Lecture #1 - Mendelian Genetics Flashcards

Question

Phenotypic analysis – Antimorphs

Answer 1

Overall - Antimorphs negatively affect WT allele function --> MEANS the more copies of WT allele can improve the phenotype (WT allele can dilute out the antimorph) Worse off - WT/m < WT/m;dup(WT)

Answer 2

- Because antimorphs function by impairing WT allele function they will behave as if they were independent LOF allele when the WT allele is absent --> Del/m or m/m phenotypes can mimic amorphic allele ONLY when include WT allele in analysis can you see the difference between the amorphic and the antimorphic

Answer 3

Better off = C because WT dilutes the antimorph

Answer 4

Need to look at entire spectrum when assigning morphs

Answer 5

Because neomorphs gain a new function - function can vary between genes or different mutations of the same gene --> makes it hard to place them on a spectrum with WT and deletion alleles All mutant genotypic combinations have the phenotype because mutations causes the gene to act in a new way (equally mutant in all situations) Hard to infer Wt function because the mutant is adopting an extra function that is not related to WT

Answer 6

The best way to characterize a suspected neomorph is to experiment with the allele to determine its GOF phenotype Exmple – Antenopedia in flies - Normally – Flies grow antena on their head - Neomorph – grows legs in place of antenei

Answer 7

Loss of function - Amoprhs and Hypomorphs (recssive) Gain of function - Hypermoprh + Antimoprh + Neomorph (dominant)

Answer 8

Exception to the difference between hypomorphic and amoprhic There are some enzymes where knocking out the allele in hypomoprhic means there wil not be enough protein from the dcerased effcicney/remaining WT = the null and the hypomorph have the same phenotype BUT when add extra WT copies then the phenotype can go away REALLY.- 1 copy if hypomorph or morph vel dies = have phenotype of the ahypomorph or the amorph even though they are usually recssive Haploinsufficincey = have phenotype present in 1 copy even though it is a LOF mutation CHECK THIS

Answer 9

Answer – Use a conditional Allele In cases where we are interested in essential genes conditional alleles of the gene might provide a way to study it

Answer 10

Alleles whose protein products function as WT under permissive conditions but become unstable under restrictive conditions - Alleles have additional utility because allow us to control gene expression or function in other types of experiments Example of conditions used – Temperature + Light + prescence of chemical compounds + induction of other genetic elements

Answer 11

Foward vs. Reverse genetics Tell you about what a gene does+ what genes are responsible for a phenotype

Answer 12

Start with the gene and observe he phenotypes that result from manipulating it - NOT identifying new genes from phenotypes BUT instead taking information about what we know about the gene and working backwards to learn more about that gene Might look at what happens when a gene is deleted or might want to characterize a disease causing mutation

Answer 13

1. CRISPR 2. RNAi 3. TALENs/Zinc Finger nucleases ALL make specific point mutations or deletion or insertions into a gene of interest

Answer 14

Take a phenotype and work to identify the genes responsible for it - Used before full genomes were sequenced - Usually trying to find new parts of a known pathway or pin down the cause of a phenotype Tools used = Chemical mutagens is EMS or MMS

Answer 15

Mutations occur in population BUT at a impractically slow rate ALSO mutations occur randomly across the genome = if we intersted in a particular gene the odds that a mutaton lands in 1 of them is slim Solution - Mutagens are used in forward genetics because they increase the probability that a mutation will occur in an organism

Answer 16

Mutagens = increase the rate of mutations --> applying mutagen to system will reduce the amount of time required to find mutation that we are interested in Issue - High concentration of a mutations will reduce survival

Answer 17

Need to balance the rate of mutation against the percent survival --> get enough survivor organisms that carry a limited number of mutations (ideally want 1 mutation per organism) IF concentration of mutagen is too high = most organism die and survivors are riddled with mutations - If the survivors have too many mutations = hard to single out 1 mutation that is cause of the phenotype IF concentration is too low = requires looking at many more organisms to find a phenotype

Answer 18

1. Even with ideal concentration mutagen it is possible that a single organsim displaying a phenotype will be harboring multiple mutations that could be the causal mutation 2. There are some classes of genes that are infrequently recovered from mutagenesis experiments

Answer 19

To ensure that a phenotype is caused by 1 mutation – need to backcross the mutant organism and cross it with the WT strain for several generations) --> eventually you can isolate a single causative mutation in an otherwise WT background

Answer 20

1. Essential genes are frequently missed because mutations in them produce infertile or dead organisms 2. Short genes are less likely to be hit by a mutations 3. Redundant genes (similar genes that preform the same function) - To identify them in mutagenesis = need simultaneous mutations in both paralogs in a single organism (otherwise organism looks WT)

Answer 21

Screening = sifting through organisms and picking those out that have a phenotype

Answer 22

Overall - producing mutation and looking for phenotypes

Answer 23

To address the question - can plot the number of mutant alleles that we found in the given gene against the number of genes containing that many allele Start of screen (found few alleles) - expect that distribution leans toward the right skew (most genes we identify are represented by 1 mutant allele (grey bars) As screen progresses (reach saturation) --> As we find more alleles and their associated genes --> the number of alleles per gene will increase and the distrubution will be more normal curve - Every time you do a screen you get more alleles (number of alleles is infinite) BUT eventually you start getting alleles in the same gene

Answer 24

Process of diminishing returns (identifying fewer new genes as we find more new alleles as the screen progresses) Once hit a saturation point – tells us that we have reached a good stopping point (can stop screen/selection)

Answer 25

Second type of mutogensis experiment that helps identfy genes responsible for a phenotype Requirements: 1. Need to know what the phenotype is that we want to select for beforehand 2. Need to impose conditions on the organisms such that only organisms that display the phenotype are able to survive Issue - NOT all phenotypes can be selected for - Example of selectable phenotype – Drug resistance + Metabolic phenotype

Answer 26

Selection = faster because only have organisms with the phenotype you are intrested in = fewer organisms to look through - BUT limited to certain phenotypes and need prior knowledge of phenotype of interest - Selection = only thing survives the treatment is the mutant you are intersted in - Look sat more specific mutants in a subste of genes in a pathway - Selection = good if you already know the gene of interest then it is faster - Can screen more EMS mutants (not looking indvidual) - Less work intensive - Selection = Fast way to get many alleles of specific pools of genes of a specific genotypes Screens = more unbiased (target all the genes involoved in a pathway) + don’t resuire a starting phenotype BUT require more time + requires more attention to detail and resources - Screen = look at al EMS colony and ask if it it grows on a second plate - Screen = more open ended and can recover more different types of genes

Answer 27

Unicellular Eukaryote Many mammalian processes + their components +their regulation are very conserved in yeast

Answer 28

Yeast = grow by dividing (yeast divide by budding) - One divsion takes 90 minutes Haploids = divide to make a new haploid cell that is genetically idetical to the mother cells - Each haploid has a mating type (a or alpha) Diploids can divide to make new diploid cells OR can go through meiosis - Meoisis = makes 2 mating type a and 2 mating type alpha haploids (process = called sporulation)

Answer 29

Yeast can exist in both haploid and diploid stages (goes between 2n and n haploid state) - Haploid = have a single set of 16 chromosomes - Diploids = have two sets of 16 --> have 32 total chromosomes Goes to diploid and haploid by mating - 2 haploid can mate to form a diploid (ONLY different types of halpoids can mate --> Only alpha and a can mate) - Diploid goes back to haploid by meiosis

Answer 30

Yeast = fully sequenced in 1996 (First Eukryotic genome to be fully sequenced) Yeast = Have 16 chromsomes (15 mb) ; Have 1 gene every 2 kb Have 6,617 predicted ORFs (80% of ORFs have been verified ; 20% of known ORFs but don’t know if they are expressed) 20% of yeast genes are required for growth (majority of yeast genes are doing something else)

Answer 31

Lots of genes are required for basic Eukaryotic maintainance (Ex. Translation has many genes) Have many genes for response to chemicals --> because respond to chemicals is how yeast responds to environment

Answer 32

WT gene names = italicized and uppercase (Ex. ROG1) Mutant alleles = italicized and lower case (often followed by an alleles number to differentiate mutant allele types) (ex. rog1-3) Gene deletion = italicized + lowercase + followed by a delta (Ex.rog1d) WT proteins = only have teh first letter capital and are often followed by a p (p = protein) Mutant proteins = all lowercase BUT are also followed by a p

Answer 33

1. Auxotrophic 2. Antibiotic Marker

Answer 34

2 – Antibiotic marker --> conferes resustence to Antibiotic Any yeast strain can use these markers (no endogenous genes offers resistance)

Answer 35

Auxotrophic marker --> Lack gene or genes that are needed for producing an essential metabolite - Mutants can't make a specific molecule (yeast would only survive if that molecule is added to the media)

Answer 36

Prototroph = WT yeast --> MEANS the yeast generates its own food from salts + minerals + sugar Phototrophs can grow on minimal media --> yeast makes all Amino acids and bases that it needs to make proteins + DNA + RNA

Answer 37

1. Minmal media - Has no amino acids or bases (more expensive) - WT prototraphs an grow on minimal media 2. YPD Yeast extract + epptone + dextose - Made up of ground up yeast cells + additional amino acids + sugar sources - Less expensive - More standard way to grow yeast

Answer 38

Selectable markers are often assembled into constructs with a gene of interest --> construct is introduced into yeast through transformation In the construct the marker is placed close to the gene of intrest (ensures that the marker and gene remain linked) - Constructs ALSO have 50-500 bp homology arms upstream and downstream of the gene and the marker

Answer 39

Homology arms – Help direct insertion of the construct into a target locus by taking advantage of endogenous machinery that is usually used for homologous recombination - Use diploid cells when transfroming constructs because have fewer off traget insertions compared to halpoid cells

Answer 40

Mammalian cells = occurs in 1 in every 20,000 cells (recombination is more rare) Yeast = recombination is more common (1 in 100 cell divisions)

Answer 41

Once we recombined the gene of interest and selectable marker into diploid cell --> CAN select for cells that are carrying the marker and therefore the gene of interest by plating in selective media Example – use uracil synthesis deficient strain (Uracil auxotroph and URA3 marker) --> plate cells on media lacking uracil - No URA3 marker the cells can’t make their own uracil = only the ones with the URA3 marker (and therefore the gene of interest) survive

Answer 42

Need haploid cells WHY - Need haploid cells because most mutatiion will be reccessive --> chance of having both mutations in the diploid is low (likley will only hit 1 copy BUT to see the phenotype to see the phenotype you want to use haploid because only need 1 mutation) - Need to force yeast to sporulate to obtain haploid cells

Answer 43

Each dot on the plate is a colony derived from 1 yeast cell Colony = clones that are genetically identical to the first cell

Answer 44

Sporulation - Yeast undergo meiosis and put each meiotic product into an ascus membrane (the product in the ascus membrane in now haploid) - Whole structure (haploid + ascuse) = tetrad Sporulation = occurs when yeast are placed in nutrient poor condition

Answer 45

Using tetrad dissection - tetrads can be separated and moved onto non-selective plate AFTER a few days the haploid cells will form clonal colonies --> NOW have haploid yeast where each has the gene of interest Issue - because plated in a non-selective media = don’t know which colonies ave the gene of interest or if meitic segregation is occurring as expected --> TO ANSWER you can use replica plating

Answer 46

Purpose - replica plating = genotypes haploids Process – Use a specialized pad to transfer some of each colony form the master plate onto a new plate of selective media and a copy plate of non-selective media - Stamping the colonies onto media then stamping onto a second plate END - Compare the two plates to observe if the expected 2:2 ratio of the tetrad is produced

Answer 47

Answer – NO Image – shows the Uracil pathway to make UTP --> IF have a mutation in pathway then the cell would need uracil added to the media to be able to grow

Answer 48

Goal – screen for genes that are required to build UTP

Answer 49

EMS = used as a random mutagen EMS – chemcial that he yeast absorbs --> EMS modifiees the DNA so that when DNA is replicated the polymerase will introduce a mutation - Depending on the dose of EMS = introduce a certain number of mutations randomly in the genome

Answer 50

Mutogensis with EMS --> Grow haploid yeast on YPD to spread out single cells (yeast can grow because all of the nutrients are provided) --> let the cells grow into colonies--> replica plate onto minimal media + all AA and bases – URA --> look for colonies that grow on master plate (YPD plate) but not on replica plate (minmal plate that lacks URA) - Auxotrophs can survive on YPD (YPD has everything that is needed to grow) = grow all haploids including mutants and WT after mutogensis on YPD because YPD has the uracil that they needed - Grow replica on minimal media + all AA and bases – URA --> because want to mutants that can’t make uracil to die but need other amino acids and bases because could have mutations in other pathways and wouldn’t know which amino acid impacts growth Image – the red arrpw colony failed to grow on replica plate because the minmal media is not proding te nutrinet that the colony needs

Answer 51

FOA – 5-fluroorotic acid is converted to a toxic compound by URA3 gene product Answer – Mutants LIVE - FOA only becomes toxic when have active uracil pathway - WT = die because they use URA3 to turn it to a toxic compound but ones that are defective in the pathway will survive

Answer 52

Mutagenizie yeast culture with EMS --> Spread out on minimal media + all amino acids/bases + FOA --> Colonies that grow are FOA resistant (things that grow are mutants in URA3 gene) Selection = does NOT use replica plating - Select for yeast colonies that survive (Only survivors are the mutants you are interested in) Can use a master plate as a control to make sure FOA selection is working

Answer 53

Answer – Cross haploid yeast (to make diploid offspring) and look at the phenotype - Mate URA- with WT yeast and look at the phenotype of the offspring

Answer 54

Answer – mutation is recessive When URA- is mated with URA+ --> get diploid --> IF the diploid is URA+ THEN the URA- mutation is recessive

Answer 55

Use a complementation test --> determines if two mutants are in the same gene or diffferent genes Example - Generate 20 independent lines that cause small wings BUT do not know if this represents 20 different genes or 20 alleles of a single gene

Answer 56

Can only use recessive alles (should exclude any alleles that we know are dominant)

Answer 57

Complementation implies that 2 alleles when crossed will restore the WT phenotype in the F1 generation Example – want to determine if allele a and allele b are in the same gene controlling wing size Steps - Cross a/a X b/b (cross homozygous flies from each line together and scrore the F1 generation for wing size) - IF the mutations is in the same gene for both alleles --> then allele a and allele b would expect to see progeny with short wing (because progeny would have 2 non-functional copies of the gene and still be homozygous for the defective copy of that gene) --> HERE IS NO COMPLENTATION between these alleles because the WT phenotype is not rescued - IF the mutations are in seperate genes --> Expect to only have flies with WT wing length in the F1 generation bceause each allele is complementaed/compensated for by a WT copy received from the opposite parent If we make crosses for more of the mutants identified we can organize the outcomes into a complenetartion table

Answer 58

Each intersection of complementation table represents the phenotype of the F1 generation + = means complementation (have rescue of the WT phenotype) - = no complementation (Still see small wings) 0 = self cross (Already know what the phenotype should be) Purpose - Table lets us visualize complementation group --> gives an estimate for how many genes are represented among the alleles Example (Red box) - aa and bb --> have neither allele complement the other = can assign a and b to the same complementation group

Answer 59

Each complementation group should correspond to a singe gene Example - Look at a, b, c --> can see that c complements both a and b - Tells us that c must be in its own gene = belongs in its own compmentation group WHILE a and b are still in the same gene - IF we anlyzye the whole table = get 4 different complementation groups In image - Notice that in e is part of every complementation group --> suggests that e is not a recessive allele because it can’t be a mutation in 4 seperate genes

Answer 60

1. Intragenic complementation 2. Non- allelelic non- complementation (aka secon site non-complenentation)

Answer 61

Overall - Some mutations within the same gene can complement one another Need to consider how a partiuclar mutation will affect the gene procuct Can occur through different mechasnims: 1. Different domains 2. Reduced dosage 3. Stabilization

Answer 62

Example – protein with a protease domain + dimerization domain + transmembrane domian If have a separate mutation affect the protease domain and the transmembrane domain but the dimer can still form it is possible that this protein can function close to normal

Answer 63

Reduces the occurrence of improper interaction caused by a mutation Example – protein that functions as a dimer - Mutations that disrupts the dimerization domain may not be complemented by a WT allele because the not enough dimer is formed BUT if the same mutant allele can dimerize with itself and the dimer is still functional THEN the mutant may be able to complement a null allele because ow a enough functional dimer can form

Answer 64

Two distinct mutations that are capable of interacting with one another and neither of the mutations on their own can interact with the WT protein - Complementary changes in structure allow complementation between the two mutant alleles

Answer 65

Overall – Exception to complementation that comes when mutations in seperate genes can’t rescue one another Example – Halpoinsuddiciency --> occurs when having 1 WT copy of a gene is not enough to produce the WT phenotype - Can affect complementation where 2 genes with only 1 WT copy preforms poorly - Another example of dosage BUT in this case we are below the required threshold to produce the WT phenotype

Answer 66

Occurs with Dominant negative alleles (aka antimorphs/poison alleles) Dominant negative alleles – can sequester WT protein and therefore reduce its function even when only 1 copy of the dominant negative alleles is present

Answer 67

Take haploid mutants --> cross haploid mutants to make a diploid --> IF the two different mutants are in two different genes then the WT copy of each gene should compensate and the offspring should display the WT phenotype - Easier and faster than sequencing Example - Cross URA mutants --> make diploid cells and get WT alleles for each mutations = diploid can grow on media without any uracil

Answer 68

Issue – EMS generates more than 1 mutation per yeast = when you do sequencing you don’t know which mutation is actually causing the phenotype TO potentially resolve - Sequence 100s of independent mutants and find 1 gene where mutations arise in more than 1 strain then you can do “in silico complementation” --> guess that when that gene is mutanted it causes URA-) - Hard to know if you have few mutants Overall – sequencing is not economical because ned you sequence many strains

Answer 69

WATCH class video

Answer 70

ANSWER - don't know how many genes Know 1 and 2 = + --> they are on different genes (means they complement each other) - Two alleles complement that each other are on different genes Know 1 and 1 = - --> fails to complement to self - Logical - Mutations that fail to complement = on the same gene 3 is likley dominant because get - with 1 and 2 BUT can't be on 1 and 2 because 1 and 2 have to be different genes - Mutant 3 = dominant because over WT it shows the phenotype in 1 and 2 - Could be teh same gene as 1 or 2 buyt wouldn’t be able to know from this

Answer 71

When you do a screen = do complementation tests to ask if the alles that you are getting are in new genes or if you are getting the same gene

Answer 72

Black bars = show the number of single alleles in a single complementation group White bars = idealized distribution when reach saturation (hit all of the genes in uracil biosynthesis pathway once - Have a normal distrubution across genes Need to compare the observed allele frequencies and the idealizes frequencies and conclude if you need to keep going to get more genes or if you can stop Image - Have 45 complnementations defined by 1 alelle ; 15 comeplementaions defined by 2 alleles ; 8 groups defined by 3 alelles

Answer 73

Answer – YES keep going (because you are not at saturation yet – not at a normal distribution) The distribution in black does not match the distribution in the white --> means you need more screening to get more alleles of loci in a allele and push to 2 aleles and get new mutants in genes with 1 alelles - Need to keep going to idetofy more genes

Answer 74

1. Non-essnetial vs. Essential genes - 0 = essential genes (because null would die ; hypomorphs would survive) - 8 = non-essnetial (can get a mutation in gene and yeast would survive) 2. Short genes = less alleles Vs. longer genes have 8 alleles because EMS is more likely to hit a longer gene 3. Duplicated or redundant genes

Lecture #1 - Mendelian Genetics Flashcards

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