Using the two-hybrid system to detect interactions between proteins, you obtained the following results: A clone encoding gene A gave positive results with clones B and C; clone B gave positive results with clones A, D, and E but not C; and clone E gave positive results only with clone B. Another clone F gave positive results with clone G but not with any of A–E. Can you explain these results? To follow up your two-hybrid results, you isolate null loss-of-function mutations in each of the genes A–G. Mutants of genes A, B, C, D, and E grow at only 80% of the rate of the wild type, whereas mutants of genes F and G are phenotypically indistinguishable from the wild type. You construct several double-mutant strains: The ab, ac, ad, and ae double mutants all grow at about 80% of the rate of the wild type, but af and ag double mutants exhibit lethality. Explain these results. How do the two-hybrid system and genetic interaction results complement one another? Can you reconcile your two-hybrid system and genetic interaction results in a single model?

Verified step by step guidance

1. The two-hybrid system is a molecular biology technique used to study protein-protein interactions. In this system, proteins are tested for their ability to physically interact by reconstituting a functional transcription factor. Positive results indicate a direct interaction between the proteins encoded by the tested genes. Begin by summarizing the two-hybrid results: Gene A interacts with B and C, Gene B interacts with A, D, and E, Gene E interacts only with B, and Gene F interacts only with G.
2. Analyze the genetic interaction results: Null mutations in genes A, B, C, D, and E reduce growth to 80% of the wild-type rate, suggesting these genes are involved in a shared pathway or process essential for optimal growth. In contrast, null mutations in genes F and G do not affect growth, indicating these genes are not essential under the tested conditions. Double mutants af and ag exhibit lethality, suggesting a synthetic lethal interaction between gene F or G and the pathway involving A–E.
3. Integrate the two-hybrid and genetic interaction data: The two-hybrid results suggest that proteins encoded by A, B, C, D, and E form a network of physical interactions, likely contributing to a shared biological function. The genetic interaction results support this, as mutations in these genes lead to reduced growth. The interaction between F and G, and their synthetic lethality with A, suggests that F and G are part of a separate pathway that is functionally redundant with the A–E pathway.
4. Propose a model to reconcile the data: Proteins A, B, C, D, and E likely form a complex or participate in a single pathway critical for growth. Proteins F and G may form a separate complex or pathway that compensates for the loss of the A–E pathway under normal conditions. The lethality of af and ag double mutants indicates that the two pathways are essential for survival when one is disrupted.
5. Explain how the two-hybrid and genetic interaction results complement each other: The two-hybrid system identifies direct physical interactions between proteins, providing insight into the molecular mechanisms of the pathway. The genetic interaction results reveal functional relationships between genes, highlighting redundancies and dependencies. Together, these approaches provide a comprehensive understanding of the roles of these genes and their interactions in cellular processes.

Key Concepts

Two-Hybrid System

The two-hybrid system is a molecular biology technique used to study protein-protein interactions. It involves the use of two hybrid proteins, one containing a DNA-binding domain and the other a transcriptional activation domain. When the two proteins interact, they bring these domains together, activating the transcription of a reporter gene. This system allows researchers to identify and confirm interactions between proteins in a cellular context.

Loss-of-Function Mutations

Loss-of-function mutations are genetic alterations that result in the reduced or abolished function of a gene product, typically a protein. These mutations can provide insights into gene function by revealing the phenotypic consequences when the gene is inactive. In the context of the question, the mutants of genes A–E show reduced growth rates, indicating their roles in essential biological processes, while F and G mutants show no significant phenotypic changes, suggesting they may not be critical for growth under the tested conditions.

Genetic Interactions

Genetic interactions occur when the effects of one gene are modified by one or more other genes. These interactions can be classified as epistatic, where one gene's effect masks another's, or synergistic, where combined mutations lead to a more severe phenotype. The results from the double mutants in the question illustrate how certain combinations of mutations can lead to lethality, indicating that genes F and G may have essential roles that are not compensated for by other genes, highlighting their importance in the genetic network.