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. <insert step 1> Identify the protein interactions from the two-hybrid system results. Note that gene A interacts with B and C, B interacts with A, D, and E, E interacts only with B, and F interacts only with G.>
2. <insert step 2> Analyze the growth rates of the single mutants. Genes A, B, C, D, and E mutants grow at 80% of the wild type rate, indicating these genes are likely involved in a common pathway or process essential for optimal growth. Genes F and G mutants grow at the wild type rate, suggesting they are not essential under the conditions tested.>
3. <insert step 3> Examine the double-mutant growth results. The ab, ac, ad, and ae double mutants grow at 80% of the wild type rate, similar to the single mutants, indicating no additional synthetic effects. However, the af and ag double mutants are lethal, suggesting a synthetic lethal interaction between A and F, and A and G.>
4. <insert step 4> Consider how the two-hybrid and genetic interaction results complement each other. The two-hybrid results suggest direct physical interactions between proteins, while the genetic interactions (e.g., synthetic lethality) suggest functional relationships that may not involve direct contact.>
5. <insert step 5> Propose a model that reconciles both sets of results. One possibility is that genes A, B, C, D, and E are part of a pathway or complex that is essential for growth, while F and G are part of a separate pathway that becomes essential when A is non-functional, explaining the synthetic lethality observed in af and ag double mutants.>

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 reduced or abolished gene function. These mutations can lead to phenotypic changes, such as decreased growth rates or lethality, depending on the gene's role in cellular processes. In the context of the question, the mutants of genes A, B, C, D, and E show reduced growth, indicating their involvement in essential pathways, while F and G mutants are phenotypically normal, suggesting they may not be critical for survival 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 two-hybrid system and the genetic interaction experiments can be reconciled by analyzing how the proteins interact and how these interactions influence the overall function and viability of the organism, providing insights into the pathways involved.