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Ch. 14 - Analysis of Gene Function via Forward Genetics and Reverse Genetics
Sanders - Genetic Analysis: An Integrated Approach 3rd Edition
Sanders3rd EditionGenetic Analysis: An Integrated ApproachISBN: 9780135564172Not the one you use?Change textbook
All textbooksSanders 3rd EditionCh. 14 - Analysis of Gene Function via Forward Genetics and Reverse GeneticsProblem 14a
Chapter 14, Problem 14a

When the S. cerevisiae genome was sequenced and surveyed for possible genes, only about 40% of those genes had been previously identified in forward genetic screens. This left about 60% of predicted genes with no known function, leading some to dub the genes fun (function unknown) genes. As an approach to understanding the function of a certain fun gene, you wish to create a loss-of-function allele. How will you accomplish this?

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1
Identify the target gene sequence: Obtain the DNA sequence of the fun gene from the S. cerevisiae genome database. This will allow you to design tools for gene disruption.
Design a disruption construct: Create a DNA construct that contains a selectable marker (e.g., an antibiotic resistance gene or a nutritional marker) flanked by sequences homologous to the regions upstream and downstream of the fun gene. These homologous regions will guide the construct to the correct location in the genome via homologous recombination.
Transform yeast cells: Introduce the disruption construct into S. cerevisiae cells using a transformation method such as electroporation or the lithium acetate method. This will allow the construct to enter the yeast cells.
Select for successful transformants: Grow the transformed yeast cells on a medium that selects for the presence of the selectable marker. Only cells where the disruption construct has integrated into the genome will survive.
Confirm the loss-of-function allele: Use molecular techniques such as PCR or Southern blotting to verify that the fun gene has been replaced by the disruption construct. Additionally, you can perform functional assays to confirm the loss of the gene's activity.

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Key Concepts

Here are the essential concepts you must grasp in order to answer the question correctly.

Loss-of-Function Alleles

Loss-of-function alleles are mutations that result in the complete or partial inactivation of a gene. These alleles can be generated through various methods, such as gene editing techniques like CRISPR-Cas9, which allows for precise modifications to the DNA sequence. By creating a loss-of-function allele, researchers can study the resulting phenotypic changes to infer the gene's role in biological processes.
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Gene Editing Techniques

Gene editing techniques, such as CRISPR-Cas9, enable scientists to make targeted changes to an organism's genome. This technology utilizes a guide RNA to direct the Cas9 enzyme to a specific DNA sequence, where it creates a double-strand break. The cell's repair mechanisms then attempt to fix this break, often leading to insertions or deletions that disrupt gene function, thus facilitating the creation of loss-of-function alleles.
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Functional Genomics

Functional genomics is the field of study that aims to understand the function of genes and their interactions within the genome. It employs various techniques, including gene knockout and overexpression studies, to elucidate the roles of specific genes in cellular processes. By investigating fun genes through functional genomics, researchers can uncover their biological significance and potential implications in health and disease.
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Related Practice
Textbook Question

Genetic counseling has not been discussed in this chapter, but it is a service provided by trained professional counselors who also have detailed knowledge of medical genetics, as described in Application Chapter A. Genetic counselors provide details about gene mutations and have knowledge of most of the details of diseases associated with genetic abnormalities. With regard to genetic testing to identify one's personal risk of cancer, what are the three or four topics you think are most important to be able to discuss with a genetic counselor?

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Textbook Question

The CBF genes of Arabidopsis are induced by exposure of the plants to low temperature. How would you examine the temporal and spatial patterns of expression after induction by low temperature?

Textbook Question

The CBF genes of Arabidopsis are induced by exposure of the plants to low temperature. Can you design a method that would reveal these changes in gene expression in a way that a farmer could recognize them by observing plants growing in the field?

Textbook Question

When the S. cerevisiae genome was sequenced and surveyed for possible genes, only about 40% of those genes had been previously identified in forward genetic screens. This left about 60% of predicted genes with no known function, leading some to dub the genes fun (function unknown) genes. You wish to know the physical location of the encoded protein product. How will you obtain such information?

Textbook Question

Translational fusions between a protein of interest and a reporter protein are used to determine the subcellular location of proteins in vivo. However, fusion to a reporter protein sometimes renders the protein of interest nonfunctional because the addition of the reporter protein interferes with proper protein folding, enzymatic activity, or protein–protein interactions. You have constructed a fusion between your protein of interest and a reporter gene. How will you show that the fusion protein retains its normal biological function?

Textbook Question

In humans, Duchenne muscular dystrophy is caused by a mutation in the dystrophin gene, which resides on the X chromosome. How would you create a mouse model of this genetic disease?