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...

The release of Ca2+ from internal stores is a major source of signal Ca2+ in almost all cell types. The internal Ca2+ pools are activated via two main families of intracellular Ca2+-release channels, the ryanodine and the inositol 1,4,5-trisphosphate (InsP3) receptors. Among multicellular organisms these channel types are ubiquitous, whereas in most unicellular eukaryotes the identification of orthologs is impaired probably due to evolutionary sequence divergence. However, the ciliated protozoan Paramecium allowed us to prognosticate six groups, with a total of 34 genes, encoding proteins with characteristics typical of InsP3 and ryanodine receptors by BLAST search of the Paramecium database. We here report that these Ca2+-release channels may display all or only some of the characteristics of canonical InsP3 and ryanodine receptors. In all cases, prediction methods indicate the presence of six trans-membrane regions in the C-terminal domains, thus corresponding to canonical InsP3 receptors, while a sequence homologous to the InsP3-binding domain is present only in some types. Only two types have been analyzed in detail previously. We now show, by using antibodies and eventually by green fluorescent protein labeling, that the members of all six groups localize to distinct organelles known to participate in vesicle trafficking and, thus, may provide Ca2+ for local membrane-membrane interactions. Whole genome duplication can explain radiation within the six groups. Comparative and evolutionary evaluation suggests derivation from a common ancestor of canonical InsP3 and ryanodine receptors. With one group we could ascertain, to our knowledge for the first time, aberrant splicing in one thoroughly analyzed Paramecium gene. This yields truncated forms and, thus, may indicate a way to pseudogene formation. No comparable analysis is available for any other, free-living or parasitic/pathogenic protozoan.

In the ciliate Paramecium, a variety of well characterized processes are regulated by Ca2+, e.g. exocytosis, endocytosis and ciliary beat. Therefore, among protozoa, Paramecium is considered a model organism for Ca2+ signaling, although the molecular identity of the channels responsible for the Ca2+ signals remains largely unknown. We have cloned - for the first time in a protozoan - the full sequence of the gene encoding a putative inositol (1,4,5)-trisphosphate (Ins(1,4,5)P3) receptor from Paramecium tetraurelia cells showing molecular characteristics of higher eukaryotic cells. The homologously expressed Ins(1,4,5)P3-binding domain binds [3H]Ins(1,4,5)P3, whereas antibodies unexpectedly localize this protein to the osmoregulatory system. The level of Ins(1,4,5)P3-receptor expression was reduced, as shown on a transcriptional level and by immuno-staining, by decreasing the concentration of extracellular Ca2+ (Paramecium cells rapidly adjust their Ca2+ level to that in the outside medium). Fluorochromes reveal spontaneous fluctuations in cytosolic Ca2+ levels along the osmoregulatory system and these signals change upon activation of caged Ins(1,4,5)P3. Considering the ongoing expulsion of substantial amounts of Ca2+ by the osmoregulatory system, we propose here that Ins(1,4,5)P3 receptors serve a new function, i.e. a latent, graded reflux of Ca2+ to fine-tune [Ca2+] homeostasis.

A database search of the Paramecium genome reveals 34 genes related to Ca 2؉ -release channels of the inositol-1,4,5-trisphosphate (IP 3 ) or ryanodine receptor type (IP 3 R, RyR). Phylogenetic analyses show that these Ca 2؉ release channels (CRCs) can be subdivided into six groups (Paramecium tetraurelia CRC-I to CRC-VI), each one with features in part reminiscent of IP 3 Rs and RyRs. We characterize here the P. tetraurelia CRC-IV-1 gene family, whose relationship to IP 3 Rs and RyRs is restricted to their C-terminal channel domain. CRC-IV-1 channels localize to cortical Ca 2؉ stores (alveolar sacs) and also to the endoplasmic reticulum. This is in contrast to a recently described true IP 3 channel, a group II member (P. tetraurelia IP 3 R N -1), found associated with the contractile vacuole system. Silencing of either one of these CRCs results in reduced exocytosis of dense core vesicles (trichocysts), although for different reasons. Knockdown of P. tetraurelia IP 3 R N affects trichocyst biogenesis, while CRC-IV-1 channels are involved in signal transduction since silenced cells show an impaired release of Ca 2؉ from cortical stores in response to exocytotic stimuli. Our discovery of a range of CRCs in Paramecium indicates that protozoans already have evolved multiple ways for the use of Ca 2؉ as signaling molecule.Ca 2ϩ is an important component of cell activity in all organisms, from protozoa to mammals. Thereby Ca 2ϩ may originate from the outside medium and/or from internal stores (7, 18). Ca 2ϩ release from internal stores is mediated by various Ca 2ϩ release channels (CRCs), of which the inositol-1,4,5-trisphosphate receptor (IP 3 R) and ryanodine receptor (RyR) families have been studied most extensively (8,9,29,63 (14). IP 3 generates and maintains a Ca 2ϩ gradient in the hyphal tip of Neurospora crassa and the IP 3 -sensitive channels have been reconstituted and characterized with the planar bilayer method (87). In summary, these publications suggest that IP 3 -dependent signaling pathways are conserved among unicellular organisms, including protozoa.Despite these data, the molecular characterization of IP 3 or ryanodine receptors in low eukaryotes is currently a challenge since the identification of orthologues has not been possible thus far, probably because of evolutionary sequence divergence (66). Traynor et al. (96) identified an IP 3 receptor-like protein, IplA, in Dictyostelium discoideum, which possesses regions related to IP 3 R sequences, but thus far no evidence for IP 3 interaction exists. We have recently described an IP 3 R in the ciliated protozoa Paramecium tetraurelia (referred to here as P. tetraurelia IP 3 R N ) (53), with features characteristic of mammalian IP 3 Rs in terms of topology and ability for IP 3 binding. The expression level of P. tetraurelia IP 3 R N is modulated by extracellular Ca 2ϩ concentrations ([Ca 2ϩ ] o ) and immunofluorescence studies reveal an unexpected localization to the contractile vacuole complex (CVC), the major organelle involved in osmoregulation...

The importance of Ca2+-signaling for many subcellular processes is well established in higher eukaryotes, whereas information about protozoa is restricted. Recent genome analyses have stimulated such work also with Alveolates, such as ciliates (Paramecium, Tetrahymena) and their pathogenic close relatives, the Apicomplexa (Plasmodium, Toxoplasma). Here we compare Ca2+ signaling in the two closely related groups. Acidic Ca2+ stores have been characterized in detail in Apicomplexa, but hardly in ciliates. Two-pore channels engaged in Ca2+-release from acidic stores in higher eukaryotes have not been stingently characterized in either group. Both groups are endowed with plasma membrane- and endoplasmic reticulum-type Ca2+-ATPases (PMCA, SERCA), respectively. Only recently was it possible to identify in Paramecium a number of homologs of ryanodine and inositol 1,3,4-trisphosphate receptors (RyR, IP3R) and to localize them to widely different organelles participating in vesicle trafficking. For Apicomplexa, physiological experiments suggest the presence of related channels although their identity remains elusive. In Paramecium, IP3Rs are constitutively active in the contractile vacuole complex; RyR-related channels in alveolar sacs are activated during exocytosis stimulation, whereas in the parasites the homologous structure (inner membrane complex) may no longer function as a Ca2+ store. Scrutinized comparison of the two closely related protozoan phyla may stimulate further work and elucidate adaptation to parasitic life. See also "Conclusions" section.