Deborah Court | University of Manitoba (original) (raw)

Papers by Deborah Court

Biochimica et biophysica acta, Jan 18, 2016

A novel feature of the voltage-dependent anion channel (VDAC, mitochondrial porin), is the barrel... more A novel feature of the voltage-dependent anion channel (VDAC, mitochondrial porin), is the barrel, comprising an odd number of β-strands and closed by parallel strands. Recent research has focused on the N-terminal segment, which in the available structures, resides in the lumen and is not part of the barrel. In this review, the structural data obtained from vertebrate VDAC are integrated with those from VDAC in artificial bilayers, emphasizing the array of native and tagged versions of VDAC used. The data are discussed with respect to a recent gating model (Zachariae et al. (2012) Structure 20:1-10), in which the N-terminus acts not as a gate on a stable barrel, but rather stabilizes the barrel, preventing its shift into a partially collapsed, low-conductance, closed state. Additionally, the role of the N-terminus in VDAC oligomerization, apoptosis through interactions with hexokinase and its interaction with ATP are discussed briefly.

Nucleic Acids Research, 1991

Nucleic Acids Research, 1990

The nucleotide sequence of the mitochondrial (mt) large subunit (LSU) rRNA of Neurospora crassa s... more The nucleotide sequence of the mitochondrial (mt) large subunit (LSU) rRNA of Neurospora crassa strain 74-0R23-1A (74A) has been completed by sequencing three fragments of the large exon, HindIII-19a, -15 and -14 and joining them to previously published sequences (1, 2). The composite sequence, without the intron which is located between nucleotides (nt) 2900 and 2901 (2), contains regions of similarity to fungal (3, 4) and plant mt LSU rRNAs (5). Although extensive homology exists between the N. crassa rRNA and that of Aspergillus nidulans (3), the Neurospora sequence has a 351 nt 5' extension. Furthermore, internal regions of the N. crassa sequence show homology to the chloroplast LSU rRNAs from both monocots (e.g. maize; 6) and dicots (e.g. N. tabacum; 7).

Current Genetics, 1992

The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neuros... more The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-I codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasraids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a tysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.

Current Genetics, 1991

The nucleotide sequence of kalilo, a linear plasmid that induces senescence in Neurospora by inte... more The nucleotide sequence of kalilo, a linear plasmid that induces senescence in Neurospora by integrating into the mitochondrial chromosome, reveals structural and genetic features germane to the unique properties of this element. Prominent features include: (1) very long perfect terminal inverted repeats of nucleotide sequences which are devoid of obvious genetic functions, but are unusually GC-rich near both ends of the linear DNA;

Cell, 1986

In the Mile strains of N. intermedia, senescence is initiated by insertion of a 9.0 kb foreign nu... more In the Mile strains of N. intermedia, senescence is initiated by insertion of a 9.0 kb foreign nucleotide sequence, kalDNA, into mitochondrial DNA. A 9.0 kb linear DNA plasmid that is structurally homologous to the mitochondrial kalDNA insertion sequences exists in high copy numbers in close association with the nuclei of presenescent and senescent kalilo cells, but is not present in cells of long-lived normal strains. The free karlilo plasmid has not been detected in mitochondria, suggesting that the element does not contain a mitochondrial origin of replication. Unexpectedly, the ear plasmid, like the mitochondrial insertion elet, follows a strict pattern of maternal inheritance. We surmise that the extramitochondrial plasmid is the etiological precursor of the kalDNA insertion sequences that appear in the mtDNAs of senescent cell lines and conclude that the kalilo element induces senescence because it is a mutator of mitochondrial genes.

Genetics

Many group I introns encode endonucleases that promote intron homing by initiating a double-stran... more Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage A model system was developed to analyze exon homology effects on intron homing and determine the role of the A 5'-3' exonuclease complex (Redap) in the repair event. Efficient intron homing depended on exon lengths in the 35-to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type X, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology. RADDING 1971), or bubble migration (FORMOSA and ALBERTS 1986). In either event, processing of the Corresponding authm:

Viruses, 2015

The current disease outbreak caused by the Ebola virus Makona variant (EBOV/Mak) has led to unpre... more The current disease outbreak caused by the Ebola virus Makona variant (EBOV/Mak) has led to unprecedented morbidity and lethality given its geographic reach and sustained transmission. Sodium hypochlorite and ethanol are well-accepted decontamination agents, however little published evidence supports the selection of appropriate concentrations and contact times. The present study addresses the environmental robustness of EBOV/Mak and evaluates the effectiveness of sodium hypochlorite and ethanol as disinfectants. EBOV/Mak was suspended in a simulated organic soil load and dried onto surfaces. Viability was measured at 1 hour, 24 hours, 72 hours, and 192 hours. For the evaluation of disinfectants, EBOV/Mak in a simulated organic soil was dried onto stainless steel carriers and disinfected with 0.01% (v/v), 0.1% (v/v), 0.5% (v/v) and 1% (v/v) sodium hypochlorite solutions or 67% (v/v) ethanol at contact times of 1, 5 or 10 minutes. EBOV/Mak persisted longer on steel and plastic surfac...

Molecular and cellular biology, 1996

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitocho... more Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix...

Yeast (Chichester, England), 1995

The nucleotide sequence of yeast chromosome III encompassing the previously the previously descri... more The nucleotide sequence of yeast chromosome III encompassing the previously the previously described open reading frames (ORFs) YCR80w, YCR77c and YCR78c (Oliver et al., 1992) has been updated. In the corrected sequence, these ORFs are replaced by two new ORFs, YCR80w (453 bp) and YCR77c (2391 bp). In addition, the orientation of Ycr79c is reversed to give ORF Ycr79w, which has an unaltered nt sequence. The predicted translation products do not exhibit significant homology to known proteins. ORF Ycr77p encodes an 88 kDa, cytosolic protein. A fraction of the protein is associated with small membranous structures in a salt-sensitive fashion. Initial characterization revealed that the protein is not essential for yeast viability, growth on non-fermentable carbon sources, mating and sporulation. The chromosome III DNA sequence that was used for the analysis has the Accession Number X59720 in the GenBank/EMBL database.

Virus Research, 2012

Kyasanur Forest Disease Virus (KFDV) is a tick-borne, hemorrhagic fever-causing member of the Fla... more Kyasanur Forest Disease Virus (KFDV) is a tick-borne, hemorrhagic fever-causing member of the Flaviviridae virus family. With infections annually ranging from 50 to 1000 people in south-west India and the lack of effective treatments, a better understanding of this virus is needed. The development of a reverse genetics system (RGS) for KFDV would provide the opportunity to address these issues. The KFDV genome sequence was elucidated and the RGS was created. Utilizing this system, live infectious KFDV particles were produced from mammalian cell culture, thereby validating the success of the RGS. Flaviviruses have the ability to suppress the type 1 interferon response and indications are that the non structural (NS) proteins serve this role. Using luciferase bioassays, the NS5 protein of KFDV was determined to be the primary antagonist of the IFN response when compared to the other NS proteins, specifically NS4B and NS4B-2k. Moreover, our results indicate that this is attributed to a region, beginning before and including the RNA-dependent RNA polymerase (RdRp). With evasion of the interferon response by KFDV established, the further implementation of the reverse genetics system will enable investigation into pathogenesis and disease progression of KFDV with respect to the innate immune response, at the IFN and the NS5 protein levels.

Yeast, 2008

Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Thro... more Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Throughout its development as a research tool, several strain backgrounds have been utilized and different combinations of auxotrophic marker genes have been introduced into them, creating a useful but non-homogeneous set of strains. The ade2 allele was used as an auxotrophic marker, and for 'red-white' screening for respiratory competence. his3 alleles that influence the expression of MRM1 have been used as selectable markers, and the MIP1[S] allele, found in the commonly used S228c strain, is associated with mitochondrial DNA defects. The focus of the current work was to examine the effects of these alleles, singly and in combination, on the maintenance of mitochondrial function. The combination of the ade2 and MIP1[S] alleles is associated with a slight increase in point mutations in mitochondrial DNA. The deletion in the his3 200 allele, which removes the promoter for MRM1, is associated with loss of respiratory competence at 37 • C in the presence of either MIP1 allele. Thus, multiple factors can contribute to the maintenance of mitochondrial function, reinforcing the concept that strain background is an important consideration in both designing experiments and comparing results obtained by different research groups. ; Stuart et al. (2006) EM93 MATa/MATα SUC2/SUC2 GAL2/ga12 MAL/MAL mel/mel CUP1/cup1 FLO1/flo1, heterozygous for MIP1 A and G at position 1981 MIP1 [contributes ∼90% of the S288c genome] Mortimer and Johnston (1986); Baruffini et al. Median values are reported; ranges are shown in parentheses. Respiratory competence experiments were carried out for 4 days and repeated with at least three independent isolates of each strain. Erythromycin-resistance data were obtained from 3-13 replicates Strain background and mitochondrial DNA function in S. cerevisiae 909

Mitochondrion, 2007

Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Thro... more Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Throughout its development as a research tool, several strain backgrounds have been utilized and different combinations of auxotrophic marker genes have been introduced into them, creating a useful but non-homogeneous set of strains. The ade2 allele was used as an auxotrophic marker, and for 'red-white' screening for respiratory competence. his3 alleles that influence the expression of MRM1 have been used as selectable markers, and the MIP1[S] allele, found in the commonly used S228c strain, is associated with mitochondrial DNA defects. The focus of the current work was to examine the effects of these alleles, singly and in combination, on the maintenance of mitochondrial function. The combination of the ade2 and

Mitochondrion, 2012

Porin, the voltage-dependent anion-selective channel (VDAC) in the mitochondrial outer membrane, ... more Porin, the voltage-dependent anion-selective channel (VDAC) in the mitochondrial outer membrane, contributes to metabolism and apoptosis. VDAC function was investigated in Neurospora, an obligate aerobe with a single porin. Porinless strains are viable, with cold-sensitive growth, cytochrome deficiencies and overexpression of alternative oxidase. iTRAQ labeling of mitochondria from a porinless strain and its progenitor revealed a small group of proteins with altered expression levels in the mutant organelles. Porinless Neurospora appears to compensate not by inducing alternative pores, but by altering electron flow and nucleotide metabolism. Transcriptional and post-transcriptional mechanisms contribute to the response, reflecting the extent of porin influence.

Journal of Biological Chemistry, 1996

Journal of Biological Chemistry, 1996

We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes ... more We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes for an ABC transporter of Saccharomyces cerevisiae mitochondria. The suppressor, termed BAT1, encodes a protein of 393 amino acid residues with an NH2-terminal extension that directs Bat1p to the mitochondrial matrix. A highly homologous protein, Bat2p, of 376 amino acid residues was found in the cytosol. Both Bat proteins show striking similarity to the mammalian protein Eca39, which is one of the few known targets of the myc oncogene. Deletion of a single BAT gene did not impair growth of yeast cells. In contrast, deletion of both genes resulted in an auxotrophy for branched-chain amino acids (Ile, Leu, and Val) and in a severe growth reduction on glucose-containing media, even after supply of these amino acids. Mitochondria and cytosol isolated from bat1 and bat2 deletion mutants, respectively, contained largely reduced activities for the conversion of branched-chain 2-ketoacids to their corresponding amino acids. Thus, the Bat proteins represent the first known isoforms of yeast branched-chain amino acid transaminases. The severe growth defect of the double deletion mutant observed even in the presence of branched-chain amino acids suggests that the Bat proteins, in addition to the supply of these amino acids, perform another important function in the cell.

Journal of Biological Chemistry, 1998

TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane ... more TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The protein is anchored to the lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The orientation of the protein is not influenced by the charges flanking its trans-membrane segment, in contrast to observations regarding proteins of other membranes. In vitro import studies utilizing TOM22 preproteins harboring deletions or mutations in the cytosolic domain revealed that the combination of the trans-membrane segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment of the cytosolic domain was found to be essential for the import and assembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in which the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 contains physically separate import and membrane anchor sequences.

Journal of Biological Chemistry, 1996

The protein Tom71 is encoded by the open reading frame YHR117w (yeast chromosome VIII) and shares... more The protein Tom71 is encoded by the open reading frame YHR117w (yeast chromosome VIII) and shares 53% amino acid sequence identity with Tom70, a protein import receptor of the mitochondrial outer membrane. We investigated the cellular function of Tom71 and addressed the question of whether Tom71 and Tom70 fulfill similar functions. Like Tom70, Tom71 is anchored to the mitochondrial outer membrane via its N terminus, thereby exposing a large C-terminal domain to the cytosol. Tom71 is associated with the protein import complex of this membrane and can be cross-linked to a protein with a molecular mass of 30-35 kDa. Disruption of the TOM71 gene does not reduce cell growth, except on nonfermentable carbon sources at elevated temperatures. Deletion of both the TOM71 and TOM70 genes does not acerbate this growth defect. In vitro import studies demonstrated no functional requirement for Tom71 in the import of several preproteins destined for each of the mitochondrial subcompartments. In particular, the import of Tom70-dependent preproteins is minimally affected by the deletion of Tom71, irrespective of the presence or absence of the Tom70 receptor. Thus, despite their strikingly similar biochemical properties, Tom71 and Tom70 do not perform identical functions.

FEBS Letters, 1996

The signals for targeting and assembly of porin, a protein of the mitochondrial outer membrane, h... more The signals for targeting and assembly of porin, a protein of the mitochondrial outer membrane, have not been clearly defined. Targeting information has been hypothesized to be contained in the N-terminus, which may form an amphipathic a-helix, and in the C-terminal portion of the protein. Here, the role of the extreme N-and C-termini of porin from Neurospora crassa in its import into the mitochondrial outer membrane was investigated. Deletion mutants were constructed which lacked the N-terminal 12 or 20 residues or the C-terminal 15 residues. The porins truncated at their N-termini were imported in a receptordependent manner into the outer membrane of isolated mitochondria. When integrated into the outer membrane, these preproteins displayed an increased sensitivity to protease as compared to wild-type porin. In contrast, mutant porin truncated at its C-terminus did not acquire protease resistance upon incubation with mitochondria. Thus, unlike most other mitochondrial preproteins, porin appears to contain important targeting and/or assembly information at its C-terminus, rather than at the N-terminus.

Biochimica et biophysica acta, Jan 18, 2016

A novel feature of the voltage-dependent anion channel (VDAC, mitochondrial porin), is the barrel... more A novel feature of the voltage-dependent anion channel (VDAC, mitochondrial porin), is the barrel, comprising an odd number of β-strands and closed by parallel strands. Recent research has focused on the N-terminal segment, which in the available structures, resides in the lumen and is not part of the barrel. In this review, the structural data obtained from vertebrate VDAC are integrated with those from VDAC in artificial bilayers, emphasizing the array of native and tagged versions of VDAC used. The data are discussed with respect to a recent gating model (Zachariae et al. (2012) Structure 20:1-10), in which the N-terminus acts not as a gate on a stable barrel, but rather stabilizes the barrel, preventing its shift into a partially collapsed, low-conductance, closed state. Additionally, the role of the N-terminus in VDAC oligomerization, apoptosis through interactions with hexokinase and its interaction with ATP are discussed briefly.

Nucleic Acids Research, 1991

Nucleic Acids Research, 1990

The nucleotide sequence of the mitochondrial (mt) large subunit (LSU) rRNA of Neurospora crassa s... more The nucleotide sequence of the mitochondrial (mt) large subunit (LSU) rRNA of Neurospora crassa strain 74-0R23-1A (74A) has been completed by sequencing three fragments of the large exon, HindIII-19a, -15 and -14 and joining them to previously published sequences (1, 2). The composite sequence, without the intron which is located between nucleotides (nt) 2900 and 2901 (2), contains regions of similarity to fungal (3, 4) and plant mt LSU rRNAs (5). Although extensive homology exists between the N. crassa rRNA and that of Aspergillus nidulans (3), the Neurospora sequence has a 351 nt 5' extension. Furthermore, internal regions of the N. crassa sequence show homology to the chloroplast LSU rRNAs from both monocots (e.g. maize; 6) and dicots (e.g. N. tabacum; 7).

Current Genetics, 1992

The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neuros... more The nucleotide sequence of maranhar, a senescence-inducing linear mitochondrial plasmid of Neurospora crassa, was determined. The termini of the 7-kb plasmid are 349-bp inverted repeats (TIRs). Each DNA strand contains a long open reading frame (ORF) which begins within the TIR and extends toward the centre of the plasmid. ORF-I codes for a single-subunit RNA polymerase that is not closely related to that encoded by another Neurospora plasmid, kalilo. The ORF-2 product may be a B-type DNA polymerase resembling those encoded by terminal protein-linked linear genetic elements, including linear mitochondrial plasmids and linear bacteriophages. A separate coding sequence for the terminal protein could not be identified; however, the DNA polymerase of maranhar has an amino-terminal extension with features that are also present in the terminal proteins of linear bacteriophages. The N-terminal extensions of the DNA polymerases of other linear mitochondrial plasraids contain similar features, suggesting that the terminal proteins of linear plasmids may be comprised, at least in part, of these cryptic domains. The terminal protein-DNA bond of maranhar is resistant to mild alkaline hydrolysis, indicating that it might involve a tyrosine or a tysine residue. Although maranhar and the senescence-inducing kalilo plasmid of N. intermedia are structurally similar, and integrate into mitochondrial DNA by a mechanism thus far unique to these two plasmids, they are not closely related to each other and they do not have any nucleotide sequence features, or ORFs, that distinguish them clearly from mitochondrial plasmids which are not associated with senescence and most of which are apparently non-integrative.

Current Genetics, 1991

The nucleotide sequence of kalilo, a linear plasmid that induces senescence in Neurospora by inte... more The nucleotide sequence of kalilo, a linear plasmid that induces senescence in Neurospora by integrating into the mitochondrial chromosome, reveals structural and genetic features germane to the unique properties of this element. Prominent features include: (1) very long perfect terminal inverted repeats of nucleotide sequences which are devoid of obvious genetic functions, but are unusually GC-rich near both ends of the linear DNA;

Cell, 1986

In the Mile strains of N. intermedia, senescence is initiated by insertion of a 9.0 kb foreign nu... more In the Mile strains of N. intermedia, senescence is initiated by insertion of a 9.0 kb foreign nucleotide sequence, kalDNA, into mitochondrial DNA. A 9.0 kb linear DNA plasmid that is structurally homologous to the mitochondrial kalDNA insertion sequences exists in high copy numbers in close association with the nuclei of presenescent and senescent kalilo cells, but is not present in cells of long-lived normal strains. The free karlilo plasmid has not been detected in mitochondria, suggesting that the element does not contain a mitochondrial origin of replication. Unexpectedly, the ear plasmid, like the mitochondrial insertion elet, follows a strict pattern of maternal inheritance. We surmise that the extramitochondrial plasmid is the etiological precursor of the kalDNA insertion sequences that appear in the mtDNAs of senescent cell lines and conclude that the kalilo element induces senescence because it is a mutator of mitochondrial genes.

Genetics

Many group I introns encode endonucleases that promote intron homing by initiating a double-stran... more Many group I introns encode endonucleases that promote intron homing by initiating a double-strand break-mediated homologous recombination event. A td intron-phage A model system was developed to analyze exon homology effects on intron homing and determine the role of the A 5'-3' exonuclease complex (Redap) in the repair event. Efficient intron homing depended on exon lengths in the 35-to 50-bp range, although homing levels remained significantly elevated above nonbreak-mediated recombination with as little as 10 bp of flanking homology. Although precise intron insertion was demonstrated with extremely limiting exon homology, the complete absence of one exon produced illegitimate events on the side of heterology. Interestingly, intron inheritance was unaffected by the presence of extensive heterology at the double-strand break in wild-type X, provided that sufficient homology between donor and recipient was present distal to the heterologous sequences. However, these events involving heterologous ends were absolutely dependent on an intact Red exonuclease system. Together these results indicate that heterologous sequences can participate in double-strand break-mediated repair and imply that intron transposition to heteroallelic sites might occur at break sites within regions of limited or no homology. RADDING 1971), or bubble migration (FORMOSA and ALBERTS 1986). In either event, processing of the Corresponding authm:

Viruses, 2015

The current disease outbreak caused by the Ebola virus Makona variant (EBOV/Mak) has led to unpre... more The current disease outbreak caused by the Ebola virus Makona variant (EBOV/Mak) has led to unprecedented morbidity and lethality given its geographic reach and sustained transmission. Sodium hypochlorite and ethanol are well-accepted decontamination agents, however little published evidence supports the selection of appropriate concentrations and contact times. The present study addresses the environmental robustness of EBOV/Mak and evaluates the effectiveness of sodium hypochlorite and ethanol as disinfectants. EBOV/Mak was suspended in a simulated organic soil load and dried onto surfaces. Viability was measured at 1 hour, 24 hours, 72 hours, and 192 hours. For the evaluation of disinfectants, EBOV/Mak in a simulated organic soil was dried onto stainless steel carriers and disinfected with 0.01% (v/v), 0.1% (v/v), 0.5% (v/v) and 1% (v/v) sodium hypochlorite solutions or 67% (v/v) ethanol at contact times of 1, 5 or 10 minutes. EBOV/Mak persisted longer on steel and plastic surfac...

Molecular and cellular biology, 1996

Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitocho... more Tom22 is an essential component of the protein translocation complex (Tom complex) of the mitochondrial outer membrane. The N-terminal domain of Tom22 functions as a preprotein receptor in cooperation with Tom20. The role of the C-terminal domain of Tom22, which is exposed to the intermembrane space (IMS), in its own assembly into the Tom complex and in the import of other preproteins was investigated. The C-terminal domain of Tom22 is not essential for the targeting and assembly of this protein, as constructs lacking part or all of the IMS domain became imported into mitochondria and assembled into the Tom complex. Mutant strains of Neurospora expressing the truncated Tom22 proteins were generated by a novel procedure. These mutants displayed wild-type growth rates, in contrast to cells lacking Tom22, which are not viable. The import of proteins into the outer membrane and the IMS of isolated mutant mitochondria was not affected. Some but not all preproteins destined for the matrix...

Yeast (Chichester, England), 1995

The nucleotide sequence of yeast chromosome III encompassing the previously the previously descri... more The nucleotide sequence of yeast chromosome III encompassing the previously the previously described open reading frames (ORFs) YCR80w, YCR77c and YCR78c (Oliver et al., 1992) has been updated. In the corrected sequence, these ORFs are replaced by two new ORFs, YCR80w (453 bp) and YCR77c (2391 bp). In addition, the orientation of Ycr79c is reversed to give ORF Ycr79w, which has an unaltered nt sequence. The predicted translation products do not exhibit significant homology to known proteins. ORF Ycr77p encodes an 88 kDa, cytosolic protein. A fraction of the protein is associated with small membranous structures in a salt-sensitive fashion. Initial characterization revealed that the protein is not essential for yeast viability, growth on non-fermentable carbon sources, mating and sporulation. The chromosome III DNA sequence that was used for the analysis has the Accession Number X59720 in the GenBank/EMBL database.

Virus Research, 2012

Kyasanur Forest Disease Virus (KFDV) is a tick-borne, hemorrhagic fever-causing member of the Fla... more Kyasanur Forest Disease Virus (KFDV) is a tick-borne, hemorrhagic fever-causing member of the Flaviviridae virus family. With infections annually ranging from 50 to 1000 people in south-west India and the lack of effective treatments, a better understanding of this virus is needed. The development of a reverse genetics system (RGS) for KFDV would provide the opportunity to address these issues. The KFDV genome sequence was elucidated and the RGS was created. Utilizing this system, live infectious KFDV particles were produced from mammalian cell culture, thereby validating the success of the RGS. Flaviviruses have the ability to suppress the type 1 interferon response and indications are that the non structural (NS) proteins serve this role. Using luciferase bioassays, the NS5 protein of KFDV was determined to be the primary antagonist of the IFN response when compared to the other NS proteins, specifically NS4B and NS4B-2k. Moreover, our results indicate that this is attributed to a region, beginning before and including the RNA-dependent RNA polymerase (RdRp). With evasion of the interferon response by KFDV established, the further implementation of the reverse genetics system will enable investigation into pathogenesis and disease progression of KFDV with respect to the innate immune response, at the IFN and the NS5 protein levels.

Yeast, 2008

Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Thro... more Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Throughout its development as a research tool, several strain backgrounds have been utilized and different combinations of auxotrophic marker genes have been introduced into them, creating a useful but non-homogeneous set of strains. The ade2 allele was used as an auxotrophic marker, and for 'red-white' screening for respiratory competence. his3 alleles that influence the expression of MRM1 have been used as selectable markers, and the MIP1[S] allele, found in the commonly used S228c strain, is associated with mitochondrial DNA defects. The focus of the current work was to examine the effects of these alleles, singly and in combination, on the maintenance of mitochondrial function. The combination of the ade2 and MIP1[S] alleles is associated with a slight increase in point mutations in mitochondrial DNA. The deletion in the his3 200 allele, which removes the promoter for MRM1, is associated with loss of respiratory competence at 37 • C in the presence of either MIP1 allele. Thus, multiple factors can contribute to the maintenance of mitochondrial function, reinforcing the concept that strain background is an important consideration in both designing experiments and comparing results obtained by different research groups. ; Stuart et al. (2006) EM93 MATa/MATα SUC2/SUC2 GAL2/ga12 MAL/MAL mel/mel CUP1/cup1 FLO1/flo1, heterozygous for MIP1 A and G at position 1981 MIP1 [contributes ∼90% of the S288c genome] Mortimer and Johnston (1986); Baruffini et al. Median values are reported; ranges are shown in parentheses. Respiratory competence experiments were carried out for 4 days and repeated with at least three independent isolates of each strain. Erythromycin-resistance data were obtained from 3-13 replicates Strain background and mitochondrial DNA function in S. cerevisiae 909

Mitochondrion, 2007

Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Thro... more Saccharomyces cerevisiae is a valuable model organism for the study of eukaryotic processes. Throughout its development as a research tool, several strain backgrounds have been utilized and different combinations of auxotrophic marker genes have been introduced into them, creating a useful but non-homogeneous set of strains. The ade2 allele was used as an auxotrophic marker, and for 'red-white' screening for respiratory competence. his3 alleles that influence the expression of MRM1 have been used as selectable markers, and the MIP1[S] allele, found in the commonly used S228c strain, is associated with mitochondrial DNA defects. The focus of the current work was to examine the effects of these alleles, singly and in combination, on the maintenance of mitochondrial function. The combination of the ade2 and

Mitochondrion, 2012

Porin, the voltage-dependent anion-selective channel (VDAC) in the mitochondrial outer membrane, ... more Porin, the voltage-dependent anion-selective channel (VDAC) in the mitochondrial outer membrane, contributes to metabolism and apoptosis. VDAC function was investigated in Neurospora, an obligate aerobe with a single porin. Porinless strains are viable, with cold-sensitive growth, cytochrome deficiencies and overexpression of alternative oxidase. iTRAQ labeling of mitochondria from a porinless strain and its progenitor revealed a small group of proteins with altered expression levels in the mutant organelles. Porinless Neurospora appears to compensate not by inducing alternative pores, but by altering electron flow and nucleotide metabolism. Transcriptional and post-transcriptional mechanisms contribute to the response, reflecting the extent of porin influence.

Journal of Biological Chemistry, 1996

Journal of Biological Chemistry, 1996

We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes ... more We have isolated a high copy suppressor of a temperature-sensitive mutation in ATM1, which codes for an ABC transporter of Saccharomyces cerevisiae mitochondria. The suppressor, termed BAT1, encodes a protein of 393 amino acid residues with an NH2-terminal extension that directs Bat1p to the mitochondrial matrix. A highly homologous protein, Bat2p, of 376 amino acid residues was found in the cytosol. Both Bat proteins show striking similarity to the mammalian protein Eca39, which is one of the few known targets of the myc oncogene. Deletion of a single BAT gene did not impair growth of yeast cells. In contrast, deletion of both genes resulted in an auxotrophy for branched-chain amino acids (Ile, Leu, and Val) and in a severe growth reduction on glucose-containing media, even after supply of these amino acids. Mitochondria and cytosol isolated from bat1 and bat2 deletion mutants, respectively, contained largely reduced activities for the conversion of branched-chain 2-ketoacids to their corresponding amino acids. Thus, the Bat proteins represent the first known isoforms of yeast branched-chain amino acid transaminases. The severe growth defect of the double deletion mutant observed even in the presence of branched-chain amino acids suggests that the Bat proteins, in addition to the supply of these amino acids, perform another important function in the cell.

Journal of Biological Chemistry, 1998

TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane ... more TOM22 is an integral component of the preprotein translocase of the mitochondrial outer membrane (TOM complex). The protein is anchored to the lipid bilayer by a central trans-membrane segment, thereby exposing the amino-terminal domain to the cytosol and the carboxyl-terminal portion to the intermembrane space. Here, we describe the sequence requirements for the targeting and correct insertion of Neurospora TOM22 into the outer membrane. The orientation of the protein is not influenced by the charges flanking its trans-membrane segment, in contrast to observations regarding proteins of other membranes. In vitro import studies utilizing TOM22 preproteins harboring deletions or mutations in the cytosolic domain revealed that the combination of the trans-membrane segment and intermembrane space domain of TOM22 is not sufficient to direct import into the outer membrane. In contrast, a short segment of the cytosolic domain was found to be essential for the import and assembly of TOM22. This sequence, a novel internal import signal for the outer membrane, carries a net positive charge. A mutant TOM22 in which the charge of the import signal was altered to -1 was imported less efficiently than the wild-type protein. Our data indicate that TOM22 contains physically separate import and membrane anchor sequences.

Journal of Biological Chemistry, 1996

The protein Tom71 is encoded by the open reading frame YHR117w (yeast chromosome VIII) and shares... more The protein Tom71 is encoded by the open reading frame YHR117w (yeast chromosome VIII) and shares 53% amino acid sequence identity with Tom70, a protein import receptor of the mitochondrial outer membrane. We investigated the cellular function of Tom71 and addressed the question of whether Tom71 and Tom70 fulfill similar functions. Like Tom70, Tom71 is anchored to the mitochondrial outer membrane via its N terminus, thereby exposing a large C-terminal domain to the cytosol. Tom71 is associated with the protein import complex of this membrane and can be cross-linked to a protein with a molecular mass of 30-35 kDa. Disruption of the TOM71 gene does not reduce cell growth, except on nonfermentable carbon sources at elevated temperatures. Deletion of both the TOM71 and TOM70 genes does not acerbate this growth defect. In vitro import studies demonstrated no functional requirement for Tom71 in the import of several preproteins destined for each of the mitochondrial subcompartments. In particular, the import of Tom70-dependent preproteins is minimally affected by the deletion of Tom71, irrespective of the presence or absence of the Tom70 receptor. Thus, despite their strikingly similar biochemical properties, Tom71 and Tom70 do not perform identical functions.

FEBS Letters, 1996

The signals for targeting and assembly of porin, a protein of the mitochondrial outer membrane, h... more The signals for targeting and assembly of porin, a protein of the mitochondrial outer membrane, have not been clearly defined. Targeting information has been hypothesized to be contained in the N-terminus, which may form an amphipathic a-helix, and in the C-terminal portion of the protein. Here, the role of the extreme N-and C-termini of porin from Neurospora crassa in its import into the mitochondrial outer membrane was investigated. Deletion mutants were constructed which lacked the N-terminal 12 or 20 residues or the C-terminal 15 residues. The porins truncated at their N-termini were imported in a receptordependent manner into the outer membrane of isolated mitochondria. When integrated into the outer membrane, these preproteins displayed an increased sensitivity to protease as compared to wild-type porin. In contrast, mutant porin truncated at its C-terminus did not acquire protease resistance upon incubation with mitochondria. Thus, unlike most other mitochondrial preproteins, porin appears to contain important targeting and/or assembly information at its C-terminus, rather than at the N-terminus.