Reverse transcription of the pFOXC mitochondrial retroplasmids of Fusarium oxysporum is protein primed

BACKGROUND: The pFOXC retroplasmids are small, autonomously replicating DNA molecules found in mitochondria of certain strains of the filamentous fungus Fusarium oxysporum and are among the first linear genetic elements shown to replicate via reverse transcription. The plasmids have a unique clothespin structure that includes a 5'-linked protein and telomere-like terminal repeats, with pFOXC2 and pFOXC3 having iterative copies of a 5 bp sequence. The plasmids contain a single large open reading frame (ORF) encoding an active reverse transcriptase (RT). The pFOXC-RT is associated with the plasmid transcript in a ribonucleoprotein (RNP) complex and can synthesize full-length (-) strand cDNA products. In reactions containing partially purified RT preparations with exogenous RNAs, the pFOXC3-RT has been shown to initiate cDNA synthesis by use of snapped-back RNAs, as well as loosely associated DNA primers. RESULTS: The complete sequence of the distantly related pFOXC1 plasmid was determined and found to terminate in 3-5 copies of a 3 bp sequence. Unexpectedly, the majority of (-) strand cDNA molecules produced from endogenous pFOXC1 transcripts were attached to protein. In vitro experiments using partially purified pFOXC3-RT preparations having a single radiolabeled deoxyribonucleotide triphosphate (dNTP) generated a nucleotide-labeled protein that migrated at the size of the pFOXC-RT. The nucleotide preference of deoxynucleotidylation differed between pFOXC3 and pFOXC1 and showed complementarity to the respective 3' terminal repeats. In reactions that include exogenous RNA templates corresponding to the 3' end of pFOXC1, a protein-linked cDNA product was generated following deoxynucleotidylation, suggesting that reverse transcription initiates with a protein primer. CONCLUSIONS: The finding that reverse transcription is protein primed suggests the pFOXC retroplasmids may have an evolutionary relationship with hepadnaviruses, the only other retroelement family known to initiate reverse transcription via a protein primer. Moreover, the similarity to protein-primed linear DNA elements supports models in which the terminal repeats are generated and maintained by a DNA slideback mechanism. The ability of the pFOXC-RT to utilize RNA, DNA and protein primers is unique among polymerases and suggests that the pFOXC plasmids may be evolutionary precursors of a broad range of retroelements, including hepadnaviruses, non-long terminal repeat (non-LTR) retrotransposons and telomerase

The Genome of Nectria haematococca: Contribution of Supernumerary Chromosomes to Gene Expansion

The ascomycetous fungus Nectria haematococca, (asexual name Fusarium solani), is a member of a group of >50 species known as the “Fusarium solani species complex”. Members of this complex have diverse biological properties including the ability to cause disease on >100 genera of plants and opportunistic infections in humans. The current research analyzed the most extensively studied member of this complex, N. haematococca mating population VI (MPVI). Several genes controlling the ability of individual isolates of this species to colonize specific habitats are located on supernumerary chromosomes. Optical mapping revealed that the sequenced isolate has 17 chromosomes ranging from 530 kb to 6.52 Mb and that the physical size of the genome, 54.43 Mb, and the number of predicted genes, 15,707, are among the largest reported for ascomycetes. Two classes of genes have contributed to gene expansion: specific genes that are not found in other fungi including its closest sequenced relative, Fusarium graminearum; and genes that commonly occur as single copies in other fungi but are present as multiple copies in N. haematococca MPVI. Some of these additional genes appear to have resulted from gene duplication events, while others may have been acquired through horizontal gene transfer. The supernumerary nature of three chromosomes, 14, 15, and 17, was confirmed by their absence in pulsed field gel electrophoresis experiments of some isolates and by demonstrating that these isolates lacked chromosome-specific sequences found on the ends of these chromosomes. These supernumerary chromosomes contain more repeat sequences, are enriched in unique and duplicated genes, and have a lower G+C content in comparison to the other chromosomes. Although the origin(s) of the extra genes and the supernumerary chromosomes is not known, the gene expansion and its large genome size are consistent with this species' diverse range of habitats. Furthermore, the presence of unique genes on supernumerary chromosomes might account for individual isolates having different environmental niches

Tissue-Specific Target Analysis of Disease-Associated MicroRNAs in Human Signaling Pathways

MicroRNAs are a large class of post-transcriptional regulators that bind to the 3′ untranslated region of messenger RNAs. They play a critical role in many cellular processes and have been linked to the control of signal transduction pathways. Recent studies indicate that microRNAs can function as tumor suppressors or even as oncogenes when aberrantly expressed. For more general insights of disease-associated microRNAs, we analyzed their impact on human signaling pathways from two perspectives. On a global scale, we found a core set of signaling pathways with enriched tissue-specific microRNA targets across diseases. The function of these pathways reflects the affinity of microRNAs to regulate cellular processes associated with apoptosis, proliferation or development. Comparing cancer and non-cancer related microRNAs, we found no significant differences between both groups. To unveil the interaction and regulation of microRNAs on signaling pathways locally, we analyzed the cellular location and process type of disease-associated microRNA targets and proteins. While disease-associated proteins are highly enriched in extracellular components of the pathway, microRNA targets are preferentially located in the nucleus. Moreover, targets of disease-associated microRNAs preferentially exhibit an inhibitory effect within the pathways in contrast to disease proteins. Our analysis provides systematic insights into the interaction of disease-associated microRNAs and signaling pathways and uncovers differences in cellular locations and process types of microRNA targets and disease-associated proteins

Relaxed Primer Specificity Associated with Reverse Transcriptases Encoded by the pFOXC Retroplasmids of Fusarium oxysporum

The pFOXC mitochondrial retroplasmids are small, autonomously replicating linear DNAs that have a telomere-like repeat of a 5-bp sequence at their termini. The plasmids are possible evolutionary precursors of the ribonucleoprotein complex telomerase, as they encode an active reverse transcriptase (RT) that is involved in plasmid replication. Using an in vitro system to study reverse transcription, we show that the pFOXC RT is capable of copying in vitro-synthesized RNAs by use of cDNA primers or extension of snapped-back RNA templates. The ability of the pFOXC RT to use base-paired primers distinguishes it from the closely related RTs encoded by the Mauriceville and Varkud mitochondrial retroplasmids of Neurospora spp. Reaction products are similar, but not identical, to those obtained with conventional RTs, and differences reflect the ability of the pFOXC RT to initiate cDNA synthesis with loosely associated primers. The pFOXC RT can also copy DNA templates and extend 3′ mismatched DNA oligonucleotide primers. Analysis of pFOXC in vivo replication intermediates suggests that telomeric repeats are added during reverse transcription, and the ability to extend loosely associated primers could play a role in repeat formation by mechanisms similar to those associated with telomerase and certain non-long-terminal-repeat retrotransposons

Model-based Reactive Programming of Cooperative Vehicles for Mars Exploration

In the future webs of unmanned vehicles will act together to robustly achieve elaborate missions within uncertain environments. This web may be a distributed satellite system forming an interferometer, or may be a heterogenous set of rovers and blimps exploring Mars. We coordinate these systems by introducing a reactive model-based programming language (RMPL) that combines within a single unified representation the flexibility of embedded programming and reactive execution languages, and the deliberative reasoning power of temporal planners. To support fast mission planning as graph search, the KIRK planner compiles an RMPL program into a temporal plan network (TPN), similar to those used by temporal planners, but extended for symbolic constraints and decisions. To robustly coordinate air vehicle or rover maneuvers we combine the Kirk planning algorithm with randomized algorithms for kinodynamic path planning. Finally, we describe our Mars exploration testbed, including four RWI ATRV vehicles

Proteogenomics produces comprehensive and highly accurate protein-coding gene annotation in a complete genome assembly of Malassezia sympodialis

Complete and accurate genome assembly and annotation is a crucial foundation for comparative and functional genomics. Despite this, few complete eukaryotic genomes are available, and genome annotation remains a major challenge. Here, we present a complete genome assembly of the skin commensal yeast Malassezia sympodialis and demonstrate how proteogenomics can substantially improve gene an-notation. Through long-read DNA sequencing, we obtained a gap-free genome assembly for M. sympodi-alis (ATCC 42132), comprising eight nuclear and one mitochondrial chromosome. We also sequenced and assembled four M. sympodialis clinical isolates, and showed their value for understanding Malassezia reproduction by confirming four alternative allele combinations at the two mating-type loci. Importantly, we demonstrated how proteomics data could be readily integrated with transcriptomics data in standard annotation tools. This increased the number of annotated protein-coding genes by 14% (from 3612 to 4113), compared to using transcriptomics evidence alone. Manual curation further increased the number of protein-coding genes by 9% (to 4493). All of these genes have RNA-seq evidence and 87% were confirmed by proteomics. The M. sympodialis genome assembly and annotation presented here is at a quality yet achieved only for a few eukaryotic organisms, and constitutes an important reference for future host-microbe interaction studies

Music, Power, and Propaganda in Julio\u2010Claudian Imperial Rome (27 BC\u201368 AD)

Under the aegis of Apollo Citharoedus, Augustus undertook a program to restore public order and religious tradition that also employed music. The present article demonstrates how this transformation involved all the forms of expression and portrayal of the Empire, including public spectacles, technological development and representation of the emperor. Studies on a ritualized language of a mainly sacred sphere, on public spectacles and on individual musical instruments have shown a similar function of music and of its representation, aimed at favouring actions of control over society and of propaganda by the Empire and the single emperor. In continuity with several studies begun in the last fifteen years, the article sheds further light on the role of music in amplifying the cultural and political identity of leaderships based on the concept of pre-eminence