Molecular Biology Reports 31: 165–169, 2004. © 2004 Kluwer Academic Publishers. Printed in the Netherlands. 165 Identification of a cDNA clone encoding DIP1-binding protein in Drosophila melanogaster Bruna De Felice 1 , Loredana F. Ciarmiello 1 & Robert R. Wilson 2 1 Department of Life Sciences, University of Naples II, Via Vivaldi 43, 81100 Caserta, Italy 2 NOAA, 325 Broadway, Boulder, CO, USA (Phone: ++39-823-274543; Fax: ++39-823-274571; E-mail: bruna.defelice@unina2.it) Accepted 1 March 2004 Key words: two-hybrid system assay, DIP1 protein, L27a protein Abstract The Drosophila melanogaster L27a gene encodes a ribosomal protein which is a member of the L15 family of ribosomal proteins. D.m. L27a is closely related to the mammalian protein that has been found differentially expressed in lung cancer tissues and therefore could be involved in the control of cell proliferation such as the ribosomal protein S6. Our work elucidates the role of DIP1 which is a novel protein that we found in Drosophila. We performed a two-hybrid system assay and identified the L27a protein as an interactor of DIP1. The interaction was then validated by in vitro binding assays. DIP1, similar to other nuclear proteins in eukaryotes, is localized to the nuclear periphery and chromatin domain in all nuclei, but disappears at the metaphase. It is possible that in D.m. L27a protein, via interaction with DIP1, could be involved in protein synthesis as well as in cell cycle regulation. Introduction DIP1 is a novel protein in D.m. that, similar to other nuclear proteins in eukaryotes [1–5], is localized to the nuclear periphery and chromatin domain in all nuclei, but disappeared at the metaphase [6]. DIP1 has been found at the nuclear envelope during interphase. This association is not likely to be transient since it sur- vives the strong extraction procedure used in isolating nuclear envelope. The significance of this finding is unclear, but it suggests that DIP1 may play different functions in the cell cycle, because the localization of a protein within a cell plays an important role in the function of that protein [1]. Alternatively, DIP1 could have a dichotomous role, because it might be involved in cell cycle and/or regulating the nuclear transport of proteins required for entry into mitosis. Protein-protein interactions played important roles in almost all events that take place in a cell. Because proteins assembled into large complexes to perform discrete activities, the characterization of the interac- tion pattern of a protein could provide assistance in the elucidation of the functions of that protein [7]. To elucidate DIP1 function in D.m. we looked for DIP1 interactor proteins using the yeast two-hybrid system assay and we identified L27a protein as an interactor with DIP1. Material and methods Yeast two-hybrid system assay The two hybrid system assay was performed using Matchmaker GAL4 two-hybrid system 3 and vec- tor pACT2 containing the Drosophila embryo cDNA library obtained from Clontech. Yeast strain AH109 (MATa, trp 1-901, leu 2-3,112, ura 3-52, his 3- 200, gal4, gal80, LYS2:GAL1 UAS -GAL1 TATA - HIS3,GAL2 UAS -GAL2 TATA -ADE2, URA3:MEL1 UAS - MEL1 TATA -LacZ) was used to screen the library and to verify protein-protein interactions, which could eliminate false positives by using three reporters-