Construction of a vector for site-specific frameshift mutagenesis containing the mutable hotspot of Salmonella typhimurium TA98 on an M13 bacteriophage.
Frameshift mutations demonstrate a high degree of sequence specificity. In order to provide a vector for site-specific frameshift mutagenesis experiments, a recombinant M13 phage (M13MB102) was constructed by substitution of 27 base pairs of the Salmonella typhimurium hisD3052 sequence for 27 base pairs of the polylinker region of M13mp19. The inserted sequence contains most of the hotspot for frameshift mutations in hisD3052 and its derivative strain TA98. Structural elements of the insert include reiterated bases, direct repeats, and palindromes, and four unique restriction endonuclease cleavage sites. The recombinant phage produced blue plaques when grown in Escherichia coli strain JM105 on X-Gal indicator plates and exhibited a spontaneous mutation frequency similar to that of M13mp19. Methodology is described for preparation, isolation, and purification of closed circular duplex M13MB102 genomes containing an adduct between the SphI and BssHII cleavage sites in the (-)-strand and uracil residues in the (+)-strand. The latter modification decreases replication of the (+)-strand by 4 orders of magnitude and maximizes use of the adducted (-)-strand for in vivo replication. The structure of M13MB102 and the procedures described for introducing adducts at defined positions in its hisD3052 insert provide a convenient approach for evaluating the potential of individual carcinogen-DNA adducts to induce frameshift mutations.
Benamira
M
M
A. B. Hancock, Jr., Memorial Laboratory for Cancer Research, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-0146.
Marnett
L J
LJ
D017104:Bacteriophage M13 / Q000235:genetics*; D001483:Base Sequence; D004269:DNA, Bacterial / Q000737:chemistry; D004270:DNA, Circular / Q000737:chemistry; D004279:DNA, Viral / Q000737:chemistry; D016368:Frameshift Mutation; D005822:Genetic Vectors; D016679:Genome, Viral; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009692:Nucleic Acid Heteroduplexes / Q000737:chemistry; D012486:Salmonella typhimurium / Q000235:genetics*; D014498:Uracil / Q000737:chemistry
D004269:DNA, Bacterial; D004270:DNA, Circular; D004279:DNA, Viral; D009692:Nucleic Acid Heteroduplexes; D014498:Uracil