Transcript Slide 1

Raghav Ramachandran
Ambhi Ganesan
September 9,2008
Test out various hypotheses
on a smaller scale.
 Saves time and money.
 Practically impossible to
carry out certain research
directly on intended targets.
 Learn from mistakes and go
back to the drawing board.
 Insights into the complex
mechanisms of Life, that are
conserved over different
levels.
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Neurodegenerative disorders:
• Ease of expt’al manipulation, high conservation of mechanisms,
well defined genome
• Studying mechanisms in yeast provide insights into human
system.
• Designing therapeutics/drugs against homologous protein
aggregates in yeast.
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Aging:
• Calorie restriction affects life span in worms, flies, and rodents,
apart from yeast (reducing glucose or amino acid concns can
increase life spans)
• Life span extension from Sir2-overexpression, TOR-inhibition,
Sch9/Akt or DR has been observed in yeast, worms, and flies
• Nutrient responsive protein TOR speculated to play conserved
role.
 The
S.cerevisiae yeast has one of the
smallest genomes of eukaryotes, being
unicellular.
 Its genome contains 12, 495,682 base
pairs and 5770 genes as opposed to 3.3
*109 base pairs and ~20500 genes in
humans.
 Also, S.cerevisiae (baker’s yeast) is viable
with numerous markers and available in
large quantities making it cheap to study.
 Perhaps, the
most striking feature of
S.cerevisiae is its existence in both
haploid and diploid forms.
 This makes it easy to isolate recessive
mutations in haploids.
 Also, DNA transformed in S.cerevisiae
can undergo homologous recombination
readily, into the S.cerevisiae genome.
 By
analyzing the S.cerevisiae mutants
observed from homologous
recombination of foreign DNA, the
functions of several proteins in vivo can
be discerned.
 The entire genome of S.cerevisiae was
sequenced in 1996 and since then, it has
been used as a eukaryotic model for the
study of protein interactions and
infectious diseases.
 Each Yeast
Cell undergoes four phases in
its life cycle: G1,S,G2,M (growth,
synthesis, mitosis)
 In S. cerevisiae, arrangement of
microtubules and duplication of spindle
pole bodies takes place early in the life
cycle to allow for bud formation.
 Thus, budding S.cerevisiae lacks clear
distinction between S, G2 and M phases.
 In
the G1 phase of the cycle, the yeast
cell has three options
• It can complete the cycle and divide
• It can leave the cycle, if nutritionally starved,
where it is resistant to heat and chemical
treatment
• It can mate with a cell of opposite sex, if haploid,
after a transient arrest in G1.
• It can undergo meiosis to produce four haploid
cells under nutritional starvation, if diploid
A
gene is a cell cycle regulator if its
mutant causes inappropriate progression
through the cycle.
 CDC 28-p34 protein kinase in G1/S and
G2/M
 CLN1, CLN2, WHI1-G1 cyclins in G1/S
 MIH1- inducer of mitosis in G2/M
 CKS 1, CDC 37, CDC 36 (haploids), CDC
39 (haploids)
 The
START phase occurs during G1 and
after this phase, the cell is committed to
DNA replication and cell division.
 Before passing START, cells must obtain a
critical mass.
 CDC28 is a critical START p34 protein
kinase whose mutants can block cells at
G1/S or G2 phases.
 While
START commits the cell to its life
cycle, mitosis can only take place after
the S phase thus requiring a second
checkpoint in the life cycle, namely the
G2/M phase.
 The MIH1 gene speeds up the entry of
cells into mitosis. The role of other genes
in regulating G2/M in S.cerevisiae is
unclear.
 Initiation
of M phase depends upon
successful completion of DNA replication
in S phase. The RAD9 gene performs this
function in S.cerevisiae. If DNA
replication is delayed, cells undergo
mitosis with lethal effects.
 The p34 kinase increases in activity on
the onset of mitosis and its activity can be
regulated by tyrosine dephosphorylation
at the G2/M stage.
 START-specific genes, like CDC 28, act
after DNA replication in G1/S meiosis,
whereas they have an indirect affect on
DNA replication in meiosis.
 However, CDC28 is required for the
second G2/M meiosis, where the M and S
phases are uncoupled from each other.
 Under
nutritional starvation, yeast cells
stop growing and exit the life cycle (G0
phase).
 Unlike in mammalian cells, growth factors
may not play a role in growth control in
yeast cells and such cells in the
stationary (G0 phase) are metabolically
dormant.
 Chemical
mutagen 
grow colonies 
replica plate 
identify isolates
 Complementation to
identify recessive
lethal mutants
 Clone WT gene and
sequence it
 Linkage analysis
using Meiotic
analysis.
 The
usual methods:
• Random mutagenesis – rapid but matching
phenotypes is slower
• Genetic footprinting – simultaneous testing but
mutant strains can not be recovered
 The
new method:
• Delete entire ORFs using PCRs and homologous
recombination
• Direct and simultaneous analysis
• Rapid and increased sensitivity
 Deletions of ‘essential’ genes:
• Essential for viability and lacking human
homologues => targets for antifungal drugs
• 356 ORFs identified as essential – failed to grow
(YEPD, 30 oC) as haploid deletants.
• Only 56 % of these previously shown to be essential
for viability.
 1620
non-essential genes identified.
 Additional homozygous and 2 haploid
deletants constructed.
 Non-essential
genes:
• Relatively more complicated to analyze than
essential genes; may require complicated growth
conditions to observe the effects for some.
• 558 homozygous deletion mutants pooled and grown
in Rich (R) and Minimal (M) media.
• Aliquots from both pools  Amplify tags 
Hybridize to complements on array  Hybrid. Data,
measure of growth rate.
• Correlation of UPTAG and DOWNTAG growth rates
(<0.6 of WT for the growth-impaired strains)
 New
findings:
• Genes whose inactivation affects growth are not
necessarily the ones induced during growth
under the same particular conditions.
 Caveats:
• Neomycin phosphatase (product of KanMX4)
may affect fitness
• Composition of pool, culture conditions
• Complementation (?)
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Native GAL4 protein (881aa) contains 2
distinct domains: DNA binding and
Activation Domains
Fuse DB (1-147) with protein X
Fuse portion of AD (768-881) with protein
Y
If X and Y interact with each other in vivo,
DB and AD will be brought together
sufficient enough to activate the AD.
This recruits the transcription machinery
LacZ product is formed.
Caveats:
• Interactions need to occur within yeast
nucleus
• GAL4 Activation region is accessible to
transcription machinery
• BD-X hybrid is itself not an activator
http://www.biologicalprocedures.com/bpo/arts/1/16/m16f1lg.gif