Promoter analysis and characteristics of the 5'-untranslated region of the mouse glial cell line-derived neurotrophic factor gene.
We have cloned the mouse GDNF cDNA and genomic DNA to study the molecular mechanism of gene expression. Primer extension and RT-PCR analyses indicated that the mouse gene contains 1086 bp of 5'-untranslated region (5'-UTR) [Gene 203 (1997) 149]. In this report, we identified the core promoter region of mouse GDNF and examined the role of the 5'-UTR in gene expression. Promoter deletion analyses indicated that the proximal region (-81 to +28), which includes a TATA-box, is necessary for high-level expression of GDNF. Using reporter constructs encoding luciferase or fusion gene of GDNF to enhanced green fluorescent protein that were transiently transfected to mouse astroglial cell-line TGA-3 cells and rat glioma C6 cells, we investigated effects of the 5'-UTR on promoter activity. Luciferase reporter assay indicated that a region downstream of the transcription initiation site may include a positive regulatory element, while two more distal regions appear to contain negative regulatory elements, which was correlated to the mRNA level based on RNase protection assay. Both negative regulatory elements attenuated promoter activity in a position-dependent manner. Nuclear proteins from C6 glioma cells were shown to interact with several regions (+65/+105, +233/+265, and +554/+582) including each of the regulatory elements, suggesting that regulation of GDNF expression by the 5'-UTR occurred mainly at the transcriptional level.
Tanaka
M
M
Laboratory for Genes of Motor Systems, Bio-Mimetic Control Research Program, The Institute of Physical and Chemical Research (RIKEN), Moriyama, Nagoya 463-0003, Japan.
Ito
S
S
Matsushita
N
N
Mori
N
N
Kiuchi
K
K
D016475:3T3 Cells; D020121:5' Untranslated Regions / Q000235:genetics*; D000818:Animals; D001483:Base Sequence; D015252:Deoxyribonucleases, Type II Site-Specific; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation; D017930:Genes, Reporter; D051100:Glial Cell Line-Derived Neurotrophic Factor; D005910:Glioma; D008156:Luciferases / Q000235:genetics; D051379:Mice; D008969:Molecular Sequence Data; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins / Q000235:genetics*; D015345:Oligonucleotide Probes; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012333:RNA, Messenger / Q000032:analysis; D012260:Ribonucleases; D014407:Tumor Cells, Cultured
D020121:5' Untranslated Regions; C495040:Gdnf protein, mouse; D051100:Glial Cell Line-Derived Neurotrophic Factor; D009414:Nerve Growth Factors; D009419:Nerve Tissue Proteins; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D008156:Luciferases; D012260:Ribonucleases; C025651:CCCGGG-specific type II deoxyribonucleases; D015252:Deoxyribonucleases, Type II Site-Specific; C038485:GGGCCC-specific type II deoxyribonucleases