Lecture 9 One Gene One enzyme

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Lecture 9One Gene One enzyme

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Genes DNA sequences that code for RNA- prm, RBS, ORF, Term

---TTGACAT------TATAAT-------AT-/-AGGAGGT-/-ATG CCC CTT TTG TGA---AACTGTA------ATATTA-------TA-/-TCCTCCA-/-TAC GGG GAA AAC ATT

(-10)(-35)

PROMOTER

5’3’

3’ 5’

antisense

sense

RIBOSOMEBINDING

SITE

U-/-AGGAGGU-/-AUG CCC CUU UUG UGA

5’ 3’

Met Pro leu leu stp

Prokaryotic Genes

When ALL OF THESE RULES ARE SATISFIED THEN AND ONLY THEN WILL A PIECE OF DNA GENERATE A PROTEIN.EUKARYOTES ARE EVEN MORE COMPLICATED.

Is there a ribosome binding site upstream of the ATG

Is there a promoter upstream of the ribosome binding site

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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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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.

Formal genetics provides little information about these intermediary steps.

Since the 1970’s, a set of techniques have been developed that enable us to elucidate each step in a pathway

These techniques are generally placed under the rubric of Molecular Biology or Molecular Genetics

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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 interdisciplinary approach.

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

DNAGeneticistBioinformatics

RNAMolecular biologist

ProteinBiochemist

Protein complexBiochemist

Cellular phenotypeCell biologists

Organism phenotypePhysicians

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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.

1909 Garrod termed the defect- inborn error (genetic) 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.

Garrods results and his explanation were ignored

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

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The work of biochemists showed that chemical compounds in the cell are synthesized through a series of intermediates-a biochemical pathway

Ornithine Citruline Arginine

Enzyme1 Enzyme2

Glutamic acid-

How do you link genes to enzymes

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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:

1 Products are synthesized as a series of steps2 Each step is catalyzed by an unique enzyme3 Each enzyme is specified by a unique gene

The logic:

Precursor Int1 Int2 Product

EnzA EnzB EnzC

GeneA GeneB GeneC

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Consequences of mutations

Precursor Int1 Int2 Product

EnzA EnzB EnzC

GeneA GeneB GeneC

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(Red bread mold)

It had the advantage in that it could be grown on a defined growth medium.

Given salts like Na3 citrate,KH2PO4,NH4NO3,MgSO4,CaCl2

and sugars like sucrose

Neurospora can synthesize the amino acids, vitamins etc required and grow to form colonies on agar plates.

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Prototroph: a strain that

utilizes sugar, salt and water to grow.

Auxotroph: Mutant strain that

needs a specific amino acid or vitamin along with sugar, salt and water to grow.

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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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Logic of experiment

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.

Conclusion: Enzyme for making arginine was missing

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The method

To identify mutants

Transfer mutants to minimal media (water, sugar, salts)

Strain1 and 7 can grow on complete media but not minimal media.They have a mutation in a gene required for growth on minimal media!!!

Complete

All mutants grow

minimal

1 2 3 4 5 6 7 8 9 10

Irradiate spores.

Take mutant spores. Plate individual spores on complete media(sugar, salts and water, AND vitamins AND all 20 amino acids).

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Analogy

In the class:

There are two kinds of students:

Students who can climb treesStudents that cannot climb trees.

Under normal growth conditions when supermarkets are open, both kinds of students live happily

When supermarkets are closed

Students who can climb trees grow happily because they can climb trees and eat fruit

Students who cannot climb trees do not grow. They cannot climb trees, and go hungry.

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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


Conclusion: strain1 is defective in the production ofVitamins and the mutant is rescued by adding backvitamins

Take Strain 1

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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)


+ 20 amino acids

Minimal media(salt+sugar)+ vitamins


Conclusion: strain7 is defective in the production ofAmino acids and the mutant is rescued by adding backamino acids

Which of the 20 amino acids does strain7 fail to produce

complete media(salt+sugar)

Vitamin + amino acids

Take strain 7

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Which amino acid

Minimal media + vitamin + all 20 amino acidGrowthMinimal media + vitamin + lysineNo growthMinimal media + vitamin + glutamineNo growthMinimal media + vitamin + arginineGrowthMutant7 is in a gene required for the production of Arginine.

Beadle and Tatum found that three mutants could not produce arginine

Arg1 Arg2 Arg3

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

enz1 enz2 enz3

The biochemical pathway for arginine synthesis was kind of known. Ornithine and citrulline are closely related to arginine and were thought to be precursorsThe pathway for arginine biosynthesis is :

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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


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 mutant gene codes for which enzyme????

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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 - -+


enz1 enz2 enz3

Arg1 Arg2 Arg3

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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.

The Results Also show that THREE different Genes/enzymes are necessary for ONE phenotype- synthesis of ARG!

This would affect phenotype ratios in a cross

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Analogy

UCSC

Walnut Laurel Bay

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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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CompoundE B N A

Mut3 + - + +

Mut1 - - + +

Mut2 - - + -

B----> E----> A----> N

mut3 mut1 mut2

CompoundB E A N

Mut3 - + + +

Mut1 - - + +

Mut2 - - - +

CompoundE B N A

Mut1 - - + +

Mut2 - - + -

Mut3 + - + +

Rearrange the mutants

Rearrange the compounds

Another example

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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----> S-----> A----> N

This process might also identify multiple mutants for the steps in the pathway!

B----> E----> A----> N

Mut3mut5

Mut1mut4

Mut2

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This rationale currently is being used in many laboratories to elucidate more complex pathways in multicellular organisms

ｷ Development- formation of the body axis

ｷ Behavior- courtship and mating

ｷ Biological clocks

ｷ Aging

ｷ Cell cycle and Cancer

Review

ｷ Biochemical processes occur as a series of discrete stepwise reactions

ｷ Each reaction is catalyzed by a single enzyme

ｷ Each enzyme is specified by a unique gene

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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Temperature-sensitive mutations

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

Temperature-sensitive mutations

Some mutations exhibit a phenotype at one temperatures (the restrictive temperature) but function normally at another temperature (permissive temperature).

Reasons: Slight destabilization/alteration of the 3D conformation of the enzyme or its ability to interact with other proteins

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.

Heat sensitive mutants

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Cold sensitive

Protein is functional at high temp and inactive at low tempActive at 30C but inactive at 15C

Temperature sensitiveProtein is functional at low temperature but inactive at high temperatureActive at 23C but inactive at 32C

K253E

Cs

Interacts very stably with RFC

C752T

Ts

Mis folding

PCNA

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An example of a Ts mutation:

Dogs and cats that are white with black feet or vice versa

The genes for coat color are normal at one temperatures but are inactive at another temperatures

One of the genes for coat color is Albino - in cats

This gene affects melanin production. The normal or dominant form, C, is 'full color'. Various mutant alleles. These mutants are temperature sensitive -

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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 is temperature sensitive.

c is the most recessive form, also known as albino. In the homozygote cc this causes complete absence of any pigment and white fur.

Cb is incompletely dominant over Cs; the heterozygote (Cb/Cs) gives a phenotype intermediate between Burmese and Siamese, known as Tonkinese.

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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.

The biosynthetic pathway for corn kernel color is as follows:

Precursor-----> Chalcone ----> Flavanone ---->Anthocyanins

(white) (yellow) (white) (blue)

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

Beadle/Tatum Results Also show that THREE different Genes/enzymes are necessary for ONE phenotype- synthesis of ARG!

Similarly for blue corn multiple genes/enzymes are required.

Let’s see how this would affect phenotype ratios in a cross

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Mutants and Genetic pathways

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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Multiple genes affecting a single phenotype


ｷ A, B = normal alleles

ｷ a, b= nonfunctional mutant alleles

Parental cross: AAbb x aaBB

white yellow

F1 AaBb (blue)

x

AaBb (blue)

F2

EnzA EnzB

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Multiple genes affecting a single phenotype

AB Ab aB ab

AB

Ab

aB

ab

AABB AABb AaBB AaBb

AAbB AabB

aABB

aAbB

aABb

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

Precursor----> intermediate----> productyellow white blueEnzA EnzB

F2

AAbb Aabb

aaBB aaBb

aAbb aabBaabb

4:3:9Y:W:B

Parental cross: AAbb x aaBB

white yellow

F1 AaBb (blue) x AaBb (blue)

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Labradors recessive Epistasis give 9:4:3 ratio

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

EEBB EEBb EeBB EeBb

EEBb EeBb

EeBB

EeBb

EeBb

EEbb Eebb

Eebb eebb

eeBB eeBb

eeBb

4:3:9Y:Br:Bl

Recessive epistasisHomozygous ee gene alleles mask effect of B gene allelese is epistatic to B

Epistasis= When the Alleles of One Gene Mask the Expression of Alleles of a Second Gene

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Gene interactions give 9:7

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

EnzA EnzB

AB Ab aB ab

AB

Ab

aB

ab

AABB AABb AaBB AaBb

AAbB AabB

aABB

aAbB

aABb

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

AAbb Aabb

aaBB aaBb

aAbb aabBaabb

AaBb x AaBb

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Enz V+

Precursor -----Brown pigment \(white) \ transporter W+

--------- Red

/Precursor ----- Vermilion pigment /(white) Enz B+

WT -- Brown

WT -- Vermilion

WT -- White

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Gene Interaction: A range of Phenotypes Arise From Combined Action of Alleles of Two Genes

The 9:3:3:1 ratio in the F2 suggests two genes control coat color.

Dominant Epistasis give 13:3 ratio or 12:3:1 ratio

In white leghorn and white wyandotte chickens, a dominant B allele masks color production associated with the dominant A allele of a second gene.

Altered 9:3:3:1 ratios are a hallmark of epistasis involving two genes.

Dominant allele of one gene hides effects of both alleles of second gene= 12:3:1 ratioDominant allele of one gene hides effect of dominant allele of second gene = 13:3 ratio

B/b is the epistatic gene. Any chicken with a dominant B in their genome will have white feathers. Being homozygous recessive bb at this locus enables the expression of genes coded for at the hypostatic locus (A).At the hypostatic locus A/a the dominant allele A codes for colored feathers while the recessive a codes for no color. Hence, a chicken that is homozygous recessive aa will also be white giving you a 13:3 ratio

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Summer squash color

GeneW determines pigment productionW No pigmentw Pigment

GeneY determines color of pigmentY yellowy green

9 W-Y- white3 W-yy white3 wwY- yellow1 wwyy green

Multiple genes regulate a single phenotype

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Pepper Color

Gene 1: R=red r=yellow

Gene 2: Y=absence of chlorophyll (no green)

y=presence of chlorophyll (green)

Possible genotypes:

R-/Y- : red (red/white no chlorophyll) R-/yy : brown/orange (red/green chlorophyll) rr/Y- : yellow (yellow/white no chlorophyll) rr/yy : green (yellow/green chlorophyll)

Two genes affect Chicken Combs

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4 different chicken comb phenotypes result:

Rose Combs (R-pp) Walnut Combs (R-P-) Pea Combs (rrP-) Single Combs (rrpp)

Multi-gene traits

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Many human traits, such as height, skin color etc are determined by multiple genes. Multigenic ("many gene") traits exhibit a mode of inheritance that would have surprised Gregor Mendel. Complex interactions between genes.

Number of traits result from mutations in single genes- MONOgenic trait.

Multigenic diseases result from less severe mutations in more than one gene. Any of these mutations alone might not affect a trait, but together, they can lead to significant phenotypic differences.

Interaction among the phenotypic effects of different genes, called epistasis, adds a layer of complexity to the study of genetic disease.Genes don't function alone; rather, they constantly interact with one another. These gene-gene interactions result in an output phenotype. Certain genes are known to modify the phenotype of other genes.This implies that multiple genes may interact to increase or decrease disease susceptibility. If the effect of the disease-bearing gene is masked or altered by the effects of a second gene (by say altering expression level of the disease bearing gene), then identifying the first gene can be complicated. In addition, if more than one genetic interaction occurs to cause a disease, then identifying the multiple genes involved and defining their relationships becomes even more difficult.

Alzheimers

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Alzheimer's disease, is a progressive neurodegenerative disorder that causes memory loss and dementia.A gene called apolipoprotein E4 was associated with a higher risk of developing Alzheimer's. While having one or two copies of mutant apolipoprotein E4 increase one's risk of Alzheimer's, not all carriers of apolipoprotein E4 develop the disease. This suggested that gene-gene interactions were involved.They confirmed 27 different genetic interactions in 4 different biochemical pathways: cholesterol metabolism, beta-amyloid production, inflammation, oxidative stress. Some interactions were synergistic, while others were antagonistic. The synergistic interactions indicate that the pair of involved genes together increase the risk of Alzheimer’s. The strongest interactions involved the pairing of apolipoprotein E4 mutation with mutations in three different genes: alpha(1)-antichymotrypsin, β-secretase, and butyrylcholinesterase K. These genes are not acting alone, but in a pathway that affect one another.

The APP gene produces a transmembrane protein that is modified, cleaved by secretase and then inserted into membranes. Function of APP is not fully known- could be membrane or Ca+ trafficking in neurons. APP interacts with chymotrypisn and Apolipoprotein. Alterations in these interactions lead to formation of APP plaques (amyloid fibers) leading to Alzheimers. ApoE4 mutation leads to alterations in cholesterol in membranes. The ApoE4 allele causes greater aggregation of APP and greater amyloid deposition.

Four genes for hair color

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Hair Color Hair color is controlled by multiple genes on

chromosomes 3, 6, 10, and 18. The more dominant alleles that appear in the

genotype, the darker the hair!

Multiple genes affect a single phenotype- additive effects

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Additive Gene Interaction for Continuous Variation

Continuously varying traits are also called quantitative traits.

Additive Gene Interaction Model for Continuous Variation

Continuously varying traits are also called quantitative traits.

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The height of plants is controlled by 4 pairs of alleles. Alleles A, B, and C contribute 3 cm to the plant's height. Alleles that are recessive do not contribute to the height. In addition Gene L is always found in a homozygous dominant condition and always contributes 40 cm to the height.

a) What would be the height of a plant with the genotype AABBCCLL? 3+3+3+3+3+3+40

b) What would be the height of a plant with a genotype aabbccLL? 0+0+0+0+0+0+40

c) What would be the height of the offspring produced from a cross between the plants in a) and b)? AaBbCcLL 3+0+3+0+3+0+40

d) What would be the heights of the offspring produced from a cross between AaBbCcLL and AaBbCcLL?

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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


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


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.

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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: val his leu thr pro *glu *glu

ｷ Hb S: val his leu thr pro *val* glu

ｷ Hb C: val his leu thr pro *lys* glu

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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Lecture 9 One Gene One enzyme