title: Influence of the polymorphisms of the alpha-major regulatory element HS-40 on in vitro gene expression.
abstract: The alpha-MRE is the major regulatory element responsible for the expression of human alpha-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 alpha-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 alpha-MRE enhancer activity. We analyzed and compared the expression of luciferase of nine constructs containing different alpha-MRE elements as enhancers. Genomic DNA samples from controls with A (wild-type alpha-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 alpha-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 alpha-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.
authors: Departamento de Patologia Clínica, Universidade Estadual de Campinas, SP, Brasil.
authors: D M
authors: DM
authors: Ribeiro
authors: T R
authors: TR
authors: Zaccariotto
authors: M N N
authors: MN
authors: Santos
authors: F F
authors: FF
authors: Costa
authors: M F
authors: MF
authors: Sonati
mesh_terms: D005786:Gene Expression Regulation / Q000235:genetics*; D005914:Globins / Q000235:genetics*; D006239:Haplotypes / Q000235:genetics*; D006801:Humans; D020014:K562 Cells; D008156:Luciferases / Q000235:genetics; D009154:Mutation / Q000235:genetics*; D011110:Polymorphism, Genetic / Q000235:genetics*; D050436:Regulatory Elements, Transcriptional / Q000235:genetics*
chemical_list: D005914:Globins; D008156:Luciferases