Download (X) is one desirable mutation

UV Mutagenesis in Yeast
Geneticists need variation to study the function of gene products.
We create variation in the laboratory by mutagenesis
Fig. 7.2
Fig. 7.6
Fig. 7.12b1
Fig. 7.12b2
By choosing the correct mutagens,
we can control the type of mutations we make
Fig. 7.7
Photoreactivation
requires photolyase enzyme
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.
Survival curve
Mutation curve
Mutation in a diploid self fertilizing organism:
How to generate useful mutants to study?
Arabidopsis:
Genome size:
124,000 kb
30,000 genes
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.
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
Number of seeds to mutagenize:
Depends on number of cells affected and the experimentally
determined rate of mutations per cell.
P = fn
P is probability of getting at least one desirable mutation,
f is complement of the mutation rate, fraction of seeds without a
mutation if the mutation rate is X.
n is seed number.
Say rate (X) is one desirable mutation per 104 seeds
F = (9999/10000)
0.99 = (9999/10000)n
n = log (1/100)/log (9999/10000) = 46,050
Number of M1 plants: Each seed contains effectively 2 cells
that will make gametes, so 46,050/2 seeds will give a 99%
probability of finding a desired mutation at this mutation rate.
Number of M2 plants to screen
M1 plants are chimeric but only 2 cells contribute to germ line,
From one plant, mutant and wild type gametes will be made.
Allow M1 mutant plants t self-fertilize, Collect M2 seeds in lots.
If one of the germ cells in the M1 plant got the mutation. ½ of its
flower will be haploid for the desired mutation.
If and the mutant is recessive, ½ the progeny of the M1 plant will
segregate 3:1 for the mutant phenotype.
To ensure we see these mutants with a probability of 0.99%, we
need to screen 20 progeny of each M1 plant.
Mutagenise 10,000 seeds
Grow M1 plants up in 100 lots of 100 plants each.
Plant at least 2000 plants per lot.
Select or screen phenotypes.
Fig. B.14
Select or screen phenotypes.
Fig. B.17
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 –> 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 F1’s and 23,221 F3 mutants at time of
publication of first mutants.
Number of mouse families to screen
Mouse genome is 2600,000 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. 17:36-43.
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
Targeted mutagenesis to create a mouse model
for human disease
Fig. E.14 a-c
(Cont’d next slide)
ES culture
(cont’d next slide)
Fig. E.14 d,e
Fig. E.14 f
(cont’d next slide)
Inheritance pattern