A method for selection of forward mutations in supF gene carried by shuttle-vector plasmids

Area of Science:

• Molecular Biology
• Genetics
• Microbiology

Background:

• The supF gene in Escherichia coli is a common target for mutagenesis studies using shuttle-vector plasmids.
• Existing screening methods, like colony color assays, are insufficient for detecting low mutation frequencies in prokaryotes.
• This limitation hinders detailed analysis of mutational spectra and DNA repair mechanisms.

Purpose of the Study:

• To develop a novel, highly sensitive method for selecting forward mutations in the supF gene on shuttle-vector plasmids.
• To enable the study of low-frequency mutations and mutational spectra in bacterial systems.
• To facilitate comparative studies of DNA repair and mutagenesis between prokaryotic and eukaryotic cells.

Main Methods:

• Construction of a specialized Escherichia coli strain (MBL50) with an araD- araC(Am) mutation for L-arabinose resistance selection.
• Transformation of the MBL50 strain with supF+ and supF- shuttle-vector plasmids to establish the selection principle.
• In vivo mutagenesis experiments using N-methyl-N-nitrosourea (MNU) on a shuttle-vector-bearing strain deficient in O6-methylguanine (O6meG) alkyltransferase repair.
• DNA sequencing of induced supF mutants to determine mutation types and locations.

Main Results:

• The L-arabinose resistance selection method successfully detected supF- mutant fractions significantly lower (up to three orders of magnitude) than traditional colony color assays.
• Mutagenesis with MNU in a DNA repair-deficient strain resulted in G:C-->A:T transitions, consistent with O6meG lesion mispairing.
• A preference for 5'-GG-3' sequences was observed, indicating a 5'-flanking base influence on mutagenesis.
• All MNU-induced mutations clustered in three specific sites within the supF gene, potentially due to the lack of repair capacity.

Conclusions:

• The L-arabinose resistance method provides a sensitive and rapid approach for selecting and analyzing supF forward mutations in bacteria.
• This method significantly enhances the utility of the supF gene as a target for bacterial mutagenesis and DNA repair studies.
• The ability to use shuttle-vectors that replicate in both bacteria and mammalian cells opens avenues for comparing prokaryotic and eukaryotic mutational spectra.