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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
Blanco eye
Forked
bristle
Shaven body
White eye
yellow
chromosome
Many genes
Genes on
DNA
w1
w2
w3
Mutations in white
b1
b2
1
Mutations in blanco
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.
-
Ornithine
Citruline
Enzyme1
Arginine
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 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
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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
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White and Blanco-one or two genes?
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)
Enzyme1
Gene1
White
Product
(red pigment)
Enzyme2
Gene2
Blanco
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Easy way!!!
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:
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True breeding Blanco x true breeding white
The actual Cross
Cross
white (w)
x
blanco (b)
w/w
x
b/b
White
Female
Blanco
male
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If White and Blanco are the same gene ….
Precursor
(white)
Product
(red pigment)
Enzyme1
White= Blanco
wwww
(white eye)
x
wbwb
(white blanco eye)
F1
wwwb
Phenotype= ?????
In the F1 will the flies be red eyed or white eyed?
All white
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The cross
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
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Precursor
White
------->
enzymeA
product
red
White x white
aa
aa
F1 aa
White
What happens if you do a self cross with the F1
a
F2
a
aa
(white)
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If White and Blanco are two genes……
Precursor
(white)
Intermediate
(blanco)
Enzyme1
Product
(red pigment)
Enzyme2
Gene1
White
Gene2
Blanco
wwwwBB
(white eye)
x
WWbb
F1
Www Bb
phenotype= ?????
(blanco eye)
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
(white eye)
x
WWbb
F1
Www Bb
phenotype= RED
(blanco eye)
In the F1 what percentage of flies would be red eyed and
what percentage would be white eyed?
All red
ww
B
W
bb
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Gene interactions give 9:7
Precursor----> intermediate---->
white
white
EnzA
product
red
EnzB
White
aaBB
x
white
AAbb
F1
AaBb (phenotype= red)
What happens if you do a self cross with the F1
AB
F2
Ab
aB
ab
AB
AABB
AABb
AaBB
AaBb
Ab
AAbB
AAbb
AabB
Aabb
aB
ab
aABB
aAbB
aABb
aAbb
aaBB
aabB
aaBb
aabb
9 A-B3A-bb
3aaB1aabb
red
white
white
white
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Map Genes
singed
forked
Shaven body
blanco
white
yellow
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!
singed
Blanco
forked
Shaven body
white
yellow
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!
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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.
What if the w and b mutations disrupt the same gene?
w
b
w
b
w
F1
white=blanco
b
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+
W+
b
F1
W+
b
w
B+
If the F1 w/b flies are red-eyed, we know that the white
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and blanco mutations disrupt two genes.
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.
singed
forked
Shaven body
blanco
white
yellow
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
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Two genes
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)
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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
W+
b
forked
blanco
white
singed
B+
yellow
blanco
w
Shaven body
white
gene2
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Precursor
white
Product
red
Intermediate
white
Enzyme1
Enzyme2
gene1
gene2
OR
Precursor
white
Product
red
Intermediate
white
Enzyme1
gene2
Enzyme2
gene1
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Molecular basis of mutations
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.
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Mutant genes
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--20
Two White Genes
Lets make things more complicated.
w1 and w2 disrupt one gene (geneA).
w3 disrupt a second gene (geneB)
Precursor
white
Product
red
Intermediate
white
Enzyme1
Enzyme2
Gene A
Gene B
Aw1
Aw2
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
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Genes
Precursor
white
Product
red
Intermediate
white
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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Intermediate
white
Blanco
enzyme
Product
red
blanco
White
enzyme
white
Precursor
white
w1/w1 B/B
w2/w2 B/B
F1=
w1/w2 B/B
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Intermediate
white
Blanco
enzyme
Product
red
blanco
White
enzyme
white
Precursor
white
w1/w1 B/B
W/W b(w3)/b(w3)
F1=
W/w1 b(w3)/B
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Complementation analysis
Genotype
eye color
Aw1/Aw2
Aw1/Bw3
Aw2/Bw3
white
red
red
complementation
N
Y
Y
w1, w2 = geneA= complementation groupA (multiple alleles)
w3,= geneB= complementation groupB
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Suppose we isolate 5 delta wing mutations
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
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Complementation crosses
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
Mutation complement
Different genes
- is delta wing
mutation don’t complement
same gene
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Complementation crosses
You construct a complementation table
+ is flat wing
Mutation complement
Different genes
d1
d2
d3
d4
d5
d1
+
+
-
- is delta wing
mutation don’t complement
same gene
d2
+
+
-
d3
+
+
+
d4
+
+
+
d5
+
+
-
Gene1= alleles (d1, d2, d5)
Gene2= alleles (d3, d4)
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The pathway
Precursor
delta
Product
flat
Intermediate
delta
Enzyme1
Enzyme2
Gene1
Gene2
(allele d1, d2, d5)
(allele d3, d4)
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