GENOMICS

11,317-323

(19%)

Fine Structure Physical Mapping of the Region of Mouse Chromosome 10 Homologous to Human Chromosome 21 GREG MACDONALD,*

MON.LI Ct+u,t AND DAVID R. Cox*

*Departments of Pediatrics and Neurology, University of California, San Francisco, California 94143; tDepartments of Biochemistry, Molecular Biology, and Dermatology, Thomas Jefferson University, Philadelphia, Pennsylvania 79 107; and *Departments of Psychiatry, Biochemistry/Biophysics, and Pediatrics, University of California, San Francisco, California 94 143 Received

December

7, 1990;

revised

May 30, 1991

number of regions of genetic homology between the human and mouse genomes (Nadeau, 1989; Searle et al., 1989). Comparative mapping provides the cornerstone for understanding chromosomal evolution in mammals and for the recognition of synteny groups and identification of new genes within such groups, and ultimately forms the basis for mouse models of complex human diseases. Human Chromosome 21 has long been known to bear genetic homology to mouse Chromosome 16. A segment of the long arm of Chromosome 21 spanning the region from 21q11.2 to 21q22 has been shown to bear at least 10 markers whose murine homologs lie on Chromosome 16: D21S16h, D21S13h, D21S52h (anonymous markers), App (amyloid precursor protein), Sod-l (copper-zinc superoxide dismutase), Mx1 (influenza virus resistance), Ets-2 (cellular protooncogene), Prgs (phosphoribosylglycineamide synthetase), and Ifnar (interferon-o receptor) (Cox and Epstein, 1985; Lovett et al., 1987; MacDonald and Cox, 1989; Reeves et aZ., 1987a,b; 1988). A second small region of human Chromosome 21 carries at least three sequences whose mouse homologs have been mapped to mouse Chromosome 17: D21S56h, Crya-l (o-crystalline), and Cbs (cystathionine /3-synthetase) (Munke et al., 1988; Skow and Danner, 1985). It is now known from radiation hybrid and pulsed-field gel electrophoresis mapping studies that the Chromosome 21 sequences homologous to mouse Chromosome 16 lie centromeric to the small region carrying sequences homologous to mouse Chromosome 17 (Burmeister et al., 1990; Cox et al., 1990). In addition, these studies have shown that the segment of human Chromosome 21 extending from the region of mouse Chromosome 17 homology to the 21q telomere encompasses some 3000 kb of DNA and includes the genes PFKL (liver-type phosphofructokinase), CD18 (/3 subunit of the Mac-l leukocyte surface glycoprotein family), COL6Al and COL6A2 (a1 and a2 chains of collagen type VI), and SlOOB (B subunit of the glial calcium-binding protein SlOO). These five sequences had also been independently assigned to

Comparative mapping of human and mouse DNA for regions of genetic homology between human Chromosome 2 1 and the mouse genome is of interest because of the possibility of developing mouse models of human trisomy 21 (Down syndrome), understanding chromosome evolution, and isolating novel sequences conserved between the two species. At least two mouse chromosomes are known to carry sequences homologous to those on human Chromosome 21: mouse Chromosome 16 (D21S16h, D21S13h, D21S52h, App, Sod-l, Mx-1, Ets-2, Prgs, Ifnar) and mouse Chromosome 17 (D21S56h, Crya-1, and Cbs). Recently, five additional genes have been mapped within region 21q22 of human Chromosome 21: PFKL, CDlS, COLGAl, COLBAB, and 51008. To assign these sequences to specific mouse chromosomes, we used human cDNA probes for COL6A1, COL6A2, CDlS, and PFKL and a rat brain cDNA probe for SlOOB in conjunction with a panel of seven Chinese hamster-mouse somatic cell hybrids segregating mouse chromosomes. The specific chromosome complements of the hybrid cell lines and the presence or absence of hybridizing mouse sequences in their DNAs allow us to assign all five sequences to mouse Chromosome 10, with the assignment of Pfkl reported here for the first time. Analysis of genomic mouse DNA fragments produced by digestion with rare-cutting restriction enzymes and separated using pulsed-field gel electrophoresis allows us to construct a fine-structure physical map of two segments of the region of Chromosome 10 containing these five markers. The five loci span at least 1900 kb of mouse DNA and are consistent with the human order: Pfkl-Cd-1%ColGa-l-Co16a-2SlOOb. These genes define a region of the mouse genome homologous to the most distal 3000 kb of the long arm of human Chromosome 21, with evidence of conservation of probe order and relative distance between the species. These results have implications for further efforts to construct mouse models of human Down syndrome and to isolate additional shared sequences in this region. o 1991 Academic Press, Inc.

INTRODUCTION

Recent advances in genetic linkage and physical mapping techniques have led to the elucidation of a 317

08&3-7543/91$3.00 Copyright 0 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.

318

MACDONALD,

CHU,

Pfkl 1

2

AND

Cd-18 3 4

COX

SlOOb 123456

5

Ms-

Ha+

MsHaMS-

ColGa-I 1

Co16a-2 2 3 4

5

Ms-

c-

c

HaMs-

FIG. 1. Autoradiograms of Southern blots assigning Pfkl, Cd-18, ColGa-1, Co16a-2, and SlOO-b to mouse chromosome 10. See Table 1 for the chromosome complements of these somatic cell hybrid clones. For these autoradiograms, band sizes were estimated from standard size markers run on each gel (not shown). Upper left: Pfkl probe hybridizes to a 1.5-kb hamster (Ha) band (lane 1) and 2.3- and 1.8-kb mouse (MS) bands (lane 2) in TqI-digested genomic DNAs. The mouse bands are absent in clone III-16 (lane 3) but present in clones I-2 (lane 4) and I-7B-4 (lane 6). A hamster polymorphism for the probe explains the absence of the genomic hamster band in the hybrids. Upper center: Cd-18 probe detects a 6.6-kb Ha (lane 1) and a 5.3-kb MS (lane 2) band in BarnHI-digested DNAs. The mouse band is present in clones I-7B-4 (lane 3) and I-2 (lane 4) but not in III-16 (lane 6). Lower left: ColGa-1 probe shows 12-kb Ha (lane 1) and 9.4-kb MS (lane 2) bands in Hi&II-digested DNAs. The mouse band is present in I-7B-4 (lane 3) and I-2 (lane 4), but is absent from III-16 (lane 5). Lower center: Co16a-2 probe hybridizes to a 25-kb Ha (lane 1) and an 18kb MS (lane 2) band in HindIII-digested DNAs. The mouse band is present in I-7B-4 (lane 3) and I-2 (lane 4), but is absent from III-16 (lane 5). Right: SlOOb probe detects an 8-kb Ha (lane 1) and a 2.2-kb MS (lane 2) band in BarnHI-digested DNAs. The mouse band is absent from I-3A-2 (lane 3), present in I-7B-4 (lane 4) and I-2 (lane 6), and absent from III-16 (lane 6). These and additional data, summarized in Table 1, assign all five genes to mouse Chromosome 10.

band 21q22 of human Chromosome 21 in the past (Burmeister et aZ., 1990; Chu et al., 1987; Kishimoto et al., 1987; Levanon et al., 1987; Marlin et al., 1986; Weil et cd, 1987). We reported initial assignment of ColGa-1, Co16a-2, SlOOb, and Cd-18, the murine homologs of four of these latter five human genes, to mouse Chromosome 10 using a panel of somatic cell hybrids segregating mouse chromosomes (MacDonald et al., 1988). Subsequent linkage studies by another group, using a single interspecific backcross of (C57B/6J X Mus spretus)Fl X C57B/6J mice showed no recombination between SlOOb, ColGa-1, and Co16a-2 in 97 progeny, giving a 95% upper confidence limit that the three genes are separated by no more than 3 CM (Justice et al, 1990). This same study showed linkage of these three genes to other mouse Chromosome 10 markers, placing them 0.9 CM distal to Bcr (breakpoint cluster region, human homolog on Chromosome 22q) and 7.4 CM proximal to Pah (phenylalanine hydroxylase, human homolog on Chromosome 12q22-q24), in the middle of mouse Chromosome 10. We report here the new assignment of the gene Pfkl, as well as the details of the assignment of the

four genes Cd-18, ColGa-1, Co16a-2, and SlOOb to mouse Chromosome 10 using a panel of seven Chinese hamster-mouse somatic cell hybrids segregating known mouse chromosomes. We also show physical mapping data for these five markers derived from pulsed-field gel electrophoresis. Our results support conservation of gene order and spacing between these markers in the two species and define regions of syntenic homology between mouse Chromosome 10 and the most distal 3000 kb of human Chromosome 21q. This finding has significance for further efforts to construct mouse models of human trisomy 21 (Down syndrome) and suggests novel approaches to the identification of conserved genes in this region. MATERIALS

AND

METHODS

Somatic Cell Hybrid DNAs Chinese hamster-mouse somatic cell hybrids segregating mouse chromosomes were made by polyethylene glycol-mediated fusion of mouse spleen cells or peritoneal macrophages to an established Chinese hamster cell line (380-6). Seven independent cell

MOUSE

CHROMOSOME

10: HUMAN

CHROMOSOME

TABLE Assignment Mouse Somatic cell hybrid clone III-16 I-3A-2 I-7B-4 I-2 VI-6 I-8-5 ADCT-25

ColGa-1

1

of ColGa-1, Co16a-2, SlOO-b, Cd-l& and Pfkl to Mouse Chromosome Using Somatic Cell Hybrids bands

present0

Mouse

Co16a-2

Cd-18

SlOOb

Pfkl

1

2

3

-

-

-

-

-

(+)

+ +

+

+ + -

+ + -

+ +

+ +

-

319

21 HOMOLOGY

+++ + -

-

4

5

6

7

9

10

+++++ +

+ +

(+) +

8

chromosomes

+

+ +

11

10

present’

12

13

14

15

16

17

18

19

X

+ +

+ +

+

(+) + +

+ +

+

+ + +

+ + +

+

+ +

+

2”

+ + +

16’

+

+ +

Note. The distribution of mouse-specific restriction fragments among these Chinese hamster-mouse somatic cell hybrids of known mouse chromosome complements assigns five loci to mouse Chromosome 10. ’ A + indicates the presence and a - the absence of mouse-specific restriction fragments in the corresponding somatic cell hybrid DNA. The scored fragments are noted in Fig. 1. * The mouse chromosome complements of these Chinese hamster-mouse somatic cell hybrid cell lines were determined by isozyme and/or karyotype analysis and by analysis of hybrid DNAs with DNA probes specific for 15 different mouse chromosomes (Refs. (57)). A + indicates the presence of an intact chromosome; a (+) indicates the presence of a partial or fragmented chromosome. Hybrid VI-6 retains a T(16;2)28H mouse translocation chromosome, while hybrid ADCT-25 retains the reciprocal T(2;16)28H translocation chromosome.

lines, previously described (Cox and Epstein, 1985; Cox and Palmiter, 1983; Cox et al., 1982; Joyner et al., 1985), were used in this study. Together, these hybrid lines contain the full haploid complement of murine TABLE

2

Mouse DNA Fragment Sizes Recognized by Probes on PFGE

Not1 MluI EagI Sal1

Cd-18

ColGa-1

Co16a-2

SlOOb

--1400 --1100 L.M. 540 260

1400 1100 L.M. 175

L.M. N.D. 575 590 440 370 -650

L.M. L.M. 150 625

L.M.

L.M. N.D. 575 590 440 400 650

L.M. N.T.

50 N.T.

85 33 35

ss 85 50

90

BssHII WI SfiI

Pfkl

+ x/m1

L.M. N.T.

350 150

Note. The approximate sizes, in kb, of bands recognized by each of five probes in genomic mouse (A9) DNA digested with seven enzymes are shown. Underlined bands are shown in Fig. 2 and are the fragments used to construct the mapped segments shown in Fig. 3. L.M. denotes limiting mobility; hybridizing bands at or above this molecular size were not considered in the data analysis. N.T. denotes not tested, N.D. denotes that no discernible distinct bands were seen with the particular probe. A listing of the same size band for two probes for a given enzyme means that exactly superimposable bands were seen on autoradiograms when the two probes were sequentially hybridized to the same PFGE filter. Multiple bands are seen in partial digests. Sizes were estimated from the known sizes of yeast chromosomes and X multimers run as well-resolved markers on each gel.

chromosomes except for Chromosome 11 and the Y Chromosome. At the time of DNA preparation, the cell lines were characterized by isozyme analysis and/ or karyotype. Additionally, DNA from each hybrid was analyzed for the presence or absence of mouse chromosome-specific DNA sequences representing 15 different mouse chromosomes (Cox and Epstein, 1985; Cox et al., 1982). DNA was prepared from newly confluent tissue culture dishes of the seven hybrid lines as well as from genomic hamster (380-6) fibroblasts and genomic mouse (A9) fibroblasts grown in Dulbecco’s modified Eagle’s medium with 10% fetal calf serum and penicillin-streptomycin in a 5% CO, atmosphere. Cells were lysed and digested with proteinase K and extracted with phenol-chloroform, and the DNA was precipitated with ethanol. DNAs were digested with HindHI, BamHI, or TaqI restriction endonuclease (BRL) according to the manufacturer’s directions, and 10 pg of digested DNA per lane was subjected to agarose gel electrophoresis on 0.8% gels in 1X TBE (SeaKem ME agarose). DNA was depurinated with HCl, hydrolyzed with NaOH, transferred to nylon membranes (MS1 Magnagraph) by capillary blotting, and immobilized by ultraviolet cross-linking. Hybridization

Probes

A 2.0-kb EcoRI insert specific for ColGa-1 and a 2.5-kb EcoRI insert specific for Co16a-2 were originally derived from a human placental cDNA library (Weil et aZ., 1987). A 700-bp EcoRI/HindIII fragment specific for the SlOOb gene was isolated from a rat

320

MACDONALD,

Not-l

1

- MILI-I

2

34

~Eag-I . ,-,I

56

-

Sal-l cl,

7

a

BssH-2 ,.- c\I

9

IO

Sfi-I c\I

11 12

FIG. 2. Physical linkage of marker pairs by PFGE. Genomic mouse (AS) DNA was digested with rare-cutting enzymes, separated by PFGE, and blotted to nylon membranes which were then sequentially hybridized to the probes shown. Several membranes were used in this figure, with each pair of lanes showing sequential hybridization of two probes to the identical blot. Sizes were estimated from yeast chromosome (YP 148 and S. pombe) and X multimers run as markers on each gel. In all cases, the markers were well resolved in the region of the depicted bands. Partial digests give multiple bands. Pfkl (lane 1) and Cd-18 (lane 2) hybridize to a 1400-kb Not1 fragment (4) and to an llOO-kb MluI fragment (4, lanes 3 and 4). ColGa-1 and Co16a-2 share a 575 kb EagI fragment (4, lanes 5 and 6) and 590-kb (4) and 440-kb (4) SalI partial digest fragments (lanes 7 and 8). ColGa-1 hybridizes to a 400-kb SaZI fragment in lane 7 that is distinct from the 370-kb Sal1 fragment detected by Co16a-2 in lane 8. A 650-kb BssH2 fragment (4, lanes 9 and 10) is also shared by ColGa-1 and Co16a-2. Co16a-2 and SlOOb share an 85-kb SfiI fragment (4, lanes 11 and 12) which upon double digestion with XhoI gives a 35-kb Co16a-2 fragment and a 50-kb SlOOb fragment (Table 2, data not shown). Conditions of PFGE varied for the individual lane pairs; see text for details.

brain cDNA clone (Allore et al., 1988). A l.O-kb EcoRI insert for Cd-18 and corresponding to the 5’ end of the gene (Mp-9) was originally derived from a human tonsil cDNA library (Kishimoto et al., 1987). A 2.9-kb EcoRI/BamHI fragment specific for Pfkl was derived from a human cDNA library (Levanon et al., 1987). Each of these DNA inserts was radiolabeled with [a32P]dCTP to a specific activity of at least 2 X lo* cpm/microgram DNA using a nick-translation kit (BRL) or random hexamer primed labeling (Pharmacia) . Pulsed-Field

Gel Electrophoresis

Low-melt agarose blocks (BRL) containing 1 X lo6 cells in an 80-~1 block were prepared from cultured mouse fibroblasts (A9) and from spleens of littermate C57B/6J male mice. Cells in the blocks were lysed and digested with proteinase K and SDS in 0.5 M EDTA, treated with PMSF to inactivate the protease, and stored in 0.5 M EDTA. Blocks were rinsed in TE prior to digestion with restriction endonucleases. Digestion was carried out with 20 units per block of rare-cutting

CHU,

AND

COX

restriction enzymes (NotI, SalI, SfiI, BssHII, XhoI, EagI, and MZuI, all New England Biolabs) with the appropriate buffers. Partial digests ranged from 30 min to 4 h; complete digests were 18-20 h. Blocks were then loaded into gels of 1.0% agarose (BRL Ultrapure) in 0.5~ TBE and subjected to OFAGE or CHEF pulsed-field electrophoresis (Pharmacia/LKB Pulsaphor Plus System). Two general size ranges of fragments were separated; one from 50 to 1100 kb and one from 500 to 2500 kb, in comparison with standard markers (Schwartz and Cantor, 1984). In general, electrophoresis was carried out at 12°C 70-165 V, for 36 h to 11 days with stepped programs of switch intervals. DNA was transferred after depurination and alkaline hydrolysis to a nylon membrane (MS1 Magnagraph) and immobilized by ultraviolet cross-linking. Several filters were prepared in each size range, but probes were considered to share a same-sized fragment only if they sequentially hybridized to an identical band on the same filter. Probe Hybridization Somatic cell hybrid and pulsed-field gel filters were prehybridized for 6-12 h at 53-58°C in hybridization buffer (200 mM NhHPO, pH 7.2,l mhf EDTA, 1% BSA, 7% SDS, 15% formamide) and then hybridized with the radiolabeled probes (>2 X 10’ cpm/pg; 2-5 X lo5 cpm/ml) in the same buffer for 18-24 h at 5358°C depending on the probe. Filters were then washed at a final stringency of 0.2X SSC and 0.1% SDS at the temperature of hybridization for 1 h. Hybridized filters were exposed to X-ray film at -70°C with intensifying screens for 2-10 days. Probes were removed from filters by rinsing in 0.1X SSC at 85°C for 20 min, and removal of probe was ensured by blank X-ray film exposure of the stripped filters before hybridization of the next probe. RESULTS Assignment of Five Genes to Mouse Chromosome Using Somatic Cell Hybrids

10

The data used to map ColGa-1, Co16a-2, Cd-18, Pfkl, and SlOOb to mouse Chromosome 10 are shown in Fig. 1 and Table 1. The ColGa-1 probe hybridizes to a 12-kb fragment of HindIII-digested genomic Chinese hamster DNA and fragments of 9.4- and 5.5-kb in HindIII-digested genomic mouse DNA. The 9.4-kb mouse band is seen in somatic cell hybrid clones I-7B4 and I-2, localizing this mouse-specific sequence to one or more of mouse Chromosomes 1,2,5,7,10,14, 15, 18, 19, or X retained in these two hybrids. Of these, only Chromosomes 2 and 10 are present in both hybrids. Neither mouse band is seen in any of the remaining hybrid DNAs, three of which carry an in-

MOUSE

Mouse

Chromosome

CHROMOSOME

10:

lo:

HUMAN

Pfkl

CHROMOSOME

Col6a-1

Cd-18

321

21 HOMOLOGY

Co16a-2

1400 KB Not-I

85

1100 KB Mlu-I

KB Sfi-I

35 KB Sal-I+Xho-I 650

SlOOb

KB Sal-I+Xho-I

50

KB BssH-2

590 KB Sal-I 440 KB Sal-I 575 KB Eag-I Human Chiromosome 21:

CRYAl PFKL

CD18

2500

1500

2000

COL6Al 800

COL6A2 600

SlOOB 200

telomere 0 (KB)

FIG. 3. Mouse PFGE map and comparison to human Chromosome 21. The top line shows the order and minimum intermarker distances in kb, as determined by PFGE, for the five mouse Chromosome 10 loci listed in Table 2. Below the composite map of mouse Chromosome 10 are the key DNA fragments from Table 2 used to construct the map. The total size of the regions of mouse Chromosome 10 homologous to human Chromosome 21 encompasses approximately 1966 kb of DNA. The orientation of the Pfkl-Cd-18 segment with respect to the Co16a-l-Co16a-2-SlOOb segment, as well as physical linkage of the two mapped segments, is not determined with certainty from the data, and the orientation of the entire region on mouse Chromosome 10 cannot be stated. The homologous region of human Chromosome 2lq, consisting of the most telomeric 3066 kb (2), is displayed below the mouse map for comparison.

tact Chromosome 2 and none of which carry any of Chromosome 10, eliminating all possible chromosomes among these except mouse Chromosome 10. The expected hamster band is seen with equal intensity in all hybrid clones. The 5.5-kb mouse band, light in the genomic mouse DNA, is too faint in the hybrid cell lines to permit its unequivocal assignment to a particular mouse chromosome, but in no case is it seen to segregate independently of the 9.4-kb band. A similar analysis of the data for Co16a-2, SlOOb, Cd-l& and Pfkl, all of which share the same pattern of hybridization to the somatic cell hybrid DNAs, assigns these sequences to mouse Chromosome 10 as well. PFGE Map of Pfk-L, Cd-18, ColGa-1, Col6a-2, and SlOOb on Mouse Chromosome 10 Because of their proximity on human Chromosome 21 (Cox et al., 1990) and their colocalization to mouse Chromosome 10, the five markers Cd-18, Pfkl, ColGa1, Co16a-2, and SlOOb, seemed good candidates for physical mapping using PFGE techniques. PFGE mapping requires that markers be colocalized to large DNA fragments produced by digesting DNA with rare-cutting endonucleases. Such DNA fragments were prepared from genomic mouse DNA (A9 fibroblasts and C57B/6J spleens) digested with enzymes giving average DNA sizes of 50 to 2000 kb. Enzymes used in this study were NotI, &%I, BssHII, SalI, EagI, MU, and XhoI. Large DNA fragments were separated using PFGE in either CHEF or OFAGE modes to produce gels separating DNA in two size ranges: 50

to 1100 and 500-2500 kb. The gels were blotted onto nylon membranes and successively hybridized to the labeled probes. Table 2 lists the size bands obtained for each DNA probe with the six enzymes used in this study. Several enzymes gave partial digests, resulting in multiple bands. In such cases, colocalization of probes was inferred only if the same bands were recognized by both probes and if the relative intensities of the bands were the same for the two probes. Two probes are definitely linked and ordered as adjacent if they hybridize to identical fragments on the same PFGE filter in digests with each of two different enzymes (making comigration of nonidentical fragments less likely), if they produce the same pattern and relative intensities of bands in a partial digest with a single enzyme, and if the other probes tested do not hybridize to the same band. Possible rearrangements in the cultured cell line A9 were excluded by testing C57B/6J spleen DNA in selected cases. In all cases tested, no differences were seen between the two sources of DNA (data not shown). Figure 2 shows the bands shared by pairs of markers from Table 2. ColGa-1 and Co16a-2 share DNA fragments from several different restriction digests. Six hundred fifty-kilobase BssHII, 575-kb EugI, and 590- and 440-kb SaZI bands are shared by ColGa-1 and Co16a-2, placing these markers no greater than 440 kb apart. None of the other probes hybridize to these bands, excluding them from the region between the two collagen probes. Thus ColGa-1 and Co16a-2 are physically linked by strict criteria. Co16a-2 is

322

MACDONALD,

linked to SlOOb by an 85kb SfiI band shared by no other probes. This band, while near the lower size limit of the gel, was distinct and was seen on two different gels probed with Co16a-2 and SlOOb. In addition, double digestion with XhoI and SfiI gave a 35kb band hybridizing only to Co16a-2 and a 50-kb band hybridizing only to SlOOb. The sum of these two bands is the original 85-kb SfiI band shared by the two markers, adding evidence that these two are physically linked (data not shown). Pfkl and Cd-18 share a 1400-kb Not1 fragment and an llOO-kb MuI fragment; no smaller piece was found to bear both markers among the six enzymes tested. The other three probes did not hybridize to either of these fragments on the same filter, excluding a location for them between Pfkl and Cd-18. These data are consistent with two segments demonstrating physical linkage: Pfkl-Cd-18 and ColGa-lCo16a-2-SlOOb. Although an indistinct 175-kb Sal1 partial digest fragment is shared by Cd-18 and ColGa1 and not by the other probes (data not shown), suggesting that the overall order is Pfkl-Cd-18-Co16a-lCo16a-2-SlOOb, the possibility that this represents coincidental migration of two similar-sized pieces of DNA is too great to permit assignment of order based on this piece of data alone. Thus the map shown in Fig. 3 is displayed with a break between Cd-18 and ColGa-1. While these PFGE studies provided evidence for linkage of markers on two segments: ColGa-1-ColGa2-SlOOb and Pfkl-Cd-18, the intermarker distances determined by these studies are less certain. The sum of both regions encompasses about 1900 kb of mouse Chromosome 10, but the exact distances within these regions and the sum of both regions with the segment that connects them are unknown. DISCUSSION The pattern of hybridization of cDNA probes for the o1 and (Yechains of Type VI collagen, the ~3subunit of the glial calcium-binding protein SlOO, the /3 subunit of the Mac1 family of leukocyte surface glycoproteins, and the liver isoform of phosphofructokinase to a panel of seven somatic cell hybrids segregating known mouse chromosomes allows us to assign all five sequences to mouse Chromosome 10. PFGE mapping of these five probes permits construction of a partial physical map, with these five loci spanning approximately 1900 kb of DNA on mouse Chromosome 10. Comparison of the proposed mouse PFGE mapped segments to the human PFGE map (Burmeister, et aZ., 1990) in Fig. 3 is consistent with relative conservation of order and physical distance where data can be compared for the two species. In the mouse, Co16a-2 and SlOOb seem to be somewhat

CHU,

AND

COX

closer and Pfkl and Cd-18 somewhat farther apart than in the human map, but these differences may not be significant given the accepted size variations in comparing different PFGE maps. This conserved region, studied at high resolution, defines a region of homology between mouse Chromosome 10 and the most telomeric 3000 kb of human Chromosome 21q. The PFGE map of mouse Chromosome 10 presented here is partial and does not define with certainty the relative orientation of the two linked segments with one another, nor does it define the orientation of either segment on mouse Chromosome 10. The region surrounding the CD18 locus on human Chromosome 21 proved difficult to map using PFGE (Burmeister et al., 1990) because of the CpG-rich regions surrounding the locus. The human map was constructed by using partial digests with enzymes that are less specific for such CpG-rich sequences. It is interesting to note that the evidence in the current study suggesting linkage of Cd-18 to ColGa-1 in the mouse is a 175-kb SaZI partial digest fragment, and Sal1 is relatively insensitive to CpG “islands.” Efforts are continuing to link the two segments using partial digests with enzymes such as CZuI and NruI, under the assumption that CpG-rich areas may surround Cd-18 in the mouse as they do in human. Efforts are also underway to clone this entire region of mouse Chromosome 10 and the homologus region of human Chromosome 21 to confirm and complete the physical map, isolate new probes, and identify new sequences that are shared between the two species. Comparison of our mapping data to published linkage data for ColGa-1, Co16a-2, and SlOOb on mouse Chromosome 10 confirms the chromosomal assignment of these markers and is consistent with their localization to a small DNA segment, as determined by meiotic mapping (Justice et al., 1990). This also underscores the complementarity of different mapping techniques, using somatic cell hybrids and meiotic linkage to generate large-scale maps and techniques such as radiation hybrid mapping (4) and PFGE to produce finer structure maps. Such highresolution physical maps are valuable in developing more detailed comparisons between regions of mouse-human homology and in defining the exact extent of the synteny. Future efforts to develop mouse models of Down syndrome will need to consider this third region of genetic homology between the long arm of human Chromosome 21 and the mouse genome. If the human Chromosome 21 genes whose homologs lie on mouse Chromosome 10 contribute to the pathophysiology of Down syndrome, their omission from models based solely on mouse Chromosome 16 may be significant. Conversely, models incorporating this small region of

MOUSE

CHROMOSOME

lo:

HUMAN

mouse Chromosome 10 may provide useful insights into the pathogenesis of Down syndrome. ACKNOWLEDGMENTS Our thanks to Margit Burmeister for helpful comments and review of the data, to Suwon Kim and E. Royden Price for technical assistance, and to the following investigators who generously made probes available to us: R. Dunn for SlOOb, T. Kishimoto for CD-18 and Y. Groner for Pfkl. This work was supported by Clinical Investigator Development Award K08 NS01331 from the NINDS of the NIH (G.P.M.), by Grant HD 24610 from the NIH (D.R.C.), and by Grant AR 38912 from the NIH (M.-L.C.).

CHROMOSOME

2.

3.

4.

ALLORE, R., O’HANLON, D., NEILSON, K., WILLARD, H. F., COX, D. R., MARKS, A., AND DUNN, R. J. (1988). SlOO protein beta subunit gene on chromosome 21: Implications for Down syndrome. Science 260: 1311-1313. BLISTER, M., KIM, S. W., DELANGE, T., TANTRAVAHI, U., MYERS, R. M., AND COX, D. R. (1990). A map of the distal long arm of human chromosome 21, constructed using radiation hybrids and pulsed field gel electrophoresis. Genomics 9: 19-30. CHU, M.-L., MANN, K., DJXUTZMANN, R., PFUBULA-CONWAY, D., HSU-CHEN, C.-C., BERNARD, M. P., AND TIMPL, R. (1987). Characterization of three constituent chains of collagen type VI by peptide sequences and cDNA clones. J. Biathem. 168: 309-317. Cox, D. R., BURMEISTER, M., PRICE, E. R., KIM, S., AND MYERS, R. M. (1990). Radiation hybrid mapping: A somatic cell genetic method for constructing high resolution maps of mammalian chromosomes. Science 250: 245-250.

5.

COX, D. R., AND EPSTEIN, C. J. (1985). of human chromosome 21 and mouse N. Y. Acad. Sci. 460: 169-177.

6.

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MACDONALD, G. P., AND Cox, D. R. (1989). The mouse T28H translocation breakpoint occurs in a region of mouse chromosome 16 homologus to human chromosome 21, separating the sequences D21S13 and D21S52 from App, Sod-l, and Ets-2. Am. J. Hum. Genet. 45(Suppl.): A149. [abstract]

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Fine structure physical mapping of the region of mouse chromosome 10 homologous to human chromosome 21.

Comparative mapping of human and mouse DNA for regions of genetic homology between human Chromosome 21 and the mouse genome is of interest because of ...
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