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--- chapter_13 [2024/08/21 17:15] – [Genetic analysis of Gal mutants] mike
+++ chapter_13 [2025/04/29 11:54] (current) – [Galactose metabolism in yeast] mike
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-<typo fs:x-large>Chapter 13. Gene regulation in eukaryotes</typo>
+<-chapter_12|Chapter 12^table_of_contents|Table of Contents^chapter_14|Chapter 14->
+<typo fs:x-large>Chapter 13. %%Gene regulation in eukaryotes%%</typo>
 ===== Introduction =====
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-Once a gene (such as $gal1$) has been identified as being inducible under certain conditions (in this case by the addition of galactose), we can begin to dissect its regulatory mechanism by isolating mutants that defective in the regulatory process, i.e., mutants that constitutively express the $GAL$ genes even in the absence of galactose, and mutants that have lost the ability to induce the $GAL$ genes in the presence of galactose. If we were studying galactose regulation today, we would probably use a $lacZ$ reporter system similar to what we discussed in [[chapter_12|Chap. 12]].
+Once a gene (such as $gal1$) has been identified as being inducible under certain conditions (in this case by the addition of galactose), we can begin to dissect its regulatory mechanism by isolating mutants that are defective in the regulatory process, i.e., mutants that constitutively express the $GAL$ genes even in the absence of galactose, and mutants that have lost the ability to induce the $GAL$ genes in the presence of galactose. If we were studying galactose regulation today, we would probably use a $lacZ$ reporter system similar to what we discussed in [[chapter_12|Chap. 12]].
 <figure Fig2>
 {{ :gal_induction_mutants.png?400 |}}
 <caption>
-Using the mini-Tn7 strategy to find regulatory Gal mutants.
+Using the mini-Tn7 strategy to find regulatory Gal mutants. Glycerol is a carbon source for yeast that does not induce Gal genes.
 </caption>
 </figure>
@@ Line 74: / Line 76: @@
 </figure>
-$gal80$ mutant: The next useful regulatory mutant isolated was $gal80$, in which the $GAL1$-encoded galactokinase is expressed even in the absence of galactose and is not further induced in its presence. In other words, $gal80$ mutants are constitutive. Again, heterozygous diploids ($gal80$/$GAL80$) showed that $gal80$ is recessive, and mapping by tetrad analysis showed that $gal80$ is not linked to $gal1$, $gal4$ or any other $gal$ genes. If a mutant $gal80$ results in constitutive Gal1p expression, the simplest model to explain the data is that $GAL80$ negatively regulates Gal1p expression. Since $GAL4$ positively regulates and $GAL80$ negatively regulates Gal1p expression, we have to figure out how these two gene products work together to achieve such regulation. Assuming that $GAL4$ and $GAL80$ act in series (that is, in a linear genetic pathway), there are two formal possibilities:
+$gal80$ mutant: The next useful regulatory mutant isolated was $gal80$, in which the $GAL1$-encoded galactokinase is expressed even in the absence of galactose and is not further induced in its presence. In other words, $gal80$ mutants are constitutive. Again, heterozygous diploids ($\frac{gal80}{GAL80}$) showed that $gal80$ is recessive, and mapping by tetrad analysis showed that $gal80$ is not linked to $gal1$, $gal4$ or any other $gal$ genes. If a mutant $gal80$ results in constitutive Gal1p expression, the simplest model to explain the data is that $GAL80$ negatively regulates Gal1p expression. Since $GAL4$ positively regulates and $GAL80$ negatively regulates Gal1p expression, we have to figure out how these two gene products work together to achieve such regulation. Assuming that $GAL4$ and $GAL80$ act in series (that is, in a linear genetic pathway), there are two formal possibilities:
 <figure Fig4>
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   * On the other hand, assume that Model 2 is correct; in this case, if there is loss of function in both $gal4$ and $gal80$ then Gal80p can never be inhibited by Gal4p, but this is irrelevant because there won't be any functional Gal80p to begin with. Therefore, Gal1p will constitutively be expressed.
-We could make the $gal4$; $gal80$ double mutant strain using molecular engineering approaches (such as by first cloning and then knocking out $gal4$ and $gal80$; see below), but an easier way is to let yeast meiosis do the job for you (cloning and knocking out genes is not super hard but tetrad analysis is much easier). If we mate the $gal4$; $GAL80$ haploid strain with the $GAL4$; $gal80$ haploid strain we should obtain double mutants among the tetratype and non-parental ditype tetrads that result from this cross (remember that this chapter assumes a basic familiarity with tetrad analysis; see [[Appendix_A|Appendix A]] for details).
+We could make the $gal4$; $gal80$ double mutant strain using molecular engineering approaches (such as by first cloning and then knocking out $gal4$ and $gal80$; [[chapter_14#Making_targeted_gene_knockouts_in_yeast|see Chapter 14]]), but an easier way is to let yeast meiosis do the job for you (cloning and knocking out genes in yeast is not super hard but tetrad analysis is much easier). If we mate the $gal4$; $GAL80$ haploid strain with the $GAL4$; $gal80$ haploid strain we should obtain double mutants among the tetratype and non-parental ditype tetrads that result from this cross (remember that this chapter assumes a basic familiarity with tetrad analysis; see [[Appendix_A|Appendix A]] for details).
 <table Tab2>
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-Now let's consider a new class of mutant that turned out to be quite informative. gal81 mutants, like gal80 mutants, are constitutive for Gal1 expression. But unlike gal80, gal81 is dominant. That is, gal81/GAL81 heterozygotes are constitutive. An obvious question is whether gal81 mutants are still constitutive in a gal4 background, as it was already established that Gal4 positively regulates Gal1 (and the other Gal genes). To test this, we can attempt epistasis analysis by trying to make a gal4, gal81 double mutant by crossing the single mutants:
+Now let's consider a new class of mutant that turned out to be quite informative. $gal81$ mutants, like $gal80$ mutants, are constitutive for Gal1 expression. But unlike $gal80$, $gal81$ is dominant. That is, $\frac{gal81}{GAL81}$ heterozygotes are constitutive. An obvious question to ask is whether $gal81$ mutants are still constitutive in a $gal4$ background, as it was already established that Gal4p positively regulates Gal1p (and the other Gal genes). In other words, what is the epistatic relationship between $gal81$ and $gal4$? To test this, we can attempt to make a $gal4$·$gal81$ double mutant by crossing the single mutants:
-MATa; gal81, GAL4 x MATα; gal4, GAL81
+$$ gal81 \cdot GAL4 \times gal4 \cdot GAL81 $$
-A surprising finding from this cross was that all the tetrads were of the parental ditype; in other words, there were no tetratypes or nonparental ditypes, indicating that gal81 and gal4 are very tightly linked (another reminder to study tetrad analysis). Indeed, it turns out that gal81 maps to the coding region of the gal4 gene. In other words, gal81 is an allele of gal4; we say that "gal81 is allelic to gal4". The gal81 mutation was therefore renamed as gal481. Essentially gal481 behaves as a super-activator that is impervious to the negative effects of gal80. gal481 thus activates independently of galactose and Gal80. After cloning and sequencing gal481 and lots of other biochemical experiments, scientists learned that the gal481 mutation is a missense mutation, which alters the amino acid sequence of the region of the Gal4 protein that normally interacts with Gal80. This mutation prevents Gal80 from interacting with Gal4.
+A surprising finding from this cross was that all the tetrads were of the parental ditype (PD); there were no tetratypes (TT) or nonparental ditypes (PD), indicating that $gal81$ and $gal4$ are very tightly linked((Another reminder to study tetrad analysis in [[Appendix_A|Appendix A]]!)). Indeed, it turns out that $gal81$ maps to the coding region of the $gal4$ gene. In other words, $gal81$ is an allele of $gal4$; we say that "$gal81$ is allelic to $gal4$". The $gal81$ mutation was therefore renamed as $gal4^{81}$. Essentially $gal4^{81}$ behaves as a super-activator that is impervious to the negative effects of $gal80$. $gal4^{81}$ thus activates Gal1p expression independently of galactose and Gal80p.
-Discussion Box: In the above paragraph, we discuss that gal81 was shown to be allelic to gal4 by linkage, followed by cloning and sequencing. Could scientists have established that gal81 is allelic to gal4 using the complementation test? Why or why not?
+After cloning and sequencing $gal4^{81}$ and lots of other biochemical experiments, scientists learned that the $gal4^{81}$ mutation is a missense mutation that alters the amino acid sequence of the region of the Gal4p protein that normally interacts with Gal80p. This mutation prevents Gal80p from interacting with Gal4p. This was a very satisfying result, as genetics (a discipline concerned with abstract ways of thinking about biology) was used to learn  biochemical knowledge about how two gene products (proteins) physically interact with each other to regulate gene expression.
-A general model for regulation of eukaryotic gene expression
-These and many other genetic, molecular, and biochemical experiments led to the following model to explain Gal gene regulation. Upstream of the GAL1 gene (and other Gal genes), two cis-acting elements are needed for transcriptional activation. First, the TATA-binding protein (TBP) binds to the TATA consensus site  in the GAL1 promoter and provides a scaffold for a very large RNA polymerase complex (RNAP). The area of DNA immediately upstream of the transcription start site of GAL1 that contains the TATA consensus site is also called the promoter; the promoter is usually around 40-50 bp of DNA in size. Note that the same word is used to describe cis-regulatory sequences in E. coli (Chap. 10); however, prokaryotic promoters and eukaryotic promoters are different from each other.
+===== A general model for regulation of eukaryotic gene expression =====
-RNAP binding to TBP alone does not enable transcription; the complex must be activated by a transcriptional activator (transactivator), which in this case is the Gal4 protein. The Gal4 protein binds to another cis-acting element in the Gal1 promoter region called the upstream activator sequence (UAS) that physically binds the Gal4 protein. The region of DNA that contains UAS is usually several hundred bp upstream from the promoter (see Chap. 14) and is commonly called an enhancer. In the absence of galactose, Gal80 physically prevents Gal4 from recruiting and activating RNAP. In the presence of galactose, the Gal80 protein changes conformation and binds to a different region of Gal4, unveiling the ability of Gal4 to recruit and activate RNAP. The mutation in the gal481 allele interferes with Gal80 binding, thereby allowing mutant Gal481 protein to recruit and activate RNAP all the time, even in the absence of galactose.
-Discussion Box: How does regulation of Gal genes in yeast compare to regulation of Lac operon genes in E. coli (seen in Chap. 10)? This is an important discussion to have!
+These and many other genetic, molecular, and biochemical experiments led to the following model (Figure {{ref>Fig5}}) to explain Gal gene regulation. Upstream of the $GAL1$ gene (and other Gal genes), two cis-acting DNA elements are needed for transcriptional activation: the promoter, and upstream activator sequences (UASs).
+First, TATA binding protein (TBP) binds to a DNA sequence called the TATA consensus site (also called a TATA box), which is located just in front (upstream((In the context of gene expression, we use the terms upstream and downstream to describe directions on a gene. Relative to the transcription start site, any DNA positioned against the direction of transcription is described as upstream, and any DNA positioned with the direction of transcription is described as downstream.))) of the $GAL1$ transcription start site. TBP bound to the TATA box forms a scaffold for a very large RNA polymerase complex. The area of DNA immediately upstream of the transcription start site of GAL1 that contains the TATA box is also called the promoter; the promoter is usually around 40-50 bp of DNA in size. Note that the word "promoter" is also used to describe cis-regulatory sequences in //E. coli// ([[chapter_10|Chap. 10]]); however, prokaryotic promoters and eukaryotic promoters do not work in the same way.
+RNA polymerase binding to TBP alone does not enable transcription; the complex must be activated by a kind of protein called a transcriptional activator (or transactivator), which in this case is Gal4p. Gal4p binds to another DNA sequence further upstream of the $GAL1$ transcription start site called the upstream activator sequence (UAS). The region of DNA that contains UAS is usually several hundred bp upstream from the promoter (see [[chapter_14|Chap. 14]]) and is more generally called an enhancer. In the absence of galactose, Gal80p physically prevents Gal4p from activating RNAP. In the presence of galactose, the Gal80 protein changes conformation and binds to a different region of Gal4p, unveiling the ability of Gal4p to activate RNAP. The mutation in the $gal4^{81}$ allele interferes with Gal80p binding, thereby allowing mutant Gal4<sup>81</sup>p protein to recruit and activate RNAP all the time even in the absence of galactose.
+<figure Fig5>
+{{ :transcription_general_model.png?400 |}}
+<caption>
+Model of $GAL1$ regulation, based on genetic and biochemical analysis of Gal mutants. See text for details.
+</caption>
+</figure>
-Figure 13.5. Model of Gal1 regulation, based on genetic and biochemical analysis of Gal mutants.
+It is important to know that, while minor details will be different in different genes and organisms, the model shown in Fig. {{ref>Fig5}} generally holds true for all eukaryotic genes. This includes mammalian and human genes that may have biomedical or economic relevance.
-It is important to know that, while minor details will be different in different genes and organisms, the model shown in Fig. 13.5 generally holds true for all eukaryotic genes. This includes mammalian and human genes that may have biomedical or economic relevance.
-A final comment about the model for induction of the Gal genes by galactose: For many years it was assumed that galactose binds directly to the Gal80 protein, thus preventing it from inhibiting the Gal4 protein from activating Gal1 transcription. However, it now seems that one extra protein is involved in this chain of events. The Gal3 protein turns out to directly bind galactose. This allows Gal3 to move from the cytoplasm into the nucleus, upon which the galactose/Gal3 moiety binds to Gal80 to facilitate moving Gal80 to a different site on the Gal4 protein, thus allowing Gal4 to activate transcription. While the model as written in Fig. 13.5 does not include Gal3, the models are still formally correct.
+===== Closing thoughts =====
-In the next chapter we will be looking at structures of proteins and cis-regulatory elements, and how to genetically analyze them in more detail.
+A final comment about the model for induction of the Gal genes by galactose: For many years it was assumed that galactose binds directly to Gal80p, thus preventing it from inhibiting Gal4p from activating $GAL1$ transcription. However, one extra protein is involved in this chain of events. The Gal3 protein turns out to directly bind galactose. This allows Gal3p to move from the cytoplasm into the nucleus, upon which the galactose/Gal3p moiety binds to Gal80p to facilitate moving Gal80p to a different site on Gal4p, thus allowing Gal4p to activate transcription. While the model as written in Fig. {{ref>Fig5}} does not include Gal3p, the models are still formally correct.
+In the next chapter we will be looking at structures of some of these regulatory proteins and cis-regulatory elements, and how to genetically analyze them in more detail.
 ===== Questions and exercises =====
+Conceptual question: If you were one of the original scientists studying Gal gene regulation and you had a $gal4$ mutant, how could you clone $gal4$ by complementation? Your only tool you have for measuring phenotypes is measuring galactokinase activity (see footnote 2). Similarly, how could you clone $gal80$ by complementation?
-Exercise 13.1: If you were one of the original scientists studying Gal gene regulation, and you had a gal4 mutant, how could you clone gal4 by complementation?
-Exercise 13.2: Similarly, how could you clone gal80 by complementation?
+Exercise 1: In the chapter, we discuss that $gal81$ was shown to be allelic to $gal4$ by linkage, followed by cloning and sequencing. Could scientists have established that gal81 is allelic to gal4 using the complementation test? Why or why not?
+Exercise 2: $gal4^{81}$ and $gal80$ are both constitutive mutants (although one is dominant and one is recessive). Devise an experiment to show that $gal4^{81}$ and $gal80$ are not alleles of the same gene.
+Conceptual question: How does regulation of Gal genes in yeast compare to regulation of Lac operon genes in //E. coli// (seen in [[chapter_10|Chap. 10]])? This is an important thing to think about!
-Exercise 13.3: How could you show that gal4 and gal80 are not alleles of the same gene?