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Open Access Publications from the University of California

Scholarly Works (52 results)

  • Article
  • Peer Reviewed
UC Riverside Previously Published Works (2005)

To enable investigation of nematode mitochondrial DNA evolution, methodology has been developed to amplify intact nematode mitochondrial genomes in preparative yields using a rolling circle amplification strategy. Successful reactions were generated from whole cell template DNA prepared by alkaline lysis. Rolling circle amplification products can be used as templates for standard PCR reactions, restriction enzyme reactions, and direct DNA sequencing.

    Cover page: Rolling circle amplification of complete nematode mitochondrial genomes
    • Thesis
    • Peer Reviewed
    UC Riverside Electronic Theses and Dissertations (2013)

    Eukaryotic cells are divided into many compartments, enabling spatial and temporal regulation in higher cells. Mitochondria are unique among these compartments in animal cells, as they harbor a DNA genome separate from the nuclear genome, the mitochondrial DNA (mtDNA). Defects in the replication, transcription, translation or assembly of mtDNA encoded subunits cause severe diseases in humans. Deleterious mtDNA mutations may be introduced by several routes, particularly replication error. A better understanding of the mechanism of mtDNA replication is required to generate hypotheses as to how to remedy the introduction of mtDNA mutations as a possible avenue to reduce the incidence of mitochondrial myopathies. To further understand the mechanism of mtDNA replication as well as the spectrum of mtDNA mutations present across eukaryotic species, I initiated computational and biochemical studies addressing mtDNA replication processes using the genetic nematode model, Caenorhabditis elegans. I used a computational approach to analyze transition and transversion mutations which alter the distribution of nucleotides across mtDNA strands. The development of custom scripts allowed an expansion of this work to include all metazoan mitochondrial genomes publicly available. I identified lineage-specific variation in mtDNA mutation patterning that suggested differences in the mechanism of mtDNA replication between animal phyla. Furthermore, I examined mtDNA topoisomers isolated from the nematode C. elegans. A detailed analysis by two-dimensional agarose gel electrophoresis and transmission electron microscopy revealed that the mtDNA of C. elegans is not replicated by these mechanisms, but rather rolling circle replication, a fourth mechanism novel among animals. My results indicate that generalizations regarding mtDNA replication modes cannot be derived by studying model organisms alone. Multiple mtDNA replication mechanisms have evolved to protect replicating mtDNA from a vulnerable, single-stranded state which is thought to be highly mutable.

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    Cover page: Replication and Recombination of Nematode Mitochondrial DNA
    • Article
    • Peer Reviewed
    UC Riverside Previously Published Works (2011)
    Rare syntenic conservation, sequence duplication, and the use of both DNA strands to encode genes are signature architectural features defining mitochondrial genomes of enoplean nematodes. These characteristics stand in contrast to the more conserved mitochondrial genome sizes and transcriptional organizations of mitochondrial DNAs (mtDNAs) derived from chromadorean nematodes. To address the frequency of gene rearrangement within nematode mitochondrial DNA (mtDNA), mitochondrial genome variation has been characterized within a more confined enoplean taxonomic unit, the family Mermithidae. The complete nucleotide sequences of the mosquito parasitic nematodes Romanomermis culicivorax, R. nielseni, and R. iyengari mtDNA have been determined. Duplicated expanses encompassing different regions of the mitochondrial genomes were found in each of these congeners. These mtDNA shared few rRNA and protein gene junctions, indicating extensive gene rearrangement within the Romanomermis lineage. Rapid structural changes are also observed at the conspecific level where no two individual nematodes carry the same haplotype. Rolling circle amplification was used to isolate complete mitochondrial genomes from individuals in local populations of Thaumamermis cosgrovei, a parasite of terrestrial isopods. Mitochondrial DNA length variants ranging from 19 to 34 kb are observed, but haplotypes are not shared between any two individuals. The complete nucleotide sequences of three haplotypes have been determined, revealing a constant region encoding most mitochondrial genes and a hypervariable segment that contains intact and pseudogene copies of several mitochondrial genes, duplicated to different copy numbers, resulting in mtDNA size variation. Constant rearrangement generates new T. cosgrovei mtDNA forms.
      Cover page: Rampant gene rearrangement and haplotype hypervariation among nematode mitochondrial genomes
      • Article
      • Peer Reviewed
      UC Riverside Previously Published Works (2003)

      Evolutionary relationships based on nucleotide variation within the D3 26S rDNA region were examined among a collection of seven Meloidogyne hapla isolates and seven isolates of M. arenaria, M. incognita, and M. javanica. Using D3A and D3B primers, a 350-bp region was PCR amplified front genomic DNA and double-stranded nucleotide sequence obtained. Phylogenetic analyses using three independent clustering methods all provided support for a division between the automictic M. hapla and the apomictic M. arenaria, M. incognita, and M. javanica. A nucleotide sequence character distinguishing M. hapla from the three apomictic species was a 3-bp insertion within the interior of the D3 region. The three apomictic species shared a common D3 haplotype, suggesting a recent branching. Single M. hapla individuals contained two different haplotypes, differentiated by a Sau3AI restriction site polymorphism. Isolates of M. Javanica appeared to have only one haplotype, while M. incognita and M. arenaria maintained more than one haplotype in air isolate.

        Cover page: Nucleotide substitution patterning within the Meloidogyne rDNA D3 region and its evolutionary implications
        • Article
        • Peer Reviewed
        UC Riverside Previously Published Works (2015)
        Mitochondrial DNA (mtDNA) encodes respiratory complex subunits essential to almost all eukaryotes; hence respiratory competence requires faithful duplication of this molecule. However, the mechanism(s) of its synthesis remain hotly debated. Here we have developed Caenorhabditis elegans as a convenient animal model for the study of metazoan mtDNA synthesis. We demonstrate that C. elegans mtDNA replicates exclusively by a phage-like mechanism, in which multimeric molecules are synthesized from a circular template. In contrast to previous mammalian studies, we found that mtDNA synthesis in the C. elegans gonad produces branched-circular lariat structures with multimeric DNA tails; we were able to detect multimers up to four mtDNA genome unit lengths. Further, we did not detect elongation from a displacement-loop or analogue of 7S DNA, suggesting a clear difference from human mtDNA in regard to the site(s) of replication initiation. We also identified cruciform mtDNA species that are sensitive to cleavage by the resolvase RusA; we suggest these four-way junctions may have a role in concatemer-to-monomer resolution. Overall these results indicate that mtDNA synthesis in C. elegans does not conform to any previously documented metazoan mtDNA replication mechanism, but instead are strongly suggestive of rolling circle replication, as employed by bacteriophages. As several components of the metazoan mitochondrial DNA replisome are likely phage-derived, these findings raise the possibility that the rolling circle mtDNA replication mechanism may be ancestral among metazoans.
          Cover page: A Rolling Circle Replication Mechanism Produces Multimeric Lariats of Mitochondrial DNA in Caenorhabditis elegans
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2020)
          The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau has a central role in the pathogenesis of several forms of dementia known as tauopathies-including Alzheimer's disease, frontotemporal dementia and chronic traumatic encephalopathy1. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity2. This observation and complementary experimental studies3,4 have suggested that tau can spread in a prion-like manner, by passing to naive cells in which it templates misfolding and aggregation. However, although the propagation of tau has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein receptor-related protein 1 (LRP1) controls the endocytosis of tau and its subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and in induced pluripotent stem cell-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule-binding repeat region of tau. Furthermore, downregulation of LRP1 in an in vivo mouse model of tau spread was found to effectively reduce the propagation of tau between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain, and therefore a potential target for the treatment of diseases that involve tau spread and aggregation.
            Cover page: LRP1 is a master regulator of tau uptake and spread
            • Article
            • Peer Reviewed
            UC San Francisco Previously Published Works (2020)
            The spread of protein aggregates during disease progression is a common theme underlying many neurodegenerative diseases. The microtubule-associated protein tau has a central role in the pathogenesis of several forms of dementia known as tauopathies-including Alzheimer's disease, frontotemporal dementia and chronic traumatic encephalopathy1. Progression of these diseases is characterized by the sequential spread and deposition of protein aggregates in a predictable pattern that correlates with clinical severity2. This observation and complementary experimental studies3,4 have suggested that tau can spread in a prion-like manner, by passing to naive cells in which it templates misfolding and aggregation. However, although the propagation of tau has been extensively studied, the underlying cellular mechanisms remain poorly understood. Here we show that the low-density lipoprotein receptor-related protein 1 (LRP1) controls the endocytosis of tau and its subsequent spread. Knockdown of LRP1 significantly reduced tau uptake in H4 neuroglioma cells and in induced pluripotent stem cell-derived neurons. The interaction between tau and LRP1 is mediated by lysine residues in the microtubule-binding repeat region of tau. Furthermore, downregulation of LRP1 in an in vivo mouse model of tau spread was found to effectively reduce the propagation of tau between neurons. Our results identify LRP1 as a key regulator of tau spread in the brain, and therefore a potential target for the treatment of diseases that involve tau spread and aggregation.
              Cover page: LRP1 is a master regulator of tau uptake and spread
              • Article
              • Peer Reviewed
              UC San Francisco Previously Published Works (2018)
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2019)
                The NIH Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative is focused on developing new tools and neurotechnologies to transform our understanding of the brain, and neuroethics is an essential component of this research effort. Coordination with other brain projects around the world will help maximize success.
                  • Article
                  • Peer Reviewed
                  UC San Diego Previously Published Works (2014)

                  Importance

                  Converging evidence indicates that clusterin, a chaperone glycoprotein, influences Alzheimer disease neurodegeneration. However, the precise role of clusterin in Alzheimer disease pathogenesis is still not well understood.

                  Objective

                  To elucidate the relationship between clusterin, amyloid-β (Aβ), phosphorylated tau (p-tau), and the rate of brain atrophy over time among nondemented older individuals.

                  Design, setting, and participants

                  This longitudinal cohort included cognitively normal older participants and individuals with mild cognitive impairment assessed with baseline lumbar puncture and longitudinal structural magnetic resonance imaging. We examined 241 nondemented older individuals from research centers across the United States and Canada (91 participants with a Clinical Dementia Rating score of 0 and 150 individuals with a Clinical Dementia Rating score of 0.5).

                  Main outcomes and measures

                  Using linear mixed-effects models, we investigated interactions between cerebrospinal fluid (CSF) clusterin, CSF Aβ1-42, and CSF p-tau at threonine 181 (p-tau181p) on the atrophy rate of the entorhinal cortex and hippocampus.

                  Results

                  Across all participants, we found a significant interaction between CSF clusterin and CSF Aβ1-42 on the entorhinal cortex atrophy rate but not on the hippocampal atrophy rate. Cerebrospinal fluid clusterin was associated with the entorhinal cortex atrophy rate among CSF Aβ1-42-positive individuals but not among CSF Aβ1-42-negative individuals. In secondary analyses, we found significant interactions between CSF Aβ1-42 and CSF clusterin, as well as CSF Aβ1-42 and CSF p-tau181p, on the entorhinal cortex atrophy rate. We found similar results in subgroup analyses within the mild cognitive impairment and cognitively normal cohorts.

                  Conclusions and relevance

                  In nondemented older individuals, Aβ-associated volume loss occurs in the presence of elevated clusterin. The effect of clusterin on Aβ-associated brain atrophy is not confounded or explained by p-tau. These findings implicate a potentially important role for clusterin in the earliest stages of the Alzheimer disease neurodegenerative process and suggest independent effects of clusterin and p-tau on Aβ-associated volume loss.
                    Cover page: The Role of Clusterin in Amyloid-β–Associated Neurodegeneration
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