Lecture #2 - Yeast Genetics Flashcards

Question

Synthetic Genetic Array - Interpreting the interactions Example

Answer 1

WT colony (AB) --> growth is 1 Mutant colony b (Ab) --> growth is 0.75 (slight growth defect) Mutant colony a (aB) --> growth is 0.5 (bigger growth defcet) To get the predicted size of the double mutants --> we multiply the sizes of the individual mutants --> 0.75 X 0.5 --> predict the double mutant to have a 0.375 growth size IF there is no intercation - Uses multiplicative modeling --> model assumes that these two mutants are indepentdent BUT if the two genes are involved in the same pathway then the actual growth of the double mutant will be a different value -

Answer 2

In exmaple - predicted growth rate of double mutant is 0.375 IF the double mutant grows better than 0.375 then there is a positive interaction - Positive interactions may mean that the genes are in the same pathway IF the double mutant grows worse than 0.375 --> then there is a negative interaction IF the double mutants growth rate is the product of the two single mutant growth rates = there is no interaction IF the double mutant is dead (growth rate is 0) = the interaction is syntehtic leathl

Answer 3

Image: Positive interaction = green Negative inteaction = Red No intreaction = Black Gene 1 = has 5,000 data points (1 for each double mutant) - Do this for every gene (Ex. Gene 2 also have 5,000 data points) --> computer will show the pattern and compare the pattern of 1 gene to all other patterns of other genes and find the ones that are more similar - Doing an unsupervised cluster analysis

Answer 4

Image – every row is 1 gene and it is show next to the gene that has the most similar pattern of interaction - NOT looking at the gene in order 1-5000 INSTAED places genes next to each other than have the most similar pattern of interactions

Answer 5

Image: 3 genes at the top --> shows that the analysis puts genes next to each other because they have similar patterns - Image – the first 3 genes have similar patterns and are likely to have similar functions Negative genetic interactions = red --> shows synthetic leathity on a large scale If we know the function of gene 467 and gene 7 then now know the function of 1273 because it is likley a SU

Answer 6

Genes of similar functions are found in SGA because when you delete 1 gene and there is a gene that functions to that gene it will show a similar pattern of interactions Example – delete the alpha SU then that won't be able to form a dimer OR delete the beta SU then you also can’t form the dimer = have similar genetic interactions with other genes --> alpha and beta tubulin would be next to each other on the map

Answer 7

Because looking at deletions of genes you won’t find proteins but you can find parraell pathways Red genes (negative interactions) = show parrallel pathways

Answer 8

Positive interactions report on protein complexes and gene networks without knowing anything about gene function Example 6 SU complex (6 genes that makes 6 proteins in 1 complex) - IF you remove any gene then you lose the function of the complex (Need all 6 genes for the complex to work) IF remove 1 gene = cells grow at half of the rate vs. IF you delete a different SU then the cells grow at 0.5 IF you delete both SU (double mutant) you expect growth to be 0.25 (0.5 X 0.5) if there is no genetic interactions BUT when you have a double mutant you actualy get 0.5 - Get 0.5 because each mutant will remove the function of the whole complex = when remove the SU you will always grow at 0.5 (doesn't matter id you delete 1 or 2 SU) - Since expected 0.25 but grows at 0.5 = observed growth rate is higher = positive interaction

Answer 9

Gene X likley functions with genes that have similar patterns (functions with 405 etc) Gene X has positive intercation with some of the genes in the columns it might have functinos with those (could be in the same complex)

Answer 10

For SGA – don't need to only do KO mutants Instead of making double mutants --> NOW look at how combining a drug on WT cells vs. Mutants Take WT and add drug --> grow at 0.5 Take Drug and add to each of the single mutant --> look at growth - Already know what the rate of growth of the single mutant without the drug is END - get genetic pattern for teh drug --> have it cliuster - Look at the patterns from the drug and see what genes it clusters in from the SGA analysis Example - say the genetic pattern of the drug clusters with genes 301, 405, and 2894 --> The genes that it clusters with are good candidates for the genes/proteins that are being targeted by the drug

Answer 11

SGA is not limited to yeast – can also do in mammalian cells In mammalian cells NOT mating cells to make double mutants BUT instead have 1 gene that is repressed or KO because of CRIPSR or shRNA - Look at phenotype of cells that have 1 gene repressed or KO and then also do that for double mutant (viruses that will code to knocking out double mutants) - Can do this in cultured cells

Answer 12

Example of a gene network created based on SGA data - Each single mutant will have a different set of results, or signature, based on its growth scores with the 5000 non-essential mutants - The growth signature of each gene, cross-referenced with every other gene, will be different --> These mutants are then subject to cluster analysis to learn more about gene functions - Clustering is based on negative and positive interactions --> if a and b negatively interact AND b and c negatively interact, then all three genes are likely functionally related

Answer 13

Purpose - Set up a screen to identify synthetic interactions with a specific mutant that we are interested in - Used if we are interested in a particular gene

Answer 14

Start with a plasmid that has he WT gene of interest and a URA3 marker --> transform this plasmid into the yeast strain that has the mutation/deletion in the gene of interst --> NOW the cell will be phenotypicallt normal bevyase they have a WT copy of the gene of interst on the plasmid --> Once tranform the cel --> mutaginize the yeast with EMS to cause point mutations across the genome --> plate the mutogenized yeast onto non-slecetive medium so that eveyrthing will grow --> To find the synthetic leath genes --> replica plate onto 5-FOA plates - 5-FOA = chemical that turns into a toxic metabolite when URA3 gene is present --> cells thay have the plasmid will die on 5-FOA END – colonies that have synthetic lethal interactions will grow on non-selective meidum but NOT 5-FOA - Once we have these colonies we can map their mutations to find the genes that are synthetic lethal with our gene of interest CHECK THIS Because have URA3 on plasmid --> cells that keep the plasmid have synthetic lethal --> Cells have to keep the plasmid = the cells grow on 5-FOA - Looking for synthetically lethal mutant in the cell that can grow

Answer 15

When yeast cells are not placed under selection --> the yeast will lose the plasmidsBUT cells that have synthetic lethal mutations MUST maintain the plasmid in order to grow Results - Synthetic lethal mutant will grow on non-selective mediuam and not on 5-FOA because it must retain the plasmid carrying the URA3 gene

Answer 16

Can perform a similar screen in bacteria to identify synthetic lethal interactions Start - we have a deletion or mutant cell and a WT copy of this gene of interest on a plasmid (plasmid also has amplicilin resistence + LacZ reproter) --> THEN preform transposon mutogensis --> a kanamycin resistent gene is insterted randonly through the genome --> TEHn screen the bacteria on kanamycin + IPTG + X-gal plates End – Bacteria that inserted a transposon will be able to grow on kanalycin and IPTG will cause the LacZ from the plasmid to be expressed --> LAcZ will metabolize the X-Gal = make blue product - In cases where the transposon insertion and gene of interst are synthetic lethal + THEN the cell must mainatin the plasmid which has the LacZ gene --> colony truns blue

Answer 17

Plasmid = has a WT copy of the gene + has a LacZ reporter + ampicillin resistance Plasmid = needs to be highly unstable so that in the absence of ampicillin, the bacteria only maintain the plasmid if it is required for growth. (only keep the plasmid if it is needed for growth)

Answer 18

Because of whole genome sequencing --> now have another way to screen for synthetic lethal interactions in bacteria Process: 1. Create a saturated libration of Transposon inserted mutants in both a WT and mutant bacteria strain 2. Sequence the whole genome to identify the transposon insertion sites in Wt and mutants END - The frequency of insertion in the mutant will be reduced compared to WT if there is a synthetic lethal interaction between the mutant and the transposon insertion mutations

Answer 19

Example – reads mapping back to each gene (image) Gene B is synthetic lethal to our gene of interest because the cells with a transposon that interrupts gene B can only grow in Wt but not in the mutant cells

Answer 20

We can learn a lot about the function of different genes by studying synthetic lethal interactions

Answer 21

1. Parallel pathways 2. Physically interacting proteins

Answer 22

Synthetic lethal interacts can arise in genes in parrallel pathways Example - redundant genes - In the WT – both pathways function - In mutant a - red pathway does not fucntion BUT gene b can funcion - In mutant b – the blue pathway does not function BUT gene a can function so the cell survives - The cell needs at least 1 of these pathways to survive --> SO if you mutate both pathways the cell dies When you see synthetic lethality --> tells you 2 genes function in the same pathway

Answer 23

Mainly seen in non-deletion mutants If have a mutation in gene a --> then protein can still bind to protein b (get complex but it functions less effcicetley) ; If have a mutation in gene b --> then protein can still bind to protein BUT when you mutate both a and b (both interacting oartners are mutanted) then you can’t make a complex and the cells can’t grow --> cell dies - Synthetic interaction tells you there are interacting proteins - In image - when mutaate a or mutate b --> the change from circle the square still allows for intercation and function BUT when both a and b are mutated in the same cell the interaction is compelley lost

Answer 24

Problem with identifying genes in mitosis = genes are likley essnetial genes Solution – Screen a collection of temperature sensative mutants

Answer 25

Temperature sensitive mutants = conditional mutants that grow at 25 degrees but not 36 degrees - Gene with point mutation --> mutants grow at 25 degrees but they don't grow at 36 degrees Temperature sensitive mutants allow you to study essential genes in haploid yeast - 1,000 of the total 6,000 genes in yeast are essential

Answer 26

Purpose – Temperature sensatice mutant librarieres to permit study of essential processes Want to study all 1,000 essential genes = need to generate a lot of mutants To make the temperature sensitive mutant libraries = select for mutants that fail to grow at 36 degrees - Process – Mutagenize WT yeast on rich medium (YPD) at 25 degrees --> cells will grow to colinies --> replica plate using velva pad onto rich medium at 36 degrees --> Select for mutants that do not grow - Don't actualy know what the muation is at this point (just collect genes that you know have mutations in essential genes that you wnat to study)

Answer 27

Interphase yeast cells have 1C DNA content when use flow and stain for DNA S phase cells have 2C DNA content when use flow and stain for DNA

Answer 28

Overall - Growth is blocked at 2C = accumulate 2C DNA --> Use 2C DNA as phenotype of cells that are blocked in mitosis Process – Pool mutants --> grow at 25 degrees --> synchronize the cells in S phase --> shift to 36 degrees --> release from S phase --> stain and sort for cells with 2C DNA content --> Retest single mutants - High DNA content = means that the mutant is blocked in S phase - Cells with high 2C content = stuck in mitosis vs. mutants that are not stuck will go through and go to 1C DNA

Answer 29

Supressors and supressor screens = how you use mutants to find other mutants in the same pathway/function with gene of interest - Find genes related to the mutated gene of interest - ALSO find genes related to mutated gene using synthetic lethality screens Example - Start with having temperature sensitive mutants and the mutated genes is involved in mitosis --> want to find other genes that function with the already mutated gene and also likley function in mitosis

Answer 30

Have WT cells with WT red and WT blue --> do mutant hunt and find a temperature sensitive mutant in the red gene - YOU want to find other genes that function with the red gene --> do a supressor screen To do supressor screen - Start with temperature sensative mutants that can’t grow at 36 degrees (starting with mutant cels) --> mutogenize the cells --> NOW look at cells that can grow at 36 degrees --> cells that grow at 36 degrees have a supresor if the first mutation - Asking what restores the phenotype - First mutation causes the cells to be unable to grow at 36 degrees THEN have another mutation that allows the cells to grow at 36 degeres

Answer 31

Compensatry mutations that restores WT phenotype - Use suppressor genetics to find supressors Supressor = often genes that function in the same or related pathways

Answer 32

In Supressor screen – look for a new mutant to appear such that there is a supressor with the WT phenotype New mutation is literally supressing the mutant phenotype

Answer 33

1. Intragenic - Revertant + Second site mutation 2. Extragenic - Intercation + Bypass + Epistatsic + Dosage + Mass action

Answer 34

Intragenic – mutation occur within the same gene - Includes true revertant and second site mutation Intragenic Supressors do NOT help us find genes in pathway

Answer 35

True revertant --> The mutant nucleotide chnages back to the WT nucleotides - Red gene has a mutatiion --> then have a true reveratnt Example (in temperature sensitive mutants) – After revertant --> NOW WT sequence and can grow at 36 degrees

Answer 36

Second site mutation (image shows in blue) --> Second mutation in the same gene that restores the phenotype - Can restore the WT phenotype by affecting splice sites or amino acid seqeunce Example – salt bridge – get mutation that allows the protein to fold again

Answer 37

Extragenic – Have a second mutation in other genes Includes – Interactions + Bypass + epistatic + Dosage + Mass action

Answer 38

Allele specific for the mutant and the supressor (In example - Blue mutant only works when have red triangle) Does not rescue null mutations --> example – a mutaion in the blue protein would not suppress a KO of the red protein and vica versa

Answer 39

Example – WT red and blue proteins interact with each other (in temoature sensative example to grow at 36 degrees you need the square to bind to the blue square to form a complex ) - Mutate Red = blue and red can't interact = cells can’t grow at 36 degrees - Blue protein can have an allele specific conformational mutations that allows red and blue to interact again --> restores the WT = cells grow at 36 degrees - Mutant allele of the blue protein = the supressor

Answer 40

Overall - have mutation in alpha tubulin that matches beta tubulin mutation --> allows them to bind - Alpha tubulin is the supressor - The mutaion is at the interface between alpha and beta tubulin --> specific that it will help that particular mutant allele of tubulin grow Shows that interaction supressor won't rescue null - changing alpha won’t help if there is no beta tubulin

Answer 41

Can rescue a null mutant because when a cell has a bypass supressor it no longer needs the function of the gene of interest Bypass supressor can ALSO confer a novel function to a protein that allow sit to perform the function lost in the mutant Parrallel pathways can be mutated to have bypass function

Answer 42

Red protein is a maltose channel and the blue protein is a lacotse chanel - When the red chanel is mutated --> maltose can’t be transproted - When there is a bypass supressr in the blue lactose chanel --> allows the chanel to move BOTH maltose and lactose (NOW the functinoal red protein is no longer needed for the WT phenotype) Start --> Mutate maltose = cells can’t grow on matose --> Find supressor in the mutates and now they can grow on maltose

Answer 43

Type of bypass Supressor Can rescue a null - When the cell has a bypass suppressor, it no longer needs the function of the gene of interest - Supressor itself can be a null mutant

Answer 44

Red gene makes a circle that typically supresses the blue protein --> give WT phenotype Blue protein normally prevents the cell from growing (Ex. Blue has a checkpoint function – when it is active it blocks the cycle and cells won’t grow) When have Red = blocks the blue – cells grow Mutate Red = Not blocking blue = blue is active = cells don’t grow (mutant phenotype) Mutant blue (Blue and Red are both mutated) = Blue is not active = cells grow because you remove the checkpoint = restores WT phenotoe

Answer 45

Dosage compensation worse by restoring balance in a system - Modulates the balance of proteins Does not rescue a null Rare

Answer 46

Start – blue and red are equally expressed (Red and blue proteins need to be in equal balance) In the mutant --> less red is made --> the unbalance amount of blue protein causes a mutant phenotye THEN A compensatory muations in the blue = restores the stoicheometry of red:blue proteins --> brings balance back t the system --> get WT phenotype --> cells grow - Blue = supressor

Answer 47

Example #2 – Have too much alpha tubulin (because have less beta tubulin in the mutant = have a lot of alpha tubulin) --> THEN have a mutation that lowers the amount of alpha tubulin = allows the amount of alpha tubulin to match the amount of beta tubulin = can get interaction

Answer 48

Type of dosage supressor Does not rescue null + the supressor muation cant be a null Overall - Can stabilize mutant by intreaction based on the level of protein

Answer 49

Example – Blue and red proteins normally interact with each other (circle and square need to form a complex for the cells to grow) Have mutation --> causes a conformational change which causes red protein to be misfoloded --> blue and red can't interact --> cells don't grow BUT those mutated red traingles are always going between triangle and circle in equillibrium --> over experess blue square = increase binding of the blue to the porperly folded red circle and stabilityzes the blue bindinig to the red = protein can fold and fucntion - Overexpress blue protein = restores WT phenotype - Blue mutation that leads to over expression = supressor muation Example #2 – overexpress alpha tubulin = can form the alpha-beta tubulin dimer

Answer 50

Muatant has 2 phenotypes: 1. Temperature sensative for growth 2. Accumulates 2C DNA at 36 degrees

Answer 51

Purpose – Identify mutants that restore the WT phenotype (identify suppressors) - New mutation is literally suppressing the mutant phenotype

Answer 52

1. Duplicated genes 2. Essential genes 3. Small genes 4. Genes with mistaken assumptions about the phenotype 5. If Supressor has 2 copies of it = might miss it - Example – 2 copies of alpha tubulin THEN might not find alpha tubulin genes)

Answer 53

Grow a large culture of mutant in rich medium (ex. Mutant that can’t make uracil) --> spontenous random and rare mutaions will occur during cell growth due to errors in DNA replicatiion --> Plate the cells onto sleective medium (Ex. Grow in media that alcks uracil) --> after a few days only the mutants that have a supress muation will grow and form colonies - Suppressors = genes we are intersted in to learn more about the uracil synthesis patwhays --> care about colonies with supressor muations that survive in the selective medium

Answer 54

Process – Start with WT cells with Wt Tub --> mutogenize to get mutated Tub 2 --> now have mutant cells (temperature senstaive mutants with mutanted Tub2) -> grow temperature sensative mutants at 25 degrees --> Mutaginzie them (Add EMS) --> Grow cells at 36 degrees --> Look for mutants that can now grow at 36 degrees (only Tub2 mutants with supressor will grow) --> Sequence --> Look at which genes are most commonly muatted - End - Have cells that have a supressor and also Tub 2 mutation - KNOW that the supressor mutation will be in genes that are related to tubulin

Answer 55

1. Integenic supressors --> Tub2-1 --> reertant to TW 2. Second site - Salt bridge and have mutation within Tub2 that allows that to fold again and restore WT 3. Interaction supressor --> allele specific mutant - Example - muation in alpha tubulin that allows it to bind to mutated beta tubulin (alpha tubuloin is the supressor) 4. Dosage supressor --> having too much alpha tubulin is a problem --> if lower alpha tubulin then matches again 5. Mass action supressor --> over express alpha tubulin so can form the dimer again

Answer 56

Issue - Have 1 supressor mutant (mutants have Tub2 mutation and supressor muation) BUT the cells have more than just the supressor mutations (have hundreds of mutations) Solution – sequence the genome of 30 supressor mutants --> the other non-supressor muations won't align across the mutants - Look for mutations among suppressor strains that are in the same gene = find the gene that is the supressor - If have 3 genes that are suppressors = have cluster of 3 genes IF cells have their own phenotype = can follow phenotype and see what the gene is

Answer 57

START - we have mutant that will NOT grow when plated on galactose Can grow up mutant in LB (rich media) --> plate on selective medium (which contains galactose) --> then see if the colonies with suppressor mutations will form

Answer 58

NEXT step in supresor screen = actually identify the supressor gene Uses Linkage analyss (in yeast) + whole genome sequecing + Tn—seq for transon mapping (Bacteria)

Answer 59

Yeast – can use yeast genetics and linkage analysis to determine the different number of genes and the differet number of suppressors from the screen) Yeast + bacteria - Whole genome sequencing is now affordable = we can also sequence the genome of independent isolates to find these suppressor mutations - Bacteria = whole genome sequencing before and after is often used to identify the suppressor mutations Bacteria - use transposon insertion mutagenesis to induce mutations --> If any of these mutants are suppressors, we can identify the transposon insertion site using PCR and Sanger sequencing

Answer 60

Overall - perform a plasmid screen to identify suppressors Purpose - Find redundant genes + dosage suppressors + mass action suppressors - This screen leverages dosage for suppression - Don't get bypass or epistatic supressors because not a nul - Don't get interaction because not generating an allele specific mutant

Answer 61

1. Transform our mutant with the high copy genomic library - We do this so that each mutant cell gets 1 plasmid ; Each plasmid overexpresses one gene - Each plasmid in the library has a piece of the yeast genome (test function of DNA that is on the 1 plasmid) - Plasmids are overexpressed because in high copy number in the cells 2. Then plate the yeast or bacteria at restrictive temperature or on selective medium so that only the suppressors will grow 3. Using the power of selection, we can determine which plasmids allowed for growth to see which genes are suppressors of our mutant - Need to determine the plasmids that allowed for growth

Answer 62

Mutation in Tub 2 at the interface of alpha and beta SU tubulin - Temperature sensitive mutant with Tub 2 --> high temperature = no spindle = stuck in mitosis Do high copy number plasmid supressor screen – Have a library of plasmid --> Add plasmids to mutant yeast cell (done for the whole library) --> plate cells at 36 degrees --> some cells with grow - Cells grow even though started with mutants that don’t normally grow at 36 degrees because they now have plasmid that codes for a peice of DNA codes for a supressor when it is overexpressed that allows them to grow --> when that plasmid is over expressed it acts as a supressor - Interested in gene in plasmid that is in the cells that can grow at 36 degrees

Answer 63

Get both intragenic and extragenic - Have temperature sensative Tub2 alelle that now have a plasmid with WT tub2 alele --> intragenic supressor - Extregnic = WT porteins that can rescue the mutations

Answer 64

1. Mass actions – because plasmid is overexpressing the WT gene 2. Dosage supressor - Mass action and dosage supressor BOTH restore teh WT phenoype by chnaging gene dosage which is done by the high copy number of the plasmids 3. Could maybe get bypass – supress a null - Yeast have many paralogs --> Maybe there is a diferent Tubulin gene taht is not normally used because normally use Tub2 gene BUT in mutants with the plasmid there could be overepxression of the second gene (paralog of tub2) --> get beta tubulin --> restore growth of cells + rescues null

Answer 65

Suppressors allow us to find new components in pathways

Lecture #2 - Yeast Genetics Flashcards

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