Chromosomal mapping of the human annexin IV (ANX4) gene

12, 313-318

CENOMICS

(19%)

Chromosomal JONATHAN

Medicine,

tpathology,

and *Biochemistry, ReceivedAugust26,

Annexin

IV (placental

anticoagulant

protein

II) is a member

of the annexin or lipocortin family of calcium-dependentphospholipid-binding proteins. A cDNA for human annexin IV was isolated from a placental library that is 675 bases longer in the 3’ untranslated region than previously reported, indicating the existence of alternative mRNA processing for this gene. Genomic Southern blotting with a cDNA probe indicated a gene size of 18-56 kb. Primers developed for polymerase chain reaction (PCR) allowed amplification of a 1.6-kb portion of the ANX4 gene. DNA sequence analysis showed that this PCR product contained a single intron with exon-intron boundaries in exactly the same position as in the mouse annexin I and annexin II genes. PCR analysis of a somatic cell hybrid panel mapped the ANX4 gene to chromosome 2, and in situ hybridization with a cDNA probe showed a unique locus for ANX4 at 2~13. This study provides further evidence that genes for the annexins are dispersed throughout the genome but are similar in size and exon-intron organization. o 1992 Academic

Press, Inc.

University of Washington,

EMBL/GenBank

from Data

this article have been deposited with Libraries under Accession No. M82809.

AND METHODS

Cell lines and DNA. Purified genomic DNA samples from a panel of 25 human-hamster hybrid cell lines were obtained from the BIOS Corp. (New Haven, CT) and were exactly the same as described in two previous studies (Tait et al., 1991a,b). In addition, purified DNA from three human-mouse hybrid cell lines was obtained from BIOS. These three additional cell lines were originally derived in the laboratory of Dr. Thomas Shows (Roswell Park Memorial Institute, Buffalo, NY) as described (Owerbach et al., 1980; Shows et al., 1984); their chromosome content (determined by isozyme analysis) is listed in Table 1. Human genomic DNA was prepared from peripheral blood samples of unrelated Caucasians. Mouse genomic DNA was from a male mouse, strain C57BL/6. Isolation of annexin ZV(PAP II) cDNA. Annexin IV (PAP’ II) was purified from human placenta as described (Tait et al., 1988), and polyclonal rabbit antiserum was prepared. A Xgtll cDNA library prepared from human placental poly(A) RNA (Clontech, Palo Alto, CA) was screened with affinity-purified radiolabeled anti-PAP-II by standard methods (Ausubel et aZ., 1989). A total of seven clones were isolated and sequenced. The structure of the cDNA was established by sequencing both strands of overlapping clones with methods described below with universal or specific primers. One of these clones, pPAP-

1 Abbreviations polymerase chain morphism.

the

98195

and bovine [endonexin (Hamman et uZ., 1988)] cDNA clones for annexin IV have been reported. Annexin IV has 4559% identity with other members of the annexin family (Hauptmann et al., 1989). Identification of a genetic or neoplastic disease involving one of the annexins would provide a strong indication of function. Thus, we have been mapping the genes for mammalian annexins as part of a study of their structure and function and to provide the means for identifying their possible involvement in disease. In this study, we have mapped the human ANX4 gene to a unique locus at 2~13 by PCR analysis of somatic cell hybrids and in situ hybridization with a cDNA probe. We also report the sequence of a cDNA encoding human annexin IV that is much longer at the 3’ end than previously reported, providing evidence for alternative processing of the annexin IV mRNA. Genomic Southern blot and PCR data indicate that the human ANX4 gene is similar in size and exon-intron organization to the mouse annexin I and annexin II genes. MATERIALS

The annexins (Crumpton and Dedman, 1990) or lipocortins are a family of calcium-dependent phospholipidbinding proteins whose function in normal physiology is uncertain (reviewed by Crompton et al., 1988; Klee, 1988; Haigler et al., 1989). We previously isolated four annexins from human placenta based on their in vitro anticoagulant activity, including annexin IV (placental anticoagulant protein II) (Funakoshi et al., 1987a; Tait et aZ., 1988). Other investigators have isolated the same protein in studies of other physiological processes: as a potential mediator of exocytosis [32.5 kDa calelectrin (Walker 1982; Sudhof et al., 1984) or endonexin I (Geisow et uZ., 1986) or chromobindin 4 (Creutz et uZ., 1987)]; as a potential cytoskeletal component in intestinal epithelium [protein II (Gerke and Weber, 1984)]; and as a potential mediator of intracellular calcium signals [35P-calcimedin (Kaetzel et al., 1989)]. The porcine protein has been completely sequenced [protein II (Weber et al., 1987)], and human [PP4-X (Grundmann et aC, 1988)]

Seattle, Washington

1991

INTRODUCTION

Sequencedata

IV (ANX4) Gene

F. TAtT,*,t CHRISTINA SMITH,* D. ALAN FRANKENBERRY,* CAROL H. MIAO,$ DAVID A. ADLER,t AND CHRISTINE M. DlSTECHEt

of *Laboratory

Departments

Mapping of the Human Annexin

used: PAP, placental anticoagulant protein; reaction; RFLP, restriction fragment length

313 All

Copyright 0 1992 rights of reproduction

PCR, poly-

osw7543/92 $3.00 by Academic Press, Inc. in any form reserved.

314

TAIT

TABLE

1

Percentage Chromosome Lines

Discordance for the ANX4 Gene Content of Mouse-Human Hybrid

Human chromosome

Percentage discordance”

Mouse-human

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 x Y

17.8 3.6 21.4 19.3 85.7 21.4 14.3 25.0 21.4 17.8 17.8 17.8 28.3 32.1 21.4 21.4 10.7 25.0 32.1 21.4 25.0 28.5 14.2 25.0

’ Calculated from results of Fig. cell lines. The chromosome content is the same as previously described

DG-L-10

+

and Cell

cell line 38L- 19

+ +

38L-3

+

ET

AL.

quantitated visually from an ethidium-stained gel and diluted as needed. An overnight ligation of 50 ng of vector and 85 ng of insert (a 1:3 molar ratio) was followed by transformation, colony confirmation by PCR, and overnight culture. Double-stranded sequencing of the miniprep isolated plasmid was performed as described (Kraft et al., 1988) with Ml3 forward and reverse sequencing primers. In situ hybridization. The whole plasmid pPAP-II-B6 was labeled by nick-translation with 3H-labeled nucleotides to a specific activity of 1.3 X 107cpm/rg and hybridized as described (Marth etal, 1986) at a concentration of 0.08 rig/al to metaphase chromosomes prepared from lymphocytes of a normal male donor. The slides were exposed for 25 days and the chromosomes were identified by Q-banding.

+ + +

RESULTS + +

Isolation of cDNA Clones for Annexin IV + + +

+ + f +

+

+

+ +

+

+

+

3 for a total of 28 human-rodent of the 25 human-hamster hybrids (Tait et al., 1991a,b).

II-B6 (see Fig. l), was cloned into the EcoRI a genomic hybridization probe.

site of pUC18

and used as

PCR and Southern blotting. Oligonucleotides were designed with 19 bases at the 3’end matching the annexin IV cDNA sequence and an EcoRI or Hind111 restriction site at the 5’ end. The following primers were used for chromosomal mapping (see Fig. 1): 5’-GGG AAT TCG ACG CCC ACG GTG CTG TAT-3 (sense primer) and 5’-AAA AGC TTA GGT TTG GCT TAT GCG CCG-3’ (antisense primer). PCR was performed as described (Mullis and Faloona, 1987; Saiki er al., 1988). Each 50-pl reaction was set up as described (Tait et al., 1991a), except that the concentration of Taq DNA polymerase was 1.25 U and the gelatin was 0.01 g/liter. The samples were amplified in a DNA thermal cycler (Perkin-Elmer, Norwalk, CT) in the step-cycle mode. The thermal profile included an initial 6-min denaturation step at 94°C; 30 cycles of 94°C for 1 min, 55°C for 1 min, 72°C for 3 min; and finally a lo-min extension step at 72°C. Genomic Southern blotting was performed as previously described (Tait et al., 1991b) with a final wash step at 65°C in 0.1X SSC/l% SDS. Cloning and sequence analysis of PCR products. Two vectors were used for cloning the PCR product prior to sequencing. For Ml3 cloning, the PCR product (1400 ~1) was extracted with phenohchloroform (1:l) and chloroform, ethanol-precipitated, cleaved with EcoRI and HindIII, and subjected to preparative agarose-gel electrophoresis, electroelution, extraction, and precipitation. Half of the redissolved PCR product was then ligated with 0.1 pg M13mp18 RF cleaved with the same enzymes. DNA sequencing was performed by the dideoxy chain termination method according to standard methods with modified T7 DNA polymerase (Sequenase, USB) and universal primers (Ausubel et al., 1989). In some experiments, the PCR product, after the initial extraction, was cloned into the pCRlOO0 vector (TA Cloning Kit, Invitrogen, San Diego, CA). Extracted PCR product was

cDNA clones encoding annexin IV were isolated and sequenced as described under Materials and Methods. The composite sequence (Fig. 1) contains 73 bases of 5’ untranslated sequence, 963 bp of protein-coding sequence, and 940 bp of 3’ untranslated sequence. The predicted protein sequence agrees completely with 143 residues of protein sequence data previously reported (Tait et al., 1988). Although the mature protein has a blocked N-terminus, predicted residue Met-3 must be present in the mature protein because protein sequence data were obtained from a CNBr peptide starting at residue 4 of the predicted sequence (Tait et al., 1988). It is likely that the initiator Met is removed by proteolytic processing (indicated by parentheses in Fig. 1) and the resulting N-terminal Ala is then acetylated, as occurs for annexin V (Funakoshi et al., 1987b). Thus, the indicated residue Met-l is the most likely site for initiation of translation. The clones that we isolated contain a much longer 3 untranslated region than that previously reported for the human cDNA (Grundmann et al., 1988). Instead of a poly(A) sequence beginning after base 1301, we observed another 675 bases in two independent clones. The difference in length indicates alternative processing of the annexin IV transcript (see Discussion). The first 1301 bases are identical to bases 2 to 1301 previously reported by Grundmann et al. (1988) with two exceptions. We found a G instead of an A at base 366, encoding an Arg instead of a Glu residue at codon 98. Our sequence is confirmed independently by PCR analysis of this region of the ANX4 gene (see Fig. 2). However, it is still possible that this sequence difference is due to a polymorphism. A second difference was noted in the 5’ untranslated region: an extra base (C) at position 17 of our sequence. Genomic Southern Blot and PCR Analysis of the Human ANX4 Gene Southern blots of human genomic DNA were probed with the pPAP-II-B6 cDNA clone to estimate the size of the ANX4 gene and to assessthe feasibility of somatic

CHROMOSOMAL

MAPPING

OF

ANX4

315

GENE

16

(M,AMAlKGGTVKAASGF --> CCAGAGGAGGAGCGCACGCCGGCCTCGAAGMCTTCTGCTTGGGTGGCTGMCTCTGATCTTGACCTAGAGTCATGGCCATGGCMCCAAAGGAGGTACTGTCAAAGCTGCTTCAGGATT

120

NAMEDAPTLRKAMKGLGTDEDAIISVLAYRNTAQRQEIRT CMTGCCATGGMWITGCCCAGACCCTGAGGMGGCCATGMAGGGCTCGGCACCGATGMGACGCCATTATTAGCGTCCTTGCCTACCGCMCACCGCCCAGCGCCAGGA~TCAGGAC

56 240

AYKSTlGRDLIDDLKSELSGNFEPVlVGnHTPTVLYDVQE AGCCTACMGAGCACCATCGGCAGGGACTTGATAGACGACCTGMGTCAGMCTGAGTGGCMCTTCGAGCAGGTGATTGTGGGGATGATGACGCCCACGGTGCTGTATGACGTGCMCA

96 360

L~RAMKGAGTDEGCLIEILASRTPEElRRlSPTYPPPYCR GCTGCGMGGGCCATGAAGGGAGCCGGCACTGATGAGGGCTGCCTMTTGAGATCCTGGCCTCCCGGACCCCTGAGGA~TCCGGCGCATMGCC~CCTACCAGCAGCMTATGGACG e .

136 480

SLEDDIRSDTSFMFQRVLVSLSAGGRDEGNYLDDALVRQD GAGCCTTGAAGATGACATTCGCTCTGACACATCGTTCATGTTCCAGCGAGTGCTGGTGTCTCTGTCAGCTGGTGGGAGG~TGMGGAMTTATCTGGAC~TGCTCTCGTGAGACAGGA

176 600

AQDLYEAGEKKUGTDEVKFLTVLCSRNRNHLLHVFDEYKR TGCCCAGGACCTGTATGAGGCTGGAGAGMGAAATGGGGGACAGATGAGGTG~TTTCTMCTGTTCTCTGTTCCCGGMCCGAAATCACCTGTTGCATGTGTTTGATGMTACMMG

216 720

ISPKDIEPSIKSETSGSFEDALLAIVKCMRNKSAYFAEKL GATATCACAGMGGATATTGMCAGAGTATTAMTCTGAAACATCTGGTAGCTTTGMGATGCTCTGCTGGCTATAGTAMGTGCATGAGGMCAAATCTGCATATTTTGCTGMMGCT

256 840

YKSMKGLGTDDNTLIRVMVSRAEIDMLDIRAHFKRLYGKS CTATMATCGATGMGGGCTTGGGCACCGATGATMCACCCTCATCAGAGTGATGGTTTCTCGAGCAG~TTGACATGTTGGATATCCGGGCACACTTCMGAGACTCTATGGAMGTC

296 960

L Y S F I KG D T S G D Y R KV L L V L C G G D D stop TCTGTACTCGTTCATCAAGGGTGACACATCTGGAGACTACAGGMAGTACTGCTTGTTCTCTGTGGAGGAGATGATTAMATAAAAATCCCAGMGGACAGGAGGATTCTCMCACTTTG

321 1080

AATTTTTTTMCTTCATTTTTCTACACTGCTATTATCATTATCTCAGAATGCTTATTTCCAATTAAAACGCCTACAGCTGCCTCCTAGMTATAGACTGTCTGTATTATTATTCACCTAT

1200

MTTAGTCATTATGATGCTTTAAACCTGTACTTGCATTTC~GCTTATAAGATAT~TGGAGATTTTMAGTAGAAATA~TATGTATTCCATGTTTTTMMGATTACTTTCTACTT .*

1320

TGTGTTTCACAGACATTGAATATATTAMTTATTCCATATTTTCTTTTCAGTGMAAATTTTTT~TGGMGACTGTTCTAAAATCACTTTTTTCCCTMTCCMTTTTTAGAGTGGCT

1440 1560 1680

1800 CTAGGTATTCTGGGMTGATGTMTGCTCTGMTTTAGTATGATATMAGAAAACTTTTTTGTGCTMAAATACTTTTTAAAATCMTTTTGTTGATTGTAGTMTTTCTATTTGCACTG <-. TGCCTTTCMCTCCAGAAACATTCTMGATGTACTTGGATTTAATTAAAAAGTTCA

1920

1976

FIG. 1. Sequence of annexin IV cDNA. The nucleotide sequence was determined by analysis of overlapping clones. The sequence of the pPAP-II-B6 clone used as a probe for in situ hybridization and Southern blotting is delineated by arrows. The locations of the primers used for genomic PCR are underlined. The amino acid sequence is given in single-letter code above the first base of the corresponding codon; amino acids are numbered starting with the initiator Met, although it is probably removed by proteolytic processing (see text). Lowercase letters indicate the sequence of Grundmann et al. (1988) where it differs from the nresent sequence in the coding region, and the asterisk indicates the 3’ limit of the sequence reported by Grundmann et al. (1988).

cell hybrid analysis by this technique. The sum of the detected fragments ranged from 18 to 56 kb for the enzymes tested: BamHI-12.5, 11.0, 8.2, 4.5, 4.2 (total 40.4); BglII-12.0, 9.0, 6.9, 3.9, 2.3, 1.6, 1.5 (total 37.2); EcoRI-23.0,13.0,9.0,7.2,3.6 (total55.8);HindIII-9.4, 4.4, 2.8, 2.5, 2.1, 1.9, 1.6, 0.6, to.6 (total 25.9); KpnI20.0, 11.5 (total 31.5); NcoI-12.5, 5.8 (total 18.3); P&I -4.4, 3.7, 3.3, 2.5, 2.3, 2.0, 1.1 (total 19.3); PuuII-6.4, 4.4, 3.0, 2.6, 2.3, 1.7 (total 20.4); SucI-23.0, 8.0 (total 31.0); StuI-7.9,6.1,5.0 (total 19.0). The observedbands are likely to be due to the ANX4 gene only, because other known annexins are sufficiently dissimilar (>40%) to prevent cross-hybridization under stringent conditions (Tait et aZ., 1991a,b). However, because of the multiple bands observed, PCR analysis was judged to offer a better means for unambiguous detection of the human gene in a rodent background. We used the cDNA sequence (Fig. 1) to design primers that would amplify putative intron-containing regions of the ANX4 gene, guided in part by the known exon-intron structure of the mouse annexin II gene (Amiguet et aZ., 1990). Different primer sets were tested with the human and rodent genomic DNA templates, and the primer set that amplified a single human-specific band

most efficiently was selected. Amplification of human genomic DNA gave a single 1.6-kb band, while mouse and hamster DNA gave no amplification (see Fig. 3). The sequence of this PCR product confirmed that it was derived from the ANX4 gene (Fig. 2), as it exactly matched the cDNA sequence from bases 331 to 461. In addition, an exon-intron junction was found between cDNA bases 379 and 380, at the boundary between codons 102 and 103. The observed splice donor and acceptor sites are consistent with established consensus sequences (Mount, 1982). Chromosomal

Mapping

of the ANX4

Gene

A panel of 28 human-rodent hybrid cell lines was then tested for amplification of the 1.6-kb ANX4-specific band. Cell lines DG-L-10,38L-19, and 38L-3 were positive for this band. Analysis indicated that chromosome 2 had 3.6% discordancy; the next lowest discordancy was 10.7% for chromosome 17. The single discordancy for chromosome 2 was due to a negative result for cell line 854, which contained chromosome 2 by cytogenetic analysis. Repeated tests with the same and different PCR primers failed to show consistent evidence for presence

316

TAIT AA

a1

cDNA PCR

331

T G ACG G ACG

P T CCC ACG CCC ACG

V

GTG GTG

L Y CTG TAT CTG TAT

D V GAC GTG GAC GTG

ET

AL.

Q E L CAA GAG CTG CAA GAG CTG

R R A CGA AGG GCC CGA AGG GCC

M ATG ATG

K AAG AAG

102

379 gtctgtgct

cttcctctcgtgctcttggtgctgttggtgcaaacgctgatgtgctttcttaagaaactgtcacccaataagaaagacat.......... . . . . . . . . . . . . . . ..INTRON

(1.5

kb)

. . . . . . . . . . . . . . . ..attctctgcagattcatgacagatgtattccttctgtgtctg 103G 380

ggcctcagctttgtaaactggctcatatagccctgtcctctggtttcttgtttag GCLIEILASRTPEEIRRISQT GGC TGC CTA ATT GGC TGC CTA ATT

GAG GAG

ATC ATC

CTG GCC CTG GCC

TCC CGG ACC TCC CGG ACC

CCT GAG GAG ATC CCT GAG GAG ATC

GGA GGA

A G GCC GGC GCC GGC

CGG CGC CGG CGC

ATA ATA

T ACT ACT

D E GAT GAG GAT GAG

AGC AGC

CAA ACC CAA ACC

108 391

T T

129 461

FIG. 2. Sequence of amplified portion of human ANX4 gene. Human genomic DNA from a single individual was amplified, cloned, and sequenced as described under Materials and Methods. The sequence is compared with the annexin IV cDNA sequence from Fig. 1; the cDNA is in capital letters and organized by codons, with the predicted amino acid indicated in single-letter code. The PCR product sequence is below the cDNA, exon sequence is identified by capital letters and intron sequence is in small letters. The PCR primers are underlined. The reported sequence is the consensus of three independent clones.

of the ANX4 gene in this cell line. The explanation for this discrepancy is uncertain, but it is possible that there has been a microdeletion of the region containing the ANX4 gene in this cell line. In situ hybridization with the pPAP-II-B6 probe provided an independent mapping assignment of the gene to chromosome 2 (Fig. 4a). A total of 68 cells were examined. Of 144 sites of hybridization, 42 (29% of the sites) were between 2~12 and 2~15, with a peak at 2~13 (Fig. 4b). There were no peaks of hybridization on other chromosomes. Search for RFLP in the ANX4

Gene

Southern blots of 10 to 12 Caucasian genomic DNA specimens were hybridized with the pPAP-II-B6 cDNA probe. No RFLPs were detected with 15 different enzymes (BumHI, BgZII, DdeI, EcoRI, HindIII, KpnI, MspI, NcoI, P&I, PuuII, RsuI, SucI, StuI, TaqI, and

X&I). In addition, the 1.6-kb ANX4 PCR product from 10 different genomic samples was digested with each of 24 different restriction enzymes. No RFLP were detected with any of these enzymes (AccI, A&, BanI, BcZI, CZaI, DdeI, EcoRV, HueIII, HhuI, HindII, Hi&I, KprzI, NcoI, NruI, SucI, Suu3A1, ScuI, SmuI, Sphl, SstII, StuI, XbuI, and XhoI). DISCUSSION

We have found that the human ANX4 gene maps to a single locus on chromosome 2 at band 2~13. Results for in situ hybridization and somatic cell hybrid analysis agree. This mapping adds to the evidence that the annexin gene family is widely dispersed in the genome. Five other human annexin genes have been mapped to unique loci as follows: ANXl to 9qll-q22 (Huebner et al., 1988); ANX2 to 15q21-q22 (Huebner et al., 1988;

2.3 2.0 1.4 1.1 0.9 0.6

FIG. 3. PCR analysis of human-rodent hybrid cell lines. Genomic DNA fmm human, hamster, mouse, or hybrid cell lines was amplified with annexin IV-specific primers as described under Materials and Methods. PCR product (25 ~1) was electrophoresed at 70 V for 4 h in a 1.4% agarose gel (11 X 14 X 0.8 cm) in Tris-borate-EDTA buffer; gels were then stained with ethidium bromide. The hyphenated numbers refer to human-mouse hybrid cell lines; the other numbers identify human-hamster hybrid cell lines. The molecular weight standard is X DNA (Hind111 digest) plus @X174 DNA (Hue111 digest).

CHROMOSOMAL

MAPPING

OF

ANX4

317

GENE

24 2322 25-4 21

. .. . l .

2

a FIG. 4. distribution

b

Regional location of the ANX4 gene to 2p12-~15. (a) Distribution of sites of hybridization on a diagram of human chromosome 2.

of 144 sites

of hybridization

on human

(b)

chromosomes;

Spano et CL, 1990); ANX3 to 4q21 (Tait et aZ., 1991a); ANX5 to 4q26-q28 (Modi et al., 1989; Tait et al., 1991b); and ANX6 to 5q32-q34 (Davies et al., 1989). In addition, three annexin II pseudogenes have been mapped to chromosomes 4,9, and 10 (Huebner et al., 1988; Spano et al., 1990). The human annexin IV cDNA that we have isolated largely confirms the sequence of the protein-coding region previously reported by Grundmann et al. (1988). However, we observed a single amino acid difference at residue 98, which was confirmed by PCR analysis of the gene. In addition, the cDNA we isolated has a much

annexin family at the protein level will also be reflected the structure of their genes.

longer 3’ untranslated

Amiguet, P., D’Eustachio, P., Kristensen, T., Wetsel, R. A., Saris, C. J. M., Hunter, T., Chaplin, D. D., andTack, B. F. (1990). Structure and chromosome assignment of the murine p36 (calpactin I heavy chain) gene. Biochemistry 29: 1226-1232. Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K. (1989). In “Current Protocols In Molecular Biology,” Wiley, New York.

region. It seems most likely

that

alternate transcripts are produced from the same gene, since Northern blots of placental poly(A) RNA probed with the annexin IV cDNA show two bands of approximately 1400 and 2500 nucleotides (Grundmann et d., 1988). This may be due either to alternative splicing or to use of alternative polyadenylation signals within the same transcript. Among the annexins, only the mouse annexin I gene (Horlick et al., 1991) and the mouse (Am&et et al., 1990) and human (Spano et al., 1990) annexin II genes have been characterized in detail so far. These genes have 12 introns and span 17-40 kb, and their exon-intron structure is closely conserved. Interestingly, the intron-containing region of the ANX4 gene that we have amplified has exon-intron boundaries in exactly the same position as the homologous regions of the annexin I and II genes. Genomic Southern blots indicate that the ANX4 gene is also similar in size, spanning 18-56 kb. Thus, it seemslikely that the similarities observed in the

ACKNOWLEDGMENTS We thank Don Gibson for oligonucleotide synthesis, and Drs. Dominic Chung and Kazuo Fujikawa for valuable discussions. This work was supported by NIH Grants HL-40801 (to J.F.T.) and AG-01751 and March of Dimes l-1019 (to C.M.D.).

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