Forward GeneticsPhenotype(Function)

Genetics Gene A Gene B Gene C

Proteins A B C

P

Survey

In classical or forward genetics, we commonly use chemicals or radiation to generate mutations in model organisms such as yeast, worm, fly or mouse.

In doing so, we typically try to generate mutations in

A: somatic cells B: germline cells (gametes, sperm or oocytes)C: not sure

Mutagenesis-sex chromosome

Oocyte

gametes X

XY

50%

XX

meiosis

female

Sperm

YX

50% 50%

XY

Male

fertilization

Oocyte

X

2n

XX

50%

Mutagenesis-sex chromosome

Oocyte Sperm

gametes x YX

XX XY

meiosis

female Male

fertilization

Oocyte

X

2n

Mutagen

xX xY

heterozygote mutant

Mutagenesis-autosome

Oocyte Sperm

gametes A AA

50% 50%

AA

50%

AA

50%

A A A A

meiosis

female Male

fertilization

Oocyte

A

2n

x y

Mutagenesis-autosome

Oocyte

gametes A

50% 50%

AA

50%

A A

meiosis

female

fertilization

Oocyte

A

2n

AA

50%

X-ray

a

Aa

some

Heterzygousmutant

Sperm

AA

A A

Male

x y

Your opinionsWhen you mutagenize the gametes of P0 animals, you usually do not get homozygous mutants in the F1 generation.

A: YesB: NoC: not sure.

Do you usually get homozygous mutants in the F2?

A: YesB: NoC: not sure.

F1 screens vs. F2 screens

++

Po WT

m+

X++

F1 mutant

++

Po WT

m+

X++

F1 WT Xm+

mm

F2 mutant

But, how do we get the same m in two F1 and let them mate?

Balancer chromosomes

Chromosomes that suppress crossover. Homozygous of the chromosome is either lethal or with a visible phenotype.

Usually contain inversions and translocations

A B

B AA B

B A Neither can be paired with WT

wt

balancer

Resulting abnormalchromosomes

Traditional F2 screen in flyX-ray

X

TM

TM

*

* XTM

TMF1

* *X

**

F3 homozygotes

F2 screens in the worm

Sperm Oocyte

gametes X X

100%100%

XX

100%

Self-progeny

XX

meiosis

Hermaphrodite

fertilization

x

xX

Po

F1

Worm F2 screen

xX F1

Sperm Oocyte

gametes X X

meiosis

X x

xx F2

Homozygous mutant

1/4

summary++

Po WT

m+

X++

F1 WT X++

mm

F3 mutant

m+

F1 WT Xm+

Fly, mouse, …

++

Po WT

m+

F1 WT

mm

F2 mutant

worm

mutagen mutagen

Basic mutagenesis in the worm

Treat with 50mM EMSfor 4 hrs

Several L4 or young adult worms/plate.Multiple large plates

P0

F1 eggs

F1

Remove P0 parents after laying ~100 eggs

Recovering for a few hours

each F1 carries two mutagenized chromosomes

m/+ heterozygotes for each mutation

F2 eggs

Remove F1 worms after laying ~ 2000 eggs for 20 hrs

F2

Worms with m/m genotype for each mutation are mixed with m/+ and +/+ animals

mutant worms (m/m) are individually picked on to a fresh small plate.

Total genomes screened = 2X # of F1 animals x # of F1 plates

Question

If you plan to mutagenize and screen for a mutation in a tumor suppressor gene that may leads to tumorigenesis, would you do F1 screen or F2 screen?

A: F1

B: F2

Genetic and physical map

rme-2

1.8 2.0 2.2

unc-5 skn-1him-3

unc-77

fem-1sup-41

mor-2sup-23

evl-7

Genetic map

map unit

Cloned genes

Non-cloned genes

mDf4nDf41

rme-2unc-5 fem-1 skn-1

Physical map

cosmids

YACs

Named genes eP14 nhr-48

Genetic map and physical map will completely unified when every gene has been mutated. A: yes. B: No. C: not sure.

A few Dumpy (Dup)

dpydpy

X

A few wild type

Uncoordinate (Unc)

uncunc X dpy

+

Wild type

Pick several male progeny

Segregate no dumpy progeny, discard

If the two genes are linked unc + + +

unc + + dpy

Segregate dumpy progeny, continue mapping with the plates

dpy +

unc +

; + +

unc +

;

Pick several wild-type hermaphroditesand place each to one pate

If the two genes are unlinked

50%50%

Figure 8.15. Linkage analysis between two mutations.

+; dpy +; +

unc; +

unc; +

+ dpy + +

unc +

unc +

Wild type 9/16

genotypes unc +

dpy +

;

+ +

dpy +

;

unc +

+ +

;

4/16

2/16

2/16

+ +

+ +

; 1/16

Unc 3/16

uncunc

dpy +

; 2/16

uncunc

+ +

; 1/16

Dpy 3/16

unc +

dpydpy

; 2/16

+ +

dpydpy

; 1/16

Dpy-and-Unc 1/16

uncunc

dpydpy

; 2/16

Situation 1. The two genes are unlinked

unc +

dpy +

;

Phenotypes of wormsIn the plate

A single plate

Pick 20 Unc animals

If ~ 2/3 of these Unc worm segregate Dpy-and-Unc animals, non-linkage between the two genes is deduced.

Situation 2. The two genes are linked on one of the six chromosomes

A single plate + dpy unc +

Phenotypes

Wild type 1/2

Non-recombinantGenotypes

+ dpy unc +

Dpy-and-Unc 0

unc dpy unc dpy

Unc 1/4

unc + unc +

Dpy 1/4

+ dpy + dpy

Pick 20 Unc animals

linkage between the genes is indicated by segregation of no Dpy animals from all or the majority of Unc animals.

majority

unc + unc +

Self fertilizing

The frequency of rare recombinants that segregate Dpy animals is correlated with the genetic distance between the two genes.

unc dpy unc dpy

Rare

unc + + dpy

unc +

+ dpy

unc dpy unc +

unc dpy + dpy

Rare recombinants

unc dpy

Figure 8.16. Example of genetic three point mapping

unc dpy

egl

mapping strain

phenotype

Wild type hermaphrodite

genotype

unc dpy

egl

meiosis

Sperms or eggs

Most of the gametes Rare gametes from a recombination event

dpyegl

unc

unc + dpp unc + dpy

Progeny from combination of the common gametes

unc + dpp + egl +

+ egl + + egl +

Progeny from combination between common gametes and recombinant gametes

unc + dpp + egl dpy

unc + dpp unc + +

Self-fertilizing

Wild type

Unc and Dpy

Egl

Dpy non-Unc

Unc non-Dpy

Wild typeunc + + + egl +

+ egl dpy + egl +

Egl

Sperms or eggs

unc dpy

eglto the right of the egl

dpyegl

unc

dpy

eglunc

unc + dpp + egl dpy

unc + dpp unc + +

unc + dpp + + dpy

unc + dpp unc egl +

Dpy non-Unc No Unc non-Dpy Yes

Dpy non-Unc YesUnc non-Dpy No

Progeny with Egl phenotype

Recombination occurs to the left of egl

Recognizable recombinants

Map position

unc dpyegl

a b

a b

= # of recombinations occurred to the left of egl # of recombinations occurred to the right of egl

eglunc

Xeglunc

Genetic mutant derived from the strain from Bristol, England.The egl mutation is being mapped.

A C. elegans strain from Hawaii. SNPs between this strain and the Bristol strain have been determined.

eglunc

* * * * * *1 2 3 4 5 6

The hybrid strain. Stars indicate SNPs in the region

Select Unc but non-Egl recombinants

X X X

eglunc

* * * * * *2 3 4 5 6unc

eglunc

* * * * * *4 5 6unc

eglunc

* * * * * *6unc

Determine SNP #4 for all recombinant worms by sequencing or digestion.

A B C

Determine SNP #5 and #6 for those that have lost SNP#4 (worm C only)

Worm C has SNP #6 but not #5: the egl gene maps to the right of SNP#5

Worms A and B have #4 SNP from the Hawaii strain

Figure 8.17. An example of genetic mapping using SNPs

Genetic mutation

Injection of subclones

sequencing mutant DNA

Mapping using marker mutations

SNP mapping

RNAi of candidate genesMicroinjection of cosmid/YAC clones

Common steps involved in cloning C. elegans genes defined by mutations.

What would be the flow chart for cloning in yeast?Fly?Human?

Which is the strongest evidence for claiming the cloning of the gene defined by the mutation?

A. The transgene put back into the animal can rescue the mutant phenotype.

B. You find a missense mutation in this gene by sequencing.

C. Reducing the gene activity by RNAi mimics the mutant phenotype.

D. The gene is expressed in the tissue with the mutant phenotype.

Figure 8.19. Microinjection transformation in C. elegans.

DNA solution is injected to the distal arms of the gonad

unc-119(+) gene as a marker

Select F1 transgenic animals, most are unstable

F2 transgenic animals, stable lines

Injection

Three types of markers

a strongly expressed GFP gene as a marker

A dominant rol-6 mutant gene as a marker

unc-119(-) mutant

Wild type

Wild type

Roller

Wild type

Green worm

Figure 8.21. The prevailing model for the mechanism of RNAi

dsRNA

Introduced into cells

Dicer Bind to Dicer-RDE-1enzyme complex

dsRNA is cut to ~22 nt siRNA

Incorporated into RISC nuclease complex

Unwinding siRNAs, activation of RISC

AAAAAAAA

5’

Target mRNA

Cleavage of target mRNA

Multiple-protein components of RISC

Figure 8.22. RNAi methods in C. elegans.

dsRNAintestine

A. Injecting ds RNA into intestine or gonad

B. Soak worms with dsRNA solution

Observe phenotype in progeny

Observe phenotype in progeny

Soak for 24 hoursTransfer to plates

Transfer to plates

Feed worms the bacterial strain

Observe phenotype in progeny

Grow the bacterial strain containing the vector expressing dsRNA

C. Feed the worms a bacterial strain that expresses dsRNA