Linkage of a gene for neural cell adhesion molecule, L1 (CamL1) to the Rsvp region of the mouse X chromosome

GENOMICS

8,

113-118

(1990)

Linkage of a Gene for Neural Cell Adhesion Molecule, Ll (CamLI) to the Rsvp Region of the Mouse X Chromosome VERNE M. CHAPMAN,’

BERNADETTE T. KEITZ, DENNIS A. STEPHENSON, LINDA J. MULLINS,* MARION Moos,t AND MELITTA SctiAcHNwt

Molecular and Cellular Biology Department, Roswell Park Cancer Institute, Buffalo, New York 14263; *Department of Pharmacology, University of Heidelberg, Federal Republic of Germany; and tDepartment of Neurobiology, University of Heidelberg, Federal Republic of Germany Received

December

27, 1989;

Prws,

April 4, 1990

linked to the induction of fascicle formation (Schachner et al., 1985). Ll interacts with itself via a so-called homophilic binding mechanism (Grumet and Edelman, 1988; Kadmon et al., 1990a,b) and is potentiated in its binding capacity by a molecular association with NCAM (Kadmon et al., 1990a,b). Ll is a glycoprotein that belongs to the immunoglobulin superfamily in that it contains six C2 domains and shares three type III domains with fibronectin (Moos et al., 1988; Tacke et al., 1987). Ll is similar if not identical to the nerve growth factor-inducible large external glycoprotein NILE in the rat (Bock et al., 1985) and the neuronglia cell adhesion molecule Ng-CAM, G4, or 8D9 in the chicken (Grumet and Edelman, 1984; Rathjen and Schachner, 1984). Available data indicate that the Ll gene product is encoded by a single structural gene (Tacke et al., 1987). In situ hybridization data on human chromosomes indicated that the Ll gene was located on the human X chromosome at Xq27-Xq28 (Djabali et al., 1989). To verify the X chromosome linkage of the Ll gene in other mammalian species,we have identified restriction fragment variation between diverse Mus species and used that variation to map the position of Ll near the red-sensitive visual pigment locus (Z&up) in a multilecus genetic analysis of backcross progeny. The use of diverse Mus speciesas a source of genetic variation to analyze the segregation and linkage of genesin the mouse genome has proven to be a powerful tool, especially for multipoint linkage analysis of gene order. Our laboratory has applied these methods to the analysis of more than 25 genes on the X chromosome that have been analyzed in the same backcross series of mice. As a consequence,these resources are available to analyze additional genes on the mouse X chromosome in a cumulative manner which establishes the order of genes relative to a large number of markers as well as estimates of intergenic recombinational distances (Mullins et al., 1988, 1990; Chamberlain et al.,

Ll is a glycoprotein with an apparent molecular weight of 200 kDa in the developing fetus and adult central nervous system. In the peripheral nervous system, it has a molecular weight of 230 kDa. The Ll protein appears to be encoded by a single gene that has been located on the human X chromosome by in situ hybridization. In this paper we describe restriction variation in genomic DNA Southern analysis between Mus species for the K13 cDNA probe for the Ll neural cell adhesion molecule. We have designated the locus described by this variation as cell adhesion molecule L 1, CamL 1. The X chromosome linkage and the relative position on the X chromosome coincident with the genes Rsvp/GGpd/Cf-8 were defined in backcross matings involving M. spretus and M. musculus. Q ISSO Academic

revised

Inc.

INTRODUClION

The neural cell adhesion molecule Ll has been shown to mediate aggregation and adhesion between neurons (Keilhauer et al., 1985; Rathjen and Schachner, 1984), granule cell migration in the early postnatal mouse cerebellum (Lindner et al., 1983), fasciculation of neurites (Fischer et al., 1986), and neurite extension on other neurites (Chang et al., 1987) and on Schwann cells (Bixby et al., 1988; Seilheimer and, Schachner, 1988). Because of its inducibility by nerve growth factor in neurons and glial cells of the peripheral mammalian nervous system, it has been hypothesized to be involved in axon regrowth during regeneration (Seilheimer and Schachner, 1987,1988). Ll is specifically expressed by regions of axons undergoing fasciculation (Schachner et al., 1985), suggesting that Ll expression is causally 1 To whom correspondence should be addressed at Roswell Park Cancer Institute, Elm and Carlton Streets, Buffalo, NY 14263. 113

0333-x543/90 $3.00 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.

114

CHAPMAN

1987; Disteche et al., 1989). The methods employed in these studies are essentially the same as those reported by other laboratories using similar Mus species crosses (Amar et aZ., 1985; Avner et al., 1987; Brockdorff et aZ., 1987a,b, 1988; Ryder-Cook et al., 1988). Most importantly, the relative order of X chromosome genes is the same in each of these studies, which increases the validity of the Mu.s species backcrosses for gene mapping. In this report we describe restriction variation in genomic DNA between Mus species for the K13 cDNA probe for the Ll neural cell adhesion molecule. We have designated the locus described by this variation as cell adhesion molecule Ll, CamLl. The X chromosome linkage and the relative position on the X chromosome are defined in a series of Fi and backcross matings, respectively. MATERIALS

AND

METHODS

Mice The mice used in this study are the same as the backcrosses originally described by Mullins et al. (1988). They include the inbred strain C57BL/6Ros, M. musculus from an outbred colony of mice maintained at Roswell Park Cancer Institute since 1973 which were originally trapped in Northern Jutland, Denmark, and M. spretus from an outbred colony maintained in our laboratory since 1979. The latter were derived from mice originally trapped in eastern Spain. In each cross, we used C57BL/6Ros females mated with either M. spretus or M. musculus males. The resulting Fi females are backcrossed to the inbred male, C57BL/6. Thus, the inbred strain dam of the F1 progeny and the inbred sire of the backcross represent a constant genetic background so that the phase of linkage of various alleles is always known in the backcross progeny. Only backcross maleswere used for these analyses to minimize the complexity of restriction fragment patterns seen in segregating progeny. Molecular Analysis Genomic DNA was digested with restriction endonucleases (listed in Table 1). The resulting fragments were separated by electrophoresis on 0.8% agarose gels and transferred to either Zetabind (CUNO) membranes or nitrocellulose according to the method of Southern (1975). The membranes were hybridized with probes that were isotopically labeled by the random priming method (Feinberg and Vogelstein, 1984). Hybridization conditions used for these analyses were 5X Denhardt’s, 4X SSC, 0.1% SDS, and 0.1% sodium pyrophosphate (Sigma) at 60°C. The K13 Ll probe used in this study is an insert of approximately 2.5 kb in length that is found at the EcoRI site of the pGEM2 vector and rep-

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AL.

resents the 5’ end of the Ll transcript. The origin and characterization of this clone are described in Moos et al. (1988). Rsvp is identified by the clone hs-7. The polymorphisms for this gene have been previously described (Mullins et al., 1988). The clone for human FIX, pKT218, is a 3.1-kb insert in the PstI site of a unique pBR322 derivative (McGraw et al., 1985). The variation for Cf-9 was previously defined in Mullins et al. (1988). Genomic variation in the dystrophin locus (Dmd) was defined by the probe MC2-6 (Chamberlain et al., 1987). RESULTS

Restriction fragment variation between the inbred laboratory mouse (BL/6) and different Mus specieswas observed for three of the six endonucleasestested using Southern analysis (Table 1). M. musculus differs from both M. spretus and BL/6 by a BamHI fragment while M. spretus is variant for PstI and PuuII. These results indicate that M. spretus is slightly more diverged from BL/6 than is M. musculus and that these two species have acquired different variants for this locus. The X chromosome linkage of the locus described by PstI variation was ascertained by comparing the restriction patterns of the parental mice, BL/6 and M. spretus, with those of backcross male progeny. We observed that backcross males had fragment sizes that were either BL/6 or M. spretus-like and that these two patterns segregated among males (Fig. 1, lanes 1 and 2). These patterns were uniformly observed for all backcross males and these results indicate that the original parental differences were expressed in a manner consistent with the hemizygous genotype of X chromosome inheritance in males.

TABLE

1

Fragment Sizes (kb) Detected with the CumL.1 cDNA Probe K13Ll in Southern Analysis of C57BL/6Ros, M. spretus, and M. musculus Genomic DNA Digested with the Restriction Endonucleases EcoRI, PstI, PuuII, BgZII, HindIII, and BarnHI Genotype

BarnHI

BglII

EcoRI

HindI

M. muaculus

16 6.6

16 3.9

12 5.9

9.4

M. spretus

16 3.4

16 3.9

12 6.9

9.4

C57BL/6Ros

16 3.4

16 3.9

12 5.9

9.4

Psi9

PuuII

8 3.7 .9 .7 8 4.6 .6 8 3.7 .9 .7

4.4 4.1 3.4 2.5 5.8 3.9 4.4 4.1 3.4 2.5

MOUSE

2.3 -

X CHROMOSOME

LINKAGE

-

PstI FIG. 1. Southern analysis of genomic DNA digested with P&I from M. spretw (S), C57BL/6 (B/6), and (BL/6 X M. spretus) F1 X BL/6 backcross males (1) and (2). Lane 1 is a B/6 allele and lane 2 is an S allele.

The X chromosome inheritance of the variation between M. musculus and BL/6 was confirmed in segregation of BamHI variation in reciprocal F, male progeny from BL/6 and M. musculus dams, respectively (data not sho.wn). We also established that the F1 female progeny of both crosses have identical pheno-

TABLE

CamLl S B B B S S S S B S S B Concordance

Progeny identity no. 85 3 14 80 2 32 4 5 6 24 7 30

6112

2

Genes in a Series of (BL/6 X M. spretus) F1 X BL/6 at Various Sites across the Chromosome Map Where

DXWasM B S S S B B S S B S S B

115

CamLl

types, which were the same as a mixture of the BL/6 and M. muscuhs parentals (data not shown). Thus, the variation observed in both crosses indicated that the CumLl locus identified by the cDNA probe LlK13 was inherited in an X-linked fashion. The position of CamLl on the mouse X chromosome map was established in two steps as outlined previously (Mullins et al., 1990). In brief, the regional localization was initially determined by analyzing the segregation of variation for CamLl in a series of 12 backcross male progeny which were recombinant for one of five intergenie regions spanning more than 75 CM of the X chromosome. The localization was determined from the highest concordance of cosegregation with six loci in these recombinant males (Table 2). We observed that the concordance of segregation with CamLl increased from 6/12 to lo/l2 between the centromeric marker DXWAS70 and Hprt, respectively, and that there was total concordance with the next locus Rsvp (12/12). Cosegregation of CamLl and the more distal X chromosome genesdecreasedto 8/12 and 6/12, respectively. These results indicate that CamLl maps to the Rsvp region of the mouse X chromosome. The specific order of CamLl relative to other genes in this region was further established by analyzing the segregation of CamLl in backcross male progeny that had been previously scored as being recombinant between Cf-9 and RSVP. Overall, 9/100 progeny from crosseswith M. spretus and 7/103 progeny from crosses involving M. musculus were potentially informative about the relative order of CamLl in this region (Table 3). We observed that CamLl cosegregated with Rsvp in 9/9 of the progeny from the (BL/6 X M. spretus) F1 mothers and with 6/6 in the progeny of (BL/6 X M. musculus) F1 females; DNA from one of the seven

2.0 -

Segregation of Car&J with X Chromosome Progeny Which Have Single Recombinations Allele and B Is the BL/6 Allele

OF

Timp X X

S B S S B B S S B S S B S/12

x X

Hprt

Rsvp

A@

S B B B B B S S B S S B

S B B B S S S S B S S B

S B B B S S B B S B s B

10/12

X X

12/12

X X X X

S/12

Backcross Male S Is the Spretus

DXWa.931

x X

S B B B S S B B S B B S 6/12

116

CHAPMAN

TABLE Segregation of Cf-9 and Progeny from [C57BL/6 X C57BL/6 (Cross 1) and musculus-Denmark (D)]F, Chromosome

type

Parent 1 (BL/6) Parent 2 (spretus/musculus)” Recombinant 1, 2’ Recombinant 2, 1

3 RSVP in Backcross Male (B) X M. spretus (S)]F, from [C57BL/6 (B) X M. X C57BL/6 (Cross 2)

Cf-9

RSip

B S/D B S/D

B SD S/D B

Total

CFOSS

1

39 52 6 3 loo

&FOSS 2 41 55 3 4 103

a Where spretus~muscdus refers to either the spretus parent 2 (cross 1) or the musculus parent 2 (cross 2) and the S/D indicates that the male progeny are either S or D at the indicated locus. b 1,2-Cf-9 allotype parent 1, Rsvp allotype parent 2.

progeny was not available for analysis (Table 4). These results indicate a gene order of Cf-9,CamLl/Rsvp with an estimated distance of 7.9 f 1.9 CM between Cf-9 and CamLlIRsvp. In this instance, we have pooled the frequencies of recombination across the region from both crosses and used the combined total of 203 progeny as the total sample size. We also examined the segregation of CamLl among progeny that had recombined between Rsvp and the adjacent locus for dystrophin (Dmd) (Ryder-Cook et aZ., 1988; Chamberlain et aZ., 1987; Brockdorff et.al., 1987a; Heilig et al., 1987). We observed that all four progeny that were recombinant between Rsvp and Dmd had CamLl and Rsvp alleles that were the same strain types. Thus, these data are consistent with an overall order of Cf-9, (CamLl/Rsvp/GGpd/Cf-8), Dmd. The additional genesG6pd and Cf-8 are added because they cosegregate with Rsvp in all 203 progeny. DISCUSSION We have used a panel of 203 backcross progeny previously typed for segregation of more than 25 X chromosome genes to map the locus CamLl for the cell adhesion protein, Ll, identified with the cDNA clone K13. The methodology involved (1) the identification of informative restriction variation among the original parental mice used in the cross, (2) the localization of the CamLl locus to the Rsvp region in a set of 12 progeny that were single recombinants at one of five regions on the X chromosome, and (3) the ordering of CamLl in the indicated region by analyzing all of the available crossovers between Cf-9 and Rsvp for segregation of CamLl. The work required to map CamLl essentially involved five Southern blots and some of these were available for rehybridization from previous analyses. The relative positions of Cf-9, CamLl, RSVP, Dmd were established from the crossover events that we had

ET

AL.

previously identified in these backcross progeny. The coincident segregation of CamLl with the Rsvp/GGpd/ Cf-8 gene cluster was based upon 15 backcross progeny that were recombinant between Cf-9 and the cluster and upon 4 progeny that were recombinant between the cluster and Dmcl out of a total of 203 progeny. Work reported by other laboratories has identified a recombination between Rsvp and GGpd/Cf-8 (Ryder-Cook et al., 1988). Thus, the combined results of the mouse recombination data from this work and the work of others suggest a potential X chromosome gene order of RSVP,CamLl,4Xpd/Cf-8, Dmd where recombination between CamLl, G@d, and Cf-8 is not observed in these data or reported by others. We are currently analyzing additional backcross progeny that will increase the probability of recovering a recombination of genes separated by less than 1 CM to 95% or better. The comparative gene maps of X chromosomes in mouse and humans suggest that the region between Hprt and the cluster, Rsvp/GGpd/Cf-8, is conserved on the human X chromosome at the distal end of Xq from Xq26-28 (Avner et al., 1988; Amar et al., 1988; Mullins et al., 1988; Searle et aZ., 1987). The linkage with Cf-9 (F9) is particularly interesting in light of the conserved order of genes between mouse and human for F9 and F8. These results are consistent with the Xq27-Xq28 localization of the CamLl locus on the human X chromosome and physical mapping information using large fragment electrophoretic characterization of CamLl. These results indicate that Ll hybridizes with CZoI fragments that are the same as the fragment sizes observed with cloned probes for the CBB and GGPD loci (Djabali et al., 1989). Furthermore, the data from the physical mapping of the human gene order suggest a gene order of CBB, CAMLl, GGPD, F8. It is of particular interest that this is the region that has been identified with the fragile X syndrome which has mental retardation as a prominent phenotype in

TABLE

4

Analysis of CamLl Segregation in Backcross Progeny (N) That Were Recombinant between Cf-9 and RSVP from (C57BL/6 X M. spretus) F1 Females (Cross 1) and from (CS?BL/G X M. muaxdus) F1 Females (Cross 2) Source of recombinant Cross

1

Cross

2

N

Cf-3

3 6 3 3

S B B D

Note. DNA from 1 recombinant, was not available for Ll analysis.

CamLl X x X X

Table

RSVP

B S D B 3, between

B S D B Cf-9 and

Rsvp

MOUSE

X CHROMOSOME

approximately 30% of the affected males. The linkage analysis of the fragile X syndrome places it 12.5-25% recombination from F9 with an average recombination of 15% (Nussbaum and Ledbetter, 1986; Oberle et al., 1985). Conversely, cytogenetic analysis of the fragile X against GGPD places the GGPD locus distal to the fragile X site (Szabo et al., 1984). More recently, a combined genetic and physical mapping of the fragile X site has confirmed the relative position of fragile X (FRAXA) proximal to F8 at a distance greater than 2 Mb (Patterson et al., 1989). These data indicate that the gene for Ll is within the fragile X domain and it raises the possibility that mental retardation may be in part a consequence of perturbing the expression of Ll either partially in all cells or entirely in a subset of cells of individuals with the fragile X phenotype and who manifest mental retardation. The K13 Ll cDNA sequences have been studied for relative levels of expression in various tissues and the available data suggest that they have the highest levels of expression in the brain (Tacke et al., 1987). A search of the established genetic map of the mouse X chromosome for possible candidate mutations for this gene indicates that a recessive neurological defect, trembly (ty), mapped near the gene Bent-tail (Bn) and approximately 12 CM proximal to Tu (Taylor et al., 1978). Affected ty males exhibited tremors at 2 weeks and seizures somewhat later. The mutant males did not survive beyond weaning. Unfortunately, this mutation has been lost and a direct test of Ll expression in these mutant mice is not possible (Taylor et al., 1978). Lacking other mutant models the CamLl locus may be a good candidate for targeted recombination mutagenesis to establish the developmental consequencesof a defect at this locus (Hooper et al., 1987; Kuehn et al., 1987; Doetschman et al., 1988; Thompson et al., 1989; Koller et al., 1989). ACKNOWLEDGMENTS The authors acknowledge the primary characterizations done on the M. spretus backcross by Dr. Stephen Grant. The authors also thank Nancy Holdsworth for her assistance in preparing the manuscript. This work was supported in part by Grants GM 33160 and GM 24125 from the National Institutes of Health to V.M.C. and from the Bundesministerium fiir Forschung und Technologie to M.L.S.

LINKAGE

3.

4.

5.

6.

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