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LECTURE 3 Learning points. 1. How does one do a high copy screen and what types of things do you get out. 2. Starting with a mutant or gene in hand, you should understand how you would go about performing a second site suppressor screen and a synthetic lethal screen. 3. You should understand the different ways one mutation might suppress the phenotype of another mutation. You should also understand how you might distinguish among these. 4. You should understand why two mutations could generate a synthetic lethal phenotype. 5. You should understand what one can learn from epistasis. References An example of a high copy screen. Hadwiger JA, Wittenberg C, Richardson HE, de Barros Lopes M, Reed SI. A family of cyclin homologs that control the G1 phase in yeast. Proc Natl Acad Sci U S A. 1989 Aug;86(16):6255-9. An example of an allele specific second site suppressor analysis. Pennock E, Buckley K, Lundblad V. Cdc13 delivers separate complexes to the telomere for end protection and replication. Cell. 2001 Feb 9;104(3):387-96. Other examples of second site suppressors. Fantes PA. Control of timing of cell cycle events in fission yeast by the wee 1+ gene. Nature. 1983 Mar 10;302(5904):153-5. Russell P, Nurse P. Negative regulation of mitosis by wee1+, a gene encoding a protein kinase homolog. Cell. 1987 May 22;49(4):559-67. Schneider BL, Yang QH, Futcher AB. Linkage of replication to start by the Cdk inhibitor Sic1. Science. 1996 Apr 26;272(5261):560-2. Tyers M. The cyclin-dependent kinase inhibitor p40SIC1 imposes the requirement for Cln G1 cyclin function at Start. Proc Natl Acad Sci U S A. 1996 Jul 23;93(15):7772-6. A large scale synthetic lethal screen. Tong AH, ………..and Boone C. Global mapping of the yeast genetic interaction network. 1 Science. 2004 Feb 6;303(5659):808-13. 2 C. How do you start with essential gene and generate a conditional allele? 1) Can use a regulatable promoter. -This is easy, but slow for stable gene products. Mnaimneh et al (Cell 118, pg 31) integrated titratable promoters for most of the essential yeast genes and characterized them by size, microarray, etc. 2) One can also screen for a ts allele by first mutagenizing your gene 3) One can make a “degron”- a portable temperature sensitive domain. Better than transcriptional shut-off because of speed. -Varshavsky lab D. Newer ways to do screen looking at non-essential genes. 1) All nonessential genes have been deleted in a complete series. a) These can be tested directly for straightforward phenotypes b) If one needs one or two other mutations in order to do their screen, it is harder. These need to be crossed into every strain using a robot (not that hard). Example of such a screen: You want to find the genes required to induce transcription after heat shock, or DNA damage, or during mating. -You put a marker (e.g. ADE2) under the control of a known inducible promoter. You induce the pathway and look for mutants that don’t produce ADE2. You would have to cross this into the deletion collection. c) Big advantage- You go through EVERY gene and don’t have to clone your mutants 2) Example: Cell size in yeast: a) Cell size in yeast, a primer: Budding yeast must hit a size threshold before they commit to start. This is affected by ploidy (diploids bigger than haploids) and by nutrients. Cells in rich media grow bigger before they hit start!! b) Mutants had been isolated which affect cell size in yeast. Most famously an allele of the G1 cyclin CLN3, originally called WHI1-1, later renamed CLN3-1 3 -What do you know about the allele based on the designation above? It is dominant!! c) These mutants are difficult to isolate, but it can be done. They are even harder to clone because they cannot be easily screened. d) The Tyers lab Screen: They went through the entire deletion collection and individually determined the average size of each mutant. Found small and large mutants How do you size cells? A coulter counter is a machine that will count particles and give you their approximate diameter. What about the essential genes? Some of these might be involved in sizing. What if these are rate limiting components; a two-fold decrease in dose could yield a phenotype. Therefore, the Tyers lab also examined diploids with heterozygous deletions of each of the essential genes. Science 297, 395 (2002) -But, maybe cells are just small when the mutants are slow growing, because they spend a lot of time in G1 trying to get big enough to reach the size threshold. One way to look at this is to compare growth rate with size, and see if there are mutants that have the same growth rate, but very different sizes. 4 E. Screens that start with a gene, not a phenotype. That is, “find me more genes in this pathway” type screens. 1) high copy suppressors This is what we talked about wanted to avoid when we were cloning our gene. a) Very often a gene on a 2 plasmid will suppress the phenotype of other mutants in other genes in the same pathway. This has the great advantage of NO CLONING. Very fast analysis. Example 1- a screen: Could you find genes that activate the Cdc28 kinase by overexpressing random genes and selecting for those that allow a strain with a temperature sensitive allele of CDC28 to grow at the non-permissive temperature (36°C). The screen: 5 But, if you just plate cdc28-4 strains on 36°C plates, you will also see that a few colonies grow. What are these? How can you avoid them? One could get a mutation in the strain, such as reversion of the cdc28-4, that allows the strain to grow at 36°C. To identify these: What did the authors find? 3 Genes: - They isolated CDC28 -They isolated two genes that were similar to each other and similar to cyclins found in pombe and Sea Urchins. They called these CLN1 and CLN2. They deleted each separately, no phenotype. However, the cln1∆ cln2∆ double mutants were slow growing. (they weren’t dead because there is a third cyclin gene, CLN3, which they didn’t find). But, many cln1∆ cln2∆ cells were large and elongated, just like cdc28-4 mutants at the permissive temperature. Interesting note: many human cyclin genes were subsequently identified in yeast by their ability to rescue cdc28 mutants. 2) second site suppressor screens. 6 a) Just remutagenize your mutant and look for strains that have gone back to wildtype. For example, temperature sensitive mutants that can grow at 36°C again. These may be mutations in genes that encode proteins that physically interact with the original mutant protein. One can also get reversions or tRNA suppressors. These are less interesting. b) Three interesting classes of second site suppressors. There are many scenarios i) imagine two proteins that bind each other. If the original mutation is a glu lysine in protein 1, the suppressor may be a lysine glu in the binding partner to restore the salt bridge. Example: Intro: Cdc13 binds telomeres. Loss of Cdc13 can cause the ends of chromosomes to be degraded within one cell cycle. There are also mutations in CDC13 that lead to the inability to generate new telomeres, causing a senescence phenotype. This allele is called cdc13-2. This means that new telomeres can’t be added such that telomeres get shorter and shorter and cells die after 40 or so cell divisions. 7 ii) The original gene and the suppressor could have opposing activities. Example Introduction. The Cdc28 kinase, and it homologue in S. pombe called Cdc2, is regulated by phosphorylation. There is an activating phosphorylation, which we will ignore here. There is also an inhibitory phosphorylation, performed by a kinase called Wee1 (cells are small when they lack this kinase). There is a dedicated phosphatase, called Cdc25, whose sole known job is to remove this phosphate. Thus: mutation of cdc25 can lead to a temperature sensitive arrest, due to loss of Cdc2 activity. But, this temperature sensitivity is of cdc25 mutants is suppressed by an additional mutation in wee1 8 iii) Similarly, there are several ways in which suppression can arise when two genes function in opposite directions in a single, linear pathway. one possibility: A---| B---| C Loss of A rescued by loss of B Example Intro: Deletion of the genes encoding all three G1 cyclins (Cln1, Cln2, and Cln3) is lethal. The most important role of the these cyclins is to inhibit S phase by targeting an inhibitor (called Sic1) for degradation. This inhibitor of S phase functions by inhibiting the B type cyclins! What would be some of the characteristics of these types of suppressors that you could use to distinquish between them? a) allele vs bypass suppression A bypass suppressor is a suppressor that will allow suppress a null allele. In the previous three examples, which of these suppressors could act as bypass suppressors? -If a suppressor can act as a bypass suppressor, it suggests that it is not restore the biochemical activity lacking in the mutant, but is obviating the need for it. Epistasis. Definition. Historically, it means that one mutation “masked the expression” of another allele. This has come to be a little vague, so I’ll define it in a way that is most useful, and most commonly used in yeast genetics: If the phenotype of yfg1∆ and yfg2∆ differ, and the yfg1∆yfg2∆ double mutant phenotype is the same as the yfg1∆ phenotype, one says that yfg1∆ is epistatic to yfg2∆. If the phenotypes or yfg1∆ and yfg2∆ are the same, and the phenotype of the yfg1∆yfg2∆ double mutant is the same as the single mutants, one says that yfg1∆ and yfg2∆ are epistatic. 9 Does this word have uses beyond trying to confuse biochemists?? What does it mean? There are no simple formulas for interpretation. But epistasis experiments serve, mostly, to eliminate models, which is often the way models are proven. Lets take the following example WT (ADE2 ADE3) strains are white ade2∆ mutants are RED ade3∆ mutants are WHITE ade2∆ ade3∆ double mutants are …… WHITE. First, how does one refer to the epistasis here? ade3∆ is epistatic to ade2∆ Provide an interpretation, both in terms in genetic diagrams and actual biology? Genetic X ADE3 Y ADE 2 adenine Y is an intermediate in adenine biosynthesis called phospho-ribosylamino imidazole (AIR) NOTE There are actually many more genes and intermediates than this What about the following (Genes Dev. 1996 10: 2025-2037) rad51∆ are defective in recombinational DNA repair: 19 ± 2 % of WT (RAD51) rad52∆ are very defective in recombination: 0.022 ± 0.009 of WT (RAD52) rad51∆ rad52 doubles are like rad52∆ mutants: 0.028 ± .007 of WT (RAD51 RAD52). That is, rad52∆ is epistatic to rad51∆. What is an explanation of this???? RAD52 is required for “normal” recombination, and also other, less frequent events. Rad51 is not required for these other events 10 RAD6 is a DNA repair gene. rad6∆ are very sensitive to DNA damage. This can be partially suppressed by deletion of SRS2. SRS stands for Suppressor of Rad Six What models can you come up with for this? What could SRS2 being doing? 11