Experiment 1B: Complementation analysis in Yeast Complementation is a way of determining whether two mutants that cause the same

or similar phenotype (e.g. his-) are due to mutations in the same or different genes. Indeed, in the absence of other information, complementation group is our operational definition of a gene. Complementation analysis requires that both the mutant alleles be phenotypically recessive. Complementation is essentially a description of the phenotype observed when a diploid organism is produced from parents containing independently acquired mutations that have the same or similar phenotype or affect the same process. Complementation is due to the interaction of gene products produced by different genes and is not due to interaction of the genes themselves (by recombination or strand exchange, for example). In sexually reproducing eukaryotes, complementation is tested by producing offspring from two mutant strains (see Figure 1). If the F1 offspring are wild-type, we say that the two mutant alleles are in different complementation groups and infer that they are due to mutations in different genes. If the F1 offspring exhibit a mutant phenotype, then we say that the two mutant alleles cannot complement each other and are in the same complementation group. We infer that they are mutations of the same gene.

Figure 1: A. Two individuals are homozygous for different independently acquired recessive mutations (m-1 and m) that cause the same phenotype (e.g. his-). The mutations reside in different genes on different chromosomes. Wild-type allele for each gene is indicated by a +. B. Two individuals are homozygous for different independently acquired recessive mutations (m and m) that cause the same phenotype (e.g. his-). However the mutations reside in the same gene on the same chromosome.

The process is similar in determining complementation in viruses. In this case the two strains of mutant viruses (neither able to replicate in a non-permissive host cell) are allowed to infect the non-permissive host at a high MOI (multiplicity of infection). This produces a situation similar to that in eukaryotes. Many cells will have both mutant viral genomes in the same cytoplasm. Consequently the products of their respective wild-type genes (if the mutations were on different genes) can supply both viruses with all the requisite materials for replication; i.e. the mutations complement each other and are in different complementation groups. If both mutations were in the same gene, there would be no normal gene product present, and the viruses would not be able to replicate; i.e. the mutations do not complement each other and are in the same complementation group. In bacteria, the same principle applies. If you have an unknown mutant that does not synthesize histidine and you introduce into the mutant bacteria a plasmid that produces the wild-type hisD product, you can ask whether that plasmid complements the mutation or not (e.g. does it make histidine). If it complements, the unknown mutant is a hisD mutant. If the wild-type hisD product produced by the plasmid does not complement the mutant, we can infer that our unknown mutant is not a hisD mutant. However, unlike the case of the sexually reproducing organisms and the viruses, we cannot produce a diploid state in bacteria. We can only produce partial diploids. Because of this, the usual procedure in bacteria is to introduce a functional wild-type copy of a gene into a mutant strain via a plasmid. In each of these examples, complementation (or the lack of complementation) requires only the presence of two genes in the same cell. No second event (recombination) or rare process (reversion) is required. A complementation group is usually the same as a gene (in fact, complementation group is used as an operational definition of a gene, a unit of function). However, like most operational definitions, there are exceptions and complications. Occasionally, a mutation (like a deletion or a polar mutation in a polycistronic operon) can affect more than one related gene. This would mislead one into thinking that the two genes are really one complex gene. This is more common, in prokaryotes, where there are polycistronic operons that link together genes that produce enzymes of a single biological pathway. In the lac operon, for example, some mutations that are solely within the lacZ gene can lead to early termination of mRNA transcription (before the mRNA transcribes the lacY gene). Because the lacY gene is not transcribed, there will be no lacY gene product. Thus, this mutation, which is solely in the lacZ gene and does not affect the lacY structural gene, still causes a lacZ- and lacY- phenotype. And, occasionally, specific mutations in a single gene can be complemented by a different specific mutation in the same gene. Again, although relatively infrequent, genes can have multiple functions and have complex interactions (e.g. his4 in S. cerevisiaeis). It is important to remember that these types of genes have more than one region of importance or unique function (e.g. the enzyme coded by the his4 gene is involved in three different steps of the histidine biosynthetic pathway -steps 2, 3 and 10 see Figure 2). This complexity of function can lead to intragenic complementation. Knowledge of the type of mutation you have (e.g. point mutation versus polar mutation) and the availability of several different kinds of known mutants will help you figure out these complex events or at least detect them genetically. In our yeast complementation analysis, we will be checking for such intragenic complementation within the his4 gene, and intergenic complementation between the other his genes that are involved in histidine biosynthesis.

Figure 2: The metabolic pathway of histidine biosynthesis in yeast S. cerevisiae. The encircled number is the reaction number. The italicized symbol is the gene encoding that enzyme activity. X indicates an unstable intermediate. Table 1 provides the full names of the enzyme activities and the chromosomal location of the genes encoding these activities.

Table 1: Histidine Biosynthesis in S. cerevisiae Reaction 1 2 3 4 Enzyme Activity Names ATP PR-transferase 1-[5'-PR]-ATP: pyro-P PR-transferase PR-ATP pyrophosphohydrolase PR-AMP cyclohydrolase 1-N-[5'-phospho-D-ribosyl]-AMP 1,6-hydrolase BBMII isomerase N-[5'-phospho-D-ribosylformimino]-5-amino1-[5''-PR]-4-imidazolecarboxamide ketolisomerase glutamine amidotransferase catalyzing conversion of BBMII to imidazoleglycerol-P imidazoleglycerol-P dehyratase D-erythro-imidazoleglycerol-P hydro-lyase histidinol-P aminotransferase L-histidinol-P:2-oxyglutarate aminotransferase histidinol-P phosphatase L-histidinol-P P-hydrolase histidinol dehydrogenase L-histidinol: NAD oxidoreductase 4.2.1.19 3.5.4.19 EC # 2.4.2.17 Chromosomal Gene locus Location

his1 his4B his4A his6

5.3.1.16

V III III IX*

56 7 8 9 10

his7 his3 his5

2.6.1.9

II XV IX* VI III

his2
3.1.3.15

his4C
1.1.1.23

These enzyme activities are due to a single multifunctional enzyme encoded by the his4 gene. * Although both of these genes are on chromosome IX, they are unlinked (>>50 cM)

Experiment 1B: Complementation analysis in yeast The yeast S. cerevisiae has different phases to its life cycle that ensures the perpetuation and survival of the species. You have isolated histidine auxotrophs from a typical laboratory yeast strain TD28 (Mat a, ino1-13). This strain contains one copy of each of the 16 different chromosomes (haploid = 1n = 16; n = the # of unique chromosomes) that compose the yeast haploid genome. A single cell of this strain grows vegetatively by budding to produce a daughter cell. During the budding division each of the 16 chromosomes is replicated and one copy of each is distributed to the mother and daughter cell during mitosis in the nucleus (Figure 3). Thus, each daughter cell is a genetic duplicate of the mother cell containing a single copy of each of the 16 chromosomes in its nucleus. Yeast can also exist as a diploid organism (Figure 3). Haploid yeast strains are either of the a mating type or the mating type. Only a and strains can mate with each other. During mating the two cells fuse. The two nuclei of each strain also fuse. This results in a yeast cell that is virtually indistinguishable from the haploid yeast cell. However, the cell produced by mating is now diploid for its genetic content having 2 copies of each of the 16 different chromosomes (2n = 32; n = 16); one set from one parent and the other set from the other parent. The diploid cell, like the haploid cell, grows vegetatively, budding a genetically identical daughter cell during each division by mitosis. However, in contrast to the haploid daughter cell that contains 1 copy of each of 16 chromosomes, the diploid daughter receives 1 copy of each of the mother's 32 chromosomes during mitosis. This represents a homologous pair of each of the 16 different chromosomes

Figure 3: The life cycle of yeast. Yeast can grow vegetatively as either haploid or diploid cells. Histidine biosynthesis is a complex pathway that requires 10 enzymatic steps (Figure 2). In yeast there are 7 genes that have been identified by mutation that result in a His phenotype (Figure 2 and Table 1). These genes encode proteins (enzymes) that catalyze the 10 biosynthetic steps. Most of these genes encode a single polypeptide (protein) that performs only one of the catalytic

steps in the pathway. However, two of the genes are complex. The his4 gene encodes a single protein that is multifunctional and catalyzes the 2nd, 3rd and 10th steps in histidine biosynthesis. The his7 gene also encodes a multifunctional protein that catalyzes the 5th and 6th steps in the pathway (Figure 2). Because the His4 and His7 proteins are biochemically complex, the his4 and his7 genes are also genetically complex. The his4 gene, for example, is divided into 3 genetic subregions, his4A, his4B, and his4C. Each subregion corresponds to the enzymatic domain that catalyzes the 3rd (his4A), the 2nd (his4B) and the 10th (his4C) step in histidine biosynthesis (Figure 2). Each enzymatic activity of the His4 protein behaves as an independent domain unaffected by mutations in one of the other domains (like 3 beads on a string). For example, a missense mutation in the his4A region only results in the loss of the catalytic activity required for the 3rd step and has no effect on the encoded his4B or C catalytic activity (the 2nd and 10th steps are intact). Only certain types of mutations, like a deletion of the entire his4 gene, can destroy all three enzymatic activities. A nonsense or a frameshift mutation in the early coding region of his4 that disrupts the translation of the rest of the protein will also knock-out all 3 activities of the HIS4 protein (Why?). Mutations affecting downstream functions of a gene or transcriptional unit are called polar mutations. We will see a similar phenomenon in bacterial genetics later in this course. The chromosomal map position has been determined for each of the 7 his genes encoded in the yeast genome (Table 1). The genes are dispersed throughout the genome, being genetically unlinked and located on a number of different chromosomes. This situation is typical of eukaryotic genomes. In contrast, genes in a common biochemical pathway are often found to be tightly linked in prokaryotes and, in fact, are often transcribed as part of a single mRNA transcript (e.g. lac operon). We will use complementation analysis to determine if any of the different histidine auxotrophs that you isolated in Experiment 1A are specific histidine biosynthetic genes. There are a number of other approaches that could be used to determine what gene was mutated. For example, you could assay each his auxotroph, biochemically, for the presence or absence of the 10 different activities required for histidine biosynthesis. Because it is already established which genes are responsible for catalyzing each step, if enzyme extracts made from one of your his mutants was only defective in the ability to catalyze the 8th step of histidine biosynthesis you would know that it was a his5 mutant (Figure 2). The same reasoning would apply to mutants in other steps. This was obviously the procedure used to initially arrive at the gene-enzyme assignments. A second approach uses a physiological analysis. For example, let's say that you have a his5 mutant (but don't know it). If you made nine SD w/o histidine plates, each containing one of the 9 different intermediates in histidine biosynthesis (Figure 2), a his5 mutant could only grow on plates that contained histidinolP or histidinol, the two intermediates in the pathway after the 8th step. It could not grow on any intermediates involved in steps 18 (Why is this?). Thus, by inference, the mutant must be defective in step 8 and contain a mutation in the his5 gene. Unfortunately, both the biochemical and the physiological approach to analyze his- auxotrophs are labor intensive and require expensive substrates which are not easily obtained nor stable for routine use. Furthermore, in some cases these substrates can not be used by cells when added externally.

However, a direct and easy way to arrive at the desired information (which his gene is mutated) is by performing a simple genetic complementation test or cis/trans test. The usefulness of a complementation test is that many phenotypes (like histidine auxotrophy) can be caused by mutations in one of a number of genes. As part of the genetic analysis of a defect, we want to describe how many genes may contribute to and are involved in a complex pathway. The complementation test can only be performed with mutations that are recessive to the wild-type allele. Mutations that cause histidine auxotrophy in yeast are almost always recessive. The basis of the complementation test is that, in general, mutations in the same gene cannot complement each other. Hence, if you mated a his5 mutant of the a mating type to a his5 mutant of the mating type, the diploid formed would still be a histidine auxotroph; both copies of the his5 gene in this diploid strain are defective as derived from the parent strains and therefore the diploid strain is similarly defective in the 8th step in the histidine biosynthetic pathway (see Figure 2). We would say that these strains do not complement each other and that both of these mutations are in the same complementation group or cistron (cis/trans test). Thus, the inability of two mutants to complement tells you that they each contain a mutation in the same gene. However, if you mated a his5 mutant of the a mating type to a his2 mutant of the mating type, the phenotype of the diploid would be His+ despite the fact that both parent strains were Hisauxotrophs. We would say that his2 mutants complement his5 mutants (or vice versa) as each parent strain supplies the missing function in trans (cis/trans test). The genotype of the diploid explains this, being his5-/His2+ , His5+/his2- with the a parent providing a wild-type copy of the his2 gene and the parent contributing a wild-type copy of the his5 gene to the diploid. Both wildtype copies are dominant to the recessive mutant alleles and therefore supply normal proteins that enable catalysis of the 8th and 9th steps of histidine biosynthesis (Figure 2). Thus, the ability of two mutants to complement usually tells you that each contains a mutation in a different gene (but not always, see below). The above is an example of intergenic complementation (complementation between genes). In addition to intergenic complementation, there can be intragenic complementation (complementation within a gene or complementation between alleles of the same gene). This type of complementation is observed with genes that are complex. The his4 gene mentioned above is such an example. If we mate a his4A mutant of the a mating type to a his4A mutant of the mating type the resulting diploid (genotype his4A/his4A) would be a histidine auxotroph like the parent strains. The diploid strain would be unable to catalyze the 3rd step in histidine biosynthesis (Figure 2). Thus, the inability to complement tells us that each histidine mutation is in the same gene. However, the his4 gene is biochemically complex, that is, a single gene encoding a single polypeptide that contains 3 enzymatic functions in the pathway, the 2nd, 3rd, and 10th steps in histidine biosynthesis (Figure 2). Because missense mutations in his4A only affect the 3rd step in the pathway and do not affect the enzymatic activity encoded in the his4B or his4C regions of this gene (2nd and 10th steps) this is reflected in a complementation test. That is, a his4A mutant will complement a his4B mutant or a his4C mutant. This is because a his4A mutant would supply normal HIS4B and HIS4C protein function and a his4B mutant would supply normal HIS4A and HIS4C protein function to a diploid, complementing each of the defects observed in each parent strain. Therefore, the ability to complement doesn't always tell us that mutations are in different genes. Complementation occurs at the level of phenotype.

Given this complication, how can we determine that a his4A mutation and a his4B mutation are really in the same gene, namely, his4? We can determine this using a complementation test, by testing these mutations against mutations at his4 that alter all three enzymatic activities (polar or null mutations). For example, a deletion of the entire his4 gene would be lacking enzymatic activities for the 2nd, 3rd and 10th steps in the histidine biosynthetic pathway (his4ABC). This mutant would not be able to complement any mutation in the his4A, his4B or his4C region (Why?). A similar result would also be observed by using early polar mutations that knock out the ability to transcribe or translate the his4 gene (Why?). Thus, the inability to complement defines the cistron (his4) and says that his4A, his4B and his4C are part of the his4 gene (we refer to them as genetic sub-regions of his4 or sub-complementation groups). This is what is meant when we refer to his4 as being genetically complex. That is, a genetically complex gene shows complementation between different alleles (intragenic complementation). These complementation responses are distinguished from intergenic complementation responses by non-complementing mutants (deletions or polar mutations) at his4. However, not all genetically complex genes contain multiple enzymatic activities like his4. There are a number of ways you can get complementation of function with two non-identical mutant alleles. Some genes contain a single enzymatic function but show intragenic complementation because the protein functions only by forming a multimeric complex (dimer, tetramer, etc.). The basis of intragenic complementation under these conditions is believed to occur through protein-protein interactions whereby the defective part of the protein expressed from one mutant gene gets corrected (at least partially) by interacting with the protein produced by the other mutant allele when the multimeric complex is formed. There are many examples of genetically complex genes and they are important in defining functional regions of the protein. However, most genes are not genetically complex and therefore most mutants do not exhibit intragenic complementation or other complex complementation patterns. Complementation in Higher Eukaryotes: In Drosophila and other higher eukaryotes, complementation analysis is performed in exactly the same way as in yeast in order to answer the question of whether or not two mutations are in the same or different genes. In these tests, gametes (haploid cells) from organisms with the two independently obtained mutations fuse to form a diploid zygote; i.e. the two mutant organisms mate. If the mutations were in the same gene, the new zygote will (usually) have a mutant phenotype the mutations do not complement each other. If the mutations were in different genes, the new zygote will (usually) have a non-mutant phenotype the mutations complement each other.

Experimental Protocol for Experiment 1B Experimental Organism: Mutants were isolated from the strain TD28 (Mat a ura3-52 ino1-13) of the yeast S. cerevisiae in Experiment 1A. We will mate these mutants to known histidine mutants his1-7) from the strain TD28 (Mat ura3-52 inol-13). A. One day before class: 1. We will be provided you with a plate streaked with your putative his- auxotrophs identified from Experiment 1A in lines on a YEPD plate (~6 auxotrophs/plate) equidistant from and parallel to each other. 2. For intergenic complementation: We will steak out six of the 7 his- tester strains on a YEPD plate (Tester 1). These strains represent haploid yeast strains of the mating type (each of which contains a mutation in one of the 7 histidine biosynthetic genes) as follows: his1 (F57), his2 (F77), his3(F79), his5 (F83), his6(F85) , and his7 (F87). 3. For intragenic complementation: We will streak out four of the his4- tester strains on a YEPD plate (Tester 2). These strains represent mating type strains that contain an early polar mutation in the his4 gene, his4ABC (F89), and missense mutations in the his4A (F91), his4B (F406) and his4C (F99) subregions, respectively. 4. Each plate will be grown overnight at 30. B. Week 1: (See Figure 4 for details) 1. Replica plate your mutant plate (M1, M2, M3, U1, U2, U3) to two fresh YEPD plates (Mutant 1 and Mutant 2 plates -label the lines of the mutants as well as the mutant designations). 2. Replica plate Tester 1 plate to a fresh velvet. 3. Take Mutant 1 plate and place on velvet perpendicular to Tester Plate 1 lines (label mutant and tester lines). 4. Replica plate Tester 2 plate to a fresh velvet. 5. Take Mutant 2 plate and place on velvet perpendicular to Tester Plate 2 lines (label mutant and tester lines). 6. Wrap all five plates together and incubate at room temperature for two days. 7. Refrigerate the YEPD plates. C. Week 2: 1. Examine your two mating plates (see Thought Questions 1). 2. Replica plate each mating plate to a SD-his plate (see Thought Questions 2). 3. Wrap all four plates together (YEPD and SD-his) and incubate for 2 days at 30C. 4. Refrigerate plates. D. Week 3: 1. Score the points of intersections for their ability to grow or not grow. 2. Record your results in Table 1

Figure 4: Method of creating mating plates for experiment 1B. We will go over this in class. The his mutant tester of the strains are designated his1, his2, his3, his5, his6, and his7 on one tester plate and his4ABC, his4A, his4B and his4C on the other tester plate. Thought Question 1: What will happen at the intersections of the lines containing the known hismutants of the mating type and the lines containing the unknown his- mutants of the a mating type? Thought Question 2: After replica plating the two mating plates on to SD-his plates, which mating plate will identify intergenic genes and which one will identify intragenic genes? Thought Question 3: Would you expect growth of the haploid his- mutants along the line? How would you interpret the result if one of your putative his- mutants does grew along the whole line?

Final Report: The Final Report for Experiment 1B will be handed in along with the Final Report for Experiment 1A. Class data and specific instructions for analyzing the data and writing the final report are found on our class web site under Experiment 1B.

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Experiment 1B: Complementation Analysis in Yeast Your Name: Lab Day:

Data Table 1: Scoring Complementation Analyziz in Yeast Unknowns Intergenic Knowns Intragenic Knowns

his1 his2 his3 his5 his6 his7

his4ABC

his4A

his4b

his4c

Mutant Is?

M1 M2 M3 U1 U2 U3 Score the intersections of the lines as: ++ = good complementation growth + = a few colonies - = no complementation (no growth) = unclear If your result does not make sense, check with the A.I. or demonstrator If the entire line grows, the putative mutant is his+ If no mating occurred, put No Mat in "Mutant Is?" box.

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