Transcript Genetic Approaches to Studying Genome Function

E. coli = E. lephant ?
1965
1966
F. Jacob
J. Monod
A. Pardee
D. Hawthorne
H. Douglas
Y. Oshima
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Analogy and homology as tools in
genetic investigation
Animal
Mandibular Arch
(ventral)
Mandibular Arch
(dorsal)
Hyoid Arch
(dorsal)
Shark
Meckel's cartilage
Palatoquadrate
cartilage
Hyomandibular
cartiliage
Amphibian
Articular (bone)
Quadrate (bone)
Stapes
Mammal
Malleus
Incus
Stapes
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a cells produce a pheromone and a receptor
a cells produce a pheromone and a receptor
diploid (a/a) cells produce none of the above
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Shmoo
Al Capp (1948) – Li’l Abner
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Marsh and Rose diagram
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The phenotype of a haploid yeast cell with
respect to mating is determined by
transcription factors
An a cell produces two transcription factors,
Mata1p and Mata2p, that ensure expression of
a specific genes, including the pheromone and
receptor, and repress expression of a specific
genes.
In an a cell, Mata1p and Mata2p are not
expressed, and a different transcription factor is
expressed, Mata1p. The a genes are off, and
the a genes (pheromone and receptor) are on.
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A.9
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Amazing but true
A wild-type haploid yeast cell contains THREE
copies of mating type-determining genes:
• Copy #1: the a1 and a2 genes (silent).
• Copy #2: the a1 and a2 genes (also silent).
• Copy #3: An additional copy of genes in item 1,
or of the genes in item 2, but active.
Whichever genes are contained in copy #3
determines the mating type.
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A.11
A.12
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“An easily understood, workable falsehood is more useful than an incomprehensible truth.”
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a2 a1
a cell
HMLa
silent
cen
a2 a1
a2 a1
MAT
HMRa
active
silent
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Loss of silencing at the silent mating type
cassettes creates a “nonmater” – a haploid
that is a/a and that thinks it’s a diploid.
a2 a1
a cell
HMLa
active
cen
a2 a1
a2 a1
MAT
HMRa
active
active
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Screen for silencing mutants
A sample “screen”:
1. Take haploid cells.
2. Mutate them.
3. Screen for those that don’t mate.
Problem: mating is so much more than
proper silencing of mating type loci!!
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The mating
pheromone
response
Also see Fig. A.13.
Jeremy Thorner
Thorner diagram
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How to screen for silencing mutants
a2 a1
a cell
HMLa
silent
a2 a1
a2 a1
MAT
HMRa
active
silent
cen
Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.
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How to screen for silencing mutants
a2 a1
HMLa
silent
cen
a2 a1
a2 a1
mata1-1
HMLa
active
silent
Note: mata1-1 is a special allele of the a gene – it is recessive to a
Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.
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Rine schematic
mate to a cells
Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.
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The data
• Colonies screened: 675,000
• Colonies that mated to a: 295
• Major complementation groups: 4
silent information regulators:
SIR1, SIR2, SIR3, SIR4
Jasper Rine and Ira Herskowitz (1987) Genetics 116: 9-22.
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Question
What molecular mechanisms are responsible for
silencing at the mating type loci?
 heterochromatin formation in metazoa
prostate cancer
 breast cancer
 ageing
 “normal” gene regulation in mammals
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Homework
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How can one explain the
evolution of two distinct mating
types in budding yeast?
Surely a pathway could have just
evolved for the fusion of two
identical haploid cells?
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Two mating types have evolved under
selective pressure to avoid inbreeding
One evolutionary advantage of mating is the production
of novel genotypic combinations via the fusion of two
genomes with different life histories.
D1
D1
x
M
D2
D2
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Granddaughters of any given
mother can switch mating type
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[email protected]
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a2 a1
a cell
a cell
a2 a1
a2 a1
MAT
HMRa
silent
active
silent
a2 a1
a2 a1
a2 a1
MAT
HMRa
HMLa
HMLa
cen
cen
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Epigenetic inheritance
• In an a strain, the genetic information at MAT
and at HMLa is identical.
• The one at MAT is expressed, but the one at
HML is not – it is epigenetically silenced.
Epigenetic: mitotically stable (persists through cell
division) change in gene expression state that is
not associated with a change in DNA sequence.
Examples: X chromosome inactivation; imprinted
genes; transgene silencing in gene therapy.
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Compaction into chromatin brings
the eukaryotic genome to life
15,000x
compaction
< 10-5 metres
> 1 metre
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“Beads on a string”
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The Nucleosome Core Particle:
8 histones, 146 bp of DNA
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Histones: Conserved and
Charged
H.s. = Lycopersicon esculentum
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“Extremely conserved histone H4 N terminus is
dispensable for growth but essential for repressing
the silent mating loci in yeast” (M. Grunstein)
Fig. 3 kayne
Kayne et al. (1988) Cell 55: 27-39.
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Fig. 6 and 7 of Kayne.
Kayne et al. (1988) Cell 55: 27-39.
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Kayne et al. (1988) Cell 55: 27-39.
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Acetylation of lysine in histone tail
neutralizes its charge (1964)
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“Genetic evidence for an interaction between SIR3
and histone H4 in the repression of the silent
mating loci in Saccharomyces cerevisiae”
Reverse genetics: introduce point mutations in H4 tail!!
Johnson et al. (1990) PNAS 87: 6286-6290.
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Table 2
Johnson et al. (1990) PNAS 87: 6286-6290.
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And 5 years later …
Sir3p and Sir4p bind H3 and H4 tails
Hecht et al. (1995) Cell 80: 583.
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Houston, we have a …
Every nucleosome in the cell has an H3 and
H4 tail (two of each, actually).
Why do the SIRs bind only where they bind?
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The silencers
“Hawthorne deletion” (1963) and onwards:
two silencers flank the mating type loci:
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The key question
How do the SIRs
spread from the
silencer and over the
mating type loci
genes?
= how do the SIRs
actually silence txn?
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Roy Frye (Pitt)
“Characterization of five human cDNAs with homology to
the yeast SIR2 gene: Sir2-like proteins (sirtuins)
metabolize NAD and may have protein ADPribosyltransferase activity” BBRC 260: 273 (1999).
1. Bacteria have proteins homologous to Sir2.
2. So do humans (>5).
3. The bacterial proteins are enzymes, and use NAD to
ADP-ribosylate other proteins.
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J. Denu: Sir2p is a NAD-dependent
histone deacetylase (HDAC)
Sir2p
Tanner et al., PNAS 97: 14178 (2000)
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Rusche L, Kirchmaier A, Rine J (2002) Mol. Biol. Cell 13: 2207.
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Histone tail acetylation promotes
chromatin unfolding (somehow)
acetylation
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Next time:
the genetics of heterochromatin
in metazoa
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