Genetika Molekuler (3)

1. Genetika Molekuler (3) Sutarno

2. 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 cycle cell cycle blocked (Cdc-) divides faster (wee-) • GAL transcription no transcription constitutive transcription • signaling pathway no signaling constitutive signal • Drug action resistant hypersensitive • 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 test test of FUNCTION looking at phenotype of a diploid • Linkage test of LOCATION looking 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).