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The Study of Subcellular Localization of TERE1/UBIAD1 and Gene Regulation of Its Drosophila Ortholog HEIX

Author: ZhaoWei
Tutor: HongLing
School: Huazhong University of Science and Technology
Course: Genetics
Keywords: subcellular localization prenyltransferase TERE1/UBIAD1 HEIX microRNAs
CLC: Q75
Type: Master's thesis
Year: 2010
Downloads: 20
Quote: 0
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Abstract


Prenyltransferase is a class of important protein regulating intracellular signal transduction network. It has profound impact on cancer cells. Following protein synthesis, prenyltransferase transfers the isopentene radical to the cysteine residue in the C terminal of protein. So the modified protein can attach to the cell membrane. Many proteins of eukaryotic cells need prenylation to promote cell growth and differentiation, and to maintain cell morphology and cell division cycle. The most proteins modified by prenyltransferase are Ras family proteins, such as Ras, Rab.Human TERE1/UBIAD1 protein is a new class of prenyltransferase. It has been proven that TERE1/UBIAD1 is a novel human MK-4 biosynthetic enzyme, which is related to human bladder cancer, prostate cancer and other cancers. TERE1/UBIAD1 is a candidate gene for Schnyder crystalline corneal dystrophy (SCCD) disease, an autosomal dominant disorder involving deposition of cholesterol in the corneal stroma. Drosophila heix and human TERE1/UBIAD1 are homologous genes. In Drosophila, heix is a tumor suppressor gene. It regulates the proliferation and differentiation of Drosophila blood cells. At present, the research on the TERE1/UBIAD1 has gradually become one of the focuses in the cancer research field. In this paper, we have studied the subcellular localization, transmembrane structure and molecular evolution of TERE1/UBIAD1 using bioinformatics, molecular biology, cell biology and fluorescence imaging methods. Meanwhile we have predicted and identified the activity of Drosophila heix gene promoter, and the microRNAs regulating the expression of heix.Our bioinformatics results indicate that TERE1/UBIAD1 protein has multiple hydrophobic regions. TERE1/UBIAD1 is an eight transmembrane protein located on the cell membrane, endoplasmic reticulum and Golgi apparatus. Phylogenetic analysis showed that TERE1/UBIAD1 is a class of prenyltransferase or menaquinone synthase. TERE1/UBIAD1 protein has a barrel structure surrounded by eight transmembrane segments.Live cell fluorescence imaging results indicate that TERE1/UBIAD1 is co-localized with endoplasmic reticulum and Golgi apparatus. Also a small amount of TERE1/UBIAD1 protein is located in the other organelles besides the cell membrane, endoplasmic reticulum and Golgi apparatus. The main distribution of TERE1/UBIAD1 is on the Golgi apparatus. The immunofluorescence result showed that the N-terminal of TERE1/UBIAD1 resides in the cell cytoplasm, and its Loop1 structure is closer to the inner membrane. The truncated TERE1/UBIAD1 experiments indicate that the N-terminal of TERE1/UBIAD1 plays an important role in the subcellular localization of TERE1/UBIAD1.The human Hras gene has been successfully cloned. And the blue fluorescent protein(BFP) gene has been connected to the 5` end of Hras gene, to construct pBFP-hras DNA recombinant plasmid, which could express the fusion protein BFP-hras in eukaryotic cell. These have provided solid grounds for the study of the relationship between TERE1/UBIAD1 and Hras.The results of Drosophila heix gene regulation study indicate that the Drosophila heix gene promoter is located between the upstream 170bp and downstream 350bp of the transcription initiation site. The Drosophila heix gene promoter contains several transcriptional regulatory elements, and has high transcriptional activity. There are specific microRNA target sites in the 3 `UTR region of Drosophila heix gene. Heix gene expression may be post-transcriptional regulated by several Drosophila microRNAs, such as dmiRNA3,dmiRNA309,dmiRNA318,dmiRNA4,dmiRNA133 and dmiRNA79.Our research results have laid a good foundation for the further study of the function and expression regulation of heix gene.

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