GENOMICS zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA 26, 543-549 (19%) A YAC Contig Containing the reeler Locus with Preliminary Characterization of Candidate Gene Fragments I. BAR,* zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA C. LAMBERT DE ROUVROIT,* I. ROYAUX,* D. B. KRt.zww,t C. DERNONCOURT,* D. RUELLE,* M. C. BECKERS,’ AND A. M. GOFFINET*,* *Department of Physiology, FUNDP School of Medecine, Namur, Belgium; and tNational Center for Human Genome Research, National Institutes of Health, Bethesda, Maryland zyxwvutsrqponmlkjihgfedcbaZYXW ReceivedOctober 14, 1994; acceptedJanuary31, 1995 zyxwvutsrqponmlkjihgfedcbaZYXWVUT The zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA reeler mutation in the mouse maps to proximal chromosome 5 and defines a key gene involved in brain development and evolution. No gene product is known, and the locus is currently being characterized by posi- tional cloning. YAC clones corresponding to the closest markers &I&fit61 and D5iWt72 have been isolated. Cloned extremities of the YAC inserts were used to construct a 1.1.Mb contig, a 700.kb fragment of which was shown to contain the reeler locus. The integrity of the contig was verified by physical mapping on geno- mic DNA. The classical allele of the reeler mutation was associated with a 150.kb deletion between D52Mit61 and D5Mit72, while no gross chromosomal anomaly was found in the Orleans allele. Candidate coding sequences were isolated to construct a prelimi- nary transcriptional map of the reeler region. Cosmid clones mapping within the rl deletion revealed a large transcript of more than 11 kb, which was present in normal embryonic brain but barely detectable in ho- moxygous rP1lrZorl embryonic brain, suggesting strongly that it corresponds to the reeler transcript. D 1~15 zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFED Academic Press, Inc. INTRODUCTION The zyxwvutsrqponmlkjihgfedcbaZYXWVUTSRQPONMLKJIHGFEDCBA reeler mutation in the mouse maps to proximal chromosome 5 and defines a key gene involved in brain development. Numerous morphological studies (re- viewed in Goffinet, 1984, 1990) have shown that the reeler gene affects the development of orderly cell pat- terns at almost every location in the central nervous system by acting at the end of neuronal migration. Comparative studies of brain development in reptiles suggest that the reeler gene played a significant role during brain evolution in the synapsid lineage leading from stem reptiles to mammals. Inasmuch as the prod- 1 Present address: Department of Biochemistry, McGill University, Montreal, Quebec, Canada. ’ To whom correspondence should be addressed at the Department of Physiology, FUNDP School of Medecine, 61, rue de Bruxelles, B- 5000 Namur, Belgium. Telephone: 32-81-724277. Fax: 32-81-230391 or 32-81-724280. uct of this biologically interesting gene is unknown, its characterization by positional cloning was undertaken. Mapping studies using intra- and interspecific crosses between reeler and normal mice (Goffinet and Dernon- court, 1991; Dernoncourt et al., 1991; Beckers et al., 1994) showed that two microsatellite markers, D5Mit61 and D5Mit72, are located between 0.0 and 1.0 CM from reeler (95% confidence interval), a distance considered amenable to cloning in YACs. The present study reports the construction of a 1.1.Mb YAC contig containing D5Mit61 and D5Mit72, which is shown ge- netically to span the reeler locus, and the physical map- ping of the region in the classical and Orleans allele of reeler. Several candidate gene fragments encoded by this genomic region have been isolated, two of which belong to a large RNA thought to be the reeler tran- script. MATERIALS AND METHODS YAC isolation and characterization. The Princeton YAC libraries were screened for the presence of the microsatellite loci using PCR, as described (Rossi et al., 1992; Dietrich et al., 1992). Yeast agarose plugs were prepared and analyzed by pulsed-field gel electrophoresis (PFGE) on a CHEF-DRII (Bio-Rad) apparatus, following established protocols (Birren and Lai, 1993). YAC size was determined by PFGE by reference to yeast chromosomes and after hybridization with ra- diolabeled mouse DNA. YAC insert ends were isolated and cloned using either inverse PCR (Arveiler and Porteous, 1991) or vectorette (bubble) PCR (Riley et al., 1990; Green, 1993). Restriction mapping of YACs. YAC DNA was embedded in low- melting agarose plugs (Sea Plaque, FMC) and digested with various rare-cutting enzymes (New England Biolabs), and fragments were separated by PFGE. Both complete and partial digestions were per- formed (Birren and Lai, 1993; Hamvas et al., 1994). Restriction frag- ments were transferred to Hybond N’ (Amersham) membranes and hybridized with segments of pBR322 corresponding to the left and right extremities of the pYAC4 vector. Southern hybridization was carried out as described (Cox et al., 1994). Restriction map of genomic DNA. High-molecular-weight DNA from normal BALB/c and reeler (classical rl and Orleans r1O” alleles) mice was prepared from spleen cells embedded in agarose (Sea Plaque, FMC) (Herrmann et al.,1987). After digestion with rare- cutting enzymes, fragments were separated using PFGE, transferred to nylon membranes, and hybridized with various probes (see Re- sults). Genetic mapping. Some end clones and anonymous DNA frag- 543 0888-7643/95 $6.00 Copyright 0 1995 by Academic Press, Inc. All rights of reproduction in any form reserved.