A comprehensive mechanism for 5-carboxylcytosine-induced transcriptional pausing revealed by Markov state models.

RNA polymerase II (Pol II) surveils the genome, pausing as it encounters DNA lesions and base modifications and initiating signals for DNA repair among other important regulatory events. Recent work suggests that Pol II pauses at 5-carboxycytosine (5caC), an epigenetic modification of cytosine, because of a specific hydrogen bond between the carboxyl group of 5caC and a specific residue in fork loop 3 of Pol II. This hydrogen bond compromises productive NTP binding and slows down elongation. Apart from this specific interaction, the carboxyl group of 5caC can potentially interact with numerous charged residues in the cleft of Pol II. However, it is not clear how other interactions between Pol II and 5caC contribute to pausing. In this study, we use Markov state models (a type of kinetic network models) built from extensive molecular dynamics simulations to comprehensively study the impact of 5caC on Pol II translocation. We describe two translocation intermediates with specific interactions that prevent the template base from loading into the Pol II active site. In addition to the previously observed state with 5caC constrained by fork loop 3, we discovered a new intermediate state with a hydrogen bond between 5caC and fork loop 2. Surprisingly, we find that 5caC may curb translocation by suppressing kinking of the helix bordering the active site (the bridge helix) because its high flexibility is critical to translocation. Our work provides new insights into how epigenetic modifications of genomic DNA can modulate Pol II translocation, inducing pauses in transcription.

Phylogenetic assessment of Chromocyphellaceae (Agaricineae, Basidiomycota) and a new lamellate species of Chromocyphella.

Cyphelloid fungi represent a polyphyletic assemblage of reduced agarics, including the brown-spored family Chromocyphellaceae. In order to elucidate the phylogenetic position of Chromocyphellaceae, newly generated sequences of Chromocyphella were included in a multigene alignment of the Agaricineae and phylogenetically analyzed. The current analyses show that the Chromocyphella muscicola specimen used to phylogenetically place Chromocyphellaceae in its original description was misidentified and that the Chromocyphellaceae nests in the Hymenogastraceae, Chromocyphella being sister to Flammula. Chromocyphella is emended, including now a new species with lamellate and stipitate basidiomata, C. lamellata. The name Cymbella crouanii, type species of Chromocyphella, is lecto- and epitypified. Our analyses support a new origin of cyphelloid fungi. The shift to a cyphelloid basidioma from an agaric ancestor is suggested to have occurred in two evolutionary steps in Chromocyphella, an initial reduction in basidioma size and a subsequent loss of lamellae and stipe.

An RNA polymerase II-driven Ebola virus minigenome system as an advanced tool for antiviral drug screening.

Ebola virus (EBOV) causes a severe disease in humans with the potential for significant international public health consequences. Currently, treatments are limited to experimental vaccines and therapeutics. Therefore, research into prophylaxis and antiviral strategies to combat EBOV infections is of utmost importance. The requirement for high containment laboratories to study EBOV infection is a limiting factor for conducting EBOV research. To overcome this issue, minigenome systems have been used as valuable tools to study EBOV replication and transcription mechanisms and to screen for antiviral compounds at biosafety level 2. The most commonly used EBOV minigenome system relies on the ectopic expression of the T7 RNA polymerase (T7), which can be limiting for certain cell types. We have established an improved EBOV minigenome system that utilizes endogenous RNA polymerase II (pol II) as a driver for the synthesis of minigenome RNA. We show here that this system is as efficient as the T7-based minigenome system, but works in a wider range of cell types, including biologically relevant cell types such as bat cells. Importantly, we were also able to adapt this system to a reliable and cost-effective 96-well format antiviral screening assay with a Z-factor of 0.74, indicative of a robust assay. Using this format, we identified JG40, an inhibitor of Hsp70, as an inhibitor of EBOV replication, highlighting the potential for this system as a tool for antiviral drug screening. In summary, this updated EBOV minigenome system provides a convenient and effective means of advancing the field of EBOV research.

Annotation of genomics data using bidirectional hidden Markov models unveils variations in Pol II transcription cycle.

DNA replication, transcription and repair involve the recruitment of protein complexes that change their composition as they progress along the genome in a directed or strand-specific manner. Chromatin immunoprecipitation in conjunction with hidden Markov models (HMMs) has been instrumental in understanding these processes, as they segment the genome into discrete states that can be related to DNA-associated protein complexes. However, current HMM-based approaches are not able to assign forward or reverse direction to states or properly integrate strand-specific (e.g., RNA expression) with non-strand-specific (e.g., ChIP) data, which is indispensable to accurately characterize directed processes. To overcome these limitations, we introduce bidirectional HMMs which infer directed genomic states from occupancy profiles de novo. Application to RNA polymerase II-associated factors in yeast and chromatin modifications in human T cells recovers the majority of transcribed loci, reveals gene-specific variations in the yeast transcription cycle and indicates the existence of directed chromatin state patterns at transcribed, but not at repressed, regions in the human genome. In yeast, we identify 32 new transcribed loci, a regulated initiation-elongation transition, the absence of elongation factors Ctk1 and Paf1 from a class of genes, a distinct transcription mechanism for highly expressed genes and novel DNA sequence motifs associated with transcription termination. We anticipate bidirectional HMMs to significantly improve the analyses of genome-associated directed processes.

Fitness cost, gyrB mutation, and absence of phosphotransferase system fructose specific IIABC component in novobiocin-resistant Streptococcus iniae vaccine strain ISNO.

To understand the fitness cost of novobiocin-resistance in an attenuated Streptococcus iniae vaccine strain ISNO compared to its virulent parent strain ISET0901, cell proliferation rate of the two strains were compared to each other. Our results revealed that the cell proliferation rates of ISNO were significantly (P<0.05) smaller than that of ISET0901. To understand whether there was any mutation at the target site of novobiocin, DNA gyrase subunit B (gyrB) was sequenced from both strains. Sequencing results revealed a point mutation of AGA to AGC, resulting in a deduced amino acid substitution of R635S. To determine whether any unique DNA sequence was present in ISET0901 but absent in ISNO, PCR-select bacterial genome subtractive hybridization was performed. A phosphotransferase system fructose specific IIABC component sequence was confirmed to be present in ISET0901 but absent in ISNO. Using genomic DNAs from ten field-strains of S. iniae as templates, the phosphotransferase system fructose specific IIABC component sequence was found to be present in five highly virulent strains, but absent in five avirulent strains. Taken together, our results suggest that: (1) As fitness cost of novobicin resistance, ISNO had significantly smaller cell proliferation rate; (2) point mutation at target site gyrB resulting in R635S substitution was associated with novobiocin resistance in ISNO; and (3) phosphotransferase system fructose specific IIABC component was associated with virulence of S. iniae.

The devil in the details: interactions between the branch-length prior and likelihood model affect node support and branch lengths in the phylogeny of the Psoraceae.

In popular use of Bayesian phylogenetics, a default branch-length prior is almost universally applied without knowing how a different prior would have affected the outcome. We performed Bayesian and maximum likelihood (ML) inference of phylogeny based on empirical nucleotide sequence data from a family of lichenized ascomycetes, the Psoraceae, the morphological delimitation of which has been controversial. We specifically assessed the influence of the combination of Bayesian branch-length prior and likelihood model on the properties of the Markov chain Monte Carlo tree sample, including node support, branch lengths, and taxon stability. Data included two regions of the mitochondrial ribosomal RNA gene, the internal transcribed spacer region of the nuclear ribosomal RNA gene, and the protein-coding largest subunit of RNA polymerase II. Data partitioning was performed using Bayes' factors, whereas the best-fitting model of each partition was selected using the Bayesian information criterion (BIC). Given the data and model, short Bayesian branch-length priors generate higher numbers of strongly supported nodes as well as short and topologically similar trees sampled from parts of tree space that are largely unexplored by the ML bootstrap. Long branch-length priors generate fewer strongly supported nodes and longer and more dissimilar trees that are sampled mostly from inside the range of tree space sampled by the ML bootstrap. Priors near the ML distribution of branch lengths generate the best marginal likelihood and the highest frequency of "rogue" (unstable) taxa. The branch-length prior was shown to interact with the likelihood model. Trees inferred under complex partitioned models are more affected by the stretching effect of the branch-length prior. Fewer nodes are strongly supported under a complex model given the same branch-length prior. Irrespective of model, internal branches make up a larger proportion of total tree length under the shortest branch-length priors compared with longer priors. Relative effects on branch lengths caused by the branch-length prior can be problematic to downstream phylogenetic comparative methods making use of the branch lengths. Furthermore, given the same branch-length prior, trees are on average more dissimilar under a simple unpartitioned model compared with a more complex partitioned models. The distribution of ML branch lengths was shown to better fit a gamma or Pareto distribution than an exponential one. Model adequacy tests indicate that the best-fitting model selected by the BIC is insufficient for describing data patterns in 5 of 8 partitions. More general substitution models are required to explain the data in three of these partitions, one of which also requires nonstationarity. The two mitochondrial ribosomal RNA gene partitions need heterotachous models. We found no significant correlations between, on the one hand, the amount of ambiguous data or the smallest branch-length distance to another taxon and, on the other hand, the topological stability of individual taxa. Integrating over several exponentially distributed means under the best-fitting model, node support for the family Psoraceae, including Psora, Protoblastenia, and the Micarea sylvicola group, is approximately 0.96. Support for the genus Psora is distinctly lower, but we found no evidence to contradict the current classification.

Critical assessment of the Lactarius gerardii species complex (Russulales).

This paper investigates species delimitation within the Lactarius gerardii species complex and explores its taxonomic and geographical extent. A combined molecular phylogeny based on ITS, LSU and rpb2 gene sequences is constructed and morphological characters are evaluated. While L. gerardii was originally described from North America, it has later been reported from all over Asia. Therefore a worldwide sampling range was aimed at, including species exhibiting morphological affinities with L. gerardii. The phylogenetic analyses indicate that intercontinental conspecificity in L. gerardii is absent. Thirty strongly supported clades are retrieved of which 18 are morphologically identifiable species. The group is elevated to Lactarius subg. Gerardii stat. nov. It includes, apart from L. gerardii s.l., L. atrovelutinus, L. bicolor, L. ochrogalactus, L. petersenii, L. reticulatovenosus, L. sepiaceus, L. subgerardii and L. wirrabara, as well as the pleurotoid L. uyedae. The paraphyletic nature of the genus Lactarius is confirmed. Lactarius subg. Gerardii appears not affiliated with L. subg. Plinthogalus and this can be substantiated morphologically. No representatives are known from Europe, Africa or South America. The high frequency of intercontinental sister relationships observed between America, Asia and the Australian region, suggests multiple migration and speciation events have occurred across continents.

A multigene molecular phylogenetic assessment of true morels (Morchella) in Turkey.

A collection of 247 true morels (Morchella spp.) primarily from the Mediterranean and Aegean Regions of Southern Turkey, were analyzed for species diversity using partial RNA polymerase I (RPB1) and nuclear ribosomal large subunit (LSU) rDNA gene sequences. Based on the result of this initial screen, 62 collections representing the full range of genetic diversity sampled were subjected to multigene phylogenetic species recognition based on genealogical concordance (GCPSR). The 62-taxon dataset consisted of partial sequences from three nuclear protein-coding genes, RNA polymerase I (RPB1), RNA polymerase II (RPB2), translation elongation factor (EF1-alpha), and partial LSU rDNA gene sequences. Phylogenetic analyses of the individual and combined datasets, using maximum parsimony (MP) and maximum likelihood (ML), yielded nearly fully resolved phylogenies that were highly concordant topologically. GCPSR analysis of the 62-taxon dataset resolved 15 putative phylogenetically distinct species. The early diverging Elata (black morels) and Esculenta Clades (yellow morels) were represented, respectively, by 13 and two species. Because a Latin binomial can be applied with confidence to only one of the 15 species (Morchella semilibera), species were identified by clade (Mel for Elata and Mes for Esculenta) followed by a unique Arabic number for each species within these two clades. Eight of the species within the Elata Clade appear to be novel, including all seven species within the Mel-20-to-31 subclade and its sister designated Mel-25. Results of the present study provide essential data for ensuring the sustainability of morel harvests through the formulation of sound conservation policies.

DNA structure in human RNA polymerase II promoters.

The fact that DNA three-dimensional structure is important for transcriptional regulation begs the question of whether eukaryotic promoters contain general structural features independently of what genes they control. We present an analysis of a large set of human RNA polymerase II promoters with a very low level of sequence similarity. The sequences, which include both TATA-containing and TATA-less promoters, are aligned by hidden Markov models. Using three different models of sequence-derived DNA bendability, the aligned promoters display a common structural profile with bendability being low in a region upstream of the transcriptional start point and significantly higher downstream. Investigation of the sequence composition in the two regions shows that the bendability profile originates from the sequential structure of the DNA, rather than the general nucleotide composition. Several trinucleotides known to have high propensity for major groove compression are found much more frequently in the regions downstream of the transcriptional start point, while the upstream regions contain more low-bendability triplets. Within the region downstream of the start point, we observe a periodic pattern in sequence and bendability, which is in phase with the DNA helical pitch. The periodic bendability profile shows bending peaks roughly at every 10 bp with stronger bending at 20 bp intervals. These observations suggest that DNA in the region downstream of the transcriptional start point is able to wrap around protein in a manner reminiscent of DNA in a nucleosome. This notion is further supported by the finding that the periodic bendability is caused mainly by the complementary triplet pairs CAG/CTG and GGC/GCC, which previously have been found to correlate with nucleosome positioning. We present models where the high-bendability regions position nucleosomes at the downstream end of the transcriptional start point, and consider the possibility of interaction between histone-like TAFs and this area. We also propose the use of this structural signature in computational promoter-finding algorithms.