Lecture 4 Mosaic analysis Maternal/zygotic screens Non-complementation screen Cytogenomics to...
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Transcript of Lecture 4 Mosaic analysis Maternal/zygotic screens Non-complementation screen Cytogenomics to...
Lecture 4
• Mosaic analysis
• Maternal/zygotic screens
• Non-complementation screen
• Cytogenomics to genomics
• Designer deletions
• Gene disruption in Drosophila
• Systematic RNAi screens
Lab 4 schedule• MONDAY SECTION: Sunday 9:30-11:30
AM come in to start your in situ. WSC 339
• TUESDAY SECTION: Monday 10-12AM come in to start your in situ. B&G 3015
• On Monday and Tuesday the lab B&G 3015 will be open at 10AM to start your washes.
Biology 4540g Midterm Examination
Tuesday February 28, 2012
Room UCC 63
7-10 PM
Mosaic Analysis
What is a genetic mosaic?
Mitotic recombination
w-
w+
+
-
- +
Mitotic recombination
w-
w+
+
-
- +
Mitotic recombination
+
-
- +
Mitotic recombination
+
-
- +
w-
w+
w-
w+
Mitotic recombination
+
-
- +
w-
w+
w-
w+
Eye
w-
w+
Mitotic recombination
Clones of cells
X-rays
Indiscriminant crossing over
Flip mediated site specific recombination
Yeast two micron plasmid
A
B
C
D
A
B C
DFlip
FRT
FRT FRT
FRT
Flip mediated mitotic recombination
FRT
FRT
Flip mediated mitotic recombination
FRT
FRT
+
Sb
Flip mediated mitotic recombination
FRT
FRT
+
Sb
+
FRT
Sb
FRT
Flip mediated mitotic recombination
FRT
FRT
+
Sb
+
FRT
Sb
hspFLP
FRT
Flip mediated mitotic recombination
FRT
FRT
+
Sb
+
FRT
Sb
hspFLP
FRT
FLP
Flip mediated mitotic recombination
FRT
FRT
+
Sb
+
FRT
Sb
hspFLP
FRT
FLP
Flip mediated mitotic recombination
FRT
FRT
+
Sb
+
FRT
Sb
FRT
Flip mediated mitotic recombination
FRT
FRT
+
Sb
FRT
+
FRT
Sb
Flip mediated mitotic recombination
FRT
FRT
+
Sb
FRT
+
FRT
Sb
Mosaic analysis
• Assay adult phenotype of an embryonic lethal.
• Determine the site of action of a gene product.
• Analyze Zygotic lethal/Maternal effect mutants.
+Bal
+Bal
Zygote Oogenesis
Pure maternal and zygotic screens
+Bal
+Bal
Zygote Oogenesis
Pure zygotic screens
Assume pure expression of a genefrom the zygotic genome only.
+Bal
+Bal
Zygote Oogenesis
Pure maternal screens
Assume pure maternal expression of a gene
Exceptions to the strict assumptions
1. Maternal rescue
2. Zygotic lethal/Maternal effect
+Bal
Zygote
Oogenesis
Maternal Rescue
The maternally suppliedtranscript rescues the zygotic loss of function.
Maternal Rescue
Hide an essential role for a gene in embryonic pattern formation.
Pure zygotic screens may miss important functions.
+Bal
+Bal
Zygote Oogenesis
Pure maternal screens
Assume pure maternal expression of a gene
+Bal
+Bal
Zygote Oogenesis
Zygotic lethal/Maternal effect
Dies atlatelarval stageor pupal stage
We can not assay if the gene hasa maternal effect phenotype.
Zygotic lethal/Maternal effect
Since a homozygous adult mothercan not be established, we can notassay whether the gene contributesinformation to the egg.
Screen for maternal rescue and zygoticlethal/maternal effect
1/2 to 2/3 of lethal mutations are notembyonic lethal.
Norbert Perrimon collected a large #of late larval and pupal lethals.
Properties of OvoD
OvoD dominant female sterile germ line specific cell autonomous
OvoD
No eggs
Mosaic analysis with OvoD
OvoD
No eggs
l
+
+
Irradiate with X-rays
OvoD
l
+
+
Mosaic analysis
OvoD
l
+
+
Mosaic analysis
OvoD
l
+
+
Mosaic analysis
OvoD
l
+
+
OvoD
l
+
+
OvoD
l
+
+
Mosaic analysis
OvoD
l
+
+
OvoD
l
+
+
Eggmaker
Mosaic analysis
Maternal rescue vs. Maternal effect
Maternal rescue can be rescued by thepaternal allele.
Maternal effect is independent of thegenotype of the sperm.
l
l
Egg chamber
Maternal effect
l
+
Maternal effect
l
l
l
+
Embryonic phenotype
Embryonic phenotype
l
l
Egg chamber
Maternal rescue
l
+
Maternal rescue
l
l
l
+
Embryonic phenotype
No phenotyperescued by the paternal allele
Non-complementation screen
Isolation of more independent mutantalleles in a gene.
Non-complementation screen
wild-type en1/en1
Non-complementation screen
en1/en1 engrailed phenotype
en1/deletion engrailed phenotype
en1/+ wild-type phenotype
en1/enx
Non-complementation screen
CyO/L virgin females X b pr cn males
en1/en1 virgin females X b pr cn*/CyO
Screen vials for flies with the en phenotype
F0
F1
F2
EMS
4,000 crosseswith individual males
Vials with no engrailed flies
Vials with engrailed flies
en1
CyO
en1
CyO
b pr en+cn* en1
b pr enxcn en1
50% 50%
50% 50%
Cytogenomics
Salivary gland chromosome: high resolutioncytogenetics.
Cytogenomics
Obsolete
Drosophila genome
• 2000 first draft of genome published
• More than 200,000 expressed sequence tags available
• Large scale transposon mutagenesis
• Flybase: computer database and resource
Scaffold
Human PP2A Holoenzyme
Flybase and Modern Drosophila Research
1. No longer have to clone and sequence a gene- reagents readily available.
2. Store of characterized mutants. RNAi
3. Large scale genome screens like 2 hybrid analysis and insitu hybridization are available.
4. Allows researchers to propose and formulate questions with data available.
Cytogenomics
Salivary gland chromosome: high resolutioncytogenetics.
Designed deletions
FRT
FRT
Designed deletions
FRT
FRT
Designed deletions
FRT FRT
Deletion with known breakpoints
Gene disruption via homologous recombination
Marker
Marker
Marker
Marker
Ends out
Ends in
In vivo recombinant molecules
Ends in
FRT FRTMarker
Rare restrictionenzyme site
Gene fragment
In vivo recombinant moleculesEnds in
FRT
FRT
Marker
Rare restrictionenzyme site
Gene fragment
In vivo recombinant moleculesEnds in
FRT
Marker
Rare restrictionenzyme site
Gene fragment
In vivo recombinant moleculesEnds in
FRT
Marker
Gene fragment
In vivo recombinant moleculesEnds in
FRT Marker
RNAi in Drosophila
1.) Cell culture RNAi. Any Drosophila gene’s expression can be reduced in cell culture.
2.) RNAi transgenes in flies. 88% of the annotated genes of Drosophila represented in this collection of fly stocks.
RNAi in Drosophila
RNAi in Drosophila is cell autonomous ie where the RNAi is produced is where the effect will be.
C. elegans RNAi is systemic--non cell autonnomous. Can enter the worm via diet.
Binary or two component system
Fly stock A: UAS line Fly stock B: Driver line
RNAi
GAL4 binding sitesUASGAL4
GAL4Promoter
or Enhancer
RNAi GAL4
Progeny
GAL4
Dietzl et al., 2007 Nature 448, 151
Dietzl et al., 2007 Nature 448, 151