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Genomics 59, 1–5 (1999)Article ID geno.1999.5812, available online at http://www.idealibrary.com on

Integration of Cytogenetic with Recombinational and PhysicalMaps of Mouse Chromosome 16

Clara S. Moore,* Jennifer S. Lee,† Bruce Birren,† Gail Stetten,*Laura L. Baxter,‡ and Roger H. Reeves‡ ,1

*Department of Gynecology and Obstetrics and ‡Department of Physiology, Johns Hopkins University School of Medicine, Baltimore,Maryland 21205; and †Whitehead Institute/Center for Genome Research, Cambridge, Massachusetts 02139-1561

Received December 23, 1998; accepted March 4, 1999

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To link the cytogenetic map for mouse chromosome6 (Chr 16) to the more detailed recombinational andhysical maps, multiple probes were mapped by fluo-escence in situ hybridization (FISH). Sixteen largensert clones (YACs, BACs, and PACs) containing

arkers that have been assigned to the Chr 16 recom-inational map were localized to a cytogenetic band orubband by high-resolution FISH. This linkage of theytogenetic and recombinational maps provides a use-ul tool for assigning new probe locations and for de-ning breakpoints in mice with chromosomal rear-angements. A direct application of these probes isemonstrated by identifying mice trisomic for distalhr 16 using FISH analysis of interphase nuclei. © 1999

cademic Press

INTRODUCTION

A well-defined recombinational map exists for mousehromosome 16 (Chr 16) (Reeves et al., 1997). Markershat have been assigned to Chr 16 are continuouslyntegrated into a report available on-line and pub-ished annually [see Chromosome 16 at http://www.nformatics.jax.org/bin/ccr/index and (Cabin andeeves, 1998)]. Contiguous physical maps are also be-

ng constructed so that the entire chromosome may beequenced. Although many markers have been as-igned to these maps, few have been studied by high-esolution cytogenetic mapping to assign a specificand or subband location on the G-banded chromo-ome.Assignment of expressed genes to Chr 16 by fluores-

ence in situ hybridization (FISH) has extended infor-ation about regions of conserved synteny betweenhr 16 and several human chromosomes (Fig. 1). How-ver, only seven markers have been previously mappedy high-resolution FISH to a single cytogenetic band or

1 To whom correspondence should be addressed at Physiology 205,ohns Hopkins University School of Medicine, 725 N. Wolfe Street,altimore, MD 21205. Telephone: (410) 955-6621. Fax: (410) 955-461. E-mail: rreeves@welchlink.welch.jhu.edu.

1

ubband, and six of these are in the proximal half ofhr 16 [Mouse Genome Database (MGD), URL: http://ww.informatics.jax.org, December, 1998]. The distalortion of Chr 16 includes genes located on humanhromosomes 3 and 21, demonstrating perfect con-erved linkage with most of human chromosome 21HSA 21). Prior to this work, little correlation existedetween the cytogenetic map and other maps of thisegion of mouse Chr 16.Trisomy 21, or Down syndrome, is the most frequent

ive-born aneuploidy in human beings, and a number ofouse models have been developed to study the devel-

pmental consequences of the dosage imbalance ofSA 21 genes (Kola and Hertzog, 1998). Ts65Dn mice

nherit two normal chromosomes 16 plus a smallarker chromosome resulting from a reciprocal trans-

ocation between Chr 16 and Chr 17, T(16C3-4;7A2)65Dn (Davisson et al., 1990). These mice are atosage imbalance for genes derived from Chr 16 thatorrespond to the region of HSA 21 from APP to MX1Reeves et al., 1995). A variety of phenotypes corre-ponding to those in DS have been observed in Ts65Dnice, which represent a widely used model for studies

f the most frequent live-born human aneuploidy.In the work presented here, markers localized to Chr

6 by recombinational and/or physical mappingReeves, 1997; Cabin et al., 1998; http://carbon.wi.mit.du:8000/cgi_bin/mouse/sts_by_chrom) were used todentify large-insert mouse clones. These clones wereybridized by FISH to normal mouse chromosomes andssigned to single cytogenetic bands, allowing align-ent of the recombinational, physical, and cytogeneticaps. Two clones were used in an interphase FISH

ssay for rapid identification of mice carrying the65Dn marker chromosome.

MATERIALS AND METHODS

BAC clones were identified by PCR from a 129 SV mouse BACibrary (B. Birren, unpublished results) or hybridized to arrayed PACibraries (bacpac.med@buffulo.edu). All BACs described can be ob-ained from Research Genetics (Huntsville, AL). After identification

0888-7543/99 $30.00Copyright © 1999 by Academic Press

All rights of reproduction in any form reserved.

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2 MOORE ET AL.

f a positive address, bacteria from the well of the library plates weretreaked to obtain individual colonies and PCR was repeated toerify STS content. DNA was prepared from cultures grown to sat-ration in LB containing the appropriate antibiotic using an alkaline

ysis procedure. YACs were prepared as described (Cabin et al.,998).Probes were mapped by FISH (Pinkel et al., 1986). One microgram

f DNA was labeled with biotin (BioNick kit, Bethesda Researchaboratories), digoxigenin (Boehringer Mannheim), Spectrum Or-nge, or Spectrum Green (Vysis) in a nick-translation reaction. La-eled probe (200 ng) was precipitated with 5–103 excess mouseot-1 DNA, resuspended in 10 ml of solution containing 50% form-mide, 23 SSC, and 10% dextran sulfate, denatured for 5 min at5°C, and preannealed for 1–2 h. Metaphase spreads were preparedrom colchicine-treated mouse J1 ES cells by standard methodsDavisson and Akeson, 1987). For prometaphase chromosomes, 5

FIG. 1. Maps of mouse Chr 16 showing regions of conservedecombinational map (map units based on MGD map), and markreviously on the cytogenetic map are shown in small type. The reg

g/ml of ethidium bromide was added to the cell culture medium for0 min prior to colchicine treatment.For interphase analysis, slides were prepared according to Paris et

l. (1996) with slight modifications. The hypotonic solution used was5 mM KCl, and slides were placed in steam for 10 s prior to drying.lides were pretreated in 23 SSC at 37°C for 30 min, dehydrated,nd denatured for 5 min at 75°C before hybridization at 37°C for–18 h. The slides were washed twice in 50% formamide, 23 SSCnd twice in 23 SSC at 42°C. Interphase slides were washed in 23SC at 72°C for 5 min. Probes were detected with FITC-avidin orhodamine anti-digoxigenin according to the manufacturer’s instruc-ions (Oncor). Chromosomes were counterstained with DAPI (0.1g/ml) and viewed with a Zeiss Axioskop equipped with a SenSysooled CCD camera (Photometrics) and Smart Capture imaging soft-are (Vysis). DAPI images were inverted to produce G-bandingatterns viewed simultaneously with hybridization signals.

nteny with human chromosomes, positions of key genes on thelocations on the high-resolution cytogenetic map. Genes mappedof Chr 16 at dosage imbalance in Ts65Dn mice is indicated.

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3MOUSE CHROMOSOME 16 CYTOGENETIC MAP

RESULTS

Fourteen newly identified BAC/PAC and two YAClones were isolated from regions of conserved syntenyetween mouse Chr 16 and HSA 16 (1 clone), HSA 221 clone), HSA 3 (2 clones), or HSA 21 (12 clones).

arkers were selected to provide a high-density map ofistal Chr 16 as predicted from recombinational mapistances and to overlap with previously mapped cyto-enetic markers. DNA from isolated BACs was labeledy incorporation of biotin, digoxigenin, or directlyinked fluorochromes. After hybridization and detec-ion, 10 or more metaphase spreads with signals onoth Chr 16 homologs were viewed to determine theytogenetic band localization of each marker. Hybrid-zation on prometaphase chromosomes allowed local-zation of each probe to a single band or subband (Table, Fig. 2).A composite cytogenetic map is presented in Fig. 1.

his map includes all known markers previouslyapped with high resolution on the cytogenetic map ofouse Chr 16 (MGD, December, 1998) plus the 16arkers mapped in this study. The compilation ofarkers allows accurate alignment of the recombina-

ional and cytogenetic maps. A clone containing Tnp2,hich is tightly linked to Prm1 and Prm2, maps to6B1 and is among the most proximal known genes onhr 16. BAC 6D3-6 (GenBank Accession No. 000096;und et al., 1999), which contains mouse orthologs ofarkers on HSA 22 within the critical region for Di-eorge/velocardiofacial syndrome, localizes to 16B2.reviously, the region of conserved synteny to HSA 3as defined by localization of Cd86 and Cd80 (mapnits 26.9 and 28, respectively) to band 16B5. Twodditional markers, BAC 367A9/D16Mit39 and BAC11G23/D16Mit48, map within the known conservedynteny with HSA 3 and localize to bands 16C1.1 and6C3.1, respectively (Fig. 1).

Cytogenetic Band Location of 16 Mapped Probes

Marker Clone nameMap

positionaBand

assignment

np2 PAC 55J21 3.4 16B1iGeorge/VCFS BAC 6D3-6 10.9 16B216Mit39 BAC 367A9 29 16C1.116Mit48 BAC 411G23 43 16C3.116Mit93 YAC 24G7 46 16C3.2tch PAC 60L4 47.2 16C3.216Mit49 YAC 140D8 53 16C3.316Mit19 BAC 365A4 54 16C3.316Mit153 BAC 426P3 56.8 16C3.316Mit70 BAC 409J8 57 16C3.316Mit191 BAC 393K7 57.8 16C3.316Mit128 BAC 401C2 66.3 16C416Mit52 BAC 43D12 66.8 16C416Mit204 BAC 433G17 67.1 16C416Mit205 BAC 456J11 71.1 16C4x1 PAC 452K24 71.2 16C4

a From Mouse Genome Database.

Stch is the most proximal known gene on HSA 21nd is found on 16C3.2 (Fig. 2). This establishes thenown boundary of conserved linkage between thesehromosomes. The remaining 11 markers saturateands 16C3.2–16C4. The 16C3.3–C4 boundary is de-ned by localization of these markers and comparisono orders determined on the recombinational and phys-cal maps. The breakpoint of the T65Dn marker chro-

osome, at map unit 55.8, can now be placed in sub-and 16C3.3.An interphase FISH assay would greatly facilitate

dentification of segmentally trisomic, Ts65Dn micerom among their euploid littermates. A screening as-ay was developed using simultaneous hybridization ofwo differentially labeled BAC clones. BAC 401C2, con-aining D16Mit128, was directly labeled with Spec-rum Orange, while BAC 433G17, containing16Mit204, was labeled with Spectrum Green for two-

olor cohybridization. Interphase cells were preparedrom a single drop of tail blood. Slides were routinelyybridized overnight, although signal was visible afterh of hybridization. Cells carrying the T65Dn marker

hromosome in addition to two normal copies of Chr 16ere readily distinguished by the presence of three

ignals of each color per nucleus, while normal micehowed only two signals per nucleus (Fig. 3). In alinded study, peripheral bloods from nine adult prog-ny of a Ts65Dn mother were analyzed by FISH withACs 401C2 and 433G17 and by standard karyotyping

Davisson and Akeson, 1987). Both procedures identi-ed the same five segmentally trisomic and four eu-loid animals.

FIG. 2. FISH mapping localizes PAC 60L4, which containsTCH (red), to 16C3.2, establishing the known border of homologyetween HSA21 and Chr 16.

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DISCUSSION

More than 700 markers have been mapped to chro-osome 16 in the mouse, but only 7 were previously

ocalized with high resolution on the cytogenetic map.hese fall mainly in the proximal half of the chromo-ome. Thus, the cytogenetic map has been poorlyligned with recombinational and physical maps of Chr6. Using high-resolution cytogenetic mapping ofarkers well positioned on recombinational and phys-

cal maps, close alignment of the maps is now possible.he four bands containing well-localized markers arexpanded to include coverage of 9 cytogenetic bands orubbands. Genes mapped relative to Chr 16 referencearkers anywhere on the chromosome may now be

ssigned to a cytogenetic band by inference from thesestablished landmarks.Thirty-three genes and markers mapped both toSA 21q11.2–q22.3 and to mouse Chr 16 show perfect

onserved linkage between the species (Reeves et al.,997). HSA 21 represents about 1.8% of the humanaryotype, and the long arm is estimated to be 40 MbKorenberg et al., 1997). The region of conserved link-ge with Chr 16, from STCH to MX1, represents about0% of the long arm. Chr 16 constitutes about 4% of theouse genome (120 Mb), and 16C3.2 3 ter comprises

0–15% of the chromosome. These estimates of the sizef conserved regions are sufficiently different to sug-est that although marker order is perfectly conservedetween human and mouse, the mouse region is phys-cally compressed relative to that of human. This com-ression is consistent with higher resolution andmaller scale comparisons of mouse and human phys-cal maps for the HSA21 homologous segments (Cabint al., 1998; Cole et al., 1999).

The markers mapped here provide useful tools fortudies of translocation breakpoints and mouse modelsor human disease. For example, mice carrying the65Dn marker chromosome are trisomic for many ofhe genes triplicated in Down syndrome. Along with

FIG. 3. Interphase analysis of Ts65Dn mice using multicolor FISeadily recognized by the presence of three copies of each gene (A), wnd green signals appear yellow.

lear definition of the cytogenetic breakpoint (Fig. 1),his work provides probes for the study of these mice.nterphase FISH analysis from a single drop of bloodrovides rapid, accurate identification of partially tri-omic mice. Due to the limited number of cells, cohy-ridization with two colors provides more reliable datarom each nucleus. This FISH method is less invasive,ess labor-intensive, and faster than traditional-banding of metaphase spreads. Because screening

an be performed on younger pups, earlier develop-ental and behavioral studies are facilitated by this

pproach.In human beings, the relatively high frequency of

riploidy might necessitate additional analyses to dis-riminate triploidy from trisomy. However, analysis of651 mouse embryos across more than a dozen strainsemonstrated a hyperploidy frequency of only 0.8%,nd all of these were trisomies (Mailhes, 1987). Trip-oid embryos have been reported only from the LT/Svtrain, which carries a mutation predisposing to theormation of diploid oocytes (West et al., 1993). Triploidouse embryos arrest by the forelimb bud stage of

mbryogenesis (embryonic day 9–9.5) and are obvi-usly dysmorphic by E10 (Henery and Kaufman, 1993).hus, the FISH approach described here for postnatalyping of Ts65Dn mice should be useful for most pre-atal typing, as well.

ACKNOWLEDGMENTS

This work was supported by PHS Awards HG00934 (B.B.),D24605 (G.S. and R.R.), and HG00405 (R.R.).

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