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Evidence that the intron open reading frame of the phage T4 td gene encodes a specific endonuclease
D K West1, L M Changchien, G F Maley
1Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Abstract:
The phage T4 thymidylate synthase (td) gene contains an intron open reading frame that encodes a 245-amino acid-long basic protein (Chu, F. K., Maley, G. F., West, D. K., Belfort, M., and Maley, F. (1986) Cell 45, 157-166). The open reading frame (Irf) has been cloned as a fusion protein behind a phage T7 promoter and overexpressed in Escherichia coli. The amplified Irf protein is associated with insoluble inclusion bodies and migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis about 7 kDa smaller than expected. Data obtained from DNA sequencing, amino acid sequencing of the fusion protein, and carboxypeptidase Y digestion suggest that although the cloned gene is not altered and the protein is made from the expected start codon, it appears to terminate about 90 amino acids before the encoded stop codon. Proteolytic cleavage during or soon after synthesis appears to be responsible for the truncated Irf. The expressed protein is solubilized in guanidine HCl and renatured by dialysis against high salt. This partially purified preparation has been found to contain a DNA endonuclease activity specific for the td delta I gene, which contains a precise deletion of the intron.
Insights
The intron open reading frame (Irf) from phage T4 thymidylate synthase (td) gene was overexpressed in E. coli. The resulting truncated Irf protein exhibits specific DNA endonuclease activity against the td delta I gene.
Area of Science:
- Molecular Biology
- Enzymology
- Bacteriophage Genetics
Background:
- The thymidylate synthase (td) gene in phage T4 contains an intron with an open reading frame (Irf).
- This Irf encodes a 245-amino acid basic protein.
- Understanding the function and expression of this Irf is crucial for phage genetics and DNA repair mechanisms.
Purpose of the Study:
- To clone, overexpress, and characterize the Irf protein from the phage T4 td gene.
- To investigate the enzymatic activity of the expressed Irf protein.
- To determine the cause of the observed truncated protein product.
Main Methods:
- Cloning of the Irf as a fusion protein using a phage T7 promoter.
- Overexpression in Escherichia coli and isolation of inclusion bodies.
- Protein analysis via SDS-PAGE, DNA sequencing, amino acid sequencing, and carboxypeptidase Y digestion.
- Solubilization, renaturation, and partial purification of the expressed protein.
- Assay for DNA endonuclease activity.
Main Results:
- Overexpressed Irf protein formed insoluble inclusion bodies in E. coli.
- The expressed protein was truncated, migrating ~7 kDa smaller than expected on SDS-PAGE.
- DNA and protein sequencing indicated the protein terminated prematurely, likely due to proteolytic cleavage.
- The partially purified Irf preparation displayed specific DNA endonuclease activity targeting the td delta I gene (intron deletion).
Conclusions:
- The Irf protein from phage T4 td gene is expressed as a truncated form in E. coli due to proteolytic cleavage.
- The expressed Irf protein possesses specific DNA endonuclease activity.
- This activity is directed against a specific deletion within the td gene's intron, suggesting a role in intron processing or DNA repair.