Figures & data
Figure 1 Synthetic lethality between [PSI+] and mutant alleles of SUP45. [PSI+]S, [PSI+]W and [psi-] derivatives of the strain 1A-D1628 [pRS316/SUP45] were transformed with the plasmids pRS315/SUP45 or pRS315/sup45. Resulting transformants were replica plated to 5-FOA medium to select against the URA3 plasmid pRS316/SUP45, and photographed after five days of incubation. Strong variant of [PSI+] ([PSI+]S) reveals synthetic lethality with sup45 missense (m) and nonsense (n) alleles whereas weak variant of [PSI+] ([PSI+]W) demonstrates lethality with some missense alleles and sublethality with all tested nonsense alleles of SUP45 gene.
![Figure 1 Synthetic lethality between [PSI+] and mutant alleles of SUP45. [PSI+]S, [PSI+]W and [psi-] derivatives of the strain 1A-D1628 [pRS316/SUP45] were transformed with the plasmids pRS315/SUP45 or pRS315/sup45. Resulting transformants were replica plated to 5-FOA medium to select against the URA3 plasmid pRS316/SUP45, and photographed after five days of incubation. Strong variant of [PSI+] ([PSI+]S) reveals synthetic lethality with sup45 missense (m) and nonsense (n) alleles whereas weak variant of [PSI+] ([PSI+]W) demonstrates lethality with some missense alleles and sublethality with all tested nonsense alleles of SUP45 gene.](/cms/asset/e491e0df-6615-4f47-9763-c460001fed73/kprn_a_10904533_f0001.gif)
Figure 2 Combination of two sup45 alleles in diploid [PSI+] strain is lethal. The [PSI+] and [psi-] variants of haploid strains 90-D201 (wild-type SUP45) and K62-90-D201 (sup45-k62) (vertical lines) were mated to a haploid strain 1A-D1628 bearing either pRS315/SUP45 or pRS315/sup45 plasmid (horizontal lines). Diploids were selected by incubation on the medium lacking uracil and phenylalanine for five days. Combination of two different mutant alleles of SUP45 gene is lethal in diploid [PSI+] strain but not in [psi-].
![Figure 2 Combination of two sup45 alleles in diploid [PSI+] strain is lethal. The [PSI+] and [psi-] variants of haploid strains 90-D201 (wild-type SUP45) and K62-90-D201 (sup45-k62) (vertical lines) were mated to a haploid strain 1A-D1628 bearing either pRS315/SUP45 or pRS315/sup45 plasmid (horizontal lines). Diploids were selected by incubation on the medium lacking uracil and phenylalanine for five days. Combination of two different mutant alleles of SUP45 gene is lethal in diploid [PSI+] strain but not in [psi-].](/cms/asset/fd6245d4-462d-436a-bde1-e37fa39e8374/kprn_a_10904533_f0002.gif)
Figure 3 Heterozygous mutant alleles of SUP45 exhibit synthetic lethality with [PSI+]S in SUP45 dose-dependent manner. (A) Isogenic strains OT55 [PSI+]W (W) and OT56 [PSI+]S (S) and their [psi-] (-) derivatives (vertical lines) were mated to derivatives of the strain 1A-D1628 bearing the wild-type (WT), missense (m) or nonsense (n) allele of the SUP45 gene (horizontal lines). Plates were replica plated onto the medium selective for diploids, and incubated for four days. Synthetic lethality was observed in all [PSI+]S SUP45/sup45 combinations, except two weak sup45 missense mutations (sup45–113 and sup45–115). (B) The cells of the [PSI+]S strain OT56 were individually mated to the cells of the 1A-D1628 derivatives, bearing the wild-type (WT), missense (sup45–103) or nonsense (sup45–105) allele of the SUP45 gene on a plasmid. Photographs were made before the incubation (0) and after two hours (2h), four hours (4h) and two days (2d) of the incubation on YPD medium. At least ten individual zygotes were analyzed for each combination. Synthetic lethality in the individual zygotes was observed for [PSI+]S SUP45/sup45 combinations after 2–6 cell divisions. (C) Plasmid loss assay was performed on diploids obtained in the cross of OT56 [PSI+]S bearing pRS316/SUP45 plasmid with derivatives of 1A-D1628 (sup45Δ) transformed with pRS315/SUP45 (WT) or pRS315/sup45–103 (103) or pRS315/sup45–105 (105) plasmids. Diploids were tested by frequency of spontaneous plasmid loss at non-selective conditions. The mean values and standard deviations, calculated on the basis of three independent experiments, are presented in each case. [PSI+]S diploids heterozygous for mutation in the SUP45 gene are viable only in the presence of additional copy of the SUP45 gene on the plasmid.
![Figure 3 Heterozygous mutant alleles of SUP45 exhibit synthetic lethality with [PSI+]S in SUP45 dose-dependent manner. (A) Isogenic strains OT55 [PSI+]W (W) and OT56 [PSI+]S (S) and their [psi-] (-) derivatives (vertical lines) were mated to derivatives of the strain 1A-D1628 bearing the wild-type (WT), missense (m) or nonsense (n) allele of the SUP45 gene (horizontal lines). Plates were replica plated onto the medium selective for diploids, and incubated for four days. Synthetic lethality was observed in all [PSI+]S SUP45/sup45 combinations, except two weak sup45 missense mutations (sup45–113 and sup45–115). (B) The cells of the [PSI+]S strain OT56 were individually mated to the cells of the 1A-D1628 derivatives, bearing the wild-type (WT), missense (sup45–103) or nonsense (sup45–105) allele of the SUP45 gene on a plasmid. Photographs were made before the incubation (0) and after two hours (2h), four hours (4h) and two days (2d) of the incubation on YPD medium. At least ten individual zygotes were analyzed for each combination. Synthetic lethality in the individual zygotes was observed for [PSI+]S SUP45/sup45 combinations after 2–6 cell divisions. (C) Plasmid loss assay was performed on diploids obtained in the cross of OT56 [PSI+]S bearing pRS316/SUP45 plasmid with derivatives of 1A-D1628 (sup45Δ) transformed with pRS315/SUP45 (WT) or pRS315/sup45–103 (103) or pRS315/sup45–105 (105) plasmids. Diploids were tested by frequency of spontaneous plasmid loss at non-selective conditions. The mean values and standard deviations, calculated on the basis of three independent experiments, are presented in each case. [PSI+]S diploids heterozygous for mutation in the SUP45 gene are viable only in the presence of additional copy of the SUP45 gene on the plasmid.](/cms/asset/a027c464-7aab-4bab-9014-57f98139efdd/kprn_a_10904533_f0003.gif)
Figure 4 Mutant suppressor tRNA has a codon-specific effect on the synthetic lethality. [PSI+] and [psi-] variants of BSC783/4c strain (vertical lines) carrying suppressor tRNA SUQ5 were mated with 1A-D1628 derivatives bearing missense or nonsense sup45 alleles (horizontal lines) on pRS315 plasmid and replica-plated on the medium selective for hybrids. Pictures were taken after four days of incubation.
![Figure 4 Mutant suppressor tRNA has a codon-specific effect on the synthetic lethality. [PSI+] and [psi-] variants of BSC783/4c strain (vertical lines) carrying suppressor tRNA SUQ5 were mated with 1A-D1628 derivatives bearing missense or nonsense sup45 alleles (horizontal lines) on pRS315 plasmid and replica-plated on the medium selective for hybrids. Pictures were taken after four days of incubation.](/cms/asset/ff4c6d3c-b2a4-49da-9196-22a8c61bbb07/kprn_a_10904533_f0004.gif)
Figure 5 Synthetic lethality of [PSI+]S and mutant sup45 alleles is compensated by Sup35C. (A) OT56 [PSI+] strain and its [psi-] derivative carrying centromeric or multicopy (*) plasmid that encodes C-terminal domain of Sup35 (vertical lines) were mated to derivatives of the strain 1A-D1628 (designed the same as in ). Plates were replica plated onto the medium selective for diploids, and incubated for four days. Presence of Sup35C on centromeric or multicopy plasmid neutralize the synthetic lethality of [PSI+]S and mutations in the SUP45 gene in diploid strain. (B) Levels of Sup45 are increased in the presence of C-terminal domain of Sup35. [PSI+]S strain OT56 and its [psi-] derivative were transformed with the plasmid pYX242/SUP35MC or control vector pYX242. Crude cell extracts was prepared, run on SDS-PAGE, transferred to nitrocellulose filter and reacted to antibodies against Sup45, Sup35 and tubulin. Blots were analyzed by densitometry, and Sup45 amounts were normalized by using tubulin as a loading control. Sup45/tubulin ratio in the control sample in the strain bearing an empty plasmid is taken as one in each case.
![Figure 5 Synthetic lethality of [PSI+]S and mutant sup45 alleles is compensated by Sup35C. (A) OT56 [PSI+] strain and its [psi-] derivative carrying centromeric or multicopy (*) plasmid that encodes C-terminal domain of Sup35 (vertical lines) were mated to derivatives of the strain 1A-D1628 (designed the same as in Fig. 2). Plates were replica plated onto the medium selective for diploids, and incubated for four days. Presence of Sup35C on centromeric or multicopy plasmid neutralize the synthetic lethality of [PSI+]S and mutations in the SUP45 gene in diploid strain. (B) Levels of Sup45 are increased in the presence of C-terminal domain of Sup35. [PSI+]S strain OT56 and its [psi-] derivative were transformed with the plasmid pYX242/SUP35MC or control vector pYX242. Crude cell extracts was prepared, run on SDS-PAGE, transferred to nitrocellulose filter and reacted to antibodies against Sup45, Sup35 and tubulin. Blots were analyzed by densitometry, and Sup45 amounts were normalized by using tubulin as a loading control. Sup45/tubulin ratio in the control sample in the strain bearing an empty plasmid is taken as one in each case.](/cms/asset/3cbe59bd-4a3a-47bd-b4bc-72614787b593/kprn_a_10904533_f0005.gif)
Table 1 Yeast strains
Table 2 Yeast plasmids
Table 3 Frequency of zygote formation in various strain combinations