Lecture 10 what is a gene
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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.

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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

Lecture 10: What is a gene?

chromosome

Forked

bristle

Many genes

yellow

Blanco eye

Shaven body

White eye

Genes on

DNA

b1

w1

w2

b2

w3

Mutations in white

Mutations in blanco


Complementation

Ornithine

Citruline

Arginine

Enzyme1

Enzyme2

Complementation

The 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.

Glutamic acid-

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 arginine

Mutant2+citruline=cell cant make arginine- mutant

It is not often this easy.

The wildtype eye color in flies is red

Say two different laboratories isolated mutants in that had white eyes. You cant feed flies eye color precursor to figure things out!


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

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

Mut3

Mut1

mut4

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

mut4

Mut2


White and blanco one or two genes
White and Blanco-one or two genes? steps in the pathway!

QUESTION

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

Precursor

(white)

Product

(red pigment)

Enzyme1

Gene1

Blanco=White????

OR

Precursor

(white)

Intermediate

(white)

Product

(red pigment)

Enzyme1

Enzyme2

Gene1

White

Gene2

Blanco


Easy way
Easy way!!! steps in the pathway!

There is an easier way

QUESTION: IS BLANCO THE SAME GENE AS WHITE or

IS BLANCO AND WHITE TWO DIFFERENT GENES

The following cross is performed:

True breeding Blanco x true breeding white


The actual cross
The actual Cross steps in the pathway!

Cross

white (w) x blanco (b)

White Blanco

Female male

w/w x b/b


If white and blanco are the same gene
If White and Blanco are the same gene …. steps in the pathway!

Precursor

(white)

Product

(red pigment)

Enzyme1

White= Blanco

wwww x wbwb

(white eye) (white blanco eye)

F1 wwwb

Phenotype= ?????

In the F1 will the flies be red eyed or white eyed? All white


The cross
The cross steps in the pathway!

WHITE EYE

What are genotypes and phenotypes of the cross

ww x bb

b

w

w b

(?)

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

?White or Red????

ww

wb


Precursor -------> product steps in the pathway!

White enzymeA red

White x white

aa aa

F1 aa

White

What happens if you do a self cross with the F1

a

a

aa

(white)

F2


If white and blanco are two genes
If White and Blanco are two genes…… steps in the pathway!

Precursor

(white)

Intermediate

(blanco)

Product

(red pigment)

Enzyme1

Enzyme2

Gene1

White

Gene2

Blanco

wwwwBBxWWbb

(white eye) (blanco eye)

F1 Www Bb

phenotype= ?????

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


w steps in the pathway!wwwBBxWWbb

(white eye) (blanco eye)

F1 Www Bb

phenotype= RED

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

ww

B

bb

W


Gene interactions give 9 7

aB steps in the pathway!

Ab

ab

AB

AaBb

AB

AABB

AABb

AaBB

AAbB

AabB

Ab

aABb

aABB

aB

ab

aAbB

Gene interactions give 9:7

Precursor----> intermediate----> product

white white red

EnzA

EnzB

White x white

aaBB AAbb

F1 AaBb (phenotype= red)

What happens if you do a self cross with the F1

AAbb

Aabb

F2

aaBb

aaBB

aabB

aAbb

aabb

9 A-B- red

3A-bb white

3aaB- white

1aabb white


Map genes

singed steps in the pathway!

forked

yellow

Shaven body

blanco

white

singed

forked

yellow

Shaven body

Blanco

white

Map 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!

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.

Mapping genes takes lots of crosses and is time consuming

There is an easier way!


How many genes

W+ steps in the pathway!

b

w

W+

b

w

b

B+

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.

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

w

b

w

b

F1

white=blanco

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

What if w and b mutations disrupt two genes?

W+

b

w

B+

w

B+

F1

If the F1 w/b flies are red-eyed, we know that the white

and blanco mutations disrupt two genes.


Single gene

singed steps in the pathway!

forked

yellow

Shaven body

blanco

white

Single gene

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.

If there is a single gene then

****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


Two genes
Two genes steps in the pathway!

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

Say in the complementation test you get red eyed females

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)


Two genes1

W+ steps in the pathway!

w

b

B+

white

singed

forked

yellow

Shaven body

blanco

blanco

white

Two genes

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

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)

Precursor

white

Product

red

Intermediate

white

Enzyme1

Enzyme2

gene1

gene2


Precursor steps in the pathway!

white

Product

red

Intermediate

white

Enzyme1

Enzyme2

gene1

gene2

OR

Precursor

white

Product

red

Intermediate

white

Enzyme1

Enzyme2

gene2

gene1


Molecular basis of mutations
Molecular basis of mutations steps in the pathway!

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

ATGCCCCCCCCCCCCCCCCCCCTAA = GeneW = proteinW

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


Mutant genes
Mutant genes steps in the pathway!

Nucleotide sequence of the normal W gene:

---ATGCCCCCCCCCCCCCCCCCCCCCCCCCCCCCCTAA---

---TACGGGGGGGGGGGGGGGGGGGGGGGGGGGGGGATT---

The sequence of the w1 mutation of gene W

---ATGCCCCCCCCCCCCCCCCCCCCCCCCCCTCCCTAA---

---TACGGGGGGGGGGGGGGGGGGGGGGGGGGAGGGATT---

The sequence of the w2 mutation of gene W

---ATGCCCCACCCCCCCCCCCCCCCCCCCCCCCCCTAA---

---TACGGGGTGGGGGGGGGGGGGGGGGGGGGGGGGATT---

So at the molecular level, the w1/w2 white female fly

would be depicted as:

---ATGCCCCCCCCCCCCCCCCCCCCCCCCCCTCCCTAA---

---TACGGGGGGGGGGGGGGGGGGGGGGGGGGAGGGATT---

---ATGCCCCACCCCCCCCCCCCCCCCCCCCCCCCCTAA---

---TACGGGGTGGGGGGGGGGGGGGGGGGGGGGGGGATT---


Two white genes

Precursor steps in the pathway!

white

Product

red

Intermediate

white

Enzyme1

Enzyme2

Gene B

Bw3

Gene A

Aw1

Aw2

Two White Genes

Lets make things more complicated.

w1 and w2 disrupt one gene (geneA).

w3 disrupt a second gene (geneB)

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


Genes

Precursor steps in the pathway!

white

Product

red

Intermediate

white

Enzyme1

Enzyme2

Gene B

Gene A

Genes

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?


white steps in the pathway!

blanco

Precursor

white

Product

red

Intermediate

white

White

enzyme

Blanco

enzyme

w1/w1 B/B

w2/w2 B/B

w1/w2 B/B

F1=


white steps in the pathway!

blanco

Precursor

white

Product

red

Intermediate

white

White

enzyme

Blanco

enzyme

w1/w1 B/B

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

W/w1 b(w3)/B

F1=


Complementation analysis
Complementation analysis steps in the pathway!

Genotype eye color complementation

Aw1/Aw2 white N

Aw1/Bw3 red Y

Aw2/Bw3 red Y

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

w3,= geneB= complementation groupB


Suppose we isolate 5 delta wing mutations steps in the pathway!

d1

d2

d3

d4

d5

We want to know how many genes are disrupted in

these mutations and which mutations are in the

same complementation group


Complementation crosses
Complementation crosses steps in the pathway!

We systematically perform crosses

First 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 perform

d1/d1 x d3/d3

F1 d1/d3

wing=flat or delta

You construct a complementation table

+ flat wing - is delta wing

Mutation complement mutation don’t complement

Different genes same gene


Complementation crosses1
Complementation crosses steps in the pathway!

You construct a complementation table

+ is flat wing - is delta wing

Mutation complement mutation don’t complement

Different genes same gene

d1 d2 d3 d4 d5

d1 - - + + -

d2 - - + + -

d3 ++ - - +

d4 + + - - +

d5 - - + + -

Gene1= alleles (d1, d2, d5)

Gene2= alleles (d3, d4)


The pathway
The pathway steps in the pathway!

Precursor

delta

Product

flat

Intermediate

delta

Enzyme1

Enzyme2

Gene2

(allele d3, d4)

Gene1

(allele d1, d2, d5)


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