Features of the immune response to DNA in mice. I. Genetic control.
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The genetic control of the immune response to DNA was studied in various strains of mice F1 hybrids and corresponding back-crosses immunized with single stranded DNA complexed to methylated bovine serum albumin. Anti-DNA antibody response was measured by radioimmuno-logical technique. High responder, low responder, and intermediate responder strains were found and the ability to respond to DNA was characterized as a dominant genetic trait which is not linked to the major locus of histocompatibility. Studies in back-crosses suggested that this immune response is under multigenic control. High responder mice produce both anti-double stranded DNA and anti-single stranded DNA 7S and 19S antibodies, while low responder mice produce mainly anti-single stranded DNA 19S antibodies. (+info)
Prolonged eosinophil accumulation in allergic lung interstitium of ICAM-2 deficient mice results in extended hyperresponsiveness.
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ICAM-2-deficient mice exhibit prolonged accumulation of eosinophils in lung interstitium concomitant with a delayed increase in eosinophil numbers in the airway lumen during the development of allergic lung inflammation. The ICAM-2-dependent increased and prolonged accumulation of eosinophils in lung interstitium results in prolonged, heightened airway hyperresponsiveness. These findings reveal an essential role for ICAM-2 in the development of the inflammatory and respiratory components of allergic lung disease. This phenotype is caused by the lack of ICAM-2 expression on non-hematopoietic cells. ICAM-2 deficiency on endothelial cells causes reduced eosinophil transmigration in vitro. ICAM-2 is not essential for lymphocyte homing or the development of leukocytes, with the exception of megakaryocyte progenitors, which are significantly reduced. (+info)
Phenotype of mice and macrophages deficient in both phagocyte oxidase and inducible nitric oxide synthase.
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The two genetically established antimicrobial mechanisms of macrophages are production of reactive oxygen intermediates by phagocyte oxidase (phox) and reactive nitrogen intermediates by inducible nitric oxide synthase (NOS2). Mice doubly deficient in both enzymes (gp91(phox-/-)/NOS2(-/-)) formed massive abscesses containing commensal organisms, mostly enteric bacteria, even when reared under specific pathogen-free conditions with antibiotics. Neither parental strain showed such infections. Thus, phox and NOS2 appear to compensate for each other's deficiency in providing resistance to indigenous bacteria, and no other pathway does so fully. Macrophages from gp91(phox-/-)/NOS2(-/-) mice could not kill virulent Listeria. Their killing of S. typhimurium, E. coli, and attenuated Listeria was markedly diminished but demonstrable, establishing the existence of a mechanism of macrophage antibacterial activity independent of phox and NOS2. (+info)
Enhanced Th1 activity and development of chronic enterocolitis in mice devoid of Stat3 in macrophages and neutrophils.
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We have generated mice with a cell type-specific disruption of the Stat3 gene in macrophages and neutrophils. The mutant mice are highly susceptible to endotoxin shock with increased production of inflammatory cytokines such as TNF alpha, IL-1, IFN gamma, and IL-6. Endotoxin-induced production of inflammatory cytokines is augmented because the suppressive effects of IL-10 on inflammatory cytokine production from macrophages and neutrophils are completely abolished. The mice show a polarized immune response toward the Th1 type and develop chronic enterocolitis with age. Taken together, Stat3 plays a critical role in deactivation of macrophages and neutrophils mainly exerted by IL-10. (+info)
Localization and properties of a silencing element near the mat3-M mating-type cassette of Schizosaccharomyces pombe.
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Transcription is repressed in a segment of Schizosaccharomyces pombe chromosome II that encompasses the mat2-P and mat3-M mating-type cassettes. Chromosomal deletion analysis revealed the presence of a repressor element within 500 bp of mat3-M. This element acted in synergy with the trans-acting factors Swi6, Clr1, Clr2, Clr3, and Clr4 and had several properties characteristic of silencers: it did not display promoter specificity, being able to silence not only the M mating-type genes but also the S. pombe ura4 and ade6 genes placed on the centromere-distal side of the mat3-M cassette; it could repress a gene when placed further than 2.6 kb from the promoter and it acted in both orientations, although with different efficiencies, the natural orientation repressing more stringently than the reverse. Following deletion of this element, two semistable states of expression of the mat3-M region were observed and these two states could interconvert. The deletion did not affect gene expression in the vicinity of the mat2-P cassette, 11 kb away from mat3-M. Conversely, deleting 1.5 kb on the centromere-proximal side of the mat2-P cassette, which was previously shown to partially derepress transcription around mat2-P, had no effect on gene expression near mat3-M. A double deletion removing the mat2-P and mat3-M repressor elements had the same effect as the single deletions on their respective cassettes when assayed in cells of the M mating type. These observations allow us to refine a model proposing that redundant pathways silence the mating type region of S. pombe. (+info)
RAD53 regulates DBF4 independently of checkpoint function in Saccharomyces cerevisiae.
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The Cdc7p and Dbf4p proteins form an active kinase complex in Saccharomyces cerevisiae that is essential for the initiation of DNA replication. A genetic screen for mutations that are lethal in combination with cdc7-1 led to the isolation of seven lsd (lethal with seven defect) complementation groups. The lsd7 complementation group contained two temperature-sensitive dbf4 alleles. The lsd1 complementation group contained a new allele of RAD53, which was designated rad53-31. RAD53 encodes an essential protein kinase that is required for the activation of DNA damage and DNA replication checkpoint pathways, and that is implicated as a positive regulator of S phase. Unlike other RAD53 alleles, we demonstrate that the rad53-31 allele retains an intact checkpoint function. Thus, the checkpoint function and the DNA replication function of RAD53 can be functionally separated. The activation of DNA replication through RAD53 most likely occurs through DBF4. Two-hybrid analysis indicates that the Rad53p protein binds to Dbf4p. Furthermore, the steady-state level of DBF4 message and Dbf4p protein is reduced in several rad53 mutant strains, indicating that RAD53 positively regulates DBF4. These results suggest that two different functions of the cell cycle, initiation of DNA replication and the checkpoint function, can be coordinately regulated through the common intermediate RAD53. (+info)
Efficient homologous and illegitimate recombination in the opportunistic yeast pathogen Candida glabrata.
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The opportunistic pathogen Candida glabrata causes significant disease in humans. To develop genetic tools to investigate the pathogenicity of this organism, we have constructed ura3 and his3 auxotrophic strains by deleting the relevant coding regions in a C. glabrata clinical isolate. Linearized plasmids carrying a Saccharomyces cerevisiae URA3 gene efficiently transformed the ura3 auxotroph to prototrophy. Homologous recombination events were observed when the linearized plasmid carried short terminal regions homologous with the chromosome. In contrast, in the absence of any chromosomal homology, the plasmid integrated by illegitimate recombination into random sites in the genome. Sequence analysis of the target sites revealed that for the majority of illegitimate transformants there was no microhomology with the integration site. Approximately 0.25% of the insertions resulted in amino acid auxotrophy, suggesting that insertion was random at a gross level. Sequence analysis suggested that illegitimate recombination is nonrandom at the single-gene level and that the integrating plasmid has a preference for inserting into noncoding regions of the genome. Analysis of the relative numbers of homologous and illegitimate recombination events suggests that C. glabrata possesses efficient systems for both homologous and nonhomologous recombination. (+info)
Fus3p and Kss1p control G1 arrest in Saccharomyces cerevisiae through a balance of distinct arrest and proliferative functions that operate in parallel with Far1p.
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In Saccharomyces cerevisiae, mating pheromones activate two MAP kinases (MAPKs), Fus3p and Kss1p, to induce G1 arrest prior to mating. Fus3p is known to promote G1 arrest by activating Far1p, which inhibits three Clnp/Cdc28p kinases. To analyze the contribution of Fus3p and Kss1p to G1 arrest that is independent of Far1p, we constructed far1 CLN strains that undergo G1 arrest from increased activation of the mating MAP kinase pathway. We find that Fus3p and Kss1p both control G1 arrest through multiple functions that operate in parallel with Far1p. Fus3p and Kss1p together promote G1 arrest by repressing transcription of G1/S cyclin genes (CLN1, CLN2, CLB5) by a mechanism that blocks their activation by Cln3p/Cdc28p kinase. In addition, Fus3p and Kss1p counteract G1 arrest through overlapping and distinct functions. Fus3p and Kss1p together increase the expression of CLN3 and PCL2 genes that promote budding, and Kss1p inhibits the MAP kinase cascade. Strikingly, Fus3p promotes proliferation by a novel function that is not linked to reduced Ste12p activity or increased levels of Cln2p/Cdc28p kinase. Genetic analysis suggests that Fus3p promotes proliferation through activation of Mcm1p transcription factor that upregulates numerous genes in G1 phase. Thus, Fus3p and Kss1p control G1 arrest through a balance of arrest functions that inhibit the Cdc28p machinery and proliferative functions that bypass this inhibition. (+info)