Molecular Cloning, Characterization And Functional Analysis Of A New Motor Protein Gene, Oocyte-G1

Reproductive system is recognized as a source of regulatory molecules that influence animal development. A complicated signal network is formed by these molecules; through an array of complex interaction, the development process is under control. Recently, more and more hormones and signal molecules were identified; however, function of the majority of members of the network and the detailed mechanism are still unclear. During efforts to clone the follicular developmental related genes, we screened and identified a new kinesin superfamily gene, Oocyte-G1 by DDRT-PCR and cDNA library screening. This gene shows different expression levels in mouse ovaries during development. Oocyte-G1 belongs to Kinesin N-8 subfamily. The open reading frame of Oocyte-G1 is 2,994 bp long, which encodes a 997-residue predicted protein. Northern blot analysis revealed the presence of`~3.6 kb Oocyte-G1 mRNA in ovary, lung, kidney, testis and brain. Oocyte-G1 was weakly expressed on day 5 and was expressed at moderate level on day 10. Thereafter, on day 15 or day 40, there was an increase in expression, followed by a decline on day 20 or day 120. Furthermore, we studied the Ooctye-G1 protein by using the antiserum against a peptide sequence unique to this gene in Western blot analysis and immunolocalization. The antiserum recognized a prominent band of ~110 kD in immunoblots, Oocyte-G1 were dispersed in oocytes and some surrounding granulose cells in mouse ovaries.To study the function of Oocyte-G1, a series of experiments in vitro and in vivo was performed. By using TUNEL and flow cytometry methods, increased apoptosis caused by over-expression of Oocyte-G1 was observed in cultured cells. Oocyte-G1 transgenic mouse model was then constructed to verify this result, an increased apoptosis rate of the germ cells was observed in the Oocyte-G1 transgenic mice. In addition, the majority of the Oocyte-G1 transgenic mice experienced developmental retardation releated aspects, such as the body weight or the organ significantly smaller than wild type ones; in adult mouse testis, seminiferous tubules were underdevelopment hence the spermatogenesis was impaired, in female transgenic mice, most follicles are in early developmental stage of folliculogenesis and no mature follicles were detected in 6-week-old age.Immunoprecipitation were then used to screen potential candidates, which can interact with Oocyte-G1. The results revealed a potential interaction exist between Oocyte-G1 and Caspase-3. The results of co-localization show that this interaction was located at the surrounding area of the nucleus. Increased expression level of Caspase-3 was observed in Oocyte-G1 transgenic mice. By using over-expression and RNAi strategy in vitro, we found that expression level of Caspase-3 was up-regulated by Oocyte-G1.In summary, this dissertation describes the cloning, identification, characterization and functional analysis of a new mouse Kinesin N-8 member gene, Oocyte-G1. All results suggest the potential roles of Oocyte-G1 in apoptosis: it may promote apoptosis through act on some members of the apoptotic signaling pathways. These data may provide several clues to further understanding of the animal reproductive system development and the mechanism or physiological roles of Oocye-G1 in apoptosis.