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<Original Article> Identification of Microsatellite Repeat Polymorphisms in the Placental Alkaline Phosphatase Gene 利用統計を見る

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Yamanashi Med. J. 7(4), 169--I72, 1992

Identification of Microsatellite Repeat Polymorphisms

in the Placental Alkaline Phosphatase Gene

Zen£aro YAMAGATAi)2), Kathy HANDA2) Moyra SMiTx2), and Akie AsAKAi)

J)Department ofHealth Sciences, Yamanashi Medical Universdy, Tamaho, Yamanashi 409-38,laPan

and 2)Department ofPediatrics, Univemsdy ofCalijbrnia,

Irvine, CA. 927I7, U.S.A.

Abstract: Examinationofthepublishedsequenceofplacentaialkalinephosphatase(PLAP)gene reveaied the presence o{' (CA). repeats at position 542-565. We utilized sequeRce iRformation to

design primers to amplify a DNA fragment of about l12 bases・ by rr}eaRs of PCR. Use of this primer set led £o the identifica£ion of PLAP polymorpkisms with six alleles. We used the DNA from human hamster somatic cell hybrids to confirm that the polymorphism was dependenton the presence of human chromosome 2. Use ofthis primer set also led to the amplification ofthe DNA

present in the intestinal alkaline phosphatase (IALP) geRe, using the clone IAP8, which coRtains

the IALP gene. The product derived from the IALP gene migrated differently to the polymorphic alleles, in denaturing acrylamide gels. This polymorphism will be useful for linkage analysis.

Key words: Microsatellite repeat, Polymorphism, Polymerase chain reaction, Placental alkaline phosphatase, Chromosome 2

INTRODUCTION

Human alkaline phosphatases' (ALP,

orthophosphoric-moneester

phosphohydre-lase, EC 3.li3.1) are enzymes that hydroly a variety of phosphate esters at high pH optima. The three main classes-placental alkaline phos-phatase (PLAP), intestinal alkaline phospha-tase (IALP) and liverlbone!kidney alkaline phosphatase (LIBIK ALP)int4) are c}assified by differences iR thermostability, immunolegical properties and electrophoretic mobility. The

human PLAP gene and the IALP gene map to

chremesome 2q34-q375)6), aRd £he LIBIK ALP geRe is located in chromosome lp36.l-p347). The PLAP and IALP genes have been isolated by Knell et al.8), Millalt et al.9), Henthorn et

al.ie) aRd Weiss et al,ii>

There have been many studies of PLAP, for example, the existence ofa PLAP-like gene, or

germ cell PLAP9)i2), genetic

po}y-morphismi3)i4) and its use as a tumor

markeri5>. We were interested in genetic poly-morphism of the PLAP gene and we examined the PLAP microsatellite repeat polymerphism

using of a pelymerase chain reactioR

(pcR)i6mi8) in this present study.

Received December Accepted January 8,

22, l992 l993

MATERIALS AND METHODS

Two 20-mer oligonucleotide primers

(PLAPI : GGCCTGAGGCTACAGCTCTC

and

PLAP2:GGTCCGGGTTCTCCTCCT-CA) were designed from the region fianking

the (CA)n repeat iR the human PLAP gene

(locus:H[UMALPHA9)).

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170 Z. Yamagata

Cyclic Reactor Instrument (Ericomp) and the

GeneAmp DNA Amplification Reagent Kit

(Perkin Elmer Cetus). PCR primers were end labeled with 82P-7-ATPi9). The PCR reaction mixture in a total volume of 20 ul contained 1

pt (2000 ng) human genomic DNA, 3.0 paM

labeled PLAPI primer, 2.0 paM

non-labeled PLAP2, 1.0 ptM non-labeled PLAP2 primer,

200 uM each of dATP, dCTP, dGTP, dTTP,

reaction buffer and 1.0 unitiassay Taq

polymerase. The PCR protocol was as follows: 1 minute denaturation at 940C, 25 cycles of 1 minute at 940C, 1 minute at 550C, 1 minute at 720C and a final step of 10 minutes at 720C.

Aliquots of PCR products containing an

equal volume of stop solution (25% forma-mide, 20 mM ethylenediaminetetraacetic acid, O.05% bromophenol blue, O.05% xylene cyanol FF) were heated at 950C and loaded onto 8%

polyacrylamide DNA sequence gels

(acrylamide:bis acrylamide =19: 1) including 7 M urea and run for 4 hours at 45 W. The gel

was covered with cellophane posed to X-ray film at -600C

wrap and

ex-for 18 hours. REsuLTS AND DIscuSSION

The size of the expected PCR product

generated from the PLAP gene was about 112 bp. The PCR product was also generated from

plasmid IAP8 which contains the 5' gene

region of IALP, using the PLAP primers

described above. The PCR product generated

from IAP8 was not polymorphic in the

de-naturing gel.

Our results show that the PCR product of the same size as the polymorphic product is

generated from a somatic cell hybrid HHW

1164 (provided by Dr. J. Wasmuth, Depart-ment of Biochemistry, University of Califor-nia, Irvine, U.S.A.) in which only chromosome 2 is present (Fig. I).

Amplified DNA from 55 individuals of 9

different families was analyzed. Mendelian

"Lfsti$l,res);ee}hsgii>>Z,e)}e:s・s"XKiillg>)iestNsliK'XSiill"r,sK"lj,eSs3e,i`sss 'X"liltl/fses

y..nj?.:Ntit'ssr't,fillSlil '

', iue- wawt"Y" l' :lt,Sl・ r' ii・・'

' 'eree.

pt

tll: ttt ,i,-Slliitel

t・・

i:,t$- kslis. , geexg-.tt-"kit'.Vt"k-¥E

PLAP

IALP

Fig. I. Electrophoresis of PLAP (CA)n repeat PCR products on a denaturing gel. The band at the bottom is the PCR product derived from the IAP8 gene and the bands above the bottom band are the PCR products from PLAP gene. HHW 342, HHW 107 and HHW 105 are human hamster somatic cell hybrids which do not include human chromosome 2. HHW 1164, HHW 1068 are human hamster somatic cell hybrids which include human chromosome 2.

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Polymorphisms in the PLAP Gene 3

2

4/4

3B

3

t

4

x

l

3f4

eV6

314

9・

3Y33f4

t.

3f6

m

m・

mim

memmwmmm"ttiu

3/4

MNllilliliWWWntl

co-m

<

celt$tawt

lLApa

171 ・" ' '

l.-..-.,.V t

-.. t

-.

'

・・・l '','la:-'"t':k・・; ';

' ..

tt tt- tt t. t.

t tt. ' '

Fig. 2. Electrophoresis of PLAP (CA)n repeat PCR products in a denaturing gel. The pedigree is shown above the gel photograph. The genotypes are shown below.

inheritanceofthePLAPmicrosatelliterepeat Table1.Allelefrequencies,heterozygosityandthe PIC value of the (CA)n repeat in the polymorphisms is shown in Fig. 2. Each allele

contains one or two (relatively intense) major PLAP gene in 25 unrelated individuals

bands and some shadow bands which are

Allele N % Heterozygosity PIC

presumed to be due to template-primer

slip-pageoftheTaqpolymerasei7)20),Therefore 1 3 6

22

4 we consider that one of the intense bands

82 O.76 O.64

3

16

indicatesanalleleproduct.Additionally,six 4 s lo

allelesofthemicrosatelliterepeatpoly- 5 2 4

morphismwereidentifiedin25unrelated 6 22 44

individuals. The frequencies of alleles,

Total 50 100

zygosity and the polymorphism information

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172 Z. Yamagata

content (PIC) value are shown in Table 1. This primer set alse led te £he amplification of DNA present in the IALP gene as evidenced by amplificatioR of DNA from the clone IAP8, which contains the IALP gene. 'I'he product derived from the IALP gene rnigrated dif-ferently from that ofthe polymorphic alleles in denaturing acrylamide gels. These resu}ts indi-cate that the microsatellite repeat poly-morphism will be useful for linkage studies ir} diseases lecalized to chromosome 2q35-q37.

REFERENCES

}) mental change in httman intestinal alkaline phospha£ase. Proc Natl Acad Sci USA l978; 75: 3909--3912.

2) McKenna MJ, HamiltoR TA, Sussman HH. Comparison of human alkaline phosphatase isozymes: Structural evidence for three protein

classes. Biochem J l979; 1881: 67--73. 3) Seargent LE, Stlnson RA. Evidence that three structural genens code fer human alkaline phosphatases. Na£ure l979; 281: 152-l54. 4> HarrisH.MultiplocusenzymesystemsaRdthe evolution of gene expression. The harvey }ectures; series 76. New York Academic Press 1982; 95---124.

5) Mar£iR D, Tucker DF, Gorman P, Sheer D, Spurr NK, Trowsdale J. The human placentai alkaline phosphatase gene and related eRces map to chromosome 2 band q87. Ann Hum Genet l987; 51: l45-l52.

6) GriMn CA, Smith M, Henthorn PS, Harris H, Weiss MJ, Raducha M, Emanuel BS. Human p}acental and iR{estinal alkaline phosphatase genes map te 2q34-qS7. Am J Hum Genet 1987・ 41: I025-1034.

,

7) Smith M, Weiss MJ, Gri£fin CA, MurrayJC, Buetwo KH, Emanuel BS, Henthorn PS, ris H. Regional assignrnen£ of the gene for human liverlbone/kidney alkaline phospkatase to chromosome lpS6.I-p34. Genomics 1988; 2: l39-148.

8) KRollBJ,Rothb}umKN,LongleyIV{.Twogene duplication events iR the evo}ution of the human heat-stable alkaline phosphatases. Gene (Amst> l987; 60: 267-276.

9) MillanJL,MaResT.Seminopaa-derivedNagao isozyme is encoded by a germ-cell alkaline phosphatase gene. Proc Natl Acad Sci USA. I991; 85: 3024-3028.

10) HeRthom PS, Raducha M, Kadesch T, Welss MJ, Harris H. Sequence and characterization of the human intes£inal alkaline phosphatase gene. J Biol Chem i988; 263: l201Fl2019. 11) Weiss MJ, Ray K, HeRtkorn PS, Lamb B, Harris, H. Structure ef the human llverl bonefkidney alkaline phosphatase geRe. J Biol Chem 1988; 26S: 12002-l2010.

12) Knoll BJ, Rothblum KN, LoRgley M. leo£ide sequence of the humaR placental liRe phosphatase gene.J Biol Ckem 1988; 168: 12020--l2027.

I3) MartinD,SpurrNK,TrowsdaleJ.RFLPofthe human placental alkaline phosphatase gene (PLAP). Nucleic Acids res 1987; !5: 91e4. 14) Beckman G, BeckmaR L, Kivela A, MillanJL, Sikstrom C. A new Pstl restriction fragmeBt length polymorphism (RFLP) of placental line phosphatase. Hum Hered l991; 41: l22-128.

15)

lar c}oning of corr}plementary DNAs encoding alkaijne phosphatase in human colon cancer ce}ls. Cancer Res 1990' 5e: le85-1091. ,

16) Saiki RK, Scharf Sg, Faloona F, Mullis KB, Horn GT, Arnheim N. Enzymatlc tion ef beta globin genomic sequences aRd restriction site analysis for diagnosis of sickle

cell anemia. Science l985' 230: 1350-1354. ,

I7) LlttM,LutyJA.Ahypervariablemicrosatellite revealed by in vivo amplification of a }eotide repeat within the cardiac muscle actin gene. Am J Hum Genet 1989; 16: 397-401. 18) WeberJL, May PE. Abundant class of human DNA po}ymorphisms which can be typed using the po}yrnerase chain reaction, Am J Hum Genct l989; 44: 388-396.

19) SambreokJ,FritschEF,ManiatisT.Molecular cloning: a Iaboratory maxxual, 2nd ed. Cold SpriRg Harbor laboratory, Cold Spring bor, NY. 1989; IO.51-IO.67.

20) bi HY. Linkage mapping and fluorescenge in

situ hybridization of TCTEI on human

chromosome 6p: Ana}ysis of dinucleotide morphisms on native ge}s. Genomics 1991; !O: 921-926.

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