Transcript Prof. Kamakaka`s Lecture 9 Notes

Lecture 9
One gene One enzyme hypothesis
In the next few lectures, the following questions will be
Addressed:
1
DMD
Duchene muscular dystrophy
is a human disease caused by
an X-linked recessive
mutation.
DMD affects one in 3500
males.
Age of onset is between one
and six years.
Affected individuals are
often initially identified
because they rise from the
prone position in an unusual
manner.
2
Pathways
Biologists and clinicians want to address the question of how
altering a particular set of base pairs that make up the 3
billion base pairs in the human genome led to this phenotype.
3
DMD
Through these techniques, it was found that DMD patients have
a mutation in a single gene.
The normal function of the gene is to enable muscle fibers to
make a protein called dystrophin.
Dystrophin localizes to the plasma membrane in muscle cells.
The normal dystrophin protein stabilizes the muscles during
muscle contractions.
Muscle fibers in people affected with DMD are extremely
deficient in dystrophin.
Without this protein, the plasma membrane ruptures during
muscle contraction and degeneration of the muscle tissue
occurs.
4
Huntington's Disease
Huntington's disease (HD) results from degeneration of
neurons, in certain areas of the brain. This degeneration
causes uncontrolled movements, loss of intellectual faculties,
emotional disturbance and early death
This disease is caused by a single dominant mutation on the
forth chromosome.
Each child of an HD parent has a 50-50 chance of inheriting
the mutation.
A person who inherits the mutation will sooner or later develop
the disease!
What is the normal function of the Huntington gene?
What happens in the mutant?
Can it be blocked?
To understand this disease we need an interdisciplinary
Approach.
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Alkaptonuria
Degenerative disease. Darkening of connective tissue, arthritis
Darkening of urine
1902
Garrod characterized the disorder-
using Mendels rules- Autosomal recessive.
Affected individuals had normal parents and normal offspring.
1908
Garrod termed the defect- inborn error of metabolism
Homogentisic acid is secreted in urine of these patients.
This is an aromatic compound and so Garrod suggested that it
was an intermediate that was accumulating in mutant individuals
and was caused by lack of enzyme that splits aromatic rings of
amino acids.
1958
La Du showed that accumulation of homogentistic acid
is due to absence of enzyme in liver extracts
1994
Seidman mapped gene to chromosome 3 in human
1996
Gene cloned and mutant identified P230S &V300G
2000
Enzyme principally expressed in liver and kidneys
6
How does a gene generate a phenotype?
The experiments of Beadle and Tatum in the 1940’s
provided the first insight into gene function.
They developed the one gene/one enzyme hypothesis
This hypothesis has three tenets:
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Consequences of mutations
Lets say we know the biochemical pathway.
With this pathway, what are the consequences of a
mutation in geneB?
Would the final product be produced?
Would intermediate2 be produced?
Would intermediate1 be produced?
What happens if we add intermediate1 to the media?
What happens if we add intermediate2 to the media?
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Neurospora
Beadle and Tatum analyzed
biosynthetic mutations in the
haploid fungus Neurospora.
It had the advantage in that it could be grown on a
defined growth medium.
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Prototroph: wildtype strain
Utilizes sugar, salt and water to grow.
Auxotroph: Mutant strain
Needs a specific amino acid or vitamin along with sugar, salt
and water to grow.
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Beadle and Tatum set out to identify genes involved in the
biosynthetic pathway that led to the production of the amino
acid arginine.
Neurospora has approximately 15,000 genes and only 4-5 of
these genes are involved in synthesizing arginine.
How do you identify five genes from 15,000?
The POWER OF GENETICS!!!!!!
Typically the organism is exposed to a strong mutagen.
This randomly mutagenizes genes.
Then you look for a mutant in the pathway of interest.
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ARGININE BIOSYNTHESIS PATHWAY
Irradiate (mutagenize) spores.
Grow on medium containing arginine
Transfer to medium lacking arginine
DO THEY GROW OR NOT?
If the cells cannot grow on medium lacking arg,
then they must have a mutation in a gene
required for making ARGININE
Mutant needed arginine to grow.
Enzyme for making arginine was missing
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The method
complete
1
2
3
4
5
6
7
8
9 10
minimal
13
Conclusion- strain1
Strain1 and 7 are defective in either amino acid
production or Vitamin production
Complete media
(salt+sugar+
Vitamin + amino acids)
Minimal media
Minimal media
(salt+sugar)
(salt+sugar)
+ 20 amino acids
+ vitamins
Minimal media Complete media
(salt+sugar)
(salt+sugar)
Vitamin + amino acid
Conclusion:
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Conclusion- strain7
Strain1 and 7 are defective in either amino acid
production or Vitamin production
Complete media
(salt+sugar+
Vitamin + amino acids)
Minimal media Minimal media Minimal media
complete media
(salt+sugar)
(salt+sugar)
(salt+sugar)
(salt+sugar)
+ 20 amino acids + vitamins
Vitamin + amino acids
Conclusion:
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Which amino acid?
Mutant7 is in a gene required for the production of Arginine.
16
Beadle and Tatum found that three mutants could not produce
arginine
Arg1
Arg2
Arg3
The biochemical pathway for arginine synthesis was kind of
known. Ornithine and citrulline are closely related to arginine
and were thought to be precursors
Precursor -----> ornithine -----> citrulline -----> arginine
enz1
enz2
enz3
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Add back
Precursor -----> ornithine -----> citrulline -----> arginine
enz1
enz2
enz3
There are three different enzymes required for arginine
synthesis
Enz1, enz2 and enz3
Beadle and Tatum isolated three different mutations in genes
(three genes)
Arg1
Arg2
Arg3
?????Which mutation codes for which enzyme????
Instead of arginine, if they added ornithine or citrulline to the
media, some mutants were rescued and others were not
Ornithine
Citrulline
Arginine
Mutant1
Mutant2
Mutant3
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Mutant in Arg1- only precursor made
Add ornithine or citrulline to media, downstream enzymes are
functional and pathway continues---> arginine synthesized
Mutant in Arg2You need to supplement media with citrulline for the pathway
to continue. Adding the precursor or ornithine does not help.
Mutant in Arg3You need to supplement media with arginine. Adding the
precursor, ornithine or citrulline does not help.
These experiments demonstrated that a single gene (mutation)
coded for a single enzyme.
In addition, the combination of appropriate mutations and
intermediates enabled Beadle and Tatum to define the
biochemical pathway leading to Arginine synthesis.
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This rationale currently is being used in many laboratories
to elucidate more complex pathways in multicellular
organisms
ｷ
Review
Solving biochemical pathways:
The more mutations that a compound rescues, the later in
the pathway the compound is located
Conversely, the later a mutation is in a pathway, the fewer
compounds will rescue it:
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Another example
I get three mutants for a particular pathway
I add back various intermediates in this pathway and determine
the results
Mut1
Compound
E
B
-
N
+
A
+
Mut2
-
-
+
-
Mut3
+
-
+
+
What is the order of the compounds and mutations in the
pathway?
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The steps in a biochemical pathway identified by this
procedure are dependent on the available intermediates and
mutations.
This procedure does not identify every step in the pathway
This process does not identify every step in the pathway!
B----> E----> A----> N
B----> E----> X-----> A----> N
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Temperature-sensitive mutations
The one gene/one enzyme concept explains a number of genetic
phenomena
A) Temperature-sensitive mutations
Some mutations exhibit a phenotype at high temperatures (the
restrictive temperature) but function normally at lower
temperatures (permissive temperature). The mutation results in
a slight destabilization and alteration of the 3D conformation
of the enzyme
Low temp- structure of enzyme- normal- activity normal
High temp- structure of enzyme-altered- No activity
These kinds of conditional mutants allow you to turn on and off
a function of a protein.
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An example of a Ts mutation:
Dogs and cats that are white with black feet or vice versa
The gene for coat color is normal at cold temperatures but is
inactive at higher temperatures or Vice Versa
Albino - C gene in cats
This gene affects the intensity of melanin production.
The normal or dominant form, C, is 'full color'.
Various incompletely dominant mutant alleles. These mutants are
temperature sensitive The higher the temperature, the more effective they are at
producing melanin
In order of decreasing dominance we have C, Cb, Cs and c.
C is wild-type or full color. It is dominant to all other alleles.
Cb- 'Burmese' factor- it causes a slight lightening of color and is
slightly temperature sensitive.
Cs- 'Siamese' factor; it has a much greater lightening effect and
appears more temperature sensitive.
Cb is incompletely dominant over Cs; the heterozygote (Cb/Cs) gives
a phenotype intermediate between Burmese and Siamese, known as
Tonkinese.
The most recessive form is c, also known as albino. In the
homozygote cc this causes complete absence of any pigment and
white fur.
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Dominance versus Recessive
The one gene/one enzyme concept explains
Dominance versus Recessive
Precursor------> product
= phenotype
^
|
enzymeA
ｷA allele produces functional enzyme
^
|
ｷa allele produces nonfunctional enzyme
geneA
Genotype
enzyme activity
phenotype
A/A
2X
normal
A/a
1X
normal
a/a
0X
mutant
Usually substrate is limiting, enzyme is in excess
By saying that a mutation is recessive, we are saying that
1 unit of enzyme (or 50% of the normal activity) is enough to
produce a normal phenotype
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Genetic Ratios
Altered PHENOTYPE RATIOS
The one gene/one enzyme helps explain altered phenotype ratios
observed in a standard dihybrid cross: (2 genes segregating
independently)
If the Two genes being analyzed affect the same genetic
pathway
Precursor---->
yellow
intermediate---->
white
EnzA
Parental cross
product
blue
EnzB
white
x
yellow
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Precursor---->
yellow
intermediate---->
white
EnzA
product
blue
EnzB
F2
AB
Ab
aB
ab
AB
Ab
aB
ab
9 A-B3A-bb
3aaB1aabb
blue
white
yellow
yellow
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Labradors
Parental Cross:
black
x
yellow
BBEE
bbee
BbEe (black)
x
BbEe (black)
Yellow------->
E
brown--------> black
B
Given the pathway show above, what phenotypic ratios would be
produced in progeny from the dihybrid cross: BbEe x BbEe
EB
Eb
eB
eb
EB
9:3:4
Eb
eB
eb
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Precursor---->
white
intermediate---->
white
EnzA
AB
product
blue
EnzB
Ab
aB
ab
AB
Ab
aB
ab
9 A-B3A-bb
3aaB1aabb
blue
white
white
white
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Biochemical Pathways and Linked Genes
Precursor---->
yellow
intermediate---->
white
EnzC
GeneC
Parental C-D
C-D
product
blue
EnzD
GeneD
x
c-d
c-d
F1
The F1 is testcrossed
The following F2 progeny are produced:
50 yellow
40 blue
10 white
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Biochemical Pathways and Linked Genes
Precursor---->
yellow
intermediate---->
white
EnzC
product
blue
EnzD
GeneC
GeneD
Parental C-D
C-D
F1
x
C-D
c-d
x
c-d
c-d
c-d
c-d
The following F2 progeny are produced:
50 yellow 40 blue 10 white
Parental
Recomb
C-D
c-d
blue
40
c-d
c-d
yellow
40
C-d
c-d
white
10
c-D
c-d
yellow
10
What is the map distance between these two genes?
Map Distance+#Recombinants/Total Progeny x 100%
2(10)/100= 20 Map Units
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One gene: one polypeptide
The concept of 1 gene/enzyme was modified to the concept
of: 1 gene/ 1 protein
Almost all enzymes are proteins but not all proteins are
enzymes. Many proteins provide structural rather than
enzymatic roles.
For example polymers of the protein actin provide structural
integrity to the eukaryotic cell.
Perhaps the most notable example of this comes from studies
of Hemoglobin.
Hemoglobin is an iron carrying protein found in the red blood
cells and is responsible for transporting oxygen from the lungs
to the cells of the body.
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Hb
Hemoglobin consists of four polypeptides (proteins) each
associated with a specific Heme group (Heme is a small iron
containing molecule to which oxygen can attach) Adults contain 2
alpha polypeptides and 2 beta polypeptides
Alpha polypeptide = 141 amino acids
Beta polypeptide= 146 amino acids
Over 300 known hemoglobin variants are known and each is the
result of a specific mutation
Most of these are the result of a single amino acid
substitution
ｷ
Hb A:
ｷ
Hb S:
ｷ
Hb C:
These results demonstrate that:
1. Genes specify proteins that are not enzymes
2. Mutations can disrupt a single amino acid out of the many
that make up the protein.
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