We investigated the binding regions of components of the origin recognition complex (ORC) in the human genome. For this purpose, we performed chromatin immunoprecipitation assays with antibodies against human Orc1 and Orc2 proteins. We identified a binding region for human Orc proteins 1 and 2 in a <1-kbp segment between two divergently transcribed human genes. The region is characterized by CpG tracts and a central sequence rich in AT base pairs. Both, Orc1 and Orc2 proteins are found at the intergenic region in the G 1 phase, but S-phase chromatin contains only Orc2 protein, supporting the notion that Orc1p dissociates from its binding site in the S phase. Sequences corresponding to the intergenic region are highly abundant in a fraction of nascent DNA strands, strongly suggesting that this region not only harbors the binding sites for Orc1 protein and Orc2 protein but also serves as an origin of bidirectional DNA replication.The origin recognition complex (ORC) was first identified in Saccharomyces cerevisiae as a multiprotein factor that binds to yeast origins of replication in an ATP-dependent manner (4). ORC is composed of six protein subunits, Orc1p to Orc6p, encoded by essential yeast genes whose deletions result in lethality. Components of ORC interact with the Cdc6 protein as an early step in the assembly of prereplicative complexes that are subsequently completed by the loading of Mcm protein hexamers (3,18,43,64).The binding sites for ORC in yeast DNA are known as ARS (for autonomously replicating sequences) because they direct the extrachromosomal replication of ARS-bearing plasmids. Chromosomal ARS elements serve as origins of bidirectional DNA replication in yeast and consist of about 150 bp of DNA with an essential 11-bp AT-rich ARS consensus sequence A and two or three short stimulatory sequences, B1 to B3, which are functionally important but divergent in sequence (36,40). The ORC binding site in yeast origins is a bipartite DNA sequence that includes the consensus A element and the adjacent B1 element (4, 47, 54). The B3 element is the binding site for a transcription factor, Abf1 (19). Protein-DNA crosslinking studies show that subunits Orc1p, Orc2p, Orc4p, and Orc5p contact a DNA strand in the major groove of the ARS binding site and may determine the specificity of the reaction, whereas subunits Orc3p and Orc6p appear to form proteinprotein contacts (33). Bidirectional DNA replication is initiated in the immediate neighborhood of the ARS B1 element (8), suggesting that DNA-bound ORC determines the start points for DNA replication in yeast.Proteins homologous to the yeast ORC, as well as to other proteins required for prereplicative complex formation, have been detected in all eukaryotes examined, suggesting that the manner of prereplicative complex formation may be highly conserved. Experiments with Xenopus egg extracts have clearly shown that chromatin-bound ORC serves as a landing pad for the subsequent binding of Cdc6 which, together with the Cdt1 protein, is required for the recruitment...

Central aspects in the pathogenesis of scrub typhus, an infection caused by Orientia (O.) tsutsugamushi, have remained obscure. Its organ and cellular tropism are poorly understood.The purpose of this study was to analyze the kinetics of bacterial dissemination and associated inflammatory responses in infected tissues in an experimental scrub typhus mouse model, following infection with the human pathogenic strain Karp. We provide a thorough analysis of O. tsutsugamushi infection in inbred Balb/c mice using footpad inoculation, which is close to the natural way of infection. By a novel, highly sensitive qPCR targeting the multi copy traD genes, we quantitatively monitored the spread of O. tsutsugamushi Karp from the skin inoculation site via the regional lymph node to the internal target organs. The highest bacterial loads were measured in the lung. Using confocal imaging, we also detected O. tsutsugamushi at the single cell level in the lung and found a predominant macrophage rather than endothelial localization. Immunohistochemical analysis of infiltrates in lung and brain revealed differently composed lesions with specific localizations: iNOS-expressing macrophages were frequent in infiltrative parenchymal noduli, but uncommon in perivascular lesions within these organs. Quantitative analysis of the macrophage response by immunohistochemistry in liver, heart, lung and brain demonstrated an early onset of macrophage activation in the liver. Serum levels of interferon (IFN)-γ were increased during the acute infection, and we showed that IFN-γ contributed to iNOS-dependent bacterial growth control.Our data show that upon inoculation to the skin, O. tsutsugamushi spreads systemically to a large number of organs and gives rise to organ-specific inflammation patterns. The findings suggest an essential role for the lung in the pathogenesis of scrub typhus. The model will allow detailed studies on host-pathogen interaction and provide further insight into the pathogenesis of O. tsutsugamushi infection.

T cells are known to contribute to immune protection against scrub typhus, a potentially fatal infection caused by the obligate intracellular bacterium Orientia (O.) tsutsugamushi. However, the contribution of CD8+ T cells to protection and pathogenesis during O. tsutsugamushi infection is still unknown. Using our recently developed BALB/c mouse model that is based on footpad inoculation of the human-pathogenic Karp strain, we show that activated CD8+ T cells infiltrate spleen and lung during the third week of infection. Depletion of CD8+ T cells with monoclonal antibodies resulted in uncontrolled pathogen growth and mortality. Adoptive transfer of CD8+ T cells from infected animals protected naïve BALB/c mice from lethal outcome of intraperitoneal challenge. In C57Bl/6 mice, the pulmonary lymphocyte compartment showed an increased percentage of CD8+ T cells for at least 135 days post O. tsutsugamushi infection. Depletion of CD8+ T cells at 84 days post infection caused reactivation of bacterial growth. In CD8+ T cell-deficient beta 2-microglobulin knockout mice, bacterial replication was uncontrolled, and all mice succumbed to the infection, despite higher serum IFN-γ levels and stronger macrophage responses in liver and lung. Moreover, we show that CD8+ T cells but not NKT cells were required for hepatocyte injury: elevated concentrations of serum alanine aminotransferase and infection-induced subcapsular necrotic liver lesions surrounded by macrophages were found in C57Bl/6 and CD1d-deficient mice, but not in beta 2-microglobulin knockout mice. In the lungs, peribronchial macrophage infiltrations also depended on CD8+ T cells. In summary, our results demonstrate that CD8+ T cells restrict growth of O. tsutsugamushi during acute and persistent infection, and are required to protect from lethal infections in BALB/c and C57BL/6 mice. However, they also elicit specific pathologic tissue lesions in liver and lung.

The locations of the origin recognition complex (ORC) in mammalian genomes have been elusive. We have therefore analyzed the DNA sequences associated with human ORC via in vivo cross-linking and chromatin immunoprecipitation. Antibodies specific for hOrc2 protein precipitate chromatin fragments that also contain other ORC proteins, suggesting that the proteins form multisubunit complexes on chromatin in vivo. A binding region for ORC was identified at the CpG island upstream of the human TOP1 gene. Nascent strand abundance assays show that the ORC binding region coincides with an origin of bidirectional replication. The TOP1 gene includes two well characterized matrix attachment regions. The matrix attachment region elements analyzed contain no ORC and constitute no sites for replication initiation. In initial attempts to use the chromatin immunoprecipitation technique for the identification of additional ORC sites in the human genome, we isolated a sequence close to another actively transcribed gene (TOM1) and an alphoid satellite sequence that underlies centromeric heterochromatin. Nascent strand abundance assays gave no indication that the heterochromatin sequence serves as a replication initiation site, suggesting that an ORC on this site may perform functions other than replication initiation.Origins of DNA replication are the chromosomal regions where DNA replication forks for bidirectional duplication of replicons are established. The large and discontinuous genomes of eukaryotes require a large number of origins that are distributed throughout the genome to guarantee a complete replication within the limited time of the S phase in a cell division cycle (for reviews, see Refs. 1-9. The best characterized eukaryotic origins are those of the budding yeast Saccharomyces cerevisiae because they function as sites of replication initiation in extrachromosomal plasmid DNA and are, therefore, amenable to detailed molecular analyses (10, 11). Prototypic budding yeast origin (autonomously replicating sequences (ARSs) 1 ) are composed of 100 -200 base pairs and contain several essential sequence elements including a domain A with the AT-rich ARS consensus sequence and three short stimulatory elements, B1-B3, which are functionally important but divergent in sequence (12). The ARS consensus sequence and the adjacent B1 domain element constitute a binding site for proteins of the origin recognition complex (ORC), whereas the B3 domain element forms a binding site for the transcription factor Abf1 in some, but not all yeast origins (13).ORC is a multimeric protein complex composed of six essential subunits (Orc1p-Orc6p) that associate in an ATP-dependent manner with ARSs (13-15). The major known function of ORC appears to be the recruitment of factors such as Cdc6, Mcm proteins, and others for the formation of functional prereplication complexes (16 -19).Origins of replication in multicellular organisms can usually not be investigated by ARS assays. Therefore, biochemical procedures such as two-dimensional gel electrop...

Previous work with yeast cells and with Xenopus egg extracts had shown that eukaryotic pre-replication complexes assemble on chromatin in a step-wise manner whereby specific loading factors promote the recruitment of essential Mcm proteins at pre-bound origin recognition complexes (ORC with proteins Orc1p-Orc6p). While the order of assembly--Mcm binding follows ORC binding--seems to be conserved in cycling mammalian cells in culture, it has not been determined whether mammalian Mcm proteins associate with ORC-bearing chromatin sites. We have used a chromatin immunoprecipitation approach to investigate the site of Mcm binding in a genomic region that has previously been shown to contain an ORC-binding site and an origin of replication. Using chromatin from HeLa cells in G1 phase, antibodies against Orc2p as well as antibodies against Mcm proteins specifically immunoprecipitate chromatin enriched for a DNA region that includes a replication origin. However, with chromatin from cells in S phase, only Orc2p-specific antibodies immunoprecipitate the origin-containing DNA region while Mcm-specific antibodies immunoprecipitate chromatin with DNA from all parts of the genomic region investigated. Thus, human Mcm proteins first assemble at or adjacent to bound ORC and move to other sites during genome replication.