[The effect of gene polymorphism in promoter and intron 1 on human ApoA I expression].
OBJECTIVE
To construct pGL2-ApoA I luciferase reporter vector containing ApoA I gene regulation area, and to investigate the effect of G --> A and C --> T substitution in ApoA I promoter -75 bp and intron 1 +83 bp region respectively on ApoA I gene expression.


METHODS
Human chromosome DNA fragments containing ApoA I gene were amplified by PCR, and the DNA fragments consisting of ApoA I AA/CC, GG/TT and GG/CC genetypes were selected separately, then pUC vector including above three different DNA fragments was constructed. After digesting pUC vector with Sac I and Bgl II, ligate the different DNA fragments to basic pGL2 vector that containing luciferase reporter gene. Recombinant and PRL-null vector were cotransfected into HepG2 cells by using cationic liposome method. Cells were cultured for 48 h, activity of firefly and renills luciferase was measured.


RESULTS
Three vectors with pGL2-ApoA I-L(-2500 to +289 bp) long fragment vectors and 3 with pGL2-ApoA I-S(-145 to +289 bp) short fragment vectors were combinated successfully. Relative activity of luciferase for ApoA I AA/CC or GG/TT was lower than that for GG/CC significantly.


CONCLUSIONS
-75 bp G --> A and +83 bp C --> T substitution in ApoA I gene may inhibit ApoA I gene transcription and expression. It may be the reason why subjects containing -75 bp A and +83 bp T have lower high density lipoprotein cholesterol (HDL-C) concentration.
Cheng
Ai-juan
AJ
Department of Cardiovascular Surgery, Tianjin Chest Hospital, Tianjin, 300051, P. R. China. ytcaj@sina.com
Mao
Yong-min
YM
Cui
Rang-zhuang
RZ
D016632:Apolipoprotein A-I / Q000235:genetics* / Q000378:metabolism; D045744:Cell Line, Tumor; D008076:Cholesterol, HDL / Q000378:metabolism; D005822:Genetic Vectors; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D017354:Point Mutation; D020641:Polymorphism, Single Nucleotide; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D014162:Transfection
D016632:Apolipoprotein A-I; D008076:Cholesterol, HDL; D011993:Recombinant Fusion Proteins; D008156:Luciferases