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Cloning of two novel forms of human acidic fibroblast growth factor (aFGF) mRNA
R A Payson1, H Canatan, M A Chotani
1Department of Internal Medicine, Ohio State University, Davis Medical Research Center, Columbus 43210.
Abstract:
We have previously isolated two different aFGF cDNA clones from kidney and brain. The two corresponding mRNA, designated aFGF 1.A and 1.B, are the predominant species in kidney and brain, respectively. During the characterization of aFGF mRNA in glioblastoma cells, we demonstrated that aFGF mRNA in U1242MG and D65MG glioblastoma cells contain 5'-untranslated sequences different from those of 1.A and 1.B. Through a strategy combining chromosome walking, identification and sequencing of evolutionarily conserved DNA regions, and a reverse transcription and polymerase chain reaction (RT-PCR)-based assay for RNA expression, we have isolated two novel aFGF cDNA clones. The cDNA clone representing aFGF mRNA 1.C was isolated from U1242MG cells; another aFGF cDNA, designated 1.D, was isolated from D65MG cells. Promoter 1C has extensive sequence homology to the hamster aFGF gene promoter that was shown to respond to testosterone stimulation by chloramphenicol acetyltransferase reporter gene assays. Using RT-PCR, we showed that normal, benign and cancerous prostate tissues do not express aFGF 1.C mRNA. In contrast, a prostate carcinoma cell line (PC-3) expresses 1.C mRNA. RT-PCR using 1.D-specific primers showed that kidney, brain and prostate do not express 1.D mRNA even though kidney and brain are the most abundant source for aFGF protein. RNase protection analysis further showed that 1.D mRNA is the predominant aFGF transcript in D65MG glioblastoma cells and in NFF-6 neonatal foreskin fibroblast cells. The genomic DNA corresponding to these two cDNA clones and the 5'-flanking regions were also isolated and their sequences determined. These DNA clones will provide important reagents for studying the regulatory elements of aFGF gene expression.
Insights
Researchers identified novel acidic fibroblast growth factor (aFGF) mRNA variants, 1.C and 1.D, in glioblastoma cells. These findings offer new insights into aFGF gene regulation and expression in various tissues.
Area of Science:
- Molecular Biology
- Genetics
- Cancer Research
Background:
- Previous isolation of two acidic fibroblast growth factor (aFGF) cDNA clones (1.A and 1.B) from kidney and brain.
- Identification of unique 5'-untranslated sequences in aFGF mRNA from glioblastoma cell lines (U1242MG and D65MG).
Purpose of the Study:
- To isolate and characterize novel aFGF cDNA clones and their corresponding mRNA species from glioblastoma cells.
- To investigate the expression patterns of these novel aFGF variants in different normal and cancerous tissues.
- To isolate and sequence the genomic DNA and 5'-flanking regions of the novel aFGF clones for regulatory element analysis.
Main Methods:
- Chromosome walking, DNA sequencing, and identification of evolutionarily conserved regions.
- Reverse transcription and polymerase chain reaction (RT-PCR) for RNA expression analysis.
- RNase protection assays and reporter gene assays (chloramphenicol acetyltransferase).
Main Results:
- Isolation of two novel aFGF cDNA clones, 1.C from U1242MG and 1.D from D65MG glioblastoma cells.
- Promoter 1C shows homology to the testosterone-responsive hamster aFGF gene promoter.
- aFGF 1.C mRNA was not detected in normal, benign, or cancerous prostate tissues but was present in the PC-3 prostate carcinoma cell line.
- aFGF 1.D mRNA was not detected in kidney, brain, or prostate, but was the predominant transcript in D65MG glioblastoma and NFF-6 fibroblast cells.
- Genomic DNA and 5'-flanking regions for both clones were isolated and sequenced.
Conclusions:
- Discovery of two new aFGF mRNA variants (1.C and 1.D) with distinct tissue expression profiles.
- The isolated genomic DNA clones provide valuable tools for studying the regulatory mechanisms of aFGF gene expression.
- Potential implications for understanding aFGF's role in glioblastoma and other cellular processes.