Structure and regulatory expression of rice proliferating cell nuclear antigen (PCNA) gene

Proliferating cell nuclear antigen (PCNA) is an essential replication factor in eukaryotic cells.The structure of the PCNA gene isolated from rice genomic DNA as well as cDNAs for ...Thesis (Ph.D. in Agriculture)--University of Tsukuba, (A), no. 927, 1991.9.3

Endogenization and excision of human herpesvirus 6 in human genomes

Sequences homologous to human herpesvirus 6 (HHV-6) are integrated within the nuclear genome of about 1% of humans, but it is not clear how this came about. It is also uncertain whether integrated HHV-6 can reactive into an infectious virus. HHV-6 integrates into telomeres, and this has recently been associated with polymorphisms affecting MOV10L1. MOV10L1 is located on the subtelomere of chromosome 22q (chr22q) and is required to make PIWI-interacting RNAs (piRNAs). As piRNAs block germline integration of transposons, piRNA-mediated repression of HHV-6 integration has been proposed to explain this association.In vitro, recombination of the HHV-6 genome along its terminal direct repeats (DRs) leads to excision from the telomere and viral reactivation, but the expected "solo-DR scar" has not been describedin vivo. Here we screened for integrated HHV-6 in 7,485 Japanese subjects using whole-genome sequencing (WGS). Integrated HHV-6 was associated with polymorphisms on chr22q. However, in contrast to prior work, we find that the reported MOV10L1 polymorphism is physically linked to an ancient endogenous HHV-6A variant integrated into the telomere of chr22q in East Asians. Unexpectedly, an HHV-6B variant has also endogenized in chr22q; two endogenous HHV-6 variants at this locus thus account for 72% of all integrated HHV-6 in Japan. We also report human genomes carrying only one portion of the HHV-6B genome, a solo-DR, supporting in vivo excision and possible viral reactivation. Together these results explain the recently-reported association between integrated HHV-6 and MOV10L1/piRNAs, suggest potential exaptation of HHV-6 in its coevolution with human chr22q, and clarify the evolution and risk of reactivation of the only intact (non-retro)viral genome known to be present in human germlines

Somatic cell reprogramming-free generation of genetically modified pigs

Genetically modified pigs for biomedical applications have been mainly generated using the somatic cell nuclear transfer technique; however, this approach requires complex micromanipulation techniques and sometimes increases the risks of both prenatal and postnatal death by faulty epigenetic reprogramming of a donor somatic cell nucleus. As a result, the production of genetically modified pigs has not been widely applied. We provide a simple method for CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 gene editing in pigs that involves the introduction of Cas9 protein and single-guide RNA into in vitro fertilized zygotes by electroporation. The use of gene editing by electroporation of Cas9 protein (GEEP) resulted in highly efficient targeted gene disruption and was validated by the efficient production of Myostatin mutant pigs. Because GEEP does not require the complex methods associated with micromanipulation for somatic reprogramming, it has the potential for facilitating the genetic modification of pigs

Comparative evaluation of SNVs, indels, and structural variations detected with short- and long-read sequencing data

Abstract Short- and long-read sequencing technologies are routinely used to detect DNA variants, including SNVs, indels, and structural variations (SVs). However, the differences in the quality and quantity of variants detected between short- and long-read data are not fully understood. In this study, we comprehensively evaluated the variant calling performance of short- and long-read-based SNV, indel, and SV detection algorithms (6 for SNVs, 12 for indels, and 13 for SVs) using a novel evaluation framework incorporating manual visual inspection. The results showed that indel-insertion calls greater than 10 bp were poorly detected by short-read-based detection algorithms compared to long-read-based algorithms; however, the recall and precision of SNV and indel-deletion detection were similar between short- and long-read data. The recall of SV detection with short-read-based algorithms was significantly lower in repetitive regions, especially for small- to intermediate-sized SVs, than that detected with long-read-based algorithms. In contrast, the recall and precision of SV detection in nonrepetitive regions were similar between short- and long-read data. These findings suggest the need for refined strategies, such as incorporating multiple variant detection algorithms, to generate a more complete set of variants using short-read data

Constitutive E2F Expression in Tobacco Plants Exhibits Altered Cell Cycle Control and Morphological Change in a Cell Type-Specific Manner

The E2F family plays a pivotal role in cell cycle control and is conserved among plants and animals, but not in fungi. This provides for the possibility that the E2F family was integrated during the development of higher organisms, but little is known about this. We examined the effect of E2F ectopically expressed in transgenic tobacco (Nicotiana tabacum) plants on growth and development using E2Fa (AtE2F3) and DPa from Arabidopsis. E2Fa-DPa double transgenic lines exhibited altered phenotypes with curled leaves, round shaped petals, and shortened pistils. In mature but not immature leaves of the double transgenic lines, there were enlarged nuclei with increasing ploidy levels accompanied by the ectopic expression of S phase- but not M phase-specific genes. This indicates that a high expression of E2F promotes endoreduplication by accelerating S phase entry in terminally differentiated cells with limited mitotic activity. Furthermore, mature leaves of the transgenic plants contained increased numbers of small cells, especially on the palisade (adaxial) side of the outer region toward the edge, and the leaf strips exhibited hormone-independent callus formation when cultured in vitro. These observations suggest that an enhanced E2F activity modulates cell cycle in a cell type-specific manner and affects plant morphology depending on a balance between activities for committing to S phase and M phase, which likely differ between organs or tissues

NESmapper: Accurate Prediction of Leucine-Rich Nuclear Export Signals Using Activity-Based Profiles

<div><p>The nuclear export of proteins is regulated largely through the exportin/CRM1 pathway, which involves the specific recognition of leucine-rich nuclear export signals (NESs) in the cargo proteins, and modulates nuclear–cytoplasmic protein shuttling by antagonizing the nuclear import activity mediated by importins and the nuclear import signal (NLS). Although the prediction of NESs can help to define proteins that undergo regulated nuclear export, current methods of predicting NESs, including computational tools and consensus-sequence-based searches, have limited accuracy, especially in terms of their specificity. We found that each residue within an NES largely contributes independently and additively to the entire nuclear export activity. We created activity-based profiles of all classes of NESs with a comprehensive mutational analysis in mammalian cells. The profiles highlight a number of specific activity-affecting residues not only at the conserved hydrophobic positions but also in the linker and flanking regions. We then developed a computational tool, NESmapper, to predict NESs by using profiles that had been further optimized by training and combining the amino acid properties of the NES-flanking regions. This tool successfully reduced the considerable number of false positives, and the overall prediction accuracy was higher than that of other methods, including NESsential and Wregex. This profile-based prediction strategy is a reliable way to identify functional protein motifs. NESmapper is available at <a href="http://sourceforge.net/projects/nesmapper" target="_blank">http://sourceforge.net/projects/nesmapper</a>.</p></div

Positive and negative NES datasets obtained from four different data resources.

<p>(<b>A</b>) Artificial NES datasets. (<b>B</b>) DUB NES datasets. (<b>C</b>) Valid NES datasets. (<b>D</b>) Sp-protein datasets. The positive and negative datasets (B-P2 and B-N2) of the DUB datasets and the negative training dataset (D-N2) of the Sp-protein datasets were always included in the training data for the profile optimization, whereas the other training datasets were used only when they were not contained in a test dataset to be used. For example, when we conducted the prediction test with the test datasets, A-P1 and A-N1, we used the optimized profiles for NESmapper, that were trained with C-N2, in addition to B-P2, B-N2, and D-N2.</p

Nuclear export activity of class 1a and class 1c NES mutants.

<p>(<b>A</b>) Class 1a NESs carrying mutations at two hydrophobic positions and three spacer positions between Φ2 and Φ4. (<b>B</b>) Class 1c NESs carrying mutations at three positions within the spacer region between Φ2 and Φ3. These NES mutants were assayed for their nuclear export activity in NIH3T3 cells, and the activities were classified as scores from 1 to 10, as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003841#pcbi.1003841.s001" target="_blank">Figure S1</a>. The scores are indicated at the right columns of the corresponding sequences. Altered bases are highlighted in blue.</p

Activity-based profiles of CRM1-dependent NES.

<p>(<b>A</b>) Activity-based profile of class 1a/3 NES. Class 1a/3 NES is an extension of class 1a NES, in which the N-terminal region of class 1a and the C-terminal region of class 3 overlap. A single amino acid residue of a class 1a/3 NES template sequence, indicated at the top of the matrix, was replaced with the various other residues indicated in the left column. The nuclear export activity of the NES mutant was assayed in NIH3T3 cells. The indicated activity scores were determined as in <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003841#pcbi.1003841.s001" target="_blank">Figure S1</a>. This template NES has an activity score of 8. Scores with higher, slightly higher, and lower activities than the average value for each position are shown in red, orange, and blue, respectively. At several mutational positions, modified templates with a different level of basal activity were used to obtain more dispersed scores. The conserved hydrophobic positions (Φ0–Φ4) are marked on the template sequence. The scores at the Φ0 position (P^a) were estimated based on the data of Güttler et al <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003841#pcbi.1003841-Gttler1" target="_blank">[16]</a>. Blanks represent undetermined scores. (<b>B</b>) NES profile of the spacer region between the Φ1 and Φ2 positions of a class 1b NES. The template sequence has a standard activity score of 4. (PSSELAKLAGLDLN) (<b>C</b>) NES profile for the spacer regions between Φ1 and Φ3 positions of the class 1c NES. The template sequence (SELAEKLQAGLDLN) has an activity score of 8. (<b>D</b>) Activity-based profile of class 2 NESs. The template NES sequence, indicated at the top of the matrix, has a standard activity score of 3.</p