The Xer site‐specific recombination system acts at ColE1 cer and pSC101 psi sites to ensure that these plasmids are in a monomeric state prior to cell division. We show that four proteins, ArgR, PepA, XerC and XerD are necessary and sufficient for recombination between directly repeated cer sites on a supercoiled plasmid in vitro. Only PepA, XerC and XerD are required for recombination at psi in vitro. Recombination at cer and psi in vitro requires negative supercoiling and is exclusively intramolecular. Strand exchange at cer produces Holliday junction‐containing products in which only the top strands have been exchanged. This reaction requires the catalytic tyrosine residue of Xer C but not that of XerD. Recombination at psi gives catenated circular resolution products. Strand exchange at psi is sequential. XerC catalyses the first (top) strand exchange to make a Holiday junction intermediate and XerD catalyses the second (bottom) strand exchange.

Systemic cyclosporin A and tacrolimus are effective treatments for psoriasis. Cyclosporin A and tacrolimus block T cell activation by inhibiting the phosphatase calcineurin and preventing translocation from the cytoplasm to the nucleus of the transcription factor nuclear factor of activated T cells (NFAT). Inhibition of T cell activation is thought to account for their therapeutic action in psoriasis. We investigated whether nonimmune cells in human skin express calcineurin and NFAT1 and whether cyclosporin A and tacrolimus block activation of calcineurin/NFAT in epidermal keratinocytes. The expression patterns of the principal components of calcineurin/NFAT signaling pathway in normal human skin and psoriasis were determined by immunohistochemistry. We assessed calcineurin/NFAT activation in cultured keratinocytes by measuring the degree of nuclear localization of calcineurin and NFAT1 using immunofluorescence/confocal microscopy and assessed if cyclosporin A and tacrolimus blocked nuclear translocation of these proteins. A variety of cell types in normal and psoriatic skin expressed calcineurin and NFAT1, but expression was particularly prominent in keratinocytes. The principal cyclosporin A and tacrolimus binding proteins cyclophilin A and FKBP12 were also expressed by keratinocytes and nonimmune cells in skin. NFAT1 was predominantly nuclear in normal basal epidermal keratinocytes. Increased nuclear localization of NFAT1 was observed in suprabasal keratinocytes within lesional and to a lesser extent nonlesional psoriatic epidermis compared to normal skin (p = 0.001 and p = 0.03, respectively), suggesting increased activation of calcineurin in psoriatic epidermal keratinocytes. Agonists that induce keratinocyte differentiation, specifically 12-0-tetradecanoyl-phorbol-13-acetate (TPA) plus ionomycin, TPA, and raised extracellular calcium, induced nuclear translocation of NFAT1 and calcineurin in keratinocytes that was inhibited by pretreatment with cyclosporin A or tacrolimus. In contrast in human dermal fibroblasts, TPA plus ionomycin or TPA did not significantly alter the proportion of nuclear-associated NFAT1. These data provide the first evidence that calcineurin is functionally active in human keratinocytes inducing nuclear translocation of NFAT1 and also indicate that regulation of NFAT1 nuclear translocation in skin is cell type specific. Inhibition of this pathway in epidermal keratinocytes may account, in part, for the therapeutic effect of cyclosporin A and tacrolimus in skin diseases such as psoriasis.

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The Xer site-specific recombination system functions in Escherichia coli to ensure that circular plasmids and chromosomes are in the monomeric state prior to segregation at cell division. Two recombinases, XerC and XerD, bind cooperatively to a recombination site present in the E. coli chromosome and to sites present in natural multicopy plasmids. In addition, recombination at the natural plasmid site cer, present in ColEl, requires the function of two additional accessory proteins, ArgR and PepA. These accessory proteins, along with accessory DNA sequences present in the recombination sites of plasmids are used to ensure that recombination is exclusively intramolecular, converting circular multimers to monomers. Wild-type and mutant recombination proteins have been used to analyse the formation of recombinational synapses and the catalysis of strand exchange in vitro. These experiments demonstrate how the same two recombination proteins can act with different outcomes, depending on the organization of DNA sites at which they act. Moreover, insight into the separate roles of the two recombinases is emerging.

SUMMARYThe molecular mechanisms underlying B-and T-cell development are, as yet, poorly understood. However, as G proteins regulate a diverse range of biological responses including growth, proliferation and diÂerentiation, we have investigated diÂerential expression of G proteins during B-and T-cell development with the aim of identifying key signals involved in lymphocyte maturation. DiÂerential expression of b 1/2 and a-subunits of the Gs-, i-and q-families was found throughout lymphoid development. Most strikingly, Ga i1 and Ga 11 were very weakly, or not expressed in pre-, immature and mature B cells, thymocytes or mature T cells, but strongly induced in mature B-lymphoblastoid cell lines, some of which have been used as models of germinal centre B cells, suggesting that expression of these G proteins may correlate with the later stages of B-cell development. In contrast, Ga 16 expression was highest in T cells and pre-B cells and progressively declined with B-cell maturation. These findings suggest that G proteins, and the signals they regulate, such as ion channels and/or adenylate cyclase (Ga s/i) and phospholipase C (Gbc and Ga 11/16) are diÂerentially regulated in lymphoid cells in a maturationand lineage-dependent manner.