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Linear tetrapyrrole compounds (bilins) are chromophores of the phytochrome and cyanobacteriochrome classes of photosensors and light-harvesting phycobiliproteins. Various spectroscopic techniques, such as resonance Raman, Fourier transform-infrared and nuclear magnetic resonance, have been used to elucidate the structures underlying their remarkable spectral diversity, in which the signals are experimentally assigned to specific structures using isotopically labeled bilin. However, current methods for isotopic labeling of bilins require specialized expertise, time-consuming procedures and/or expensive reagents. To address these shortcomings, we established a method for pressurized liquid extraction of phycocyanobilin (PCB) from the phycobiliprotein powder Lina Blue and also the cyanobacterium Synechocystis sp. PCC 6803 (Synechocystis). PCB was efficiently cleaved in ethanol with three extractions (5 min each) under nitrogen at 125°C and 100 bars. A prewash at 75°C was effective for removing cellular pigments of Synechocystis without PCB cleavage. Liquid chromatography and mass spectrometry suggested that PCB was cleaved in the C3 -E (majority) and C3 -Z (partial) configurations. 15N- and 13C/15N-labeled PCBs were prepared from Synechocystis cells grown with NaH13CO3 and/or Na15NO3, the concentrations of which were optimized based on cell growth and pigmentation. Extracted PCB was reconstituted with a recombinant apoprotein of the cyanobacteriochrome-class photosensor RcaE. Yield of the photoactive holoprotein was improved by optimization of the expression conditions and cell disruption in the presence of Tween 20. Our method can be applied for the isotopic labeling of other PCB-binding proteins and for the commercial production of non-labeled PCB for food, cosmetic and medical applications. [ABSTRACT FROM AUTHOR]

Yeast-based genotoxicity testing systems can sensitively detect DNA damaging agents in the environment. We have developed a novel “indirect” reporter assay system based on a recombinant yeast containing both a sensor and a reporter plasmid. The sensor plasmid contains a gene encoding the artificial transcription factor of the Escherichia coli LexA DNA binding domain fused to the transcriptional activation domain of yeast Gal4p, which is regulated by the DNA damage–inducible RNR2 promoter. The reporter plasmid contains the E. coli lacZ gene with the LexA binding site in the 5′-upstream region, allowing transcriptional activation by the induced LexA-GAL4 protein. The activity of DNA damage–dependent β-galactosidase (β-gal) in the “indirect” reporter assay system was compared with that of a current yeast-based “direct” reporter system. The “indirect” system exhibited 1.5- to 5-fold greater β-gal activity upon induction by alkylating agents or camptothecin. To increase the sensitivity of the new reporter system further, several deletion yeast strains were tested, and enhanced induction of reporter activity was observed in DNA repair-deficient mag1Δ cells. The “indirect” 96-well microtiter plate assay system is a potentially inexpensive and sensitive method for detecting genotoxic activities in a wide range of compounds, and in polluted environmental samples. [ABSTRACT FROM PUBLISHER]

Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid–based reporter assays in budding yeast using a RNR3 promoter–driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid–based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid–based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. Key points: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast. [ABSTRACT FROM AUTHOR]

Microorganism-based genotoxicity assessments are vital for evaluating potential chemical-induced DNA damage. In this study, we developed both chromosomally integrated and single-copy plasmid–based reporter assays in budding yeast using a RNR3 promoter–driven luciferase gene. These assays were designed to compare the response to genotoxic chemicals with a pre-established multicopy plasmid–based assay. Despite exhibiting the lowest luciferase activity, the chromosomally integrated reporter assay showed the highest fold induction (i.e., the ratio of luciferase activity in the presence and absence of the chemical) compared with the established plasmid-based assay. Using CRISPR/Cas9 technology, we generated mutants with single- or double-gene deletions, affecting major DNA repair pathways or cell permeability. This enabled us to evaluate reporter gene responses to genotoxicants in a single-copy plasmid–based assay. Elevated background activities were observed in several mutants, such as mag1Δ cells, even without exposure to chemicals. However, substantial luciferase induction was detected in single-deletion mutants following exposure to specific chemicals, including mag1Δ, mms2Δ, and rad59Δ cells treated with methyl methanesulfonate; rad59Δ cells exposed to camptothecin; and mms2Δ and rad10Δ cells treated with mitomycin C (MMC) and cisplatin (CDDP). Notably, mms2Δ/rad10Δ cells treated with MMC or CDDP exhibited significantly enhanced luciferase induction compared with the parent single-deletion mutants, suggesting that postreplication and for nucleotide excision repair processes predominantly contribute to repairing DNA crosslinks. Overall, our findings demonstrate the utility of yeast-based reporter assays employing strains with multiple-deletion mutations in DNA repair genes. These assays serve as valuable tools for investigating DNA repair mechanisms and assessing chemical-induced DNA damage. Key points: • Responses to genotoxic chemicals were investigated in three types of reporter yeast. • Yeast strains with single- and double-deletions of DNA repair genes were tested. • Two DNA repair pathways predominantly contributed to DNA crosslink repair in yeast. [ABSTRACT FROM AUTHOR]

Mutagens and oxidative agents damage biomolecules, such as DNA; therefore, detecting genotoxic and oxidative chemicals is crucial for maintaining human health. To address this, we have developed several types of yeast-based reporter assays designed to detect DNA damage and oxidative stress. This study aimed to develop a novel yeast-based assay using a codon-optimized stable or unstable NanoLuc luciferase (yNluc and yNluCP) gene linked to a DNA damage- or oxidative stress-responsive promoter, enabling convenient sensing genotoxicity or oxidative stress, respectively. End-point luciferase assays using yeasts with a chromosomally integrated RNR3 promoter (PRNR3)-driven yNluc gene exhibited high levels of chemiluminescence via NanoLuc luciferase and higher fold induction by hydroxyurea than a multi-copy plasmid-based assay. Additionally, the integrated reporter system detected genotoxicity caused by four different types of chemicals. Oxidants (hydrogen peroxide, tert-butyl hydroperoxide, and menadione) were successfully detected through transient expressions of luciferase activity in real-time luciferase assay using yeasts with a chromosomally integrated TRX2 promoter (PTRX2)-linked yNlucCP gene. However, the luciferase activity was gradually induced in yeasts with a multi-copy reporter plasmid, and their expression profiles were notably distinct from those observed in chromosomally integrated yeasts. The responses of yNlucCP gene against three oxidative chemicals, but not diamide and zinc oxide suspension, were observed using chromosomally integrated reporter yeasts. Given that yeast cells with chromosomally integrated PRNR3-linked yNluc and PTRX2-linked yNlucCP genes express strong chemiluminescence signals and are easily maintained and handled without restrictive nutrient medium, these yeast strains with NanoLuc reporters may prove useful for screening potential genotoxic and oxidative chemicals. [ABSTRACT FROM AUTHOR]

Crop rotation is an important agricultural practice for homeostatic crop cultivation. Here, we applied high-throughput sequencing of ribosomal RNA gene amplicons to investigate soil biota in two fields of central Japan with different histories of maize–cabbage rotation. We identified 3086 eukaryotic and 17,069 prokaryotic sequence variants (SVs) from soil samples from two fields rotating two crops at three different growth stages. The eukaryotic and prokaryotic communities in the four sample groups of two crops and two fields were clearly distinguished using β-diversity analysis. Redundancy analysis showed the relationships of the communities in the fields to pH and nutrient, humus, and/or water content. The complexity of eukaryotic and prokaryotic networks was apparently higher in the cabbage-cultivated soils than those in the maize-cultivated soils. The node SVs (nSVs) of the networks were mainly derived from two eukaryotic phyla: Ascomycota and Cercozoa, and four prokaryotic phyla: Pseudomonadota, Acidobacteriota, Actinomycetota, and Gemmatimonadota. The networks were complexed by cropping from maize to cabbage, suggesting the formation of a flexible network under crop rotation. Ten out of the 16 eukaryotic nSVs were specifically found in the cabbage-cultivated soils were derived from protists, indicating the potential contribution of protists to the formation of complex eukaryotic networks. [ABSTRACT FROM AUTHOR]

By damaging DNA molecules, genotoxicants cause genetic mutations and also increase human susceptibility to cancers and genetic diseases. Over the past four decades, several assays have been developed in the budding yeast Saccharomyces cerevisiae to screen potential genotoxic substances and provide alternatives to animal-based genotoxicity tests. These yeast-based genotoxicity tests are either DNA alteration-based or DNA stress-response reporter-based. The former, which came first, were developed from the genetic studies conducted on various types of DNA alterations in yeast cells. Despite their limited throughput capabilities, some of these tests have been used as short-term genotoxicity tests in addition to bacteria- or mammalian cell-based tests. In contrast, the latter tests are based on the emergent transcriptional induction of DNA repair-related genes via activation of the DNA damage checkpoint kinase cascade triggered by DNA damage. Some of these reporter assays have been linked to DNA damage-responsive promoters to assess chemical carcinogenicity and ecotoxicity in environmental samples. Yeast-mediated genotoxicity tests are being continuously improved by increasing the permeability of yeast cell walls, by the ectopic expression of mammalian cytochrome P450 systems, by the use of DNA repair-deficient host strains, and by integrating them into high-throughput formats or microfluidic devices. Notably, yeast-based reporter assays linked with the newer toxicogenomic approaches are becoming powerful short-term genotoxicity tests for large numbers of compounds. These tests can also be used to detect polluted environmental samples, and as effective screening tools during anticancer drug development.By damaging DNA molecules, genotoxicants cause genetic mutations and also increase human susceptibility to cancers and genetic diseases. Over the past four decades, several assays have been developed in the budding yeast Saccharomyces cerevisiae to screen potential genotoxic substances and provide alternatives to animal-based genotoxicity tests. These yeast-based genotoxicity tests are either DNA alteration-based or DNA stress-response reporter-based. The former, which came first, were developed from the genetic studies conducted on various types of DNA alterations in yeast cells. Despite their limited throughput capabilities, some of these tests have been used as short-term genotoxicity tests in addition to bacteria- or mammalian cell-based tests. In contrast, the latter tests are based on the emergent transcriptional induction of DNA repair-related genes via activation of the DNA damage checkpoint kinase cascade triggered by DNA damage. Some of these reporter assays have been linked to DNA damage-responsive promoters to assess chemical carcinogenicity and ecotoxicity in environmental samples. Yeast-mediated genotoxicity tests are being continuously improved by increasing the permeability of yeast cell walls, by the ectopic expression of mammalian cytochrome P450 systems, by the use of DNA repair-deficient host strains, and by integrating them into high-throughput formats or microfluidic devices. Notably, yeast-based reporter assays linked with the newer toxicogenomic approaches are becoming powerful short-term genotoxicity tests for large numbers of compounds. These tests can also be used to detect polluted environmental samples, and as effective screening tools during anticancer drug development.By damaging DNA molecules, genotoxicants cause genetic mutations and also increase human susceptibility to cancers and genetic diseases. Over the past four decades, several assays have been developed in the budding yeast Saccharomyces cerevisiae to screen potential genotoxic substances and provide alternatives to animal-based genotoxicity tests. These yeast-based genotoxicity tests are either DNA alteration-based or DNA stress-response reporter-based. The former, which came first, were developed from the genetic studies conducted on various types of DNA alterations in yeast cells. Despite their limited throughput capabilities, some of these tests have been used as short-term genotoxicity tests in addition to bacteria- or mammalian cell-based tests. In contrast, the latter tests are based on the emergent transcriptional induction of DNA repair-related genes via activation of the DNA damage checkpoint kinase cascade triggered by DNA damage. Some of these reporter assays have been linked to DNA damage-responsive promoters to assess chemical carcinogenicity and ecotoxicity in environmental samples. Yeast-mediated genotoxicity tests are being continuously improved by increasing the permeability of yeast cell walls, by the ectopic expression of mammalian cytochrome P450 systems, by the use of DNA repair-deficient host strains, and by integrating them into high-throughput formats or microfluidic devices. Notably, yeast-based reporter assays linked with the newer toxicogenomic approaches are becoming powerful short-term genotoxicity tests for large numbers of compounds. These tests can also be used to detect polluted environmental samples, and as effective screening tools during anticancer drug development. [ABSTRACT FROM AUTHOR]

Crop rotation is an important agricultural practice for homeostatic crop cultivation. Here, we applied high-throughput sequencing of ribosomal RNA gene amplicons to investigate soil biota in two fields of central Japan with different histories of maize–cabbage rotation. We identified 3086 eukaryotic and 17,069 prokaryotic sequence variants (SVs) from soil samples from two fields rotating two crops at three different growth stages. The eukaryotic and prokaryotic communities in the four sample groups of two crops and two fields were clearly distinguished using β-diversity analysis. Redundancy analysis showed the relationships of the communities in the fields to pH and nutrient, humus, and/or water content. The complexity of eukaryotic and prokaryotic networks was apparently higher in the cabbage-cultivated soils than those in the maize-cultivated soils. The node SVs (nSVs) of the networks were mainly derived from two eukaryotic phyla: Ascomycota and Cercozoa, and four prokaryotic phyla: Pseudomonadota, Acidobacteriota, Actinomycetota, and Gemmatimonadota. The networks were complexed by cropping from maize to cabbage, suggesting the formation of a flexible network under crop rotation. Ten out of the 16 eukaryotic nSVs were specifically found in the cabbage-cultivated soils were derived from protists, indicating the potential contribution of protists to the formation of complex eukaryotic networks. [ABSTRACT FROM AUTHOR]

Nematodes are abundant metazoans that play crucial roles in nutrient recycle in the pedosphere. Although high-throughput amplicon sequencing is a powerful tool for the taxonomic profiling of soil nematodes, polymerase chain reaction (PCR) primers for amplification of the 18S ribosomal RNA (SSU) gene and preparation of template DNAs have not been sufficiently evaluated. We investigated nematode community structure in copse soil using four nematode-specific (regions 1–4) and two universal (regions U1 and U2) primer sets for the SSU gene regions with two DNAs prepared from copse-derived mixed nematodes and whole soil. The major nematode-derived sequence variants (SVs) identified in each region was detected in both template DNAs. Order level taxonomy and feeding type of identified nematode-derived SVs were distantly related between the two DNA preparations, and the region U2 was closely related to region 4 in the non-metric multidimensional scaling (NMDS) based on Bray-Curtis dissimilarity. Thus, the universal primers for region U2 could be used to analyze soil nematode communities. We further applied this method to analyze the nematodes living in two sampling sites of a sweet potato-cultivated field, where the plants were differently growing. The structure of nematode-derived SVs from the two sites was distantly related in the principal coordinate analysis (PCoA) with weighted unifrac distances, suggesting their distinct soil environments. The resultant ecophysiological status of the nematode communities in the copse and field on the basis of feeding behavior and maturity indices was fairly consistent with those of the copse- and the cultivated house garden-derived nematodes in prior studies. These findings will be useful for the DNA metabarcoding of soil eukaryotes, including nematodes, using soil DNAs. [ABSTRACT FROM AUTHOR]

The xeroderma pigmentosum variant (XP-V) is one of the most common forms of this cancer-prone syndrome. XP groups A through G are characterized by defective nucleotide excision repair, whereas the XP-V phenotype is proficient in this pathway. The XPV gene encodes DNA polymerase η, which catalyzes an accurate translesion synthesis, indicating that the XPV gene contributes tumor suppression in normal individuals. Here we describe the genomic structure and chromosomal localization of the XPV gene, which includes 11 exons covering the entire coding sequence, lacks a TATA sequence in the upstream region of the transcription-initiation, and is located at the chromosome band 6p21.1-6p12. Analyses of patient-derived XP-V cell lines strongly suggested that three of four cell lines carried homozygous mutations in the XPV gene. The fourth cell line, XP1RO, carried heterozygous point mutations in the XPV gene, one of which was located at the splice acceptor site of exon 2, resulting in the omission of exon 2 from the mature mRNA. These findings provide a basis for diagnosis and therapy of XP-V patients. Oncogene (2000) 19, 4721–4728 [ABSTRACT FROM AUTHOR]

Quantitative taxonomic compositions of nematode communities help to assess soil environments due to their rich abundance and various feeding habitats. DNA metabarcoding by the 18S ribosomal RNA gene (SSU) regions were preferentially used for analyses of soil nematode communities, but the optimal regions for high-throughput amplicon sequencing have not previously been well investigated. In this work, we performed Illumina-based amplicon sequencing of four SSU regions (regions 1–4) to identify suitable regions for nematode metabarcoding using the taxonomic structures of nematodes from uncultivated field, copse, and cultivated house garden soils. The fewest nematode-derived sequence variants (SVs) were detected in region 3, and the total nematode-derived SVs were comparable in regions 1 and 4. The relative abundances of reads in regions 1 and 4 were consistent in both orders and feeding groups with prior studies, thus suggesting that region 4 is a suitable target for the DNA barcoding of nematode communities. Distinct community structures of nematodes were detected in the taxon, feeding habitat, and life-history strategy of each sample; i.e., Dorylamida- and Rhabditida-derived plant feeders were most abundant in the copse soil, Rhabditida-derived bacteria feeders in the house garden soil, and Mononchida- and Dorylamida-derived omnivores and predators and Rhabditida-derived bacteria feeders in the field soil. Additionally, low- and high-colonizer–persister (cp) groups of nematodes dominated in the house garden and copse soils, respectively, whereas both groups were found in the field soil, suggesting bacteria-rich garden soil, undisturbed and plant-rich copse soil, and a transient status of nematode communities in the field soil. These results were also supported by the maturity indices of the three sampling sites. Finally, the influence of the primer tail sequences was demonstrated to be insignificant on amplification. These findings will be useful for DNA metabarcoding of soil nematode communities by amplicon sequencing. [ABSTRACT FROM AUTHOR]

Nematodes are representative soil metazoans with diverged species that play crucial roles in nutrient recycling in the pedosphere. Qualitative and quantitative information on nematode communities is useful for assessing soil quality, and DNA barcode-mediated taxonomic analysis is a powerful tool to investigate taxonomic compositions and changes in nematode communities. Here, we investigated four regions (regions 1–4) of the 18S small subunit ribosomal RNA (SSU) gene as PCR targets of deep amplicon sequencing for the taxonomic profiling of individual soil nematodes. We determined the sequence variants (SVs) of 4 SSU regions for 96 nematodes (total 384 amplicons) isolated from copse soils and assigned their taxonomy using the QIIME2 software with dada2 or deblur algorithm and the SILVA database. Dada2 detected approximately 2-fold more nematode-derived SVs than deblur, and a larger number of SVs were obtained in regions 1 and 4 than those in other regions. These results and sufficient reference sequence coverage in region 4 indicated that DNA barcoding using a primer set for region 4 followed by dada2-based analysis would be most suitable for soil nematode taxonomic analysis. Eighteen SSU-derived operational taxonomic units (rOTUs) were obtained from 68 isolates, and their orders were determined based on the phylogenetic trees built by four regional sequences of rOTUs and 116 nematode reference species as well as the BLASTN search. The majority of the isolates were derived from three major orders Dorylaimida (6 rOTUs, 51.5% in 68 isolates), Rhabditida (4 rOTUs, 29.4%), and Triplonchida (7 rOTUs, 17.6%). The predicted feeding types of the isolates were fungivores (38.2% in total nematodes), plant feeders (32.4%), and 14.7% for both bacterivores and omnivores/predators. Additionally, we attempted to improve the branch structure of phylogenetic trees by using long nucleotide sequences artificially prepared by connecting regional sequences, but the effect was limited. [ABSTRACT FROM AUTHOR]

Cyanobacteria have evolved various photoacclimation processes to perform oxygenic photosynthesis under different light environments. Chromatic acclimation (CA) is a widely recognized and ecologically important type of photoacclimation, whereby cyanobacteria alter the absorbing light colors of a supermolecular antenna complex called the phycobilisome. To date, several CA variants that regulate the green-absorbing phycoerythrin (PE) and/or the red-absorbing phycocyanin (PC) within the hemi-discoidal form of phycobilisome have been characterized. In this study, we identified a unique CA regulatory gene cluster encoding yellow–green-absorbing phycoerythrocyanin (PEC) and a rod-membrane linker protein (CpcL) for the rod-shaped form of phycobilisome. Using the cyanobacterium Leptolyngbya sp. PCC 6406, we revealed novel CA variants regulating PEC (CA7) and the rod-shaped phycobilisome (CA0), which maximize yellow–green light-harvesting capacity and balance the excitation of photosystems, respectively. Analysis of the distribution of CA gene clusters in 445 cyanobacteria genomes revealed eight CA variants responding to green and red light, which are classified based on the presence of PEC, PE, cpcL , and CA photosensor genes. Phylogenetic analysis further suggested that the emergence of CA7 was a single event and preceded that of heterocystous strains, whereas the acquisition of CA0 occurred multiple times. Taken together, these results offer novel insights into the diversity and evolution of the complex cyanobacterial photoacclimation mechanisms. Certain cyanobacteria alter components of their light-harvesting complex, the phycobilisome, via a process called chromatic acclimation (CA). We discovered novel types of CA that regulate yellow–green-absorbing phycoerythrocyanin and an atypical rod-shaped phycobilisome. By analyzing the genomes of 445 cyanobacteria strains, we revealed a total of eight CA variants responding to green and red light in the cyanobacteria phylum and inferred how these variants evolved. [ABSTRACT FROM AUTHOR]

We aimed to develop the bioassays for genotixicity and/or oxidative damage using the recombinant yeast. A genotoxicity assay was developed using recombinant Saccharomyces cerevisiae strain BY4741 with a green fluorescent protein (GFP) reporter plasmid, driven by the DNA damage-responsive RNR3 promoter. Enhanced fluorescence induction was observed in DNA repair-deficient strains treated with methyl methanesulfonate, but not with hydrogen peroxide. A GFP reporter yeast strain driven by the oxidative stress-responsive TRX2 promoter was newly developed to assess oxidative damage, but fluorescence was poorly induced by oxidants. In place of GFP, yeast strains with luciferase gene reporter plasmids ( luc2 and luc2CP, encoding stable and unstable luciferase, respectively) were prepared. Transient induction of luciferase activity was clearly detected only in a TRX2 promoter-driven luc2CP reporter strain within 90 min of oxidant exposure. However, luciferase was strongly induced by hydroxyurea in the RNR3 promoter-driven luc2 and GFP reporter strains over 8 h after the exposure, suggesting that the RNR3 promoter is continuously upregulated by DNA damage, whereas the TRX2 promoter is transiently activated by oxidative agents. Luciferase activity levels were also increased in a TRX2-promoter-driven luc2CP reporter strain treated with tert-butyl hydroperoxide and menadione and weakly induced with diamide and diethyl maleate. Weakly enhanced luciferase activity induction was detected in the sod1Δ, sod2Δ, and rad27Δ strains treated with hydrogen peroxide compared with that in the wild-type strain. In conclusion, tests using GFP and stable luciferase reporters are useful for genotoxicity, and oxidative damage can be clearly detected by assay with an unstable luciferase reporter. [ABSTRACT FROM AUTHOR]

Soil nematodes play crucial roles in the soil food web and are a suitable indicator for assessing soil environments and ecosystems. Previous nematode community analyses based on nematode morphology classification have been shown to be useful for assessing various soil environments. Here we have conducted DNA barcode analysis for soil nematode community analyses in Japanese soils. We isolated nematodes from two different environmental soils of an unmanaged flowerbed and an agricultural field using the improved flotation-sieving method. Small subunit (SSU) rDNA fragments were directly amplified from each of 68 (flowerbed samples) and 48 (field samples) isolated nematodes to determine the nucleotide sequence. Sixteen and thirteen operational taxonomic units (OTUs) were obtained by multiple sequence alignment from the flowerbed and agricultural field nematodes, respectively. All 29 SSU rDNA-derived OTUs (rOTUs) were further mapped onto a phylogenetic tree with 107 known nematode species. Interestingly, the two nematode communities examined were clearly distinct from each other in terms of trophic groups: Animal predators and plant feeders were markedly abundant in the flowerbed soils, in contrast, bacterial feeders were dominantly observed in the agricultural field soils. The data from the flowerbed nematodes suggests a possible food web among two different trophic nematode groups and plants (weeds) in the closed soil environment. Finally, DNA sequences derived from the mitochondrial cytochrome oxidase c subunit 1 (COI) gene were determined as a DNA barcode from 43 agricultural field soil nematodes. These nematodes were assigned to 13 rDNA-derived OTUs, but in the COI gene analysis were assigned to 23 COI gene-derived OTUs (cOTUs), indicating that COI gene-based barcoding may provide higher taxonomic resolution than conventional SSU rDNA-barcoding in soil nematode community analysis. [ABSTRACT FROM AUTHOR]

In the course of systematic RNA interference (RNAi)-based screens with helicase-like genes in Caenorhabditis elegans, we have identified the drh-3( D2005.5) gene as a candidate gene for protection against X-ray irradiation. This gene encodes a novel RNA helicase-like protein that is similar to two nematode Dicer-related helicases (DRH). Here, we have showed the increased expression of drh-3 transcripts during maturation of larvae to adults, and characterized the phenotype of drh-3-interferred nematodes using feeding RNAi method. RNAi-mediated depletion of the drh-3 transcripts caused embryonic lethality of F1 progeny and temperature-sensitive reproductive capacity but did not affect the nematode life span. F1 progeny from drh-3( RNAi) animals exhibited increased lethality after X-ray irradiation or exposure to camptothecin. In drh-3( RNAi) worms, aggregated chromosomes were observed in diakinesis oocyte nuclei. In developing early zygotic embryos from drh-3( RNAi) worms, abnormally segregated chromosomes were observed and embryonic development was largely arrested at the mid-stages of embryogenesis. Finally, examination of checkpoint responses in mitotic germ cells with regards to replication arrest by hydroxyurea and X-ray-induced DNA damage suggested that both checkpoints function normally under these genotoxic stress conditions. Taken together, these results indicate that the drh-3 gene is essential for the development of germ-lines by maintaining chromosomal integrity in C. elegans. [ABSTRACT FROM AUTHOR]

Cdt1 is an essential component for the assembly of a pre-replicative complex. Cdt1 activity is inhibited by geminin, which also participates in neural development and embryonic differentiation in many eukaryotes. Although Cdt1 homologues have been identified in organisms ranging from yeast to human, geminin homologues had not been described for Caenorhabditis elegans and fungi. Here, we identify the C. elegans geminin, GMN-1. Biochemical analysis reveals that GMN-1 associates with C. elegans CDT-1, the Hox protein NOB-1, and the Six protein CEH-32. GMN-1 inhibits not only the interaction between mouse Cdt1 and Mcm6 but also licensing activity in Xenopus egg extracts. RNA interference-mediated reduction of GMN-1 is associated with enlarged germ nuclei with aberrant nucleolar morphology, severely impaired gametogenesis, and chromosome bridging in intestinal cells. We conclude that the Cdt1-geminin system is conserved throughout metazoans and that geminin has evolved in these taxa to regulate proliferation and differentiation by directly interacting with Cdt1 and homeobox proteins. [ABSTRACT FROM AUTHOR]

The yeast Dbp9p is a member of the DEAD box family of RNA helicases, which are thought to be involved in RNA metabolism. Dbp9p seems to function in ribosomal RNA biogenesis, but it has not been biochemically characterized. To analyze the enzymatic characteristics of the protein, we expressed a recombinant Dbp9p in Escherichia coli and purified it to homogeneity. The purified protein exhibited RNA unwinding and binding activity in the absence of NTP, and this activity was abolished by a mutation in the RNA-binding domain. We then characterized the ATPase activity of Dbp9p with respect to cofactor specificity; the activity was found to be severely inhibited by yeast total RNA and moderately inhibited by poly(U), poly(A), and poly(C) but to be stimulated by yeast genomic DNA and salmon sperm DNA. In addition, Dbp9p exhibited DNA-DNA and DNA-RNA helicase activity in the presence of ATP. These results indicate that Dbp9p has biochemical characteristics unique among DEAD box proteins. [ABSTRACT FROM AUTHOR]

The Dicer-related helicases (DRHs) are members of a helicase subfamily, and mammalian DRHs such as retinoic acid-inducible gene-I (RIG-I), are involved in antiviral immunity. Caenorhabditis elegans DRH-1 and DRH-3 play crucial roles in antiviral function and chromosome segregation, respectively. Although intrinsic double-stranded RNA-dependent ATP-hydrolyzing activity has been observed in the recombinant DRH-3 protein prepared from Escherichia coli, there are no reports of biochemical studies of the nematode RIG-I homolog DRH-1. In this study, the secondary structure prediction by JPred4 revealed that DRH-1 and DRH-3 had distinct N-terminal regions and that a 200-amino acid N-terminal region of DRH-1 could form a structure very rich in α-helices. We investigated expressions and purifications of a codon-optimized DRH-1 with four different N-terminal tags, identifying poly-histidine (His)-small ubiquitin-like modifier (SUMO) as a suitable tag for DRH-1 preparation. Full-length (isoform a) and a N-terminal truncated (isoform b) of DRH-1 were purified as the His-SUMO-tagged fusion proteins. Finally, the nucleic acid-dependent ATPase activities were investigated for the two His-SUMO-tagged DRH-1 isoforms and His-tagged DRH-3. The tagged DRH-3 exhibited dsRNA-dependent ATPase activity. However, detectable dsRNA dependency of ATPase activities was not found in either isoform of tagged DRH-1 and a tag-free DRH-1 (isoform a) treated with SUMO protease. These observations suggest that DRH-1 and its short isoform have no or poor nucleic acid-dependent ATPase activity, unlike DRH-3 and mammalian DRHs. [ABSTRACT FROM AUTHOR]

Cyanobacterial phytochrome-class photosensors are recently emerging optogenetic tools. We isolated Fischerella sp. strain NIES-3754 from hotspring at Suwa-shrine, Suwa, Nagano, Japan. We determined complete genome sequence of the NIES-3754 strain, which is composed of one chromosome and two putative replicons (total 5,826,863 bp containing no gaps). We identified photosensor genes of 5 phytochromes and 9 cyanobacteriochromes, which will facilitate optogenetics of thermophile. [ABSTRACT FROM AUTHOR]

To explore the diverse photoreceptors of cyanobacteria, we isolated Nostoc sp. strain NIES-3756 from soil at Mimomi-Park, Chiba, Japan, and determined its complete genome sequence. The Genome consists of one chromosome and two plasmids (total 6,987,571 bp containing no gaps). The NIES-3756 strain carries 7 phytochrome and 12 cyanobacteriochrome genes, which will facilitate the studies of phytochrome-based bioengineering. [ABSTRACT FROM AUTHOR]