A molecular genetic linkage map of mouse chromosome 13 anchored by the beige (bg) and satin (sa) loci

GENOMICS

6,341-351

(1990)

A Molecular Genetic Linkage Map of Mouse Chromosome Anchored by the Beige (bg) and Satin (sa) Loci’ MONICA

13

J. JUSTICE, COLLEEN M. SILAN, JEFFREY D. CECI, ARTHUR M. BUCHBERG, NEAL G. COPELAND, AND NANCY A. JENKINS’ Mammalian Genetics Laboratory, BRI-Basic Research Program, NCI-Frederick Cancer Research Facility, P. 0. Box B, Frederick, Maryland 2 170 1 Received July 18, 1989; revised September 28, 1989

in the other species. The utility of comparative mapping can be demonstrated by the mouse muscular dystrophy (mdx) mutation, a model of human Duchenne’s muscular dystrophy (Ryder-Cook et al., 1988). In addition, comparative mapping within mammalian species can reveal rates and modes of chromosomal evolution (reviewed by Nadeau, 1989). As molecular markers are oriented on mouse chromosomes relative to phenotypic markers, relationships between cloned probes and phenotypic mutations can be identified. For example, mapping studies initially established close linkage of the c-kit proto-oncogene and the dominant white-spotting locus (IV) (Chabot et al., 1988). Subsequent studies revealed alterations of the c-kit protooncogene in some W mutations (Geissler et al., 1988), suggesting that the c-kit proto-oncogene is the wildtype homolog of W. The utility of murine genetic linkage maps can be expanded by positioning additional molecular markers on the mouse linkage map. Mapping molecular markers requires finding a restriction fragment length variant between the two strains used in the segregation analysis. Although recombinant inbred (RI) strain analysis and intraspecific crosseshave been useful for mapping many phenotypic, molecular, and biochemical markers in the mouse, it is often difficult to find restriction fragment length polymorphisms (RFLPs) for cloned genes among the progenitors of the inbred strains (Klein, 1975; Ferris et al., 1982; Robert et al., 1985; Avner et al., 1988). Interspecific backcross analysis provides a powerful method for mapping large numbers of cloned genes using the same backcross DNA panel (reviewed by Avner et al., 1988). The strength of interspecific backcrosses for mapping molecular markers lies in the high degree of DNA sequence diversity between inbred laboratory mouse strains and wild-derived mice (Avner et al., 1988). The wild mouse speciesMus spretus has been widely used for interspecific backcross

A molecular genetic linkage map of mouse chromosome 13 was constructed using cloned DNA markers and interspecific hackcross mice from two independent crosses. The map locations of Ctlu-3, Dhfr,

Fim-1, 4/12, Hexb, Hilda, Inhba, Lamb-1.13, Ral, Rrm2-pe3, and Tcrg were determined with respect to the beige (bg) and satin (sa) loci. The map locations of these genes confirm and extend regions of homology between mouse chromosome 13 and human chromosomes 5 and 7, and identify a region of homology between mouse chromosome 13 and human chromosome 6. The molecular genetic linkage map of chromosome 13 provides a framework for establishing linkage relationships between cloned DNA markers and known mouse mutations and for identifying homologous genes in mice and humans that may be involved in disease PrOCeSSeS.

Q IS90

Academic

Pra*.

Inc.

INTRODUCTION

Genetic linkage maps are necessary for understanding the organization and evolution of mammalian genomes. Murine genetic linkage maps can be useful for comparative mapping studies, for correlating molecular markers with phenotypic mutations, and for determining linkages of new markers. Comparative mapping of human and mouse genomes can be used to identify candidate genes for mouse mutations that may represent useful models of human disease.The identification of conserved chromosomal segments in mouse and human allows one to predict the locations of mutations or syndromes in one species based on their locations 1 The U.S. Government’s right to retain a nonexclusive royaltyfree license in and to the copyright covering this paper, for governmental purposes, is acknowledged. * To whom reprint requests should be addressed.

341 Copyright 0 1990 AU rights of reproduction

o&38-7543/w $3.00 by Academic Press, Inc. in any form reserved.

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mapping because it will interbreed with laboratory mice (Mus musculus and Mus domesticus) to produce fertile F1 females, even though it is evolutionarily distantly related to laboratory mice (Bonhomme et al., 1984). This evolutionary distance can be a disadvantage in determining recombination frequencies and gene order if DNA rearrangements have accumulated between the Mw species during their divergence. However, the only rearrangement detected thus far between M. spretus and M. domesticus is a small inversion in the proximal region of chromosome 17 (Hammer et al., 1989). We used interspecific backcross analyses to construct a molecular genetic linkage map of mouse chromosome 13. Prior to this mapping study, no anchor loci identified by molecular markers had been placed on mouse chromosome 13. Anchor loci are necessary to orient newly mapped loci relative to the existing mouse genetic linkage map. Loci whose positions are well known from three-point crosses and extensive data are considered anchor loci (Davisson et al., 1988). The T-cell receptor y-chain (Tcrg) locus was placed on the mouse genetic linkage map (Davisson et al., 1988) (Fig. 3), but was not mapped accurately enough to be considered an anchor locus. For example, a map distance between Tcrg and beige (bg) was obtained in a two-point backcross (Holcombe et al., 1987), but gene order could not be determined. In addition, Tcrg was mapped in RI strains to the proximal end of chromosome 13 near a spermatocyte-specific histone locus (Hid), but again, gene order could not be determined (Owen et al., 1986). Several other molecular markers have been mapped to mouse chromosome 13, but not accurately enough to be considered anchor loci. Interspecific backcrosses were used to map a common site of viral integration in Friend murine leukemia virus-induced myeloblastic leukemias (Fim-I), a hepatitis B virus transgenic integration site (4/12), and the cytotoxic T-cell lymphoid antigen-3 (C&-3), as well as Tcrg, on chromosome 13 (Sola et al, 1988). Dihydrofolate reductase (Dhfr), inhibin PA (Inhba), /3-hexosaminidase, P-chain (Hexb), and a ribonucleotide reductase M2 subunit pseudogene (Rrm2-ps3) have been placed on chromosome 13 by somatic cell hybrid analysis (Killary et al., 1986; Barton et al., 1986; Yang-Feng et al., 1987). Thus, only loci identified by a phenotypic change can be used as anchor loci to create a map of mouse chromosome 13. To generate a molecular genetic map of chromosome 13, we oriented the eight molecular markers described above relative to the anchor loci bg and satin (sa). The bg and sa loci can be identified by their effect on the mouse coat (see Materials and Methods). Both bg and sa have been mapped extensively in intraspecific crosses with respect to each other and to other loci on chromosome 13, and thus are considered anchor loci (Lyon and Meredith, 1969; Davisson et al., 1988; Lyon,

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AL.

1988). In our effort to create a comprehensive molecular genetic map of the mouse genome, we also mapped a RAS-like protein (Ral), a human interleukin in DA cells (Hilda), and laminin Bl-chain sequences (Lamb1.13) to chromosome 13. The results of these studies confirm and extend regions of homology of synteny between mouse chromosome 13 and human chromosomes 5 and 7 (Killary et al, 1986), as well as confirm a region of homology between mouse chromosome I3 and human chromosome 6 (Sola et al, 1988; Van Cong et al., 1989). MATERIALS

AND

METHODS

Mice The interspecific backcross [(C57BL/6J X M. spretus) X C57BL/6J] was performed at the NCI-Frederick Cancer Research Facility, Frederick, Maryland, as described previously (Buchberg et al., 1988, 1989). Inbred SB/Le mice homozygous for the bg and sa loci were obtained from The Jackson Laboratory, Bar Harbor, Maine. Homozygous bg/bg mice have pale ears and tail and a light undercoat (M. C. Green, 1981). Homozygous sa/sa mice have a silky coat with a high sheen (M. C. Green, 1981). The interspecific backcross [(SB/Le-bg sa/bg sa X M. spretus) X SB/Le-bg sa/bg sa] was performed at the NCI-Frederick Cancer Research Facility. Mice from this backcross were examined for the beige and satin coat phenotypes. DNA samples were prepared from each animal for subsequent linkage analyses. DNA

Isolation

and Southern

Blot Analyses

High-molecular-weight genomic DNAs were prepared from frozen mouse tissues as described (Jenkins et al., 1982). Restriction endonuclease digestions, agarose gel electrophoreses, Southern blot transfers, and hybridizations were also performed as described (Jenkins et al., 1982), except that Zetabind (CUNO, Inc.) membrane was used for Southern blotting. The pXM6R (Hilda) probe was hybridized using the following low stringency procedure (Maniatis et al., 1982) as modified: Blots were prehybridized in 40% formamide, 5X SSCP, 5X Denhardt’s, 1% SDS at 37°C for at least 2 h; then hybridized with an [a-32P]dCTP-labeled probe in 40% formamide, 5X SSCP, 1X Denhardt’s, 1% SDS, 10% dextran sulfate for 2 days at 37°C; and washed twice in 2X SSCP, 1% SDS for 30 min/wash at room temperature, twice in 0.2~ SSCP, 0.1% SDS for 1 h/wash at 42°C and twice in 0.2X SSCP, 0.1% SDS for 1 h/wash at 65°C. Probes were labeled with [a-32P]dCTP (Amersham) using a nick-translation kit (Amersham) for whole plasmids or a multiprime DNA labeling kit (Amersham) for gel-purified fragments as indicated in the

LINKAGE

MAP

OF

MOUSE

TABLE Molecular

Markers

Mapped

CHROMOSOME

1

in Interspecific

Backcross Restriction

Locus

Gene

name

Tcrg

T-cell

receptor

Znhba

Inhibin

@A

Ral

RAS-like

Rrm2-ps3

Ribonucleotide reductase subunit pseudogene-

Fim-1

Friend integration myeloblastic leukemia-l

4112

Hepatitis integration

B virus

transgenic

Lamb-1.13

Laminin

Bl-chain

homolog

Dhfr

Dihydrofolate

Hilda

Human cells

y-chain

protein

reductase

interleukin

Hexb

/3-Hexosaminidase,

ctkl-3

Cytotoxic T-cell antigen-3

in DA

j3-chain lymphoid

M2

343

13

Mice

fragment

sizes (kb)

Enzyme

C57BL16J

SB/Le

Mus

PstI TacrI

4.5, 4.0, 3.1 ND

ND 10.0, 6.2

4.9, 4.0 10.0, 6.2, 4.7

TaqI

12.5, 8.5, 1.9, 1.6, 0.6

12.5, 8.5, 1.9, 1.6, 0.6

11.0, 9.4, 7.8, 7.1, 6.6, 5.7, 1.9, 1.6, 1.1

Hind111 TwI

10.0, 8.0, 6.4, 1.9, 1.5 ND

ND 4.9, 3.4, 2.0

12.0 ,L,LI 7 7 2 6 1.9, 1.5 7.J, 4.9, 2.0

Tag1

4.6, 2.8, 2.5, 2.3, 1.7, 1.0, 0.8, 0.7, 0.6, 0.5

ND

15.5, 3.1, 2.8, 2.5, 2.1, 1.9,l2, 1.0,0.7, 0.6, 0.5

BamHI

17.0

17.0

20.0

1.1

1.1

15 -L

PVUII HincII Tad

3.4, 3.2, 1.9, 1.2 9.0, 6.2, 4.6, 2.5 ND

ND ND 2.5, 1.5, 0.9

3.2, 1.9,l5, 1.2 10.1,4.0*, 2.1*, 1.7* a, 2.5, 1.5, 0.9

PUUII TaqI

4.8, 3.2, 1.9 ND

ND 7.6, 1.7, 0.6

3.2 , 2 .,9 2 .,5 2.0 14.0, 6.1, 2.1, 0.6

Hind111 EcoRI

6.3, 5.8, 3.7 10.0, 7.7, 6.3,5.5,

ND ND

TapI

ND

9.4, 7.6, 3.8, 2.9, 1.9, 1.7, 1.6, 1.0

629 0 4A, 1 2.8’ 7.1, 6.3, 5.5, 5.4, 4.0, 3.5, 2.8 10.3, 9.4, 7.6, 4.9, 3.8, 2.9, 1.9, 1.7, 1.6, 1.0

BamHI

10.2, 7.6

10.2, 7.6

105 ;*A

BamHI

8.0

8.0

2’10 0 if9 0 8.0

3.6, 2.6

spretus’

51

Note. The 11 loci identified by molecular probes are shown with the RFLPs used for determining-segregation in the backcross progeny. Where two enzymes are given, one RFLP was mapped in the (C57BL/6J X M. spretw) X C57BL/6J backcross and the other was mapped in the (SB/Le-bg sa/bg sa X M. spretus) X SB/Le-bg sa/bg sa backcross, with the following exceptions. The segregation of Lamb-1 was followed in the C57BL/6J backcross with two enzymes, PuuII and HincII. The segregation of Hilda was followed in the C57BL/6J backcross with two enzymes, Hind111 and EcoRI. a The underlined restriction fragment(s) indicates the segregating M. spretus allele(s) typed in the analysis. ND, not determined. * These RFLPs segregated with markers on chromosome 12.

probe description. Southern blot filters hybridized with 32P-labeled probes were routinely washed three times in 0.5X SSCP, 0.1% SDS for 30 min/wash at 65°C. Filters hybridized with the Inhba and Ral probes were washed three times in 1X SSCP, 0.1% SDS for 30 min/ wash at 65°C. Southern blot filters were erased in 0.05 N NaOH, 0.1X SSC, 1% SDS at 65°C for 30 min, followed by neutralization in two washes of 0.2 h4 Tris (pH 7.5), 0.1X SSC, 0.1% SDS at room temperature. Probes The RAS-like cDNA AurII-PstI

protein (Ral) probe was a simian fragment cloned in pGEM3 (pGra/

AP; Chardin and Tavitian, 1986). The T-cell receptor y-chain (Tcrg) probe was a gel-purified 180-bp EcoRI mouse cDNA fragment encoding a portion of the ychain constant region (pGEMIC; Heilig et al., 1985). The inhibin /?A (Inhba) probe was a human EcoRIXbaI cDNA fragment (Hu-Inhib-DA, Mason et al., 1986). The ribonucleotide reductase M2 subunit (Rrm2ps3) probe was a mouse P&I cDNA fragment cloned in pUC18 (Thelander and Berg, 1986). The Friend integration myeloblastic leukemia (Fim-I) probe was a mouse genomic PuuII fragment cloned in pBR322 (167~~15; Sola et al., 1988). The hepatitis B virus transgenic integration site (4/12) probe was a mouse

344

JUSTICE

28 25

ET

AL.

1

2

9

9

9

10 8

G

1

0

FIG. 1.

Segregation of loci in 108 (C57BL/6J X M. spretus) X C57BL/6J interspecific backcross progeny. Genes mapped in the analysis on the left. Each column represents the chromosome identified in the Nz progeny that was inherited from the (C57BL/6J X M. spretus)F1 parent. The shaded boxes represent the presence of a C57BL/6J allele, and the white boxes represent the presence of a M. spretus allele. The number of offspring inheriting each type of chromosome is shown at the bottom. Only 108 chromosomes were typed for all 11 markers. are shown

genomic &u&A fragment cloned in Bluescribe (pBCu412; Hadchouel et al., 1987). The laminin Bl-chain probe (Lamb-l) was a mouse BstEII-EcoRI cDNA fragment cloned in pUC9 (pPE386; Barlow et al., 1984). The dihydrofolate reductase (Dhfr) probe was a gel-purified 1.65kb PstI fragment from a mouse partial cDNA clone (pDHFR11; Chang et al., 1978). The &hexosaminidase p-subunit probe (Herb) was a mouse EcoRI cDNA fragment cloned in Bluescript (pflHEX54; O’Dowd et al., 1985). The human interleukin in DA cells probe (Hi&) was a human cDNA XhoI fragment cloned in pXM (pXM6R; Moreau et al., 1988). The cytotoxic T-cell lymphoid antigen-3 probe (Ctlu-3) was a gel-purified 0.4-kb EcoRI fragment from a mouse cDNA clone (p9AlO; Brunet et al., 1986). Statistical Analysis Standard errors of the recombination frequencies from the results of interspecific backcrosses were calculated as described by E. L. Green (1981). Gene order was determined by minimizing the number of multiple crossovers over the length of the chromosome and confirmed by the maximum likelihood analysis (Bishop, 1985). The differences in recombination frequencies between various interspecific backcrosses described below were analyzed using a 2 X 2 table which compares proportions in independent samples (Snedecor and Cochran, 1980). The segregation patterns of SB/Le and M. spretus and of C57BL/6J and M. spretus alleles were analyzed by the x2 test for variations from Mendelian ratios in transmission of alleles; however, none of the segregants exhibited significantly different transmission ratios. Such differences have been noted for loci on other chromosomes in interspecific backcross analyses (Seldin et al., 1989; Siracusa et al., 1989; Ceci et al., 1989). RESULTS

The (C57BL/6J X M. spretus) X C57BL/6J Interspecific BUC~CFOSS We mapped 11 loci on an existing DNA panel of N2 animals from a (C57BL/6J X M. spretus) X C57BL/6J

backcross (Buchberg et al., 1989). C57BL/6J and M. spretw DNAs were digested with several restriction enzymes and analyzed by Southern blot hybridization with each of the 11 probes listed in Table 1. At least one informative RFLP was identified for each probe. The segregation of the M. spretus allele(s) detected by each probe was followed in 2108 N2 progeny by Southern blot analysis (Fig. 1). Each backcross animal was either homozygous for the C57BL/6J allele or heterozygous for the M. spretus and C57BL/6J alleles at each locus. Although only 108 animals from the C57BL/6JM. spretus backcross were analyzed for every marker and are shown in the segregation analysis (Fig. l), up to 187 animals were typed for somemarkers. Each locus was analyzed in pairwise combinations for recombination frequencies between loci using the additional data. The total number of mice exhibiting crossovers/ the total number of mice analyzed for each pair of loci and the determined gene order are [Tcrg-O/117-Inhbu0/130-Ru1-0/117-Rrm2-ps3]-3/122-Fim-1-32/182[Lamb-l.13 - O/163 - 41121- 21/124 - [Dhfr - O/118 Hilda -O/118-Hexb]-16/124-C&3. Brackets indicate loci that were not separated by crossovers. The map distance + the standard error (+ SE) between each group or pair of loci is centromere-[Tcrg, (Inhbu), (Ral), (Rrm.%p&)]-2.5 + 1.4 CM-Fim-l-17.6 f 2.8 CM[Lamb-1.13, (4/12)]-16.9 f 3.4-[Dhfr, (Hexb), (Hi&z)]12.9 & 3.0 CM-C&S. Map distances are given from the first locus within brackets; other loci are placed within parentheses. The (SB/Le-bg sa/bg su X M. spretus) X SB/Le-bg su/ bg sa Interspecific Bachcross To orient our molecular map with existing genetic maps of mouse chromosome 13, we analyzed a second interspecific backcross in which the markers bg and sa were segregating (Table 1 and Fig. 2). All of the loci mapped in the first interspecific backcross except Rrm2-ps3 were oriented relative to the bg and su loci using this panel of 64 animals. The number of mice

LINKAGE

MAP

2712 FIG. 2. Segregation of loci in 64 in the analysis are shown on the left. Le-bg sa/bg sa X M. spretus)Fi parent. of a M. spretus allele. The number of

OF

1

MOUSE

10

CHROMOSOME

3

0

1

4

345

13

3

4

4

1

0

4

4

(SB/Le-bg sa/bg sa X M. spretus) X SB/Le-bg safbg sa interspecific backcross progeny. Genes mapped Each column represents the chromosome identified in the NP progeny that was inherited from the (SB/ The shaded boxes represent the presence of an SB/Le allele, and the white boxes represent the presence offspring inheriting each type of chromosome is shown at the bottom.

exhibiting crossovers/the total number of mice for each pair of loci and the determined gene order are centromere - bg - 2164 - Tcrg - O/64 - Inhba - O/64 - Ral- 3164 Fim-1-l/64-sa-7/64-[Lamb-1.13-O/64-4/121-8/64Dhfr-O/64-H&a-l/64-Hexb-8/64-C&-3. The map distance (+ SE) between each group or pair of loci is centromere-bg-3.1 f 2.2 CM-[Tcrg, (Inhba), (Ral)]4.7 _+ 2.6 CM-Fim-l-l.6 f 1.6 CM-sa-10.9 + 3.9 CM[Lamb-1.13, (4/12)]-12.5 + 4.1 CM-Dhfr-1.6 + 1.6 CMHexb-12.5 + 4.1 CM-Ctla-3. Combined Results The data from the two interspecific backcrosses showed no statistically significant differences in recombination frequencies or gene order. All results from the C57BL/6J-M. spretus and SB/Le-bg sa-M. spretus backcrosses were combined (Table 2) to establish map distances (&SE) between markers as follows: centromere-bg-3.1 + 2.2 CM-[Tcrg, (Inhba), (Ral), (Rrm2ps3)]-3.3 +- 1.3 CM-Fim-l-1.6 f 1.6 CM-sa-10.9 f 3.9 CM-[Lamb-1.13, (4/12)]-15.4 + 2.6 CM-[Dhfr, (Hilda)]-0.5 + 0.5 CM-Hexb-12.8 f 2.4 CM-Ctla-3 (Table 2 and Fig. 3). No crossovers separated Hilda, Dhfr, and Hexb in the C57BL/6J-M. spretus backcross. One crossover in the SB/Le-bg sa backcross separated Hexb from Dhfr and Hilda, allowing us to place Hexb distal to Dhfr/HiZda. No crossovers were detected between Hilda and Dhfr in 182 Nz progeny, giving an upper 95% confidence limit of 1.6 CM between the two loci. No crossovers were detected between (1) the Tcrg and Inhba loci in 181 Nz progeny, giving an upper 95% confidence limit of 1.6 CM between the two loci, (2) the Tcrg and Rat loci in 176 progeny, giving an upper 95% confidence limit of 1.7 CM, or (3) the Tcrg and Rrm2ps3 loci in 117 progeny, giving an upper 95% confidence limit of 2.5 CM. No crossovers were detected between Lamb-l.13 and 4112 in 227 Nz progeny, giving an upper 95% confidence limit of 1.3 CM between the two loci.

DISCUSSION

The mapping results from the two interspecific backcrosses described here provide the orientation of 11 molecular markers relative to the bg and sa loci on mouse chromosome 13. The molecular and phenotypic markers mapped in this study span 48 CM of chromosome 13, and thus extend over much of the predicted 68 CM length of the chromosome (Green et al., 1972; Roderick and Davisson, 1981). The placement of molecular markers on chromosome 13 now gives molecular access to discrete regions along the entire length of chromosome 13. For example, the Tcrg, Inhba, and Ral markers map between bg and sa in the region that contains extra-toes (Xt), a mutation that affects head and limb development (Johnson, 1967). In addition, the 4/l 2 and Lamb-l. 13 markers map in the central region of chromosome 13 that contains the muted (mu), dumpy (dpy), and flexed-tail (f) mutations. The mu mutation affects coat color and otoliths (Lyon and Meredith, 1969), the dpy mutation affects bone development and reproduction (Hollander, 1981), and the f mutation affects erythroid hematopoiesis (Gruneberg, 1942). This molecular genetic linkage map will be useful for placing additional molecular markers on chromosome 13 using the (C57BL/6J X M. spretus) X C57BL/ 6J interspecific backcross. Additional molecular markers may be candidates for mutations on chromosome 13; among these are bg, which has been proposed as a model of the human Chediak-Higashi syndrome (Owen et al., 1986, Holcombe et al., 1987). Thus, the molecular genetic linkage map of chromosome 13 provides a framework for establishing linkage relationships between additional cloned DNA markers and known mouse mutations and for identifying homologous genes in mice and humans that may be involved in disease processes.

346

JUSTICE

ET

TABLE

TV&!

k Tcrg

Genetic

Distances

Inhba

Rd

3.1 + 2.2

3.1 f 2.2

3.1 + 2.2

(Z/64)

W’W

(2/W

0.0-1.6

0.0-1.7

-

@/181) (O/176) Inhba

Rd

between from

0.0-2.5 (O/117)

0.0-1.5 W194)

0.0-2.4

-

0.0-2.5

Kwm (O/117)

Rrm%ps3

-

2

Markers on Mouse Chromosome Two Interspecific Backcrosses

Rrm2-ps3 ND

AL.

Firm1

sa

+ 3.4 W’W 3.3 t 1.3 w1m

9.4 k 3.6

7.8

6/W 6.3 t 3.0

(4/W 3.6 k 1.3 6.3 + 3.0 U/193) (4/W 3.4 f 1.2 6.3 +-3.0 W23.9 M/W 2.5 + 1.4

ND

W122) 1.6 f 1.6

Fim-I

U/64)

Lamb-l.13 + 5.0

20.3

W/W 18.2 i 2.9 (33/181)

19.0 f 2.8

4112

* 5.0 (13/W 18.2 i 2.9

20.3

(33/181)

34.4 * 5.9 (22/64)

46.9 k 6.2

k 4.6 (41/112)

34.3 T 3.5 (62/181)

45.9 * 3.7

46.1 * 3.6 (89/193)

36.6

41.4 + 3.7 (721174)

35.2 f 3.5 (64/182)

48.8 + 3.5 (101/207)

18.9 k 3.5 (23/122)

18.9 k 3.5 (23/122)

35.6 + 4.4 (42/118)

36.5 + 4.5 (42/115)

39.0 + 4.5

46.1 + 4.5 (57/122)

16.3 + 2.4

14.6 f 2.4 (31/212)

30.9 * 3.4

37.7 If- 3.8 (63/167)

31.4

k 3.4 (59/188)

44.1

23.4

25.0 k 5.4 (16/64)

37.5

(15/W 19.4 + 2.9 (35/180)

16.0 k 2.7 (30/188)

31.8

20.3 IL 3.3 (30/148)

15.8 f 2.7

(29/183)

30.0 f 3.2 (62/207)

0.5 + 0.5

12.8

(40/246)

4112

-

-

0.0-1.3

68/1tW + 5.3

(W.W k 2.6

15.4

(O/227) cw1w 15.3 + 2.7 CWlW

-

-

23.4

+ 5.3

O-l.6

KWW -

-

(62/183)

(46/118)

(w38) 0.5 f 0.5

(l/182) Herb

-

-

-

-

(83/181)

34.6 f 3.6 (63/182)

(43/220)

-

-

(30/64)

19.5 f 2.7

Lamb-I.13

-

f 5.9

cww

20.7 f 2.6 (52/251)

U/W

HiMa

_t 3.5 (61/181)

33.7

32.8

ctkl-3

33.9 + 3.5

10.9 + 3.9

-

@l/W

Hexb

36.8 + 4.3 (46/125)

U/W -

-

f 5.9

32.8

Hilda

33.3 f 3.5 (61/183)

10.9 f 3.9

Dhfr

Dhfr

18.8 + 2.8 (37/197)

(38/200)

-

sll

13 as Determined

-

-

-

-

f 3.4 (94/213) f 6.1

W/W + 3.2 (69/217)

k 2.4

CW1W 13.3 f 2.8 (19/143) 12.8 zk 2.4

WWW Note. Data from the (C57BL/6J X M. spretus) X C57BL/6J and (SB/Le-bgsa/bg sa X M. spretus) X SB/Le-bg sa/bg sa interspecific backcrosses were combined to establish map distances between loci. For each pairwise combination of genes, recombination frequencies were calculated as the number of backcross animals that exhibited crossovers between the two loci divided by the total number of animals typed for both loci (numbers in parentheses). The recombination frequencies are reported as genetic distances in centiMorgans (&SE). When no crossovers were detected, the distance is reported from zero to the upper 95% confidence limit. ND, not determined.

Comparison with Previous Mapping Studies An important consideration in interspecific backcross mapping studies is whether the data are consistent with mapping results obtained by recombinant inbred strain analyses and intraspecific crosses,as well as by cy-togenetic and somatic cell hybrid analyses. Although few molecular markers had been oriented on chromosome 13 prior to this study, the data obtained in our studies are consistent with previous mapping results. For example, the map distance between bg and Tcrg obtained in this study (3.1 f 2.2 CM) is similar to the map distance obtained using an intraspecific mouse backcross (2.5 f 1.4 CM, Holcombe et aZ., 1987). The map distance between bg and sa obtained in our studies (9.4 f 3.6 CM) is also consistent with map distances reported previously (6.2 CM f 1.4, Lyon and Meredith, 1969; 9.0 + 2.8 CM, St. Amand and Cupp, 1958). In addition, CtZa-3 has been localized to the distal end of

chromosome 13, band D, by in situ analysis (Sola et al., 1988; Lyon, 1988), consistent with our interspecific backcross map location. Although these data suggest that no major rearrangements involving these markers have occurred on chromosome 13 during the evolutionary divergence of SB/Le, C57BL/6J, and M. spretus, other more subtle rearrangements cannot be ruled out. Ctla-3, Fim-1,4/12, and Tcrg have been mapped previously in interspecific backcross mice, giving map distances WE) and gene order as follows: centromereTcrg-14.5 + 5.0 CM-Fim-l-20.4 + 5.7 cM4/12-33.3 f 6.8 CM-CtZu-3 (Sola et al., 1988). Our results are consistent with this gene order, although our data place Fim-1 much closer to Tcrg (3.3 f 1.3 CM). Analysis of the difference in recombination frequencies between Tcrg and Fim-1 in the two mapping studies using a 2 X 2 table (Snedecor and Cochran, 1980) indicates that the recombination frequencies are significantly differ-

LINKAGE

MAP

OF

MOUSE

CHROMOSOME

13

347

3.1

,Gz?h&a,h&l, Rrm.+x?

3.3 1.6

Lamb-J.f3.

4/Z

128

FIG. 3. Linkage maps of mouse chromosome 13. The chromosome on the left shows the loci mapped in the current study, with distances between loci given in centiMorgans. The chromosome on the right shows the January 1989 version of the chromosome 13 linkage map compiled by M. T. Davisson, T. H. Roderick, A. L. Hillyard, and D. P. Doolittle and provided from GBASE, a computerized database maintained at The Jackson Laboratory, Bar Harbor, Maine. This map is based largely on genetic crosses among laboratory mouse strains and RI strain analyses. The two linkage maps were aligned at the Tcrg locus. Boxed loci indicate genes previously placed on the mouse map that were included in the interspecific backcross analysis. Mouse genes that have been mapped in humans are underlined. Locations of these genes on human chromosomes are shown in the middle; arrowheads point to the locus that has been mapped in humans. Parentheses around the human location indicate that the location of the structural locus in the mouse has not been determined. 9n humans, TCRG has been mapped to 7~15, INHBA to 7~15-13, and RAL to 7~22-15.

ent (P < 0.05). Only 48 animals were analyzed for the Tcrg and Fim-1 loci in the Sola et al. (1988) study, whereas 181 animals were analyzed for the two loci in this study. The Sola et al. (1988) study and one interspecific backcross in this study used C57BL/6J and M. spretus. However, the two M. spretus strains used in the two studies may be different, since they are derived from two independent M. spretus isolates (Sola et al., 1988; Buchberg et al., 1988, 1989). It is possible that a chromosomal rearrangement involving Tcrg was present in the M. spretus isolate used previously. Alternatively, the Sola et al. (1988) study used a T-cell receptor y-chain variant region probe, whereas this study used a T-cell receptor -y-chain constant region probe. The T-cell receptor variant and constant chain regions are believed to be closely linked, as are other immu-

noglobulin and T-cell receptor loci (Kranz et al., 1985); however, the map distance has not been determined. In previous studies, Elliott (1987) used somatic cell hybrids and a mouse Lamb-l probe to map laminin Bl-chain-related sequences to mouse chromosome 12 and a second unidentified mouse chromosome. Seldin et al. (1989) have subsequently mapped Lamb-l to mouse chromosome 12. We detected several RFLPs in HincII digests of the interspecific backcross mice (Table 1). Three of these RFLPs segregate to the same locus on chromosome 12, which we presume is the Lamb-l locus identified by Seldin et al. (C. M. Silan, B. C. Cho, N. G. Copeland, and N. A. Jenkins, unpublished results). One of the polymorphisms segregates to chromosome 13. We also detected TuqI and PuuII RFLPs that map to chromosome 13 (Table 1). We have

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designated the Lamb-l-related sequences that map to chromosome 13 Lamb-1.13. At this time, it is unclear which is the structural Lamb-l locus. It is also unclear whether the additional locus is a pseudogene or a Lambl-related structural gene. A laminin gene closely related to Lamb-l (s-luminin) has recently been described (Hunter et al., 1989). The chromosomal location of sluminin is distinct from the two loci detected by the mouse Lamb-l probe (M. J. Justice, J. P. Merlie, N. G. Copeland, and N. A. Jenkins, unpublished results). Therefore the polymorphisms detected by the Lamb-l probe do not identify the s-laminin locus. Comparative Mapping Mouse Chromosome

of the Proximal 13

Region of

Two of the loci mapped previously to the proximal region of chromosome 13, Tcrg and Hid, have been mapped to human chromosome 7~15 and 7q22, respectively (Murre et al., 1985; Chandler et al., 1979). Two of the loci mapped in this study to the proximal region of chromosome 13, Inhba and Ral, have been mapped to human chromosome 7~15-13 and 7~22-15, respectively (Barton et al., 1986; Rousseau-Merck et al., 1988). The human homolog of Lamb1 has been mapped to human chromosome 7, band q22-31 (Modi et al., 1986; Pikkarainen et al., 1987). If Lamb-l.13 is the mouse homolog of human LAMBl, the region of mouse chromosome 13 exhibiting homology with human chromosome 7 spans nearly 20 CM. However, this potential region of homology would be interrupted by the Fim-1 locus, which maps to human chromosome 6~23 (Van Cong et al., 1989). The new map locations of Ral and Inhba near Tcrg (upper 95% confidence limits, 1.6 and 1.7 CM, respectively) identify a region of synteny on the proximal end of chromosome 13 that is conserved on the short arm of human chromosome 7 (see Fig. 3). Several investigators have suggested that the mouse mutation Xt is the homolog of the human Greig cephalopolysyndactyly syndrome (GCPS) (Winter, 1988; Winter and Huson, 1988, Brueton et al., 1988). This hypothesis is based on linkage of Xt and GCPS with Tcrg in mouse and humans and on morphological similarities between the mouse mutation and the human disease. GCPS maps to human chromosome 7~13 (Brueton et al., 1988). Humans affected by the dominant GCPS have extra digits and cranial malformations (Winter and Huson, 1988). Heterozygous Xt/+ mice have extra digits on the feet and many have an extra interfrontal bone in the skull; homozygous Xt/Xt mice die either shortly before or at birth and have paddle feet, abnormalities of the extremities, brain, and spinal column, and edema (Johnson, 1967). The identification of this syntenic group on chromosome 13 that is conserved on

ET

AL.

human chromosome 7p gives strong support to the suggestion that GCPS and the Xt mutation are homologous. Although Ral, Inhba, and Tcrg have not been mapped directly with Xt, their locations between bg and sa indicate that they may be useful markers to gain molecular access to Xt and possibly to the human GCPS locus. Comparative Mapping Mouse Chromosome

of the Distal Region of 13

The map locations of Dhfr and Hexb on the distal region of chromosome 13 identify another region of synteny that is conserved in mouse and human. Arylsulfatase B (As-l) has also been mapped to the distal region of chromosome 13 (Elliott et al., 1985) (Fig. 3). The human homologs of these loci are syntenic on the long arm of chromosome 5. DHFR maps to human 5qll.l-13.2, HEXB maps to 5q13, and arylsulfatase B (ARSB) maps to 5qll-13 (Anagnou et al., 1988; Dana and Wasmuth, 1982; Fidzianska et al., 1986). Notably, DHFR, diphtheria toxin sensitivity (DTS), chromate resistance (CRS ) , emetine resistance (EMTB) , and leucyl t-RNA synthase (LARS) are included in a syntenic group in both humans and Chinese hamsters (Killary et al., 1986; Hellkuhl and Grzeschik, 1978; Gilbert et al., 1975; Dana and Wasmuth, 1982; Creagan et al., 1975). Hexb and As-l have not been mapped in Chinese hamsters. It will be interesting to determine if these seven loci define a conserved syntenic group in all three species. HILDA is a human hemopoietic growth factor that has been identified independently as leukemia inhibitory factor (LIF) that induces macrophage differentiation of the murine Ml myeloid leukemia cell line (Gearing et al., 1987; Gough et al., 1988; Moreau et al., 1988). The evidence that HILDA is identical to LIF is based on cDNA sequence analysis (Moreau et al., 1988). LIF has also been proposed to be identical to a differentiation inhibitory activity (DIA) in embryonic stem (ES) cells (Williams et al., 1988). LIF and DIA are believed to be identical because of biochemical similarities; in addition, both inhibit the differentiation of ES cells in vitro (Williams et al., 1988). The localization of a murine homolog of Hilda near Dhfr would suggest that HILDA or HILDA-related sequences will map to human chromosome 5q. However, a probe for LIF has recently been mapped to human chromosome 22qll12 near the immunoglobulin X 1, light chain (IGL-l), and platelet-derived growth factor-p (PDGFBISIS proto-oncogene; Sutherland et al., 1989). Note in Table 1 that numerous RFLPs are detected by the Hilda probe in both C57BL/6J and M. spretus. Although EcoRI, HindIII, and TaqI detect strongly hybridizing RFLPs that cosegregate with markers on chromosome

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MAP

OF

MOUSE

13, other weakly hybridizing polymorphisms were detected with the Hilda probe. One of these segregated to mouse chromosome 12 (M. Justice, N. Copeland, and N. Jenkins, unpublished results). It is clear that the Hilda probe recognizes more than one locus in the mouse. These loci could be Hilda-related structural genes or pseudogenes. If the Hilda locus that we mapped to chromosome 13 is the murine homolog of the locus mapped to human chromosome 22q, it would suggest that Hildu maps proximal to Dhfr on mouse chromosome 13. Consequently, the region between Dhfr and Hiida would define a breakpoint in mouse between human chromosome 22q and human chromosome 5q homology. In this regard, it is interesting to note that homologs of loci on human chromosome 22 mapping near LIF are widely dispersed in the mouse genome. For example, Igl-l maps to mouse chromosome 16 (Epstein et al., 1986) and PDGFB/sis maps to mouse chromosome 15 (Huppi et al., 1988). Thus, the homologs of genes in this region of human chromosome 22 may be widely dispersed in the mouse genome, and the map location of Hi&z may identify a new region of homology with human chromosome 22 on mouse chromosome 13.

CHROMOSOME

the loci for the three subunits of inhibin, man. Cytogenet. Cell Genet. 46: 578.

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ACKNOWLEDGMENTS We thank R. Barth for the Tcg probe, P. Seeburg and J. Kaumeyer for the In&x probe, P. Chardin for the Ral probe, L. Thelander for the Rrm2 probe, P. Tambourin for the Fim-I probe, C. Pourcel for the 4112 probe, M. F. Seldin for the Lamb-l probe, R. T. Schimke for the Dhfr probe, B. Triggs-Raine for the Herb probe, G. G. Wong for the H&&I probe, and P. Golstein for the Ctla-3 probe. We also thank D. A. Swing, M. B. Cybulski, B. Cho, J. Diem, B. Eagleson, and M. Bodamer for providing excellent technical assistance. D. M. Kingsley, L. F. Lock, and L. D. Siracusa are thanked for critically reviewing the manuscript. This research was supported by the National Cancer Institute, DHHS, under Contract NOl-CO-74101 with BRI. The NCI-Frederick Cancer Research Facility is fully accredited by the American Association for Accreditation of Laboratory Animal Care.

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CHROMOSOME

64. 65.

66.

67.

68.

69. 70.

71.

13

351

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