A researcher has a Trp ^- auxotrophic strain of E . coli...

A researcher has a Trp $^{-}$ auxotrophic strain of $E .$ coli with a mutation in a single gene. To identify that mutant gene, she uses a genomic library made from a wild-type version of that same strain to find plasmids that rescue the mutant phenotype. The result is surprising. She recovers 10 plasmids that provide a Trp $^{+}$ phenotype, but six of the plasmids contain gene $X,$ while the other four contain gene $Y$. Our scientist has encountered a phenomenon called multicopy suppression, related to the fact that plasmids are usu- ally present in several copies per bacterium. Because the genes in the plasmids are present in more than their usual single copy in the bacterial chromosome, more than the usual amount of Protein $X$ or Protein $Y$ is being produced from the plasmids. Sometimes, over expression of one protein can rescue the mutant phenotype caused by loss of a different protein. Suggest at least two ways that our scientist could determine which of the two genes, gene $X$ or gene $Y$, actually corresponds to the mutant gene causing the Trp $^{-}$ phenotype.

A researcher has a Trp $^{-}$ auxotrophic strain of $E .$ coli with a mutation in a single gene. To identify that mutant gene, she uses a genomic library made from a wild-type version of that same strain to find plasmids that rescue the mutant phenotype. The result is surprising. She recovers 10 plasmids that provide a Trp $^{+}$ phenotype, but six of the plasmids contain gene
$X,$ while the other four contain gene $Y$. Our scientist has encountered a phenomenon called multicopy suppression, related to the fact that plasmids are usu-
ally present in several copies per bacterium. Because the genes in the plasmids are present in more than their usual single copy in the bacterial chromosome, more than the usual amount of Protein $X$ or Protein $Y$ is being produced from the plasmids. Sometimes, over expression of one protein can rescue the mutant phenotype caused by loss of a different protein. Suggest at least two ways that our scientist could determine which of the two genes, gene $X$ or gene $Y$, actually corresponds to the mutant gene causing the Trp $^{-}$ phenotype.

Labs

Want to see this concept in action?

NEW

Explore this concept interactively to see how it behaves as you change inputs.

View Labs

Key Concepts

Multicopy Suppression

Multicopy suppression involves the rescue of a mutant phenotype by increasing the dosage of a gene through the introduction of multiple copies, typically via a plasmid. This phenomenon can mask a mutation by overexpressing another gene product that compensates for the lost function, even if the gene overexpressed isn’t directly part of the same metabolic pathway or complex.

Genetic Complementation Testing

A genetic complementation test determines whether two mutations affecting the same phenotype are in the same gene or in different genes. By introducing a wild-type copy of each candidate gene individually into the mutant and observing whether the mutant phenotype is rescued, researchers can identify the gene that is originally mutated. This testing is fundamental for assigning gene function when multiple candidates exist.

Gene Subcloning and Deletion Analysis

Gene subcloning and deletion analysis involve fragmenting a genomic library and inserting individual gene fragments or systematically deleting portions of cloned fragments to pinpoint the minimal region that confers rescue of the mutant phenotype. This method allows researchers to conclusively determine which gene product is necessary for complementation by isolating the specific gene responsible from among several candidates.

Transcript

Please give Ace some feedback