Source was embryonic extract of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.

Identical results were obtained with ovary nuclear extract. IPs were either performed with or without ethidium bromide in the extracts before precipitation to demonstrate that DNA was unnecessary for interaction.

Source was embryonic extract of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.

Identical results were obtained with ovary nuclear extract. IPs were either performed with or without ethidium bromide in the extracts before precipitation to demonstrate that DNA was unnecessary for interaction.

Interaction in vitro; protein produced in a baculovirus and insect cell system.

Binding of Orc6 to preassembled Orc1-5 complex captured as 5 pairwise interactions.

Interaction in vitro; protein produced in a baculovirus and insect cell system.

Crystal structure of hexameric complex captured as 15 pairwise interactions.

Source was adult female ovaries of transgenic fly line; bait produced from endogenous gene; prey produced from transgenic construct.

When expressed at endogenous levels using the Orc6 promoter, the Orc6 mutations Y225F and D224A,Y225A disrupt immunoprecipitation of GFP-tagged Orc6 with endogenous Orc2. When mutant GFP-Orc6 is overexpressed using tubulin-GAL4, a small amount of mutant Orc6 can immunoprecipitate with Orc2.

Embryonic nuclear extract was treated with DNase and RNase.

Source was embryos of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.

Embryonic nuclear extract was treated with DNase and RNase.

Source was embryos of wild-type fly line; bait produced from endogenous gene; prey produced from endogenous gene.

Source was adult female ovaries of transgenic fly line; bait produced from endogenous gene; prey produced from tagged transgenic construct.

The Y225S mutation corresponds to the Meier-Gorlin-Syndrome mutation.

His-Orc1, Orc2, Orc3, Orc4, Orc5 and MBP-Orc6 were co-infected, then purified by sequential His-Orc1 then MBP-Orc6 pull down with additional preparative gel filtration purification.

The Drosophila origin of replication complex (ORC) forms a stable, hexameric complex containing subunits Orc1, Orc2, Orc3, Orc4, Orc5 and Orc6. Binding of Orc6 to Orc2 is probably indirect as it is only observed when all ORC subunits are present.

Source was cell extract of baculovirus-infected High5 cell line; bait produced from transfected construct; prey produced from transfected construct.

The ORC complex visualized by EM was captured as 15 pairwise interactions. The addition of ATP caused the complex to adopt an active/open conformation.

Interaction in vitro; proteins produced as a recombinant fusion proteins in baculovirus and High5 cell system.

Comparison of EM structures of ORC complex, ORC complex bound to DNA and a ternary complex consisting of ORC, DNA and Cdc6 curated as a series on binary protein-protein interactions.

Interaction in vitro; proteins produced as recombinant fusion proteins in a baculovirus and insect cell system.

A hybrid Orc6 protein containing the human N-terminal TFIIB-like domain and the Drosophila C-terminal domain was also IPed with Orc2, whereas the reciprocal Orc6 hybrid with a Drosophila N-terminus and human C-terminus and full length human Orc6 were not.

Source was ovaries of transgenic, Orc6 mutant fly line; bait produced from endogenous gene; prey produced from tagged transgenic construct.