An activator element within the preprotachykinin-A promoter.
The rat Preprotachykinin-A promoter (PPT) directs high levels of expression in dorsal root ganglia (DRG) neurons in culture either endogenously or when linked to a receptor construct. It is not active in any of the established tissue culture cell lines which we have analyzed. To search for transcriptional regulators within this promoter we have started to dissect the promoter into individual elements to determine their function. A DNA element which had previously been suggested to regulate transcription from DNA sequence analysis of the rat PPT promoter occurs at position -200 relative to the major start of transcription within the PPT promoter. The equivalent element from the bovine PPT promoter had previously been proposed to be a cAMP responsive element (CRE). The sequence of this enhancer has similarities with both the AP1 and CRE DNA consensus sequences. We have demonstrated that one copy of this rat PPT element linked to a heterologous basal promoter will enhance transcription in HeLa and PC12 cell lines as well as adult rat DRG neurons grown in culture. It is also demonstrated that the rat PPT element will bind proteins in HeLa nuclear extract distinct from those binding to the well-characterized Gibbon Ape Leukemia Virus (GALV) AP1 or somatostatin CRE sites by gel retardation analysis. This PPT element, when cloned in a heterologous reporter construct, although showing properties of both AP1 and CRE elements, was functionally distinguished from both the somatostatin CRE element and the GALV AP1 enhancer when these elements were tested in the same reporter construct. This PPT element has a constitutive level of activity in adult rat DRG neurons, which is fivefold higher than that driven by the reporter construct promoter. It is also significantly different from the same reporter construct linked to the somatostatin CRE and analyzed in DRG neurons.
MRC Brain Metabolism Unit, Royal Edinburgh Hospital, United Kingdom.
C F
CF
Morrison
J
J
McAllister
S P
SP
Dobson
P K
PK
Mulderry
J P
JP
Quinn
D000818:Animals; D001483:Base Sequence; D005576:Colforsin / Q000494:pharmacology; D017362:Cyclic AMP Response Element-Binding Protein / Q000235:genetics*; D004247:DNA / Q000235:genetics*; D004268:DNA-Binding Proteins / Q000235:genetics*; D005727:Ganglia, Spinal / Q000378:metabolism; D015870:Gene Expression; D006367:HeLa Cells; D018398:Homeodomain Proteins; D006801:Humans; D015778:Minor Histocompatibility Antigens; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes / Q000235:genetics; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D011498:Protein Precursors / Q000235:genetics* / Q000378:metabolism; D011506:Proteins / Q000378:metabolism; D019253:Proto-Oncogene Proteins c-bcl-2; D051818:Replication Protein C; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015320:Tachykinins / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000502:physiology
C096816:BCL2-related protein A1; D017362:Cyclic AMP Response Element-Binding Protein; D004268:DNA-Binding Proteins; D018398:Homeodomain Proteins; C096063:MATA1 protein, S cerevisiae; D015778:Minor Histocompatibility Antigens; D015345:Oligonucleotide Probes; D011498:Protein Precursors; D011506:Proteins; D019253:Proto-Oncogene Proteins c-bcl-2; D012097:Repressor Proteins; D029701:Saccharomyces cerevisiae Proteins; D015320:Tachykinins; D014157:Transcription Factors; C044206:preprotachykinin; D005576:Colforsin; D004247:DNA; D051818:Replication Protein C