Announcements
-First midterm exam will be in this room on Friday (4-25) from 10:30AM-12:20PM
-Exam will cover material presented in lecture and quiz section through the end of last week, however…
-this weeks material will reinforce some of the previous concepts you have learned
-this weeks material WILL be covered on the next midterm exam
Leo Pallanck’s office hours: Friday afternoons by appointment ([email protected])
Today
Inheritance
Mutant analysis
Genomicswhat can we learn by studying whole genomes?
how are biological processes studied by analyzing mutants?
Throughout the quarter . . .how and why are model organisms used in genetics?
how does that information apply to humans?
What this course is about
how are unique physical traits determined by genes?
how are traits transmitted to progeny?
how is genetic information read within cells?
From Lecture 1
More to come!
Mutant analysis (AKA Genetic Analysis)
The use of mutants to understand how a biological process normally works*
Very powerful - can be used to study metabolic pathways, animal development, neurobiology, cell division, etc.
A simple analogy…
*See the Salvation of Doug article at the following site:http://bio.research.ucsc.edu/people/sullivan/savedoug.html
Analysis of pizza synthesis
No red sauce?!
Analagous to a mutation
The mutant phenotype
Analagous to genes
What is a model organism?A species that one can experiment with to ask a biological question
Why bother with model organisms?- All organisms are related at the molecular
level
- Not always possible to do experiments on the organism you want
- If the basic biology is similar, it may make sense to study a simple organism rather than a complex one
Mutant analysis involves model organisms
Features of a good model organism• Short generation time
• Small, easy to maintain
Telomeres
96 million telomeres per cell!
• Large numbers of progeny
• Well-studied life cycle, biology
• Appropriate for the question at hand• Mendel used a model organism—the garden pea- relatively short generation time—one
per year- lots of progeny per cross- self-pollination and out-crossing possible
- true-breeding varieties readily available from local merchant
Some commonly used model organisms
- Bacteria — Escherichia coli
- Budding yeast — Saccharomyces cerevisiae
- Fruit fly — Drosophila melanogaster
- Nematode — Caenorhabditis elegans
- Mouse — Mus musculus
QuickTime™ and aTIFF (Uncompressed) decompressorare needed to see this picture.
Mutagenesis is easier in single-cell organisms with haploid lifestyles
Budding yeast—a single-celled fungus that divides by buddingYeast cells can exist as haploids…
Haploid life cycle:
Yeast as a model “genetic” organism
mitosis
cytokinesis
The haploid life cycle (1n)
mutation
mating
diploid zygote
haploid
a haploid
1n
The haploid life cycle (1n)
The life cycle of “budding” yeast
Yeast cells can also exist as diploids…
meiosis
A tetrad with 4 haploid spores(“gametes”)
Mendelian segregation occurs here
2 cells
2 a cells
1n
The life cycle of “budding” yeast (cont)
a/ diploid life cycle (2n)
Wild-type yeast can survive on ammonia, a few vitamins, a few mineral salts, some trace elements and sugar…
They synthesize everything else they need, including adenine
What genes does yeast need to synthesize
adenine?
Case study: analyzing the adenine biosynthetic pathway by generating and studying “ade” mutants
(Why might we care about adenine?)
Conducting a mutant analysis with yeast
-adenine plate
“complete” plate
sterile piece of velvet
Adenine-requiring colonies
(ade mutants)
m2
m1m3
“Replica-plating”
plate cells
Treat wt haploid cells with a mutagen:
Identifying yeast mutants that require adenine
m1 wild-type
“complete” plate
-adenine plate
replica-plate using velvet
That is, are they LOF mutations? Why do we care?
What do you conclude?
Genotypes:
ADEade
diploids
ADE is dominant over ade
“” mating type“a” mating type
Are the adenine-requiring mutants recessive?
What would you predict if…
• only one enzyme is needed for synthesis of adenine?
• many enzymes are needed for synthesis of adenine?
How to find out how many different genes we have mutated?
Are m1 and m2 alleles of the same gene?
Do complementation test to ask: are the mutations alleles of the same gene or of different genes?
All mutants would be alleles of the same gene.
Different genes might be mutant.
Are all of the mutations in one gene?
m1 m2
“complete”
diploids
-adenine
replica-plate
“” mating type“a” mating type
Do m1 and m2 complement, or fail to complement?
Are m1 and m2 alleles of the same gene, or alleles of different genes?
Performing a complementation test
What do you conclude from the pair-wise crosses shown below?
m1
m2
m3
m4
m5
m6
m7m
1m2m3m4m5m6m7
o
o
o
o
o
o
o
x
o+ + + + o
Complementation tests with ade mutants
m1, m5, m7 are mutations in one gene
Conclusion?
o = no growth on -ade+ = growth on -ade
m1
m2
m3
m4
m5
m6
m7m
1m2m3m4m5m6m7
x
+ + + +o+
o
+ + +
+ + + +
+ + +
+
+
m1, m5, m7 are mutations in one gene
Conclusion?
m2, m4 are in one gene
Four complementation groups
o
o
o
o
o
o
o
o o
What do you conclude from the pair-wise crosses shown below?
o = no growth on -ade+ = growth on -ade
m3
m6
Usually means four genes
Complementation tests with ade mutants
Yeast cells can normally grow on a sugar called galactose as the sole carbon source. Seven mutant “” haploid yeast strains have been isolated that are unable to grow on galactose (“gal”) plates.
Six of these mutant strains were each cross-stamped on a gal plate with a wild type “a” strain. The resulting pattern of growth on the gal plates is depicted below (shading = growth). In all plates, the wild type strain is in the horizontal streak.
On the leftmost plate, mark the location of the a/ diploid with a circle.What is the mode of inheritance of mutant phenotype in mutants 1-6? How can you tell?
Diploids grow on gal plate… so, wild type is dominant
Practice Question
Each of the seven “” mutant strains was cross-stamped on gal plates against “a” versions of the seven mutants. The results are depicted below:
Looking just at mutants 1–6 for now… group these six mutants by complementation group.
Mutant 1 Mutant 2 Mutant 3 Mutant 4 Mutant 5 Mutant 6 Mutant 7
Mutant1
Mutant2
Mutant3
Mutant4
Mutant5
Mutant6
Mutant7
m1, m2, m5
m3, m6
m4
Practice question (continued)
Now consider mutant 7. What is surprising about the result in the complementation table?
Mutant 7 was cross-stamped on gal plate with wild type as you saw with the other six mutants earlier:
What do you conclude about the mode of inheritance of mutant 7? How does that help you explain the complementation test result for mutant 7?
What can you conclude about how many genes are represented in this collection of seven mutants?
Fails to complement any of the others… how could it be an allele of 3 different genes?
Complementation test fails with a dominant mutation… heterozygote will always show the mutant phenotype
At least 3 genes (can’t tell about m7)
Practice question (continued)
Complementation is relevant to humans
Family A Family B= deaf
Within each family, does deafness look like it’s dominant or recessive?
Assign genotypes (A, B, etc.) to the deaf individuals in these pedigrees.
recessive
aaBB AAbb
AaBb
Complementation is relevant to humans
Niemann Pick Type C disease (NPC): a recessive human neurodegenerative disease resulting in premature death
Cellular cholesterol accumulation accompanies the disease (can be detected using a chemical called ‘filipin’ which fluoresces upon binding cholesterol)
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.normal NPC
QuickTime™ and aTIFF (Uncompressed) decompressor
are needed to see this picture.
NPC1:NPC2 NPC1:NPC3
Do NPC1 & NPC2 complement?Do NPC1 & NPC3 complement?
Practice Question
Hearing mice… independently assorting genes A and B, both needed for hearing:
AaBb AaBbx
WITHOUT drawing a Punnett square, predict the progeny phenotypes and proportions with respect to hearing ability.
a, b: complete LOF, recessive
N
R
CH
NHC
C
H2N
N
R
CH
NC
C
H2N
-OOCN
R
CH
NC
C
H2N
CHCHN
OCH2
COO-
COO-
C
O
NHC
HN
NH
CH
NC
C
using mutants to order the steps in a pathway
Adenine
(AIR)
(CAIR)
(SAICAR)
...
For example, this molecule accumulates in an ade13 mutant.
ADE13
encodes the enzyme that carries out the next step
...
ADE4 ADE5*ADE8 ADE6 ADE7 ADE2 ADE1
ADE13
ADE17ADE16
ADE17ADE16
ADE12
ADE13
AMP
ADE3*
X
redpigment
The Yeast Adenine Biosynthetic Pathway
A B
CD E F G
H
I
J
K
Y
A second phenotype of some ade mutants…
plate cellsMutagenize:
-adenine plate
Replica-plate
complete plate
Some of the adenine-requiring mutants are red!
Are the red ade mutations recessive?
m8 wild-type
complete plate
-adenine plate
replica-plate using velvet
Genotypes:
ADEade
White color is
dominant
Ability to make adenine is dominant
How can one LOF mutation generate two very different phenotypes?
some intermediate
“X”adenine
ADE1
ade1
LOF mutation
Xred
pigment
UNK1*
*not a real gene name! This gene has not yet been identified
X
Hypothesis: Two phenotypes/one LOF mutation
another intermediat
e “Y”
adenine
Y
Suppose we isolate LOTS of independent red mutants:
Are all red mutants defective in the SAME GENE?
How to tell?
m9 m10
mutations fail to complement = same gene
mutations complement = different genes
Must modify the hypothesis.
All pairwise combinations reveal two complementation groups.
diploids
m9 m11
white diploids
Complementation tests of red mutants
XADE2
ade2
X
YADE1
ADE1
red pigmen
t
“UNK1”
Modified hypothesis for red phenotype
adenine
adenine
X Y
But how do mutations in ADE1 result in a build-up of X?
YXADE2 ade1
YX
Mutations in either ADE1 or ADE2 lead to a defect in adenine biosynthesis and lead to the build-up of intermediates in the pathway. Excess “X” is converted to a red pigment.
red pigmen
t
“UNK1”
adenine
Modified hypothesis for red phenotype
YXade2
ade1
YXred
pigment
Same as ade2 single mutation! Red and adenine-requiring.
“UNK1”
1. Phenotype of ade1 ade2 double mutation?
Test your understanding
adenine
ADE7
ADE13
2. Phenotype of ade2 ade7 double mutation?
3. Phenotype of ade2 ade13 double mutation?Same as ade7 single mutation! White and adenine-requiring.
Same as ade2 single mutation! Red and adenine-requiring.4. Phenotype of unk1?White and able to grow on -ade plates.
Practice Questions
YXADE2 ADE1
YX
red pigment
“UNK1”
adenineADE3
MATa ade2 ADE3MAT 2 3ADE adeDiploid
:strain color on growth on complete - ?adenine plate ?plate ( )yes or no1A. A MATa ade2 ADE3 mutant was mated to a MAT ADE2 ade3 mutant to create a diploid. What are the phenotypes of the three strains? Assume all other genes are wild type.
MATa ade2 ADE3MAT 2 3ADE adeDiploid
:strain color on growth on complete - ?adenine plate ?plate ( )yes or nored no
white no
white yes
Practice Questions
YXADE2 ADE1
YX
red pigment
“UNK1”
adenineADE3
1A. A MATa ade2 ADE3 mutant was mated to a MAT ADE2 ade3 mutant to create a diploid. What are the phenotypes of the three strains? Assume all other genes are wild type.
1B. ADE2 and ADE3 assort independently. Draw the chromosomes at metaphase of meiosis I such that the two WILD TYPE alleles face the same pole. Place a crossover on the other chromosome arm relative to the ADE2 and ADE3 genes.
A
CD
B
Tetrad on complete plates
1C. Recall that each tetrad contains the products of a single meiosis. Predict the genotypes and growth properties of each spore resulting from this meiosis.1D. Analysis of many tetrads demonstrates that three types are found, depending on the behavior of the chromosomes in meiosis. Which tetrad best fits the meiosis you just drew? Letter the spores below to match the genotypes in your table.
#1 #2 #3
red
Spore grow without adenine? complete genotype?
A
B
C
D
noade2 ade3
yesADE2 ADE3 yesADE2 ADE3
noade2 ade3
ADE2
ADE2ade2
ade2
ADE3
ADE3ade3
ade3
A
BC
D
1E. Now draw the chromosomes at metaphase of meiosis I such that one wild type and one mutant allele face each pole. Place a crossover on the other chromosome arm relative to the Adenine genes.
Spore grow without adenine?complete genotype?
A
B
C
D
Tetrad on complete plates
1F. Predict the genotypes and growth properties of each spore resulting from this meiosis.
1G. Which tetrad best fits the meiosis you just drew? Letter the spores below to match the genotypes in your table.
#1 #2 #3
red
ADE2
ADE2
ade3
ade3
A
B
1E. Now draw the chromosomes at metaphase of meiosis I such that one wild type and one mutant allele face each pole. Place a crossover on the other chromosome arm relative to the Adenine genes.
Spore grow without adenine?complete genotype?
A
B
C
D
Tetrad on complete plates
1F. Predict the genotypes and growth properties of each spore resulting from this meiosis.
1G. Which tetrad best fits the meiosis you just drew? Letter the spores below to match the genotypes in your table.
#1 #2 #3
red
ADE2
ADE2ade2
ade2
ade3
ade3
ADE3
ADE3
nonono
noade2 ADE3ADE2 ade3ade2 ADE3
ADE2 ade3
A
CB
D
A
CD
B
1H. Now draw the chromosomes at metaphase of meiosis I such that one wild type and one mutant allele face each pole. On one chromosome, place a crossover on the other chromosome arm relative to the Adenine gene. On the other chromosome, place a crossover BETWEEN the centromere and the Adenine gene.
Spore grow without adenine? complete genotype?
A
B
C
D
Tetrad on complete plates
1I. Predict the genotypes and growth properties of each spore resulting from this meiosis.
#1 #2 #3
red
1J. Which tetrad best fits the meiosis you just drew? Letter the spores below to match the genotypes in your table.