Evidence that the intron open reading frame of the phage T4 td gene encodes a specific endonuclease.
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.
West
D K
DK
Wadsworth Center for Laboratories and Research, New York State Department of Health, Albany 12201-0509.
Changchien
L M
LM
Maley
G F
GF
Maley
F
F
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D004720:Endonucleases / Q000235:genetics*; D004926:Escherichia coli / Q000235:genetics*; D005796:Genes; D005814:Genes, Viral; D007438:Introns; D008969:Molecular Sequence Data; D009154:Mutation; D010957:Plasmids; D015183:Restriction Mapping; D013604:T-Phages / Q000201:enzymology / Q000235:genetics*; D013940:Thymidylate Synthase / Q000235:genetics*
D013940:Thymidylate Synthase; D004720:Endonucleases