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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