The two-hybrid system was used to identify interactions between proteins, and the succeeding results were obtained: A clone coding gene A gave affirmative results with clones B and C, while clone B gave affirmative results with clones A, D, and E but not C, and clone E gave positive results only with clone B. Alternative clone F gave helpful results with clone G but not with any of A–E . Can you explain these results? To follow up the two-hybrid results, null loss-of-function mutations in each of genes A–G were isolated. 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 impossible to differentiate from the wild type. Several double-mutant strains are constructed: 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 bear lethality. Provide the explaination of these results. Determine the approach by which two-hybrid system and genetic interaction results complement one another. whether reconcile two-hybrid system and genetic interaction results in a single model can reconcile. Introduction: The two- hybrid system is a method used to determine the interaction between two proteins. The system is based on the modular nature of the transcription factor Gal4 from yeast. The triggering of the downstream reporter gene by binding of transcription factor onto upstream activating sequence (UAS), is the idea behind this method. For the Two-Hybrid system, the transcription factor distributes into two domains i.e. DNA binding domain (DBD or BD) and activating domain (AD). Two fusion proteins are systematized – Gal4BD + Bait and Gal4AD+ prey. Transcription of the gene follows when both the fusion proteins are produced and bait interacts with the prey. Both BD and AD have different functions. BD domain boosts the binding to UAS, while AD is responsible for the activation of the transcription. Y2H is a protein- fragment complementation assay.

Question

Want to see the full answer?

Answer 1

Question

Chapter 16, Problem 28P

Summary Introduction

To analyze:

The two-hybrid system was used to identify interactions between proteins, and the succeeding results were obtained:

A clone coding gene A gave affirmative results with clones B and C, while clone B gave affirmative results with clones A, D, and E but not C, and clone E gave positive results only with clone B. Alternative clone F gave helpful results with clone G but not with any of A–E. Can you explain these results? To follow up the two-hybrid results, null loss-of-function mutations in each of genes A–G were isolated. 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 impossible to differentiate from the wild type. Several double-mutant strains are constructed: 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 bear lethality.

Provide the explaination of these results. Determine the approach by which two-hybrid system and genetic interaction results complement one another. whether reconcile two-hybrid system and genetic interaction results in a single model can reconcile.

Introduction:

The two- hybrid system is a method used to determine the interaction between two proteins. The system is based on the modular nature of the transcription factor Gal4 from yeast. The triggering of the downstream reporter gene by binding of transcription factor onto upstream activating sequence (UAS), is the idea behind this method.

For the Two-Hybrid system, the transcription factor distributes into two domains i.e. DNA binding domain (DBD or BD) and activating domain (AD). Two fusion proteins are systematized – Gal4BD + Bait and Gal4AD+ prey. Transcription of the gene follows when both the fusion proteins are produced and bait interacts with the prey.

Both BD and AD have different functions. BD domain boosts the binding to UAS, while AD is responsible for the activation of the transcription. Y2H is a protein- fragment complementation assay.

Expert Solution & Answer

Want to see the full answer?

Check out a sample textbook solution

See solution

Students have asked these similar questions

In DNA-hybridization experiments on six species of plants in the genus Vicia, DNA was isolated from each of the six species, denatured by heating, and sheared into small fragments (W. Y. Chooi. 1971. Genetics 68:213–230). In one experiment, DNA from each species and from E. coli was allowed to renature. The graph shows the results of this renaturation experiment. Q. Notice that, for the Vicia species, the rate of renaturation is much faster in the first hour and then slows down. What might cause this initial rapid renaturation and the subsequent slowdown?

Below is an EMSA showing four different reactions, A-D. In each tube there is some combination of labelled DNA probe, Protein X (the protein you are studying), and an antibody for Protein X. Identify which combination of components are found in each of the four reactions and explain how you determined that based on the molecular interactions being studied and your knowledge of gel electrophoresis. It is possible that multiple lanes have the same component(s). A B C D EMSA

In DNA-hybridization experiments on six species of plants in the genus Vicia, DNA was isolated from each of the six species, denatured by heating, and sheared into small fragments (W. Y. Chooi. 1971. Genetics 68:213–230). In one experiment, DNA from each species and from E. coli was allowed to renature. The graph shows the results of this renaturation experiment. Q. Can you explain why the E. coli DNA renatures at a much faster rate than does DNA from any of the Vicia species?