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Cloning of a cDNA encoding phosphofructokinase from Haemonchus contortus
R D Klein1, E R Olson, M A Favreau
1Molecular Biology Research, Upjohn Laboratories, Kalamazoo, MI 49001.
Molecular and Biochemical Parasitology
|September 1, 1991
Summary
Researchers cloned the phosphofructokinase (PFK) gene from the parasitic nematode Haemonchus contortus. This enzyme is crucial for glycolysis and shows high similarity to mammalian PFKs.
Area of Science:
- Parasitology
- Molecular Biology
- Biochemistry
Background:
- Phosphofructokinase (PFK) is a critical regulatory enzyme in glycolysis.
- Haemonchus contortus is a pathogenic parasitic nematode impacting livestock.
- Understanding PFK in H. contortus can reveal metabolic vulnerabilities.
Purpose of the Study:
- To clone and characterize the phosphofructokinase (PFK) gene from Haemonchus contortus.
- To enable functional analysis of H. contortus PFK in a heterologous system.
- To investigate the potential of PFK as a drug target in parasitic nematodes.
Main Methods:
- Functional complementation of an Escherichia coli PFK-deficient strain (DF1020) using a Haemonchus contortus cDNA library.
- Selection of transformants on minimal medium with mannitol as the sole carbon source.
- Plasmid isolation, DNA sequencing, and protein sequence analysis.
Main Results:
- Successfully cloned a 2.7-kb cDNA insert encoding H. contortus PFK.
- Complemented E. coli strain DF1020 exhibited restored mannitol metabolism (PFK phenotype).
- The predicted 789-amino acid protein showed ~70% similarity to mammalian PFKs, with conserved catalytic site residues.
Conclusions:
- The cloned gene encodes a functional phosphofructokinase from H. contortus.
- The high similarity to mammalian PFKs suggests conserved function and structure.
- This work provides a foundation for exploring PFK as a potential therapeutic target against H. contortus infections.