Synthesis of 18O-labeled RNA for application to kinetic studies and imaging

Radioisotopes and fluorescent compounds are frequently used for RNA labeling but are unsuitable for clinical studies of RNA drugs because of the risk from radiation exposure or the nonequivalence arising from covalently attached fluorophores. Here, we report a practical phosphoramidite solid-phase synthesis of ¹⁸O-labeled RNA that avoids these disadvantages, and we demonstrate its application to quantification and imaging. The synthesis involves the introduction of a nonbridging ¹⁸O atom into the phosphate group during the oxidation step of the synthetic cycle by using ¹⁸O water as the oxygen donor. The ¹⁸O label in the RNA was stable at pH 3–8.5, while the physicochemical and biological properties of labeled and unlabeled short interfering RNA were indistinguishable by circular dichroism, melting temperature and RNA-interference activity. The ¹⁸O/¹⁶O ratio as measured by isotope ratio mass spectrometry increased linearly with the concentration of ¹⁸O-labeled RNA, and this technique was used to determine the blood concentration of ¹⁸O-labeled RNA after administration to mice. ¹⁸O-labeled RNA transfected into human A549 cells was visualized by isotope microscopy. The RNA was observed in foci in the cytoplasm around the nucleus, presumably corresponding to endosomes. These methodologies may be useful for kinetic and cellular-localization studies of RNA in basic and pharmaceutical studies.     

Clerodane diterpenoids from the roots of Portulaca pilosa

Three trans-clerodane diterpenoids, pilosanol A, B and C, the last compound being a glucoside, have been isolated from the roots of Portulaca pilosa. They show a marked contrast in skeletal type with the constituents of aerial part. Evolutionary changes in the biosynthetic abilities of Portulaca species is discussed.     

Formation of Chlorophyll-Protein Complexes in Greening Cucumber Cotyledons in Light and then in Darkness

The changes in chlorophyll-protein complexes (CPs) in cucumbercotyledons during illumination and subsequent dark incubationwere studied by SDS-polyacrylamide gel electrophoresis. Whenetiolated cucumber seedlings were illuminated, chlorophyll wassynthesized and CPs were formed. In the early phase of greening(6 h of illumination), light-harvesting chlorophyll a/b-proteincomplex (LHCP) was the main GP. As the greening proceeded, P700chlorophyll a-protein complex (CP1) accumulated. When 6-h illuminatedseedlings were transferred to darkness, CP1 accumulated concomitantlywith a decrease in LHCP without new chlorophyll synthesis. Thechanges in the amounts of CPs in the dark became smaller withthe progress of greening and were not observed after 72 h ofillumination. These changes were confirmed by examining thechlorophyll/P700 ratio and the low temperature absorption spectrumof cotyledons. These results suggest that in the early phaseof greening, CPs were unstable and their chlorophyll moleculeseasily exchanged with those of other kinds of CPs. (Received October 14, 1982; Accepted December 1, 1982)     

Phylogeography of the temperate grassland plant Tephroseris kirilowii (Asteraceae) inferred from multiplexed inter-simple sequence repeat genotyping by sequencing (MIG-seq) data

Journal of Plant Research - A group of temperate grassland plant species termed the “Mansen elements” occurs in Japan and is widely distributed in the grasslands of continental East...     

Integrative genomic phylogeography reveals signs of mitonuclear incompatibility in a natural hybrid goby population

Hybridization between divergent lineages generates new allelic combinations. One mechanism that can hinder the formation of hybrid populations is mitonuclear incompatibility, that is, dysfunctional interactions between proteins encoded in the nuclear and mitochondrial genomes (mitogenomes) of diverged lineages. Theoretically, selective pressure due to mitonuclear incompatibility can affect genotypes in a hybrid population in which nuclear genomes and mitogenomes from divergent lineages admix. To directly and thoroughly observe this key process, we de novo sequenced the 747‐Mb genome of the coastal goby, Chaenogobius annularis, and investigated its integrative genomic phylogeographics using RNA‐sequencing, RAD‐sequencing, genome resequencing, whole mitogenome sequencing, amplicon sequencing, and small RNA‐sequencing. Chaenogobius annularis populations have been geographically separated into Pacific Ocean (PO) and Sea of Japan (SJ) lineages by past isolation events around the Japanese archipelago. Despite the divergence history and potential mitonuclear incompatibility between these lineages, the mitogenomes of the PO and SJ lineages have coexisted for generations in a hybrid population on the Sanriku Coast. Our analyses revealed accumulation of nonsynonymous substitutions in the PO‐lineage mitogenomes, including two convergent substitutions, as well as signals of mitochondrial lineage‐specific selection on mitochondria‐related nuclear genes. Finally, our data implied that a microRNA gene was involved in resolving mitonuclear incompatibility. Our integrative genomic phylogeographic approach revealed that mitonuclear incompatibility can affect genome evolution in a natural hybrid population.     

Effects of double rice cropping with irrigation on the diversity of herbaceous plants and their utilization as a food source in paddy fields of southern Lao People’s Democratic Republic

Herbaceous plant diversity including rare aquatic species has been lost in many countries by agricultural intensification and abandonment. In paddy fields of the Lao People’s Democratic Republic (Lao PDR), irrigation facilities have been constructed rapidly since 1997. The aim of this study was to clarify the impacts of double rice cropping accompanied by the introduction of irrigation systems on herbaceous plant diversity and utilization in paddy fields of southern Lao PDR. Ground vegetation surveys and interviews were conducted in Kok Deau and Lak 30 villages in Champasak Province, and propagule bank survey was conducted in Kok Deau village. The species richness and species diversity, measured by the Shannon’s diversity index, were not significantly different between the irrigated and rainfed paddies (p?>?0.05), when compared in both the wet and dry seasons. However, double rice cropping with irrigation systems affected herbaceous plant species composition in paddy fields. Increased use of chemical fertilizers in irrigated paddies resulted in predominance of tall undesirable species, such as Fimbristylis miliacea (L.) Vahl and Echinochloa colona (L.) Link. Small and frequently submerged species were dominant in the ground vegetation and propagule banks of the rainfed paddies. Since small submerged species are often sensitive to environmental changes, increase of irrigated paddy area may lead to a decrease in the variety of aquatic herbaceous plants in Lao PDR. While a total of 9 herbaceous plant species were utilized as foods in the villages, no change was recognized by farmers in species composition and frequency of utilization of paddy plants as food before and after the development of the irrigation systems.

     

Different patterns of allopreening in the same-sex and opposite-sex interactions of juvenile large-billed crows (Corvus macrorhynchos)

Allogrooming, where an individual grooms another, has been extensively studied in various social animals to understand its role in the evolution of cooperation/prosociality. In existing studies in mammals, allogrooming has been suggested to exhibit not only a hygiene but also a social function. Allopreening, a topic of increasing interest in mammals but recently also in birds, has been studied mostly with mature animals. However, in some species immature individuals also show allopreening and its function remains poorly understood. Crows, Corvus spp., are an ideal model to study this phenomenon, because juveniles form year-round aggregates during their long juvenile stage (e.g., throughout 3–4 years). Here, we investigated the function of allopreening in juvenile groups of wild-caught large-billed crows (C. macrorhynchos). Allopreening frequency and duration for three groups of wild-caught juveniles were analysed to determine whether there was a symmetrical (i.e., reciprocal) or asymmetrical allopreening pattern, and if sex composition of the dyad and/or relative dominance of donor and recipient had an effect. We found that both the frequency and duration of male allopreening correlated with frequency of aggression. Allopreening between both males and females occurred unidirectionally from dominants to subordinates but not in the opposite direction. On the contrary, allopreening between a male and a female was found to be reciprocated, though the absolute frequency and duration were both greater in males than in females. These results suggest that the social function of allopreening in juvenile crows differs depending on the sex composition of the dyad, functioning as a dominance signal for same-sex dyads, and serving a social bonding function for opposite-sex dyads. These findings may reflect the potentially crucial roles of allopreening in within-sex competition and opposite-sex attraction during the 3 year-long juvenile stage affecting future mate choice in lifelong monogamy.     

Evolutionary Changes in Chlorophyllide a Oxygenase (CAO) Structure Contribute to the Acquisition of a New Light-harvesting Complex in Micromonas

Chlorophyll b is found in photosynthetic prokaryotes and primary and secondary endosymbionts, although their light-harvesting systems are quite different. Chlorophyll b is synthesized from chlorophyll a by chlorophyllide a oxygenase (CAO), which is a Rieske-mononuclear iron oxygenase. Comparison of the amino acid sequences of CAO among photosynthetic organisms elucidated changes in the domain structures of CAO during evolution. However, the evolutionary relationship between the light-harvesting system and the domain structure of CAO remains unclear. To elucidate this relationship, we investigated the CAO structure and the pigment composition of chlorophyll-protein complexes in the prasinophyte Micromonas. The Micromonas CAO is composed of two genes, MpCAO1 and MpCAO2, that possess Rieske and mononuclear iron-binding motifs, respectively. Only when both genes were introduced into the chlorophyll b-less Arabidopsis mutant (ch1-1) was chlorophyll b accumulated, indicating that cooperation between the two subunits is required to synthesize chlorophyll b. Although Micromonas has a characteristic light-harvesting system in which chlorophyll b is incorporated into the core antennas of reaction centers, chlorophyll b was also incorporated into the core antennas of reaction centers of the Arabidopsis transformants that contained the two Micromonas CAO proteins. Based on these results, we discuss the evolutionary relationship between the structures of CAO and light-harvesting systems.