TNBGGA: The No Bullshit Guide to Genetic Analysis

First, let us look at how the genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) and genomesplugin-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 of some eukaryoticplugin-autotooltip__default plugin-autotooltip_bigeukaryote: organism whose cells have membrane bound organelles, including the nucleus. organisms compare 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). at one extreme, and humans at the other (Fig. 1).

Let’s first think about the number of genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) in an organism and the size of the organism’s 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. The average 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 about 300 amino acidsplugin-autotooltip__default plugin-autotooltip_bigAmino acid: molecules that are polymerized to form proteins. long, requiring 300 triplet codonsplugin-autotooltip__default plugin-autotooltip_bigCodon: a three nucleotide sequence that is read by the ribosome and specifies an amino acid that is added to a growing poplypeptide chain based on the genetic code., or 900 bp of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.. Let's just round up to 1 Kb of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. to make the math easier. Thus, it makes sense that to encode 4,200 genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), 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). requires a 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 of 5 million base pairsplugin-autotooltip__default plugin-autotooltip_bigBase pair: a term used to describe how nitrogenous bases (G, A, T/U, and C) in nucleic acids interact with each other via hydrogen bonds to form double-stranded molecules (including dsDNA, dsRNA, and DNA/RNA hybrids). G always pairs with C, and T/U always pairs with A. (4,200 x 1000 = 4.2 million; that's pretty close to 5 million). Most of 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 consists of geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) 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). (using our rough estimates, 84% of 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 is geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) 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).); 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). 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 is “geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) dense”.

By comparison, the human 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 has about 22,500 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 :-), so by the same logic the human 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 should be about 22 million (2.2×10⁷) bp (let's temporarily ignore the fact that the average size of a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) may be different, since we're just doing back of the envelope math here). In fact, humans have a haploidplugin-autotooltip__default plugin-autotooltip_bigHaploid: a term that describes a cell or organism that has only one copy of genetic information. Haploid cells typically arise from meiosis (or mitosis of a haploid mother cell). 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 that is ~3,000 million bp (~3,000 Mbp, or ~3 billion (3×10⁹) bp). In other words, there is about 100-fold more DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. in the human 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 than is required for 22,500 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 :-). The human 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 is far less “geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) dense” than 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). 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; and in fact, most vertebrate genomesplugin-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 are similar. What is all this extra DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. doing? Some of it constitutes 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. usually 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 each geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), and some is structural DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. around centromeresplugin-autotooltip__default plugin-autotooltip_bigCentromere: a locus on a chromosome that marks where spindle fibers attach during mitosis and meiosis. and telomeres (the end of chromosomesplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins.). Most of it is simply intergenic regionsplugin-autotooltip__default plugin-autotooltip_bigIntergenic region: a region of DNA that sits between genes and does not contain other genes or coding sequences. Integenic regions usually contain various cis-acting regulatory elements that control the expression of nearby genes. (non-coding regions between genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-)). Some of the DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. is present as intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns..

Intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns. are 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. inside genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that do not encode 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. but interrupt the 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).. This represents one of the fundamental organizational differences between prokaryoticplugin-autotooltip__default plugin-autotooltip_bigProkaryote: an organism that does not have membrane bound organelles. In this book prokaryotes refer to bacteria. and 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 :-)¹⁾. The DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. segments that are ultimately 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. as 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., i.e., 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. that contains triplet codonplugin-autotooltip__default plugin-autotooltip_bigCodon: a three nucleotide sequence that is read by the ribosome and specifies an amino acid that is added to a growing poplypeptide chain based on the genetic code. information, are called exonsplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons.. Using an enzymeplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. called the spliceosomeplugin-autotooltip__default plugin-autotooltip_bigSpliceosome: a large and complex enzyme located in the nucleus of eukaryotic cells that catalyzes intron splicing., the intronplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns. 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. are removed from the pre-mRNAplugin-autotooltip__default plugin-autotooltip_bigpre-mRNA: In eukaryotes, genes are transcribed as a longer RNA molecule called pre-mRNA, which is then processed via splicing, 5'-capping, and 3' polyadenylation before it is transported into the cytoplasm and becomes a mature mRNA. by splicingplugin-autotooltip__default plugin-autotooltip_bigIntron splicing: the act of remove introns and joining exons from pre-mRNA to form an mRNA. This occurs in the nucleus of eukaryotic cells and is catalyzed by an enzyme called the spliceosome. (Fig. 2).

A major consequence of this arrangement is the potential for alternative splicingplugin-autotooltip__default plugin-autotooltip_bigAlternative splicing: intron splicing where the splicing patterns are different from a default pattern, e.g., different exons can be skipped to produce spliced mRNAs with different sequences to produce different proteins. to produce different 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. from the same geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) and primary transcript. Alternative splicingplugin-autotooltip__default plugin-autotooltip_bigAlternative splicing: intron splicing where the splicing patterns are different from a default pattern, e.g., different exons can be skipped to produce spliced mRNAs with different sequences to produce different proteins. allows different mRNAsplugin-autotooltip__default plugin-autotooltip_bigmessenger RNA (mRNA): an RNA molecule that codes for protein. to be formed by joining different combinations of exonsplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. during splicingplugin-autotooltip__default plugin-autotooltip_bigIntron splicing: the act of remove introns and joining exons from pre-mRNA to form an mRNA. This occurs in the nucleus of eukaryotic cells and is catalyzed by an enzyme called the spliceosome.. This gives the potential for increasing the complexity of mammals and other eukaryotesplugin-autotooltip__default plugin-autotooltip_bigeukaryote: organism whose cells have membrane bound organelles, including the nucleus. through many more thousands of possible 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. from a relatively fixed number of genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (around 20,000 for most metazoanplugin-autotooltip__defaultMetazoan: of Kingdom Animalia. species). For example, Figure 2 shows a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) with 3 exonsplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. and 2 intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns. where the two intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns. are spliced out to give an mRNAplugin-autotooltip__default plugin-autotooltip_bigmessenger RNA (mRNA): an RNA molecule that codes for protein. that is formed by joining exonsplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. 1, 2, and 3. If this transcript were alternatively spliced, one possibility would be that exonplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. 2 is skipped so that exonsplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons. 1 and 3 are joined together after splicingplugin-autotooltip__default plugin-autotooltip_bigIntron splicing: the act of remove introns and joining exons from pre-mRNA to form an mRNA. This occurs in the nucleus of eukaryotic cells and is catalyzed by an enzyme called the spliceosome.. This results in a different mRNAplugin-autotooltip__default plugin-autotooltip_bigmessenger RNA (mRNA): an RNA molecule that codes for protein. 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 therefore a different 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 is translated; this different 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. likely has a different function than 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. made from the “fully” spliced mRNAplugin-autotooltip__default plugin-autotooltip_bigmessenger RNA (mRNA): an RNA molecule that codes for protein..

Note that lower eukaryotesplugin-autotooltip__default plugin-autotooltip_bigeukaryote: organism whose cells have membrane bound organelles, including the nucleus. such as 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 S. cerevisiae only have ~ 5% of their genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) interrupted by intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns., but for multicellular organisms like humans, >90% of all genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) are interrupted by anywhere between 2 and 60 intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns., with most genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) having between 5 and 12 intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns.. Intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns. tend to be longer than exonsplugin-autotooltip__default plugin-autotooltip_bigExon: the portion of a pre-mRNA that contains codons. pre-mRNAs usually contain multiple exons that are interspersed with introns. Formally, exons are defined as a component of pre-mRNA, but the DNA sequence that codes for exons can also be informally referred to as exons.. Thus, having intronsplugin-autotooltip__default plugin-autotooltip_bigIntron: sequences in a pre-mRNA that sit between exons and are removed by splicing. Formally speaking, introns are defined as being part of pre-mRNA, but the DNA sequence that codes for introns can also be informally described as introns. tends to make genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) much larger than what you might expect based on the size of 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.

In this chapter, we are interested in identifying 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 genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) for which their 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. changes in response to changes in the environment; we are also interested in identifying 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 genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that regulate this process. Although 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 is a eukaryoteplugin-autotooltip__default plugin-autotooltip_bigeukaryote: organism whose cells have membrane bound organelles, including the nucleus., it is also a unicellular microbe like 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).. Therefore, many experimental strategies for identifying 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 genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that work for 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). can also work 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. It helps that 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 also have plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors.²⁾! For example, the 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. approach (Chap. 09) works just as well 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 as it does 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..

However, in this chapter we will introduce a different way of 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 and identifying regulated genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) and their regulators 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. It is a slightly more complex strategy than vanilla 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., but it also allows us to find things that might be more difficult to find if we were to use just 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.. We are going to combine a few neat genetic tools for this, some of which you learned about in earlier chapters:

• A libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. 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 genomicplugin-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 fragments cloned into a bacterialplugin-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. plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors.. We learned about the concept of genomicplugin-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 libraries in Chap. 09.
• 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 :-). We learned about the $lacZ$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) in Chapters 09 and 10. In this experiment the $lacZ$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) is going to be used 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 as 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 :-) (sometimes just called 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.) 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. activity 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 genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). The $lacZ$ 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). works 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 because 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). 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 both use the exact same universal genetic codeplugin-autotooltip__default plugin-autotooltip_bigGenetic code: the code that matches codons with specific amino acids. Since each codon is 3 nucleotides long (i.e., the genetic code is a triplet code) and there are 4 different RNA nucleotides (G, A, U, and C), the genetic code could in theory specify up to $4^3=64$ different amino acids. But since there are only 20 different for converting triplet codonplugin-autotooltip__default plugin-autotooltip_bigCodon: a three nucleotide sequence that is read by the ribosome and specifies an amino acid that is added to a growing poplypeptide chain based on the genetic code. 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. into amino acidsplugin-autotooltip__default plugin-autotooltip_bigAmino acid: molecules that are polymerized to form proteins..
• A modified bacterialplugin-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. transposonplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus. called mini-Tn7 (Fig. 3). Transposonsplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus. are naturally occurring pieces of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. that can transpose, or jump around, to random locations in genomesplugin-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. We can modify transposonsplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus. such that we can experimentally control when they jump around, and we can also construct them to carry genetic 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). that help us track the transposonplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus.. In this experiment, we have engineered mini-Tn7 to contain the $lacZ$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (but without any 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. such as $lacO$ or $lacP$), 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 geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) called $URA3$ required for 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. prototrophyplugin-autotooltip__default plugin-autotooltip_bigPrototroph: an organism that can synthesize all nutrients it needs to survive and proliferate (such as amino acids or nucleotides) from inorganic molecules. (in this case we are including native 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. necessary 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 cells to express $URA3$), and 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). geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (plus appropriate bacterialplugin-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. 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.) that confers drug resistance to the antibioticplugin-autotooltip__default plugin-autotooltip_bigAntibiotics: naturally produced compounds, usually found in molds and fungi but are typically commercially available, that either kill bacteria or prevent their replication. In genetics research, they are often used to select for the presence of an episome that carries an antibiotic resistance tetracyclineplugin-autotooltip__default plugin-autotooltip_bigTetracycline: a common type of antibiotic used in genetics research. ($tet^R$).

The mini-Tn7 is introduced into a population of 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). that already contains a plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. of the S. cerevisiae 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. Each 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). cell in this libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. contains a plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. that contains a different segment of the S. cerevisiae 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, such that the 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 is represented many times over in this population of 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).. The mini-Tn7 is allowed to transpose by integrating into either the plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. or the bacterialplugin-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. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth.³⁾. The original DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. that carries the mini-Tn7 cannot replicate, but cells that have integrated the mini-Tn7 into a plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. or 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). chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. are selected as tetracyclineplugin-autotooltip__default plugin-autotooltip_bigTetracycline: a common type of antibiotic used in genetics research. resistant (Tet^R) coloniesplugin-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; cells that do not contain mini-Tn7 are killed by tetracyclineplugin-autotooltip__default plugin-autotooltip_bigTetracycline: a common type of antibiotic used in genetics research..

Plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. is purified from these transformantsplugin-autotooltip__default plugin-autotooltip_bigTransformant: a bacterial or yeast cell that has been transformed with a foreign piece of DNA. and retransformed into tetracyclineplugin-autotooltip__default plugin-autotooltip_bigTetracycline: a common type of antibiotic used in genetics research. sensitive 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)., and we use tetracyclineplugin-autotooltip__default plugin-autotooltip_bigTetracycline: a common type of antibiotic used in genetics research. selectionplugin-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 again. The resulting tetracyclineplugin-autotooltip__default plugin-autotooltip_bigTetracycline: a common type of antibiotic used in genetics research.-resistant 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. contain only plasmidsplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. that have an integrated mini-Tn7 transposonplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus.; clonesplugin-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 in which mini-Tn7 integrated into 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). chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. are removed by this step. Plasmidplugin-autotooltip__default plugin-autotooltip_bigPlasmid: a circular episome found in bacteria and yeast. Plasmids are commonly used as cloning vectors. DNAs are isolated from these cells and 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 genomicplugin-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 fragments are isolated by digestion with an appropriate restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences.. Most of these 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 genomicplugin-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 fragments should contain mini-Tn7 insertions (Fig. 4).

Now we have a libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. 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 genomicplugin-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 fragments each of which has the transposonplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus. inserted; these genomicplugin-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 fragments can be transformedplugin-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. into S. cerevisiae cells that are 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. for the $ura3$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (these cells are 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. auxotrophsplugin-autotooltip__default plugin-autotooltip_bigAuxotroph: a mutant that cannot synthesize one more essential nutrient from minimal media needed for survival or proliferation.). The method for 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. and preparing competent 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 is similar to that of preparing competent 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)., except that LiCl is used instead of CaCl₂ to treat the cells. Importantly, the fragments cannot replicate on their own, but they can integrate into the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. 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. that targets 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. on the chromosomeplugin-autotooltip__default plugin-autotooltip_bigChromosome: a structure that organizes dsDNA in a cell through interactions with various DNA binding proteins. that match the transformedplugin-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. fragments. After 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., we grow the cells on plates that lack 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 are 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 cells that have taken up a $URA3$-containing DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. fragment somewhere in its 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. Each Ura⁺ transformantplugin-autotooltip__default plugin-autotooltip_bigTransformant: a bacterial or yeast cell that has been transformed with a foreign piece of DNA. colonyplugin-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 that grows will have recombined a mini-Tn7-containing genomicplugin-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 DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. fragment into its 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. This essentially gives us a libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. 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 with transposonsplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus. randomly integrated into its 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.

Note that the $lacZ$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) in the transposonplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus. contains only its amino acidplugin-autotooltip__default plugin-autotooltip_bigAmino acid: molecules that are polymerized to form proteins. 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).. It does not have its own 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. from 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). such as $lacO$ and $lacP$ (and even if $lacO$ and $lacP$ were present, it would have no effect on $lacZ$ 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 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; think about that!). But if the transposonplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus. inserts 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 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 geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) 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 (somewhat rare) and in the correct orientation (1 in 2 chance), and in the correct triplet codonplugin-autotooltip__default plugin-autotooltip_bigCodon: a three nucleotide sequence that is read by the ribosome and specifies an amino acid that is added to a growing poplypeptide chain based on the genetic code. reading frame (1 in 3 chance), the $lacZ$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) would then come under the control of that 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. When 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. is activated from that 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, a LacZ fusion 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 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., and most LacZ 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. have robust β-galactosidase activity.

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 expressing β-galactosidase activity can easily be detected by growth in the presence of X-galplugin-autotooltip__default plugin-autotooltip_bigX-gal: an abbreviation for 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, a colorless chemical that turns blue when it is hydrolyzed by β-galactosidase. (we first saw X-galplugin-autotooltip__default plugin-autotooltip_bigX-gal: an abbreviation for 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, a colorless chemical that turns blue when it is hydrolyzed by β-galactosidase. in Chap. 08); recall that LacZ (β-galactosidase) cleaves X-galplugin-autotooltip__default plugin-autotooltip_bigX-gal: an abbreviation for 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, a colorless chemical that turns blue when it is hydrolyzed by β-galactosidase. to release a chemical moiety that has that has a brilliant blue color. 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 express LacZ (or a LacZ fusion 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.) will turn bright blue when stained with X-galplugin-autotooltip__default plugin-autotooltip_bigX-gal: an abbreviation for 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, a colorless chemical that turns blue when it is hydrolyzed by β-galactosidase.. Normal 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 do not have β-galactosidase, so normal 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 stained with X-galplugin-autotooltip__default plugin-autotooltip_bigX-gal: an abbreviation for 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, a colorless chemical that turns blue when it is hydrolyzed by β-galactosidase. remain white (the natural color 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).

In both theory and practice, you can create 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. using mutagensplugin-autotooltip__default plugin-autotooltip_bigMutagen: a chemical or source of ionizing radiation that has the potential to damage DNA such that the DNA sequence will be altered. directly 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 without going through all the trouble described above 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 them 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.. However, there are at least three useful things to come out a libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. of 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. created in this way:

1. Any transposonplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus. that integrated into a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) will essentially disrupt that geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) and is likely to generate a 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. 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. (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.). 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. 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 very useful!
2. For transposonsplugin-autotooltip__default plugin-autotooltip_bigTransposon: also called a “transposable element”, “jumping gene”, or “mobile genetic element”, a transposon is a gene that has the ability to move around a genome. Thus, unlike most genes, transposons do not have a fixed locus. that integrate such that the $lacZ$ geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) is in frame with the coding region of 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 geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-), the level of β-galactosidase (LacZ) activity in these cells therefore becomes an indicator) for the level of 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 that geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-). We call $lacZ$ in this context 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 :-) (sometimes we abbreviate that to just “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.”).
3. This kind of insertion libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. approach allows you to use tricks like inverse 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. (see Fig. 8) to help make 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 your geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) easier (this method is much easier than 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.).

Here are two examples of how such a libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. can be used:

1. to identify genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that protect cells against a DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. damaging agent that causes cancer. Let's take the example of one of the many compounds found in tobacco smoke; and
2. to identify genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) whose 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. is upregulated in response to being exposed to this tobacco smoke chemical.

The chemical we will use as an example is 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK). 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 random mini-Tn7 insertions are first plated out on Petri dishesplugin-autotooltip__default plugin-autotooltip_bigPetri dish: a round dish, usually 5-10 cm in diameter, that can contain growth media to grow cells in vitro. at a low density so that individual cells each give rise to single coloniesplugin-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 on the agar surface. Each colonyplugin-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 represents a 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 that contains a unique insert. To 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. the libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. for genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that protect against NNK-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. cell killing, the coloniesplugin-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 are replica platedplugin-autotooltip__default plugin-autotooltip_bigReplica plating: a technique in microbiology that allows you to duplicate colony patterns on different Petri dishes such that clones can be easily tested on different kinds of selective media. onto agar medium that either does or does not contain a high dose of NNK. Replica platingplugin-autotooltip__default plugin-autotooltip_bigReplica plating: a technique in microbiology that allows you to duplicate colony patterns on different Petri dishes such that clones can be easily tested on different kinds of selective media. is a technique where coloniesplugin-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 on one Petri dishplugin-autotooltip__default plugin-autotooltip_bigPetri dish: a round dish, usually 5-10 cm in diameter, that can contain growth media to grow cells in vitro. are copied to a different Petri dishplugin-autotooltip__default plugin-autotooltip_bigPetri dish: a round dish, usually 5-10 cm in diameter, that can contain growth media to grow cells in vitro. while maintaining their positions in the dish, so that we can the observe the same 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 under different growth conditions. To 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. the libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. for genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that are transcriptionallyplugin-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. regulated in the presence of NNK, the coloniesplugin-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 are replica platedplugin-autotooltip__default plugin-autotooltip_bigReplica plating: a technique in microbiology that allows you to duplicate colony patterns on different Petri dishes such that clones can be easily tested on different kinds of selective media. onto agar medium containing either X-galplugin-autotooltip__default plugin-autotooltip_bigX-gal: an abbreviation for 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, a colorless chemical that turns blue when it is hydrolyzed by β-galactosidase. alone or X-galplugin-autotooltip__default plugin-autotooltip_bigX-gal: an abbreviation for 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside, a colorless chemical that turns blue when it is hydrolyzed by β-galactosidase. plus a low dose of NNK.

Interesting coloniesplugin-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 can be retrieved from the master plate for further study and for identification and subsequent 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 of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) responsible for the interesting phenotypeplugin-autotooltip__default plugin-autotooltip_bigPhenotype: an observable feature or property of an organism.. If you used an insertion libraryplugin-autotooltip__default plugin-autotooltip_bigLibrary: in genetics research, a library refers to a collection of clones representing a set of genetic information, e.g., a genomic library or a cDNA library. to generate your 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 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., you can use various molecular biologyplugin-autotooltip__default plugin-autotooltip_bigMolecular biology: the study of nucleic acids, specifically DNA and RNA. tricks to 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 your genesplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (instead of 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.).

One such trick is called inverse 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., which doesn't give you a full length 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 of your geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) of interest the way 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. does, but gives you a way to quickly determine the molecular identity of the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) (see Chap. 07 to review 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.). To do inverse 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., you would purify genomicplugin-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 DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. from an insertion 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. and cut it with a restriction enzymeplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. that does not cut anywhere inside the insertion (the red segment in Figs. 5 and 6). Since restriction enzymesplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. cut at predictable intervals of DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth., this must mean that the restriction enzymesplugin-autotooltip__default plugin-autotooltip_bigRestriction enzyme: an type of enzyme, usually commercially available, that is used to cleave (restrict) dsDNA at specific 4, 6, or 8 (less common) bp recognition sequences. will cut somewhere outside of the insertion (the blue portions in Figs. 5 and 6). If you take the digested genomicplugin-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 DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. and ligate (join) the ends using an enzymeplugin-autotooltip__default plugin-autotooltip_bigEnzyme: a macromolecule, usually a protein (but sometimes an RNA), that functions as a catalyst of some kind of biochemical reaction. called DNA ligaseplugin-autotooltip__default plugin-autotooltip_bigDNA ligase: an commercially available enzyme used to form phosphodiester bonds between the 3' end of one strand of dsDNA and the 5'end of another. Used for molecular cloning experiments. Its natural function in cells relates to lagging strand DNA synthesis and various DNA repair mechanisms., then you will have created DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. circles. You can then use primersplugin-autotooltip__default plugin-autotooltip_bigPrimer: (1) A short (usually 15-30 nt long, depending on application) piece of ssDNA that is synthesized in vitro and used in applications such as DNA sequencing and PCR. In this context, primers can also be called oligonucleotides. (2) a free 3'-OH end on a nucleic acid that can be used for initiating that start in the middle of the insertion (for which you know the 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.) but point away from each other rather towards each other. This allows you to 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. amplify 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 flanks the insertion site, which includes the 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 the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that was disrupted. You can then 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. this 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. product using Sanger or Nanopore 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 (Chap. 07). This allows you to at least partially determine 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 the geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that is disrupted by your insertion (Fig. 9). Since there is already a 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 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. 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, even a partial 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 your disrupted geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) will give you enough information to find the full-length 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 your geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) just by searching through 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 database.

Once we have identified a geneplugin-autotooltip__default plugin-autotooltip_bigGene: read Chapters 02, 03, 04, 05, and 06 for a definition of gene :-) that is transcriptionallyplugin-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. upregulated in response to an environmental change (such as the presence of NNK), how can we use genetics to figure out how regulation is achieved? This is the topic of the next chapter, although we will study galactose 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 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. instead of NNK.

Conceptual question: should the $URA3$ DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. fragment within the mini-Tn7 construct contain its own 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? How about the $tet^R$ DNAplugin-autotooltip__default plugin-autotooltip_bigDNA: deoxyribonucleic acid. The genetic material for nearly all life on Earth. fragment? If they did contain their own promotersplugin-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, would they 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 or 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). promotersplugin-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?

Conceptual question: why is ligation into circles necessary for reverse 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.?

Exercise 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 cells are killed by high concentrations of NNK. Design an experiment to 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 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 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 are resistant to high concentrations of NNK.