Gene targeting in yeast is performed just as in bacteria...

Gene targeting in yeast is performed just as in bacteria (review Fig. 14.31 ). Because the sequence of the yeast genome is available, researchers can easily use gene targeting to create a yeast strain with a knockout mutation in any gene. a. Draw a gene targeting construct that you could introduce into yeast in order to generate a strain with a knockout of the histone subunit $H 2 A$ gene. Your construct should contain a gene that confers resistance to kanamycin $\left(k a n^{r}\right)$ b. List the steps that you would perform to generate the mutant yeast. c. Recall that yeast can grow as diploids or haploids. Would you perform the gene targeting in a haploid or a diploid strain? Explain. d. Suppose you knock out the $H 2 A$ gene in a diploid strain to generate a heterozygote, sporulate the diploid, and find that all four haploid spores are viable (they produce haploid colonies). What would you conclude? Is this the result that you expect for the H2A gene? How can you explain the result?

Gene targeting in yeast is performed just as in bacteria (review Fig. 14.31 ). Because the sequence of the yeast genome is available, researchers can easily use gene targeting to create a yeast strain with a knockout mutation in any gene.
a. Draw a gene targeting construct that you could introduce into yeast in order to generate a strain with a knockout of the histone subunit $H 2 A$ gene. Your construct should contain a gene that confers resistance to kanamycin $\left(k a n^{r}\right)$
b. List the steps that you would perform to generate the mutant yeast.
c. Recall that yeast can grow as diploids or haploids. Would you perform the gene targeting in a haploid or a diploid strain? Explain.
d. Suppose you knock out the $H 2 A$ gene in a diploid strain to generate a heterozygote, sporulate the diploid, and find that all four haploid spores are viable (they produce haploid colonies). What would you conclude? Is this the result that you expect for the H2A gene? How can you explain the result?

Key Concepts

Gene Targeting

Gene targeting is a method used to modify or disrupt a specific gene within an organism's genome by introducing a designed DNA construct that contains regions homologous to the target gene. This process allows for precise genetic alterations, such as gene knockouts, by taking advantage of the cell's natural DNA repair mechanisms. It is a pivotal technique in functional genomics for studying gene function and regulation.

Homologous Recombination

Homologous recombination is a cellular mechanism for repairing DNA that uses a homologous sequence as a template, and it is the basis for gene targeting. In this process, a DNA construct designed with sequences flanking the desired mutation aligns with the corresponding genomic region, allowing the cell's machinery to swap the targeted gene with the modified sequence. This accurate exchange underlies the success of gene knockout experiments.

Selectable Marker Genes

Selectable marker genes, such as antibiotic resistance genes, are incorporated into gene targeting constructs to facilitate the identification and selection of successfully modified cells. When the introduced marker confers resistance to a specific antibiotic, only cells that have integrated the construct survive under selective conditions. This strategy streamlines the identification of cells with the intended genetic alteration.

Yeast Genetic Background (Haploid vs Diploid)

Yeast genetics involves understanding the differences between haploid and diploid states. Haploid yeast carry a single copy of each gene, making any genetic modification immediately evident, whereas diploid yeast possess two copies, which can mask the effects of a knockout if the remaining allele compensates. The choice between using haploid or diploid strains depends on the experimental objectives, particularly when assessing gene function or essentiality.

Gene Essentiality

Gene essentiality refers to whether a particular gene is critical for survival or proper functioning of an organism. By generating targeted knockouts, researchers can observe the resulting phenotype to determine if the loss of a gene leads to viable or lethal outcomes. When the knockout of an expected essential gene results in viable cells, it suggests possible redundancy, compensatory mechanisms, or that the gene may not be as critical as originally thought.

Please give Ace some feedback