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"Cloning"
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Accumulation of Alkaloids in Different Tall Fescue KY31 Clones Harboring the Common Toxic IEpichloë coenophiala/I Endophyte under Field Conditions
Tall fescue (Lolium arundinaceum) is a highly adaptable forage, pasture and turf grass that is grown on over 14 M ha in the eastern half of the United States and in other temperate regions of the world. A significant factor in adaptability, productivity and stand persistence is in part due to the presence of an intercellular, seed-transmissible, endophytic fungus, Epichloë coenophiala. Epichloë endophytes have been shown to produce a number of alkaloid compounds only in planta, some that are beneficial in repelling insects, while others are toxic to animals. The goal of this work was to monitor the level of the ergot and loline (classified as pyrrolizidine) alkaloid accumulation in individual plants to determine the plant genotype contribution to alkaloid concentrations. The experimental design consisted of sixteen tall fescue KY31 clones in a space-planted, replicated trial over three years. Our results demonstrated that while changes in the alkaloid concentrations for each plant/endophyte genotype were observed over the three years, the overall alkaloid levels remained relatively constant when compared to other plant/endophyte genotypes combinations in the field. Additionally, overall levels of the ergot and loline alkaloid accumulation did not vary in the same way over the three years. Since the E. coenophiala endophyte genotype was the same across all clones, our results indicate that it is the plant genotype that is responsible for determining alkaloid levels in each plant, and suggest that the signal(s) from the plant to the endophyte may not be the same for ergot and loline alkaloid production.
Journal Article
Correction: A MultiSite Gateway Toolkit for Rapid Cloning of Vertebrate Expression Constructs with Diverse Research Applications
(B) Schematic of lentiviral destination vectors pEpic and pEpic_Lite. attR3 and 4 sites flanking the ccdB/CmR selection cassette are positioned in an anti-sense orientation to viral RNA expression driven by a Rous sarcoma virus (RSV) promoter. pEpic_Lite lacks puromycin resistance (PuroR).LTR = long terminal repeat; RRE = Rev response element; cPPT = central polypurine tract; ccdB = E. coli ccdB toxin; CmR = chloramphenicol resistance; mPGK = mouse phosphoglycerate kinase promoter; WPRE = woodchuck hepatitis virus posttranslational regulatory element. https://doi.org/10.1371/journal.pone.0176543.g001 The correct plasmids and their sequences have been deposited with Addgene (www.addgene.org), with the following catalog numbers: pEpic #84372; pEpic_Lite #84373.
Journal Article
The ABCs of gene cloning
Clear and concise, this easy-to-use book offers an introductory course on the language of gene cloning, covering microbial, plant, and mammalian systems. It presents the nuts and bolts of gene cloning in a well-organized and accessible manner. Part I of this book outlines the essentials of biology and genetics relevant to the concept of gene cloning. Part II describes common techniques and approaches of gene cloning, ranging from the basic mechanics of DNA manipulation, vector systems, process transformation, to gene analysis. Part III & IV present application technologies of major impact in agriculture, biomedicine, and related areas. The ABCs of Gene Cloning, Third Edition contains updates including a tutorial chapter on gene-vector construction, methodologies on exome sequencing in finding disease genes, revised topics on gene therapy and whole genome sequencing, new developments for gene targeting and genome editing, as well as the current state of next generation sequencing. With more than 140 illustrations, this new edition provides an invaluable text for students and anyone who have interest in gaining proficiency in reading and speaking the language of gene cloning.
Book
EasyClone: method for iterative chromosomal integration of multiple genes Saccharomyces cerevisiae
Abstract
Development of strains for efficient production of chemicals and pharmaceuticals requires multiple rounds of genetic engineering. In this study, we describe construction and characterization of EasyClone vector set for baker's yeast Saccharomyces cerevisiae, which enables simultaneous expression of multiple genes with an option of recycling selection markers. The vectors combine the advantage of efficient uracil excision reaction-based cloning and Cre-LoxP-mediated marker recycling system. The episomal and integrative vector sets were tested by inserting genes encoding cyan, yellow, and red fluorescent proteins into separate vectors and analyzing for co-expression of proteins by flow cytometry. Cells expressing genes encoding for the three fluorescent proteins from three integrations exhibited a much higher level of simultaneous expression than cells producing fluorescent proteins encoded on episomal plasmids, where correspondingly 95% and 6% of the cells were within a fluorescence interval of Log10 mean ± 15% for all three colors. We demonstrate that selective markers can be simultaneously removed using Cre-mediated recombination and all the integrated heterologous genes remain in the chromosome and show unchanged expression levels. Hence, this system is suitable for metabolic engineering in yeast where multiple rounds of gene introduction and marker recycling can be carried out.
EasyClone genetical toolbox allows faster development of yeast strains for biotechnological applications.
Journal Article
De-extinction : the science of bringing lost species back to life
In the twenty-first century, because of climate change and other human activities, many animal species have become extinct, and many others are at risk of extinction. Once they are gone, we cannot bring them back or can we?
Book
The Arabidopsis Transcription Factor LUH/MUM1 Is Required for Extrusion of Seed Coat Mucilage1WOA
During differentiation, the Arabidopsis (Arabidopsis thaliana) seed coat epidermal cells secrete mucilage composed primarily of rhamnogalacturonan I that is extruded from the seed coat upon imbibition. The mucilage of the mucilage modified1 (mum1) mutant contains rhamnogalacturonan I that is more highly branched and lacks the ability to be extruded when exposed to water. Our cloning of the MUM1 gene shows that it encodes a putative transcription factor, LEUNIG_HOMOLOG (LUH). Cellular localization and transcriptional assay results suggest that LUH/MUM1 is a nucleus-localized transcriptional activator. LUH/MUM1 is expressed in all the tissues examined, including the seed coat. Quantitative reverse transcription-polymerase chain reaction data suggest that LUH/MUM1 is expressed throughout seed coat development, reaching peak expression late in differentiation. LUH1/MUM1 expression in plants homozygous for mutations in several genes encoding regulators of seed coat mucilage was unchanged. Thus, LUH/MUM1 expression appears to be independent of other transcription factors known to regulate aspects of seed coat mucilage biology. The expression in the luh/mum1 mutant of three genes encoding enzymes needed for mucilage extrusion, MUM2, SUBSILIN PROTEASE1.7, and β-XYLOSIDASE1, was reduced relative to that of the wild type. Overexpression of MUM2 could partially rescue the mum1 phenotype. These data suggest that LUH/MUM1 is a positive regulator of all three genes.
Journal Article
Cloning and genetic engineering
This enlightening volume offers arguments for both sides of the cloning and genetic engineering debate.
Book
Reacción a la inoculacion artificial con Phytophthora palmivora de frutos desprendidos de clones de cacao seleccionados
La podredumbre negra de la mazorca (BPR), causada por varias especies del género Phytophthora, es una de las enfermedades más limitantes para la producción de cacao ya que se presenta en todas las regiones productoras del mundo y genera importantes pérdidas. El objetivo de este estudio fue establecer la respuesta a esta infección en seis clones de cacao (EET8, IMC67, TSH565, PA46, ICS95 y CCN51), mediante una prueba de inoculación en frutos desprendidos utilizando cinco aislados de P. palmivora de cinco regiones productoras. Se evaluó la incidencia y severidad de la enfermedad en las vainas desprendidas a los seis y diez días después de la inoculación (DAI). El clon CCN51 se clasificó como susceptible y los clones IMC67 y PA46 como moderadamente susceptibles a los seis DAI. Todos los clones evaluados fueron categorizados como susceptibles a los diez DAI. Los aislamientos HURV19 de P. palmivora mostraron la mayor agresividad en comparación con ANYA228, que resultó ser el menos agresivo.
Journal Article