1. Expression And Distribution Of Trihydrophobin 1 In Postnatal Developing Mouse Testis 2. Identification Of The Association Of SGT With CD99 In Yeast Cells

Parti: Expression and Distribution of Trihydrophobin 1 in Postnatal Developing Mouse TestisObjective To study the expressions and varieties of trihydrophobin1 (TH1) mRNA and protein levels during postnatal development in mouse testis, and evaluate the potential role of TH1 as a binding partner of A-Raf in mouse testis.Methods Preparing TH1 mRNA and protein of different time points, and the expression levels of TH1 mRNA in the developing mouse testis were detected by SYBR Green Real-time PCR. The protein levels were detected by Western blotting. We confirmed that TH1 was a binding partner of A-Raf in the physiological situation. Using immunohistochemistry analysis, we detected the temporal and spatial expression patterns of TH1 protein in testis sections. Immunofluorescence staining showed the cellular distribution of TH1 and co-location of TH1 and A-Raf.Results (1) TH1 associated with A-Raf in mouse testis using co-immunoprecipitation.(2) Realtime PCR analysis and statistical analysis indicated that the individual TH1 gene was not expressed differentially during mouse testicular development (P>0.05).(3) Western Blotting analysis indicated that TH1 protein expression levels were lower in testis of postnatal 4, 6-week-old mice as compared to testis of postnatal 1, 2, 8 and 10-week-old mice during development (P<0.05).(4) TH1 was found to localize in different cells of mouse testis at different ages. For postnatal 2-week-old mice, TH1 was present in primary spermatocytes, which has bigger nuclei than the surrounding cells. Sertoli cell (sustentacular cell) and Leydig cells also showed affinity for TH1 staining. For postnatal 8-week-old mice TH1 was most prominently expressed in spermatogonia, but in other cells there was no positive staining of TH1. An interesting transition of TH1 immunostaining that was observable in the testis sections of postnatal 4-week-old mice. An intense immunostaining was evident in Leydig cells throughout postnatal testicular development.(5) We analyzed TH1 distribution in cultured rat Leydig cells, the immunofluorescence staining was then performed on permeabilized Leydig cells from adult rat testis with rabbit polyclonal antiserum against human TH1. The high expression of TH1 was mainly found in the cytoplasm.(6) For mLTC-1 cells and the testis sections, the strong positive signals in yellow color were visualized in the merged images, representing the colocalization of TH1 and A-Raf in the Leydig cellsConclusions As a partner of A-Raf, TH1 may play an important role in the testicle development. The expressing changes of TH1 establish a basis to further study their functions and mechanisms in regulating mammalian testicular development. Part II: Identification of the Association of SGT with CD99in Yeast Cells.Objective To identify the association of SGT and CD99 in the yeast cells.Method The EGY48 yeast strain cells cotransformed with plasmids were transferred to selective medium lacking tryptophan, leucine, uracil andhistidine and supplemented with X-b-gal. The colonies can grow and turnblue (LEU2~+ /LacZ~+), indicating the association in yeast cells. The interacting proteins, p53 and SV40 large T-antigen, fused with the BD and the AD vectors, respectively, were used as positive controls. Co-transformation with empty vector plexA and pB42AD plasmid, pLexA-SGT and pB42AD, or pLexA and pB42AD-CD99 were used as negative controls.Results Only we co-transformed into yeast cells with SGT and CD99 plasmids, the colonies could grow and turn blue. The degrees of turning blue and the growth of colonies accorded with the positive control. It was confirmed when we changed the vectors of BD and AD, the same results were obtained.Conclusion Our results revealed that SGT associated CD99 in the yeast cells.