Alterations of RNA Editing for the Mitochondrial ATP9 Gene in a New orf220-type Cytoplasmic Male-sterile Line of Stem Mustard （Brassica juncea var. tumida）

RNA editing for the mitochondrlal ATP9 gene of encoding regions has been observed in both cytoplasmic malesterile and maintainer lines of stem mustard, where its editing capacity varied spatially and temporally in the cytoplasmic male sterility （CMS） line. There were four RNA editing sites for the mitochondrial ATP9 gene according to Its normal editing sites in mustard, of which three sites occurred as C-to-U changes and one as a U-to-C change. As a result, the hydrophobicity of deduced ATP9 protein was reduced due to the conversions at its 17th, 45th and 64th positions. Meanwhile, the conservation of deduced ATP9 protein was enhanced by changes at the 56th position. Loss of a specific editing site for ATP9 was observed in juvenile roots, senile roots, senile leaves and floret buds of the CMS line. Comparatively, complete RNA editing for ATP9 gene was retained in juvenile roots, juvenile leaves and floret buds of its maintainer line; however, the loss of a specific editing site for ATP9 gene occurred at senile roots and senile leaves in its maintainer line. These observations allow us to produce a hypothesis that the dysfunction of a specific mitochondrial gene arising from RNA editing could probably be a factor triggering CMS and organ senescence through unknown cross-talk pathways during development.     

How Complex Are the Editosomes in Plant Organelles？

In chloroplasts and mitochondria of most land plants, RNA editing modifies specific nucleotides. The individual nucleotides are identified in the complex RNA molecule population of either organelle by their sequence context. The biochemical reaction is in effect a C-to-U deamination in flowering plants, in addition a U-to-C amination reaction occurs in ferns and hornworts.     

Longevity of animals under reactive oxygen species stress and disease susceptibility due to global warming

The world is projected to experience an approximate doubling of atmospheric CO_2 concentration in the next decades. Rise in atmospheric CO_2 level as one of the most important reasons is expected to contribute to raise the mean global temperature 1.4 ℃-5.8 ℃ by that time. A survey from 128 countries speculates that global warming is primarily due to increase in atmospheric CO_2 level that is produced mainly by anthropogenic activities. Exposure of animals to high environmental temperatures is mostly accompanied by unwanted acceleration of certain biochemical pathways in their cells. One of such examples is augmentation in generation of reactive oxygen species(ROS) and subsequent increase in oxidation of lipids, proteins and nucleic acids by ROS. Increase in oxidation of biomolecules leads to a state called as oxidative stress(OS). Finally, the increase in OS condition induces abnormality in physiology of animals under elevated temperature. Exposure of animals to rise in habitat temperature is found to boost the metabolism of animals and a very strong and positive correlation exists between metabolism and levels of ROS and OS. Continuous induction of OS is negatively correlated with survivability and longevity and positively correlated with ageing in animals. Thus, it can be predicted that continuous exposure of animals to acute or gradual rise in habitat temperature due to global warming may induce OS, reduced survivability and longevity in animals in general and poikilotherms in particular. A positive correlation between metabolism and temperature in general and altered O_2 consumption at elevated temperature in particular could also increase the risk of experiencing OS in homeotherms. Effects of global warming on longevity of animals through increased risk of protein misfolding and disease susceptibility due to OS as the cause or effects or both also cannot be ignored. Therefore, understanding the physiological impacts of global warming in relation to longevity of animals will become very crucial challenge to biologists of the present millennium.     

Construction of an efficient genomic editing system with CRISPR/Cas9 in the vector mosquito Aedes albopictus

Aedes (Stegomyia) albopictus, also known as the Asian tiger mosquito, is a mosquito which originated in Asia. In recent years, it has become increasingly rampant throughout the world. This mosquito can transmit several arboviruses, including dengue, Zika and chikungunya viruses, and is considered a public health threat. Despite the urgent need of genome engineering to analyze specific gene functions, progress in genetical manipulation of Ae. albopictus has been slow due to a lack of efficient methods and genetic markers. In the present study, we established targeted disruptions in two genes, kynurenine hydroxylase (kh) and dopachrome conversion enzyme (yellow), to analyze the feasibility of generating visible phenotypes with genome editing by the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated protein 9 (Cas9) system in Ae. albopictus. Following Cas9 single guide RNA ribonucleoprotein injection into the posterior end of pre-blastoderm embryos, 30%-50% of fertile survivors produced alleles that failed to complement existing kh and yellow mutations. Complete eye and body pigmentation defects were readily observed in GI pupae and adults, indicating successful generation of highly heritable mutations. We conclude that the CRISPR/Cas9-mediated gene editing system can be used mAe. albopictus and that it can be adopted as an efficient tool for genome-scale analysis and biological study.     

RNA silencing and its application in plants

<正>RNA silencing, also known as gene silencing, is a sequence-specific RNA degradation mechanism that is highly conserved in eukaryotic organisms, including plants, animals, nematodes, and fungi. In eukaryotes, RNA silencing has a pivotal role in the regulation of development, genome stability, and responses to biotic and abiotic stresses through     

Plant base editing and prime editing: The current status and future perspectives

Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement.Base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates,respectively.Since their discoveries,various strate...     

Perspectives of pluripotent stem cells in livestock

The recent progress in derivation of pluripotent stem cells(PSCs)from farm animals opens new approaches not only for reproduction,genetic engineering,treatment and conservation of these species,but also for screening novel drugs for their efficacy and toxicity,and modelling of human diseases.Initial attempts to derive PSCs from the inner cell mass of blastocyst stages in farm animals were largely unsuccessful as either the cells survived for only a few passages,or lost their cellular potency;indicating that the protocols which allowed the derivation of murine or human embryonic stem(ES)cells were not sufficient to support the maintenance of ES cells from farm animals.This scenario changed by the innovation of induced pluripotency and by the development of the 3 inhibitor culture conditions to support na?ve pluripotency in ES cells from livestock species.However,the long-term culture of livestock PSCs while maintaining the full pluripotency is still challenging,and requires further refinements.Here,we review the current achievements in the derivation of PSCs from farm animals,and discuss the potential application areas.     

Hoarding habitat selection of squirrels (Sciurus vulgaris) in Liangshui Nature Reserve, China

This study was conducted during October 2003–April 2004 in the Liangshui National Nature Reserve of the Xiao Xing'an Mountains of Northeast China. Results showed that hoarding behavior of squirrels exhibited selectivity. The preference order of hoarding habitat selection of squirrels is as follows: original Korean pine forest, secondary natural fir forest, artificial fir forest, mix-conifer leaf forest, artificial fallen leaves pine forest, mix-conifer-broadleaf forest, mix-broadleaf forest birch forest, and artificial Korean pine forest. Compared with the existing results, using the cache spots as an index, the order of habitat selection changed, revealing that this research should include more factors, such as pilferage from other animals and secondary dispersal by the squirrels or other species. The Vanderploeg and Scavia selectivity indexes, Wi and Ei, were used to evaluate the use of microhabitat by squirrels in the original Korean pine forest. Results indicated that the squirrels exhibited a significant microhabitat utilization pattern: (1) Squirrels prefer to use the microhabitat in original Korean pine forest with high canopies, the medium shrubby density, and the medium stub density, where pilferage animals are few (Ei > 0.2); (2) Squirrels did not prefer to use the microhabitat in the original Korean pine forest with a low slope degree, high density of fallen logs and stumps, a high herbage coverage, and an abundance of pilferage animals (Ei < ?0.2), and they showed an aversion for a shaded slope (Ei = ?0.5368). The selectivity of microhabitat utilization for the hoarding behavior of squirrels plays an important role in determining the spatial pattern of Korean pine seedlings.     

Increasing the efficiency of CRISPR/Cas9-based gene editing by suppressing RNAi in plants

<正>Dear Editor,CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR-associated)-based genome editing is a powerful and widely adopted technology for introducing specific mutations in animal and plant systems. Typically, a CRISPR/Cas9 system is composed of the endonuclease protein Cas9 and an engineered single-guide RNA (sgRNA)that specifically recognizes its target DNA sequence in the genome. In plants, strong constitutive promoters, such as     

Hoarding habitat selection of squirrels (Sciurus vulgaris) in Liangshui Nature Reserve, China

This study was conducted during October 2003–April 2004 in the Liangshui National Nature Reserve of the Xiao Xing'an Mountains of Northeast China. Results showed that hoarding behavior of squirrels exhibited selectivity. The preference order of hoarding habitat selection of squirrels is as follows: original Korean pine forest, secondary natural fir forest, artificial fir forest, mix-conifer leaf forest, artificial fallen leaves pine forest, mix-conifer-broadleaf forest, mix-broadleaf forest birch forest, and artificial Korean pine forest. Compared with the existing results, using the cache spots as an index, the order of habitat selection changed, revealing that this research should include more factors, such as pilferage from other animals and secondary dispersal by the squirrels or other species. The Vanderploeg and Scavia selectivity indexes, Wi and Ei, were used to evaluate the use of microhabitat by squirrels in the original Korean pine forest. Results indicated that the squirrels exhibited a significant microhabitat utilization pattern: (1) Squirrels prefer to use the microhabitat in original Korean pine forest with high canopies, the medium shrubby density, and the medium stub density, where pilferage animals are few (Ei > 0.2); (2) Squirrels did not prefer to use the microhabitat in the original Korean pine forest with a low slope degree, high density of fallen logs and stumps, a high herbage coverage, and an abundance of pilferage animals (Ei < ?0.2), and they showed an aversion for a shaded slope (Ei = ?0.5368). The selectivity of microhabitat utilization for the hoarding behavior of squirrels plays an important role in determining the spatial pattern of Korean pine seedlings.     

Induction of apoptosis in purified animal and plant nuclei by Xenopus egg extracts

We have developed a cell-free system that can trigger the nuclei purified from mouse liver and suspensioncultured carrot cells to undergo apoptosis as defined by the formation of apoptotic bodies and nucleosomal DNA fragments.The effects of different divalent cations and cycloheximide on DNA cleavage in this system were assessed.The fact that nuclei of plant cells can be induced to undergo apoptosis in a cell-free animal system suggests that animals and plants share a common signal transduction pathway triggering in the initiation stage of apoptosis.     

Yeast KEOPS complex regulates telomere length independently of its t6A modification function

In Saccharomyces cerevisiae, the highly conserved Sua5 and KEOPS complex(including five subunits Kae1,Bud32, Cgi121, Pcc1 and Gon7) catalyze a universal t RNA modification, namely N~6-threonylcarbamoyladenosine(t~6A), and regulate telomere replication and recombination. However, whether telomere regulation function of Sua5 and KEOPS complex depends on the t~6A modification activity remains unclear. Here we show that Sua5 and KEOPS regulate telomere length in the same genetic pathway.Interestingly, the telomere length regulation by KEOPS is independent of its t~6A biosynthesis activity.Cytoplasmic overexpression of Qri7, a functional counterpart of KEOPS in mitochondria, restores cytosolic t RNA t~6A modification and cell growth, but is not sufficient to rescue telomere length in the KEOPS mutant kae1△ cells, indicating that a t~6A modification-independent function is responsible for the telomere regulation. The results of our in vitro biochemical and in vivo genetic assays suggest that telomerase RNA TLC1 might not be modified by Sua5 and KEOPS. Moreover, deletion of KEOPS subunits results in a dramatic reduction of telomeric G-overhang, suggesting that KEOPS regulates telomere length by promoting G-overhang generation. These findings support a model in which KEOPS regulates telomere replication independently of its function on t RNA modification.     

Silicon deprivation decreases collagen formation in wounds and bone,and ornithine transaminase enzyme activity in liver

We have shown that silicon (Si) deprivation decreases the collagen concentration in bone of 9-wk-old rats. Finding that Si deprivation also affects collagen at different stages in bone development, collagen-forming enzymes, or collagen deposition in other tissues would have implications that Si is important for both wound healing and bone formation. Therefore, 42 rats in experiment 1 and 24 rats in experiment 2 were fed a basal diet containing 2 or 2.6 μg Si/g, respectively, based on ground corn and casein, and supplemented with either 0 or 10 μg Si/g as sodium metasilicate. At 3 wk, the femur was removed from 18 of the 42 rats in experiment 1 for hydroxyproline analysis. A polyvinyl sponge was implanted beneath the skin of the upper back of each of the 24 remaining rats. Sixteen hours before termination and 2 wk after the sponge had been implanted, each rat was given an oral dose of ¹⁴C-proline (1.8 μCi/100 g body wt). The total amount of hydroxyproline was significantly lower in the tibia and sponges taken from Si-deficient animals than Si-supplemented rats. The disintegrations per minute of ¹⁴C-proline were significantly higher in sponge extracts from Si-deficient rats than Si-supplemented rats. Additional evidence of aberrations in proline metabolism with Si deprivation was that liver ornithine aminotransferase was significantly decreased in Si-deprived animals in experiment 2. Findings of an increased accumulation of ¹⁴C-proline and decreased total hydroxyproline in implanted sponges and decreased activity of a key enzyme in proline synthesis (liver ornithine aminotransferase) in Si-deprived animals indicates an aberration in the formation of collagen from proline in sites other than bone that is corrected by Si. This suggests that Si is a nutrient of concern in wound healing as well as bone formation. The U.S. Department of Agriculture, Agricultural Research Service, Northern Plains Area is an equal opportunity/affirmative action employer, and all agency services are available without discrimination. Mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may be suitable.     

Effects of Acetylcholine, Cytochalasin B and Amiprophosmethyl on Phloem Transport in Radish （Raphanus sativas）

We investigated the role of the ＂sieve tube-companion cell complex＂ lining the tube periphery, particularly the microfilament and microtubule, in assisting the pushing of phloem sap flow. We made a simple phloem transport system with a living radish plant, in which the conducting channel was exposed for local treatment with chemicals that are effective in modulating protoplasmic movement （acetylcholine, （ACh） a neurotransmitter in animals and insects; cytochalasin B, （CB） a specific inhibitor of many cellular responses that are mediated by microfilament systems and amiprophos-methyl, （APM） a specific inhibitor of many cellular responses that are mediated by microtubule systems）. Their effects on phloem transport were estimated by two experimental devices： （i） a comparison of changes in the amount of assimilates in terms of carbohydrates and ^14C-labeled photosynthetic production that is left in the leaf blade of treated plants; and （ii） distribution patterns of ^14C-labeled leaf assimilates in the phloem transport system. The results indicate that CB and APM markedly inhibited the transfer of photosynthetic product from leaf to root via the leaf vein, while ACh enhanced the transfer of photosynthetic product in low concentrations （5.0×10^-4 mol/L） but inhibited it in higher concentrations （2.0×10^-3 mol/L） from leaf to root via the leaf vein. Autoradiograph imaging clearly reveals that ACh treatment is more effective than the control, and both CB and APM treatments effectively inhibit the passage of radioactive assimilates. All of the results support the postulation that the peripheral protoplasm in the sieve tube serves not only as a passive semi-permeable membrane, but is also directly involved in phloem transport.     

Morphophysiological and molecular evidence supporting the augmentative role of Piriformospora indica in mitigation of salinity in Cucumis melo L.

Salinity is one of the major limiting factors in plant growth and productivity.Cucumis melo L.is a widely cultivated plant,but its productivity is significantly influenced by the level of salinity in soil.Symbiotic colonization of plants with Piriformospora indica has shown a promotion in plants growth and tolerance against biotic stress.In this study,physiological markers such as ion analysis,antioxidant determination,proline content,electrolyte leakage and chlorophyll measurement were assessed in melon cultivar under two concentrations(100 and 200 mM)of NaCl with and without P.indica inoculation.Results showed that the endophytic inoculation consistently upregulated the level of antioxidants,enhanced plants to antagonize salinity stress.The expression level of an RNA editing factor(SLO2)which is known to participate in mitochondria electron transport chain was analyzed,and its full mRNA sequence was obtained by rapid amplification of cDNA ends(RACE).Under salinity stress,the expression level of SLO2 was increased,enhancing the plant’s capability to adapt to the stress.However,P.indica inoculation further elevated the expression level of SLO2.These findings suggested that the symbiotic association of fungi could help the plants to tolerate the salinity stress.     

The Arabidopsis thaliana RNA Editing FactorSLO2, which Affects the Mitochondrial ElectronTransport Chain,Participates in Multiple Stressand Hormone Resoonses

Recently, we reported that the novel mitochondrial RNA editing factor SLO2 is essential for mitochondrialelectron transport, and vital for plant growth through regulation of carbon and energy metabolism. Here, we show thatmutation in SL02 causes hypersensitivity to ABA and insensitivity to ethylene, suggesting a link with stress responses.Indeed, slo2 mutants are hypersensitive to salt and osmotic stress during the germination stage, while adult plantsshow increased drought and salt tolerance. Moreover, slo2 mutants are more susceptible to Botrytis cinerea infection.An increased expression of nuclear-encoded stress-responsive genes, as well as mitochondrial-encoded NAD genes ofcomplex I and genes of the alternative respiratory pathway, was observed in slo2 mutants, further enhanced by ABAtreatment. In addition, H202 accumulation and altered amino acid levels were recorded in slo2 mutants. We conclude thatSLO2 is required for plant sensitivity to ABA, ethylene, biotic, and abiotic stress. Although two stress-related RNA editingfactors were reported very recently, this study demonstrates a unique role of SLO2, and further supports a link betweenmitochondrial RNA editing events and stress response.     

Epidermal growth factor and insulin-like growth factor-1 protect MDA-231 cells from death induced by actinomycin D: The involvement of growth factors in drug resistance

Summary In the present study, we investigated the ability of epidermal growth factor (EGF), insulin-like growth factor-1 (IGF-1), and insulin to protect the human breast cancer cell line MDA-231 from death induced by the antitumor drug actinomycin D (ACT-D). ACT-D is an inhibitor of RNA and protein synthesis, and its cytotoxicity may result due to continuous depletion in some vital protein molecules. Cell death was induced in the MDA-231 cells by either continuous exposure to a low dose of ACT-D (0.2μg/ml), or by a short-time exposure to a high dose of ACT-D (2μg/ml) and further culturing in the absence of the drug. Cell death was evaluated by the trypan blue dye exclusion test, the release of lactic dehydrogenase into the culture medium, and the depletion in the cellular ATP content. EGF and IGF-1, each at an optimal concentration of 20 ng/ml, enhanced substantially survival of cells exposed either to a low or a high dose of ACT-D. The combination of EGF (10 ng/ml) and IGF-1 (10 ng/ml) had an additive survival effect, which proposes that each of the growth factors enhanced survival by a distinct pathway. Insulin up to 40 ng/ml had no effect on cell survival. Pretreatment of the cells for 1 to 5 h with EGF and IGF-1 protected cells from the cytotoxic effect of ACT-D. Exposure of the cells to 2μg/ml of ACT-D for 1 h resulted in a drastic inhibition in uridine incorporation and only in a slight inhibition in leucine incorporation. Further incubation in the absence of ACT-D resulted in a continuous decrease in uridine and in leucine incorporation, either in the absence or presence of the growth factors. However, EGF and IGF-1, but not insulin, attenuated significantly this continuous decrease. We assume that EGF and IGF-1 protect cell viability by a mechanism that maintains a critical level of some vital protein molecule above the critical level at which cells die. Our finding that EGF and IGF-1 induced resistance to ACT-D suggests that growth factors may be involved in the mechanism of drug resistance.