Genetics according to Mendel and Morgan, a reminder:

XY X X

A A A A

♂ = ♀

♂ ♀

A: autosome

X ou Y: sex-associated chromosomes

♂ ♀

Our chromosomes:

+ = wild-type, dominant ;

m = mutation,recessive

♂♀

A1

A2

A2A1

A1 + / A1 + A1 + / A2 m

A1 + / A2 m A2 m / A2 m

♀ = ♂ : ¾ [+], ¼ [mutant]

♀, ♂ ♀, ♂

♀, ♂ ♀, ♂

For the meiosis of Autosomes:

+

+ m

m

[+]

[+] [mutant]

[+]

[+]

[+]

♂♀

X1

X2

YX1

X1/ X1 X1/ Y

X1/ X2 X2/ Y

¾ [+], ¼ [mutant] but! ♀ ♂

♀

♀

♂

♂

A similar – but not identical - situation holds for sex chromosomes :

[+][+]

+

m

[m]

[+]

[+]

[+]

+

Daltonism, or Red-Green color blindness: a syndrome associated with the X chromosome

X A

Color perception and photoreceptors:Red green and blue

A A

Y X

Color perception and photoreceptors:Red green and blue

♂♀

X1 A

Y AX1 A

X1/ X2; A/A

♀ ♂

Sex Chromosomes vs. Autosomes

X2 A

[+]

[+]

[+]X1/ X1; A/A X1/ Y; A/A

X2/ Y; A/A [Red-green Color Blind]

Red

[+]

[+]

Woman normal

Woman carrier

Man normal

Man Color blind

X

A

R+ V+ B+ R+ V+ B+ R+ V+ B+ V+ B+

X X X

A A A A A A A

XX Y Y

Color perception and photoreceptors:Red green and blue

Where do nearly identical DNAs for Red and Green come from?

X A

Color perception and photoreceptors:Red green and blue

* ** * * * *

Duplication then Divergence

Common ancestor

Recent duplication

Divergence

Color perception and photoreceptors:Red green and blue

More ancient duplication

divergence

duplication

divergence

Origin of photoreceptors:Red Green Blue

High frequency of Daltonism. Where do so many mutations come from???

X A

One example: illegitimate recombination … And the loss of normale function.

Normalpairing

Inappropriatepairing

Recombination by error

X

New mutations

in the nucleus after spreading

Giant chromosomes of Drosophila

Traditional karyotype for a human cell (e.g. amniocentesis)

Trisomy for chromosome 21with associated defects (mongolism)

2 copies = normal

Fluorescent In Situ hybridization: FISH

Fluorescent DNAs, different colors for different chromosomes

T+

Chr21

Karyotype by FISHFluorescent In Situ hybridization

=>Trisomy / Chromosome 21

E.g. Burkitt’s Lymphoma

Regulatory DNA Sequences Coding DNA Sequences

A normal gene :

function

mRNA

proteinWhere?When?

How much?

FISH applied to disease diagnosis

Strong immune expression IgH protein

One normal gene :

General expression C-myc oncogene

Another normal gene :Breakage/repair with error

Strong immune expression IgH protein

General expression C-myc oncogene

Fluorescent In Situ hybridization: FISH

Normal :

General expression C-myc oncogene

Also normal :

Strong immune expression c-myc oncogene

Strong immune expression IgH protein

Abnormal :

Fluorescent DNA-1

Fluorescent DNA-2

And what about the lab?

(XX)

(XO)

The nematode C. elegans as a laboratory model

~1 mm

Eating E. coli

Hermaphrodite

+

mutagen

+ m

+ +/ + +/ m

+ m

+ +/ + +/ m

m +/ m m/m

New, recessive mutation m

Screening for mutants

+ m

+ +/ + +/ m

m +/ m m/m

m m

m

m m/mm/m

m/mm/m

Inbreeding in the lab is helpful

A new mutant line

2 x ‘2n’

A

a

2n

A

a

4n

a

A

A

A

a

a

4 x 1n (gametes)

Meiosis: one mother cell becomes four gametes

Here, we only look at one pair of chromosomes among several…

2 x ‘2n’

A

a

4n

4 x 1n (gametes)

Linkage of DNA sequences on the same chromosome (Mendel I);Independent transmission of different chromosomes (Mendel II)

A

a

2n

A/a = forms of a gene

= transposable element insertions

a

A

OU

a

A

A

a

A

a

1/4

1/4

1/4

1/4

1/2

1/2

Additional variation from physical exchanges (recombination)

A

a

4n

4 x 1n (gametes)

Recombination generates still more diversity

A

a

2n

A/a = forms of a gene

= transposable element insertions

A

a

A

a

<1/4

<1/4

<1/4

<1/4

xa

a

A

A

4xParental 4xRecombinant

f < 0.5

11 22 33 44 55 XX

X

(XX)

(XO)

Bristol

Autosomes Sex chromosome

dpy-6 lin-14 sma-5

X

Vulva: muscles, nerves, skin…

+

lin-14

= [Bag of worms]

Defective vulva

Isolating mutants that affect vulval development: the bag of worms screen

Defective vulva

Egg-laying

11 22 33 44 55 (XX)

(XO)

Bristol

(XX)

(XO)

Bergerac

Autosomes Sex chromosome

lin-14-

lin-14-

lin-14- lin-14 -

lin-14+

lin-14+

lin-14 +

(but otherwise +)

(& otherwise +)

lin-14+

Tc1 elements

Berg

Brislin-14-

lin-14 -

lin-14 +

X(effect female-specific)

lin-14-

lin-14+

[lin-14+]

Brislin-14-

lin-14 -X

« Backcross » N°1

Meiosis + recombination

lin-14+

lin-14+

lin-14-

lin-14+

[lin-14+]

Brislin-14-

lin-14 -X

« Backcross » N°1

Meiosis + recombination

lin-14-

lin-14+

[lin-14+]

Brislin-14-

lin-14 -X

« Backcross » N°2

x x x x x x x x x x x

Serial dilutions, genetic-style

Meiosis + recombinationlin-14-

lin-14+

[lin-14+]

Brislin-14-

lin-14 -X

« Backcross » N°2

lin-14-

lin-14+

[lin-14+]

Brislin-14-

lin-14 -X

« Backcross » N°3

lin-14-

lin-14+

[lin-14+]

« Backcross » N°9

Self-fertilize, select normal hermaphrodites, giving only normal offspring

=> lin-14+ / lin-14+, pure homozygous stock.

Associating the repeated elements closest to lin-14+

Dilution = 29

dpy-6+ lin-14+ sma-5+

X

lin-14+

lin-14+

lin-14+

Tc1 Tc2

The approximate location is identified by the Tc1 repeats. Candidate genes in the known genome sequence can be tested.

Genetic analysis of Aniridia, a rare eye syndromecaused by a Dominant mutation

Dominant

+/+ An/+

Iris reduced=> pupil open

≥ 1 base pair changed out of 3 billion (3x109)

Aniridia (human) : a dominant autosomal syndrome caused by mutation of a single gene, Pax6

Pax-6 Autosomal

and dominant:

An/+ x +/+

↓

½ [An], ½ [+]

♀ = ♂

=> Aniridia results from dominant loss-of-function mutations of Pax6 (haploinsufficient: not enough active protein)

Pax6a (Faux-sens)*

Pax6Δ (deletion)

Pax6+ Pax6a/Pax6+/ Pax6Δ == [Aniridia]

Pax6+

+

pf

pf

Pax6+

+

Small eye (Sey) :Small eye, reduced iris, cranio-facial defects (as for Aniridia)

Origin: dominant, haploinsufficient mutations of the mouse Pax6 gene

+Sey/+

Small eye, a mouse version of Aniridia

Sey/+

♂♀

A1

A2

A2A1

A1 + / A1 + A1 + / A2 Sey

A1 + / A2 Sey

Expected : ♀, ♂ equivalent: 1/4 [+], 2/4 [Sey], ¼ [?]

♀, ♂ ♀, ♂

♀, ♂

Genotype/phenotype for Sey

+

+ Sey

Sey

[+]

[Sey]

[Sey]

[Sey]

[Sey]

?

( and ¼ Sey/Sey [dead] )

Genotype/phenotype for Sey

♂♀

A1

A2

A2A1

A1 + / A1 + A1 + / A2 Sey

A1 + / A2 Sey A2 Sey / A2 Sey

♀, ♂ ♀, ♂

♀, ♂ ♀, ♂

+

+ Sey

Sey

[+]

[Sey] [dead]

[Sey]

[Sey]

[Sey]

Observed : living ♀ = ♂ : 1/3 [+], 2/3 [Sey]

Sey/Sey+/+

Dead how? As eyeless embryos…=> Pax6 / Sey required for normal eye differentiation

Localised expressionof Pax6(mRNA)

Normal eye development follows a genetic program

and requires Pax6+

Normal Pax6 expressionis necessary for eye development

Localised gene expression

= normalNo expression

= absence of function

The Drosophila eyeless (ey) gene encodes dPax6

+ ey-

Pax6 is necessary for normal Drosophila eye development - as in man and mouse -

+

+mPax6 -/-

-/-dPax6+

dPax6 gf

Human,mouse

Drosophila

Very different eyes

with a common origin?

humain

calamar

mouche

Can mis-expression of Pax6 re-program development?

localised gene expression= normal

No expression= loss of function

Novel expression

=> ???

The Test :

Cross appropriate lines …

Transgenic driver Line :

Fly regulatory sequences Yeast GAL4 Protein

Transgenic expresser Line :

UAS (GAL4 target) dPax6 (or other)

Regulatory DNA Sequences Coding DNA Sequences

A normal gene :

functionWhere? When? How much?

Fly regulation GAL4

UAS dPax6 x

?

dPax6 induces supplementary eyes

*

**

mPax6, expressed in the fruit fly, does the same

Frog Pax6 makes new eyes in Xenopus

Hence the idea that Pax6 is an eye selector, since early in evolution

●A DNA binding motif in many selector genes:

The Homeodomain (60 amino acids long)

● proteins are composed of 20 different amino acids (a.a.)

● For a given homeodomain (60 a.a.), p = 1 / 2060 ~ 0

lof gof

mx

lab

proboscipedia (pb) is a conserved selector gene

The fly and human pb homeodomains are 57/60 identical; Probability that this happened by chance: ~ 0

* ** * * * *

Divergence of homologous genes

Common ancestorEmergence of new species

Eye Selector

Leg Selector

Wing Selector

We possess the same genes

These selector genes encode Transcription factors,that regulate other genes in developing eyes, legs, wings…

DNA

Pax6 Transcription of target genes

Pax6

-- DNA of which target genes? Their functions? -- Functional organisation of gene networks?

mRNA

Gene expression involves transcription followed by translation

Looking at gene expression for 15,000 genes:

The fly transcriptome

Systems biology:

dPax6 can induce supplementary eyes

*

**

Such mutations help to look at genetic programs : Including via the transcriptome

Mutant cell mRNA Normal cell mRNA

Using mutations to look at whole-genomes:

The transcriptome

Mutant cell mRNANormal cell mRNA +

Hybridize with miniaturized plates carrying DNA sequences for each gene

Most mRNAfrom normal cells

Most mRNAfrom mutant cells

Equal amounts

Mostly normal

Mostly mutant

Mutant = Normal

Mutant cell mRNANormal cell mRNA +

Different gene in each spot

Tests of a genetic program in vivo

-/-

+/- +/-

Confocalmicroscope

genotype differentiation

Systems biology (e.g. transcriptome) to generate predictions

Genetics to test them in vivo