The Drosophila latheo (lat) gene was identified in a behavioral screen for olfactory memory mutants. The original hypomorphic latP1 mutant (Boynton and Tully, 1992) shows a structural defect in adult brain. Homozygous lethal lat mutants lack imaginal discs, show little cell proliferation in the CNS of third instar larvae, and die as early pupae. latP1 was cloned, and all of the above mentioned defects of hypomorphic or homozygous lethal lat mutants were rescued with a lat+ transgene. lat encodes a novel protein with homology to a subunit of the origin recognition complex (ORC). Human and Drosophila LAT both associate with ORC2 and are related to yeast ORC3, suggesting that LAT functions in DNA replication during cell proliferation.
A new member of human origin recognition complex (ORC) has been cloned and identified as the human homologue of Saccharomyces cerevisiae ORC4. HsORC4 is a 45-kDa protein encoded by a 2.2-kilobase mRNA whose amino acid sequence is 29% identical to ScORC4. Hs-ORC4 has a putative nucleotide triphosphate binding motif that is not seen in ScORC4. HsORC4P also reveals an unsuspected homology to the ORC1-Cdc18 family of proteins. HsORC4 mRNA expression and protein levels remain constant through the cell cycle. HsORC4P is coimmunoprecipitated from cell extracts with another subunit of human ORC, HsORC2P, consistent with it being a part of the putative human origin recognition complex.Initiation of eukaryotic DNA replication involves the controlled and simultaneous firing of numerous sites of initiation. In the budding yeast Saccharomyces cerevisiae, these sites are defined by specific sequences recognized by a multisubunit complex, the origin recognition complex (ORC) 1 (1-5). All six members of ORC identified in yeast are essential for cell viability (3, 6 -11). ORC, in its pre-replicative or in its post-replicative form, is bound to DNA throughout the cell-cycle (1, 2) and could act as a platform for the recruitment of other proteins involved in the replication machinery.One of the proteins believed to be recruited by ORC before the initiation of DNA replication is the CDC6/Cdc18 (S. cerevisiae or Schizosaccharomyces pombe) protein. CDC6/Cdc18 is closely related in sequence to one of the subunits of ORC, ORC1, over a region that includes a putative nucleotide binding motif. Yeast ORC has been demonstrated to utilize ATP for binding to DNA and to have an ATPase activity that is modulated by binding to the origin of DNA replication (1, 12), suggesting that like DNA replication initiator proteins in Escherichia coli (DnaA) or the simian virus 40 (T antigen), ATP binding and hydrolysis by the eukaryotic initiator protein will be an important regulator of the initiation process.Although DNA sequences defining an origin of replication have not yet been identified in higher eukaryotes, two members of a putative ORC complex homologous to yeast ORC1 and ORC2 have been identified so far in mammals, both in humans and in mice (13,14), suggesting a universal mechanism of initiation of DNA replication in eukaryotes. We report here the identification of a novel member of human ORC homologous to S. cerevisiae ScORC4. Cloning the gene for a third member of the human origin recognition complex is an important step toward the ultimate goal of reconstituting the entire human ORC in vitro. EXPERIMENTAL PROCEDURESCloning and Sequencing-In the Expressed Sequence Tag (EST) Data base (National Center for Biotechnology Information), the partial sequence of a mouse cDNA (AA168456) was deposited with significant homology to a portion of ScORC4 from S. cerevisiae. A BLAST search with the AA168456 sequence revealed a homologous sequence human EST W23942, which in its turn identified a mouse EST AA110785. A BLAST search with the latter identified ...
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