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