The Gene Stard4 Expression In Fat, Stard6, Rnpc3 The Study

Subtracted cDNA library of adipose tissue of C57 mouse(Mus musculus) wasconstructed under high-cholesterol diet using suppression substractivehybridization (SSH) method in order to study the gene involved inbiosynthesis and metabolism of cholesterol. After screening andsequencing we get several clones with differentially express cDNA, twoof them are StARD4 and StARD6 genes after blast search in NCBI againstnon redundant GeneBank database.Previous report revealed StARD4 was expressed only in liver and decreasedin mice fed a high-cholesterol diet and StARD6 was expressed only in testis.We first find both StARD4 and StARD6 expressed in adipose tissue and theydecreased in adipose tissue of mice fed a high-cholesterol diet. StARD4has one new splice variant and StARD6 have two new splice variants inadipose tissue. All of them destroy START domains which can bind sterol.StARD4 and StARD6 expressed in 3T3L1 pre-adipose cell, however, there areno evident expression alteration of StARD4 and StARD6 when 3T3L1pre-adipose cell differentiates. Recombinant Adenovirus encoding mouseStARD4 gene was successfully established and confirmed by PCR and westernblot. The 3T3L1 pre-adipose cell infected by recombinant Adenovirusencoding mouse StARD4 gene have produce much cholesterol.We predicted the putative human StARD4, human StARD6 and mouse StARD4promoters by online promoter prediction biosoftware and cloned them.These DNA fragments were inserted in plasmid pGLBasic luciferase reportervector which provide a basis for the analysis of promoter potentiallyregulate gene expression. The result revealed that some of them havepromoter property.In mouse adipose tissue library constructed under cholesterol feedingusing suppression subtractive hybridization (SSH) method we found a cDNAfragment.This cDNA fragment corresponds to a mouse gene whose accessionnumber is NM_ 026043 in GeneBank and these clones in the gene library复旦大学博士学位论文 脂肪中表达的基因 StARD4,StARD6 和 RNPC3 的研究established in our lab. But there is no human homolog gene reported. Wefound this cDNA corresponds to a human gene numbered 1147b09 in human genelibrary established in our lab, thus we first reported this gene andsubmitted it to GeneBank(accession number AY099329). We named it RNPC3(ribonucleoprotein containing 3) in agreement with HUGO Gene NomenclatureCommittee. This novel human RNPC3 gene is 1870 bp in length and encodesa protein with 517 amino acids containing two RNA recognition Motifs. Italso contains two bipartite nuclear targeting sequences, which raeimportant for nuclear targeting for proteins, especially those functionin the cell nucleus. GFP Location of RNPC3 Gene Product shows this proteinlocated in the cell nucleus. RT-PCR revealed that it is ubiquitouslyexpressed and abundantly in kidney and pancreas.