Systematic overrepresentation of DNA termini and underrepresentation of subterminal regions among sequencing templates prepared from hydrodynamically sheared linear DNA molecules

<p>Abstract</p> <p>Background</p> <p>Analysis of fungal genome sequence assemblies reveals that telomeres are poorly represented even though telomeric reads tend to be superabundant. We surmised that the problem might lie in the DNA shearing conditions used to create clone libraries for genome sequencing.</p> <p>Results</p> <p>A shotgun strategy was used to sequence and assemble circular and linear cosmid DNAs sheared using conditions typical for a genome project. The DNA sheared in circular form assembled into a single sequence contig. However, the linearized cosmid produced an incomplete assembly because the two DNA termini, though greatly overrepresented in the clone library used for sequencing, were separated from neighboring sequences by gaps of ~1.4 and 1.8 kb. These gap sizes were reduced, but not eliminated, by shearing the linear cosmid into smaller fragments. Mapping of shearing breakpoints revealed a paucity of breaks in the subterminal regions of the linearized cosmid and also near chromosome ends of the fungus <it>Neurospora crassa</it>.</p> <p>Conclusion</p> <p>Together, our data indicate that the ends of linear DNA molecules are recalcitrant to hydrodynamic shearing. We propose that this causes DNA termini to be overrepresented in the resulting fragment population but ultimately prevents their incorporation into sequence assemblies.</p

Restriction maps of telomeres from Magnaporthe grisea

At least five genetic maps exist for the rice blast fungus, Magnaporthe grisea (Romao and Hamer 1991 Proc. Natl. Acad. Sci. USA 89:5316-5320; Sweigard et al. 1993 in Genetic Maps, O\u27Brien ed. Cold Spring Harbor Press pp. 3.112-3.115; Skinner et al. 1993 Theor. Appl. Genet. 87:545-557; Hayashi and Naito 1993 in Abstracts for International Symposium on Rice Blast Disease, Madison, WI; and Tharreau et al. 1994 in Abstracts of 7th meeting of International Program on Rice Biotechnology, Bali, Indonesia). We have been integrating three of these maps by placing markers used in other laboratories on the map constructed in our laboratory (Skinner et al.. 1993, ibid.). The integrated map will be a primary reference map for this fungus and the Guy11 and 2539 parents used to generate the mapping progeny will become standard strains. Guy11 is a natural isolate; therefore this map provides information on the genome organization of a wild, rice-infecting isolate

Two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 (Polygyridae) and gene order evolution in Helicoidea (Mollusca, Gastropoda)

Helicoidea is a diverse group of land snails with a global distribution. While much is known regarding the relationships of helicoid taxa, comparatively little is known about the evolution of the mitochondrial genome in the superfamily. We sequenced two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 representing the first such data from the helicoid family Polygyridae, and used them in an evolutionary analysis of mitogenomic gene order. We found the mitochondrial genome of P. mexicana to be 14,008 bp in size, possessing the typical 37 metazoan genes. Multiple alternate stop codons are used, as are incomplete stop codons. Mitogenome size and nucleotide content is consistent with other helicoid species. Our analysis of gene order suggested that Helicoidea has undergone four mitochondrial rearrangements in the past. Two rearrangements were limited to tRNA genes only, and two involved protein coding gene

Two Complete Mitochondrial Genomes from \u3cem\u3ePraticolella mexicana\u3c/em\u3e Perez, 2011 (Polygyridae) and Gene Order Evolution in Helicoidea (Mollusca, Gastropoda)

Helicoidea is a diverse group of land snails with a global distribution. While much is known regarding the relationships of helicoid taxa, comparatively little is known about the evolution of the mitochondrial genome in the superfamily. We sequenced two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 representing the first such data from the helicoid family Polygyridae, and used them in an evolutionary analysis of mitogenomic gene order. We found the mitochondrial genome of P. mexicana to be 14,008 bp in size, possessing the typical 37 metazoan genes. Multiple alternate stop codons are used, as are incomplete stop codons. Mitogenome size and nucleotide content is consistent with other helicoid species. Our analysis of gene order suggested that Helicoidea has undergone four mitochondrial rearrangements in the past. Two rearrangements were limited to tRNA genes only, and two involved protein coding genes

Two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 (Polygyridae) and gene order evolution in Helicoidea (Mollusca, Gastropoda)

Helicoidea is a diverse group of land snails with a global distribution. While much is known regarding the relationships of helicoid taxa, comparatively little is known about the evolution of the mitochondrial genome in the superfamily. We sequenced two complete mitochondrial genomes from Praticolella mexicana Perez, 2011 representing the first such data from the helicoid family Polygyridae, and used them in an evolutionary analysis of mitogenomic gene order. We found the mitochondrial genome of P. mexicana to be 14,008 bp in size, possessing the typical 37 metazoan genes. Multiple alternate stop codons are used, as are incomplete stop codons. Mitogenome size and nucleotide content is consistent with other helicoid species. Our analysis of gene order suggested that Helicoidea has undergone four mitochondrial rearrangements in the past. Two rearrangements were limited to tRNA genes only, and two involved protein coding gene

Chromosome-End Knockoff Strategy to Reshape Alkaloid Profiles of a Fungal Endophyte

Molecular genetic techniques to precisely eliminate genes in asexual filamentous fungi require the introduction of a marker gene into the target genome. We developed a novel strategy to eliminate genes or gene clusters located in subterminal regions of chromosomes, and then eliminate the marker gene and vector backbone used in the transformation procedure. Because many toxin gene clusters are subterminal, this method is particularly suited to generating nontoxic fungal strains. We tested this technique on Epichloë coenophiala, a seed-transmissible symbiotic fungus (endophyte) of the important forage grass, tall fescue (Lolium arundinaceum). The endophyte is necessary for maximal productivity and sustainability of this grass but can produce ergot alkaloids such as ergovaline, which are toxic to livestock. The genome sequence of E. coenophiala strain e19 revealed two paralogous ergot alkaloid biosynthesis gene clusters, designated EAS1 and EAS2. EAS1 was apparently subterminal, and the lpsB copy in EAS2 had a frame-shift mutation. We designed a vector with a fungal-active hygromycin phosphotransferase gene (hph), an lpsA1 gene fragment for homologous recombination at the telomere-distal end of EAS1, and a telomere repeat array positioned to drive spontaneous loss of hph and other vector sequences, and to stabilize the new chromosome end. We transformed E. coenophiala with this vector, then selected “knockoff” endophyte strains, confirmed by genome sequencing to lack 162 kb of a chromosome end including most of EAS1, and also to lack vector sequences. These ∆EAS1 knockoff strains produced no detectable ergovaline, whereas complementation with functional lpsB restored ergovaline production

A Comparative Genomic Analysis of Putative Pathogenicity Genes in the Host-Specific Sibling Species \u3cem\u3eColletotrichum graminicola\u3c/em\u3e and \u3cem\u3eColletotrichum sublineola\u3c/em\u3e

Background: Colletotrichum graminicola and C. sublineola cause anthracnose leaf and stalk diseases of maize and sorghum, respectively. In spite of their close evolutionary relationship, the two species are completely host-specific. Host specificity is often attributed to pathogen virulence factors, including specialized secondary metabolites (SSM), and small-secreted protein (SSP) effectors. Genes relevant to these categories were manually annotated in two co-occurring, contemporaneous strains of C. graminicola and C. sublineola. A comparative genomic and phylogenetic analysis was performed to address the evolutionary relationships among these and other divergent gene families in the two strains. Results: Inoculation of maize with C. sublineola, or of sorghum with C. graminicola, resulted in rapid plant cell death at, or just after, the point of penetration. The two fungal genomes were very similar. More than 50% of the assemblies could be directly aligned, and more than 80% of the gene models were syntenous. More than 90% of the predicted proteins had orthologs in both species. Genes lacking orthologs in the other species (non-conserved genes) included many predicted to encode SSM-associated proteins and SSPs. Other common groups of non-conserved proteins included transporters, transcription factors, and CAZymes. Only 32 SSP genes appeared to be specific to C. graminicola, and 21 to C. sublineola. None of the SSM-associated genes were lineage-specific. Two different strains of C. graminicola, and three strains of C. sublineola, differed in no more than 1% percent of gene sequences from one another. Conclusions: Efficient non-host recognition of C. sublineola by maize, and of C. graminicola by sorghum, was observed in epidermal cells as a rapid deployment of visible resistance responses and plant cell death. Numerous non-conserved SSP and SSM-associated predicted proteins that could play a role in this non-host recognition were identified. Additional categories of genes that were also highly divergent suggested an important role for co-evolutionary adaptation to specific host environmental factors, in addition to aspects of initial recognition, in host specificity. This work provides a foundation for future functional studies aimed at clarifying the roles of these proteins, and the possibility of manipulating them to improve management of these two economically important diseases

Structure and Specificity of a Permissive Bacterial C-prenyltransferase

This study highlights the biochemical and structural characterization of the L-tryptophan C6 C-prenyltransferase (C-PT) PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including daptomycin. Two additional PTs also produced novel prenylated daptomycins with improved antibacterial activities over the parent drug

Structure and Specificity of a Permissive Bacterial C-Prenyltransferase

This study highlights the biochemical and structural characterization of the L-tryptophan C6 C-prenyltransferase (C-PT) PriB from Streptomyces sp. RM-5-8. PriB was found to be uniquely permissive to a diverse array of prenyl donors and acceptors including daptomycin. Two additional PTs also produced novel prenylated daptomycins with improved antibacterial activities over the parent drug

Telomere Roles in Fungal Genome Evolution and Adaptation

Telomeres form the ends of linear chromosomes and usually comprise protein complexes that bind to simple repeated sequence motifs that are added to the 3′ ends of DNA by the telomerase reverse transcriptase (TERT). One of the primary functions attributed to telomeres is to solve the “end-replication problem” which, if left unaddressed, would cause gradual, inexorable attrition of sequences from the chromosome ends and, eventually, loss of viability. Telomere-binding proteins also protect the chromosome from 5′ to 3′ exonuclease action, and disguise the chromosome ends from the double-strand break repair machinery whose illegitimate action potentially generates catastrophic chromosome aberrations. Telomeres are of special interest in the blast fungus, Pyricularia, because the adjacent regions are enriched in genes controlling interactions with host plants, and the chromosome ends show enhanced polymorphism and genetic instability. Previously, we showed that telomere instability in some P. oryzae strains is caused by novel retrotransposons (MoTeRs) that insert in telomere repeats, generating interstitial telomere sequences that drive frequent, break-induced rearrangements. Here, we sought to gain further insight on telomeric involvement in shaping Pyricularia genome architecture by characterizing sequence polymorphisms at chromosome ends, and surrounding internalized MoTeR loci (relics) and interstitial telomere repeats. This provided evidence that telomere dynamics have played historical, and likely ongoing, roles in shaping the Pyricularia genome. We further demonstrate that even telomeres lacking MoTeR insertions are poorly preserved, such that the telomere-adjacent sequences exhibit frequent presence/absence polymorphism, as well as exchanges with the genome interior. Using TERT knockout experiments, we characterized chromosomal responses to failed telomere maintenance which suggested that much of the MoTeR relic-/interstitial telomere-associated polymorphism could be driven by compromised telomere function. Finally, we describe three possible examples of a phenomenon known as “Adaptive Telomere Failure,” where spontaneous losses of telomere maintenance drive rapid accumulation of sequence polymorphism with possible adaptive advantages. Together, our data suggest that telomere maintenance is frequently compromised in Pyricularia but the chromosome alterations resulting from telomere failure are not as catastrophic as prior research would predict, and may, in fact, be potent drivers of adaptive polymorphism