Dissertation > Biological Sciences > Molecular Biology > Genetic engineering (genetic engineering)

Clone and Identification and of a New Isoform of Human ITSN2 Gene

Author LiuZuo
Tutor ZhangJian;XiangShuangLin
School Hunan Normal University
Course Biochemistry and Molecular Biology
Keywords intersectin2 gene alternative splicing isoform RT-PCR
Type Master's thesis
Year 2010
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The family members of Intersectin, ITSN1 and ITSN2, are all widely expressed proteins. They all contain two EH domains, one coiled-coil domain and five SH3 domains. Through the alternative splicing, they produced two major transcripts. They were a short transcript of S and an additional C terminal respectively, which increased the long transcript L of DH, PH and C2 structural domains. In contrast to ITSN1, ITSN2 major transcripts have a more ubiquitous tissue expression pattern. Their biochemical and functional characterization has revealed these multimodular proteins to play an integral role in clathrin-mediated endocytosis.TNFAIP1 gene is induced rapidly and transiently by TNFa. To identify potential TNFAIP-interacting partners, a yeast two-hybrid screen was performed in human brain cDNA library. One of the identified clones encodes partial of ITSN2, including the EH2 domain and coiled coil region of ITSN2. For further research we cloned ITSN2 gene. Three parts of ITSN2 (front, middle, back) were cloned seperately by using different primers in the process of cloning ITSN2, and then we linked the three parts together to get the full-length of ITSN2. The sequencing result showed that a part has missed relative to the ITSN2-L, sequence analysis showed that the missing parts were 17 exon and a part of 36 exon of ITSN2-L. We named this new splice variant as ITSN2-M. Analysis showed that the open reading frame of ITSN2-M has not changed by the lack of 17 exon. But the miss of 36 exon made a stop codon. Because of the deletion of 17 exon, the middle CC domain was destroyed; and the deletion of 36 exon resulted the termination of translation, thus affecting the PH domain and C2 domain. It showed that this transcript is different from the ITSN2-L transcript, it has different 2domains which were important in the interaction of protein. Maybe they can make ITSN2-M combine with different proteins, thus exercise different functions.We designed validated primers, and used RT-PCR to demonstrate that the 17 exons and 36 exon were missing at the same time in the ITSN2-M. There exists no reports about the two parts, this is a new transcript of ITSN2.We cultivated HeLa,293FT, MCF7 and Pancl celllines, extracted their RNA, and then reversely transcripte them into cDNA. According to reverse transcripte PCR we tested the expression of exon lacking in these celllines. In order to test the expression of ITSN2-M protein, we used INSN2’s EH and CC domain segments as antigen, received antibodies of EC by injecting rabbits. We tested the specificity of antibody through immunofluorescence and Western Bolt separately. The pEGFP-ITSN1 and pEGFP-ITSN2-M were transfected in HeLa cell and tested by GFP antibody and EC antibody separately. The results showed that the EC antibody could only test ITSN2-M, but not ITSN1. We uesd the same method in Western Blot, EC antibody could only test ITSN2-M.We used EC antibody in 293FT, HeLa, SK-SN-SH cells and human normal brain and brain stem tissue to test ITSN2. We extracted cells and the total protein of tissues and used EC antibody for Western Blot,The expression of ITSN2-M was high in normal brain and brain stem, however, its expression was quite low in the neuroblastoma cell line SK-N-SH. This may indicates that ITSN2-M plays an important physiological role in the nerve cells.

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