γδ T cells are considered to be innate lymphocytes that play an important role in host defense against tumors and infections. We recently reported that IL-18 markedly amplified γδ T cell responses to zoledronate (ZOL)/IL-2. In an extension of this finding, we analyzed the mechanism underlying the IL-18–mediated expansion of γδ T cells. After incubation of PBMCs with ZOL/IL-2/IL-18, the majority of the cells expressed γδ TCR, and the rest mostly exhibited CD56brightCD11c+ under the conditions used in this study. CD56brightCD11c+ cells were derived from a culture of CD56intCD11c+ cells and CD14+ cells in the presence of IL-2 and IL-18 without the addition of ZOL. They expressed IL-18Rs, HLA-DR, CD25, CD80, CD83, CD86, and CD11a/CD18. In addition, they produced IFN-γ, TNF-α, but not IL-12, when treated with IL-2/IL-18, and they exerted cytotoxicity against K562 cells, thus exhibiting characteristics of both NK cells and dendritic cells. Incubation of purified γδ T cells with CD56brightCD11c+ cells in the presence of ZOL/IL-2/IL-18 resulted in the formation of massive cell clusters and led to the marked expansion of γδ T cells. However, both conventional CD56−/intCD11chigh dendritic cells induced by GM-CSF/IL-4 and CD56+CD11c− NK cells failed to support the expansion of γδ T cells. These results strongly suggest that CD56brightCD11c+ cells play a key role in the IL-18–mediated proliferation of γδ T cells.

The EcoO109I restriction-modification system, which recognizes 5'-(A/G)GGNCC(C/T)-3', has been cloned, and contains convergently transcribed endonuclease and methylase. The role and action mechanism of the gene product, C.EcoO109I, of a small open reading frame located upstream of ecoO109IR were investigated in vivo and in vitro. The results of deletion analysis suggested that C.EcoO109I acts as a positive regulator of ecoO109IR expression but has little effect on ecoO109IM expression. Assaying of promoter activity showed that the expression of ecoO109IC was regulated by its own gene product, C.EcoO109I. C.EcoO109I was overproduced as a His-tag fusion protein in recombinant Escherichia coli HB101 and purified to homogeneity. C.EcoO109I exists as a homodimer, and recognizes and binds to the DNA sequence 5'-CTAAG(N)(5)CTTAG-3' upstream of the ecoO109IC translational start site. It was also shown that C.EcoO109I bent the target DNA by 54 +/- 4 degrees.

A DNA fragment carrying the genes coding for EcoO109I endonuclease and EcoO109I methylase, which recognize the nucleotide sequence 5′-(A/G)GGNCC(C/T)-3′, was cloned from the chromosomal DNA of Escherichia coli H709c. TheEcoO109I restriction-modification (R-M) system was found to be inserted between the int and psu genes from satellite bacteriophage P4, which were lysogenized in the chromosome at the P4 phage attachment site of the corresponding leuX gene observed in E. coli K-12 chromosomal DNA. Thesid gene of the prophage was inactivated by insertion of one copy of IS21. These findings may shed light on the horizontal transfer and stable maintenance of the R-M system.

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EcoO109I is a type II restriction endonuclease that recognizes seven base pairs of the degenerate and discontinuous sequence RGGNCCY. The enzyme and its complex with DNA were successfully crystallized by the hanging-drop vapour-diffusion method using polyethylene glycols as precipitants. The crystal of EcoO109I belongs to space group I4, with unit-cell parameters a = b = 175.5, c = 44.6 angstoms, and that of the DNA complex belongs to space group P2(1)2(1)2(1), with unit-cell parameters a = 49.1, b = 71.8, c = 203.2 angstroms. Full sets of X-ray diffraction data from the enzyme and its complex with DNA were collected to 2.4 and 1.9 angstroms resolution, respectively.