Cloning, expression, and functional analysis of rat liver cytosolic inorganic pyrophosphatase gene and characterization of its functional promoter.
Inorganic pyrophosphate (PPi) is formed in several metabolic processes and its hydrolysis by the ubiquitously expressed enzyme inorganic pyrophosphatase (iPPase) is essential for the reactions to proceed in the direction of biosynthesis. Recently, we have reported differential expression and activity of cytosolic iPPase in rat liver with aging. In this article we report the cloning of the coding region of rat liver cytosolic iPPase gene in a bacterial expression vector, its expression, purification, and functional analysis by in-gel enzyme assay. SDS-PAGE and Western blot analysis of this expressed protein revealed that its molecular weight (MW) is approximately 33 kDa, while in-gel assay showed that it is functionally active just as the liver cytosolic iPPase. We have determined the genomic organization of this gene by genome blast approach. We have also cloned and characterized its proximal approximate 1 kb functional promoter (-1009 to +82) by transient transfection and luciferase assay of different 5'-deleted iPPase promoter-luciferase constructs and also established its transcription start site by primer extension analysis, along with protein-DNA interaction studies for a few putative transcription factor binding sites.
Panda
Harekrushna
H
Institute of Life Sciences, Bhubaneswar, India.
Pandey
Ravi S
RS
Debata
Priya R
PR
Supakar
Prakash C
PC
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D015153:Blotting, Western; D003001:Cloning, Molecular; D003600:Cytosol / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics; D015870:Gene Expression; D017930:Genes, Reporter; D043564:Inorganic Pyrophosphatase / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008099:Liver / Q000201:enzymology*; D008156:Luciferases / Q000378:metabolism; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D024363:Transcription Initiation Site
D011994:Recombinant Proteins; D008156:Luciferases; D043564:Inorganic Pyrophosphatase