Microcytic anaemia mice have a mutation in Nramp2, a candidate iron transporter gene.

Although disorders of iron metabolism are prevalent, iron transport remains poorly understood. To address this problem, we undertook a positional cloning strategy to identify the causative mutation in mice with microcytic anaemia (mk). Homozygous mk/mk mice have microcytic, hypochromic anaemia due to severe defects in intestinal iron absorption and erythroid iron utilization. We report the identification of a strong candidate gene for mk, and suggest that the phenotype is a consequence of a missense mutation in Nramp2 (ref. 5), a previously identified gene of unknown function. Nramp2 is homologous to Nramp1, a gene activa in host defense. If Nramp2 is mk, as the cumulative evidence suggests, our findings have broad implications for the understanding of iron transport and resistance to intracellular pathogens.

Identification of a candidate modifying gene for spinal muscular atrophy by comparative genomics.

Spinal muscular atrophy (SMA) is a common recessive disorder characterized by the loss of lower motor neurons in the spinal cord. The disease has been classified into three types based on age of onset and severity. SMA I-III all map to chromosome 5q13 (refs 2,3), and nearly all patients display deletions or gene conversions of the survival motor neuron (SMN1) gene. Some correlation has been established between SMN protein levels and disease course; nevertheless, the genetic basis for SMA phenotypic variability remains unclear, and it has been postulated that the loss of an additional modifying factor contributes to the severity of type I SMA. Using comparative genomics to screen for such a factor among evolutionarily conserved sequences between mouse and human, we have identified a novel transcript, H4F5, which lies closer to SMN1 than any previously identified gene in the region. A multi-copy microsatellite marker that is deleted in more than 90% of type I SMA chromosomes is embedded in an intron of this gene, indicating that H4F5 is also highly deleted in type I SMA chromosomes, and thus is a candidate phenotypic modifier for SMA.

Multigenic control of Listeria monocytogenes susceptibility in mice.

We have used a novel quantitative trait locus model to study the genetics of survival of F2 progeny of susceptible BALB/cByJ and resistant C57BL/6ByJ mice that have been infected with Listeria monocytogenes. This allowed us to map modifiers of L. monocytogenes susceptibility to chromosomes 5 and 13.

A genetic map of the mouse with 4,006 simple sequence length polymorphisms.

We have constructed a genetic map of the mouse genome containing 4,006 simple sequence length polymorphisms (SSLPs). The map provides an average spacing of 0.35 centiMorgans (cM) between markers, corresponding to about 750 kb. Approximately 90% of the genome lies within 1.1 cM of a marker and 99% lies within 2.2 cM. The markers have an average polymorphism rate of 50% in crosses between laboratory strains. The markers are distributed in a relatively uniform fashion across the genome, although some deviations from randomness can be detected. In particular, there is a significant underrepresentation of markers on the X chromosome. This map represents the two-thirds point toward our goal of developing a mouse genetic map containing 6,000 SSLPs.

A YAC-based physical map of the mouse genome.

A physical map of the mouse genome is an essential tool for both positional cloning and genomic sequencing in this key model system for biomedical research. Indeed, the construction of a mouse physical map with markers spaced at an average interval of 300 kb is one of the stated goals of the Human Genome Project. Here we report the results of a project at the Whitehead Institute/MIT Center for Genome Research to construct such a physical map of the mouse. We built the map by screening sequenced-tagged sites (STSs) against a large-insert yeast artificial chromosome (YAC) library and then integrating the STS-content information with a dense genetic map. The integrated map shows the location of 9,787 loci, providing landmarks with an average spacing of approximately 300 kb and affording YAC coverage of approximately 92% of the mouse genome. We also report the results of a project at the MRC UK Mouse Genome Centre targeted at chromosome X. The project produced a YAC-based map containing 619 loci (with 121 loci in common with the Whitehead map and 498 additional loci), providing especially dense coverage of this sex chromosome. The YAC-based physical map directly facilitates positional cloning of mouse mutations by providing ready access to most of the genome. More generally, use of this map in addition to a newly constructed radiation hybrid (RH) map provides a comprehensive framework for mouse genomic studies.

Legionella pneumophila is internalized by a macropinocytotic uptake pathway controlled by the Dot/Icm system and the mouse Lgn1 locus.

The products of the Legionella pneumophila dot/icm genes enable the bacterium to replicate within a macrophage vacuole. This study demonstrates that the Dot/Icm machinery promotes macropinocytotic uptake of L. pneumophila into mouse macrophages. In mouse strains harboring a permissive Lgn1 allele, L. pneumophila promoted formation of vacuoles that were morphologically similar to macropinosomes and dependent on the presence of an intact Dot/Icm system. Macropinosome formation appeared to occur during, rather than after, the closure of the plasma membrane about the bacterium, since a fluid-phase marker preloaded into the macrophage endocytic path failed to label the bacterium-laden macropinosome. The resulting macropinosomes were rich in GM1 gangliosides and glycosylphosphatidylinositol-linked proteins. The Lgn1 allele restrictive for L. pneumophila intracellular replication prevented dot/icm-dependent macropinocytosis, with the result that phagosomes bearing the microorganism were targeted into the endocytic network. Analysis of macrophages from recombinant inbred mouse strains support the model that macropinocytotic uptake is controlled by the Lgn1 locus. These results indicate that the products of the dot/icm genes and Lgn1 are involved in controlling an internalization route initiated at the time of bacterial contact with the plasma membrane.

A genetic linkage map of the mouse: current applications and future prospects.

Technological advances have made possible the development of high-resolution genetic linkage maps for the mouse. These maps in turn offer exciting prospects for understanding mammalian genome evolution through comparative mapping, for developing mouse models of human disease, and for identifying the function of all genes in the organism.

Genetic susceptibility to Leishmania: IL-12 responsiveness in TH1 cell development.

The genetic background of T lymphocytes influences development of the T helper (TH) phenotype, resulting in either resistance or susceptibility of certain mouse strains to pathogens such as Leishmania major. With an in vitro model system, a difference in maintenance of responsiveness of T cells to interleukin-12 (IL-12) was detected between BALB/c and B10.D2 mice. Although naive T cells from both strains initially responded to IL-12, BALB/c T cells lost IL-12 responsiveness after stimulation with antigen in vitro, even when cocultured with B10.D2 T cells. Thus, susceptibility of BALB/c mice to infection with L. major may derive from the loss of the ability to generate IL-12-induced TH1 responses rather than from an IL-4-induced TH2 response.

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.

Allelotype analysis of mouse lung carcinomas reveals frequent allelic losses on chromosome 4 and an association between allelic imbalances on chromosome 6 and K-ras activation.

We generated allelotypes of 38 methylene chloride-induced lung carcinomas from female C57BL/6J x C3H/6J F1 (hereafter called B6C3F1) mice. Two or more polymorphic markers per autosome, most of them microsatellites, were examined for loss of heterozygosity. Allelic losses throughout the genome were generally infrequent except for markers on chromosome 4, which were lost in approximately one-half of the carcinomas. Analysis of lung adenomas indicated that chromosome 4 loss was associated with malignant conversion. In addition, chromosome 4 loss were specific for lung carcinomas based on comparison to methylene chloride-induced liver tumors and additional studies of lung tumors from a variety of treatment protocols and different mouse strains. Preferential loss of the maternal chromosome 4 was observed in B6C3F1 carcinomas. Analyses of additional tumors induced in mice from two reciprocal crosses, A/J x C3H/HeJ F1 (hereafter called AC3F1) and C3H/HeJ x A/J F1 (hereafter called C3AF1), provided evidence for the inactivation of one allele of the putative chromosome 4 tumor suppressor gene by parental imprinting. Most B6C3F1 tumors lost all chromosome 4 markers examined, suggesting nondisjunction events. In contrast, several C3AF1 and AC3F1 tumors appeared to have interstitial deletions that defined the smallest region of overlap as a 9-cM interval between Ifa-2 and D4Nds2. The homologous region on human chromosome 9p21-22 is frequently lost in a variety of tumors including lung cancers. A candidate tumor suppressor gene, MTS1, is located in this region, which is homozygously deleted or mutated in cell lines derived from a variety of human tumors. Finally, an association between K-ras gene activation and allelic imbalances on chromosome 6 was observed for B6C3F1 lung tumors.

Mom1 is a semi-dominant modifier of intestinal adenoma size and multiplicity in Min/+ mice.

The intestinal tumor multiplicity in mice heterozygous for ApcMin is strongly modulated by genetic background. On the sensitive C57BL/6J (B6) background, mice develop large numbers of intestinal adenomas. The AKR/J (AKR) strain carries alleles that correlate with a strong reduction in tumor multiplicity. To study the effect of one of these modifiers, Mom1, we have generated a mouse line in which the AKR allele of Mom1 is carried on the sensitive B6 genetic background. This strain was produced by using a marker-assisted selection method to eliminate unlinked AKR alleles more rapidly. The application and efficiency of this method are discussed. We used this strain to determine that Mom1 affects both tumor multiplicity and tumor size in a semi-dominant fashion.

Genetic evaluation of candidate genes for the Mom1 modifier of intestinal neoplasia in mice.

As genetic mapping of quantitative trait loci (QTL) becomes routine, the challenge is to identify the underlying genes. This paper develops rigorous genetic tests for evaluation of candidate genes for a QTL, involving determination of allelic status in inbred strains and fine-structure genetic mapping. For the Mom1 modifier of intestinal adenomas caused by ApcMin, these tests are used to evaluate two candidate genes: Pla2g2a, a secretory phospholipase, and Rap1GAP, a GTPase activating protein. Rap1GAP passes the first test but is excluded by a single fine-structure recombinant. Pla2g2a passes both tests and is a strong candidate for Mom1. Significantly, we also find that ApcMin-induced adenomas remain heterozygous for the Mom1 region, consistent with Mom1 acting outside the tumor lineage and encoding a secreted product.

IMP-8 observations of the spectra, composition, and variability of solar heavy ions at high energies relevant to manned space missions.

In more than 25 years of almost continuous observations, the University of Chicago's Cosmic Ray Telescope (CRT) on IMP-8 has amassed a unique database on high-energy solar heavy ions of potential relevance to manned spaceflight. In the very largest particle events, IMP-8/CRT has even observed solar Fe ions above the Galactic cosmic ray background up to approximately 800 MeV/nucleon, an energy sufficiently high to penetrate nearly 25 g/cm2 of shielding. IMP-8/CRT observations show that high-energy heavy-ion spectra are often surprisingly hard power laws, without the exponential roll-offs suggested by stochastic acceleration fits to lower energy measurements alone. Also, in many solar particle events the Fe/O ratio grows with increasing energy, contrary to the notion that ions with higher mass-to-charge ratios should be less abundant at higher energies. Previous studies of radiation hazards for manned spaceflight have often assumed heavy-ion composition and steeply-falling energy spectra inconsistent with these observations. Conclusions based on such studies should therefore be re-assessed. The significant event-to-event variability observed in the high-energy solar heavy ions also has important implications for strategies in building probabilistic models of solar particle radiation hazards.

Genetic analysis of susceptibility to Chlamydia trachomatis in mouse.

Chlamydia trachomatis is a bacterial pathogen that is a major cause of blindness and infertility in diverse populations across the world. In an effort to model genetic complexities that are observed in human populations and to identify novel genes involved in susceptibility to C. trachomatis, we have adapted a murine model of systemic infection for use in genetic analysis. In this model, chlamydial colonization and replication is measured in the spleens of mice shortly after intravenous delivery of C. trachomatis L2. Here, we show that C57BL/6J and C3H/HeJ inbred mice are differentially susceptible to this systemic infection. Additionally, fibroblasts cultured from C57BL/6J and C3H/HeJ embryos are differentially permissive for chlamydial replication. We have taken advantage of this natural variation to map quantitative trait loci on Chromosomes 2, 3, and 11 that segregate with the bacterial load in F2 cross progeny during the acute phase of C. trachomatis infection in vivo. To validate our mapping results, we also generated mice that are congenic for a portion of Chromosome 11 from the susceptible parent. This congenic interval confers increased susceptibility to C. trachomatis, both in vivo and in vitro, suggesting that our screen identified at least one gene that is involved in cellular resistance to C. trachomatis replication.

High-resolution genetic and physical map of the Lgn1 interval in C57BL/6J implicates Naip2 or Naip5 in Legionella pneumophila pathogenesis.

Prior genetic and physical mapping has shown that the Naip gene cluster on mouse chromosome 13D1-D3 contains a gene, Lgn1, that is responsible for determining the permissivity of ex vivo macrophages to Legionella pneumophila replication. We have identified differences in the structure of the Naip array among commonly used inbred mouse strains, although these gross structural differences do not correlate with differences in L. pneumophila permissiveness. A physical map of the region employing clones of the C57BL/6J haplotype confirms that there are fewer copies of Naip in this strain than are in the physical map of the 129 haplotype. We have also refined the genetic location of Lgn1, leaving only Naip2 and Naip5 as candidates for Lgn1. Our genetic map suggests the presence of two hotspots of recombination within the Naip array, indicating that the 3' portion of Naip may be involved in the genomic instability at this locus.

Genetic, physical, and transcript map of the Ltxs1 region of mouse chromosome 11.

Lethal factor (LF) is a toxin secreted by Bacillus anthracis that plays an important role in the pathogenesis of anthrax. Intoxication with LF results in a macrophage-specific cytolysis that is not well understood. Interestingly, inbred mouse strains exhibit dramatic differences in the susceptibility of their cultured macrophages to killing by LF, and a gene that influences this phenotype, called Ltxs1, has been mapped to mouse chromosome 11. Here we report a high-resolution genetic map that confines the Ltxs1 region to a 0.51-cM interval between D11Mit90 and D11Die37/D11Die38. We have also constructed a complete physical map of YAC and BAC clones covering the Ltxs1 region. In conjunction with synteny homology searching, BLAST searches of sequences obtained from the clones in the physical map have revealed 14 known genes and five ESTs that reside in the critical interval. Additionally, a region of 100 kb or more is deleted in the Ltxs1 interval of some strains. Our genetic, physical, and transcript map provides an important resource for the molecular cloning of Ltxs1.

Genetic control of resistance to experimental infection with virulent Mycobacterium tuberculosis.

Over 2 billion people are estimated to be infected with virulent Mycobacterium tuberculosis, yet fewer than 10% progress to clinical tuberculosis within their lifetime. Twin studies and variations in the outcome of tuberculosis infection after exposure to similar environmental risks suggest genetic heterogeneity among individuals in their susceptibility to disease. In a mouse model of tuberculosis, we have established that resistance and susceptibility to virulent M. tuberculosis is a complex genetic trait. A new locus with a major effect on tuberculosis susceptibility, designated sst1 (susceptibility to tuberculosis 1), was mapped to a 9-centimorgan (cM) interval on mouse chromosome 1. It is located 10-19 cM distal to a previously identified gene, Nramp1, that controls the innate resistance of mice to the attenuated bacillus Calmette-Guérin vaccine strain. The phenotypic expression of the newly identified locus is distinct from that of Nramp1 in that sst1 controls progression of tuberculosis infection in a lung-specific manner. Mice segregating at the sst1 locus exhibit marked differences in the growth rates of virulent tubercle bacilli in the lungs. Lung lesions in congenic sst1-susceptible mice are characterized by extensive necrosis and unrestricted extracellular multiplication of virulent mycobacteria, whereas sst1-resistant mice develop interstitial granulomas and effectively control multiplication of the bacilli. The resistant allele of sst1, although powerful in controlling infection, is not sufficient to confer full protection against virulent M. tuberculosis, indicating that other genes located outside of the sst1 locus are likely also to be important for controlling tuberculosis infection.