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Amplification and sequencing of mRNA encoding acidic fibroblast growth factor (aFGF) from porcine heart
M Schmidt1, H S Sharma, R J Schott
1Max-Planck-Institute for Physiological and Clinical Research, Department of Experimental Cardiology, Bad Nauheim, Germany.
Abstract:
Progredient stenosis of coronary arteries can induce angiogenic processes, which are probably regulated by polypeptide growth factors like aFGF. Using applications of reverse transcription-polymerase chain reaction, we amplified and sequenced an mRNA encoding aFGF in the porcine myocardium. A DNA fragment of expected size encoding aFGF was amplified with human and bovine aFGF specific oligonucleotide primers in porcine heart. Identity of amplified PCR product to aFGF sequence was confirmed by internal reamplification, Southern hybridization and sequencing of asymmetrically amplified PCR products. The nucleotide sequence analysis of porcine aFGF revealed a homology of 94% to the human and 92% to the bovine cDNA sequences respectively. The amino acid sequence was homologous to the known sequences except for three alterations in the human and thirteen in the bovine aFGF sequences.
Insights
Researchers identified acidic fibroblast growth factor (aFGF) in pig hearts using molecular techniques. This finding is crucial for understanding blood vessel growth in coronary artery disease.
Area of Science:
- Cardiovascular Biology
- Molecular Biology
- Genetics
Background:
- Coronary artery stenosis can trigger angiogenesis, a process potentially mediated by growth factors like acidic fibroblast growth factor (aFGF).
- Understanding the role of aFGF in porcine models is relevant for cardiovascular research.
Purpose of the Study:
- To amplify and sequence the messenger RNA (mRNA) encoding aFGF in porcine myocardium.
- To investigate the genetic homology of porcine aFGF to human and bovine sequences.
Main Methods:
- Reverse transcription-polymerase chain reaction (RT-PCR) was employed to amplify aFGF mRNA from porcine heart tissue.
- Sequence identity was confirmed through internal reamplification, Southern hybridization, and sequencing of amplified products.
Main Results:
- A DNA fragment encoding aFGF was successfully amplified from porcine myocardium using human and bovine specific primers.
- Nucleotide sequence analysis showed 94% homology to human and 92% homology to bovine aFGF cDNA.
- Amino acid sequence analysis revealed minor variations compared to human and bovine aFGF.
Conclusions:
- The study successfully identified and characterized porcine aFGF mRNA.
- The high sequence homology suggests conserved function of aFGF in mammals, relevant for angiogenesis studies.