TNBGGA: The No Bullshit Guide to Genetic Analysis

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chapter_02 [2024/08/26 20:53] – [The yeast lifecycle] mikechapter_02 [2026/10/25 00:54] (current) – [Growing yeast in the lab] mike
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-<typo fs:x-large>**Chapter 02: Defining genes by function**</typo>+<- chapter_01|Chapter 01^table_of_contents|Table of Contents^chapter_03|Chapter 03 -> 
 +<typo fs:x-large>**Chapter 02: Defining %%genes%% by function**</typo>
  
 ===== What is a gene? Why do we care? ===== ===== What is a gene? Why do we care? =====
  
-Generally speaking, the answer to "what is a gene" has already largely been answered by scientists. Starting from [[wp>Gregor_Mendel|Gregor Mendel's]] famous pea plant experiments in the 1860s to define the patterns of how observable traits (i.e., phenotypes) are inherited from parents to offspring, and culminating in the discovery of the structure of DNA by [[wp>James_Watson|James Watson]], [[wp>Francis_Crick|Francis Crick]], [[wp>Rosalind_Franklin|Rosalind Franklin]], and [[wp>Maurice_Wilkins|Maurice Wilkins]] in the 1950s, there was a scientific golden age in the second half of the 20th century that not only led to a clear understanding of the physical nature of a gene, it further allowed scientists to fully catalogue all the genes (i.e., the genome) of many organisms, including humans. While 20th century geneticists typically studied only a few genes at a time, new 21st century technologies allow scientists to study thousands of genes simultaneously in a single experiment - sometimes from a single cell! Genes are talked about even in casual conversation among non-scientists. As a student of biology, you probably already have at least a general idea of what a gene is - it's instructive for students to think about your prior knowledge on genes before reading further.+Generally speaking, the question "What is a gene?" has already largely been answered by scientists. Starting from [[wp>Gregor_Mendel|Gregor Mendel's]] famous pea plant experiments in the 1860s to define the patterns of how observable traits (i.e., phenotypes) are inherited from parents to offspring, and culminating in the discovery of the structure of DNA by [[wp>James_Watson|James Watson]], [[wp>Francis_Crick|Francis Crick]], [[wp>Rosalind_Franklin|Rosalind Franklin]], and [[wp>Maurice_Wilkins|Maurice Wilkins]] in the 1950s, there was a scientific golden age in the second half of the 20th century that not only led to a clear understanding of the physical nature of a gene, it further allowed scientists to fully catalogue all the genes (i.e., the genome) of many organisms, including humans. While 20th century geneticists typically studied only a few genes at a time, new 21st century technologies allow scientists to study thousands of genes simultaneously in a single experiment - sometimes from a single cell! Genes are talked about even in casual conversation among non-scientists. As a student of biology, you probably already have at least a general idea of what a gene is - it's instructive for students to think about your prior knowledge on genes before reading further.
  
 <WRAP center round tip 60%> <WRAP center round tip 60%>
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-The fact that scientists already know the answer to "what is a gene" begs the next question: "why do we care?" The "we" in this question is referring to you, the reader, who presumably is a student of biology. From that perspective, there are several answers to this question. First, it is useful for students of biology not just to know what something is; it is much more important to know how we know. In some fields of biology, you may learn about experimental techniques, such as how to use a microscope or how to dissect a specimen. In comparison, genetics involves thought experiments that give a great deal of insight into how biologists (especially geneticists) conceptually think about problems. Second, genetics can be used as a tool for studying biology. Even though the laboratory technologies have changed, the ideas that classical geneticists of the 20th century used to study and define the gene can still be applied to answering new questions about how things work in biology. Many modern advances in medicine are built upon knowledge gained from studying the genetics of diseases.+After asking "What is a gene?" the next obvious question is: "Why do we care?" The "we" in this question is referring to you, the reader, who presumably is a student of biology. From that perspective, there are several answers to this question. First, it is useful for students of biology not just to know what something is; it is much more important to know how we know. In some fields of biology, you may learn about experimental techniques, such as how to use a microscope or how to dissect a specimen. In comparison, genetics involves thought experiments that give a great deal of insight into how biologists (especially geneticists) conceptually think about problems. Second, genetics can be used as a tool for studying biology. Even though the laboratory technologies have changed, the ideas that classical geneticists of the 20th century used to study and define the gene can still be applied to answering new questions about how things work in biology. Many modern advances in medicine are built upon knowledge gained from studying the genetics of diseases.
  
-The answer to the question "what is a gene?" will take some effort to answer because there are actually several different definitions that are appropriate in different contexts. The definitions we use in this book are first given here:+Explaining "What is a gene?" will take some effort because there are actually several different definitions that are appropriate in different contexts. The definitions we use in this book are first given here:
  
   - Genes are sources of information that provide some observable biological function.   - Genes are sources of information that provide some observable biological function.
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   - Genes are defined sequences of DNA that code for RNA or protein.   - Genes are defined sequences of DNA that code for RNA or protein.
  
-We first think about genes in the context of their function.+We will first think about genes in the context of their function.
  
 ===== Bakers' yeast as a model genetic organism ===== ===== Bakers' yeast as a model genetic organism =====
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 ==== The yeast lifecycle ==== ==== The yeast lifecycle ====
  
-In the laboratory, we can grow yeast either on a Petri dish (Fig. {{ref>Fig1}}) or in some kind of liquid media in a container such as an Erlenmeyer flask or test tube. Yeast can reproduce sexually, but instead of saying they have two sexes (such as male and female) we say there are two mating types. Yeast cells can exist as haploids of either mating type α ($MATα$) or mating type a ($MATa$). Haploid cells of different mating types when mixed together will fuse to form a diploid cell. Both haploid and diploid cells can undergo mitosis to make more clones of themselves. Diploid cells can also undergo meiosis and form four ascospores that can germinate and become four haploid cells. Two of these cells will be $MATα$ and the other two will be $MATa$ (Fig. {{ref>Fig2}}). +In the laboratory, we can grow yeast either on a Petri dish (Fig. {{ref>Fig1}}) or in some kind of liquid media in a container such as an Erlenmeyer flask or test tube. Yeast can reproduce sexually, but instead of saying they have two sexes (such as male and female) we say there are two mating types. Yeast cells can exist as haploids of either mating type α ($MATα$) or mating type a ($MATa$). Haploid cells of different mating types when mixed together will fuse to form a diploid cell. Both haploid and diploid yeast cells can undergo mitosis to make more clones of themselves. Diploid cells can also undergo meiosis and form four ascospores that can germinate and become four haploid cells. Two of these cells will be $MATα$ and the other two will be $MATa$ (Fig. {{ref>Fig2}}). 
 <figure Fig2> <figure Fig2>
  
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 In yeast, haploids and diploids are isomorphic – meaning that if there is a change in a gene (i.e., a mutation ) that affects some function of the yeast cells, then essentially the same change will be observed in both haploid and diploid cells. This allows us to look at the effect of having two different alleles (i.e., versions) of the same gene in the same diploid cell. For instance, a haploid yeast cell might carry a mutant allele of the $his3$ gene, whereas a diploid might carry two identical $his3$/$his3$ mutant alleles or two different alleles such as $his3$/$HIS3$.   In yeast, haploids and diploids are isomorphic – meaning that if there is a change in a gene (i.e., a mutation ) that affects some function of the yeast cells, then essentially the same change will be observed in both haploid and diploid cells. This allows us to look at the effect of having two different alleles (i.e., versions) of the same gene in the same diploid cell. For instance, a haploid yeast cell might carry a mutant allele of the $his3$ gene, whereas a diploid might carry two identical $his3$/$his3$ mutant alleles or two different alleles such as $his3$/$HIS3$.  
  
-All yeast needs to grow in the lab (other than water and warmth) are salts, minerals, and glucose; growth media that contains only these things is called minimal media. Using the compounds contained in minimal media, normal yeast cells can synthesize all the molecules that are needed to construct a cell, such as amino acids (including histidine) and nucleotides. Organisms that are able to do this are called prototrophs. In a laboratory context, we define normal yeast cells that can grow on minimal media as wildtype. Note that "wildtype" and "wild" do not mean the same thing!+All that yeast needs to grow in the lab (other than water and warmth) are salts, minerals, and an organic carbon source (usually a sugar, but even glycerol can work!); growth media that contains only these things is called minimal media. Using the compounds contained in minimal media, normal yeast cells can synthesize all the molecules that are needed to construct a cell, such as amino acids (including histidine) and nucleotides. Organisms that are able to do this are called prototrophs. In a laboratory context, we define normal yeast cells that can grow on minimal media as wildtype. Note that "wildtype" and "wild" do not mean the same thing!
    
 ===== Genes are functionally defined by mutations that alter their function ===== ===== Genes are functionally defined by mutations that alter their function =====
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 </caption> </caption>
 </figure> </figure>
-Each intermediate compound in the pathway is converted to the next compound through a chemical reaction catalyzed by an enzyme (A is converted to B, B is converted to C, etc.). If there is a mutation that somehow affects the function of enzyme 3, then intermediate C cannot be converted to intermediate D and ultimately the cell cannot make histidine. Such a mutant will only grow if histidine is provided in the growth medium. This type of mutant is known as an auxotroph or auxotrophic mutant. This type of mutation is known as an auxotrophic mutation. A strain refers to a collection of individual organisms of a particular genotype (in the case of yeast, individual cells) that can be propagated in perpetuity. It's easy to maintain most yeast strains – all you need to do is provide them with food (sugars) and they will go through mitosis to make more copies of themselves. +Each intermediate compound in the pathway is converted to the next compound through a chemical reaction catalyzed by an enzyme (A is converted to B, B is converted to C, etc.). If there is a change in a gene (i.e., a mutation) that somehow affects the function of enzyme 3, then intermediate C cannot be converted to intermediate D and ultimately the cell cannot make histidine. Such a mutant will only grow if histidine is provided in the growth medium. This type of mutant is known as an auxotroph or auxotrophic mutant. This type of mutation is known as an auxotrophic mutation. A strain refers to a particular genetic variant of an organism that can be propagated in perpetuity. It's easy to maintain most yeast strains – all you need to do is provide them with food (sugars) and they will go through mitosis to make more copies of themselves. 
  
 ==== Defining dominant and recessive alleles ==== ==== Defining dominant and recessive alleles ====
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 </table> </table>
  
-Having performed this cross: if the two mutants don't complement, we conclude that they are mutant in the same gene.  ((In rare cases, just because two mutants do not complement each other does not automatically mean that the mutants have mutations in the same gene. There can be situations called "non-allelic non-complementation" where mutations in different genes do not complement each other. You can show that non-complementing mutations are in different genes by mapping their position ([[chapter_05|Chapter 05]]). When two different genes show non-allelic non-complementation, that can be an indication that the gene products of the two genes (usually the proteins encoded by the genes) have some sort of physical interaction.)) Another way we say this is that "$hisX$ is allelic to $his3$". Conversely, if they do complement, we conclude that they are mutant in different genes. For instance, $hisX$ might be a mutation in the $his4$ gene. To understand the reasoning behind this conclusion, look at the genotypes of the diploid in the two possible outcomes of this experiment.+Having performed this cross: if the two mutants don't complement, we conclude that they are mutant in the same gene.  ((In rare cases, just because two mutants do not complement each other does not automatically mean that the mutants have mutations in the same gene. There can be situations called "non-allelic non-complementation" where mutations in different genes do not complement each other. You can show that non-complementing mutations are in different genes by mapping their position ([[chapter_05|Chapter 05]]). When two different genes show non-allelic non-complementation, that can be an indication that the gene products of the two genes (usually the proteins encoded by the genes) have some sort of physical interaction.)) Another way we say this is that "$hisX$ is allelic to $his3$". Conversely, if they do complement, we conclude that they are mutant in different genes. For instance, $hisX$ might be a mutation in the $his4$ gene. 
  
-This works because in row 2 of Table {{ref>Tab4}}, the $his3$ haploid parent is presumed to be mutant only in the $his3$ gene and therefore implicitly carries the wildtype $HISX$ allele, and similarly the $hisX$ haploid parent is presumed to be mutant only in this one gene and thus implicitly carries the wildtype $HIS3$ allele. Therefore, the diploid produced from the cross will be heterozygous for both the $his3$ and $hisX$ genes and therefore provide normal function for both genes. It is important to know that this test only works for recessive mutations.+To understand the reasoning behind this conclusion, look at the combination of alleles (i.e., the genotype) of the diploid in the two possible outcomes of this experiment. In row 2 of Table {{ref>Tab4}}, the $his3$ haploid parent is presumed to be mutant only in the $his3$ gene and therefore implicitly carries the wildtype $HISX$ allele, and similarly the $hisX$ haploid parent is presumed to be mutant only in this one gene and thus implicitly carries the wildtype $HIS3$ allele. Therefore, the diploid produced from the cross will be heterozygous for both the $his3$ and $hisX$ genes and therefore provide normal function for both genes. It is important to know that this test only works for recessive mutations.
  
 The beauty of the complementation test is that the trait can serve as a read-out of gene function even without knowledge of what the gene is doing. We can simply define a gene based on its function, and we can distinguish between different genes (assuming we have recessive mutant alleles of those genes) using the complementation test. In fact, you can even use the complementation test for mutants that have different phenotypes! The only requirement for the complementation test is that the two mutants must be recessive.  The beauty of the complementation test is that the trait can serve as a read-out of gene function even without knowledge of what the gene is doing. We can simply define a gene based on its function, and we can distinguish between different genes (assuming we have recessive mutant alleles of those genes) using the complementation test. In fact, you can even use the complementation test for mutants that have different phenotypes! The only requirement for the complementation test is that the two mutants must be recessive. 
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 <table Tab5> <table Tab5>
 <columns 100% *100%*> <columns 100% *100%*>
-|     ^  wildtype  ^  $leu1$  ^  $leu2$  ^  $leu3$  ^  $leu4$  ^  $leu5$  ^  $leu6$  ^ +|     ^  %%wildtype%%  ^  $leu1$  ^  $leu2$  ^  $leu3$  ^  $leu4$  ^  $leu5$  ^  $leu6$  ^ 
-^  wildtype  |  Leu<sup>+</sup>  |     |     |     |     |     |     |     +^  %%wildtype%%  |  Leu<sup>+</sup>  |     |     |     |     |     |     |     
 ^  $leu1$  |  Leu<sup>+</sup>  |  Leu<sup>-</sup>  |     |     |     |     |     | ^  $leu1$  |  Leu<sup>+</sup>  |  Leu<sup>-</sup>  |     |     |     |     |     |
 ^  $leu2$  |  Leu<sup>+</sup>  |  Leu<sup>+</sup>  |  Leu<sup>-</sup>  |     |     |     |     | ^  $leu2$  |  Leu<sup>+</sup>  |  Leu<sup>+</sup>  |  Leu<sup>-</sup>  |     |     |     |     |
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