Genetika Molekuler (8)

1. Genetika Molekuler (8) Sutarno

2. 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 don’t / can’t sequence it, is full length protein being made? (by antibodies) • If it’s 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 doesn’t 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 • O’Farrell (Drosophila) and Reed and Beach and Roberts (human) identify cyclins C, D, and E as suppressors of either cdc28 (O’Farrell) 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 don’t 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 don’t need all the components of the pathway • Don’t need any information about the gene products, other than their phenotypes • The genes don’t 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? • Don’t 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 • 1 2 3 4 • Metabolic A  B  C  D  E • In a metabolic pathway, the epistatic mutation is upstream • Example: mutationaccumulates • D gene2 C • D gene4 D • D gene2 D gene4 B • In a regulatory/switch pathway, the epistatic mutation is downstream • Bet understood by giving examples (from the mating pathway) • First identified mutants that don’t mate or don’t 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 sterile ste7 is epistatic to STE4cSTE4 requires STE7 47 • ste11 STE4c sterile ste11 is epistatic to STE4cSTE4 requires STE11 47, 11 • ste2 STE4c signals STE4c is epistatic to ste2STE4 does not require STE2 247, 11 • ste7 ste11c sterile ste7 is epistatic to STE11c STE11 requires STE7 24711 • Also: 2m STE12 (constitutive signaling) is epistatic to ste2, ste4, ste7, ste11 • (Doesn’t require any of the STE gene, and is therefore the furthest downstream) • 2471112 • 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…..