Investigating The Conservation Pattern Of A Putative Second Terpene Synthase Divalent Metal Binding Motif In Plants, Ke Zhou, Reuben J. Peters
Reuben J. Peters
Terpene synthases (TPS) require divalent metal ion co-factors, typically magnesium, that are bound by a canonical DDXXD motif, as well as a putative second, seemingly less well conserved and understood (N/D)DXX(S/T)XXXE motif. Given the role of the Ser/Thr side chain hydroxyl group in ligating one of the three catalytically requisite divalent metal ions and the loss of catalytic activity upon substitution with Ala, it is surprising that Gly is frequently found in this ‘middle’ position of the putative second divalent metal binding motif in plant TPS. Here we report mutational investigation of this discrepancy ...
A Functional Genomics Approach To Tanshinone Biosynthesis Provides Stereochemical Insights, 2017 Chinese Academy of Chinese Medical Science
A Functional Genomics Approach To Tanshinone Biosynthesis Provides Stereochemical Insights, Wei Gao, Matthew L. Hillwig, Luqi Huang, Guanghong Cui, Xueyong Wang, Jianqiang Kong, Bin Yang, Reuben J. Peters
Reuben J. Peters
Tanshinones are abietane-type norditerpenoid quinone natural products that are the bioactive components of the Chinese medicinal herb Salvia miltiorrhiza Bunge. The initial results from a functional genomics-based investigation of tanshinone biosynthesis, specifically the functional identification of the relevant diterpene synthases from S. miltiorrhiza, are reported. The cyclohexa-1,4-diene arrangement of the distal ring poises the resulting miltiradiene for the ensuing aromatization and hydroxylation to ferruginol suggested for tanshinone biosynthesis.
Strand-Specific Libraries For High Throughput Rna Sequencing (Rna-Seq) Prepared Without Poly(A) Selection, 2017 University of Massachusetts Medical School
Strand-Specific Libraries For High Throughput Rna Sequencing (Rna-Seq) Prepared Without Poly(A) Selection, Zhao Zhang, William E. Theurkauf, Zhiping Weng, Phillip D. Zamore
BACKGROUND: High throughput DNA sequencing technology has enabled quantification of all the RNAs in a cell or tissue, a method widely known as RNA sequencing (RNA-Seq). However, non-coding RNAs such as rRNA are highly abundant and can consume >70% of sequencing reads. A common approach is to extract only polyadenylated mRNA; however, such approaches are blind to RNAs with short or no poly(A) tails, leading to an incomplete view of the transcriptome. Another challenge of preparing RNA-Seq libraries is to preserve the strand information of the RNAs. DESIGN: Here, we describe a procedure for preparing RNA-Seq libraries from 1 ...
Pirna Biogenesis And Transposon Silencing In Drosophila: A Dissertation, 2017 University of Massachusetts Medical School
Pirna Biogenesis And Transposon Silencing In Drosophila: A Dissertation, Zhao Zhang
piRNAs guide PIWI proteins to silence transposons in animal germ cells. In Drosophila, the heterochromatic piRNA clusters transcribe piRNA precursors to be transported into nuage, a perinuclear structure for piRNA production and transposon silencing. At nuage, reciprocal cycles of piRNA-directed RNA cleavage—catalyzed by the PIWI proteins Aubergine (Aub) and Argonaute3 (Ago3) in Drosophila—destroy the sense transposon mRNA and expand the population of antisense piRNAs in response to transposon expression, a process called the Ping-Pong cycle. Heterotypic Ping-Pong between Aub and Ago3 ensures that antisense piRNAs predominate. My thesis research mainly focuses on two fundamental questions about the piRNA ...
An Approach To Identify Mycobacteriophage Diversity Prior To Dna Sequencing, 2017 Western Kentucky University
An Approach To Identify Mycobacteriophage Diversity Prior To Dna Sequencing, Charles Gregory
Honors College Capstone Experience/Thesis Projects
Over 6,869 Mycobacteriophages have been isolated and purified. Of these, 1,367 genomes have been sequenced at the DNA level and more are added each year through the SEA-PHAGES program. Sequenced mycobacteriophages are grouped into clusters based on a 50% or greater nucleotide identity. The number and breadth of these clusters represents the diversity present in the environment. Each year, as new phages are discovered by students in the SEA-PHAGES program, the question arises, “Which isolates should we sequence?” In order to sequence phages that represent the greatest possible diversity, and thus broaden under-represented clusters and identify new singletons ...
Unique Genetic Differences In Responses Of Chicken Immune Cells To An Inflammatory Stimulus And Heat Stress, 2017 Iowa State University
Unique Genetic Differences In Responses Of Chicken Immune Cells To An Inflammatory Stimulus And Heat Stress, Angelica Van Goor, Anna Slawinska, Carl Schmidt, Susan J. Lamont
Susan J. Lamont
Bone marrow antigen presenting cells (BM-APC), from Fayoumis (disease resistant and heat tolerant) and Leghorn (disease susceptible) chicken lines were evaluated for response to an inflammatory stimulus and heat stress. BM-APC from Fayoumis produced more nitric oxide (NO) and had higher Major Histocompatibility Complex (MHC) class II cell surface expression compared to those from Leghorn, indicating that BM-APC studied in vitro may be a useful tool to evaluate molecular effects of disease and/or heat tolerance in chickens.
Infection Of Commercial Laying Hens With Newcastle Disease Virus: Differing Responses Between Birds Provide Potential For Genetic Improvement Through Selection, Kaylee Rowland, Huaijun Zhou, Rodrigo Gallardo, David Bunn, Susan J. Lamont
Susan J. Lamont
Exotic Newcastle Disease Virus (ENDV) cause extremely rapid mortality in chickens after exposure to the virus. People rely heavily on poultry to provide protein and income in many places where NDV is not effectively controlled through vaccination and biosecurity. Losses from NDV contribute to worldwide hunger and poverty. It may be possible to use genetic selection to produce chickens that have a stronger immune response in the face of NDV challenge. For genetic selection to be successful, two major elements are required: differences in immune response among chickens and genetic control of these differences. This study clearly demonstrated the existence ...
Effects Of Diet And Genetics On Production Traits In Response To Repeated Exposure To Heat Stress In Pigs, 2017 Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria
Effects Of Diet And Genetics On Production Traits In Response To Repeated Exposure To Heat Stress In Pigs, Wendy Rauw, Johana Mayorga Lozano, Soi Lei, John Patience, Nicholas Gabler, Steven Lonergan, Jack Dekkers, Lance Baumgard
Steven M. Lonergan
Hot temperatures negatively impact global livestock production and are a serious financial burden to pig producers. Trends predict that global temperatures will continue toincrease, making this a pertinent issue now and in the future. Study objectives were to investigate the effects of genetics and dietary fiber content on growth,feed intake, and feed conversion efficiencyin response to repeated exposure to heat stress.Atotal of 103 finishing barrows from a contemporary commercial line and Yorkshire linesdivergently selected for high and low feed efficiency based on residual feed intake (RFI) were used in this experiment. All pigs were subjected three consecutivetimes to ...
Results Of Survey Of Stakeholders Regarding Knowledge Of And Attitudes Towards Feed Intake, Efficiency And Genetic Improvement Concepts, R. L. Weaber, J. E. Beever, H. C. Freetly, D. J. Garrick, S. L. Hansen, K. A. Johnson, M. S. Kerley, D. D. Loy, E. Marques, H. L. Neibergs, E. J. Pollak, R. D. Schnabel, C. M. Seabury, D. W. Shike, M. L. Spangler, J. F. Taylor
Stephanie L. Hansen
Individual animal feed efficiency plays a key role in the profitability and sustainability of the US beef industry. During the growing and finishing phase of production, a 10% improvement in feed efficiency has a two-fold greater impact on profit than a 10% increase in rate of gain (Fox et al., 2001). The traits that beef producers routinely record are outputs which determine the value of product sold and not the inputs defining the cost of beef production. The inability to routinely measure feed intake and feed efficiency on large numbers of cattle has precluded the efficient application of selection despite ...
National Program For Genetic Improvement Of Feed Efficiency In Beef Cattle, 2017 University of Missouri
National Program For Genetic Improvement Of Feed Efficiency In Beef Cattle, Jerry Taylor, Monty Kerley, Robert Schnabel, Daniel Pomp, Dorian Garrick, Stephanie Hansen, Dan Loy, J. R. Tait, Robert Weaber, Chris Seabury, Jon Beever, Dan Faulkner, Dan Shike, Scott Fahrenkrug, Matt Spangler, Tad Sonstegard, Harvey Freetly, John Pollak, Kris Johnson, Holly Neibergs
Stephanie L. Hansen
Our goal is to sustainably reduce feed resources required to produce beef via the rapid development and deployment of novel nutritional, genomic and genetic improvement technologies. We will strengthen the international competitiveness of US agriculture and enable increased food production by increasing the animal protein produced without additional feed inputs and with a reduced greenhouse gas footprint.
Engineering And Verifying Requirements For Programmable Self-Assembling Nanomachines, 2017 Iowa State University
Engineering And Verifying Requirements For Programmable Self-Assembling Nanomachines, Robyn Lutz, Jack Lutz, James Lathrop, Titus Klinge, Eric Henderson, Davita Mathur, Dalia Abo Sheasha
We propose an extension of van Lamsweerde's goal-oriented requirements engineering to the domain of programmable DNA nanotechnology. This is a domain in which individual devices (agents) are at most a few dozen nanometers in diameter. These devices are programmed to assemble themselves from molecular components and perform their assigned tasks. The devices carry out their tasks in the probabilistic world of chemical kinetics, so they are individually error-prone. However, the number of devices deployed is roughly on the order of a nanomole (a 6 followed by fourteen 0s), and some goals are achieved when enough of these agents achieve ...
Requirements Analysis For A Product Family Of Dna Nanodevices, 2017 Iowa State University
Requirements Analysis For A Product Family Of Dna Nanodevices, Robyn R. Lutz, Jack H. Lutz, James I. Lathrop, Titus H. Klinge, Divita Mathur, D. M. Stull, Taylor G. Bergquist, Eric R. Henderson
DNA nanotechnology uses the information processing capabilities of nucleic acids to design self-assembling, programmable structures and devices at the nanoscale. Devices developed to date have been programmed to implement logic circuits and neural networks, capture or release specific molecules, and traverse molecular tracks and mazes. Here we investigate the use of requirements engineering methods to make DNA nanotechnology more productive, predictable, and safe. We use goal-oriented requirements modeling to identify, specify, and analyze a product family of DNA nanodevices, and we use PRISM model checking to verify both common properties across the family and properties that are specific to individual ...
Automated Requirements Analysis For A Molecular Watchdog Timer, 2017 Iowa State University
Automated Requirements Analysis For A Molecular Watchdog Timer, Samuel J. Ellis, Eric R. Henderson, Titus H. Klinge, James I. Lathrop, Jack H. Lutz, Robyn R. Lutz, Divita Mathur, Andrew S. Miner
Dynamic systems in DNA nanotechnology are often programmed using a chemical reaction network (CRN) model as an intermediate level of abstraction. In this paper, we design and analyze a CRN model of a watchdog timer, a device commonly used to monitor the health of a safety critical system. Our process uses incremental design practices with goal-oriented requirements engineering, software verification tools, and custom software to help automate the software engineering process. The watchdog timer is comprised of three components: an absence detector, a threshold filter, and a signal amplifier. These components are separately designed and verified, and only then composed ...
Increasing Diterpene Yield With A Modular Metabolic Engineering System In E. Coli: Comparison Of Mev And Mep Isoprenoid Precursor Pathway Engineering, Dana Morrone, Luke Lowry, Mara K. Determan, David M. Hershey, Meimei Xu, Reuben J. Peters
Reuben J. Peters
Engineering biosynthetic pathways in heterologous microbial host organisms offers an elegant approach to pathway elucidation via the incorporation of putative biosynthetic enzymes and characterization of resulting novel metabolites. Our previous work in Escherichia coli demonstrated the feasibility of a facile modular approach to engineering the production of labdane-related diterpene (20 carbon) natural products. However, yield was limited (<0.1 mg/L), presumably due to reliance on endogenous production of the isoprenoid precursors dimethylallyl diphosphate and isopentenyl diphosphate. Here, we report incorporation of either a heterologous mevalonate pathway (MEV) or enhancement of the endogenous methyl erythritol phosphate pathway (MEP) with our modular metabolic engineering system. With MEP pathway enhancement, it was found that pyruvate supplementation of rich media and simultaneous overexpression of three genes (idi, dxs, and dxr) resulted in the greatest increase in diterpene yield, indicating distributed metabolic control within this pathway. Incorporation of a heterologous MEV pathway in bioreactor grown cultures resulted in significantly higher yields than MEP pathway enhancement. We have established suitable growth conditions for diterpene production levels ranging from 10 to >100 mg/L of E. coli culture. These amounts are sufficient for nuclear magnetic resonance analyses, enabling characterization of enzymatic products and hence, pathway elucidation. Furthermore, these results represent an up to >1,000-fold improvement in diterpene production from our facile, modular platform ...0.1>
Genetic Basis And Functional Consequences Of Differential Expression Of The Cmeabc Efflux Pump In Campylobacter Jejuni Isolates, Tara Grinnage-Pulley, Qijing Zhang
The CmeABC multidrug efflux transporter of Campylobacter jejuni plays a key role in antimicrobial resistance and is suppressed by CmeR, a transcriptional regulator of the TetR family. Overexpression of CmeABC has been observed in laboratory-generated mutants, but it is unknown if this phenotype occurs naturally in C. jejuni isolates and if it has any functional consequences. To answer these questions, expression of cmeABC in natural isolates obtained from broiler chickens, turkeys and humans was examined, and the genetic mechanisms and role of cmeABC differential expression in antimicrobial resistance was determined. Among the 64 C. jejuni isolates examined in this study ...
Rv0989c Encodes A Novel (E)-Geranyl Diphosphate Synthase Facilitating Decaprenyl Diphosphate Biosynthesis In Mycobacterium Tuberculosis, Francis M. Mann, Jill A. Thomas, Reuben J. Peters
Reuben J. Peters
Mycobacterium tuberculosis (Mtb) has a highly complex cell wall, which is required for both bacterial survival and infection. Cell wall biosynthesis is dependent on decaprenyl diphosphate as a glyco-carrier, which is hence an essential metabolite in this pathogen. Previous biochemical studies indicated (E)-geranyl diphosphate (GPP) is required for the synthesis of decaprenyl diphosphate. Here we demonstrate that Rv0989c encodes the “missing” GPP synthase, representing the first such enzyme to be characterized from bacteria, and which presumably is involved in decaprenyl diphosphate biosynthesis in Mtb. Our investigation also has revealed previously unrecognized substrate plasticity of the farnesyl diphosphate synthases from ...
Magnesium Depletion Triggers Production Of An Immune Modulating Diterpenoid In Mycobacterium Tuberculosis, 2017 Iowa State University
Magnesium Depletion Triggers Production Of An Immune Modulating Diterpenoid In Mycobacterium Tuberculosis, Francis M. Mann, Brian C. Vanderven, Reuben J. Peters
Reuben J. Peters
Mycobacterium tuberculosis (Mtb) is the causative agent of the human disease Tuberculosis, and remains a worldwide health threat responsible for ~1.7 million deaths annually. During infection, Mtb prevents acidification of the engulfing phagosome, thus blocking endocytic progression and eventually leading to stable residence. The diterpenoid metabolite isotuberculosinol (isoTb) exhibits biological activity indicative of a role in this early arrest of phagosome maturation. Presumably, isoTb production should be induced by phagosomal entry. However, the relevant enzymatic genes are not transcriptionally upregulated during engulfment. Previous examination of the initial biosynthetic enzyme (Rv3377c/MtHPS) involved in isoTb biosynthesis revealed striking inhibition by ...
Two Rings In Them All: The Labdane-Related Diterpenoids, 2017 Iowa State University
Two Rings In Them All: The Labdane-Related Diterpenoids, Reuben J. Peters
Reuben J. Peters
In his original exposition of the biogenetic isoprenoid rule, L. Ruzicka noted the structural identity between the fused A/B rings of triterpenoids/sterols and certain multicyclic diterpenoids as part of the impetus leading to that profound insight. His prescient hypothesis that this chemical structure relationship reflects similarities in the initial cyclization of these diterpenoids with that occurring in triterpenoid biosynthesis has since been verified. However, this chemical structure relationship does not continue to hold true for the additional rings found in many of these di- and tri- terpenoid natural products. This is now appreciated to arise from differences in ...
Diterpenoid Biopolymers: New Directions For Renewable Materials Engineering, 2017 Iowa State University
Diterpenoid Biopolymers: New Directions For Renewable Materials Engineering, Matthew L. Hillwig, Francis M. Mann, Reuben J. Peters
Reuben J. Peters
Most types of ambers are naturally occurring, relatively hard, durable resinite polymers derived from the exudates of trees. This resource has been coveted for thousands of years due to its numerous useful properties in industrial processes, beauty, and purported medicinal properties. Labdane diterpenoid based ambers represent the most abundant and important resinites on earth. These resinites are a dwindling, non-renewable natural resource, so a new source of such materials needs to be established. Recent advances in sequencing technologies and biochemical engineering are rapidly accelerating the rate of identifying and assigning function to genes involved in terpenoid biosynthesis, as well as ...
Diterpene Cyclases And The Nature Of The Isoprene Fold, 2017 University of Illinois at Urbana-Champaign
Diterpene Cyclases And The Nature Of The Isoprene Fold, Rong Cao, Yonghui Zhang, Francis M. Mann, Cancan Huang, Dushyant Mukkamala, Michael P. Hudock, Matthew Mead, Sladjana Prisic, Ke Wang, Fu-Yang Lin, Ting-Kai Chang, Reuben Peters, Eric Oldfield
Reuben J. Peters
The structures and mechanism of action of many terpene cyclases are known, but there are no structures of diterpene cyclases. Here, we propose structural models based on bioinformatics, sitedirected mutagenesis, domain swapping, enzyme inhibition and spectroscopy that help explain the nature of diterpene cyclase structure, function, and evolution. Bacterial diterpene cyclases contain ～20 α-helices and the same conserved “QW” and DxDD motifs as in triterpene cyclases, indicating the presence of a βγ barrel structure. Plant diterpene cyclases have a similar catalytic motif and βγ-domain structure together with a third, α-domain, forming an αβγ structure, and in H+-initiated cyclases, there ...