DNA RESEARCH 5, 103-113 (1998)

Physical Map of the Human Chromosome 8pl2-p21Encompassing Tumor Suppressor and Werner's Syndrome GeneLoci

Koji ICHIKAWA,1 Akira SHIMAMOTO,1 Osamu IMAMURA,1 Yoshiki TOKUTAKE,1 Yukako YAMABE,1

Saori KITAO,1 Noriyuki SUZUKI,1 Kahori SUGAWARA,1 Takehisa MATSUMOTO,1 Winston THOMAS,2'*

Dennis DRAYNA,2^ Makoto GOTO,3 Masanobu SUGIMOTO,1 Minoru SUGAWARA,1 and

Yasuhiro FURUICHI1-*

AGENE Research Institute, 200 Kajiwara, Kamakura, Kanagawa, 247-0063, Japan,1 Mercator GeneticsInc., 4040 Campbell Avenue, Menlo Park, CA, 94025 USA,2 and Tokyo Metropolitan Otsuka Hospital,Minami Otsuka, Toshima-ku, Tokyo, 170-0005, Japan3

(Received 6 February 1998)

Abstract

Detailed physical maps of the human genome are important resources for identification and isolationof genes responsible for diseases and for the study of their structure and function. We constructed a2.0-Mb high-resolution physical map within the human chromosome 8pl2-p21 region extending from markerD8S131 to D8S283. The map comprises a series of contigs mostly Pl/PAC clones, which span the loci ofpotential tumor suppressor genes and the Werner's syndrome gene. Each Pl/PAC DNA was defined byits size, restriction sites, terminal sequences, intermarker distances and location relative to major genesand markers. The genes on these Pl/PAC DNAs were analyzed by an exon amplification method todetermine their locations. The genes newly found by the exon amplification method together with otherknown genes, including those of glutathion reductase, a general transcription factor, protein phosphatase2A (3 subunit and Werner's syndrome, were precisely mapped within the contigs. These Pl/PAC DNAsare useful reagents for the generation of new microsatellite markers to narrow the candidate region of thetumor suppressor gene(s) and/or genes responsible for other diseases, which are believed to exist in thisregion by linkage analysis.Key words: physical map; Pl/PAC; exon amplification; 8pl2-p21

1. Introduction der associated with premature aging. Furthermore, thecentromere-proximal region in the short arm of chromo-

Human chromosome 8 contains various genes related to s o m e 8 is believed to contain other important gene(s),diseases and disorders of medical importance.1 Its short s u c h as a tumor suppressor gene(s).4-5

arm, 8pl2-p21, contains the following genes: glutathion T h e pUtative tumor suppressor genes on the short armreductase (GSR), which is a causative gene for hemolytic of human chromosome 8 are considered to be relatedanemia due to glutathione reductase deficiency; clusterin t o carcinomas of the bladder, breast, colon, liver, lung,which is involved in atherosclerosis; lutenizing hormone a n d prostate.4'5 These speculations are based on the lossreleasing hormone (LHRH) for hypogonadotropic hypog- of constitutional heterozygosity,5 which has been usedonadism due to LHRH deficiency; and plasminogen ac- a s a genetic marker in this region.6 Detailed deletiontivator tissue type responsible for plasminogen activator mapping allows identification of two distinct subregionsdeficiency.1 This region includes also the gene responsi- w i t n a proximal site located at 8pll.2-p21.3 and a distalble for Werner's syndrome {WRN),2'3 a recessive disor- s j t e a t 8p22-pter, which are commonly deleted.4'5 These

Communicated by Dai Ayusawa findings are consistent with the idea that at least two* To whom correspondence should be addressed. Tel. +81-467- tumor suppressor genes exist on the short arm of the

46-9590, Fax. +81-467-48-6595, E-mail: furuichi@agene.co.jp human chromosome 8.4 Hereditary forms of breast can-t Present address: Winston Thomas, Projenitor, Inc. of the same c e r a r e associated with recently identified Susceptibility

address with Mercator Genetics Inc.; Dennis Drayna, National /r,ns~tt\ s\ <• i r>r>n»n7 • < • iCenter for Human Genome Research, National Institutes of S e n e S (BRCA). One of these genes, BRCA3,' was inferredHealth, Room 2C36, 49 Convent Drive, MSC 4431, Bethesda, from the linkage analysis of the 8pl2-p21 region in inher-MD 20892-4431 ited breast cancer families, and is unlinked to two other

104 Physical Map of Human Chromosome 8pl2-p21 Locus. [Vol. 5,

genes BRCA18 and BRCA2? The BRCA3 gene showsa positive, cumulative, multipoint lod score of 2.51 withtwo markers of the 8pl2-p21 region,7 suggesting that theBRCA3 gene is in the region of 8pl2-p21.

Cohen et al.10 constructed a contig comprised of CEPHyeast artificial chromosomes (YACs) extending the en-tire region of human chromosome 8. In general, however,YACs are often found to contain chimeric DNA sequencesthat make it difficult to construct a complete contig.11

Consequently, a contig generated in vectors other thanYACs is needed to provide additional information on thisregion of chromosome 8 to accurately locate and order theknown genes, as well as to search for new genes. Accord-ingly, the Pl/PAC phage, BAC, or cosmid clones fromchromosome specific libraries were chosen to construct acontig map.

In this study, we constructed a 2.0-Mb high-resolutionphysical map of the human chromosome 8pl2-p21 lo-cus made by overlapping Pl/PAC DNAs that extendfrom the stanniocalcin (STC)12 gene to WRN. We alsosearched for new genes in this region by an exon amplifi-cation method,13'14 and then integrated these new genesand known genes, as well as currently available geneticmarkers, and transcribed the sequences into a physicalmap.

2. Materials and Methods

2.1. Pl/PAC clones and purificationPl/PAC DNA clones were used for the construction of

contigs unless otherwise mentioned. The Pl/PAC librarywas screened using a sequence tag site (STS) marker bya polymerase chain reaction (PCR)-based strategy andpositive clones were isolated by Genome System, Inc.,(St. Louis, MO). The Pl/PAC DNAs were isolated bythe alkaline lysis procedure of Sternberg15 with a slightmodification, and were further purified by equilibriumcentrifugation in CsCl-ethidium bromide gradients forsubsequent analysis.

2.2. Characterization of Pl/PAC and STSsDNA sequences of Pl/PAC inserts were determined by

PCR-based cycle sequencing with T7 and SP6 primersusing a Prism Sequencing Kit (Perkin Elmer). The re-action mixture was run and analyzed by an automatedDNA sequencer (Model ABI 373, Applied Biosystems,Inc.). The STSs at the ends of Pl/PAC were devel-oped from the end fragment sequences determined bythe TAIL-PCR method.16 End-specific STSs were usedto screen the Pl/PAC libraries.

2.3. OligonucleotidesOligonucleotides were purchased from Sawaday Tech-

nology Inc. (Tokyo, Japan). The oligonucleotide primerswere designed, and the optimal annealing temperature

for each set of primers was determined using the OLIGOprimer analysis software program (National Biosciences).Table 1 lists the oligonucleotide primer sequences cor-responding to the STSs of each Pl/PAC end fragmentand the amplicon sizes of the PCR reaction. The chro-mosomal origins of the end sequences and other STSswere assessed by PCR using the genomic DNA from amouse-human somatic hybrid cell line containing humanchromosome 8 described in the following sections.

2.4. Cell linesThe following cells were used in our experiments.

Mouse A9 (neo 8) cell line contains a neo-tagged chromo-some 8 derived from normal human fibroblasts;17 SCVA2cell lines were originally derived from SV40-transformedSCID fibroblasts: SC(+8)-2 is a SCVA2 microcell hybridcontaining a neo-tagged entire human chromosome 8 andSC(R8)-2 is a SCVA2 microcell hybrid containing a neo-tagged short arm of human chromosome 8; B16F10(+8)is a melanoma microcell hybrid containing a neo-taggedentire human chromosome 8. To maintain a human chro-mosome tagged with pSV2neo, 800 /ig/ml of the antibi-otic G418 sulfate (GIBCO, Grand Island, NY) was addedto the growth medium. COS-7 cells were used for exonamplification.

All of these cell lines were maintained in Dulbecco'smodified Eagle's medium supplemented with 100 U/mlpenicillin, 100 /xg/ml streptomycin, 2 mM L-glutamineand 10% heat-inactivated fetal calf serum. All cell lineswere cultured in an incubator with 5% CO2 and 95% air.Cells in the logarithmic phase of growth were used toisolate the genomic DNA.

2.5. DNA labelingThe probes for Southern blot hybridization were made

either by the rando;n hexamer method or by ran-dom primed incorporation of [a-32P]dCTP (3,000 or6,000 Ci/mmol) with a DNA fragment generated byPCR.18 The amplified DNA product was purified by elec-trophoresis in a 3% agarose gel (NuSeive GTG, FMCCorporation) followed by extraction from the gel. Unin-corporated 32P was removed by Sephadex-G25 or -G50spin column chromatography. The specific activity ofprobes ranged from 108 to 109 cpm//Ltg DNA. The probeswere denatured by boiling for 1 min followed by chillingin ice. They were preannealed at 65 °C for 90 min with a5000-fold excess (w/w) of sonicated salmon sperm DNAbefore hybridization.

2.6. Pulse-field gel electrophoresis (PFGE)The Pl/PAC DNA clones were purified by CsCl gradi-

ent centrifugation, and were characterized by restrictiondigestion analysis with Not I, 5a/1, Sma I, Sac II, andBssHIl. The electrophoresis of Pl/PAC DNA digestedwith these restriction endonucleases was performed on

No. 2] K. Ichikawa et al. 105

Table 1. Pl/PAC Clones and Specific Oligonucleotide Primer Sequences clone Name; PI; no mark, PAC;*, BAC;

Clone Mine (GSI No)

Clone Addrcw7641*

PAC-98-10J7340*

PAC-257-1P6720*

PAC-98-16J5979*

PAC-10-6G5980*

PAC-158-23K6734

DMPC-HFFM-1219-G107016*

PAC-135-12K7650

DMPC-HFFH 4973-A6

7557*PAC-203-11M

5453*PAC-22-19J

4976*PAC-231-SB

4974*PAC-49-24D

4478DMPC-HFF#1-816-3E

5476*PAC-946047*

PAC-232-A196044*

PAC-95-026472*

PAC-68-14N6473*

PAC-2S0-10K6788*

PAC-60-8L7108

DMPCHFFH -1216-H87723*

PAC-32I-16I8333*

PAC-122-N11

8336*PAC-188-G2

7831*PAC-59-I5K

7832*PAC-157-13G

7345DMPC-HHFWl-0960-Dl

6758"PAC-305-19I

6461DMFC-HFFM -0636-Dl 2

6207*PAC-S4-9E

5331*PAC-37-23K

4895*PAC-304-8G

4386DMPC-HFTW1-434-3F

4387DMPC-HFFI1-245-5H

2588DMPOHFFtl -1006-SD

2589DMPC-HFF#l-502-9B

2587DMPC-HFFH-743-7C

4497DMPC-HFFH-475-9D

3349DMPC-HFFM -909-12B

3350DMPC-HFF#1-1391-4B

4370DMPC-HFFM-1243-A7

4369DMPC-HFFM-I19-E7

4117DMPC-HFF41-S04-9B

insert(kbp)NJ).

NJ>.

NJ).

100

95

NJ).

NJ).

NJ>.

NJ).

134

82

165

65

157

110

130

90

140

NJ).

76

NJ).

NJ).

143

NJ).

95

82

110

73

140

140

97

80

90

85

80

65

82

90

90

65

75

82

clone endT7/SP6

T7SP6T7

SP6T7

SP6T7

SP6T7

SP6T7SP6T7

SP6T?

SP6

17SP6T7

SP*T7

SP6T7

SP6n

SP6T7

SP6T7

SP6T7

SP6T7

SP6T7

SP6T7

SP6T7

SP6T7

SP6T7

SP6

T7SP6T7speT7

SP6T7

SP6T7

SP6T7

SP6T7

SP6T7

SP6T7

SP6T7SP6T7

SPti17SP6T7SP6•n

SP6Tl

SP6T7

SP6T7

SP6T7

SP6T7

SP6T7

SP6

forward primer : 5' to 3'

CAC AGG TAA AAT TTT CCT TTC CCCA TCA TAG AAT ACT ACA AAC

AGA TGC AGA AAG ACC CTT TGG C

CTA ACT CTG AAT TTA CAT GCTAAG GGA GCA GGT ATG AGT GAC

CCA TAG TGC TAG CAC ATG ACT

ATG AGA GAG AAT CCT AGT TCTGCA ACC TAG ATC CCT TGC ATGCGG ACT ACA GGC AAG CAC CCAC CAT CTC CAT TCA CCG CC

GCA AGA AAT TCT AAC AAA GCCCTG GAT GGC CTG ATT CAT GAT G

AAA CTC ACA TCC ACC TCT TACCTA CTA ATG ATC AAA AAG CCC

GCC AAC TCC ATC TGC ACA GAG

ACA CCT GGC CCC ACA GAA AGCTC TCT TCA TCT CCC AGA GA

CTG GTT TAT AAG GGA AAA CAT

GAC CCA CCG GAG GTT AGG TAAGG CCC CAC TTC TAA CGT TG

GCT CAA GCG ATC CTC CCA CTTCT GGA GTT AGA GAA TTC CAA

TCC TAG TGT TTT CTT TGT GGGCTC AAA CTC CAG ACC TCC GGTAGG TGA TCC ACC CAC CTC GT

GTT CTA ACC CAG GGC TCC TGGGC CAA TGT GGC GTA ACC CTG

CCC AAA GTG CTG GGA TTA TAG G

CCT CAA ACC TAG TTA CAA CAT TCGAA TGT CTG ACT CTT TTG CTA GCTA CGA TGA GCT AAA GGA GC

CAT GAG AAT CAC AGC AGA AGTTC AAC ATG TCT TTT GGA GGG

CCA ACA TCA TCT AAT TCA TCT C

CAA AGG CCA AGT TTA AAT GGC

GGA AGG AGA CAC CTC CTC TCTCCC AGT AAA GAA GAC CCA GGT

repetitive

CCA CAT TAC TGA TTG CTG AGA GCAGC TGG GAT CTC ACT AAT TGCCT CTT GGG CTC AAG CGA TCCTC CCC ACC CTT AGT CCA TCGGT AGA ATC CAT ATC ACA ATC CCCC TTC CAA GTT TAA TCC TGGTC TAG AAA TTG GGT CCT AGTAA GAC AGA ACT CAT GCC TCTGA AAC ATC CTT CAT ATG CCAAC AAA CAC TGA GCA CCT GCTAG GAA AGA TTT GTA AGG AGAGT GAA CAG GCA ACC TAC AGATG AAA GAA TGA ATG AGA GGGGAA CTT ATT TAT GGA GTT AGCTT CTG ACC TTT AAG CTA ATT TGCTA CAC CTT GTT CTT TTC ACTGC AAC CTT GAA CTC CCA AGAGT ATC CCA GCT ACT CAG GAG

CTA GAG TGC CCT TTC ATC TGGATC TGC AAG TCA CAT AAC ATT TCCAAC ACT ACT ACT CCT ATT CTGGCC CAG AAG TCA GAG GTT GCA

reverse primer : 5' to 3'

GCA AGT AGC AAT TTG TTA GCGTG AAT CTG TCT GGT CCT GG

TTC CAG TGC TAT GTT GAA AAG C

TCT CCA TCT CCT GAC CTT GTGCCA GCA TAT CAC ACG AGC TG

CTG TCC CAA AGC ACT CCC AC

CTA ACC AGT CCA CTC TGA ACCCAT CTG TAT TTA CAG CCA CTCCTG CCA GCT TAA ATA ATC TCCGTA CTA AGG CGC GTG ACT TTT G

CTC CCT GAT GAG CTA AGT AGGCTT TCA TCC TAG ATC TGC GC

CTA GGC ATT GTC CTA AGC ACAGG AGT GTA TGT GGA TTC CAG

ATG CAG CAT ACC TGA AGT GCC

GTG CTG AAG GAA AGA CTA TGGCCT GAT CTG CTT GAA CTC G

GAT AAG ACT GGA GAT TGG ATT

TGA CTT CTA CAG TAA TGA TTG CGAC TTT CTC TTT CCA ATA CCC

GGA GGC CAA GGT GGG TGG AGAAA TAC GTT CTT TTG CCT AAA

AAA TAT CAA GAC AGA ATC AAG GGTG AGC CAT GAT CAC GCC ATTTGC ACA AAT TAT TGT GGA CTT G

GTT CTT GGA TGC CAC GTG ACCTA GAG TGC AAT GGC TCA GTC

GAG GCA GAG GTA AAA AGC GCA TGG

GCA GCA AGT TCT GTG TAC CAA GCCTG GGA CTA CCT AAG CCA ACCGTA ATG CAA GAA CAT GTG CTG TG

CAC ATT CTC TTT TGA ACT TTGTTG GGG AAA GTT TAG TAA AGG

TTT CCA AGG TCA CAC ATA AGA G

CCA AAC ACT GGA GTC AGA GAC

CAG TAG AAA GTG ATT TGT GGGGGT TCA TTC AGG ATC CAC TGC

repetitive

TGA GTT GAA TAA CTT GCC CTG GTAG CAT ATT GCT GAA AGT GGAGC CAA GAT GGT GCC ATT GCGCC TCA GCC TCC TAA AGT GCACA GCC TCA GTT ATT ATC TGGGGA TGT GCC AAT TCA TTA TCTCA CCA GAA GAG ACC CAA TGTTG AGT ATG CTA AAA GCC AGACC TGT GAA TGA GAA TAC AGGGA GTT CCA TTT CCA AAT GGCAA TGA AAG TTG AAT AGA CGAGC AAT TTT TCA TGT GTC TGCTC AAA ACT AAA CAT GCT CCCAAT TAC TGT GAG ATG TGA TCCCT GCA CTA CTG GAA ATA TTTTA ATG AGC TAT TCT TCT GGGAG ACC AGC AGT TCG AGA CCATC TCA GCT CAC TGC TAC CTC

TGC TCT TAC CAT TAG GAA ACCGCT GTC TCT GGG TAG GCA TAA GAGT GAG CCG AGA TTG TGC CA

Opt.TaCO

5052

48

48

55

5248

48

48

55

55

555552

5352

55

504865

45

52

55

55

50

53

55

4456545556565656565649524050434648

525348

size

<bp)106200

166

160

119

150

156

135165100

110107

150

175

163

138150

122

147

174

160

83

9917119]

170160

94

165137130

110

146

113

162

130

176

244

52151

12285721549690n9837

75200119144

144

Mapped Genes[STS, D8S#]

STC

STC

FAK2

FAK2,EHH31]EB11311EU.ApoJ

WS-3

TUBBP1liossiTUBBPI, RBP-MS{1055}RBP-MS

RBP-MS

RBP-MS13391RBP-MS13391RBP-MS13391RBP-MSGTF2E2GTF2E2, GSR{540}GSR{540}Rtp-8

top*

Rep-8, PPP2CB

106 Physical Map of Human Chromosome 8pl2-p21 Locus.

Table 1. Continued.

[Vol. 5,

4624*PAC-23-23N

4625*PAC-31-15C

9965*PAC-243-21O

10468DMPC-HFFM -1239-F3

14391**BAC255-7F

12406DMPC-HFFfl-W0-10A

11865DMPC-HFFH -1256-1C

12339*PAC-261-4B

11187DMPC-HFPH 4162-D8

11188DMPC-HFFM-0745-E3

11402DMPC-HFFfl -O596-B7

11867DMPCHFFfl-876-9B

12494DMPC-HFFM-115-10G

4030DMPC-HFFfl-172-JD

7556*PAC-106-21O

8015*PAC-25-10D

8482*PAC-289-F16

8805DMPC-HFFW1 -1195-4E

97

125

100

95

130

95

50

100

80

75

97

70

NJ).

50

82

ND.

NJ>.

ND.

T7SP6T7

SWT7

SP6T7

SP6T7SP«T7

SF6T7

SP6T7

SF617

SPtiT7

SP6T7

SP6T7

SP6T7

SP6

T7SP6T7

SP6T7

SP6T7

SP6T7SP6

ACA GTA GGG TCT TCA GAA GGrepetitive

TTA AAG TTA TCA CAG GGA GAT

GAT AAC TGT GCA TGC CTG AGG ACrepetetive

CAA TTG AGA GTA CAA AGT CTC Crepetitive

CAC ACA ACA GAG CCG ATC ATA G

GTC AGG AGT TCG AGA CCA GCGAT AGC AAC TGG GAG CAG CTC

CTA GAC CAT CAT TCT GGA CTGCTG GGC AAC AAG AGC GAA ACGAA CCT CTG ACT ACA AAT GTGCTT AAA GCA CTG GGA TTA CAGCTT GCT GGT ACA ACG ATG CC

CTG TGG TTT CTG AAC TCT CTA G

CCT AGG AAT CTA AAG AAT CAGGCT AAT ACG ACT CAC TAT AGG GAG AGG ATC

repetitiveGCT GTG CAG AAG CTT TTT AGCGTG ATG CCT GTT CCC TAT TG

repetitiveCGC CAG GAA TGC AAA TAG GGCCC TCA TGG CTA ATG TTG TTGCAT TAG GTT AAC TGA AGT TAA GGT GGAT AAG TAT CTA GAA CTT CCC C

CCC ACT GCT AGG TAT ATA AC

CAG AGC AGC TTT TAG TCA TGrepetitive

GCA GGG TGA GCA ACA AAG

GAG GTT GCA GTG AGC CAA GAT Crepetitive

GTT TGA CAC CAG TTT GGC AACrepetitive

GTG CTG CTG AAG CGA GCA CTA C

GAG TGC AGT GGT GTG ATC TCGCA TGA TCA CAT CCC ACT GAA G

CAC ATT GTA TCG GAG AAT GGGGCG TGC ATG TGT CTT TGT AACGCT TTA GTA CCG TGT TTC TGGGA ACT ACA CAG ATC AAC AAGCAA AAG GCA AAG ACT TAC CAGCTC ATT AAA GAT GGT GGG ATG

GAC ATC TTT GGA CTT TTA GGG C

CTA CTC GAG AGG CTG ATA CCT CCAA TGT TAG CAA AAA GGA GCC C

repetitiveACA TCA GAA AAC CTC TTC TGGGCA AAC AGA GAA CCA ATG AAA

repetitiveGGC AGG CTC TTA GGA GAA GCGTC AAC GAA GAG CTC GTA CCCTG CTG TTA ATC TGT TAA TCT GGTA CAT TAT TCA CAA GGT GCC

GTC TAT GAA CAC TTA TGT TGC

46

45

55

55524848

45

70

62

185230216209

266

2732 »

2272631772111S5228

266

246244

3112

»750980

142

PPP2CB12144}ppncB12144}12144, 2138]

{2144,2138,2156, 21681

12174,2150]

WRW

WRN[21(21WRN[21621

1283]

{2831

N.D.; not determined

standard horizontal agarose gels (0.8-1.0%) containing1 x TBE buffer (89 mM Tris, 89 mM boric acid, 0.2 mMEDTA, pH 8.0). Contour-clamped homologous elec-tric field (CHEF) gel electrophoresis was performed tocharacterize the Pl/PAC DNAs under the conventionalmethod using a CHEF DRII system (Bio-Rad, Rich-mond, CA). All gels were made up in a 0.5 x TBE elec-trophoresis buffer with 1% agarose. Gels were run us-ing parameters obtained from the Bio-Rad CHEF DRIICHEFMAPPER program to separate fragments smallerthan 1.2 Mb. The electrophoresis was performed at a cir-culating temperature of 14 °C using the Bio-Rad CHEFIIsystem. The Pl/PAC DNA fragments were transferredto a nylon membrane by capillary blotting. Hybridiza-tion of membranes was performed using as a probe eitherDNA 32P-labeled PCR products obtained by the randompriming method or 5'-end 32P-labeled oligonucleotides asdescribed by Feinberg and Vogelstein.19

2.7. andPreparation of high-molecular-weight DNASouthern blot analysis

High molecular weight DNA was isolated from the hu-man placenta and human- mouse hybrid cell lines by sar-cosyl disruption of the cell membranes followed by threephenol and two phenol/chloroform extractions. PurifiedDNA was dialyzed against four changes of 10 mM Tris(pH 8.0) and 1 mM EDTA. Southern blot hybridizationwas carried out by standard procedures as described bySambrook et al.20 Ten micrograms of genomic DNA or

1 /x of Pl/PAC DNA were digested overnight or for 2 hat 37 °, respectively, with a five fold excess of restrictionendonuclease. The cleaved DNAs were then separated on0.7% agarose gels in 1 x TBE buffer. After visualizationon an ultraviolet transilluminator, the gels were dena-tured and neutralized and then were transferred to nylonmembranes by capillary blotting. The membranes werehybridized with a radiolabeled DNA fragment as a probein a solution of 50% formamide, 10 x Denhardt's solution,5 x SSPE (20 x SSPE is 3 M NaCl, 0.2 M NaH2PO4,and 0.02 M EDTA adjusted to pH 7.4), 1% SDS, and0.6 mg/ml sonicated salmon sperm DNA. The filter waswashed at high stringency (0.2 x SSC, 0.5% SDS, 65 °C),and was exposed to X-ray film with intensifying screensat -70 °C.

2.8. Exon amplificationExon sequences were amplified by the exon amplifica-

tion method13-14 from the Pl/PAC DNAs that comprisedthe contig. Individual Pl/PAC DNAs were digested tocompletion with a combination of 5am HI and Bgl II, andthe resultant fragments were cloned into pSPL3 (GibcoBRL, Gaithersburg, MD). The DNAs from the transfor-mants were purified using Qiagen columns (Qiagen, Ger-many) and were used to transfect COS-7 cells by elec-troporation. The cells were grown for 48 h and the totalcytoplasmic RNA was extracted, which was then con-verted into cDNA with reverse transcriptase. The frag-ments containing the exons were amplified by PCR with

No. 2] K. Ichikawa et al. 107

specific primer pair sets, Next, they were subcloned intothe pGEM-T vector and their DNA sequences were de-termined.

2.9. Somatic cell hybrid mappingThe genomic DNAs from SC(+8)-2 and SC(R8)-2 cells

were used for the preparation of template genomic DNAand cDNA for the PCR analysis. PCR was performedin a 10 (A mixture containing 1.5 mM MgCl2, 20 mMdNTP, lx of Perkin-Elmer Cetus buffer, 0.3 mM of eachprimer, 0.25 units of Taq polymerase, and 50-ng of ge-nomic DNA from mouse-human somatic hybrid cells orcDNA as prepared below. The PCR conditions were asfollows: initial denaturation at 94 °C for 5 min; 35 cyclesof denaturation at 94 °C for 30 s, annealing at 55 °Cfor 30 s, elongation at 72 °C for 1 min; and a final elon-gation at 72 °C for 5 min. Human and mouse genomicDNAs were included as controls. The amplified prod-ucts were visualized on 1-3% agarose gels. One micro-gram of mRNA was used to prepare cDNA. Reverse tran-scription was made by Superscript™ RT II (BRL) witholigo d(T)12~18 and random hexamers as primers. Afterthe reaction was completed, the mixture was diluted 500times in distilled water. All the samples were run withoutreverse transcriptase as a control to check for contamina-tion with genomic DNA. None of these control samplesyielded products resulting from genome DNA contam-ination. Electrophoresis was performed in 3% agarosegels and the DNA was visualized by ethidium bromide-staining.

2.10. Database search of DNA sequenceSequences obtained from analyses of exons were

searched for homology or identity with any known se-quences listed in the public databases such as FASTAand BLASTN from the GCG database search programpackage.21

3. Results and Discussion

3.1. The Pl/PAC contig map of human chromosome8pl2-p21

The Pl/PAC libraries were first screened with primersto obtain PI DNA clones that contain the D8S339(=WT251) marker. The marker D8S339 was previouslyshown to be very near the WRN locus.22 This initialstrategy identified three PI clones: Pl-#2587, -#2588,and -#2589. The same Pl/PAC libraries were alsoscreened with D8S540 (=GSR2), which is also a markervery close to the WRN locus.23 Two PI clones, Pl-#3349and -#3350, containing D8S540 were isolated by this ef-fort. Similar screenings permitted the isolation of Piscontaining the other STS markers, including D8S131,D8S1055, GSR1, D8S2144, D8S2138, D8S2156, D8S2168,

D8S2174, D8S2150, D8S2162, and D8S283. The termi-nal sequences of all the Pl/PAC DNAs were analyzed bydirect sequencing. These Pl/PAC DNAs were confirmedto be on chromosome 8 using somatic cell hybrid map-ping, and were used to construct a contig. These effortsresulted in construction of a contig (WS contig) consist-ing of more than 14 Pl/PAC DNAs which represent aphysical map of WS locus (Fig. 1).

Similarly, another two contigs were made that were onthe telomeric side of the above contig: a contig containingthe STC gene (STC contig) and a contig containing theD8S131 marker (D8S131 contig). Gaps exist betweencontigs STC, D8S131 and WRN. We could not completethe contig by covering these gaps because appropriatePl/PAC DNAs could not be isolated from the Pl/PAClibraries.

We used two methods to determine the overlap amongPl/PAC clones. First, PCR was used to find overlap-ping Pl/PAC clones and to determine the overlap ofthe clones. We examined the terminal end sequences ofPl/PAC clones by the TAIL-PCR method13 to identifytheir overlap with each other (Fig. 1 and Table 1). Theterminal sequence of each Pl/PAC clone provided an STSwhich permitted us to search for new clones adjacent tothe terminal sequence. Repeating these procedures al-lowed us to group the Pl/PAC clones and to extend thecontig. Second, we compared the profiles of restrictionenzyme digestion of overlapping Pl/PAC clones to assessthe degree of their overlap: each Pl/PAC clone was di-gested with Not I, SssHII and Kpn I, and the restrictionfragment patterns were compared by PFGE (Fig. 2). Theoverlapping clones typically shared three or more DNAfragments of identical size. In addition, we determinedthe relative distances between the Pl/PAC clones andtheir order by fluorescence in situ hybridization (FISH)as reported previously by us (Fig. I).24

All of the STS markers listed in Table 1 and thePCR conditions are available by e-mail from fsug-awar@agene.co.jp. Most of the STSs were obtained bysequencing the termini of Pl/PAC clones. To use STSsto screen the Pl/PAC libraries or to map the STS, thePCR conditions were optimized to amplify specificallythe sequences from human genomic DNA.

3.2. Mapping of newly developed microsatellite markersThe polymorphic CA-repeat markers are important

milestones in the map to search for new genes of medicalimportance. To make the map more versatile, new CA-repeat markers were developed from the Pl/PAC clonesconstituting the contig (Table 2). Identification of suchmarkers was carried out by the method of Hudson25 andtheir genotypes were assessed according to Weber andMay.6 A total of 12 new markers were obtained from 22Pl/PAC DNAs that extend through the 1.0-Mb WRNregion, yielding an average of one marker per 100 kb.

o 00

m k

fi-m

t

Teh

.15

0,

AG

25

2/2

53

AG

25

2/2

53

AG

25O

/251

D8S

131]

• A

G36

8/36

9 1

AG

595/

596

AO

854/

855

AO

864/

865

AG

59

1/5

92

I A

G59

3/59

4 |A

G85

6/85

7^A

G85

0/85

AG

862/

863

ITP

AC

#597

9 (1

00kb

)

PA

C#4

974

(165

kb)

#5

45

3(1

34

kb

) 1

#765

0*

PA

C#

83

34

,J5

*

GA

P

PA

C#5

476

(157

kb)

g •}-«

PA

C#6

047/

6374

(11

0kb)

PA

C#7

557*

o- i-

PA

C#8

153-

56«

'

PA

C#

60

44

(13

0k

b)t

>P

AC

#647

2 (9

0kb)

PA

C#

64

73

(140

kb)

PA

C#6

2O8

( 80k

b)

PA

C#6

207

( 140

kb)

#646

1*

"

PA

C#

67

57

1

#710

8*

PA

C#7

O17

*'

PA

C#

67

58

«

#734

5*—

oP

AC

#783

2*(#

7017

)

B

en o' I B Q cr o B o a 0

0 o n C

AG

366/

367

AG

254/

255

AG

AG

589/

59(J

O

«S

20

55

AG

64/6

5,1

\ (M

S8

-13

4)!

A

G60

/6<

j

AG

62.

DSS

3J9

168/

69

,AG

23S

/236

AG

19

2/1

91

„„

„,

D8S

2150

U8S

2174

; D

8S21

62D

8S28

3C

ent.

I #25

87 (

65kb

)#3

349

(90k

b)!

1

#2

58

9(8

0k

b)'

#2

58

8(8

5k

b);

#438

7 (9

0kb)

I #

43

86

(80

kb

)

PA

C#4

895

(97k

b)

PA

C#5

331

(140

kb)

#335

0 (9

0kb)

T

#411

7 (8

2kb;

• #4

388/

4496

(55

kb)

#4

49

7/4

38

9 (8

2kb)

^>

«

o #4

030

(50k

b)

[Rj

>

f P

AC

#755

6

- PA

C#6

208

(80k

b)

PAC

#620

7 (1

40kb

)

—

PI

and

PAC

clo

nes

I O

T

7-en

d /

• S

Pt-

end]

* lu

iow

n ST

S «

size

-unk

now

i

O n

ew S

TS

R r

epet

itiv

e

No. 2] K. Ichikawa et al. 109

100kb— 1

TVBBP1

N

S1 1 1

I i 1 H

f rft,

NB

Nil",

1 II 1 1

1

RBP-MS

"A A 4"l ^ K B N

N SII mi

S # 2 5 8 8NI I I II 1*1187 I n

NI I I #4386

SI J M B I U

22kb

C^87

589

A

T

57kb

S1 1 1

s1

18kb

K 1

#4388

GTF2E2

22kb,18kb

40kb

L N # 4 w

B ^ B K N^

V ,., f

GSR

fs

K

, 32kb

S

N1 i i

S_J #3350

1

Rep-8 PPP2CB

t-f rim—<

9kb

K K K J K B ^

s

NI i | #4170

B l f l#4624

SJ#4117

10kb

Figure 2. High-density physical and transcription maps near the D8S339 and D8S540 STS markers. Detailed profiles of restrictionenzyme sites of overlapping Pl/PAC clones from TUBBP1 to PPP2CB are shown. The orientation of genes was determined bySouthern blot analysis using as probes the PCR products from the 5' and 3' ends of the genes. Human genomic inserts were clonedbetween Not I and Sal I sites of Pl/PAC vector. Each Pl/PAC clone was digested with Not I (N), BssHII (B), Kpn I (K), Apa I(A) and 5a/ I (S) and the restriction fragment patterns were analyzed by PFGE.

These markers were used in the linkage analysis of theJapanese population to narrow the candidate region ofthe WRN locus. The details of these CA-repeat markersand the genotypic data of Werner's syndrome patientsare described by Matsumoto et al.26

3.3. Construction of transcription map by exonamplification

From the physical map obtained, the exact locationsof known genes on the 8pl2-p21 region were determined.Multiple sets of primers were designed from the se-quences of exons obtained by exon amplification fromthese Pl/PAC clones, as well as on the known markersequences. The nucleotide sequences of these exons weredetermined, and they were classified into known andnovel exons by searching GenBank/EMBL Data Bases.We also confirmed by PCR using as a template genomicDNA or cDNA from mouse somatic hybrid cells contain-ing human chromosome 8 that these exons were, in fact,located on chromosome 8.

Table 3 lists the genes relevant to the exons obtainedfrom Pl/PAC DNAs by an exon amplification methodin this study. From these results, the exact locations onthe contigs of the genes relevant to the exons were de-termined. Human STC protein is a glycoprotein homol-ogous to the fish STC protein that may be an importantregulator of calcium uptake from the aquatic environ-ment. Two exons of human STC gene were obtainedfrom PAC-#5980, which is at about 487 kb of the telom-ere of the D8S131 marker.12 One exon of the focal adhe-sion kinase gene (FAK)27 was obtained from PAC-#4974and PAC-#4976. Eleven exons of the epoxide hydro-lase gene (EH)28 were obtained from PAC-#4974 andPAC-#5476. Two exons of apolipoprotein J (ApoJ)29

were obtained from PAC-#5476. These three genes arenear to each other and are within 200 kb of the D8S131marker. We obtained a total of nine exons (AG1-AG5)whose sequences corresponded to those of the expressedsequence tag (EST) of the public databases. The location

Figure 1. A 2.0-Mb Pl/PAC physical and transcription maps of the chromosome 8pl2-p21 region from STC to WRN. ThePl/PAC clones assembled in a contig. Pl/PAC DNAs are drawn to scale, and positions of markers are shown with the approximatesizes and physical distances. Open circles indicate the confirmed markers in Pl/PAC clones. The rough order of Pl/PAC cloneswas confirmed by dual-color FISH experiments.24 The contigs were divided into three parts, the STC, D8S131 and WRN contigs,from telomere to centromere. In the shaded boxes at the top, the names of the known genes and their approximate span areas areindicated with their transcriptional direction shown by 5' and 3'. The top line in the figure is a scale graduated in 100 kb. Thebold line indicates the human genomic DNA. The numbers between the scale line and the bold line, such as D8S1055 and D8S339,indicate known STS markers with their approximate positions. The short solid lines are the position and approximate relativesize of Pl/PAC DNAs. Under the human genomic DNA (bold line), the numbers with # indicate the serial numbers for Pl/PACDNAs. The numbers in parentheses are the approximate sizes of Pl/PAC DNAs determined by PFGE. The open squares andopen circles at the ends of each Pl/PAC clone show the SP6 and T7 promoter site, respectively, which determine the orientation.The orientation of genes was determined by Southern blot analysis using the PCR products from 5' and 3' proximal ends of genesas probes.

Physical Map of Human Chromosome 8pl2-p21 Locus. [Vol. 5,

Table 2. Distribution of the New Polymorphic Markers in the WRN Region

(CA)n Markers in WS region

Marker

6207-4

438«-l4386-2

438«-34386-5 m4497-1

4369-1 i i m4624-2 m = 4369-14030-2

7556-1 = D8S283

Published MarkersD8S1055

D8S339

D8S540

GSR1D8S283

- - m m -- -

-m lite

-

H ---

mM m

--

- - Mi M m -

= GSR2

= 7556-1

Table 3. The list of exons obtained by amplification from the P l / P A C DNAs of the contigs

Pl/PACPAC-#59S0PAC -#4974/4976PAC-#4974/5476PAC-#5476PAC-#5476/6044PAC-#6044PAC-#6<)44PAC-#6463/6207PAC-#6463/6207PAC-#6207PAC-#4895PAC-#6207P1-#4497/4386/2588/2587PI .#4497/3349/3350PI-#3350PI-#4370PI-#4625

No. of exon2-1

11-)-)1i

31

1189i

31

Genehuman STCFAKEHApoJCyioiysis I/SP-40/TRPM-2

human AG1human AG2human AG3human AG4human AG5TL'BBPlWS-3RBP-MSGTF2E2GSRRep-8PPP2CB

References and databaseRef. 12Ref. 27Ref 28: L05779Ref. 29: JO3536M25915/X14723/M64722F13626N'40395T71480A78953/T86448/TS6895L44514H78562Ref. 32Ref. 30Ref. 31Ref. 33Ref. 34Ref. 35Ref. 36

of ESTs are: F13626 in PAC-#6044; N40395 was in PAC-#6044; T71480, T78953, T86448, T86895 and L44514were in PAC-#6463/PAC-#6207; and H78562 was inPAC-#6207. Of these, two exons from PAC-#6044(AG2) were found to belong to one gene, because RT-PCR by primers set up on these exons produced twokinds of DNA fragments containing both of these exons.This gene is reported in the database as a hypotheticalprotein in the 5' region of RC114 (an early meiotic recom-bination gene of yeast, database; Z47071). PAC-#6207contained an exon of the WS-3 gene,30 which was clonedand characterized by us. The WS-3 protein has an R-G-D(Arg-Gly-Asp) motif in the N-terminal region that seems

to confer adhesive properties to macromolecular proteinslike fibronectin. Eight exons of a large gene encodingan RNA-binding protein (RBP-MS)31 were found in Pl-#4386, -#4387, -#2588, -#2589, -#2587 and -#4497covering about 200 kb. An exon with a partial sequenceof the human beta-tubulin pseudogene (TUBBP1)32 wasfound in PAC-#4895 just telomeric to the RBP-MS gene.Nine exons of the GTF2E233 gene were found in Pl-#4497, -#3349 and -#3350, and two exons of GSR34

were found in Pl-#3350. Three exons of a new gene,Rep-835 were found in Pl-#4370 and one exon of pro-tein phosphatase 2A (3 subunit (PPP2CB)36 was foundin Pl-#4624 and Pl-#4625.

No. 2] K. Ichikawa et al. I l l

We also assessed the efficiency of exon amplificationin this study. The open reading frame of GSR cDNAhas 1,440 bp and the total length of exons amplified was448 bp, indicating that the efficiency was 31.1%. Simi-larly, 76.6% efficiency was obtained for EH cDNA: a to-tal of 1,276-bp of exons was amplified from a 1,665-bpfull-length cDNA. These results indicate that the exonamplification method used in this experiment providesa powerful method to obtain new genes present in thisPl/PAC contig.

Thus, our present physical map consisting of contigsof Pl/PAC DNAs offers an effective means to discovernew genes and to understand the genomic organizationof genes in this region.

3.4- Discussion and conclusionWe constructed a 2.0-Mb high-resolution physical map

of 8pl2-p21 extending from the STC gene to WRN. Thisphysical map is mostly consistent with the high-densitygenetic maps,2'37"39 a radiation hybrid map,40 FISHmaps,21 polymorphic STSs41'42 and EST43 content maps,which have so far been partially characterized. This con-struction may help to analyze various interesting genesincluding tumor suppressor genes, that were reported tobe in this region.

Recently, an integrated map composed of YACs includ-ing the chromosome 8pl2-p21 region was constructed byImbert et al.44 According to their map, the GTF2E2genewas assigned to a location quite different from that as-signed by our contig map. This discrepancy in the as-signment of the GTF2E2 location may be due to thechimerism and instability of the YAC clones used byImbert et al. We confirmed that the GTF2E2 gene islocated in PI clone #3349 together with the proximalmarker D8S540.23'43 Our experiment confirmed the sta-bility of Pl/PAC clones and their usefulness to constructa contig covering a relatively large genomic area.

The physical map of this study clarified the orderof the STS markers: telomere-D8S131-D8S1055-D8S339-D8S540-D8S2144-D8S2138-D8S2156-D8S2168-D8S2174-D8S2150-D8S2162-D8S283-D8S87-ANKl-centromere;and those of both new and known genes: telomere- [STC]-[FAK\-[EH\-[ApoJ\-[WS-3]-[TUBBPl]-[RBP-MS]-[GT-F2E2]-[GSR]-[Rep-8]-[PPP2CB]-[WRN]-centiomeie(Figs. 1 and 2, and Table 1).

These results may facilitate the following genetic anal-yses. First, newly developed polymorphic markers, to-gether with known ones, are expected to contribute to thefurther narrowing of the tumor suppressor gene(s) can-didate region. Second, the Pl /PAC DNAs forming thecontig map make it possible to obtain exons and to iso-late new cDNAs. Indeed, based on this strategy, we havecloned the RBP-MS, Rep-8, and WS-3 genes. Third, thedistribution of the unique sites of restriction enzymes,including BssHII, Sac II, Not I and 5a/ I, suggests the

presence of multiple CpG islands,45 which are often asso-ciated with transcribed regions of vertebrate genomes.46

Evidence suggests that CpG islands may participate inepigenetic mechanisms regulating gene expression.47'48

Therefore, this information may also help us to locatenew transcription units in this region.

Acknowledgments: This work was supported byThe Organization for Drug ADR Relief, R k D Promo-tion and Product Review of the Japanese Government.

References

1. Spurr, N. K., Blanton, S., Bookstein, R. et al. 1995, Re-port and abstracts of the second international workshopon human chromosome 8 mapping 1994, Cytogenet. CellGenet. 68, 148-164.

2. Goto, M., Rubenstein, M., Weber, J., Woods, K., andDrayna, D. 1992, Genetic linkage of Werner's syndrometo five markers on chromosome 8, Nature, 355, 735-738.

3. Yu, C.-E., Oshima, J., Fu, Y.-H. et al. 1996, Positionalcloning of the Werner's syndrome gene, Science, 272,258-262.

4. Fujiwara, Y., Emi, M., Ohata, H. et al. 1993, Evidencefor the presence of two tumor suppressor genes on chro-mosome 8p for colorectal carcinoma, Cancer Res., 53,1172-1174.

5. Trapman J., Sleddens, H. F., van der Weiden, M. M. et al.1994, Loss of heterozygosity of chromosome 8 microsatel-lite loci implicates a candidate tumor suppressor genebetween the loci D8S87 and D8S133 in human prostatecancer, Cancer Res., 54, 6061-6064.

6. Weber, J. L. and May, P. E. 1989, Abundant class ofhuman DNA polymorphisms which can be typed usingthe polymerase chain reaction, Am. J. Hum. Genet., 44,388-396.

7. Karangueven, F., Essioux, L., Dib, A. et al. 1995, Loss ofheterozygosity and linkage analysis in breast carcinoma:Indication for a putative third susceptibility gene on theshort arm of chromosome 8, Oncogene, 10, 1023-1026.

8. Miki, Y., Swensen, J., Shattuck-Eidens, D. et al. 1994, Astrong candidate for the breast and ovarian cancer sus-ceptibility gene BRCA1, Science, 266, 66-71.

9. Wooster, R., Neuhausen, S. L., Mangion, J. et al.1994, Localization of a breast cancer susceptibility gene,BRCA2, to chromosome 13ql2-13, Science, 265, 2088-2090.

10. Cohen, D., Chumakov, I., and Weissenbach, J. 1993, Afirst-generation physical map of the human genome, Na-ture, 366, 698-701

11. Couch, F. J., Castilla?, L. H., Xu, J. et al. 1995, A YAC-,P1-, and cosmid-based physical map of the BRCA1 regionon chromosome 17q21, Genomics, 25, 264-273.

12. Chang, C.-M., Jeffrey, K. J., Tokutake, Y. et al. 1998,Human stanniocalcin (STC): genomic structure, chromo-somal localization, and the presence of CAG trinucleotiderepeats. Genomics (in press).

13. Buckler, A. J., Chang, D. D., Graw, S. L., Brook, J. D.,Haber, D. A., Sharp, P. A., and Housman, D. E. 1991,Exon amplification: A strategy to isolate mammaliangenes based on RNA splicing, Proc. Natl. Acad. Sci. USA,

112 Physical Map of Human Chromosome 8pl2-p21 Locus. [Vol. 5,

88, 4005-4009.14. Church, D. M., Stotler, C. J., Rutter, J. L.,

Murrell, J. R., Trofatter, J. A., and Buckler, A. L. 1994,Isolation of genes from complex sources of mammaliangenomic DNA using exon amplification, Nature Genet.6, 98-105.

15. Sternberg, N. 1990, Bacteriophage PI cloning system forthe isolation, amplification, and recovery of DNA frag-ments as large as 100 kilobase pairs, Proc. Natl. Acad.Sci. USA., 87, 103-107.

16. Liu, Y-G. and Whittier, R. F. 1995, Thermal AsymmetricInterlaced PCR: Automatable amplification and sequenc-ing of insert end fragments from PI and YAC clones forchromosome walking, Genomics, 25, 674-681.

17. Kurimasa, A., Nagata, Y., Shimizu, M., Emi, M.,Nakamura, Y., and Oshimura, M. 1994, A human genethat restores the DNA-repair defect in SCID mice is lo-cated on 8pll.l-qll. l , Hum. Genet., 93, 21-26.

18. Schowalter, D. B. and Sommer, S. S. 1989, The gener-ation of radiolabeled DNA and RNA probes with poly-merase chain reaction, Anal. Biochem. 177, 90-94.

19. Feinberg, A. P. and Vogelstein, B. A. 1983, A techniquefor radiolabeling DNA restriction endonuclease fragmentsto high specific activity, Anal. Biochem., 132, 6-13.

20. Sambrook, J., Frietsch, E. F., and Maniatis, T. 1989,Molecular cloning: A Laboratory Manual. 2nd Edition,Cold Spring Harbor Laboratory: New York, Vol. 1,2,3.

21. Altschul, S. F., Gish, W., Webb, M., Myers, W., andLipman, D. J. 1990, Basic local alignment search tool, J.Mol. Biol. 215, 403-410.

22. Thomas, W., Rubenstein, M., Goto, M. et al. 1993, Agenetic analysis of the Werner syndrome region on humanchromosome 8p, Genomics, 16, 685-690.

23. Yu, C.-E., Anderson, L., Oshima, J., andSchellenberg, G. D. 1994, Dinucleotide repeat polymor-phisms at the GSR gene, Hum. Mol. Genet. 3, 212.

24. Tokutake, Y., Satoh, M., Suzuki, N. et al. 1996, Esti-mation of the physical distance between major genomicmarkers in the Werner syndrome locus (8pll.2-12) bydual-color FISH analysis, Jpn. J. Human Genet., 41,291-297.

25. Hudson, T. J. 1994, Colony hybridization to screen formicrosatellites. In: Dracopoli, N. et al. editors, Cur-ren Protocols in Human Genetics. Unit 2.3.1, Wiley andSons, New York.

26. Matsumoto, T., Imamura, O., Yamabe, Y. et al. 1997,Mutation and haplotype of the Werner's syndrome genebased on its genotype: Genetic epidemiology in theJapanese population, Human Genet., 100, 123-130.

27. Herzog, H., Nicholl, J , Hort, Y. J , Sutherland, G. R.,and Shine, J. 1996, Molecular cloning and assignment ofFAK2, a novel human focal adhesion kinase, to 8pll.2-p22 by nonisotopic in situ hybridization, Genomics, 32,484-486.

28. Beetham, J. K. and Hammock, B. D. 1993, cDNA cloningand expression of a soluble epoxide hydrolase from humanliver, Arch. Biochem. Biophys., 305, 197-201.

29. De Silva, H. V., Harmony, J. A., Stuart, W. D.,Gil, C. M., and Robbins, J. 1990, Apolipoprotein J:structure and tissue distribution, Biochemistry, 29,5380-5389.

30. Ichikawa, K., Yamabe, Y., Imamura, O. et al. 1997,Cloning and characterization of a novel gene, WS-3, inhuman chromosome 8pll-pl2, Gene, 189, 277-287.

31. Shimamoto, A., Kitao, S., Ichikawa, K. et al. 1996, Aunique human gene that spans over 230 kb in the humanchromosome 8pll-12 and codes multiple family proteinssharing RNA-binding motifs, Proc. Natl. Acad. Sci. USA,93, 10913-10917.

32. Wilde, D. C, Crowther, C. E., Cripe, T. P., Lee, M. G-S.,and Cowan, N. J. 1982 Evidence that a human ,3-tubulinpseudogene is derived from its corresponding mRNA, Na-ture, 297, 83-84.

33. Purrello, M., Pietro, C. D., Rapisarda, A. et al. 1994,Localization of the human genes encoding the two sub-units of general transcription factor TFIIE, Genomics,23, 253-255.

34. Tutic, M., Lu, X., Schirmer, H. R., and Werner, D. 1990,Cloning and sequencing of mammalian glutathione re-ductase cDNA, Eur. J. Biochem., 188, 523-528.

35. Yamabe, Y., Ichikawa, K., Sugawara, K. et al. 1997,Cloning and characterization of Rep-8 (D8S2298E) in thehuman chromosome 8pll.2-pl2, Genomics, 39, 198-204.

36. Jones, T. A., Barker, H. M., da Cruz e Silva, E. F. etal. 1993, Localization of the genes encoding the catalyticsubunits of protein phosphatase 2A to human chromo-some bands 5q23-q31 and 8pl2-pll.2, respectively, Cyto-genet Cell Genet, 63, 35-41.

37. Emi, M., Takahashi, E., Koyama, K., Okui, K.,Oshimura, M., and Nakamura, Y. 1992, Isolation andmapping of 88 new RFLP markers on human chromo-some 8, Genomics, 13, 1261-1266.

38. Thomas, W., Rubenstein, M., Goto, M., and Drayna, D.1993, A genetic analysis of the Werner syndrome regionon human chromosome 8p, Genomics, 16, 685-690.

39. Nakura, J., Wusman, E. M., Miki, T. et al. 1994,Homozygosity mapping of the Werner syndrome locus(WRN), Genomics, 23, 600-608.

40. Oshima, J., Yu, C.-E., Boehnke, M. et al. 1994, Inte-grated mapping analysis of the Werner syndrome regionof chromosome 8, Genomic, 23, 100-113.

41. Emi, M., Fujiwara, Y., and Nakamura, Y. 1993, A pri-mary genetic linkage map of 14 polymorphic loci for theshort arm of human chromosome 8, Genomics, 15, 530-534.

42. Tomfohrde, J., Wood, S., Schertzer, M. et al. 1992, Hu-man chromosome 8 linkage map based on short tandemrepeat polymorphisms: Effect of genotyping errors, Ge-nomics, 14, 144-152.

43. Koyama, K., Sudo, K., and Nakamura, Y. 1995, Isolationof 115 human chromosome 8-specific expressed sequencetags by exon amplification, Genomics, 26, 245-253.

44. Imbert, A., Chaffanet, M., Essioux, L. et al. 1996, Inte-grated map of the chromosome 8pl2-p21 region, a regioninvolved in human cancers and Werner syndrome, Ge-nomics, 32, 29-38.

45. Bickmore, W. and Bird, A. 1992, The use of restric-tion endonucleases to detect and isolate genes from mam-malian cells, Methods Enzymoi, 216, 224-244.

46. Lindsey, S. and Bird, A. 1987, Use of restriction enzymesto detect potential gene sequences in mammalian DNA,Nature, 327, 336-338.

No. 2] K. Ichikawa et al. 113

47. Bird, A. P. 1987, CpG islands as gene markers in the DNA methylation depends on CpG density and promotervertebrate nucleus, Trends. Genet., 3, 342-347. strength: Evidence for involvement of a methyl-CpG

48. Boyes, J. and Bird, A. 1992, Repression of genes by binding protein, EMBO J., 11, 327-333.