Prions!Other molecules besides organelle DNA areinherited in non-Mendelian patterns.Examples of non-Mendelian patterns of inheritanceextend beyond the inheritance of organelle DNA.!Certain DNA and RNA plasmids, for example, areinherited in non-Mendelian patterns, but thestrangest cases of non-Mendelian inheritance is theinheritance of endogenous proteins with alteredstructures; these proteins are known as prions.Prions can be inherited in yeast and thefilamentous fungus Podospera anserina, and most ofour discussion will focus on two elements in yeastknown as [URE3] and [PSI+].!Prions were first proposed to cause spongiformencephalopathies in mammalsPrions were first discovered as the etiologicalagents in transmissible spongiformencephalopathies such as scrapie in sheep andgoats, bovine spongiform encephalopathy (BSE ormad cow disease) in cattle, and Creutzfeldt-Jacobdisease (CJD), Gerstmann-Straussler-Schneinkersyndrome (GSS), fatal familial insomnia and kuruin humans. Collectively, these diseases have beenexplained by the prion hypothesis. This hypothesisposits that the infectious agent is an abnormal formof a cellular protein known as PrP. During thecourse of the prion disease, a protease resistant,aggregated form of PrP, designated PrPSc,accumulates in the brain. The model proposes thatthe presence of PrPSc, which has the samesequence as PrP but a different structure, willrecruit normal PrP into the abnormal structure tospread the disease. The disease is thought tospread when animals are fed food containing brainmaterial. Alternatively, with rare genetic forms ofthe disease, individuals inherit a mutant form ofPrP that has a higher probability of spontaneouslyfolding into a protease resistant prion form. In1997, Stanley Prusiner, a neurologist at UCSF, wonthe Nobel Prize in Physiology and Medicine for hiswork leading to the prion hypothesis. Below areseveral results for this class of diseases that areconsistent with the prion hypothesis. (1) Nonucleic acid has ever been shown to de associatedwith the purified Scrapie infectious agent. PurifiedPrPSc can cause Scrapie in many animals includingmice. (2) Familial spongiform encephalopathies inhumans are associated with mutations in the PrPgene. About 10% of human prion diseases arehereditary, and these are associated withmutations in the PrP gene. Individuals affected byGSS, for example, contain a Pro to Leu mutation intheir PrP gene. Transgenic mice that carry a mousePrP gene with the GSS lesion will spontaneouslydevelop spongiform disease. Normal mice carryingthe wild-type! PrP gene do not. (3) Mice lacking thePrP gene are normal, but are resistant to Scrapie.!All of these results are consistent with the prionhypothesis.!Further evidence for the prion hypothesisEvidence of a different type supporting the prionhypothesis has come from experiments in fungi.!The yeast element [URE3], for example, can nowbe explained by the prion hypothesis. In yeast,mutations in the nuclear gene for aspartatetranscarbamylase, an enzyme in the pyrimidinebiosynthetic pathway that producesureidosuccinate from carbamyl phosphate andaspartate, cause an auxotrophic phenotype that canbe rescued by growing on exogenousureidosuccinate in low nitrogen.!!Yeast growing on a rich nitrogen source, such asammonia, repress transcription of enzymes andtransporters needed for the utilization of poornitrogen sources. The Ure2 protein senses thepresence of a rich nitrogen source and blocks theaction of the Gln3 protein, a positivetranscriptional regulator of many genes whoseproducts facilitate the utilization of poor nitrogensources. One of the genes that Gln3p regulates isDAL5, which encodes a protein that can importureidosuccinate into the cell. Thus, cells mutant!for aspartate transcarbamylase will not grow onexogenous ureidosuccinate in the presence ofammonia because Gln3 is inactivated by Ure2protein. In the absence of active Gln3, the Dal5transporter is not produced.!Aspartate transcarbamylase mutant grown onexogenous ureidosuccinate!URE2 genotype nitrogen growth + -NH3 + + + NH3 - ure2- + NH3 +[URE3] + NH3 +!!In the 1970s, Lacroute identified mutations in thegene URE2 on the basis of this phenotype: mutantsthat would now grow in the absence of theaspartate transcarbamylase gene and the presenceof ammonia and ureidosuccinate. ure2 mutationssegregate 2:2 as a Mendelian trait.! Other "mutants"with the same phenotype called [URE3] were alsoisolated, but these elements are not inherited in aMendelian pattern.! For example, crosses betweenwild type and [URE3] mutants generated diploidsthat were [URE3].! Hence, the mutant appeareddominant. Sporulation of the [URE3] diploids,however, usually generated 4 [URE3] ; 0 wild-typespores, although other inheritance patterns werealso seen.! This inheritance pattern of [URA3] isclearly non-Mendelian. These experimentssuggested that [URE3] was mitochondrial, butwhen mitochondrial DNA was lost in these strains,[URE3] was still present. Lacroute carried out theseexperiments in the early 1970s and didn’t have agood explanation for [URE3].In the 1990s, Reed Wickner tested the idea that[URE3] was a prion form of the Ure2 protein.Several experiments supported the prionhypothesis. First, increasing the amount of Ure2protein by 10 fold increases the frequency ofspontaneous [URE3] mutants generated by 100fold.! Second, yeast cells containing [URE3] can becured of this element by growth in lowconcentrations of guanidine, a protein denaturant.Third, the normal Ure2 protein is susceptible toprotease, but resistant in a [URE3] background.Fourth, the URE2 gene, and thus the Ure2 protein,was also shown to be required for the activity of[URE3].! [URE3] is lost when placed in a ure2mutant background.Additional evidence for the prion hypothesiscomes from [PSI], which is likely to be a prion formof the Sup35 protein, a translational terminator.Like [URE3], [PSI] is a non-Mendelian element thatrequires the presence of the SUP35 gene for itseffects. The [PSI] phenotype occurs at a muchhigher frequency in yeast that overexpress Sup35protein, similar to the relationship between [URE3]and the Ure2 protein.! Removal of the geneencoding the chaperone protein Hsp104 cures yeastof [PSI]. The involvement of a protein-foldingchaperone in the maintenance of [PSI] arguesstrongly that [PSI] is a protein structure.These genetic a biochemical results are allconsistent with [URE3] and [PSI] being prion formsof the Ure2 and Sup35 proteins. It
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