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A yeast prion provides a mechanism for genetic
variation and phenotypic diversity
Heather L. True & Susan L. LindquistDepartment of Molecular Genetics and Cell Biology
Howard Hughes Medical InstituteUniversity of Chicago
Nature – 28 September 2000
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A biological problem• Individual spontaneous
genetic changes may be deleterious
Happy, living cells
Dead cells
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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
+ +
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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?
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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?
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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?
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STOP
RNA transcript
RNA transcript
polypeptide
ribosome
Sup45Sup35
Sup35 = yeast translation termination factor• forms complex with Sup45• Sup45 recognizes STOP codon• Sup35 facilitates release of nascent polypeptide
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Sup35 ([psi-])
Has a prion form
[PSI+][PSI+] reduces fidelity of translation termination → causes ribosomes to read through stop codons→ suppresses nonsense mutations
produces heritable changes in phenotype with no changes in nucleic acids
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N M C
N-terminalregion
Middleregion
C-terminalregion
Sup35 has three distinct regions
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
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Diverse growth phenotypesproduced by [PSI+]
Compared growth characteristics of [PSI+] and [psi-] cells in >150 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
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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
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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 NM
33G-D373 NM
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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
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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 experiments2) letters indicate growth rates:
R = rapid; M = medium; S = slowV = very slow; NG = no growth
Change in growth in corresponding DNM strainIndicated 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
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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 experiments2) letters indicate growth rates:
R = rapid; M = medium; S = slowV = very slow; NG = no growth
Change in growth in corresponding DNM strainIndicated 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
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•[PSI+] does not alter growth on YPD at pH 6.8
• [PSI+] causes strain-specific changes at pH 6.0
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•[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
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[PSI+] affects growth in different ways in different genetic backgrounds
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Colony morphology changes
Cells grown in YPD, spotted onto medium with potassium acetate as sole carbon source
[PSI+] colonies [psi-] colonies Spontaneous appearance of [PSI+] colony in a group of [psi-] cells
Cells can switch from [PSI+] to [psi-], and vice versa
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Stress tolerance
Cells spotted on plates with ethanol gradient 0 10%
Cells in log phase were incubated at 37ºC for 30 min. then transferred to 50ºC for the times indicated
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[PSI+] negatively affected growth of all strain backgrounds when cells were plated with 5mM ZnCl2
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Divergent range of phenotypes
Strong enhancement, but only at high concentration of metal
Both these strains affected similarly by [PSI+]
Same strains affected very differently by [PSI+]
[PSI+] has little effect on most strains, but strongly affects one
[PSI+] enhances growth on one strain, inhibits on others, under same conditions
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Divergent range of phenotypes
• in nearly half the conditions tested, [PSI+] had a substantial impact in growth of at least some strains
• in > 25% of tests, the impact on the strain was beneficial
• each strain exhibited a unique combination of phenotypes in response to [PSI+]
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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
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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
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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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