Evolutionary history of Phakopsora pachyrhizi (the Asian soybean rust) in Brazil based on nucleotide sequences of the internal transcribed spacer region of the nuclear ribosomal DNA

Phakopsora pachyrhizi has dispersed globally and brought severe economic losses to soybean growers. The fungus has been established in Brazil since 2002 and is found nationwide. To gather information on the temporal and spatial patterns of genetic variation in P. pachyrhizi, we sequenced the nuclear internal transcribed spacer regions (ITS1 and ITS2). Total genomic DNA was extracted using either lyophilized urediniospores or lesions removed from infected leaves sampled from 26 soybean fields in Brazil and one field in South Africa. Cloning prior to sequencing was necessary because direct sequencing of PCR amplicons gave partially unreadable electrophoretograms with peak displacements suggestive of multiple sequences with length polymorphism. Sequences were determined from four clones per field. ITS sequences from African or Asian isolates available from the GenBank were included in the analyses. Independent sequence alignments of the ITS1 and ITS2 datasets identified 27 and 19 ribotypes, respectively. Molecular phylogeographic analyses revealed that ribotypes of widespread distribution in Brazil displayed characteristics of ancestrality and were shared with Africa and Asia, while ribotypes of rare occurrence in Brazil were indigenous. The results suggest P. pachyrhizi found in Brazil as originating from multiple, independent long-distance dispersal events.

Re-evaluating the taxonomic status of Phaeoisariopsis griseola, the causal agent of angular leaf spot of bean.

Angular leaf spot of Phaseolus vulgaris is a serious disease caused by Phaeoisariopsis griseola, in which two major gene pools occur, namely Andean and Middle-American. Sequence analysis of the SSU region of nrDNA revealed the genus Phaeoisariopsis to be indistinguishable from other hyphomycete anamorph genera associated with Mycosphaerella, namely Pseudocercospora and Stigmina. A new combination is therefore proposed in the genus Pseudocercospora, a name to be conserved over Phaeoisariopsis and Stigmina. Further comparisons by means of morphology, cultural characteristics, and DNA sequence analysis of the ITS, calmodulin, and actin gene regions delineated two groups within P. griseola, which are recognised as two formae, namely f. griseola and f. mesoamericana.

Purification of supercoiled plasmids from crude cell lysates using high performance anion exchange chromatography.

High performance liquid chromatography is of increasing importance in the purification of nucleic acids. Recently, a new anion exchange column called Gen-Pak FAX has been introduced for this purpose. Previously, it has been used in the purification of restriction fragments and oligonucleotides. In this paper we present the use of the Gen-Pak FAX column for the purification of plasmids from crude E. coli lysates. The different conformational forms of the plasmid can be well separated and collected with high recoveries of both mass and activity. Up to 50 micrograms of supercoiled plasmid can be purified in a single 30 min run with up to 98% purity.

High-resolution chromatography of nucleic acids on the Gen-Pak FAX column.

High-performance liquid chromatography (HPLC) on a Gen-Pak FAX column has been used to separate and purify microgram amounts of single- and double-stranded DNA and RNA molecules. HPLC of mixtures of DNA restriction fragments showed that fragments within the size range 0.125-23.1 kilobase were easily resolved. Supercoiled (form I) plasmid DNA molecules were readily separated from single-stranded circular DNA of the same length and from various DNA conformational isomers including nicked (form II) and linear (form III) species. Topological isomers generated from supercoiled plasmid DNA molecules by DNA topoisomerase I exhibited different retention times than supercoiled molecules. Supercoiled (form I) DNA molecules were resolved from fully relaxed (form IV) molecules. Synthetic oligonucleotides of 74 and 128 nucleotides in length were separated from failure sequences, as well as from other contaminating synthesis products. Single-stranded circular M13mp18 DNA molecules sufficiently pure for use in automated DNA sequencing systems were prepared by HPLC on a Gen-Pak FAX column. HPLC was also used to fractionate linear double-stranded porcine rotavirus genomic RNA fragments into size classes between 0.3 and 3 kilobase. Finally, HPLC of unfractionated Escherichia coli tRNA molecules resolved multiple species. In all cases, HPLC on Gen-Pak FAX was carried out in phosphate or Tris buffers at neutral pH in the presence of sodium chloride. Columns were not damaged by repeated exposure to impure samples, provided they were cleaned frequently with sodium hydroxide and acetic acid. Although procedures for resolution of the various size ranges for each class of DNA and RNA molecules require further optimization, our preliminary data on the separations obtained, the moderate salt concentrations employed, and the durability of the matrix suggest that this column merits further study.

Defective acidification of endosomes in Chinese hamster ovary cell mutants "cross-resistant" to toxins and viruses.

Like many physiological ligands, several viruses and toxins enter mammalian cells through receptor-mediated endocytosis. Once internalized, the nucleic acids of several viruses and the toxic subunit of diphtheria toxin gain access to the cytosol of the host cell through an acidic intracellular compartment. In this report, we present evidence that one class of mutants of Chinese hamster ovary (CHO)-K1 cells, which is "cross-resistant" to Pseudomonas exotoxin A, diphtheria toxin, and several animal viruses, has a defect in acidification of the endosome. Cells were allowed to internalize fluorescein isothiocyanate-conjugated dextran before subcellular fractionation. Fluorescence measurements on subcellular fractions permitted measurement of the internal pH of the isolated endosomes and lysosomes. Our results show that (i) endosomes and lysosomes from CHO-K1 cells maintain an acidic pH, (ii) acidification of both endosomes and lysosomes is mediated by a Mg2+/ATP-dependent process, (iii) GTP can satisfy the ATP requirement for acidification of lysosomes but not of endosomes, and (iv) at least one class of mutants that is cross-resistant to toxins and animal viruses has a defect in the ATP-dependent acidification of their endosomes. These studies provide biochemical and genetic evidence that the mechanisms of acidification of endosomes and lysosomes are distinct and that a defect in acidification of endosomes is one biochemical basis for cross-resistance to toxins and viruses.

Subfractionation of rat liver Golgi apparatus: separation of enzyme activities involved in the biosynthesis of the phosphomannosyl recognition marker in lysosomal enzymes.

A highly purified Golgi apparatus preparation from rat liver was subfractionated on a Percoll gradient into two major protein peaks of similar size that migrated at densities of 1.028 and 1.051 g/ml. The lighter protein peak contained 70--80% of the total activities of the oligosaccharide-processing enzymes alpha-1,2-mannosidase and mannosidase II and of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosaminyl-1-phosphotransferase (alpha-N-acetylglucosaminylphosphotransferase), an enzyme involved in the biosynthesis of the mannose 6-phosphate recognition marker of lysosomal enzymes. These enzyme activities were enriched 2-fold in specific activity over that of the heavy protein peak. In contrast, 80% of the alpha-N-acetylglucosaminylphosphodiesterase, an enzyme that exposes 6-phosphomonoesters of mannose on the oligosaccharide chains of lysosomal enzymes, migrated in a region of slightly higher density than did the protein peak of density 1.051 g/ml. Sialyltransferase (SiaTase) and galactosyltransferase (Gal-Tase) activities distributed almost equally among the two protein peaks. Controls rule out that the two protein peaks were the result of aggregation/deaggregation and that enzyme activities were altered by Percoll per se. Lysosomal enzyme activities migrated in a region essentially devoid of Golgi apparatus-associated enzyme activities. These results suggest a physical separation within the Golgi apparatus of some of the enzymes involved in the biosynthesis and processing of the oligosaccharides on glycoproteins, including those responsible for the formation of the mannose 6-phosphate recognition marker on lysosomal enzymes.

The role of intermediate vesicles in the adsorptive endocytosis and transport of ligand to lysosomes by human fibroblasts.

Recent work from several laboratories has suggested the participation of intermediate structures in the delivery of adsorbed ligands from the plasma membrane to lysosomes. This report presents subcellular fractionation studies bearing on the role of these structures in adsorptive pinocytosis of epidermal growth factor (EGF), beta-hexosaminidase, and low density lipoprotein (LDL) by human fibroblasts. Using a two-step Percoll density gradient fractionation, we identified newly internalized (5 min) EGF in two intermediate density structures that are essentially negative for plasma membrane marker, and more bouyant than secondary lysosomes. Continued incubation for 20 min resulted in transfer to (or conversion to) vesicles sedimenting with secondary lysosomes. Internalized beta-hexosaminidase and LDL behaved similarly, appearing first in structures of intermediate density, and later appearing in association with secondary lysosomes. Two drugs, NH4Cl and monensin, were found to inhibit ligand transfer to the secondary lysosome peak, although they did not inhibit entry of bound ligands into intermediate density structures. Upon removal of both inhibitors, internalized ligands were quickly transferred to the secondary lysosome peak. This "transfer process" was faster for EGF, than for the other two ligands studied. We interpret these data to indicate that the endocytosis of these three ligands, and their delivery to lysosomes in fibroblasts, proceeds through a common pathway, involving intermediate nonlysosomal structures.

The resolution of two populations of lysosomal organelles containing endocytosed Wistaria floribunda agglutinin from murine fibroblasts.

Subcellular fractionation of Balb/c 3T3 fibroblasts exposed to Wistaria floribunda agglutinin was performed to localize fractions containing internalized lectin. Employing two sequential self-generating silica sol density gradients, the postnuclear supernatant of cell homogenates was resolved into five distinct cellular components. Lysosome enzyme activities were displayed by two populations of vesicles, each separated from plasma membrane, golgi, and mitochondria markers. The more dense of these fractions exhibited morphological and biochemical properties ascribed to secondary lysosomes. The more buoyant population was similar to that reported by Rome et al [11] who noted that it may be a product of vesiculated golgi endoplasmic reticulum lysosome (GERL). Treatment with W floribunda agglutinin of cells, surface radioiodinated demonstrated that plasma membrane proteins were localized within both the buoyant and dense lysosome populations in as little as 10 min after exposure to lectin. Prolonged incubation of the cells with W floribunda agglutinin resulted in maintenance of this distribution. However, when nonradiactive cells were exposed to 125I-labeled W floribunda agglutinin for 10 min, radioactivity was detected only in the buoyant population of lysosomes as well as the plasma membrane/golgi fraction. Treatment of cells with W floribunda agglutinin for 30 min resulted in appearance of lectin associated radioactivity in the dense lysosome fraction in addition to those populations containing radioactivity seen after a 10-min incubation. These data indicate that the endocytosis of W floribunda agglutinin differed substantially from the internalization of a portion of the plasma membrane proteins. Furthermore, we found radioactivity associated with both plasma membrane proteins W floribunda agglutinin in regions of the density gradient fractionation that virtually lacked golgi and lysosome markers. These fractions may have represented populations of nonlysosome vesicles formed during the process of endocytosis.