Identification and analysis of the promoter region of the human methionine sulphoxide reductase A gene.
MsrA (methionine sulphoxide reductase A) is an antioxidant repair enzyme that reduces oxidized methionine to methionine. Moreover, the oxidation of methionine residues in proteins is considered to be an important consequence of oxidative damage to cells. To understand mechanisms of human msrA gene expression and regulation, we cloned and characterized the 5' promoter region of the human msrA gene. Using 5'-RACE (rapid amplification of cDNA ends) analysis of purified mRNA from human cells, we located the transcription initiation site 59 nt upstream of the reference MsrA mRNA sequence, GenBank accession number BC 054033. The 1.3 kb of sequence located upstream of the first exon of msrA gene was placed upstream of the luciferase reporter gene in a pGL3-Basic vector and transfected into different cell lines. Sequentially smaller fragments of the msrA promoter region were generated by PCR, and expression levels were monitored from these constructs within HEK-293 and MCF7 human cell lines. Analysis of deletion constructs revealed differences in promoter activity in these cell lines. In HEK-293 cells, the promoter activity was constant from the minimal promoter region to the longest fragment obtained. On the other hand, in MCF7 cells we detected a down-regulation in the longest fragment. Mutation of a putative negative regulatory region that is located between -209 and -212 bp (the CCAA box) restored promoter activity in MCF7 cells. The location of the msrA promoter will facilitate analysis of the transcriptional regulation of this gene in a variety of pathological contexts.
De Luca
Antonella
A
Department of Biomedical Sciences, University of Chieti G. D'Annunzio School of Medicine, and Center of Excellence on Aging, G. D'Annunzio University Foundation, Chieti, Italy.
Sacchetta
Paolo
P
Di Ilio
Carmine
C
Favaloro
Bartolo
B
D001483:Base Sequence; D001665:Binding Sites; D002460:Cell Line; D003001:Cloning, Molecular; D017353:Gene Deletion; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D051150:Methionine Sulfoxide Reductases; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000235:genetics*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D024363:Transcription Initiation Site
D010088:Oxidoreductases; D051150:Methionine Sulfoxide Reductases; C027219:methionine sulfoxide reductase