Proc. Natl. Acad. Sci. USA Vol. 83, pp. 3257-3261, May 1986 Biochemistry

Characterization, mapping, and expression of the human ceruloplasmin gene* (intragenic triplication/dissimilar cDNAs/human chromosome 3/lymphocyte expression/DNA polymorphisms)

FUNMEI YANG, SUSAN L. NAYLOR, JEAN B. LUM, STEPHEN CUTSHAW, JEROME L. MCCOMBS, KATHLEEN H. NABERHAUS, JOHN R. MCGILL, GWEN S. ADRIAN, CHARLEEN M. MOORE, DON R. BARNETT, AND BARBARA H. BOWMAN The University of Texas, Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284

Communicated by Eloise R. Giblett, January 21, 1986

causing this inherited disease, however, was recently mapped to chromosome 13 in a large Israeli-Arab kindred (13), making it unlikely that a mutation in the CP gene is responsible for Wilson disease. Results from the deduced sequence of clotting factor VIII (14) demonstrated a striking homology of this X-linked protein and CP, the CP-like sequence being repeated three times in the coding regions of the factor VIII (F8) gene. The sequence homology observed in human CP, porcine factor VIII, and bovine factor V (15) showed that the three structurally related proteins constitute a family whose genes are located on at least two chromosomes. CP is an acute-phase reactant and its concentration increases during inflammation. In addition to its role in copper transport, the glycoprotein is said to function also as an antioxidant (16) and as an oxidase that converts iron(II) to iron(III) (17). Specific receptors for CP have been found in membrane fragments from aortic and cardiac tissues (18). In the study described here, two slightly dissimilar cDNAs corresponding to the CP gene were identified, characterized, and mapped to chromosome 3q25; the presence of CP mRNA in T lymphocytes, macrophages, and liver was detected by in situ histohybridization with CP cDNA.

ABSTRACT Ceruloplasmin (CP) is a copper-binding protein in vertebrate plasma. It is the product of an intragenic triplication and is composed of three homologous domains. Oligonucleotide probes constructed according to published amino acid sequences were used to identify cDNA clones encoding human CP. Two clones, CP-1 and CP-2, differed from each other by the presence or absence, respectively, of a deduced sequence of four amino acids. The two clones provided 81% of the sequence encoding CP. Comparison of the nucleotides of the three domains of the CP coding sequence revealed internal domain homology with identity of sequences ranging from 50.1% to 56%. The nucleotide sequence of CP-2 cDNA was compared to that of a homologous human protein, clotting factor VIII, and was found to be 48% identical overall. The CP gene was mapped to human chromosome 3 by somatic-cellhybrid analysis and to 3q25 by in situ hybridization; however, sites of hybridization to DNA on other chromosomal sites suggested additional CP-like DNA sequences in the human genome. A DNA polymorphism was detected with CP cDNA after endonuclease digestion of human DNA by Pst I. CP mRNA was detected in human liver, macrophages, and lymphocytes by in situ histohybridization.

Ceruloplasm (CP) is an a2-globulin in human plasma that binds six copper atoms. It has a molecular weight of 132,000, is composed of 1046 amino acids, and is the evolutionary product of an ancient intragenic triplication (1). CP is functionally similar to two small blue proteins, plastocyanin and azurin, from plants and bacteria, respectively (2). Inherited alterations have been found in human CP (3-5). None of these has been extensively analyzed. However, two apparent sites of amino acid interchange at positions 79 and 449 were identified in the amino acid sequence analysis of pooled CP. Both involved single-point interchanges of glycine and lysine (1). It is not known whether these alterations are correlated with known CP variants. The CP and transferrin (TF) genes were reported to be genetically linked in cattle (6). Weitkamp (7) demonstrated linkage between the TF and CP loci in human families at a recombination frequency of 10-15%. Therefore, when the TF gene was mapped to human chromosome 3q21-25 (8), a heretofore unmapped linkage group consisting of pseudocholinesterase-1 (9, 10), CP, and the gene for a2-HS-glycoprotein (A2HS) (11) were also mapped to the same chromosomal region. Cox and Francke (12) have confirmed the presence of the (A2HS) gene on human chromosome 3 by analysis of somatic-cell hybrids. CP levels are decreased in many cases of Wilson disease, an inherited defect in copper transport. The defective gene

MATERIALS AND METHODS Synthesis of Oligonucleotides of Mixed Sequence. Oligodeoxynucleotide probes were constructed on the basis of amino acid sequences of human CP reported by Takahashi et al. (1). For the first sequence, residues 538-543 (Met-LysIle-Cys-Lys-Lys), 24 mixed 17-mer oligonucleotides were synthesized: 5' ATG-AAR-ATH-TGY-AAR-AA 3'. For the second sequence, residues 667-672 (His-Met-Trp-Pro-AspThr), 16 mixed 17-mer oligonucleotides were synthesized: 5' CAY-ATG-TGG-CCN-GAY-AC 3'. (Single-letter abbreviations are as follows: R is A or G; Y is C or T; H is A, C, or T; N is A, G, C, or T.) The probes were synthesized by P-L Biochemicals. Purified oligonucleotides were labeled at the 5' end with [y-32P]ATP and polynucleotide kinase (19). Isolation of CP cDNA. A human liver cDNA library (20), kindly provided by S. H. Orkin (Harvard Medical School, Boston), was screened with the oligonucleotide probes. The hybridization conditions and plasmid DNA purification and isolation were as described (8). DNA Sequence Determination. Both strands of the CP cDNAs were sequenced by the chemical-cleavage method (21) and by the dideoxy chain-termination method (22). Template DNAs for dideoxy sequencing were prepared by inserting the restriction enzyme fragments of the CP cDNA

The publication costs of this article were defrayed in part by page charge

Abbreviations: PBMCs, peripheral blood mononuclear cells; kb, kilobase(s). *A preliminary report of this research has been presented (32).

payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Proc. Natl. Acad. Sci. USA 83 (1986)

199 210 220 VaIVal Met PheSeoVal ValAsp lu AenPhe Se Trp Tyr Lou GluAsp As le Lyse2hreMr qeSer CGC ATG T17 TCT GTG GTG CAT GMA MT TTC MGC TGG TAC CTA GMA GAC MAC AT MAA ACC TAC TOG TCA

230

GluPPO GULimVal Aap LfeAsp GMA CCA GAG MAA OTT GAG AMA GAG

C GYG

260 250 260 Aen Clu Aep The Cln Glu See Au. Arg Net L1ye Ser Val Aen Cly LyIf T% u he Cly See Lou Pro Cly Ge. See Net Cy. AZa Cl. Asp Aeg Val Lye lop Lye Lo. AAC GMA GAC TTC CMG GAG ACT MC MGA LTG TAT TCT GTG MT GGA TAG ACT TTT GM MCT CTC CCA OGA CTC TCC ATO TOT OCT GMA GAG AMA OTA AMA TOG TAC CTT 260 290 300 Au. Cl, Val Asp Val Hi. Ala Ala The The lie GZv Cl, Ala Geu The Ae. Lye Ae. lyr Aeg flU Aep The fle Ae. Ge. The Pro AlZa The Ge. The Asp Ala Tyr MAT GMA OTT CAT OTO CAC GCA GOT TTC TOT CMC GOG CMA GCA CTC ACT MAC MA AAC TAG COT ATT MAC A06 ATC AAG CTC ITT COT OCT ACC OTC TOT MAT OCT TAT

270

The Cly Net Gl0 ITT GOCT LTG GoT

310

Not Val Ala Cl, ATG GTG 0CC CAC

320 330 340 350 Aen Pro Gly CT. Top Net Ge. See Cye Cl, Aey' Lou Au. Hie Ge. Lye Ala ClY Lou Clm Ala The The Cl., Val GClGCl.ye Au. Lye Ser Her Her Lye Aep Au. flu Arg Cly Life MAC COT GMA GMA TOG ATO OTC ACC TOT CMG MT CTA MAC CAT TG MA" GCC GOT ITO CMA OCC TOT TTC CMG OG CM GMW TOT MAC MC TOT TCA TCA MOG CAT MT ATC COT 000 MAA

370

3,60

no0

390

Hie Val Aop Hie Lr Tyr flu Ala Ala Cl. Cl. flu Ile Top Au. lye Ato Pmo See Cly Ila Asp Ilu The The Lyfe Ctu Au. Le. The Ala Pro Cly Her Asp See Ala Val Phe The CAT GTT MGA COG TAG TAG ATT 0CC OCT GMG GMA ATC ATC TOG MGC TAT GOT CCC TOT GOT AlA MAC LC TIC MCT A" GMA AMC ITO AMA CA COT 006 MCT GMC TCA 0CG TGC TTT T1T

400 420 430 410 Aep Ala Her The The Au. Aop Lyfe Cl. Aop Cl, Cln Cly The The Aeg Ite Cl0f Cly Ser lye Lye Lye Ge. PalI Tyr Aopg Cl, Lyr The Cly Pro Clu Cl, Cl. Hie Ge. Cziy fle Lo, GMA CMA OCT MCC ACA AMA OTT GMA GOC TOT TAT AAA MA~ CTG OTT TAT COT GAC TAG AMA MT 0CC TCC TIC AM MT CM AM GAG MGA Cc COT GM GM" GMG CAT CIT GOC ATC CTC 440 430 460 470 Cly Pro Val Ile ?orp AlZa CO. Vol Cly~ Asp The flu Aop Vol The The Die Au. Lye C~y Ala lye Pro Ge. see 11* Cl., pro flu Cly Val Aop The Au. Lye Aen Au. Cl. Gly The HOT COT OTC OTT TOG OCA GAG GIG GMA MC ACC LOG AMA OTA LCC TIC CAT Mc MAA GM 006 TOT ccc cTc McT OTT GAc CcOG OTT 000 OTG AMA rTC OAT AMc Mc MC GAG C~C AGO '480

300

490

Tyr lye Her Pro Au. Tyr Au. Pro Cln Her Aop Her VolI Pro Pro Her Ala Her Die Vol Ala Pro The Cl. The The The lye Cl. lop The VolI Pro TAG TOT TCC CCL MT TAC MC CCC CMG AC AMA MT GIG COT COT TCA 0CC TCC CAT GIG CCL CCC 006 GAA ACA TOG 0CC TOT GMA TOG LOT GTC CCC 520 530 540 Ala Aep Pro Vol Lye Ge. Ala Lye Net L~ye lye Her Ala Val Aep Pro The Lye Aep flu The The Cly Ge. flu CIy Pro Neot Lye Ilu Lye Lyfe Lye 006 MAT COT OTG TOT OTA GOT MAG ATG TAT TAT TOT OCT GTG MAT CCC MCT A" MAT ATA TIC ACT 00 CIT LIT 00 CCL LOG AAA ATA TOG MAA A" 360

570

Lye Cl. Vol Cly MAA GAA OTA GMA

510

Pro The Au.

LOT MAT 330 Cly Sue Geu Nie Ala Au. Gly GMA MT ITO CAT OGO MT GGC CCC

360

590

o Ge. Ge.Cl Aep Au. rZ. Arp Net The Thr The Ala Pro, Aep CMm Vol Aep Lye Arg Cl. Lye Aep Vol Aep Lye CO. The Lye Ge. The Pro The Vol The Asp Clu Au. Clu See Ge. AMA CM MA" CAT GIL MAC MOG GM TIC TAT ITO ITT COT AMA 1TA ITT MAT MCG MT GMG MCT ITO OTC OTG GM MAT MAT LIT AMA LOG TTT AMA LOT OCA COT CAT CMG GIC CAT MC

600

610

Cl, Aep GO. Aej The Cln Cl. Ser Au. Lye Net Hie Her Net Au. Cly The Net Tyr Cly Au. Cl, Pro Cly Ge. The Net Cye GAA CAT GMA MC TTT CMG GMA TOT MT "A ATG CAC TCC ATO MLT GMA TIC LOG TOT 000 MAT CMG CCG OCT CTC MCT LOG TOG 640 610 Lie Cly flLye The Her Cly Au. The MLye Ge. lp Arg Cly Gl. Avg Org Aep The Ala Cly Au. Gl. Ala Aep VolHi GGM MT MCG 0CC OAT OTA CAT 000 ATA TAG TTT 106 GMA "C 006 TAT OTG TOO MGA GMA GM COG MGA MC AGO GCL 680 690 C LyewGe The The Aep Lie ?yr The Cly Cly Net Lye Cl.t Lye Lyr T1he Vol Aep Thr Clu Cly The The Au. Vo lu GMC AAGO MCG 00 CLT TOT MT OIT GMA TOG CIT AGO ACT MAT CAT TLC AM OGG GOC LTG AM CM MA" TOT AOT GIG

720 Tyr Tyr flu Ala Ala Vola Cl. Val Cl. lop Aep Lye oro Pro GL. C~y Cl, Arg Thr TC TOT ATC OGO OGO GTO C OATG GM TOO MAT TAT TCC CCL CMA GG CMALG AGO AC 760

620

630

Lyfe Cly Aep See Vol Vol lo2p Tyr Lo. The SereAla

"AM A GM AT

TCG

GIC OTOG TOG TLC ITA TIC LOG GCC

660

670

700

710

730 A rg Lt.

740

750

770

760

790

Au. Ge. The Pro Cl, The Ser Ge. The Ge. eie Net Top Pro MC COG TIC COT CMA 006 MT CIT ACG CTC CMC LTG TOG COT

Arg Aeg Cl. Her Cl., Aep See The The Tyr Lou MC CM TOG MGG COG CMG TOT MC MAT TCC ACC TIC TAG OTG

Au.G~ClLye

7To Cl. Lye Cl, Le. Hie His e GeCl.v Cl. Cln Au. Vol Her Au. Ala The Lou Asp ACC 'AC TOG GM% MA GAG COG CAT CAT ITLACGMAM CMG MT OTT OGL MT GCA ITT TIA OAT

AMG GM GMG TTT TAG ATA OGG TCA AMG TAAAGMA"MAOTTGG TLT COG CMG TAT ACT MAT MGC AM TIC COT GIT CCA00CMG MAAA MA" OCT GAA GMA GM GOT OTG GMA LIT CIA OCT 600 810 620 630 Pro Cln La. Nie Ala Aep Vol Cly Aep Lye Vol Lyfe fle flu The Lyfe Au. Net Ala Thr Arg Pro Lye Ser flu Nie Ala Die, Cly Vol Cl. The Cl. Ser Her 2The Vol Thr Pro The CCA C" CIT CAT GCA MAT OTT GGM MC MA" TC MA" LIT ATC TT MA" AM ATO 0CC AMA A00 CCC TAG T06 ATA CAT GCC CAT GCM OTA CM AMAGA MCMT TOT 006 OT OCT CCA ACA 660

650

880

690

660

670

900

910

940

950

Cly Cl, The Ge, The Lye Vol lo2p Lye flu Pro Cl. Aeg See Cly AlZa C1y The Gl. Aep See Ala Lye flu Pro Ilop A la Lye Ser The Vol Aep Cl, Vol Lye Asp Ge. TTL CCL OCT GMA LOT CTC MCT TACGOTA TOO AM AOG CMA GM AMA TOT GMA GOT 000 AM MCG MAT TOT OCT TOT LIT CMA TOO OCT Lyer ULT TAT TCA LOT OTG MAT CCM CIT AMG GAG CTC

Ge. Pro

Lye See Clyf Ge. flu Cly Pro Ge. flu Vol Lye Aeg Aop Pro Lye Ge. Lyfe VolI The Au. TAG MGT GMA ITA LIT GCO CCC OTO LIT OTT TOT CG4 AM COT TAG ITO MA OTL TIC MT 920

Pro CCC

Avg A~rg Lye Le. CG. The Ala Ge. Ge, The Ge. Vol The Aep GT, Au. Clu Ser Top AMA MA AM CTG GM TTT 0CC CTT COG TIT OTA O"TT TT MAT GAG MT GMA TOT TOG

930

He Asp Die Pro Clu Lye VoI Au. Lyfe Aep Aep Cl. Cl. FPe fl~ uClSer Au. Lye Net Lie Ala flu Au. Cly Aeg Net Phe CMy Au Ge. L1yr Ge. Aep Aep Aur. fluLe The Lye Sr o TAG ITO CAT MA C AM LOG MALCA TAG TOT MAT GAG CCC GMG MA OT MC AAA MAT MAT GM GM TIC LTA GMA MC MT "A LOG CAT OCT AIT MT GMA AMA LOG TTT GMA MC OTA 960 970 980 990 Cl Cvt G e.Lo The Net Lie Vol Cly Aep Cl. VoI Au. lop Lye Ge, Net Cly Net Cly Au. Cl. flu Aep Ge. Die The Vol DUs The Die CMy Lie Ser he Cl. Lye Lyfe Die Are Cy CM GGC CTC LCA LOG CAC OTG GMA GAT GMA GIG AMC TOG TAT CTO LOG GMA LOG OGG MT GMA 010 GC ITO AC ACT GUA CAT TTT MAC GOC CAT MGC IT CM TAC MAC CAC LOG 000

1010 1600 1020 1030 Vol Lyer Her Seo Aep Vol The Aep flu The Poe C~y The Lye Cl. TheLoGe. C Met The ?ro Avg The Pee Cly flu lop Le. Ge. Die Lye Die Vol The Aep Hie fle Die Al. Cly OTT TLT MCT TOT MAT CTC ITT MGA OTT TIC COT GMA LCA TAG CM LOG CTA GMA LOG TrT CUAGMA AMA COT GM LIT TOO TTA CTC CAC TOG CAT OT LCC GM CMC LIT CAT GOT GMA

The Vol LOTG GMA AGC LOT TLG ACC OT 01 MA UTOTG01 TMA MT WAM ATC AM AMA OTA TAT MT MAT

Net Cl. The The Lye

1040 Ge. Cl. Au.

1042

GU. U4GI L TyrF~ jAsp The Lye Her Cly CTA C" MTGAIWTM GA ATCC LACC MA" TOT GOC TM LOG AM TM LIT GOT MT AMG TOG MA" MAA GAG AM AMC CM TM TIC ATL O ITO TOG MT MCc TAT "AM CAT TM AMG AMc MT GM Mcc ATA cA CIT TOT AMA TOT 0 00 GM AMc TOT TAAM TTlGC CCA MT 7ACIT 006 TMA CTO AM CIT? OTA CAC TMG OT 010 AAA COG LIT CAT AMAOTM I poly At2ll

inserts into M13 cloning vectors. The complex CP cDNA sequences were assembled from sequences of overlapping restriction enzyme fragments. Homology of nucleotide sequences in the three domains of CP was evaluated by calculation of "accident probability," Pa, the probability that a homology equal to, or greater than, that being considered might arise accidentally (23). Gene Mapping. Chromosome mapping of CP was performed using human-mouse somatic-cell hybrids (24). DNAs from the cell hybrids were isolated and digested with EcoRI and HindIII and were analyzed by Southern filter hybridization as described (25). The cDNA inserts, CP-1 and CP-2, were isolated by Pst I digestion, agarose electrophoresis, and electroelution of the human fragments. Each nick-translated probe was hybridized to the cell-hybrid DNA as described (25). In situ hybridization of 3H-labeled CP-1 cDNA was carried out on human chromosomal spreads, following a modification (8) of the procedure of Harper and Saunders (26). In Situ Histohybridization. Human peripheral blood mononuclear cells (PBMCs) were obtained by Ficoll/Hypaque (Sigma) separation of heparin-treated venous blood drawn from healthy volunteers. Populations of adherent cells (macrophages and monocytes) and nonadherent cells (T and B

lymphocytes) were obtained as described by Lum et al. (27).

AMA LIG

GMA AM

CP-2 A C G T

FIG. 1. Nucleotide sequence of the cDNA and deduced amino acid sequence of human CP revealed by CP-1 and CP-2 clones. The amino acid sequence corresponding to the entire human CP is numbered according to that reported (1). The four additional amino acids found in CP-1 are boxed.

CP-1 C G

A

T

c C

,.,

M_,O

A

_l_

c

IlowW40wo

Thr

v

G

A T A

omwmw

act 1040

-

-1.

AL

A

-_

Asr

Asp

1042

G

C T

-

G T

1043

}A

40wmvm

Glu

Thy,

A

C

VW

9-

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_

2ftf

mom"m I...

Tgi r

G

I

ud

mm .1.1

i'm .0-,

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ul

1~

A:;>.

1040

T v . A

."I"

A

FIG. 2. Autoradiograph of the DNA sequencing gels showing the nucleotide sequences of clones CP-1 and CP-2 that differ by a region in CP-1 that encodes four additional amino acids.

~2.4

Proc. Natl. Acad. Sci. USA 83 (1986)

Biochemistry: Yang et al.

3259

Table 1. Nucleotide sequence comparisons between CP and factor VIII and between CP domains Amino acid residues compared Comparison Nucleotide identity (%) Gaps* 196-2025 vs. 199-1046 14 Factor VIII/CP 1143/2383 (48.0) CP internal domains 199-350 vs. 560-710 3 I/II 219/433 (50.6) 6 351-710 vs. 710-1046 II/III 502/1002 (50.1) 2 199-350 vs. 998-1046 I/III 246/439 (56.0) *Introduced to maximize homology (31).

PBMCs were stimulated with phytohemagglutinin as described (27). Cells were collected and deposited on pretreated glass slides (28). Fixation was carried out for 7 min in phosphate-buffered 2-3% glutaraldehyde. The CP cDNA probe was 3H-labeled, using three radiolabeled nucleoside triphosphates, by nick-translation (19) to a specific activity of 2.6 x 107 cpm/gg of DNA. The hybridization mixture contained 50% (vol/vol) formamide, 10 mM Tris Cl (pH 7.5), 1 mM EDTA, 600 mM NaCl, 5% (wt/vol) dextran sulfate, and 100 pug of denatured Escherichia coli DNA per ml. Hybridization and autoradiography were carried out as described (28). Southern Blot Analysis of DNA Polymorphisms. High molecular weight DNAs were isolated from human lymphocyte nuclei (29). Filter hybridization of lymphocyte DNA with human CP cDNA was carried out at 420C in 50% formamide/5x SSPE/10% dextran sulfate/5x Denhardt's solution/0.1% NaDodSO4. Filters were washed with 2x SSC at room temperature and then with 0.1 x SSC/0.1% NaDodSO4 at 550C. (1 x SSPE is 0.18 M NaCl/10 mM sodium phosphate, pH 7.4/1 mM EDTA; lx Denhardt's solution is 0.02% polyvinylpyrrolidone/0.02% Ficoll/0.02% bovine serum albumin; 1 x SSC is 0.15 M NaCl/15 mM sodium citrate, pH 7.0.)

RESULTS AND DISCUSSION Of 2 x 106 cDNA clones isolated from a human liver cDNA library (20), 3 contained sequences of the CP gene as judged by their deduced amino acid sequences (1) and the presence of sequences corresponding to two synthesized oligonucleotide probes. One cDNA clone, CP-1, contained 2557 bases encoding 81% of the entire CP sequence, a stop codon, and 244 bases in the 3' untranslated region. This clone contained the sequence corresponding to amino acid residues 199-1046, with an additional sequence of four amino acids, previously unreported, between residues 1041 and 1042. The new amino acid sequence, -Gly-Glu-Tyr-Pro-, found in CP domain III is boxed in the sequence of CP-1 shown in Fig. 1. The second clone, CP-2, identified by screening the same cDNA library, contained the coding sequence of amino acid residues 202-1046 and 124 bases in the 3' untranslated region. It matched the amino acid sequence reported by Takahashi et al. (1) exactly and did not contain the four extra codons found in CP-1. The nucleotide sequence determination for amino acid residues 1040-1043 in CP-1 and CP-2 is shown in Fig. 2. The entire sequences of the two clones were compared and, with the exception of the four additional amino acid codons, were identical in all 2449 nucleotides, including those of the 3' noncoding regions. Clone CP-1 identified all genomic DNA fragments that hybridized with CP-2 in Southern blot analysis with four different restriction enzymes (EcoRI, Pst I, Pvu II, and HindIII). Although the origin of the two dissimilar CP clones must await examination of the corresponding region of genomic CP DNA, there are several explanations that can be considered. Examination of the factor VIII genomic sequence (30) reveals the presence of an intron between exons 19 and 20, a region homologous to the CP cDNA encoding residues 1041-1042 (14). If a similar intron arrangement

occurs in CP, the additional amino acids in CP-1 could have arisen from alternative splicing of an intron during maturation of CP mRNA in some liver cells, with the 12 additional nucleotides of CP-1 cDNA originating from an intronic sequence. Other explanations include coexistence of two CP alleles in the donor's genome or the existence of two independent CP loci. It seems unlikely that the CP-1 sequence could result from a cloning error, since the 12 new nucleotides constitute an internal sequence within the cDNA insert and therefore are unlikely to have arisen during reverse transcription or ligation. Amino acid sequence analysis of pooled CP preparations by Takahashi et al. (1) have revealed two sites, positions 79 and 449, where both Lys and Gly were present in a ratio of 8:2. The sequence deduced from clones CP-1 and CP-2 has Lys in position 449, but neither clone included sequences

encoding residue 79. A study of intragenic homology of the CP cDNA was carried out by comparing the nucleotide sequences of each of the three domains (1). These data are shown in Table 1. Identities of the CP domains ranged from 50.1% to 56.0%. When the cDNA sequences of the three CP domains were compared by statistical analysis to identify regions of extensive internal nucleotide sequence identity (23), three paired sequences were found to have homologous regions at least 219 base pairs long (data not shown). A comparison of the nucleotide sequence encoding residues 204-276 with that encoding residues 903-975 reveals 66.2% identity, while A

B

1

4

1

2

3

4

5

6

14-- __

-11.1 -~~

-*

4-~7.9

.4-5.7 4.8 ---o1

_

2.4--

16=40

.~~~~~~~~~~~~~~~~~~~~~~~~~~~~~. _~~~~~~ 7. 1. 4

U.9--

-'-0.9

FIG. 3. Chromosome mapping by somatic-cell-hybrid analysis. (A) HindIII digest of DNA from human (lane 3), mouse (lane 4), and somatic-cell hybrids (lanes 1 and 2) hybridized to CP cDNA. The 11.1-, 7.9-, and 5.7-kilobase (kb) fragments segregated with chromosome 3 (q21- qter). (B) EcoRI digest of DNA from human (lane 1), mouse (lane 2), and cell hybrids (lanes 3-6). The 14-, 4.8-, and 4.4-kb bands segregated with chromosome 3 (q21-qter). The 0.9-kb fragment contains a CP-related sequence that is located on chromosome 11 (pll--qter).

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Proc. Natl. Acad. Sci. USA 83 (1986)

Table 2. Segregation of the CP gene with human chromosomes in human-mouse somatic-cell hybrids Chromosome Hybrid CP CP-like 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Translocation* -+ - - - - + - - + + - ICL-15 -+--+ + ---+ -+ - - - - - + - + + - + 17/3 TSL-2 + - + + - + + + + + + + - + + + -+ ATR-13 5/X + + +- + - + --+ + + + + + + + + + + - + + +- 7qJSR-17S ---++ + - - + + WIL-5 + + + -+ + - + -+ +++++ + + + WIL-6 + - + + NS - - + + WIL-2 + + + + + -+ + - -+ ++++ + + + + + WIL-8X + -+ + + + + + + - + + - + -+ + - + + WIL-7 + + -+ + + -+ + + + + + - + + + + - + + + + + lpJWR-26C 17/9 + + + ----+ - - + -+ - + + + + + + NSL-9 2/1 ---+ - + - - -+ + - - + - - + + -+ + - JWR-22H + + + + + + + + -+ + + + + + - + + + + + + + + + REW-7 .+ + ... + --+ ----+ -+ + WIL-14 ----++ + + WIL-13 ---+ + + + + + + NS REW-ll + + ++ - + + + + + + + + - 11/X, X/11 + + + + -+ + XER-11 + +++++++++ + + + + + + + + - - - + 11/X XER-7 ... 3/X + - - + ... XTR-3BSAGB+ + + + + + + - X/3 -+ -+ + + -+ -+ + XTR-22 The WIL-2 hybrid, which contains human chromosome 3 fragments, contained the CP 7.9-kb HindIII fragment but not other CP fragments. The 0.9-kb EcoRI fragment resides in the p21-+qter region of chromosome 11. One hybrid, JWR-22H, was discordant for the 0.9-kb EcoRI fragment. NS, not scored. *Translocation chromosomes from the human parent retained by some hybrid cells: 11/X (llqter-.llpll: :Xqll-*Xqter), X/11 (Xpter--+Xq1l::l1p13--l 1pter), 3/X (3pter--3q21::Xq28--+Xqter), and X/3 (Xpter--Xq28::3q21--*3qter).

204-276 compared with 565-637 shows 61.2% identity and 565-637 with 903-975, 59.4% identity. The basis of the evolutionary constraints is unknown but usually reflects conservation of functional sites. It is of interest that the homology observed here does not reflect conservation of the sites thought to be important in type-1 copper binding (14, 31), possibly indicating evolutionary conservation of an alternative CP function. A comparison of the nucleotide sequences of the three homologous domains in CP and factor VIII yielded a 48% identity of sequence when CP nucleotides encoding amino acids 199-1046 were compared to the nucleotides encoding factor VIII residues 196-2025 (Table 1). Chromosomal mapping of the CP cDNA was accomplished by somatic-cell analysis and in situ hybridization. The CP gene was mapped' to human chromosome 3 by Southern blot analysis of human-mouse somatic-cell hybrids after human and mouse DNAs were cleaved with a variety of restriction endonucleases. CP-1 and CP-2 gave similar patterns on Southern blot analysis. Fig. 3 illustrates Southern analysis of EcoRI and HindIII digests of genomic DNA ofhuman-mouse somatic-cell hybrids. These two enzymes distinguished human from mouse DNA fragments hybridizing to CP cDNA. The most intensely hybridizing human fragments [11.1, 7.9, and 5.7 kb (HindIII) and 14, 4.8, and 4.4 kb (EcoRI)] segregated with chromosome 3 and with the q21-*qter region of chromosome 3 contained in the XTR-22 hybrid (Table 2). It was not possible to score the 2.4-', 1.7-, and 1.6-kb EcoRI fragments or the 2.4-, 2.0-, 1.4-, and 0.9-kb HindIII fragments (Fig. 3) in the cell hybrids; however, the 0.9-kb EcoRI fragment was found to segregate with chromosome 11. The short CP-homologous sequence detected on chromosome 11 may be a pseudogene or part of an expressed gene that contains a CP-like coding sequence. In situ hybridization of 3H-labeled CP cDNA on chromosomal spreads revealed significant labeling (30%) on chromosome 3, with 59% of the silver grains on chromosome 3 at 3q25 (Fig. 4). Although the coding region of the factor VIII gene contains three CPhomologous sequences, there was no hybridization detected

on the X chromosome by somatic-cell analysis or by in situ hybridization with CP cDNA. DNA polymorphisms consisting of 11.5- and 9.6-kb bands were observed after hybridization of the CP cDNA probes

20

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.

.

*

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FIG. 4. Gene mapping by in situ hybridization on 100 chromosomal spreads. Chromosome 3 contained 30% of the labeled sites (Upper); 59o of the labeled sites on chromosome 3 were on

3

3q25 (Lower).

q

Biochemistry: Yang et al.

Proc. Natl. Acad. Sci. USA 83 (1986)

including the liver as well as circulating macrophages and lymphocytes.

F M 1 2'

kb W-r

I--8

Ad

~S4qLF

9.4-

FIG. 5. CP DNA polymorphism within a family, detected with Pst I digestion. Arrowhead designates a 9.6-kb band that is present in the father's DNA (lane F) and is inherited in one of three offspring (lane 3). Markers at left represent size (in kb) and positions of HindIII fragments of phage X DNA.

Bi 6.6

-

4.4--

with human lymphocyte DNA digested with Pst I. Fig. 5 shows the segregation of the polymorphism in a family. The gene frequency of the allele associated with the 9.6-kb band is 0.34 in a panel of 16 unrelated individuals (data not shown). By use of in situ histohybridization techniques with radiolabeled human CP cDNA as a probe, a panel of human tissues and peripheral blood cells was examined for sites of CP mRNA. CP mRNA was observed, as expected, in liver tissue (data not shown). In addition, 10-15% of the PBMCs contained CP mRNA. Fig. 6 shows lymphocytes and macrophages labeled with silver grains denoting CP mRNA. After phytohemagglutinin stimulation, CP mRNA was observed in 25-35% of the PBMCs in 10 random microscopic fields. This increase was seen predominantly within the lymphocyte population. Gene expression of both the transferrin gene (TF) (27) and CP in lymphocytes has now been observed, suggesting coordinated functional roles of these two metalbinding and genetically linked plasma proteins. It has been suggested that the oxidase activity of CP is required to convert iron(II) to iron(III), which is required for transferrin's function in iron transport (17). In summary, the CP gene has been mapped to human chromosome 3q25. Two slightly dissimilar CP cDNAs have been characterized in the same cDNA library; one contains codons for an additional four amino acids. Hybridization of both cDNA clones produces the same restriction pattern with genomic DNA. In situ histohybridization with the CP cDNA probe identified the presence of mRNA in human tissues

L

M

Amklb.

w

FIG. 6.

3261

Localization of CP mRNA in PBMCs

by in situ

histohybridization with the CP cDNA probe. Presence of silver grains indicates CP mRNA in lymphocytes (L) and a macrophage (M). (x 830.)

We thank Dr. S. H. Orkin (Harvard Medical School) for the human liver cDNA library; Victor Luna, Rod Cupples, and Judith Gardella for technical help; James Buchanan for photography; and Betty Russell for preparation of the manuscript. This work was supported in part by grants from the National Institutes of Health (GM33298, HD16584, and DE07418), The American Cancer Society (NP-470), March of Dimes Birth Defects Foundation (MOD 1-927), and the Council for Tobacco Research, USA Inc. (CTR-1620). 1. Takahashi, N., Ortel, T. L. & Putnam, F. W. (1984) Proc. Natl. Acad. Sci. USA 81, 390-394. 2. Ryden, L. & Eaker, D. (1983) Eur. J. Biochem. 132, 241-247. 3. Shreffler, D. C., Brewer, G. J., Gall, J. C. & Honeyman, M. S. (1967) Biochem. Genet. 1, 101-115. 4. McCombs, M. L. & Bowman, B. H. (1969) Tex. Rep. Biol. Med. 27, 769-772. 5. Shokeir, M. H. K. & Shreffler, D. C. (1970) Biochem. Genet. 4, 517-528. 6. Larsen, B. (1977) Anim. Blood Groups Biochem. Genet. 8, 111-113. 7. Weitkamp, L. R. (1983) Ann. Hum. Genet. 47, 293-297. 8. Yang, F., Lum, J. B., McGill, J. R., Moore, C. M., Naylor, S. L., van Bragt, P. H., Baldwin, W. D. & Bowman, B. H. (1984) Proc. Natl. Acad. Sci. USA 81, 2752-2756. 9. Robson, E. B., Sutherland, I. & Harris, H. (1966) Ann. Hum. Genet. 29, 325-336. 10. Eiberg, H. & Mohr, J. (1979) Cytogenet. Cell Genet. 25, 149-150. 11. Eiberg, H., Mohr, J. & Nielsen, L. S. (1983) Cytogenet. Cell Genet. 37, 461. 12. Cox, D. W. & Francke, U. (1985) Hum. Genet. 70, 109-115. 13. Frydman, M., Bonnd-Tamir, B., Farrer, L. A., Conneally, P. M., Magazanik, A., Ashbel, S. & Goldwitch, Z. (1985) Proc. Natl. Acad. Sci. USA 82, 1819-1821. 14. Vehar, G. A., Keyt, B., Eaton, D., Rodriguez, H., O'Brien, D. P., Rotblat, F., Oppermann, H., Keck, R., Wood, W. I. & Harkins, R. N. (1984) Nature (London) 312, 337-342. 15. Church, W. R., Jernigan, R. L., Toole, J., Hewick, R. M., Knopf, J., Knutson, G. J., Nesheim, M. E., Mann, K. G. & Fass, D. N. (1984) Proc. Natl. Acad. Sci. USA 81, 6934-6937. 16. Goldstein, I. M., Kaplan, H. B., Edelson, H. S. & Weissmann, G. (1979) J. Biol. Chem. 254, 4040-4045. 17. Frieden, E. & Hsieh, H. S. (1976) Adv. Enzymol. 44, 187-236. 18. Stevens, M. D., DiSilvestro, R. A. & Harris, E. D. (1984) Biochemistry 23, 261-266. 19. Maniatis, T., Fritsch, E. F. & Sambrook, J. (1982) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory, Cold Spring Harbor, NY), pp. 122-127. 20. Prochownik, E. V., Markham, A. F. & Orkin, S. H. (1983) J. Biol. Chem. 258, 8389-8394. 21. Maxam, A. M. & Gilbert, W. (1980) Methods Enzymol. 65, 499-560. 22. Sanger, F., Nicklen, S. & Coulson, A. R. (1977) Proc. Natl. Acad. Sci. USA 74, 5463-5467. 23. Sargent, T. D., Yang, M. & Bonner, J. (1981) Proc. Natl. Acad. Sci. USA 78, 243-246. 24. Shows, T. B., Naylor, S. L. & Sakaguchi, A. Y. (1982) in Advances in Human Genetics, eds. Hirschhorn, K. & Harris, H. (Plenum, New York), Vol. 12, pp. 341-452. 25. Naylor, S. L., Sakaguchi, A. Y., Szoka, P., Hendy, G. N., Kronenberg, H. M., Rich, A. & Shows, T. B. (1983) Somatic Cell Genet. 9, 609-616. 26. Harper, M. E. & Saunders, G. F. (1981) Chromosoma 83, 431-439. 27. Lum, J. B., Infante, A. J., Makker, D. M., Yang, F. & Bowman, B. H. (1986) J. Clin. Invest., in press. 28. Lum, J. B. (1986) BioTechniques, in press. 29. Bell, G. I., Karem, J. H. & Rutter, W. J. (1981) Proc. Natl. Acad. Sci. USA 78, 5759-5763. 30. Gitschier, J., Wood, W. I., Goralka, T. M., Wion, K. L., Chen, E. Y., Eaton, D. H., Vehar, G. A., Capon, D. J. & Lawn, R. M. (1984) Nature (London) 312, 326-330. 31. Ortel, T. L., Takahashi, N. & Putnam, F. W. (1984) Proc. Natl. Acad. Sci. USA 81, 4761-4765. 32. Bowman, B. H., Yang, F., Brune, J. L., Naylor, S. L., Barnett, D. R., McGill, J. R., Moore, C. M., Lum, J. B. & McCombs, J. (1985) in Colloquium on Protides of the Biological Fluids, ed. Peeters, H. (Pergamon, Oxford), Vol. 33, pp. 15-20.