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Lecture 10: What is a gene?

Each chromosome has one DNA molecule

Each chromosome has many genes

A gene produces a protein that give rise to a phenotype

A gene has many forms- alleles

Different alleles are caused by different changes in the same gene

Mutations in different genes CAN give you the same phenotype

chromosome

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nco e

ye

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b

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Wh

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ye Many genes

Genes on DNA

w1 w2 w3

Mutations in white

b1 b2

Mutations in blanco

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ComplementationThe complementation test is a rapid method of determining whether two independently isolated mutants with the same phenotype (in the same pathway) are in one or two (or more) genes.

Ornithine Citruline Arginine

Enzyme1 Enzyme2

Both mutant1 and mutant2 cannot make arginine.If you did not know the pathway you would wonder if these two mutants were mutations in the same gene or mutations in two different genes

If you are working with Neurospora, you can feed the intermediate (Citruline) to the mutants and see if they can now make arginine.You are “complementing” the mutants with intermediates

Mutant1+ citruline=cell makes arginineMutant2+citruline=cell cant make arginine- mutant

It is not often this easy. The wildtype eye color in flies is redSay two different laboratories isolated mutants in that had white eyes. You cant feed flies eye color precursor to figure things out!

Glutamic acid-

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Side bar: Naming mutants

The wildtype eye color in flies is redSay two mutants are ISOLATED BY TWO DIFFERENT LABS Mutant flies have white eyes.

The researcher who identified the first white eyed mutant lived in the US and named it white.Small case w designates the recessive mutant allele Upper case W designates normal (dominant) wildtype

The researcher who identified the second mutant lived in Spain and named it blanco. Small case b designates recessive mutant allele Upper case B designates normal (dominant) wildtype allele

The researcher who isolates the mutant names it!!!!!

CheapdateCockeyeking tubbySevenlessBride of sevenlessDaughter of sevenless

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

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

Mut3 Mut1mut4

Mut2

mut1 and mut4 might be two different mutations in the same gene.

OR

Mutations in two different genes that work in the same pathway

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

Mut3 Mut1 Mut2mut4

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White and Blanco

Precursor(white)

Intermediate(white)

Product(red pigment)

Enzyme1 Enzyme2

Gene1White

Gene2Blanco

Precursor(white)

Product(red pigment)

Enzyme1

Gene1Blanco=White????

OR

QUESTIONAre the two independently isolated mutations THAT HAVE THE SAME WHITE EYE PHENOTYPE disrupting the same or different genes?

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White gene

Mapping genes takes lots of crosses and is time consumingThere is an easier way!

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If on the other hand the two mutations map to different regions of the chromosome (or different chromosomes) then that would indicate that they are two different genes.

You could map each mutation. If Blanco = white then the two mutations WILL map to the same spot on the chromosome. That would indicate that they are the SAME GENE -two different alleles!

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Easy way!!!

There is an easier way

QUESTION: IS BLANCO THE SAME GENE AS WHITE orIS BLANCO AND WHITE TWO DIFFERENT GENES

The following cross is performed:

True breeding Blanco x true breeding white

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The actual Cross

Cross white (w) x blanco (b)White BlancoFemale male

w/w x b/b

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If White and Blanco are the same gene ….

Precursor(white)

Product(red pigment)

Enzyme1

White= Blanco

yellow

fork

ed

wh

ite

bla

nco

wwww x wbwb

(white eye) (white blanco eye)

F1 wwwb

Phenotype= ?????

In the F1 what percentage of flies would be red eyed and what percentage would be white eyed? All white

ww

wb

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wwww x wbwb

(white eye) (white blanco eye)

F1 wwwb

Phenotype= ?????

In the F1 what percentage of flies would be red eyed and what percentage would be white eyed? All white

ww

wb

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If White and Blanco are two genes……

Precursor(white)

Intermediate(blanco)

Product(red pigment)

Enzyme1 Enzyme2

Gene1White

Gene2Blanco

yellow

bla

nco

fork

ed

wh

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wwwwBB x WWbb(white eye) (blanco eye)

F1 Www Bbphenotype= ?????

In the F1 what percentage of flies would be red eyed and what percentage would be white eyed? All red

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wwwwBB x WWbb(white eye) (blanco eye)

F1 Www Bbphenotype= ?????

In the F1 what percentage of flies would be red eyed and what percentage would be white eyed? All red

ww

bbW

B

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The cross

b

w w b(?)

What is the eye-color of the w/b fly?

?White or Red????

WHITE EYE

What are genotypes and phenotypes of the cross

ww x bb

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Precursor -------> productWhite enzymeA red

White x whiteaa aa

F1 aaWhite

What happens if you do a self cross with the F1

a

a aa(white)

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Gene interactions give 9:7

Precursor---->intermediate----> productwhite white red

EnzA EnzB

AB Ab aB ab

AB

Ab

aB

ab

AABB AABb AaBB AaBb

AAbB AabB

aABB

aAbB

aABb

9 A-B- red3A-bb white3aaB- white1aabb white

AAbb Aabb

aaBB aaBb

aAbb aabB aabb

White x whiteaaBB AAbb

F1 AaBb (phenotype= red)

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How many genes?

The answer to the question (What is the eye-color of the w/b fly?) depends on whether the w and b mutations disrupt the same gene or two different genes.

If the F1 w/b flies are red-eyed, we know that the whiteand blanco mutations disrupt two genes.

What if the w and b mutations disrupt the same gene?

white=blancow

b

w

w

b

b

W+ b

w B+F1

w B+

w B+

W+ b

W+ b

What if w and b mutations disrupt two genes?

If the F1 w/b flies are white-eyed, we know that the white and blanco mutations disrupt one gene.

F1

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If the F1 flies are white-eyed, the mutations disrupt the same gene. A geneticist would say the two mutations do not complement one another because normal function is not restored.

Single gene

If there is a single gene then

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****Nomenclature**** If the researchers discover that blanco (b) and white (w) are mutations within the same gene, there is a problem. The same gene has two names. One would like to have names that indicate that these are two alleles of the same gene.

These are renamed

White becomes w1

Blanco becomes w2 or wb

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a

a aa

Precursor----> productWhite Red

EnzA

P aa x aa

F1 aa

F2

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Two genes

Say in the complementation test you get red eyed femalesThere are two genes --- W and B.

If there are two genes then:How do these two genes relate to one another?(one gene one enzyme)

If on the other hand the w/b F1 flies are red-eyed, we know that the white and blanco mutations disrupt two genes.

Geneticist would say that these two mutations complement one another. They complement because normal function is restored

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Two genes

There are two genes --- W and B.

If there are two genes then:How do these two genes relate to one another(one gene one enzyme)

Precursorwhite

Intermediatewhite

Productred

Enzyme1 Enzyme2

gene1 gene2

white

blanco

w B+

W+ b

If the F1 w/b females are red-eyed, then white and blanco mutations disrupt two genes. You say that these two mutations complement one another. They complement because normal function is restored

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Gene interactions give 9:7

Precursor---->intermediate----> productwhite white red

EnzA EnzB

AB Ab aB ab

AB

Ab

aB

ab

AABB AABb AaBB AaBb

AAbB AabB

aABB

aAbB

aABb

9 A-B- red3A-bb white3aaB- white1aabb white

AAbb Aabb

aaBB aaBb

aAbb aabB aabb

White x whiteaaBB AAbb

F1 AaBb (phenotype= red)

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Enzyme1 Enzyme2

Precursorwhite

Intermediatewhite

Productred

gene1 gene2

Enzyme1 Enzyme2

Precursorwhite

Intermediatewhite

Productred

gene2 gene1

OR

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Now what is the molecular basis for two mutations within the same gene?

Lets say that w1 and w2 both disrupt geneW

What is a gene?

Molecular basis of mutations

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Now what is the molecular basis for two mutations within the same gene?

Lets say that w1 and w2 both disrupt geneW

What is a gene?

It’s a piece of DNA with a specific sequence

DNA consists of a linear array of the four nucleotides

Adenine- Cytosine- Guanine- Thymine

ACGT

Specific DNA sequence = gene = protein

CCCCCCCCCCCCCCCCCCC = GeneW = proteinW

The nucleotides in the sequence of the gene is critical for its proper function.

Molecular basis of mutations

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Mutant genes

Nucleotide sequence of the normal W gene:

---CCCCCCCCCCCCCCCCCCCCCCCCCCCCCC------GGGGGGGGGGGGGGGGGGGGGGGGGGGGGG---

The sequence of the w1 mutation of gene W

---CCCCCCCCCCCCCCCCCCCCCCCCCCTCCC------GGGGGGGGGGGGGGGGGGGGGGGGGGAGGG---

The sequence of the w2 mutation of gene W

---CCCCGCCCCCCCCCCCCCCCCCCCCCCCCC------GGGGCGGGGGGGGGGGGGGGGGGGGGGGGG---

So at the molecular level, the w1/w2 white female flywould be depicted as:

w1:---CCCCCCCCCCCCCCCCCCCCCCCCCCTCCC------GGGGGGGGGGGGGGGGGGGGGGGGGGAGGG---

w2:---CCCCGCCCCCCCCCCCCCCCCCCCCCCCCC------GGGGCGGGGGGGGGGGGGGGGGGGGGGGGG---

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Two White Genes

Precursorwhite

Intermediatewhite

Productred

Enzyme1 Enzyme2

Gene A

Aw1

Aw2

Gene B

Bw3

Disruptions in geneA (Aw1 and Aw2) and geneB (Bw3) give rise to white eyes.

HOW DO YOU FIGURE OUT THAT w1 and w2 disrupt gene A and w3 disrupts geneB

Lets make things more complicated.w1 and w2 disrupt one gene (geneA). w3 disrupt a second gene (geneB)

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wh

ite

bla

nco

Whiteenzyme

Blancoenzyme

Precursorwhite

Intermediatewhite

Productred

w1/w1 B/B

w2/w2 B/B

F1= w1/w2 B/B

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wh

ite

bla

nco

Whiteenzyme

Blancoenzyme

Precursorwhite

Intermediatewhite

Productred

w1/w1 B/B

W/W b(w3)/b(w3)

F1= W/w1 b(w3)/B

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Genes

Precursorwhite

Intermediatewhite

Productred

Enzyme1 Enzyme2

Gene A Gene B

Disruptions in geneA and geneB both give rise to white eyes.

Cross a Aw1 fly with a Aw2 fly and see if you get red eyes.

If w1 and w2 disrupt geneA, they will/will not complement .

What about Bw3?

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Complementation analysis

Genotype eye color complementation

Aw1/Aw2 white NAw1/Bw3 red YAw2/Bw3 red Y

w1, w2 = geneA= complementation groupA (multiple alleles)

w3,= geneB= complementation groupB

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Suppose we isolate 5 delta wing mutations

d1d2d3d4d5

We want to know how many genes are disrupted in these mutations and which mutations are in the same complementation group

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Complementation crosses

We systematically perform crossesFirst we perform the cross

d1/d1 x d2/d2

F1 d1/d2 are produced

wing= flat or delta

If they are flat, they disrupt -------- gene

Then we performd1/d1 x d3/d3F1 d1/d3wing=flat or delta

You construct a complementation table

+ flat wing - is delta wingMutation complement mutation don’t complementDifferent genes same gene

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Complementation crosses

You construct a complementation table

+ is flat wing - is delta wingMutation complement mutation don’t complementDifferent genes same gene

Gene1= alleles (d1, d2, d5)Gene2= alleles (d3, d4)

d1 d2 d3 d4 d5d1 - - + + -d2 - - + + -d3 + + - - +d4 + + - - +d5 - - + + -

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The pathway

Enzyme1 Enzyme2

Precursordelta

Intermediatedelta

Productflat

Gene1

(allele d1, d2, d5)

Gene2

(allele d3, d4)

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