TNBGGA: The No Bullshit Guide to Genetic Analysis

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chapter_11 [2024/08/25 12:19] – [Stable regulatory circuits] mikechapter_11 [2025/04/07 20:58] (current) – mike
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-<typo fs:x-large>Chapter 11. Gene circuits and epistasis</typo>+<-chapter_10|Chapter 10^table_of_contents|Table of Contents^chapter_12|Chapter 12-> 
 + 
 +<typo fs:x-large>Chapter 11. %%Gene circuits and epistasis%%</typo>
  
 In [[chapter_10|Chapter 10]], we studied regulatory mechanisms in well-known //E. coli// operons to see how mutations in different elements of the system would behave in dominance tests and cis/trans tests. We also presented the information in reverse - we told you the answer first, then discussed how mutant phenotypes were interpreted.  In [[chapter_10|Chapter 10]], we studied regulatory mechanisms in well-known //E. coli// operons to see how mutations in different elements of the system would behave in dominance tests and cis/trans tests. We also presented the information in reverse - we told you the answer first, then discussed how mutant phenotypes were interpreted. 
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 </figure> </figure>
  
-Although loss of function mutations in genes for repressors or activators are generally the most common type of regulatory mutation, Table {{ref>Tab1}} will help you to interpret mutations in sites or more complicated mutations in proteins. With mutants in hand, you can potentially clone them by complementation as discussed in [[chpater_09|Chap. 09]]. You can then sequence your clones as discussed in [[chapter_08|Chap. 08]]. This will allow you identify the amino acid sequence of the protein/enzyme that carries out the function of the gene that is mutated in your mutants. This approach of discovering protein/enzyme function based on random mutants with interesting phenotypes is called forward genetics. +Although loss of function mutations in genes for repressors or activators are generally the most common type of regulatory mutation, Table {{ref>Tab1}} will help you to interpret mutations in sites or more complicated mutations in proteins. With mutants in hand, you can potentially clone them by complementation as discussed in [[chapter_09|Chap. 09]]. You can then sequence your clones as discussed in [[chapter_08|Chap. 08]]. This will allow you to  
 +identify the amino acid sequence of the protein/enzyme that carries out the function of the gene that is mutated in your mutants. This approach of discovering protein/enzyme function based on random mutants with interesting phenotypes is called forward genetics. 
    
 <table Tab1> <table Tab1>
 <columns 100% *100%*> <columns 100% *100%*>
 ^  type of mutation  ^  phenotype  ^  dominant/recessive?  ^  cis/trans-acting?  ^ ^  type of mutation  ^  phenotype  ^  dominant/recessive?  ^  cis/trans-acting?  ^
-|  repressor loss-of-function  |  constitutive  |  recessive  |  trans-acting  | +|  repressor loss of function  |  constitutive  |  recessive  |  trans-acting  | 
-|  activator loss-of function  |  uninducible  |  recessive  |  trans-acting  | +|  activator loss of function  |  uninducible  |  recessive  |  trans-acting  | 
-|  operator loss-of-function  |  constitutive  |  dominant*  |  cis-acting  |+|  operator loss of function  |  constitutive  |  dominant*  |  cis-acting  |
 |  promoter (or initiator) loss-of-function  |  uninducible  |  recessive*  |  cis-acting  | |  promoter (or initiator) loss-of-function  |  uninducible  |  recessive*  |  cis-acting  |
 |  repressor dominant negative or super activator  |  constitutive  |  dominant  |  trans-acting  | |  repressor dominant negative or super activator  |  constitutive  |  dominant  |  trans-acting  |
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 </columns> </columns>
 <caption> <caption>
-Analysis of regulatory mutants. *note: operator/promoter mutants need to be analyzed in the context of the coding sequence they regulate, e.g., $lacO$/$lacP$ need to be analyzed in the context of a functioning lacZ. In a sense, $lacO$/$lacP$ and $lacZ$ are different portions of the same gene since they don't complement each other. By comparison, $lacI$ (at least in principle) can be analyzed as a separate entity away from $lacZ$ since $lacI$ and $lacZ$ complement each other and therefore are not in the same gene.  +Analysis of regulatory mutants. *note: operator/promoter mutants need to be analyzed in the context of the coding sequence they regulate, e.g., $lacO$/$lacP$ need to be analyzed in the context of a functioning $lacZ^+$. In a sense, $lacO$/$lacP$ and $lacZ$ are different portions of the same gene since they don't complement each other. By comparison, $lacI$ (at least in principle) can be analyzed as a separate entity away from $lacZ$ since $lacI$ and $lacZ$ complement each other and therefore are not in the same gene.  
 </caption> </caption>
 </table> </table>
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 After an initial unstable period immediately after infection, either $cro$ expression or $cI$ expression will dominate. After an initial unstable period immediately after infection, either $cro$ expression or $cI$ expression will dominate.
  
-  * Mode 1: High cro expression blocks cI expression. In this state, all of the genes for lytic growth are made and the phage enters the lytic program. +  * Mode 1: High $cro$ expression blocks $cI$ expression. In this state, all of the genes for lytic growth are made and the phage enters the lytic program. 
-  * Mode 2: High cI expression blocks cro expression. In this state, none of the genes except for cI are expressed. This produces a stable lysogen.+  * Mode 2: High $cI$ expression blocks $cro$ expression. In this state, none of the genes except for $cI$ are expressed. This produces a stable lysogen.
  
 In gene regulation, as in good circuit design, stability is achieved by feedback. The result is a bi-stable switch that is similar to a “flip-flop”, one of the basic elements of digital electronic circuits. Other genes participate in the initial period to bias the decision to one mode or the other. These genes act so that the lytic mode is favored when //E. coli// is growing well and there are few phage per infected cell, whereas the lysogenic mode is favored when cells are growing poorly and there are many phage per infected cell. In gene regulation, as in good circuit design, stability is achieved by feedback. The result is a bi-stable switch that is similar to a “flip-flop”, one of the basic elements of digital electronic circuits. Other genes participate in the initial period to bias the decision to one mode or the other. These genes act so that the lytic mode is favored when //E. coli// is growing well and there are few phage per infected cell, whereas the lysogenic mode is favored when cells are growing poorly and there are many phage per infected cell.
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 ===== Questions and exercises ===== ===== Questions and exercises =====
  
-Discussion Box: As a general principle, making double mutants in obligate diploids is much easier than making double mutants in bacteria. Why? +Exercise 1 (challenge question): Revisit [[chapter_02|Chapter 02]] Figure 3. Is it possible to analyze yeast $his$ mutants using epistasis? Why or why not? What question would you be answering with epistasis? What additional information might you need to know first? You may want to look up some classic genetic experiments by [[wp>One_gene–one_enzyme_hypothesis|Beadle and Tatum]] using the bread mold Neurospora to help with answering this question. 
- +
-Exercise 11.1: Revisit Figure 2.3. Is it possible to analyze yeast his mutants using epistasis? Why or why not? What question would you be answering with epistasis? What additional information might you need to know first? You may want to look up some classic genetic experiments by Beadle and Tatum using the bread mold Neurospora to help with answering this question. +
  
  
chapter_11.1724613599.txt.gz · Last modified: 2024/08/25 12:19 by mike