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A yeast prion provides a mechanism for genetic variation and phenotypic diversity

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A yeast prion provides a mechanism for genetic variation and phenotypic diversity Heather L. True & Susan L. Lindquist Department of Molecular Genetics and Cell Biology – PowerPoint PPT presentation

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Title: A yeast prion provides a mechanism for genetic variation and phenotypic diversity


1
A yeast prion provides a mechanism for genetic
variation and phenotypic diversity
  • Heather L. True Susan L. Lindquist
  • Department of Molecular Genetics and Cell Biology
  • Howard Hughes Medical Institute
  • University of Chicago
  • Nature 28 September 2000

2
A biological problem
  • Individual spontaneous genetic changes may be
    deleterious

Happy, living cells
Dead cells
3
A biological problem
  • Individual spontaneous genetic changes may be
    deleterious
  • But what if several independent changes could
    occur, that would work together to produce a
    beneficial new phenotype?

Happy, living cells


Dead cells
4
What if there is a way to accumulate genetic
changes behind closed doors and then allow them
to express together to produce a new form or
functions?
5
What if there is a way to accumulate genetic
changes behind closed doors and then allow them
to express together to produce a new form or
functions?
6
A proposed mechanism
  • Gene duplication is a common event
  • A duplicated gene that is retained in an
    inactive state could accumulate variations
  • Inactive genes reactivated all at once cell
    benefits from combinatorial changes

HOW?
7
  • Sup35 yeast translation termination factor
  • forms complex with Sup45
  • Sup45 recognizes STOP codon
  • Sup35 facilitates release of nascent polypeptide

RNA transcript
ribosome
STOP
Sup45
Sup35
RNA transcript
polypeptide
8
Sup35 (psi-)
Has a prion form
produces heritable changes in phenotype with no
changes in nucleic acids
  • PSI reduces fidelity of translation
    termination
  • causes ribosomes to read through stop codons
  • suppresses nonsense mutations

PSI
9
Sup35 has three distinct regions
N
M
C
N-terminal region
Middle region
C-terminal region
  • C-terminal region responsible for translation
  • termination essential for viability
  • N-terminal Middle regions
  • allow Sup35 to acquire stable prion conformation
  • allow cell to switch between PSI and psi-
    states
  • can be deleted in psi- cells with no apparent
    effect
  • deletion results in loss of prion, restoration
    of
  • translation termination fidelity

10
Diverse growth phenotypesproduced by PSI
  • Compared growth characteristics of PSI and
    psi- cells in gt150 phenotypic assays
  • fermentable non-fermentable carbon sources
  • simple complex nitrogen sources in presence of
    salts metals
  • with inhibitors of diverse cellular processes
  • DNA replication
  • Signalling
  • Protein glycosylation
  • Microtubule dynamics
  • general stress conditions
  • different temperatures

11
Diverse growth phenotypesproduced by PSI
  • Examined cells from seven different genetic
    backgrounds
  • to distinguish traits acquired from
    PSI state
  • from traits arising in specific genomes

Strains
74-D694
33G-D373
SL1010-1A
D1142-1A
5V-H19
10B-H49u
BSC783/4c
  • used guanidine hydrochloride curing to create
    stable, isogenic pairs (PSI psi-
  • phenotypic differences would be due to PSI
    effects on gene expression, not genetic changes
    from long-term culture
  • each strain had a different intrinsic level of
    PSI-mediated nonsense suppression
  • low- to high- levels of PSI nonsense
    suppression activity represented in selection of
    strains

12
Diverse growth phenotypesproduced by PSI
  • CONTROL Examined NM deletion strains in two
  • genetic backgrounds
  • To detect phenotypes that may be due to loss of
    NM region function (which may occur with prion
    formation due to occlusion), in the absence of
    PSI activity

Strains
74-D694 DNM
33G-D373 DNM
13
Carefully controlled experimental procedures
  • Strain pairs examined at same growth stage
  • Mid-log phase cells grown in rich medium (YPD)
  • Serially diluted 5X, spotted onto test plates
  • Tested amino acid supplementation in parallel
    experiments
  • No changes in growth patterns phenotypic
    differences were not owing to effects of
    auxotrophic markers the strains contain
  • Most strains carried adenine mutations, which
    turn red in the absence of adenine (PSI
    suppressed these mutations some of the time)
  • YPD control plates were periodically interspersed
    with test plates
  • Assured consistent spotting of cells, with same
    densities
  • Performed repeat experiments
  • Same phenotypic variances were observed each time

14
Marked growth differences observed between
isogenic PSI psi- derivatives on many test
plates
Change in colony morphology
Enhanced growth in psi- derivative


Enhanced growth in PSI derivative
Stronger color stronger effect

Growth scored in two ways 1) numbers designate
the highest dilution that produced significant
growth in repeat experiments 2) letters indicate
growth rates R rapid M medium S
slow V very slow NG no growth
Change in growth in corresponding DNM
strain Indicated by superscript a growth
similar to psi- b growth similar to
PSI c different from both psi- PSI
For stress assays Growth rates H high M
medium L low slightly increased
tolerance
15
Change in colony morphology
Growth scored in two ways 1) numbers designate
the highest dilution that produced significant
growth in repeat experiments 2) letters indicate
growth rates R rapid M medium S
slow V very slow NG no growth

Enhanced growth in psi- derivative

Enhanced growth in PSI derivative
Stronger color stronger effect

Change in growth in corresponding DNM
strain Indicated by superscript a growth
similar to psi- b growth similar to
PSI c different from both psi- PSI
For stress assays Growth rates H high M
medium L low slightly increased
tolerance
16
  • PSI does not alter growth on
  • YPD at pH 6.8
  • PSI causes strain-specific
  • changes at pH 6.0

17
  • PSI does not alter growth on
  • YPD at pH 6.8
  • PSI causes strain-specific
  • changes at pH 6.0

psi- grew better here
PSI grew better here
18
PSI affects growth in different ways in
different genetic backgrounds
19
Colony morphology changes Cells grown in YPD,
spotted onto medium with potassium acetate as
sole carbon source
psi- colonies
Spontaneous appearance of PSI colony in a
group of psi- cells
PSI colonies
Cells can switch from PSI to psi-, and vice
versa
20
Stress tolerance
Cells in log phase were incubated at 37ºC for 30
min. then transferred to 50ºC for the times
indicated
Cells spotted on plates with ethanol gradient 0
10
21
PSI negatively affected growth of all strain
backgrounds when cells were plated with 5mM ZnCl2
22
Divergent range of phenotypes
Both these strains affected similarly by PSI
Same strains affected very differently by PSI
Strong enhancement, but only at high
concentration of metal
PSI enhances growth on one strain, inhibits on
others, under same conditions
PSI has little effect on most strains, but
strongly affects one
23
Divergent range of phenotypes
  • in nearly half the conditions tested, PSI had
    a substantial impact in growth of at least some
    strains
  • in gt 25 of tests, the impact on the strain was
    beneficial
  • each strain exhibited a unique combination of
    phenotypes in response to PSI

24
Are there common biological effects of PSI ?
  • Enhance stress tolerance?
  • no significant difference between PSI psi-
    in either ethanol tolerance or thermotolerance
    conditions
  • Affect all strains in the same way?
  • only once ZnCl2
  • affected all six strains capable of growth in
    presence of calcofluor white

Possible biological effect of PSI on zinc
metabolism or cell wall biosynthesis
25
NM specific phenotypes?
PSI had both enhancing and inhibiting effects
on growth in the presence of benomyl no common
biological effect
  • DNM mutants grew better in the presence of
    benomyl than either PSI or psi- variants
  • DNM phenotypes were mostly different from both
    PSI psi-

The NM region of Sup35 may play a role in yeast
biology in addition to its role in PSI
formation
26
CONCLUSIONS
  • PSI element of S. cerevisiae provides a means
    to activate silent genetic information to produce
    new heritable phenotypes
  • as a suppressor of nonsense mutations, PSI
    provides mechanism for read-through of
    naturally-occuring STOP codons, providing
    potential access to genetic variability in
    duplicate genes or in 3 non-coding regions
  • allows alternative heritable phenotypes to be
    encoded by a single genome
  • provides possibility of survival in fluctuating
    environment
  • provides a means for evolution of new traits
  • Yeast cells spontaneously switch between PSI
    psi- states
  • both phenotypes are available, for adaptation to
    changing environment

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