The p47 GTPases are essential for interferon-γ-induced cell-autonomous immunity against the protozoan parasite, Toxoplasma gondii, in mice, but the mechanism of resistance is poorly understood. We show that the p47 GTPases, including IIGP1, accumulate at vacuoles containing T. gondii. The accumulation is GTP-dependent and requires live parasites. Vacuolar IIGP1 accumulations undergo a maturation-like process accompanied by vesiculation of the parasitophorous vacuole membrane. This culminates in disruption of the parasitophorous vacuole and finally of the parasite itself. Over-expression of IIGP1 leads to accelerated vacuolar disruption whereas a dominant negative form of IIGP1 interferes with interferon-γ-mediated killing of intracellular parasites. Targeted deletion of the IIGP1 gene results in partial loss of the IFN-γ-mediated T. gondii growth restriction in mouse astrocytes.

The insertion of adenovirus type 12 (Ad12) DNA into the hamster genome and the transformation of these cells by Ad12 can lead to marked alterations in the levels of DNA methylation in several cellular genes and DNA segments. Since such alterations in DNA methylation patterns are likely to affect the transcription patterns of cellular genes, it is conceivable that these changes have played a role in the generation or the maintenance of the Ad12-transformed phenotype. We have now isolated clonal BHK21 hamster cell lines that carry in their genomes bacteriophage λ and plasmid pSV2neo DNAs in an integrated state. Most of these cell lines contain one or multiple copies of integrated λ DNA, which often colocalize with the pSV2neo DNA, usually in a single chromosomal site as determined by the fluorescent in situ hybridization technique. In different cell lines, the loci of foreign DNA insertion are different. The inserted bacteriophage λ DNA frequently becomes de novo methylated. In some of the thus-generated hamster cell lines, the levels of DNA methylation in the retrotransposon genomes of the endogenous intracisternal A particles (IAP) are increased in comparison to those in the non-λ-DNA-transgenic BHK21 cell lines. These changes in the methylation patterns of the IAP subclone I (IAPI) segment have been documented by restriction analyses with methylation-sensitive restriction endonucleases followed by Southern transfer hybridization and phosphorimager quantitation. The results of genomic sequencing experiments using the bisulfite protocol yielded additional evidence for alterations in the patterns of DNA methylation in selected segments of the IAPI sequences. In these experiments, the nucleotide sequences in >330 PCR-generated cloned DNA molecules were determined. Upon prolonged cultivation of cell lines with altered cellular methylation patterns, these differences became less apparent, perhaps due to counterselection of the transgenic cells. The possibility existed that the hamster BHK21 cell genomes represent mosaics with respect to DNA methylation in the IAPI segment. Hence, some of the cells with the patterns observed after λ DNA integration might have existed prior to λ DNA integration and been selected by chance. A total of 66 individual BHK21 cell clones from the BHK21 cell stock have been recloned up to three times, and the DNAs of these cell populations have been analyzed for differences in IAPI methylation patterns. None have been found. These patterns are identical among the individual BHK21 cell clones and identical to the patterns of the originally used BHK21 cell line. Similar results have been obtained with nine clones isolated from BHK21 cells mock transfected by the Ca2+-phosphate precipitation procedure with DNA omitted from the transfection mixture. In four clonal sublines of nontransgenic control BHK21 cells, genomic sequencing of 335 PCR-generated clones by the bisulfite protocol revealed 5′-CG-3′ methylation levels in the IAPI segment that were comparable to those in the uncloned BHK21 cell line. We conclude that the observed changes in the DNA methylation patterns in BHK21 cells with integrated λ DNA are unlikely to preexist or to be caused by the transfection procedure. Our data support the interpretation that the insertion of foreign DNA into a preexisting mammalian genome can alter the cellular patterns of DNA methylation, perhaps via changes in chromatin structure. The cellular sites affected by and the extent of these changes could depend on the site and size of foreign DNA insertion.

Integration of foreign DNA into an established host genome can lead to changes in methylation in both the inserted DNA and in host sequences and potentially alters transgene and cellular transcription patterns. This work addresses the questions of what factors influence de novo methylation, and whether the integration site or inserted DNA can affect de novo methylation. Homologous recombination was used to integrate foreign DNA into a specific gene, B lymphocyte kinase (BLK), in mouse embryonic stem (ES) cells. Two plasmids were chosen for integration; one contained the adenovirus type 2 E2AL promoter upstream of the luciferase reporter gene, and the second carried the early SV40 promoter. The methylation patterns were analyzed using HpaII and MspI restriction endonucleases for both homologously recombined and randomly integrated foreign DNA in the ES cell clones.Upon homologous reinsertion of the BLK gene into the genome of mouse ES cells, methylation patterns in this gene were reestablished. In DNA segments adjoined to the BLK gene, the de novo patterns of DNA methylation depended on the viral sequences in these clones and on the locations of the inserts, i.e. on whether the insertions resulted from homologously recombined or randomly integrated foreign DNA. In homologously recombined DNA, sequences carrying the adenovirus type 2 promoter were heavily methylated, and those with an SV40 promoter and an SV40 enhancer element remained unmethylated or hypomethylated. Upon removal of the enhancer element, these inserted constructs also became heavily methylated. In addition, all randomly integrated constructs were heavily methylated independently of the promoter and enhancer element present in the construct. These results indicate that modes and sites of integration as well as the inserted nucleotide sequence, possibly promoter strength, are factors affecting de novo methylation.

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Indrikis Muiznieks 1,5The state of methylation of the 5Ј-CpG-3Ј sites is known to be linked to the regulation of Birgit Schmitz 1 promoter function by modulating DNA-protein interactions and to the structure of chromaGudrun Schell 1 tin. As part of a project to determine methylation patterns in the human genome, the Yoshihito Yawata 2 methylation profiles were examined in genes for the human erythroid membrane proteins; Walter Doerfler 1 protein 4.2 (P4.2), gene (ELB42), band 3 (B3), gene (EPB3), and β-spectrin (β-Sp), gene 1 Institute of Genetics, University (SPTB). The bisulfite protocol of the genomic sequencing method was applied.(1) In the of Cologne, Köln, Germany DNA from peripheral white blood cells, the promoter regions of EPB3 and ELB42 were 2 Division of Hematology, extensively methylated, but the promoter of SPTB was totally unmethylated. (2) During Department of Medicine, erythroid differentiation, (i) ELB42 was unmethylated in DNAs from the cell line UT-7/EPO, Kawasaki Medical School, but became methylated (55Ϫ93%) in cultured erythroblasts from peripheral BFU-E. The Kurashiki City, Japan mRNA from ELB42 was first detected in early erythroblasts and protein 4.2 was expressed Present addresses: in late erythroblasts. (ii) EPB3 was consistently methylated in UT-7/EPO and also in cultu-3 Institute of Human Genetics red erythroblasts from burst forming unit erythroid (BFU-E) from peripheral blood. EPB3 and Anthropology, Heinrichand ELB42 were efficiently transcribed in UT-7 cells only after erythropoietin stimulation. Heine-University, Duesseldorf, (iii) SPTB remained unmethylated in DNAs from UT-7/EPO and cultured erythroblasts. (3) Germany 4 Institute of Human Genetics, We also investigated methylation profiles in peripheral white blood cells from patients with University of Essen Medical erythroid diseases, like complete P4.2 deficiency due to ELB42 mutations, hereditary School, Essen, Germany spherocytosis with EPB3 mutations, and hereditary elliptocytosis with SPTB mutations. 5 Department of Microbiology,The methylation profiles of the promoter regions of these three genes were essentially Latvian University, Riga, Latvia identical to those in healthy individuals.