HIV encephalitis is the common pathologic correlate of HIV-dementia (HAD). HIV-infected brain mon... more HIV encephalitis is the common pathologic correlate of HIV-dementia (HAD). HIV-infected brain mononuclear phagocytes (MP) (macrophages and microglia) are reservoirs for persistent viral infection. When activated, MP contribute to neuronal damage. Such activated and virus-infected macrophages secrete cellular and viral factors, triggering neural destructive immune responses. Our Center's laboratories have begun to decipher the molecular and biochemical pathways for MP-mediated neuronal damage in HAD. This review will discuss the salient clinical and pathological features of HAD and highlight the recent advances made, by our scientists and elsewhere, in unraveling disease mechanisms, including the role of chemokines and their receptors in the neuropathogenesis of HIV-1 encephalitis.
In vivo propagation of [PSI ؉ ], an aggregation-prone prion isoform of the yeast release factor S... more In vivo propagation of [PSI ؉ ], an aggregation-prone prion isoform of the yeast release factor Sup35 (eRF3), has previously been shown to require intermediate levels of the chaperone protein Hsp104. Here we perform a detailed study on the mechanism of prion loss after Hsp104 inactivation. Complete or partial inactivation of Hsp104 was achieved by the following approaches: deleting the HSP104 gene; modifying the HSP104 promoter that results in low level of its expression; and overexpressing the dominant-negative ATPase-inactive mutant HSP104 allele. In contrast to guanidine-HCl, an agent blocking prion proliferation, Hsp104 inactivation induced relatively rapid loss of [PSI ؉ ] and another candidate yeast prion, [PIN ؉ ]. Thus, the previously hypothesized mechanism of prion dilution in cell divisions due to the blocking of prion proliferation is not sufficient to explain the effect of Hsp104 inactivation. The [PSI ؉ ] response to increased levels of another chaperone, Hsp70-Ssa, depends on whether the Hsp104 activity is increased or decreased. A decrease of Hsp104 levels or activity is accompanied by a decrease in the number of Sup35 PSI؉ aggregates and an increase in their size. This eventually leads to accumulation of huge agglomerates, apparently possessing reduced prion forming capability and representing dead ends of the prion replication cycle. Thus, our data confirm that the primary function of Hsp104 in prion propagation is to disassemble prion aggregates and generate the small prion seeds that initiate new rounds of prion propagation (possibly assisted by Hsp70-Ssa).
HIV encephalitis is the common pathologic correlate of HIV-dementia (HAD). HIV-infected brain mon... more HIV encephalitis is the common pathologic correlate of HIV-dementia (HAD). HIV-infected brain mononuclear phagocytes (MP) (macrophages and microglia) are reservoirs for persistent viral infection. When activated, MP contribute to neuronal damage. Such activated and virus-infected macrophages secrete cellular and viral factors, triggering neural destructive immune responses. Our Center's laboratories have begun to decipher the molecular and biochemical pathways for MP-mediated neuronal damage in HAD. This review will discuss the salient clinical and pathological features of HAD and highlight the recent advances made, by our scientists and elsewhere, in unraveling disease mechanisms, including the role of chemokines and their receptors in the neuropathogenesis of HIV-1 encephalitis. ß
Tuberculosis has plagued humankind since prehistoric times, as is evident from characteristic les... more Tuberculosis has plagued humankind since prehistoric times, as is evident from characteristic lesions on human skeletons dating back to the Neolithic period. The disease in man is due predominantly to infection with either Mycobacterium tuberculosis or Mycobacterium bovis, both members of the M. tuberculosis (MTB) complex. A number of studies have shown that when conditions permit, surviving mycobacterial DNA may be amplified from bone by PCR. Such ancient DNA (aDNA) analyses are subject to stringent tests of authenticity and, when feasible, are invariably limited by DNA fragmentation. Using PCRs based on single-nucleotide polymorphic loci and regions of difference (RDs) in the MTB complex, a study was made of five Iron Age individuals with spinal lesions recovered from the cemetery of Aymyrlyg, South Siberia. A sensitive screening PCR for MTB complex mycobacteria was positive in four out of the five cases. Genotyping evidence indicated that all four cases were due to infection with M. bovis rather than M. tuberculosis and the data were consistent with the proposed phylogenetic model of the MTB complex. This is believed to be the first report of M. bovis causing Pott's disease in archaeological human remains. The study shows that genotyping of ancestral strains of MTB complex mycobacteria from contexts of known date provides information which allows the phylogeny of the model to be tested. Moreover, it shows that loss of DNA from RD4, which defines classic M. bovis, had already occurred from the genome over 2000 years before the present.
This is a brief summary of the animal models session held during the 12th Annual Meeting of the S... more This is a brief summary of the animal models session held during the 12th Annual Meeting of the Society on NeuroImmune Pharmacology, Santa Fe, NM, USA. This session provided important information for participants on availability and utility of animal models for the studies of HIV-1 central nervous system infection and drug abuse. It highlighted animal model relevance to human disease/condition, its utility for the studies of pathogenesis, potential importance for the development of therapeutics, and demonstrated limitations/pitfalls.
Injection of human immunodeficiency virus type 1 (HIV-1)infected human monocyte-derived macrophag... more Injection of human immunodeficiency virus type 1 (HIV-1)infected human monocyte-derived macrophages (MDMs) into the basal ganglia of severe combined immunodeficient mice recapitulates histopathologic features of HIV-1 encephalitis (HIVE). Here, we show that the neural damage in HIVE mice extends beyond the basal ganglia and is associated with cognitive impairment. Morris water maze tests showed impaired spatial learning 8 d after MDM injection. Moreover, impaired synaptic potentiation in the hippocampal CA1 subregion was demonstrated at 8 and 15 d. By day 15, post-tetanic, shortterm, and long-term potentiation were reduced by 14.1, 29.5, and 45.3% in HIVE mice compared with sham-injected or control animals. Neurofilament (NF) and synaptophysin (SP) antigens were decreased significantly in the CA2 hippocampal subregion of HIVE mice with limited neuronal apoptosis. By day 15, the CA2 region of HIVE mice expressed 3.8-and 2.6-fold less NF and SP than shams. These findings support the notion that HIV-1-infected and immune-competent brain macrophages can cause neuronal damage at distant anatomic sites. Importantly, the findings demonstrate the value of the model in exploring the physiological basis and therapeutic potential for HIV-1-associated dementia.
Alterations in hippocampal physiology affect cognition in human immunodeficiency virus type 1 (HI... more Alterations in hippocampal physiology affect cognition in human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD). The mechanism for how this occurs is not well understood. To address this, we investigated how changes in synaptic transmission and plasticity are affected by viral infection and macrophage activation using a severe combined immunodeficiency mouse model of human HIV-1 encephalitis (HIVE). HIVE was induced in mice by stereotactic injection of HIV-1-infected human monocyte-derived macrophages (MDM) into the striatum. Animals were sacrificed after 3, 7 and 15 days. Hippocampal slices were prepared from HIV-1, MDM-and sham-injected animals. Electrically evoked field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. Neuronal physiology was assessed by input-output and by long-term potentiation (LTP) assays. We observed that a higher stimulation intensity (mA) was required to induce a 1-mV response in the HIVE mice 0.32؎0.06) compared with shams (0.17؎0.01) at day 7. The stimulation intensities at day 15 were 0.44؎0.07 and 0.23؎0.05 in the HIVE and shams, respectively. An impairment of synaptic function was detected through measuring synaptic responses induced by stimuli with different intensities. Paired-pulse facilitation (PPF) showed deficits in HIVE mice at days 3, 7, and 15. At day 3, PPF ratios were 1.13؎0.02 and 1.24؎0.04 in HIVE and sham. The induction and maintenance of LTP was also impaired in HIVE mice. The average magnitude of LTP was 131.23؎15.26% of basal in HIVE as compared with sham animals of 232.63؎24.18%. MDM-injected mice showed an intermediate response. Taken together, the results show a range of neuronal synaptic transmission and plasticity changes in HIVE mice that may reflect the mechanisms of cognitive dysfunction in human HAD.
Secretory products from HIV-1-infected immune-competent mononuclear phagocytes (MP) damage neuron... more Secretory products from HIV-1-infected immune-competent mononuclear phagocytes (MP) damage neuronal dendritic arbor (Zheng et al., 2001). The mechanism behind neuronal injury and whether it is species and/or viral strain dependent is not fully understood. To these ends, we investigated whether HIV-1-infected and lipopolysachharide (LPS)-activated MDM elicit neuronal injury in primary human neurons. Neuronal damage was compared to that seen in rat neurons. Utilizing a spectrum of HIV-1 strains to infect human monocyte-derived macrophages (MDM), productive viral replication proved necessary, but not sufficient, for neuronal injury. Neuronal demise was induced by virion-free HIV-1-infected and immune-activated MDM culture supernatants. Maximal alterations in glutamate mediated neuronal signaling, resulted from exposure to secretory products from HIV-1-infected and immune-activated MDM. Apoptosis was the predominant mechanism of cell death induced by HIV-1-infected and LPS-treated MDM. Importantly, neuronal injury and increases in calcium influx mediated by HIV-1-infected and immune-activated MDM culture supernatants was partially blocked by the N-methyl D-aspartate (NMDA) receptor antagonist, MK 801. These data support a primary role for immune-activation in MP neurotoxic activities. The upregulation of NMDA receptor sensitive soluble factors and neuronal apoptosis by HIV-1-infected and immune-activated MDM provide unique insights into links between soluble factors, produced as a consequence of MP immunity, and neuronal demise in HAD.
, a prion-like form of the release factor Sup35, was shown to be regulated by the interplay betwe... more , a prion-like form of the release factor Sup35, was shown to be regulated by the interplay between chaperone proteins Hsp104 and Hsp70. While overproduction of Hsp104 protein cures cells of [PSI], overproduction of the Ssa1 protein of the Hsp70 family protects [PSI] from the curing effect of Hsp104. Here we demonstrate that another protein of the Hsp70 family, Ssb, previously implicated in nascent polypeptide folding and protein turnover, exhibits effects on [PSI] which are opposite those of Ssa. Ssb overproduction increases, while Ssb depletion decreases, [PSI] curing by the overproduced Hsp104. Both spontaneous [PSI] formation and [PSI] induction by overproduction of the homologous or heterologous Sup35 protein are increased significantly in the strain lacking Ssb. This is the first example when inactivation of an unrelated cellular protein facilitates prion formation. Ssb is therefore playing a role in protein-based inheritance, which is analogous to the role played by the products of mutator genes in nucleic acid-based inheritance. Ssb depletion also decreases toxicity of the overproduced Sup35 and causes extreme sensitivity to the [PSI]-curing chemical agent guanidine hydrochloride. Our data demonstrate that various members of the yeast Hsp70 family have diverged from each other in regard to their roles in prion propagation and suggest that Ssb could serve as a proofreading component of the enzymatic system, which prevents formation of prion aggregates. on September 5, 2014 by guest http://mcb.asm.org/ Downloaded from MATERIALS AND METHODS Yeast strains. The S. cerevisiae strains used in this study are described in Table 1. The haploid [PSI ϩ ] strains OT55 and OT56, also called [PSI ϩ ]1-1-74-D694 and [PSI ϩ ]7-74-D694, respectively (21, 22, 39), and isogenic [psi Ϫ PIN ϩ ] strain OT60, also called [psi Ϫ ]-74-D694 (11, 22), were described earlier. The haploid [PSI ϩ ] strains GT81-1C and GT81-1D were obtained by A. Galkin as a result of sporulating and dissecting the diploid strain GT81, constructed via HO-mediated self-homozygotization of the haploid strain GT56-34D (12). The [PSI ϩ ] strains GT128, containing the ssb1⌬::HIS3 disruption, and GT127, containing the double ssb1⌬::HIS3 ssb2⌬::URA3 disruption (ssb1/2⌬), are derivatives of strain GT81-1C, constructed as described below. Strain GT146 is a spontaneous Ura Ϫ derivative of strain GT127, selected on medium containing 5-fluoroorotic acid (30). The [psi Ϫ PIN ϩ ] derivatives of strains GT81-1C and GT146 were obtained by transforming these strains with plasmid pYS-GAL104 (see below), which bears the HSP104 gene under control of the GAL promoter, and curing [PSI] as a result of galactose-induced overproduction of Hsp104 as described earlier . The resulting [psi Ϫ ] derivatives were subsequently cured of plasmid pYS-GAL104. The [psi Ϫ pin Ϫ ] derivatives of strains GT81-1C, GT127, and GT146 were obtained by curing cells of [PSI] and [PIN] after growth on YPD medium containing 5 mM guanidine hydrochloride (GuHCl) (22). The haploid strain GT84-5A is isogenic to GT81-1C except that it contains the hsp104⌬::URA3 disruption, which eliminates [PSI]. This strain was constructed by sporulating and dissecting diploid strain GT84, which is a GT81 derivative with one of the HSP104 copies disrupted via direct transplacement with the hsp104⌬::URA3 allele as described above (11). All strains contain the [PSI]-suppressible UGA mutation ade1-14, so that [PSI ϩ ] strains are white and Ade ϩ , while [psi Ϫ ] strains are red and Ade Ϫ , as described previously .
HIV encephalitis is the common pathologic correlate of HIV-dementia (HAD). HIV-infected brain mon... more HIV encephalitis is the common pathologic correlate of HIV-dementia (HAD). HIV-infected brain mononuclear phagocytes (MP) (macrophages and microglia) are reservoirs for persistent viral infection. When activated, MP contribute to neuronal damage. Such activated and virus-infected macrophages secrete cellular and viral factors, triggering neural destructive immune responses. Our Center's laboratories have begun to decipher the molecular and biochemical pathways for MP-mediated neuronal damage in HAD. This review will discuss the salient clinical and pathological features of HAD and highlight the recent advances made, by our scientists and elsewhere, in unraveling disease mechanisms, including the role of chemokines and their receptors in the neuropathogenesis of HIV-1 encephalitis.
In vivo propagation of [PSI ؉ ], an aggregation-prone prion isoform of the yeast release factor S... more In vivo propagation of [PSI ؉ ], an aggregation-prone prion isoform of the yeast release factor Sup35 (eRF3), has previously been shown to require intermediate levels of the chaperone protein Hsp104. Here we perform a detailed study on the mechanism of prion loss after Hsp104 inactivation. Complete or partial inactivation of Hsp104 was achieved by the following approaches: deleting the HSP104 gene; modifying the HSP104 promoter that results in low level of its expression; and overexpressing the dominant-negative ATPase-inactive mutant HSP104 allele. In contrast to guanidine-HCl, an agent blocking prion proliferation, Hsp104 inactivation induced relatively rapid loss of [PSI ؉ ] and another candidate yeast prion, [PIN ؉ ]. Thus, the previously hypothesized mechanism of prion dilution in cell divisions due to the blocking of prion proliferation is not sufficient to explain the effect of Hsp104 inactivation. The [PSI ؉ ] response to increased levels of another chaperone, Hsp70-Ssa, depends on whether the Hsp104 activity is increased or decreased. A decrease of Hsp104 levels or activity is accompanied by a decrease in the number of Sup35 PSI؉ aggregates and an increase in their size. This eventually leads to accumulation of huge agglomerates, apparently possessing reduced prion forming capability and representing dead ends of the prion replication cycle. Thus, our data confirm that the primary function of Hsp104 in prion propagation is to disassemble prion aggregates and generate the small prion seeds that initiate new rounds of prion propagation (possibly assisted by Hsp70-Ssa).
HIV encephalitis is the common pathologic correlate of HIV-dementia (HAD). HIV-infected brain mon... more HIV encephalitis is the common pathologic correlate of HIV-dementia (HAD). HIV-infected brain mononuclear phagocytes (MP) (macrophages and microglia) are reservoirs for persistent viral infection. When activated, MP contribute to neuronal damage. Such activated and virus-infected macrophages secrete cellular and viral factors, triggering neural destructive immune responses. Our Center's laboratories have begun to decipher the molecular and biochemical pathways for MP-mediated neuronal damage in HAD. This review will discuss the salient clinical and pathological features of HAD and highlight the recent advances made, by our scientists and elsewhere, in unraveling disease mechanisms, including the role of chemokines and their receptors in the neuropathogenesis of HIV-1 encephalitis. ß
Tuberculosis has plagued humankind since prehistoric times, as is evident from characteristic les... more Tuberculosis has plagued humankind since prehistoric times, as is evident from characteristic lesions on human skeletons dating back to the Neolithic period. The disease in man is due predominantly to infection with either Mycobacterium tuberculosis or Mycobacterium bovis, both members of the M. tuberculosis (MTB) complex. A number of studies have shown that when conditions permit, surviving mycobacterial DNA may be amplified from bone by PCR. Such ancient DNA (aDNA) analyses are subject to stringent tests of authenticity and, when feasible, are invariably limited by DNA fragmentation. Using PCRs based on single-nucleotide polymorphic loci and regions of difference (RDs) in the MTB complex, a study was made of five Iron Age individuals with spinal lesions recovered from the cemetery of Aymyrlyg, South Siberia. A sensitive screening PCR for MTB complex mycobacteria was positive in four out of the five cases. Genotyping evidence indicated that all four cases were due to infection with M. bovis rather than M. tuberculosis and the data were consistent with the proposed phylogenetic model of the MTB complex. This is believed to be the first report of M. bovis causing Pott's disease in archaeological human remains. The study shows that genotyping of ancestral strains of MTB complex mycobacteria from contexts of known date provides information which allows the phylogeny of the model to be tested. Moreover, it shows that loss of DNA from RD4, which defines classic M. bovis, had already occurred from the genome over 2000 years before the present.
This is a brief summary of the animal models session held during the 12th Annual Meeting of the S... more This is a brief summary of the animal models session held during the 12th Annual Meeting of the Society on NeuroImmune Pharmacology, Santa Fe, NM, USA. This session provided important information for participants on availability and utility of animal models for the studies of HIV-1 central nervous system infection and drug abuse. It highlighted animal model relevance to human disease/condition, its utility for the studies of pathogenesis, potential importance for the development of therapeutics, and demonstrated limitations/pitfalls.
Injection of human immunodeficiency virus type 1 (HIV-1)infected human monocyte-derived macrophag... more Injection of human immunodeficiency virus type 1 (HIV-1)infected human monocyte-derived macrophages (MDMs) into the basal ganglia of severe combined immunodeficient mice recapitulates histopathologic features of HIV-1 encephalitis (HIVE). Here, we show that the neural damage in HIVE mice extends beyond the basal ganglia and is associated with cognitive impairment. Morris water maze tests showed impaired spatial learning 8 d after MDM injection. Moreover, impaired synaptic potentiation in the hippocampal CA1 subregion was demonstrated at 8 and 15 d. By day 15, post-tetanic, shortterm, and long-term potentiation were reduced by 14.1, 29.5, and 45.3% in HIVE mice compared with sham-injected or control animals. Neurofilament (NF) and synaptophysin (SP) antigens were decreased significantly in the CA2 hippocampal subregion of HIVE mice with limited neuronal apoptosis. By day 15, the CA2 region of HIVE mice expressed 3.8-and 2.6-fold less NF and SP than shams. These findings support the notion that HIV-1-infected and immune-competent brain macrophages can cause neuronal damage at distant anatomic sites. Importantly, the findings demonstrate the value of the model in exploring the physiological basis and therapeutic potential for HIV-1-associated dementia.
Alterations in hippocampal physiology affect cognition in human immunodeficiency virus type 1 (HI... more Alterations in hippocampal physiology affect cognition in human immunodeficiency virus type 1 (HIV-1)-associated dementia (HAD). The mechanism for how this occurs is not well understood. To address this, we investigated how changes in synaptic transmission and plasticity are affected by viral infection and macrophage activation using a severe combined immunodeficiency mouse model of human HIV-1 encephalitis (HIVE). HIVE was induced in mice by stereotactic injection of HIV-1-infected human monocyte-derived macrophages (MDM) into the striatum. Animals were sacrificed after 3, 7 and 15 days. Hippocampal slices were prepared from HIV-1, MDM-and sham-injected animals. Electrically evoked field excitatory postsynaptic potentials were recorded in the CA1 region of the hippocampus. Neuronal physiology was assessed by input-output and by long-term potentiation (LTP) assays. We observed that a higher stimulation intensity (mA) was required to induce a 1-mV response in the HIVE mice 0.32؎0.06) compared with shams (0.17؎0.01) at day 7. The stimulation intensities at day 15 were 0.44؎0.07 and 0.23؎0.05 in the HIVE and shams, respectively. An impairment of synaptic function was detected through measuring synaptic responses induced by stimuli with different intensities. Paired-pulse facilitation (PPF) showed deficits in HIVE mice at days 3, 7, and 15. At day 3, PPF ratios were 1.13؎0.02 and 1.24؎0.04 in HIVE and sham. The induction and maintenance of LTP was also impaired in HIVE mice. The average magnitude of LTP was 131.23؎15.26% of basal in HIVE as compared with sham animals of 232.63؎24.18%. MDM-injected mice showed an intermediate response. Taken together, the results show a range of neuronal synaptic transmission and plasticity changes in HIVE mice that may reflect the mechanisms of cognitive dysfunction in human HAD.
Secretory products from HIV-1-infected immune-competent mononuclear phagocytes (MP) damage neuron... more Secretory products from HIV-1-infected immune-competent mononuclear phagocytes (MP) damage neuronal dendritic arbor (Zheng et al., 2001). The mechanism behind neuronal injury and whether it is species and/or viral strain dependent is not fully understood. To these ends, we investigated whether HIV-1-infected and lipopolysachharide (LPS)-activated MDM elicit neuronal injury in primary human neurons. Neuronal damage was compared to that seen in rat neurons. Utilizing a spectrum of HIV-1 strains to infect human monocyte-derived macrophages (MDM), productive viral replication proved necessary, but not sufficient, for neuronal injury. Neuronal demise was induced by virion-free HIV-1-infected and immune-activated MDM culture supernatants. Maximal alterations in glutamate mediated neuronal signaling, resulted from exposure to secretory products from HIV-1-infected and immune-activated MDM. Apoptosis was the predominant mechanism of cell death induced by HIV-1-infected and LPS-treated MDM. Importantly, neuronal injury and increases in calcium influx mediated by HIV-1-infected and immune-activated MDM culture supernatants was partially blocked by the N-methyl D-aspartate (NMDA) receptor antagonist, MK 801. These data support a primary role for immune-activation in MP neurotoxic activities. The upregulation of NMDA receptor sensitive soluble factors and neuronal apoptosis by HIV-1-infected and immune-activated MDM provide unique insights into links between soluble factors, produced as a consequence of MP immunity, and neuronal demise in HAD.
, a prion-like form of the release factor Sup35, was shown to be regulated by the interplay betwe... more , a prion-like form of the release factor Sup35, was shown to be regulated by the interplay between chaperone proteins Hsp104 and Hsp70. While overproduction of Hsp104 protein cures cells of [PSI], overproduction of the Ssa1 protein of the Hsp70 family protects [PSI] from the curing effect of Hsp104. Here we demonstrate that another protein of the Hsp70 family, Ssb, previously implicated in nascent polypeptide folding and protein turnover, exhibits effects on [PSI] which are opposite those of Ssa. Ssb overproduction increases, while Ssb depletion decreases, [PSI] curing by the overproduced Hsp104. Both spontaneous [PSI] formation and [PSI] induction by overproduction of the homologous or heterologous Sup35 protein are increased significantly in the strain lacking Ssb. This is the first example when inactivation of an unrelated cellular protein facilitates prion formation. Ssb is therefore playing a role in protein-based inheritance, which is analogous to the role played by the products of mutator genes in nucleic acid-based inheritance. Ssb depletion also decreases toxicity of the overproduced Sup35 and causes extreme sensitivity to the [PSI]-curing chemical agent guanidine hydrochloride. Our data demonstrate that various members of the yeast Hsp70 family have diverged from each other in regard to their roles in prion propagation and suggest that Ssb could serve as a proofreading component of the enzymatic system, which prevents formation of prion aggregates. on September 5, 2014 by guest http://mcb.asm.org/ Downloaded from MATERIALS AND METHODS Yeast strains. The S. cerevisiae strains used in this study are described in Table 1. The haploid [PSI ϩ ] strains OT55 and OT56, also called [PSI ϩ ]1-1-74-D694 and [PSI ϩ ]7-74-D694, respectively (21, 22, 39), and isogenic [psi Ϫ PIN ϩ ] strain OT60, also called [psi Ϫ ]-74-D694 (11, 22), were described earlier. The haploid [PSI ϩ ] strains GT81-1C and GT81-1D were obtained by A. Galkin as a result of sporulating and dissecting the diploid strain GT81, constructed via HO-mediated self-homozygotization of the haploid strain GT56-34D (12). The [PSI ϩ ] strains GT128, containing the ssb1⌬::HIS3 disruption, and GT127, containing the double ssb1⌬::HIS3 ssb2⌬::URA3 disruption (ssb1/2⌬), are derivatives of strain GT81-1C, constructed as described below. Strain GT146 is a spontaneous Ura Ϫ derivative of strain GT127, selected on medium containing 5-fluoroorotic acid (30). The [psi Ϫ PIN ϩ ] derivatives of strains GT81-1C and GT146 were obtained by transforming these strains with plasmid pYS-GAL104 (see below), which bears the HSP104 gene under control of the GAL promoter, and curing [PSI] as a result of galactose-induced overproduction of Hsp104 as described earlier . The resulting [psi Ϫ ] derivatives were subsequently cured of plasmid pYS-GAL104. The [psi Ϫ pin Ϫ ] derivatives of strains GT81-1C, GT127, and GT146 were obtained by curing cells of [PSI] and [PIN] after growth on YPD medium containing 5 mM guanidine hydrochloride (GuHCl) (22). The haploid strain GT84-5A is isogenic to GT81-1C except that it contains the hsp104⌬::URA3 disruption, which eliminates [PSI]. This strain was constructed by sporulating and dissecting diploid strain GT84, which is a GT81 derivative with one of the HSP104 copies disrupted via direct transplacement with the hsp104⌬::URA3 allele as described above (11). All strains contain the [PSI]-suppressible UGA mutation ade1-14, so that [PSI ϩ ] strains are white and Ade ϩ , while [psi Ϫ ] strains are red and Ade Ϫ , as described previously .
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