Characterization of a promoter for the human glial cell line-derived neurotrophic factor gene.
To address the regulation of glial cell line-derived neurotrophic factor (GDNF) gene expression, we have isolated 5' extended cDNAs, cloned the human GDNF gene, and characterized the promoter. GDNF-encoding 5' extended cDNAs containing a novel exon were isolated via reverse transcription-polymerase chain reaction (RT-PCR) of mRNA from human fetal kidney and adult skeletal muscle. The GDNF gene was isolated from a human genomic library in a P1 bacteriophage vector. Analysis of the 5' flanking sequence revealed a promoter that lacks a CCAAT-box motif and is GC rich. Consensus binding sites for a variety of transcription factors have been identified in the promoter. Promoter/reporter plasmids have been constructed by fusion of the promoter and a portion of exon I to a luciferase gene. The promoter/reporter construct and a number of promoter deletions were transiently transfected into two human cell lines known to express GDNF. Multiple enhancer and silencer regions were revealed as well as a minimal promoter sufficient for basal transcription. Finally, a RT-PCR assay, specific for transcripts originating from this GDNF promoter, was developed and used to show that this promoter is active in vivo. The results suggest GDNF is regulated in a complex manner.
Baecker
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Department of Molecular and Cellular Biochemistry, Center for Biological Research, Neurobiology Unit, Roche Bioscience, M/S R2-101, 3401 Hillview Ave., Palo Alto, CA, 94304-1397, USA. preston.baeker@roche.com
Lee
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Verity
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Eglen
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Johnson
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D000328:Adult; D001483:Base Sequence; D002872:Chromosome Deletion; D003001:Cloning, Molecular; D005314:Embryonic and Fetal Development / Q000502:physiology; D004742:Enhancer Elements, Genetic; D018507:Gene Expression Regulation, Developmental / Q000502:physiology*; D017930:Genes, Reporter; D051100:Glial Cell Line-Derived Neurotrophic Factor; D006801:Humans; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured
C495039:GDNF protein, human; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins