Lecture #9 - Drosophilla 1 Flashcards

1
Q

Why is Drosophilla a good model

A
  1. Many genes and biological processes are conserved in flies (Ex. 75% of human disease causing genes have homologs in Drosophila meaning we can swap human homologs)
  2. Inexpensive
  3. Easy to maintain
  4. Small
  5. Short life cycle
  6. Produce many offspring (females lay 100 eggs per day)
  7. Simple genome (Have four chromosomes = designing genetic crosses is simple)
  8. Array of sophisticated genetic tools are available
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2
Q
A

Answer - E

Can store dead embypres in the freezer BUt they would not be alive after

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3
Q

Drosophila genome

A

15k protein-coding genes

Higher gene density than humans

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4
Q

Drosophilla Life cycle

A

Have a short life span (40-50 days)

Four stages in Drosophila development - egg + larva + pupa + adult

Life cycle - Egg is fertilized –> after 24 hours it will undergo embryonic development and hatch into a larva –> The larva subsequently undergoes three molting phases called instars (instar 1 –> 2 –> 3) –> Next, the pupa enclosed in a pupal case undergoes metamorphosis and the body structure is rearranged and the adult structure
- Instar 1 and 2 = last one day vs. the third instar = last two days
- Adult flies begin mating about ten hours after eclosion (emergence from pupal case)
- Life cycle is dependent in temperature

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5
Q

What type of females are used in genetic crosses

A

Need to use female virgins when setting up a genetic cross to ensure parentage of the progeny
- Important because females can store sperm from males

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6
Q

Drosophila – Tissues + development + brain functions

A

There is a lot of conservation of development in drosophila and humans –> A lot of the foundational understanding of development came from drosophilla

Example - Can stain for different features of the embyroe
- Top image – patterning genes that set up the body plan (early embryo)
- 2nd image – Trachea
- 3rd image – Muscles (Figured out how cell-cell fusion works by studying flies )
- 4th image – Nervous system in flies
- Image in middle – Larave neuromuscular junction (study in flies)
- Right image – dopamine neurons in brain in adult (study brain using flies)

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7
Q

Circadian rythms + flies

A

Circadian rhythms = discovered in flies –> work in fruit fly led to nobel prize in understanding the central clock that regulates fly and human circadium rhythms (molecularly conserved)
- Molecular conservation = led to understanding of basic biology

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8
Q

Polytene chromosomes

A

Polytene chromosmes = Giant chromosomes formed by multiple rounds of DNA replication without cell division (DNA is replicated 1000s of times)

Polytene chromosomes are found in certain cells in flies

Use - Early fly geneticists used polytene chromsomes to decipher the structure and arrangement of the genome
- PolyT chrosomes = how poeple made maps of chromosomes

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9
Q

Drosophilal chrosome cytological maps

A

Able to look at PolyTene chromosomes by squashing the cells and flattening the nucleus content (including the chromsomes)
- Looking at PolyT chromosome = gave people link between chromsomes and where genes are in position along the chromosome

Bottom part of image – see PolyTene chromosome
- See dark and light areas –> shows areas with more or less DNA condensation (The pattern is reproducible across flies)
- Image shows bands 20-40 on second chromosome left arm
- Have pictures for all chromosomes in flies

TOP picture = shows the bands on all arms of the chromosome (X has 0-20 ; 2 has 21-60 ; 3 has 61-100 )

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10
Q
A

ANSWER – 3R (because 94 is between 81 and 100 – look at last slide)

Can do FISH for all genes and they will map to band on chromosome (green signal)
- Upper micrograph = have 94e1 THEN do FISH and see a fluorescent band

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11
Q

Sexing Flies

A

Male (XY):
1. Smaller
2. Black and rounded abdomen
3. External genitalia
4. Have sex combs (patches of dark bristles on the foreleg for mating)

Female (XX):
1. Larger than males (BUT could have mutations that change body size)
2. Internal genitalia (no Sex combs)
3. Striped and pointed abdomen

Y does NOT determine maleness –> INSTEAD the lack of a second X chromosome determines maleness

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12
Q

Drosophila chromosomes

A

Drosophila have 4 pairs of chromosomes
- Chromosome 1 = the sex chromosome
- Chromosomes 2-4 = autosomes

Chromosome 4 – dot chromosomes (very small ; 2% of genome)

Chromosome 2 and 3 = have their centromere positioned in the center which seperates each chromosome into a right arm and a left arm (Each arm carries about 20% of the fly’s genes)

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13
Q

Drosophilla genome

A

132 million base pairs (1/20 the size of humans)

15k gene

Gene density is much higher than the human genome

Karyotype - Shows have 2 pairs of chromsome 2 + shows chrosmome 3 i+ shows the X chrosmome in orange + shows chromsome 4 as two dots

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14
Q

Do we care about chromosome 4

A

Often don’t care about chromsome 4 in crosses because has so few genes
- Often don’t include when writting genotypes of flies

BUT Chromosme 4 does HAVE essential genes
- Hard to resrecah things on chromosome 4 because it has a lot of heterochromatin

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15
Q

Genetic Nomenclature

A

Wild type = wt or +

Gene name and genetic elements is Italicized (Curly)
- Genes in flies are given the name of the mutant phenotype

Gene symbole is itallized (abreaviate name of gene) - Ex. Cy

Dominant mutations are capitalized (Ex. Dr or Cy)

Recessive mutations are written in lowercase (Ex. vg or hh )

Allele of gene uses a super script (allele name is written as a super script)
- Example - W^a –> allele of white gives rise to a pale orange eye color (allele = apricot)
- Example 2 – hh^8 –> 8th allele of hh gene (allele name is superscript)

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16
Q

Writting a genotype

A

Overall - the genotype of each chromosome is indicated separately

Chromosomes are separated by semicolons
- Chr 1 ; Chr 2 ; Chr 3

Each homologous chromosomes are separated by a line
- Maternal chr 1 / paternal Chr 1 ; Maternal chr 2 / paternal Chr 2 ; Maternal chr 3 / paternal Chr 3

Different alelles on the same chromosme are seperarated by commas
- +/+ ; Allele 1, Allele 2/ + ; +/+ (Allele 1 and 2 both on chromosome 2)

The sex chromosomes can be written in multiple ways
- Female = +/+ or x/x
- Male = +/ weird symbol or x/y

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17
Q
A

Answer – C –> has 2 domiannt mutations on chromsome 2 and a recessive mutation on chromsome 3 and it is male

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18
Q
A

ANSWER – B

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19
Q
A

Answer – C

w- = means have 2 X chromosomes (w-/w-)
Chr 2 = lowercase = recessive
Ch 3 = uppercase = Dominant

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20
Q
A

Answer E (Know it is E because hh is on the second chromsome (based on A) AND NOT D – because gig is on the 3rd chrosome (Based on A

w is mutant
- w- is an allele of w mutant

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21
Q

Shortcuts to write fly genotypes

A

Might not write out all of the chromosomes (if one chromosome does not have any alleles of note on it it won’t be written)

If a pair of chromosomes are homozygous THEN only one chromosome will be shown

Sometimes alleles are not separated with a comma

Sometimes allele names are not written out

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22
Q

Example – Cross a male that is heterzygous for Cy (on 2nd chromosome) and Heterzygous for dr (on the 3rd chromsome) TO females that is homozygoyz for eb (recceisve mutation on the 3rd chromsome)

A

Each offspring receives one chromosome from each homologous pair from each parent

Because the female is homozygous for every chromosome, there is only one combination of maternally inherited chromosomes –> +; + ; eb

Male can give 8 possible combinations (Can give + or Y on chromsome 1 ; can give Cy or + on chromsome 2 ; can give Dr or + on chrosome 3)

END - There are eight possibilities for the genotypes of the offspring (because 1 possible from female and 8 possible form male)

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23
Q
A
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24
Q

Calculating offspring ratios

A

Maternal gametes – w or +
Paternal gametes – w or Y

SLIDES = shows possible offspring
Punnet square = shows you the distribution of gamete

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25
Q
A

ANSWER – ¼

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26
Q

How to assign sex to the parent genotypes

A

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

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27
Q

How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes

A

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

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28
Q

How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes - If there needs to be meiotic recombination to produce the desired F1 progeny

A

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.

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29
Q

How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes - If one of the parent genotypes has a limited amount of flies

A

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

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30
Q

How to assign sex to the parent genotypes - Chromosomal location of genes within the parental genotypes - If you are going to mutagenize the parents

A

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

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31
Q

Fowards genetics in Drosophilla

A

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

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32
Q

Reverse genetics in Drosophila

A

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)

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33
Q

Generating Mutations in Flies

A
  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
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34
Q

Foward genetic Screen

A

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

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35
Q

Example Fowards Genetics Screen

A

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

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36
Q

Example Fowards Genetics Screen - What method would you use to generate mutation

A

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

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37
Q

Example Fowards Genetics Screen:
1. Will you mutagenize males or females
2. 2 – What Drosophilla genetic tool will allow you to establish a mutant stock

A

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

38
Q

Fowards genetic Screen process

A
  1. Mutagenize male with EMS –> generates mutations across all chromosomes
39
Q

Issue when mutagenize with EMS

A

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

40
Q

Drosophilla Bodies

A

Drosophilla have complex bodies and every morphical feature is fair game

Body structures and morpholigical features can exhibit chnages due to mutations

41
Q

Drosophila Mutant Examples

A

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

42
Q

Rerousces for fly genes

A

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”

43
Q

Issue in fly crosses

A

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

44
Q

Balancer Chromosome Use

A

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

45
Q

Nomenclature of balancers

A

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)

46
Q

Use of Recessive visible marker

A

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)

47
Q

Mainatining balancers

A

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

48
Q

Why does PIN maintain balancers in stock

A

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

49
Q

Features on balancer chromosome

A

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
50
Q

Dominant marker on Balancers

A

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

51
Q

Recessive lethal mutations on Balancers

A

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)

52
Q

Inversions in Balancers

A

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)

53
Q

WHY is it important that the inversion suppress recombination

A

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

54
Q

How do balancers prevent you from losing mutants of interest

A

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 )

55
Q

Balancer Notation

A

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

56
Q
A
57
Q
A
  1. True
  2. True (Has domiannt marker)
  3. False (has recessive lethal)
  4. True
  5. Generally True
  6. False
58
Q

Why can you use PIN in males

A

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

59
Q

Forward screen process:

A

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

60
Q

Crossing mutagenized males to a balancer female

A

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)

61
Q

Foward Screen - Single male crosses

A

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)

62
Q

Foward Screen - Sibling cross

A

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

63
Q

Foward Screen - Analyze the homozygous phenotype

A

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

64
Q

Example - Sequencing of the 2nd chromosome shows that a gene called eos is mutated in flies with defecteive Photorecptors (Mutant allele = eos^23 )

EMS results in mutations all over the genome –> how can you show that the mutations in eos are responsible for the defective PRs

A

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

65
Q

How do you do a phenotypic rescue

A
  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

66
Q

2nd method for expressing transgenes

A

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)

67
Q

GAL4-UAS - Overall

A

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

68
Q

Why might we want temperal and spatial control over the gene expression pattern

A

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

69
Q

Components of GAL4-UAS binary gene expression system

A
  1. GAL4
  2. UAS
70
Q

GAL4

A

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

71
Q

UAS (Upstream activating sequence)

A

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)

72
Q

Is GAL4 endogenous to flies

A

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

73
Q
A

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

74
Q
A

Answer - 4

75
Q

Scenerio – have GAL4 that is in eos gene –> Cross GAL4 with UAS FP reproter

How would verify that the expression pattern you go relfects the real expression pattern of gene itself because using transgene to report the expression of genes but need to valdiate

A

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

76
Q

Rescueing a mutant with GAL4-UAS

A

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)

77
Q

Results from cross when rescueing a mutant with GAL4-UAS

A

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

78
Q

Steps after rescue the mutant pheotype

A

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

79
Q

ACTIVITY – phenotypic rescue of eos with GAL4/UAS

A

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

80
Q

Using GAL4-UAS to find other genes in the pathway

A

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

81
Q

Using GAL4-UAS to find other genes in the pathway - Experiment

A

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

82
Q

What type of gene can rescue the mutant phenotype

A

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

83
Q

Other uses of GAL4

A
  1. Tissue specific knockdown using UAS-RNAi
  2. Temperal control of expression usning GAL80TS

Overall - Many RNAi and GAL80 fly transgenic lines are availible

84
Q

Tissue specific knockdown using UAS-RNAi

A

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

85
Q

Temperal control of expression using GAL80TS

A

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

86
Q
A

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

87
Q

Necessary

A

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

88
Q

Sufficient

A

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

89
Q

Neccessary Vs. Sufficient

A

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

90
Q
A
90
Q
A
91
Q
A