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Home , Epistasis, Phenotype

Genetic Techniques for Biological Research Corinne A. Michels Copyright q 2002 John Wiley & Sons, Ltd ISBNs: 0-471-89921-6 (Hardback); 0-470-84662-3 (Electronic)

6 Analysis

OVERVIEW

The Random House Dictionary of the English Language-Unabridged Edition (1 966) defines epistasis asa genetic term describing the‘ between nonallelic in which one combination of such genes has a dominant effect over other combinations’.The key word to remember is ‘nonallelic’, i.e. different genes. Epistasis (from the Greek meaning ‘stand above’) is the masking of the of a in one by the phenotype of a mutation in another gene (Huang & Sternberg, 1995). One gene is said to be epistatic to another when the double strain exhibits the phenotype of that mutantgene. This is in clear contrast to the terms dominant and recessive, which describe the relationship between different of the same gene. It is very important not to confuse these concepts. Epistasis analysis is used to determine if genes with related mutant act in the same or different pathways, and, if in the same pathway, to place them in a linear order relative to one another based on the step in the pathway controlled by that gene. In otherwords, one uses epistasis analysis to construct an order-of- function map that reflects the sequence of events in a pathway controlled by several genes. The use of epistasis analysis for the study of complex pathways was suggested moreor less simultaneously by twoindependent research groups working in different fields. Jarvik & Botstein (1973) reportedthe isolation of temperature- sensitive and cold-sensitive mutationsthat block phage P22 , i.e. assembly of the phage particle. They used a combination of double mutant studies and reciprocal temperature shifts (made possible by their use on conditional ts and CS )to determine the order of events in phage assembly. Theirwork demonstrated that head and tail assembly were independent processes but that both were dependent on phage DNA replication. Hereford & Hartwell (1974) used epistasis analysis to order events in the Saccharomyces cell division . They used temperature-sensitive mutants that blocked the cellcycle at morphologically dis- tinguishable points. The execution points of these genes were ordered relative to the block produced by the cell cycle inhibitor a-factor by temperature-shift experiments and relative to each other by double mutant studies. To determine the epistatic relationshipbetween two genes, in these genes must have distinguishable phenotypes. Epistasis analysis is undertaken only after the initial steps of genetic analysis. Mutationsare isolated and placed into com- plementation groups. Then, representative alleles are selected for a detailed charac- terization of phenotype so as to reveal subtle differences in phenotype not obvious from initial characterization. This could be a complete morphological analysis, such as in secretory pathway mutants or cell division cycle mutants, or intensive bio- chemical characterization, such as for DNA replication mutants or mutantsaffecting metabolicpathways. Occasionally, different alleles exhibit somewhat different 80 GENETICTECHNIQUESBIOLOGICAL FOR RESEARCH phenotypes. The researcher can now capitalize on these phenotypic differences in the epistasis analysis. For the purposes of epistasis analysis, there are two types of pathway in living systems, substrate-dependentpathways and switchregulatory pathways (Huang & Sternberg, 1995). A substrate-dependent pathway consists of an obligate series of steps or reactions that are required to produce a final outcome. The outcome can be as simple as the synthesis of a nutrient such as an or a macromolecule, or can be as complex as the formation of aribosome. The substrate-dependent pathway can be thought of as a progression of events or even as a river flowing downstream with separate tributaries joining at different points and finally flowing into the lake. Another view is as a series of positive reactions each dependent on a source of substrate and a functional gene product for the successful completion of each step in the pathway. Moreover, the product of the more upstream step is used as the substrate of the downstream step. If there is no substrate available or if any one of the is missing or inactive, then the pathway will be blocked at that step. A production line at a factory would be considered to be this type of pathway. It is dependent on the input of parts (substrates) and workers (gene products) to assemble these parts. A switch regulatory pathway consists of a series of genes or gene products that alternate between two states, ‘on’ and ‘off’. The components of this pathway are usually acting directly on each other as opposed to on substrates, as occurs in a substrate-dependent pathway. The activity of a switch regulatory pathway is regu- lated by an upstream signal that stimulates the pathway and produces the down- stream response. Environmental changes, cell-cell , zygote formation, and mitogenic signals are only a few of the signals that can act as initiators of a switch regulatory pathway. The downstreamresponse can be altered , cell division, or the initiation of a developmental process such as . Mutations in the genes encoding components of a switch regulatory pathway can lock the component into a permanently ‘on’ or permanently ‘off’ state. This has the effect of separating the downstream response from the initiating signal. Mutations that allow the response to be produced even in the absence of a stimulatory signal or despite the presence of an inhibitory signal are referred to as constitutive mutations. The isolation of constitutive mutations is a strong indicator that one is dealing with a switch regulatory pathway. In a switch regulatory pathway the actionof a particular component,or regulatory factor, can be either positive or negative. The function of a positive regulatory factoris to activate the next component in the pathway (its downstream component) when it is in its active form. A recessive (loss of function) mutation in a gene encoding a positive regulatory factor blocks the pathway. A dominant (gain of function) muta- tion in a gene encoding a positive regulatory factor produces a capable of functioning constitutively even in the absence of upstream activation of the pathway. A negative regulatory factor in the activated state inactivates the next downstream component in the pathway. Therefore, arecessive, loss of function mutation in a gene encoding a negative regulatory factor allows the next step in the pathway to be constitutively active. A dominant, gain of function mutation in a gene encoding a negative regulatory factor produces a gene product capable of constitutively inhibit- ing thepathway evenin the absence of the signal. By determining whether the EPISTASIS ANALYSIS81 mutation is dominant or recessive, constitutive or blocks the response, the geneticist can decide whether the gene product is a negative or positive regulatory factor. Determining whether the pathway under investigation is a substrate-dependent or switch regulatory pathway is not a simple task. Nonetheless, as will be seen below, it is important because the interpretation of the results of double mutant analysis depends on the type of pathway. The characterization of mutant alleles of pathway genes canprovide some clues. As discussed above, if constitutivemutants are obtained in any of the pathway genes, then one can conclude that the pathway is a switch regulatory pathway.More often thannot, a switch regulatorypathway controls more than one downstream response. As a result mutations in a switch regulatory pathway affect a number of phenotypic traits, such as the expression of several genes, andare said to be pleiotropic. The identification of pleiotropic mutants is suggestive of a switch regulatory pathway. If no constitutive mutations in pathwaycomponents have been identified, onecan proceed with an epistasis analysis underthe assumption that one is dealing with asubstrate-dependent pathway. For complex processes this is unlikely to be the case. More likely, the initial characterization of mutations has not uncovered all the genes in the pathway or isolated a sufficiently varied array of mutant alleles. As the genetic analysis of the pathway proceeds newgenes and/or newalleles willbe identified and thetrue character of the pathway will be revealed in full detail. This will become clearer as examples from the literature are discussed.

EPISTASIS ANALYSIS OF A SUBSTRATE-DEPENDENT PATHWAY

Let us say that GENl and GEN2 are related because mutations in both genes decrease the production of Z. Mutations in GENl give phenotype A, and mutations in GEN2 give phenotype B. Only recessive loss of function allelesof GENl and GEN2 have been isolated. No constitutive allelesof GENl or GEN2 have been identified and mutations in these genes alter Z production but appear not to affect otherphenotypes. We assumethat this is asubstrate-dependent pathway and proceed with the epistasis analysis. The four mechanisms of genetic interaction between GENl and GEN2 are given in Table 6.1 (based on Hereford & Hartwell, 1974) and the resulting phenotype of the single or double mutation with regard to production of Z is indicated. Models 1 and2: Genlp andGen2p participate in different steps of the same pathway and protein Genlp acts in a step that is upstream (Model 1) or down- stream (Model 2) of the step catalyzed by protein Gen2p. Model 3: Proteins Genlp andGen2p are components of two independent and parallel pathways for Z production. Model 4: Proteins Genlp and Gen2p act at the same step and in conjunction with one another. To determine the epistatic relationship between these two genes, one constructs a strain that is mutant at bothgenes and observes the phenotype of the double mutant 82 GENETIC TECHNIQUESBIOLOGICAL FORRESEARCH Table 6.1 Epistasisanalysis of asubstrate-dependent pathway Phenotype of Phenotype of Phentotype of genl gen2 genl gen2 single single double mutation mutation mutationmutation mutation

~~ ~ Model 1 GEN2GENI A B A Ad Model 2 GENIGEN2 A B B Model 3 GENI -----W A B Unique GEN2- Model 4 GENI, GEN2 A B =B =A or P unique strain. If the double mutant phenotype is A, then GENI is epistatic to GEN2 and GENI encodes the upstream component in the pathway. Alternately, if the double mutant phenotype is B, then GEN2 is epistatic to GENI and GEN2 encodes the upstream component. In summary, in a substrate-dependent pathway, if the double mutant exhibits a phenotype identical to one or the other mutant genes, then that gene is epistatic and encodes the more upstream component in the pathway. If Model 3 or 4 describes the relationship, the results can be more difficult to interpret. The term ‘unique’ used in Table 6.1 indicates that thephenotype is different from either phenotype A or B. It can be qualitatively related to phenotypes A and B but quantitatively more extreme. For example, mutations in two different RAD genes might partially reduce the rate of recombination but to different extents, while thedouble mutant completely blocks all recombination.This type of interaction is called enhancement and will be discussed in detail in Chapter 9. Another classic example of two parallel pathways affecting a single trait comes from . The reddish brown results from a mixture of two pigments synthesized by parallel pathways. Mutations in one of these pathways that blocks red pigment productionproduces brown eyeswhile mutations in thealternate pathway that blocks brown pigment production produces red eyes. Flies defective for the production of both pigments, that is double mutants, have white eyes. White eyes is a unique phenotype and could not have been predicted from observing the phenotypes of the single mutants. The double mutant in Model 4 might exhibit a unique phenotype or could be phenotype A or B. What will distinguish Model 4 from Models 1 and 2 is that the phenotype of the double mutant is likely to vary with the alleles. Thus, epistasis analysis has limitations. The researcher will have to proceed to other methods, such as suppressor and analysis or coimmuno- precipitation, to support a proposed model.

EPISTASIS ANALYSIS OF A SWITCH REGULATORY PATHWAY

In a switch regulatory pathway, the epistatic gene encodes the downstream com- ponent. Because thistype of pathway involves both negative and positive EPISTASIS ANALYSIS 83 components, it is not possible to come up with a simple table describing all possible results. Instead, several examples of hypothetical switch regulatory pathways will be presented for the reader to ponder. Then some sample results will be described and the reader can practice analytical skills. In the pathways given below, an arrowhead indicates thatthe action of theprotein or signal is positive anda vertical line indicates that theaction of theprotein or signal is negative. For each pathway the reader should determine the phenotype of mutants in each protein component. The classes of mutationsto consider are alleles thatput the component in the permanently ‘on’ state and thosethat put it in the permanently ‘off’ state.The possible phenotypes are constitutive or no response produced in the presence of signal.

Regulatory pathway 1 Signal -Protein A -Protein B -Protein C -Response Regulatory pathway 2 Signal -Protein X +Protein Y +Protein Z -Response

The experiments listed below present the results of epistasis analysis of mutations in genes GENl, GEN2, GEN3,and GEN4. Mutations in these genes affect the same process and may be in a common pathway.A number of alleles of each are available including constitutive alleles. Experiments 1-5 provide an example of an epistasis analysis of a switch regulatory pathway. Use these results as a practice exercise. Determine whether each gene encodes a positive or negative regulator and the order-of-function of the genes in thepathway. As a guide, the results of each experiment are followed by their interpretation. Regulatory pathway 3 synthesizes these conclusions into an order-of-function map of the pathway.

Experiment 1: A recessive mutation in GENl does not respond to the signal. A recessive mutation in GEN2 is constitutive. A strain carrying both mutations (genl gen2) is constitutive. (Conclusions: Genl protein is a positive regulator.Gen2 protein is a negative regulator. GEN2 is epistatic to GENI and the product of GEN2 acts downstream of the product of GENI.)

Experiment 2: A recessive mutation in GEN2 is constitutive. A recessive mutation in GEN4 blocks the response to the signal. A strain carrying both mutations (gen2 gen4) doesnot respond to the signal. (Conclusions: Gen4protein is a positive regulator. GEN4 is epistatic to GEN2 and the product of GEN4 acts downstream of the product of GEN2.)

Experiment 3: A dominantconstitutive alleleof GENl is identified. A recessive mutation in GEN3 blocks the response to the signal. A strain carrying both muta- tions (GENI-c gen3) does not respond to the signal. (Conclusions: Genl-c protein is an activated form of the protein that is locked in the ‘on’ state. Gen3 protein is a positive regulator. GEN3 is epistatic to GENl indicating that the product of GEN3 acts downstream of the product of GENI.) 84 GENETICTECHNIQUESBIOLOGICAL FOR RESEARCH Experiment 4: A dominantconstitutive of GEN4 is isolated. A recessive mutation in GEN3 blocks the response to the signal. A strain carrying both muta- tions (gen3GEN4-c) is unable to respond tothe signal. (Conclusion:GEN3 is epistatic to GEN4 and the product of GEN3 acts downstream of the product of GEN4.)

Experiment 5: A dominantconstitutive allele of GEN3 is isolated. A recessive mutation in GEN4 blocks the response to the signal. A strain carrying both muta- tions (GEN3-c gen4) does not respond to the signal. (Conclusion: GEN4 is epistatic to GEN3. This result taken together with the results of Experiment 4 suggests that the products of GEN3 and GEN4 could act at the same step.

Regulatory pathway 3 Signal -GENl -GEN2 -GEN3, GEN4 -Response

EPISTASIS GROUP

Genes encoding proteins that function in the same pathway or process will exhibit epistasis, as defined in the discussion presented above. Such genes are said to be members of an epistasis group. A well-studied example of an epistasis group is the RAD52 epistasis group (Paques & Haber, 1999). Mutations ingenes such as RADSO, RAD51, RAD52, RAD54, RAD.55, RAD57, RAD59, SPO11, and MREll exhibit defects in recombination and double-strand break repair. Construction of doublemutant strainsdemonstrates epistatic relationships among the various members of this group of genes. Mutations in RAD3 or RAD6, which like RAD52 were originally isolated because of their increased sensitivity to X-ray radiation, do not exhibit epistasis with the RAD52 epistasis group or with each other. When paired with RAD52 or other members of the RAD52 epistasis group, the results are consistent with Model 3 in Table 6.1 and clearly indicate that RAD3 and RAD6 are in distinct pathways. Thus, despite certain similarities in phenotype, RAD52, RADS, and RAD6 are members of different epistasis groups.

REFERENCES AND FURTHER READING

Avery, L. & S. Wasserman (1992) Ordering gene functions: the interpretation of epistasis in regulatory hierarchies. Trends Genet. 8: 3 12-316. Botstein, D. & R.Maurer (1982) Genetic approaches to the analysis of microbial development. Ann. Rev. Genet. 16: 61-83. Hereford, L.M. & L.H. Hartwell (1974) Sequential gene function in theinitiation of Succhuromyces cerevisiae DNA synthesis. J. Mol. Biol. 84: 445-461. Huang, L.S. & R.W. Sternberg (1995) Genetic dissection of developmental pathways. Methods Cell Biol. 48: 97-122. Jarvik, J. & D. Botstein (1973) A genetic method for determining the order of events in a biological pathway. Proc. Nut1 Acad. Sci. USA 70: 2046-2050. Paques, F. & J.E. Haber (1999) Multiple pathways of recombination induced by double- stranded breaks in Saccharomyces cerevisiae. Microbiol. Mol. Biol. Rev. 63: 349-404.