Lecture #2 Notes (Yeast Genetics) LECTURE 2: MUTANT ISOLATION STRATEGIES, BASIC GENETICS TESTS If we want to do genetics the most important step is ISOLATING MUTANTS Genetics is a conceptual science, (people like Mendel and Morgan and McClintock didn’t know what a gene was, but they were still able to identify and map genes). Mutants can be isolated with no prior knowledge of what their specific function is. The goal...to identify ALL the genes that affect a given process otherwise it's like trying to solve a puzzle without having all the pieces will take multiple selections to achieve this One choice that has to be made: General vs. specific Cast a wide net? Sometimes specific selections are needed, but pre- conceived notions can limit your findings; you might find what you want, but can miss more interesting things (example: mating defective vs. mutants defective for response to pheromone signaling, or pheromone production, or nuclear fusion, etc.) Look for different classes of mutants. Some ideas on ways to change the selection: Typically the first mutations that are found are those that block a process. But they are not the only kind of mutants….also important are mutations that accelerate or de-regulate a pathway. (opposite phenotype) accelerate a pathway (block a pathway)or when it shouldn't process doesn't occuror more rapidly cell cyclecell cycle blocked (Cdc-)divides faster (wee-) GAL transcriptionno transcriptionconstitutive transcription signaling pathwayno signalingconstitutive signal Drug actionresistanthypersensitive Both classes are informative! These can also be combined with other changes to the selection: dominant vs. recessive (having the ability to do haploid genetics in yeast allows the easy isolation of recessive mutations.) cis-acting (chromosome segregation, replication, transcription, etc.) conditionals overexpression phenotypes others (suppressors, synthetic lethals, dominant negative, unlinked non-complementers) Combined, this results in a HUGE variety of possible selections. Experience has to be used to judge which will give the most interesting class. _____________________________________________ _________ OK, let’s say we set up a selection, BUT we don’t get any mutants...why? 1. essential gene…rarer mutations needed that allow survival, yet cause mutant phenotype 2. dominant mutations...same reason, rare changes may be necessary to satisfy selection 3. redundant function (histones on C-II and C- XIV)(cyclins) 4. pleiotropic phenotypes mask your phenotype (Drosophila example..a screen for eye mutants may not identify a gene if it is required earlier in development) 5. practical reasons not enough cells screened (use mutagen or a new one to increase frequency) too stringent (if too much drug used, won't get resistance) not stringent enough (looking for Ts- at 32o vs 30o) Basic Genetic Tests: Dominance and Complementation (including exceptions) We have mutants: now what do we do??? The first thing that I want to know is: are the mutations dominant or recessive. Why? Three good reasons: NOT JUST A FORMALITY !! 1. need to know this before doing complementation tests 2. the cloning strategy differs depending on whether the mutation is D or R. 3. it gives important clues to interpret the WT gene function How? Cross the mutant with a WT parent, look at the phenotype of the diploid. Ts- / Ts+ -->if diploid grows at NPTo, then recessive if diploid doesn't grow at NPTo, then dominant What does it mean? Recessive mutations are most commonly due to loss of function. (= hypomorph) It could be partial loss or complete loss, but gene activity is lowered or eliminated. Receptor example: Dominant mutations can be due to several types of mutations. A common one is due to a gain of function (= hypermorph) Receptor example, deleted for an intracellular inhibitory domain, that signals in the absence of ligand. Other possibilities for dominant mutations: 1. Haploinsufficiency loss of function results in dominant phenotype (e.g. a haploid level of WT gene product is insufficient for WT phenotype in the diploid) example (assume no feedback regulation, etc.): if a kinase is produced in just barely enough levels to give WT phenotype (Needs ~90% of WT levels to be WT, then a heterozygous null over a diploid might be expected to produce only ~50% of the WT kinase activity. That mutant would be dominant due to haploinsufficiency. Another good example: histone genes Essential and duplicated in most eukaryotes, only two copies of each in yeast D of one copy causes some mutant phenotypes, due to fewer nucleosomes The determining factors are how much activity is lost due to the mutation and how much activity is needed to result in the WT phenotype. 2. Dominant negative (= Antimorph) when a protein has two functions, loses one due to mutation, and the other function competes with the WT protein in the diploid. Give DNA binding protein example: DB domain and dimerization domain. DB mutation, results in dimerization domain competing with WT protein. Two characteristics of dominant negatives: Typically, a dominant negative mutation over WT causes a more severe phenotype than a null over WT. Also, dominant negative mutations are typically dosage sensitive: overexpression of the allele may cause a stronger phenotype than single copy. Greater excess of the mutant exacerbates the phenotype. Dominant negatives are especially useful in mammalian systems to identify the pathways that they are involved in (see the Herskowitz review). 3. Gain of abnormal function (= Neomorph) This is the class to avoid, since the phenotype is misleading. examples: DNA binding protein that recognizes a new sequence, resulting in mutant phenotype a protein kinase that gains a new/different substrate specificity a fusion protein mis-localized protein inappropriately regulated protein (either in wrong cell type or developmental stage....e.g. Antp, which turns antenna into leg, but normally functions in the thorax) To distinguish this class from the more interesting classes of dominant mutations, need to create nulls. If the null has a dramatically different/unrelated phenotype to the dominant mutation, give it up. Overexpression studies, combined with creating nulls can distinguish the classes of dominant mutations. _____________________________________________ _________ ** Based on this info, it should be clear that doing a good mutant hunt is not just a matter of aimlessly looking for something that doesn't grow. Good geneticists keep these ideas in mind when setting up a hunt, and are continuously successful. Bad ones do aimless genetics, and waste a lot of time. Knowledge of these concepts allows us to find exactly the most interesting mutations to us. Examples: if you suspect that there is a repressor of GAL gene transcription, how would you set up a selection to identify that repressor? NOT Gal-! Recessive constitutive or dominant non-inducible (Gal-) would be the best choices. To find proteins that inhibit/block the cell cycle, don't look for recessive Cdc-, but more likely, dominant Wee-, dominant non-cycling cells, recessive larger cells, or overexpressors that block cell division. Keep these thoughts in mind when reading genetics papers: why did they set up the mutant hunt this way? Why did they select for this class of mutants instead of another? What would we expect to get out of that particular selection? _____________________________________________ _________ Once we know whether our mutations are dominant or recessive. What's next? Try to group them. Most important grouping: find out how many genes are represented in the mutant collection. Why? Can't work with 500 mutants...we need to identify how many genes are involved, then we can pick a small number of representative mutants to work with. To determine how many genes are represented in a mutant collection, both complementation tests and linkage analysis are needed. Complementation testtest of FUNCTIONlooking at phenotype of a diploid Linkagetest of LOCATIONlooking at progeny from a diploid _____________________________________________ _________ Complementation test How?cross 2 recessive haploid mutants that have the same phenotype, and look at the phenotype of the diploid Ts-#1 x Ts-#2 if the diploid is Ts-, then the mutations don't complement each other, they are said to be in the same complementation group, and are likely to be in the same gene if the diploid is Ts+, then the mutations complement each other, are said to be in different complementation groups, and are likely to be in different genes. Notice the difference between complementation test and dominance test. Both are looking at the phenotype of a diploid, but for dominance test we are looking at mutant over WT, in a comp test we are looking at a heterozygous double mutant diploid. _____________________________________________ _________ Interpretation:generally, mutations in the same complementation group are in the same gene, and mutations in different complementation groups are in different genes. This needs to be confirmed by linkage analysis. WHY? Because there are exceptions. Exceptions: Intragenic complementation (mutations in the same gene complement each other) usually implies a multimeric or multifunctional protein examples: a-complementation in LacZ omega fragment of b-gal is an N-terminal deletion (D(lacZ)M15 allele) the a-complementing fragment just produces the N-terminal 146 amino acids each fragment is inactive by itself, but when produced in the same cell, they complement each other b-gal is a tetramer yeast examples: HIS4, CMD1 (Sci 263:963), TUB2, ACT1 Unlinked (extragenic) non-complementation When mutations in 2 different genes don't complement each other (not as expected) usually implies subunits of a multi-protein complex yeast examples: TUB1 and TUB2, SPTs, SIRs, ANC1 and ACT1 _____________________________________________ _________ Let’s say you have identified 15 genes in a mutant hunt. You need some way to figure out which ones to study first. One easy way is to simply test whether those mutations cause any other (non-selected) phenotypes. More specific or additional phenotypes: Cdc- mutations grouped according to stage of cell cycle (examine microscopically) Ste- (mating defective) for what step is blocked (using simple assays) Gal- transcription mutants looked at for whether they affect transcription of other genes by testing for other mutant phenotypes. Advantages of having other phenotypes: Gives a logical basis for deciding which mutants to study first clues to other processes the gene may be involved with (e.g. cse1- is Met-) often makes cloning easier Ts- or Cs- might provide clues as to which are essential allows grouping of the most closely related genes If you have mutants within a complementation group that have subsets of phenotypes, then you can next ask if those mutations cluster (perhaps defining a functional domain).