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Lecture 9

One gene One enzyme hypothesis

In the next few lectures, the following questions will be Addressed:

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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.

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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.

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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.

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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!

To understand this disease we need an interdisciplinaryApproach.

What is the normal function of the Huntington gene?What happens in the mutant?Can it be blocked?

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Alkaptonuria

Degenerative disease. Darkening of connective tissue, arthritisDarkening 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 metabolismHomogentisic 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 individualsand was caused by lack of enzyme that splits aromatic rings of amino acids.

1958 La Du showed that accumulation of homogentistic acidis 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

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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

1 2 3 4 5 6 7 8 9 10

complete

minimal

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Conclusion- strain1

Strain1 and 7 are defective in either amino acid production or Vitamin production

Complete media(salt+sugar+

Vitamin + amino acids)

Minimal media(salt+sugar)

+ 20 amino acids

Minimal media(salt+sugar)+ vitamins

Minimal media(salt+sugar)

Conclusion:

Complete media(salt+sugar)

Vitamin + amino acids

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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(salt+sugar)

+ 20 amino acids

Minimal media(salt+sugar)+ vitamins

Minimal media(salt+sugar)

Conclusion:

complete media(salt+sugar)

Vitamin + amino acids

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Which amino acid?

Mutant7 is in a gene required for the production of Arginine.

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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

Instead of arginine, if they added ornithine or citrulline to the media, some mutants were rescued and others were not

Ornithine CitrullineArginine

Mutant1

Mutant2

Mutant3

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????

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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 Arg2- You need to supplement media with citrulline for the pathway to continue. Adding the precursor or ornithine does not help.

Mutant in Arg3-You 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

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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

CompoundE B N A

Mut1 - - + +

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

Genotype enzyme activityphenotype

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

Precursor------> product = phenotype

enzymeA

geneA

^|

^|

ｷ A allele produces functional enzyme

ｷ a allele produces nonfunctional enzyme

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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----> intermediate----> productyellow white blue

EnzA EnzB

Parental cross white x yellow

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AB Ab aB ab

AB

Ab

aB

ab

9 A-B- blue3A-bb white3aaB- yellow1aabb yellow

Precursor----> intermediate----> productyellow white blue

EnzA EnzB

F2

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Labradors

Parental Cross: black x yellow

BBEE bbee

BbEe (black)

x

BbEe (black)

Given the pathway show above, what phenotypic ratios would be

produced in progeny from the dihybrid cross: BbEe x BbEe

Yellow-------> brown--------> blackE B

EB Eb eB eb

EB

Eb

eB

eb

9:3:4

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Precursor----> intermediate----> productwhite white blue

EnzA EnzB

AB Ab aB ab

AB

Ab

aB

ab

9 A-B- blue3A-bb white3aaB- white1aabb white

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Biochemical Pathways and Linked Genes

The F1 is testcrossed

The following F2 progeny are produced:

50 yellow

40 blue

10 white

Precursor----> intermediate----> productyellow white blue

EnzC EnzD

GeneC GeneD

Parental C-D x c-dC-D c-d

F1

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Biochemical Pathways and Linked Genes

The following F2 progeny are produced:

50 yellow 40 blue 10 white

Precursor----> intermediate----> productyellow white blue

EnzC EnzD

GeneC GeneD

Parental C-D x c-dC-D c-d

F1 C-D c-dc-d x c-d

Parental C-D blue 40c-d

c-d yellow 40c-d

Recomb C-d white 10c-d

c-D yellow 10c-d

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.