MadSci Network: Molecular Biology
Query:

Re: what is the need of amber suppressor strains in molecular cloning experiments?

Date: Thu Aug 10 05:40:16 2006
Posted By: Dr Jim Caryl, Research Scientist, University of Leeds
Area of science: Molecular Biology
ID: 1154068452.Mb
Message:

Hello there,

Thank you for your question. Basically, the use of suppressor mutant strains, most commonly E. coli K-12 derivatives, permits a rather simple form of in vivo mutagenesis. Rather than using site- directed mutagenesis to introduce multiple mutations within your protein of interest, a researcher could just introduce an amber (TAG/UAG) codon at the position(s) of interest.

In a non-suppressor strain, the protein would prematurely terminate at this stop codon, however in a strain containing amber suppressor mutations, the stop codon will be tolerated and depedning on the type of suppression genes present, will introduce one of several other amino acids at this position.

The supression strains are able to do this as they contain altered tRNA molecules that read the UAG codon as containing an amino acid. Different amber supressors produce different species of altered tRNAs resulting in a number of different possible amino acids being inserted at the site of the amber mutation (Person & Osborn, 1968).

Amber mutations are particularly successful as UAG codons can be generated in almost any gene by single-base substitutions in any of eight triplets that code for seven different amino acids. The efficiency of amber supression tRNAs can be quite high, accommodating up to 60% amino acid insertion at the amber codon. Not the case with ochre mutations, which are poorly suppressed (Belin, 2003).

The technology has been manipulated such that any one of 12 amino acids can be inserted at the position of TAG. Thus only a single mutation is required and by using the appropriate suppressor mutation, encoded either on a plasmid or chromosomally, the site can be mutated with oyour choice of alternate amino acid (Promega).

Hope this helps,

Jim Caryl
MAD Scientist

Person & Osborn (1968) The conversion of amber suppressors to ochre suppressors. Proc Natl Acad Sci USA. 60(3): 10301037. [Free via PubMed]

Belin D. (2003) Why Are Suppressors of Amber Mutations So Frequent Among Escherichia coli K12 Strains?: A Plausible Explanation for a Long-Lasting Puzzle. Genetics 165: 455-456. [URL]

Mutational Analysis Using in vivo Suppression of Amber Stop Codons: The INTERCHANGE(TM) in vivo Amber Suppression Mutagenesis System. Promega [Note: links to a PDF file].


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