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The Study of Epistasis and Complementation led to

the Genetic Analysis of Metabolic Pathways


Normal ABO blood groups of humans is best characterized by which


of the following allelic interactions?



a) Dominant recessive.

b) Codominant.

c) Dominant -recessive and codominant.

d) Recessive lethal.

e) All of the above

If individuals of genotype A/a; B/b; C/c are mated, how many


different phenotypes may appear in their offspring, if all allelic
interactions are dominant-recessive ?



a) 3

b) 4

c) 6

d) 8

e) 16

If an trait X has codominant allelic interactions and trait Y has


dominant- recessive allelic interactions. In a large sample of seeds
how many different phenotypes are expected from inbreeding two
heterozygotes (XxYy) ?



a) 2

b) 4

c) 6

d) 8

e) None of the above

L 8: Epistasis, Complementation
and Biochemical Pathways

Epistasis is used in 3 different ways, variations
on interactions between genes:

(1) classical epistasis: how an allele of one gene
blocks or masks the effect of an allele of another
gene, altering classical expected ratios

(2) functional epistasis: relationships between the
expression of different genes (enzymes) operating
in the same pathway

(3) statistical epistasis: a nonadditive effect in
quantitative genetics

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Visible phenotypes show full dominance, round is dominant, wrinkled


recessive. But the biochemical cause of wrinkled & round, SBE1
protein appears to be codominant with respect to starch grain size
which affects the pattern of drying, seed shrinkage.

SBE1- starch branching


enzyme, required to produce
amylopectin

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Dominance may be due to!


(1) Codominant biochemical interactions produce dominant
phenotypes most genes interact with others.!
(2) Modifier gene(s)- genes that are not associated with specific
phenotypes but have epistatic or pleiotropic effects on the
expression of other genes.!
E.g. Modifier genes of hereditary hearing loss!
Recent functional studies of modifier genes of hearing-loss loci
have begun to refine our understanding of hearing processes and will
guide the rational design of medical therapies for hearing loss. Thomas
Friedman et al. 2000 Curr. Opin. Neurobiol.10(4):487-93.!
"

(3) Dominance explained by biochemical interactions: Nonfunctional mutations are recessive, haplosufficient alleles are
dominant. Note there are 2 alleles affecting dominance in the
heterozygote:

(1) functional or haplosufficient allele, (2) non- functional allele


Scale distinctions


dominance, incomplete dominance and codominance are somewhat
arbitrary. these categories are designed for the convenience of
analysis.pp 215




This could suggest to you that the authors think classical analysis models are
arbitrary, but what they are saying is genetic analysis, must :

(1) define the scale of analysis and (2) the scale of the causal mechanisms.



Thus, (1) dominance, incomplete dominance and codominance are
observations, empirical explanations that can be repeatedly confirmed
by careful breeding studies. These categories are used in classical
models to interpret visible phenotype ratios, signifying underlying
genotype ratios and a class of allelic interactions.



But a mechanistic explanation of dominance on a phenotypic level may
involve:

1) biochemical interactions (physiology) 2) haplosufficiency (level of gene
expression), or 3) modifier genes (gene interaction).



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Functional epistasis and the complementation test



(1) The complementation test and the geneticphysiological mechanism causing duplicate
recessive epistasis. This is a functional test for
gene identity and difference.



(2) Using the complementation test in a mutant
screen of diploid organisms.



(3) Using mutant screens to characterize
biochemical pathways in haploid organisms.



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Definitions vary between textbooks, indicating a


different scale and focus but they all refer to to the same
interaction between recessive mutations affecting the
same phenotype
(A)

Complementation is the production of a wild type phenotype


when two haploid genomes bearing different recessive mutations are
united in the same cell (pp 223).

and (B) Complementation happens when different homozygous


recessive parental strains for the same Mendelian character have
offspring expressing a dominant phenotype.

or (C) Complementation testing asks whether two recessive
mutations affecting the same character are in the same gene or in
different genes in one cell, individual or phenotype.

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The blue pigment in


Lobelia is Delphinidin-3glucoside. The mutation for
white color is recessive. With
only one active copy of the gene
there is sufficient pigment
produced to have blue flowers
(haplosufficient).

Observation: There are two inbred white (mutant)


strains.
Breeders question: are these mutations in the same
or different genes ?
X
or
Same- same strain

different -different strain


F1
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In other words, more than 1 gene may be


governing character or phenotype expression.

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Complementation (trans) test: Do two mutants


have mutations in the same gene (A), or (B) in genes
in another location (= different genes)?

X
or
A

F1
Same (ww)
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different
(complementation)

White phenotype (similar to the parentals)



Explanation: (A) both mutations are in the same gene - same
expression, same chromosome location (F1 homozygous (w1/ w2=
ww)
(B) both mutations are recessive loss-of function
mutations in different genes affecting the same character.

X
or

F1
Same (ww)
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(B) The simplest explanation for a blue F1 hybrid.

Gene W1+ ,
Gene W1+ ,

Gene w2
-

Gene w2-

(W1+ / W1+ , w2- / w2-)


Gene w1- , Gene W2+



Gene w1- , Gene W2+



(w1- / w1-, W2+ / W2+)

Trans and

haplosufficient

They complement

Gene W1+ . Gene w2-

Gene w1- . Gene W2+

Note this is the expression of maternal and paternal chromosomes


in F1 individuals.

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The F1 indicates the strains 1, and 3 are complementary, How


do you test for the independence of the 2 genes?

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(W1+/W1+, w2-/w2-)

(w1-/w1-, W2+/W2+)

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(1)Both genes affect the same function



(1b) These mutations complement each other, they are in different
w1-/W1+ w2-/W2+
X
w1-/W1+ w2-/W2+

9 W1+__ W2+__

3 w1-w1- W2+__

3


1

W1+__ w2-w2-

w1-w1- w2-w2-

genes.



9
recessive epistasis

without a cumulative

effect

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(2) Independent chromosome
assortment but an epistatic
interaction

Review To determine if two recessive mutants in


inbred strains with the same phenotype are in different genes
we do a trans or complementation test.

Mechanism model: (A) Loss of function mutations inactivate the protein
encoded by the gene. If both mutations are in alleles of a single gene, then the 2
non-functional alleles block the pathway producing the blue pigment in a
homozygous recessive individual - white.



(B) If loss of function mutations are in different genes that
both produce enzymes necessary to complete a pigment pathway, then
haplosufficient heterozygotes complement or RESCUE each other producing pigment.


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Gene w1-

Gene W2+

Gene w1-

Gene W2+

Homozygous loss of function alleles


Enzyme 1

Compound A

(white)

Enzyme 2

Compound B

(white)

Compound C

(blue - not expressed)


A mutation in W1 blocks the pathway


no blue pigment is produced.
the flower is white.
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Gene W1+
Gene W1+

Gene w2-

Gene w2-

Enzyme W1+

Compound A

(white)

Enzyme2

Compound B

(white)

2 strains, 2 genes evidence ?


Compound C

(blue- not expressed)

A mutation in W2 blocks the


pathway.No blue pigment is
produced. The flower is white.
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If the trans test indicates complementation, then (1) there must


be at least 2 different genes in the pathway

White 1

White 2

Gene W1-

Gene W2-

Gene W1+

Gene W2+

Enzyme 1
Compound A

(white)

Compound B

(white)

Enzyme2

Compound C

(blue- expressed)

In this case, the genes are epistatic, affecting the


same phenotype character, but they are different,
genes.

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A mutant screen, a large-scale experiment testing the


genetic nature of multiple mutants affecting a character

# of genes ?

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An easy way to figure


out how many different,
(non-duplicated) genes
there are in a pathway
using a mutant screen

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Genetic Analysis of a Biochemical Pathway


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The one-gene-one-enzyme hypothesis was developed by



George Beadle and Edward Tatum in the 1940s based on the

study of mutants of Neurospora.


(1)They selected haploid mutants that required arginine (an


amino acid) for growth.

(2)They identified three mutants which could grow (phenotype or
character grow or not grow) on different supplements (suggesting
they are biochemical precursors of arginine).

(3)They hypothesized that each mutation caused an inactivation
of a different enzyme that acted in the biosynthetic pathway of
arginine.


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Neurospora Life Cycle


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(1)Induce mutation
variation, make
heterozygotes, promote
haplotype spore
variation..

(2) Screen on minimal media for
nutritional mutants (no growth).

(3) Select for arginine requiring
mutants of Neurospora from the
original haploid individuals
inoculated on complete media

Arginine - deficient mutants
grow when arginine (one of
many amino acids) is added to
minimal media, or arginine
rescues these individuals.
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arg-

Some
mutants
could grow when
related compounds
were added to the
media
instead of arginine,
suggesting a
biosynthetic
pathway.

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Supplement

The different classes have different phenotypes determined by


which compounds could rescue them. The mutations were mapped
and were found to be on different chromosomes.
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Beadle and Tatum proposed this biosynthetic pathway and


hypothesized that the mutations were in genes encoding the
three enzymes of the pathway. Mutations cause the loss of
active enzymes , blocking a biosynthetic pathway. The
blocks are rescued by adding downstream products.

arg-1+
Enzyme 1

precursor

arg-2+
Enzyme 2

ornithine

arg-3+
Enzyme 3

citrulline

arginine
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precursor

ornithine

citrulline

arginine

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Genetic Analysis of a Metabolic Pathway - Through a trial by sampling and best chemical
guess process, four compounds appear required to make compound 4, this is a measurable
product, a metabolic character or phenotype. You have also found by testing different
bacterial populations there are mutants (eg B) for specific enzymes catalyzing a specific
compound transformations. Rather than beginning a long biochemical analysis, you can use a
genetic analysis of the mutations and participating compounds to order the metabolic pathway

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(+) indicates that the product phenotype (wild type) was expressed. (-) indicates the phenotype
has not been produced or expressed, indicating an enzyme mutant.

(1)What is the order of supplement compounds (1-6)-look at the columns

Start here- the supplement at the beginning of the pathway never rescues the pathway. The final
supplement in the pathway always rescues the pathway.

(2) What is the order of enzymes (A-E) in the pathway -look at the rows

Start here- which enzyme catalyzes the first reaction means which mutant enzyme is rescued by
all but 1 compound - which must be the precursor substrate. One mutant enzyme is only rescued
by 1 compound , indicating it is at the end of the pathway.

Supplement

1
2
3
4
5

-

-

-

-

+

+

+

+

+

+

Enzyme

A

B

C

D

E

-

+

-

+

+

-

-

-

+

+

+

+

-

+

+

-

-

-

-

-

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Genetic (mutation) analysis of a pathway



Supplement

1
2
3
4
5
6

Enzyme

A

B

+
+
+
+
-
+

-
+
-
+
-
+

C

D

E

-
+
+
+
-
+

-
-
-
-
-
+

-
-
-
+
-
+

Order of compounds in the pathway ?



Order of enzyme action in the pathway ?

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