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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
ww+
- +
+
Mitotic recombination
ww+
- +
+
Mitotic recombination
-
- +
+
Mitotic recombination
ww-
-
- +
+
w+
w+
Mitotic recombination
w-
Eye
w-
-
- +
+
w+
w+
ww+
Mitotic recombination
Clones of cells
X-rays
Indiscriminant crossing over
Flip mediated site specific recombination
Yeast two micron plasmid
A
B
FRT C
FRT
D
Flip
A
B
FRT
FRT
D
C
Flip mediated mitotic recombination
FRT
FRT
Flip mediated mitotic recombination
+
FRT
Sb
FRT
Flip mediated mitotic recombination
+
+
FRT
FRT
Sb
Sb
FRT
FRT
Flip mediated mitotic recombination
hspFLP
+
+
FRT
FRT
Sb
Sb
FRT
FRT
Flip mediated mitotic recombination
hspFLP
+
+
FRT
FRT
Sb
Sb
FLP
FRT
FRT
Flip mediated mitotic recombination
hspFLP
+
+
FRT
FRT
Sb
Sb
FLP
FRT
FRT
Flip mediated mitotic recombination
+
+
FRT
FRT
Sb
Sb
FRT
FRT
Flip mediated mitotic recombination
+
FRT
+
FRT
Sb
FRT
Sb
FRT
Flip mediated mitotic recombination
+
FRT
+
FRT
Sb
FRT
Sb
FRT
Mosaic analysis
• Assay adult phenotype of an embryonic
lethal.
• Determine the site of action of a gene
product.
• Analyze Zygotic lethal/Maternal effect
mutants.
Pure maternal and zygotic screens
Bal
Bal
+
+
Zygote
Oogenesis
Pure zygotic screens
Bal
Bal
+
+
Zygote
Oogenesis
Assume pure expression of a gene
from the zygotic genome only.
Pure maternal screens
Bal
Bal
+
+
Zygote
Oogenesis
Assume pure maternal expression of a
gene
Exceptions to the strict assumptions
1. Maternal rescue
2. Zygotic lethal/Maternal effect
Maternal Rescue
Zygote
Bal
+
Oogenesis
The maternally supplied
transcript 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.
Pure maternal screens
Bal
Bal
+
+
Zygote
Oogenesis
Assume pure maternal expression of a
gene
Zygotic lethal/Maternal effect
Bal
Bal
+
+
Zygote
Oogenesis
Dies at
late
larval stage
or pupal stage
We can not assay if the gene has
a maternal effect phenotype.
Zygotic lethal/Maternal effect
Since a homozygous adult mother
can not be established, we can not
assay whether the gene contributes
information to the egg.
Screen for maternal rescue and zygotic
lethal/maternal effect
1/2 to 2/3 of lethal mutations are not
embyonic 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
Mosaic analysis
OvoD +
+
l
Mosaic analysis
OvoD +
+
l
Mosaic analysis
OvoD +
+
l
Mosaic analysis
OvoD +
OvoD +
OvoD +
+
+
l
+
l
l
Mosaic analysis
OvoD +
OvoD +
+
l
+
l
Egg
maker
Maternal rescue vs. Maternal effect
Maternal rescue can be rescued by the
paternal allele.
Maternal effect is independent of the
genotype of the sperm.
Maternal effect
Egg chamber
l
l
l
+
Maternal effect
l
l
Embryonic phenotype
l
+
Embryonic phenotype
Maternal rescue
Egg chamber
l
l
l
+
Maternal rescue
l
l
Embryonic phenotype
l
+
No phenotype
rescued by the
paternal allele
Non-complementation screen
Isolation of more independent mutant
alleles in a gene.
Non-complementation screen
wild-type
en1/en1
Non-complementation screen
en1/en1 engrailed phenotype
en1/+ wild-type phenotype
en1/deletion engrailed phenotype
en1/enx
Non-complementation screen
EMS
F0 CyO/L virgin females X b pr cn males
F1
en1/en1 virgin females X b pr cn*/CyO
4,000 crosses
with individual males
F2 Screen vials for flies with the en phenotype
Vials with no engrailed flies
50%
en1
CyO
50%
b pr en+cn*
en1
Vials with engrailed flies
50%
en1
CyO
50%
b pr enxcn
en1
Cytogenomics
Salivary gland chromosome: high resolution
cytogenetics.
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
Human PP2A Holoenzyme
Scaffold
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 resolution
cytogenetics.
Designed deletions
FRT
FRT
Designed deletions
FRT
FRT
Designed deletions
FRT
FRT
Deletion with known breakpoints
Gene disruption via homologous
recombination
Marker
Ends out
Marker
Marker
Ends in
Marker
In vivo recombinant molecules
Ends in
FRT
Marker Gene fragment
Rare restriction
enzyme site
FRT
In vivo recombinant molecules
Ends in
Rare restriction
enzyme site
Gene fragment
FRT
Marker
FRT
In vivo recombinant molecules
Ends in
Marker
FRT
Gene fragment
Rare restriction
enzyme site
In vivo recombinant molecules
Ends in
Marker
FRT
Gene fragment
In vivo recombinant molecules
Ends 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
Promoter
RNAi
GAL4
or Enhancer
GAL4 binding sites
UASGAL4
GAL4
GAL4
RNAi
Progeny
Dietzl et al., 2007 Nature 448, 151
Dietzl et al., 2007 Nature 448, 151