Cloning and promoter analysis of the human B-50/GAP-43 gene
- 54 Downloads
We here report isolation of exon 1 and analysis of the human B-50 promoter. A human genomic λEMBL3 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.
Index EntriesHuman B-50/GAP-43 gene promoter P19-EC cells
Unable to display preview. Download preview PDF.
- Eggen B. J. L., Kleijnen M. F., Verhaagen J., and Schrama L. H. (1995a) Neuron-specificity of rat B-50/GAP-43 promoter P2 in P19-EC cells.Neurosci. Res. Commun. 17, 19–26.Google Scholar
- Graff J. M., Stumpo D. J., and Blackshear P. J. (1989) Characterization of the phosphorylation sites in the chicken and bovine myristoylated alaninerich C kinase substrate protein, a prominent cellular substrate for protein kinase C.J. Biol. Chem. 264, 11,912–11,919.Google Scholar
- Jap Tjoen San E. R. A., Mercken M., Oestreicher A. B., Schotman P., De Laat S. W., and Gispen W. H. (1991) Expression of B-50 (GAP-43) during differentiation of P19 embryonal carcinoma cells.Soc. Neurosci. Abstr. 21, 222. 9Google Scholar
- Liu Y. C. and Storm D. R. (1989) Dephosphorylation of neuromodulin by calcineurin.J. Biol. Chem. 264, 12,800–12,804.Google Scholar
- Neve R. L., Perrone-Bizzozero N. I., Finklestein S. P., Zwiers H., Bird E., Kurnit D. M., and Benowitz L. I. (1987) The neuronal growth-associated protein GAP-43 (B-50, F1): specificity, developmental regulation and regional expression of the human and rat genes.Mol. Brain. Res. 2, 177–183.CrossRefGoogle Scholar
- Nielander H. B., Schrama L. H., Van Rozen A. J., Kasparaitis M., Oestreicher A. B., De Graan P. N. E., Gispen W. H., and Schotman P. (1987) Primary structure of the neuron-specific phosphoprotein B-50 is identical to growth-associated protein GAP-43.Neurosci. Res. Commun. 1, 163–172.Google Scholar
- Rosenthal A., Chan S. Y., Henzel W., Haskell C., Kuang W.-J., Chen E., Wilcox J. N., Ullrich A., Goeddel D. V., and Routtenberg A. (1987) Primary structure and mRNA localization of protein F1, a growth-related protein kinase C substrate associated with synaptic plasticity.EMBO J. 6, 3641–3646.PubMedGoogle Scholar
- Rudnicki M. A. and Mcburney M. W. (1986) Cell culture methods and induction of differentiation of embryonal carcinoma cell lines.IRL Press 19–49.Google Scholar
- Sambrook J., Fritsch E. F., and Maniatis T. (1989)Molecular Cloning: a Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
- Schrama L. H., Heemskerk F. M. J., and De Graan P. N. E. (1989) Dephosphorylation of protein kinase C phosphorylated B-50/GAP-43 by the calmodulin-dependent phosphatase calcineurin.Neurosci. Res. Commun. 5, 141–147.Google Scholar