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1.
J Microbiol Biol Educ ; 16(1): 34-40, 2015 May.
Article in English | MEDLINE | ID: mdl-25949755

ABSTRACT

Four laboratory modules were designed for introductory biology students to explore the field of metagenomics. Students collected microbes from environmental samples, extracted the DNA, and amplified 16S rRNA gene sequences using polymerase chain reaction (PCR). Students designed functional metagenomics screens to determine and compare antibiotic resistance profiles among the samples. Bioinformatics tools were used to generate and interpret phylogenetic trees and identify homologous genes. A pretest and posttest were used to assess learning gains, and the results indicated that these modules increased student performance by an average of 22%. Here we describe ways to engage students in metagenomics-related research and provide readers with ideas for how they can start developing metagenomics exercises for their own classrooms.

2.
Virology ; 442(2): 122-31, 2013 Aug 01.
Article in English | MEDLINE | ID: mdl-23639873

ABSTRACT

O-GlcNAcylation is a dynamic protein modification which has been studied mainly in metazoans. We reported previously that an Arabidopsis thaliana O-GlcNAc transferase modifies at least two threonine residues of the Plum pox virus (PPV) capsid protein (CP). Now, six additional residues were shown to be involved in O-GlcNAc modification of PPV CP. CP O-GlcNAcylation was abolished in the PPV CP7-T/A mutant, in which seven threonines were mutated. PPV CP7-T/A infected Nicotiana clevelandii, Nicotiana benthamiana, and Prunus persica without noticeable defects. However, defects in infection of A. thaliana were readily apparent. In mixed infections of wild-type arabidopsis, the CP7-T/A mutant was outcompeted by wild-type virus. These results indicate that CP O-GlcNAcylation has a major role in the infection process. O-GlcNAc modification may have a role in virion assembly and/or stability as the CP of PPV CP7-T/A was more sensitive to protease digestion than that of the wild-type virus.


Subject(s)
Acetylglucosamine/metabolism , Capsid Proteins/metabolism , Plum Pox Virus/pathogenicity , Protein Processing, Post-Translational , Arabidopsis/virology , Capsid Proteins/chemistry , DNA Mutational Analysis , Plant Diseases/virology , Plum Pox Virus/chemistry , Prunus/virology , Nicotiana/virology
3.
FEBS Lett ; 580(25): 5822-8, 2006 Oct 30.
Article in English | MEDLINE | ID: mdl-17014851

ABSTRACT

A large number of O-linked N-acetylglucosamine (O-GlcNAc) residues have been mapped in vertebrate proteins, however targets of O-GlcNAcylation in plants still have not been characterized. We show here that O-GlcNAcylation of the N-terminal region of the capsid protein of Plum pox virus resembles that of animal proteins in introducing O-GlcNAc monomers. Thr-19 and Thr-24 were specifically O-GlcNAcylated. These residues are surrounded by amino acids typical of animal O-GlcNAc acceptor sites, suggesting that the specificity of O-GlcNAc transferases is conserved among plants and animals. In laboratory conditions, mutations preventing O-GlcNAcylation of Thr-19 and Thr-24 did not have noticeable effects on PPV competence to infect Prunus persicae or Nicotiana clevelandii. However, the fact that Thr-19 and Thr-24 are highly conserved among different PPV strains suggests that their O-GlcNAc modification could be relevant for efficient competitiveness in natural conditions.


Subject(s)
Capsid Proteins/chemistry , Plum Pox Virus/chemistry , Amino Acid Sequence , Amino Acid Substitution , Base Sequence , Binding Sites , Capsid Proteins/genetics , Capsid Proteins/metabolism , DNA, Viral/genetics , Glycosylation , Molecular Sequence Data , Mutagenesis, Site-Directed , Plant Diseases/virology , Plum Pox Virus/genetics , Plum Pox Virus/pathogenicity , Protein Processing, Post-Translational , Prunus/virology , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/metabolism , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Threonine/chemistry , Nicotiana/virology
4.
FEBS Lett ; 580(25): 5829-35, 2006 Oct 30.
Article in English | MEDLINE | ID: mdl-17027982

ABSTRACT

The capsid protein of Plum pox virus (PPV-CP) is modified with O-linked GlcNAc (O-GlcNAc). While Arabidopsis has two O-GlcNAc transferases, SECRET AGENT (SEC) and SPINDLY (SPY), previous work suggests that SEC modifies PPV-CP and that the modification plays a role in the infection process. Here, we show that when co-expressed in Escherichia coli SEC modifies PPV-CP. Deletion mapping and site-directed mutagenesis identified three threonine and a serine located near the N-terminus of PPV-CP that are modified by SEC. Two of these threonines have recently been shown to be modified in virus from plants suggesting that SEC has the same specificity in plants and E. coli.


Subject(s)
Arabidopsis Proteins/metabolism , Capsid Proteins/metabolism , N-Acetylglucosaminyltransferases/metabolism , Plum Pox Virus/metabolism , Amino Acid Substitution , Arabidopsis/enzymology , Base Sequence , Binding Sites , Capsid Proteins/chemistry , Capsid Proteins/genetics , DNA, Viral/genetics , Glycosylation , Mutagenesis, Site-Directed , Plum Pox Virus/genetics , Protein Processing, Post-Translational , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Sequence Deletion , Serine/chemistry , Threonine/chemistry
5.
Genetics ; 161(3): 1279-91, 2002 Jul.
Article in English | MEDLINE | ID: mdl-12136030

ABSTRACT

The Arabidopsis SECRET AGENT (SEC) and SPINDLY (SPY) proteins are similar to animal O-linked N-acetylglucosamine transferases (OGTs). OGTs catalyze the transfer of N-acetylglucosamine (GlcNAc) from UDP-GlcNAc to Ser/Thr residues of proteins. In animals, O-GlcNAcylation has been shown to affect protein activity, stability, and/or localization. SEC protein expressed in Escherichia coli had autocatalytic OGT activity. To determine the function of SEC in plants, two tDNA insertional mutants were identified and analyzed. Although sec mutant plants did not exhibit obvious phenotypes, sec and spy mutations had a synthetic lethal interaction. This lethality was incompletely penetrant in gametes and completely penetrant postfertilization. The rate of both female and male sec spy gamete transmission was higher in plants heterozygous for both mutations than in plants heterozygous for sec and homozygous for spy. Double-mutant embryos aborted at various stages of development and no double-mutant seedlings were obtained. These results indicate that OGT activity is required during gametogenesis and embryogenesis with lethality occurring when parentally derived SEC, SPY, and/or O-GlcNAcylated proteins become limiting.


Subject(s)
Arabidopsis/genetics , N-Acetylglucosaminyltransferases/genetics , Amino Acid Sequence , Arabidopsis/enzymology , Arabidopsis/growth & development , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cloning, Molecular , Crosses, Genetic , Genetic Linkage , Maltose-Binding Proteins , Molecular Sequence Data , Mutagenesis, Insertional , N-Acetylglucosaminyltransferases/chemistry , N-Acetylglucosaminyltransferases/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Seeds/physiology , Sequence Alignment , Sequence Homology, Amino Acid
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