UV Mutagenesis in Yeast

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UV Mutagenesis in Yeast
Geneticists need variation to study the function
of gene products. We create variation in the
laboratory by mutagenesis
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Fig. 7.2
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Fig. 7.6
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Fig. 7.12b1
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Fig. 7.12b2
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By choosing the correct mutagens, we can control
the type of mutations we make
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Fig. 7.7
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Formation of Thymidine Dimer
sugar
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Photoreactivation requires photolyase enzyme
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Mutagenesis of yeast
haploid
Irradiate with UV. Calculate survival
curve Select optimal dose for isolation of
mutations. Select on appropriate selective
media Replica plating to identify nutrient
deficiencies.
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Survival curve
In yeast, 50-90 killing is used in mutagenesis
experiments
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Mutation curve
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DNA damage is more lethal to haploids
Haploids
Diploid cells
Multicellular diploids
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Mutation in a diploid self fertilizing organism
How to generate useful mutants to study?
Arabidopsis
Genome size 124,000 kb 30,000 genes
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Arabidopsis Expose seeds to chemical
mutagenesis EMS Dosage high enough to induce
multiple mutations per nucleus. 2 cells in each
seed will give rise to germline-gametes.
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Generate homozygotes for new mutations
EMS treat seed. 2 cells per seed give rise to
M1 plants. M1 plants are potential
heterozygotes for any mutation. M1 plants are
self fertilized . M2 plants will segregate wild
type and homozygous mutations
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Number of seeds to mutagenize Depends on genome
size, number of cells affected and the
experimentally determined rate of mutations per
cell. Arabidopsis has a genome size of
approximately 130 X 106 bp 2 Cells are affected
per seed We use doses of EMS that give 10-30
mutations per seed. If we mutagenize 10,000
seeds, we expect over 105 mutations. That
represents one mutation for every 1300 bp of DNA
in the genome. The average gene is 2,500 bp.
Therefore we expect to hit each gene at least
twice.
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Embryo lethal mutations reveal mutation rate
M1 plants are expected to be haploid for any
induced mutation
They are self fertilized to make seeds. The seeds
in a pod will segregate 31 for any recessive
mutation
We examine seed pods for embryo lethal
mutations We look at 100 seed pods for ¼ embryo
lethal phenotypes 10 with embryo lethal mutants
is the goal. When you have a minimum of three
alleles for any one locus, you can assume you
have saturation.
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Fig. B.14
Select or screen phenotypes.
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Fig. B.17
Seedling phenotypes Make powerful screens
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Mutation in a diploid sexually dimorphic species
mouse
Chemical mutagenesis of males leads to sperm with
multiple mutations. Beutler lab at Scripps. Du
et al 2004 Genetics 166 332-340
Mate treated males to wild type females gt 20
offspring F1 animals are potentially
heterozygotes for the gene of interest. F1
animals are mated together to get F2. Since
every F1 is from a different sperm, they are
unlikely to have the same mutation. To get
homozygotes for recessive alleles Mate F2
females with their F1 father, Screen 6 male and 6
female F3 progeny for phenotypes and embryo
lethality. Beutler lab had made 16,000 F1s and
23,221 F3 mutants at time of publication of first
mutants.
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Number of mouse families to screen
Mouse genome is 2,600,000 kb Number of predicted
genes 30,000 How many mutants to screen for
saturation? At the rate of chemical exposure
used each F1 mouse gets 45 genes
mutated. Beutler lab has screened 1513 families
from F1 males They calculate that they are 8.6
of the way to 95 probability of finding any
mutations. Would need 1513/0.086 to have 95
probability of finding any mutation. 17600 F1
families.
Beutler et al., 2005 Curr. Op. Immunol. 1736-43.
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Alternative Directed mutations or selectable
mutations in transgenic mice
Availability of Genome Sequence allows us to
choose a gene of interest. We can make a mouse
with a deletion in that gene and observe the
phenotype of homozygous progeny
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Targeted mutagenesis to create a mouse model for
human disease
(Contd next slide)
Fig. E.14 a-c
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ES culture
(contd next slide)
Fig. E.14 d,e
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Fig. E.14 f (contd next slide)
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Inheritance pattern
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Summary
Geneticists use mutagenesis to get variation in
an otherwise Uniform genetic background. Differen
t chemical and radiation treatments will lead to
Predictable changes in DNA sequences Mutagenesis
protocols depend on the biology of the
organism Yeast, plants, mammals Directed
mutations can be made in some model organisms
using transformation and homologous
recombination.