Lecture #9 - Drosophilla 1 Flashcards

Question

Answer 1

ANSWER – ¼

Answer 2

Need to consider many things when thinking about which parent genotype will be male or female in the cross: 1. The chromosomal location of genes within the parental genotypes 2. 2 - If there needs to be meiotic recombination to produce the desired F1 progeny 3. If one of the parent genotypes has a limited amount of flies 4. If you are going to mutagenize the parents

Answer 3

If the gene to be inherited by the F1 generation is on the X --> the parent with the X containing gene must be female Example – IF you want progenicy with baltimore in genotypes - When cross a fly that is homozygous for the gene Baltimore on the X chr and WT on the other chromosomes to a fly that is homozygous for the gene Maryland on the 2nd chr but WT on the other chromosomes --> the homozygous Baltimore fly must be the female to generate progeny with 1 copy of Baltimore in their genotype (see genotype on slide) - WHY - If we were to make the homozygous Baltimore fly male, then only the the female progeny (NOT the male progeny) will have 1 copy of Baltimore in their genotype

Answer 4

If there needs to be meiotic recombination to produce the desired F1 progeny Only females can do meiotic recombination = you must use females parents BOTH males and females can do mitotic recombination.

Answer 5

If one of the parent genotypes has a limited amount of flies --> should make that the male parent WHY - males do not have to be virgins prior to mating --> means it is easier to use males when there is a limited number of flies with that genotype

Answer 6

If you are going to mutagenize the parents you should mutagenize males WHY – because males generate more gametes than females --> means that there is higher potentoal for more uniquley mutagenized progeny

Answer 7

Fowards genetics – Go from phenotype to genotype Uses: 1. Random point mutatiions (EMS or radiations) 2. Random Insertions (P-elements) 3. Enhancer or supressor screens 4. Deficicey kits

Answer 8

Reverse Genetics – Go from genotype to pheotype Uses: 1. Gene Knockdown (RNAi) 2. Knockins/deletions (CRIPSR) 3. Overexpression (GAL-4-UAS) 4. Cell types specific expression (GAL-4-UAS and clonal analysis)

Answer 9

1. Chemical mutagens (EMS) --> Makes Point mutations - Pro – random + saturation + different types of muatnts - Con – Slow gene identifications + large scale 2. Ionizing radiation (X-ray and gamma rays) --> Makes chromosome rearangments - Pro – Random + gene deletions + duplication - Con – Slow gene identification + no real saturatiion + inefficnet 3. Transposons --> Makes DNA insertiions (mostly hypomorphic in UTRs) - Pro – fast gene identification + flexible scale - Con – Non-random (hot spot) + no saturation 4. Homologous Recombination and CRIPSr/cas9 --> Makes trageted mutogensis of specfic - Pro - Very efficient - Con – Currentley ineffcincet for screening

Answer 10

Foward genetic Screen = unbiased screening for mutations producing a phenotype of interest

Answer 11

Goal – Screen for genes on second chromosome essential for photoreceptor function - Fundemental for studying RAS – all unraveled by studying fly eye

Answer 12

ANSWER – EMS – want point mutations (most common) - NOT CRSIP because not efficinet - NOT trasnpons because would get large deletions/rearngemnmts

Answer 13

1 – Will you mutagenize males or females - ANSWER – Mutagenize males because males make more gametes + recombination is only in females --> means the mutant male won’t recombine and move the mutant around on the chromsome 2 – What Drosophilla genetic tool will allow you to establish a mutant stock - Answer – Balancers

Answer 14

1. Mutagenize male with EMS --> generates mutations across all chromosomes

Answer 15

Issue – EMS will cause a recessive mutation that we can’t visualize when the fly is heterozygous (looks WT) --> how do we keep track of a mutaion that we can’t see Solution - Uuse balancers to keep track of recessive mutations (allows you to follow recessive mutations) - Because EMS is genreating recessive mutations that we can’t see in heteroygous fly = need to cross a balancer chromsome to recover the mutant

Answer 16

Drosophilla have complex bodies and every morphical feature is fair game Body structures and morpholigical features can exhibit chnages due to mutations

Answer 17

Eye color is complex and ranges widley Fly genetics can take advtange of this by linkning eye color to a gene (Ex. Link to GFP) --> NOW if eye color is present then that means GFP is also present Image - shows mutations that cause chnages in eye shape

Answer 18

The most popular public database is FlyBase (flybase.org) Use - Use it to search for your gene of interest using the search bar Example – serach for hedgehog (hh) - Results on flybase show that hedgehog is a protein coding gene (it is a singlaing pathway ligand) ; located on the right arm of chromosome 3 - Website also tells us that - If we are interested in studying hedgehog further (Ex. perhaps knocking it down or overexpressing it) --> we can find 39 publicly available stocks - Can also see Molcular fuction + Expression patterns + Alleles + Phenotypes resulting for LOF or a mutation of a gene - Can get the DNA sequence of a gene by clicking on “get decorated FASTA”

Answer 19

Issue – How do you miantain a lethal mutaion in the heterozygous state Example – Cross parents that each carry a recessive lethal mutations (on 2nd chromosome) - Half of the progencey will be heterozygous for the mutatinos ; ¼ of progencey will be WT ; ¼ of the progeney will be homozygous for leathal mutations (¼ will die) Solution - Balancer chromosomes

Answer 20

Overall prevent reccesive mutations from being lost over time + prevent recombination between homologous chrosomsomes Use – Balancer chromosomes allow us to maintain a mutations (ex. lethal mutations) in the heterozygous state and trace a mutation through the generations - Balancers are important IF the mutation is homozygous lethal - Useful if flies with the mutations can’t be phenotyoically distiguished from the WT fly (Used to trace a mutation that cannot be phenotypically distinguished from a wildtype fly) --> because the flies carrying a balancer chrosome can be phenotypically distigushed from flies without a balancer chromosme

Answer 21

Image – show shows how you write balancers + how you maintain in stock In(2lR) = inversion of the 2nd chromsome Cy = dominant marker dp = recessive lethal (don’t see in heterozygous state) pr^1cn^2 = recessive visible marker (don’t see in heterozygous state)

Answer 22

Recessive visible marker – used to show ressive lethal works in marker (because if recessivle lethal stops working then you would get homozygous and would be able to see the recesive visible marker)

Answer 23

Overall - Balancers need to be kept in a heterozygous state = they are kept opposite of a difefrent dominant marker Balancer chromsomes are miantained as stocks with a dominan viable lethal mutation (ex. Pin - maintain flies as Pin1/SM6c) - When flies cross for a while they will keep the Pin1/SM6c genotype - Dominant visible lethal mutation = visible phenotype is domiant but lethality is recessive - Need PIN to stay heterozygous PIN/SM6c

Answer 24

PIN is dominant for visble phenotype BUT recesive for lethality (can’t have Pin/PIN) and can’t have SM6/SM6 = can only have Pin/SM6 END - PIN shows you the flies that are heterozygous for SM6c and Pin1

Answer 25

Balancers have 3 features that distiguish them from a WT chromosome 1. Dominant marker 2. Recessive lethal mutations 3. Inversions over the length of the chromosome to supress recombination

Answer 26

Use - Allows us to see which flies have the balancer Dominant marker is NOT lethal Example (in image) - curly and Pin - Flies with a balancer that has the curly marker have curly wings OR Flies with a balancer containing Pin have shortened bristles that look like pins

Answer 27

Use - Have recessive lethal mutations so that flies can NEVER be homozygous for the balancer chromosomes AND they can never be homozygous for the mutant chromsome --> MEANS that the mutations of interst (which is on the chromosome that is being balancers) can be maintained in the heterozygous state END - Flies have to be heterozygous balancer/chromosome with mutation (flies have to have the recessive mutation of interest and balancer chromosome) - Means that the balancers can never outcomepete the mutation of interest (limites the propagation of chromosomes)

Answer 28

Use - Supress Recombination - Inversion breakpoints occur at the same points where the recessive lethal mutations map The inverted regions are largely WT except for their orientation --> means that with a balancer you are supply a nearly WT version of the chromosome = keeps the heterozygous healthy How do the balancers suppress recombination - When a balancer and WT recombine = creates an acentric and dicentric chromosome --> Unhealthy --> leads to death = eliminates recombination (because cells would die if they had recombination)

Answer 29

IF you want to study a double mutant (double mutant exist as a heterozygous) --> during meiosis when recombination occurs you could end up with what you want (have no recombination and get m1 and m2 on the same chromsome) or can get recombination and have chromoeosme with just m1 or just m2 THEREFORE balancers can suppress this recombination due to heir inversion and maintain the double mutant that you want

Answer 30

IF have parents that is m/+ X +/+ --> progeny will be m/+ or +/+ - IF these offspring flies mate THEN over time you will lose m mutations because m is homozygous lethal --> homozygous lethal mutatons usually give disadvantage even when heterozygous IF have parents that have 2 homozygous lethal mutants (m1 and m2) - Cross m1, + / + , m2 X m1, + / + , m2 - Some of offspring is m1,+ / m1, + OR m2,+ / m2, + = dead - 1/3 of offsrping ate m1,+ / +,m2 = heterozygous (alive = allows you to maintain the mutations) - IN reality – female flies can recombine chromosmes = you won't mainatin the two mutations on that chromsomes - To solve issue of recombination = put inversions on chrosmomes = supress recombination --> allows you to make balance stock (Lose ability to do recombination = won’ be able to lose m1 or m2 = maintain mutations )

Answer 31

Balancers usually start with the letters F, S, or T --> indicates if they balance the first, second, or third chromosomes Example - SM6b = second chromosome balancer and contains dominant markers for rough eyes and curly wings

Answer 32

1. True 2. True (Has domiannt marker) 3. False (has recessive lethal) 4. True 5. Generally True 6. False

Answer 33

Can use Pin to balance a gene in males because they don’t do recombination (don't need inversion to prevent recombination if there is no recombination BUT could not use in females) - PIN = does not have inversion = it is NOT a balancer - Need SM6c in females because it is a true balancer

Answer 34

Foward screen process: EMS males to make mutations across genome --> Different mutations are made --> Cross to balancer females (cross to virgin females of balancer stock) --> Single male cross --> sibling cross --> Anylyze homozygous phenotype - When chromosomes are mutated --> each chromosmes will have a spectrum of mutations --> can have many alleles of a gene = each fly needed to be handled individually because different flies will ahve unique spectruum of mutatons + some chromosmes might not have mutations

Answer 35

After EMS --> Cross mutagenized males to a balancer female - Get a mix of genotypes and phenotypes AFTER cross - Select for flies with SM6c (curly wings) --> mut1/SM6c or +/SM6c (select for flies that don’t have PIN) - Because mutations are recessive – you would not be able to tell mut1/SM6c and +/SM6c apart (ALSO both would look the same as mut2/SM6C - could also not tell apart from +/SM6c)

Answer 36

Once have mut1/SM6c or +/SM6c or mut2/SM6c --> do single male crosses Have to seperate out the males and cross each single male to many virgin balancers (Pin/SM6C) - NOW we are just dealing with 1 mutation per cross - Done so that you only have 1 mutation per fly (ex. only have mut1)

Answer 37

From EACH single male cross you select for progeny (both male and virgin females) that have SM6c but not Pin THEN do sibling cross Once have SM6c flies = set up sibling cross = create a balanced stock

Answer 38

From the F2 siblings you select the progeny that do NOT have SM6c = NOW have mut1/mut1 or mut2/mut2 - You can look if any of the mutants have the phenotype you are looking for - Many of the homozygous mutant flies will not have the phenotype we want = this type of screen requires thousands of flies and hundreds of crosses - Could screen for any phenotype

Answer 39

To show that mutations in eos are respinsible for defective photoreceptors you do a phenotypic resuce Phenotypic analysis – adults without SM6c are homozygous mutants --> look for phenotypic rescue in non-SM6c flies

Answer 40

1. Establish transgenic line carrying heatshock inducible WT eos (eos+) - Add transgene under induucible promoter to restore gene function - Cross in image = shows how to do this - Apply heat shock to induce transgene function 2. Cross the transgene into your mutant background 3. Look for phenotypic rescue in homozygous mutants after heatshock Application - Can determine developmental time period when eos is needed by heat shocking at different times

Answer 41

SAY you have a hard time rescuing eos using hs-eos+ since expressing eos+ throughout the body is bad and INSTEAD you want to express eos+ specifcially in photoreceptors NOW you would use the tissue specifc expression of GAL4/UAS binary expression system (NOW you can express specifically in photo receptors)

Answer 42

Use – Directed gene expression with spatial (in tissue/cell type) and temperal control (life stage) System is adapted from yeast – creates specificty in the fly

Answer 43

Control is good IF you want to study gene’s function in a specific tissue at a specific point in development BUT over expression or reduction of the gene in the whole organism may be lethal + don’t want to over complicate the experiment by altering gene expression in more tissues than desired Solution – Use GAL-4-UAS to overexpress the gene of interest in the specific tissue you are interested in

Answer 44

1. GAL4 2. UAS

Answer 45

GAL4 = Transcription factor Gene encoding GAL4 is inserted using P-elements downstream of enhancers --> determines where GAL4 will be transcribed - Enhnacers can be tissue specifc = tissue specifc GAL4 expression Example - Enhancer can only be expressed in the wings = creates a fly line that only expressed GAL4

Answer 46

UAS = Enhancer than GAL4 binds to and affects expression of the downstream gene - UAS is inserted into the fly genome In a tissue where enhancer is active --> GAL4 is expressed THEN GAL4 binds to UAS --> Activates trancrtiption of the downstream gene (get expression of the gene in tissue specific manner)

Answer 47

GAL4-UAS is NOT enodgenous to flies --> means a fly with ONLY UAS or ONLY GAL4 expression would be otherwise WT (won’t get expression of the downstream gene) Important controls because if there is an effect in the flies with only GAL4 or only UAS transgene it would change your interpretation of the experiment

Answer 48

Answer - B The gene downstream of the UAS is expressed only when/where the GAL4 is expressed, which is dictated by the enhancer element(s) located nearby the GAL4 insertion site

Answer 49

Answer - 4

Answer 50

ANSWER – Look for GFP expression and verify for anti-eos antbody (immunostain for protein) , FISH or other reproter in the same gene

Answer 51

Can learn about mutations/KO with GAL4 UAS system in order to ask questions about resucing a phenotype Example – Mutants (hopkin) --> cause a malformation of eyes - GOAL – want to express the WT version of the gene in the eye in the mutant background How to do this – make a fly line with the mutant allele and GAL4 (GAL4 would be under and enhnacer that is expressed ONLY in the eye) THEN you can idetify the desired progeny using a balancer chromosome - CyO helps us follow the mutant alelle (mutant = maryland allele) because if have no curly wings then you have to be homozyous for mutant - TM3 = helps us follow GAL4 Cross – males (have mutant hopkins with Cyo and eye-GAL4 balanced by TM3) to females that have WT hopkins donwstream of UAS (being balanced by TM6B)

Answer 52

AFTER make the cross = we will ONLY collect flies that do not have a balancer to ensure that the homozygous mutants have both GAL4 and UAS transgenes WHY no balancer (Because on chromsome 2 need eye-GAl4 and UAS hopkins = the offspring with no balancer would have eye-GAl4 and UAS:Hopkins) NOW we can see if overexpressing the WT hopkins resuces the mutant phenotype in the eye

Answer 53

AFTER Overexpress hopkins to see if it can resuce the mutant phenotype --> Might want to link structure to function and understand what part of the hopkins gene is doing a function To Study – replace the UAS hopkins transgene with the UAS transgenes that are mutants of hopkins that ONLY disrupt parts of the gene - Example - Looking at a kinase dead allele of the hopkins gene --> Now can ask – does expressing the kinase dead domain version of the gene rescue the mutant - Instead of adding the full WT gene downstream of UAS you only add part of it to see which parts of the gene is needed End - Using GAL4-UAS we can see how much and which part of the gene is needed for the rescue

Answer 54

Slides = shows the cross you would preform to use GAL4/UAS + Slide shows progeny from cross - Cross = mutant/balancer and eos-GAL4 X homozygous mutant and UAS/balancer

Answer 55

Can use GAL4 system in screen to see if overexpression of other genes can rescue a KO of the gene of interest - NOW see if overexpressing a different gene in the eye can resuce the mutant phenotype Overall - CROSS the hopkins mutants with UAS upstream of different genes

Answer 56

Experiment – Start with fly that has gene of interst mutated that ALSO expressed GAL4 in the eye --> mate these flies to library of UAS flies --> Look for phenotype of interest in offspring

Answer 57

IF the mutant phenotype is rescued then you may have found a supressor gene of your gene of interest CAN also find gens that make the phenotype worse than the KO alone

Answer 58

1. Tissue specific knockdown using UAS-RNAi 2. Temperal control of expression usning GAL80TS Overall - Many RNAi and GAL80 fly transgenic lines are availible

Answer 59

Asking what is the phenotype of a fly that is missing the protein of interst ONLY 1 tissue (Ex. in the eye) Here the shRNA is expressed under the UAS promoter --> expresion of shRNA decreases protein expression

Answer 60

Asking what is the phenotype of a fly that expresses the protein at a certain time GAL80TS = temeprature sensative version of GAL4 inhibtor - GAL80 = GAL4 inhibitor

Answer 61

Answer - B and C A fly expressing only the GAL4 should not have any way of expressing a marker to indicate which cells it is expressed in. Therefore, you must cross your GAL4 flies to a UAS marker line, but not UAS:GFP RNAi as this would result in knockdown of GFP RNAi would result in a Knockdown of GFP

Answer 62

Necessary – Deleting your gene of interest creates mutant phenotype Example – If you study fly development and find that flies without the eyeless gene have no eyes --> Shows that eyeless is necessary for eye development - No eyeless gene = have no eyes --> eyeless gene is necessary for eyes

Answer 63

Sufficient – Rescuing mutant phenotype by introducing your gene Example – Studying fly development and start with fly that has no eyes THEN express the gene eyeless --> flies NOW have eyes = shows that the gene is sufficient for development - Add back in eyeless and get eyes = eyeless is sufficient

Answer 64

GAl4-UAS is a good tool for studying necessity and sufficiency of gene of interest Sometimes you can have one without the other = need to test both

Lecture #9 - Drosophilla 1 Flashcards

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