UV Mutagenesis in Yeast
Geneticists need variation to study the function of gene products.
We create variation in the laboratory by mutagenesis
Features of a good model organism
•Ease of Genetic Manipulation
•Conserved cellular processes
•Cell biology
•Biochemistry
4
proteins
2
3
5
1
DNA
6
Important lab properties
• Genetics: introduce mutations (UV,
chemical, Xray) and design screens to
identify mutations in process you are
interested in
• Isolate the products of meiosis
• Recover mutations: stable haploid and
diploid lifecycles.
• Easy molecular manipulations-clonability
(few introns), high rate of homologous
recombination
Fig. 7.2
Mutations are
classified
by what they do to
DNA
Fig. 7.6
Fig. 7.12b1
Fig. 7.12b2
By choosing the correct mutagens,
we can control the type of mutations we make.
Because most of the chemicals are nasty,
you will be using UV light to generate mutations
Formation of Thymidine Dimer
sugar
sugar
sugar
sugar
Fig. 7.7
Photoreactivation
requires photolyase enzyme
Not present in humans
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
In yeast, 50-90% killing is used in mutagenesis experiments
Mutation curve
DNA damage is more lethal to haploids
Haploids
Diploid cells
Multicellular diploids
Identifying mutants in adenine biosynthesis
What can you do with yeast if you
are not a geneticist?
• Take advantage of the genes identified in
genetic screens
• Cell biology
PCR can generate fusions or point
mutations
Fusions to GFP allow live cell analysis
of protein function
Aequoria victoria
Green Fluorescent Protein
Visualization of chromosome and microtubule
dynamics to elucidate mechanisms of nuclear
movement and chromosome segregation
• lacI-GFP binding to
tandem repeat lacO
sequences integrated at
specific loci.
• Dynamics of GFP-fusions
to tubulin, spindle pole and
mt-based motor proteins.
• Specific genetic
perturbations to establish
cause and effect
Yeast Mitotic Spindle
Structure
EM
Tub-GFP
Kubai, 1978
Tomographic reconstruction
16 kinetochore microtubules and 4 interpolar microtubules
emanate from each spindle pole: 40 mts/1.5 m spindle
Architecture of the kinetochore
Maiato et al. (2004)
CENTROMERE
KINETOCHORE
MT PLUS ENDS
FIBROUS CORONA
OUTER PLATE
CENTROMERIC
HETEROCHROMATIN
McEwen et al. (1998)
CENP-B
MCAK
INCENP
Aurora B
Survivin
Borealin/Dasra B
ICIS
chromatid pairing,
structural support,
& MT attachment
error correction
INNER PLATE
INTERZONE
CENP-A
CENP-C
CENP-G
CENP-H
CENP-I/hMis6
CeKNL-1
hMis12
3F3/2 antigens
tension receptors &
checkpoint signalling
Ndc80/Hec1
Nuf2
Spc24
Spc25
BUB1
BUBR1
BUB3
MPS1
MAD1
MAD2
Cdc20
CENP-E
KinI,Kip3?
kinetochore assembly
& size determination
MT attachment,
regulation of MT dynamics,
& checkpoint signalling
INNER
KINETOCHORE
OUTER
KINETOCHORE
Kinetochores are clustered at the ends
the cohesin cylinder
Ndc80RFP
Spc29RFP
Smc3GFP
DIC
DIC
Smc3GFP
Ndc80RFP
overlay
overlay
Interstrand cohesin
Inflection
point
Kinetochore sleeve
Intrastrand cohesin
C-loop
Proposed Path of Centromere DNA in a Eukaryotic Kinetochore: C-loop
How do we dissect the function of the kinetochore?
Pericentric looping requires NDC10 and not MT binding
Patterns observed at 37oC and
25oC in temperature sensitive
mutants
Disruption of Centromeric
Attachment via Ndc10
Effects of Centromeric Attachment:
ndc10-1 at Restrictive Temperature
Centromere
Microtubule
Disruption of Microtubule
Attachment via Nuf2
Effects of Microtubule Attachment:
nuf2-60 at Restrictive Temperature
Centromere
Microtubule
Permissive
Temperature
Restrictive
Temperature
Effects of Microtubule Attachment on
the Inner and Outer Kinetochore
Using genetics and cell biology we have
dissected functional elements of the
kinetochore
Inner kinetochore complex CBF3 bends centromere DNA .
Clustering of 16 kinetochores requires both inner (COMA, MIND) and outer
kinetochore (Ndc80) complexes.
Outer kinetochore complexes essential for plus end microtubule interactions.
Anderson et al. MCB 2009