Aashiq H Kachroo | Concordia University (Canada) (original) (raw)

Papers by Aashiq H Kachroo

ABSTRACTThiabendazole (TBZ) is an FDA-approved benzimidazole widely used for its antifungal and a... more ABSTRACTThiabendazole (TBZ) is an FDA-approved benzimidazole widely used for its antifungal and antihelminthic properties. We showed previously that TBZ is also a potent vascular disrupting agent and inhibits angiogenesis at the tissue level by dissociating vascular endothelial cells in newly formed blood vessels. Here, we uncover TBZ’s molecular target and mechanism of action. Using human cell culture, molecular modeling, and humanized yeast, we find that TBZ selectively targets only 1 of 9 human β-tubulin isotypes (TUBB8) to specifically disrupt endothelial cell microtubules. By leveraging epidemiological pesticide resistance data and mining chemical features of commercially used benzimidazoles, we discover that a broader class of benzimidazole compounds, in extensive use for 50 years, also potently disrupt immature blood vessels and inhibit angiogenesis. Thus, besides identifying the molecular mechanism of benzimidazole-mediated vascular disruption, this study presents evidence r...

Many gene families have been expanded by gene duplications along the human lineage, relative to a... more Many gene families have been expanded by gene duplications along the human lineage, relative to ancestral opisthokonts, but the extent to which the duplicated genes function similarly is understudied. Here, we focused on structural cytoskeletal genes involved in critical cellular processes including chromosome segregation, macromolecular transport, and cell shape maintenance. To determine functional redundancy and divergence of duplicated human genes, we systematically humanized the yeast actin, myosin, tubulin, and septin genes, testing ∼85% of human cytoskeletal genes across 7 gene families for their ability to complement a growth defect induced by deletion of the corresponding yeast ortholog. In 5 of 7 families—all but α-tubulin and light myosin, we found at least one human gene capable of complementing loss of the yeast gene. Despite rescuing growth defects, we observed differential abilities of human genes to rescue cell morphology, meiosis, and mating defects. By comparing phe...

Gene duplication is seen as a major source of structural and functional divergence in genome evol... more Gene duplication is seen as a major source of structural and functional divergence in genome evolution. Under the conventional models of sub- or neofunctionalizaton, functional changes arise in one of the duplicates after duplication. However, we suggest here that the presence of a duplicated gene can result in functional changes to its interacting partners. We explore this hypothesis by in-silico evolution of a heterodimer when one member of the interacting pair is duplicated. We examine how a range of selection pressures and protein structures leads to differential patterns of evolutionary divergence. We find that a surprising number of distinct evolutionary trajectories can be observed even in a simple three member system. Further, we observe that selection to correct dosage imbalance can affect the evolution of the initial function in several unexpected ways. For example, if a duplicate is under selective pressure to avoid binding its original binding partner, this can lead to c...

Despite over a billion years of evolutionary divergence, several thousand human genes possess cle... more Despite over a billion years of evolutionary divergence, several thousand human genes possess clearly identifiable orthologs in yeast, and many have undergone lineage-specific duplications in one or both lineages. The ortholog conjecture postulates that orthologous genes between species retain ancestral functions despite divergence over vast timescales, but duplicated genes will be free to diverge in function. However, the retention of ancestral functions among co-orthologs between species and within gene families has been difficult to test experimentally at scale. In order to investigate how ancestral functions are retained or lost post-duplication, we systematically replaced hundreds of essential yeast genes with their human orthologs from gene families that have undergone lineage-specific duplications, including those with single duplications (one yeast gene to two human genes, 1:2) or higher-order expansions (1:>2) in the human lineage. We observe a variable pattern of replac...

Molecular Biology and Evolution

Gene duplication is seen as a major source of structural and functional divergence in genome evol... more Gene duplication is seen as a major source of structural and functional divergence in genome evolution. Under the conventional models of sub or neofunctionalization, functional changes arise in one of the duplicates after duplication. However, we suggest here that the presence of a duplicated gene can result in functional changes to its interacting partners. We explore this hypothesis by in silico evolution of a heterodimer when one member of the interacting pair is duplicated. We examine how a range of selection pressures and protein structures leads to differential patterns of evolutionary divergence. We find that a surprising number of distinct evolutionary trajectories can be observed even in a simple three member system. Further, we observe that selection to correct dosage imbalance can affect the evolution of the initial function in several unexpected ways. For example, if a duplicate is under selective pressure to avoid binding its original binding partner, this can lead to c...

eLife, Jun 29, 2017

Eukaryotes and prokaryotes last shared a common ancestor ~2 billion years ago, and while many pre... more Eukaryotes and prokaryotes last shared a common ancestor ~2 billion years ago, and while many present-day genes in these lineages predate this divergence, the extent to which these genes still perform their ancestral functions is largely unknown. To test principles governing retention of ancient function, we asked if prokaryotic genes could replace their essential eukaryotic orthologs. We systematically replaced essential genes in yeast by their 1:1 orthologs from Escherichia coli. After accounting for mitochondrial localization and alternative start codons, 31 out of 51 bacterial genes tested (61%) could complement a lethal growth defect and replace their yeast orthologs with minimal effects on growth rate. Replaceability was determined on a pathway-by-pathway basis; codon usage, abundance, and sequence similarity contributed predictive power. The heme biosynthesis pathway was particularly amenable to inter-kingdom exchange, with each yeast enzyme replaceable by its bacterial, huma...

Briefings in functional genomics, Jan 13, 2015

Despite a billion years of divergent evolution, the baker's yeast Saccharomyces cerevisiae ha... more Despite a billion years of divergent evolution, the baker's yeast Saccharomyces cerevisiae has long proven to be an invaluable model organism for studying human biology. Given its tractability and ease of genetic manipulation, along with extensive genetic conservation with humans, it is perhaps no surprise that researchers have been able to expand its utility by expressing human proteins in yeast, or by humanizing specific yeast amino acids, proteins or even entire pathways. These methods are increasingly being scaled in throughput, further enabling the detailed investigation of human biology and disease-specific variations of human genes in a simplified model organism.

Journal of Biological Chemistry, 2015

Background: Anti-microbial hexapeptides trap Holliday junctions and inhibit junction-processing e... more Background: Anti-microbial hexapeptides trap Holliday junctions and inhibit junction-processing enzymes. Results: Hexapeptides induce multiple conformations and dynamic fluctuations of two Holliday junctions that differ in core sequence. Conclusion: Destabilization of the functional junction conformation likely contributes to inhibition of enzymes that process Holliday junctions. Significance: Ligand-induced conformational dynamics may contribute generally to the action of anti-microbial agents that target specialized DNA structures.

Science (New York, N.Y.), Jan 22, 2015

To determine whether genes retain ancestral functions over a billion years of evolution and to id... more To determine whether genes retain ancestral functions over a billion years of evolution and to identify principles of deep evolutionary divergence, we replaced 414 essential yeast genes with their human orthologs, assaying for complementation of lethal growth defects upon loss of the yeast genes. Nearly half (47%) of the yeast genes could be successfully humanized. Sequence similarity and expression only partly predicted replaceability. Instead, replaceability depended strongly on gene modules: Genes in the same process tended to be similarly replaceable (e.g., sterol biosynthesis) or not (e.g., DNA replication initiation). Simulations confirmed that selection for specific function can maintain replaceability despite extensive sequence divergence. Critical ancestral functions of many essential genes are thus retained in a pathway-specific manner, resilient to drift in sequences, splicing, and protein interfaces.

Mobile DNA III, 2015

Tyrosine site-specific recombinases (YRs) are widely distributed among prokaryotes and their viru... more Tyrosine site-specific recombinases (YRs) are widely distributed among prokaryotes and their viruses, and were thought to be confined to the budding yeast lineage among eukaryotes. However, YR-harboring retrotransposons (the DIRS and PAT families) and DNA transposons (Cryptons) have been identified in a variety of eukaryotes. The YRs utilize a common chemical mechanism, analogous to that of type IB topoisomerases, to bring about a plethora of genetic rearrangements with important physiological consequences in their respective biological contexts. A subset of the tyrosine recombinases has provided model systems for analyzing the chemical mechanisms and conformational features of the recombination reaction using chemical, biochemical, topological, structural, and single molecule-biophysical approaches. YRs with simple reaction requirements have been utilized to bring about programmed DNA rearrangements for addressing fundamental questions in developmental biology. They have also been ...

Nucleic Acids Research, 2015

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl... more Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The S P preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or R P-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5 to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

Microbiology Spectrum, 2014

The multicopy 2-micron plasmid of Saccharomyces cerevisiae , a resident of the nucleus, is remark... more The multicopy 2-micron plasmid of Saccharomyces cerevisiae , a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely to efficient replication, equal segregation, and copy number maintenance. A partitioning system comprised of two plasmid-coded proteins, Rep1 and Rep2, and a partitioning locus, STB , is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep- STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining a stea...

Brenner's Encyclopedia of Genetics, 2013

ABSTRACT The widespread existence of selfish genetic elements in biological kingdoms is the resul... more ABSTRACT The widespread existence of selfish genetic elements in biological kingdoms is the result of their ability for efficient replication and propagation during host cell division. A subset of these elements can also transmit horizontally, thereby modulating the fitness of cell populations within an ecological niche and influencing selection under specific conditions. Multiple mechanisms have evolved for the spread of distinct classes of selfish elements. Some involve the duplication of an element during its movement, others not necessarily so. Retrotransposons and retrohoming introns mediate their mobility through RNA intermediates. Certain extreme forms of selfish elements ensure their persistence in a host by killing cells that fail to acquire them. Not all of the elements grouped under the ‘selfish’ label may be exclusively selfish. Some of these elements may serve important functions in gene regulation, confer selective advantages to the host under stress conditions, and contribute in general to genome organization, dynamics, and evolution.

Journal of Biological Chemistry, 2014

Background: Mutations in the human mitochondrial DNA polymerase (Pol-␥) have been linked to disea... more Background: Mutations in the human mitochondrial DNA polymerase (Pol-␥) have been linked to diseases with varying severity and age of onset. Results: Yeast cells expressing human Pol-␥ reveal a correlation of Pol-␥ fidelity with human disease onset. Conclusion: Humanized yeast provides an efficient system to correlate biochemical defects in Pol-␥ with physiological consequences. Significance: The Pol-␥-associated diseases may be caused by the low accuracy of Pol-␥ mutants, not low rates of replication.

Journal of Bacteriology, 2012

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5&... more Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on dl-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on dl-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on dl-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on dl-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of dl-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by dl-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by dl-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.

ABSTRACTThiabendazole (TBZ) is an FDA-approved benzimidazole widely used for its antifungal and a... more ABSTRACTThiabendazole (TBZ) is an FDA-approved benzimidazole widely used for its antifungal and antihelminthic properties. We showed previously that TBZ is also a potent vascular disrupting agent and inhibits angiogenesis at the tissue level by dissociating vascular endothelial cells in newly formed blood vessels. Here, we uncover TBZ’s molecular target and mechanism of action. Using human cell culture, molecular modeling, and humanized yeast, we find that TBZ selectively targets only 1 of 9 human β-tubulin isotypes (TUBB8) to specifically disrupt endothelial cell microtubules. By leveraging epidemiological pesticide resistance data and mining chemical features of commercially used benzimidazoles, we discover that a broader class of benzimidazole compounds, in extensive use for 50 years, also potently disrupt immature blood vessels and inhibit angiogenesis. Thus, besides identifying the molecular mechanism of benzimidazole-mediated vascular disruption, this study presents evidence r...

Many gene families have been expanded by gene duplications along the human lineage, relative to a... more Many gene families have been expanded by gene duplications along the human lineage, relative to ancestral opisthokonts, but the extent to which the duplicated genes function similarly is understudied. Here, we focused on structural cytoskeletal genes involved in critical cellular processes including chromosome segregation, macromolecular transport, and cell shape maintenance. To determine functional redundancy and divergence of duplicated human genes, we systematically humanized the yeast actin, myosin, tubulin, and septin genes, testing ∼85% of human cytoskeletal genes across 7 gene families for their ability to complement a growth defect induced by deletion of the corresponding yeast ortholog. In 5 of 7 families—all but α-tubulin and light myosin, we found at least one human gene capable of complementing loss of the yeast gene. Despite rescuing growth defects, we observed differential abilities of human genes to rescue cell morphology, meiosis, and mating defects. By comparing phe...

Gene duplication is seen as a major source of structural and functional divergence in genome evol... more Gene duplication is seen as a major source of structural and functional divergence in genome evolution. Under the conventional models of sub- or neofunctionalizaton, functional changes arise in one of the duplicates after duplication. However, we suggest here that the presence of a duplicated gene can result in functional changes to its interacting partners. We explore this hypothesis by in-silico evolution of a heterodimer when one member of the interacting pair is duplicated. We examine how a range of selection pressures and protein structures leads to differential patterns of evolutionary divergence. We find that a surprising number of distinct evolutionary trajectories can be observed even in a simple three member system. Further, we observe that selection to correct dosage imbalance can affect the evolution of the initial function in several unexpected ways. For example, if a duplicate is under selective pressure to avoid binding its original binding partner, this can lead to c...

Despite over a billion years of evolutionary divergence, several thousand human genes possess cle... more Despite over a billion years of evolutionary divergence, several thousand human genes possess clearly identifiable orthologs in yeast, and many have undergone lineage-specific duplications in one or both lineages. The ortholog conjecture postulates that orthologous genes between species retain ancestral functions despite divergence over vast timescales, but duplicated genes will be free to diverge in function. However, the retention of ancestral functions among co-orthologs between species and within gene families has been difficult to test experimentally at scale. In order to investigate how ancestral functions are retained or lost post-duplication, we systematically replaced hundreds of essential yeast genes with their human orthologs from gene families that have undergone lineage-specific duplications, including those with single duplications (one yeast gene to two human genes, 1:2) or higher-order expansions (1:>2) in the human lineage. We observe a variable pattern of replac...

Molecular Biology and Evolution

Gene duplication is seen as a major source of structural and functional divergence in genome evol... more Gene duplication is seen as a major source of structural and functional divergence in genome evolution. Under the conventional models of sub or neofunctionalization, functional changes arise in one of the duplicates after duplication. However, we suggest here that the presence of a duplicated gene can result in functional changes to its interacting partners. We explore this hypothesis by in silico evolution of a heterodimer when one member of the interacting pair is duplicated. We examine how a range of selection pressures and protein structures leads to differential patterns of evolutionary divergence. We find that a surprising number of distinct evolutionary trajectories can be observed even in a simple three member system. Further, we observe that selection to correct dosage imbalance can affect the evolution of the initial function in several unexpected ways. For example, if a duplicate is under selective pressure to avoid binding its original binding partner, this can lead to c...

eLife, Jun 29, 2017

Eukaryotes and prokaryotes last shared a common ancestor ~2 billion years ago, and while many pre... more Eukaryotes and prokaryotes last shared a common ancestor ~2 billion years ago, and while many present-day genes in these lineages predate this divergence, the extent to which these genes still perform their ancestral functions is largely unknown. To test principles governing retention of ancient function, we asked if prokaryotic genes could replace their essential eukaryotic orthologs. We systematically replaced essential genes in yeast by their 1:1 orthologs from Escherichia coli. After accounting for mitochondrial localization and alternative start codons, 31 out of 51 bacterial genes tested (61%) could complement a lethal growth defect and replace their yeast orthologs with minimal effects on growth rate. Replaceability was determined on a pathway-by-pathway basis; codon usage, abundance, and sequence similarity contributed predictive power. The heme biosynthesis pathway was particularly amenable to inter-kingdom exchange, with each yeast enzyme replaceable by its bacterial, huma...

Briefings in functional genomics, Jan 13, 2015

Despite a billion years of divergent evolution, the baker's yeast Saccharomyces cerevisiae ha... more Despite a billion years of divergent evolution, the baker's yeast Saccharomyces cerevisiae has long proven to be an invaluable model organism for studying human biology. Given its tractability and ease of genetic manipulation, along with extensive genetic conservation with humans, it is perhaps no surprise that researchers have been able to expand its utility by expressing human proteins in yeast, or by humanizing specific yeast amino acids, proteins or even entire pathways. These methods are increasingly being scaled in throughput, further enabling the detailed investigation of human biology and disease-specific variations of human genes in a simplified model organism.

Journal of Biological Chemistry, 2015

Background: Anti-microbial hexapeptides trap Holliday junctions and inhibit junction-processing e... more Background: Anti-microbial hexapeptides trap Holliday junctions and inhibit junction-processing enzymes. Results: Hexapeptides induce multiple conformations and dynamic fluctuations of two Holliday junctions that differ in core sequence. Conclusion: Destabilization of the functional junction conformation likely contributes to inhibition of enzymes that process Holliday junctions. Significance: Ligand-induced conformational dynamics may contribute generally to the action of anti-microbial agents that target specialized DNA structures.

Science (New York, N.Y.), Jan 22, 2015

To determine whether genes retain ancestral functions over a billion years of evolution and to id... more To determine whether genes retain ancestral functions over a billion years of evolution and to identify principles of deep evolutionary divergence, we replaced 414 essential yeast genes with their human orthologs, assaying for complementation of lethal growth defects upon loss of the yeast genes. Nearly half (47%) of the yeast genes could be successfully humanized. Sequence similarity and expression only partly predicted replaceability. Instead, replaceability depended strongly on gene modules: Genes in the same process tended to be similarly replaceable (e.g., sterol biosynthesis) or not (e.g., DNA replication initiation). Simulations confirmed that selection for specific function can maintain replaceability despite extensive sequence divergence. Critical ancestral functions of many essential genes are thus retained in a pathway-specific manner, resilient to drift in sequences, splicing, and protein interfaces.

Mobile DNA III, 2015

Tyrosine site-specific recombinases (YRs) are widely distributed among prokaryotes and their viru... more Tyrosine site-specific recombinases (YRs) are widely distributed among prokaryotes and their viruses, and were thought to be confined to the budding yeast lineage among eukaryotes. However, YR-harboring retrotransposons (the DIRS and PAT families) and DNA transposons (Cryptons) have been identified in a variety of eukaryotes. The YRs utilize a common chemical mechanism, analogous to that of type IB topoisomerases, to bring about a plethora of genetic rearrangements with important physiological consequences in their respective biological contexts. A subset of the tyrosine recombinases has provided model systems for analyzing the chemical mechanisms and conformational features of the recombination reaction using chemical, biochemical, topological, structural, and single molecule-biophysical approaches. YRs with simple reaction requirements have been utilized to bring about programmed DNA rearrangements for addressing fundamental questions in developmental biology. They have also been ...

Nucleic Acids Research, 2015

Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl... more Tyrosine site-specific recombinases, which promote one class of biologically important phosphoryl transfer reactions in DNA, exemplify active site mechanisms for stabilizing the phosphate transition state. A highly conserved arginine duo (Arg-I; Arg-II) of the recombinase active site plays a crucial role in this function. Cre and Flp recombinase mutants lacking either arginine can be rescued by compensatory charge neutralization of the scissile phosphate via methylphosphonate (MeP) modification. The chemical chirality of MeP, in conjunction with mutant recombinases, reveals the stereochemical contributions of Arg-I and Arg-II. The S P preference of the native reaction is specified primarily by Arg-I. MeP reaction supported by Arg-II is nearly bias-free or R P-biased, depending on the Arg-I substituent. Positional conservation of the arginines does not translate into strict functional conservation. Charge reversal by glutamic acid substitution at Arg-I or Arg-II has opposite effects on Cre and Flp in MeP reactions. In Flp, the base immediately 5 to the scissile MeP strongly influences the choice between the catalytic tyrosine and water as the nucleophile for strand scission, thus between productive recombination and futile hydrolysis. The recombinase active site embodies the evolutionary optimization of interactions that not only favor the normal reaction but also proscribe antithetical side reactions.

Microbiology Spectrum, 2014

The multicopy 2-micron plasmid of Saccharomyces cerevisiae , a resident of the nucleus, is remark... more The multicopy 2-micron plasmid of Saccharomyces cerevisiae , a resident of the nucleus, is remarkable for its high chromosome-like stability. The plasmid does not appear to contribute to the fitness of the host, nor does it impose a significant metabolic burden on the host at its steady state copy number. The plasmid may be viewed as a highly optimized selfish DNA element whose genome design is devoted entirely to efficient replication, equal segregation, and copy number maintenance. A partitioning system comprised of two plasmid-coded proteins, Rep1 and Rep2, and a partitioning locus, STB , is responsible for equal or nearly equal segregation of plasmid molecules to mother and daughter cells. Current evidence supports a model in which the Rep- STB system promotes the physical association of the plasmid with chromosomes and thus plasmid segregation by a hitchhiking mechanism. The Flp site-specific recombination system housed by the plasmid plays a critical role in maintaining a stea...

Brenner's Encyclopedia of Genetics, 2013

ABSTRACT The widespread existence of selfish genetic elements in biological kingdoms is the resul... more ABSTRACT The widespread existence of selfish genetic elements in biological kingdoms is the result of their ability for efficient replication and propagation during host cell division. A subset of these elements can also transmit horizontally, thereby modulating the fitness of cell populations within an ecological niche and influencing selection under specific conditions. Multiple mechanisms have evolved for the spread of distinct classes of selfish elements. Some involve the duplication of an element during its movement, others not necessarily so. Retrotransposons and retrohoming introns mediate their mobility through RNA intermediates. Certain extreme forms of selfish elements ensure their persistence in a host by killing cells that fail to acquire them. Not all of the elements grouped under the ‘selfish’ label may be exclusively selfish. Some of these elements may serve important functions in gene regulation, confer selective advantages to the host under stress conditions, and contribute in general to genome organization, dynamics, and evolution.

Journal of Biological Chemistry, 2014

Background: Mutations in the human mitochondrial DNA polymerase (Pol-␥) have been linked to disea... more Background: Mutations in the human mitochondrial DNA polymerase (Pol-␥) have been linked to diseases with varying severity and age of onset. Results: Yeast cells expressing human Pol-␥ reveal a correlation of Pol-␥ fidelity with human disease onset. Conclusion: Humanized yeast provides an efficient system to correlate biochemical defects in Pol-␥ with physiological consequences. Significance: The Pol-␥-associated diseases may be caused by the low accuracy of Pol-␥ mutants, not low rates of replication.

Journal of Bacteriology, 2012

Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5&... more Diaminopropionate ammonia lyase (DAPAL) is a pyridoxal-5'phosphate (PLP)-dependent enzyme that catalyzes the conversion of diaminopropionate (DAP) to pyruvate and ammonia and plays an important role in cell metabolism. We have investigated the role of the ygeX gene of Escherichia coli K-12 and its ortholog, STM1002, in Salmonella enterica serovar Typhimurium LT2, presumed to encode DAPAL, in the growth kinetics of the bacteria. While Salmonella Typhimurium LT2 could grow on dl-DAP as a sole carbon source, the wild-type E. coli K-12 strain exhibited only marginal growth on dl-DAP, suggesting that DAPAL is functional in S. Typhimurium. The expression of ygeX in E. coli was low as detected by reverse transcriptase PCR (RT-PCR), consistent with the poor growth of E. coli on dl-DAP. Strains of S. Typhimurium and E. coli with STM1002 and ygeX, respectively, deleted showed loss of growth on dl-DAP, confirming that STM1002 (ygeX) is the locus encoding DAPAL. Interestingly, the presence of dl-DAP caused a growth inhibition of the wild-type E. coli strain as well as the knockout strains of S. Typhimurium and E. coli in minimal glucose/glycerol medium. Inhibition by dl-DAP was rescued by transforming the strains with plasmids containing the STM1002 (ygeX) gene encoding DAPAL or supplementing the medium with Casamino Acids. Growth restoration studies using media lacking specific amino acid supplements suggested that growth inhibition by dl-DAP in the absence of DAPAL is associated with auxotrophy related to the inhibition of the enzymes involved in the biosynthetic pathways of pyruvate and aspartate and the amino acids derived from them.