A high-resolution physical map of human chromosome 11

Vol. 93,pp.3149-3154,April1996 Genetics

A high-resolution physical map of human chromosome 11

SHIZHEN QIN*t,

NORMAJ.

NOWAK*t$, JIALU ZHANG*,

SHEILA N. J.

SAIT*,

PETERG.

MAYERS*§,

MICHAEL J. HIGGINS*, YI-JUN CHENG*, LI LI*,

DAVID

J. MUNROE¶, DANIELA S. GERHARDII,

BERNHARD H. WEBER**, EVA BRIC¶,

DAVID

E. HOUSMAN¶, GLEN

A.

EVANStt,

ANDTHOMAS B.

SHOWS*#$

*DepartmentofHumanGenetics,Roswell Park CancerInstitute, Buffalo,NY14263;

¶Center

for CancerResearch,Massachusetts Institute ofTechnology, Cambridge,MA02139;IIDepartmentofGenetics,Washington UniversitySchoolofMedicine,St.Louis,MO63110;**JuliusMaximiliansUniversitateWurzberg, Institute forHumangenetik,D-97074Wurzberg, Germany;andttTheEugeneMcDermott Center forGrowth andDevelopment,UniversityofTexasSouthwestern MedicalCenter, Dallas,TX75235

ContributedbyDavidE.Housman, December14, 1995

ABSTRACT The

development

ofa

highly

reliable

physical

mapwithlandmarksites

spaced

anaverageof100

kbp

apart has been a central

goal

of the Human Genome

Project.

We have

approached

the

physical mapping

ofhumanchromosome 11 with this

goal

as a

primary

target. We have focused on

strategies

that would utilize yeast artificial chromosome

(YAC) technology,

thus

permitting long-range

coverage of hundreds of kilobases of

genomic DNA,

yet we

sought

to minimize the

ambiguities

inherent in theuseofthis technol- ogy,

particularly

the occurrence of chimeric

genomic

DNA clones. This was achieved

through

the

development

of a

chromosome

11-specific

YAC

library

from ahuman somatic cell

hybrid

line that has retained chromosome 11 as its sole humancomponent.Tomaximize the

efficiency

of YAC

contig

assembly

and

extension,

wehave

employed

anAlu-PCR-based

hybridization screening

system.Thissystemeliminatesmany of the more

costly

and

time-consuming

stepsassociatedwith sequence

tagged

site content

mapping

such as

sequencing, primer production,

and hierarchical

screening, resulting

in greater

efficiency

with increased

throughput

and reduced cost.

Using

these

approaches,

wehaveachieved YAC coverage for >90% of human chromosome

11,

with an averageinter- marker distance of <100

kbp. Cytogenetic

localization has beendetermined for each

contig by

fluorescent in situ

hybrid-

ization

and/or

sequence

tagged

sitecontent.The YAC

contigs

thatwehave

generated

should

provide

arobust frameworkto moveforwardto

sequence-ready templates

for the

sequencing

efforts of the Human Genome

Project

aswellas morefocused

positional cloning

^on chromosome 11.

High-fidelity physical

mapsofeach chromosome will facilitate the

sequencing

efforts of the Human Genome

Project

aswell

astheidentification and localization of human disease genes.

Construction of such maps has been

simplified by

recent

technological

advances such as yeast artificial chromosome

(YAC) cloning

and the

widespread

useof PCR-basedscreen-

ing

systems for

arrayed

libraries

(1). Application

of these methods has resulted in the construction of low-order

physical

maps,in the form ofYAC

contigs,

forchromosome

21q (2)

and the euchromatic

region

of the Y chromosome

(3).

These

contigs

^were ordered and

developed largely

^on the basis of sequence

tagged

site

(STS)

^content.

Assembly

of these

contigs

was facilitated

by prior knowledge

of STS order across the target

regions, obtained,

inthecaseof the

21q

map,

by

aset of well-characterized chromosome

21-specific

somatic cell

hybrid mapping panels integrated

with adensesetof ordered

genetic

markers

(2). Similarly,

a

large

collection of

naturally occurring

Ychromosome

breaks,

used in

conjunction

withaY chromosome-enriched YAC

library,

^were vital to the

rapid development

of theYmap

(3).

The

production

of YAC

contigs

Thepublicationcostsof thisarticleweredefrayedinpartbypagecharge payment.Thisarticlemusttherefore beherebymarked"advertisement"in accordancewith 18U.S.C.§1734solelytoindicate this fact.

spanning

other chromosomesorchromosomearmshas

proved

to be more difficult. The difficulties encountered with the

development

of suchmapscan

largely

be attributedto

(i)

the

comparative

lack of similarordered

mapping

reagents avail- able for other

chromosomes, (ii)

the relative

inefficiency

of STS-content

mapping,

and

(iii)

the inherent

physical

and technicallimitationsof whole genome YAC

library screening including

their

large

size and

high

rate of chimerism. We

sought

to

directly

addressthese limitations

during

thedevel-

opment

ofaYAC

contig-based physical

map of chromosome 11. Inanefforttominimizemany ofthe

ambiguities

associated with the

screening

ofwhole genome YAC

libraries,

wehave

developed

an

arrayed

chromosome

11-specific

YAC

library

fromasomatic cell

hybrid

line that has retained chromosome 11 as its sole human component

(4).

The small size and

essentially

nonchimeric natureof this

library

has accelerated

contig assembly

and

greatly

increasedthe

sensitivity

ofscreen-

ing

in

comparison

to that of whole genome libraries. In

addition,

^{as an} alternative to STS-content

mapping,

wehave

employed

an Alu-PCR-based

hybridization

system for the

assembly

of

large

YAC clone

contigs (5, 6).

Thissystemoffers several

advantages

overSTS-content

mapping

with

respect

to increased

throughput, efficiency,

andcostreduction

(5, 6).

As

a

result,

we have achieved YAC coverage for '130

Mbp,

or

>90% of chromosome

11,

with an average intermarker dis- tanceof<100

kbp. Furthermore,

since each of the 1824 clones in the

library

has been sized and

they

are

largely

devoid of

chimeras,

anaccurate assessmentofintermarkerdistancecan

be estimated from these

contigs.

MATERIALS

AND METHODS

YAC Libraries. The 4X chromosome

11-specific

YAC li-

brary

was

prepared

from the Jl monochromosomal

hybrid,

screened

against

hamster Cot-1 DNAtoeliminate

interspecies chimeras,

and

arrayed

into 19 96-well microtiter

plates

^as described

(4).

Each YAC has been

assigned

anaddress based upon its location within a

plate,

row, and column. All 1824 clones have been sized

by pulsed-field gel electrophoresis.

The average insert size is 350 kb.The CEPHmega YAC

library

was

constructed, arrayed,

^andcharacterizedas described

(7).

YAC

Library Pooling

Schemes and DNA

Preparation.

The chromosome

11-specific

YAC

library

was

arranged

into three blocks of six microtiter

plates

each. Individual YACs from within each blockweregrowntosaturation and combined into

aseries of

pools corresponding

torows,

columns,

and"half- Abbreviations:FISH,fluorescent in situhybridization;STS,sequence taggedsite; YAC, yeast artificial chromosome.

tS.Q.and N.J.N. contributedequallytothis work.

*Forquestions regardingthe data base andresources,direct e-mailto

nowak@shows.med.buffalo.edu.

§Forquestionsregardingtheserver,directe-mailtomayers@shows.

med.buffalo.edu.

t$Towhomreprintrequestsshould be addressed.

3149

Proc. Natl. Acad. Sci. USA 93

(1996) plates." Row,

column and

half-plate pools

contained

24, 24,

and 48 individual YAC

clones, respectively. High-purity

DNAs were

prepared

from each of the 182

pools

inagarose

plugs by

the

lyticase/LiCl dodecyl

sulfatemethod

(7).

The CEPHmega YAC

library

was

arranged

into

pools

as described

(7).

Alu-PCR

Amplification

ofYACDNAs. Alu-PCR

amplifica-

tion from all

templates

was directed from theAlu S/Alu

J, Alu-end,

and 47-23

primer

sets

(8).

Alu-PCR

amplification

of YAC DNA

pools

was

performed

ina100-,ulreaction mixture

containing

10 mMTris

(pH 8.3),

50 mM

KCl,

1.5mM

MgCl2,

200 ,uMeach

dNTP,

and2.5 units of

AmpliTaq

DNA

polymerase (Perkin-Elmer).

Alu-PCR

amplifications

from the Alu-end

primer

were

performed

with3.5 mM

MgCl2.

The

thermocycling parameters

usedwere94°C for 1

min,

58°C for 1

min,

and72°C for 45secfor 35

cycles

followed

by

72°Cfor 4 min.

Preparation

and

Screening

ofAlu-PCR

Hybridization

Mem- branes. YAC DNA

pools

wereAlu-PCR

amplified

with each individualAlu

primer

set. Alu-PCR

amplification products

from each

pool

were

visually inspected

onethidiumbromide- stainedagarose

gels

and immobilizedonto 8 x 12 cm

nylon

membranes

using

amanualoffset

spotting

device

(John

Kriet-

ler, Washington University

machine

shop, Washington

Uni-

versity,

St.

Louis).

Filterswere

processed by baking

for 1-2 hr at

80°C,

denaturation in0.4 M

NaOH/0.5

MNaClfor 10

min,

and neutralization in0.5 M

Tris, pH 8.0/0.5

MNaCl for5 min.

Alu-PCR

product probes

for

hybridization

were

generated

STS

51

3

35 18

31 4

1

1233 1

|12

³¹

3 11 2 3

p

15.5 15.3

> .

f;

I

9| 181

⁷

4131 | 14|

10

331

5

36

3

|

⁸⁸

21 5

2 31

312

8151

17 13.1

13.3

23.3

====

I 2525

q

from

cosmid, phage,

or YAC

template

DNAs as described above. The entire set of PCR

amplification products

corre-

sponding

^{to each}

template

was ethanol

precipitated

and la- beled

by

random

priming.

Probeswere

preannealed

with an

equal

volume ofhuman

placental

DNA

(10 ,ug/ml)

and 0.3 volumeof 1 Msodium

phosphate (pH 8.0)

for 2 hr at 65°C.

Nylon

filters were

prehybridized

for 2-18 hr at 42°C in 5x Denhardt's

solution,

5x

SSC,

0.1%

SDS,

50%

formamide,

and salmon spermDNAat100

mg/ml.

After

overnight hybridiza-

tion at

42°C,

filters were rinsed twice in 2x SSC at room

temperature

for 5 min and thenwashed twice in 0.1x

SSC/

0.5%SDSat65°C for 30min.

Exposure

times variedbetween 2 hr and 2

days.

STS

Screening.

The chromosome

11-specific

YAC

library

wasscreened fora total of278 STSs. Established STSswere

obtainedeither

through

theGenomeData Base

(Baltimore)

or asdescribedinSmithetal.

(9).

In

addition,

several

unique

^STSs

were

generated

fromYACinsertends

(10).

All PCR reactions weredone ina 15-,ulreaction volume

using

a

Perkin-Elmer/

Cetus9600 thermal

cycler

asdescribed

(9).

All

positives

were

verified

by

PCR

using

DNA

prepared

fromindividual clones.

Fluorescent in Situ

Hybridization (FISH) Mapping.

FISH

analysis

was

performed

asdescribed

(4).

Data

Analysis

and

Contig Assembly. Contigs

wereassem-

bled

using

SEGMAP

(10),

an interactive

graphical

tool for

analyzing

and

displaying physical mapping

data.The follow-

FISH

26

10

138

2

3

4

33

16 17

21

29

112 18

16

⁴

13

^I

1

I

1 4

3 5

4

4 1

17 4

130

²

1 4

2 3

13

^{I 18}

I

^.

11121

12 ^{1 22} 12 ⁴ ~

12 23

112

|11

^{9 5 1}

3 1 2

6

7

I

¹

FIG. 1.

Approximately

500YAC cloneswerelocalizedto

specific

binsindicated

by

thevertical barsonchromosome11

by

STScontent

(Left)

and FISH

(Right).

I

3150

Gntc:Qne

l

ing

nomenclature has been used:

(i)

YACclones from the

chromosome-specific library

havethe

prefix yRP

followed

by

the

plate address, (ii)

YAC clones from the CEPH mega YAC collection have the

prefix yMega

followed

by

the

plate address, (iii)Alu-PCR probes

havethe

prefixes ySJ (S/J), y47 (47-23),

and

y3 (Alu-end)

followed

by

the

plate

address of the YAC clone from which

they

were

derived, (iv)

STSs derived fromthe YAC clone insert endsare

designated yRP

ormegafollowed

by

the

plate

address withREorLEfor the

right

or left end of the insert,

respectively,

and

(v)

STSs derived from anonymous DNA sequences and genes have nomenclature

assigned

tothem

by

the Genome Data Base.

Restriction

Map Analysis

of

Contigs.

Restriction enzyme

digests

ofYAC DNAs

prepared

inagarosewerecarriedoutas

recommended

by

the

supplier (New England BioLabs);

sper-

midine^wasadded

(to

afinalconcentration of 5

mM)

tobuffers with >50 mM NaCl. Partial

digests

wereachieved

using

serial dilutions of theenzymewithincubations

ranging

from 30min to4hr.Allreactionswereallowedto

equilibrate

onice forat least 1hr

prior

toincubationatthe

appropriate

temperature.

Pulsed-field

gel electrophoresis

wascarriedoutonthe CHEF- DRIIsystem

(Bio-Rad).

The DNA

samples

were

analyzed

on

1% agarose

gels

in 0.5x TBE

(lx

TBE is 89 mM Tris-

borate/89

^mMboric

acid/2

mMEDTA,

pH 8.0)

at200Vfor 22 hr with

ramping

from 10sto50sor20s to60s. Transfer of

pulsed-field gels

to

nylon

membranes and

hybridization

were as described

(12).

The membranes were

sequentially hybridized

with three sets of

probes: (i)

2.6-kb and 1.7-kb

fragments

from

pBR322 digested

with BamHI and Pvu II, whichare

homologous

tothe

right

andleftarmof the

pYAC4

vector,

respectively; (ii)

human Cot-1 DNA; or

(iii)

inter-Alu

probes generated

from the individual YAC clones.

RESULTS

GenerationofInter-AluPCR Product

Hybridization

Probes.

Inter-Alu PCR

product hybridization probes

^were

generated

from individual YAC clones

using Alu-specific primers.

Six hundred

fifty-four probes generated

with

primer S/J,

404

probes generated

with

primer

47-23,and 50

probes generated

withthe Alu-end

primer

^wereutilized inthe final

phase

ofthis

study.

Thelow number of

probes generated

fromtheAlu-end

primers

reflectsthe

degree

of

contig assembly

thatwas

already

achieved

by

the timethis

primer

setwasused for

hybridization

and notits failure to generate successful

hybridizing probes.

Each YACclone

yielded

from 4 to >10 PCR

products

when visualizedon1.5%

agarose/ethidium

bromide

gels irrespective

of the

primer

set.Thisincludes YACclones thathad

previously

been

mapped

to Giemsadarkbands.The

products ranged

in sizefrom <100

bp

to >1kb. Greater than95%of the

pooled products proved

tobesuccessfulas

probes.

Alu-PCR

products

were also

generated

from a smaller set of chromosome 11-

specific phage

andcosmidclones.

Approximately

1100inter-Alu

probes, generated

from indi- vidual YAC clones, were

hybridized

to filters

stamped

with inter-Alu

products generated

with the

corresponding primer

from YAC clone

pools (see

Materials and

Methods). Screening ambiguities

wereresolved

by examining half-plate pools

or

by

Southernblot

analysis

ofindividual YAC cloneinter-AluPCR

fingerprints.

STSContent

Mapping.

YAC clones have been identified for 278 STSs

representing

62 genes, 171 anonymous DNA seg- ments, and45 YAC clone insert ends. Onaverage,each PCR assayidentifiedthreeorfour individual YACclones,aswould be

expected

with a fourfold

library.

However, the STSs

generally

identified

contigs previously

assembled

by

Alu-PCR producthybridization, andonly twoSTSs successfullyjoined

separate contigs.

Contig Anchoring. Approximately

500YAC clones

(27%

of the

library)

have been localizedto

specific

bandsonchromo-

some 11

by

FISH

(Fig. 1).

An additional200clones

(11%

of the

library)

areanchored

by

^{virtue of}

containing

a

mapped

STS

(Fig. 1) (ref. 9;

GenomeData

Base).

Asa

result,

every

contig

containsatleastone, and

usually several,

clonesanchored

by

FISH

and/or

STScontent.The localizationdata is

presented

both

graphically

and in a tabular

(pter-qter)

format

(Figs.

2 and

3;

WWW server

URL, http://shows.med.buffalo.edu/

home.html).

Verification of

Contigs.

Over 85% of the 119

contigs

as-

sembled

by

Alu-PCR

product hybridization

and STScontent have been verified

by

Southern blot

analysis

of Alu-PCR

fingerprints.

These data have also servedtoconfirm all

single

YAC

linkages

as well as resolve

ambiguities

inherent in the

pooling

scheme. Further verification of YAC

contig integrity

has been

provided by pulsed-field

restriction

mapping.

YAC clones

comprising

four

contigs,

chosenat

random,

have been

subjected

to

pulsed-field

restriction

analysis.

Asdemonstrated in

Fig. 2,

these restriction maps confirm

contig integrity

with respecttorelative order andextentof

overlap

between clones.

Data

Analysis. Analysis

of both the

hybridization

and STS content data

by

SEGMAP has resulted in the

assembly

of 119

contigs ranging

in size from 275 kbto6100 kband

containing

from 2 to 97 YAC

clones, respectively,

with 61

singletons (single

YAC with at least ^one

probe).

An

example

ofa map

generated by

SEGMAP for

contig ySJ-la2

is shown in

Fig.

3.

SEGMAP utilizes the YAC clone size

data,

determined

by pulsed-field gradient analysis

of each clone

(depicted

in pa- rentheses below the

clones),

and also

incorporates

localization information either

directly

above the

clones,

if thelocalization

wasdetermined

by

FISH

analysis,

orabove the STScontained inthe clones for

assembling

the

contig

maps. Restrictionmap

analysis

ofindividualYAC clones

making

upthe shortest

tiling path through

the

contig

demonstrates that therelativeorder of YAC

clones,

as well as intermarker distances

predicted by

SEGMAP, are

essentially

correct

(Fig. 2). Thus,

while this programwas

originally designed

^to

analyze

STScontentdata

defining single points,

it is also

capable

of

handling complex mapping

information

generated by

^inter-Alu PCR

product probes,

which mayrepresenttheentire

length

ofaYAC clone.

The

predicted

size estimates of individual

contigs

are

generally

within 10% of the actual size.

Chromosome11DataBase.Thedata basecanbe searched

using

a YACclone addressor

probe,

and each of the corre-

sponding contigs

^canbe

graphically

viewed.

Contig

mapsand tables

containing

all screened STSsand FISHlocalizationsare

availableand can be searchedeither

directly

fora

particular probe

or YAC clone or in a pter-qter mode for

regional

information

(http://shows.med.buffalo.edu/home.html).

DISCUSSION

A central

goal

of the Human Genome

Project

has been the

development

ofa

highly

reliable

physical

mapwithlandmark sites

spaced

^anaverageof 100

kbp

apart.The rationale for this

goal

is the

ability

^{to use}such amap as aframework for the

development

of

sequence-ready genomic

DNA clonesets as

wellastheidentificationand

cloning

of human disease genes.

We have

approached

the

physical mapping

of human chro-

mosome11with this

goal

asa

primary

target.Wefocusedon

strategies

that wouldutilize YAC

technology,

thus

permitting long-range

coverage of hundreds of kilobases of

genomic DNA,

yet

sought

to minimize the

ambiguities

inherent in the

useof this

technology, particularly

theoccurrenceofchimeric

genomic

DNA clones. To achieve this

goal,

^we

developed

a

chromosome

11-specific

YAC

library

froma human somatic cell

hybrid

line that has retained chromosome 11 as its sole human component(4).Thereduced

complexity

and

essentially

nonchimericnature ofthis

library

hastranslatedinto

signifi- cantly

increased

efficiency

and

sensitivity

of

screening

when

compared

tothat ofwhole genome libraries. At thesame

time,

3152 Genetics:

Qin

etal. Proc. Natl.Acad. Sci. USA 93

(1996)

inanefforttomaximize the

efficiency

of

contig assembly

and increased

throughput

and reduced cost

(5, 6). Using

these

extension,

we

employed

^an Alu-PCR-based

hybridization approaches,

we have assembled the 1824 clone chromosome

screening system (5, 6).

This

system

eliminates many of the

11-specific

YAC

library

into119

contigs representing >90%

of

more

costly

and

time-consuming steps

associated with STS- the chromosome withanaverageintermarker

spacing

of<100 content

mapping

such as

sequencing, primer production,

and

kbp. Significantly,

because the chromosome

11-specific

YAC hierarchical

screening, resulting

in

greater efficiency

with

library

is

largely

devoid of chimeric clones

(4), sizing

and

ySJ-1 a2 A

y y y yySSyy y y

S S 4 yJJSSyy S y S y y

J J 7 4--JJSS J S J S S

711--JJ - J J J

Total 1 1 1 -341 1-- 1 - 5

Contig 2 7 1 7ab4525 0 1 f 9 1

Length h g d d11dhhe g a 1 f a

1486 o 7 2 5004 5221329 5 210002 2 6 1490kb

yRP-17g2 I 11P5.p15.4

(525) *

(150) 7 '(575) ;

yRP-11'8 .L yRP-19h10.

(375) 5h3 ---

.:"i

(350)

'

^t

yRP-2h27y,,,,, yRP-9f2' ' '

0'(3745) T . ' .

yRP-7c5l' , __'R- yRP-1a6I,

(375) ³ ( ',,,(, (175)*

yRP-1ld24,y(3275) 3a27W7i j,-(100)yRP-912, . Z

yRPy15h34-c

-_

yRP-7d5 Li.&'

'R

(375) ( i

yRP-14b12 1 .

(3500) ,

I yRP1

yRP-13a12 ' ' '

(325) T(-22), yRP-14dl,

'J"~-'-'.

I

(325) 'TTT7T

yRP-18c6412',"- '' (300)

yRP-10b5

L,,

L B

(275) I'

yRP-1ea29

(400) a I I 1

yRP-14b12. | S

(350) R -,-i T T S

yRP-19a61

"''

yRP-6d5' (225)^;i

YRP-12e8. I

(375)

...

B

F B

FBS N N FSFB B S B S FSNSF F 1675kb

500 1000 1500 1800kb

y

RP-

^BS

N N F

^SFB

yRP-17g2a1

I

yRP-7c51 F SFB B R

yRP-5e91

'I

yRP-1a21

I I

L S F R

yRP-5f12

t 1 I

R

RB

B S F S NSB Ll

yRP-8h21 I i II

L BB R

yRP-12e8L sNS F F R

yRP-12e81

FIG. 2. Restrictionmap ofcontig ySJ-la2usingYACclonesmakingupthe shortest

path through

the

contig. High

molecular

weight

DNAfrom YACclones

(appearing

inboldonthe

map)

yRP-17g2,yRP-7c5, yRP-5e9,yRP-la2,

yRP-5f12, yRP-8h2,

and

yRP-12e8

^was

digested

asdescribed inMaterialsand Methods withNot I

(N), Sfi

I

(F),

SalI

(S),

and BssHII

(B)

and

separated by electrophoresis

onCHEF

gels

followed

by

Southern blotanalysis. Land Rrepresentthe leftandrightendsof theclones, respectively.

Proc.Natl.Acad. Sci. USA 93

(1996)

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Proc. Natl. Acad. Sci. USA 93

(1996)

restriction

mapping

information

garnered

from these YAC

contigs closely

reflects that found in

genomic

DNAs

(Fig.

2and datanot

shown).

The average size of the assembled

contigs (-1 Mbp)

is somewhat smaller thanwould be

predicted (1-2 Mbp)

con-

sidering

the number of inter-Alu

probes (-1100)

used to assemble the

contigs (13).

This difference may be a conse- quence ofsome

degree

ofunevennessinthe distribution ofAlu elements in thegenome

(5, 6, 8).

The

density

of Alu elements

was

clearly

sufficient to

support contig assembly by

the Alu- PCR-based

hybridization. However, clustering

^ofAlu

probes

couldleadtoa

significant degree

of undetected

overlap

among the

contigs.

Thelocalization information for each of the

contigs suggests

the existence of several

interesting

structural features of chromosome 11

including duplications,

low-order

repetitive elements, chromosome-specific repetitive elements,

and ho-

mologous regions

onother chromosomes.Several YAC

clones,

from the

chromosome-specific library, consistently mapped

to two different

regions

on the chromosome

by

FISH

analysis.

CEPH mega YAC clones

covering

the same area

similarly

exhibit this dual localization. Endclones and anonymous DNA

segments

from one such chromosome

11-specific

YACwere

sequenced,

converted to

STSs,

and

mapped by

PCR on a

chromosome

11-specific

somatic cell

hybrid mapping panel.

These STSs

mapped simultaneously

toboth the p and qarms of chromosome

11, suggesting

the presence ofanintrachro- mosomal

duplication (data

^not

shown). Approximately

10%of the

chromosome-specific

YAC clones map to two or more locations onchromosome

11,

consistent with thepresenceof

low-copy number, chromosome-specific, repetitive

elementsas hasbeen

suggested

forchromosomes5 and 7

(14, 15).

Simi-

larly,

15% of theclones also detected

specific

loci on other

chromosomes, suggesting

the presence of

homologous regions

or low-order

repetitive

elements.

The

general principles

thatwe have

exploited

in the con-

struction of this

physical

mapcan

easily

betransferredtoother chromosomes orchromosomal arms. The YAC

contigs

pre- sented here should

provide

a robust framework to move

forward to

sequence-ready genomic templates

as

part

of the

sequencing

efforts of the Human Genome

Project

aswell as morefocused

positional cloning

on chromosome 11.

We acknowledge the excellenttechnical assistance of RogerEddy,

LindaHaley,W. MichaelHenry, andStacey Simpson.This workwas

supported byNational Institutes ofHealth GrantsHG00359, HG00333, CA63333, and EY10514 and The Retinitis Pigmentosa Foundation (T.B.S.);National Cancer Institute Grant HL41486(D.J.M.);American CancerSocietyGrantCN64 and The RetinitisPigmentosaFoundation (D.S.G.); National Institutes of Health Grant HG00299(D.E.H.);Na- tionalCenterforHumanGenomeResearch HG00102 andDepartment ofEnergyGrantDEEG03-88ER60694(G.A.E.).

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3154

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