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Farnesoid X receptor (FXR) agonism is emerging as an important potential therapeutic mechanism of action for multiple chronic liver diseases. The bile acid-derived FXR agonist obeticholic acid (OCA) has shown promise in a phase 2 study in patients with nonalcoholic steatohepatitis (NASH). Here, we report efficacy of the novel nonbile acid FXR agonist tropifexor (LJN452) in two distinct preclinical models of NASH. The efficacy of tropifexor at <1 mg/kg doses was superior to that of OCA at 25 mg/kg in the liver in both NASH models. In a chemical and dietary model of NASH (Stelic animal model [STAM]), tropifexor reversed established fibrosis and reduced the nonalcoholic fatty liver disease activity score and hepatic triglycerides. In an insulin-resistant obese NASH model (amylin liver NASH model [AMLN]), tropifexor markedly reduced steatohepatitis, fibrosis, and profibrogenic gene expression. Transcriptome analysis of livers from AMLN mice revealed 461 differentially expressed genes following tropifexor treatment that included a combination of signatures associated with reduction of oxidative stress, fibrogenesis, and inflammation. Conclusion : Based on preclinical validation in animal models, tropifexor is a promising investigational therapy that is currently under phase 2 development for NASH.

Spinal muscular atrophy (SMA), a rare neuromuscular disorder, is the leading genetic cause of death in infants and toddlers. SMA is caused by the deletion or a loss of function mutation of the survival motor neuron 1 (SMN1) gene. In humans, a second closely related gene SMN2 exists; however it codes for a less stable SMN protein. In recent years, significant progress has been made toward disease modifying treatments for SMA by modulating SMN2 pre-mRNA splicing. Herein, we describe the discovery of LMI070/branaplam, a small molecule that stabilizes the interaction between the spliceosome and SMN2 pre-mRNA. Branaplam (1) originated from a high-throughput phenotypic screening hit, pyridazine 2, and evolved via multiparameter lead optimization. In a severe mouse SMA model, branaplam treatment increased full-length SMN RNA and protein levels, and extended survival. Currently, branaplam is in clinical studies for SMA.

Spinal muscular atrophy (SMA), which results from the loss of expression of the survival of motor neuron-1 (SMN1) gene, represents the most common genetic cause of pediatric mortality. A duplicate copy (SMN2) is inefficiently spliced, producing a truncated and unstable protein. We describe herein a potent, orally active, small-molecule enhancer of SMN2 splicing that elevates full-length SMN protein and extends survival in a severe SMA mouse model. We demonstrate that the molecular mechanism of action is via stabilization of the transient double-strand RNA structure formed by the SMN2 pre-mRNA and U1 small nuclear ribonucleic protein (snRNP) complex. The binding affinity of U1 snRNP to the 5' splice site is increased in a sequence-selective manner, discrete from constitutive recognition. This new mechanism demonstrates the feasibility of small molecule-mediated, sequence-selective splice modulation and the potential for leveraging this strategy in other splicing diseases.

Angiogenesis is the process by which new blood vessels are formed from existing vessels. Mammalian populations, including humans and mice, harbor genetic variations that alter angiogenesis. Angiogenesis-regulating gene variants can result in increased susceptibility to multiple angiogenesis-dependent diseases in humans. Our efforts to dissect the complexity of the genetic diversity that regulates angiogenesis have used laboratory animals due to the availability of genome sequence for many species and the ability to perform high volume controlled breeding. Using the murine corneal micropocket assay, we have observed more than ten-fold difference in angiogenic responsiveness among various mouse strains. This degree of difference is observed with either bFGF or VEGF induced corneal neovascularization. Ongoing mapping studies have identified multiple loci that affect angiogenic responsiveness in several mouse models. In this study, we used F2 intercrosses between C57BL/6J and the 129 substrains 129P1/ReJ and 129P3/J, as well as the SJL/J strain, where we have identified new QTLs that affect angiogenic responsiveness. In the case of AngFq5, on chromosome 7, congenic animals were used to confirm the existence of this locus and subcongenic animals, combined with a haplotype-based mapping approach that identified the pink-eyed dilution mutation as a candidate polymorphism to explain AngFq5. The ability of mutations in the pink-eyed dilution gene to affect angiogenic response was demonstrated using the p-J allele at the same locus. Using this allele, we demonstrate that pink-eyed dilution mutations in Oca2 can affect both bFGF and VEGF-induced corneal angiogenesis.

Inflammasomes are cytosolic multiprotein complexes that sense microbial infection and trigger cytokine production and cell death. However, the molecular components of inflammasomes and what they sense remain poorly defined. Here we demonstrate that 35 amino acids of the carboxyl terminus of flagellin triggered inflammasome activation in the absence of bacterial contaminants or secretion systems. To further elucidate the host flagellin-sensing pathway, we generated mice deficient in the intracellular sensor Naip5. These mice failed to activate the inflammasome in response to the 35 amino acids of flagellin or in response to Legionella pneumophila infection. Our data clarify the molecular basis for the cytosolic response to flagellin.

Chlamydiae are obligate intracellular bacterial pathogens that exhibit a broad range of host tropism. Differences in host tropism between Chlamydia species have been linked to host variations in IFN-gamma-mediated immune responses. In mouse cells, IFN-gamma can effectively restrict growth of the human pathogen Chlamydia trachomatis but fails to control growth of the closely related mouse pathogen Chlamydia muridarum. The ability of mouse cells to resist C. trachomatis replication is largely dependent on the induction of a family of IFN-gamma-inducible GTPases called immunity-related GTPases or IRGs. In this study we demonstrate that C. muridarum can specifically evade IRG-mediated host resistance. It has previously been suggested that C. muridarum inactivates the IRG protein Irga6 (Iigp1) to dampen the murine immune response. However, we show that Irga6 is dispensable for the control of C. trachomatis replication. Instead, an effective IFN-gamma response to C. trachomatis requires the IRG proteins Irgm1 (Lrg47), Irgm3 (Igtp), and Irgb10. Ectopic expression of Irgb10 in the absence of IFN-gamma is sufficient to reduce intracellular growth of C. trachomatis but fails to restrict growth of C. muridarum, indicating that C. muridarum can specifically evade Irgb10-driven host responses. Importantly, we find that Irgb10 protein intimately associates with inclusions harboring C. trachomatis but is absent from inclusions formed by C. muridarum. These data suggest that C. muridarum has evolved a mechanism to escape the murine IFN-gamma response by restricting access of Irgb10 and possibly other IRG proteins to the inclusion.

Macrophages from the C57BL/6 (B6) mouse strain restrict intracellular growth of Legionella pneumophila, whereas A/J macrophages are highly permissive. The mechanism by which B6 macrophages restrict Legionella growth remains poorly understood, but is known to require the cytosolic microbe sensors Naip5 (Birc1e) and Ipaf. We hypothesized that Naip5 and Ipaf may act in partnership with other antimicrobial signalling pathways in macrophages. Indeed, we found that macrophages lacking either tumour necrosis factor (TNF)-alpha or type I interferon (IFN) signalling are permissive for growth of L. pneumophila, even in the presence of functional Naip5 and Ipaf alleles. Similarly, macrophages lacking Naip5 and/or Ipaf signalling were permissive even though we found that Naip5 or Ipaf were not required for induction of TNF-alpha and type I IFN. Therefore, our data suggest that the mechanism by which B6 macrophages restrict intracellular replication of L. pneumophila is more complex than previously appreciated, and involves the concerted action of cytokine and intracellular microbe sensor signalling pathways.

We undertook a quantitative trait locus (QTL) analysis in mice to identify modifier genes that might influence the severity of human iron disorders. We identified a strong QTL on mouse chromosome 9 that differentially affected macrophage iron burden in C57BL/10J and SWR/J mice. A C57BL/10J missense allele of an evolutionarily conserved gene, Mon1a, cosegregated with the QTL in congenic mouse lines. We present evidence that Mon1a is involved in trafficking of ferroportin, the major mammalian iron exporter, to the surface of iron-recycling macrophages. Differences in amounts of surface ferroportin correlate with differences in cellular iron content. Mon1a is also important for trafficking of cell-surface and secreted molecules unrelated to iron metabolism, suggesting that it has a fundamental role in the mammalian secretory apparatus.

Infections caused by the bacteria Chlamydia trachomatis contribute to diverse pathologies in a variety of human populations. We previously used a systemic model of C. trachomatis infection in mice to map three quantitative trait loci that influence in vivo susceptibility differences between the C57BL/6J and C3H/HeJ inbred strains of mouse. One of these quantitative trait loci, Ctrq-3, influences an IFN-gamma-dependent susceptibility difference in primary embryonic fibroblasts isolated from these strains. Here we use fine structure mapping in congenic fibroblasts carrying DNA from the susceptible parent to localize the effect of Ctrq-3 to a 1.2-megabase interval of genomic DNA that contains Irgb10 and Igtp, two members of the IFN-gamma-inducible p47 family of GTPases. This class of proteins has been widely implicated in resistance to intracellular pathogens in mice. We analyzed expression of Irgb10 and Igtp in parental and congenic embryonic fibroblasts treated with IFN-gamma and found that relatively resistant fibroblasts express more Irgb10 than relatively susceptible fibroblasts. However, we also found that abolishing the expression of either Irgb10 or Igtp increases susceptibility of embryonic fibroblasts to C. trachomatis. Thus, we conclude that, although a difference in Irgb10 expression is likely responsible for the effect of Ctrq-3 on susceptibility to C. trachomatis, both genes play a role in intracellular resistance to C. trachomatis.

Macrophages from C57BL/6J (B6) mice restrict growth of the intracellular bacterial pathogen Legionella pneumophila. Restriction of bacterial growth requires caspase-1 and the leucine-rich repeat-containing protein Naip5 (Birc1e). We identified mutants of L. pneumophila that evade macrophage innate immunity. All mutants were deficient in expression of flagellin, the primary flagellar subunit, and failed to induce caspase-1-mediated macrophage death. Interestingly, a previously isolated flagellar mutant (fliI) that expresses, but does not assemble, flagellin did not replicate in macrophages, and induced macrophage death. Thus, flagellin itself, not flagella or motility, is required to initiate macrophage innate immunity. Immunity to Legionella did not require MyD88, an essential adaptor for toll-like receptor 5 (TLR5) signaling. Moreover, flagellin of Legionella and Salmonella induced cytotoxicity when delivered to the macrophage cytosol using Escherichia coli as a heterologous host. It thus appears that macrophages sense cytosolic flagellin via a TLR5-independent pathway that leads to rapid caspase-1-dependent cell death and provides defense against intracellular bacterial pathogens.

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.

Baculovirus inhibitor of apoptosis repeat-containing 1 (Birc1) proteins have homology to several germline-encoded receptors of the innate immune system. However, their function in immune surveillance is not clear. Here we describe a Birc1e-dependent signaling pathway that restricted replication of the intracellular pathogen Legionella pneumophila in mouse macrophages. Translocation of bacterial products into host-cell cytosol was essential for Birc1e-mediated control of bacterial replication. Caspase-1 was required for Birc1e-dependent antibacterial responses ex vivo in macrophages and in a mouse model of Legionnaires' disease. The interleukin 1beta converting enzyme-protease-activating factor was necessary for L. pneumophila growth restriction, but interleukin 1beta was not required. These results establish Birc1e as a nucleotide-binding oligomerization-leucine-rich repeat protein involved in the detection and control of intracellular L. pneumophila.

The pathogenesis of Bacillus anthracis, the bacterium that causes anthrax, depends on secretion of three factors that combine to form two bipartite toxins. Edema toxin, consisting of protective antigen (PA) and edema factor (EF), causes the edema associated with cutaneous anthrax infections, whereas lethal toxin (LeTx), consisting of PA and lethal factor (LF), is believed to be responsible for causing death in systemic anthrax infections. EF and LF can be transported by PA into the cytosol of many cell types. In mouse macrophages, LF can cause rapid necrosis that may be related to the pathology of systemic infections. Inbred mouse strains display variable sensitivity to LeTx-induced macrophage necrosis. This trait difference has been mapped to a locus on chromosome 11 named Ltxs1 (refs. 7,8). Here we show that an extremely polymorphic gene in this locus, Nalp1b, is the primary mediator of mouse macrophage susceptibility to LeTx. We also show that LeTx-induced macrophage death requires caspase-1, which is activated in susceptible, but not resistant, macrophages after intoxication, suggesting that Nalp1b directly or indirectly activates caspase-1 in response to LeTx.

Epidemiological, clinical, and experimental approaches have convincingly demonstrated that host resistance to infection with intracellular pathogens is significantly influenced by genetic polymorphisms. Using a mouse model of infection with virulent Mycobacterium tuberculosis (MTB), we have previously identified the sst1 locus as a genetic determinant of host resistance to tuberculosis. In this study we demonstrate that susceptibility to another intracellular pathogen, Listeria monocytogenes, is also influenced by the sst1 locus. The contribution of sst1 to anti-listerial immunity is much greater in immunodeficient scid mice, indicating that this locus controls innate immunity and becomes particularly important when adaptive immunity is significantly depressed. Similar to our previous observations using infection with MTB, the resistant allele of sst1 prevents formation of necrotic infectious lesions in vivo. We have shown that macrophages obtained from sst1-resistant congenic mice possess superior ability to kill L. monocytogenes in vitro. The bactericidal effect of sst1 is dependent on IFN-gamma activation and reactive oxygen radical production by activated macrophages after infection, but is independent of NO production. It is possible that there is a single gene that controls common IFN-dependent macrophage function, which is important in the pathogenesis of infections caused by both MTB and L. monocytogenes. However, host resistance to the two pathogens may be controlled by two different polymorphic genes encoded within the sst1 locus. The polymorphic gene(s) encoded within the sst1 locus that controls macrophage interactions with the two intracellular pathogens remains to be elucidated.

The B10.Q/J strain of mice was serendipitously discovered to be highly susceptible to infection by the intracellular protozoan parasite, Toxoplasma gondii but markedly resistant to induction of autoimmune arthritis. We have previously shown that the B10.Q/J phenotype is controlled by a single recessive locus and is associated with lymphocyte hyporesponsiveness to IL-12. Using genetic approaches, we have now localized the B10.Q/J locus to chromosome 9 and established its identity as Tyk2, a Janus kinase essential for IL-12 and IFN-alpha/beta cytokine signaling. The B10.Q/J Tyk2 gene contained a single missense mutation resulting in a nonconservative amino acid substitution (E775K) in an invariant motif of the pseudokinase (Janus kinase homology 2) domain. This mutation appeared to result in the absence of the B10.Q/J-encoded Tyk2 protein, despite presence of Tyk2-specific transcripts. Phenotypically, B10.Q/J cells were indistinguishable from Tyk2-deficient cells, showing impaired signaling and biologic responses to IL-12, IL-23, and type I IFNs. The analogous E782K mutant of human Tyk2 failed to restore IFN-alpha responsiveness in Tyk2 null 11,1 cells. Our results indicate a crucial role for Tyk2 in T helper 1-mediated protective and pathogenic immune responses. An additional implication of our findings is that naturally occurring mutations in the Tyk2 gene may underlie altered susceptibilities to infectious or autoimmune diseases in human and animal populations.

Background: Legionella pneumophila is a gram-negative bacterial pathogen that is the cause of Legionnaires' Disease. Legionella produces disease because it can replicate inside a specialized compartment of host macrophages. Macrophages isolated from various inbred mice exhibit large differences in permissiveness for intracellular replication of Legionella. A locus affecting this host-resistance phenotype, Lgn1, has been mapped to chromosome 13, but the responsible gene has not been identified., Results: Here, we report that Naip5 (also known as Birc1e) influences susceptibility to Legionella. Naip5 encodes a protein that is homologous to plant innate immunity (so-called "resistance") proteins and has been implicated in signaling pathways related to apoptosis regulation. Detailed recombination mapping and analysis of expression implicates Naip5 in the Legionella permissiveness differences among mouse strains. A bacterial artificial chromosome (BAC) transgenic line expressing a nonpermissive allele of Naip5 exhibits a reduction in macrophage Legionella permissiveness. In addition, morpholino-based antisense inhibition of Naip5 causes an increase in the Legionella permissiveness of macrophages., Conclusions: We conclude that polymorphisms in Naip5 are involved in the permissiveness differences of mouse macrophages for intracellular Legionella replication. We speculate that Naip5 is a functional mammalian homolog of plant "resistance" proteins that monitor for, and initiate host response to, the presence of secreted bacterial virulence proteins.

Spt3 of Saccharomyces cerevisiae is required for the normal transcription of many genes in vivo. Past studies have shown that Spt3 is required for both mating and sporulation, two events that initiate when cells are at G(1)/START. We now show that Spt3 is needed for two other events that begin at G(1)/START, diploid filamentous growth and haploid invasive growth. In addition, Spt3 is required for normal expression of FLO11, a gene required for filamentous growth, although this defect is not the sole cause of the spt3Delta/spt3Delta filamentous growth defect. To extend our studies of Spt3's role in filamentous growth to the pathogenic yeast Candida albicans, we have identified the C. albicans SPT3 gene and have studied its role in C. albicans filamentous growth and virulence. Surprisingly, C. albicans spt3Delta/spt3Delta mutants are hyperfilamentous, the opposite phenotype observed for S. cerevisiae spt3Delta/spt3Delta mutants. Furthermore, C. albicans spt3Delta/spt3Delta mutants are avirulent in mice. These experiments demonstrate that Spt3 plays important but opposite roles in filamentous growth in S. cerevisiae and C. albicans.

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.

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.

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., (Copyright 2001 Academic Press.)

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 mouse locus called Lgn1 determines differences in macrophage permissiveness for the intracellular replication of Legionella pneumophila. The only regional candidate genes for this phenotype difference lie within a cluster of closely linked paralogs of the Neuronal Apoptosis Inhibitory Protein (Naip) gene. Previous genetic and physical mapping of the Lgn1 phenotype narrowed it to an interval containing only Naip2 and Naip5, suggesting that there is not complete functional overlap among the mouse Naip loci. In order to gather more information about polymorphisms among the Naip genes of the 129 mouse haplotype, we have determined the genomic sequence of a substantial portion of the 129 Naip gene array. We have constructed an evolutionary model for the expansion of the Naip gene array from a single progenitor Naip gene. This model predicts the presence of two distinct families of Naip paralogs: Naip1/2/3 and Naip4/5/6/7. Unlike the divergences among all the other Naip paralogs, the splits among Naip4, Naip5, Naip6, and Naip7 occurred relatively recently. The high degree of sequence conservation within the Naip4/5/6/7 family increases the likelihood of functional overlap among these genes.

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.

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.

The orthologous genomic segments on mouse chromosome 13D1-D3 and human chromosome 5q11.2-q13.3 have been extensively studied because of their involvement in two distinct disease phenotypes, spinal muscular atrophy (SMA) in human and susceptibility to Legionella pneumophila (determined by Lgn1) in mice. The overlapping intervals in both species contain genomic amplifications of distinct structure, indicating an independent origin. We have endeavored to construct a comprehensive comparative gene map of the mouse and human Lgn1/SMA intervals in the hopes that the origins and maintenance of the genomic amplifications may become clear. Our comparative gene map demonstrates that the only regional gene in common between the amplified segments in mouse and human is the Lgn1 candidate Naip/NAIP. We have also determined that mice of the 129 haplotype harbor seven intact and three partial Naip transcription units arranged in a closely linked direct repeat on chromosome 13. Several, but not all, of these Naip loci are contained within the Lgn1 critical interval. We present a model for the origins of the mouse and human repetitive arrays from a common ancestral haplotype., (Copyright 2000 Academic Press.)

Human chromosome 5q11.2-q13.3 and its ortholog on mouse chromosome 13 contain candidate genes for an inherited human neurodegenerative disorder called spinal muscular atrophy (SMA) and for an inherited mouse susceptibility to infection with Legionella pneumophila (Lgn1). These homologous genomic regions also have unusual repetitive organizations that create practical difficulties in mapping and raise interesting issues about the evolutionary origin of the repeats. In an attempt to analyze this region in detail, and as a way to identify additional candidate genes for these diseases, we have determined the sequence of 179 kb of the mouse Lgn1/SMA interval. We have analyzed this sequence using BLAST searches and various exon prediction programs to identify potential genes. Since these methods can generate false-positive exon declarations, our alignments of the mouse sequence with available human orthologous sequence allowed us to discriminate rapidly among this collection of potential coding regions by indicating which regions were well conserved and were more likely to represent actual coding sequence. As a result of our analysis, we accurately mapped two additional genes in the SMA interval that can be tested for involvement in the pathogenesis of SMA. While no new Lgn1 candidates emerged, we have identified new genetic markers that exclude Smn as an Lgn1 candidate. In addition to providing important resources for studying SMA and Lgn1, our data provide further evidence of the value of sequencing the mouse genome as a means to help with the annotation of the human genomic sequence and vice versa., (Copyright 1999 Academic Press.)

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.

Recently, the human orthologue to the cell cycle checkpoint genes rad17 (Schizosaccharomyces pombe) and RAD24 (Saccharomyces cerevisiae), called HRAD17, has been isolated and localized to chromosome 4. Independently, we have isolated the HRAD17 transcript and mapped it to chromosome 5q13 between the CCNB1 and BTF2p44cen genes. Furthermore, we have identified the complete exon-intron structure of HRAD17. The gene is organized into 14 exons, the translation initiation site lies within exon 2, and the stop codon within exon 14. Two further HRAD17 pseudogenes, HRAD17P1 and HRAD17P2, were identified on chromosomes 7p21 and 13q14.3, respectively, encompassing exons 3-14 and bearing 84% and 93% homology, respectively. Additionally, we have isolated the coding region of the mouse orthologue, Mrad17, and mapped it on chromosome 13 between Ccnb1 and Btf2p44, the same two genes between which it maps in human. The predicted Mrad17 polypeptide encompasses 687 amino acids and shows 89% similarity to HRAD17. Both genes are most highly expressed in testis compared to all other tissues, as shown by Northern blot hybridization. Histological studies, based on in situ hybridization with radioactively labeled antisense HRAD17 riboprobes, showed a strong expression within the germinal epithelium of the seminiferous tubuli in normal testis whereas in testicular tumors (seminomas) only weak, diffuse signals were seen. In light of the known function of the yeast orthologue at meiotic and mitotic checkpoints, as well as the strong expression in testis and weak expression in seminomas, we suggest a putative involvement of HRAD 17 in testicular tumorigenesis.

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.

Th phenotype development is controlled not only by cytokines but also by other parameters including genetic background. One site of genetic variation between murine strains that has direct impact on Th development is the expression of the IL-12 receptor. T cells from B10.D2 and BALB/c mice show distinct control of IL-12 receptor expression. When activated by Ag, B10.D2 T cells express functional IL-12 receptors and maintain IL-12 responsiveness. In contrast, under the same conditions, BALB/c T cells fail to express IL-12 receptors and become unresponsive to IL-12, precluding any Th1-inducing effects if subsequently exposed to IL-12. Previously, we identified a locus, which we termed T cell phenotype modifier 1 (Tpm1), on murine chromosome 11 that controls this differential maintenance of IL-12 responsiveness. In this study, we have produced a higher resolution map around Tpm1. We produced and analyzed a series of recombinants from a first-generation backcross that significantly narrows the genetic boundaries of Tpm1. This allowed us to exclude from consideration certain previous candidates for Tpm1, including IFN-regulatory factor-1. Also, cellular analysis of F1(B10.D2 x BALB/c) T cells demonstrates that Tpm1 exerts its effect on IL-12 receptor expression in a cell-autonomous manner, rather than through influencing the extracellular milieu. This result strongly implies that despite the proximity of our locus to the IL-13/IL-4 gene cluster, these cytokines are not candidates for Tpm1.

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.

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.

We have examined the effects of inactivation of the p53 tumor suppressor gene on the incidence of apoptotic cell death in two stages of the adenoma-to-carcinoma progression in the intestine: in early adenomas where p53 mutations are rare and in highly dysplastic adenomas where loss of p53 occurs frequently. Homozygosity for an inactivating germ-line mutation of p53 had no effect on the incidence or the rate of progression of ApcMin/+-induced adenomas in mice and also did not affect the frequency of apoptosis in the cells of these adenomas. To examine the effect of p53 loss on apoptosis in late-stage adenomas, we compared the incidence of apoptotic cell death before and after the appearance of highly dysplastic cells in human colonic adenomas. The appearance of highly dysplastic cells, which usually coincides during colon tumor progression with loss of heterozygosity at the p53 locus, did not correlate with a reduction in the incidence of apoptosis. These studies suggest that p53 is only one of the genes that determine the incidence of apoptotic in colon carcinomas and that wild-type p53 retards the progression of many benign colonic adenoma to malignant carcinomas by mechanism(s) other than the promotion of apoptosis.

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.

Comparative genomic hybridization (CGH) provides a method of surveying the entire tumor genome for regional variations in DNA sequence copy number. Such variations, if found recurrently, may indicate the locations of genes that contribute to tumor development through upregulation of oncogenes (copy number increase), inactivation of tumor-suppressor genes (copy number decrease), or changes in the level of expression through gene dosage effects. Thus, CGH is a powerful tool for screening for new cancer genes. Although CGH is widely applied to human genome analysis, application to the mouse is only beginning. The present study is designed to compare results obtained by CGH with those obtained by other techniques used for analysis of the murine genome. We report CGH analysis of several control cell lines with cytogenetically established regional copy number changes, as well as analysis of 16 primary insulinomas, four tumor-derived cell lines, and three hyperplasia-derived cell lines from transgenic mice expressing the SV40 large T antigen under control of the rat insulin promoter. Loss of heterozygosity (LOH) on chromosomes 9 and 16 had previously been found to be frequent in primary insulinomas, and specimens were selected for the present study based on the LOH status of these chromosomes. We found complete concordance of the CGH results with the cytogenetically described copy number changes in the control cell lines and with the LOH on chromosomes 9 and 16 in the tumors. Thus, CGH can provide accurate data in murine systems, and it reveals that the LOH in these islet cell tumors most frequently results from deletion of one of the alleles.

Spinal muscular atrophy (SMA) is a relatively common, autosomal recessively inherited neurodegenerative disorder that maps to human chromosome 5q13. This region of the human genome has an intricate genomic structure that has complicated the evaluation of SMA candidate genes. We have chosen to study the mouse region syntenic for human SMA in the hope that the homologous mouse interval would contain the same genes as human 5q13 on a simpler genomic background. Here, we report the mapping of such a region to mouse chromosome 13 and to the critical interval for Lgn1, a mouse locus responsible for modulating the intracellular replication and pathogenicity of the bacterium Legionella pneumophila. We have generated a mouse YAC contig across the Lgn1/Sma interval and have mapped the two flanking gene markers for the human SMA locus, MAP1B and CCNB1, onto this contig. In addition, we have localized the two SMA candidate genes, SMN and NAIP, to the Lgn1 critical region, making these two genes candidates for the Lgn1 phenotype. Upon subcloning of the YAC contig into P1s and BACs, we have detected a large, low copy number repeat that contains at least one copy of Naip exon 5. Identification of the Lgn1 gene will either provide a novel function for SMN or NAIP or reveal the existence of another, yet uncharacterized gene in the SMA critical region. Mutations in such a gene might help to explain some of the phenotypic variability among the human SMAs.

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.

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.

Genetic background of the T cell can influence T helper (Th) phenotype development, with some murine strains (e.g., B10.D2) favoring Th1 development and others (e.g., BALB/c) favoring Th2 development. Recently we found that B10.D2 exhibit an intrinsically greater capacity to maintain interleukin 12 (IL-12) responsiveness under neutral conditions in vitro compared with BALB/c T cells, allowing for prolonged capacity to undergo IL-12-induced Th1 development. To begin identification of the loci controlling this genetic effect, we used a T-cell antigen receptor-transgenic system for in vitro analysis of intercrosses between BALB/c and B10.D2 mice and have identified a locus on murine chromosome 11 that controls the maintenance of IL-12 responsiveness, and therefore the subsequent Th1/Th2 response. This chromosomal region is syntenic with a locus on human chromosome 5q31.1 shown to be associated with elevated serum IgE levels, suggesting that genetic control of Th1/Th2 differentiation in mouse, and of atopy development in humans, may be expressed through similar mechanisms.

The availability of dense genetic linkage maps of mammalian genomes makes feasible a wide range of studies, including positional cloning of monogenic traits, genetic dissection of polygenic traits, construction of genome-wide physical maps, rapid marker-assisted construction of congenic strains, and evolutionary comparisons. We have been engaged for the past five years in a concerted effort to produce a dense genetic map of the laboratory mouse. Here we present the final report of this project. The map contains 7,377 genetic markers, consisting of 6,580 highly informative simple sequence length polymorphisms integrated with 797 restriction fragment length polymorphisms in mouse genes. The average spacing between markers is about 0.2 centimorgans or 400 kilobases.

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.

EL/Suz (EL) mice experience recurrent seizures that are similar to common partial complex epilepsy in humans. In the mice, seizures occur naturally at 90-100 days of age, but can be induced in younger mice and analyzed as a semi-quantitative trait after gentle rhythmic stimulation. A previous genetic mapping study of EL backcrosses to the strains ABP/LeJ or DBA/2J showed two quantitative trait loci (QTL) with large effects on seizure frequency (El1, Chr 9; El2, Chr 2) and implied the existence of other QTL with lesser effects. To further the understanding of EL-derived seizure alleles, we examined intercross progeny of EL and the strains ABP/LeJ and DDY/Jcl, and also a backcross of (EL x DDY)F1 hybrids to DDY. A new large-effect seizure frequency QTL was found (El5, Chr 14), a more minor QTL confirmed (El3, Chr 10), and two additional QTL proposed (El4, Chr 9; El6, Chr 11). The serotonin receptor gene, Htr2a, maps near and is a candidate for El5, and linkages of other serotonin receptor genes to seizure frequency QTL are noted. In addition, a strong gender effect was revealed, and epistasis was found between Chr 9 and Chr 14 markers. Despite this progress, however, our results revealed a more complex determinism of epilepsy in EL mice than previously described. In particular, no single El locus or pair was essential for frequent seizures, as QTL with large effects, such as El5, El2, and El1, were highly dependent on genetic context. Our studies highlight the importance of gene interaction in some complex mammalian traits defined by natural variation.

The mouse is the best model system for the study of mammalian genetics and physiology. Because of the feasibility and importance of studying genetic crosses, the mouse genetic map has received tremendous attention in recent years. It currently contains over 14,000 genetically mapped markers, including 700 mutant loci, 3500 genes, and 6500 simple sequence length polymorphisms (SSLPs). The mutant loci and genes allow insights and correlations concerning physiology and development. The SSLPs provide highly polymorphic anchor points that allow inheritance to be traced in any cross and provide a scaffold for assembling physical maps. Adequate physical mapping resources--notably large-insert yeast artificial chromosome (YAC) libraries--are available to support positional cloning projects based on the genetic map, but a comprehensive physical map is still a few years away. Large-scale sequencing efforts have not yet begun in mouse, but comparative sequence analysis between mouse and human is likely to provide tremendous information about gene structure and regulation.

The intracellular pathogen Legionella pneumophila is unable to replicate in macrophages derived from most inbred mouse strains. Here, we report the mapping of a gene, called Lgn1, that determines whether mouse macrophages are permissive for the intracellular replication of L. pneumophila. Although Lgn1 has been previously reported to map to mouse chromosome 15, we show here that it actually maps to chromosome 13, between D13Mit128 and D13Mit70. In the absence of any regional candidates for Lgn1, this map position will facilitate positional cloning attempts directed at this gene.