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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
yellow
Bla
nco e
ye
Sh
aven
bod
y
Fork
ed
b
ristl
e
Wh
ite e
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!
yellow
sin
ged
Sh
aven
bod
y
fork
ed
wh
ite
Bla
nco
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!
yellow
sin
ged
Sh
aven
bod
y
fork
ed
wh
ite
bla
nco
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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
ite
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
yellow
sin
ged
Sh
aven
bod
y
fork
ed
wh
ite
bla
nco
****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
yellow
sin
ged
Sh
aven
bod
y
fork
ed
wh
ite
bla
nco
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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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