Genetic transformation of Sulfolobus acidocaldarius by a multiply marked pyrE gene provided a highresolution assay of homologous recombination in a hyperthermophilic archaeon. Analysis of 100 Pyr ؉ transformants revealed that this recombination system could transfer each of 23 nonselected base pair substitutions to the recipient chromosome along with the selected marker. In 30% of the recombinants, donor markers were transferred as multiple blocks. In at least 40% of the recombinants, donor markers separated by 5 or 6 bp segregated from each other, whereas similar markers separated by 2 bp did not segregate. Among intermarker intervals, the frequency of recombination tract endpoints varied 40-fold, but in contrast to other recombination systems, it did not correlate with the length of the interval. The average length of donor tracts (161 bp) and the frequent generation of multiple tracts seemed generally consistent with the genetic properties observed previously in S. acidocaldarius conjugation. The efficiency with which short intervals of diverged pyrE sequence were incorporated into the genome raises questions about the threat of ectopic recombination in Sulfolobus spp. mediated by this apparently efficient yet permissive system.All cells and some viruses encode systems of homologous recombination (HR) which support the successful replication of their genomes. In eukaryotic cells, HR systems repair double-strand breaks and ensure proper chromosome segregation during meiosis (1, 2, 17). Double-strand break repair by eukaryotic HR has been studied intensively in yeast, where it has been shown to cause the net transfer of a short section of sequence from the intact DNA to the broken DNA in a unilateral, i.e., nonreciprocal, manner. Outside this central zone, the flanking segments of the two DNAs may also be exchanged, generating a crossover. The relative yields of noncrossover versus crossover events vary in different situations, and this appears to reflect the different ways in which displacement loops formed by strand invasion are ultimately resolved (1,17,36).In bacteria, HR helps reassemble replication forks disrupted by encounters with various DNA lesions (6,20,27). For practical reasons, however, genetic assays of bacterial HR typically follow the process of replacing a segment of a recipient chromosome or plasmid with a corresponding (i.e., homologous) donor DNA segment introduced into the cell. This "ends-out" mode of HR underlies the classical techniques of genetic mapping and strain construction of Escherichia coli and other bacteria. It also occurs in natural populations and contributes to genome evolution, as indicated by the "mosaic" patterns of sequence polymorphisms documented in various E. coli lineages (28). Thus, the functional properties of the host HR system combine with those of the DNA transfer systems to influence the rate of genetic exchange and the nature (including the abundance and average length) of the homologous DNA segments incorporated as a result.The importance of HR for genetic e...

TRPM7 is a cation channel-protein kinase highly expressed in T lymphocytes and other immune cells. It has been proposed to constitute a cellular entry pathway for Mg 2+ and divalent metal cations such as Ca 2+ , Zn 2+ , Cd 2+ , Mn 2+ and Ni 2+ . TRPM7 channels are inhibited by cytosolic Mg 2+ , rendering them largely inactive in intact cells. Dependence of channel activity on extracellular Mg 2+ is less well studied. Here, we measured native TRPM7 channel activity in Jurkat T cells maintained in external Mg 2+ concentrations varying between 400 nM and 1.4 mM for 1-2 days, obtaining an IC 50 value of 54 μM. Maintaining the cells in 400 nM or 8 μM [Mg 2+ ] o resulted in almost complete activation of TRPM7 in intact cells, due to cytosolic Mg 2+ depletion. 1.4 mM [Mg 2+ ] o was sufficient to fully eliminate the basal current. Submillimolar concentrations of amiloride prevented cellular Mg 2+ depletion, but not loading. We investigated whether the cytotoxicity of TRPM7 permeant metal ions Ni 2+ , Zn 2+ , Cd 2+ , Co 2+ , Mn 2+ , Sr 2+ and Ba 2+ requires TRPM7 channel activity. Mg 2+ loading modestly reduced cytotoxicity of Zn 2+ , Co 2+ , Ni 2+ and Mn 2+ but not of Cd 2+ . Channel blocker NS8593 reduced Co 2+ and Mn 2+ but not Cd 2+ or Zn 2+ cytotoxicity and interfered with Mg 2+ loading as evaluated by TRPM7 channel basal activity. Ba 2+ and Sr 2+ were neither detectably toxic, nor permeant through the plasma membrane. These results indicate that in Jurkat T cells entry of toxic divalent metal cations primarily occurs through pathways distinct from TRPM7. By contrast, we found evidence that Mg 2+ entry requires TRPM7 channels.

House dust mite extracts used for diagnostic tests and immunotherapy contain bioreactive molecules including proteins and endotoxin. These extracts can influence the cytokine secretion and adhesion molecule expression by cells in the skin and lung airways. The aim of this study was to determine the role of proteins and endotoxin in mite extracts in modulating gene expression and cytokine secretion by human dermal fibroblasts. Cultured normal human dermal fibroblasts were stimulated with whole mite extracts, mite extracts boiled to denature proteins, or mite extracts treated with polymyxin B to inactivate lipopolysaccharide. Gene expression and secretion of interleukin-6 (IL-6), IL-8, and monocyte chemoattractant protein-1 (MCP-1) were determined after 6 h of stimulation. Whole Dermatophagoides farinae, D. pteronyssinus and Euroglyphus maynei extracts induced dose-dependent IL-6 and IL-8 secretion. In addition, D. farinae and E. maynei induced secretion of MCP-1. D. farinae and E. maynei also induced parallel cytokine gene expression. Cells stimulated with boiled D. farinae extract showed moderate to marked reductions in IL-6 and IL-8 secretion. In contrast, boiled D. pteronyssinus and E. maynei extracts induced equal or greater cytokine secretions than untreated extracts. The stimulating properties were reduced for all three extracts following treatment with polymyxin B. Our data suggest that both endotoxin and proteins in mite extracts modulate the secretion of cytokines by dermal fibroblasts. The biological activities of D. farinae, D. pteronyssinus, and E. maynei extracts are not equivalent. There appears to be a lipopolysaccharide-binding protein in some mite extracts.

Although homologous recombination (HR) is known to influence the structure, stability, and evolution of microbial genomes, few of its functional properties have been measured in cells of hyperthermophilic archaea. The present study manipulated various properties of the parental DNAs in high-resolution assays of Sulfolobus acidocaldarius transformation, and measured the impact on the efficiency and pattern of marker transfer to the recipient chromosome. The relative orientation of homologous sequences, the type and position of chromosomal mutation being replaced, and the length of DNA flanking the marked region all affected the efficiency, linkage, tract continuity, and other parameters of marker transfer. Effects predicted specifically by the classical reciprocal-exchange model of HR were not observed. One analysis observed only 90 % linkage between markers defined by adjacent bases; in another series of experiments, sequence divergence up to 4 % had no detectable impact on overall efficiency of HR or on the co-transfer of a distal non-selected marker. The effects of introducing DNA via conjugation, rather than transformation, were more difficult to assess, but appeared to increase co-transfer (i.e. linkage) of relatively distant non-selected markers. The results indicate that HR events between gene-sized duplex DNAs and the S. acidocaldarius chromosome typically involve neither crossing over nor interference from a mismatch-activated anti-recombination system. Instead, the donor DNA may anneal to a transient chromosomal gap, as in the mechanism proposed for oligonucleotidemediated transformation of Sulfolobus and other micro-organisms.

Transient receptor potential melastatin 7 (TRPM7) is a unique protein functioning as a cation channel as well as a serine/threonine kinase and is highly expressed in immune cells such as lymphocytes and macrophages. TRPM7 kinase-dead (KD) mouse model has been used to investigate the role of this protein in immune cells; these animals display moderate splenomegaly and ectopic hemopoiesis. The basal TRPM7 current magnitudes in peritoneal macrophages isolated from KD mice were higher; however, the maximum currents, achieved after cytoplasmic Mg 2+ washout, were not different. In the present study, we investigated the consequences of TRPM7 kinase inactivation in splenic and peritoneal macrophages. We measured the basal phagocytic activity of splenic macrophages using fluorescent latex beads, pHrodo zymosan bioparticles, and opsonized red blood cells. KD macrophages phagocytized more efficiently and had slightly higher baseline calcium levels compared to WT cells. We found no obvious differences in store-operated Ca 2+ entry between WT and KD macrophages. By contrast, the resting cytosolic pH in KD macrophages was significantly more alkaline than in WT. Pharmacological blockade of sodium hydrogen exchanger 1 (NHE1) reversed the cytosolic alkalinization and reduced phagocytosis in KD macrophages. Basal TRPM7 channel activity in KD macrophages was also reduced after NHE1 blockade. Cytosolic Mg 2+ sensitivity of TRPM7 channels measured in peritoneal macrophages was similar in WT and KD mice. The higher basal TRPM7 channel activity in KD macrophages is likely due to alkalinization. Our results identify a novel role for TRPM7 kinase as a suppressor of basal phagocytosis and a regulator of cellular pH.