New Human Gene Pp5644 Functional Studies As Well As Yeast Two-hybrid Vector Transformation And Library Construction

This thesis is composed of two separate parts: the study of a human novel gene pp5644 which is possibily related to hepatocellular carcinema; the reconstruction of vector plasmids and library constructions of yeast two-hybrid system.In Part I , we studied pp5644 and its biological functions deeply. The pp5644 gene encodes a protein consisting of 124 amino acids. MTC-PCR researched the tissue expression pattern of pp5644 and the result showed that pp5644 was expressed in almost all tested tissues, except skeletal muscle and pancreas. The highest level of pp5644 mRNA expression was observed in brain, kidney and ovary. Subcellur localization assay displayed that pp5644 accumulated in cytoplasm and exhibited a punctuate distribution. Cell growth curve test and colony formation assay showed that the overexpression of pp5644 could inhibit tumor cell growth. Through yeast two-hybrid screening, we obtained three known proteins to interacting with pp5644: FAPP1(phosphatidylinositol-four-phosphate adaptor proteinl) associated protein-1, named as FASP1; NFκ B essential modulater, named as NEMO or IKKγ; bZIP transcription factor MafF. Yeast mating assay, GST Pull-down assay in vitro and co-immunoprecipitation assay in vivo confirmed the specificity of these interactions. The immunofluorescent staining assay indicated that pp5644 and FASP1 co-localized in cytoplasm primarily. But the subcellular colocalization assay showed that the subcellular distribution of pp5644 and MafF were both drastically altered when they co-expressed in cells. Pp5644 translocated partially from the cytoplasm to the nucleolus, and MafF led to a prominent increase in the nucleolus compared with that of MafF alone. As a result, pp5644 and MafF could co-localize in the nucleolus.We further researched the interaction between pp5644 and MafF. Series truncated mutants assay displayed that the coiled-coil domain of pp5644 and the leucine zipper domain of MafF are sufficient and necessary for this specific interaction. The ONPG liquid semi-quantitative assay suggested that the interaction between these two domains (pp5644Δ3 and MafFΔ4) was the strongest and the most directly. Through amino acid sequences analysis and computer simulation analysis on these two domains,we found that the composing and the form on amino acids of them are very similar. They are both atypical heptad-repeats: seven residue repeat pattern, denoted abcdefg, in which residues at positions d are generally hydrophobic, and form a hybrophobic stripe along the surface when this domain is in a helical conformation after protein was fold. Therefore, in an aqueous environment, it is favorable for the coiled-coil domain of pp5644 to interact with the Leu-zipper domain of MafF via their hydrophobic residues, because in such a way the hydrophobic residues of two domains are buried in the interface of the complex, leading to steady exist of this protein complex. The trans-activation assays in yeast and mammalian cells revealed that the interaction between pp5644 and MafF could activate the transcription driven by US2 DNA element that MafF could specifically recognize and bind to, but MafF alone could not trans-activate. And the truncated mutant pp5644Δ3 had almost the same functions of pp5644 in cooperateing trans-activation. These results indicated that pp5644 could act as a co-activator of transcription factor MafF. Then pp5644 and MafF together carried out trans-activation to regulate some specific genes on transcription level.Considering the interactions between pp5644 and FASP1 and NEMO in cytoplasm or MafF in nucleolus, we surmised that pp5644 could take part in various signaling pathways through these different interactions to affect the transcriptions of some important genes, which finally lead to the ability of pp5644 to inhibit the growth of tumor cells or other results. pp5644 may be an important and multi-functions gene. These studies throw a highlight on pp5644's biological functions, such as the transcription regulation function, the possible mechanism and detailed process in inhibiting cell growth and so on.In Part II of the thesis, we summarized the partial work of Human Liver Proteome Project(HLPP) which our group joined in, including the reconstruction of vector plasmids and library constructions of yeast two hybrid system. HLPP hope to use large-scale yeast two-hybrid screening to establish a linkage map of protein-protein interaction network in human liver. For this work, we firsrly need a set of vectors and methods to large-scale gene cloning. What's more, in order to fully utilize resourcesand research the specific gene library of Chinese in the future, we also hope to construct the yeast two-hybrid libraries of Chinese liver cDNA. According to these needs, we reconstructed the vector plasmids of two common yeast two-hybrid systems, including pPC86, pDBLeu and pGADT7, pGBKT7. They were all inserted a novel multi-cloning sites, which contain Sfi I A and Sfi I B digested sites. After enzyme digestion test and DNA sequencing, the reconstructed plasmids were indetified and named as NpPC86, NpDBLeu, NpGADT7 and NpGBKT7. These reconstruncted vector plasmids had been widly used to large-scale and high-flux gene cloning and yeast two-hybrid screening.Using Clontech SMART technology, we carried out RT-PCR using mRNA of Chinese liver or purchased mRNA as templates. The obtained dscDNA were digested with Sfi I enzyme and reclaimed, then were ligated with Sfi I -digested NpPC86 or NpDBLeu vector. The ligation mixtures were transformed into XL10-Gold high efficiency competent cells. Harvest all the transformants and NpPC86-Chinese liver cDNA library or NpDBleu-liver cDNA library was obtained. The library was titered and the percentage of recombinant clones was determined, and the size of the inserts was identified by PCR. The constructed umplified cDNA library consists of more than 10° independent clones with a recombinant rate of above 95%, and the titering of amplified library was more than 10~8cells/ml. The insert's size ranged from 0.4 to 2.0 kb. The libraries sufficed the requirements of library construction qualitatively. The initial usage result also indicated that the constructed libraries can be widely used to yeast two-hybrid screening. Our work not only made up the limitations of commercial yeast two-hybrid libraries, such as the sources of dscDNA are limited, there are only AD-library and without BD-library, but also layed a solid foundation for the future study of HLPP.