Identification of two promoter regions in the rat B-50/GAP-43 gene.
To determine cis-acting elements controlling the rat B-50/GAP-43 gene expression, the genomic DNA encoding exon 1 and the 5' flanking sequence was isolated. Sequence analysis of 1 kb 5' untranslated region (UTR) revealed the presence of a (GA)-repeat and a (GT)-repeat. The size of the (GA)-repeat varied due to both an instability of phage lambda lambda DNA in E. coli and genomic variation between rats. Transcription initiation sites were mapped in 8-day-old rat brain poly(A)+ mRNA. Primer extension indicated multiple transcription start sites at -159 and -339/-342 nt upstream of the translation start site; reverse transcriptase coupled PCR showed that the most 5' transcription start site is located between -465 and -440. Northern blotting demonstrated that approximately 90% of the B-50 mRNAs initiates at approximately -50. Promoter analysis by transient transfection assays in undifferentiated and retinoic acid-differentiated P19-EC cells revealed that the rat B-50 gene contains two promoters. P1 (located between -750 and -407) contains commonly observed promoter elements such as a TATA box and CCAAT boxes. P2 (located between -233 and -1) neither contains TATA boxes, CCAAT boxes nor consensus sequences of house-keeping gene promoters like GC-boxes. The activity of P1 is inhibited at neuroectodermal differentiation of P19-EC cells whereas the activity of P2 is stimulated. In 8 day old rat brain the majority of the B-50 mRNA transcripts are derived from P2. It is concluded that at this developmental stage P2 is the most important promoter.
Eggen
B J
BJ
Laboratory for Physiological Chemistry, University of Utrecht, The Netherlands.
Nielander
H B
HB
Rensen-de Leeuw
M G
MG
Schotman
P
P
Gispen
W H
WH
Schrama
L H
LH
D000818:Animals; D001483:Base Sequence; D018236:Carcinoma, Embryonal; D019922:GAP-43 Protein; D005796:Genes; D008562:Membrane Glycoproteins / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000235:genetics*; D016716:PC12 Cells; D011401:Promoter Regions, Genetic; D051381:Rats / Q000235:genetics*; D017207:Rats, Sprague-Dawley; D012045:Regulatory Sequences, Nucleic Acid; D012091:Repetitive Sequences, Nucleic Acid; D016385:TATA Box; D014162:Transfection; D014407:Tumor Cells, Cultured
D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins