Trapping DNA Replication Origins from the Human Genome

Synthesis of chromosomal DNA is initiated from multiple origins of replication in higher eukaryotes; however, little is known about these origins’ structures. We isolated the origin-derived nascent DNAs from a human repair-deficient cell line by blocking the replication forks near the origins using two different origin-trapping methods (i.e., UV- or chemical crosslinker-treatment and cell synchronization in early S phase using DNA replication inhibitors). Single-stranded DNAs (of 0.5–3 kb) that accumulated after such treatments were labeled with bromodeoxyuridine (BrdU). BrdU-labeled DNA was immunopurified after fractionation by alkaline sucrose density gradient centrifugation and cloned by complementary-strand synthesis and PCR amplification. Competitive PCR revealed an increased abundance of DNA derived from known replication origins (c-myc and lamin B2 genes) in the nascent DNA fractions from the UV-treated or crosslinked cells. Nucleotide sequences of 85 and 208 kb were obtained from the two libraries (I and II) prepared from the UV-treated log-phase cells and early S phase arrested cells, respectively. The libraries differed from each other in their G+C composition and replication-related motif contents, suggesting that differences existed between the origin fragments isolated by the two different origin-trapping methods. The replication activities for seven out of 12 putative origin loci from the early-S phase cells were shown by competitive PCR. We mapped 117 (library I) and 172 (library II) putative origin loci to the human genome; approximately 60% and 50% of these loci were assigned to the G-band and intragenic regions, respectively. Analyses of the flanking sequences of the mapped loci suggested that the putative origin loci tended to associate with genes (including conserved sites) and DNase I hypersensitive sites; however, poor correlations were found between such loci and the CpG islands, transcription start sites, and K27-acetylated histone H3 peaks

Taxonomic profiling of individual nematodes isolated from copse soils using deep amplicon sequencing of four distinct regions of the 18S ribosomal RNA gene.

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

Profiling nematode communities in unmanaged flowerbed and agricultural field soils in Japan by DNA barcode sequencing.

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

Distinct prokaryotic and eukaryotic communities and networks in two agricultural fields of central Japan with different histories of maize–cabbage rotation

Abstract 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

An unrooted phylogram of rOTUs from soil nematodes isolated from the flowerbed and agricultural field with predicted trophic types.

<p>Twenty-nine SSU rDNA barcode sequences of K01rOTUs (flowerbed samples) and H01rOTUs (agricultural field samples) were aligned for preparing a phylogenetic tree. The numbers of soil nematodes belonging to each rOTU correspond to the numbers of open (flowerbed samples) and closed (agricultural field samples) squares at the right. Numbers on nodes are bootstrap values (>50%). Trophic types indicated in the rOTUs were derived from those of the nematode species with the highest homology in SSU rDNA barcode sequences (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0051785#pone-0051785-t001" target="_blank">Table 1</a>), and the feeding types for the rOTUs containing a large number of nematode members are shown in boldface. Bar: 0.1 substitutions per site.</p

Photograph of the flowerbed and agricultural field sites for soil sampling.

<p>The unmanaged flowerbed framed by concrete walls (A) and the soybean-cultivated field (B) in the campus of Toyohashi University of Technology from autumn till winter in 2010.</p