scholarly journals Characterization of a novel NADP+-dependent D-arabitol dehydrogenase from the plant pathogen Uromyces fabae

2005 ◽  
Vol 389 (2) ◽  
pp. 289-295 ◽  
Author(s):  
Tobias Link ◽  
Gertrud Lohaus ◽  
Ingrid Heiser ◽  
Kurt Mendgen ◽  
Matthias Hahn ◽  
...  
Keyword(s):  
Plant Pathogen ◽  
Broad Bean ◽  
Rust Fungus ◽  
Plant Defence ◽  
In Vitro System ◽  
Uromyces Fabae ◽  
Factor Specificity ◽  
Defence Reactions

We have identified and characterized a novel NADP+-dependent D-arabitol dehydrogenase and the corresponding gene from the rust fungus Uromyces fabae, a biotrophic plant pathogen on broad bean (Vicia faba). The new enzyme was termed ARD1p (D-arabitol dehydrogenase 1). It recognizes D-arabitol and mannitol as substrates in the forward reaction, and D-xylulose, D-ribulose and D-fructose as substrates in the reverse reaction. Co-factor specificity was restricted to NADP(H). Kinetic data for the major substrates and co-factors are presented. A detailed analysis of the organization and expression pattern of the ARD1 gene are also given. Immunocytological data indicate a localization of the gene product predominantly in haustoria, the feeding structures of these fungi. Analyses of metabolite levels during pathogenesis indicate that the D-arabitol concentration rises dramatically as infection progresses, and D-arabitol was shown in an in vitro system to be capable of quenching reactive oxygen species involved in host plant defence reactions. ARD1p may therefore play an important role in carbohydrate metabolism and in establishing and/or maintaining the biotrophic interaction in U. fabae.

scholarly journals Expression and Characterization of the Flocculin Flo11/Muc1, a Saccharomyces cerevisiae Mannoprotein with Homotypic Properties of Adhesion

2007 ◽  
Vol 6 (12) ◽  
pp. 2214-2221 ◽  
Author(s):  
Lois M. Douglas ◽  
Li Li ◽  
Yang Yang ◽  
A. M. Dranginis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Biofilm Formation ◽  
In Vitro System ◽  
Glycosylphosphatidylinositol Anchor ◽  
Fungal Adhesion ◽  
Very High ◽  
Molecular Weight Form

ABSTRACT The Flo11/Muc1 flocculin has diverse phenotypic effects. Saccharomyces cerevisiae cells of strain background Σ1278b require Flo11p to form pseudohyphae, invade agar, adhere to plastic, and develop biofilms, but they do not flocculate. We show that S. cerevisiae var. diastaticus strains, on the other hand, exhibit Flo11-dependent flocculation and biofilm formation but do not invade agar or form pseudohyphae. In order to study the nature of the Flo11p proteins produced by these two types of strains, we examined secreted Flo11p, encoded by a plasmid-borne gene, in which the glycosylphosphatidylinositol anchor sequences had been replaced by a histidine tag. A protein of approximately 196 kDa was secreted from both strains, which upon purification and concentration, aggregated into a form with a very high molecular mass. When secreted Flo11p was covalently attached to microscopic beads, it conferred the ability to specifically bind to S. cerevisiae var. diastaticus cells, which flocculate, but not to Σ1278b cells, which do not flocculate. This was true for the 196-kDa form as well as the high-molecular-weight form of Flo11p, regardless of the strain source. The coated beads bound to S. cerevisiae var. diastaticus cells expressing FLO11 and failed to bind to cells with a deletion of FLO11, demonstrating a homotypic adhesive mechanism. Flo11p was shown to be a mannoprotein. Bead-to-cell adhesion was inhibited by mannose, which also inhibits Flo11-dependent flocculation in vivo, further suggesting that this in vitro system is a useful model for the study of fungal adhesion.

Regeneration and characterization of adult mouse hippocampal neurons in a defined in vitro system

2009 ◽  
Vol 177 (1) ◽  
pp. 51-59 ◽  
Author(s):  
Kucku Varghese ◽  
Mainak Das ◽  
Neelima Bhargava ◽  
Maria Stancescu ◽  
Peter Molnar ◽  
...  
Keyword(s):  
Hippocampal Neurons ◽  
Adult Mouse ◽  
In Vitro System

scholarly journals An In Vitro System Recapitulates Chromatin Remodeling at the PHO5 Promoter

1999 ◽  
Vol 19 (4) ◽  
pp. 2817-2827 ◽  
Author(s):  
Elizabeth S. Haswell ◽  
Erin K. O’Shea
Keyword(s):  
Chromatin Remodeling ◽  
Chromatin Structure ◽  
Nuclear Extract ◽  
In Vitro System ◽  
Chromatin Remodeling Complex ◽  
Promoter Dna ◽  
Pho5 Promoter

ABSTRACT The Saccharomyces cerevisiae gene PHO5 is an excellent system with which to study regulated changes in chromatin structure. The PHO5 promoter is packaged into four positioned nucleosomes under repressing conditions; upon induction, the structure of these nucleosomes is altered such that the promoter DNA becomes accessible to nucleases. We report here the development and characterization of an in vitro system in which partially purified PHO5 minichromosomes undergo promoter chromatin remodeling. Several hallmarks of thePHO5 chromatin transition in vivo were reproduced in this system. Chromatin remodeling of PHO5minichromosomes required the transcription factors Pho4 and Pho2, was localized to the promoter region of PHO5, and was independent of the chromatin-remodeling complex Swi-Snf. In vitro chromatin remodeling also required the addition of fractionated nuclear extract and hydrolyzable ATP. This in vitro system should serve as a useful tool for identifying the components required for this reaction and for elucidating the mechanism by which the PHO5promoter chromatin structure is changed.

Repair of deletions and double-strand gaps by homologous recombination in a mammalian in vitro system

1991 ◽  
Vol 11 (1) ◽  
pp. 445-457
Author(s):  
R Jessberger ◽  
P Berg
Keyword(s):  
Dna Sequences ◽  
Kanamycin Resistance ◽  
Double Strand Breaks ◽  
Strand Transfer ◽  
In Vitro System ◽  
Strand Breaks ◽  
Nuclear Extracts ◽  
Dna Strand

We have designed an in vitro system using mammalian nuclear extracts, or fractions derived from them, that can restore the sequences missing at double-strand breaks (gaps) or in deletions. The recombination substrates consist of (i) recipient DNA, pSV2neo with gaps or deletions ranging from 70 to 390 bp in the neo sequence, and (ii) donor DNAs with either complete homology to the recipient (pSV2neo) or plasmids whose homology with pSV2neo is limited to a 1.0- to 1.3-kbp neo segment spanning the gaps or deletions. Incubation of these substrates with various enzyme fractions results in repair of the recipient DNA's disrupted neo gene. The recombinational repair was monitored by transforming recA Escherichia coli to kanamycin resistance and by a new assay which measures the extent of DNA strand transfer from the donor substrate to the recipient DNA. Thus, either streptavidin- or antidigoxigenin-tagged beads are used to separate the biotinylated or digoxigeninylated recipient DNA, respectively, after incubation with the isotopically labeled donor DNA. In contrast to the transfection assay, the DNA strand transfer measurements are direct, quantitative, rapid, and easy, and they provide starting material for the characterization of the recombination products and intermediates. Accordingly, DNA bound to beads serves as a suitable template for the polymerase chain reaction. With appropriate pairs of oligonucleotide primers, we have confirmed that both gaps and deletions are fully repaired, that deletions can be transferred from the recipient DNA to the donor's intact neo sequence, and that cointegrant molecules containing donor and recipient DNA sequences are formed.

scholarly journals The role of haustoria in sugar supply during infection of broad bean by the rust fungus Uromyces fabae

2001 ◽  
Vol 98 (14) ◽  
pp. 8133-8138 ◽  
Author(s):  
R. T. Voegele ◽  
C. Struck ◽  
M. Hahn ◽  
K. Mendgen
Keyword(s):  
Broad Bean ◽  
Rust Fungus ◽  
Uromyces Fabae
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