechanistic point of view, these small genetic alterations are deletion occurred in an Mu element(Mu 11 in thep53 gene), whereas the different from those causing larger deletions or even a completely rear 3' breakpoint was 110 bp downstream from a second Mu element (Mu ranged p53 gene. Gross genetic changes in p5.3 have been reported in 12) withinp53. In addition,a 175-bpMu fragmentwasinsertedbetween several human cell lines and primary human tumors, especially from lung the breakpoints of the deleted area. This additional Mu fragment showed and osteosarcomas, and there is one report indicating a large deletion of a high homology with another reported intron 1 Mu element (Mu 8). p53 in a patient from a Li-Fraumeni syndrome family (2, 3, 11). Such Further sequence analysis revealed that the l75-bp Mu insert was actu cases ofp53 rearrangement are of interest for mechanistic,evolutionary, ally identical to Mu 8 in intron 1 of p53. and physiological reasons. Homologous recombination that might occur There are two possible explanations for the observed p53 deletion/ between repeated elements in the genome is an obvious source of dde insertion event in Hs766T involving Mu elements. The first would tions. Mu elements are good potential substrates because of the large involve retrotransposition, by which multiplication of an Mu element

12598 12882 Fig. 4. Model indicating the three Alu elements and their possible involvement leading to the rearrangement and inactivation of the p53 tumor suppressor gene observed in the pancreas carcinoma cell line Hs766T. The DNA sequences present in Hs766T are indicated by thick lines, whereas the regions that became deleted (including exons 2—4andAlu 12) @ ateindicatedbythinlines.ThethreeMuelements0andtherecombinationhotspotswithinthem areindicated. 5335

Downloaded from cancerres.aacrjournals.org on September 27, 2021. © 1996 American Association for Cancer Research. ALU RECOMBINATION IN p.S.3 occurs in a fashion similar to that seen for retroviruses. Retrotransposition mammalian cells (20).2 Whereas DNA divergence might be cx is thought to be responsible for the generation of new, evolutionary pected to reduce the interaction between Alu repeats, a DNA “young―Muelements, for instance, those of the Mu-Sb2 class (15). At double break has been shown to greatly stimulate recombination present, however, there is no evidence for retrotransposition of Mu between highly divergent direct repeats (5). elements in somatic cells. In addition, if retrotransposition were respon In conclusion, we analyzed a deletion/insertion event in p53 in a sible for the deletion/insertion in Hs766T, one would expect at least some pancreas cancer cell line at the molecular level and demonstrated that degree of target site duplication associated with the retrotransposition the recombination event involved at least two separate Mu elements. event. However, no evidence for such a mechanism is present in either Mthough the inactivation ofp53 by genetic recombination appears to the Hs766T p53 DNA sequence or in the sequence differences found in be infrequent in most human cancers, the true frequency of p53 this cell line. A more attractive explanation of the observed deletion/ inactivation may be underestimated when only traditional assays for insertion event would be through homology-driven recombination in p53 mutation are used. which the homology between different Mu elements leads to an cx Acknowledgments change of genetic material. Under this model, the p53 deletion/insertion in Hs766T would have been a complex rearrangement because it in We thankDr.Dmmtr@Gordeninforsuggestionson the manuscriptandGreg volved at least two separate Mu elements within the p53 gene. A Solomon for technical support. schematic representation of this rearrangement is shown in Fig. 4. References Several properties of the Hs766T deletion/insertion sequence are of I. Hollstein, M., Sidransky, D.. Vogelstein, B., and Harris, C. C. p53 mutations in interest. The 5' part of the inserted Alu fragment starts with the last 13 human cancers. Science (Washington DC), 253: 49—53,1991. bp of a 26-bp core sequence that was reported to be a hot spot for 2. Masuda,H., Miller,C., Koeffler,H. P., Battifora,H., and Cline, M. 3. Rearrangementof thep53 genein humanosteogenicsarcomas.Proc.Nail. Aced. Sci. USA. 84: 7716-7719, Alu-associated recombination (16). This sequence includes the 5'- 1987. GCTGG-3' motif that forms the first 5 bases of the 5'-GC'FG 3. Takahashi, T., Nau, M. M., Chiba, I., Birrer, M. I., Rosenberg, R. K., Vinocour, GTGG-3' prokaryotic recombination hot spot c/ti sequence (17). The M., Levitt, M., Pass, H., Gazdar, A. F., and Minna, J. D. p53: a frequent target for genetic abnormalities in lung cancer. Science (Washington DC), 246: 491—494, actual 5 ‘breakpoint does not fall within this consensus sequence but 1989. rather at the 3 ‘endof a short GA dinucleotide repeat. There are three 4. Gordenin, D. A., Lobachev, K. S., Degtyareva, N. P., Malkova, A. L., Perkins, E., and Resnick, M. A. inverted DNA repeats: a source of eukaryotic genomic instability. occurrences of another 4-bp motif of chi (5'-CCAG-3') at or near the Mol. Cell. Biol., 13: 5315—5322,1993. breakpoints of the Hs766T deletion/insertion. This motif has been 5. Tran, H., Degtyareva, N., Gordenin, D., and Resnick, M. A. Altered replication and reported to be located near nonhomologous recombination break inverted repeats induce mismatch repair-independent recombination between highly diverged DNAs in yeast. Mol. Cell. Biol., 17: 1027—1036,1997. points of the immunoglobulin loci. Also, one-ended homology-driven 6. Mighell, A. J., Markham, A. F., and Robinson, P. A. Alu sequences. FEBS Lett., 417: recombination has been demonstrated for Escherichia coli and mdi 1-5,1997. 7. Lehman, M. A., Schneider, W. J., Sudhof, 1. C., Brown, M. S., Goldstein, I. L, and rectly in mammalian cells (18). Russell, D. W. Mutation in LDL receptor: Alu-Alu recombination deletes exons Although the position of the 5' Alu 8 breakpoint falls within the encoding transmembrane and cytoplasmic domains. Science (Washington DC), 227: 140—146,1985. 26-bp core sequence, there is no clear evidence that any chi-related 8. Marshall, B., lsidro, G., and Boavida, M. G. Insertion of a short Alu sequence into the sequence was directly involved in the observed deletion/insertion of hMSH2 gene following a double cross over next to sequences with chi homology. the Hs766T p53 gene. We also did not find any homology between the Gene (Amst.), 174: 175—179,1996. 9. Schichman, S. A., Caligiuri, M. A., Strout, M. P., Carter, S. L., Gu, Y., Canaani, E., Alu elements and the intron 4 sequence starting 3' from the deletion/ Bloomfield, C. D., and Ct-ace, C. M. ALL-l tandem duplication in acute myeloid insertion breakpoint. leukemia with a normal karyotype involves homologous recombination between Alu elements. CancerRes., 54: 4277—4280,1994. In addition to the larger genetic alterations, it has been reported that 10. Onno, M., Nakamura, T., Hillova, I., and Hill, M. Rearrangement of the human tre Hs766T harbors a mutation in p53 exon 5 at codon 181 (CGC to CAC, oncogeneby homologousrecombinationbetweenAlu repeatsof nucleotidesequences from two different chromosomes. Oncogene, 7: 2519—2523,1992. changing an Arg to His; Ref. 13). Such a mutation would be of interest 11. Plummer, S. J., Santibanez-Koref, M., Kurosaki, T., Liao, S., Noble, B., Fain, P. R., because p53 inactivation has recently been linked to increased intra Anton-Culver, H., and Casey, G. A germline 2.35 kb deletion of p53 genomic DNA chromosomal homologous recombination (19). However, we could creatinga specific loss of the oligomerizationdomain inheritedin a Li-Fraumeni syndrome family. Oncogene. 9: 3273—3280,1994. not confirm this exon S mutation in Hs766T, a result that is in 12. Albertoni, M., Daub, D. M., Arden, K. C., Viars. C. S., Powell, C., and Van Mew, agreement with another report on the status of p53 in this pancreas E. G. Genetic instability leads to loss of both p53 alleles in a human glioblastoma. Oncogene. 16: 321—326,1998. cancer cell line (14). From a theoretical point of view, a point 13. Ruggen, B., Zhang, S. Y., Caamano. J.. DiRado, M., Flynn, S. D., and Klein-Szanto, mutation in p53 would eliminate the need to inactivate p53 through A. J. Human pancreatic carcinomas and cell lines reveal frequent and multiple other mechanisms. alterations in the p53 and Rb-l tumor-suppressor genes. Oncogene, 7: 1503—1511, 1992. One additional property of the sequences around the p53 dde 14. Redston,M. S., Caldas,C., Seymour,A. B., Hruban,R. H., da Costa,L., Yea. C. I., tion/insertion is that the breakpoint occurred at the 3' end of an and Kern, S. E. p53 mutationsin pancreaticcarcinomaand evidence of common involvement of homocopolymertracts in DNA microdeletions.Cancer Res., 54: almost perfect l2-bp-long GA dinucleotide repeat. It has been 3025—3033.1994. reported that pancreas cancer has an increased incidence of small 15. Jurka,J. A new subfamilyof recentlyretroposedhumanAlu repeats.Nucleic Acids Res.,21:2252,1993. deletions and insertions at homocopolymer tracts (repeats of pu 16. Rudiger. N. S., Gregersen, N., and Kielland-Brandt, M. C. One short well conserved rines or pyrimidines of at least 4 bases; Ref. 14). Such tracts may region of Alu-sequences is involved in human gene rearrangements and has homology form dinucleotide repeats such as the GA repeat located at the p53 with prokaryotic chi. Nucleic Acids Res., 23: 256—260,1995. 17. Stahl, F. W. Special sites in generalized recombination. Annu. Rev. Genet., 13: 7—24, breakpoint in Hs766T. In a recent report on p53 inactivation in a 1979. case of glioblastoma, it was shown that a CA dinucleotide repeat 18. Belmaaza, A., and Chartrand, P. One-sided invasion events in homologous recom bination at double-strand breaks. Mutat. Res., 314: 199—208,1994. (STS D17S96O) was located close to the breakpoint of a somatic 19. Bertrand, P., Rouillard, D., Boulet, A., Levalois, C.. Soussi, T., and Lopez, B. S. rearrangement that inactivated the p5.3 gene (12). It would be of Increase of spontaneous intrachromosomal homologous recombination in mammalian cells expressing a mutant p53 protein. Oncogene. 14: 1117—1122,1997. interest to determine whether such motifs provide structures that 20. Gordenin, D. A., and Resnick, M. A. Yeast ARMs (DNA at-risk motifs) can reveal might initiate recombination. Motifs referred to as at-risk motifs sources of genome instability. Mutat. Rca., 41X1:45—58,1998. have been identified that stimulate recombination including large inverted repeats comprised of genes and Alu elements in yeast and 2 Unpublished data. 5336

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A Human Placenta-specific ATP-Binding Cassette Gene (ABCP) on Chromosome 4q22 That Is Involved in Multidrug Resistance1

Rando Allikmets, Lynn M. Schriml, Amy Hutchinson, Vincenzo Romano-Spica, and Michael Dean2

Intramural Research Support Program, Science Applications international Corporation-Frederick National Cancer Institute-Frederick Cancer Research and Development Center, Frederick@Marjland21702-1201(R.A., A. H.): National CancerInstitute,Laboratory of GenomicDiversity, Frederick Maryland 21702-1201fL M. S., M. DI: and institute of Hygiene and Public Health, Faculty ofMedicine ofthe Catholic University, 00168 Rome, Italy (V. R-S.J

Abstract albicans (13—15).However, the endogenous substrates for these pro teins have not been established. We characterized a new human ATP-binding cassette (ABC) tram porter gene that Is highly expressed In the placenta. The gene, ABCP, Materials and Methods produces two transcripts that differ at the 5' end and encode the same 655-amino acid protein. The predicted protein Is closely related to the Sequence Analysis. Searches of the EST database (http:llwww.ncbi.nlm. Drosophila white and yeast ADPJ genes and is a member of a subfamily mh.gov) were performed with the BLAST program (16) using the that includes several multidrug resistance transporters. ABCP, white, and HuESTJ5748J sequence. Phylogenetic analysis was performed using the ADPI all have a single ATP-bindlng domain at the NH2 te@ninus and a PHYLIP package (http:llevolution.genetics.wasinngton.edu/phylip.html). thigle COOH-termlnal set of transmembrane segments. ABCP maps to cDNA Ooth@. Primerswere designed from the sequences of the EST human chromosome 4q22, between the markers D4S2462 and D4S1557, clones from 5' and 3' regions of the gene and used to link the EST cDNA and the murine gene (Abcp) is located on chromosome 6 28-29 cM from sequences by RT-PCR with placenta QUICK-Clone cDNA (Clontech) as a the centroinere. ABCP defines a new syntenic segment between human template. Primers ABCP-Rl (5'-CCAGCAAGTIIIIGAATGAACGCT chromosome4 and mouse chromosome 6. The abundant expression of this TOG) and ABCP-R2 (5'-AGGTGGTGTAGCTGATCTCCTFGAAGACTG) gene in the placenta suggests that the protein product has an important were used for 5' RACE reactions using RACE-ready cDNA (Clontech). PCR role in transport of specific molecule(s) Into or out of this tissue. products were cloned into the pGEM-T vector (Promega). Sequencing was performedwiththe Taq Dyedeoxy TerminatorCycleSequencingkit (Applied Introduction Biosystems), according to the manufacturer's instructions. Sequencing reac tions were resolved on an ABI 373A automated sequencer. The sequence of the A universal requirement for all organisms is the transport of mol ABCP cDNA has been deposited.4 ecules across cellular membranes. The ABC3 genes comprise a large Northern Hybridization. DNA fragments used as probes were purified on superfamily, the protein products of which carry out the transport of a 1% low-melting temperature agarose gel. DNA was labeled directly in many specialized compounds in prokazyotes, eukaryotes, and archae agarose with the Random Primed DNA labeling kit (Boehringer Mannheim) bacteria. ABC transporters are one of the few superfamilies abundant and hybridized to a multiple tissue Northern blot and a Master blot (Clontech), in all three kingdoms and are characterized by an extensive conser according to the manufacturer's instructions. vation of the AlP-binding domains throughout evolution (1, 2). A Genetic Mapping. The human ABCP (also designated ABCJ5) was mapped in the GeneBridge 4 radiation hybrid panel. A murine ABCP-related functional ABC transporter consists of two ATP-binding domains and sequence was identified through searches of the mouse EST database. A two sets of TM domains. In eukaryotes, most ABC genes either have representative clone (AA008579) was obtained and sequenced from both the 5' all four domains in a single open reading frame (full transporters) or and 3' ends.4 Primers to amplify a portion of the 3' untranslated region were represent half transporters with a single TM and single AlP-binding designed (AbcpFl, 5'-AATCAGGGCATCGAACTGTC; AbcpRl, 5'-GGTA segment (3, 4). Half transporters typically form heterodimers to pro ATCAAAGTGCCCAT) using the PRIMER program (http://www-genome. duce a functional protein (5). Structurally, most eukaryotic transport wi.mit.edu) and used to screen a mouse radiation hybrid panel (Research era have the TM segments NH2-terminal to the AlP-binding domains Genetics). The panel was constructed from irradiated mouse embryo primary (TM-ATP-TM-ATP). cells (l29aa) fused with a hamster cell line (A23). Chromosome location was ThewhitelocuswasthefirstgeneticmarkerdescribedinDrosoph determinedby The JacksonLaboratoryMappingGroup. ila and encodes an ABC half transporter (6). The white protein forms Results complexes with two related transporters, brown and scarlet, to trans port guanine and tryptophan, the precursors to eye pigments (7—9). Previously, we identified over 25 new human ABC genes from the white is related to the Saccharomyces cerevisiae ADPJ gene and a human DNA sequence databases (Ref. 17; data not shown). One of these human locus on chromosome 21q22.3, ABC8 (10—12).Altogether, genes, formerly designated HuEST157481, is highly expressed in the these genes form an ABC subfamily that includes both half and full placenta and is found at low to undetectable levels in some other tissues transporters (3, 4). In addition, this subfamily includes several drug (Fig. lA). Hybridization of a Masterblot (Clontech) confirmed the very resistance genes described in S. cerevisiae, S. ponthe, and Candida high placental expression, about 100 times more than in other detectable tissues (data not shown) such as heart, ovary, kidney, and fetal liver (Fig. Received 10l2@8;accepted 10/19/98. 1B). This gene, ABCP, is represented in the database by over 30 EST The costs of publication of this article were defrayed in part by the payment of page clones from placenta, infant brain, fetal liver/spleen, and uterus. charges. This article must therefore be hereby marked advertisement in accordance with Sequencing of cDNA clones, reverse transcription-PCR and RACE 18 U.S.C. Section 1734 solely to indicate this fact. I This projectwas funded in part with Federal funds from the National Cancer Institute, products revealed two transcripts that differ by —200bp at the 5' end. NIH. under Contract No. NO1-CO-56000. These data are in agreement with the Northern analysis (Fig. IA), 2To whomrequestsfor reprintsshouldbe addressed.Phone:(301)846-5931;Fax: (301) 846-1909; E-mail: [email protected]. 3 The abbreviations used: ABC, ATP-binding cassette; ABCP, placenta-specific ABC 4 The nucleotide sequence data reported in this study have been deposited at the transporter, TM, transmembrane; EST, expressed sequence tag; RACE, rapid amplifica National Center for Biotechnology Infonnation/Genilank Data Library under accession tioii of cDNA ends. numbers AF103796 and AF103875. 5337

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A B hybrid panel (Fig. 4). No other ABC gene has previously been mapped to human chromosome 4. A partial mouse Abcp cDNA was sequenced and used to map Abcp in murine radiation hybrids. Abcp mapped to HBPILuLIMKPa I 2345678 chromosome 6, 28—29cM from the centromere. Only one other gene, 7.5- A- the delta 2 ionotropic glutamate receptor (GRID2), has been localized B— to the human 4q22 region and to mouse chromosome 6. Thus, Abcp 4.4 — C- and Grid2 define a new syntenic group between human chromosome D 4 and murine chromosome 6. 2.4 — E— F— . Discussion 1.35- I H- Here we describe a new human ABC gene that is expressed at a very high level in the placenta. ABCP belongs to a subfamily of ABC Fig. I. Expression of the ABCP gene. In A, a fragment of the ABCP gene was hybridized to a Northern blot containing RNA from adult human heart (H), brain (B), proteins that includes transporters of guanine and tryptophan, as well placenta (P1), lung (Lu), liver (LI), muscle (M), kidney (K), and pancreas (Pa). A longer as several fungal multidrug resistance genes. However, the transmem exposure revealed a weak hybridization signal in the heart but no detectable expression in brane domain of ABCP is rather distinct from its paralogs. In addition, the other tissues (data not shown). B, hybridization ofABCP to a dot blot containing RNA from 43 adult (rows A—F)and7 fetal (row G) tissues. Row H contains negative controls. different members of the same ABC subfamily often transport very Significant hybridization was seen to placenta (F4), and to lesser extent heart (CI), ovary different substrates. Identification of the endogenous substrate of (D2). kidney (El), and fetal liver (G4). ABCP will require additional biological data. In situ hybridization to placental tissue at different stages of development should yield some

1 MSSSNVEVFI PVSQGNTNGF PATVSNDLKA FTEGAVLSFH NICYRVKLKS clues to ABCP location and function. To date, all known ABC half transporters are localized to the membrane of intracellular organelles

S 1 GFLPCRKPVE KEILSNINGI MKPGLNAILG PTGGGKSSLL DVLAARKDPS (5, 9, 18). It will be important to determine whether ABCP is also located inside the cell. 101 GLSGDVLING APRPANFX@N SGTVVQDDVV )SGTLTVRENL QFSAALRL&T Many ABC genes play a role in human inherited diseases, including cystic fibrosis, adrenoleukodystrophy, diabetes, and retinal degener 15 1 TI4TNHEXNER INRVIEELGL DKVADSKVGT QFIRGVSGGB RKRTSI@4EL ation. In addition, mammalian ABC proteins play important roles in 201 ITDPSILSLD EPTTGLDSST ANAVLLLLXR NSKQGRTIIF SIHQPRYSIF drug resistance and peptide and ion transport (1, 19). No obvious candidate phenotypes are found in the regions that ABCP maps to in 251 KLFDSLTLL& SGRLMFHGPAQEALGYFESAGYHCEAYNNPADFFLDIING either human or mouse genomes. Curiously, a genome-wide scan of preeclampsia, a major complication of pregnancy, revealed linkage to 301 DSTAVALNRE EDFKATEIIE PSKQDKPLIE XLAEIYVNSS FYKETKAELH chromosome 4, but to a region that would appear to exclude ABCP 351 QLSGGEKKKK ITVFKEISYT TSFCHQLRWV SKRSFKNLLG [email protected] (20).It is feasiblethatABCPcanplaya rolein theplacentalbarrier in vivo, either by protecting the fetus from harmful compounds or by 401 VTVVLGLVIGAIYFGLKNDS TGIQNR.AGVLFFLTTNOCFS SVSAVELFVV transporting some important, not yet identified, substrate. Most ABC proteins transport very specific molecules, so it is likely that the 451 EKKLFIHEYI SGYYRVSSYF LGKLLSDLLP MRMLPSIIFT CIVYFMLGL1( ABCP pumpsa substance(s)thatplays an importantrolein the 501 PKADAFFVMM FTL?O1VAYSASSMALAIAAG QSVVSVATLL MTICFVF@t@ homeostasis of the placenta. The placenta does contain transporters for some specific substrates, such as glucose (21). 551 FSGLLVNLTT IASWLSWLQY FSIPRYGFTA LQHNEFLGQN FCPGLNATGN

601 NPCNYATCTG EEYLVKQGID LSPWGLWK@fiVALACMIVIF LTIAThKLLF

651 LKKYS Fig. 2. Open reading frame of ABCP. The predicted open reading frame of the ABCP gene is shown with the ATP-binding domain in boldface and the six putative TM segments underlined. where two equally strong transcripts are present. Both transcripts contain the same open reading frame of 658 amino acids (Fig. 2) that consists of an ATP-binding domain at the NH2 terminus and six predicted TM segments in the COOH-terminal portion of the mole cule. The amino acid sequence of ABCP is 31% identical to the Drosophila white protein, and it represents a member of an ABC gene subfamily that also contains the Drosophila brown and scarlet genes, as well as the yeast ADPJ and two other human white-related genes (Fig. 3). Phylogenetic analysis revealed that ABCP is most closely related to ADP1 and YOLO75 and is outside the ABC8 and white! brown/scarlet groups. However, given the sparse knowledge of sub strates for these transporters and the considerable divergence in their Fig. 3. Phylogenetictreeof ABCP-relatedproteins.An aminoacidalignmentof ABCP andseveralrelatedgenes was generatedusingPILEUP(GeneticsComputingGroup).The TM domains, it is hard to speculate on the possible function(s) of sequence was trimmed to a 673-residue, minimally overlapping segment and used for ABCP based on phylogenetic studies. neighbor-joininganalysis. Bootstap analysis of both neighbor-joiningand maximum parsimony trees demonstrated that the ABC8/AbC8/ABC8L and whitelbrown/scarlet The ABCP gene was mapped to human chromosome 4q22, between clustersaresignificantlyrelated(datanot shown). ABC8, human;Abc8, mouse;ABC8L, the markers D4S2462 and D4S1557, with the GeneBridge 4 radiation human ABC8-like;Y0L075, yeast open reading frame; bfrlC, COOH-terminalhalfof bfrl. 5338

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1C 15.3

Fig.4. LocationoftheABCPandAbcpgenes. The location of the ABCP gene on human chromo some 4q22 and Abcp on mouse chromosome 6, 28—29centimorgans (cM) from the centromere, is @ shownalongwithseveralflankingmarkers.Both ,v' Hox:1@ 26 ABCP and GPJD2 (glutamate receptor, ionotropic, delta-2)have been localizedto 4q22 and mouse N q11-q21@@‘— ABCP chromosome6.Thesemarkersdefinea shortsyn :@:@ Abcp 28-29 @ tenic Interval that is flanked in the human by SPPI q22 ‘@@‘%@ GRID2 (secreted phosphoprotein-l) and ADHI (alcohol Grid2 —.29.7 @ dehydregenase 1), which map to mouse chromo q21-q23 somes 6 and 3, respectively. The mouse chromo @ some 6 segment is flanked by Hoxa (homeo box 31.1 ADHI Igk Ia) and Igk (immunoglobulinkappa chain com 31.2 plex)located on chromosomes 7plS-pl4 and 2pl2, 31.3 respectively. 32 33 Mu6 34 35 Hu4

Recently, Miyake et a!.5 demonstrated that the ABCP gene is 9. Ewart, 0. D., Cannell, D., Cox, G. B., and Howells, A. J. Mutational analysis of the overexpressed and amplified in certain human breast and colon cancer traffic ATPase (ABC) transporters involved in uptake of eye pigment precursors in Drosophila melanogaster. Implications for structure-function relationships. J. Biol. cell lines (22) resistant to the chemotherapeutic drugs mitoxantrone Chem., 269: 10370—10377,1994. and, to a lesser extent, daunorubicin. Mitoxantrone is used in the 10. Croop, i. M., Tiller, G. E., Fletcher, J. A., Lux, M. L., Raab, E., Goldenson, D., Son, treatment of acute leukemias and has shown promise in the treatment D., Arciniegas, 5, and Wu, R. L. Isolation and characterization of a mammalian homolog of the Drosophila white gene. Gene (Amst.), 185: 77—85,1997. of breast and ovarian cancers (23, 24). These data suggest that ABCP 11. Savary, S., Denizot, F., Luciani, M., Matte, I. M., and Chimini, G. Molecular cloning is a transporter for some chemotherapeutic compounds and that over of a mammalian ABC transporter homologous to Drosophila white gene. Mamm. expression of this transporter may play a role in drug resistance in Genome, 7: 673—676,1996. 12. Chen, H., Rossier, C., Lalioti, M. D., Lynn, A., Chakravarti, A., Perrin, G., and some cancers. The ABC gene subfamily that includes ABCP contains Antonarakis, S. E. Cloning of the cDNA for a human homologue of the Drosophila a number of full transporters that are involved in yeast multidrug whitegeneandmappingto chromosome21q22.3.Am. J. Hum. Genet.,59: 66—75, resistance. These pumps (PDR5, SNQ2, CDR1, and CDR2) confer 1996. resistance to a wide variety of compounds including cycloheximide, 13. Prasad, R., Dc Wergifosse, P., Goffeau, A., and Balzi, E. Molecular cloning and characterization of a novel gene of Candida albicans, CDRI, conferring multiple chioramphenicol, brefeldin A, and several antifungal agents (25). resistance to drugs and antifungals. Curr. Genet., 27: 320—329,1995. Further understanding of the function and regulation of ABCP may be 14. Sanglard, D., lscher, F., Monod, M., and Bille, J. Cloning of Candida albicans genes important to effective chemotherapy. conferring resistance to azole antifungal agents: characterization of CDR2, a new multidrug ABC transporter gene. Microbiology, 143: 405—416,1997. 15. Nagao, K., Taguchi, Y., Arioka, M., Kadokura, H., Takatsuki, A., Yoda, K., and Acknowledgments Yamasaki, M. bfrl +, a novel gene of Schizosaccharomyces pombe which confers brefeldin A resistance, is structurally related to the ATP-binding cassette superfamily. We thank Susan Bates and Tito Fojo for helpful discussions and Stan J. Bacteriol., 177: 1536—1543,1995. Cevario for oligonucleotide synthesis and assistance with DNA sequencing. 16. Altschul, S. F., Gish, W., Miller, W., Myers, E. W., and Lipman, D. J. Basic local Computing resources were provided by the Frederick Biomedical Supercom alignment search tool. J. Mol. Biol., 215: 403—410, 1990. puting Center. 17. Allikmets, R., Hutchinson, A., Gerrard, B., and Dean, M. Characterization of the human ABC superfamily: isolation and mapping of 21 new genes using the EST database. Hum. Mol. Genet., 5: 1649—1655, 1996. References 18. Lehner, P. J., and Trowsdale, J. Antigen presentation: coming out gracefully. Curr. 1. Dean, M., and Allikmets, R. Evolution of ATP-binding cassette transporter genes. Biol., 8: R605-R608, 1998. CulT. Opin. Genet. Dcv., 5: 779—785, 1995. 19. Allikmets, R., Singh, N., Sun, H., Shroyer, N. F., Hutchinson, A., Chidambaram, 2. Tatusov, R. L., Koonin, E. V., and Lipman, D. J. 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Elizabeth E. Mannick, Luis E. Bravo, Guillermo Zarama, et al.

Cancer Res 1996;56:3238-3243.

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