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Rat liver carbamoyl phosphate synthetase I gene expression in mammalian cells

Author: GuoYongQing
Tutor: XuRuiLing;NiuBo
School: Shanxi Medical
Course: Pathophysiology
Keywords: Mammalian cells Rat liver Acid synthase Mammalian cell expression Restriction endonuclease Vectors expressing Carbamic acid Liver cells Gene structure Chromatin structure
CLC: R346
Type: Master's thesis
Year: 2002
Downloads: 38
Quote: 0
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Abstract


The carbamyl phosphate synthetase Ⅰ (carbamyl phosphate synthetase Ⅰ, CPS Ⅰ) is the initial enzyme of the urea cycle, mainly present in the liver cell mitochondria, and its gene expression characteristics are significant liver tissue-specific and developmental stage, has a close relationship with the differentiation of the liver cells. Studies have shown that abnormal regulation of cell proliferation and differentiation of the cells to become cancerous is closely related to abnormal differentiation plays a more important role in the carcinogenesis process. The experiment proved that the induced process of the rat hepatoma found the CPS I the activity decreases, which may be due to the result of a reduction of the content of enzyme protein. Hepatoma cells the mRNA quantity of mRNA in chromatin structure analysis confirmed the carcinogenic process CPS Ⅰ reduce the CPS I transcribed region of the chromatin structure by loose change for high transcriptional activity of the low activity of compact nucleosome structure. That CPS I gene expression during carcinogenesis abnormal regulation may occur at the transcriptional level. In vitro transcription, oligonucleotide competition and DNA-binding proteins further prompts the CPS I upstream sequence present in the liver cell-specific DNA-binding protein regulatory sequence, transcription of organized specific and differentiation-specific. Above, our research shows that the CPS the Ⅰ's expression and regulation of the process, the LIVER and differentiation process have a close relationship, so in-depth study of the CPS I gene expression and regulation, to elucidate the mechanism of hepatocarcinogenesis, rat liver carbamic acid phosphate synthase enzyme! Experimental study of the gene expression in mammalian cells Thesis liver cell differentiation processes and pathological and clinical diagnostic significance. Mammalian cell gene expression is the recent development of molecular biology tools and cell biology means the combination of a new technology, in terms of the production of the valuable function of proteins and gene therapy applications, or in the to clarify gene structure, function The basic theoretical research in their mutual relations have a unique value and prospects. Elucidate the CPS gene structure and function, in-depth understanding of the process of differentiation and carcinogenesis of liver cells, a large number of in vitro production of CPS of natural protein, to provide the necessary materials for basic research and the clinical and pathological diagnosis, using the rat CPS cDNA gene as a gene source in CHO cells, NIH / 3 T3 cells, 7402 cells of mammalian cells in the form of vectors expressing constructed three kinds of the \CPS adjustment mechanism. At home and abroad with the carrier more than 10 kinds of gene expression are low molecular weight protein gene, this article also explored the possibility of large molecular weight CPSI gene expression PSVZ carrier results 5.6Kb long CPSI sequence genes successfully in expressed in different cell lines. The above work have not been reported. The following summary of this paper, methods and results. First the CPSI GDNA gene fragment was modified and modified fragments were subcloned to adapt the gene A carrier recombination and gene expression of exogenous gene structure requirements. Bal31 and S; deoxyribonuclease puncturing tangent method, Experimental Study Thesis rat liver carbamic acid phosphate synthase 1 gene expression in mammalian cells are deleted from the 5 'end of the CPSI cDNA recombinant gene directional cut The obtained modified fragment with PBS plasmid recombination, restriction endonuclease method and double-stranded DNA sequencing analysis, the results are screened PB Sm-CPS. . (PS, two subclones, with after the modification of the length of the CPSI CDNA fragment contain 0.587Kb and 0.712, respectively, of the two C ** A fragments retain the original ** a gene AT G translation starting from signal; *] 2 * be ** A retains the complete leader sequence doors * the blood of rs ** enCe) back 00.587Kb CPSI GDNA retained only 8 mononuclear acid leader sequence structure, their 'terminal structure did not change, the two fragments 5' Hindlll restriction sites are introduced prior to an end. The same time, we also carried out several carrier * S transformation. Blunt end-modified by the method, eliminating the carrier no effect on function an ECORI locus. And then again in the vector at the cloning site, the introduction of two BalnHI coRI adaptation sub. After transformation, the formation of two new carrier with plasmid the the PSV plant DFHR 'and PSV factory DFHR'', with a wide range of restructuring adaptability. Identification of mammalian cell expression, the above transformation vector function multi-l D * referral based on gene and vector modifications and alterations from PBS \, subcloned recycled the 0.7Kb, and 0.SKb CPSICDNA fragment and directed reorganization with PSV. carrier, built the PSV-CPS., and PSV.-CPS.., two mammalian cell Expression qualitative 4 the rat liver carbamic acid phosphate synthase! Experimental study of the gene expression in mammalian cells Thesis tablets, by restriction endonuclease analysis, and that the direction of the inserted fragment and the structure to meet the design requirements, the recombinant plasmid of each component complete DNA calcium phosphate coprecipitation-mediated gene transfection method-CPS will PSVZ. s Wo PSV-CPS, respectively adopt the PSV. chanting eo total transfected NIH/3T3 cells, cells indirect immunofluorescence and enzyme linked immunosorbent assay identification to prove PSVZ-CPSS. SAIH/3T3, PSVZ-CPS507-N'IH / 3T3 transformed cells have specific CPSI gene expression of two, through the G418 resistant screening, has been the establishment of the the two stable expression system strains PSV-CPSs the PSV-CPS -NH/3T3 Wo. ** a fragment of the-N'IH / 3T3 expression was transformed succeeded in demonstrating that the two forms can be expressed sexual whereby we choose 0 corpse 12Kb CPSI fragment as full sequence CPSIcDNA a fragment of another fragment with BalnHI and EcoRI restriction endonuclease enzymatic PHN3491 obtained by recycling the fragments in the BamHI recombination sites at docking the composition contains the complete CPSI leader sequence and the structural gene CPSI cDNA, but without the original gene polyadenylation signal by the carrier, the signal derived from SV40 PO

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