TNBGGA: The No Bullshit Guide to Genetic Analysis

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 with the $gal4^{81}$mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. - this mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. 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 DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. of upstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:

* In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of regulatory sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. of a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of 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 proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.. We will start to think about a new genetic approach to studying biological function: reverse geneticsplugin-autotooltip__default plugin-autotooltip_bigReverse genetics: an approach to studying genes wherein a researcher starts with knowledge of the physical identity of a gene (i.e., the DNA sequence of the gene) but does not know its function. In reverse genetics, the researcher uses various molecular genetic tools to create modified alleles that are reintroduced into an organism, with the goal of trying to deduce the function of the.

Reverse geneticsplugin-autotooltip__default plugin-autotooltip_bigReverse genetics: an approach to studying genes wherein a researcher starts with knowledge of the physical identity of a gene (i.e., the DNA sequence of the gene) but does not know its function. In reverse genetics, the researcher uses various molecular genetic tools to create modified alleles that are reintroduced into an organism, with the goal of trying to deduce the function of the is the opposite of what we have been studying so far. In Chapters 1-13, we discussed mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. with interesting phenotypesplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism., and used mappingplugin-autotooltip__default plugin-autotooltip_bigGenetic mapping: a term describing a variety of different experimental approaches used to determine the physical locations of genes on chromosomes. and cloningplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of by complementationplugin-autotooltip__default plugin-autotooltip_bigComplementation: a concept where an additional allele of a gene (usually a wildtype allele) can provide normal function to an organism with a recessive loss of function mutation in that gene. The concept of complementation underlies the complementation test. to try to identify the proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. coding sequenceplugin-autotooltip__default plugin-autotooltip_bigCoding sequence: refers to the portion of DNA or mRNA in a gene that contains direct information on the gene product. In most cases, this means a portion of DNA or mRNA that correlates to codons. Note that not all parts of a gene will necessarily be coding sequence (e.g., intron sequences). of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that is mutated - this approach starts with function (as defined by mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference.) and ends with identifying a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), and is called forward geneticsplugin-autotooltip__default plugin-autotooltip_bigForward genetics: an approach to studying genes wherein a researcher starts with mutants with interesting phenotypes and uses mapping and cloning methods to try and identify the physical identity of the gene. Compare to reverse genetics.. In reverse geneticsplugin-autotooltip__default plugin-autotooltip_bigReverse genetics: an approach to studying genes wherein a researcher starts with knowledge of the physical identity of a gene (i.e., the DNA sequence of the gene) but does not know its function. In reverse genetics, the researcher uses various molecular genetic tools to create modified alleles that are reintroduced into an organism, with the goal of trying to deduce the function of the, we already have a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that has been cloned. We might know of this geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) from a whole genomeplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of sequencingplugin-autotooltip__default plugin-autotooltip_bigSequencing: the procedure used to determine the sequence of a biological polymer such as DNA, RNA, or protein. Although there are indeed biochemical techniques that can be used to directly sequence RNA or protein, these methods are almost never used in modern molecular genetics research - instead, RNA project; or we might have found a mouse geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) based on sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. similarity to a Drosophilaplugin-autotooltip__default plugin-autotooltip_bigDrosophila melanogaster: a fruit fly species used in genetics research. geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that was found by forward geneticsplugin-autotooltip__default plugin-autotooltip_bigForward genetics: an approach to studying genes wherein a researcher starts with mutants with interesting phenotypes and uses mapping and cloning methods to try and identify the physical identity of the gene. Compare to reverse genetics.. We are interested in studying the function of this newly discovered geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). In some cases, the function of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) is completely unknown. In other cases, we may already know the normal function of a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), but we want to modify it in some way to further understand details of how the gene productplugin-autotooltip__default plugin-autotooltip_bigGene product: the molecule that is produced based on information contained within a gene and provides function to the organism. Most of the time, a gene product is a protein. Sometimes gene products can also be an RNA molecule. In forward genetic analysis, we can't formally tell if a gene product is works. In essence, reverse geneticsplugin-autotooltip__default plugin-autotooltip_bigReverse genetics: an approach to studying genes wherein a researcher starts with knowledge of the physical identity of a gene (i.e., the DNA sequence of the gene) but does not know its function. In reverse genetics, the researcher uses various molecular genetic tools to create modified alleles that are reintroduced into an organism, with the goal of trying to deduce the function of the starts with a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) and ends with identifying a function.

In this chapter we will discuss three examples of reverse geneticplugin-autotooltip__default plugin-autotooltip_bigReverse genetics: an approach to studying genes wherein a researcher starts with knowledge of the physical identity of a gene (i.e., the DNA sequence of the gene) but does not know its function. In reverse genetics, the researcher uses various molecular genetic tools to create modified alleles that are reintroduced into an organism, with the goal of trying to deduce the function of the strategies in yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 to study geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) function: (1) using reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) to study cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting regulatory sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids.; (2) using the yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 two hybrid systemplugin-autotooltip__default plugin-autotooltip_bigTwo hybrid system: an experimental procedure, usually performed in yeast, that leverages our knowledge of how transcription works in the $GAL4$-UAS system. It is a general test that can be used to determine if two proteins physically interact with each other. to study proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.-proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. interactions; and (3) making targeted geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics..

In Chapter 13 we considered a classic case of how genetic analysis could be used to dissect a regulatory mechanism. This analysis was contingent upon having “clean” phenotypesplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism. (i.e., phenotypesplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism. that are clear and unambiguous) associated with the isolated mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference.. For example, mutationsplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. in the $gal80$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) result in a phenotypeplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism. of constitutiveplugin-autotooltip__default plugin-autotooltip_bigConstitutive: a term describing a pattern of gene expression, wherein the gene is always expressed no matter what. For some genes, constitutive expression is normal (see housekeeping gene). For inducible genes or operons, constitutive is a mutant state. Gal1p expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places.. However, in many cases it is difficult to identify regulatory proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. by isolating mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference.. For example, regulatory proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that influence the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of a large number of genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) might be essential genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), and mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. in these genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) might be lethal and you would never be able to isolate such a mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference., much less observe its phenotypesplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism.. Or, mutationsplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. in genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that code for important regulatory genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) may show pleiotropyplugin-autotooltip__default plugin-autotooltip_bigPleiotrophy: describes a state wherein a mutation in a single gene causes multiple, seemingly unrelated mutant defects. This usually indicates that a gene may have more than one function. (see below) and phenotypesplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism. may be difficult to interpret.

One solution to this has been to work backwards from the cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. for particular genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) of interest. Let’s take the $GAL1$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) as an example. We learned in Chap. 13 that galactose inducesplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of the $GAL1$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (along with other Gal genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-)). What we did not mention is that if glucose is present in addition to galactose, the inductionplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. of the Gal genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) does not occur! This is known as glucose repressionplugin-autotooltip__default plugin-autotooltip_bigGlucose repression: a phenomenon in many different organisms wherein genes that are normally induced by alternate carbon sources (such as lactose or galactose) become mostly uninducible when glucose is present.. Glucose repressionplugin-autotooltip__default plugin-autotooltip_bigGlucose repression: a phenomenon in many different organisms wherein genes that are normally induced by alternate carbon sources (such as lactose or galactose) become mostly uninducible when glucose is present. also happens in bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably.; for instance, when glucose is present, the Lac and Mal operonsplugin-autotooltip__default plugin-autotooltip_bigOperon: two or more genes that are transcribed together on an mRNA from a single promoter. Most commonly seen in bacteria; very rare in eukaryotes. in E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). will not be expressedplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. even though lactose or maltose is present (see Chap. 10. The details of how bacteriaplugin-autotooltip__default plugin-autotooltip_bigBacteria: Single-celled organisms that also utilize DNA and the standard genetic code as all organisms on earth, but unlike eukaryotes do not have intracellular membranes and membrane-bound organelles. In this book we use bacteria and prokaryote interchangeably. and yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 do it are very different; here, we focus on yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02.

Glucose repressionplugin-autotooltip__default plugin-autotooltip_bigGlucose repression: a phenomenon in many different organisms wherein genes that are normally induced by alternate carbon sources (such as lactose or galactose) become mostly uninducible when glucose is present. makes physiological sense because glucose is a far more efficient energy source than galactose for yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 and is therefore the preferred carbon source. Why bother to use extra steps to metabolize galactose when glucose is present? In fact, glucose represses a very large number of genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) whose products metabolize a wide range of different carbon sources (sucrose, maltose, galactose, etc.) that are less energy efficient than glucose, as well as repressing a whole host of other genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-).

It seems reasonable to expect that there is a proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that functions as a transcriptionalplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. repressorplugin-autotooltip__default plugin-autotooltip_bigRepressor: a DNA binding protein that binds to negatively acting cis-acting elements such as operators in bacteria to inhibit transcription. Compare to negative regulator. and responds to glucose levels; this repressorplugin-autotooltip__default plugin-autotooltip_bigRepressor: a DNA binding protein that binds to negatively acting cis-acting elements such as operators in bacteria to inhibit transcription. Compare to negative regulator. proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. would be inactive when glucose is low or absent, and active when glucose is present. It also seems reasonable that one could isolate transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. that fail to repress galactose-inducedplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. Gal geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. in the presence of glucose. However, because glucose represses a large number of different genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), this made it difficult to identify such mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference.. Such mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. usually have pleiotropicplugin-autotooltip__default plugin-autotooltip_bigPleiotrophy: describes a state wherein a mutation in a single gene causes multiple, seemingly unrelated mutant defects. This usually indicates that a gene may have more than one function. phenotypesplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism. (multiple defects that may not directly relate to the primary one you are interested in) and are difficult to work with (they are usually very sick and difficult to grow in the lab).

Instead of looking for mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. that fail to execute glucose repressionplugin-autotooltip__default plugin-autotooltip_bigGlucose repression: a phenomenon in many different organisms wherein genes that are normally induced by alternate carbon sources (such as lactose or galactose) become mostly uninducible when glucose is present. at the $GAL1$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), studies of the $GAL1$ upstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:

* In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of regulatory region itself provided the key to dissecting the mechanism of glucose repressionplugin-autotooltip__default plugin-autotooltip_bigGlucose repression: a phenomenon in many different organisms wherein genes that are normally induced by alternate carbon sources (such as lactose or galactose) become mostly uninducible when glucose is present.. Specifically, the GAL1 upstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:

* In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. region was cloned and fused to the E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). $lacZ$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), on a plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. that can replicate autonomously in S. cerevisiae. It was first important to establish that regulation of LacZ (β-galactosidase) from the plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. mirrored the regulation of Gal1p (galactokinase) from its chromosomalplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. locusplugin-autotooltip__default plugin-autotooltip_bigLocus (plural form: loci): a physical location of a gene; often used as a synonym for a gene., i.e., that β−galactosidase was inducedplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. by galactose in the absence of glucose, but not in its presence (Fig. 2).

Having established that our reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. worked the way we needed it to, it was possible to go on and examine fragments of the $GAL1$ regulatory region for their role in inductionplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. of Gal1p expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. by galactose, as well as repression of Gal1p expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. by glucose. The minimal length of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. stretching upstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:

* In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of from the $GAL1$ transcriptionplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. start site that supported galactose inductionplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. of the $lacZ$ reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) was 400 bp of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. (Fig. 3).

Once this functional region was delineated, systematic deletions of 50 bp or so could be made across the 400 bp region; this is easy to do with some laboratory recombinantplugin-autotooltip__default plugin-autotooltip_bigRecombinant: (adj.) Describing something that has undergone recombination, e.g., recombinant DNA or recombinant offspring. “Non-parental” is a synonym when referring to organisms. (n.) Something that has undergone recombination, e.g., “This fly is a recombinant.” DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. tricks that are not important to know about here. Suffice to say that this deletion analysis revealed two regions critical for transcriptionalplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. control, as well as the location of the TATA sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. that is required for loading of the basal transcriptionplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. machinery.

From the data in Fig. 4, we can deduce the location of important cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting regulatory sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. for the $GAL1$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-):

• Deletions 7 and 8 do not express the $lacZ$ reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. under any conditions because the deletions have removed some of the TATA boxplugin-autotooltip__default plugin-autotooltip_bigTATA box: a short DNA sequence (usually just 6 bp long) that is part of most eukaryotic promoters located around 35-40 bp upstream of the transcription start site. It is the binding site for TATA binding protein (TBP). sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. that is required for RNA polymeraseplugin-autotooltip__default plugin-autotooltip_bigRNA polymerase: the enzyme that carries out RNA transcription. There are many different types of RNA polymerase, but in this book we collectively refer to them as just “RNA polymerase” for simplicity. binding.
• Deletions 1 and 2 eliminate the ability of galactose to increase expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of the reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements., and since expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. is not inducedplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. there is nothing for glucose to repress. It turns out that the 75 bp sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. between -310 and -385 is the DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. binding site for Gal4p. This kind of region is generally called a UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.
  
  Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$ (upstream activator sequenceplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.
  
  Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$) and in this case it is specifically called UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.
  
  Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$_GAL. This was alluded to in Chap. 13. We will come back to thinking about Gal4p binding to the UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.
  
  Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$ recognition sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. later.
• Deletions 3, 5, and 6 have no effect on the ability of galactose to induceplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. because the UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.
  
  Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$ remains intact. Note that shortening the distance between the UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.
  
  Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$ and the TATA boxplugin-autotooltip__default plugin-autotooltip_bigTATA box: a short DNA sequence (usually just 6 bp long) that is part of most eukaryotic promoters located around 35-40 bp upstream of the transcription start site. It is the binding site for TATA binding protein (TBP). region is not detrimental to inductionplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive.. Indeed, other experiments not shown here demonstrated that increasing the distance by inserting extra DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. between the UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.
  
  Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$ and the TATA boxplugin-autotooltip__default plugin-autotooltip_bigTATA box: a short DNA sequence (usually just 6 bp long) that is part of most eukaryotic promoters located around 35-40 bp upstream of the transcription start site. It is the binding site for TATA binding protein (TBP). also has little effect on inducibility. This has led to the idea that UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.
  
  Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$ sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. can work at long distances (1,000 – 10,000 bp) away from the TATA boxplugin-autotooltip__default plugin-autotooltip_bigTATA box: a short DNA sequence (usually just 6 bp long) that is part of most eukaryotic promoters located around 35-40 bp upstream of the transcription start site. It is the binding site for TATA binding protein (TBP). and the transcriptionplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. start site. This winds up being generally true for upstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:
  
  * In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of regulatory elements of eukaryoticplugin-autotooltip__default plugin-autotooltip_bigeukaryote: organism whose cells have membrane bound organelles, including the nucleus. genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-).
• Deletion 4 reveals important information about glucose repressionplugin-autotooltip__default plugin-autotooltip_bigGlucose repression: a phenomenon in many different organisms wherein genes that are normally induced by alternate carbon sources (such as lactose or galactose) become mostly uninducible when glucose is present.. While galactose inducesplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. in this construct, glucose is unable to repress that expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places.. The deleted region therefore defines the position of a DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. element that is required for glucose repressionplugin-autotooltip__default plugin-autotooltip_bigGlucose repression: a phenomenon in many different organisms wherein genes that are normally induced by alternate carbon sources (such as lactose or galactose) become mostly uninducible when glucose is present.. A DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. element that behaves this way is generally called a URSplugin-autotooltip__default plugin-autotooltip_bigUpstream repressor sequence: In a general sense, a URS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which repressors bind to silence transcription of nearby genes.
  
  Although the term “URS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term silencer is used more often to describe these $GAL1$ (upstream repressor sequenceplugin-autotooltip__default plugin-autotooltip_bigUpstream repressor sequence: In a general sense, a URS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which repressors bind to silence transcription of nearby genes.
  
  Although the term “URS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term silencer is used more often to describe these $GAL1$), and in this case it is specifically called URSplugin-autotooltip__default plugin-autotooltip_bigUpstream repressor sequence: In a general sense, a URS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which repressors bind to silence transcription of nearby genes.
  
  Although the term “URS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term silencer is used more often to describe these $GAL1$_GAL. URSplugin-autotooltip__default plugin-autotooltip_bigUpstream repressor sequence: In a general sense, a URS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which repressors bind to silence transcription of nearby genes.
  
  Although the term “URS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term silencer is used more often to describe these $GAL1$_GAL is found in the upstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:
  
  * In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of regulatory regions of many genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) besides Gal genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-).

After determining that there was a URSplugin-autotooltip__default plugin-autotooltip_bigUpstream repressor sequence: In a general sense, a URS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which repressors bind to silence transcription of nearby genes.

Although the term “URS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term silencer is used more often to describe these $GAL1$ element controlling glucose repressionplugin-autotooltip__default plugin-autotooltip_bigGlucose repression: a phenomenon in many different organisms wherein genes that are normally induced by alternate carbon sources (such as lactose or galactose) become mostly uninducible when glucose is present. at the $GAL1$ promoterplugin-autotooltip__default plugin-autotooltip_bigPromoter: has multiple closely related but subtly different meanings depending on context:

* In bacteria, a promoter is a cis-acting DNA sequence near the transcription start site of a gene or operon that binds to bacterial RNA polymerase. * In eukaryotes, the formal definition of a promoter (also called a basal promoter) is a RNA, it was possible to go on to identify a proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. called Mig1p that binds the URSplugin-autotooltip__default plugin-autotooltip_bigUpstream repressor sequence: In a general sense, a URS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which repressors bind to silence transcription of nearby genes.

Although the term “URS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term silencer is used more often to describe these $GAL1$_GAL sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids.. The Snf1 complex is a proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. kinase that actively phosphorylates the Mig1 repressorplugin-autotooltip__default plugin-autotooltip_bigRepressor: a DNA binding protein that binds to negatively acting cis-acting elements such as operators in bacteria to inhibit transcription. Compare to negative regulator. under low glucose conditions; this prevents it from entering the nucleusplugin-autotooltip__default plugin-autotooltip_bigNucleus: in eukaryotes, the membrane-bound organelle in cells that contains the chromosomes.. Low glucose concentration is permissive for galactose inductionplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. of $GAL1$ expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. via the UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.

Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$. In high glucose conditions the Snf1 kinase is inactivated, so Mig1 is not phosphorylated, and unphosphorylated Mig1 enters the nucleusplugin-autotooltip__default plugin-autotooltip_bigNucleus: in eukaryotes, the membrane-bound organelle in cells that contains the chromosomes. where it binds to the URSplugin-autotooltip__default plugin-autotooltip_bigUpstream repressor sequence: In a general sense, a URS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which repressors bind to silence transcription of nearby genes.

Although the term “URS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term silencer is used more often to describe these $GAL1$ sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. and recruits two other proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that together achieve repression of $GAL1$ expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. (Fig. 5).

All these results were achieved using other genetic and biochemical experiments too numerous to be listed here. But mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. in $mig1$, $snf1$, and other related genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) with functions in glucose repressionplugin-autotooltip__default plugin-autotooltip_bigGlucose repression: a phenomenon in many different organisms wherein genes that are normally induced by alternate carbon sources (such as lactose or galactose) become mostly uninducible when glucose is present. would likely not have been discovered using traditional forward geneticplugin-autotooltip__default plugin-autotooltip_bigForward genetics: an approach to studying genes wherein a researcher starts with mutants with interesting phenotypes and uses mapping and cloning methods to try and identify the physical identity of the gene. Compare to reverse genetics. approaches without having first identified URSplugin-autotooltip__default plugin-autotooltip_bigUpstream repressor sequence: In a general sense, a URS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which repressors bind to silence transcription of nearby genes.

Although the term “URS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term silencer is used more often to describe these $GAL1$_GAL using reverse geneticsplugin-autotooltip__default plugin-autotooltip_bigReverse genetics: an approach to studying genes wherein a researcher starts with knowledge of the physical identity of a gene (i.e., the DNA sequence of the gene) but does not know its function. In reverse genetics, the researcher uses various molecular genetic tools to create modified alleles that are reintroduced into an organism, with the goal of trying to deduce the function of the.

We will segue briefly back to E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). genetics to make a point. In Chap. 10, we discussed the classic cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different/transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different experiments by Jacob and Monod. They showed that dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. mutationsplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. in $lacO$ (such as $lacO^c$) were only dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. when they were located on the same piece of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. as $lacZ^+$ (i.e., when $lacO^c$ is in cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different with $lacZ^+$). When $lacO^c$ was placed on an episomeplugin-autotooltip__default plugin-autotooltip_bigEpisome: a piece of DNA that exists separately from chromosomes and that can replicate and segregate during cell division. While all episomes are natural in origin, they are commonly exploited by researchers for genetics research. (such as an F' plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors.) by itself and the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. contained $lacZ^+$ (i.e., when $lacO^c$ is in transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different with $lacZ^+$), it could not affect the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of $lacZ^+$. By comparison, dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. mutationsplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. in $lacI$ (such as $lacI^s$) appeared to be dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. no matter whether it was located together with $lacZ^+$ on an episomeplugin-autotooltip__default plugin-autotooltip_bigEpisome: a piece of DNA that exists separately from chromosomes and that can replicate and segregate during cell division. While all episomes are natural in origin, they are commonly exploited by researchers for genetics research. (in cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different) or whether it was on a separate piece of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. as $lacZ^+$ (e.g., $lacI^s$ on a F' plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. and $lacZ^+$ on the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.).

The Jacob and Monod experiments are important but are a little challenging to understand conceptually, so it is helpful to review Chap. 10 carefully. But let's just review and summarize their interpretation and conclusions:

• If a dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. that affects the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of another geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) only acts dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. in cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different but not in transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different, we abbreviate this as saying it is a cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant.. We can also say this geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (or more accurately, this mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant.) acts in cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different. An example is $lacO^c$. We interpret cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting mutationsplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. to say that the normal (non-mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference.) function of the “geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-)” defined by that mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. is a DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. that does not itself code for proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. but rather regulates the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of nearly proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.-coding genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). In the case of Jacob and Monod's Lac operonplugin-autotooltip__default plugin-autotooltip_bigLac operon: an operon in E. coli that contains the cis-acting regulatory elements $lacP$ and $lacO$, and the protein coding genes $lacZ$, $lacY$, and $lacA$. The Lac operon is an example of a negatively-regulated operon. experiments, we would say that $lac^O$ is a cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. that regulates the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of $lacZ$.
• By comparison, if a dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. that affects the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of another genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) acts dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. both in cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different and in transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different, we abbreviate this as saying it is a transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant.. We can also say that this geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (or more accurately, this mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant.) acts in transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different. An example is $lacI^s$. We interpret transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting mutationsplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. to say that the normal function of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) defined by that mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. is that it codes for a proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that regulates the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of its target genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). This proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.'s effect is not limited to the same piece of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. from which it was encoded – proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. are separate molecules from the DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. and therefore are free to bind to any DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. molecule it encounters inside a cell. In the case of Jacob and Monod's Lac operonplugin-autotooltip__default plugin-autotooltip_bigLac operon: an operon in E. coli that contains the cis-acting regulatory elements $lacP$ and $lacO$, and the protein coding genes $lacZ$, $lacY$, and $lacA$. The Lac operon is an example of a negatively-regulated operon. experiments, we would say that $lacI$ codes for a transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting factor that regulates the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of $lacZ$.

Jacob and Monod used very formal and abstract genetic concepts to interpret and describe their findings; as such, the language can be a little difficult to parse. Nonetheless, the important concept here is that their studies were the first to suggest that geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. is regulated by proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that bind to DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. located physically near the transcriptionplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. start site of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). This was a Big Deal in the 1970s; they won the Nobel Prize, after all! Remember that they had to figure this out from scratch and very few tools with which to work with, except genetics.

It turns out that all eukaryotesplugin-autotooltip__default plugin-autotooltip_bigeukaryote: organism whose cells have membrane bound organelles, including the nucleus., including yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02, regulate geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. in the same basic way (although the details are different). Let's return to our discussion of Gal geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) regulation in yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02. Using similar experiments as Jacob and Monod, yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 geneticists would go on to show that mutationsplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. in UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.

Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$ act in cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different (Table 1). Along the same lines, dominantplugin-autotooltip__default plugin-autotooltip_bigDominant: used to describe an allele, usually in comparison to wildtype. Dominant alleles will express their phenotype when combined with a wildtype allele. mutationsplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. such as $gal4^{81}$ act in transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different. Using the same genetic logic as Jacob and Monod, we can interpret these results to say that UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.

Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$ is a DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. that does not code for proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. itself but instead regulates the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of nearby proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.-coding target genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-); we can also say that GAL4 likely codes for a proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that regulates the expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of target genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-).

A more modern take on the naming of these elements is that we call cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting sequencesplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. such as UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.

Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$ and URSplugin-autotooltip__default plugin-autotooltip_bigUpstream repressor sequence: In a general sense, a URS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which repressors bind to silence transcription of nearby genes.

Although the term “URS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term silencer is used more often to describe these $GAL1$ cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different regulatory elements; cisplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different regulatory elements that activate geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. are called enhancersplugin-autotooltip__default plugin-autotooltip_bigEnhancer: a more inclusive term for a UAS. One reason this is a more inclusive term is because not all enhancers are located upstream of genes; some enhancers are located downstream of a gene, and in some cases can even be located inside a gene., and those that repress geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. are called silencersplugin-autotooltip__default plugin-autotooltip_bigSilencer: a more inclusive term for a URS. One reason this is a more inclusive term is because not all silencersare located upstream of genes; some silencers are located downstream of a gene, and in some cases can even be located inside a gene.. Transplugin-autotooltip__default plugin-autotooltip_bigCis and trans: In genetics, cis and trans are terms used to describe the relative physical locations of genes or genetic elements. If two genes are in cis, this means that they are physically located on the same DNA molecule. If two genes are in trans, this means that they are physically located on two different-acting factors are usually just generically called transcription factorsplugin-autotooltip__default plugin-autotooltip_bigTranscription factor: a generic term that describes any DNA binding protein that affects transcription (can havea positive or negative effect on transcription).; those that activate geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. are called transactivatorsplugin-autotooltip__default plugin-autotooltip_bigTransactivator (activator): a DNA binding protein that binds to promoter sequences to activate the transcription of nearby genes. In the context of gene expression, “activator” is usually a synonym for transactivator. Compare with positive regulator., and those that repress geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. are called repressorsplugin-autotooltip__default plugin-autotooltip_bigRepressor: a DNA binding protein that binds to negatively acting cis-acting elements such as operators in bacteria to inhibit transcription. Compare to negative regulator..  

The Gal4p transactivatorplugin-autotooltip__default plugin-autotooltip_bigTransactivator (activator): a DNA binding protein that binds to promoter sequences to activate the transcription of nearby genes. In the context of gene expression, “activator” is usually a synonym for transactivator. Compare with positive regulator. is one of the most well-studied proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that carries out transcriptionalplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. activation. To study Gal4p, a $lacZ$ reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. was used in a creative way to establish that the Gal4p proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. has two functional domains that are separated by a flexible region in the proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.. This time, we generate a yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. where the $GAL1$ upstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:

* In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of regulatory region (including the TATA boxplugin-autotooltip__default plugin-autotooltip_bigTATA box: a short DNA sequence (usually just 6 bp long) that is part of most eukaryotic promoters located around 35-40 bp upstream of the transcription start site. It is the binding site for TATA binding protein (TBP). and the UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.

Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$) remains intact upstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:

* In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of of the $lacZ$ reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements., but deletions are made across the proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. coding region of the $GAL4$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (Fig. 6). This is the inverse of keeping $GAL4$ intact and making deletions along the $GAL1$ promoterplugin-autotooltip__default plugin-autotooltip_bigPromoter: has multiple closely related but subtly different meanings depending on context:

* In bacteria, a promoter is a cis-acting DNA sequence near the transcription start site of a gene or operon that binds to bacterial RNA polymerase. * In eukaryotes, the formal definition of a promoter (also called a basal promoter) is a RNA 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 $GAL4$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (see Questions and exercises below), you can insert it into another plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. and transformplugin-autotooltip__default plugin-autotooltip_bigTransformation: in microbiology (including for yeasts), transformation is the alteration of phenotype due to uptake of external DNA into a cell. Not to be confused with “transformation” in the context of cell biology, where the definition is the alteration of a normal cell to a cancerous cell. it into $gal4$ mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 cells. This should complementplugin-autotooltip__default plugin-autotooltip_bigComplement: used to describe the relationship between two recessive mutants. If a diploid created by mating the two mutants has a wildtype phenotype, we say the two mutants complement each other. the $gal4$ mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant.; in other words, the plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. is providing the only functional Gal4p in the cell. You then separately make in-frame deletions of the $GAL4$ cloneplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of at different parts of the proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. coding region, transformplugin-autotooltip__default plugin-autotooltip_bigTransformation: in microbiology (including for yeasts), transformation is the alteration of phenotype due to uptake of external DNA into a cell. Not to be confused with “transformation” in the context of cell biology, where the definition is the alteration of a normal cell to a cancerous cell. those constructs into $gal4$ mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference., and assess the function of those deletion constructs.

The data in Fig. 6 show if the N-terminalplugin-autotooltip__default plugin-autotooltip_bigN-terminus: the end of a polypeptide chain that contains the first amino acid that was translated. portion of Gal4p is deleted, the proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. cannot bind to the UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.

Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$_GAL sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids., and so is unable to activate transcriptionplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. of the reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). This suggests that the N terminal portion of the proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. is a DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. binding domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. (DB). But when the C-terminalplugin-autotooltip__default plugin-autotooltip_bigC-terminus: the end of a polypeptide chain that contains the last amino acid that was translated. portion of Gal4p is deleted, the proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. can still bind DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. but can no longer activate the reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements.. This was interpreted to mean that Gal4p must have a region near its C-terminalplugin-autotooltip__default plugin-autotooltip_bigC-terminus: the end of a polypeptide chain that contains the last amino acid that was translated. end that is responsible for recruiting and activating the RNA polymeraseplugin-autotooltip__default plugin-autotooltip_bigRNA polymerase: the enzyme that carries out RNA transcription. There are many different types of RNA polymerase, but in this book we collectively refer to them as just “RNA polymerase” for simplicity., thus allowing expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. of the reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). This is called an activation domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. (AD). The most remarkable thing of all was that a large region in the center of Gal4p can be deleted without loss of functionplugin-autotooltip__default plugin-autotooltip_bigLoss of function: a general term used to describe mutant alleles that have less activity than wildtype. Amorphic and hypomorphic mutations are loss of function mutations.; as long as the DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. binding domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. is present at the N-terminusplugin-autotooltip__default plugin-autotooltip_bigN-terminus: the end of a polypeptide chain that contains the first amino acid that was translated. and the activating domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. is present at the C-terminusplugin-autotooltip__default plugin-autotooltip_bigC-terminus: the end of a polypeptide chain that contains the last amino acid that was translated., Gal4p can activate transcriptionplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. from the UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.

Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$_GAL sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids.(Fig. 6).

This remarkable separation of function between these two domains of Gal4p was dramatically demonstrated by a series of experiments called domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. swapping (Fig. 7). Using recombinantplugin-autotooltip__default plugin-autotooltip_bigRecombinant: (adj.) Describing something that has undergone recombination, e.g., recombinant DNA or recombinant offspring. “Non-parental” is a synonym when referring to organisms. (n.) Something that has undergone recombination, e.g., “This fly is a recombinant.” DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. techniques, the Gal4 transcriptionplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. activation domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. (AD) was fused to the DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. binding (DB) domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. of an E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs). proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. called LexA, which is a repressorplugin-autotooltip__default plugin-autotooltip_bigRepressor: a DNA binding protein that binds to negatively acting cis-acting elements such as operators in bacteria to inhibit transcription. Compare to negative regulator. that binds to a known DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. called the LexA operatorplugin-autotooltip__default plugin-autotooltip_bigOperator: in bacterial genetics, an operator is a cis-acting genetic element that negatively regulates the expression of nearby genes. $lacO$ of the Lac operon is an example of an operator. ($lexA \ \ OP$). Also, the Gal4 DB domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. was fused to the AD transcriptionplugin-autotooltip__default plugin-autotooltip_bigRNA transcription: the process of RNA polymerase using the DNA sequence of a gene as a template to form an mRNA (in prokaryotes) or pre-mRNA (in eukaryotes). In most cases, “transcription” implies RNA transcription. activation domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. of a viral proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. called VP16, which was known to be a strong activatorplugin-autotooltip__default plugin-autotooltip_bigTransactivator (activator): a DNA binding protein that binds to promoter sequences to activate the transcription of nearby genes. In the context of gene expression, “activator” is usually a synonym for transactivator. Compare with positive regulator.. These chimeric proteinsplugin-autotooltip__default plugin-autotooltip_bigChimeric protein: a protein that is the result of artificially joining together parts of two or more unrelated proteins. Also called a fusion protein. (also called fusion proteinsplugin-autotooltip__default plugin-autotooltip_bigChimeric protein: a protein that is the result of artificially joining together parts of two or more unrelated proteins. Also called a fusion protein.) were introduced into yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 cells with the appropriate $lacZ$ reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) constructs and the results of these domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. swapping experiments were dramatic.

Two derivatives of a $gal4$ mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and were created: one containing the $lacZ$ reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. construct downstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:

* In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of of UASplugin-autotooltip__default plugin-autotooltip_bigUpstream activator sequence (UAS): In a general sense, a UAS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which transactivators bind to activate transcription of nearby genes.

Although the term “UAS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term enhancer is used more often to describe these $GAL1$_GAL, and the other containing the $lacZ$ reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. construct downstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:

* In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of of the $lexA \ \ OP$. The two different chimeric proteinsplugin-autotooltip__default plugin-autotooltip_bigChimeric protein: a protein that is the result of artificially joining together parts of two or more unrelated proteins. Also called a fusion protein. were expressedplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places. in each strainplugin-autotooltip__default plugin-autotooltip_bigStrain or line: refers to a pool or colony of individuals or cultured cells of a desired genotype or phenotype that is mostly homogeneous and can be bred and/or produced in perpetuity for research or commercial purposes. “Strain” tends to be used more for microorganisms and and the ability to induceplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive. LacZ activity was monitored. In addition, constructs designed to express the following proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. were also introduced into the two strains: the wildtypeplugin-autotooltip__default plugin-autotooltip_bigWildtype: a reference strain of an organism that scientists operationally define as “normal” to which mutants are compared. Not to be confused with wild organisms. Gal4 proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. and a third chimeric proteinplugin-autotooltip__default plugin-autotooltip_bigChimeric protein: a protein that is the result of artificially joining together parts of two or more unrelated proteins. Also called a fusion protein. with the activation domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. of the Gal4⁸¹ (the super-inducerplugin-autotooltip__default plugin-autotooltip_bigInducer: a molecule that triggers the expression of a gene or genes. Examples in E. coli include lactose and IPTG, which induce expression of the Lac operon in E. coli, and maltose, which induces expression of the Mal operon. An example in yeast is galactose, which induces expression of Gal genes.) mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. fused to the LexA DB domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein.. The results from these experiments (Table 2) clearly show that the AD and the DB domains function independently of one another.

These domainplugin-autotooltip__default plugin-autotooltip_bigDomain: in biochemistry, a domain is a portion of a protein structure that can function independently or semi-independently from other portions of the protein. swapping experiments had a larger impact than just on our understanding of geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) regulation in yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02; they provided some of the first lines of evidence that proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. generally have a modular structure, and their function can be determined by combinations of different domains, each which can function semi-independently of the other but in combination result in new functions.

Studies on Gal4p and USA_GAL have turned out to have a profound effect on all biological research because it contributed to the development of a widely used technology called the yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 two hybrid assayplugin-autotooltip__default plugin-autotooltip_bigTwo hybrid system: an experimental procedure, usually performed in yeast, that leverages our knowledge of how transcription works in the $GAL4$-UAS system. It is a general test that can be used to determine if two proteins physically interact with each other. (Fig. 9). This assay makes it possible to determine whether two proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. interact with each other as a complex with long-lived interaction, and sometimes even when two proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. only interact transiently.

To determine whether proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. X interacts with either proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. Y or proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. Z one can do the following: fuse proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. X to the Gal4 DB; this chimeric proteinplugin-autotooltip__default plugin-autotooltip_bigChimeric protein: a protein that is the result of artificially joining together parts of two or more unrelated proteins. Also called a fusion protein. is known as the bait, and it will attach to the UASGAL that lies upstreamplugin-autotooltip__default plugin-autotooltip_bigUpstream/downstream: These descriptors have different meanings depending on context:

* In genetics, these are terms used to describe directions on DNA, usually relative to the transcription start site of a gene. DNA sequences that are located in the same direction as the direction of of a reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), usually a selectableplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a markerplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). or LacZ, or both. This bait lies in wait for an interaction with another proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.. The GAL4 AD is fused to either proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. Y or proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. Z. Should either one of these proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. be able to interact with proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. X then the Gal4 AD region will become tethered to the UASGAL region and will recruit and activate RNA polymeraseplugin-autotooltip__default plugin-autotooltip_bigRNA polymerase: the enzyme that carries out RNA transcription. There are many different types of RNA polymerase, but in this book we collectively refer to them as just “RNA polymerase” for simplicity..

Note that the proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. X, Y and Z do not have to be yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.; the only requirement is that the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) coding for the proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. has been cloned (or can be easily generated via PCRplugin-autotooltip__default plugin-autotooltip_bigPolymerase chain reaction (PCR): An experimental technique invented by Kary Mullis used to exponentially amplify DNA in vitro. PCR made obtaining large quantities of DNA for analysis much faster and easier than using traditional cloning methods.); these genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) are then modified such that they produce the appropriate Gal4p chimeric proteinsplugin-autotooltip__default plugin-autotooltip_bigChimeric protein: a protein that is the result of artificially joining together parts of two or more unrelated proteins. Also called a fusion protein.. Many yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 two hybridplugin-autotooltip__default plugin-autotooltip_bigTwo hybrid system: an experimental procedure, usually performed in yeast, that leverages our knowledge of how transcription works in the $GAL4$-UAS system. It is a general test that can be used to determine if two proteins physically interact with each other. experiments use libraries instead of a specific “proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. Y” or “proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. Z” so that one can screenplugin-autotooltip__default plugin-autotooltip_bigScreen: a screen is a process through which a researcher looks through a population of individuals in an attempt to find rare individuals with certain phenotypes, usually with no obvious way to enrich for the rare individuals. contrast to a selection. for previously unknown proteinsplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. that interact with your bait “proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. X”. Systematic yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 two hybrid assaysplugin-autotooltip__default plugin-autotooltip_bigTwo hybrid system: an experimental procedure, usually performed in yeast, that leverages our knowledge of how transcription works in the $GAL4$-UAS system. It is a general test that can be used to determine if two proteins physically interact with each other. have been performed for all possible proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. pairs for various organisms, including E. coliplugin-autotooltip__default plugin-autotooltip_bigEscherichia coli: an enteric bacterium used both as a model organism and as a utility organism in genetics research. E. coli is commonly used to host various cloning vectors, such as plasmids, cosmids, F factors, and bacterial artificiak chromosomes (BACs)., yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02, Drosophilaplugin-autotooltip__default plugin-autotooltip_bigDrosophila melanogaster: a fruit fly species used in genetics research., and mouse, among others. This “map” of proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.-proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. interactions is often referred to as the interactome. (Other experimental techniques are also used to generate interactome maps in addition to two hybrid assaysplugin-autotooltip__default plugin-autotooltip_bigTwo hybrid system: an experimental procedure, usually performed in yeast, that leverages our knowledge of how transcription works in the $GAL4$-UAS system. It is a general test that can be used to determine if two proteins physically interact with each other..)

One of the most important tricks in the toolbox of a yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 geneticist is the ability to create targeted geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. for almost any geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) in the genomeplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of. Geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. in multicellular organisms (mice) are discussed in Chapter 16. The general principles are similar, but it is much easier to make geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knockoutsplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. in yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 for two reasons: (1) yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 are unicellular microbes, which makes growing large numbers of yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 cells and screeningplugin-autotooltip__default plugin-autotooltip_bigScreen: a screen is a process through which a researcher looks through a population of individuals in an attempt to find rare individuals with certain phenotypes, usually with no obvious way to enrich for the rare individuals. contrast to a selection. or selectingplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a for rare events very easy to do; and (2) DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. recombinationplugin-autotooltip__default plugin-autotooltip_bigRecombination: Recombination can have slightly different meanings depending on context:

* In the context of genetic crosses (usually a dihybrid cross or a test cross), recombination refers to the phenomena where the phenotype of the F2 offspring is different than either parent (P generation). $lox$ rates are very high in yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02.

Let's say that you are interested in how Gal80p functions. Using the yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 two hybrid assayplugin-autotooltip__default plugin-autotooltip_bigTwo hybrid system: an experimental procedure, usually performed in yeast, that leverages our knowledge of how transcription works in the $GAL4$-UAS system. It is a general test that can be used to determine if two proteins physically interact with each other., 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 geneticsplugin-autotooltip__default plugin-autotooltip_bigForward genetics: an approach to studying genes wherein a researcher starts with mutants with interesting phenotypes and uses mapping and cloning methods to try and identify the physical identity of the gene. Compare to reverse genetics.. However, you also discover that Gal80p binds to a new proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. you have not studied closely yet. You temporarily name this proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes. Gal3p, and you identify and cloneplugin-autotooltip__default plugin-autotooltip_bigClone: Depending on the context, this word can have a few different meanings:

* In the context of genes, cloning means that the physical identity of a gene has been found, and the gene has been sequenced. * In the context of DNA, a cloned DNA fragment is one that has been inserted into some kind of the physical geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) $gal3$ ($gal3$ is briefly mentioned in Chapter 13). Although your two hybridplugin-autotooltip__default plugin-autotooltip_bigTwo hybrid system: an experimental procedure, usually performed in yeast, that leverages our knowledge of how transcription works in the $GAL4$-UAS system. It is a general test that can be used to determine if two proteins physically interact with each other. experiments indicate that Gal3p binds to Gal80p, you don't have a mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. in the $gal3$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), and that prevents you from doing further genetic analysis. How can I make a mutationplugin-autotooltip__default plugin-autotooltip_bigMutation: a change in the DNA of a gene that results in a change of phenotype compared to a reference wildtype allele. See also: mutant. in the $gal3$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) if all I know is the DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. sequenceplugin-autotooltip__default plugin-autotooltip_bigSequence: the precise order of monomers in a polymer. In DNA, it refers to the order of G, A, T, and C nucleotides. In RNA, it refers to the order of G, A, U, and C nucleotides. In proteins, it refers to the order of amino acids. of $gal3$?

We first build a targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. made from dsDNAplugin-autotooltip__default plugin-autotooltip_bigDouble-stranded DNA (dsDNA): DNA that consists of two complementary strands of ssDNA paired together via hydrogen bonds between the nitrogenous bases G, A, T, and C. in vitroplugin-autotooltip__default plugin-autotooltip_bigIn vitro: taking place outside of a living organism, usually in a test tube or Petri dish. (Fig. 10). Importantly, the targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. will not be part of a plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors.; therefore, it cannot replicate or segregate in yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 cells going through mitosisplugin-autotooltip__default plugin-autotooltip_bigMitosis: a type of cell division that generates two genetically identical daughter cells.. This targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. will consist of the cloned $gal3$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), but with only the start and the end of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) present (usually around 30 bp on either end is sufficient for yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02); the middle of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) will have been replaced with a selectableplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a markerplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs).. There are many different kinds of selectableplugin-autotooltip__default plugin-autotooltip_bigSelection: There are two distinct but somewhat related definitions for this term:

In model organism research, a selection is a process through which a researcher is attempting to find rare individuals with certain phenotypes and has some way of enriching for the rare individuals by killing off all other individuals that do not match the search criteria. Contrast to a markersplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). one can choose; we will use the yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 $URA3$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) in our example. $URA3$ is an auxotrophicplugin-autotooltip__default plugin-autotooltip_bigAuxotroph: a mutant that cannot synthesize one more essential nutrient from minimal media needed for survival or proliferation. markerplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs).; $URA3$ is required to synthesize uracilplugin-autotooltip__default plugin-autotooltip_bigNitrogenous bases: ringed chemical structures that are part of nucleotides. They include adenine (A), guanine (G), thymine (T), and cytosine (C) in DNA, and uracil (U) that substitutes for thymine in RNA., which is an essential metabolite for yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02. $ura3$ mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. cannot grow on minimal mediaplugin-autotooltip__default plugin-autotooltip_bigMinimal media: growth media that only contains (other than some kind of carbon source as food) inorganic nutrients, usually some nitrogen and phosphorous containing salts. unless it is supplemented with uracilplugin-autotooltip__default plugin-autotooltip_bigNitrogenous bases: ringed chemical structures that are part of nucleotides. They include adenine (A), guanine (G), thymine (T), and cytosine (C) in DNA, and uracil (U) that substitutes for thymine in RNA.. We will transformplugin-autotooltip__default plugin-autotooltip_bigTransformation: in microbiology (including for yeasts), transformation is the alteration of phenotype due to uptake of external DNA into a cell. Not to be confused with “transformation” in the context of cell biology, where the definition is the alteration of a normal cell to a cancerous cell. $ura3$ mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 with our targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. and grow the cells from the transformationplugin-autotooltip__default plugin-autotooltip_bigTransformation: in microbiology (including for yeasts), transformation is the alteration of phenotype due to uptake of external DNA into a cell. Not to be confused with “transformation” in the context of cell biology, where the definition is the alteration of a normal cell to a cancerous cell. experiment on media that lacks uracilplugin-autotooltip__default plugin-autotooltip_bigNitrogenous bases: ringed chemical structures that are part of nucleotides. They include adenine (A), guanine (G), thymine (T), and cytosine (C) in DNA, and uracil (U) that substitutes for thymine in RNA..

In principle, only cells in which the targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. has integrated into the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. somehow will be Ura⁺. Transformantsplugin-autotooltip__default plugin-autotooltip_bigTransformant: a bacterial or yeast cell that has been transformed with a foreign piece of DNA. (i.e., yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 cells that started as Ura^- but wound up as Ura⁺) might have the targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. integrated into a random location in the yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 genomeplugin-autotooltip__default plugin-autotooltip_bigGenome: a dataset that contains all DNA information of an organism. Most of the time, this also includes annotation and curation of that information, e.g., the names, locations, and functions of genes within the genome. As an adjective (“genomic”), this usually is used in the context of, or they could have the targeting constructplugin-autotooltip__default plugin-autotooltip_bigTargeting construct: a piece of DNA engineered in vitro that is used to make a knockout allele. integrate into the $gal3$ locusplugin-autotooltip__default plugin-autotooltip_bigLocus (plural form: loci): a physical location of a gene; often used as a synonym for a gene. through homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species.. You can distinguish between these two outcomes using PCRplugin-autotooltip__default plugin-autotooltip_bigPolymerase chain reaction (PCR): An experimental technique invented by Kary Mullis used to exponentially amplify DNA in vitro. PCR made obtaining large quantities of DNA for analysis much faster and easier than using traditional cloning methods.; one benefit to using yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 for this kind of experiment is that homologous recombinationplugin-autotooltip__default plugin-autotooltip_bigHomologous recombination: DNA recombination between two pieces of DNA that have a high degree of homology (i.e., nearly but not necessarily perfectly identical to each other). The length of DNA sequence homology needed for homologous recombination varies from species to species. is very efficient in yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02, so most transformantsplugin-autotooltip__default plugin-autotooltip_bigTransformant: a bacterial or yeast cell that has been transformed with a foreign piece of DNA. will be from targeting events. Integration into the $gal3$ locusplugin-autotooltip__default plugin-autotooltip_bigLocus (plural form: loci): a physical location of a gene; often used as a synonym for a gene. would result in the replacement of most of the $gal3$ coding sequenceplugin-autotooltip__default plugin-autotooltip_bigCoding sequence: refers to the portion of DNA or mRNA in a gene that contains direct information on the gene product. In most cases, this means a portion of DNA or mRNA that correlates to codons. Note that not all parts of a gene will necessarily be coding sequence (e.g., intron sequences). with URA3, effectively deleting $gal3$ and creating a knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. alleleplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence.. We usually write these kinds of mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. allelesplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence. as $\Delta gal3::URA3$ to indicate that it is a deletion ($\Delta$) where the original geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) ($gal3$) has been replaced with a markerplugin-autotooltip__default plugin-autotooltip_bigMarker: an allele of a gene that provides an easily observable phenotype. Markers are usually cloned or least well mapped. They are used as genetic landmarks in various genetic experiments. In some cases, markers do not have easily observable phenotypes and can only be detected using molecular methods (e.g., SNPs or SSRs). ($URA3$). In the vast majority of cases, knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. allelesplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence. are considered to be nullplugin-autotooltip__default plugin-autotooltip_bigAmorphic mutation: assuming a wildtype allele has 100% gene function, an amorphic mutation is a mutant allele that has 0% gene function. Also called a null mutation or a complete loss of function mutation. allelesplugin-autotooltip__default plugin-autotooltip_bigAllele: a version of a gene. Alleles of a gene are different if they have differences in their DNA sequence. (i.e., a complete loss of functionplugin-autotooltip__default plugin-autotooltip_bigLoss of function: a general term used to describe mutant alleles that have less activity than wildtype. Amorphic and hypomorphic mutations are loss of function mutations.). Once you have a knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics. mutantplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference., you can examine its phenotypeplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism. to see if there are any defects. In this case, you would probably want to see if $gal3$ mutantsplugin-autotooltip__default plugin-autotooltip_bigMutant: an individual that has a different phenotype than wildtype and likely contains one more mutations that cause this difference. are defective for $GAL1$ inductionplugin-autotooltip__default plugin-autotooltip_bigInducible: a term describing a pattern of gene expression, wherein genes or operons are not expressed until some kind of condition is met, e.g., inducer is present. Not all genes are naturally inducible; some genes are naturally constitutive.; you could measure this directly, or you could use a $lacZ$ reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. described earlier this chapter.  

In the last few chapters, we have looked at one particular regulatory network in S. cerevisiae and have employed a wide range of tools to understand this network. Although different organisms have different biological properties and therefore are studied using different techniques, these basic ideas and strategies can be used to genetically study almost any phenomenon in other eukaryoticplugin-autotooltip__default plugin-autotooltip_bigeukaryote: organism whose cells have membrane bound organelles, including the nucleus. organisms.

Conceptual question: Why is it advantageous to use a $lacZ$ (or similar) reporterplugin-autotooltip__default plugin-autotooltip_bigReporter gene: a gene whose gene product has an easily observed function and that can be used to indirectly measure the level of transcription determined by a promoter or other cis-acting regulatory elements. to study the regulation of geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) expressionplugin-autotooltip__default plugin-autotooltip_bigExpression: a term used to describe the idea that the function of a gene is apparent and can be observed. Genes may not always be expressed all the time in all places.?

Conceptual (challenge) question: Having learned about the various tools that can be used to study geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) function in this chapter, can you come up with an idea as to how Mig1 was discovered as a GAL<sub>URSplugin-autotooltip__default plugin-autotooltip_bigUpstream repressor sequence: In a general sense, a URS is a cis-acting regulatory element, usually located significantly upstream of the promoter and TATA box, to which repressors bind to silence transcription of nearby genes.

Although the term “URS” can in principle be used to describe any cis-acting upstream sequence that activates transcription, in practice the term silencer is used more often to describe these $GAL1$</sub> binding proteinplugin-autotooltip__default plugin-autotooltip_bigProtein: a molecule that is formed by the translation of messenger RNAs (mRNAs). Functions that proteins provide are what usually give organisms their phenotypes.?

Exercise 1: We discussed three different experimental strategies in this chapter: deletion mappingplugin-autotooltip__default plugin-autotooltip_bigGenetic mapping: a term describing a variety of different experimental approaches used to determine the physical locations of genes on chromosomes., yeastplugin-autotooltip__default plugin-autotooltip_bigYeast: in this book, refers to Saccharomyces cerevisiae, a single-celled eukaryotic microbe used as a model genetic organism. See Chapter 02 two hybridplugin-autotooltip__default plugin-autotooltip_bigTwo hybrid system: an experimental procedure, usually performed in yeast, that leverages our knowledge of how transcription works in the $GAL4$-UAS system. It is a general test that can be used to determine if two proteins physically interact with each other., and geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) knockoutplugin-autotooltip__default plugin-autotooltip_bigKnockout: a knockout allele is a null allele generated through reverse genetics.. In your own words, write what the purpose is for each of these strategies: what can you learn with these experimental approaches?