Cloning and promoter analysis of the human B-50/GAP-43 gene.
We here report isolation of exon 1 and analysis of the human B-50 promoter. A human genomic lambda EMBL3 library was screened with a homologous PCR probe. Two independent clones were analyzed and partially sequenced: They contained up to 5 kb sequence upstream of the translation start site and approx 13 kb of intron 1 sequence. There was a high degree of homology between the rat and the human gene with 100% homology from -504 to -427, with respect to the translation start codon. However, relatively long GT and GA repeats as seen in the rat gene were absent. Various promoter-reporter constructs, containing 5.0 to 0.12 kb of the upstream region, were transfected into undifferentiated and neuroectodermally differentiated P19-EC. Two promoter activities were found. The minimal fragment with promoter activity still responsive to differentiation was the 0.22 kb construct, similar to rat promoter P2. We conclude that the human B-50 gene is expressed in a similar way to the rat B-50 gene, based on the presence of two transcripts, the high degree of homology between the rat and the human sequence, and the two promoter activities found in P19-EC cells.
de Groen
P C
PC
Division of Gastroenterology, Mayo Clinic and Foundation, Rochester, MN, USA.
Eggen
B J
BJ
Gispen
W H
WH
Schotman
P
P
Schrama
L H
LH
D000818:Animals; D010582:Bacteriophage lambda / Q000235:genetics; D001483:Base Sequence; D002454:Cell Differentiation; D002460:Cell Line; D003001:Cloning, Molecular; D017931:DNA Primers; D005091:Exons; D019922:GAP-43 Protein; D006801:Humans; D007438:Introns; D008156:Luciferases / Q000096:biosynthesis; D008562:Membrane Glycoproteins / Q000096:biosynthesis* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis* / Q000235:genetics*; D009474:Neurons; D009838:Oligodeoxyribonucleotides; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D014176:Protein Biosynthesis; D051381:Rats; D011994:Recombinant Proteins / Q000096:biosynthesis; D012091:Repetitive Sequences, Nucleic Acid; D015183:Restriction Mapping; D012689:Sequence Homology, Nucleic Acid; D016385:TATA Box; D014162:Transfection
D017931:DNA Primers; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D008156:Luciferases