Genetika Molekuler (8) Sutarno

Lecture #6 Notes (Yeast Genetics) LECTURE 6: HOW TO DISTINGUISH SUPPRESSION MECHANISMS Know as much as possible about the original allele The most critical decision is the starting allele(its not always predictable which allele will be best, but you can at least stack the odds in your favor) missense, nonsense, or frameshift allele? Promoter mutation? Null? (different major suppressor classes expected for each of these) If you dont / cant sequence it, is full length protein being made? (by antibodies) If its a Ts- allele, is the protein stable? Is it in a known domain? If the structure is known, is it on the surface? Is the suppressor tightly linked to the original mutation? If so, then intragenic. Ballgame. Does it suppress a null allele? If so, then it is by definition a bypass allele. Rules out some models (interaction, increasing the amount or activity of the original protein) Specificity Is it allele-specific suppression? (does it suppress a null, mutations only in one domain, weak vs strong alleles, or only the original allele?) Frequetly used as an argument for direct interaction, but not necessarily true Is it gene-specific? Does it suppress other mutations with similar phenotype? If it is part of a complex, does it suppress other mutations in that complex? The more tested, and the closer the phenotype, the better example: can test whether a suppressor of a cdc mutant suppresses other cdc mutants, but better if those mutants are defective in the same part of the cell cycle Dominant or recessive? Allows some interpretation of the mechanism (likely due to gain or loss of function), just as with any other mutation What is the phenotype of the suppressor by itself? Critical test to determine the relationship between the two genes. Expect the suppressor by itself (in a wild type background for the original locus) to have a phenotype related to that of the original gene. That phenotype could be the same or opposite to the original mutant. The possible outcomes: Related phenotype……………………best case scenario No phenotype………………………..not necessarily a problem Completely unrelated phenotype……scary…start considering another project Does a null mutation in the suppressor suppress? Then suppression is due to a loss of function. Ballgame. These tests are extremely important, because when you clone SUP+ (especially if it doesnt look like anything), your major clue to what it is doing is going to come from these tests! Another type of suppressor hunt strategy: Suppression by overexpression Transform with a high copy number library, and select plasmids that suppress when present at high copy number Hadwiger…..suppressors of cdc28 (kinase), got CDC28, CLN1, CLN2 (cyclins) Transform with a library that contains genes expressed from an inducible promoter GAL1p OFF on glucose, ON in galactose Main advantages FAST…The gene is already cloned; just need sequencing and subclones to confirm. (should get the WT gene too) You already know part of the suppression mechanism…overexpression causes it Transform with a cDNA library containing genes from another organism Nurse….human Cdk1 isolated as complementing pombe cdc2 OFarrell (Drosophila) and Reed and Beach and Roberts (human) identify cyclins C, D, and E as suppressors of either cdc28 (OFarrell) or cln mutations (Reed, Roberts, and Beach) Next: Ask many of the same questions as with other suppressors to uncover the mechanism Allele- and gene-specificity of suppression Does it suppress a null? (bypass?) KEY: What is the phenotype of a mutation in the suppressor? ______________________________________________________ ENHANCERS / SYNTHETIC LETHALS When one mutation enhances the phenotype of a pre-existing mutation Two mutations that separately dont cause a phenotype, but together, they do Often identifies genes with similar/overlapping/redundant functions Sometimes found by accident (select for a phenotype, then find that phenotype is due to two mutations) Sometimes found by directly testing: cross two mutations with similar phenotypes, find a more severe or lethal phenotype (but need tests of specificity to interpret well, to avoid the sick plus sick equals dead argument) Can be screened for: (Q: how to you find synthetic lethal pairs if they are dead?) Background: ade2- = red ade3- = white ade2- ade3- = white The selection: URA3 YFG+ADE3 ade2- ade3- ura3- yfgD This starting strain will be red, with white sectors, and 5-FOAr, since the plasmid can be lost (assuming YFG is not essential) Mutagenize, and look for red non-sectoring colonies, which should also be 5-FOAs Synthetic lethal mutants can be studied just like any other mutation: Dominant or recessive Complementation groups Linkage Cloning Also need to test: Specificity of the synthetic phenotype Phenotype of the mutation in a WT background (related to the original mutant?) (common in flies…enhancer of zeste, enhancer of hairy wing, etc) ORDERING GENES INTO A PATHWAY USING EPISTASIS ANALYSIS When you have mutations that cause opposite phenotypes you can use a technique called epistasis analysis to order those components. The advantages: You dont need all the components of the pathway Dont need any information about the gene products, other than their phenotypes The genes dont have to be cloned Epistasis (my definition): when two mutation cause opposite or distinguishable phenotypes, the phenotype observed in the double mutant is epistatic to the other one Epistasis establishes dependencies of two gene products: which phenotype requires the other? Dont confuse this with dominance or complementation! In a dominance test, a WT and a mutant strain are crossed, ask if the phenotype of the diploid is WT or mutant (only 1 mutation is involved) In complementation test, two recessive mutants with the same phenotype are crossed, asking if the phenotype of the heterozygous diploid is WT or mutant In epistasis, a double mutant haploid is generated, ask which phenotype is expressed in the haploid Ordering genes: First determine which gene is upstream and which is downstream Then determine what the downstream gene is doing (stimulate or inhibit the pathway) Finally, determine what the upstream gene is doing (stimulate or inhibit the pathway) Ordering is different depending on the type of pathway 1234 MetabolicA B C D E In a metabolic pathway, the epistatic mutation is upstream Example: mutationaccumulates D gene2C D gene4D D gene2 D gene4B In a regulatory/switch pathway, the epistatic mutation is downstream Bet understood by giving examples (from the mating pathway) First identified mutants that dont mate or dont signal (sterile):ste2, 4, 5, 7, 11, 12, 20 Next identified mutants that signal constitutively:gpa1, STE11c, STE4c Now they could put these genes into a pathway simply by creating double mutants: Double mutant mutantsphenotypeconclusionin Englishpathway ste7 STE4c sterileste7 is epistatic to STE4cSTE4 requires STE74 7 ste11 STE4csterileste11 is epistatic to STE4cSTE4 requires STE114 7, 11 ste2 STE4csignalsSTE4c is epistatic to ste2STE4 does not require STE22 4 7, 11 ste7 ste11csterileste7 is epistatic to STE11cSTE11 requires STE Also: 2m STE12 (constitutive signaling) is epistatic to ste2, ste4, ste7, ste11 (Doesnt require any of the STE gene, and is therefore the furthest downstream) Once we had the mutants with opposite phenotypes, we went from simply knowing that these genes had a role in the mating pathway to now having a strong genetic model for the order that they function. This was then tested biochemically. This was especially important, since this pathway contains a kinase cascade (Ste11 phosphorylates and activates Ste7). This would never have been possible if the mutants all had the same phenotype. Potential problem: correct interpretation requires knowledge of the alleles (nulls are the most reliably interpreted) (an example of interpreting partial loss of function alleles is in the suppression article) This is not THE answer, but generates a good model for further study. On to Drosophila and other larger (not necessarily higher) eukaryotes…..