Download The white gene

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 mutations in the same gene
Mutations in different genes can give you the same phenotype
blanco
Forked
bristle
Shaven body
white
yellow
chromosome
Many genes
Genes on
DNA
w1
w2
w3
Mutations in a gene
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Complementation
The complementation test is a rapid method of determining
whether two independently isolated mutants with
the same phenotype are in the same or different genes.
Ornithine
Citruline
Enzyme1
Arginine
Enzyme2
Both mutant1 and mutant2 cannot make arginine.
Are these two different 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.
Mutant1+ citruline=arginine
Mutant2+citruline=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
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to figure things out!
Naming mutants
It is not often this easy.
The wildtype eye color in flies is red
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!!!!!
Cheapdate
Cockeye
king tubby
Sevenless
Bride of sevenless
Daughter of sevenless
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White and Blanco
QUESTION
Are the two independently isolated mutations 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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White gene
You could map each mutation. If the two mutations map to
The same regions of the X-chromosome then that would
indicate that they are the SAME GENE
If the two mutations map to different regions of the Xchromosome 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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Easy way!!!
There is an easier way
The following cross is performed:
QUESTION: IS BLANCO THE SAME GENE AS WHITE
or
TWO DIFFERENT GENES
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True breeding Blanco x true breeding white
Mono and Di hybrid Crosses review
ww
x
(white)
F1
WW
(red)
Ww
(red)
ww
x
(white)
F1
ww
(white)
ww
(white)
-------------------------------------------wwBB x
(white)
(Normal)
F1
WWbb
(red)
(forked)
WwBb
(red eye)
(normal bristle)
wwbb x
(white)
(forked)
wwbb
(white)
(forked)
wwbb
(white eye)
(forked bristle)
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The actual Cross
Cross
white (w)
x
blanco (b)
w/w
x
b/b
White
Female
Blanco
male
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White, Blanco, same gene?
Precursor
(white)
Product
(red pigment)
Enzyme1
forked
blanco
white
yellow
Gene1
White=
Blanco
wwww
(white eye)
x
F1
wwwb
?????
wbwb
(white blanco eye)
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In a self cross what percentage of flies would be red eyed
and what percentage would be white eyed? All white
White, Blanco, two genes?
Precursor
(white)
Intermediate
(blanco)
Enzyme1
Enzyme2
wwwwBB
(white eye)
x
blanco
forked
Gene2
Blanco
white
yellow
Gene1
White
F1
Product
(red pigment)
WWbb
(blanco eye)
wwWbB
?????
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In a self cross what percentage of flies would be red eyed
and what percentage would be white eyed? 9red:7white
The cross
WHITE EYE IS X-LINKED
What are genotypes and phenotypes of the cross
ww
x
bY
Y
b
w
F1
w
What is the eye color of the w/Y males
What is the eye-color of the w/b females?
?
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How many genes?
The answer to the second question depends on whether
the w and b mutations disrupt the same gene or
different genes
What if the w and b mutations disrupt the same gene?
What if w and b mutations disrupt different genes?
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How many genes?
The answer to the second question depends on whether
the w and b mutations disrupt the same gene or
different genes
What if the w and b mutations disrupt the same gene?
w
b
w
b
w
white=blanco
b
If the w/b females 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 w/b females are red-eyed, we know that the white
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and blanco mutations disrupt two genes.
Single gene
If the females 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.
What are two mutations in the same gene called???
(allele)
There is a problem. The same gene has two names.
These are then renamed
White becomes w1
Blanco becomes
w2 or wb
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Two genes
If the w/b females 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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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?
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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
---CCCCACCCCCCCCCCCCCCCCCCCCCCCCC-----GGGGTGGGGGGGGGGGGGGGGGGGGGGGGG---
So at the molecular level, the w1/w2 white female fly
would be depicted as:
w1:
---CCCCCCCCCCCCCCCCCCCCCCCCCCTCCC-----GGGGGGGGGGGGGGGGGGGGGGGGGGAGGG--w2:
---CCCCACCCCCCCCCCCCCCCCCCCCCCCCC-----GGGGTGGGGGGGGGGGGGGGGGGGGGGGGG--17
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
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
Lets make things more complicated
w1 and w2 disrupt one gene (A). w3 disrupts the second gene
(B).
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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Complementation analysis
Genotype
eye color
phenotype
complementation
Aw1/Aw2
Aw1/Bw3
Aw2/Bw3
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Suppose we isolate 5 curly wing mutations
c1
c2
c3
c4
c5
We want to know how many genes are disrupted by
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
c1/c1 x
c2/c2
F1
c1/c2 are produced
wing= flat or curly
If they are flat, they disrupt -------- gene
Then we perform
c1/c1 x
c3/c3
F1
c1/c3
wing=flat or curly
You construct a complementation table
+ flat wing
Mutation complement
Different genes
c1
c2
c3
c4
c5
c1
- is curly wing
mutation don’t complement
same gene
c2
c3
c4
c5
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Intragenic recombination
Up until this point, genes have been viewed as a linear array
Of indivisible functional units on a chromosome
The tenets of the model are
1
Genes are fundamental units of function
Parts of a gene cannot function
2
Genes are fundamental units of mutation
The gene changes as a whole from one form to
another
3
Genes are fundamental units of structure
The gene is indivisible by recombination
We will go through experiments that show that
recombination does occur within a gene
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Recombination and genes
If recombination occurs within a gene,
how do you detect it?
How do we detect recombination between
two different genes?
Drosophila: Two X-linked genes
forked (f) is a recessive mutation that gives rise to
forked bristles
F=normal bristles
f=forked bristles
carnation is a recessive mutation that alters the
normal bright Red-eyes to a dull red color
carnation
Forked bristles
C=normal red eyes
c=dull carnation eyes
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Recombination
How do you detect recombination between these genes?
f
c
F C
f c
F
f
x
F C
Y
C
c
(X-linked)
F
C
Y
The presence of individuals with recombinant phenotypes
indicates that recombination has occurred between these
two genes
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What about recombination within a gene
Does recombination occur within a gene?
Is there something special about a gene that makes it
indivisible by recombination
If recombination occurs within a gene how
would you detect it?
Recombination rates are a function of the
distance between genes
Greater the distance between genes, higher the
recombination frequency
For forked and carnation, Rf was approximately 10%
These two genes are 240,000 bp apart
To find out if recombination occurs within a gene we can
look for recombination between two mutations
within a gene
f
c
C
F
f1
f2
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Recombination
frequency
Looking for recombination between two mutations within
the same gene, we are dealing with extremely small
rates of Recombination
For example
The Drosophila X chromosome is approximately
10 megabases (10,000,000 bps)
The white gene (w) is about 1000 bp
The white gene represents about 1/10,000th the length
of the X-chromosome
To look for recombination within a gene we perform
the identical set of crosses used to look for
recombination between genes
w1
w1
w2
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w2
Recombination
frequency
To look for recombination within a gene we perform
the identical set of crosses used to look for
recombination between genes
For example we isolate two independent mutations in the
white gene
w1 and w2
(How do we know that w1 and w2 are in the same gene?)
w1
w2
w1
w2
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Intragenic recombination cross
w1
w2
w1
w2
To detect rare recombinants between w1 and w2
We perform the following cross:
w1/w2
w1
x
W/Y
W
w2
Y
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Normal gene
ATG GGG GGG TTT CCC TTT AAA
Mutant1
ATG CGG GGG TTT CCC TTT AAA
Mutant2
ATG GGG GGG TTT CCC ATT AAA
mut1 mut2
x
normal/Y
Parental classes
1
ATG CGG GGG TTT CCC TTT AAA
2
ATG GGG GGG TTT CCC ATT AAA
Recombinant classes
1
ATG CGG GGG TTT CCC ATT AAA
4
ATG GGG GGG TTT CCC TTT AAA
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IC
Definitions
Intragenic recombination: Recombination occurring within
a gene
Intergenic recombination: Recombination occurring
between genes
Fine structure Analysis: Mapping a large number of
mutations within a single gene through recombination
CCCCCCCCCCCCCCCCCCCCTCCCC
GGGGGGGGGGGGGGGGGGGGAGGGG
CCACCCCCCCCCCCCCCCCCCCCCC
GGTGGGGGGGGGGGGGGGGGGGGGG
Analysis in the bacteriophage T4 reveals that
recombination can occurs between single nucleotides
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