Although overall Idas and Geminin show low series similarity, they exhibit significant conservation between their coiled-coil domains, indicating that they are evolutionarily related. shown to interact with transcriptional regulators of differentiation, such as Six3 (16), Hox and Polycomb family members (17), the catalytic Ellagic acid subunit of the SWI/SNF chromatin remodeling complex, Brg1/Brm (18), and regulators of Sox2 gene transcription (19), thereby modulating proliferation-differentiation decisions during development. Geminin balanced interactions with its multiple binding Ellagic acid partners are central to its function in the coordination of proliferation and differentiation. The central region of Geminin, made up of the Geminin coiled-coil, is sufficient for interactions with Cdt1 and inhibition of licensing and mediates homodimerization of Geminin (20C23). In this study we introduce a previously uncharacterized human protein that is similar to Geminin in KSHV K8 alpha antibody its central coiled-coil region. We name this protein Idas (Idas being a cousin of the Gemini in Ancient Greek Mythology). We show that Idas binds to the Geminin coiled-coil region and can modulate Geminin ability to bind Cdt1. Our data spotlight Idas as a novel Geminin binding partner and regulator. EXPERIMENTAL PROCEDURES Bioinformatics Analysis Tblastn (NCBI) was used to search expressed sequences from the human genome using the hGeminin protein sequence as query. mRNAs deriving from 5q11.2, on chromosome 5, were identified as encoding a protein with Ellagic acid significant similarity to Geminin. This region is currently annotated as LOC345643. Gene2EST was used to identify Expressed Sequence Tags from this locus. The expression of LOC345643 is usually supported by 1 full-length cDNA (clone CS0DK002YL21) and 10 Expressed Sequence Tags that represent sequence reads from 5 cDNAs from HeLa cells (clone CS0DK002YL21), Jurkat cells (clone CS0DJ001YB09), melanotic melanoma (clone IMAGE:3916292) and mammary adenocarcinoma cell lines (clone IMAGE:5406358), and pooled primary tissues. (“type”:”entrez-nucleotide”,”attrs”:”text”:”DR007866.1″,”term_id”:”66271956″,”term_text”:”DR007866.1″DR007866.1). Alignment of available Expressed Sequence Tags and cDNA sequences was used to generate a predicted mRNA of 2087 nucleotides. This is longer Ellagic acid than the automatically predicted mRNA present in the databases (Locus “type”:”entrez-nucleotide”,”attrs”:”text”:”XR_040412″,”term_id”:”239742543″,”term_text”:”XR_040412″XR_040412, 1158 nucleotides) because of the presence of 5-UTR (178 nucleotides) and 3-UTR (929 nucleotides) sequences. The intron-exon boundaries were defined by aligning the predicted mRNA to human genomic sequences. Fragments of the predicted mRNA were amplified by polymerase chain reaction (PCR) and sequenced to verify expression of this locus in HeLa cells. Real time PCR was used to detect expression of Idas in different human cell lines. The full predicted open reading frame (ORF) was amplified from HeLa cDNA and sequenced. The hIdas protein sequence was derived from the ORF. Idas orthologues in mouse (LOC622408) and (LOC100158359) were identified by Blast using the human Idas protein sequence as query. For sequence analysis and alignments, the following programs were used: Gene2EST (24), Coils (25), ELM (Eukaryotic Linear Motif) resource for functional sites in proteins (26), and ClustalW (27). Plasmids Total HeLa cDNA was used to amplify the hIdas ORF by nested PCR, introducing NheI and KpnI sites at the ends of the predicted hIdas ORF. The PCR product was cloned into the mammalian expression vector cDNA3.1EGFP (Invitrogen) at the Ellagic acid NheI and KpnI restriction sites to produce a protein C-terminally fused to green fluorescent protein (GFP) under the control of the constitutive CMV promoter. N-Idas (amino acids 1C127) and C-Idas (amino acids 131C385) were cloned into the NheI and HindIII sites of pcDNA3.1EGFP (Invitrogen) after PCR amplification from full-length Idas to introduce restriction sites. IdasHA and Idas-Cherry were generated by replacing GFP from the IdasGFP pcDNA3.1 construct with three repeats of the human influenza hemagglutinin epitope (HA) and sequences coding for Cherry (Clontech), respectively. All products produced by PCR were fully sequenced. For experiments, the predicted folded domain name (101C284, dIdas) and the coiled-coil domain name (173C245, tIdas) were cloned for expression in the NKI-His-3C-LIC (for cleavable His-tag expression) and the NKI-LIC plasmids (for native versions). Because these plasmids are resistant to kanamycin and ampicillin, respectively, they allow for efficient co-expression experiments. The Idas sequence was analyzed using the.