A High-Resolution Genetic Map of the Nervous Locus on Mouse Chromosome 8

GENOMICS

48, 346–353 (1998) GE975193

ARTICLE NO.

A High-Resolution Genetic Map of the Nervous Locus on Mouse Chromosome 8 Philip L. De Jager, Diane Harvey,1 Alexandros D. Polydorides, Jian Zuo, and Nathaniel Heintz2 Howard Hughes Medical Institute, The Laboratory of Molecular Biology, The Rockefeller University, New York, New York 10021 Received October 24, 1997; accepted December 17, 1997

The nervous (nr) mutant mouse displays two gross recessive traits: both an exaggeration of juvenile hyperactivity and a pronounced ataxia become apparent during the third and fourth postnatal weeks. Using an intersubspecific intercross, we have established a highresolution map of a segment of mouse Chromosome 8 that places the nr locus in a genomic segment defined by D8Rck1 on the centromeric end and D8Mit3 on the telomeric end. This map position places the nr locus within the BALB/cGr congenic region of the C3HeB/ FeJ-nr strain, confirming the accuracy of our study. We used this map position to identify and evaluate three genes—ankyrin 1, cortexin, and farnesyltransferase— as candidates for the nr gene. These three genes were eliminated from consideration but allowed us to establish the conservation of synteny between the region containing the nr locus and a segment of the short arm of human chromosome 8 (8p21–p11.2). Finally, the incomplete penetrance of the nr phenotype led us to perform a screen for modifier loci, and we present evidence that such a nervous modifier locus may exist on mouse Chromosome 5. q 1998 Academic Press

INTRODUCTION

The Purkinje cell of the cerebellum stands out among other neurons by the size and complexity of its dendritic arbor. It provides a formidable integrating function, as the activity of synapses with an average of 200,000 parallel fibers from granule cells, 1 climbing fiber from an inferior olivary neuron, and many other interneurons is processed to provide the sole output of the cerebellar cortex (Kandel et al., 1991). Yet, this complexity in function is associated with a susceptibility to degeneration in response to a large variety of insults. In most cases, these insults, whether of genetic 1

Current address: Department of Human Genetics, WP26A-3000, Merck Research Laboratories, West Point, PA 19486. 2 To whom correspondence should be addressed at the Howard Hughes Medical Institute, The Laboratory of Molecular Biology, Rockefeller University, 1230 York Ave, Box 260, New York, NY 10021. Telephone: (212) 327-7956. Fax: (212) 327-7878.

0888-7543/98 $25.00 Copyright q 1998 by Academic Press All rights of reproduction in any form reserved.

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or chemical origin, have a global effect on the Purkinje cell population that is exposed. However, one mouse mutant displays an interesting neurodegenerative phenotype: the nervous (nr) mutation results in the catastrophic but selective loss of Purkinje cells. The surviving Purkinje cells are found in characteristic parasagittal stripes that correlate with histochemically defined compartments within the superficially homogeneous cerebellar cortex (Edwards et al., 1994). Since the antigen that defines these compartments, zebrin II, is known to encode aldolase C (Ah et al., 1994), a metabolic enzyme, these compartments are likely to contain a population of Purkinje cells that are metabolically and possibly functionally distinct from their neighbors. Thus, the nr mutation presents an insult to which there is a differential response among Purkinje cell compartments. nervous was first described in 1970 by Sidman and Green as a recessive mutation that arose spontaneously in the BALB/cGr inbred strain and may have an incompletely penetrant phenotype. This mutation affects the central nervous system at several different levels. The earliest behavioral abnormality is observed during the third postnatal week: there is a striking exaggeration of juvenile hyperactivity among nr mice. It is also at this time that a locomotor deficit becomes apparent: nervous mice are ataxic. This ataxia is characterized by mild hesitation during locomotion, a wobbly gait, and occasional falls onto one haunch or the other. Both the hyperactivity and the ataxia appear around postnatal day 25 (P25); however, the hyperactivity resolves quickly and only the ataxia is apparent in the adult mouse (Sidman and Green, 1970). Yet, another behavioral deficit becomes apparent during adult life: nr/nr females are poor mothers. This poor nurturing behavior was first noticed by Sidman and Green (1970), and we observed it in our cross to CAST/ Ei. In the latter cross, a nr/nr sire had no effect on pup viability, but a nr/nr mother gave a poor prognosis to her pups. The earliest histological abnormality observed in nr/ nr mice is a swelling of mitochondria to a rounded shape in certain neurons during the second and third

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postnatal weeks. This rounded mitochondrial shape has been observed in wildtype mice in other tissues such as the liver, where hepatocytes display similar changes following various metabolic stresses (Landis, 1973b). In nr/nr animals, this change appears in many different neuronal cell types in the telencephalon, diencephalon, mesencephalon, and rhombencephalon (Landis, 1973b,c). In most cases, the mitochondria assume a normal shape by the end of the first postnatal month. However, some cell types degenerate during the period in which the abnormal mitochondria are observed; in the cerebellum, approximately 90% of Purkinje cells in the lateral cerebellar hemispheres and 50% of Purkinje cells in the cerebellar vermis degenerate during the fourth postnatal week (Sidman and Green, 1970). It is interesting to note that all Purkinje cells display rounded mitochondria by P15, but that only a portion degenerate within the first postnatal month (Landis, 1973c). By P40, the mitochondria of the surviving Purkinje cells have recovered their normal appearance. Nevertheless, Sotelo and Triller (1979) observed evidence of further Purkinje cell degeneration in mice that were 232 days of age; thus, the early catastrophic loss of Purkinje cells is followed by a slowly progressive phase of neurodegeneration. Degeneration also occurs in another neuronal population: there is a biphasic loss of photoreceptor cells (Mullen and LaVail, 1975; LaVail et al., 1993). Rounded mitochondria are first observed at P6 in photoreceptor cells, but it is only at P13 that degenerating cells are observed (LaVail et al., 1993; White et al., 1993). There is an initial rapid loss of photoreceptor cells between P13 and P19, which is followed by a slower attrition in subsequent months. During the first 2 months, both rods and cones are lost to the same degree, but loss of rods is more prominent during the later phase of neurodegeneration (LaVail et al., 1993). Thus, the nr mutation appears to have several effects in different neuronal populations; the early swelling of mitochondria is associated with an exaggeration of juvenile hyperactive behavior and extensive cell death among the Purkinje cell and photoreceptor populations. A later phase of progressive neuronal degeneration is also apparent in both of these neuronal populations but is not associated with a gross change in behavior in the adult animal except for the abnormal nurturing behavior displayed by nr/nr mothers. Previous mapping efforts by Sidman and Green (1970) placed the nr locus in linkage group XVIII, now known as proximal mouse Chromosome 8. However, the limited resolution of these early mapping efforts prompted us to establish a new genetic map of the region as a prelude to a positional cloning effort. Concurrently, another group took advantage of polymorphisms between the strain of origin (BALB/cGr) and the strain (C3HeB/FeJ) in which the nr mutation has been maintained for 39 generations; these polymorphisms were used to define the BALB/cGr congenic region that contains the nr locus. The D8Mit155–

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D8Mit18 segment contains this congenic region and is estimated to be 5.6 cM in length (Campbell and Hess, 1996). Our own genetic analysis refines the characterization of the genomic region containing the nr locus; this genetic map led us to evaluate ankyrin 1 (Ank1), cortexin (Ctxn), and farnesyltransferase (Fnta) as candidate genes and to investigate this region’s synteny to human chromosomes. Finally, we have extended our genetic analysis to address the question of the incomplete penetrance of the nr phenotype; we present suggestive evidence that a modifier locus on mouse Chromosome 5 affects the expression of the nr phenotype. MATERIALS AND METHODS Mice. Mice used in this study (CAST/Ei and C3HeB/FeJ-nr) were bought from The Jackson Laboratory and maintained at the specificpathogen-free facility at the Rockefeller University Laboratory Animal Research Center under standard procedures. Genomic DNA. Genomic DNA was isolated using standard protocols involving proteinase K digestion of minced tissue followed by extraction with buffered phenol, phenol–chloroform, and chloroform–isoamyl alcohol. Genomic DNA was then recovered by ethanol precipitation. Approximately 1 mg of genomic DNA was used as template per polymerase chain reaction. PCR amplification. The DNA Engine (M. J. Research, Inc.) was used for PCR amplification in this study. The standard 101 PCR buffer (Perkin–Elmer Cetus) was used in all reactions. All PCRs were carried out in 25-ml volume. Samples were processed through an initial denaturation (947C for 4 min), then 35 cycles of denaturation (947C, 30 s), annealing (507C, 30 s), and elongation (727C, 30 s), followed by 10 min elongation at 727C and storage at 47C. Primer pairs were either purchased from Research Genetics (Huntsville, AL) (microsatellite markers from the MIT collection) or designed by our group (D8Rck1 and D8Rck2). A 507C annealing temperature was used for the latter two primer pairs. The D8Rck1 primer pair was designed using the sequence of an intron between exons 41 and 42 of the Ank1 gene (GenBank Accession No. U76758). The oligonucleotide sequences are 5*-CTTTGCTCAGGTTGCAATGC (F) and 5*-GTAGGAAATCACACAGGCAG (R). The D8Rck2 primer pair was designed using the sequence of the 3* untranslanted region of the Fnta gene (GenBank Accession No. D49744). The oligonucleotide sequences are 5*-TCCCGTAAAGGAACTACTGC (F) and 5*-GGCATTAGTTGGTTACAGTG (R). Detection of polymorphisms. For simple sequence length polymorphisms (SSLPs), a standard three-step PCR cycle was used to amplify the desired genomic segment. Amplified products were separated in a 15 1 20-cm 10% acrylamide gel (30:1 acrylamide to bis-acrylamide) in 0.51 TBE at 200 mV for 2.5 h. Single-strand conformation polymorphism analysis was used to visualize the polymorphisms at D8Mit3 and D8Mit222; the protocol described by Vidal-Puig and Moller (1994) using large acrylamide gels with glycerol was used in both cases. Statistical analysis. A standard x2 test was used to determine the significance of our observations: n

x2 Å ∑ iÅ1

(obsi 0 expi)2 . expi

The equivalent P value was derived using the Chiprobe program (Ott, 1991).

RESULTS

Mapping the nr Locus In an effort to establish a genetic map of the nr locus, we initiated a (C3HeB/FeJ-nr 1 CAST/Ei)F1 1 (C3HeB/

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FeJ-nr 1 CAST/Ei)F1 intersubspecific intercross. Since nr/nr females are poor mothers, all of the (C3HeB/FeJnr 1 CAST/Ei)F1 animals were generated using nr/nr males. The progeny of the intercross were phenotyped, and only those animals that clearly demonstrated both the hyperactive and the ataxic symptoms during the fourth postnatal week were used in the haplotype analysis. Of 1678 progeny, only 175 met our criteria and were phenotyped as nr. Thus, only 10.4% of the progeny displayed the recessive phenotype. Since we did not observe dying or dead pups in the intercross litters and a number of mice with borderline symptoms were rejected, we believe that the reduced proportion of affected mice probably reflects an incomplete penetrance of these traits in the mixed C3HeB/FeJ and CAST/Ei background of the F2 animals. The 350 meioses that we selected phenotypically were used to map the nr locus relative to a series of microsatellite markers from mouse Chromosome 8. This mapping effort resulted in the identification of five markers—D8Mit23, D8Mit143, D8Mit171, D8Mit222, and D8Mit257—that cosegregated with the nr locus in all the haplotypes (Fig. 1). In addition, we isolated markers flanking the nr locus centromerically (D8Mit159) and telomerically (D8Mit3). However, a trinucleotide repeat used to map the Ank1 gene (D8Rck1) proved to be the marker closest to the nr locus on the centromeric flank (Fig. 1). The genomic segment defined by D8Rck1 and D8Mit3 contains a total of five recombination events, yielding a genetic distance of 1.4 cM in our CAST/Ei intercross. To confirm the relative positions of the various microsatellite markers and to acquire another estimate of the genetic distance between the flanking markers, we characterized this genomic region using the subset of genomic DNA samples from the London collection of the European Collaborative Interspecific Backcross (EUCIB) that is backcrossed to Mus spretus. This analysis confirmed the relative position of the flanking and the nonrecombinant markers (Fig. 2A) and identified five recombination events between markers D8Mit21 and D8Mit3. The genetic distance between the flanking markers D8Mit21 and D8Mit3 is thus smaller in the EUCIB map (1.2 cM) than in our phenotypic cross (2.6 cM). Such variation in genetic distance occurs because of strain-, subspecies-, and speciesspecific variation in recombination frequencies in small regions of the genome. Accurate estimates of the physical size of the D8Mit21 – D8Mit3 segment will have to wait for the completion of a physical map over this segment. Another confirmation of the validity of our genetic map comes from the Whitehead/MIT Center for Genome Research’s effort to produce a physical map of the mouse genome (Copeland et al., 1993; Dietrich et al., 1994, 1996; Nusbaum et al., 1997). Many yeast artificial chromosomes (YACs) have been isolated by screening a YAC genomic library with microsatellite markers. A complete contig covering the nr locus has

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not emerged yet, but the existing contigs proved to be useful in our effort to characterize the genomic region surrounding the nr locus. They provide an independent confirmation of the relative positions of many of the markers of interest. In return, our genetic map was useful in confirming and correcting the results of the physical mapping effort and in orienting the existing contigs relative to the centromere of mouse Chromosome 8 and to one another (Fig. 2B). Evaluation of Candidate Genes Having refined the position of the nr locus, we identified a large number of genes that are candidates to be the nr gene by their position in the genetic map of mouse Chromosome 8. We have evaluated three of these genes at this time. First, we can eliminate Ctxn, a gene that has been suggested as a candidate for nr, from consideration as it maps 7.8 cM telomeric to D8Mit3 (Watson et al., 1994), placing it out of the genomic segment that contains the nr locus. These mapping data confirm the tentative conclusion of Horvath and colleagues (1996), who sequenced the Ctxn message derived from nr/nr mice and failed to find a difference from the wildtype message. Second, we mapped Fnta, which was previously mapped between D8Bir6 and D8Hun1 (Porter and Messer, 1996), using an SSLP found in the 3* untranslated region of this gene between the C57BL/6 and M. spretus alleles. The EUCIB panel was screened and yielded a map position for Fnta that is telomeric to D8Mit3 (Fig. 2A). This position invalidates Fnta as a candidate for the nr gene. Finally, we considered Ank1, which was a strong candidate since its normoblastic (Ank1nb) allele is associated with Purkinje cell degeneration (Peters et al., 1991). However, a polymorphic trinucleotide repeat located next to exon 41 of Ank1 (marker D8Rck1) maps two recombination events, or 0.6 cM, away from the nr locus (Fig. 1). Conservation of Synteny between Mouse Chromosome 8 and Human 8p in the Vicinity of the nr Locus The conservation of the D8Rck1–D8Mit3 segment’s synteny in the human genome was evaluated by using the map positions of Ank1 and Fnta. Since these genes respectively flank nr centromerically and telomerically and their human homologues are both located on the short arm of human chromosome 8, the human homologue of the nr gene can be said to reside on 8p. More specifically, it will lie between ANK1 (8p11.2) (Lux et al., 1990; Tse et al., 1990) and FNTA (8p22–q11) (Andres et al., 1993). Furthermore, since the region of 8p that is syntenic to mouse Chromosome 8 (mC 8) is bordered by a region of synteny to mC 16 centromerically—C/EBP-d maps to 8p11.2–p11.1 and mC 16 (Cleutjens et al., 1993; Jenkins et al., 1995)—and to mC 14 telomerically—gonadotropin-releasing hormone maps to 8p21–p11.2 and mC 14 (Williamson et

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FIG. 1. Linkage analysis of the nr locus 22 microsatellite markers and 1 novel marker (D8Rck1) developed from an intron of Ank1 were mapped using the 175 animals from the intersubspecific intercross that met our phenotypic criteria. The markers are presented in a column to the left of a diagram of mouse Chromosome 8. The chromosomal segment containing the nr locus is also outlined. To the right of the chromosome are the haplotypes that were used to resolve the position of the markers of interest. The number of haplotypes displaying a specific pattern is displayed above each column. Black boxes represent a CAST/Ei allele, and white boxes represent either a C3HeB/FeJ or a BALB/cGr allele.

al., 1991; Yang-Feng et al., 1986; Bruskiewich et al., 1996)—the human homologue of the nr gene will be found in the 8p21–p11.2 segment. Another Locus Modifies the Expression of the nr Mutation Having observed only 10.4% of nr/nr animals among the progeny of our intercross, we attempted to isolate loci that modify the expression of the nr phenotype. We designed a genetic screen based on the outline proposed by Silver (1995). Thus, we genotyped 36 randomly selected affected intercross animals with 46 markers distributed evenly throughout the genome. Several putative loci were identified in the initial screen. After genotyping the complete collection of phenotypically nr/nr animals at each of these loci,

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three of these were deemed to be false positives. At this time, further characterization presents evidence that there may be a modifier locus on mouse Chromosome 5 (Table 1). Near the center of mouse Chromosome 5, there is a distortion in allele frequencies away from the expected Mendelian distribution; the P values associated with this observation are 0.018 for 1 degree of freedom (df) at D5Mit239 and 0.016 for 2 df at D5Mit275 (Table 1). These P values are relatively large and thus only hint that there may be a locus on mouse Chromosome 5 that influences the expression of the nr phenotype. DISCUSSION

This study of the nr mutation provides a strong base from which to launch further efforts to clone the gene

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FIG. 2. Confirmation of the genetic map. (A) Haplotype analysis of EUCIB. 11 microsatellite markers were mapped using the LS subset of EUCIB animals. In addition, D8Rck2, a marker developed from the Fnta 3* UTR sequence, was also mapped using this panel. Marker names are displayed to the left of a diagram of mouse Chromosome 8. The haplotypes are displayed to the right of this diagram. The number of times a certain haplotype is observed in this collection of animals is displayed on top of each haplotype diagram. Black boxes represent a M. spretus allele, and white boxes represent a C57BL/6 allele. A total of 429 EUCIB animals, the subset of animals backcrossed to M. spretus in the London collection, were screened. (B) Partial physical map of the nr locus. A diagram of mouse Chromosome 8, labeled with the microsatellite markers under study, is found at the top. The genomic segment containing the nr locus is outlined below, and a diagram of three contigs of YACs is found at the bottom. Each of the microsatellite markers that we mapped genetically was used to search the Whitehead/MIT Center for Genome Research’s physical map of the mouse genome database. As a result, we identified two Whitehead YAC contigs, WC8.7 and WC8.8, that respectively flank the nr locus centromerically and telomerically. The nonrecombinant marker D8Mit171 is found on a series of YACs, but these do not overlap with either contig at this time. The relative positions and orientations of the three contigs outlined below this diagram of mouse Chromosome 8 are based on the genetically determined position of the markers that they contain. In the case of WC8.8, the genetic map clarified the relative positions of D8Mit3, D8Mit143, and D8Mit222, which were ambiguous in the physical map.

affected by this mutation. A candidate gene approach is already under way in our laboratory, but a concurrent positional cloning approach is now feasible. Five recombination events remain within the D8Rck1–D8Mit3 segment and should prove useful in refining the genomic segment that contains the nr mutation as markers are isolated from the YAC contig. The strength of the genetic map derived from the current analysis rests on its consistency among the results of various analyses of this locus. The map derived from our intercross was successively confirmed by the EUCIB genetic map and the YAC-based physical map; furthermore, its accuracy was established since it is

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almost completely contained within the BALB/cGr congenic region. Indeed, the centromeric border of the genomic segment containing the nr locus is well within the congenic region that stretches to D8Mit60 (Fig. 3) (Campbell and Hess, 1996). This result was confirmed by D8Rck1, which yielded a BALB/cGr allele when amplified from nr/nr mice. On the other side, the result is less clear since this boundary of the congenic region lies somewhere between D8Mit16 and D8Mit18, neither of which could be mapped during our analysis. However, in the Mouse Genome Database (MGD) D8Mit3 maps telomeric to D8Mit18 (Fig. 3) (MGD, 1997). Thus, a portion of the D8Rck1–D8Mit3 segment is not con-

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TABLE 1 Characterization of a Modifier Locus of the nr Mutation on Mouse Chromosome 5 Marker

Distance from centromere (cM)

Number of animals genotyped

P value with 1 df

P value with 2 df

D5Mit61 D5Mit153 D5Mit361 D5Mit92 D5Mit275a D5Mit239a D5Mit177 D5Mit292

8.0 50.0 50.0 53.0 54.0 58.0 61.0 83.0

36 173 173 173 173 173 95 36

0.76 0.032 0.053 0.053 0.032 0.018 0.042 0.50

0.64 0.043 0.043 0.025 0.016 0.017 0.098 0.19

Note. Initially, 36 nr/nr animals from the phenotypic cross were genotyped using three evenly spaced microsatellite markers (D5Mit61, D5Mit92, and D5Mit292) from mouse Chromosome 5. These data were analyzed using a standard x2 formula for deviations away from the 1:1 ratio of CAST/Ei:C3HeB/FeJ alleles; this analysis is referred to as the 1 df test. We also analyzed the data for deviations away from the expected 1:2:1 distribution of CAST/Ei (Ca) and C3HeB/FeJ (C3) alleles among the animals (Ca, Ca:Ca, C3:C3, C3). This analysis is referred to as the 2 df test. For both tests, the result of the statistical analysis is presented as a P value. When a distortion was observed at D5Mit92, additional animals and loci were investigated. The lowest P values for the 1 df and 2 df tests are outlined in bold. a The paucity of polymorphic markers in this region of mouse Chromosome 5 prevented a more precise localization of the modifier locus. However, the improvement in statistical significance would be minor at best.

tained within the congenic region. Nevertheless, the 5 locus was characterized extensively, but the associproximity of D8Mit3 in the YAC contig to D8Mit143 ated P values (Table 1) do not meet the threshold for (Fig. 2B), another marker confirmed to be of BALB/cGr ‘‘suggestive’’ results proposed by Lander and Kruglyak origin in the C3HeB/FeJ-nr strain (Campbell and Hess, (1995) (2.4 1 1003 for 1 df and 1.6 1 1003 for 2 df in 1996), indicates that at least a large portion of the seg- an intercross). Thus, we should consider our efforts as ment that we have defined as containing the nr muta- having provided a hint that a modifier locus is found tion lies within the congenic region, demonstrating the on mouse Chromosome 5. This hint is strengthened by accuracy of our mapping effort. several supporting facts. First, there is the limitation This study demonstrates that the nr mutation lies of the data set; the original intercross was not designed between D8Rck1 and D8Mit3; unfortunately, we do for the purpose of a genetic screen for modifier loci. not yet have an accurate estimate of the physical size As mentioned earlier, the severity of the nr phenotype of this genomic segment. We can provide an estimate appears to be variable, but only those animals meeting based on the genetic distance separating these two a certain phenotypic threshold were genotyped in the markers; however, such an estimate is prone to error phenotypic cross. Furthermore, within this genotyped because of the variation in recombination frequencies group, no effort was made to grade the severity of the in such small sections of the genome. From the inter- phenotype, since this would not have added any inforsubspecific intercross, D8Rck1 and D8Mit3 are sepa- mation to the mapping of the nr locus. Such a mixed rated by 1.4 cM which, given an average of 2.0 Mb/cM population—and the loss of the unaffected or less af(Silver, 1995), yields an estimated physical distance fected but genotypically nr/nr animals—reduces the of 2.8 Mb for the genomic segment containing the nr resolution of our analysis. In addition, our preliminary locus. Thus, the genomic segment of interest is likely evidence supporting the existence of other modifier loci to be large, emphasizing the need for further refine- on mouse chromosomes 1 and 16 suggests that our ment of the genetic map once markers are isolated analysis is further hampered by the possible additive from the YAC contig. Nonetheless, we have already and interactive effects of multiple loci. What is needed evaluated three candidate genes that map to this re- now is an extension of this study with a much more gion of mouse Chromosome 8, and the genetic map detailed characterization of the various traits associoffers us the opportunity to examine many more. Addi- ated with the nr mutation, both at the gross and at tional candidate genes can now also be identified by the histological level; such a study will be necessary to examining the human 8p21 – p11.2 region, which is confirm the putative locus described here and perhaps cosyntenic with the chromosomal segment that con- to identify additional ones. tains the nr mutation. The cloning of the nr gene will define an interesting In screening for modifier loci that contribute to the stimulus that results in selective degeneration reduced penetration of the nr traits, we isolated loci among the large variety of neurons that are affected. on mouse Chromosomes 1, 5, and 16 that displayed This particular phenotype may provide an insight distortions in the expected Mendelian distribution of into the response of neurons to potentially lethal alleles in our phenotypic cross (P. De Jager and A. stimuli. However, the most interesting trait associPolydorides, data not shown). The mouse Chromosome ated with the nr mutation may be the striking exag-

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FIG. 3. Composite detailing the BALB/cGr congenic region and regions of synteny with the human genome. A diagram of mouse Chromosome 8, which is labeled with a selection of markers and outlines the genomic segment containing the nr locus, is shown. To the left of the chromosome is a diagram that summarizes the information presented by Campbell and Hess (1996): the BALBcGr congenic region is outlined as well as regions that remained undetermined. To the right of the chromosome are diagrams of segments of human chromosomes 13 and 8 that are syntenic to portions of mouse Chromosome 8. Based on the map positions presented in MGD, the closest gene that has synteny to another human chromosome, coagulation factor X (Cf10/F10) (13q34), maps approximately 1 cM from the microsatellite marker that defines the nr locus (MGD; de Grouchy et al., 1984; Koizumi et al., 1995). *The markers labeled with an asterisk were not mapped in this study. Their position in the genetic map of mouse Chromosome 8 was taken from MGD (1997). Overall, only the relative positions of the markers are presented; relative distances between the markers were not taken into account. †The Ank1 allele in nr/nr mice is of BALB/cGr origin, confirming the results of Campbell and Hess (1996) (P. De Jager, data not shown).

geration of juvenile hyperactivity. It is well known that attention deficit and hyperactivity disorders (ADHD) in humans often present transiently, disappearing in adulthood in approximately one-half of cases (Andreasen and Black, 1991). nr may thus be an analog of certain rare forms of inherited ADHD or may implicate certain cellular processes in the generation of ADHD symptoms. ACKNOWLEDGMENTS We thank colleagues in our laboratory and Dr. Wendy K. Chung for their support and comments. P.L.D. and A.D.P. are M.D.–Ph.D.

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candidates supported by the National Institutes of Health/National Institute of General Medical Sciences MSTP Grants GM07739 (P.L.D.) and GM07739 (A.D.P.), while D.H. was and J.Z. is a postdoctoral associate and N.H. is an Investigator of the Howard Hughes Medical Institute.

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