Cloning and functional analysis of the swine eNOS promoter.
We have cloned the swine eNOS promoter and analyzed its function in newborn swine pulmonary artery endothelial cells (PAECs). Analysis of the 2.1 kb 5' flanking region revealed that the swine eNOS promoter is, like its counterparts in human and other species, a TATA-less promoter. The transcription start site, determined by 5' RLM-RACE, was located 62 bp upstream of the translation start codon. Promoter activity was demonstrated by transient transfection of 5' deletion promoter/luciferase constructs into swine PAECs, and indicated that the proximal region from -227 to -82 was necessary for basal promoter activity. Positive cis-regulatory elements were present from -227 to -1290, while negative cis-regulatory elements may be present from -1290 to -1926 bp. Electrophoretic mobility shift assay (EMSA) of the proximal region demonstrated that multiprotein complexes were formed in the conserved proximal region of the swine eNOS promoter and a novel Spl site at -68/-59 was involved in the formation of these complexes.
Shontz
Kimberly M
KM
Columbus Children's Research Institute, Columbus Children's Hospital and Department of Pediatrics, The Ohio State University, Columbus, OH 43205, USA.
Zhou
Bi
B
Yu
C Yung
CY
Su
Baogen Y
BY
D024506:5' Flanking Region / Q000235:genetics; D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D017124:Conserved Sequence; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D052250:Nitric Oxide Synthase Type III / Q000235:genetics* / Q000502:physiology; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017422:Sequence Analysis, DNA; D034421:Sus scrofa / Q000235:genetics*; D016385:TATA Box / Q000235:genetics; D024363:Transcription Initiation Site / Q000502:physiology
D052250:Nitric Oxide Synthase Type III