Abstract
Pituitary tumor-transforming gene (PTTG) is a recently characterized proto-oncogene that is expressed specifically in adult testis. In this study, we have used in situ hybridization and developmental Northern blot assays to demonstrate that PTTG mRNA is expressed stage-specifically in spermatocytes and spermatids during rat spermatogenic cycle. We have used the yeast two-hybrid system to identify proteins that interact with PTTG in testicular cells. Two positive clones were characterized. One of the clones is the ribosomal protein S10, the other encodes a novel human DnaJ homologue designated HSJ2. Northern blot analysis showed that testis contains higher levels of HSJ2 mRNA than other tissues examined, and the expression pattern of HSJ2 mRNA in postnatal rat testis is similar to PTTG. S10 mRNA levels do not vary remarkably among different tissues and remains unchanged during testicular germ cell differentiation. In vitro binding assays demonstrated that both S10 and HSJ2 bind to PTTG specifically and that PTTG can be co-immunoprecipitated with S10 and HSJ2 from transfected cells. Moreover, the binding sites for both proteins were located within the C-terminal 75 amino acids of the PTTG protein. These results suggest that PTTG may play a role in spermatogenesis.
Highlights
Pituitary tumor-transforming gene (PTTG)1 was originally isolated from a rat pituitary tumor cell line by RNA differential display [1]
We have used in situ hybridization and developmental Northern blot assays to determine the cell type and the differentiation stage in which PTTG mRNA is expressed during the spermatogenic cycle
Our results showed that PTTG mRNA is expressed predominantly in spermatocytes and spermatids in a stage-specific manner, suggesting that PTTG may play a role in spermatogenesis
Summary
Pituitary tumor-transforming gene (PTTG) was originally isolated from a rat pituitary tumor cell line by RNA differential display [1]. The yeast two-hybrid system developed by Fields and Song [3] was used to provide a physiological environment in which to detect potential interactions involving the PTTG protein This system has been used to screen cDNA libraries for clones encoding proteins capable of binding a protein of interest (4 – 6). Eukaryotic DnaJ proteins have been implicated in various cellular processes, including correct protein folding [7,8,9], intracellular vesicle traffic [10, 11], activity control of regulatory proteins [12], promotion of translation initiation [13], and tumor suppression [14] Another cDNA clone isolated encodes a previously characterized protein, ribosomal protein S10. The cloning and characterization of HSJ2 and S10 is described, as well as their association with PTTG and the possible functional implications of this interaction
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