Influence of the polymorphisms of the α-major regulatory element HS-40 on in vitro gene expression

Brazilian Journal of Medical and Biological Research

D.m. Ribeiro²  T.r. Zaccariotto²  M.n.n. Santos²  F.f. Costa¹  M.f. Sonati² 

[1] ,Universidade Estadual de Campinas Departamento de Patologia Clínica

关键词: Alpha-major regulatory element;    HS-40;    Alpha-globin genes;    Genetic polymorphisms;    Gene expression;   

DOI  :  10.1590/S0100-879X2009005000014

来源: SciELO

【 摘 要 】

The α-MRE is the major regulatory element responsible for the expression of human α-like globin genes. It is genetically polymorphic, and six different haplotypes, named A to F, have been identified in some population groups from Europe, Africa and Asia and in native Indians from two Brazilian Indian tribes. Most of the mutations that constitute the α-MRE haplotypes are located in flanking sequences of binding sites for nuclear factors. To our knowledge, there are no experimental studies evaluating whether such variability may influence the α-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different α-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type α-MRE) and B haplotypes were used to generate C-F haplotypes by site-directed mutagenesis. In addition, three other elements containing only the G→A polymorphism at positions +130, +199, and +209, separately, were also tested. The different α-MRE elements were amplified and cloned into a plasmid containing the luciferase reporter gene and the SV40 promoter and used to transiently transfect K562 cells. A noticeable reduction in luciferase expression was observed with all constructs compared with the A haplotype. The greatest reductions occurred with the F haplotype (+96, C→A) and the isolated polymorphism +209, both located near the SP1 protein-binding sites believed not to be active in vivo. These are the first analyses of α-MRE polymorphisms on gene expression and demonstrate that these single nucleotide polymorphisms, although outside the binding sites for nuclear factors, are able to influence in vitro gene expression.

【 授权许可】

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