Kif1C, a kinesin-like motor protein, mediates mouse macrophage resistance to anthrax lethal factor

BACKGROUND: Inbred mouse strains exhibit striking differences in the susceptibility of their macrophages to the effects of anthrax lethal toxin (LeTx). Previous data has shown that this difference in susceptibility lies downstream of toxin entry into macrophages. A locus controlling this phenotype, called Ltxs1, has been mapped to chromosome 11, but the responsible gene has not been identified. RESULTS: Here, we report the identification of the Ltxs1 gene as Kif1C, which encodes a kinesin-like motor protein of the UNC104 subfamily. Kif1C is the only gene in the Ltxs1 interval exhibiting polymorphisms between susceptible and resistant strains. Multiple alleles of Kif1C determine the susceptibility or resistance of cultured mouse macrophages to LeTx. Treatment of resistant macrophages with brefeldin-A (which alters the cellular localization of Kif1C) induces susceptibility to LeTx, while ectopic expression of a resistance allele of Kif1C in susceptible macrophages causes a 4-fold increase in the number of cells surviving LeTx treatment. We also show that cleavage of map kinase kinase 3, a target of LeTx proteolysis, occurs in resistant cells. CONCLUSIONS: We conclude that mutations in Kif1C are responsible for the differences in the susceptibility of inbred mouse macrophages to LeTx and that proper Kif1C function is required for LeTx resistance. Since the LeTx-mediated proteolysis of map kinase kinase 3 occurs even in resistant cells, Kif1C does not affect cellular entry or processing of LeTx and likely influences events occurring later in the intoxication pathway.     

A 2-Mb YAC/BAC-based physical map of the ovum mutant (Om) locus region on mouse chromosome 11

The embryonic lethal phenotype observed when DDK females are crossed with males from other strains results from a deleterious interaction between the egg cytoplasm and the paternal pronucleus soon after fertilization. We have previously mapped the Om locus responsible for this phenotype, called the DDK syndrome, to an approximately 2-cM region of chromosome 11. Here, we report the generation of a physical map of 28 yeast and bacterial artificial chromosome clones encompassing the entire genetic interval containing the Om locus. This contig, spanning approximately 2 Mb, was used to map precisely genes and genetic markers of the region. We determined the maximum physical interval for Om to be 1400 kb. In addition, 11 members of the Scya gene family were found to be organized into two clusters at the borders of the Om region. Two other genes (Rad51l3 and Schlafen 2) and one EST (D11Wsu78e) were also mapped in the Om region. This integrated map provides support for the identification of additional candidate genes for the DDK syndrome.     

Ltx1, a mouse locus that influences the susceptibility of macrophages to cytolysis caused by intoxication with Bacillus anthracis lethal factor, maps to chromosome 11

The lethal factor (LF) toxin that is produced by Bacillus anthracis plays an important role in the pathogenesis of anthrax. LF has mononuclear phagocyte-specific intoxicating effects that are not well understood. We have identified genetic differences in inbred mouse strains that determine whether their cultured macrophages are susceptible to the cytolytic effect of LF intoxication. Our identification of resistant and susceptible mouse strains enabled us to analyse crosses between these strains and to map a single responsible gene (called Ltx1 ) to chromosome 11. Ltx1 probably influences intoxication events that occur after the delivery of LF to the cytosol, as all mouse macrophages are killed by polypeptides containing the catalytic domain of Diphtheria toxin fused to the domain of LF required for cytosolic transport. Furthermore, the susceptibility phenotype is dominant to resistance, suggesting that resistance is caused by an absence of or polymorphism in a molecule that acts jointly with, or downstream of, the activity of LF. Our mapping of Ltx1 is a crucial first step in its positional cloning, which will provide more information about the mechanism of LF intoxication.     

Physical and transcriptional map of a 3-Mb region of mouse chromosome 1 containing the gene for the neural tube defect mutant loop-tail (Lp)

The Lp mouse mutant provides a model for the severe human neural tube defect (NTD), cranio-rachischisis. To identify the Lp gene, a positional cloning approach has been adopted. Previously, linkage analysis in a large intraspecific backcross was used to map the Lp locus to distal mouse chromosome 1. Here we report a detailed physical map of this region. The interval surrounding Lp has been cloned in a yeast artificial chromosome (YAC) contig consisting of 63 clones spanning approximately 3.2 Mb. Fifty sequence tagged sites (STSs) have been used to construct the contig and establish marker order across the interval. Based on the high level of conserved synteny between distal mouse chromosome 1 and human 1q21-q24, many of these STSs were designed from expressed sequences identified by cross-screening human and mouse databases of expressed sequence tags. Added to other known genes in the region, a total of 29 genes were located and ordered within the contig. Seven novel polymorphisms were identified within the region, allowing refinement of the genetic map and a reduction in the size of the physical interval containing the Lp gene. The Lp interval, between D1Mit113 and Tagln2, can be spanned by two nonchimeric overlapping YACs that define a physical distance of approximately 1 Mb. Within this region, 10 potential candidate genes have been mapped. The materials and genes described here will provide a resource for the identification and further study of the mutated Lp gene that causes this severe neural tube defect and will provide candidates for other defects known to map to the homologous region on human chromosome 1q.     

A High-Resolution Genetic, Physical, and Comparative Gene Map of the Doublefoot (Dbf) Region of Mouse Chromosome 1 and the Region of Conserved Synteny on Human Chromosome 2q35

The mouse doublefoot (Dbf) mutant exhibits preaxial polydactyly in association with craniofacial defects. This mutation has previously been mapped to mouse chromosome 1. We have used a positional cloning strategy, coupled with a comparative sequencing approach using available human draft sequence, to identify putative candidates for the Dbf gene in the mouse and in homologous human region. We have constructed a high-resolution genetic map of the region, localizing the mutation to a 0. 4-cM (±0.0061) interval on mouse chromosome 1. Furthermore, we have constructed contiguous BAC/PAC clone maps across the mouse and human Dbf region. Using existing markers and additional sequence tagged sites, which we have generated, we have anchored the physical map to the genetic map. Through the comparative sequencing of these clones we have identified 35 genes within this interval, indicating that the region is gene-rich. From this we have identified several genes that are known to be differentially expressed in the developing mid-gestation mouse embryo, some in the developing embryonic limb buds. These genes include those encoding known developmental signaling molecules such as WNT proteins and IHH, and we provide evidence that these genes are candidates for the Dbf mutation.     

A high-resolution genetic, physical, and comparative gene map of the doublefoot (Dbf) region of mouse chromosome 1 and the region of conserved synteny on human chromosome 2q35.

The mouse doublefoot (Dbf) mutant exhibits preaxial polydactyly in association with craniofacial defects. This mutation has previously been mapped to mouse chromosome 1. We have used a positional cloning strategy, coupled with a comparative sequencing approach using available human draft sequence, to identify putative candidates for the Dbf gene in the mouse and in homologous human region. We have constructed a high-resolution genetic map of the region, localizing the mutation to a 0.4-cM (+/-0.0061) interval on mouse chromosome 1. Furthermore, we have constructed contiguous BAC/PAC clone maps across the mouse and human Dbf region. Using existing markers and additional sequence tagged sites, which we have generated, we have anchored the physical map to the genetic map. Through the comparative sequencing of these clones we have identified 35 genes within this interval, indicating that the region is gene-rich. From this we have identified several genes that are known to be differentially expressed in the developing mid-gestation mouse embryo, some in the developing embryonic limb buds. These genes include those encoding known developmental signaling molecules such as WNT proteins and IHH, and we provide evidence that these genes are candidates for the Dbf mutation.     

Construction of a long-range YAC physical map spanning the 10-cM region between the markers D18Mit109 and D18Mit68 on mouse proximal chromosome 18.

Four yeast artificial chromosome (YAC) contigs, physically approximately 8 Mb, have been constructed spanning a 10-cM region on mouse proximal chromosome 18 and include the sites of 21 known genes, including those near the twirler (Tw) locus and the recently isolated Niemann-Pick type C1 (npc1) gene, formerly designated as the spm locus. This physical map consists of 49 YAC clones that cover roughly 15% of the chromosome. The physical order of 38 microsatellite sequence-tagged sites (STSs) could be assembled and confirmed based on their presence or absence in individual YACs, from proximal D18Mit109 through distal D18Mit68. These YACs provide an important resource for the further characterization and identification of known and unknown genes. The physical map has been integrated with our previously published genetic linkage map and showed an average genetic to physical distance of cM/Mb > 1.1.     

A 11 Mb YAC-Based Contig Spanning the Familial Juvenile Nephronophthisis Region (NPH1) Located on Chromosome 2q

A gene (NPH1) responsible for approximately 90% of the purely renal form of familial juvenile nephronophthisis, a progressive tubulo-interstitial kidney disorder, maps to human chromosome 2. We report the construction of a YAC-based contig spanning the critical NPH1 region and the flanking genetic markers. This physical map was integrated with a refined genetic map that restricted the NPH1 interval to about 2 cM; this interval corresponds in a maximum physical distance of 3.5 Mb. The entire contig covers 9 cM between the loci D2S135 and D2S121. The maximum physical distance between these two markers is approximately 11.3 Mb. Forty-five sequence-tagged sites, including six genes, have been located within this contig. PAX8, a member of the human paired box gene family, that is expressed in the developing kidney, was assigned outside the restricted NPH1 critical region and cannot therefore be regarded as a candidate gene. This set of overlapping clones represents a useful resource for further targeted development of genetic markers and for the characterization of candidate genes responsible for juvenile nephronophthisis.     

Mouse H2 congenic intervals: analysis and use for mapping

In this study we exploit the unique genetic resource of inbred mouse major histocompatibility complex (H2) congenic and recombinant strains to construct a high-resolution map of microsatellite loci in and around the H2 region, as well as an independent genetic map of other loci on mouse Chromosome (Chr) 17. Microsatellite loci were analyzed in 11 C57BL/10 (B10) strains to determine the size of the congenic interval in each. The length of the congenic interval found in each strain varied widely. Interestingly, the intervals were generally smaller than statistical expectations. However, the observed congenic intervals were still sufficiently long that these strains and probably wild-derived H2 congenics are an important source of genetic variability. The staggered ends of the various congenic intervals and the recombinants were used to construct the map. This map will be useful for physical cloning and to help localize novel genes. As evidence of the mapping application of congenic strains, locational information was derived about Trp53-ps and Stl.Deceased, Aug. 8, 1994.     

Localization on a physical map of the NKC-linked Cmv1 locus between Ly49b and the Prp gene cluster on mouse chromosome 6.

The Cmv1 locus controls NK cell-mediated resistance to infection with murine CMV. Our recent genetic analysis of backcross mice demonstrated that the NK gene complex (NKC)-linked Cmv1 locus should reside between the Ly49 and Prp gene clusters on distal mouse chromosome 6. We have aligned yeast artificial chromosome (YAC) inserts in a contig spanning the interval between the Ly49 and Prp gene clusters. This YAC contig includes 13 overlapping YAC inserts that span more than 2 megabases (Mb) in C57BL/6 (B6) mice. Since we have identified genomic clones that span the Ly49-Prp gene region, we hypothesize that at least one should contain the Cmv1 locus. To narrow the Cmv1 critical region, we developed novel NKC genetic markers and used these to genotype informative backcross and intra-NKC recombinant congenic mouse DNA samples. These data suggest that Cmv1 resides on a single YAC insert within an interval that corresponds to a physical distance of approximately 390 kb. This high resolution, integrated physical and genetic NKC map will facilitate identification of Cmv1 and other NKC-linked loci that regulate NK cell-mediated immunity.     

Allelic loss mapping and physical delineation of a region harboring a thymic lymphoma suppressor gene on mouse chromosome 16

Our previous mapping of allelic loss in gamma-ray induced thymic lymphomas in F(1) hybrid and backcross mice between BALB/c and MSM strains identified three regions with high frequencies of allelic loss which probably harbor a tumor suppressor gene. One region, Tlsr7, exists near the D16 Mit122 locus on chromosome 16. This study has further localized Tlsr7 by constructing a physical map and scanning a total of 587 thymic lymphomas. The map consists of 13 overlapping BAC clones and isolation of BAC-derived polymorphic probes leads to fine mapping of allelic losses. Eleven lymphomas show informative breakpoints of allelic loss regions relative to the flanking markers on the map. Pulsed-field gel electrophoresis of NotI digests of the clones shows that the commonly lost region is localized within an approximately 300 kb interval near D16Mit192. This map is invaluable to facilitate the identification of genes in the Tlsr7 region.     

An integrated physical, genetic and cytogenetic map around the sunn locus of Medicago truncatula.

The sunn mutation of Medicago truncatula is a single-gene mutation that confers a novel supernodulation phenotype in response to inoculation with Sinorhizobium meliloti. We took advantage of the publicly available codominant PCR markers, the high-density genetic map, and a linked cytogenetic map to define the physical and genetic region containing sunn. We determined that sunn is located at the bottom of linkage group 4, where a fine-structure genetic map was used to place the locus within a approximately 400-kb contig of bacterial artificial chromosome (BAC) clones. Genetic analyses of the sunn contig, as well as of a second, closely linked BAC contig designated NUM1, indicate that the physical to genetic distance within this chromosome region is in the range of 1000 -1100 kb.cM-1. The ratio of genetic to cytogenetic distance determined across the entire region is 0.3 cM.microm(-1). These estimates are in good agreement with the empirically determined value of approximately 300 kb.microm(-1) measured for the NUM1 contig. The assignment of sunn to a defined physical interval should provide a basis for sequencing and ultimately cloning the responsible gene.     

An integrated gene and SSLP BAC map framework of mouse chromosome 11

Physical maps are important resources both in sequencing and in functional analyses of large genomes. Global contig-building approaches are regarded to be more efficient relative to the cumulative outcome of scattered and more localized physical mapping studies accompanying positional cloning. This work is part of an effort to assemble a complete physical map of mouse chromosome 11 in which selection of clones containing specific genetic markers from genomic libraries is the first step in the process. Using a previously developed strategy, we identified 361 bacterial artificial chromosomes (BACs) containing 88 gene markers. Since the linkage positions of markers chosen for these studies are known, the BAC framework obtained is anchored to the genetic map and represents about 13% of the length of the entire chromosome. Together with similar assignments of BACs generated previously using D11Mit markers (Cai et al., 1988, Genomics, 54: 387-397), 36-40% of the chromosome 11 is now assembled into contigs, and these contigs correlate through 51 clones carrying both gene and simple sequence length polymorphism markers.     

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS): high-resolution physical and transcript map of the candidate region in chromosome region 13q11

Autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS or SACS) is a neurodegenerative disease frequent in northeastern Québec. In a previous study, we localized the disease gene to chromosome region 13q11 by identifying excess sharing of a marker allele in patients followed by linkage analysis and haplotyping. To create a detailed physical map of this region, we screened CEPH mega-YACs with 41 chromosome 13 sequence-tagged-sites (STSs) known to map to 13q11-q12. The YAC contig, composed of 27 clones, extends on the genetic map from D13S175 to D13S221, an estimated distance of at least 19.3 cM. A high-resolution BAC and PAC map that includes the ARSACS critical region flanked by D13S1275 and D13S292 was constructed. These YAC and BAC/PAC maps allowed the accurate placement of 29 genes and ESTs previously mapped to the proximal region of chromosome 13q. We confirmed the position of two candidate genes within the critical region and mapped the other 27 genes and ESTs to nearby intervals. Six BAC/PAC clones form a contig between D13S232 and D13S787 for sequencing within the ARSACS critical region.     

Genetic and physical mapping of the Chediak-Higashi syndrome on chromosome 1q42-43.

The Chediak-Higashi syndrome (CHS) is a severe autosomal recessive condition, features of which are partial oculocutaneous albinism, increased susceptibility to infections, deficient natural killer cell activity, and the presence of large intracytoplasmic granulations in various cell types. Similar genetic disorders have been described in other species, including the beige mouse. On the basis of the hypothesis that the murine chromosome 13 region containing the beige locus was homologous to human chromosome 1, we have mapped the CHS locus to a 5-cM interval in chromosome segment 1q42.1-q42.2. The highest LOD score was obtained with the marker D1S235 (Zmax = 5.38; theta = 0). Haplo-type analysis enabled us to establish D1S2680 and D1S163, respectively, as the telomeric and the centromeric flanking markers. Multipoint linkage analysis confirms the localization of the CHS locus in this interval. Three YAC clones were found to cover the entire region in a conting established by YAC end-sequence characterization and sequence-tagged site mapping. The YAC contig contains all genetic markers that are nonrecombinant for the disease in the nine CHS families studied. This mapping confirms the previous hypothesis that the same gene defect causes CHS in human and beige pheno-type in mice and provides a genetic framework for the identification of candidate genes.