Copyright 0 1996 by the Genetics Society of America

Genesis and Variability of [PSA Prion Factors in Saccharomyces cerewiSiae

Irina L. Derkatch,*3t Yury 0. Chernoff,*’t’x Vitaly V. Kushnir~v,~ Sergey G. Inge-Vechtomovt and Susan W. Liebman*

*Laboratory for Molecular Biology, Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60607, tLaboratory of Physiolopal Genetics, Department of Genetics, St . Petersburg State University, 199034 St. Petersburg, Russia, :School of Biology, Georgia Institute of Technology, Atlanta, Georgia 30332-0230 and sZnstitute of Experimental Cardiology,

Cardiology Research Center, 121552 Moscow, Russia Manuscript received July 10, 1996

Accepted for publication September 12, 1996

ABSTRACT We have previously shown that multicopy plasmids containing the complete SUP35 gene are able to

induce the appearance of the non-Mendelian factor [PSrJ . This result was later interpreted by others as a crucial piece of evidence for a model postulating that [ PSrJ is a self-modified, prion-like conformational derivative of the Sup35 protein. Here we support this interpretation by proving that it is the overproduc- tion of Sup35 protein, and not the excess of SUP35 DNA or mRNA that causes the appearance of [PSrJ . We also show that the “prion-inducing domain” of Sup35p is in the N-terminal region, which, like the “prion-inducing domain” of another yeast prion, Ure2p, was previously shown to be distinct from the functional domain of the protein. This suggests that such a chimeric organization may be a common pattern of some prion elements. Finally, we find that [PSrJ factors of different efficiencies and different mitotic stabilities are induced in the same yeast strain by overproduction of the identical Sup35 protein. We suggest that the different [PSZI-containing derivatives are analogous to the mysterious mammalian prion strains and result from different conformational variants of Sup35p.

C ERTAIN related neurodegenerative mammalian diseases, including mad cow disease, human Creu-

tzfeldt-Jacob disease, and sheep scrapie, appear to be transmitted by a protein, called a prion, without the aid of any nucleic acid (see PRUSINER 1994). According to this hypothesis infection depends on the prion pro- tein’s ability to convert unmodified nonprion protein (PrP”), encoded by the same host gene, into the prion conformation (PrPS‘), thereby triggering a chain reac- tion of conversion that results in both progressive accu- mulation of PrPS‘ and development of the disease. The inheritance of strain-specific properties associated with prion protein (DICKINSON and MEIKLE 1971; BRUCE and FRASER 1991) has lead to the suggestion that the prion can take on various stable conformations (BESSEN et al. 1995). Although the prion hypothesis is widely ac- cepted, alternative hypotheses involving nucleic acid transmission are still discussed (NARANG 1990, 1994; MANUELIDIS et al. 1995).

From the geneticist’s point of view, the prion model suggests a new mechanism of genetic variability, which operates at the level of protein conformation rather than at the level of nucleic acids. WICKNER (1994) has recently noted that the inheritance of the non-Mende- lian yeast factors [ URE3] and [PSI) can be explained by the prion model, suggesting that prion elements are not limited to neurodegenerative mammalian diseases, but may be a widely used mechanism of inheritance.

Corresponding author: Susan W. Liebman, University of Illinois, Mo- lecular Biology Research Facility, Laboratory for Molecular Biology (M/C 567), 900 S. Ashland Ave., Chicago, IL 60607. E-mail: suel@uic.edu

Genetics 144: 1975-1586 (December, 1996)

The [PSI) factor was originally described as a non- Mendelian element, present in some strains of Saccharo- myces cermisiae, that increases the efficiency of certain codon-specific translational suppressors (COX 1965; LIEBMAN et al. 1975). Later it was demonstrated that [ PSI) causes weak omnipotent translational suppression by itself (LIEBMAN and SHERMAN 1979; O N 0 et al. 1986; COX et al. 1988; TIKHODEEV et al. 1990). Despite inten- sive studies, [PSI) was not linked to any known extra- chromosomal DNA or RNA (for review see COX et al. 1988). It was also demonstrated that the [PSI) factor is eliminated from cells by various stress-inducing agents (SINGH et al. 1979; TUITE et al. 1981). Guanidine hydro- chloride was found to be the most efficient cure for [PSZI (TUITE et al. 1981).

Several pieces of evidence strongly suggest that [PSd is a prion form of the Sup35 protein (Sup35p) (for reviews see LINDQUIST et al. 1995; WICKNER et al. 1995; TUITE and LINDQUIST 1996), which is a translation ter- mination factor, eRF3 (STANSFIELD et al. 1995; ZHOU- RAVLEVA et al. 1995). (1) Mutations in the SUP35 gene cause omnipotent suppression, just as [PSA does (INGE- VECHTOMOV and ANDRIANOVA 1970; HAWTHORNE and LEWPOLD 1974; GERLACH 1975). (2) Multiple copies of the wild-type SUP35 gene induce [PSI) ( CHERNOFF et al. 1993). Presumably, when an excess of Sup35p is made there is a greater chance for some molecules to fold in the prion conformation and for the prion seed to am- plify, perhaps as a result of more protein/protein inter- actions. ( 3 ) The SUP35 5’ proximal region (SUP35N) is essential for [PSI) maintenance (DOEL et al. 1994;

1376 I. L. Derkatch et al.

TABLE 1

Strains of the yeast Saccharomyces cereuisiae used in this work

Name Genotype Source

33GD373 MATO pheA-lO(UAA) his7-1(UAA) lysY-A2Z(UAA) CHERNOFF et al. (1988, 1992)

74D694 MATa adel-l4(UGA) trpl-289(UAG) hzs3A-200 CHERNOFF et al. (1995) trpl-289(UAG) ade2-144,717 ura3-52 leu2-3,112

ura3-52 leu2-?,112

thr4-B15(UGA) leu2-1A ura3A dU&132-L28" MATa adel-I4(UGA) lys2-87(UGA) his7-1(UAA) 0. V. TARUNINA

2G-H19 MATa ade2-l(UAA) SUP16 his3 lysl-l ura3 M. D. TER-AVANESYAN

" The full name of the strain according to the nomenclature of the Peterhof Genetic Collection is dU8-132- Y

L28-2V-P3982.

TER-AVANESYAN et al. 1994), just as the PrPgene is essen- tial for susceptibility to prion infection (BUELER et al. 1993). Note that Sup35p's Gproximal part (Sup35Cp) is conserved in evolution and required for viability, while its N-proximal part (Sup35Np) is variable and is not essential for viability (HOSHINO et al. 1989; KUSH- NIROV et al. 1990; SAMSONOVA et al. 1991; TER-AVANES- YAN et al. 1993). (4) Sup35Np contains a region of tan- dem oligopeptide repeats that shows a remarkable similarity to the corresponding region of mammalian PrP protein (COX 1994; KUSHNIROV et al. 1995). (5) Our finding (CHERNOFF et al. 1995) that the inheritance of [PSA depends upon the concentration of Hspl04p chaperone implies that the inheritance is based on pro- tein conformation, since the only known function of Hspl04p (PARSELL et al. 1994) is to facilitate protein conformational changes.

The work presented here supports the prion hypoth- esis by proving that it is Sup35 protein overproduction, as opposed to excess SUP35 DNA or mRNA, that in- duces the de novo appearance of the [PSA factor. As found for another yeast prion-maintenance gene, URE2 (MASISON and WICKNER 1995), we now show that the minimal Sup35p "prion-inducing" region is in the N- terminal region and is apparently identical to the SUP35 region previously (TER-AVANESYAN et al, 1994) shown to be required for maintenance of [PSfl. We also show that [PSA factors of various efficiencies and mitotic sta- bilities are induced by overproduction of Sup35 protein in the same yeast strain.

MATERIALS AND METHODS

Strains and cultivation procedures: Yeast strains used in this study are listed in Table 1. Standard yeast media and cultivation procedures were used (SHERMAN et al. 1986). Un- less specifically mentioned yeast were grown in organic com- plete medium (YPD). The efficiency of suppression of the [PSA suppressible alleles his7-l (UAA), lys2-87(UGA), lysY- A21(UAA), adel-l4(UGA) and ade2-1(UAA) was estimated from the level of growth on media where suppression was required for propagation, i.e., synthetic complete glucose me- dia (SC) lacking histidine (SC-His), lysine (SGLys) and ade- nine (SGAde), respectively, at both 30" and 20". Suppression of the adel-14 and ade2-1 mutations was also scored by a color

test on WD. On WD, colonies of adel and ade2 mutants are red due to the accumulation of a red pigment. Suppression of the these mutations prevents accumulation of this pigment resulting in lighter (pink or white) colonies. The intensity of the color change reflects the efficiency of suppression.

Transformants were grown in media selective for plasmid maintenance, e.g., SC-Ura. Ura- colonies were selected in SC medium containing 1 mg/ml of 5-fluoroorotic acid (+5-FOA; BOEKE et al. 1984). W D medium containing 5 mM guanidine hydrochloride (+GuHCl) was used to eliminate [PSg. To induce the GAL promoter, organic or synthetic complete me- dia containing 20 mg/ml galactose as a single carbon source was used (YPGal and SGal, respectively). In the presence of the GAL4.ER.Wl6 activator (see below) the GAL promoter was induced on organic or synthetic complete glucose media containing @-estradiol (10 or 20 nM; purchased from Sigma).

Plasmids: A series of pEMBL-yex4 (CESARINI and MURRAY 1987) based plasmids containing either the complete SUP?5 gene (pEMBL-SUP35) or deletion alleles controlled by the original SUP35 promoter (see Figure 2) has been described earlier (TER-AVANESYAN et al. 1993). These 2p plasmids carry both the yeast URA3 and LEU2-d selection markers. While plasmids are normally maintained at 10-20 copies per cell (e.g., on SC-Ura medium), they can be overamplified to around 100 copies per cell by selecting for expression of a defective LEU2-d allele on SC-Leu medium (see copy number determination below).

The pGAL : : SUP35 plasmid (also called pVK71) is a YCp50- based CENvector that contains the promoterless SUP35 gene under the control of the inducible CYCl-GAL1 (GAL) pro- moter from pEMBL-yex4. pGAL::sup35-f20 is identical to pGAL: : SUP35 except that it contains a (+ 1) frameshift muta- tion in the 20th codon of SUP35 (GGT to GGCT). This muta- tion was constructed using the U-DNA procedure (KUNKEL 1985): a Sad-XbaI fragment containing the promoterless SUP35 gene from the pGAL: : SUP35 plasmid was inserted into the pBluescriptIISK( +) phagemid (purchased from Stra- tagene); the mutagenic oligonucleotide primer 5' TTGTTG GTTAGCCGTTCTGG 3' was purchased from Amitof; the mu- tant phagemid was identified by the disappearance of a BstEII restriction site and was confirmed by sequence analysis.

Plasmid pHCA/GAL4(1-93) .ER.\rP16 (LOUWON et al. 1994) contains a constitutively expressed fusion of the human estro- gen receptor hormone-binding domain, the GAL4 DNA-bind- ing domain and the viral WIG transcriptional activator. The fusion protein activates the GAL promoter in the presence of @-estradiol. In the absence of galactose the level of promoter induction is determined by the @-estradiol concentration in the medium (LOUVION et al. 1994).

Plasmid pYS-GAL104, kindly provided by S. LINDQUIST, is a centromeric plasmid that contains the HSP104 gene under

Genesis and Variability of [PSA 1377

the control of the GAL promoter and that carries the URA3 selective marker (LINDQUIST and KIM 1996). Overexpression of Hspl04p was shown to eliminate [PSA (CHERNOFF et al. 1995).

The CEN-ACT plasmid (constructed by T. DUNN and D. SHORTLE, Johns Hopkins University, kindly provided by J. STRATHERN), is a YCp50-based vector that contains the 3.5-kb BamHI-EcoRI insertion with the complete ACTl gene.

YEp24 (BOTSTEIN et al. 1979) andYCp50 (ROSE et al. 1987) are standard 2p and centromeric yeast vectors, respectively.

Qualitative tests for the induction of the de novo appearance of the [PSA factor: To test for [PSA induction by pEMBL- based plasmids, transformants selected on SC-Ura were patched to SC-Ura and replica plated two times on either SC- Ura (for moderate copy number induction) or on SGLeu (for high copy number induction). Plasmids were then elimi- nated on +5-FOA and loss of all plasmids was confirmed by replica plating to SC-Ura. Cells were then replica plated to score for suppression efficiency as described above (see also CHERNOFF et al. 1995). Papillation on SC-Ade, SC-His or S G Lys, or the appearance of white or pink papillae on YPD indicated the de novo appearance of [PSA .

To test for [PSA induction by pGAL: : SUP35 or pGAL: : sup35420 plasmids in 74DG94, transformants selected and patched on SGUra were replica plated to SGal-Ura or SGUra- His+p-estradiol, where the GAL promoter was induced. In the latter case a second plasmid, pHCA/GAL4( 1-93) .ER.WlG, was also present. SGUra repressing medium was used as a control. Cultures were then replica plated to YPD or SGAde where the GAL promoter is repressed. Papillation on SGAde or the appearance of white or pink papillae on YPD indicated the de novo appearance of [PSA.

In all cases, the stability of suppressor phenotypes on WD, and instability on +GuHCl, were used as criteria to verify the non-Mendelian inheritance of suppression. The presence of [PSA in 33GD373 derivatives was also confirmed by crosses to 2GH19, a strain that lacks [PSA and is marked by the ade2- 1 ochre mutation and the SUP16 tRNA mutation that can suppress ade2-l only in the [PSd background (COX 1965). The appearance of suppression in the diploids indicated the presence of [PSa in the 33GD373 derivative used in the cross.

Quantitative estimates of the induction of de novo appear- ance of the [PSA factor: To compare the de novo appearance of [PSd in cells bearing moderate and high copy number pEMBL-based plasmids (Table 2 ) transformant colonies of equal size were selected on SGUra (day 3) and SC-Leu (days 5-14). Plasmids that carried certain alleles of SUP35 caused the growth rate on SC-Leu to be dramatically slower than that on SC-Ura (see RESULTS). Therefore, transformant colonies were transferred directly to YPD to allow for plasmid loss, without additional passes on media selective for plasmid main- tenance. After two subsequent replica platings on YPD to in- crease the chance of plasmid loss, cultures were streaked for single colonies and replica plated to identify colonies that had lost the plasmid. Finally colonies that had lost the plasmid were patched on YPD and replica plated to media for the analysis of suppression of nonsense mutations and the pres- ence of [PSA as described above (qualitative test).

In previous studies (CHERNOFF et al. 1993), where direct comparisons with high copy plasmids were not involved, trans- formants were replica plated two times on media selective for plasmid maintenance prior to transfer to YPD. This increased the exposure to excess SUP35 and therefore the proportion of Psi' clones, and is the reason for the difference between the previously reported -20% of Psi+ colonies induced by moderate copy number SUP35 plasmids (CHERNOFF et al. 1993) and the 3% induction reported here (Table 2) .

To quantify the induction of [PSA by plasmids containing

SUP35 alleles under the GAL promoter, equal amounts of cells from transformants selected and grown on SC-Ura were pipetted on GAL-inducing (SGal-Ura) and -repressing (SC- Ura) media and incubated for 2 days. Individual transformant cultures were then washed off and suspensions were diluted and spread on YPD (repressing) medium where the propor- tion of Psi+ colonies was determined by the color test. The numbers of red us. white or pink colonies were counted after 7 days of incubation. A few small yellowish respiratory-deficient (petite) colonies also appeared and were not included in either group. Several white colonies were confirmed to be Psi+ using additional suppression and GuHCl tests as de- scribed above.

DNA manipulations: Routine DNA manipulations were performed according to standard protocols (SAMBROOK et al. 1989). Plasmid copy number in transformants bearing pEMBL-based plasmids was determined using a slot-blot of total DNA isolated from transformants grown on SGUra or SC-Leu and hybridized with the Hind111 fragment from YEp24 plasmid containing the complete UM? open reading frame (ORF). The amount of DNA loaded was normalized using the BamHI-EcoRI fragment from CEN-ACT plasmid con- taining the complete ACT1 ORF as a probe.

Analysis of SUP35 &A and Sup35 protein levels: Yeast were grown to late log phase and the same cultures were used for the analysis of SUP35 mRNA and Sup35 protein levels. RNA isolations and Northern hybridization experiments were performed according to standard protocols (SAMBROOK et al. 1989; SCHMITT et al. 1990). The Sad-XbaI fragment from pGAL : : SUP35 containing the complete SUP?5 ORF was used as a probe. The amount of RNA loaded was normalized to ACTl mRNA levels. Protein extract5 were made as described previously (COLE and REED 1991). Polyclonal Sup35p antibod- ies (DIDICHENKO et al. 1991) were obtained from the labora- tory of M. D. TER-AVANESYAN. Western hybridizations were performed according to the ECL (Amersham) protocol utiliz- ing ECL secondary anti-rabbit antibodies and reagents. The amount of protein loaded was normalized to Coomassie bril- liant blue staining of identical protein gels and to Ponceau staining of hybridization membranes.

RESULTS

The frequency of [PSA appearance is roughly propor- tional to the level of SUP35 amplification: The pres- ence of a moderate copy number plasmid containing the wild-type SUP35 gene has been previously shown to cause nonsense suppression and to induce the de nouo appearance of the [PSA factor (CHERNOFF et aZ. 1993). In Table 2A we show that the frequency of the [PSA factor appearance roughly correlates with the copy number of the SUP35 gene.

Transcriptional induction of a SUP35 gene induces [PSI: To determine if the induction of the de novo a p pearance of [PSA is caused by an excess of SW35 DNA us. mRNA or protein, we used a singlecopy (centromeric) plasmid, pGAL: : SUP35, containing the wild-type SW35 gene fused to the inducible GAL. promoter. Trans- formants of yeast strain 74D694 carrylng pGAL: : SUP35 or the control plasmid, YCp50, were grown on media where the GAL promoter was induced. An increase in SW35 mRNA and Sup35 protein levels was detected by Northern and Western hybridization experiments (Fig- ure 1, A and B, and Table 3). Following induction, trans-

1378 I. L. Derkatch et al.

TABLE 2 The frequency of [PSA appearance caused by different levels of amplification of SUP35 alleles

Plasmid

~ ~~~~~~

No. of transformants No. of clones

Induction Giving rise medium Total to [PSd Total [ P W "

A. pEMBL-yex4 (control)

pEMBL-SUP35

B. pEMBLABst

pEMBL-ABstAHind

pEMBL-Ll2ATG pEMBL-A3ATG pEMBL-ABal2

pEMBL-ABcl

pEMBL-ASal

pEMBL-AHpa

SC-Ura SC-Leu SC-Ura SC-Leu

SC-Ura SGLeu SC-Ura SGLeu SC-Ura SC-Ura SC-Ura SC-Leu SGUra SC-Leu SC-Ura SC-Leu SC-Ura

14 8

16 14

30 16 29 6

28 29 16 4

16 3 9 6

16

0 1 5

13

0 0 0 0 0 0

10 4 9 3 5 3 5

122 172 159 125

170 38

219 49

262 295 150 28

119 28 90 18

158

0 1 (0.6) 5 (3.1)

39 (31.2)

0 0 0 0 0 0

17 (11.3)

20 (16.9) 15 (53.6) 10 (11.1) 4 (22.2) 5 (3.2)

11 (39.3)

~~~~

Yeast strain 33GD373 was transformed with the indicated plasmids and [PSd de novo appearance in the presence of moderate (SGUra) and high (SGLeu) copy number plasmids was compared as described in MATERIALS AND METHODS. Plasmid copy number was determined to be about 20 and 100 copies per cell in transformants grown on SC-Ura and SC-Leu, respectively.

Values in parentheses are percentage.

formants were replica plated to SGAde medium where the GAL promoter was repressed and expression of SW?5 was discontinued (Figure 1C). pGAL: : SUP35, but not YCp50, transformants showed suppressor activity on this medium indicating the appearance of the [PSA fac- tor. Overexpression of SW35 is required for induction of the de novo appearance of [PSr], for this phenomena was not observed when pGAL: : SUP35 transformants were incubated exclusively under repressive conditions.

The fraction of cells that became Psi+ following SUP35 overexpression on galactose was measured quan- titatively. The progeny of 12 pGAL: : SUP35 trans- formants were analyzed as described in MATERIALS AND METHODS. Of 3633 colonies examined, 614 (or -17%) were pink or white after incubation in SGal-Ura (induc- ing) medium, while only one colony out of 2149 was white after incubation in the control SC-Ura (repress- ing) medium. However, this may be an underestimate of the efficiency of [PSd induction since we have ob- served that the pGAL: : SUP35 plasmid inhibits growth of [PSZI-containing 74D694 derivatives in the inducing SGal-Ura medium but not in the repressing SC-Ura me- dium (Figure 1D). In contrast, no growth inhibition was detected for 74D694 strain lacking [PSA. Our data confirm that the previously described incompatibility between [ PSr] and multicopy SUP?5 vectors (CHERNOFF et al. 1988,1992; DACKESAMANSKAYA and TER-AVANESYAN 1991) is indeed due to SUP35 overexpression.

Transcriptional induction of a (+ 1) frameshift mu- tant SUP35 gene fails to induce the appearance of [PSI: To determine if the observed [PSd induction was caused by an excess of SW?5 mRNA vs. protein, we introduced a (+1) frameshift mutation, sup?5-f20, into the SW?5 gene. This mutation created a UAA nonsense triplet at the 21st codon that blocked the synthesis of the Sup35 protein but not mRNA, as shown by Western and Northern analyses (Figure 1, A and B). Aliquots from each culture used for Northern and Western experiments were plated on W D to score for [PSd induction as the appearance of white or pink colonies (see MATERIALS AND METHODS). No Psi+ clones were observed in the progeny of transformants bearing pGAL::sup35-f20 or YCp50 plasmids, while -20% of the progeny from transformants bearing pGAL : : SUP35 were Psi+. The results of the qualitative test (see MATERI- ALS AND METHODS) demonstrating that the induction of the pGAL: :sup35420 plasmid does not cause the de novo appearance of the [PSd factor are presented in Figure 1C: no growth is observed on SC-Ade (repress- ing) medium following GAL promoter induction. Fur- thermore, promoter induction did not cause suppres- sion on SGal-Ade medium (data not shown). This suggests that it is the overproduction of Sup35 protein and not SUP35 mRNA that causes suppression and [PSa factor appearance.

While overexpression of SW?%pecific mRNA was ob-

Genesis and Variability of [PSA 1379

C 0 3 c1 D

Z O

+, e- d

4 8 -$

E 8 d

x S l z x 5 3 x YCpSO YCp50

pGAL:SUP35 -b -b

pGAL:SUP35

pGAL:sup35-f20 pGAL::sup35-f20

FIGURE: 1.- (A) Northern hybridization experiment. Total RNA was isolated from YCp50, pGAL::sup35-f20 and pGAL:: SUP35 transformants of 74D694 grown in SGal-Ura (indicated as +Gal) or SGUra-His+lO n u pestradio1 (in the presence of the pHC'/GAL4( 1-93).ER.W16 plasmid; indicated as +pestradiol). The blot was probed with the pGAI,::SUP35 Snd-X/mI fragment containing the complete SUIV5 coding sequence (indicated as SUP35) and with a fragment containing the complete ACT1 coding sequence (indicated as ACT]; loading control). RNA isolated from each type o f media was analyzed separately. (B) Western hybridization experiment. Proteins were extracted from the transformants listed above grown in SGal-Ura medium, and the blot was probed with Sup35pspecific antibody. Coomassie brilliant blue staining is presented as a loading control, bottom. ( C ) The induction of the & novo appearance of the [I'SII factor under conditions of Sup35 protein overproduction. Spots show growth of 74D694 transformants containing the indicated plasmids on the media listed for GAL promoter induction (SGal-Ura and SGUra-His+PO nu oestradiol) or repression (SGUra), and on SGAde medium following GALpromoter induction or repression. Arrows indicate replica plating. Incubation on medium containing P-estradiol was in the presence of the pHCA/ GAL4( 1-93).ER.W16 plasmid. (D) Inhibition of growth of the [ R ' s r ] 74D694 strain caused by overproduction of Srlp3.5 protein. Growth of transformants of [PSIjcontaining 74D694 strain with the indicated plasmids on media for GAL promoter induction (SGal-Ura; left) and repression (SGUra; right).

served in transformants with either pGAL::SUP35 or pCAL::supS5f20, it was less pronounced in pGAL:: sup35420 transformants due to nonsense-mediated mRNA decay. Disruption of UPFl (LEEDS el nl. 1991) stabilized SUP35specific mRNA in pGAL : : sup35f20 transformants but still did not permit the induction of the appearance of [RYl (data not shown). We also com- pensated for the sup35-f20 mRNA decav by utilizing dif- ferent levels of GAL promoter induction (see Table 3) and demonstrated that a sixfold increase in the amount of wild-type SUP35 mRNA in pGAL::SUP35 trans formants resulted in [PSfl induction, while even a 1 9 fold increase in the amount of mRNA in pGAL::sup35 f20 transformants did not induce [PSll.

We have also transformed a [RVJcontaining derivative of 74D694 with pGAL: : sup35f20 and demonstrated that the growth of transformants was not inhibited in G A L inducing medium selective for plasmid maintenance (Figure 1D). This indicates that. the three phenotypes caused by SUP35 gene overexpression, namely the induc-

tion of [PSll appearance, omnipotent suppression in strains lacking [RSll and growth inhibition in [RSllcon- taining strains are all due to Sup35 protein overproduc- tion.

The Sup35 region required for [PSq factor induction is localized to the N-terminal part of the protein: To localize the regions of SUP35 that are responsible for [PSll factor induction, we utilized a set of multicopy plasmids containing various SUP35 gene fragments (TER-AVANFSYAN et d . 1993). Strains 74D694 and 3%- D373, each bearing the wild-type SUP35 chromosomal copy, were transformed with the plasmids. Suppression of nhl-14, kis7-1 and lys9-A21 nonsense mutations was detected on omission media not selective for plasmid maintenance (SGAde, SGHis and SGLys) and on me- dia selective for the maintenance of the plasmids at moderate copy number (SGUra-Ade, SGUra-His and SGUra-Lys). The analysis of suppression of nonsense mrltations in our strains in the presence of plasmids containing SUP35 gene fragments (Figure 2) gave re-

1380 I. L. Derkatch et nl.

TABLE 3

Induced levels of SUP35specific mRNA

Induction medium YCp50 (control) pGAL : : SUP35 PGAL : s~p35-EO

Relative SIJP35 mRNA levels

Galactose 1 6 2 10 nM P-estradiol 1 18 1 0 20 nM 0-estradiol 1 76 19

Yeast strain 74D694 was transformed with the plasmids indicated above as well as with pHCA/GAL4(1- 93).ER.W16. Transformants were grown on SGal-Ura, SC-Ura-His + 10 nM P-estradiol or Sc-Ura-His + 20 nbl 0-estradiol media and total RNA was isolated. SUP35 mRNA levels in pGAI,:: SUP35 and pGAI,::sup35-F20 transformants were commred to those in YCD~O transformants using Northern hybridization experiments (see Figure 1A).

sults similar to those reported earlier (TER-AVANESYAN et al. 1993). However, two differences deserve mention. First, in contrast to the previous results, we observed suppression in our strains in the presence of the pEMBL-ABst plasmid. Second, the ABstAHind con- struct, which was the least efficient suppressor in the previous paper, suppresses at least as efficiently as the plasmid with the complete SUP35 gene in our strains.

[PSd induction was analyzed after the plasmids main- tained at moderate or high copy number (see MATERIAIS AND METHODS) were eliminated. The results of quantita- tive and qualitative analyses of [PSd induction in the presence of multicopy plasmids bearing SW35 fragments are presented in Table 2B and Figure 2, respectively. Plasmids capable of inducing the de novo appearance of [PSA did so at both moderate and high copy number.

While analyzing the de novo appearance of the [PSA factor in the presence of plasmids overamplified on SC-Leu, we observed that the growth of transformants bearing the complete SUP35 gene or some of its frag- ments was significantly inhibited. These data are pre- sented in Figure 2.

The results show that the region of SUP35 required for suppression of nonsense mutations, the ability to induce the de novo appearance of the [PSA factor and the inhibition of growth under overamplification can be localized to the 5' proximal part of the gene. The shortest fragment capable of causing all these pheno- types encodes the first 154 amino acids of Sup35p. The BstEII fragment encoding amino acids 21-69 in the Sup35 protein overlaps the region required for [PSr] induction and growth inhibition but is not essential for the suppression of nonsense mutations.'

' A s shown previously in other Psi' strains (TEK-AVMEX~N rt 01. 1994), we demonstrated that the BslEII SUP?5 fragment is required for the maintenance of a [PLSIj induced in our experiments. The BstEII fragment was deleted from the chromosomal copy of'the SLY35 gene in a [PSI]-containing derivative of strain 33GD373 and the elimination of [PSfl was confirmed by crossing 28 independent dele- tion isolates to the derivatives of the tester SUP16 strain, 2GHI9, containing and lacking [PSIj. In these crosses weak suppression of the 0&2-l nonsense mutation by SUP16 was observed in hybrids with the [PSI]-containing drrivative of the 2GH19 tester, but not in hy- brids with the tester lacking [PSIj. In analogous crosses of either tester to the original [PSIj 33GD373 strain containing the complete SW?5 gene a& 2-1 was suppressed in hybrids (data not shown).

Different forms of [PSa can be induced in the same yeast strain: [PSZI-containing isolates, induced in one and the same strain by Sup35p overproduction under the same conditions, varied in suppression efficiency. Furthermore, the same array of [PSA efficiencies was obtained independently of whether a pGAL : : SUP35 construct or a multicopy plasmid containing either a complete SUP35 gene or any of the [PSZI-inducing SUP35 fragments was used. Such variation was detected for all strains used in our experiments. The data for strains dU8-132-L28 and 33GD373 are presented in Ta- ble 4. Differences in suppression efficiency associated with [PSA isolates obtained in 74D694 are shown in Figure 3.

Eight independent Psi+ derivatives of 74D694 were chosen for further experiments. Four are characterized by low efficiency of adel-14 suppression leading to pink color on W D and growth on SC-Ade only after 7 days of incubation at 30". The other four are high-efficiency suppressors that are white on W D and grow on SC-Ade after 3-4 days of incubation at 30". Efficiency of suppres- sion in these isolates remained unchanged after 28 gen- erations on YPD. Interestingly, differences in efficiency of suppression appear to be accompanied by differences in mitotic stabilities. [PSIl-containing isolates character- ized as strong suppressors were mitotically very stable during growth on YPD: no derivatives with Psi- pheno- types were detected among at least 3000 colonies tested for each strain. However, [PSA-containing isolates with weak suppression spontaneously produced Psi- colonies with a frequency of 0.1 -1% (see Figure 3).

To check the efficiency of elimination of different [PSI variants, each Psi' derivative was patched and in- cubated on +GuHCl medium two or three consecutive times. The generation times on +GuHCl medium were the same for strains containing or lacking [PSfl . Cells were then streaked for single colonies on YF'D and Psi derivatives were detected as red colonies. After two con- secutive incubations (approximately 14 generations) on +GuHCl 80-93% of the colonies derived from strains with weak suppression were red, while only 55-64% of the colonies derived from strains with strong suppres- sion had lost [PLSIj. After three incubations on +GuHCl

Genesis and Variability of [PSA 1381

SUP35 allele

- (Control)

SUP35

ABst

A2ATG

A3ATG

ABa12

ABcl

ASal

M P a

-0

U -

~

[PSI1

induction

Growth

inhibition

-

+

-

-

-

-

+

+

+

+

Suppression

~

-

++

+

+++

-

-

++++

++++

+++

++

FIGURE 2.-Localization of the Sup35p region required for [PSI factor induction. The SUP35 open reading frame is boxed and the open and filled portions correspond, respectively, to the coding regions for the N-terminal and Gterminal Sup35p domains. The positions of the first three ATG codons are marked. Restriction sites and their positions relative to the beginning of the ORF are as follows: Bs, BstEII (58, 202); H, Hind111 (98,433); B, Bad (461, 1175); Bc, Bcd (713) Hp, HpaI (751, 1237); S, Sad (1444). Yeast strains 33GD.77.7 and 74-D694 were transformed with the pEMRLbased plasmids containing the indicated SUP35 alleles. [PSA induction was analyzed qualitatively as described in .MATERIAIS AND METHoI)s. Results were the same whether plasmids were at moderate or high copy number. To determine the inhibition of growth caused by plasmids at high copy number both the number of days required for the appearance of transformant colonies on SGLeu and the growth rate of transformant? originally selected on SGUra and then transferred to SGLeu were measured. A minus ( - ) indicates the absence of inhibition of growth: the appearance of transformant colonies on SGLeu after 5 days of incubation and growth of replicas to SGLeu after 3 days of incubation. A plus (+) indicates inhibition of growth: the appearance of transformants on SGLeu after 7-14 days and growth of replicas to SGLeu after 410 days. Very strong (++++), strong (+++), medium (++), weak (+) and no (-) suppression was determined at 30" by the level of growth of transformants containing the plasmids at moderate copy number on media where suppression was required for growth. For example, for 74D694 transformants + + + +, + + +, + + or + correspond to growth after 2, 3-5, 5-7 or 10-14 days on SGAde medium, respectively.

(approximately 21 generation) 98.2-99.8% of the c o b cure [PSA on galactose media due to induction of the nies derived from weak suppressors were red, while 6- GAL:: HSP104 construct (CHERNOFF et nl. 1995). Trans- 17% of the progeny of strong suppressors were still formants were grown on SGal-Ura and were then white. Analogously, one weak and one strong [PSq- streaked on YPD. While more than 90% of the deriva- containing strain were transformed with the pYS tives from weak [PS'llcontaining strains had become GAL104 plasmid, which has previously been shown to Psi-, only "50% of the derivatives from strong [PSA-

1382

TABLE 4

I. L. Derkatch et al.

A Variability of suppressor efficiencies of [PSI] factors induced in yeast strains 33GD373 and dU&132-L28

[PSI] isolates

Strong Moderate Weak Strain Total suppressors suppressors suppressors

33GD373 24* 12 3 9 39** 23 9 7

dU8-132-L28 51* 6 28 17

[PSflcontaining derivatives were obtained from trans- formants of the indicated strains with pEMBLSUP35 plasmid at moderate (*) or high (**) copy number. Nonsense suppres- sion was analyzed as described in MATERIALS AND METHODS. For strain dU8-132-L28 strong suppressors are characterized by weak suppression of the lys2-87 nonsense mutation leading to growth on SGLys medium at 20" and efficient suppression of the adel-14 nonsense mutation leading to growth on SG Ade medium at 20" and 30", moderate suppressors are charac- terized by absence of suppression of lys2-87 and efficient sup pression of ad&-1-14 leading to growth on SGAde at both at 20" and 30", weak suppressors are characterized by weak suppres- sion of ndel-14 leading to growth on SGade only at 20". For strain 33GD373 strong suppressors are analogously character- ized by suppression of both his7-1 and lys9-A21 nonsense mu- tations, medium suppressors by suppression of lysPA21 both at 20" and 30" and weak suppressors by weak suppression of lys9-A21 leading to growth on SGLys only at 20". The propor- tion of different types of [PSIJ in the two strains should not be compared since his7-l is relatively better suppressed than 4.~2-8 7.

containing strains had lost [PSA. These data indicate again that [PSA stability is correlated with [PSA sup pressor efficiency.

One might suggest that highly efficient Psi+ deriva- tives contain additional nuclear mutations that modify [ H A efficiency. Such modifiers of [PSA, that have no suppressor activity by themselves in strains lacking [PSA were described previously (ONO et al. 1986, 1991). To check this hypothesis, both GuHCl- and Hspl04in- duced Psi- derivatives of [PSA isolates with both weak and strong suppressor efficiency were transformed with the pGAL : : SUP35 plasmid. [ H A was induced on galac- tose-containing medium as described above. For each strain both weak and strong [PSA-containing derivatives were isolated. This result shows that differences in sup pressor efficiencies are controlled by inducible cyto- plasmic elements themselves, rather than by a nuclear genetic background.

DISCUSSION

Previously, we have found that multicopy plasmids, bearing the SIP35 gene, induce suppression ( C m - NOW et al. 1988,1992) and cause the de novoappearance of the non-Mendelian [PSd factor (CHEWOFF et al. 1993). Since the increase in SW35 copy number is ac- companied by proportional increases in the levels of both SUP35 mRNA (CHERNOFF et al. 1992) and Sup35

strong weak no [PSI] [PSI] [PSI]

YPD

SC-Ade

+GuHCl L

A

FIGURE 3.-Induction of [PSq factors with different prop erties. Psi+ isolates were obtained from the progeny of 7 4 D694[pGAL: : SUP351 transformants incubated on SGal-Urn. The inducing plasmid was then eliminated from isolates ex- hibiting weak and strong suppression. (A) Psi" isolates lacking the inducing plasmid1 were spotted on the indicated media. The strong [PSllcontaining strain grows better on SGAde and is lighter in color on YF'D compared with the weak [PSA - containing strain. These differences are eliminated by incuba- tion on +GuHCZ, that is known to cure [Ed. (B) Strong and weak Psi+ isolates were subcloned on YPD (top and bottom, respectively). The color difference, which reflects dif€erences in suppression efficiency, is mitotically stable. Red sectors and colonies indicate loss of [PSIJ, which occurs more frequently for weak than for strong Psi+ isolates.

protein (DIDICHENKO et al. 1991), these results are con- sistent with the idea that suppression and [PSA appear- ance are consequences of the increase in Sup35p levels. A similar observation was later made for another yeast non-Mendelian element, [ UTE31 : overexpression of the URE2 gene increased the frequency of [ W 3 ] appear-

Genesis and Variability of [PSd 1383

ance (WICKNER 1994). Partially on the basis of these findings WICKNER (1994) suggested that [PSA is a prion conformational derivative of the Sup35 protein, suppos- ing that an excess of the Sup35 protein makes it more likely for at least one molecule to take on the prion conformation. In mammals, the overexpression of the PrP gene results in neurodegenerative disease (WESTA- WAY et aZ. 1994), although the infectious nature of the disease was not confirmed.

The results described in this paper support WICK- NER’S interpretation and confirm that, indeed, overac- cumulation of Sup35 protein is responsible for suppres- sion and [PSd induction: (1) the efficiency of [PSA induction is roughly proportional to the level of SUP35 amplification; (2) transcriptional induction of the wild- type SUP35 gene induces [PSA ; (3) even higher levels of the transcript of the sup35-f20frameshift allele, which do not cause Sup35 protein overaccumulation, fail to induce the appearance of [PSA.

Our previous finding that an intermediate level of the chaperone protein Hspl04p is required for the propagation of [PSA (CHERNOFF et al. 1995) strongly supports the “protein only” model since the only known function of Hspl04p (PARSELL et al. 1994) is to facilitate protein conformational changes. The balance of chaperone proteins participating in Sup35p folding as well as the balance of other proteins forming com- plexes with Sup35p may affect [PSA maintenance and/ or induction. An excess of Sup35p relative to other cellular proteins might increase the chance of Sup35p- Sup35p interactions, which could both initiate and propagate the prion conformational change.

We show that the first 154 amino acids of Sup35p are sufficient to cause the induction of [PSfl. All “prion- inducing” constructs used in our experiments induce suppression of nonsense mutations, while some “sup- pression-inducing’’ constructs do not induce [PSA. In particular, the constructs containing SW35 or SW35N lacking the BstEIJ fragment encoding amino acids 21 - 69 cause suppression but do not induce [PSA. One possibility to explain these findings is that overaccumu- lation of Sup35p or its fragments causes improper intra- cellular partitioning of the protein, leading to a titra- tion of limited translational components and, as a result, to the malfunctioning of the translational ma- chinery and to suppression. This is supported by the previously published observation that a 10-fold overpro- duction of Sup35p results in only a twofold increase of the ribosome-associated sup35p fraction (DIDICHENKO et al. 1991). Also, when SUP45 is overexpressed together with SUP35, thus preventing titration of Sup45p, non- sense mutations are not suppressed. Instead, an antisup pressor phenotype is observed, indicating the efficient termination of translation (STANSF~ELD et al. 1995). Al- ternatively, suppression could be due to the accumula- tion of improperly folded Sup35p. The fact that HSPlO4 deletion (hsplO4-A) blocks not only [PSA induction

but also multicopy SUP35induced suppression ( CHER- NOFF et al. 1995) suggests that accumulated Sup35p iso- forms are different in HSPl04and hsplO4-A cells. Over- expression of certain SW35N fragments could induce the truncated and/or complete Sup35 protein to take on a conformation that interferes with termination fac- tor activity, but that is not [PSA because it is not self- propagating. This would mean that “prionization” is not a direct consequence of suppression, but rather it occurs as another step that requires the BstEII region.

When fragments containing the BstEII region are overexpressed, they cause the complete Sup35p to adopt the prion conformation. Apparently the frag- ments can fold in a truncated [PSA-like conformation that can work in trans. The exact mechanism of [PSA induction under conditions of overproduction of Sup35p or its fragments is not known. However, either of two possibilities, stochastic folding of Sup35p (or Sup35Np) into the prion conformation, or prion forma- tion resulting from the interaction of two non-prion Sup35p (or Sup35Np) molecules, would be more prob- able when the protein is overaccumulated and un- bound. Following the initial appearance of [PSA its maintenance is presumed to involve interactions of Sup35p with [PSA (or with truncated [PSA ). Previous work determined which SUP35Nfragments can support [PSA maintenance when expressed in trans to SW35C (TER-AVANESYAN et al. 1994). However, it was not possi- ble to predict that overproduction of the region re- quired for maintenance of preexisting [PSA would be sufficient for seed formation necessary for the de nouo appearance of [PSA. As it turns out, the “[PSA-induc- ing” region, defined in our work as the 154 N-terminal amino acids, is so far indistinguishable from the region sufficient for [PSA maintenance. Our data also suggest that the Sup35p domain overlapping the region en- coded by the BstEII fragment of the gene is required for seed formation in addition to [PSA maintenance, since we failed to detect the de nouo appearance of [PSA when SUP35 or SUP35N fragments lacking BstEII frag- ment were overexpressed in the presence of the com- plete chromosomal copy of SW35, which should have been sufficient for propagation of any prion seeds that appeared. Earlier findings that both a chromosomal deletion of the BstEII fragment and a missense muta- tion in codon 58 cause the Pnm phenotype, i.e., loss of existing [PSA factor (DOEL et al. 1994; TER-AVANESYAN et al. 1994) demonstrate the significance of this region for [PSA maintenance. The piece encoded by the BstEII fragment includes the beginning of a region of tandem oligopeptide repeats (amino acids 55-96) that shows a remarkable structural similarity to the corresponding region of mammalian PrP (COX 1994; KUSHNIROV et al. 1995). In humans, expansions of the repeats were associated with several familial cases of Creutzfeldt-Ja- cob disease (reviewed by GOLDFARB et aZ. 1994). The fact that this region is similar in the yeasts S. cermisiae

1384 I. L. Derkatch et al.

and Pichia methanolica (formerly called P. p inus ) despite the variability of other parts of Sup35Np (KUSHNIROV et al. 1990) further suggests the involvement of oligo- peptide repeats in [PSII-like prionization. However, the possibility that N-terminal repeats are not crucial for scrapie propagation has been raised by the finding that a deletion of all but a single repeat in mice does not affect susceptibility to disease (FISCHER et al. 1996). Fur- thermore, mutations affecting the development of prion diseases were found throughout the PrP gene in mammals (reviewed by COLLINGE and PALMER 1994; GOLDFARB et al. 1994; PRUSINER 1994; BALDWIN et al. 1995). This suggests that in Sup35p additional amino acids upstream of the 154th amino acid may also be essential for the prionization process. It should be noted that the whole PrP protein is only a little longer than the Sup35p “prion-inducing” domain (BOL,TON et al. 1982; OESCH et al. 1985).

While we have localized the [PSZI-inducing domain of Sup35p to Sup35Np, the functional domain of Sup35p that is required for cell viability is in Sup35Cp (TER-AVANESYAN et al. 1993) and they do not overlap. The other known yeast prion, Ure2p, shares this feature (MASISON and WICKNER 1995). Such a chimeric organi- zation including the PrP-like N-terminus and a distinct functional domain of the protein in the C-proximal part may be a common pattern of yeast prion elements.

The presence of [PSd in yeast strains does not sig- nificantly affect growth rate or viability, although the fidelity of translation is decreased (for review see COX Pt al. 1988). However, Sup35p overproduction in [PSd strains or high levels of SW35 amplification in strains lacking [PSII, both cause growth inhibition (Figures 1D and 2) . These effects are unlikely to result from an excess of translational errors since overexpression of the A B s t A H i n d allele, which causes efficient suppres- sion, does not cause growth inhibition. Rather, only overexpression of SUP35 alleles that induce [PSA has the toxic effect. Furthermore, since overexpression of the wild-type SW35 allele is among those that are toxic, the growth inhibition is unlikely to result from an insuf- ficient level of functional Sup35p. Instead, a harmful accumulation of Sup35p isoforms may cause the toxic effect by physically damaging the cell, or by binding essential proteins causing their depletion.

It is commonly known that the suppression efficiency of the [PSA factor varies in different strains. Genetic background undoubtedly contributes to such variations (see Cox et al. 1988). Nevertheless, not all variations in the properties of yeast non-Mendelian factors affecting translational suppression could be easily attributed to strain genetic background: the GuHC1-curable factor [ETA] with inheritance patterns different from those of [PSIJ, causing lethality in combination with certain sup35 alleles, was described in different yeast strains (LIEBMAN and ALL-ROBYN 1984). A detailed study of different strains in the Peterhof Genetic Collection and

selection in the same strain suggested the existence of [PSII-like non-Mendelian factors of different suppres- sor efficiencies (0. N. TIKHODEEV and S. G. INGE-VECH- TOMOV, unpublished results). In this paper we show for the first time that [PSa factors of different efficiencies are induced in one and the same strain by overproduc- tion of one and the same Sup35 protein. While other interpretations are possible, we favor the one that is based on analogy with mammalian prion “strains” (BE- SSEN et nl. 1995) and states that Sup35p can take on more than one prion conformation and that different conformations of Sup35p correspond to [PSA factors of different suppression efficiencies and stabilities. If so, one could suggest that there could also be multiple [psi-] conformations that do not result in translational readthrough. From this point of view, the appearance of [PSA may be considered as a “by-product” of the Sup35p conformational switches, which normally serve a yet unknown regulatory function.

W’e thank M. D. TER-AVANE.WAN (Institute ofExperimental Cardiol- ogy, Cardiology Research Center, Moscow, Russia) for providing Sup35pspecific antibodies and the strain 2GHI9, as well as for help- ful discussions and critical reading of the manuscript. We are also grateful to S. LINDQUIST (University of Chicago) for providing the pYSGAL104 plasmid and for helpful comments on the mannscript, to 0. V . TARUNINA (St. Petersburg State University, St. Petersburg, Russia) for the strain dU8-132-128, to 0. N. T ” x V (St. Petersburg State University, St. Petersburg, Russia) for sharing his unpublished data and to M. BRAIII.EY and G. NEU-NAM for technical assistance. This work was partially supported by a Human Frontier Sciencr Program grant (RG360/93M; S.W.L.), a Frontiers in Genetics grant (Y.0.C.) and Russian Foundation for Fundamental Research grants (S.G.I.-\’. and V.V.K.).

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Communicating editor: F. WINSTON