1. Construction ofConstruction of
double mutated progenydouble mutated progeny
from single mutation parentsfrom single mutation parents
By Ka Lo Lee and Joe Yee Wong
Chemistry Research 3913
Queens College CUNY Department of Chemistry and Biochemistry
Fall 2009
- and a series of tests for identifying their genotype -

3. ICLs repairing mechanism
• ICLs (Interstrand Cross-links) : caused by a mutagen called psoralen under UV
• Because the strands are joined together  can’t duplicate  lethal
Repair method 1 :
NER (nucleotide excision repair)
But…

4. ICLs repairing mechanism (con’t)
• Repairing method 2 : HR (Homologous Recombination)
• Template = homologous chromosome

5. ICL repairing mechanism (con’t)
• For yeast, do HR and NER work
together in repairing ICLs?
• …This can be predicted from survival
curve
• The graph the change in number of
surviving colonies with respect to
mutagen dosage
• IF working together, THEN the curve for
of NER and HR deficient will look like
the one with only one kind of repairing
mechanism missing (“wheel of a car”)
• IF working separately, THEN the
survival curve should shift down when
both mechanisms are not available… (“a
car and a bus”)
Question : Are HR and NER working together???Question : Are HR and NER working together???

6. Recombination
Recombination can switch on or off some genes by deletion
Recombination should occur spontaneously in Baker’s yeast. The
frequency of recombination will be greatly increased if exposed under
radiation. So…
How serious will the amount of radiation affectHow serious will the amount of radiation affect
recombination frequency???recombination frequency???

8. Life Cycle of S. cerevisiae
• Replication by budding: happens
in both diploids and haploids
• Mating types: MATa and MATα
• Mating: observed between
opposite mating type haploids –
mediated by phermones
• Diploid: will not mate, but can
sporulate under stressful
conditions
• Sporulation: meiosis, resulting in
four haploid spores encapsulated
in ascus

9. Exchange of genes through mating
Mating of two haploid
carrying different forms of
gene
Formation of zygote
Duplication of
chromosome during
S phase

11. Outline of experiment
Glusulase Treatment of Asci Grow the spores on YPD Replica onto YPD with grids
Growth
Tests with selective plates
Mate with known
mating type haploids
Test the mated yeast by
double selective plates
Growth
Record the colonies passed
The tests
Pick up the wanted colonies
Grow them on YPD grid

12. Glusulase Treatment
• Glusulase : a digestive enzyme
from garden snail, able to digest
the ascus
• After digestion, the free spores
can now reproduce on a nourished
environment (YPD plate)

13. Choosing colonies and replicate them onto grids
 After 2 days of
cultivation, colonies
are visible.
Pick 100 colonies, organize
them onto grids, so that we
can track how they grow 
 A sample of grid

14. Confirming Phenotypes : Selecive Plating
• Replicas : onto selective plates instead of YPD plates
• Selective plates : some nutrients may be missing OR antibiotic added

15. Mating Test : Are they haploid?
-A lawn of haploid with known mating type is
used to cover a plate with replica of the grids
on YPD
-Incubate for 2 days = allow mating
-Then the mated yeast are tested on double
selective plate
For those colonies growing on double
selective plates = diploid formed
successfully during the mating test
However, colonies mating with both testing strains indicates that they
consist of improper haploid spores formed during sporulation.

17. Crosses : the parents of the spores
• Three sets are used for sporulation in our experiment :
1. rad2 Δ (324#6)::KanMX x rad57(462)::LEU2
2. rad2 Δ (322#4)::KanMX x rad57(464)::LEU2
3. rad2 Δ (324#6)::KanMX x rad55(452)::LEU2
rad2 Δ::KanMX means there is a deletion of RAD2 gene
and its place is replace by a gene KanMX; this parent
consist of gene KanMX, his3, TRP1
While rad57::LEU2 indicates there is an insertion of
LEU2 gene into RAD57 gene with some part of RAD57
left on DNA and this strain contains LEU2, his3, TRP1

18. Remarkable genes in our experiment
• RAD2 : for Nucleoide Excision Repair (NER)
• RAD57 : for Homologous Recombination (HR)
• RAD55 : works with RAD57, helps the protein by RAD51 to load on
DNA, enhancing the efficiency of HR
• LEU2 : ability to make Leucine
• KanMX : resistance to the antibiotic G418
What exactly they do???

20. Tests with Selective Plates : -His, -Trp
• All eight plates are replicated onto 4 kinds of selective plates
• We will only discuss the situation of 4-I and 4-II
• -His plates are necessary to be observed twice in 2 days for tracing
recombination
-His plate
Day 1 – colony #36 on 4-II shows a solid growth, indicates that colonies contains His+
carrying haploids
Day 2 – All replicated colonies show a thin layer of growth, it is, these colonies are able
to undergo spontaneous recombination
-Trp plate
Day 2 – All colonies are growing = all colonies are carrying gene necessary for
tryptophan synthesis (i.e. Trp+
)

21. Tests with Selective Plates : -Leu, YPD with G418
4-I 7, 15, 21, 22, 23, 28, 33, 34, 37, 38, 39, 41, 43, 47, 50
4-II 14, 15, 18, 20, 21, 22, 28, 29, 30, 31, 32, 37, 40, 41, 48, 50
4-I 1, 3, 6, 9, 10, 15, 17, 21, 28, 31, 33, 37, 39, 46, 50
4-II 2, 5-8, 15, 21, 25, 28, 32, 34, 36, 38, 39, 46, 48, 50
-Leu Plates = testing for RAD55 disruption
This is a list of non-growing colonies after growing for 3 days; they
are auxotrophic for leucine (i.e. Leu-
)
YPD Plates with G418 = testing for RAD2 disruption
A table of absent colonies after growing for 2 days; they are
sensitive to antibiotic G418 (i.e. G418S
)

22. Mating type test on -Leu-Ura plates
Mating test = testing for haploid status
Colonies which are covered by a lawn of haploid with known mating type
and growing on these plates (i.e. Leu+Ura+) :
Covered by RY60 (MATα) lawn (i.e. MATa type) Covered by RY59 (MATa) lawn (i.e. MATα type)
4-I 3, 11, 13, 17, 27, 36 6, 8, 14, 23, 29, 31, 40, 45, 46
4-II 4, 16, 39, 44, 46, 49 10, 27, 35, 36, 38, 42

23. Picking colonies which passed all the tests
4-I : 11
4-I : 13
4-I : 27
4-I : 36
4-II : 4
4-II : 16
4-II : 44
4-II : 49
4-I : 8
4-I : 14
4-I : 29
By combining the data from the preious tests, we have colonies with phenotype
Leu+
, Trp+
, G418R
, able to recombine spontaneously and mating types confirmed:
Mating type = MATa Mating type = MATα
4-I : 40
4-I : 45
4-II : 10
4-II : 27
4-II : 35
4-II : 42
These colonies are replicated
onto new YPD grids,
assigned with new numbers.
Allowing them to grow for 2 to
3 days and they will be ready
for further investigation :
Test for DNA repair and
recombination after
crosslink treatment.

24. Citation
• Special thanks : Dr. Wilma Saffran’s lecture on setup of experiment
• Microscopic picture and some general information of yeast :
The Keogh Lab Website
http://mckeogh.googlepages.com/
• Models of ICL processing by XPF-ERCC1 complex at a stalled fork.
The Journal of Biological Chemistry
http://www.jbc.org/content/283/3/1275/F6.expansion
• Repair of DNA double-strand breaks by DSBR and SDSA.
Nature Reviews
http://www.nature.com/nrm/journal/v7/n10/fig_tab/nrm2008_F2.html

25. The EndThe End
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