Novel cell lines for the analysis of preprotachykinin A gene expression identify a repressor domain 3' of the major transcriptional start site.
Until now, no clonal cells have been identified that support the expression of a marker gene expressed from the rat preprotachykinin A (rPPT) promoter. We have analysed recently available cell lines that are candidates for supporting reporter gene expression directed by the rPPT promoter. These are the neuronal-derived cell line NF2C and the pancreatic cell lines RINm5F and a derivative RIN-1027-B2. The NF2C line was derived from the brain homogenate of a transgenic animal in which a temperature-sensitive simian virus 40 large T antigen was expressed from a neurofilament promoter. All three lines are able to support expression of a reporter gene directed by a fragment of the 5' rPPT promoter. Analysis of reporter gene expression supported by various fragments of the rPPT promoter demonstrated that although -865 to +92 bp supported expression, the addition of fragments between +92 and +447 bp led to repression of expression. Subsequent analysis of reporter gene constructs microinjected into primary cultures of dorsal root ganglion neurons (DRG) confirmed the existence of this repressor domain. This repression could be relieved totally in both RIN cell lines and partly in NF2C cells by mutating residues between +373 and +396 bp. This indicates that these cell lines support PPT promoter activity similar to that observed in DRG and determines a novel repressor domain within the promoter.
Department of Veterinary Pathology, University of Edinburgh, Summerhall, Edinburgh EH9 1QH, Scotland, U.K.
C E
CE
Fiskerstrand
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P
Newey
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B
Ebrahimi
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L
Gerrard
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Harrison
G P
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McGregor
J P
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Quinn
D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D017931:DNA Primers; D015870:Gene Expression; D009154:Mutation; D011498:Protein Precursors / Q000235:genetics*; D051381:Rats; D012097:Repressor Proteins / Q000235:genetics*; D015320:Tachykinins / Q000235:genetics*; D014158:Transcription, Genetic
D017931:DNA Primers; D011498:Protein Precursors; D012097:Repressor Proteins; D015320:Tachykinins; C044206:preprotachykinin