Question

The SOS repair genes in \(E\). coli (discussed in Chapter 15 ) are negatively regulated by the lexA gene product, called the LexA (a) Describe two different mutations that would result in a \(u v r A\) constitutive phenotype. Indicate the actual genotypes involved. (b) Outline a series of genetic experiments that would use partial diploid strains to determine which of the two possible mutations you have isolated.

Short answer

Answer: Two possible mutations that result in a uvrA constitutive phenotype are a mutation in the lexA gene (e.g., lexA1) or a mutation in the cis-regulatory region of the uvrA gene (e.g., uvrA2). Genetic experiments using partial diploid strains, such as lexA1 lexA+ and uvrA2 uvrA+, can help in determining the isolated mutation by monitoring the phenotypes of these strains and measuring the expression of the uvrA gene. The strain with the constitutive expression will indicate which mutation is responsible for the observed phenotype.

Step-by-step solution

Part (a) - Mutations that result in uvrA constitutive phenotype

In E. coli, the SOS repair genes are negatively regulated by the LexA gene product. A uvrA constitutive phenotype indicates that the uvrA gene is constantly expressed, even when it is not needed. There are two primary types of mutations that could result in a uvrA constitutive phenotype: 1. Mutation in the lexA gene: A mutation in the lexA gene can result in a LexA protein that cannot effectively repress the SOS repair genes. This would cause constitutive expression of the uvrA gene. The genotype might be lexA1, where lexA1 represents a mutant allele of the lexA gene. 2. Mutation in the cis-regulatory region of the uvrA gene: A mutation in the regulatory sequence that LexA binds to, called the operator, can result in a malfunctioning binding site for the LexA repressor. This would lead to the constitutive expression of the uvrA gene due to the inability of LexA to repress the gene. The genotype might be uvrA2, where uvrA2 represents a mutant allele of the uvrA gene, specifically in its cis-regulatory region.

Part (b) - Genetic experiments to determine the isolated mutation

To determine whether the mutation is in the lexA gene (lexA1) or the cis-regulatory region of the uvrA gene (uvrA2), we can perform a series of genetic experiments using partial diploid strains. These are bacteria strains carrying both the normal genes and the mutant genes under investigation. 1. Constructing partial diploid strains: Create partial diploid strains by introducing the wild-type lexA and uvrA genes fused to a plasmid into the mutant strain (lexA1 and uvrA2). This will result in strains with the genotypes lexA1 lexA+ and uvrA2 uvrA+. 2. Monitoring the phenotypes of the partial diploid strains: Analyze the expression of the uvrA gene in the partial diploid strains. This can be done by performing an assay to measure the enzymatic activity of UvrA protein or by using reporter genes like lacZ to measure the expression of the uvrA gene. 3. Interpreting the results: If the lexA mutation (lexA1) is responsible for the uvrA constitutive phenotype, the lexA1 lexA+ strain should show constitutive expression because the mutant lexA1 gene will not be able to repress the SOS repair genes effectively. In this case, the uvrA2 uvrA+ strain will show normal expression levels, indicating that the mutation in the cis-regulatory region of the uvrA gene is not responsible for the observed phenotype. On the contrary, if the mutation in the cis-regulatory region of the uvrA gene (uvrA2) is responsible for the phenotype, the uvrA2 uvrA+ strain will display constitutive expression, while the lexA1 lexA+ strain will show normal expression levels. The outcome of these experiments will help us determine which of the two mutations is responsible for the uvrA constitutive phenotype.

Key concepts

LexA Gene

The LexA gene holds a central role within the SOS response of Escherichia coli. It encodes for a protein called the LexA repressor, which is essential in maintaining normal cell operations by preventing unnecessary production of repair enzymes.

Under standard conditions, the LexA protein binds to specific DNA sequences known as SOS boxes, which are located in the regulatory regions of SOS genes, including the uvrA gene. When DNA damage occurs, a series of events leads to the self-cleavage of the LexA repressor, thereby initiating the SOS response and permitting the repair processes to commence.

A mutation in the LexA gene that results in a defective LexA protein might fail to repress the uvrA gene among others, leading to a uvrA constitutive phenotype where repair enzymes are continuously produced, regardless of whether they are needed or not. This mutational change is represented by a different allele, such as lexA1. Therefore understanding the LexA gene is pivotal for the comprehension of the SOS repair pathway and its regulation.

uvrA Constitutive Phenotype

The uvrA constitutive phenotype is characterized by the continuous and unregulated expression of the uvrA gene, one of the several that comprise the SOS repair system in E. coli.

In a healthy cell, the uvrA gene is tightly regulated and its expression is induced only in the presence of DNA damage. However, in cells exhibiting a uvrA constitutive phenotype, this regulation is disrupted, causing persistent activation. This could be due to mutations in either the LexA repressor (lexA1) or the SOS box in the cis-regulatory region of the uvrA gene itself (uvrA2).

To identify the cause of a constitutive phenotype, researchers analyze the genotype. For example, a genotype denoted as uvrA2 would suggest a mutation affecting the regulatory region of the uvrA gene, which prevents proper LexA binding and thus, repression. The ability to determine the underlying mutation is crucial for understanding and potentially correcting genetic errors in DNA repair pathways.

Genetic Experiments

Genetic experiments are designed to explore the function of genes, their interactions, and their effects on an organism's phenotype. They often involve the comparison between wild-type and mutant strains to identify which changes in the DNA sequence cause a particular phenotype.

In the context of identifying whether the lexA1 or uvrA2 mutations cause a uvrA constitutive phenotype, genetic experiments take on a very structured approach. Researchers create partial diploid strains, analyze gene expression, and interpret the results. These steps enable the determination of the mutation's location, whether it alters the LexA coding sequence or the DNA binding site. These well-planned experimental procedures illuminate genetic function and regulation, which is essential for advancing knowledge in molecular biology and genetics.

Partial Diploid Strains

Partial diploid strains are powerful genetic tools for dissecting gene function, often used in experiments to determine the dominance or recessiveness of mutations. These strains occur when an organism inherits or carries an extra copy of a gene or genes, which can be either the wild-type or an alternative (mutant) form.

In genetic research involving E. coli and the SOS response genes, scientists can construct partial diploids by introducing a plasmid containing the wild-type LexA or uvrA genes into a mutant strain. The resulting partial diploids, with genotypes such as lexA1 lexA+ and uvrA2 uvrA+, offer a unique comparative model. By evaluating these strains under various conditions, researchers gain insights into the functional relationships between genes and decipher whether a mutation is dominant or recessive. This understanding is essential for comprehending complex genetic networks and could be critical for developing bacterial strain modifications or therapeutic interventions.