THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1986 hy The American Society of Biological Chemists, Inc.

Vol. 261, No. 7, Issue of March 5, pp. 3112-3115 1986 Printed in lkS.A.

Molecular Cloning and Sequence Analysis of Human Placental Alkaline Phosphatase*

(Received for publication, October 4, 1985)

Jose Luis Mill& From the Cancer Research Center, La Jolla Cancer Research Foundation, La Jolla, California 92037

The complete amino acid sequence of the precursor and mature forms of human placental alkaline phos- phatase have been inferred from analysis of a cDNA. A near full-length PLAP cDNA (2.8 kilobases) was identified upon screening a bacteriophage Xgtll placen- tal cDNA library with antibodies against CNBr frag- ments of the enzyme. The precursor protein (535 amino acids) displays, after the start codon for translation, a hydrophobic signal peptide of 21 amino acids before the amino-terminal sequence of mature placental al- kaline phosphatase. The mature protein is 513 amino acids long. The active site serine has been identified at position 92, as well as two putative glycosylation sites at Asn”’ and AsnZd9 and a highly hydrophobic mem- brane anchoring domain at the carboxyl terminus of the protein. Significant homology exists between pla- cental alkaline phosphatase and Escherichia coli al- kaline phosphatase. Placental alkaline phosphatase is the first eukaryotic alkaline phosphatase to be cloned and sequenced.

Human placental alkaline phosphatase (EC 3.1.3.1) is a member of a multigene family that, in man, includes at least three isozymes (1-3). Placental alkaline phosphatase is a new evolutionary gene product detectable only in the placenta of man, orangutan, and chimpanzee (4, 5). This late appearance in evolution may correlate with the existence of an extensive allelic variation in placental alkaline phosphatase (6). Except for the Escherichia coli enzyme (7), no other alkaline phos- phatase has been sequenced to date. Therefore, the evolution- ary and functional relatedness of the different alkaline phos- phatases remains to be determined. The possible biological roles of placental alkaline phosphatase are at present only a matter of speculation. The enzyme is a cell surface glycopro- tein but it is also often found in the serum, fluids, and tumor tissue of certain cancer patients where it can be measured and its levels followed as a marker of malignancy. Placental al- kaline phosphatase levels have been measured and used for the clinical management of testicular and ovarian tumors (8- 11). The tumor expression of the enzyme is an example of inappropriately high expression of embryonic genes during

CA 30199-04 and Grant R01 CA 21967-08 (to William H. Fishman). * This project was supported by Cancer Center Support Grant P30

This work was presented at the 13th Annual Meeting of the Inter- national Society for Oncodevelopmental Biology and Medicine, Paris, September 11,1985, the 5th conference of Nature’s Update in Molec- ular Biology, San Francisco, October 7, 1985, and the Isozymes meeting, St. Croix, Virgin Islands, December 4, 1985. The costs of publication of this article were defrayed in part by the payment of page charges, This article must therefore be hereby marked “aduer- tkernent” in accordance with 18 U.S.C. Section 1734 soIely to indicate this fact.

malignancy. Elucidation of the basic mechanism of genetic control and expression of these “oncodevelopmental genes” is fundamental for a better understanding of certain features of the biology of cancer. I have undertaken the molecular cloning and structural analysis of the placental alkaline phosphatase and its related genes as a first and necessary step towards a systematic molecular approach to the study of the function, genetic variability, and tumor expression of these oncofetal antigens. In this paper, the isolation and characterization of a near full-length placental alkaline phosphatase cDNA clone from a human placental cDNA X g t l l expression library is reported. The complete amino acid sequence of the precursor and mature forms of the enzyme have been inferred from the nucleotide sequence.

MATERIALS AND METHODS

Construction of a Xgtn Placental cDNA Library-A freshly delivered premature (34-week-old) human placenta expressing the homozygous S phenotype of placental alkaline phosphatase (12) was used as source of RNA for the preparation of the cDNA library. RNA was extracted from the placental tissue by the guanidinium isothiocyanate proce- dure (13) followed by CsCl centrifugation as described (14), and total polyadenylated RNA was selected by oligo(dT)-cellulose (15). Syn- thesis of first-strand cDNA was carried out using reverse transcrip- tase, oligo(dT) as primer, and [5-’H]dCTP as tracer (16). Second- strand synthesis was carried out by the ribonuclease H procedure also using [tG3H]dCTP as tracer (16). Potential EcoRI restriction sites were blocked with EcoRI methylase (17) and the double-stranded cDNA was made blunt-ended with the Klenow fragment of DNA polymerase I (15). EcoRI linkers were ligated to the cDNA with T4 DNA and RNA ligase (15). After EcoRI endonuclease digestion and sizing by sucrose gradient centrifugation, cDNA larger than 1.4 kb’ was used for the construction of the X g t l l cDNA library.

Xgtll DNA purified from E. coli BNN 97 lysogen (17, 18) as described (15) was digested with EcoRI endonuclease, dephosphoryl- ated with calf intestinal alkaline phosphatase, and ligated to the cDNA. The recombinant DNA was packaged using the Packagenee System (Promega Biotec, Madison, WI). Recombinants were obtained at an efficiency of 17,000 plaques/ng of double-stranded cDNA. The library, consisting of 1 X lo6 independent recombinant phages and containing less than 1% nonrecombinants, was amplified on E. coli Y1088 (17, 18).

Antibody Production and Immunochemical Screening-Purified placental alkaline phosphatase of the FS heterozygous phenotype was obtained by a monoclonal antibody immunosorbent procedure (19). The purified enzyme was reduced, carboxymethylated, and subjected to CNBr cleavage (19). A rabbit (number 244) was immunized with the mixture of CNBr fragments (50 pg), emulsified with complete Freund’s adjuvant, and boosted four times at 15-day intervals. IgG from the antiserum was purified on protein A-Sepharose and absorbed with lysates of E. coli strain Y1090 and used for the screening of the X g t l l expression library (18).

Sequence Analysis-Deletion libraries of both strands of the insert were constructed using the single-stranded M13 method of Dale et al. (20) with the kit supplied by International Biotechnologies, Inc. (New

The abbreviations used are: kb, kilobase pairs; TBE, Tris/borate/ ethylenediaminotetraacetic acid; TBS, Tris-buffered saline.

3112

Cloning of Placental Alkaline Phosphatase cDNA 3113

Haven, CT). Sequencing of the deletion subclones was accomplished by the Sanger dideoxy chain termination procedure (21) using the Klenow fragment of DNA polymerase and [35S]dATP as tracer (22). Homology searches were done utilizing the BIONET” system of Intelligenetics, Inc. (Palo Alto, CA). Hydropathy analysis was carried out according to Kyte and Doolittle (22).

RESULTS AND DISCUSSION

Screening of the Agtll Library with Antibodies to Placental Alkaline Phosphatase-Preliminary experiments showed that all the polyclonal and monoclonal antibodies against placental alkaline phosphatase that we had previously characterized (23-25) reacted predominantly with conformational epitopes, as they detected poorly in immunoblottings of the denatured enzyme. Since the fusion proteins obtained in the Xgtll system may not express conformational determinants, antibodies were prepared against fragmented, carboxymethylated pla- cental alkaline phosphatase. Immunodot assays showed that such antibodies were reactive with the enzyme denatured by two different procedures, whereas antibodies prepared against native placental alkaline phosphatase were not (Fig. 1).

Screening of 1.2 x lo5 recombinant phages of the placental library with the antisera reactive with denatured placental alkaline phosphatase yielded 12 putative enzyme clones. All of these clones continued to be strongly positive through the secondary and tertiary screenings and were finally isolated from single plaques. The insert size in the 12 clones varied from 1.4 up to 2.8 kb. The nick-translated 2.8-kb fragment hybridized with all the smaller inserts after Southern blotting, indicating that the different inserts were all related (not shown). In order to obtain an independent confirmation that

Native PLAP

Denatured PLAP +a

CNBr fragments

Native PLAP

Denatured PLAP S244

CNBr fragments

5 0.5 0.05

FIG. 1. Comparison of the reactivity of antisera against intact placental alkaline phosphatase (PLAP) and denatured placental alkaline phosphatase. Different quantities (5, 0.5, and 0.05 pg) of (a ) native PLAP, (b ) PLAP after reduction with 2- mercaptoethanol and boiling, and (c) CNBr fragments of PLAP were spotted onto nitrocellulose filters. The filters were washed with TBS and saturated with 20% fetal calf serum in TBS for 1 h. Subsequently, the filters were incubated with a solution of 100 pg/ml IgG from an antiserum against native PLAP (#8) or against PLAP denatured by reduction, carboxymethylation, and CNBr cleavage (#244) in 20% fetal calf serum for 1 h followed by extensive washing with TBS, TBS/Nonidet P-40, and TBS, and incubation with 1251-protein A for 1 h. After washing and drying, the filters were exposed on Kodak XR5 using Dupont Cronex intensifying screens.

the identified clones encoded placental alkaline phosphatase, a mixed 17-mer oligonucleotide probe, i.e. 5’-TTCCA(A/ G)AA(A/G)TC(A,C,G,T)GG(A/G)TT-3’ was synthesized based on residues 8 through 13 from the known 40 amino- terminal amino acid residues of mature placental alkaline phosphatase (26). The largest insert (2.8 kb) hybridized with the oligonucleotide probe, suggesting that it encodes the entire placental alkaline phosphatase molecule (not shown).

Sequence of the Placental Alkaline Phosphatase Molecule- The full sequence of the 2.8-kb placental alkaline phosphatase cDNA clone was established from both strands by sequencing overlapping deletion subclones differing in approximately 200 base pairs each. Fig. 2 shows the DNA sequence corresponding to the coding region as well as the inferred amino acid se- quence of the placental alkaline phosphatase molecule. The cDNA clone shows a short 5’ untranslated region of 32 base pairs. In contrast, the 3‘ flanking region is close to 1100 base pairs long. The cDNA clone ends with two adjacent polyaden- ylation signals and a poly(A) tail of 19 bases. The 3’ end of the cDNA shows the sequence expected for the EcoRI linker (5’-CCCGAATTC-3’), but the 5’ end is missing the expected guanidyl residues (5’-GAATTCGGG-3’), suggesting that the 5’ end represents an intrinsic EcoRI site in the 5’ flanking region of the placental alkaline phosphatase cDNA.

After the start codon for translation, the inferred amino acid sequence reveals a hydrophobic signal peptide of 21 amino acids followed by the amino-terminal amino acid se- quence of the mature protein. The mature placental alkaline phosphatase molecule (starting at amino acid 1 in Fig. 2) is 513 amino acids long and gives a molecular weight of 55,510. There exist two putative glycosylation sites in the molecule, AsnlZ2-Thr-Thr and AsnZ4’-Arg-Thr. Studies to be published elsewhere’ indicate that only is glycosylated. The res- idue at position 92 may be the active site serine of placental alkaline phosphatase. This position contains the only Asp- Ser sequence in the molecule, and this pair of amino acids has been shown to be involved in the active site of the E. coli and calf intestinal alkaline phosphatase (27, 28). Further- more, the composition of the 32P-labeled active site tryptic fragment of placental alkaline phosphatase as determined by Whitaker et al. (29) corresponds well with that of the peptide consisting of residues 87 through 104. The location of the active site and of the glycosylation site agrees with results indicating that these sites are located on different CNBr fragments of placental alkaline phosphatase (19).

It has previously been established that trypsin cleaves na- tive placental alkaline phosphatase at a unique site causing a decrease in Mr from 67,000 to 57,000 without affecting the catalytic activity of the molecule (30). The amino-terminal amino acid sequence of the 57,000-Da fragment, kindly pro- vided to us by Dr. Ronald Jemmerson (University of Minne- sota), has been determined to be Leu-Gly-Pro-Glu-Ile-( )- Leu-Ala-Met-Asp-Arg-Phe-Pro-Tyr-Val-Ala-Leu-Ser-Lys- Thr. This sequence starts at residue 63 of the placental alkaline phosphatase sequence identifying the Lys6’-Leu bond as the site of trypsin cleavage in the native placental alkaline phosphatase molecule. Bromelain releases catalytically active placental alkaline phosphatase from the surface of cells by cleaving a 2,000-Da fragment from the carboxyl terminus of the protein (31). The sequence of the carboxyl-terminal end of the molecule as deduced from the cDNA shows a stretch of 23 predominantly hydrophobic amino acids. A hydropathy analysis shows that this segment is the most hydrophobic portion of the mature protein (Fig. 3). I t seems that this

J. L. Millan, A. Dell, and M. Fukuda, manuscript in preparation.

3114 Cloning of Placental Alkaline Phosphatase cDNA

Eco R I

5"GAATTCCTGCC

TCGCCACTGTCCTGCTGCCCTCCAGAC ATG CTG GGG CCC TGC ATG CTG CTG CTG CTG CTG CTG CTG GGC CTG AGG CTA CA6 CTC TCC CTG GGC Met Leu Gly Pro Cys Met Leu Leu Leu Leu Leu Leu Leu Gly Leu Arg Leu Gln Leu Ser Leu Gly

ATC ATC CCA GTT GAG GAG GAG AAC CCG GAC TTC TGG AAC CGC GAG GCA GCC GAG GCC CTG GOT GCC GCC AAG AAG CTG CAG CCT GCA I l e I l e Pro Val Glu Glu Glu Asn Pro Asp Phe T rp Asn Arg Glu A la A la Glu A la Leu Gly Ala A la Lys Lys Leu Gln Pro A la

CAG ACA GCC GCC AAG AAC CTC ATC ATC TTC CTG GGC GAT GGG ATG GGG GTG TCT ACG GTG ACA GCT GCC AGG ATC CTA AAA GGG CAG G ln Th r A la A la Lys Asn Leu I l e I l e Phe Leu Gly Asp Gly Met Gly Va l Se r Th r Va l Th r A la A la A rg I l e Leu Lys Gly Gln

AAG AAG GAC AAA CTG GGG CCT GAG ATA CCC CTG GCC ATC GAC CGC TTC CCA TAT GTG GCT CTG TCC AAG ACA TAC AAT GTA GAC AAA Lys Lys Asp Lys Leu Gly Pro Glu I l e Pro Leu Ala Met Asp Arg Phe Pro Tyr Val Ala Leu Ser Lys Thr Tyr Asn Val Asp Lys

CAT GTG CCA GAC AGT GGA GCC ACA GCC ACG GCC TAC CTG TGC GGG GTC AAG GGC AAC TTC CAG ACC ATT GGC TTG AGT GCA GCC GCC H i s Val Pro Asp Ser Gly A l a T h r A l a T h r A l a T y r L e u Cys Gly Val Lys Gly Asn Phe Gln T h r I l e Gly Leu Ser A la A la A la

CGC TTT AAC CAG TGC AAC ACG ACA CGC GGC AAC GAG GTC ATC TCC GTG ATG AAT CGG GCC AAG AAA GCA GGG AAG TCA GTG GGA GTG Arg Phe Asn Gln Cys Asn Th r Th r A rg Gly Asn Glu Val I l e Ser Val Met Asn Arg Ala Lys Lys Ala Gly Lys Ser Val Gly Val

-

4

GTA ACC ACC ACA CGA GTG CAG Val Thr Thr Thr Arg Val Gln

CCT GCC TCG GCC CGC CAG GAG P ro A la Se r A la A rg Gln Glu

CGA AAG TAC ATG TTT CCC ATG Arg Lys Tyr Met Phe Pro Met

CTG GTG CAG GAA TGG CTG GCG Leu Val Gln Glu Trp Leu Ala

GTG ACC CAT CTC ATG GGT CTC Val Thr H i s Leu Met Gly Leu

ATG ACA GAG GCT GCC CTG CGC Ret Thr G lu A la A la Leu Arg

CAT GAA AGC AGG GCT TA6 CGG N i s Glu Ser Arg A l a T y r A r g

GAC ACG CTG AGC CTC GTC ACT Asp Thr Leu Ser Leu Val Thr

GCC CCT GGC AAG GCC CGG GAC A la P ro Gly Lys A la Arg Asp

CCG GAT GTT ACC GAG AGC GAG Pro Asp Val Thr Glu Ser Glu

GAC GTG GCG GTG TTC GCG CGC Asp Val Ala Val Phe A la A rg

GCC GCC TGC CTG GAG CCC TAC A l a A l a Cys Leu Glu Pro Tyr

CCC GCG TTG CTT CCT CTG CTG Pro Ala Leu Leu Pro Leu Leu

TCCCGGAGTTCTCCTGCTCC-(

CAC GCC TCG CCA GCC GGC ACC TAC GCC CAC ACG GTG AAC CGC AAC TGG TAC TCG GAC GCC GAC GTG H i s A la Se r P ro A la G ly Th r Ty r A la H i s Thr Val Asn Arg Asn Trp Tyr Ser Asp A l a Asp Val

GGG TGC CAG GAC ATC GCT ACG CAG CTC ATC TCC AAC ATG GAC ATT GAC GTG ATC CTA GGT GGA GGC Gly Cys Gln Asp I l e A l a T h r G l n L e u I l e S e r Asn Met Asp I l e Asp Val I l e Leu Gly Gly Gly

GGA ACC CCA GAC CCT GAG TAC CCA GAT GAC TAC AGC CAA GGT GGG ACC AGG CTG GAC GGG AAG AAT G ly Th r P ro Asp Pro G lu Ty r P ro Asp Asp Tyr Ser G ln Gly Gly Thr Arg Leu Asp Gly Lys Asn

AAG CGC CAG GGT GCC CGG TAT GTG TGG AAC CGC ACT GP.G CTC ATG CAG GCT TCC CTG GAC CCG TCT Lys Arg Gln Gly A la A rg Ty r Va l T rp Asn Arg Thr Glu Leu Met Gln Ala Ser Leu Asp Pro Ser

TTT GAG CCT GGA GAC ATG AAA TAC GAG ATC CAC CGA GAC TCC ACA CTG GAC CCC TCC CTG ATG GAG Phe Glu Pro Gly Asp Met Lys Tyr Glu I l e H i s A rg Asp Ser Thr Leu Asp Pro Ser Leu Met Glu

CTG CTG AGC AGG AAC CCC CGC GGC TTC TTC CTC TTC GTG GAG GGT GGT CGC ATC GAC CAT GGT CAT Leu Leu Ser Arg Asn Pro Arg Gly Phe Phe Leu Phe Val Glu Gly Gly A r g I l e Asp H i s Gly H i s

GCA CTG ACT GAG ACG ATC ATG TTC GAC GAC GCC ATT GAG AGG GCG GGC CAG CTC ACC AGC GAG GAG Ala Leu Thr Glu T h r I l e Met Phe Asp Asp A l a I l e Glu Arg A la Gly Gln Leu Thr Ser Glu Glu

GCC GAC CAC TCC CAC GTC TTC TCC TTC GGA GGC TAC CCC CTG CGA GGG AGC TCC ATC TTC GGG CTG A l a Asp H i s Ser H i s Val Phe Ser Phe Gly Gly Tyr Pro Leu Arg Gly Ser Ser I l e Phe Gly Leu

AGG AAG GCC TAC ACG GTC CTC CTA TAC GGA AAC GGT CCA GGC TAT GTG CTC AAG GAC GGC GCC CGG Arg Lys Ala Tyr Thr Val Leu Leu Tyr Gly Asn Gly Pro Gly Tyr Val Leu Lys Asp Gly A la A rg

AGC GGG AGC CCC GAG TAT CGG CAG CAG TCA GCA GTG CCC CTG GAC GAA GAG ACC CAC GCA GGC GAG Ser Gly Ser Pro Glu Tyr Arg Gln Gln Ser A la Val Pro Leu Asp Glu Glu Thr H i s A l a Gly Glu

GGC CCG CAG GCG CAC CTG GTT CAC GGC GTG CAG GAG CAG ACC TTC ATA GCG CAC GTC ATG GCC TTC Gly P r o G l n ' A l a H i s Leu Val H i s Gly Val Gln Glu Gln Thr Phe I l e A l a H i s Val Met Ala Phe

ACC GCC TGC GAC CTG GCG CCC CCC GCC GGC ACC ACC GAC GCC GCG CAC CCG GGG CGG TCC GTG GTC T h r A l a Cys Asp Leu A la Pro Pro A la Gly Thr Th r Asp A l a A l a H i s Pro Gly Arg Ser Val Val

GCC GGG ACC CTG CTG CTG CTG GAG ACG GCC ACT GCT CCC TGA GTGTCCCGTCCCTGGGGCTCCTGCTTCCCCA A l a Gly Thr Leu Leu Leu Leu Glu Thr Ala Thr Ala Pro ---

Eco R I

) -TGGGCGACAGAGCGAGATTCTGCCTCAAAAATAAACAAATAAATTTTAAAAATAAA~AAAAAAAAAAA~CCCGAATTC-3 '

11

104 -1

191 29

278 58

365 87

452 1 S6

539 145

626 174

203 713

800 232

887 261

974 290

106 1 319

1148 348

1235 377

1322 406

1409 435

1496 464

1583 493

1677 513

FIG. 2. Sequence of the 5' end of the 2.8-kb placental alkaline phosphatase cDNA comprising the entire coding region of the placental alkaline phosphatase molecule. The p lacenta l a lka l ine phosphatase protein sequence consists of a 21-amino acid signal sequence and a 513-amino acid mature protein. Underlined are (a ) the s igna l pept ide from amino acids -1 to -21, (b) the act ive si te Asp-Ser*, and (c) two putat ive g lycosy lat ion sites, i.e. Asn"' and Amz4'. T h e arrow ind icates the s i te where t ryps in c leaves the nat ive prote in . Approx imate ly 1000 bp of t h e 3' flanking reg ion have been omi t ted from the f igure as ind ica ted by the parentheses.

hydrophobic segment may represent a membrane-anchoring domain and that placental alkaline phosphatase would lack a cytoplasmic domain. However, since the precise length of the membrane-anchoring domain is not known, the placement of the bromelain cleavage site is only approximate.

A computer search for homology between the placental alkaline phosphatase amino acid sequence and other known proteins has revealed significant homology only with E. coli alkaline phosphatase (7). The percentage homology of the overall proteins is only 13%, but areas of high local homology

(Table I) strongly suggest a common ancestral origin for these enzymes. Recently, the sequence of the first 39 amino-termi- nal amino acids of calf intestinal alkaline phosphatase have been determined (32). This sequence shows 69% homology with the amino-terminal sequence of placental alkaline phos- phatase. As more sequence data becomes available, it will be possible to delineate the evolutionary relationship of this multi-locus enzyme system.

It is expected that knowledge of the structure of placental alkaline phosphatase will allow us to more systematically

Cloning of Placental Alkaline Phosphatase cDNA 3115

FIG. 3. Hydropathy analysis of the inferred amino acid sequence of placental alkaline phosphatase. The amino acid sequence of placental alka- line phosphatase inferred from the cDNA sequence was analyzed for hydro- phobicity and hydrophilicity according Kyte and Doolittle (22). The main fea- tures of the sequence have been indi- cated in the figure. The numbering at the bottom refers to amino acid residues of the precursor protein.

n -1

- -' 0 -.

- -. a .

N -. I .

Signal Peptide H

Membrane Anchoring Domain

U

TABLE I Regions of high homology between placental alkaline phosphatase (PLAP) and E. coli alkaline phosphatase

Three regions of homology have been identified. The position of the first and last amino acids of each area for both protein sequences are indicated. Identical amino acids in the chain are indicated by a hyphen. An asterisk indicates a single amino acid gap in one of the sequences.

residues No.

% 22 53

PLAP AAKKLEPAETAAKNLIIFLGDGMGVSTVTAAR 32 I E . coli --LRNSLSDKP---I-LLI-----D-EI---- 50

31 62 81 * 104

PLAP KTYNVDKHVPDSGATATAYLCGVK 2 4 'I E . coli - -GKP-YVT - - A - S - - -WST- - - 50

92 1 1 4 291 358

PLAP MTEAALRLLSRNPRGFFLFVEGGRIDHGHHESRAYRALTETIMFDDAIERAGQLTS EEDTLSLVTADH 68 'I1 E . coli - -DK-IE- - -K-EK- - - -Q- - -AS- -KQD-AANPCGQIG- -WL-E-VQ- -LEFAKK-G- - -VI- - - - -

302 4 9

* 370

approach the study of the biological role of this enzyme. The 9. JePPsson, A.3 Wahren, B.9 Stigbrand, T.3 Edsmyr, F., and Andersson, L.

availability of DNA probes will enable us to answer questions 10. Fishman, W. H., Inglis, N. R., Vaitukaitis, J., and Stolbach, L. L. (1976) about the number Of genes in and 11. McDicken, I. W., McLaughlin, P. J., Tromans, P. M., Luesley, D. M., and re-expression of placental alkaline phosphatase and placental Johnson, P. M. (1985) Br. J. Cancer 52,59-64

tion. The sequence data presented in this paper can be utilized 13. Ullrich, A. Shine J. Chirgwin, R., Pictet E. Tischer W. J., Rutter, W.

for use in immunodiagnosis and immunotherapy of placental 2637 alkaline phospha~ase-produc~ng tumors. more allelic var- 15. Maniatis, T., Fritsch, E. F., and Sambrook, J. (1982) Molecular Clonin A

iants of this enzyme are sequenced in the future, we will begin Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harfor, NY

to understand the mechanism of evolution of this interesting 17. youn R. A. and ~ ~ ~ i ~ , R, W, (1983) proc. ~ ~ ~ l , ~ ~ ~ d . sei, polymorphic enzyme locus. U. L@ A. 80, i194-1198.

18. Young R. A., and Davis R. W. (1983) Science 222, 778-782 19. Millag J. L., Stigbrand,'T., and Jornvall, H. (1985) Int. J . Biochem. 10,

Achnow~edgments"y thanks to Dr. H' Fishman for his 20. Dale, R. M. K., McClure, B. A,, and Houchins, J. P. (1985) Plasmid 13, encouragement and financial support, to Dr. Erkki Ruoslahti and his group for helpful discussion, to Richard Keh for technical help, and 21. Sanger, F., Nicklen, S., and Coulson, A. R. (1977) Proc. Natl. A c ~ . Sci. U.

(1983) Br. J . Virol. 55,73-78

Natl. Cancer Inst. Momgr. 42,63-74

alkaline phosphatase-like enzymes and to study their regula- 12. Millan, J. L.3 Beckman, G., Jeppsson, A.3 and Stigbrand, T. (1982) Hum. Genet. 60, 145-149

J., and doodman, H. M. (1977) Science i96,1977-1h80 to develop anti-peptide antibodies of molY? defined specificity 14. Glfin, V., Crkvenjakov, R., and Byus, C, (1974) Biochemistry 13, 2633-

16. Gubler, U., and Hoffman, B. J. (1983) Gene 25,263-269

1033-1039

31-40

Biol. 157,105-132

~~ ~~ ~, ~. and Fishman, W. H. (1982) 1. Steargent, L. E., and Stinson, R. A. (1979) Nature 281,152-154 2. McKenna, M. J., Hamilton, T. A., and sussman, H. H. BioChem. J . 26. Ez:a, E., Blacher, R., and Udenfriend, S. (1983) Biochem. Biophys. Res.

Chem. 27,2014-2018

25. Millin, J. L., and'stigbrand, T. (1983) Eur. J. Biochern 136,l-7 Cancer Res: 4 2 -2444-2449 ~

181.67-73 . -2- ."n -1nm

4. Doellgast, G. J., and Benirshke, K. (1979) Nature 280, 601-602 5. Goldstein, D. J., and Harris, H. (1979) Nature 280,602-605 6. Donald, L. J., and Robson, E. D. (1974) Ann. Hum. Genet. 37,303-313 7. Bradshaw, R. A. Cancedda, F., Ericsson, L. H., Neumann P. A., Piccoli

S. P., SchlesiAger, M. J., Shriefer, K., and Walsh, K. k. (1981) Proc: Natl. Acad. Sci. U. S. A. 78,3473-3477

8. Lange, P. H., Millan, J. L., Stighrand, T., Vessella, R. L., Ruoslahti, E., and Fishman, W. H. (1982) Cancer Res. 42,3244-3247

U . S. A. 77, 2857-2860 ~~ .-.b"c&man, F. (1961) Proc. Natl. Acad. Sci. u. S l A: 47,1986-2005

29. Whitaker,'K. B., Byfield, P. G. l%, and Moss, D. W. (1976) Clin. Chem. 28. Engstrom L. (1961) Biochirn. Bio hys. Acta 52,49-59

Acta 71.285-291 30. Jemmerson, R., and Stighrand, T. (1984) FEBS Lett. 173,357-359 31. Jemmerson R., Millan J. L., Klier, F. G., and Fishman, W. H. (1985)

32. Besman, M., and Coleman, J. E. (1985) J. Biol. Chem. 260,11190-11193 FEBS Lek 179,316'320