TNBGGA: The No Bullshit Guide to Genetic Analysis

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chapter_12 [2024/08/21 13:00] – [Using an insertion library to find interesting mutants] mikechapter_12 [2025/04/15 06:57] (current) – [Creating an insertion library for yeast] mike
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-<typo fs:x-large>Chapter 12. Cloning regulated genes in eukaryotes</typo> +<-chapter_11|Chapter 11^table_of_contents|Table of Contents^chapter_13|Chapter 13->
  
-For the last several chapters we have been looking at how one can study and manipulate prokaryotic genomes and how prokaryotic genes are regulated. In the next several chapters we will be considering eukaryotic genes and genomes and considering how model eukaryotic organisms are used to study eukaryotic gene function. While any biological function can be studied using genetic analysis, we use the study of gene regulation as our example. +<typo fs:x-large>%%Chapter 12. Cloning regulated genes in eukaryotes%%</typo>  
 + 
 +For the last several chapters we have been looking at how one can study and manipulate prokaryotic genomes and how prokaryotic genes are regulated. In the next several chapters we will be considering eukaryotic genes and genomes and considering how model eukaryotic organisms are used to study eukaryotic gene function. While any biological function can be studied using genetic analysis, we use the study of gene regulation as our example. It can be useful to compare how eukaryotes regulate gene expression with how prokaryotes such as //E. coli// do it. 
  
 ===== Eukaryotic genomes and gene structure ===== ===== Eukaryotic genomes and gene structure =====
  
-First, let us look at how the genes and genomes of some organisms compare to //E. coli// at one extreme, and humans at the other (Fig. {{rev>Fig1}}).+First, let us look at how the genes and genomes of some eukaryotic organisms compare to //E. coli// at one extreme, and humans at the other {{anchor:genome_compare}}(Fig. {{ref>Fig1}}).
  
 ==== Genome size and gene density ==== ==== Genome size and gene density ====
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   * A library of yeast genomic fragments cloned into a bacterial plasmid. We learned about the concept of genomic libraries in [[chapter_09|Chap. 09]].   * A library of yeast genomic fragments cloned into a bacterial plasmid. We learned about the concept of genomic libraries in [[chapter_09|Chap. 09]].
   * The //E. coli// $lacZ$ gene. We learned about the $lacZ$ gene in Chapters [[chapter_09|09]] and [[chapter_10|10]]. In this experiment the $lacZ$ gene is going to be used in yeast cells as a reporter gene (sometimes just called a reporter) for transcriptional activity of yeast genes. The $lacZ$ coding sequence works in yeast because //E. coli// and yeast both use the exact same universal genetic code for converting triplet codon sequences into amino acids.    * The //E. coli// $lacZ$ gene. We learned about the $lacZ$ gene in Chapters [[chapter_09|09]] and [[chapter_10|10]]. In this experiment the $lacZ$ gene is going to be used in yeast cells as a reporter gene (sometimes just called a reporter) for transcriptional activity of yeast genes. The $lacZ$ coding sequence works in yeast because //E. coli// and yeast both use the exact same universal genetic code for converting triplet codon sequences into amino acids. 
-  * A modified bacterial transposon called mini-Tn7 (Fig. {{ref>Fig3}}). Transposons are naturally occurring pieces of DNA that can transpose, or jump around, to random locations in genomes (we also call them mobile genetic elements). We can modify transposons such that we can experimentally control when they jump around, and we can also construct them to carry genetic markers that help us track the transposon. In this experiment, we have engineered mini-Tn7 to contain the $lacZ$ gene (but without any cis-acting regulatory sequences such as $lacO$ or $lacP$), a yeast gene called $URA3$ required for uracil prototrophy (in this case we are including native upstream regulatory sequences necessary for yeast cells to express $URA3$, and an //E. coli// gene (plus appropriate bacterial regulatory sequences) that confers drug resistance to the antibiotic tetracycline ($tet^R$).+  * A modified bacterial transposon called mini-Tn7 (Fig. {{ref>Fig3}}). Transposons are naturally occurring pieces of DNA that can transpose, or jump around, to random locations in genomes. We can modify transposons such that we can experimentally control when they jump around, and we can also construct them to carry genetic markers that help us track the transposon. In this experiment, we have engineered mini-Tn7 to contain the $lacZ$ gene (but without any cis-acting regulatory sequences such as $lacO$ or $lacP$), a yeast gene called $URA3$ required for uracil prototrophy (in this case we are including native upstream regulatory sequences necessary for yeast cells to express $URA3$), and an //E. coli// gene (plus appropriate bacterial regulatory sequences) that confers drug resistance to the antibiotic tetracycline ($tet^R$).
  
 <figure Fig3> <figure Fig3>
 {{ :mini_tn5.jpg?400 |}} {{ :mini_tn5.jpg?400 |}}
 <caption>  <caption> 
-Structure of a modified mini-Tn7 transposon. The drawing represents dsDNA and different genetic elements that make up the modified transposon. TR stands for terminal repeat - these are sequences that are required for mini-Tn7 to be able to transpose into random locations in DNA. $URA3$ is a yeast gene required for uracil auxotrophy. $tet^R$ is an //E. coli// gene that makes cells resistant to the antibiotic tetracycline. Credit: M. Chao. +Structure of a modified mini-Tn7 transposon. The drawing represents dsDNA and different genetic elements that make up the modified transposon. TR stands for terminal repeat - these are sequences that are required for mini-Tn7 to be able to transpose into random locations in DNA. $URA3$ is a yeast gene required for uracil prototrophy. $tet^R$ is an //E. coli// gene that makes cells resistant to the antibiotic tetracycline. Credit: M. Chao. 
 </caption> </caption>
 </figure> </figure>
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 </figure> </figure>
  
-Now we have a library of yeast genomic fragments each of which has the transposon inserted; these genomic fragments can be transformed into //S. cerevisiae// cells that are mutant for the $ura3$ gene (these cells are uracil auxotrophs). The method for transformation and preparing competent yeast cells is similar to that of preparing competent //E. coli//, except that LiCl is used instead of CaCl<sub>2</sub> to treat the cells. Importantly, the fragments cannot replicate on their own, but they can integrate into the chromosome through homologous recombination that targets DNA sequences on the chromosome that that match the transformed fragments. After transformation, we grow the cells on plates that lack uracil - we are selecting for cells that have taken up a //URA3//-containing DNA fragment somewhere in its genome. Each Ura<sup>+</sup> transformant colony that grows will have recombined a mini-Tn7-containing genomic DNA fragment into its genome. This essentially gives us a library of yeast with transposons randomly integrated into its genome.+Now we have a library of yeast genomic fragments each of which has the transposon inserted; these genomic fragments can be transformed into //S. cerevisiae// cells that are mutant for the $ura3$ gene (these cells are uracil auxotrophs). The method for transformation and preparing competent yeast cells is similar to that of preparing competent //E. coli//, except that LiCl is used instead of CaCl<sub>2</sub> to treat the cells. Importantly, the fragments cannot replicate on their own, but they can integrate into the chromosome through homologous recombination that targets DNA sequences on the chromosome that match the transformed fragments. After transformation, we grow the cells on plates that lack uracil - we are selecting for cells that have taken up a $URA3$-containing DNA fragment somewhere in its genome. Each Ura<sup>+</sup> transformant colony that grows will have recombined a mini-Tn7-containing genomic DNA fragment into its genome. This essentially gives us a library of yeast with transposons randomly integrated into its genome.
  
-<figure>+<figure Fig6>
 {{ :tn7_insertion_possibilities.jpg?400 |}} {{ :tn7_insertion_possibilities.jpg?400 |}}
 <caption> <caption>
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 </figure> </figure>
  
-Note that the $lacZ$ gene in the transposon contains only its amino acid coding sequence. It does not have its own regulatory sequences from //E. coli// such as $lacO$ and $lacP$ (and even if $lacO$ and $lacP$ were present, it would have no effect on $lacZ$ expression in yeast; think about that!). But if the transposon inserts downstream of a yeast gene promoter (somewhat rare) and in the correct orientation (1 in 2 chance), and in the correct triplet codon reading frame (1 in 3 chance), the lacZ gene would then come under the control of that promoter. When transcription is activated from that promoter, a LacZ fusion protein is expressed, and most LacZ fusion proteins have robust β-galactosidase activity.+Note that the $lacZ$ gene in the transposon contains only its amino acid coding sequence. It does not have its own regulatory sequences from //E. coli// such as $lacO$ and $lacP$ (and even if $lacO$ and $lacP$ were present, it would have no effect on $lacZ$ expression in yeast; think about that!). But if the transposon inserts downstream of a yeast gene promoter (somewhat rare) and in the correct orientation (1 in 2 chance), and in the correct triplet codon reading frame (1 in 3 chance), the $lacZ$ gene would then come under the control of that promoter. When transcription is activated from that promoter, a LacZ fusion protein is expressed, and most LacZ fusion proteins have robust β-galactosidase activity.
  
  
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   - Any transposon that integrated into a gene will essentially disrupt that gene and is likely to generate a null mutation (complete loss of function). Null mutants are very useful!   - Any transposon that integrated into a gene will essentially disrupt that gene and is likely to generate a null mutation (complete loss of function). Null mutants are very useful!
   - For transposons that integrate such that the $lacZ$ gene is in frame with the coding region of the yeast gene, the level of β-galactosidase (LacZ) activity in these cells therefore becomes an indicator) for the level of transcription of that gene. We call $lacZ$ in this context a reporter gene (sometimes we abbreviate that to just "reporter").    - For transposons that integrate such that the $lacZ$ gene is in frame with the coding region of the yeast gene, the level of β-galactosidase (LacZ) activity in these cells therefore becomes an indicator) for the level of transcription of that gene. We call $lacZ$ in this context a reporter gene (sometimes we abbreviate that to just "reporter"). 
-  - This kind of insertion library approach allows you to use tricks like reverse PCR (see Fig. {{ref>Fig8}}) to help make cloning your gene easier (this method is much easier than cloning by complementation). +  - This kind of insertion library approach allows you to use tricks like inverse PCR (see Fig. {{ref>Fig8}}) to help make cloning your gene easier (this method is much easier than cloning by complementation). 
  
 Here are two examples of how such a library can be used:  Here are two examples of how such a library can be used: 
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   - to identify genes whose transcription is upregulated in response to being exposed to this tobacco smoke chemical.    - to identify genes whose transcription is upregulated in response to being exposed to this tobacco smoke chemical. 
  
-The chemical we will use as an example is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Yeast cells with random mini-Tn7 insertions are first plated out on Petri dishes at a low density so that individual cells each give rise to single colonies on the agar surface. Each colony represents a clone that contains a unique insert. To screen the library for genes that protect against NNK-induced cell killing, the colonies are replica-plated onto agar medium that either does or does not contain a high dose of NNK. Replica plating is a technique where colonies on one Petri dish are copied to a different Petri dish while maintaining their positions in the dish, so that we can the observe the same clone under different growth conditions. To screen the library for genes that are transcriptionally regulated in the presence of NNK, the colonies are replica-plated onto agar medium containing either X-gal alone or X-gal plus a low dose of NNK. +The chemical we will use as an example is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). Yeast cells with random mini-Tn7 insertions are first plated out on Petri dishes at a low density so that individual cells each give rise to single colonies on the agar surface. Each colony represents a clone that contains a unique insert. To screen the library for genes that protect against NNK-induced cell killing, the colonies are replica plated onto agar medium that either does or does not contain a high dose of NNK. Replica plating is a technique where colonies on one Petri dish are copied to a different Petri dish while maintaining their positions in the dish, so that we can the observe the same clone under different growth conditions. To screen the library for genes that are transcriptionally regulated in the presence of NNK, the colonies are replica plated onto agar medium containing either X-gal alone or X-gal plus a low dose of NNK. 
  
 <figure Fig7> <figure Fig7>
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 <figure Fig9> <figure Fig9>
 {{ :inverse_pcr.jpg?400 |}} {{ :inverse_pcr.jpg?400 |}}
-Figure 12.8. Inverse PCR. Obviously not drawn to scale! See text for details. +<caption> 
 +Inverse PCR. Obviously not drawn to scale! See text for details.  
 +</caption> 
 +</figure>
  
-Once we have identified a gene that is transcriptionally upregulated in response to an environmental change (such as the presence of NNK), how can we use genetics to figure out how regulation is achieved? This is the topic of the next chapter, although we will study galactose induction of gene expression instead of NKK. +Once we have identified a gene that is transcriptionally upregulated in response to an environmental change (such as the presence of NNK), how can we use genetics to figure out how regulation is achieved? This is the topic of the next chapter, although we will study galactose induction of gene expression instead of NNK. 
  
 ===== Questions and exercises ===== ===== Questions and exercises =====
  
-Discussion Box: There are also yeast transposons that we could have used for this experiment instead. Why go through the trouble of using a bacterial transposon? It may be a little easier to answer this question after you have read the entire chapter to get an idea of where we are going with this experiment. +Conceptual question: should the $URA3$ DNA fragment within the mini-Tn7 construct contain its own promoter? How about the $tet^R$ DNA fragment? If they did contain their own promoters, would they be yeast or //E. coli// promoters? 
 + 
 +Conceptual question: why is ligation into circles necessary for reverse PCR?
  
-Discussion Box: should the URA3 DNA fragment within the mini-Tn7 construct contain its own promoter? How about the tetR DNA fragment? If they did contain their own promoters, would they be yeast or E. coli promoters?+Exercise 1: Yeast cells are killed by high concentrations of NNK. Design an experiment to identify and clone yeast mutants that are resistant to high concentrations of NNK. 
chapter_12.1724270402.txt.gz · Last modified: 2024/08/21 13:00 by mike