Elementsinformalgenetics

JeanJacquesPANTHIER
InstitutPasteur
 Contents
1. Mendels experiments and laws.
2. Mendels laws apply to mammals: L. Cunot, E. Castle, C. Little.
3. Mutations are not always recessive.
4. Observed variations in phenotype are not always inherited.
5. Penetrance and expressivity.
6. Genetic linkage.
7. Epigenetic inheritance.
8. Phenocopy.
9. The complementation test.
10. Gain of function and loss of function mutations.
11. Epistasis.
12. Pleiotropy.
1. Mendels experiments and laws:
Genetics as a set of principles and analytical procedures begun in 1865
with a set of experiments by an Augustinian monk named Gregor Mendel

The city of Brno

in the Czek

republic
Genetics as a set of principles and analytical procedures begun in 1865
with a set of experiments by an Augustinian monk named Gregor Mendel

The Augustinian Abbey of St Thomas in Brno

Genetics as a set of principles and analytical procedures begun in 1865
with a set of experiments by an Augustinian monk named Gregor Mendel
Gregor Mendel did work with mice
 The three major observations of Mendel :

1) F0 F0 Started with pure seeds

disappearance of
F1 F1 one parental phenotype
in the F1s

F2 : 3/4
1/4 reappearance of that
phenotype in the F2
2) F1 F0

1/2 reappearance of that

BC1 : phenotype but with a
1/2 different ratio in the BC1
 To explain these data Mendel chose to :

transform a real characteristic (the phenotype , here the shape) to

a virtual object, the determinant (we now call this determinant allele ).

Mendel found that the data of the experiments could be explained if it was postulated
that :

1. A phenotype is controlled by two determinants/alleles.

2. The two determinants/alleles separate (segregate) equally into the gametes.

3. Each determinant/allele reassorts at random from each parent to form the first cell
(or zygote).
 Application of these principles to the F2 generation :

F1 F1

F2 : 3/4 ,

1/4

The fact that the phenotype disappears in the F1 generation may be attributed
to the dominance of to
Application of these principles to the backcross BC1 :

F1 F0

BC1: 1/2

1/2
 Mendels first law :

The two members of a gene pair segregate from each other into

the gamete so that one-half of the gamete carry one member of

the pair and the other one-half of the gametes carry the other

member of the pair.

What happens in the case of two characters, like shape and color, for example ?

F1 F1

F2 : 9/16

3/16

3/16

1/16
 Mendels second law :

During gamete formation the segregation of alleles of one gene

(here, governing the shape) is independent of the segregation of

alleles of another gene (here, governing the color).

2. Mendels laws apply to mammals: L. Cunot, E. Castle, C. Little.

Mendels laws not only apply to

the garden pea (Pisum sativum)
but also to mammals.
dilute This was demonstrated using
coat color mutations

pink-eyed

From:
Experimental Studies of the
dilute
pink-eyed Inheritance of Color in Mice
Clarence C. Little, 1913
YBrBdpA
Experiments by Lucien Cunot (1902) on a single character

Albino= c = Tyrc = Cys103 -> Ser in the tyrosinase gene

Tyr+/+ Tyrc/c Tyrc/+ F1

pigmented white all pigmented Pigmentation is dominant to
the albino phenotype

Tyrc/+ F1 X Tyrc/+ F1 - 198 pigmented (Tyr+/+ or Tyrc/+)

(74% of 270 F2, # 3/4)

- 72 white (Tyrc/c)

(26% of 270 F2, # 1/4)

Conclusion: Experimental data agree with Mendels first law.

Socit de Biologie, April 12, 1902
 Experiments by E. Castle and C. Little (1909) on two characters

p+/p+ d/d (dilute) p/p d+/d+(pink-eyed) p+/p d+/d F1

F1 with wild-type coat color: dilute and pink-eyed are recessive

p+/p d/d+ F1 p+/p d/d+ F1 F2 mice

Four types of mice were obtained in F2 :

1. wild-type (presumably p+/? d+/?)
2. pink-eyed (presumably p/p d+/?)
3. dilute (presumably p+/? d/d)
4. pink-eyed and dilute (presumably p/p d/d)

Conclusion: This is a case of Mendelian dihybridism

Castle & Little, from Science, September 3, 1909.
 3. Mutations are not always recessive.

Microphthalmia (Mitfwh) is semi-dominant : the phenotype of the

heterozygote is intermediate between the phenotypes of both
homozygotes (negative dominance).
 4. Observed variations in phenotype are not always inherited

An example: the coat color of patchwork mutant mice.

Developmental noise is a major source of the observed variations in phenotype.

 5. Penetrance and expressivity.

Penetrance :

Penetrance is the percentage of individuals, with a given genotype who exhibit

the phenotype associated with that genotype :

% [m] among m/m individuals = penetrance

m is a recessive mutation responsible for a white coat color. In the row,

all individuals are homozygotes m/m. Each circle represents an individual :

However, out of 10 m/m individuals, only 6 are white. Hence, the penetrance
of the m mutation is not complete.
 Expressivity :

Expressivity describes the extend to which a given genotype is expressed

phenotypically in an individual.

m is a recessive mutation responsible for a white coat color.

In the row, all individuals are homozygotes m/m.
Each circle represents an individual

Expressivity refers to the degree to which a particular genotype is expressed

as a phenotype.
Both penetrance and expressivity are integral to the concept of the norm
of reaction : the impact of a gene at the phenotype level depends on the
modifying effect of :
1) the rest of the genome,
2) the developmental noise,
3) and the environment.

Variable penetrance and expressivity :

does not express express partially complete expression of

the mutant phenotype the mutant the mutant phenotype
phenotype
Complete penetrance but variable expressivity shown
by five grades of piebald spotting in s/s mice :
Variable expressivity shown by six grades of brachyury in T/+ mice.
 6. Genetic linkage.

Linkage. An example of linkage : ashen and dilute

Lets examine a cross between ashen and dilute mice :

1) Intercross : ash/ash x d /d (+/ ash +/d) F1

2) Backcross : (+/ash +/d)F1 x ash/ash d/d

Gametes ash d ash + + d + +

ash d ash/ash d/d ash/ash +/d +/ ash +/ ash

d/d +/d
Phenotype [ash, d] [ash, +] [+,d] [+,+]

Expected if the
segregation is 120 120 120 120
independent
Observed 1 248 227 4
Thus, the segregation of ashen and dilute is not independent:
ash and +d, on one hand,
+ash and d, on the other hand,
are preferentially transmitted together in the gametes.

The reason for that is that ashen and dilute are linked on the
same chromosome :
chromosome 9

locus ashen

linkage
locus dilute
F1 :

ash ash + + ash ash + +

+ + d d + + d d

ash ash + + Gametes

produced
by the F1
+ d + d +/ash +/d

recombinant
The frequency of recombination allows to estimate the
distance between two linked loci.
Here, there are 5 recombinants among 480 BC1 individuals.
The frequency of crossing over between dilute and ashen is equal to :
5 recombinants/480 meiosis = 0.010 = 1 centimorgan
The corresponding unit is indeed called centimorgan (cM).

Comparison between genetic distance and physical map.

dilute is encoded by the Myo5a gene, located at genomic location
75.3 Mb on chromosome 9.
ashen is encoded by the Rab27a gene, located at genomic location:
73.3 Mb on chromosome 9.
Hence, the two loci are separated by 75.3 -73.3 = 2 x 106 pb (2Mb)

One centimorgan is equivalent on average to a physical distance of

approximately 2 megabases in the mouse genome.
 7. Epigenetic inheritance : The viable yellow (Avy) mutation

All mice have identical genotype : Avy/a, i.e. the expressivity is variable
 Inheritance of the darker and paler coat color in the germ line.

a/a x Avy/a Avy/a x a/a

Morgan et al., Nature Genetics 23, 314-318 (1999)

The darker phenotype of Avy/a mice is inherited when the Avy allele is transmitted
by the female germ line, but not by the male germ line.
 8. Phenocopy.

Phenocopy : A condition where the phenotype of an individual is altered because of

an environmental factor, and thus the individual appears to have an altered genotype,
though in fact it does not.

Example: Effect on the foetus of i.p. injection of Kit-antibody to wild-type C57BL/6J females at E14.5

Reference: Nishikawa et al. 1991. In utero manipulation of coat color formation by a monoclonal anti-c-kit antibody:
two distinct waves of c-kit-dependency during melanocyte development. EMBO J 10:2111-8.
Phenocopy

Effect on the foetus of i.p. injection of Kit-monoclonal antibody to wild-type females

at E13.5, E14.5, E15.5 and at birth

E13.5 E15.5 Newborn

E14.5 E14.5
Progeny of cross : KitW-ei/+ x Kit+/+ :

KitW-ei/+

+/+
 An additional example of a phenocopy : Effect of feeding pregnant females
with methyl-supplemented diet on the phenotype of their offspring

These mice have an identical genotype: Avy/a.

Minimum ectopic Maximum ectopic

agouti expression agouti expression

Offspring of pregnant females fed with a methyl-supplemented diets exhibit a darker coat.

Wolff et al. 1998. FASEB J. 12, 949 957

 9. The complementation test and its use

The ruby mutation has been The cocoa mutation has been
discovered by Leslie Dunn at discovered by Hope Sweet at
Columbia University in 1945 the Jackson Lab in 1985

Question : Are these variants determined by recessive alleles

of the same gene or not ?
 To test for this question, lets cross ruby and cocoa mice :

If ruby and cocoa are recessive alleles of the same gene, you should
obtain the following progeny :
ru/ru x coa/coa 100% ru/coa
Hence the progeny exhibits mutant phenotype.

If ruby and cocoa are not alleles of the same gene, you should
obtain the following progeny :
ru/ru +coa/+coa x +ru/+ru coa/coa +ru/ru +coa/coa
Hence the progeny exhibits wild-type phenotype.
The result of the complementation test for ruby and cocoa is
a wild-type F1 progeny. Hence ruby and cocoa are nonallelic
mutations.

Indeed, we now know that :

ruby maps on chromosome 19, at position 44 cM.
ruby is an in frame deletion of codons 187, 188, 189 of
the Hermansky-Pudlak syndrom 6 (Hps6) gene.

cocoa maps on chromosome 3, at position 12.5 cM.

cocoa is a A T mutation in the 3 splice site of the Hermansky-
Pudlak syndrom 3 (Hps3) gene.
 Test for allelism: the complementation test.

Complementation is the production of the wild-type phenotype

when two separate genotypes determining similar recessive
phenotypes come together in a cell.

When independently derived genotypes producing similar

recessive phenotypes fail to complement each other, we retain the
hypothesis that the genetic determinants of the phenotypes are alleles
of the same gene.

The complementation test can be used provided that the mutations

are recessive and (formally) that both mutations are located at similar
sites on two homologous chromosomes.
10. Loss of function and gain of function mutations

The Ay, A, a and ae mutations

are allelic. The are localized
at the agouti locus.

However they give widely

different phenotypes and
complementation between
these mutations is complex.

Hence agouti was long called

a complex locus.
 The agouti hair colour

black

yellow

black

Dasyprocta aguti
Black hair Agouti hair
Found in Venezuela, Guyana, Suriname, French Guyana, called nonagouti
Trinidad & Tobago, and Brazil.
Agouti affects not only the colour of the coat but also its distribution

black-and-tan (at/at)
mice exhibit a black
coat on their back, but
a white belly
Yellow phenotypes are always dominant over the darker phenotypes in heterozygous mice

yellow allele black allele yellow allele black allele yellow allele wild-type allele
Yellow phenotypes are always dominant over the darker phenotypes, even among black coats

ae/a

ae/ae
Alleles responsible for yellow colours are gain of function mutations.
They are responsible for an overexpression of the agouti protein.

Alleles responsible for black colours are loss of function mutations.

They are responsible for a lack of agouti protein expression
Mutation types: Hermann J. Muller (Nobel prize, 1946)

Amorph : complete loss of function

Hypomorph: partial loss of function, reduced function

Hypermorph : gain of function (normal), increased function

Antimorph (dominant-negative): gain of function against wild-type

Neomorph : Gain of function, new and different function

Mutation types: Hermann J. Muller (Nobel prize, 1946)

Amorph : complete loss of function, albino

Tyrc/c Tg (Tyrosinase) Tyrc/c

Tyrosinase: albino, Tyrc, Cys65 -> Ser

Mutation types: Hermann J. Muller (Nobel prize, 1946)

Hypomorph: partial loss of function, reduced function

Tyrosinase: chinchilla, Tyrc-ch, Ala464 -> Thr

Mutation types: Hermann J. Muller (Nobel prize, 1946)

Hypermorph : gain of function (normal), increased function

yellow allele yellow allele yellow

black allele black allele allele wild-type allele
Mutation types: Hermann J. Muller (Nobel prize, 1946)

Antimorph (dominant-negative): gain of function against

wild-type

KitW-ei/+

KitW-ei : Gly597 -> Ala

Mutation types: Hermann J. Muller (Nobel prize, 1946)

Neomorph : Gain of function, new and different function

Yellow Darker
and and
fat lean

Avy/a

Genetically identical 15-week-old Avy/a mouse littermates

representing five coat-color phenotypes
 11. Epistasis.

An epistatic allele of one gene eliminates expression of the

alternative phenotype of another gene, and inserts its own
phenotype instead (restrictive definition by William Bateson).

The term epistasis is often used today to describe any kind

of gene interaction.
 The discovery of epistasis by William Bateson

black (nonagouti, brown (agouti, white (albino,

a/a) mice A/A) mice Tyrc/c) mice
 Cross between albino and nonagouti mice

BALB/cJ A/A Tyrc/c C57BL/6J a/a Tyr+/+ F1 A/a Tyr+/c

white black agouti
F2 :

A Tyr+ A Tyrc a Tyr+ a Tyrc

A Tyr+ agouti agouti agouti agouti 9/16 agouti

4/16 white
A Tyrc agouti white agouti white 3/16 black
(instead of
a Tyr+ agouti agouti black black 9/16, 3/16, 3/16,
1/16 for two
a Tyrc agouti white black white characters)
 Epistasis in quantitative traits
Lets imagine a quantitative trait controlled by a least deux loci A and B

Effect of the genotype at A on the phenotype: Effect of the genotype at B on the phenotype:
3 3

Phenotype
Phenotype

2 2

1 1
A1A1 A1A2 A2A2 B1B1 B1B2 B2B2

Genotype Genotype

Effect of the genotypes at A and B on the phenotype:

A and B act independently

B1B1
A en B act additively.
Phenotype

2
B1B2

B2B2

A1A1 A1A2 A2A2

Genotype
 Epistasis in quantitative traits
Lets imagine a quantitative trait controlled by a least deux loci A and B

Effect of the genotype at A on the phenotype: Effect of the genotype at B on the phenotype:
3
3

Phenotype
Phnotype

2
2

1
1
B1B1 B1B2 B1B2
A1A1 A1A2 A2A2
Locus B
Locus A

Effect of the genotypes at A and B on the phenotype:

3

B1B1

The effect of locus A is dependent on the

Phnotype

B1B2
2
genotype at the B locus.
This shows epistatic relationship between A and B.
B2B2

1
A1A1 A1A2 A2A2
Gnotype
 12. Pleiotropism: one gene with more than one phenotypic effect.

Coat color
defect :

lethal
null allele at the
spotted (Edn3ls)
Edn3 locus

Intestine
transit
effect :
megacolon
 Pleiotropic effect of the null mutation at the Edn3 locus

Edn30/0

Wild-type
 The dominant megacolon (Sox10Dom) mutation

a white belly is seen

in heterozygotes

Insertion of a guanine residue creates a translational frameshift in

the SRY-box containing gene.
Lack of enteric neurons in Dominant megacolon mice

wild-type Sox10Dom/+
 Conclusions :

The formal rules of genetic transmission and the concepts used

in formal genetics were developped at the beginning of the XXe
century using whole organisms, mainly Drosophila.

The same concepts are still applied today, while phenomena

are generally studied at the cellular and molecular levels.

It is not surprising in such conditions that tensions occasionally

appear when these concepts are used.

Data of formal genetics will always prove stronger that data from
molecular biology if in contradiction.