Protein mapping and genome expression variations in the basidiomycete Agrocybe aegerita

Summary A procedure suitable for the extraction and mapping of total proteins from the basidiomycete, Agrocybe aegerita, was developed. A. aegerita mycelia were fragmented either with a Dangoumeau grinder, an X-press bomb or a sonicator and the efficiency of these three disruption methods were compared. The extraction buffer composition was optimized to avoid proteolytic activities. 2D-SDS-PAGE analysis of protein extracts showed that the rate of reproducibility depending on extractions and electrophoretic separations was always greater than 96% for all strains. The differences in efficiency observed between the breaking procedures indicate that the A. aegerita cell wall is more mechanically resistant than that of other basidiomycetes. The efficient action of protease inhibitors (PMSF and SDS) showed that A. aegerita mycelia contains numerous and/or highly active proteases. Reproducibility of protein extraction and separation methods allowed the establishment and the comparison of standard maps. Qualitative and quantitative variations in gene products between a wild dikaryotic strain and 11 homokaryotic strains from its progeny were examined. The genetic diversity, determined by comparing the distribution of proteic variations in 11 homokaryons from the same progeny, was comparable to that observed between co-isogenic homokaryons of another basidiomycete.     

Isolation and characterization of plasma membranes from the fungus Podospora anserina

This paper describes a method for separating and isolating plasma membranes from the septated fungus Podospora anserina. Plasma membranes were isolated from protoplasts (young cell plasma membranes) and mycelia (both young and aged cell plasma membranes). The procedure of fractionation consisted of a combination of differential and isopycnic centrifugations. Characterization of cellular membranes and enrichment of the fractions with plasmalemma were carried out by assays on enzymatic activities. A plasma membrane fraction was isolated in a buoyant density peak of 1.087 g/cm3, where three enzymatic activities bound to plasma membrane, adenylate cyclase, chitin synthase, and beta-glucan synthase at low affinity for UDP-Glc, peaked together. Good purity of this fraction was determined by the absence or the very low level of other enzymatic activities used as markers for intracellular membranes, i.e., succinate dehydrogenase, alpha-mannosidase, NADPH cytochrome c reductase, and beta-glucan synthase at high affinity for UDP-Glc activities.     

Strain-specific differences in <Emphasis Type="Italic">Neisseria gonorrhoeae</Emphasis> associated with the phase variable gene repertoire

Background  

There are several differences associated with the behaviour of the four main experimental Neisseria gonorrhoeae strains, FA1090, FA19, MS11, and F62. Although there is data concerning the gene complements of these strains, the reasons for the behavioural differences are currently unknown. Phase variation is a mechanism that occurs commonly within the Neisseria spp. and leads to switching of genes ON and OFF. This mechanism may provide a means for strains to express different combinations of genes, and differences in the strain-specific repertoire of phase variable genes may underlie the strain differences.     

Genetic islands of <Emphasis Type="Italic">Streptococcus agalactiae</Emphasis> strains NEM316 and 2603VR and their presence in other Group B Streptococcal strains

Background  

Streptococcus agalactiae (Group B Streptococcus; GBS) is a major contributor to obstetric and neonatal bacterial sepsis. Serotype III strains cause the majority of late-onset sepsis and meningitis in babies, and thus appear to have an enhanced invasive capacity compared with the other serotypes that cause disease predominantly in immunocompromised pregnant women. We compared the serotype III and V whole genome sequences, strains NEM316 and 2603VR respectively, in an attempt to identify genetic attributes of strain NEM316 that might explain the propensity of strain NEM316 to cause late-onset disease in babies. Fourteen putative pathogenicity islands were described in the strain NEM316 whole genome sequence. Using PCR- and targeted microarray- strategies, the presence of these islands were assessed in a diverse strain collection including 18 colonizing isolates from healthy pregnant women, and 13 and 8 invasive isolates from infants with early- and late-onset sepsis, respectively.     

Molecular cloning, sequence and expression of Aa-polB, a mitochondrial gene encoding a family B DNA polymerase from the edible basidiomycete Agrocybe aegerita

An ORF of 1716 nucleotides, putatively encoding a DNA polymerase, was characterized in the mitochondrial genome of the edible basidiomycete Agrocybe aegerita. The complete gene, named Aa-polB, and its flanking regions were cloned and sequenced from three overlapping restriction fragments. Aa-polB is located between the SSU rDNA (5′ region) and a gene for tRNA^Asn (3′ region), and is separated from these genes by two A+T-rich intergenic regions of 1048 (5′ region) and 3864 (3′ region) nucleotides, which lack repeated sequences of mitochondrial or plasmid origin. The deduced Aa-POLB protein shows extensive sequence similarity with the family B DNA polymerases encoded by genomes that rely on protein-primed replication (invertrons). The domains involved in the 3′→5′ exonuclease (Exo I to III) and polymerase (Pol I to Pol V) activities were localized on the basis of conserved sequence motifs. The alignment of the Aa-POLB protein (571 amino acids) with sequences of family B DNA polymerases from invertrons revealed that in Aa-POLB the N-terminal region preceding Exo I is short, suggesting a close relationship with the DNA polymerases of bacteriophages that have linear DNA. The Aa-polB gene was shown to be present in all wild strains examined, which were collected from a wide range of locations in Europe. As shown by RT-PCR, the Aa-polB gene is transcribed in the mitochondria, at a low but significant level. The likelihood of the coexistence of Aa-POLB and Pol?γ in the A. aegerita mitochondrion is discussed in the light of recent reports showing the conservation of the nucleus-encoded Pol?γ from yeast to human.     

CHANGES IN CHEMICAL, SENSORY AND RHEOLOGICAL CHARACTERISTICS OF MANCHEGO CHEESES DURING RIPENING

Manchego cheese is a high-fat pressed ewe's-milk cheese made in Castilla-La Mancha (Spain) and produced by enzymatic coagulation. The minimum ripening time before marketing required by the Regulatory Board of the Manchego Cheese Appellation of Origin is 60 days.
This paper describes the physicochemical, proteolysis, sensory and texture characteristics of Manchego cheese, and the degree of homogeneity of cheeses made under the Manchego Appellation of Origin. The data gathered in this study indicate that sensory and instrumental analysis are useful tools for detecting changes in Manchego cheese during ripening. These changes were first detected by the instrumental analysis (2 months). The panelists detected differences after 4 months' ripening in all the factories. With physicochemical analysis, on the other hand, longer ripening times (6–8 months) are required before such changes become appreciated.     

Karyological studies in Sonchus section Muritimi (Asteraceae) from the Iberian Peninsula

MEJÍAS, J. A. & VALDÉS, B., 1988. Karyologiepl studies in Sonchus section Madtimi (Asteraceae) from the Iberian Peninmula. Karyological data support the distinction of S. aquatilis Pourret and S. maritimus L. at the specific level. Karyological data and hybridization experiments support the idea that S. × novocaslcllanus Cirujano has been produced by the hybridization of S. crassifolius Pourret ex Willd. and S. maritimus L.