TNBGGA: The No Bullshit Guide to Genetic Analysis

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chapter_14 [2024/08/23 18:41] – [Questions and exercises] mikechapter_14 [2025/04/29 11:56] (current) – [Introduction] mike
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-<typo fs:x-large>Chapter 14. Using reverse genetics to study molecules</typo>+<-chapter_13|Chapter 13^table_of_contents|Table of Contents^chapter_15|Chapter 15-> 
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 +<typo fs:x-large>Chapter 14. %%Using reverse genetics to study molecules%%</typo>
  
 ===== Introduction ===== ===== Introduction =====
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 In this chapter we will see how genetics can be used to dissect molecular structure and function. We have seen one example of how genetics can achieve this in [[Chapter_13|Chapter 13]] with the $gal4^{81}$mutant - this mutant taught us something about how Gal80p functions in relation to Gal4p. In this chapter we will look at two further examples of how genetics can teach us about molecular function. In our first example, we will look at a genetic approach that can be used to analyze the DNA of upstream regulatory sequences of a gene such as $GAL1$. In our second example, we will look at how we can use genetics to assign biochemical functions to different parts of a protein. We will start to think about a new genetic approach to studying biological function: reverse genetics.  In this chapter we will see how genetics can be used to dissect molecular structure and function. We have seen one example of how genetics can achieve this in [[Chapter_13|Chapter 13]] with the $gal4^{81}$mutant - this mutant taught us something about how Gal80p functions in relation to Gal4p. In this chapter we will look at two further examples of how genetics can teach us about molecular function. In our first example, we will look at a genetic approach that can be used to analyze the DNA of upstream regulatory sequences of a gene such as $GAL1$. In our second example, we will look at how we can use genetics to assign biochemical functions to different parts of a protein. We will start to think about a new genetic approach to studying biological function: reverse genetics. 
  
-Reverse genetics is the opposite of what we have been studying so far. In Chapters 1-13, we discussed mutants with interesting phenotypes, and used mapping and cloning by complementation to try to identify the protein coding sequence of the gene that is mutated - this approach starts with function (as defined by mutants) and ends with identifying a gene, and is is called forward genetics. In reverse genetics, we already have a gene that has been cloned. We might know of this gene from a whole genome sequencing project; or we might have found a mouse gene based on sequence similarity to a Drosophila gene that was found by forward genetics. We are interested in studying the function of this newly discovered gene. In some cases, the function of the gene is completely unknown. In other cases, we may already know the normal function of a gene, but we want to modify it in some way to further understand details of how the gene product works. In essence, reverse genetics starts with a gene and ends with identifying a function. +Reverse genetics is the opposite of what we have been studying so far. In Chapters 1-13, we discussed mutants with interesting phenotypes, and used mapping and cloning by complementation to try to identify the protein coding sequence of the gene that is mutated - this approach starts with function (as defined by mutants) and ends with identifying a gene, and is called forward genetics. In reverse genetics, we already have a gene that has been cloned. We might know of this gene from a whole genome sequencing project; or we might have found a mouse gene based on sequence similarity to a Drosophila gene that was found by forward genetics. We are interested in studying the function of this newly discovered gene. In some cases, the function of the gene is completely unknown. In other cases, we may already know the normal function of a gene, but we want to modify it in some way to further understand details of how the gene product works. In essence, reverse genetics starts with a gene and ends with identifying a function. 
  
 In this chapter we will discuss three examples of reverse genetic strategies in yeast to study gene function: (1) using reporter genes to study cis-acting regulatory sequences; (2) using the yeast two hybrid system to study protein-protein interactions; and (3) making targeted gene knockouts.  In this chapter we will discuss three examples of reverse genetic strategies in yeast to study gene function: (1) using reporter genes to study cis-acting regulatory sequences; (2) using the yeast two hybrid system to study protein-protein interactions; and (3) making targeted gene knockouts. 
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 {{ :gal_lacz_reporter.png?400 |}} {{ :gal_lacz_reporter.png?400 |}}
 <caption> <caption>
-Generation of a plasmid containing a reporter gene in yeast. In this case, we have created a plasmid that contains the //E. coli// gene $lacZ$ placed downstream of cis-acting regulatory sequences of GAL1 (labeled PGAL1 in the figure. "P" stands for "promoter" and includes UAS and the TATA box region ((". The term "promoter" here is used loosely. Historically in eukaryotes, DNA sequences upstream of a transcription start site were named promoters to be consistent with similar cis-acting upstream sequences in E. coli such as lacO and lacP. However, the discovery of UAS and other upstream cis-acting sequences quite distant from the TATA box revealed that eukaryotic and prokaryotic gene regulation is quite different. UASs and sequences like them were subsequently renamed enhancers to distinguish them from close-up cis-acting sequences like the TATA box. However, the term "promoter" has somewhat stuck with general usage, and in older papers and figures, the term "promoter" is still used to describe cis-acting elements that are actually enhancers. Going by word usage is here is not always effective, and it's always good to ask yourself what the context is when you see the word "promoter". This is a good example of poor language consistency in biology, which is a general problem in our field.))). +Generation of a plasmid containing a reporter gene in yeast. In this case, we have created a plasmid that contains the //E. coli// gene $lacZ$ placed downstream of cis-acting regulatory sequences of GAL1 (labeled P<sub>GAL1</sub> in the figure. "P" stands for "promoter" and includes UAS and the TATA box region ((". The term "promoter" here is used loosely. Historically in eukaryotes, DNA sequences upstream of a transcription start site were named promoters to be consistent with similar cis-acting upstream sequences in //E. coli// such as $lacO$ and $lacP$. However, the discovery of UAS and other upstream cis-acting sequences quite distant from the TATA box revealed that eukaryotic and prokaryotic gene regulation is quite different. UASs and sequences like them were subsequently renamed enhancers to distinguish them from close-up cis-acting sequences like the TATA box. However, the term "promoter" has somewhat stuck with general usage, and in older papers and figures, the term "promoter" is still used to describe cis-acting elements that are actually enhancers. Going by word usage is here is not always effective, and it's always good to ask yourself what the context is when you see the word "promoter". This is a good example of poor language consistency in biology, which is a general problem in our field.))). 
 </caption> </caption>
 </figure> </figure>
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   * Deletions 7 and 8 do not express the $lacZ$ reporter under any conditions because the deletions have removed some of the TATA box sequence that is required for RNA polymerase binding.   * Deletions 7 and 8 do not express the $lacZ$ reporter under any conditions because the deletions have removed some of the TATA box sequence that is required for RNA polymerase binding.
-  * Deletions 1 and 2 eliminate the ability of galactose to increase expression of the reporter, and since expression is not induced there is nothing for glucose to repress. It turns out that the 75 bp sequence between -310 and -385 is the DNA binding site for Gal4p. This kind of region is generally called a UAS (upstream activation sequence) and in this case it is specifically called UAS<sub>GAL</sub>. This was alluded to in [[chapter_13|Chap. 13]]. We will come back to thinking about Gal4p binding to the UAS recognition sequence later. +  * Deletions 1 and 2 eliminate the ability of galactose to increase expression of the reporter, and since expression is not induced there is nothing for glucose to repress. It turns out that the 75 bp sequence between -310 and -385 is the DNA binding site for Gal4p. This kind of region is generally called a UAS (upstream activator sequence) and in this case it is specifically called UAS<sub>GAL</sub>. This was alluded to in [[chapter_13|Chap. 13]]. We will come back to thinking about Gal4p binding to the UAS recognition sequence later. 
-  * Deletions 3, 5, and 6 have no effect on the ability of galactose to induce expression because the UAS remains intact. Note that shortening the distance between the UAS and the TATA box region is not detrimental to induction. Indeed, other experiments not shown here demonstrated that increasing the distance by inserting extra DNA between the UAS and the TATA box also has little effect on inducibility. This has led to the idea that UAS sequences can work at long distances (1,000 – 10,000 bp) away from the TATA box and the transcription start site. This winds up being generally true for mammalian enhancers. +  * Deletions 3, 5, and 6 have no effect on the ability of galactose to induce expression because the UAS remains intact. Note that shortening the distance between the UAS and the TATA box region is not detrimental to induction. Indeed, other experiments not shown here demonstrated that increasing the distance by inserting extra DNA between the UAS and the TATA box also has little effect on inducibility. This has led to the idea that UAS sequences can work at long distances (1,000 – 10,000 bp) away from the TATA box and the transcription start site. This winds up being generally true for upstream regulatory elements of eukaryotic genes. 
   * Deletion 4 reveals important information about glucose repression. While galactose induces reporter expression in this construct, glucose is unable to repress that expression. The deleted region therefore defines the position of a DNA element that is required for glucose repression. A DNA element that behaves this way  is generally called a URS (upstream repressor sequence), and in this case it is specifically called URS<sub>GAL</sub>. URS<sub>GAL</sub> is found in the upstream regulatory regions of many genes besides Gal genes.   * Deletion 4 reveals important information about glucose repression. While galactose induces reporter expression in this construct, glucose is unable to repress that expression. The deleted region therefore defines the position of a DNA element that is required for glucose repression. A DNA element that behaves this way  is generally called a URS (upstream repressor sequence), and in this case it is specifically called URS<sub>GAL</sub>. URS<sub>GAL</sub> is found in the upstream regulatory regions of many genes besides Gal genes.
  
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 It turns out that all eukaryotes, including yeast, regulate gene expression in the same basic way (although the details are different). Let's return to our discussion of Gal gene regulation in yeast. Using similar experiments as Jacob and Monod, yeast geneticists would go on to show that mutations in UAS act in cis (Table {{ref>Tab1}}). Along the same lines, dominant mutations such as $gal4^{81}$ act in trans. Using the same genetic logic as Jacob and Monod, we can interpret these results to say that UAS is a DNA sequence that does not code for protein itself but instead regulates the expression of nearby protein-coding target genes; we can also say that GAL4 likely codes for a protein that regulates the expression of target genes. It turns out that all eukaryotes, including yeast, regulate gene expression in the same basic way (although the details are different). Let's return to our discussion of Gal gene regulation in yeast. Using similar experiments as Jacob and Monod, yeast geneticists would go on to show that mutations in UAS act in cis (Table {{ref>Tab1}}). Along the same lines, dominant mutations such as $gal4^{81}$ act in trans. Using the same genetic logic as Jacob and Monod, we can interpret these results to say that UAS is a DNA sequence that does not code for protein itself but instead regulates the expression of nearby protein-coding target genes; we can also say that GAL4 likely codes for a protein that regulates the expression of target genes.
  
-A more modern take on the naming of these elements is that we call cis-acting sequences such as UAS and URS cis regulatory elements; cis regulatory elements that activate gene expression are called enhancers, and those that repress gene expression are called silencers. Trans-acting factors are usually just generically called transcription factors; those that active gene expression area called transactivators, and those that repress gene expression are called repressors.  +A more modern take on the naming of these elements is that we call cis-acting sequences such as UAS and URS cis regulatory elements; cis regulatory elements that activate gene expression are called enhancers, and those that repress gene expression are called silencers. Trans-acting factors are usually just generically called transcription factors; those that activate gene expression are called transactivators, and those that repress gene expression are called repressors.  
    
  
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-The Gal4p transactivator is one of the most well-studied proteins that carries out transcriptional activation. To study Gal4p, a $lacZ$ reporter was used in a creative way to establish that the Gal4p protein has two functional domains that are separated by a flexible region in the protein. This time, we generate a yeast plasmid where the $GAL1$ upstream regulatory region (including the TATA box and the UAS) remains intact upstream of the $lacZ$ reporter, but deletions are made across the protein coding region of the $GAL4$ gene (Fig. 14.6). This is the inverse of keeping $GAL4$ intact and making deletions along the $GAL1$ promoter as described above.+The Gal4p transactivator is one of the most well-studied proteins that carries out transcriptional activation. To study Gal4p, a $lacZ$ reporter was used in a creative way to establish that the Gal4p protein has two functional domains that are separated by a flexible region in the protein. This time, we generate a yeast plasmid where the $GAL1$ upstream regulatory region (including the TATA box and the UAS) remains intact upstream of the $lacZ$ reporter, but deletions are made across the protein coding region of the $GAL4$ gene (Fig. {{ref>Fig6}}). This is the inverse of keeping $GAL4$ intact and making deletions along the $GAL1$ promoter as described above.
  
 Again, the precise technical details of how this is accomplished are not important here - what we care about is the idea behind the experiment and what it can teach us. We can describe the method in brief, however: Assuming you have cloned the $GAL4$ gene (see [[chapter_14#Questions_and_exercises|Questions and exercises]] below), you can insert it into another plasmid and transform it into $gal4$ mutant yeast cells. This should complement the $gal4$ mutation; in other words, the plasmid is providing the only functional Gal4p in the cell. You then separately make in-frame deletions of the $GAL4$ clone at different parts of the protein coding region, transform those constructs into $gal4$ mutants, and assess the function of those deletion constructs. Again, the precise technical details of how this is accomplished are not important here - what we care about is the idea behind the experiment and what it can teach us. We can describe the method in brief, however: Assuming you have cloned the $GAL4$ gene (see [[chapter_14#Questions_and_exercises|Questions and exercises]] below), you can insert it into another plasmid and transform it into $gal4$ mutant yeast cells. This should complement the $gal4$ mutation; in other words, the plasmid is providing the only functional Gal4p in the cell. You then separately make in-frame deletions of the $GAL4$ clone at different parts of the protein coding region, transform those constructs into $gal4$ mutants, and assess the function of those deletion constructs.
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 To determine whether protein X interacts with either protein Y or protein Z one can do the following: fuse protein X to the Gal4 DB; this chimeric protein is known as the bait, and it will attach to the UASGAL that lies upstream of a reporter gene, usually a selectable marker or LacZ, or both. This bait lies in wait for an interaction with another protein. The GAL4 AD is fused to either protein Y or protein Z. Should either one of these proteins be able to interact with protein X then the Gal4 AD region will become tethered to the UASGAL region and will recruit and activate RNA polymerase. To determine whether protein X interacts with either protein Y or protein Z one can do the following: fuse protein X to the Gal4 DB; this chimeric protein is known as the bait, and it will attach to the UASGAL that lies upstream of a reporter gene, usually a selectable marker or LacZ, or both. This bait lies in wait for an interaction with another protein. The GAL4 AD is fused to either protein Y or protein Z. Should either one of these proteins be able to interact with protein X then the Gal4 AD region will become tethered to the UASGAL region and will recruit and activate RNA polymerase.
  
-<figure>+<figure Fig9>
 {{ :yeast_two_hybrid.png?400 |}} {{ :yeast_two_hybrid.png?400 |}}
 <caption> The yeast two hybrid assay. <caption> The yeast two hybrid assay.
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 </figure> </figure>
  
-Note that the protein X, Y and Z do not have to be yeast proteins; the only requirement is that the gene coding for the protein has been cloned (or can be easily generated via PCR); these genes are then modified such that they produce the appropriate Gal4p chimeric proteins. Many yeast two hybrid experiments use libraries instead of a specific "protein Y" or "protein Z" so that one can screen for previously unknown proteins that interact with your bait "protein X". Systematic yeast two hybrid assays have been performed for all possible protein pairs for various organisms, including //E. coli//, yeast, Drosophila, and mouse, among others. This "map" of protein-protein interactions is often referred to as the interactome. (Other experimental techniques are also used to generate interactome maps in addition to two-hybrid assays.)+Note that the protein X, Y and Z do not have to be yeast proteins; the only requirement is that the gene coding for the protein has been cloned (or can be easily generated via PCR); these genes are then modified such that they produce the appropriate Gal4p chimeric proteins. Many yeast two hybrid experiments use libraries instead of a specific "protein Y" or "protein Z" so that one can screen for previously unknown proteins that interact with your bait "protein X". Systematic yeast two hybrid assays have been performed for all possible protein pairs for various organisms, including //E. coli//, yeast, Drosophila, and mouse, among others. This "map" of protein-protein interactions is often referred to as the interactome. (Other experimental techniques are also used to generate interactome maps in addition to two hybrid assays.)
  
  
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-One of the most important tricks in the toolbox of a yeast geneticist is the ability to create targeted gene knockouts for almost any gene in the genome. Gene knockouts in multicellular organisms (mice) are discussed in Chapter 16. The general principles are similar, but it is much easier to make gene knockouts in yeast for two reasons: (1) yeast are unicellular microbes, which makes growing large numbers of yeast cells and screening or selecting for rare events very easy to do; and (2) DNA recombination rates are very high in yeast.+One of the most important tricks in the toolbox of a yeast geneticist is the ability to create targeted gene knockouts for almost any gene in the genome. Gene knockouts in multicellular organisms (mice) are discussed in [[chapter_16|Chapter 16]]. The general principles are similar, but it is much easier to make gene knockouts in yeast for two reasons: (1) yeast are unicellular microbes, which makes growing large numbers of yeast cells and screening or selecting for rare events very easy to do; and (2) DNA recombination rates are very high in yeast.
  
-Let's say that you are interested in how Gal80p functions. Using the yeast two hybrid assay, you discover that Gal80p binds to Gal4p (see Chapter 13). This is neither surprising nor unexpected, given what we learned about $gal4$ and $gal80$ using forward genetics. However, you also discover that Gal80p binds to a new protein you have not studied closely yet. You temporarily name this protein Gal3p, and you identify and clone the physical gene $gal3$ ($gal3$ is briefly mentioned in Chapter 13). Although your two hybrid experiments indicate that Gal3p binds to Gal80p, you don't have a mutant in the $gal3$ gene, and that prevents you from doing further genetic analysis. How can I make a mutation in the $gal3$ gene if all I know is the DNA sequence of $gal3$?+Let's say that you are interested in how Gal80p functions. Using the yeast two hybrid assay, you discover that Gal80p binds to Gal4p (see [[chapter_13|Chapter 13]]). This is neither surprising nor unexpected, given what we learned about $gal4$ and $gal80$ using forward genetics. However, you also discover that Gal80p binds to a new protein you have not studied closely yet. You temporarily name this protein Gal3p, and you identify and clone the physical gene $gal3$ ($gal3$ is briefly mentioned in Chapter 13). Although your two hybrid experiments indicate that Gal3p binds to Gal80p, you don't have a mutant in the $gal3$ gene, and that prevents you from doing further genetic analysis. How can I make a mutation in the $gal3$ gene if all I know is the DNA sequence of $gal3$?
  
 We first build a targeting construct made from dsDNA in vitro (Fig. {{ref>Fig10}}). Importantly, the targeting construct will not be part of a plasmid; therefore, it cannot replicate or segregate in yeast cells going through mitosis. This targeting construct will consist of the cloned $gal3$ gene, but with only the start and the end of the gene present (usually around 30 bp on either end is sufficient for yeast); the middle of the gene will have been replaced with a selectable marker. There are many different kinds of selectable markers one can choose; we will use the yeast $URA3$ gene in our example. $URA3$ is an auxotrophic marker; $URA3$ is required to synthesize uracil, which is an essential metabolite for yeast. $ura3$ mutants cannot grow on minimal media unless it is supplemented with uracil. We will transform $ura3$ mutant yeast with our targeting construct and grow the cells from the transformation experiment on media that lacks uracil.  We first build a targeting construct made from dsDNA in vitro (Fig. {{ref>Fig10}}). Importantly, the targeting construct will not be part of a plasmid; therefore, it cannot replicate or segregate in yeast cells going through mitosis. This targeting construct will consist of the cloned $gal3$ gene, but with only the start and the end of the gene present (usually around 30 bp on either end is sufficient for yeast); the middle of the gene will have been replaced with a selectable marker. There are many different kinds of selectable markers one can choose; we will use the yeast $URA3$ gene in our example. $URA3$ is an auxotrophic marker; $URA3$ is required to synthesize uracil, which is an essential metabolite for yeast. $ura3$ mutants cannot grow on minimal media unless it is supplemented with uracil. We will transform $ura3$ mutant yeast with our targeting construct and grow the cells from the transformation experiment on media that lacks uracil. 
chapter_14.1724463709.txt.gz · Last modified: 2024/08/23 18:41 by mike