The Investigation Of The Transcriptional New Feature Of Tumor Metastasis Suppressor Gene1TMSG-1Protein

ObjectiveTo investigate the transcriptional activity of tumor metastasis suppressor gene1(TMSG-1) protein and identify the nuclear localization signal of TMSG-1, we constructed5truncated eukaryotic expression vectors of TMSG-1containing different functional domains of TMSG-1, and explored the subcellular localization of different truncated proteins of TMSG-1.Methods Different gene fragments containing different functional domains of TMSG-1were amplified by polymerase chain reaction (PCR) using pcDNA3-TMSG-1as the template. The gene fragments were connected with eukaryotic expression vector pcDNA3after the fragments were cut by limited incision enzymes and then the recombined vectors were transformed to competent cells DH5a. The positive colons were selected by ampicillin. The limited incision enzymes and gene sequencing were applied to identify the correct recombined vectors which had correct sequence. The eukaryotic expression vectors were then transfected into Hela cells. The expressions of these fusion proteins were detected by Western blot. Immunofluorescence was applied to identifiy the subcellular localization of the different truncated protein of TMSG-1. According to analysis the structure of TMSG-1gene,we deleted the predicted nuclear localization sequence. We constructed QST5recombinant plasmid and observed the change of its subcellular localizationResults The targeted gene fragments were successfully amplified by PCR. The eukaryotic expression vectors containing different functional domains of TMSG-1:T (aa129-aa380), T2(aa71-aa380), T3(aal-aa128), T4(aal-aa70), T5(aa71-aa128) and deletantT5(aa71-aal18), which lacked the predicated nuclear localization signal (NLS) of TMSG-1, were constructed. The limited incision enzymes cut the6eukaryotic expression vectors into fragments with expected length. The gene sequencing then certificated the correct sequence of the recombined vectors. Furthermore, Western blot showed that fusion proteins with expected length were successfully expressed in Hela cells. The results of immunofluorescence revealed that the truncated proteins of TMSG-1containing Homeodomain including T2, T3and T5could enter the nucleus. T2was mainly scattered in the nucleus, and T3and T5were mainly localized in the nucleolus. Deletant T5was still mainly scattered in the nucleus,but no longer in the nucleolus;while other fusion proteins localized in the cytoplasm.Conclusion The eukaryotic expression vectors containing different functional domains of TMSG-1were successfully constructed and expressed. The truncated proteins of TMSG-1containing Homeodomain could enter the nucleus, while the truncated protein lacked the predicated NLS in the Homeodomain could not enter the nucleolus. Our results determined that there was a nucleolar localization signal in the NLS, which could facilitate the further investigation of the transcriptional function of TMSG-1and provide new sight to the gene therapy of tumor.