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A cDNA encoding fish fibroblast growth factor-2, which lacks alternative translation initiation
1Central Research Laboratory, Nippon Suisan Kaisha Limited, 559-6 Kitanomachi, Hachioji, Tokyo 192, Japan.
The Journal of Biological Chemistry
|March 14, 1997
Summary
Researchers isolated rainbow trout fibroblast growth factor-2 (FGF-2) and found unique amino acids critical for its activity. Fish cells initiate protein synthesis differently than mammals, highlighting species-specific FGF-2 functions.
Area of Science:
- Molecular Biology
- Comparative Genomics
- Biochemistry
Background:
- Fibroblast growth factor-2 (FGF-2) is a key regulator of cell proliferation and differentiation.
- Mammalian FGF-2 exhibits alternative translation initiation sites, producing different protein isoforms.
- Understanding FGF-2 in non-mammalian species like rainbow trout provides insights into evolutionary conservation and functional divergence.
Purpose of the Study:
- To isolate and characterize the rainbow trout fibroblast growth factor-2 (FGF-2) coding region.
- To investigate the translational initiation mechanisms of rainbow trout FGF-2 in fish cells.
- To determine the functional significance of unique amino acid residues in rainbow trout FGF-2's receptor-binding domain.
Main Methods:
- cDNA cloning and sequencing of rainbow trout FGF-2.
- Transfection of human FGF-2 cDNA into fish cells to study translation initiation.
- Bacterial production and purification of recombinant rainbow trout FGF-2 protein.
- Heparin-Sepharose binding assays and fibroblast proliferation assays.
- Site-directed mutagenesis to create mutant rainbow trout FGF-2 proteins.
Main Results:
- A rainbow trout cDNA clone encoding FGF-2 was isolated, revealing a stop codon upstream of the primary methionine.
- Fish cells initiated translation at the 5' CUG codon, but not at other mammalian initiation sites.
- Recombinant rainbow trout FGF-2 exhibited heparin-binding and fibroblast proliferation activities.
- Amino acids 121-123 in rainbow trout FGF-2, located in the receptor-binding domain, were found to be critical for its activity.
Conclusions:
- Rainbow trout FGF-2 has a unique translational initiation mechanism compared to mammals.
- The identified amino acid variations at positions 121-123 are crucial for rainbow trout FGF-2's biological activity.
- This study enhances our understanding of FGF-2 evolution and functional diversity across species.