scholarly journals Field Studies Using a Recombinant Mycoinsecticide (Metarhizium anisopliae) Reveal that It Is Rhizosphere Competent

2002 ◽  
Vol 68 (12) ◽  
pp. 6383-6387 ◽  
Author(s):  
Gang Hu ◽  
Raymond J. St. Leger
Keyword(s):  
Metarhizium Anisopliae ◽  
Fluorescent Protein ◽  
Pathogenic Fungi ◽  
Field Studies ◽  
Field Conditions ◽  
Research Station ◽  
Potential Reservoir ◽  
Green Fluorescent ◽  
A Site ◽  
Transgenic Strains

ABSTRACT In the summer of 2000, we released genetically altered insect-pathogenic fungi onto a plot of cabbages at a field site on the Upper Marlboro Research Station, Md. The transformed derivatives of Metarhizium anisopliae ARSEF 1080, designated GPMa and GMa, carried the Aequorea victoria green fluorescent protein (gfp) gene alone (GMa) or with additional protease genes (Pr1) (GPMa). The study (i) confirmed the utility of gfp for monitoring pathogen strains in field populations over time, (ii) demonstrated little dissemination of transgenic strains and produced no evidence of transmission by nontarget insects, (iii) found that recombinant fungi were genetically stable over 1 year under field conditions, and (iv) determined that deployment of the transgenic strains did not depress the culturable indigenous fungal microflora. The major point of the study was to monitor the fate (survivorship) of transformants under field conditions. In nonrhizosphere soil, the amount of GMa decreased from 105 propagules/g at depths of 0 to 2 cm to 103 propagules/g after several months. However, the densities of GMa remained at 105 propagules/g in the inner rhizosphere, demonstrating that rhizospheric soils are a potential reservoir for M. anisopliae. These results place a sharp focus on the biology of the soil/root interphase as a site where plants, insects, and pathogens interact to determine fungal biocontrol efficacy, cycling, and survival. However, the rhizospheric effect was less marked for GPMa, and overall it showed reduced persistence in soils than did GMa.

scholarly journals Generation of a Recombinant Cytomegalovirus for Expression of a Hantavirus Glycoprotein

2003 ◽  
Vol 77 (22) ◽  
pp. 12203-12210 ◽  
Author(s):  
Albert A. Rizvanov ◽  
Albert G. M. van Geelen ◽  
Sergey Morzunov ◽  
Elmer W. Otteson ◽  
Charlotte Bohlman ◽  
...  
Keyword(s):  
Recombinant Virus ◽  
Fluorescent Protein ◽  
Deer Mice ◽  
Anamnestic Response ◽  
Egfp Gene ◽  
Virus Challenge ◽  
Virus Particles ◽  
Green Fluorescent ◽  
Human Cmv ◽  
A Site

ABSTRACT A cytomegalovirus (CMV) was isolated from its natural host, Peromyscus maniculatus, and was designated Peromyscus CMV (PCMV). A recombinant PCMV was constructed that contained Sin Nombre virus glycoprotein G1 (SNV-G1) fused in frame to the enhanced green fluorescent protein (EGFP) gene inserted into a site homologous to the human CMV UL33 (P33) gene. The recombinant CMV was used for expression and immunization of deer mice against SNV-G1. The results of the study indicate that P. maniculatus could be infected with as few as 10 virus particles of recombinant virus. Challenge of P. maniculatus with either recombinant or wild-type PCMV produced no overt pathology in infected animals. P. maniculatus immunized with recombinant virus developed an antibody response to SNV and EGFP. When rechallenged with recombinant virus, animals exhibited an anamnestic response against SNV. Interestingly, a preexisting immune response against PCMV did not prevent reinfection with recombinant PCMV.

scholarly journals abc3 + Encodes an Iron-Regulated Vacuolar ABC-Type Transporter in Schizosaccharomyces pombe

2009 ◽  
Vol 9 (1) ◽  
pp. 59-73 ◽  
Author(s):  
Benoît Pouliot ◽  
Mehdi Jbel ◽  
Alexandre Mercier ◽  
Simon Labbé
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Chromatin Immunoprecipitation ◽  
Transcriptional Activity ◽  
Fluorescent Protein ◽  
Dependent Manner ◽  
Ferric Reductase ◽  
Low Concentrations ◽  
Green Fluorescent ◽  
A Site

ABSTRACT Studies have shown the fundamental contribution of the yeast vacuole as a site for storage and detoxification of metals. Whereas the transmembrane proteins responsible for iron transport into and out of the vacuole have been identified in Saccharomyces cerevisiae, less information is available concerning the mobilization of vacuolar iron stores in Schizosaccharomyces pombe. In this study, we report the identification of a gene designated abc3 + that encodes a protein which exhibits sequence homology with the ABCC subfamily of ATP-binding cassette transporters. The transcription of abc3 + is induced by low concentrations of iron but repressed by high levels of iron. The iron-mediated repression of abc3 + required a functional fep1 + gene. Chromatin immunoprecipitation assays showed that Fep1 associates with the abc3 + promoter in vivo, in an iron-dependent manner. Microscopic analyses revealed that a functional Abc3-green fluorescent protein localizes to the membrane vacuole when iron levels were low. Abc3 was required for growth in low-iron medium in the absence of the transport system mediated by Fio1 and Fip1. abc3Δ cells exhibited increased levels of expression of the frp1 + -encoded ferric reductase, suggesting a loss of Fep1 repression and, consequently, the activation of Fep1-regulated genes. When abc3 + was expressed using the nmt1 + promoter system, its induction led to a reduced transcriptional activity of the frp1 + gene. Because S. pombe does not possess vacuolar membrane-localized orthologs to S. cerevisiae Fth1, Fet5, and Smf3, our findings suggested that Abc3 may be responsible for mobilizing stored iron from the vacuole to the cytosol in response to iron deficiency.

239 MAC-T CELLS AS A TOOL TO EXAMINE GENOME EDITING USING CLUSTERED REGULARLY INTERSPACED SHORT PALINDROMIC REPEATS (CRISPR)

2016 ◽  
Vol 28 (2) ◽  
pp. 251
Author(s):  
S. N. Lotti ◽  
I. Tasan ◽  
H. Zhao ◽  
M. B. Wheeler
Keyword(s):  
T Cells ◽  
Milk Production ◽  
Fluorescent Protein ◽  
The United States ◽  
The Other ◽  
Total Efficiency ◽  
Guide Rnas ◽  
Exon 1 ◽  
Green Fluorescent ◽  
A Site

In 2050, the expected size of the human population is 9 billion, the demand for food will increase, and the demand for milk will increase along with it. Genetically modifying animals is a tool that can be used to meet this growing demand. In the United States, Holstein is the leading breed for milk production and Holsteins produce on average 24 291 pounds of milk per year, whereas Jerseys, the other major dairy breed, produce on average 16 997 pounds. Their ability to produce large quantities of milk is linked to 2 mutations. These mutations are on the α-lactalbumin (α-lac) gene; the α-lac exon (+1) corresponds to the transcription start point of α-lac, (+15) and (–1689) are the positions corresponding to the single nucleotide polymorphism associated with increased milk production. Holstein cows have an adenine at both of these positions in contrast to the other cattle breeds with lower milk production, which have either a cytosine or guanine at either position. Inserting an adenine at position (+15) and (–1689) in cows without this mutation could lead to increased milk production and a better response to market demands. The purpose of this experiment was to test the cutting efficiency of candidate clustered regularly interspaced short palindromic repeats (CRISPR) that will later be used in knock-in experiments. CRISPRs were used because the CRISPR-Cas9 system is inexpensive, easily programmed, and efficient. In this preliminary study, we worked with Holstein MAC-T cells, which already contain the mutation at both positions. CRISPRs were used on this cell line to cut the DNA at a site near the mutation. Based on the genomic DNA sequence of these MAC-T cells, 3 guide RNAs were designed. Cells were then transfected with the designed CRISPRs by a variety of transfection methods, including Fugene™ (Promega, Madison, WI, USA), electroporation, and Lipofectamine (ThermoFisher Scientific, Waltham, MA, USA). Green fluorescent protein was used to determine the efficiency of transfection; 30% efficiency was seen for Fugene™, whereas electroporation and Lipofectamine™ had 70% efficiency. To select for successfully transfected cells, puromycin selection was applied. The DNA was later extracted and sent in for sequencing. Next, the website TIDE was used to compare the transfected MAC-T cells to normal MAC-T cells. The TIDE software measures the editing efficiency and looks for major insertions or deletions in pools of DNA by comparing 2 sequences to quantify the editing efficacy of CRISPR-Cas9. Our results showed that CRISPRs successfully cut the DNA near the α-lac mutation region with a total efficiency of 13.8%. The desired next step will be to insert a single-strand oligonucleotide (ssODN) donor to make a single basepair mutation. The ultimate aim of this research would be to insert these mutations into other cattle species in order to increase their milk production.

Transformation of Metarhizium anisopliae mediated by Agrobacterium tumefaciens

2006 ◽  
Vol 52 (7) ◽  
pp. 623-626 ◽  
Author(s):  
Weiguo Fang ◽  
Yan Pei ◽  
Michael J Bidochka
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Metarhizium Anisopliae ◽  
Protein Gene ◽  
Fluorescent Protein ◽  
Pathogenic Fungus ◽  
Single Copy ◽  
Transformation Method ◽  
The Other ◽  
Green Fluorescent Protein Gene ◽  
Green Fluorescent

A simple, highly efficient, and reliable Agrobacterium tumefaciens-mediated transformation method was developed for the insect pathogenic fungus Metarhizium anisopliae. Expression of the green fluorescent protein gene, egfp, and the benomyl resistance gene, benA3, were used as markers in transformed M. anisopliae. Transformation efficiencies were dependent on the strain of A. tumefaciens used. With strain AGL-1, 17.0 ± 1.4 transformants per plate could be obtained using conidial concentrations of 106 conidia/mL and a 2 day co-cultivation in the presence of 200 µmol/L acetosyringone. On the other hand, transformations using strain LBA4404 were unsuccessful. Ten transformants were tested by Southern analysis and found to contain a single copy T-DNA. Twenty transformants were subcultured for five generations on nonselective media, and 95% of the transformants were mitotically stable. Agrobacterium tumefaciens-mediated transformation of M. anisopliae can serve as a useful tool to investigate genes involved in insect pathogenicity.Key words: entomopathogenic fungi, Metarhizium anisopliae, Agrobacterium tumefaciens, genetic transformation.

Immuno-EM Localization of GFP-tagged Yolk Proteins in C. Elegans Using Microwave Fixation

2001 ◽  
Vol 49 (8) ◽  
pp. 949-956 ◽  
Author(s):  
Marie-Christine Paupard ◽  
Agnes Miller ◽  
Barth Grant ◽  
David Hirsh ◽  
David H. Hall
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Cell Types ◽  
Protein Fusion ◽  
C Elegans ◽  
High Resolution Analysis ◽  
Resolution Analysis ◽  
Microwave Fixation ◽  
Green Fluorescent ◽  
Transgenic Strains

Because of the presence of a low-permeability cuticle covering the animal, fixation of C. elegans tissue for immunoelectron microscopy has proved very difficult. Here we applied a microwave fixation protocol to improve penetration of fixatives before postembedding immunogold labeling. Using this technique, we were able to successfully localize several components of yolk (YP170) trafficking in both wild-type and transgenic strains expressing a vitellogenin::green fluorescent protein fusion (YP170::GFP). Green fluorescent protein (GFP) and its variants are commonly used as markers to localize proteins in transgenic C. elegans using fluorescence microscopy. We have developed a robust method to localize GFP at the EM level. This procedure is applicable to the characterization of transgenic strains in which GFP is used to mark particular proteins or cell types and will undoubtedly be very useful for high-resolution analysis of marked structures. (J Histochem Cytochem 49:949–956, 2001)

scholarly journals A Borna Disease Virus Vector for Expression of Foreign Genes in Neurons of Rodents

2007 ◽  
Vol 81 (13) ◽  
pp. 7293-7296 ◽  
Author(s):  
Urs Schneider ◽  
Andreas Ackermann ◽  
Peter Staeheli
Keyword(s):  
Disease Virus ◽  
Fluorescent Protein ◽  
Foreign Gene ◽  
Borna Disease Virus ◽  
Highly Active ◽  
The Central Nervous System ◽  
Foreign Genes ◽  
Green Fluorescent ◽  
A Site ◽  
Borna Disease

ABSTRACT An expression cassette for green fluorescent protein was successfully inserted at a site near the 5′ end of the genome of Borna disease virus (BDV). When introduced into a mutant virus with highly active polymerase, the foreign gene was strongly expressed in neurons of infected rats. Since BDV can establish long-term persistence in the central nervous system of rodents, it may be used to engineer efficient vectors for specific delivery of foreign genes into highly differentiated neurons.

scholarly journals Genetic Analysis of the Cell Division Protein FtsI (PBP3): Amino Acid Substitutions That Impair Septal Localization of FtsI and Recruitment of FtsN

2004 ◽  
Vol 186 (2) ◽  
pp. 490-502 ◽  
Author(s):  
Mark C. Wissel ◽  
David S. Weiss
Keyword(s):  
Amino Acids ◽  
Fluorescent Protein ◽  
Transmembrane Helix ◽  
Binding Assays ◽  
Protein Fusion ◽  
Mutant Proteins ◽  
Domain Of Unknown Function ◽  
Green Fluorescent ◽  
Septal Localization ◽  
A Site

ABSTRACT FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of several proteins that localize to the septal ring. FtsI comprises a small cytoplasmic domain, a transmembrane helix, a noncatalytic domain of unknown function, and a catalytic (transpeptidase) domain. The last two domains reside in the periplasm. We used PCR to randomly mutagenize ftsI, ligated the products into a green fluorescent protein fusion vector, and screened ∼7,500 transformants for gfp-ftsI alleles that failed to complement an ftsI null mutant. Western blotting and penicillin-binding assays were then used to weed out proteins that were unstable, failed to insert into the cytoplasmic membrane, or were defective in catalysis. The remaining candidates were tested for septal localization and ability to recruit another division protein, FtsN, to the septal ring. Mutant proteins severely defective in localization to the septal ring all had lesions in one of three amino acids—R23, L39, or Q46—that are in or near the transmembrane helix and implicate this region of FtsI in septal localization. Mutant FtsI proteins defective in recruitment of FtsN all had lesions in one of eight residues in the noncatalytic domain. The most interesting of these mutants had lesions at G57, S61, L62, or R210. Although separated by ∼150 residues in the primary sequence, these amino acids are close together in the folded protein and might constitute a site of FtsI-FtsN interaction.

scholarly journals The Transmembrane Helix of the Escherichia coli Division Protein FtsI Localizes to the Septal Ring

2005 ◽  
Vol 187 (1) ◽  
pp. 320-328 ◽  
Author(s):  
Mark C. Wissel ◽  
Jennifer L. Wendt ◽  
Calista J. Mitchell ◽  
David S. Weiss
Keyword(s):  
Escherichia Coli ◽  
Fluorescent Protein ◽  
Transmembrane Helix ◽  
Alpha Helix ◽  
Alanine Scanning Mutagenesis ◽  
Amino Terminal ◽  
Acid Fragment ◽  
Green Fluorescent ◽  
Septal Localization ◽  
A Site

ABSTRACT FtsI (also called PBP3) of Escherichia coli is a transpeptidase required for synthesis of peptidoglycan in the division septum and is one of about a dozen division proteins that localize to the septal ring. FtsI comprises a short amino-terminal cytoplasmic domain, a single transmembrane helix (TMH), and a large periplasmic domain that encodes the catalytic (transpeptidase) activity. We show here that a 26-amino-acid fragment of FtsI is sufficient to direct green fluorescent protein to the septal ring in cells depleted of wild-type FtsI. This fragment extends from W22 to V47 and corresponds to the TMH. This is a remarkable finding because it is usual for a TMH to target a protein to a site more specific than the membrane. Alanine-scanning mutagenesis of the TMH identified several residues important for septal localization. These residues cluster on one side of an alpha-helix, which we propose interacts directly with another division protein to recruit FtsI to the septal ring.

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