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Study of Experssion and Regulation of Zebrafish Thymidylate Synthase Gene

Author: SongChunXia
Tutor: ZhangJuRen;LinXiuKun
School: Shandong University
Course: Cell Biology
Keywords: zebrafish thymidylate synthase expression regulation 5-FU
Type: PhD thesis
Year: 2008
Downloads: 235
Quote: 1
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Cancer has become the leading cause of death in both municipal and rural area in our country. Study of molecule mechanism of tumor biogenesis focus on several fields: cloning and functional analysis of tumor relative genes, cell signal transduction pathway and cell cycle regulation. Thymidylate synthase (TS) is a folate-dependent enzyme that catalyzes the reductive methylation of dUMP by meTHF (N5,10 -methylenetetrahydrofolate) to generate thymidylate (dTMP) and dihydrofolate. As the reaction is the last step of the sole dTMP de novo synthesis pathway essential for DNA synthesis, and also for cell division and survival, TS is an important enzyme target in chemotherapy. Previous study suggested that in addition to its enzymatic activity, TS also fuctions as an RNA bining protein and TS can interacts with its own mRNA and thus inhibits its translation. Several reports show that the TS protein also binds to a number of other mRNAs, including c-myc, bcl-2 and p53. There are increasing evidence to show that the enzyme participates in regulation of synthesis of other proteins involved in cell cycling, DNA repair and transcription.There was a significant correlation between the TS protein expression and curative effect of anticancer chemotherapy. Studying the mechanism of TS expression regulation will help us discover the new anticancer drug and understand the mechanism of celluar resistance. A close relationship has been observed between TS translational regulation and fluoropyrimidine sensitivity by using human cancer cell model. However, little is known about TS regulation mechanism in vivo. The zebrafish is an excellent system, which is suitable for study in genetics, embryology, and developmental and cell biology. Zebrafish embryos exhibit unique characteristics, including ease of maintenance and drug administration, short reproductive cycle, and transparency that permits visual assessment of developing cells and organs. The zebrafish embryo has become an important vertebrate model for assessing drug effects in recent years. A previous report has shown that zebrafish TS is most closely related to the human TS with 76% the amino acids sequence identity and 95% the active domain sequence identity. In addition, it is interesting that the same binding site for folate is found in human and zebrafish TS, and that the binding site shows a very high homology between the two species (93%). The binding site for dUMP was identical with human TS. It suggested that similar regulation mechanisms of TS may exist in zebrafish and human.In this study, we studied the regulation, expressional regulation and expression of zebrafish TS using electrophoretic mobility shift assay (EMSA), immuno-precipitation : RT-PCR, Western Blotting, Northern Blotting and in vitro translation system. Our finding suggested that a similar mechanism existed in zebrafish for TS regulation and zebrafish may be developed to an ideal model for screening TS inhibitors.In the present study, TS protein was expressed in E. coli using the following techniques. The zebrafish TS expression vector pET28a/Z-TS was transformed into E. coli BL21 (DE3) cells. The transformed cells were cultivated in medium, and the isopropyl-β-D-thiogalactopyranoside was added. 3h later, the zebrafish TS protein would be 35% of the total celluar proteins. Using the Ni-NTA spin column, we purified the enzyme to almost homogeneity on SDS-PAGE gel.In order to determine whether TS could bind its cognate mRNA in vitro, EMSA assay was carried out with TS recombinant protein and DIG-labeled full-length TS probe. A specific RNA-binding activity was determined, and it was further confirmed by the competition assay using 100 fold unlabeled TS probe. Furthermore, using ultraviolet (UV) photochemical crosslinking to detect RNA binding proteins, the molecular mass of the RNA-protein was about 80kDa. We concluded that the protein was able to bind to RNA in a dimmer form, not a single subunit.Using zebrafish embryos, Further study revealed that zebrafish TS was able to interact with its own mRNA in vivo using immunorecipitation : RT-PCR technique. The results indicated that zebrafish TS protein interacts with human TS106 monoclonal antibody. Zebrafish embryos were immunoprecipitated by TS106, followed by RNA isolation and RT-PCR using primers which encoded zebrafish TS and c-myc gene cDNA sequence. The result demonstrated that there were zebrafish TS and c-myc mRNA in the immunoprecipitated complex.The effects of 5-fluorouracil (5-FU) on zebrafish TS protein expression of healthy embryos in 1-2 cell stages were determined by western-blotting analysis. The results showed 5-FU could significantly increase the TS expression in zebrafish embryos. However, zebrafish TS mRNA level were were remained unchanged as determined by northern-bloting analysis. To determine the effect of 5-FU and 5-FdUMP on translation of TS mRNA, a rabbit reticulocyte lysate in vitro translation system was used. Addition of 5-FU, not inhibited the translation of TS mRNA. While addition of 5-FdUMP, completely repressed the translation of TS mRNA. Therefore, induced expression of thymidylate synthase by fluoropyrimidines in zebrafish occurred in translational level, not in transcriptional level. These might be the reason to explain why TS was associated with the sensitivity and resistance to 5-FU.Using both in vitro and in vivo model systems, we have demonstrated that zebrafish TS protein was able to bind to its own cogate mRNA and the fluoropyrimidine 5-fluorouracil (5-FU) regulated TS in the translational level. This is the first time to confirm that the regulation of TS is affected by TS and its cognant mRNA interaction in the whole animal level. Our results provide evidence that zebrafish may be developed as a useful model for studying the anti-metabolism agents.

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