1 GENETICS IS A STUDY OF HOW PROTEINS INTERACT, FOLD, AND FUNCTION While classical genetics often concerned itself with the rules governing the segregation of genetic material, modern genetics is more concerned with uncovering the function of proteins (mainly) and the elucidation of pathway organization. The next few lectures will stress how defects in genes lead to discrete alterations in the functions of proteins. Through these lectures we hope to provide you with an appreciation of the molecular defects caused by particular types of mutations in genes and how they result in the phenotypes observed in the organism. We will also discuss how one can use mutations in a given gene to identify more genes that function in a given pathway and how to organize a genetic pathway. 2 Classes of mutants Recessive Loss of function, often premature termination of a polypeptide Dominant 1) Hyperactivity (Deregulated activity) A) Mutation in negative regulatory domains B) Increases function (better catalyst, stronger interaction) 2) Increased expression (Usually cis acting sequence point mutants or rearrangements) A) Mutation of negative regulatory sequences or introduction of new positive elements in cis to a structural gene (includes heterochronic and ectopic expression) B) Gene amplification C) Mutations that increase RNA or protein stability 3) Novel function A) Suppressor tRNAs (INFORMATION SUPPRESSORS) B) Altered specificity of a DNA binding protein C) Generation of a new regulatory sequence in front of a gene D) Altered enzymatic activity E) Mislocalization 4) Dominant negative A) Subunit mixing B) Blocking processive processes (drug sensitivities) 5) Haploinsufficiency (Can see more often in a sensitized background) 3 Classes of mutants Recessive -Loss of function, often premature termination of a polypeptide Many types of mutations can result in a recessive phenotype. Null alleles: 1) Deletion 2) Premature stop codons, preferably early in the protein. -Truncated proteins often contain partial activities. Partial loss of function alleles: (a.k.a. hypomorphs) Altered activity These alleles can result from partial inactivation of function such as mutating the site for a binding protein such that a lower concentration of the complex between the two proteins exists. Another example is mutations in the active site of an enzyme resulting in a reduced enzymatic activity. Altered abundance 1. 2. 3. Mutations can alter the stability of a protein to reduce its levels. Mutations can reduce the level of expression by removing positively acting sequences controlling transcription, mRNA stability, or translation. Decreasing the efficiency of folding can result in a mixture of correctly and incorrectly folded proteins. Altered localization Subcellular localization is an important aspect of protein function. Mutations in targeting sequences cause loss of function. 44 Levels at which mutations can disrupt protein function. Coding Region Enhancer Transcription + Processing mRNA AUG AAAAA Translation Regulation Factors governing the overall function of a protein -Abundance -Location -Activity Translation mRNA Abundance Translation Folding Stability Localization Activity 5 Classes of mutants Dominant Gain of function. 1) Hyperactivity A) Increased function better catalyst, stronger interaction B) Mutation in negative regulatory domains genetic switches stuck in the ON or OFF position Proteins are machines that can be turned on and off. Many of the more interesting mutations in nature affect these regulatory switches. While the vast majority of mutations we deal with as geneticists are recessive, the rare dominant mutations are often the most interesting. These are often in signaling molecules or key regulators. 6 Hyperactivity A) Increased function Increasing the activity of a protein could be accomplished by changing its specific activity. i.e. reduce the activation energy, G‡, of an enzymatic reaction. Increasing the binding affinity of one molecule for another. For example imagine a situation in which a DNA binding protein is activated by a signal to bind a particular piece of DNA. In the absence of the signal, the DNA binding protein has a low affinity for the sequence in question that together with its concentration prevents high occupancy of the cognate sequence on the chromosome. DBP + DNA Keq = DBP-DNA Complex [DBP-DNA complex] [DBP] [DNA] The signal activating the DNA binding protein does so by increasing the equilibrium constant, by increasing the affinity of the DNA binding protein for the DNA. This same outcome can be accomplished through mutations that: 1) Increasing the concentration of the protein 2) Increasing the affinity of the protein for the cognate DNA site in the absence of signal. Depending upon how the protein works, an increased occupancy for the DNA could be accomplished by increasing the form of the protein that binds DNA if for example a dimer was needed. It could also be accomplished by increasing the number of contacts or strength of existing contacts the DNA binding protein makes with the DNA sequence to which it is bound. 7 Dominant uninducible mutations in the bacterial trpR gene TrpR is a homodimeric DNA binding protein that requires tryptophan for binding to DNA and repression of tryptophan biosynthetic enzymes. Tryptophan TrpR Trp biosynthetic genes Operator Site When tryptophan levels are low, there is insufficient tryptophan to bind TrpR and stablize the homodimers association with DNA. This reduces the occupancy of promoters and results in transcriptional induction. The binding affinity for the repressor on DNA is determined by the energy derived from the sum of three interations: 1) Tryptophan-TrpR interactions, 2) TrpR-TrpR interactions, and 3) TrpR-DNA interactions. G binding = G1 + G2 + G3 In a screen for altered specificity trpR mutants, a mutant was discovered that allowed TrpR to bind to mutant operator sites. This mutant was also uninducible when bound to its native site, i.e. it bound in the absence of tryptophan. Why? 8 In response to DNA damage, ssDNA is produced, RecA is activated and together with residues on LexA catalyzes the autoproteolysis of LexA. This results in loss of LexA dimerization and DNA binding activity and causes transcriptional induction. Changes of amino acids on LexA that are involved in either recognition by RecA or in the proteolysis mechanism itself result in uninducible, dominant phenotypes. Question: Why are these mutations dominant? Under what circumstances might they be recessive? 9 Dominant constitutively acting mutants of Protein kinase C Protein kinase C (PKC)is a protein kinase that acts as the recipient of diacyl gylcerol (DAG) and Ca++ signals Kinase Domain Inhibitory Domain PKC Pseudosubstrate Site DAG Active Site Ca++ DAG Ca++ Mutants causing truncation of the inhibitory domain of PKC remove the inhibitory psuedosubstrate site and activate the kinase just as if DAG and calcium were present. 10 Dominant constitutively acting mutants of Cdc2 Cdc2 is a protein kinase that regulates cell cycle transistions. It is positively regulated by binding to cyclins and negatively regulated by phosphorylation of tyrosine 15 in its ATP binding site. The timing of phosphotyrosine removal regulates entry into mitosis. Premature activation of Cdc2 causes premature entry into mitosis. Wee1(tyrosine kinase) Perpendiculars indicate inhibitory signals Cdc2/Cyclin B Arrows indicate stimulatory signals Cdc25 (tyrosine phosphatase) Mutants in which the phosphorylation target tyrosine 15 is mutated to phenylalanine create a Cdc2 mutant that is resistant to the inhibitory actions of Wee1 and is therefore hyperactive causing premature mitotic entry. 11 Hyperactive mutants in the GTPase Ras Ras is a small GDP and GTP binding protein that acts as a molecular switch. In its GDP bound form, it is considered to be "OFF". In its GTP bound form it is considered to be "ON". Dominant acting mutations in ras are responsible for a large percentage of human cancers. It is important to understand the switch in order to learn how to turn it off. Ras is positively regulated by exchange factors, GEFs, that convert the GDP bound form into a GTP bound form. Acting in opposition to GEFs are GAPs, which are proteins that activate an intrinsic GTPase function on Ras. Activating signals GEF OFF Ras( GDP ) Ras( GTP ) ON GAP Signals Mutations that affect the equilibrium between the GDP bound state and the GTP bound state can have phenotypic consequences. The Ras mutation val19 in which amino acid 19 is changed to valine severely reduces the intrinsic GTPase activity of Ras. As a result, Ras accumulates in the GTP-bound state and is active without upstream signals. 12 Dominant Mutants -Consitutive Expression Constitutive expression usually results from cis acting mutations that remove inhibitory sequences or create new stimulatory sequences in processes that are rate limiting for production of a protein. Although at first glance it seems there should be only one rate limiting step in a given process, the production of a protein is comprised of many distinct steps each of which have rate limiting components that can in a multiplicitive way influence the eventual rate of the process. Ectopic and Heterochronic expression. Ectopic expression is the expression of a protein in a cell in which it is not normally expressed. This applies primarily to multicellular organisms (Metazoans). Heterochronic expression is the expression of a protein at the incorrect time, such as earlier than it should normally be. This applies to cell cycle regulated proteins that are periodically expressed and to developmentally regulated proteins. Inappropriate expression can have dominant phenotypic consequences. Why? 13 Increased Abundance Mechanisms to enhance the abundance of a protein. Silencer Enhancer Coding Region 1) Remove silencing elements (repressors, imprinting) 2) Add additional enhancers Transcription + Processing mRNA AUG AAAAA Translation Regulation Translation 3) Enhance transcription rate (loss of pausing sites) 4) Increase mRNA stability (i.e. abundance) 5) Enhance splicing of the differential splice of interest 6) Enhance protein stability (removal of instability elements) 7) Enhance proper localization (competition) 14 Dominant Mutants -Novel function (Neomorphs) These mutations alter the protein such that it has a novel function. Classically a dominant mutant whose phenotye is not altered by the addition or subtraction of wild-type copies of the gene can be considered to be a neomorph, although you can see that this definition is somewhat lacking in clarity. For example ectopic and heterochronic mutations fall into this definition. In fact often a particular mutant will fall into more than one category, so do not be alarmed when you discover this. Examples 1)Information suppressors. tRNA suppressors that now recognizes a stop codon and instead of terminating the polypeptide, it inserts an amino acid. 2) Altered specificity of a DNA binding protein. Mutations in the DNA recognition domain of a DNA binding protein that now allow it to recognize a novel DNA sequence, thereby placing new genes under its regulation. This can be broadened specificity as well. 3) Altered specificity of protein-protein interactions. There are many examples of members of protein families that bind in a specific way to members of other protein families, for example leucine zipper motifs. Mutations can potentially alter the specificity of such interactions leading to novel partner formation and dominant effects. 4) Altered enzymatic specificity. Example evolved -galactosidase, ebvA. This is an enzyme that normally hydrolyzes a complex sugar other than lactose. However, by mutation its specificity is altered to now include lactose. 15 Dominant Negative Mutations Dominant negative mutations are mutant forms of proteins that interfere with the function of the wild-type proteins. ---hypomorphic or a null phenotypes. This class of mutants is often used to explore protein function in systems with poor genetics. It is primarily based on the idea that proteins work by interacting with other proteins, a titratable property. Dominant negative is often improperly used. Criteria 1) Phenotype must resemble the hypomorphic phenotype 2) Additional copies of WT protein should lessen the phenotype. 16 Dominant Negative Examples Subunit mixing. A tetrameric protein in which all four subunits must be wild-type in order for the protein to function. If half of all monomers are mutant, then only 1/16th of the resulting tetramers will contain no mutant proteins and will function as wild-type. Does this mean the organism will have a mutant phenotype? Phenotypic threshold. Given the fact that equal levels of the WT and mutant protein are produced, is it necessarily the case that only 1/16th of the wild-type activity will result? Ribonuclotide reductase and feedback circuits. 17 Dominant Negative Examples Transcription factors often have separable activation and DNA binding domains. ACT DBD Expression of a mutant lacking the activation domain can compete for binding DBD Overproduction of the deletion mutant is a strategy employed to enhance the interference and achieve the phenotypic threshold . 18 Dominant Negative Examples Blocking processive proceses. DNA synthesis RNA synthesis Protein synthesis R R R S Inhibitor R R Drug resistance is recessive. Drug sensitivity is dominant. S 19 Dominant Negative Examples Topoisomerases. In the course of altering the superhelicity of DNA, topoisomerases bind particular sites and cleave DNA and remain covalently bound to the DNA, allow a topological event to take place, and then religate the DNA. Various drugs can block different steps in this process. Some drugs may prevent binding to DNA. Others may block the religation step, causing the topoisomerases to remain bound to their site of action. Mutant topoisomerases exist that do not bind the drug in question and are therefore resistant to inhibition. Would you expect these drug-resistant mutants to be dominant or recessive? 19 Dominant Negative Examples Topo I Drug A Drug B Drug resistant mutants: Dominant or Recessive? 20 Dominant Negative Examples Heterotrimeric complex ABC A C B Overproduction of Wild-type Protein B B A B B B C B This is an example in which overproduction of a wild-type protein interferes with its own function. 21 Haploinsufficiency Unusual case in which a null mutant causes a phenotype in the presence of a wild-type allele of the gene. CRT1 A repressor of transcription whose levels are very tightly controlled. Loss of one copy give a partial derepression of transcription units under its regulation. Haploinsufficiency occurs very rarely and only proteins whose levels are very critical give this phenotype. The mutation must be a null to qualify. In developmental pathways, proteins that provide quantitative effects may be prone to haploinsufficiency. i.e. morphogen gradient proteins. Other cases of gene classes that might be prone to haploinsufficiency are proteins that form higher order structures such as tetramers A+A+A+A = (A)4. If the binding constant is near the concentration of A in the cell, then the concentration of A to the fourth power governs the concentration of the tetramer. Haploinsufficiency is actually much more easy to detect in pathways that have already been sensitized by mutations that already produce a mild phenotype. This observation is the basis for one of the most useful screens employed by Drosophila geneticists. 22 Conditional Mutations What does a conditional phenotype mean at a molecular level? Types, Ts, Cs, Salt-sensitive, degron Are all ts mutations the same? Different types of ts mutants 1) ts-synthesis. 2) ts-activity. 3) ts-stability. 4) ts-process. 23 Different types of ts mutants 1. Temperature sensitive synthesis In this type of mutant, proteins made at the permissive temperature are temperature resistant. Only newly made proteins are inactive. This is generally interpreted to mean that the process of protein folding is temperature sensitive. Once this type of mutant protein is folded at the permissive temperature, it is as stable as the wild type protein and has full activity. Implications in vitro Protein Activity in vivo 0 1 2 3 4 Time after temperature shift 24 Different types of Ts mutants 2. Temperature sensitive activity In this type of mutant the activity of the protein is Ts regardless of whether it was synthesized at the permissive or non-permissive temperature. These mutants are also Ts in vitro. Implications Protein Activity in vitro and in vivo 0 1 2 3 4 Time after temperature shift 25 Different types of Ts mutants 3. Temperature sensitive stability It is thought that this type of mutant becomes partially unfolded at the non-permissive temperature and therefore accessible to the cells protease machinery. This class of mutants are unstable in vivo but retain in vitro activity when purified. Implications in vitro Protein Activity in vivo 0 1 2 3 4 Time after temperature shift 26 Different types of Ts mutants 4. Temperature sensitive process This class of mutants are actually null mutations * that result in a temperature sensitive phenotype. *Does not have to be a null, but those are the easiest to definitively demonstrate. Examples 1. SPA1 spindle pole antigen -Bridging protein stabilizing a structure 2. SSN6 - general repressor of gene expression. -Derepression of lots of genes results in pleitropic phenotypes and Ts. 3. The heat shock response. Mutations in genes that are involved in dealing with changes in temperature might produce a temperature sensitive phenotype, even if their activities are not affected by temperature. Few Ts mutant proteins fall neatly into a single category with the exception of the Ts process. Often a Ts stability mutant will also have some defect in activity as well, etc. 27 REMEMBER, when you raise the temperature, you are not only affecting the activity of your mutant protein, but also the physiological state of the cell and organism, therefore 2 variables are simultaneously varied. The control becomes critical. What are the advantages of using ts mutations? There are many advantages, especially for essential genes. "Death is not a very interesting phenotype" 1) They allow us to easily work with mutations in essential genes in a haploid organism. 2) They allow us to observe changes in the organism as we change from permissive to non-permissive conditions. 3) They allow simple isolation of suppressors which may themselves be involved in the same process. More on this later. 4) They can be useful for determining the order of function of genes in a pathway. 28 Suppressor or Pseudoreversion Analysis Suppressors are mutations that partially or totally restore a particular phenotype to a mutant strain. Suppressor analysis is an attempt to use a mutation in a gene to identify other genes relevant to a particular process to make us better equipped to solve the underlying biochemical basis of the process. True Revertants vs Suppressors 1. True revertants are mutations that reverse the original mutation back to the wild-type amino acid. 2. Intragenic revertants can be true revertants or secondary mutations in the same gene that restore function to the protein. 1) If a mutant protein is non-functional because it now lacks a particular amino acid that held an alpha helix in place, this mutation can be compensated for by a second mutation that now restore the position of the helix. 2) If a mutant protein is non-functional because it has Ts-stability, a secondary mutation that overproduces the mutant protein might be able to restore enough of the protein to restore an apparent wild type phenotype. Second site suppressors are mutations in different genes, usually genetically unlinked, that compensate for the defect of the original mutation. 29 Suppressors of a pab1 Ts mutant Alan Sachs had a Ts mutation in the yeast PAB1 gene which encodes the polyA binding protein which binds to the polyA tail of eukaryotic mRNA. The Ts mutant failed to grow at 37°C. He selected for spontaneous revertants capable of growing at 37°C. Among these he chose to study those with a Cs phenotype. 25% were in the pab1 Ts gene itself. 10% were dependent upon the pab1 Ts mutation for their Cs phenotype. 65% were able to suppress a pab1 deletion. Why Cs mutations? Why spontaneous mutations. 30 Allele-specific suppressors What is an allele specific suppressor in molecular terms? 1. Two functions in one gene. Some mutants may be defective for one function, another might be defective for both. If the suppressor was isolated for the mutant with a single defect, it may be incapable of suppressing the mutant defective in both. 2. Restoration of specific protein-protein interactions. +_ A B _ + Wild-type interaction __ A1 _ B + +_ A2 _ _ B Mutants A1 and A2 have weaker interactions with B Suppressors can restore interactions _+ ++ A1 _ B1 A2 _ _ B1 + The B1 allele can suppress A1 but not A2 mutants. 31 Allele-specific suppressors 3. Degree suppressors in an allelic series. 90% in pathway A 10% in pathway B. Need 50% activity to survive or score as a suppressor. 100% 75% Activity A5(60%) 50% A4(35%) 25% A3(25%) A2(10%) A1(5%) Allele-specific suppressors of A mutations in pathway B. Allele %Activity Suppresses B1 B2 B3 B4 B5 20% 30% 40% 45% 60% A4 A4, A3 A4, A3, A2 A4, A3, A2, A1 A null 32 Allele-specific suppressors In the pab1 Ts suppressor screen, one class of suppressors was absent -the allele-specific suppressors that retain their Cs phenotype in the presence -of wild-type PAB1. Why? 1) Impossible. 2) Rare. 3) Window of selection. Yeast were grown at 25°C prior to selection at 37°C. Would you expect a suppressor of a Ts mutation to be Cs? Do you expect to miss classes of suppressors by choosing only Cs suppressors? 33 Suppressors Are suppressors usually in the same pathway as the mutation they suppress? How does one know? If the suppressor alone, or a different mutation in it, has a phenotype similar to the mutation it suppresses. Overproduction Suppressors. Gene dosage suppression is very popular in yeast molecular genetics. Why. Examples. Cdc28 isolated Clns and Cks1 as suppressors. How can overproduction of a protein suppress a mutant? A+B AB Forcing and equilibrium to the right and increasing AB. In principle it is similar to suppression by point mutations. 34 Other methods for identification of genes in a given pathway using existing genes or mutants. Cloning by association. Two hybrid systems OFF A His UAS HIS3 G Activation Dom ain A B ON His UAS G HIS3 + 35 Unlinked non-complementors Mutations in an unlinked gene that fail to complement the original mutation. Uppercase = wildtype, lowercase = mutant A/A B/B = wt, A/a B/B =wt, A/a B/b = mutant phenotype This has been used as the basis of screens to identify interacting genes. Worked well for tubulin. How does it work? 1) Phenotypic threshold. 2) Poisoned link of a chain. Screens are performed by mutagenizing a population and mating it to an organism containing a mutation in a particular gene. Screen for lack of complementation. Screen those for absence of linkage. 37 Enhancer and Suppressor screens. This type of screen in similar in principle to the synthetic lethal screen, however it is usually performed in a diploid This screen starts with a mutant background that has a detectable phenotype. a/a X/X x (a/a x/X)* * indicates mutagenized males produces a doubly heterozygous animal a/a b/B where the small b is an allele generated by mutagenesis The "b" allele could be a null allele or a dominant mutation. This screen can detect enhancers or suppressors that alter the initial mutant phenotype and is very commonly used in Drosophila mutant hunts because it can be performed in a single generation without the need to back cross the mutants to make them homozygous. This saves significant amounts of time and allows many more organisms to be examined, i.e. rarer mutants to be discovered. 36 Synthetic phenotypes. When a combination of mutations produces a phenotype more severe than either of the mutations alone. Synthetic lethality is often used in haploid organisms. Yeast sectoring screens for synthetic lethal mutants. This screen is performed on haploid yeast. Colony Red Color Plasmid URA3 Mutant Sector (WHITE) Red Sectoring GENE X Wild-type Sector (RED) Mutant in Gene X(Ts) Non-sectoring Chromosome Screen for mutagenized colonies that fail to lose the color marked plasmid containing the wild-type gene. A percentage of such colonies will contain second mutations that in combination with the initial mutation are lethal. 38 Pathway Organization - How do you know if two genes are in the same pathway or in a different pathway that affects your phenotype? Radiation sensitivity. Many different pathways contribute to repair of DNA damage. Epistasis Groups RAD52 Group Recombinational Repair RAD9 Group Cell Cycle Coordination RAD 3 Group Excision Repair Combining mutations in genes in different groups should cause synergystic sensitivities. Double mutants among genes in the same pathway generally have the same phenotype are the mutations in each gene alone. Exceptions. Some genes are in more than one pathway. 39 Gene Order (Double Mutant Analysis) EPISTASIS Describes the relationship between genes. Epistasis Groups = Pathways Epistatic interactions between genes is used to define gene order. Definition of Epistasis: When two genes of differing phenotypes are combined together in one organism, the epistatic gene is the gene whose phenotype dominates. Interpretation: This depends on what phenotype you are considering. If you are looking at accumulation of an intermediate in a pathway or an unusual structure unique to a particular phenotype, then the epistatic gene is upstream. 40 How does it depend on the way you are looking at the system? Good examples of this are the adenine biosynthetic pathway and the cell division cycle genes. If you are looking at a downstream event in a regulatory pathway, such as transcription, the epistatic gene is downstream. Key to interpretation: Use common sense. Pathway Organization Two mutants with similar phenotypes same pathway or different? Rule of thumb - if the double mutant has the same phenotype as the single mutations alone, the two genes are thought to be in the same pathway. What about synthetic phenotypes? Don't we use double mutants that enhance each others phenotypes to identify genes in the same pathway? What gives? It depends on the kind of mutations you are using. 41 Gene Order How can we use genetics to reveal the organization of a genetic pathway? Double mutant combinations The GAL1 gene is inducible in the presence of galactose gal4 mutants fail to induce GAL1 mRNA in response to galactose gal80 mutants constitutively express GAL1 without galactose Possible orders of function. AB BA GAL4 GAL80 GAL80 GAL4 GAL1 GAL1 Separate Pathways GAL4 GAL1 gal80 gal4 double mutants are uninducible. Which pathway is correct? GAL80 42 Pathway Organization Biosynthetic Pathways Example: Hypothetical tryptophan biosynthetic pathway. Intermediate feeding experiments. C 1, C 2, and C3 are biosynthetic intermediates tryptophan C1 C3 C2 X Observations 1. Mutants in trpA can grow when supplied with C 2 and C 3 but not C1 2. Mutants in trpB can grow when suppplied with C1, C2, and C3 3. Mutants in trpC cannot grow when with any of these intermediates 4. Mutants in trpD can grow with C3. 5. Double mutants between trpB and trpD can grow with C3. D is said to be epistatic to B What are the order of function of the genes? There are differences between regulatory and biosynthetic pathways concerning the use of the word epistasis. The epistatic mutation is the one whose phenotype is observed in the double mutant. However, depending upon which property is being measure, the epistatic gene can be upstream or downstream in the pathway. 43 Adenine Biosynthesis and Epistasis C1 ADE3 ADE2 C2 Red ade3 mutants are white, ade2 mutants are red, ade2ade3 double mutants are white Adenine ADE3 is epistatic to ADE2 ade3 mutants grow on C2 or adenine, ade2 mutants grow only on adenine, ade2ade3 double mutants grow only on adenine. ADE2 is epistatic to ADE3 The epistatic mutation is the one whose phenotype is observed in the double mutant. However, depending upon which property is being measure, the epistatic gene can be upstream or downstream in the pathway. If you look at the red color, ade3 is epistatic to ade2 mutations, if you look at feeding intermediates, ade2 is epistatic to ade3, but the order of the pathway is the same in both. 44 Pathway Organization Morphological Pathways Example: Phage T4 Reciprical shift experiments. This allows the use of different types of conditional mutants to independently manipulate the functions of 2 genes. Ts and Cs mutations in Phage T4 morphogenesis Gene 1 Ts Gene 2 Cs Gene 2 Cs Gene 1 Ts T4 T4 (NPT for Gene 1) (NPT for Gene 2) 18°C (NPT for Gene 1) 37°C (NPT for Gene 2) 37°C 18°C 45 Circular Pathways - The Cell Cycle Using different types of conditional mutations and morphological markers to determine the order of gene function. F START G1 cdc15 Noc cdc28 HU M S rnr1 G2 cdc13 If cdc mutants arrest with different phenotypes, i.e. G1 vs G2, they can be ordered on the basis of morphological markers. Many different cdc Ts mutants arrest with large buds and undivided nuclei. How can they be ordered? Relative to a third marker such as DNA replication. Example cdc13 and rnr1. 46 The Power of Selection Yeast as an example. 14 Mb genome 1.4 x 107 base pairs x 3 = 4.2 x 107 possible single base changes. This does not represent every possible AA change. UAX = Tyr CCX = Pro UCX = Ser The genetic code presents constraints on evolution CUX = Leu Spontaneous Mutation 10-9 per base/generation 1 Kb gene 10-6/generation A null allele in a given gene occurs spontaneously with a frequency of 10-5 to 10-6/generation. lacI STOPS 90% of all positions can tolerate any amino acid 10% can tolerate only a few different amino acids Chemical mutagenesis can increase mutation rates several orders of magnitude. Each chemical causes a different spectrum of base pair changes, a reflection of chemistry and repair systems. 47 46 -Factor (mating pheromone) F receptor STE2 GPA1 gpa1 mutants ON ste mutants OFF STE4 STE18 STE20 (kinase) STE7, 11 (kinase) FUS3, KSS1 (kinase) STE12 (TF) Fus1-lacZ Reporter 4847 -Factor (mating pheromone) F receptor STE2 GPA1 gpa1 mutants ON ste mutants OFF gpa1 ste2 ON gpa1 ste4 OFF gpa1 ste18 OFF gpa1 ste X OFF STE4 STE18 STE20 (kinase) STE7, 11 (kinase) FUS3, KSS1 (kinase) STE12 (TF) Fus1-lacZ Reporter 4948 -Factor (mating pheromone) F receptor STE2 GPA1 Dominant mutants Activating alleles of STE20 STE7 STE11 STE4 STE18 STE20 (kinase) STE7, 11 (kinase) FUS3, KSS1 (kinase) STE20* ste2 ON STE20* ste4 ON STE20* ste18 ON STE12 (TF) STE20* ste7 OFF STE20* ste11 OFF Fus1-lacZ Reporter STE20* ste12 OFF 50 49 -Factor (mating pheromone) F receptor STE2 GPA1 Dominant mutants Activating alleles of STE20 STE7 STE11 STE7* ste2 STE7* ste4 STE7* ste18 STE7* ste20 STE7* ste11 STE7* ste12 STE4 STE18 STE20 (kinase) STE7, 11 (kinase) FUS3, KSS1 (kinase) ON ON ON STE12 (TF) ON OFF Fus1-lacZ Reporter OFF 50 51 -Factor (mating pheromone) F receptor STE2 GPA1 Dominant mutants Activating alleles of STE20 STE7 STE11 STE4 STE18 STE20 (kinase) STE7, 11 (kinase) FUS3, KSS1 (kinase) STE11* ste2 ON STE11* ste4 ON STE11* ste18 ON STE12 (TF) STE11* ste20 ON STE11* ste7 OFF Fus1-lacZ Reporter STE11* ste12 OFF One can also use biochemical events to order gene function in pathways. For example, phosphorylation of proteins can be used. 52 Genetic Screens in Yeast 1) Can you devise a selection or easy screen? 2) How tight? 3) Secondary criteria are important. 4) Mutagenize 2 haploid strains of opposite mating type with different selectable markers for future diploid selection Strain 1 MATa trp1 Strain 2 MAT his3 5) Select mutants 6) Test dominance/recessiveness 7) Place into complementation groups by mating 8) For dominant mutants test allelism by linkage 9) Organize pathway by epistasis analysis 10) Clone genes by complementation, etc. A + A/ Diploid Sporulation Haploid 53 52 Genetic screens in yeast 2 A screen for mutants in a hypothetical hormone responsive pathway that activates transcription Set up selection: Hormone + P URA3 OFF ON P lacZ OFF ON 2 strains of opposite mating type MATa his3-11 ura3-52 Trp+ + reporters MAT trp1-1 ura3-52 His+ + reporters Selection for constitutives: Ura+ Blue Selection for uninducibles: Ura- (5-FOAr) white with hormone Check Dominance/Recessiveness Place recessives in complementation groups. Dominants can be tested for allelism - linkage. Epistasis Clone, Sequence, Disrupt Molecular + Biochemical Analysis 54 Genetic screens in yeast 3 A screen for dosage suppressors of a Ts mutant Transform a genomic library on a 2 micron vector (50 copies per cell) Plate transformants on selective media at the non-permissive temperature of the Ts mutant ---- colonies come up after several days Distinguish between plasmid dependent and plasmid independent events. Isolate plasmids and retest suppression. Sequence, identify gene responsible for suppression. 55 Synthetic lethal screen Red Color Red Sectoring Wild-type Mutant(Ts) Non-sectoring Screen for mutagenized colonies that fail to lose the color marked plasmid containing the wild-type gene. Use strains of differing mating types Demonstrate that the mutant phenotype is selecting for your gene and not some other gene on the plasmid. How? Complementation grouping. Cloning by complementation. How do you know the gene you have isolated is actually the gene in which the synthetic lethal mutation lies? Two ways.