Premium
Analysis of human neuropeptide FF gene expression
Author(s) -
Nystedt Johanna M.,
Brandt Annika M.,
Mandelin Jami,
Vilim Ferdinand S.,
Ziff Edward B.,
Panula Pertti
Publication year - 2002
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2002.01035.x
Subject(s) - microbiology and biotechnology , northern blot , biology , messenger rna , gene expression , reporter gene , promoter , primer extension , complementary dna , gene , transfection , chimeric gene , transcription (linguistics) , genetics , linguistics , philosophy
As an initial step to study the function of the gene encoding the human neuropeptide FF (NPFF), we cloned a 4.7‐kb sequence from the promoter region. Primer extension and 5′‐rapid amplification of cDNA ends revealed multiple transcription initiation sites. Northern blot analysis of the mRNA expression revealed a specific signal only in poly(A) + RNA from medulla and spinal cord. Chimeric luciferase reporter gene constructs were transiently transfected in A549, U‐251 MG, SK‐N‐SH, SK‐N‐AS and PC12 cells. The promoter activity was directly comparable with the level of endogenous NPFF mRNA as determined by real‐time quantitative RT–PCR. The highest promoter activity was measured when a region from − 552 to − 830 bp of the 5′‐flanking region was fused to the constructs, and a potential silencer element waslocalized between nucleotides −220 and −551. A twofold increase in NPFF mRNA was observed after 72 h of nerve growth factor stimulation of PC12 cells and the region between − 61 and − 214 bp of the 5′‐flanking region was found to be responsive to this stimulation. We postulate that control of human NPFF gene expression is the result of both positive and negative regulatory elements and the use of multiple transcription initiation sites.