Chapter 14

• Mutagenesis

A procedure whereby a population of organisms is mutagenized and their progeny are propagated and examined for mutant specific phenotypes. See also genetic screen.

• RNA interference (RNAi)

A regulatory gene-silencing mechanism based on double-stranded RNA, which can target complementary sequences for inactivation. The machinery can be harnessed to silence gene expression in a reverse genetic approach.

What organisms are haploid?

Bacteria, some euk like yeast

CRISPR

Clustered Regularly Interspaced Short Palindromic Repeats. Transcribed repetitive sequence that is processed into unique crRNAs acting in a bacterial or archael immune system.

• How can complementation testing allow scientists to identify the number of gene isolated in a screen?

Determining the numbers of genes identified and the number of alleles for each gene • Determine the number of genes by complementation testing ---• Define complementation groups! ---• If progeny produced by crossing two mutant alleles are wild-type, the mutant alleles complement, that is, affect two different genes ---• If progeny of two mutant alleles are mutant, the mutations affect the same gene (fail to complement) • Comparing the phenotypes of multiple alleles of a gene allows determination of the range of phenotypic variation possible for the gene in question

• Reverse genetics

Genetic analysis that begins with a gene sequence, which is used to identify or introduce mutant alleles and subsequently to identify and evaluate the resulting mutant phenotype. It is the complementary approach to forward genetics.

Genome Editing

Genome Editing • Precisely changing a nucleotide sequence at a specific locus to a desired sequence • DNA endonuclease targets the location and creates a double-strand break at the target site, which is fixed through repair using nonhomologous end joining (NHEJ) or homologous recombination ---• NHEJ repair: small deletions often remain at the break site, with possible loss or gain-of-function ---• HR: an exogenously supplied DNA sequence can contain the desired nucleotides

Knock-in genetic modification

functioning gene inserted into animal cells (aka gene transfer)

• Ectopic expression

gene expression that occurs outside the cell or tissue where the gene is normally expressed

3. When mutant alleles are isolated in forward genetic screens, they must be ___.

mapped and sequenced.

• Why don't scientists typically use whole genome sequencing as the first approach when trying to identify the genes affected in mutant alleles obtained from a forward genetic screen?

• Comparing mutant and wild-type gene sequences is not always straightforward ---• May be easier if complementation performed first to find region of mutation that it might exist Genome Sequencing • Comparing mutant and wild-type gene sequences is not always straightforward ---• May be easier if complementation performed first • Causative mutations must be distinguished from polymorphisms • Mutagenesis can produce hundreds of new mutations • Many mutant strains are back crossed with wild-type strains to separate the one causative mutation from other mutations

• What different reporter genes are discussed? How is each detected?

• Depends on the purpose of the experiment • lacZ gene produces β-galactosidase, which can cleave colorless substrates ONPG and X-gal, to produce yellow and blue products, respectively • Luciferase, from fireflies, catalyzes a reaction between luciferin and ATP that results in emitted light • Green fluorescent protein, from the jellyfish Aequoria victoria, is a source of natural bioluminescence ---• Gene has been modified so that the protein will respond to lower energy wavelengths, rather than UV light ---• Variant alleles emit different colors and allow several proteins to be visualized simultaneously

• Explain how complementation using a genomic or cDNA library can help to map the genes identified in a forward genetic screen.

• Genomic libraries: collections of cloned DNA fragments that represent the entire genome of an organism, including repetitive and noncoding sequences • Complementary DNA libraries (cDNA libraries): represent only a portion of the genome and are collections of cloned DNA fragments that represent mRNA produced by an organism or cell type • In both types of libraries, fragments are carried within cloning vectors Complementation Analysis • Genes can be identified by complementation of a mutant phenotype by introduction of a wildtype gene ("rescue" complementation) ---• This is not the same as deletion mapping! • Can also be used to identify the corresponding gene from another organism ---• This type of "rescue" experiment is also used to confirm functional homology

• Transgene

A gene that has been modified in vitro by recombinant DNA technology and introduced into the genome via transformation.

• What considerations should be made when choosing an organisms to use for reverse genetics?

Have to know where gene is Maybe more

• Luciferase

enzymes that produce light

5. Transgenes can serve as ___.

reporters to study gene function.

• Balancer chromosome

A chromosome with inversions used to maintain specific allele combinations (e.g., recessive lethal alleles) in genetic stocks.

• lacZ

A gene of the bacterial lac operon; encodes β-galactosidase, which breaks down lactose into glucose and galactose. Compare with lacY gene and lacA gene.

• Chimeric gene fusion

A gene sequence composed of sequences from two or more sources. Many chimeric genes form through errors in DNA replication or DNA repair so that pieces of two different genes are inadvertently combined. Chimeric genes can also form through retrotransposition where a retrotransposon accidentally copies the transcript of a gene and inserts it into the genome in a new location.

• Reporter gene

A gene whose expression is easy to assay phenotypically. Fusion of reporter genes with heterologous sequences allows both transcriptional and translational expression patterns to be visualized.

• Enhancer trap

A transgenic construct inserted randomly into the genome that allows identification of enhancer elements controlling specific patterns of gene expression.

14.4: Transgenes Provide a Means of Dissecting Gene Function • What information can be gained from a reporter gene?

Although an almost limitless array of transgenes can be constructed for genetic analysis, many fall into one of two categories. One category consists of reporter genes, used to investigate gene regulation because they produce a visual output of gene expression patterns. Fusion of the regulatory sequences of a gene of interest to coding sequences of a reporter gene provides information about where, when, and how much a gene is expressed. Some reporter genes facilitate live imaging and monitoring of gene expression in real time.

• What are conditional alleles? What kinds of conditions are associated with the permissive vs. restrictive condition?

Are those in which the gene product is either functional or not needed under one condition—the permissive condition—but is required and either absent or inactive under another condition—the restrictive condition

cas genes

CRISPR-associated genes. Cas proteins act as the catalytic component (endonuclease) of the CRISPR-Cas complex, creating double-strand breaks in target DNA molecules. In Staphylococcus, a single protein, Cas9, is sufficient.

• Cre-lox system

Can inducibly manipulate genes at specific developmental points (eg, to study a gene whose deletion causes embryonic death).

• Knockout libray

Collections of mutants in which most or all genes of a particular organism have been mutated by inactivating (or "knocking out") their expression.

• CRISPR-Cas9

Complex of the Cas9 protein with tracrRNA and crRNA that acts to target invading nucleic acids in Staphylococcus. This system has been modified for use in gene editing.

• How is the Cre-lox system used to generate conditional/tissue-specific recombination? o Why advantage does this have over traditional/whole animal knock-out? o How is tissue-specificity achieved? MAYBE LOOKUP BUT MAY HAVE SAID IN LAST CHAPTER

Cre-Lox system: Conditional/Tissue-Specific Knockout • Cre recombinase under control of a tissue- and/or stagespecific promoter

• What information is gained from enhancer trapping? o What is the "trap" associated with this method? o What is evaluated by the detection of the reporter?

Enhancer trapping uses a variation of an insertional library to identify genes based on expression patterns. This approach combines the generation of a large number of random insertion mutants with the expression of a reporter gene (Figure 14.17). In its simplest application, a population of transgenic organisms is generated by random insertion of a transposon (or T-DNA) containing the coding sequence of a reporter gene fused with a core promoter region for RNA polymerase II transcription (see Section 13.1). If the insertion occurs near enhancer or silencer regulatory sequences that can act in conjunction with the minimal promoter of the reporter gene, the reporter can be expressed in a pattern that reflects the regulatory capability of the nearby genomic DNA sequences. The enhancers (or silencers) of the adjacent genomic DNA are co-opted, or "trapped," by the insertion to drive expression of the reporter gene. Thus, from the expression patterns of the inserted reporter gene, researchers can infer the existence of regulatory sequences, presumably from adjacent genes, that drive gene expression in the observed patterns. While reporter gene expression may not precisely reflect the expression of the adjacent gene, the expression of the reporter often at least partially reflects the normal gene expression pattern of the adjacent gene. Enhancer trapping techniques were first pioneered in Drosophila and have now been adapted to other systems. Because they identify genes by gene expression patterns, enhancer trapping techniques complement forward genetic screens. • Enhancers: cis-regulatory sequences; can be several kb away from the gene(s) they affect • Uses an insertional library to identify genes based on their expression pattern • Reporter gene is fused with a minimal promoter • If insertion occurs near enhancer and silencer regulatory sequence, the reporter will be expressed in the corresponding pattern

• Gene knockout

Loss-of-function allele of a gene usually obtained via a reverse genetic approach.

• How can reporters fused to regulatory sequences allow for refined mapping of structure-function analysis of gene expression?

Reporter genes can be used to dissect regulatory DNA sequences and identify specific sequences required for particular aspects of gene regulation. The general approach is to start with a clone in which all the regulatory sequences required for proper gene expression are present and then to assay the effects of deleting or changing specific portions of the clone. An example of such an analysis of the Drosophila even-skipped (eve) gene, which is expressed in seven stripes in the segmentation pattern of the embryo, is shown in Figure 14.16. Overlapping deletions spanning large regions are assayed first. Then regions identified as important for gene regulation are dissected with smaller deletions. The concept is similar to that described earlier for deletion mapping (see Sections 6.5 and 10.4). When specific sequences required for proper gene expression are deleted, expression of the reporter gene will be correspondingly altered. If genomic sequence is available from two or more related species, regulatory elements may be predicted by searching for sequences that are conserved between the related species, using a method known as phylogenetic footprinting (discussed in Chapter 16). Such initial genomic sequence analyses can direct subsequent experimental tests that use reporter genes to analyze expression in transgenic organisms. • Regulatory sequences can be better understood with reporter genes • Full regulatory sequence fused to the reporter gene vs. smaller segments of regulatory sequence fused to reporter • Determine the effect on the expression pattern • Structure-function analysis

• Forward genetic screen

The classical approach to genetic analysis whereby genes are first identified by mutant phenotypes caused by mutant alleles and the gene sequence is subsequently identified by recombinant DNA technologies. Also known as forward genetics.

• Replica plate

The microbial method for transferring all the growing colonies from an original growth plate to one or more new growth plates.

• Once you've decided on what model organism to use, what considerations should be made about choosing a reverse genetics approach?

The reasons for shifting toward reverse genetics are twofold. First, the enormous amount of genomic sequence available has increased by orders of magnitude the number of known gene sequences, and only a fraction of them have been assigned a function by forward genetics. For example, when the E. coli genome was fully sequenced, 4288 protein-coding genes were identified, only 1853 of which had been previously identified through forward genetic screens. Second, genomic sequencing and reverse genetic screens have uncovered a degree of gene duplication not previously suspected. Gene duplications often result in genetic redundancy. In forward genetic screens, such duplicated genes would not be identified, since mutation of only one of the genes would not usually result in a conspicuous mutant phenotype. However, reverse genetics approaches, where the functions of both duplicates can be disrupted in an individual organism, are particularly suited in these situations to provide evidence of gene function. Reverse genetics begins with the creation of a mutant allele for a gene identified only by its sequence (see Figure 14.1). The selection of mutational tools is largely dependent on the biology of the experimental organism. We describe here four technologies for reverse genetics, including one that is presently revolutionizing the field of genetics.

• Synthetic lethality

The situation where a particular double mutant results in lethality but the two respective single mutants are viable.

• Genetic redundancy

The situation where the functions of one gene are compensated for by the actions of another gene.

14.3: Reverse Genetics Investigates Gene Action by Progressing from Gene Identification to Phenotype • What are the goals associated with reverse genetics?

We know the gene that we are interested in studying and we want to make mutants that are going to allow us to assess the mutant phenotype • Begins with a gene sequence and then seeks to identify an associated mutant phenotype • Forward genetic screens may miss genes due to genetic redundancy • Large amount of genomic sequences now available • Also available in genomic libraries

• Protein fusion

a gene fusion in which two coding sequences are fused so that they share the same transcriptional and translational start sites

• Gene editing

a highly precise type of genetic engineering in which DNA is inserted, deleted or replaced in the genome of an organism using engineered nucleases

• Gene fusion

a structure created by joining together segments of two separate genes, in particular when the regulatory region of one gene is joined to the coding region of a reporter gene

• Beta-galactosidase

an enzyme that catalyzes the conversion of lactose into monosaccharides

• Conditional allele

expresses a wild-type (normal) phenotype under certain (permissive) conditions and a mutant phenotype under other (restrictive) condition

1. Forward genetic screens allow for the ___.

identification of genes not previously known to be involved in a particular process.

• Translational fusion

regulatory and coding sequences are fused to the reporter gene so that the protein produced can be visualized

• Transcriptional fusion

regulatory sequences for a gene of interest are fused to the reporter gene, which will then be expressed in the pattern dictated by the regulatory sequences

• What are the general differences (in structure and in consequence) between shRNA, siRNA, and miRNA?

shRNA folds into hairpin, initiates RNAi The siRNA is an exogeneous double-stranded RNA uptaken by the cell, generally, are viral RNAs, it is also encoded by heterochromatin regions and transposons. Whereas the miRNA are endogenous single-stranded, non-coding RNA molecule, by forming a hairpin structure, it becomes duplex. Both miRNAs and siRNAs regulate gene expression by annealing to mRNA sequence elements that are partially or fully complementary. ... In animals, that potential is manifested in multiple ways: by reductions, or sometimes increases, in translation efficiency and by diminished mRNA stability.Jan 18, 2008

• shRNA

short hairpin RNA

• Transgenic

term used to refer to an organism that contains genes from other organisms

• Transgenesis

the introduction of an exogenous or outside gene into an organism; alter genotype of an animal so that researchers can study the effect of a gene

2. Synthetic lethality and genetic redundancy allow us to ___.

understand gene interactions.

• What is the general difference between homologous recombination and illegitimate recombination?

• Integrating an exogenous DNA fragment into the genome can create a mutant allele or can be used to "tag" a gene ---• Deleting the gene of interest creates a loss-of-function allele • Illegitimate recombination: integrates at a random, nonhomologous location; more common in plants and animals • Homologous recombination: requires a significant length of DNA sequence in common between the two recombining molecules; more common in bacteria and fungi

• Green fluorescent protein (GFP)

A gene, derived from the jellyfish Aequoria victoria, that is the source of the natural bioluminescence of this species, fluorescing green (a 509-nm wavelength) when illuminated with UV light (a 395-nm wavelength). When used as a reporter gene, GFP allows a noninvasive means of visualizing gene and protein expression patterns in living organisms.

• Genetic modifier screen

A genetic screen designed to identify mutations in genes that modify, either enhance or suppress, the phenotypic effects of mutations in another gene.

• Enhancer screen

A genetic screen designed to identify mutations in genes that worsen the phenotypic effects of mutations in another gene.

• Suppressor screen

A modifier genetic screen designed to identify mutations in genes that suppress the phenotypic effects of mutations in another gene.

• What information is learned from a chimeric gene fusion? o What is learned from ectopic expression?

Chimeric Gene Fusions • Cause any gene to be driven by any regulatory sequences that will function in the host organism • This is called "ectopic expression" • Example: recessive loss-of- function mutant alleles of eyeless gene in Drosophila result no eyes ---• If the eyeless gene is expressed in leg or antennal tissues in larva, eyes will grow in place of the legs or antennae normally produced by the discs A chimeric gene, as mentioned earlier, is one in which regulatory and coding sequences derived from two or more different genes are recombined in a novel manner. For example, combining the regulatory sequences from one gene with the coding sequences from another gene often results in a gain-of-function allele due to ectopic expression of the gene represented by the coding sequences. Figure 14.18 shows one way experimenters can take advantage of this potential to obtain information on gene function. This example makes use of the eyeless gene of Drosophila, so named because recessive loss-of-function mutations in this gene result in a failure of eyes to develop in the fly. The eyeless gene is normally expressed in the eye imaginal discs during Drosophila development. Imaginal discs are groups of precursor cells that are set aside during embryonic development. They grow by mitotic proliferation during larval life and later differentiate into adult body tissues during metamorphosis. However, a gain-of-function eyeless allele can be created by constructing a chimeric gene in which expression of the eyeless coding sequences is driven by regulatory sequences active in all imaginal discs. If the eyeless gene is ectopically expressed in noneye imaginal discs, such as those that would normally give rise to the antennae or legs, the imaginal discs will differentiate as eye tissue instead. This outcome indicates that cells in any imaginal disc are capable of differentiating into eyes and that the eyeless gene product can promote the development of eyes from any imaginal disc. Thus, when the eyeless allele is ectopically expressed as a gain-of-function mutation in inappropriate imaginal discs, the resulting phenotype is the converse of the phenotype of the loss-of-function eyeless allele—ectopic eyes as opposed to an absence of eyes. In cases where the gain-of-function and loss-of-function phenotypes are complementary, interpretation of the effects of ectopic expression is straightforward. Thus, in the preceding example, eyeless is revealed to be a master control gene for the differentiation of eyes in Drosophila. However, ectopic expression of genes can also lead to enigmatic phenotypes that are more difficult to interpret. For example, ectopic expression of eyeless during embryogenesis leads to embryonic lethality, a phenotype that is not easily reconciled with the loss-of-function phenotype. Therefore, when considering gain-of-function alleles generated by ectopic expression, we must remember that the phenotypes represent what the gene is capable of doing when expressed in particular contexts and may not reflect the normal function of the gene.

• Restrictive condition

Environmental condition in which environmentally sensitive (e.g., temperature sensitive) mutants exhibit the mutant phenotype.

• Permissive condition

Environmental condition in which environmentally sensitive (e.g., temperature sensitive) mutants exhibit the wild-type phenotype or can survive.

• Homologous recombination

Exchange of genetic information between homologous DNA molecules.

• Illegitimate recombination

Exchange of genetic information between non-homologous DNA molecules.

• What considerations should be made when embarking on a forward genetic screen?

How are you going to induce mutations and what kind of phenotype he was looking for. Also what genetic system is he going to use to actually see the phenotype associated with those mutant alleles • Choose a good model organism ---• Must be able to progress through its life cycle in a laboratory, have a short generation time, produce a reasonable number of progeny, and be amenable to crosses ---• Diploid and homozygous at all loci ---• Simple = better • Choose a good mutagen ---• Depends on both the research organism and the type of mutant alleles desired ---• Chemicals allow for high saturation ---• Transposons produce fewer mutations, but can have a "tag"

14.2: Genes Identified by Mutant Phenotypes Are Cloned Using Recombinant DNA Technology • Once mutant alleles are isolated from a forward genetic screen, how does one find the genes responsible?

How can you identify which gene was affected in a forward genetic screen? • Complementation • DNA sequencing technology

knock-down gene

If a DNA of an organism is genetically modified, the resulting organism is called a "knockdown organism." If the change in gene expression is caused by an oligonucleotide binding to an mRNA or temporarily binding to a gene, this leads to a temporary change in gene expression that does not modify the chromosomal DNA, ...

• guideRNA (gRNA)

In RNA editing, the nucleic acid that directs the addition or removal of nucleotides from mRNA. Also known as guide strand. In genome editing, the RNA molecule designed to operate in a CRISPR-Cas complex to target double-strand breaks in a specific DNA molecule or genomic locus.

• What information can be learned from synthetic lethal mutants?

Modifier screens can identify double mutants that display an unexpected phenotype, one that is not simply the combination of the phenotypes of the two single mutants. In perhaps the most dramatic form of enhancement, termed synthetic lethality, the two single mutants are viable but the double mutant is inviable. Synthetic lethality, or synthetic enhancement, was first noted by Drosophila geneticists who observed that some pairwise combinations of mutant alleles were inviable. For example, when Alfred Sturtevant crossed prune (pn) mutant females (pn is on the X chromosome) with males from a stock of separate origin called S/E-S, he noted that the progeny consisted solely of pn+pn+ females and no viable males (Figure 14.5a). Sturtevant determined that the S/E-S males carried an autosomal dominant mutation, which he called Prune-killer (K-pn), that in combination with pn results in lethality, but he noted that flies homozygous for K-pn mutation alone did not have an aberrant phenotype. In his cross, all male progeny inherited a pn allele from their mother and a K-pn allele from their father, and therefore these progeny died. In contrast, the female progeny were viable, since despite inheriting a K-pn allele from their father, they also inherited a pn+pn+ allele from their father. In this example, both pn mutants and K-pn mutants are viable, but the pn, K-pn double mutant results in lethality. Figure 14.5b shows two possible mechanisms to explain synthetic lethality. In one mechanism, the two genes in question act in parallel complementary pathways. In this scenario, mutations resulting in the loss of either pathway can be compensated for by the activity of the remaining pathway. However, when both pathways are disrupted, a dramatic enhancement in mutant phenotype is observed. An alternative mechanism is possible when both genes are acting in the same pathway: A reduction in function of one component of the pathway results in a mild phenotype, but when two components are disrupted, the pathway no longer functions effectively. Note that in the latter scenario, hypomorphic alleles can result in synthetic enhancement, but null alleles cannot. • Synthetic lethality: when the combination of two viable mutations results in an inviable double mutant

4. ___ can be used to study a gene of interest, by ___.

Reverse genetic. CRISPR-Cas9, homologous recombination, or RNAi.

• siRNA

Single-stranded 21- to 24-nucleotide RNA molecules derived from either endogenous or exogenous double-stranded RNA molecules that are incorporated in RISC to mediate RNAi. Endogenously produced siRNAs are most often from nongenic regions (e.g., repetitive RNA or products of an RNA-dependent RNA polymerase). Exogenously produced siRNAs are often derived from invading nucleic acids (e.g., transposons and viruses).

• miRNA

Small (21-24 nuts) regulatory RNAs produced by Dicer and acting in a RISC complex to either repress translational or cleave target mRNA molecules. Compare with RNA interference (RNAi).

• How are gene knock-outs generated in yeast and bacteria?

Taking advantage of this tendency for homologous recombination to occur in yeast, yeast geneticists create recombinant yeast both through gene insertion and gene replacement. Loss-of-function alleles are created by replacing the target gene with heterologous DNA, often a selectable marker gene, thus eliminating the production of functional wild-type protein by the target gene. Gene insertions that result in a deletion of the entire coding region of the gene create null alleles that produce no protein product. Such insertion alleles are often called gene knockouts because the insertion "knocks out" the function of the gene (as explained above in the definition given for knockout libraries), creating a recessive loss-of-function allele. Conversely, inserting a functional gene, often creating a gain-of-function allele, is called a knock-in. The ease with which homologous recombinants are generated in S. cerevisiae has allowed the production of a large number of yeast strains for genetic analysis of biological processes in this organism. Loss-of-function alleles of every gene in the S. cerevisiae genome have been generated and can be ordered from a stock center. Such stocks have greatly facilitated genetic research by relieving scientists of the need to produce mutations in the genes of interest at the start of every new genetic experiment.

• How are gene knock-outs generated in mice?

Talked about last chapter, may need to do more

• What can a reporter gene be fused to? What different information can be gained from these different fusions?

• A gene of interest can be fused to a reporter gene and its product can be detected directly or a detectable substance produced • Transcriptional fusion: Regulatory sequences for a gene of interest are fused to the reporter gene, which will then be expressed in the pattern dictated by the regulatory sequences • Translational fusion: Regulatory and coding sequences are fused to the reporter gene so that the protein produced can be visualized

• What is the CRISPR-Cas system identified in bacteria? What is it endogenously used for?

• Acts as a defense mechanism against invading viral nucleic acids • CRISPR: clustered regularly interspaced short palindromic repeats • Spacer sequences are derived from phage genomes and guide the Cas endonuclease to a specific sequence of an invading phage • CRISPR sequences are transcribed into a noncoding RNA and processed into crRNAs (cas-encoded RNAses) • tracrRNA is transcribed from the tracer RNA gene; one region of this RNA binds to the Cas endonuclease and the other binds to a crRNA • Cas endonuclease creates a double-strand break in the DNA of the invading DNA in locations determined by the crRNA • To "edit" a genome, the crRNA sequence is replaced with one that will target the sequence to be edited

• What are some advantages and disadvantages of using haploid vs. diploid organisms in a forward genetic screen?

• An advantage of using haploid organisms in a forward genetic screen is that both recessive and dominant mutations can be identified directly • A disadvantage is that mutations that cause lethality cannot be obtained ---• This problem can be circumvented by screening for conditional mutant alleles The attributes that make an organism a good genetic model (see back endsheets) also make it a good choice for a mutagenesis experiment: The organism must be able to progress through its entire life cycle in the laboratory, have a short generation time (for eukaryotic models, the time it takes to produce sexually mature progeny and complete the sexual life cycle), and produce a reasonable number of progeny. In addition, researchers must be able to manipulate it to produce specific genetic crosses. Organisms that are diploid usually have a starting genotype (the genotype to be mutagenized) that is inbred—in other words, for the most part homozygous at all loci. Such a genotype allows newly induced mutations to be readily identified, without interference from the confounding effects of polymorphisms. Finally, it is advantageous to use the simplest organism possible for the biological process under study. Because Saccharomyces cerevisiae has a rapid life cycle and is easily manipulated in the laboratory, it is often used to investigate biological processes common to all eukaryotes. The principles elucidated in S. cerevisiae can often be extended to other eukaryotes, including humans.

• What does a range in phenotypes for alleles of a single gene suggest about those alleles (and that gene)?

• Comparing the phenotypes of multiple alleles of a gene allows determination of the range of phenotypic variation possible for the gene in question

• What repair mechanisms can be used by the cell after CRISPR-induced double-stranded DNA breaks at the target site? o Which of these repair mechanisms are error-prone? o Which of these repair mechanisms is desirable if you're trying to add donor DNA to a specific locus? MAYBE LOOK UP OTHER CHAPTER

• DNA endonuclease targets the location and creates a double-strand break at the target site, which is fixed through repair using nonhomologous end joining (NHEJ) or homologous recombination ---• NHEJ repair: small deletions often remain at the break site, with possible lossor gain-of-function ---• HR: an exogenously supplied DNA sequence can contain the desired nucleotides You may not realize it, but you are living through a revolution in genetics due to advances in technologies to manipulate DNA sequences in the genomes of living cells. A dream of geneticists for many decades was to have the ability to "edit" the genome—precisely changing the nucleotide sequence at a specific chromosomal locus to any desired sequence. Remarkably, this dream has become reality in the past few years. The general concept is to design a DNA endonuclease to target a specific genomic location. The endonuclease creates a double-strand break at the site, which can be subsequently repaired by endogenous repair mechanisms, either through nonhomologous end joining (NHEJ) or homologous recombination (see Section 11.5). If the double-strand break is repaired by NHEJ, then small deletions often remain at the site of the break, leading to possible loss- or gain-of-function alleles, depending on what sequences are lost. Alternatively, the break may be repaired by homologous recombination, either with endogenous sequence from the homologous chromosome in a diploid cell or with exogenously supplied DNA sequences. In the latter case, if the exogenously supplied DNA has been constructed in such a way that it contains the desired change, a specific sequence change in the chromosome may be accomplished.

• Why is double-stranded RNA used for RNAi? What does this look like for eukaryotic cells? o What is the consequence of dsRNA introduction? o How does a scientist target their gene of interest using RNAi? o How can dsRNA be introduced to perform RNAi?

• Double-stranded RNA (dsRNA) can act as a trigger for degradation of the dsRNA and any RNA complementary to it • dsRNA acts as a trigger for degradation mRNA from the target gene will be degraded by Dicer and Argonaute (Ago) enzymes Ago will cleave them and knock down target mRNA and prevent translation

• What are type types of genetic modifier screens? o How are they similar and/or different? o What considerations should be made regarding the starting allele used for the screen? o What information can be obtained from a genetic modifier screen that can't be obtained from a standard forward genetic screen?

• Genetic modifier screen: used to see if mutations in a second gene can modify the phenotype of the first mutation ---• Enhancer screen: identifies second-site mutant alleles that enhance a mutant phenotype ---• Suppressor screen: second-site mutations that suppress the first are isolated • May identify double mutants with an unexpected phenotype not equivalent to the combination of two individual mutant phenotypes • Synthetic lethality: when the combination of two viable mutations results in an inviable double mutant Both look at double mutants Generally, mutant phenotypes reflect the response of the organism to a loss or change of a particular gene product. However, individual genes do not act in isolation. The activity of other genes may modify, by either enhancing or suppressing, the phenotypic defects caused by the loss of a gene product. One approach to discovering genetic interactions is to carry out a genetic modifier screen to see if mutations in a second gene can enhance or suppress the phenotype of the first mutation. For example, starting with a Drosophila mutant with slightly curled wings, a modifier screen could be carried out to identify second-site mutations that result either in more severely curled wings or in a wing morphology that is restored to a wild-type phenotype. Genes identified in modifier screens are often involved in the same or closely related genetic pathways. An enhancer screen is a modifier screen in which mutations in a second site enhance the phenotype of the initial mutant. A suppressor screen is a modifier screen designed to identify second-site mutations that suppress the phenotype of the initial genotype. Note that both types of screens can be performed simultaneously. Enhancer-suppressor screening strategies are almost limitless in number and sophistication and have the potential to identify genes that function in interacting genetic pathways.

• What is the result of a gene knock-in allele?

• Knock-in: inserting a functional gene, often creating a gain-of-function allele

• What are the features of a balancer chromosome that make it useful for recovering mutants isolated from a forward genetic screen?

• Marked chromosomes can be followed through generations • Balancer chromosomes in Drosophila allow specific chromosomes to be transmitted intact through multiple generations Features: 1. One or more inverted segments to prevent transmission of chromosomes that have undergone crossing over (repress recombination in the mutant chromsome) 2. A recessive allele resulting in lethality so that individuals cannot be homozygous for the balancer 3. A dominant mutation producing a visible phenotype so that segregation of the chromosome can be followed through generations (allow to see if balancer chromosome has been inherited)

• Why is RNAi called "knock-down" and not knock-out? o What process is directly affected by RNA interference (RNAi)? MAY NEED TO LOOK UP

• Method for silencing a gene by preventing translation via mRNA degradation ---• "Knock-down" ---• Double-stranded RNA (dsRNA) can act as a trigger for degradation of the dsRNA and any RNA complementary to it ---• Endogenously, RNAi is used to silence repetitive DNA and protects against viruses with double-stranded RNA genomes • dsRNA complementary to the target gene can be introduced into cells or organisms • mRNA from the target gene will be degraded through the action of Dicer and Argonaute enzymes

14.1: Forward Genetic Screens Identify Genes by Their Mutant Phenotypes • What is the goal of a forward genetic screen?

• Mutant phenotypes provide information on the wild-type function of a gene and insight into biological processes • The design of genetic screens to identify genes involved in particular biological processes is unlimited and requires innovation Wanting to understand what genes are responsible for allowing this wildtype process to occur. +3 In a forward genetics screen, we are looking at what gene might be the cause of a particular expressed mutant phenotype. In a reverse genetics screen, we mutate a particular gene and observe the phenotype that is produced. Goal is to find genes that are important for a certain biological process • Mutagenesis: organism treated with a mutagen to create mutations randomly throughout the genome • Goal is to induce mutations in every gene of a population of mutagenized individuals = saturation mutagenesis

Conditional Mutations in Yeast

• Screens were performed in yeasts to identify temperature-sensitive alleles of genes needed for cell cycle regulation • The mutants fell into categories depending on the stage at which they were arrested at the restrictive temperature • This allowed for identification of checkpoints in the cell cycle

• How does CRISPR-Cas9 gene editing work? What components of the endogenous CRISPR-Cas system are used for gene editing? What is a gRNA?

• Staphylococcus has a single Cas protein, Cas9 ---• Its tracrRNA and cRNA sequences that can be fused into one, more simplified, guideRNA (gRNA) • guideRNA hones the complex to the target site, followed by a double-strand break and repair (typically NHEJ) ---• During repair, insertions and deletions (indels) are often introduced at the target site • Components be introduced either as transgenes or protein or RNA could be directly injected into a cell • Multiple guideRNA genes may be used to target multiple loci at the same time, while limiting the binding at "off-targets" • Homologous recombination: an exogenously- supplied DNA sequence will be incorporated

• How can transposons be used for insertional mutagenesis? o How are transposons identified phenotypically in Drosophila? o How can the genetic location of a transposon be verified/determined? MAY NEED TO LOOK UP OR IN LAST CHAPTER

• Transposons produce fewer mutations, but can have a "tag"• Transposons produce fewer mutations, but can have a "tag"

• What is genetic redundancy and how does this complicate the results obtained from a traditional forward genetic screen? o How is this uncovered in a genetic modifier screen?

• Two genes may act in parallel, complementary pathways needed for an essential function • Disruption of either pathway is partially compensated by the remaining pathway (genetic redundancy), but when both are disrupted, the essential function is lost and the organism cannot survive • In the most obvious case of genetic redundancy, two genes encode very similar proteins that can function interchangeably • Genetic redundancy may also result from the compensatory action in genes with little or no sequence similarity—these are difficult to predict, but can be easily identified in modifier screens The first scenario, where two genes act in parallel, is an example of genetic redundancy, where the loss of the function of either gene alone is compensated for by the activity of the other, nonmutant gene. Only when both genes are mutant would a conspicuous mutant phenotype be evident. In such a case, a 15:1 segregation ratio could be expected in the F2F2 of a cross between the two recessive single mutants (see the discussion of duplicate gene action in Section 4.3). In the most obvious case of genetic redundancy, two genes encode very similar proteins that can function interchangeably. In many instances, the activities of the two genes do not fully compensate for one another, so that single mutations, in either gene alone, result in a mild phenotype, while a severe phenotype is seen when both genes are mutant. Genetic redundancy caused by the presence of duplicate genes can arise in a species through small-scale duplications or through whole-genome duplications. As we explore in detail in Chapter 16, genome sequences of eukaryotes show such duplications to be very common. Genetic redundancy can also arise from the compensatory action of genes that have little or no sequence similarity and encode biochemically different activities. This type of genetic redundancy is difficult to predict on the basis of the DNA sequences of the genes, but it too can be uncovered by enhancer-suppressor screens. Enhancer-suppressor screens have been performed on many organisms, including Drosophila, C. elegans, Arabidopsis, and mice (see Section 14.3), and are extremely successful at identifying interacting genetic pathways (see Section 18.3).