記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1016/j.jcoa.2024.100134

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

When a tumor is located in the medial (S7) or posterior (S10) basal segment, it is sometimes necessary to perform a bisegmentectomy of S7 and S10 (S7+S10) to achieve a sufficient surgical margin. In this report, we present surgical techniques for uniportal thoracoscopic S7+S10 bisegmentectomy. The surgical technique consists of anterior and posterior procedures to dissect the basal pulmonary vein. This approach allows en bloc resection of S7+S10 and en bloc preservation of the remaining lobe.

DOI： 10.1016/j.atssr.2023.08.003

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

RNA-binding proteins participate in diverse cellular processes, including DNA repair, post-transcriptional modification, and cancer progression through their interactions with RNAs, making them attractive for biotechnological applications. While nature provides an array of naturally occurring RNA-binding proteins, developing de novo RNA-binding peptides remains challenging. In particular, tailoring peptides to target single-stranded RNA with low complexity is difficult due to the inherent structural flexibility of RNA molecules. Here, we developed a codon-restricted mRNA display and identified multiple de novo peptides from a peptide library that bind to poly(C) and poly(A) RNA with KDs ranging from micromolar to submicromolar concentrations. One of the newly identified peptides is capable of binding to the cytosine-rich sequences of the oncogenic Cdk6 3'UTR RNA and MYU lncRNA, with affinity comparable to that of the endogenous binding protein. Hence, we present a novel platform for discovering de novo single-stranded RNA-binding peptides that offer promising avenues for regulating RNA functions.

DOI： 10.1021/acs.biomac.3c01024

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Polycyclic aromatic hydrocarbons (PAHs) contain ≲20% of the carbon in the interstellar medium. They are potentially produced in circumstellar environments (at temperatures ≳1000 kelvin), by reactions within cold (~10 kelvin) interstellar clouds, or by processing of carbon-rich dust grains. We report isotopic properties of PAHs extracted from samples of the asteroid Ryugu and the meteorite Murchison. The doubly-13C substituted compositions (Δ2×13C values) of the PAHs naphthalene, fluoranthene, and pyrene are 9 to 51‰ higher than values expected for a stochastic distribution of isotopes. The Δ2×13C values are higher than expected if the PAHs formed in a circumstellar environment, but consistent with formation in the interstellar medium. By contrast, the PAHs phenanthrene and anthracene in Ryugu samples have Δ2×13C values consistent with formation by higher-temperature reactions.

DOI： 10.1126/science.adg6304

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Amino acids in carbonaceous chondrites may have seeded the origin of life on Earth and possibly elsewhere. Recently, the return samples from a C-type asteroid Ryugu were found to contain amino acids with a similar distribution to Ivuna-type CI chondrites, suggesting the potential of amino acid abundances as molecular descriptors of parent body geochemistry. However, the chemical mechanisms responsible for the amino acid distributions remain to be elucidated particularly at low temperatures (<50°C). Here, we report that two representative proteinogenic amino acids, aspartic acid and glutamic acid, decompose to β-alanine and γ-aminobutyric acid, respectively, under simulated geoelectrochemical conditions at 25°C. This low-temperature conversion provides a plausible explanation for the enrichment of these two n-ω-amino acids compared to their precursors in heavily aqueously altered CI chondrites and Ryugu's return samples. The results suggest that these heavily aqueously altered samples originated from the water-rich mantle of their water/rock differentiated parent planetesimals where protein α-amino acids were decomposed.

DOI： 10.1126/sciadv.adh7845

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Fe-S clusters are essential cofactors mediating electron transfer in respiratory and metabolic networks. However, obtaining active [4Fe-4S] proteins with heterologous expression is challenging due to (i) the requirements for [4Fe-4S] cluster assembly, (ii) the O2 lability of [4Fe-4S] clusters, and (iii) copurification of undesired proteins (e.g., ferredoxins). Here, we established a facile and efficient protocol to express mature [4Fe-4S] proteins in the PURE system under aerobic conditions. An enzyme aconitase and thermophilic ferredoxin were selected as model [4Fe-4S] proteins for functional verification. We first reconstituted the SUF system in vitro via a stepwise manner using the recombinant SUF subunits (SufABCDSE) individually purified from E. coli. Later, the incorporation of recombinant SUF helper proteins into the PURE system enabled mRNA translation-coupled [4Fe-4S] cluster assembly under the O2-depleted conditions. To overcome the O2 lability of [4Fe-4S] Fe-S clusters, an O2-scavenging enzyme cascade was incorporated, which begins with formate oxidation by formate dehydrogenase for NADH regeneration. Later, NADH is consumed by flavin reductase for FADH2 regeneration. Finally, bifunctional flavin reductase, along with catalase, removes O2 from the reaction while supplying FADH2 to the SufBC2D complex. These amendments enabled a one-pot, two-step synthesis of mature [4Fe-4S] proteins under aerobic conditions, yielding holo-aconitase with a maximum concentration of ∼0.15 mg/mL. This renovated system greatly expands the potential of the PURE system, paving the way for the future reconstruction of redox-active synthetic cells and enhanced cell-free biocatalysis.

DOI： 10.1021/acssynbio.3c00155

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1038/s41467-023-40871-0

Web of Science

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Whereas modern proteins rely on a quasi-universal repertoire of 20 canonical amino acids (AAs), numerous lines of evidence suggest that ancient proteins relied on a limited alphabet of 10 "early" AAs and that the 10 "late" AAs were products of biosynthetic pathways. However, many nonproteinogenic AAs were also prebiotically available, which begs two fundamental questions: Why do we have the current modern amino acid alphabet and would proteins be able to fold into globular structures as well if different amino acids comprised the genetic code? Here, we experimentally evaluate the solubility and secondary structure propensities of several prebiotically relevant amino acids in the context of synthetic combinatorial 25-mer peptide libraries. The most prebiotically abundant linear aliphatic and basic residues were incorporated along with or in place of other early amino acids to explore these alternative sequence spaces. The results show that foldability was likely a critical factor in the selection of the canonical alphabet. Unbranched aliphatic amino acids were purged from the proteinogenic alphabet despite their high prebiotic abundance because they generate polypeptides that are oversolubilized and have low packing efficiency. Surprisingly, we find that the inclusion of a short-chain basic amino acid also decreases polypeptides' secondary structure potential, for which we suggest a biophysical model. Our results support the view that, despite lacking basic residues, the early canonical alphabet was remarkably adaptive at supporting protein folding and explain why basic residues were only incorporated at a later stage of protein evolution.

DOI： 10.1021/jacs.2c12987

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

<jats:p>Phosphate, an essential ingredient in fertilizers and detergents used daily worldwide, is a finite resource that may be exhausted within 70 years, while improper phosphate waste disposal into aquatic environments will result in eutrophication.</jats:p>

DOI： 10.1039/d3gc03162d

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The earliest proteins had to rely on amino acids available on early Earth before the biosynthetic pathways for more complex amino acids evolved. In extant proteins, a significant fraction of the 'late' amino acids (such as Arg, Lys, His, Cys, Trp and Tyr) belong to essential catalytic and structure-stabilizing residues. How (or if) early proteins could sustain an early biosphere has been a major puzzle. Here, we analysed two combinatorial protein libraries representing proxies of the available sequence space at two different evolutionary stages. The first is composed of the entire alphabet of 20 amino acids while the second one consists of only 10 residues (ASDGLIPTEV) representing a consensus view of plausibly available amino acids through prebiotic chemistry. We show that compact conformations resistant to proteolysis are surprisingly similarly abundant in both libraries. In addition, the early alphabet proteins are inherently more soluble and refoldable, independent of the general Hsp70 chaperone activity. By contrast, chaperones significantly increase the otherwise poor solubility of the modern alphabet proteins suggesting their coevolution with the amino acid repertoire. Our work indicates that while both early and modern amino acids are predisposed to supporting protein structure, they do so with different biophysical properties and via different mechanisms.

DOI： 10.1098/rsob.220040

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Tubulins are critical for the internal organization of eukaryotic cells, and understanding their emergence is an important question in eukaryogenesis. Asgard archaea are the closest known prokaryotic relatives to eukaryotes. Here, we elucidated the apo and nucleotide-bound x-ray structures of an Asgard tubulin from hydrothermal living Odinarchaeota (OdinTubulin). The guanosine 5'-triphosphate (GTP)-bound structure resembles a microtubule protofilament, with GTP bound between subunits, coordinating the "+" end subunit through a network of water molecules and unexpectedly by two cations. A water molecule is located suitable for GTP hydrolysis. Time course crystallography and electron microscopy revealed conformational changes on GTP hydrolysis. OdinTubulin forms tubules at high temperatures, with short curved protofilaments coiling around the tubule circumference, more similar to FtsZ, rather than running parallel to its length, as in microtubules. Thus, OdinTubulin represents an evolutionary stage intermediate between prokaryotic FtsZ and eukaryotic microtubule-forming tubulins.

DOI： 10.1126/sciadv.abm2225

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

RNA-peptide/protein interactions have been of utmost importance to life since its earliest forms, reaching even before the last universal common ancestor (LUCA). However, the ancient molecular mechanisms behind this key biological interaction remain enigmatic because extant RNA-protein interactions rely heavily on positively charged and aromatic amino acids that were absent (or heavily under-represented) in the early pre-LUCA evolutionary period. Here, an RNA-binding variant of the ribosomal uL11 C-terminal domain was selected from an approximately 1010 library of partially randomized sequences, all composed of ten prebiotically plausible canonical amino acids. The selected variant binds to the cognate RNA with a similar overall affinity although it is less structured in the unbound form than the wild-type protein domain. The variant complex association and dissociation are both slower than for the wild-type, implying different mechanistic processes involved. The profile of the wild-type and mutant complex stabilities along with molecular dynamics simulations uncovers qualitative differences in the interaction modes. In the absence of positively charged and aromatic residues, the mutant uL11 domain uses ion bridging (K+/Mg2+) interactions between the RNA sugar-phosphate backbone and glutamic acid residues as an alternative source of stabilization. This study presents experimental support to provide a new perspective on how early protein-RNA interactions evolved, where the lack of aromatic/basic residues may have been compensated by acidic residues plus metal ions.

DOI： 10.1093/molbev/msac032

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recent developments in Origins of Life research have focused on substantiating the narrative of an abiotic emergence of nucleic acids from organic molecules of low molecular weight, a paradigm that typically sidelines the roles of peptides. Nevertheless, the simple synthesis of amino acids, the facile nature of their activation and condensation, their ability to recognize metals and cofactors and their remarkable capacity to self-assemble make peptides (and their analogues) favourable candidates for one of the earliest functional polymers. In this mini-review, we explore the ramifications of this hypothesis. Diverse lines of research in molecular biology, bioinformatics, geochemistry, biophysics and astrobiology provide clues about the progression and early evolution of proteins, and lend credence to the idea that early peptides served many central prebiotic roles before they were encodable by a polynucleotide template, in a putative 'peptide-polynucleotide stage'. For example, early peptides and mini-proteins could have served as catalysts, compartments and structural hubs. In sum, we shed light on the role of early peptides and small proteins before and during the nucleotide world, in which nascent life fully grasped the potential of primordial proteins, and which has left an imprint on the idiosyncratic properties of extant proteins.

DOI： 10.1098/rsif.2021.0641

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

One goal of origins of life research is to understand how primitive informational and catalytic biopolymers emerged and evolved. Recently, a number of sequencing techniques have been applied to analysis of replicating and evolving primitive biopolymer systems, providing a sequence-specific and high-resolution view of primitive chemical processes. Here, we review application of sequencing techniques to analysis of synthetic and primitive nucleic acids and polypeptides. This includes next-generation sequencing of primitive polymerization and evolution processes, followed by discussion of other novel biochemical techniques that could contribute to sequence analysis of primitive biopolymer driven chemical systems. Further application of sequencing to origins of life research, perhaps as a life detection technology, could provide insight into the origin and evolution of informational and catalytic biopolymers on early Earth or elsewhere.

DOI： 10.1016/j.bbadva.2022.100049

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

MOTIVATION: Current techniques of protein engineering focus mostly on re-designing small targeted regions or defined structural scaffolds rather than constructing combinatorial libraries of versatile compositions and lengths. This is a missed opportunity because combinatorial libraries are emerging as a vital source of novel functional proteins and are of interest in diverse research areas. RESULTS: Here, we present a computational tool for Combinatorial Library Design (CoLiDe) offering precise control over protein sequence composition, length and diversity. The algorithm uses evolutionary approach to provide solutions to combinatorial libraries of degenerate DNA templates. We demonstrate its performance and precision using four different input alphabet distribution on different sequence lengths. In addition, a model design and experimental pipeline for protein library expression and purification is presented, providing a proof-of-concept that our protocol can be used to prepare purified protein library samples of up to 1011-1012 unique sequences. CoLiDe presents a composition-centric approach to protein design towards different functional phenomena. AVAILABILITYAND IMPLEMENTATION: CoLiDe is implemented in Python and freely available at https://github.com/voracva1/CoLiDe. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

DOI： 10.1093/bioinformatics/btaa804

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The reconstructed in vitro translation system known as the PURE system has been used in a variety of cell-free experiments such as the expression of native and de novo proteins as well as various display methods to select for functional polypeptides. We developed a refined PURE-based display method for the preparation of stable messenger RNA (mRNA) and complementary DNA (cDNA)-peptide conjugates and validated its utility for in vitro selection. Our conjugate formation efficiency exceeded 40%, followed by gel purification to allow minimum carry-over of components from the translation system to the downstream assay enabling clean and efficient random peptide sequence screening. We chose the commercially available anti-FLAG M2 antibody as a target molecule for validation. Starting from approximately 1.7 × 1012 random sequences, a round-by-round high-throughput sequencing showed clear enrichment of the FLAG epitope DYKDDD as well as revealing consensus FLAG epitope motif DYK(D/L/N)(L/Y/D/N/F)D. Enrichment of core FLAG motifs lacking one of the four key residues (DYKxxD) indicates that Tyr (Y) and Lys (K) appear as the two key residues essential for binding. Furthermore, the comparison between mRNA display and cDNA display method resulted in overall similar performance with slightly higher enrichment for mRNA display. We also show that gel purification steps in the refined PURE-based display method improve conjugate formation efficiency and enhance the enrichment rate of FLAG epitope motifs in later rounds of selection especially for mRNA display. Overall, the generalized procedure and consistent performance of two different display methods achieved by the commercially available PURE system will be useful for future studies to explore the sequence and functional space of diverse polypeptides.

DOI： 10.1002/bit.27696

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Reconstituted cell-free protein synthesis systems (e.g., the PURE system) allow the expression of toxic proteins, hetero-oligomeric protein subunits, and proteins with noncanonical amino acids with high levels of homogeneity. In these systems, an artificial ATP/GTP regeneration system is required to drive protein synthesis, which is accomplished using three kinases and phosphocreatine. Here, we demonstrate the replacement of these three kinases with one bifunctional Cytophaga hutchinsonii polyphosphate kinase that phosphorylates nucleosides in an exchange reaction from polyphosphate. The optimized single-kinase system produced a final sfGFP concentration (∼530 μg/mL) beyond that of the three-kinase system (∼400 μg/mL), with a 5-fold faster mRNA translation rate in the first 90 min. The single-kinase system is also compatible with the expression of heat-sensitive firefly luciferase at 37 °C. Potentially, the single-kinase nucleoside triphosphate regeneration approach developed herein could expand future applications of cell-free protein synthesis systems and could be used to drive other biochemical processes in synthetic biology which require both ATP and GTP.

DOI： 10.1021/acssynbio.9b00456

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

<jats:title>Abstract</jats:title><jats:p>The <jats:italic>in situ</jats:italic> detection of organic molecules in space is key to understanding the variety and the distribution of the building blocks of life, and possibly the detection of extraterrestrial life itself. Gas chromatography mass spectrometry (GC-MS) has been the most sensitive analytical strategy for organic analyses in flight, and was used on missions from NASA's Viking, Phoenix, Curiosity missions to ESA's Rosetta space probe. While pyrolysis GC-MS revealed the first organics on Mars, this step alters or degrades certain fragile molecules that are excellent biosignatures including polypeptides, oligonucleotides and polysaccharides, rendering the intact precursors undetectable. We have identified a solution tailored to the detection of biopolymers and other biomarkers by the use of liquid-based capillary electrophoresis and electrochromatography. In this study, we show that a capillary electrochromatography approach using monolithic stationary phases with tailor-made surface chemistry can separate and identify various polycyclic aromatic hydrocarbons, nucleobases and aromatic acids that could be formed under astrophysically relevant conditions. In order to simulate flyby organic sample capture, we conducted hypervelocity impact experiments which consisted of accelerating peptide-soaked montmorillonite particles to a speed of 5.6 km s<jats:sup>−1</jats:sup>, and capturing them in an amorphous silica aerogel of 10 mg cm<jats:sup>−3</jats:sup> bulk density. Bulk peptide extraction from aerogel followed by capillary zone electrophoresis led to the detection of only two stereoisomeric peptide peaks. The recovery rates of each step of the extraction procedure after the hypervelocity impact suggest that major peptide loss occurred during the impact. Our study provides initial exploration of feasibility of this approach for capturing intact peptides, and subsequently detecting candidate biomolecules during flight missions that would be missed by GC-MS alone. As the monolith-based electrochromatography technology could be customized to detect specific classes of compounds as well as miniaturized, these results demonstrate the potential of the instrumentation for future astrobiology-related spaceflight missions.</jats:p>

DOI： 10.1017/s1473550419000065

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DOI： 10.1021/acsearthspacechem.9b00108

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Electronics waste production has been fueled by economic growth and the demand for faster, more efficient consumer electronics. The glass and metals in end-of-life electronics components can be reused or recycled; however, conventional extraction methods rely on energy-intensive processes that are inefficient when applied to recycling e-waste that contains mixed materials and small amounts of metals. To make e-waste recycling economically viable and competitive with obtaining raw materials, recovery methods that lower the cost of metal reclamation and minimize environmental impact need to be developed. Microbial surface adsorption can aid in metal recovery with lower costs and energy requirements than traditional metal-extraction approaches. We introduce a novel method for metal recovery by utilizing metal-binding peptides to functionalize fungal mycelia and enhance metal recovery from aqueous solutions such as those found in bioremediation or biomining processes. Using copper-binding as a proof-of-concept, we compared binding parameters between natural motifs and those derived in silico, and found comparable binding affinity and specificity for Cu. We then combined metal-binding peptides with chitin-binding domains to functionalize a mycelium-based filter to enhance metal recovery from a Cu-rich solution. This finding suggests that engineered peptides could be used to functionalize biological surfaces to recover metals of economic interest and allow for metal recovery from metal-rich effluent with a low environmental footprint, at ambient temperatures, and under circumneutral pH.

DOI： 10.1038/s41598-019-52778-2

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

DNA is an attractive candidate for integration into nanoelectronics as a biological nanowire due to its linear geometry, definable base sequence, easy, inexpensive and non-toxic replication and self-assembling properties. Recently we discovered that by intercalating Ag+ in polycytosine-mismatch oligonucleotides, the resulting C-Ag+-C duplexes are able to conduct charge efficiently. To map the functionality and biostability of this system, we built and characterized internally-functionalized DNA nanowires through non-canonical, Ag+-mediated base pairing in duplexes containing cytosine-cytosine mismatches. We assessed the thermal and chemical stability of ion-coordinated duplexes in aqueous solutions and conclude that the C-Ag+-C bond forms DNA duplexes with replicable geometry, predictable thermodynamics, and tunable length. We demonstrated continuous ion chain formation in oligonucleotides of 11-50 nucleotides (nt), and enzyme ligation of mixed strands up to six times that length. This construction is feasible without detectable silver nanocluster contaminants. Functional gene parts for the synthesis of DNA- and RNA-based, C-Ag+-C duplexes in a cell-free system have been constructed in an Escherichia coli expression plasmid and added to the open-source BioBrick Registry, paving the way to realizing the promise of inexpensive industrial production. With appropriate design constraints, this conductive variant of DNA demonstrates promise for use in synthetic biological constructs as a dynamic nucleic acid component and contributes molecular electronic functionality to DNA that is not already found in nature. We propose a viable route to fabricating stable DNA nanowires in cell-free and synthetic biological systems for the production of self-assembling nanoelectronic architectures.

DOI： 10.1038/s41598-019-43316-1

PubMed

researchmap

記述言語：英語  

A correction to this article has been published and is linked from the HTML and PDF versions of this paper. The error has not been fixed in the paper.

DOI： 10.1038/s41598-019-42196-9

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Four different types (α4, α'2, (αβ)2 and ϵ2) of RNA-splicing endonucleases (EndAs) for RNA processing are known to exist in the Archaea. Only the (αβ)2 and ϵ2 types can cleave non-canonical introns in precursor (pre)-tRNA. Both enzyme types possess an insert associated with a specific loop, allowing broad substrate specificity in the catalytic α units. Here, the hyperthermophilic euryarchaeon Methanopyrus kandleri (MKA) was predicted to harbor an (αβ)2-type EndA lacking the specific loop. To characterize MKA EndA enzymatic activity, we constructed a fusion protein derived from MKA α and β subunits (fMKA EndA). In vitro assessment demonstrated complete removal of the canonical bulge-helix-bulge (BHB) intron structure from MKA pre-tRNAAsn. However, removal of the relaxed BHB structure in MKA pre-tRNAGlu was inefficient compared to crenarchaeal (αβ)2 EndA, and the ability to process the relaxed intron within mini-helix RNA was not detected. fMKA EndA X-ray structure revealed a shape similar to that of other EndA types, with no specific loop. Mapping of EndA types and their specific loops and the tRNA gene diversity among various Archaea suggest that MKA EndA is evolutionarily related to other (αβ)2-type EndAs found in the Thaumarchaeota, Crenarchaeota and Aigarchaeota but uniquely represents constrained substrate specificity.

DOI： 10.1093/nar/gky003

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Theories of the origin of the genetic code typically appeal to natural selection and/or mutation of hereditable traits to explain its regularities and error robustness, yet the present translation system presupposes high-fidelity replication. Woese's solution to this bootstrapping problem was to assume that code optimization had played a key role in reducing the effect of errors caused by the early translation system. He further conjectured that initially evolution was dominated by horizontal exchange of cellular components among loosely organized protocells ("progenotes"), rather than by vertical transmission of genes. Here we simulated such communal evolution based on horizontal transfer of code fragments, possibly involving pairs of tRNAs and their cognate aminoacyl tRNA synthetases or a precursor tRNA ribozyme capable of catalysing its own aminoacylation, by using an iterated learning model. This is the first model to confirm Woese's conjecture that regularity, optimality, and (near) universality could have emerged via horizontal interactions alone.

DOI： 10.1038/s41598-018-21973-y

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Amino acid biosynthesis pathways observed in nature typically require enzymes that are made with the amino acids they produce. For example, Escherichia coli produces cysteine from serine via two enzymes that contain cysteine: serine acetyltransferase (CysE) and O-acetylserine sulfhydrylase (CysK/CysM). To solve this chicken-and-egg problem, we substituted alternate amino acids in CysE, CysK and CysM for cysteine and methionine, which are the only two sulfur-containing proteinogenic amino acids. Using a cysteine-dependent auxotrophic E. coli strain, CysE function was rescued by cysteine-free and methionine-deficient enzymes, and CysM function was rescued by cysteine-free enzymes. CysK function, however, was not rescued in either case. Enzymatic assays showed that the enzymes responsible for rescuing the function in CysE and CysM also retained their activities in vitro. Additionally, substitution of the two highly conserved methionines in CysM decreased but did not eliminate overall activity. Engineering amino acid biosynthetic enzymes to lack the so-produced amino acids can provide insights into, and perhaps eventually fully recapitulate via a synthetic approach, the biogenesis of biotic amino acids.

DOI： 10.1038/s41598-018-19920-y

PubMed

researchmap

掲載種別：研究論文（学術雑誌）  

DOI： 10.3390/life7040039

researchmap

掲載種別：研究論文（学術雑誌）   出版者・発行元：Cambridge University Press ({CUP})  

DOI： 10.1017/s1473550416000458

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Desiccation resistance and a high intracellular Mn/Fe ratio contribute to ionizing radiation resistance of Deinococcus radiodurans. We hypothesized that this was a general phenomenon and thus developed a strategy to search for highly radiation-resistant organisms based on their natural environment. While desiccation is a typical feature of deserts, the correlation between radiation resistance and the intracellular Mn/Fe ratio of indigenous microorganisms or the Mn/Fe ratio of the environment, has not yet been described. UV-C radiation is highly damaging to biomolecules including DNA. It was used in this study as a selective tool because of its relevance to early life on earth, high altitude aerobiology and the search for life beyond Earth. Surface soil samples were collected from the Sonoran Desert, Arizona (USA), from the Atacama Desert in Chile and from a manganese mine in northern Argentina. Microbial isolates were selected after exposure to UV-C irradiation and growth. The isolates comprised 28 genera grouped within six phyla, which we ranked according to their resistance to UV-C irradiation. Survival curves were performed for the most resistant isolates and correlated with their intracellular Mn/Fe ratio, which was determined by ICP-MS. Five percent of the isolates were highly resistant, including one more resistant than D. radiodurans, a bacterium generally considered the most radiation-resistant organism, thus used as a model for radiation resistance studies. No correlation was observed between the occurrence of resistant microorganisms and the Mn/Fe ratio in the soil samples. However, all resistant isolates showed an intracellular Mn/Fe ratio much higher than the sensitive isolates. Our findings could represent a new front in efforts to harness mechanisms of UV-C radiation resistance from extreme environments.

DOI： 10.1016/j.jphotobiol.2016.08.017

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Here, we report the 5.09-Mb draft genome sequence of Hymenobacter sp. strain AT01-02, which was isolated from a surface soil sample in the Atacama Desert, Chile. The isolate is extremely resistant to UV-C radiation and is able to accumulate high intracellular levels of Mn/Fe.

DOI： 10.1128/genomeA.01701-15

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We present an overhang-based DNA block shuffling method to create a customized random DNA library with flexible sequence design and length. Our method enables the efficient and seamless assembly of short DNA blocks with dinucleotide overhangs through a simple ligation process. Next generation sequencing analysis of the assembled DNA library revealed that ligation was accurate, directional and unbiased. This straightforward DNA assembly method should fulfill the versatile needs of both in vivo and in vitro functional screening of random peptides and RNA created with a desired amino acid and nucleotide composition, as well as making highly repetitive gene constructs that are difficult to synthesize de novo.

DOI： 10.1038/srep09740

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

MicroRNAs have been identified and analyzed in various model species, but an investigation of miRNAs in nonmodel species is required for a more complete understanding of miRNA evolution. In this study, we investigated the miRNAs of the nonmodel species Triops cancriformis (tadpole shrimp), a "living fossil," whose morphological form has not changed in almost 200 million years. Dramatic ontogenetic changes occur during its development. To clarify the evolution of miRNAs, we comparatively analyzed its miRNAs and the components of its RNAi machinery. We used deep sequencing to analyze small RNA libraries from the six different developmental stages of T. cancriformis (egg, first-fourth instars, and adult), and also analyzed its genomic DNA with deep sequencing. We identified 180 miRNAs (87 conserved miRNAs and 93 novel candidate miRNAs), and deduced the components of its RNAi machinery: the DICER1, AGO1-3, PIWI, and AUB proteins. A comparative miRNA analysis of T. cancriformis and Drosophila melanogaster showed inconsistencies in the expression patterns of four conserved miRNAs. This suggests that although the miRNA sequences of the two species are very similar, their roles differ across the species. An miRNA conservation analysis revealed that most of the conserved T. cancriformis miRNAs share sequence similarities with those of arthropods, although T. cancriformis is called a "living fossil." However, we found that let-7 and DICER1 of T. cancriformis are more similar to those of the vertebrates than to those of the arthropods. These results suggest that miRNA systems of T. cancriformis have evolved in a unique fashion.

DOI： 10.1261/rna.045799.114

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Transfer RNA (tRNA) is widely known for its key role in decoding mRNA into protein. Despite their necessity and relatively short nucleotide sequences, a large diversity of gene structures and RNA secondary structures of pre-tRNAs and mature tRNAs have recently been discovered in the three domains of life. Growing evidences of disrupted tRNA genes in the genomes of Archaea reveals unique gene structures such as, intron-containing tRNA, split tRNA, and permuted tRNA. Coding sequence for these tRNAs are either separated with introns, fragmented, or permuted at the genome level. Although evolutionary scenario behind the tRNA gene disruption is still unclear, diversity of tRNA structure seems to be co-evolved with their processing enzyme, so-called RNA splicing endonuclease. Metazoan mitochondrial tRNAs (mtRNAs) are known for their unique lack of either one or two arms from the typical tRNA cloverleaf structure, while still maintaining functionality. Recently identified nematode-specific V-arm containing tRNAs (nev-tRNAs) possess long variable arms that are specific to eukaryotic class II tRNA(Ser) and tRNA(Leu) but also decode class I tRNA codons. Moreover, many tRNA-like sequences have been found in the genomes of different organisms and viruses. Thus, this review is aimed to cover the latest knowledge on tRNA gene diversity and further recapitulate the evolutionary and biological aspects that caused such uniqueness.

DOI： 10.3389/fgene.2014.00142

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Cleavage of introns from precursor transfer RNAs (tRNAs) by tRNA splicing endonuclease (EndA) is essential for tRNA maturation in Archaea and Eukarya. In the past, archaeal EndAs were classified into three types (α'2, α4 and α2β2) according to subunit composition. Recently, we have identified a fourth type of archaeal EndA from an uncultivated archaeon Candidatus Micrarchaeum acidiphilum, referred to as ARMAN-2, which is deeply branched within Euryarchaea. The ARMAN-2 EndA forms an ε2 homodimer and has broad substrate specificity like the α2β2 type EndAs found in Crenarchaea and Nanoarchaea. However, the precise architecture of ARMAN-2 EndA was unknown. Here, we report the crystal structure of the ε2 homodimer of ARMAN-2 EndA. The structure reveals that the ε protomer is separated into three novel units (αN, α and βC) fused by two distinct linkers, although the overall structure of ARMAN-2 EndA is similar to those of the other three types of archaeal EndAs. Structural comparison and mutational analyses reveal that an ARMAN-2 type-specific loop (ASL) is involved in the broad substrate specificity and that K161 in the ASL functions as the RNA recognition site. These findings suggest that the broad substrate specificities of ε2 and α2β2 EndAs were separately acquired through different evolutionary processes.

DOI： 10.1093/nar/gks826

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Class II transfer RNAs (tRNAs), including tRNA(Leu) and tRNA(Ser), 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 tRNA(Leu). In vitro aminoacylation assays showed that nev-tRNA(Gly) and nev-tRNA(Ile) 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-tRNA(Gly) 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.

DOI： 10.1093/nar/gkr1226

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Studies of small noncoding RNAs (sRNAs) have been conducted predominantly using culturable organisms, and the acquisition of further information about sRNAs from global environments containing uncultured organisms now is very important. In this study, hot spring water (57°C, pH 8.1) was collected directly from the underground environment at depths of 250 to 1,000 m in Yunohama, Japan, and small RNA sequences obtained from the environment were analyzed. A phylogenetic analysis of both archaeal and bacterial 16S rRNA gene sequences was conducted, and the results suggested the presence of unique species in the environment, corresponding to the Archaeal Richmond Mine Acidophilic Nanoorganisms (ARMAN) group and three new Betaproteobacteria. A metatranscriptomic analysis identified 64,194 (20,057 nonredundant) cDNA sequences. Of these cDNAs, 90% were either tRNAs, tRNA fragments, rRNAs, or rRNA fragments, whereas 2,181 reads (10%) were classified as previously uncharacterized putative candidate sRNAs. Among these, 15 were particularly abundant, 14 of which showed no sequence similarity to any known noncoding RNA, and at least six of which form very stable RNA secondary structures. The analysis of a large number of tRNA fragments suggested that unique relationships exist between the anticodons of the tRNAs and the sites of tRNA degradation. Previous bacterial tRNA degradation studies have been limited to specific organisms, such as Escherichia coli and Streptomyces coelicolor, and the current results suggest that specific tRNA decay occurs more frequently than previously expected.

DOI： 10.1128/AEM.06811-11

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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 tRNA(Thr)(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 tRNA(Thr)(GGU), one (tRNA(Thr-I)) contains a single intron, whereas another (tRNA(Thr-II)) contains two introns. Notably, in the clone possessing tRNA(Thr-II), a 5' fragment of the tRNA(Thr-I) (tRNA(Thr-F)) gene was observed 1.8-kb upstream of tRNA(Thr-II). The composite genome contains both tRNA(Thr-II) and tRNA(Thr-F), although the loci are >500 kb apart. Given that the 1.8-kb sequence flanked by tRNA(Thr-F) and tRNA(Thr-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.

DOI： 10.1371/journal.pone.0032504

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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: α(4), homodimeric: α(2), 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 (ε(2)) 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 ε(2) 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 ε(2) endonuclease in an archaeon deeply branched within Euryarchaeota represents a new example of the coevolution of tRNA and their processing enzymes.

DOI： 10.1093/nar/gkr692

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We have developed a screening system for artificial small RNAs (sRNAs) that inhibit the growth of Escherichia coli. In this system, we used a plasmid library to express artificial sRNAs (approximately 200 bases long) containing 60 bases of random nucleotide sequence. The induced expression of the known rydB sRNA in the system reduced the amount of its possible target mRNA, rpoS, supporting the reliability of the method. To isolate clones of sRNAs that inhibited the growth of E. coli, we used two successive screening steps: (i) colony size selection on plates and (ii) monitoring E. coli growth in a 96-well plate format. As a result, 83 artificial sRNAs were identified that showed a range of inhibitory effects on bacterial growth. We also introduced nucleotide replacements into one of the highly inhibitory sRNA clones, H12, which partially abolished the inhibition of bacterial growth, suggesting that bacterial growth was inhibited in a sequence-specific manner.

DOI： 10.1093/jb/mvr055

PubMed

researchmap

掲載種別：研究論文（学術雑誌）   出版者・発行元：Archaea: Structure, Habitats and Ecological Significance  

Scopus

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Recently, diverse arrangements of transfer RNA (tRNA) genes have been found in the domain Archaea, in which the tRNA is interrupted by a maximum of three introns or is even fragmented into two or three genes. Whereas most of the eukaryotic tRNA introns are inserted strictly at the canonical nucleotide position (37/38), archaeal intron-containing tRNAs have a wide diversity of small tRNA introns, which differ in their numbers and locations. This feature is especially pronounced in the archaeal order Thermoproteales. In this study, we performed a comprehensive sequence comparison of 286 tRNA introns and their genes in seven Thermoproteales species to clarify how these introns have emerged and diversified during tRNA gene evolution. We identified 46 intron groups containing sets of highly similar sequences (>70%) and showed that 16 of them contain sequences from evolutionarily distinct tRNA genes. The phylogeny of these 16 intron groups indicates that transposition events have occurred at least seven times throughout the evolution of Thermoproteales. These findings suggest that frequent intron transposition occurs among the tRNA genes of Thermoproteales. Further computational analysis revealed limited insertion positions and corresponding amino acid types of tRNA genes. This has arisen because the bulge-helix-bulge splicing motif is required at the newly transposed position if the pre-tRNA is to be correctly processed. These results clearly demonstrate a newly identified mechanism that facilitates the late gain of short introns at various noncanonical positions in archaeal tRNAs.

DOI： 10.1093/molbev/msq111

PubMed

researchmap

記述言語：日本語   掲載種別：研究論文（学術雑誌）  

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

RNase H (ribonuclease H) is an endonuclease that cleaves the RNA strand of RNA-DNA duplexes. It has been reported that the three-dimensional structure of RNase H is similar to that of the PIWI domain of the Pyrococcus furiosus Ago (argonaute) protein, although the two enzymes share almost no similarity in their amino acid sequences. Eukaryotic Ago proteins are key components of the RNA-induced silencing complex and are involved in microRNA or siRNA (small interfering RNA) recognition. In contrast, prokaryotic Ago proteins show greater affinity for RNA-DNA hybrids than for RNA-RNA hybrids. Interestingly, we found that wild-type Pf-RNase HII (P. furiosus, RNase HII) digests RNA-RNA duplexes in the presence of Mn2+ ions. To characterize the substrate specificity of Pf-RNase HII, we aligned the amino acid sequences of Pf-RNase HII and Pf-Ago, based on their protein secondary structures. We found that one of the conserved secondary structural regions (the fourth beta-sheet and the fifth alpha-helix of Pf-RNase HII) contains family-specific amino acid residues. Using a series of Pf-RNase HII-Pf-Ago chimaeric mutants of the region, we discovered that residues Asp110, Arg113 and Phe114 are responsible for the dsRNA (double-stranded RNA) digestion activity of Pf-RNase HII. On the basis of the reported three-dimensional structure of Ph-RNase HII from Pyrococcus horikoshii, we built a three-dimensional structural model of RNase HII complexed with its substrate, which suggests that these amino acids are located in the region that discriminates DNA from RNA in the non-substrate strand of the duplexes.

DOI： 10.1042/BJ20091553

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

BACKGROUND: In many eukaryotes, microRNAs (miRNAs) bind to complementary sites in the 3'-untranslated regions (3'-UTRs) of target messenger RNAs (mRNAs) and regulate their expression at the stage of translation. Recent studies have revealed that many miRNAs are evolutionarily conserved; however, the evolution of their target genes has yet to be systematically characterized. We sought to elucidate a set of conserved miRNA/target-gene pairs and to analyse the mechanism underlying miRNA-mediated gene regulation in the early stage of bilaterian evolution. RESULTS: Initially, we extracted five evolutionarily conserved miRNAs (let-7, miR-1, miR-124, miR-125/lin-4, and miR-34) among five diverse bilaterian animals. Subsequently, we designed a procedure to predict evolutionarily conserved miRNA/target-gene pairs by introducing orthologous gene information. As a result, we extracted 31 orthologous miRNA/target-gene pairs that were conserved among at least four diverse bilaterian animals; the prediction set showed prominent enrichment of orthologous miRNA/target-gene pairs that were verified experimentally. Approximately 84% of the target genes were regulated by three miRNAs (let-7, miR-1, and miR-124) and their function was classified mainly into the following categories: development, muscle formation, cell adhesion, and gene regulation. We used a reporter gene assay to experimentally verify the downregulation of six candidate pairs (out of six tested pairs) in HeLa cells. CONCLUSIONS: The application of our new method enables the identification of 31 miRNA/target-gene pairs that were expected to have been regulated from the era of the common bilaterian ancestor. The downregulation of all six candidate pairs suggests that orthologous information contributed to the elucidation of the primordial set of genes that has been regulated by miRNAs; it was also an efficient tool for the elimination of false positives from the predicted candidates. In conclusion, our study identified potentially important miRNA-target pairs that were evolutionarily conserved throughout diverse bilaterian animals and that may provide new insights into early-stage miRNA functions.

DOI： 10.1186/1471-2164-11-101

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The following unusual tRNAs have recently been discovered in the genomes of Archaea and primitive Eukaryota: multiple-intron-containing tRNAs, which have more than one intron; split tRNAs, which are produced from two pieces of RNA transcribed from separate genes; tri-split tRNAs, which are produced from three separate genes; and permuted tRNA, in which the 5' and 3' halves are encoded with permuted orientations within a single gene. All these disrupted tRNA genes can form mature contiguous tRNA, which is aminoacylated after processing by cis or trans splicing. The discovery of such tRNA disruptions has raised the question of when and why these complex tRNA processing pathways emerged during the evolution of life. Many previous reports have noted that tRNA genes contain a single intron in the anticodon loop region, a feature common throughout all three domains of life, suggesting an ancient trait of the last universal common ancestor. In this context, these unique tRNA disruptions recently found only in Archaea and primitive Eukaryota provide new insight into the origin and evolution of tRNA genes, encouraging further research in this field. In this paper, we summarize the phylogeny, structure, and processing machinery of all known types of disrupted tRNAs and discuss possible evolutionary scenarios for these tRNA genes.

DOI： 10.1007/s00239-009-9294-6

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Transfer RNA (tRNA) is essential for decoding the genome sequence into proteins. In Archaea, previous studies have revealed unique multiple intron-containing tRNAs and tRNAs that are encoded on 2 separate genes, so-called split tRNAs. Here, we discovered 10 fragmented tRNA genes in the complete genome of the hyperthermoacidophilic Archaeon Caldivirga maquilingensis that are individually transcribed and further trans-spliced to generate all of the missing tRNAs encoding glycine, alanine, and glutamate. Notably, the 3 mature tRNA(Gly)'s with synonymous codons are created from 1 constitutive 3' half transcript and 4 alternatively switching transcripts, representing tRNA made from a total of 3 transcripts named a "tri-split tRNA." Expression and nucleotide sequences of 10 split tRNA genes and their joined tRNA products were experimentally verified. The intervening sequences of split tRNA have high identity to tRNA intron sequences located at the same positions in intron-containing tRNAs in related Thermoproteales species. This suggests that an evolutionary relationship between intron-containing and split tRNAs exists. Our findings demonstrate the first example of split tRNA genes in a free-living organism and a unique tri-split tRNA gene that provides further insight into the evolution of fragmented tRNAs.

DOI： 10.1073/pnas.0808246106

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

The analysis of archaeal tRNA genes is becoming more important to evaluate the origin and evolution of tRNA molecule. Even with the recent accumulation of complete genomes of numerous archaeal species, several tRNA genes are still required for a full complement of the codon table. We conducted comprehensive screening of tRNA genes from 47 archaeal genomes by using a combination of different types of tRNA prediction programs and extracted a total of 2,143 reliable tRNA gene candidates including 437 intron-containing tRNA genes, which covered more than 99.9% of the codon tables in Archaea. Previously, the content of intron-containing tRNA genes in Archaea was estimated to be approximately 15% of the whole tRNA genes, and most of the introns were known to be located at canonical positions (nucleotide position between 37 and 38) of precursor tRNA (pre-tRNA). Surprisingly, we observed marked enrichment of tRNA introns in five species of the archaeal order Thermoproteales; about 70% of tRNA gene candidates were found to be intron-containing tRNA genes, half of which contained multiple introns, and the introns were located at various noncanonical positions. Sequence similarity analysis revealed that approximately half of the tRNA introns found at Thermoproteales-specific intron locations were highly conserved among several tRNA genes. Intriguingly, identical tRNA intron sequences were found within different types of tRNA genes that completely lacked exon sequence similarity, suggesting that the tRNA introns in Thermoproteales could have been gained via intron insertion events at a later stage of tRNA evolution. Moreover, although the CCA sequence at the 3' terminal of pre-tRNA is added by a CCA-adding enzyme after gene transcription in Archaea, most of the tRNA genes containing highly conserved introns already encode the CCA sequence at their 3' terminal. Based on these results, we propose possible models explaining the rapid increase of tRNA introns as a result of intron insertion events via retrotransposition of pre-tRNAs. The sequences and secondary structures of the tRNA genes and their bulge-helix-bulge motifs were registered in SPLITSdb (http://splits.iab.keio.ac.jp/splitsdb/), a novel and comprehensive database for archaeal tRNA genes.

DOI： 10.1093/molbev/msn216

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

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 tRNA(iMet), tRNA(Thr), tRNA(Ile), tRNA(Gly), tRNA(Gln), tRNA(Glu), tRNA(Asp), tRNA(Lys), tRNA(Arg) and tRNA(Leu) but also phylum-specific combinations in tRNA(Pro), tRNA(Ala), and tRNA(Trp). 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.

DOI： 10.1371/journal.pone.0001622

PubMed

researchmap

掲載種別：研究論文（学術雑誌）   出版者・発行元：Oxford University Press ({OUP})  

DOI： 10.1143/ptps.173.206

Scopus

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

Proteins play a critical role in complex biological systems, yet about half of the proteins in publicly available databases are annotated as functionally unknown. Proteome-wide functional classification using bioinformatics approaches thus is becoming an important method for revealing unknown protein functions. Using the hyperthermophilic archaeon Pyrococcus furiosus as a model species, we used the support vector machine (SVM) method to discriminate DNA/RNA-binding proteins from proteins with other functions, using amino acid composition and periodicities as feature vectors. We defined this value as the composition score (CO) and periodicity score (PD). The P. furiosus proteins were classified into three classes (I-III) on the basis of the two-dimensional correlation analysis of CO score and PD score. As a result, approximately 87% of the functionally known proteins categorized as class I proteins (CO score + PD score > 0.6) were found to be DNA/RNA-binding proteins. Applying the two-dimensional correlation analysis to the 994 hypothetical proteins in P. furiosus, a total of 151 proteins were predicted to be novel DNA/RNA-binding protein candidates. DNA/RNA-binding activities of randomly chosen hypothetical proteins were experimentally verified. Six out of seven candidate proteins in class I possessed DNA/RNA-binding activities, supporting the efficacy of our method.

PubMed

researchmap

記述言語：英語   掲載種別：研究論文（学術雑誌）  

We have developed a comprehensive software suite for bioinformatics research of cDNAs; it is aimed at rapid characterization of the features of genes and the proteins they code. Methods implemented include the detection of translation initiation and termination signals, statistical analysis of codon usage, comparative study of amino acid composition, comparative modeling of the structures of product proteins, prediction of alternative splice forms, and metabolic pathway reconstruction.

PubMed

researchmap