Transposable Elements and Their Use as Genetic Tools in Arthropods

The aim of the research presented in this thesis is to examine the transposition mechanism of Class 2 transposons and to assess their employment as genetic tools in arthropod genomes. The first chapter reviews transposable genetic elements and their intimate relationship with eukaryotic genome diversity, methods of host genome control for maintaining genome integrity, including the piRNA system, and the current. Chapter two introduces a hAT transposable element, Hermes, that originates from the arthropod genome of Musca domestica. The Hermes element is an active transposon that has been shown to transform several non-homologous arthropod species. Our laboratory has participated in a collaborative research project to understand to molecular details of Hermes transposition by crystal structure and biochemical assays. The research outlined in chapter two provides a rationale for the occurrence of Hermes as an octameric protein with multiple DNA binding domains, as Hermes is the only hAT transposable element for which a co-crystal structure has been successfully produced. Chapter three introduces a newly described MULE element from the genome of mosquito Aedes aegpyti, a major global arboviral vector. The research outlined demonstrates that the Ae. aegypti Muta1 element is active in cell culture and can transform the model dipteran, Drosophila melanogaster, and can remobilize in the germline. Chapter four examines the potential of the Muta1 element as the bases for a novel forward genetics-based tool in the arbovector species, Aedes aegypti, for which no enhancer trap system has been effectively developed. This chapter also outlines potential challenges to implementing a transposon-based tool in this species due to their evolutionarily expanded piRNA pathway. Chapter five explains the ability of the Muta1 element to transform and remobilize in two Anopheles mosquito species, and explores the potential benefit of having a novel enhancer trap system to deploy in mosquito species

Transposable Elements and Their Use as Genetic Tools in Arthropods

The aim of the research presented in this thesis is to examine the transposition mechanism of Class 2 transposons and to assess their employment as genetic tools in arthropod genomes. The first chapter reviews transposable genetic elements and their intimate relationship with eukaryotic genome diversity, methods of host genome control for maintaining genome integrity, including the piRNA system, and the current. Chapter two introduces a hAT transposable element, Hermes, that originates from the arthropod genome of Musca domestica. The Hermes element is an active transposon that has been shown to transform several non-homologous arthropod species. Our laboratory has participated in a collaborative research project to understand to molecular details of Hermes transposition by crystal structure and biochemical assays. The research outlined in chapter two provides a rationale for the occurrence of Hermes as an octameric protein with multiple DNA binding domains, as Hermes is the only hAT transposable element for which a co-crystal structure has been successfully produced. Chapter three introduces a newly described MULE element from the genome of mosquito Aedes aegpyti, a major global arboviral vector. The research outlined demonstrates that the Ae. aegypti Muta1 element is active in cell culture and can transform the model dipteran, Drosophila melanogaster, and can remobilize in the germline. Chapter four examines the potential of the Muta1 element as the bases for a novel forward genetics-based tool in the arbovector species, Aedes aegypti, for which no enhancer trap system has been effectively developed. This chapter also outlines potential challenges to implementing a transposon-based tool in this species due to their evolutionarily expanded piRNA pathway. Chapter five explains the ability of the Muta1 element to transform and remobilize in two Anopheles mosquito species, and explores the potential benefit of having a novel enhancer trap system to deploy in mosquito species

EFISIENSI FITOREMEDIASI MENGGUNAKANBAMBU KUNING (Bambusa vulgaris Schrad) TERHADAP KADAR BOD DAN NITRAT LIMBAH CAIRDI SENTRA INDUSTRI KERUPUKKABUPATEN INDRAMAYUTAHUN 2017

Gene drives based on CRISPR/Cas9 have the potential to reduce the enormous harm inflicted by crop pests and insect vectors of human disease, as well as to bolster valued species. In contrast with extensive empirical and theoretical studies in diploid organisms, little is known about CRISPR gene drive in haplodiploids, despite their immense global impacts as pollinators, pests, natural enemies of pests, and invasive species in native habitats. Here, we analyze mathematical models demonstrating that, in principle, CRISPR homing gene drive can work in haplodiploids, as well as at sex-linked loci in diploids. However, relative to diploids, conditions favoring the spread of alleles deleterious to haplodiploid pests by CRISPR gene drive are narrower, the spread is slower, and resistance to the drive evolves faster. By contrast, the spread of alleles that impose little fitness cost or boost fitness was not greatly hindered in haplodiploids relative to diploids. Therefore, altering traits to minimize damage caused by harmful haplodiploids, such as interfering with transmission of plant pathogens, may be more likely to succeed than control efforts based on introducing traits that reduce pest fitness. Enhancing fitness of beneficial haplodiploids with CRISPR gene drive is also promising

Structural basis of hAT transposon end recognition by hermes, an octameric dna transposase from musca domestica

Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock\u27s archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA

Efficient CRISPR/Cas9-mediated genome modification of the glassy-winged sharpshooter Homalodisca vitripennis (Germar).

CRISPR/Cas9 technology enables the extension of genetic techniques into insect pests previously refractory to genetic analysis. We report the establishment of genetic analysis in the glassy-winged sharpshooter (GWSS), Homalodisca vitripennis, which is a significant leafhopper pest of agriculture in California. We use a novel and simple approach of embryo microinjection in situ on the host plant and obtain high frequency mutagenesis, in excess of 55%, of the cinnabar and white eye pigmentation loci. Through pair matings, we obtained 100% transmission of w and cn alleles to the G3 generation and also established that both genes are located on autosomes. Our analysis of wing phenotype revealed an unexpected discovery of the participation of pteridine pigments in wing and wing-vein coloration, indicating a role for these pigments beyond eye color. We used amplicon sequencing to examine the extent of off-target mutagenesis in adults arising from injected eggs, which was found to be negligible or non-existent. Our data show that GWSS can be easily developed as a genetic model system for the Hemiptera, enabling the study of traits that contribute to the success of invasive pests and vectors of plant pathogens. This will facilitate novel genetic control strategies

The <em>C. elegans</em> Rab Family: Identification, Classification and Toolkit Construction

<div><p>Rab monomeric GTPases regulate specific aspects of vesicle transport in eukaryotes including coat recruitment, uncoating, fission, motility, target selection and fusion. Moreover, individual Rab proteins function at specific sites within the cell, for example the ER, golgi and early endosome. Importantly, the localization and function of individual Rab subfamily members are often conserved underscoring the significant contributions that model organisms such as <em>Caenorhabditis elegans</em> can make towards a better understanding of human disease caused by Rab and vesicle trafficking malfunction. With this in mind, a bioinformatics approach was first taken to identify and classify the complete <em>C. elegans</em> Rab family placing individual Rabs into specific subfamilies based on molecular phylogenetics. For genes that were difficult to classify by sequence similarity alone, we did a comparative analysis of intron position among specific subfamilies from yeast to humans. This two-pronged approach allowed the classification of 30 out of 31 <em>C. elegans</em> Rab proteins identified here including <em>Rab31/Rab50</em>, a likely member of the last eukaryotic common ancestor (LECA). Second, a molecular toolset was created to facilitate research on biological processes that involve Rab proteins. Specifically, we used Gateway-compatible <em>C. elegans</em> ORFeome clones as starting material to create 44 full-length, sequence-verified, dominant-negative (DN) and constitutive active (CA) <em>rab</em> open reading frames (ORFs). Development of this toolset provided independent research projects for students enrolled in a research-based molecular techniques course at California State University, East Bay (CSUEB).</p> </div

A novel DPH5-related diphthamide-deficiency syndrome causing embryonic lethality or profound neurodevelopmental disorder

PurposeDiphthamide is a post-translationally modified histidine essential for messenger RNA translation and ribosomal protein synthesis. We present evidence for DPH5 as a novel cause of embryonic lethality and profound neurodevelopmental delays (NDDs).MethodsMolecular testing was performed using exome or genome sequencing. A targeted Dph5 knockin mouse (C57BL/6Ncrl-Dph5em1Mbp/Mmucd) was created for a DPH5 p.His260Arg homozygous variant identified in 1 family. Adenosine diphosphate-ribosylation assays in DPH5-knockout human and yeast cells and in silico modeling were performed for the identified DPH5 potential pathogenic variants.ResultsDPH5 variants p.His260Arg (homozygous), p.Asn110Ser and p.Arg207Ter (heterozygous), and p.Asn174LysfsTer10 (homozygous) were identified in 3 unrelated families with distinct overlapping craniofacial features, profound NDDs, multisystem abnormalities, and miscarriages. Dph5 p.His260Arg homozygous knockin was embryonically lethal with only 1 subviable mouse exhibiting impaired growth, craniofacial dysmorphology, and multisystem dysfunction recapitulating the human phenotype. Adenosine diphosphate-ribosylation assays showed absent to decreased function in DPH5-knockout human and yeast cells. In silico modeling of the variants showed altered DPH5 structure and disruption of its interaction with eEF2.ConclusionWe provide strong clinical, biochemical, and functional evidence for DPH5 as a novel cause of embryonic lethality or profound NDDs with multisystem involvement and expand diphthamide-deficiency syndromes and ribosomopathies

Comparative analysis of intron position among diverse Rab subfamily members.

<p>A) Cladogram indicating evolutionary relationships of 18 species examined here <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049387#pone.0049387-Dunn1" target="_blank">[128]</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049387#pone.0049387-Csuros1" target="_blank">[130]</a>. Ecdy. = Ecdysozoa, Chromal. = Chromalveolata. For species abbreviations see Methods. B) An ML tree of Opisthokonts created from the MSA used to map intron positions. Bootstrap support (100 replicates) is indicated for each subfamily cluster. C) For each subfamily, the number of times a <u>S</u>ubfamily <u>S</u>pecific <u>C</u>onserved <u>I</u>ntron <u>P</u>osition (SSCIP) involving the indicated number of species was observed (gray bars), compared to what is expected by chance (black diamonds). The difference between observed and expected is statistically significant where indicated. *P(Monte Carlo) <0.05. ***P(Monte Carlo)≤0.00001. The Rab<i>31, 6, 5, 22, 34, 21</i> and <i>23</i> subfamilies include 17, 18, 17, 9, 9, 10, 14 and 12 species, respectively. D) and E) Heat map indicating number of introns within <i>Rab31</i> (D) or <i>Rab6</i> (E) that match SSCIPs from <i>Rab31, 5, 22, 21, 6, 34</i> and <i>23</i>. The circled number indicates the number of introns present in the MSA for each gene. % equals the percentage of introns that are shared with the true SSCIP. <i>C56E6.2</i> (D) and <i>Y71H2AM.12</i> (E) are highlighted red. Genbank Descriptions (if any) and RABDB! classifications are included. Classification abbreviations include: HypoRabX1 (H.RabX1), HypoRabX2 (H.RabX2), HypoRabX3 (H.RabX3) and MetazoaRabX3 (M.RabX3). F) A pairwise comparison of intron position conservation between specific genes (Rab31 at left, Rab6 at right) and their corresponding set of SSCIPs. Black diamonds plot the probability that a specific number of intron matches would be expected by chance for each set of conditions. Chart 1 plots a comparison of 5 introns with 7 SSCIPs (5×7). Chart 2∶4×7. Chart 3∶2×7. Chart 4∶4×6. Observed values for a subset of genes are indicated with P values estimated from the Monte Carlo simulation data (See text and methods). Species abbreviations are as in A. C) and F) 72 protosplice sites assumed.</p

Full-length ORF sequence of <i>tag-312</i> (Rab45) isolates reveals a different splice pattern than predicted.

<p>A) A nucleotide alignment of <i>tag-312</i> (FlOCS), genomic DNA and the predicted <i>tag-312</i> ORF at two regions where exon/intron splice junction differences were found. In the top alignment, FlOCS reveals a new intron, splitting predicted exon 7 into exons7a and 7b. In the bottom alignment, FlOCS reveals an alternate 5′ splice donor and 3′ splice acceptor for intron 8. Compare FlOCS-supported 5′ and 3′ splice sites boxed in bold to predicted 5′ and 3′ splices sites (boxed, not bold). B) Two multiple sequence alignments of Rab45 subfamily members spanning the two regions described in 5A above demonstrate the impact of FLOCS-supported gene structure differences. The alignment includes proteins from the nematodes, <i>Caenhorabditis elegans</i>, <i>tag-312</i>(FlOCS) and NP508523.1, <i>Caenhorabditis brenneri, Caenhorabditis remanei</i> and <i>Ascaris suum</i> in addition to <i>Xenopus laevis</i> (frog), <i>Homo sapiens</i> (human), <i>Monodelphis domestica</i> (opossum) and <i>Anolis carolinensis</i> (lizard). The intron that splits exon 7 into two creates a 15 amino acid deletion that is conserved among all species examined (top). The alternate intron 8 creates an Indel in a region of Rab45 that is conserved among nematodes only.</p