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Cloning, Chromosome Mapping and Biological Characterization of Porcine SCAP and INSIG Family

Author: QiuHuan
Tutor: YangZaiQing
School: Huazhong Agricultural University
Course: Biochemistry and Molecular Biology
Keywords: SCAP INSIG Chromosomal localization Proposed gene Gene structure Intron loss Differences spliceosome
CLC: Q953
Type: Master's thesis
Year: 2005
Downloads: 121
Quote: 0
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Abstract


Objective: To pig fat and cholesterol metabolism-related genes , including SCAP, INSIG1 and INSIG2 cloning , chromosomal localization and identification of biological characteristics , pig fat deposition explore the molecular mechanism for the function of these genes basis for the study . Method: 1 through homologous comparison method designed for amplification of specific gene primers ; 2 by RT-PCR technique to amplify specific gene cDNA fragment ; 3 Using 5 ' and 3'RACE technology , access to specific genes 5 and 3 ' end of the cDNA sequence ; four pairs of specific gene amplification product , cloned and sequenced ; 5 by RT-PCR technique , to obtain specific gene expression profile ; 6 by genomic PCR to obtain specific gene genomic sequence ; 7 using radiation hybrid Panel technology, the specific gene chromosomal localization . Results: In this study, the INSIG1 obtained 505 bp 143 bp coding region and the INSIG1 difference spliceosome (INSIG1 variant 3). The difference from wild -type spliceosome INSIG1 (INSIG1 variant 1) occurs 292 bp deletion caused . Pig INSIG1 INSIG1 encoded by the sequence of Protein Sequence and human INSIG1 97.6 % homology . RT-PCR analysis showed that INSIG1 heart, liver , spleen, lung , kidney, intestine , testis , brain, fat and muscle were expressed . INSIG1 spliceosome difference in heart and muscle expression was not detected . Human , rat and mouse EST database search and a mouse-derived RNA for RT-PCR analysis showed that the difference in splicing phenomenon exists in humans and pigs , but did not occur in rodents .

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