Molecular Karyotype of the White Rot Fungus Pleurotus ostreatus

The white rot fungus Pleurotus ostreatus is an edible basidiomycete with increasing agricultural and biotechnological importance. Genetic manipulation and breeding of this organism are restricted because of the lack of knowledge about its genomic structure. In this study, we analyzed the genomic constitution of P. ostreatus by using pulsed-field gel electrophoresis optimized for the separation of its chromosomes. We have determined that it contains 11 pairs of chromosomes with sizes ranging from 1.4 to 4.7 Mbp. In addition to chromosome separation, the use of single-copy DNA probes allowed us to resolve the ambiguities caused by chromosome comigration. When the two nuclei present in the dikaryon were separated by protoplasting, analysis of their karyotypes revealed length polymorphisms affecting various chromosomes. This is, to our knowledge, the clearest chromosome separation available for this species.     

Computational drill down on FGF1-heparin interactions through methodological evaluation

Glycosaminoglycans (GAGs) exhibit a key role in cellular communication processes through interactions with target proteins of the extracellular matrix (ECM). The sandwich-like interaction established between Fibroblast growth factor (FGF) and heparin (HE) represents quite a peculiar protein-GAG-protein system, which has been both structurally and functionally intensively studied. The molecular recognition characteristics of this system have been exploited in various computational studies in order to deepen understanding of GAG-protein interactions. Here, we drill down on the interactions established in this peculiar macromolecular complex by analyzing the applicability of docking techniques and molecular dynamics (MD)-based approaches, and we dissect the molecular recognition properties exhibited by FGF towards a series of HE derivatives. We examine the sensitivity of MM-GBSA free energy calculations in terms of receptor conformational space sampling and changes in the ligand structures. Furthermore, we investigate its predictive power in combination with other computational methods, namely the well-established Autodock3 (AD3) and dynamic molecular docking (DMD), a targeted MD-based docking method specifically developed to account for flexibility and solvent in computer simulations of protein-GAG systems. Our results show that a site-mapping approach can be effectively combined with AD3 and DMD calculations to accurately reproduce available experimental data and, furthermore, to determine specific GAG recognition patterns. This study deepens our understanding of the applicability of available theoretical approaches to the investigation of molecular recognition in protein-GAG systems.     

Comparative genomics of the oxidative phosphorylation system in fungi

In this study, we have carried out an in silico analysis of the available mitochondrial and nuclear genomes of fungi in order to identify the oxidative phosphorylation (OXPHOS) proteome, the complete set of proteins that perform the OXPHOS in mitochondria. The presence of OXPHOS proteins has been investigated in 27 nuclear and 52 mitochondrial genomes of fungi. Comparative genomics reveals a high conservation of the OXPHOS system within each fungal phyla, and notable differences between the OXPHOS proteomes of the fungal phyla. The most striking differences concerned Complexes I and V. The absence of Complex I has been previously described in various species of Ascomycota and Microsporidia, and the NDUFB4 and NURM accessory subunits of Complex I appear to be specific of fungi belonging to the subphylum Pezizomycotina. In addition, the Complex V essential subunit ATP14 appears to be specific of two subphyla of Ascomycota: the Saccharomycotina and Pezizomycotina.     

Telomere Organization in the Ligninolytic Basidiomycete Pleurotus ostreatus

Telomeres are structural and functional chromosome regions that are essential for the cell cycle to proceed normally. They are, however, difficult to map genetically and to identify in genome-wide sequence programs because of their structure and repetitive nature. We studied the telomeric and subtelomeric organization in the basidiomycete Pleurotus ostreatus using a combination of molecular and bioinformatics tools that permitted us to determine 19 out of the 22 telomeres expected in this fungus. The telomeric repeating unit in P. ostreatus is TTAGGG, and the numbers of repetitions of this unit range between 25 and 150. The mapping of the telomere restriction fragments to linkage groups 6 and 7 revealed polymorphisms compatible with those observed by pulsed field gel electrophoresis separation of the corresponding chromosomes. The subtelomeric regions in Pleurotus contain genes similar to those described in other eukaryotic systems. The presence of a cluster of laccase genes in chromosome 6 and a bipartite structure containing a Het-related protein and an alcohol dehydrogenase are especially relevant; this bipartite structure is characteristic of the Pezizomycotina fungi Neurospora crassa and Aspergillus terreus. As far as we know, this is the first report describing the presence of such structures in basidiomycetes and the location of a laccase gene cluster in the subtelomeric region, where, among others, species-specific genes allowing the organism to adapt rapidly to the environment usually map.Pleurotus ostreatus (Jacq.: Fr) Kumm. (Dikarya, Basidiomycota, Agaricomycotina, Agaricales) (52) is an active lignin degrader that lives as a saprophyte on dead or decaying wood. P. ostreatus (oyster mushroom) has been industrially cultivated for food production because of its flavor and its nutritional (49) and health-stimulating (8) properties. In addition, it produces various secondary metabolites of medical interest (33). P. ostreatus ligninolytic activity and enzymes have been used in the bioconversion of agricultural wastes (1); in the biodegradation of organopollutants, xenobiotics, and industrial contaminants (12); and in paper pulp bleaching (65), among other applications (10).The whole genome sequence of P. ostreatus is currently being assembled at the Joint Genome Institute (California). P. ostreatus is the first edible and the second lignin-degrading basidiomycete to be sequenced. The sequences of other basidiomycetes, such as Phanerochaete chrysosporium (48), Cryptococcus neoformans (44), Ustilago maydis (38), and Laccaria bicolor (47) have been published, and others (Postia placenta, Heterobasidion annosum, Agaricus bisporus, Serpula lacrymans, etc.) are in progress.Telomeres are the protective DNA-protein complexes found at chromosome termini (6, 13, 76). In most eukaryotes, telomeric DNA consists of tandem arrays of 5- to 8-bp direct repeats where specific telomere-capping proteins bind to ensure chromosomal-end integrity. Telomeres are essential for genome stability, and their shortening (attrition) can lead to chromosome instability, replicative senescence, and apoptosis (43), while their loss causes activation of DNA damage responses (45, 66), cell cycle arrest (28), and chromosome fusions, such as nonreciprocal translocations (7, 32). Moreover, high recombination rates are frequent near telomeres (50).Telomeres and subtelomeric regions are usually gene reservoirs that permit organisms to quickly adapt to new ecological niches (60). Two types of genes participate in this adaptive process: species-specific (18) and contingency genes (5). Species-specific genes are shorter than the core genes of the genomes in which they are present, contain fewer exons, exhibit a subtelomeric bias, and arise by duplication, diversification, and differential gene loss. The avirulence genes of some phytopathogenic fungi are contingency genes that appear near telomeres (15). Furthermore, it has recently been found in Fusarium species that pathogenicity-related genes cooccur with telomeric regions. In this case, chromosomal rearrangements (fusions) have maintained these structures. The Fusarium graminearum genome revealed a link between localized polymorphism and pathogen specialization (11). Among the genes frequently found in subtelomeric regions in Magnaporthe oryzae and Aspergillus sp., the presence of transposons, telomere-linked RecQ helicases, clusters of secondary-metabolite genes, cytochrome oxidases, hydrolases, molecular transporters, and genes encoding secreted proteins, among others, has been reported (18, 56).RecQ helicases are highly conserved in evolution and are required for genome stability. Genes coding for these enzymes have been described in prokaryotes and eukaryotes (4, 9, 39, 71). There are a minimum of five RecQ helicase-like genes in humans, and three of them (BLM, WRN, and RECQL4) are mutated in the Bloom, Werner, and Rothmund-Thomson recessive autosomal syndromes, which exhibit genomic instability leading ultimately to cancer (9). Fungal RecQ helicase-like genes have been previously found associated with chromosome ends (23, 35, 56, 61).In genome-sequencing projects, telomeres and subtelomeric regions are rarely present or assembled because of problems derived from their repetitive nature; therefore, it is necessary to perform direct cloning of the subtelomeric regions. The rice pathogen M. oryzae (56) is one of the few fungi with telomeric and subtelomeric regions characterized. Telomere-associated markers provide an accurate assessment of linkage group (LG) completeness and a better estimate of genetic size and help in establishing the synteny of LGs, especially in those organisms for which genetic-linkage maps are not available (34). Moreover, these markers inform us about the genome organization and the occurrence of species-specific and contingency genes (5, 18), as well as about the chromosome rearrangements that could have occurred in the evolution of the genome.In this work, we mapped and studied the telomeric and subtelomeric regions of most of the P. ostreatus chromosomes, and we describe the main genes present in them. The study was carried out with a combination of genetic, molecular, and bioinformatics tools. The results obtained show the high complexity of these regions and confirm the presence of RecQ helicase-like, heterokaryotic incompatibility (het), and short-chain dehydrogenase genes that have also been found in other fungi. In addition, a laccase gene cluster is described for the first time in the subtelomeric region of chromosome 6. This study is the first step toward analyzing the effects that the subtelomeric positions of some fungal-species-specific genes (such as the laccases can be in white rot lignocellulolytic fungi) could have in the adaptation to new growing substrates and in the generation of large families of apparently redundant elements.     

Hormone-regulated expression and distribution of versican in mouse uterine tissues

Background  

Remodeling of the extracellular matrix is one of the most striking features observed in the uterus during the estrous cycle and after hormone replacement. Versican (VER) is a hyaluronan-binding proteoglycan that undergoes RNA alternative splicing, generating four distinct isoforms. This study analyzed the synthesis and distribution of VER in mouse uterine tissues during the estrous cycle, in ovariectomized (OVX) animals and after 17beta-estradiol (E2) and medroxyprogesterone (MPA) treatments, either alone or in combination.     

Effect of D-amino acids on structure and synthesis of peptidoglycan in Escherichia coli.

Growth of Escherichia coli in the presence of certain D-amino acids, such as D-methionine, results in the incorporation of the D-amino acid into macromolecular peptidoglycan and can be lethal at high concentrations. Previous studies suggested that incorporation was independent of the normal biosynthetic pathway. An enzymatic reaction between the D-amino acid and macromolecular peptidoglycan was proposed as the mechanism of incorporation. The application of more advanced analytical techniques, notably high-pressure liquid chromatography, revealed that the presence of a D-amino acid susceptible to incorporation induced a multiplicity of alterations in peptidoglycan metabolism. Results derived basically from the study of samples treated with D-Met, D-Trp, and D-Phe indicated that the incorporation of a D-amino acid results in the accumulation of two major new muropeptides whose general structures most likely are GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-aa and GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-Ala-GlucNAc-MurNAc-L-Ala-D-Glu-m-diaminopimelic acid-D-aa, where D-aa represents a residue of the added D-amino acid. Resting cells are proficient in the incorporation of D-amino acids and can reach peptidoglycan modification levels comparable to those in growing cells. Under our conditions, D-amino acids had no apparent effect on growth or morphology but caused a severe inhibition of peptidoglycan synthesis and cross-linking, possibly leading to a reduction in the amount of peptidoglycan per cell. The properties of the reaction support the involvement of a penicillin-insensitive LD-transpeptidase enzyme in the synthesis of modified muropeptides and a possible inhibitory action of D-amino acids on high-molecular-weight penicillin-binding proteins.