Genetics and Genomics Commons^™

The Embryonic Mir-35 Family Of Micrornas Promotes Multiple Aspects Of Fecundity In Caenorhabditis Elegans, Katherine Mcjunkin, Victor R. Ambros

Victor R. Ambros

MicroRNAs guide many aspects of development in all metazoan species. Frequently, microRNAs are expressed during a specific developmental stage to perform a temporally defined function. The C. elegans mir-35-42 microRNAs are expressed abundantly in oocytes and early embryos and are essential for embryonic development. Here, we show that these embryonic microRNAs surprisingly also function to control the number of progeny produced by adult hermaphrodites. Using a temperature-sensitive mir-35-42 family mutant (a deletion of the mir-35-41 cluster), we demonstrate three distinct defects in hermaphrodite fecundity. At permissive temperatures, a mild sperm defect partially reduces hermaphrodite fecundity. At restrictive temperatures, somatic gonad …

An Efficient And Sensitive Method For Preparing Cdna Libraries From Scarce Biological Samples, Catherine H. Sterling, Isana Veksler-Lublinsky, Victor R. Ambros

Victor R. Ambros

The preparation and high-throughput sequencing of cDNA libraries from samples of small RNA is a powerful tool to quantify known small RNAs (such as microRNAs) and to discover novel RNA species. Interest in identifying the small RNA repertoire present in tissues and in biofluids has grown substantially with the findings that small RNAs can serve as indicators of biological conditions and disease states. Here we describe a novel and straightforward method to clone cDNA libraries from small quantities of input RNA. This method permits the generation of cDNA libraries from sub-picogram quantities of RNA robustly, efficiently and reproducibly. We demonstrate …

Caenorhabditis Elegans Micrornas Of The Let-7 Family Act In Innate Immune Response Circuits And Confer Robust Developmental Timing Against Pathogen Stress, Zhiji Ren, Victor R. Ambros

Victor R. Ambros

Animals maintain their developmental robustness against natural stresses through numerous regulatory mechanisms, including the posttranscriptional regulation of gene expression by microRNAs (miRNAs). Caenorhabditis elegans miRNAs of the let-7 family (let-7-Fam) function semiredundantly to confer robust stage specificity of cell fates in the hypodermal seam cell lineages. Here, we show reciprocal regulatory interactions between let-7-Fam miRNAs and the innate immune response pathway in C. elegans. Upon infection of C. elegans larvae with the opportunistic human pathogen Pseudomonas aeruginosa, the developmental timing defects of certain let-7-Fam miRNA mutants are enhanced. This enhancement is mediated by the p38 MAPK innate immune pathway acting …

Immunopurification Of Ago1 Mirnps Selects For A Distinct Class Of Microrna Targets, Xin Hong, Molly Hammell, Victor Ambros, Stephen Cohen

Victor R. Ambros

microRNAs comprise a few percent of animal genes and have been recognized as important regulators of a diverse range of biological processes. Understanding the biological functions of miRNAs requires effective means to identify their targets. Combined efforts from computational prediction, miRNA over-expression or depletion, and biochemical purification have identified thousands of potential miRNA-target pairs in cells and organisms. Complementarity to the miRNA seed sequence appears to be a common principle in target recognition. Other features, including miRNA-target duplex stability, binding site accessibility, and local UTR structure might affect target recognition. Yet computational approaches using such contextual features have yielded largely …

Drosophila Let-7 Microrna Is Required For Remodeling Of The Neuromusculature During Metamorphosis, Nicholas S. Sokol, Peizhang Xu, Yuh-Nung Jan, Victor R. Ambros

Victor R. Ambros

The Drosophila let-7-Complex (let-7-C) is a polycistronic locus encoding three ancient microRNAs: let-7, miR-100, and fly lin-4 (miR-125). We find that the let-7-C locus is principally expressed in the pupal and adult neuromusculature. let-7-C knockout flies appear normal externally but display defects in adult behaviors (e.g., flight, motility, and fertility) as well as clear juvenile features in their neuromusculature. We find that the function of let-7-C to ensure the appropriate remodeling of the abdominal neuromusculature during the larval-to-adult transition is carried out predominantly by let-7 alone. This heterochronic role of let-7 is likely just one of the ways in which …

Effect Of Life History On Microrna Expression During C. Elegans Development, Xantha Karp, Molly Hammell, Maria C. Ow, Victor R. Ambros

Victor R. Ambros

Animals have evolved mechanisms to ensure the robustness of developmental outcomes to changing environments. MicroRNA expression may contribute to developmental robustness because microRNAs are key post-transcriptional regulators of developmental gene expression and can affect the expression of multiple target genes. Caenorhabditis elegans provides an excellent model to study developmental responses to environmental conditions. In favorable environments, C. elegans larvae develop rapidly and continuously through four larval stages. In contrast, in unfavorable conditions, larval development may be interrupted at either of two diapause stages: The L1 diapause occurs when embryos hatch in the absence of food, and the dauer diapause occurs …

Micrornas: Genetically Sensitized Worms Reveal New Secrets, Victor Ambros

Victor R. Ambros

Why do many microRNA gene mutants display no evident phenotype? Multiply mutant worms that are selectively impaired in genetic regulatory network activities have been used to uncover previously unknown functions for numerous Caenorhabditis elegans microRNAs.

Prb/Cki Pathways At The Interface Of Cell Cycle And Development, Victor Ambros

Victor R. Ambros

Comment on: The cyclin-dependent kinase inhibitors, cki-1 and cki-2, act in overlapping but distinct pathways to control cell-cycle quiescence during C. elegans development. Buck SH, et al. Cell Cycle 2009; 8:2613-20.

Mutations Of Adjacent Amino Acid Pairs Are Not Always Independent, Jyotsna Ramanan, Peter Revesz

CSE Conference and Workshop Papers

Evolutionary studies usually assume that the genetic mutations are independent of each other. This paper tests the independence hypothesis for genetic mutations with regard to protein coding regions. According to the new experimental results the independence assumption generally holds, but there are certain exceptions. In particular, the coding regions that represent two adjacent amino acids seem to change in ways that sometimes deviate significantly from the expected theoretical probability under the independence assumption.

A Unified Framework For The Prioritization Of Variants Of Uncertain Significance In Hereditary Breast And Ovarian Cancer Patients, Natasha G. Caminsky

Electronic Thesis and Dissertation Repository

A significant proportion of hereditary breast and ovarian cancer (HBOC) patients receive uninformative genetic testing results, an issue exacerbated by the overwhelming quantity of variants of uncertain significance identified. This thesis describes a framework where, aside from protein coding changes, information theory (IT)-based sequence analysis identifies and prioritizes pathogenic variants occurring within sequence elements predicted to be recognized by proteins involved in mRNA splicing, transcription, and untranslated region binding and structure. To support the utilization of IT analysis, we established IT-based variant interpretation accuracy by performing a comprehensive review of mutations altering mRNA splicing in rare and common diseases.

Custom …

Borrelia Burgdorferi Reva Significantly Affects Pathogenicity And Host Response In The Mouse Model Of Lyme Disease, Rebecca Byram, Robert A. Gaultney, Angela M. Floden, Christopher Hellekson, Brandee L. Stone, Amy Bowman, Brian Stevenson, Barbara J. B. Johnson, Catherine A. Brissette

Microbiology, Immunology, and Molecular Genetics Faculty Publications

The Lyme disease spirochete, Borrelia burgdorferi, expresses RevA and numerous outer surface lipoproteins during mammalian infection. As an adhesin that promotes bacterial interaction with fibronectin, RevA is poised to interact with the extracellular matrix of the host. To further define the role(s) of RevA during mammalian infection, we created a mutant that is unable to produce RevA. The mutant was still infectious to mice, although it was significantly less well able to infect cardiac tissues. Complementation of the mutant with a wild-type revA gene restored heart infectivity to wild-type levels. Additionally, revA mutants led to increased evidence of arthritis, …

Muscle Weakness During Aging: A Deficiency State Involving Declining Angiogenesis, Charles T. Ambrose

Microbiology, Immunology, and Molecular Genetics Faculty Publications

This essay begins by proposing that muscle weakness of old age from sarcopenia is due in large part to reduced capillary density in the muscles, as documented in 9 reports of aged persons and animals. Capillary density (CD) is determined by local levels of various angiogenic factors, which also decline in muscles with aging, as reported in 7 studies of old persons and animals. There are also numerous reports of reduced CD in the aged brain and other studies showing reduced CD in the kidney and heart of aged animals. Thus a waning angiogenesis throughout the body may be …

Flavodoxin-Like Proteins Protect Candida Albicans From Oxidative Stress And Promote Virulence, Lifang Li, Shamoon Naseem, Sahil Sharma, James B. Konopka

Department of Molecular Genetics and Microbiology Faculty Publications

The fungal pathogen Candida albicans causes lethal systemic infections in humans. To better define how pathogens resist oxidative attack by the immune system, we examined a family of four Flavodoxin-Like Proteins (FLPs) in C. albicans. In agreement with previous studies showing that FLPs in bacteria and plants act as NAD(P)H quinone oxidoreductases, a C.albicans quadruple mutant lacking all four FLPs (pst1Δ, pst2Δ, pst3Δ, ycp4Δ) was more sensitive to benzoquinone. Interestingly, the quadruple mutant was also more sensitive to a variety of oxidants. Quinone reductase activity confers important antioxidant effects because resistance …

The Cababc Operon Essential For Biofilm And Rugose Colony Development In Vibrio Vulnificus, Jin Hwan Park, Youmi Jo, Song Yee Jang, Haenaem Kwon, Yasuhiko Irie, Matthew R. Parsek, Myung Hee Kim, Sang Ho Choi

Biology Faculty Publications

A transcriptome analysis identified Vibrio vulnificus cabABC genes which were preferentially expressed in biofilms. The cabABC genes were transcribed as a single operon. The cabA gene was induced by elevated 3′,5′-cyclic diguanylic acid (c-di-GMP) and encoded a calcium-binding protein CabA. Comparison of the biofilms produced by the cabA mutant and its parent strain JN111 in microtiter plates using crystal-violet staining demonstrated that CabA contributed to biofilm formation in a calcium-dependent manner under elevated c-di-GMP conditions. Genetic and biochemical analyses revealed that CabA was secreted to the cell exterior through functional CabB and CabC, distributed throughout the biofilm matrix, and produced …

Drosophila Eye Model To Study Neuroprotective Role Of Creb Binding Protein (Cbp) In Alzheimer’S Disease, Timothy Cutler, Ankita Sarkar, Michael Moran, Andrew Steffensmeier, Oorvashi Roy Puli, Greg Mancini, Meghana Tare

Biology Faculty Publications

Background: The progressive neurodegenerative disorder Alzheimer’s disease (AD) manifests as loss of cognitive functions, and finally leads to death of the affected individual. AD may result from accumulation of amyloid plaques. These amyloid plaques comprising of amyloid-beta 42 (Aβ42) polypeptides results from the improper cleavage of amyloid precursor protein (APP) in the brain. The Aβ42 plaques have been shown to disrupt the normal cellular processes and thereby trigger abnormal signaling which results in the death of neurons. However, the molecular-genetic mechanism(s) responsible for Aβ42 mediated neurodegeneration is yet to be fully understood.

Methodology/Principal Findings: We have utilized Gal4/UAS system to …

Characterization Of The Atsr/Atst Global Regulatory Pathway In Burkholderia Ceocepacia, Maryam Khodai-Kalaki

Electronic Thesis and Dissertation Repository

Phosphorylation cascades governed by two-component signal transduction systems provide key signalling mechanisms in bacteria, simple eukaryotes and higher plants, allowing them to translate signals into adaptive responses. These regulatory pathways consist of a transmembrane sensor protein that responds to an environmental cue leading to autophosphorylation, followed by the transfer of the phosphate to a cytoplasmic response regulator. Here, I study AtsR, a membrane-bound hybrid sensor kinase of Burkholderia cenocepacia, that negatively regulates quorum sensing related virulence factors such as biofilm, type 6-secretion and protease secretion. B. cenocepacia is a Gram-negative opportunistic pathogen which causes severe, chronic respiratory infections in …

An Exploration Of The Phylogenetic Placement Of Recently Discovered Ultrasmall Archaeal Lineages, Jeffrey M. O'Brien

Honors Scholar Theses

In recent years, several new clades within the domain Achaea have been discovered. This is due in part to microbiological sampling of novel environments, and the increasing ability to detect and sequence uncultivable organisms through metagenomic analysis. These organisms share certain features, such as small cell size and streamlined genomes. Reduction in genome size can present difficulties to phylogenetic reconstruction programs. Since there is less genetic data to work with, these organisms often have missing genes in concatenated multiple sequence alignments. Evolutionary Biologists have not reached a consensus on the placement of these lineages in the archaeal evolutionary tree. There …

Determination Of The Phylogenetic Range Of Sex Combs Reduced Activity In Drosophila Melanogaster, Laura E. Garofalo

Electronic Thesis and Dissertation Repository

The homeotic selector (Hox) genes are required for body patterning in bilaterians. Sex combs reduced (SCR) is a HOX protein in Drosophila melanogaster with two activities: SCR^T1 and SCR^lab activity required for patterning the prothorax (T1) and labia, respectively. SCR^T1 is proposed to be conserved throughout bilaterians while the phylogenetic range of functional conservation of SCR^lab is comparatively unknown. The goal of this work was to elucidate the evolutionary time point at which SCR activity changed. CRISPR/Cas9 transgenesis was used to incorporate ɸC31 integrase recombination sites in Drosophila Scr. The ɸC31 integrase could …

Genetic Analysis Of A Non-Germinating Mutant Of Arabidopsis Thaliana, Md Jakir Hossan

Electronic Thesis and Dissertation Repository

Seed germination is partially controlled by plant hormone gibberellins (GAs). Chemical mutagenesis yielded an Arabidopsis thaliana mutant gm11, which has an absolute gibberellin requirement for seed germination. This mutant exhibited phenotypes of GA-rescuable dwarfs, including dark-green leaves, and reduced fertility. However, with repeated GA treatment, gm11 develops into fertile plants with a nearly wild type phenotype. Bulked-segregant analysis mapped gm11 to the bottom arm of chromosome 1, and subsequent next-generation mapping revealed that the mutation is a G → A transition in At1g79460 (GA2), creating a premature stop codon. This gene encodes an ent-kaurene synthase (KS) which catalyzes …

Three-Dimensional Confocal Microscopy Indentation Method For Hydrogel Elasticity Measurement, Donghee Lee, Md Mahmudur Rahman, You Zhou, Sangjin Ryu

Md Mahmudur Rahman

No abstract provided.

Crosstalk Between Brca-Fanconi Anemia And Mismatch Repair Pathways Prevents Msh2-Dependent Aberrant Dna Damage Responses, Min Peng, Jenny X. Xie, Anna J. Ucher, Janet Stavnezer, Sharon B. Cantor

Janet M. Stavnezer

Several proteins in the BRCA-Fanconi anemia (FA) pathway, such as FANCJ, BRCA1, and FANCD2, interact with mismatch repair (MMR) pathway factors, but the significance of this link remains unknown. Unlike the BRCA-FA pathway, the MMR pathway is not essential for cells to survive toxic DNA interstrand crosslinks (ICLs), although MMR proteins bind ICLs and other DNA structures that form at stalled replication forks. We hypothesized that MMR proteins corrupt ICL repair in cells that lack crosstalk between BRCA-FA and MMR pathways. Here, we show that ICL sensitivity of cells lacking the interaction between FANCJ and the MMR protein MLH1 is …

Application Of Β-Lactamase Reporter Fusions As An Indicator Of Effector Protein Secretion During Infections With The Obligate Intracellular Pathogen Chlamydia Trachomatis , Konrad E. Mueller, Kenneth A. Fields

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Chlamydia spp. utilize multiple secretion systems, including the type III secretion system (T3SS), to deploy host-interactive effector proteins into infected host cells. Elucidation of secreted proteins has traditionally required ectopic expression in a surrogate T3SS followed by immunolocalization of endogenous candidate effectors to confirm secretion by chlamydiae. The ability to transform Chlamydia and achieve stable expression of recombinant gene products has enabled a more direct assessment of secretion. We adapted TEM-1 β-lactamase as a reporter system for assessment of chlamydial protein secretion. We provide evidence that this system facilitates visualization of secretion in the context of infection. Specifically, our findings …

A Screen To Identify Saga-Activated Genes That Are Required For Proper Photoreceptor Axon Targeting In Drosophila Melanogaster, Kaelan J. Brennan, Vikki M. Weake, Jingqun Q. Ma

The Summer Undergraduate Research Fellowship (SURF) Symposium

The inherited human genetic disease spinocerebellar ataxia type 7 (SCA7) is characterized by progressive neurodegeneration and visual impairment that ultimately leads to blindness. SCA7 results from a mutation in the human ATXN7 gene that causes an expansion of polyglutamine tracts in this gene’s corresponding protein. Human ATXN7 protein serves as a component of the deubiquitylase (DUB) module of the large, multi-subunit complex Spt-Ada-Gcn acetyltransferase, or SAGA. SAGA is a transcriptional coactivator and histone modifier that functions to deubiquitylate histone H2B and allow for transcription of SAGA-mediated genes to occur. In Drosophila, mutations in SAGA DUB’s Nonstop and sgf11 components …

Calmodulin-Like Protein 38: A Component Of Ribonucleoprotein Particles During Hypoxic Stress Responses In Arabidopsis, Ansul Lokdarshi

Doctoral Dissertations

Waterlogging stress leads to a crisis in energy metabolism and the accumulation of toxic metabolites due to the hypoxic and/or anoxic environment associated with this condition. To respond and adapt to this situation, higher plants employ an integrated genetic program that leads to the induction of anaerobic response polypeptide genes that encode metabolic and signaling proteins involved in altering metabolic flow and other adaptive responses. The study presented here shows that the Arabidopsis thaliana calmodulin-like protein CML38 is calcium sensor protein that serves as a member of the core anaerobic response gene family and is involved in modulating the survival …

Using High Throughput Genomic Sequencing To Predict Ecological Impacts On Sea Turtle Populations, Lesley Anderson

STAR Program Research Presentations

Marine turtles are long-lived, migratory vertebrates that encounter a variety of human and natural stressors throughout their lives. Understanding the biology and threats of these animals is challenging because they are hard to observe, and can migrate across whole ocean basins. Minimally invasive sampling techniques (e.g., blood samples) allow us to learn about their physiology, genetics, and the environmental conditions they have experienced. In this project, we developed a novel method to extract the RNA from whole green and loggerhead turtle blood from animals inhabiting a variety of sites across the Pacific Ocean. Some habitats are more pristine, while others …

Intracellular Listeria Monocytogenes Comprises A Minimal But Vital Fraction Of The Intestinal Burden Following Foodborne Infection, Grant S. Jones, Kate M. Bussell, Tanya Myers-Morales, Abigail M. Fieldhouse, Elsa N. Bou Ghanem, Sarah E. F. D'Orazio

Microbiology, Immunology, and Molecular Genetics Faculty Publications

Listeria monocytogenes is a highly adaptive bacterium that replicates as a free-living saprophyte in the environment as well as a facultative intracellular pathogen that causes invasive foodborne infections. The intracellular life cycle of L. monocytogenes is considered to be its primary virulence determinant during mammalian infection; however, the proportion of L. monocytogenes that is intracellular in vivo has not been studied extensively. In this report, we demonstrate that the majority of wild-type (strain EGDe) and mouse-adapted (InlA^m-expressing) L. monocytogenes recovered from the mesenteric lymph nodes (MLN) was extracellular within the first few days after foodborne infection. In addition, …

Distinct Physiological Roles For The Two Isoforms Of The Er Chaperone Grp170 In Caenorhabditis Elegans, Yuanyuan Li

Biology Theses

GRP170 is a large molecular chaperone found in the ER of all eukaryotes. The nematode Caenorhabditis elegans has two loci encoding GRP170: T24H7.2 (grp170a) and T14G8.3 (grp170b). The phenotypes of nematodes genetically deficient for either grp170a or grp170b were compared to a standard laboratory strain with functional grp170 loci. Worms that were deficient for grp170a developed 32% slower than the control strain. The loss of grp170a had a significant but modest reduction on the life span compared to the control strain. Worms deficient for grp170a also displayed significantly increased embryonic lethality and resulted in 6.9% arrested embryos. The loss of …

Characterization Of Putative Wnt3a-Inducible Enhancers, Katelynn C. Lee, Nicholas Hum, Aimy Sebastian, Gabriela Loots

STAR Program Research Presentations

The Wnt signaling pathway has been previously shown to play a major role in regulating bone metabolism and it is emerging as a target for the therapeutic intervention of bone thinning disorders such as osteoporosis. Several Wnt proteins have been shown to be expressed in bone and mutations in Wnt pathway members such as Wnt co-receptor Lrp5 and Wnt inhibitor Sost have been shown to be associated with low or high bone mass disorders, however, very little is known about specific roles played by different Wnt ligands in bone development, repair and remodeling. To identify downstream targets of Wnt signaling …

Hippocalcin Response To Calcium: Do Conserved Tryptophans – W30 Or W103 – Matter?, Sunkesula K. Sagar

Graduate School of Biomedical Sciences Theses and Dissertations

Changes in intracellular calcium levels play a very important role in cell signaling, in turn, affecting neuronal functions such as memory, learning and cell death. A class of proteins called Neuronal Calcium Sensor (NCS) proteins serves to modulate the functioning of the neuronal cells in response to changes in calcium levels, and prevent neuronal apoptosis. Structurally, all NCS proteins have 4 calcium-binding EF hand motifs, although EF1 does not bind to calcium in many members. All NCS proteins have an acyl modification at the N- terminus – where a myristoyl group is added post-translationally. Hippocalcin (HPCA) is an NCS protein, …

Beyond Bivariate Correlations: Three-Block Partial Least Squares Illustrated With Vegetation, Soil, And Topography, Daehyun Kim, Thomas J. Dewitt, César S. B. Costa, John A. Kupfer, Ryan W. Mcewan, J. Anthony Stallins

Biology Faculty Publications

Ecologists, particularly those engaged in biogeomorphic studies, often seek to connect data from three or more domains. Using three-block partial least squares regression, we present a procedure to quantify and define bi-variance and tri-variance of data blocks related to plant communities, their soil parameters, and topography. Bi-variance indicates the total amount of covariation between these three domains taken in pairs, whereas tri-variance refers to the common variance shared by all domains. We characterized relationships among three domains (plant communities, soil properties, topography) for a salt marsh, four coastal dunes, and two temperate forests spanning several regions in the world. We …