6.1.1: The Use of Mutants to Study the lac Operon

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Learning Objectives

Describe how partial diploids can be used to examine genetic relationships in haploid organisms.
Predict whether lac operon genes will be expressed when given mutations are present.

Single mutants of the lac operon

The lac operon and its regulators were first characterized by studying mutants of E. coli that exhibited various abnormalities in lactose metabolism. Some mutants expressed the lac operon genes constitutively, meaning the operon was expressed whether or not lactose was present in the medium. Such mutant are called constitutive mutants.

The operator locus (lacO) - One example is O^c, for operator constitutive, in which a mutation in an operator sequence and reduces or precludes the repressor (the lacI gene product) from recognizing and binding to the operator sequence. Thus, in O^c mutants, lacZ, lacY, and lacA are transcribed whether or not lactose is present.

The lacI locus – One type of mutant allele of lacI (callled I^–) prevents either the production of a repressor polypeptide or produces a polypeptide that cannot bind to the operator sequence. This allele is also a constitutive expresser of the lac operon because absence of repressor binding permits transcription.

Another type of mutant of lacI, called lacI^S for super-repressor, prevents the repressor polypeptide from binding allolactose, and thus will always bind to the operator and be non-inducible. This mutant constitutively represses the lac operon whether lactose is present or not.

Two lac operons in a single cell create partial diploids in E.coli

The regulation of the lac operon became further understood by using two copies of the operon sequences in one cell. Although E. coli are haploid organisms, scientists use a plasmid called the F-factor to bring an additional copy of gene sequences into the cell, while the other copy is on the genomic E. coli chromosome. This results in a partial diploid in E. coli.

The F-factor is extra-chromosomal DNA that is capable of being either a free plasmid or integrated into the host bacterial chromosome. This switching is accomplished by IS elements where unequal crossing over can recombine the F-factor and adjacent DNA sequences (genes) in and out of the host chromosome. Researchers have used this genetic tool to create partial diploids (merozygotes) that allow them to test the regulation with different combinations of different mutations in one cell. For example, the F-factor copy may have a lacI^S mutation while the genomic copy might have an O^C mutation. How would this cell respond to the presence/absence of lactose (or glucose)? This partial diploid can be used to determine that lacI^S is dominant to lacI⁺, which in turn is dominant to lacI^–. It can also be used to show the O^C mutation only acts in cis- while the lacI mutation can act in trans- .

Example \(\PageIndex{1}\)

Consider the question posed above: A cell with a copy of the lac operon from an F-factor has the I^S mutation, whereas the genomic copy has an O^C mutation. How would this cell respond to the presence/absence of lactose (or glucose)?

Solution

The I^Smutation encodes a form of the protein cannot bind allolactose and therefore continues to recognize the operator, even when lactose is present. However, the genomic copy of the operator O^C is mutated to a sequence that is no longer recognized by the lac repressor. For this reason, the lacZYA genes in the genomic lac operon will be continuously expressed in this cell, because lacI repressor will not be able to bind this sequence.

Query \(\PageIndex{1}\)

Contributors and Attributions

Dr. Todd Nickle and Isabelle Barrette-Ng (Mount Royal University) The content on this page is licensed under CC SA 3.0 licensing guidelines.