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One gene One enzyme hypothesis

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

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One gene One enzyme hypothesis

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

What is the structure of a gene?

How does a gene function?

How is information stored on the gene?

What is the relationship between genotype and phenotype?

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PathwaysBiologists 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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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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Arginine biosynthetic mutants

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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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?

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

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

Precursor -----> ornithine -----> citrulline -----> arginine

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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

CompoundE B N A

Mut1

Mut2

Mut3

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What is the order of the compounds and mutations in the pathway?

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

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Biosynthetic pathways at the grocery store

Most of the red and blue colors found in higher plants are a result of pigments synthesized from one of two metabolic pathways, the carotenoid or the anthocyanin pathway.

Grocery store corn is usually yellow. Which step in the pathway must be mutated to produce yellow corn?

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The Flavor-Saver Tomato

Ethylene gas released by fruit accelerates the ripening process

Prevention of ethylene production would block the fruit from ripening prematurely and spoiling on the way to the market.

The ethylene biosynthetic pathway is as follows:

Precursor-----> ACC------> ethylene

Which step in the pathway must be mutated to prevent Ethylene production?

ACCsynthase

ACCoxidase

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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

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

The extremities are colder than the body, therefore the enzyme is active in the feet and produces color.

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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Dominance versus Recessive

The one gene/one enzyme concept explains a number of genetic phenomena

B) 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 (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 RatiosThe one gene/one enzyme concept explains a number of genetic phenomena

C) Genetic Ratios

The one gene/one enzyme hypothesis also explains phenotype ratios observed in a standard dihybrid cross:

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 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

What is the map distance between these two genes?

Map Distance+#Recombinants/Total Progeny x 100%

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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.