Screening and identification of proteins interacting with nucleostemin.

AIM: To identify the proteins interacting with nucleostemin (NS), thereby gaining an insight into the function of NS. METHODS: Yeast two-hybrid assay was performed to screen a human placenta cDNA library with the full length of NS as a bait. X-Gal assay and beta-galactosidase filter assay were subsequently conducted to check the positive clones and the gene was identified by DNA sequencing. To further confirm the interaction of two proteins, the DNA fragment coding NS and the DNA fragment isolated from the positive clone were inserted into the mammalian expression vector pcDNA3 and pcDNA3-myc, respectively. Then, two plasmids were cotransfected into the COS-7 cells by DEAE-dextron. The total protein from the cotransfected cells was extracted and coimmunoprecipitation and Western blot were performed with suitable antibodies sequentially. RESULTS: Two positive clones that interacted with NS were obtained from human placenta cDNA library. One was an alpha isoform of human protein phosphatase 2 regulatory subunit B (B56) (PPP2R5A) and the other was a novel gene being highly homologous to the gene associated with spondylo paralysis. The co-immunoprecipitation also showed that NS specifically interacted with PPP2R5A. CONCLUSION: NS and PPP2R5A interact in yeast and mammalian cells, respectively, which is helpful for addressing the function of NS in cancer development and progression.

[Characterization of the interaction between low density lipoprotein receptor-related protein-associated protein 1 and the cancer and embryo expression protein 65].

OBJECTIVE: To understand the mechanism of cancer and embryo expression protein 65 (CEP65) in cancer development. METHODS: CEP65 was used as a bait protein to isolate the partners of CEP65 from a human placenta cDNA library with yeast two hybrid system. The DNA fragments from positive clone were expressed prokaryotic and mammalian cells respectively, the GST pull-down experiment was performed to ascertain the binding activity. RESULTS: After screening a human placenta cDNA library, the low density lipoprotein receptor-related protein-associated protein 1 (RAP) was isolated and shown to interact with CEP65. GST pull-down assay results indicated that His-CEP65 and GST-RAP expressed by prokaryotic system were immunoprecipitated, and so were Myc-RAP and GST-P65 expressed by mammalian cells. CONCLUSION: RAP can interact with CEP65 and RAP may be a candidate to mediate the function of CEP65.

[Eukaryotic expression and intracellular distribution of cancer and embryo expression protein 65].

BACKGROUND & OBJECTIVE: Previous studies show that cancer and embryo expression protein 65 (CEP65) only expresses in various cancer cells and embryo cells. This study was to detect intracellular distribution of CEP65, and make the foundation for further CEP65 function study. METHODS: Two kinds of eukaryotic expression plasmids, which can separately express full-length CEP65-GST and fluorescent fusion protein CEP65-Red, were constructed, and transfected into COS7 cells by DEAE-Dextran method. Plasmosin and nucleoprotein of COS7 cells were separated. Intracellular distribution of CEP65 was detected by Western blot, and observed under fluorescent microscope. RESULTS: Western blot confirmed that CEP65 distributed in cytoplasm and nuclei. Fluorescent protein localization showed diffuse distribution of fluorescence in the cells transfected with pcDNA3-Red, and collective fluorescent articles in cytoplasm and nuclei of the cells transfected with pcDNA3-Red-Cep65. CONCLUSIONS: CEP65 can express efficiently in COS7 cells, and locates in cytoplasm and nuclei. It may be a kind of nucleus-associated protein.

Preparation and analysis of spermatocyte meiotic pachytene bivalents of pigs for gene mapping.

Well-spread meiotic pachytene bivalents were obtained by using the prolonged hypotonic treatment combined with high chloroform Carnory's fixative solution from cells of the testes of domestic pigs. Comparison in the division index and length of pachytene bivalents with metaphase chromosomes showed that those of the former are 5 times higher and 3.42(1.87-5.98) times longer than those of the latter. Comparative studies on chromomere maps of bivalents and mitotic chromosomal G-bands were conducted by using the chromosome 12 as a example. Sex vesicle and various shapes of synaptic sex chromosomes have been observed. Two-color PRimed IN Situ (PRINS) labeling has been conducted successfully on pachytene bivalents of pigs.