Sequence Evidence in the Archaeal Genomes that tRNAs Emerged Through the Combination of Ancestral Genes as 5′ and 3′ tRNA Halves

The discovery of separate 5′ and 3′ halves of transfer RNA (tRNA) molecules—so-called split tRNA—in the archaeal parasite Nanoarchaeum equitans made us wonder whether ancestral tRNA was encoded on 1 or 2 genes. We performed a comprehensive phylogenetic analysis of tRNAs in 45 archaeal species to explore the relationship between the three types of tRNAs (nonintronic, intronic and split). We classified 1953 mature tRNA sequences into 22 clusters. All split tRNAs have shown phylogenetic relationships with other tRNAs possessing the same anticodon. We also mimicked split tRNA by artificially separating the tRNA sequences of 7 primitive archaeal species at the anticodon and analyzed the sequence similarity and diversity of the 5′ and 3′ tRNA halves. Network analysis revealed specific characteristics of and topological differences between the 5′ and 3′ tRNA halves: the 5′ half sequences were categorized into 6 distinct groups with a sequence similarity of >80%, while the 3′ half sequences were categorized into 9 groups with a higher sequence similarity of >88%, suggesting different evolutionary backgrounds of the 2 halves. Furthermore, the combinations of 5′ and 3′ halves corresponded with the variation of amino acids in the codon table. We found not only universally conserved combinations of 5′–3′ tRNA halves in tRNAiMet, tRNAThr, tRNAIle, tRNAGly, tRNAGln, tRNAGlu, tRNAAsp, tRNALys, tRNAArg and tRNALeu but also phylum-specific combinations in tRNAPro, tRNAAla, and tRNATrp. Our results support the idea that tRNA emerged through the combination of separate genes and explain the sequence diversity that arose during archaeal tRNA evolution

Nematode-specific tRNAs that decode an alternative genetic code for leucine

Class II transfer RNAs (tRNAs), including tRNALeu and tRNASer, have an additional stem and loop structure, the long variable arm (V-arm). Here, we describe Class II tRNAs with a unique anticodon corresponding to neither leucine nor serine. Because these tRNAs are specifically conserved among the nematodes, we have called them ‘nematode-specific V-arm-containing tRNAs’ (nev-tRNAs). The expression of nev-tRNA genes in Caenorhabditis elegans was confirmed experimentally. A comparative sequence analysis suggested that the nev-tRNAs derived phylogenetically from tRNALeu. In vitro aminoacylation assays showed that nev-tRNAGly and nev-tRNAIle are only charged with leucine, which is inconsistent with their anticodons. Furthermore, the deletion and mutation of crucial determinants for leucylation in nev-tRNA led to a marked loss of activity. An in vitro translation analysis showed that nev-tRNAGly decodes GGG as leucine instead of the universal glycine code, indicating that nev-tRNAs can be incorporated into ribosomes and participate in protein biosynthesis. Our findings provide the first example of unexpected tRNAs that do not consistently obey the general translation rules for higher eukaryotes

A novel three-unit tRNA splicing endonuclease found in ultrasmall Archaea possesses broad substrate specificity

tRNA splicing endonucleases, essential enzymes found in Archaea and Eukaryotes, are involved in the processing of pre-tRNA molecules. In Archaea, three types of splicing endonuclease [homotetrameric: α4, homodimeric: α2, and heterotetrameric: (αβ)2] have been identified, each representing different substrate specificity during the tRNA intron cleavage. Here, we discovered a fourth type of archaeal tRNA splicing endonuclease (ε2) in the genome of the acidophilic archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2 and its closely related species, ARMAN-1. The enzyme consists of two duplicated catalytic units and one structural unit encoded on a single gene, representing a novel three-unit architecture. Homodimeric formation was confirmed by cross-linking assay, and site-directed mutagenesis determined that the conserved L10-pocket interaction between catalytic and structural unit is necessary for the assembly. A tRNA splicing assay reveal that ε2 endonuclease cleaves both canonical and non-canonical bulge–helix–bulge motifs, similar to that of (αβ)2 endonuclease. Unlike other ARMAN and Euryarchaeota, tRNAs found in ARMAN-2 are highly disrupted by introns at various positions, which again resemble the properties of archaeal species with (αβ)2 endonuclease. Thus, the discovery of ε2 endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes

Genomic Heterogeneity in a Natural Archaeal Population Suggests a Model of tRNA Gene Disruption

Understanding the mechanistic basis of the disruption of tRNA genes, as manifested in the intron-containing and split tRNAs found in Archaea, will provide considerable insight into the evolution of the tRNA molecule. However, the evolutionary processes underlying these disruptions have not yet been identified. Previously, a composite genome of the deep-branching archaeon Caldiarchaeum subterraneum was reconstructed from a community genomic library prepared from a C. subterraneum–dominated microbial mat. Here, exploration of tRNA genes from the library reveals that there are at least three types of heterogeneity at the tRNAThr(GGU) gene locus in the Caldiarchaeum population. All three involve intronic gain and splitting of the tRNA gene. Of two fosmid clones found that encode tRNAThr(GGU), one (tRNAThr-I) contains a single intron, whereas another (tRNAThr-II) contains two introns. Notably, in the clone possessing tRNAThr-II, a 5′ fragment of the tRNAThr-I (tRNAThr-F) gene was observed 1.8-kb upstream of tRNAThr-II. The composite genome contains both tRNAThr-II and tRNAThr-F, although the loci are >500 kb apart. Given that the 1.8-kb sequence flanked by tRNAThr-F and tRNAThr-II is predicted to encode a DNA recombinase and occurs in six regions of the composite genome, it may be a transposable element. Furthermore, its dinucleotide composition is most similar to that of the pNOB8-type plasmid, which is known to integrate into archaeal tRNA genes. Based on these results, we propose that the gain of the tRNA intron and the scattering of the tRNA fragment occurred within a short time frame via the integration and recombination of a mobile genetic element

Genomic analysis of the symbiotic marine crenarchaeon, Cenarchaeumsymbiosum

Crenarchaea are ubiquitous and abundant microbial constituents of soils, sediments, lakes and ocean waters, yet relatively little is known about their fundamental evolutionary, ecological, and physiological properties. To better describe the ubiquitous nonthermophilic Crenarchaea, we analyzed the genome sequence of one representative, the uncultivated sponge symbiont, Cenarchaeum symbiosum. C. symbiosum genotypes coinhabiting the same host partitioned into two dominant populations, corresponding to previously described a- and b-type ribosomal RNA variants. Although synthetic, overlapping a- and b-type ribotypes harbored significant genetic variability. A single tiling path comprising the dominant a-type genotype was assembled, and used to explore the biological properties of C. symbiosum and its planktonic relatives. Out of a total of 2,066 predicted open reading frames, 36% were more highly conserved with other Archaea. The remainder partitioned between bacteria (18%), eukaryotes (1.5%) and viruses (0.1%). A total of 525 open reading frames were more highly conserved with sequences derived from marine environmental genomic surveys, most probably representing orthologous genes found in free-living planktonic Crenarchaea. The remaining genes partitioned between functional RNAs (2.4%), and hypotheticals (42%) with limited homology to known functional genes. The latter category likely contains genes specifically involved in mediated archaeal-sponge symbiosis. Phylogenetic analyses placed C. symbiosum as a basal crenarchaeon, sharing specific genomic features in common with either Crenarchaea, Euryarchaea, or both. The genome sequence of C. symbiosum reflect a unique and unusual evolutionary, physiological, and ecological history, one remarkably distinct from that of any other previously known microbial lineage

Proton Beam Therapy for Large Hepatocellular Carcinoma

PurposeTo investigate the safety and efficacy of proton beam therapy (PBT) in patients with large hepatocellular carcinoma (HCC).Methods and MaterialsTwenty-two patients with HCC larger than 10 cm were treated with proton beam therapy at our institution between 1985 and 2006. Twenty-one of the 22 patients were not surgical candidates because of advanced HCC, intercurrent disease, or old age. Median tumor size was 11 cm (range, 10–14cm), and median clinical target volume was 567 cm3 (range, 335–1,398 cm3). Hepatocellular carcinoma was solitary in 18 patients and multifocal in 4 patients. Tumor types were nodular and diffuse in 18 and 4 patients, respectively. Portal vein tumor thrombosis was present in 11 patients. Median total dose delivered was 72.6 GyE in 22 fractions (range, 47.3–89.1 GyE in 10–35 fractions).ResultsThe median follow-up period was 13.4 months (range, 1.5–85 months). Tumor control rate at 2 years was 87%. One-year overall and progression-free survival rates were 64% and 62%, respectively. Two-year overall and progression-free survival rates were 36% and 24%, respectively. The predominant tumor progression pattern was new hepatic tumor development outside the irradiated field. No late treatment-related toxicity of Grade 3 or higher was observed.ConclusionsThe Bragg peak properties of PBT allow for improved conformality of the treatment field. As such, large tumor volumes can be irradiated to high doses without significant dose exposure to surrounding normal tissue. Proton beam therapy therefore represents a promising modality for the treatment of large-volume HCC. Our study shows that PBT is an effective and safe method for the treatment of patients with large HCC

COSPAR Sample Safety Assessment Framework (SSAF)

The Committee on Space Research (COSPAR) Sample Safety Assessment Framework (SSAF) has been developed by a COSPAR appointed Working Group. The objective of the sample safety assessment would be to evaluate whether samples returned from Mars could be harmful for Earth's systems (e.g., environment, biosphere, geochemical cycles). During the Working Group's deliberations, it became clear that a comprehensive assessment to predict the effects of introducing life in new environments or ecologies is difficult and practically impossible, even for terrestrial life and certainly more so for unknown extraterrestrial life. To manage expectations, the scope of the SSAF was adjusted to evaluate only whether the presence of martian life can be excluded in samples returned from Mars. If the presence of martian life cannot be excluded, a Hold & Critical Review must be established to evaluate the risk management measures and decide on the next steps. The SSAF starts from a positive hypothesis (there is martian life in the samples), which is complementary to the null-hypothesis (there is no martian life in the samples) typically used for science. Testing the positive hypothesis includes four elements: (1) Bayesian statistics, (2) subsampling strategy, (3) test sequence, and (4) decision criteria. The test sequence capability covers self-replicating and non-self-replicating biology and biologically active molecules. Most of the investigations associated with the SSAF would need to be carried out within biological containment. The SSAF is described in sufficient detail to support planning activities for a Sample Receiving Facility (SRF) and for preparing science announcements, while at the same time acknowledging that further work is required before a detailed Sample Safety Assessment Protocol (SSAP) can be developed. The three major open issues to be addressed to optimize and implement the SSAF are (1) setting a value for the level of assurance to effectively exclude the presence of martian life in the samples, (2) carrying out an analogue test program, and (3) acquiring relevant contamination knowledge from all Mars Sample Return (MSR) flight and ground elements. Although the SSAF was developed specifically for assessing samples from Mars in the context of the currently planned NASA-ESA MSR Campaign, this framework and the basic safety approach are applicable to any other Mars sample return mission concept, with minor adjustments in the execution part related to the specific nature of the samples to be returned. The SSAF is also considered a sound basis for other COSPAR Planetary Protection Category V, restricted Earth return missions beyond Mars. It is anticipated that the SSAF will be subject to future review by the various MSR stakeholders

Disrupted tRNA gene diversity and possible evolutionary scenarios(International & Interdisciplinary Symposium on What is Evolution? Bicentennial of Charles Darwin's Birth)

この論文は国立情報学研究所の電子図書館事業により電子化されました