Lac Mutations

Jacob and Monod worked out the structure and function of the lac operon by analyzing mutations that affected lactose metabolism. To help define the roles of the different components of the operon, they used partial diploid strains of

E. coli. The cells of these strains possessed two different DNA molecules: the full bacterial chromosome and an extra piece of DNA. Jacob and Monod created these strains by allowing conjugation to take place between two bacteria (see Chapter 8). In conjugation, a small circular piece of DNA (a plasmid) is transferred from one bacterium to another. The plasmid used by Jacob and Monod contained the lac operon; so the recipient bacterium became partly diploid, possessing two copies of the lac operon. By using different combinations of mutations on the bacterial and plasmid DNA, Jacob and Monod determined that parts of the lac operon were cis acting (able to control the expression of genes on the same piece of DNA only) or trans acting (able to control the expression of genes on other DNA molecules).

Structural-gene mutations Jacob and Monod first discovered some mutant strains that had lost the ability to synthesize either p-galactosidase or permease. (They did not study in detail the effects of mutations on the transacetylase enzyme, so it will not be considered here.) These mutations mapped to the lacZ or lacY structural genes and altered the amino acid sequence of the enzymes encoded by the genes. These mutations clearly affected the structure of the enzymes and not the regulation of their synthesis.

Through the use of partial diploids, Jacob and Monod were able to establish that mutations at the lacZ and lacY genes were independent and usually affected only the product of the gene in which they occurred. Partial diploids with lacZ+ lacY~ on the bacterial chromosome and lacZ~ lacY+ on the plasmid functioned normally, producing p-galactosidase and permease in the presence of lactose. (The genotype of a partial diploid is written by separating the genes on each DNA molecule with a slash: lacZ+ lacY~/lacZ~ lacY+.) In this partial diploid, a single functional p-galactosidase gene (lacZ+) is sufficient to produce p-galactosidase; it makes no difference whether the functional p-galactosidase gene is coupled to a functional (lacY+) or a defective (lacY~) permease gene. The same is true of the lacY+ gene.

Regulator-gene mutations Jacob and Monod also isolated mutations that affected the regulation of enzyme production.

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