A live imaging system to analyze spatiotemporal dynamics of RNA polymerase II modification in Arabidopsis thaliana.

Spatiotemporal changes in general transcription levels play a vital role in the dynamic regulation of various critical activities. Phosphorylation levels at Ser2 in heptad repeats within the C-terminal domain of RNA polymerase II, representing the elongation form, is an indicator of transcription. However, rapid transcriptional changes during tissue development and cellular phenomena are difficult to capture in living organisms. We introduced a genetically encoded system termed modification-specific intracellular antibody (mintbody) into Arabidopsis thaliana. We developed a protein processing- and 2A peptide-mediated two-component system for real-time quantitative measurement of endogenous modification level. This system enables quantitative tracking of the spatiotemporal dynamics of transcription. Using this method, we observed that the transcription level varies among tissues in the root and changes dynamically during the mitotic phase. The approach is effective for achieving live visualization of the transcription level in a single cell and facilitates an improved understanding of spatiotemporal transcription dynamics.

Huge solitary-infected liver cyst successfully managed by deroofing operation to remove 3.8 l of pus.

Simple liver cysts are usually asymptomatic, and complications are uncommon. However, infection of cysts can very occasionally occur, and this troublesome complication requires treatment. An 87-year-old woman admitted for dyspnea, abdominal discomfort, fever, and mild icterus underwent ultrasonographic (US) and computed tomography (CT) examinations, and a huge cystic lesion was identified in the right hepatic lobe. The diagnosis of an infected gigantic liver cyst was made by abdominal CT and percutaneous transhepatic drainage under ultrasonographic guidance. The pus viscosity was so high that pus drainage was ineffective. Bacterial culture was positive for Klebsiella pneumoniae. The cyst diameter was approximately 21 cm, and the total pus discharge was 3.8 l. Emergency operation to fenestrate the cyst wall relieved the patient's critical condition. This report is clinically significant because 3.8 l of pus may be one of the largest reported quantity of drained pus from an infected cyst. We found no reports of > 3.8 l of drained pus in an English literature search. It is also very rare that imaging identified postoperative liver regeneration filling the huge empty space previously occupied by the cyst. Large liver cysts in older people should be carefully followed, preparing for the rare possibility of infection.

Two combinatorial patterns of telomere histone marks in plants with canonical and non-canonical telomere repeats.

Telomeres, nucleoprotein structures at the ends of linear eukaryotic chromosomes, are crucial for the maintenance of genome integrity. In most plants, telomeres consist of conserved tandem repeat units comprising the TTTAGGG motif. Recently, non-canonical telomeres were described in several plants and plant taxons, including the carnivorous plant Genlisea hispidula (TTCAGG/TTTCAGG), the genus Cestrum (Solanaceae; TTTTTTAGGG), and plants from the Asparagales order with either a vertebrate-type telomere repeat TTAGGG or Allium genus-specific CTCGGTTATGGG repeat. We analyzed epigenetic modifications of telomeric histones in plants with canonical and non-canonical telomeres, and further in telomeric chromatin captured from leaves of Nicotiana benthamiana transiently transformed by telomere CRISPR-dCas9-eGFP, and of Arabidopsis thaliana stably transformed with TALE_telo C-3×GFP. Two combinatorial patterns of telomeric histone modifications were identified: (i) an Arabidopsis-like pattern (A. thaliana, G. hispidula, Genlisea nigrocaulis, Allium cepa, Narcissus pseudonarcissus, Petunia hybrida, Solanum tuberosum, Solanum lycopersicum) with telomeric histones decorated predominantly by H3K9me2; (ii) a tobacco-like pattern (Nicotiana tabacum, N. benthamiana, C. elegans) with a strong H3K27me3 signal. Our data suggest that epigenetic modifications of plant telomere-associated histones are related neither to the sequence of the telomere motif nor to the lengths of the telomeres. Nor the phylogenetic position of the species plays the role; representatives of the Solanaceae family are included in both groups. As both patterns of histone marks are compatible with fully functional telomeres in respective plants, we conclude that the described specific differences in histone marks are not critical for telomere functions.

Ectopic pancreatitis in the antral stomach causing gastric outlet obstruction: a case of successful resection.

Ectopic pancreas is a congenital developmental anomaly that may be caused by embryologic errors. The pathogenetic details of ectopic pancreas remain unclear, but it has gradually been determined to originate at the gene level. DNA errors during embryological development cause ectopic pancreas. Ectopic pancreas is generally asymptomatic and harmless throughout the patient's entire life; in very rare cases, however, it can be symptomatic and harmful, causing pain, fever, bleeding, and other adverse effects. We herein report an unusual case involving a 52-year-old Japanese woman who presented with nausea and vomiting due to inflammation of an ectopic pancreas in the gastric antrum. Ectopic pancreatitis caused severe pyloric stenosis, and she was unable to ingest food or water even after admission to the internal medicine ward. Ectopic pancreatitis is a very rare clinical condition, but clinicians should be cautious of this uncommon disease in patients who develop gastric outlet obstruction without cancer or ulcers. The present case report is clinically significant because surgical treatment (distal partial gastrectomy) relieved the patient's serious distress rapidly and successfully after unsuccessful conservative treatments for acute ectopic pancreatitis. Antrectomy (distal partial gastrectomy) might be useful for ectopic pancreas, especially in case of gastric outlet obstruction due to the ectopic pancreatitis.

Auxin decreases chromatin accessibility through the TIR1/AFBs auxin signaling pathway in proliferative cells.

Chromatin accessibility is closely associated with chromatin functions such as gene expression, DNA replication, and maintenance of DNA integrity. However, the relationship between chromatin accessibility and plant hormone signaling has remained elusive. Here, based on the correlation between chromatin accessibility and DNA damage, we used the sensitivity to DNA double strand breaks (DSBs) as an indicator of chromatin accessibility and demonstrated that auxin regulates chromatin accessibility through the TIR1/AFBs signaling pathway in proliferative cells. Treatment of proliferating plant cells with an inhibitor of the TIR1/AFBs auxin signaling pathway, PEO-IAA, caused chromatin loosening, indicating that auxin signaling functions to decrease chromatin accessibility. In addition, a transcriptome analysis revealed that several histone H4 genes and a histone chaperone gene, FAS1, are positively regulated through the TIR1/AFBs signaling pathway, suggesting that auxin plays a role in promoting nucleosome assembly. Analysis of the fas1 mutant of Arabidopsis thaliana confirmed that FAS1 is required for the auxin-dependent decrease in chromatin accessibility. These results suggest that the positive regulation of chromatin-related genes mediated by the TIR1/AFBs auxin signaling pathway enhances nucleosome assembly, resulting in decreased chromatin accessibility in proliferative cells.

An ion beam-induced Arabidopsis mutant with marked chromosomal rearrangement.

Ion beams have been used as an effective tool in mutation breeding for the creation of crops with novel characteristics. Recent analyses have revealed that ion beams induce large chromosomal alterations, in addition to small mutations comprising base changes or frameshifts. In an effort to understand the potential capability of ion beams, we analyzed an Arabidopsis mutant possessing an abnormal genetic trait. The Arabidopsis mutant uvh3-2 is hypersensitive to UVB radiation when photoreactivation is unavailable. uvh3-2 plants grow normally and produce seeds by self-pollination. SSLP and CAPS analyses of F2 plants showed abnormal recombination frequency on chromosomes 2 and 3. PCR-based analysis and sequencing revealed that one-third of chromosome 3 was translocated to chromosome 2 in uvh3-2. FISH analysis using a 180 bp centromeric repeat and 45S ribosomal DNA (rDNA) as probes showed that the 45S rDNA signal was positioned away from that of the 180 bp centromeric repeat in uvh3-2, suggesting the insertion of a large chromosome fragment into the chromosome with 45S rDNA clusters. F1 plants derived from a cross between uvh3-2 and wild-type showed reduced fertility. PCR-based analysis of F2 plants suggested that reproductive cells carrying normal chromosome 2 and uvh3-2-derived chromosome 3 are unable to survive and therefore produce zygote. These results showed that ion beams could induce marked genomic alterations, and could possibly lead to the generation of novel plant species and crop strains.

Visualization of specific repetitive genomic sequences with fluorescent TALEs in Arabidopsis thaliana.

Live imaging of the dynamics of nuclear organization provides the opportunity to uncover the mechanisms responsible for four-dimensional genome architecture. Here, we describe the use of fluorescent protein (FP) fusions of transcription activator-like effectors (TALEs) to visualize endogenous genomic sequences in Arabidopsis thaliana. The ability to engineer sequence-specific TALEs permits the investigation of precise genomic sequences. We could detect TALE-FP signals associated with centromeric, telomeric, and rDNA repeats and the signal distribution was consistent with that observed by fluorescent in situ hybridization. TALE-FPs are advantageous because they permit the observation of intact tissues. We used our TALE-FP method to investigate the nuclei of several multicellular plant tissues including roots, hypocotyls, leaves, and flowers. Because TALE-FPs permit live-cell imaging, we successfully observed the temporal dynamics of centromeres and telomeres in plant organs. Fusing TALEs to multimeric FPs enhanced the signal intensity when observing telomeres. We found that the mobility of telomeres was different in sub-nuclear regions. Transgenic plants stably expressing TALE-FPs will provide new insights into chromatin organization and dynamics in multicellular organisms.

Mapping of T-DNA and Ac/Ds by TAIL-PCR to Analyze Chromosomal Rearrangements.

Insertion mutagenesis using known DNA sequences such as T-DNA and transposons is an important tool for studies on gene function in plant sciences. The transposons Activator (Ac)/Dissociation (Ds) have been systematically used to manipulate plant chromosomes. For both of these applications, the recovery of genomic DNA sequences flanking the insertions is required to estimate the sizes and/or scales of the reconstituted chromosomes. In this chapter, we describe the protocols for thermal asymmetric interlaced PCR (TAIL-PCR) for isolation of genomic sequences flanking DNA inserts in plant genomes.

Generation of an artificial ring chromosome in Arabidopsis by Cre/LoxP-mediated recombination.

A eukaryotic chromosome consists of a centromere, two telomeres and a number of replication origins, and 'artificial chromosomes' may be created in yeast and mammals when these three elements are artificially joined and introduced into cells. Plant artificial chromosomes (PACs) have been suggested as new vectors for the development of new crops and as tools for basic research on chromosomes. However, indisputable PAC formation has not yet been confirmed. Here, we present a method for generating PACs in the model plant Arabidopsis thaliana using the Cre/LoxP and Activator/Dissociation element systems. The successfully generated PAC, designated AtARC1 (A. thaliana artificial ring chromosome 1), originated from a centromeric edge of the long arm of chromosome 2, but its size (2.85 Mb) is much smaller than that of the original chromosome (26.3 Mb). Although AtARC1 contains only a short centromere domain consisting of 180 bp repeats approximately 250 kb in length, compared with the 3 Mb domain on the original chromosome 2, centromere-specific histone H3 (HTR12) was detected on the centromeric region. This result supported the observed stability of the PAC during mitosis in the absence of selection, and transmission of the PAC to the next generation through meiosis. Because AtARC1 contains a unique LoxP site driven by the CaMV 35S promoter, it is possible to introduce a selectable marker and desired transgenes into AtARC1 at the LoxP site using Cre recombinase. Therefore, AtARC1 meets the criteria for a PAC and is a promising vector.

Proteome analysis of protein partners to nucleosomes containing canonical H2A or the variant histones H2A.Z or H2A.X.

Although the existence of histone variants has been known for quite some time, only recently are we grasping the breadth and diversity of the cellular processes in which they are involved. Of particular interest are the two variants of histone H2A, H2A.Z and H2A.X because of their roles in regulation of gene expression and in DNA double-strand break repair, respectively. We hypothesize that nucleosomes containing these variants may perform their distinct functions by interacting with different sets of proteins. Here, we present our proteome analysis aimed at identifying protein partners that interact with nucleosomes containing H2A.Z, H2A.X or their canonical H2A counterpart. Our development of a nucleosome-pull down assay and analysis of the recovered nucleosome-interacting proteins by mass spectrometry allowed us to directly compare nuclear partners of these variant-containing nucleosomes to those containing canonical H2A. To our knowledge, our data represent the first systematic analysis of the H2A.Z and H2A.X interactome in the context of nucleosome structure.

Does BLM helicase unwind nucleosomal DNA?

RecQ helicases maintain chromosome stability by resolving several highly specific DNA structures. BLM, the protein mutated in Bloom's syndrome, is a member of the RecQ helicase family, and possesses both DNA-unwinding and strand-annealing activity. In this study, we have investigated the unwinding activity of BLM on nucleosomal DNA, the natural nuclear substrate for the enzyme. We generated a DNA template including a strong nucleosome-positioning sequence flanked by forked DNA, which is reportedly one of the preferred DNA substrates for BLM. BLM did not possess detectable unwinding activity toward the forked DNA substrate. However, the truncated BLM, lacking annealing activity, unwound it partially. In the presence of the single-stranded DNA-binding protein RPA, the unwinding activity of both the full-length and the truncated BLMs was promoted. Next, the histone octamer was reconstituted onto the forked DNA to generate a forked mononucleosome. Full-length BLM did not unwind the nucleosomal DNA, but truncated BLM unwound it partially. The unwinding activity for the mononucleosome was not promoted dramatically with RPA. These results indicate that full-length BLM may require additional factors to unwind nucleosomal DNA in vivo, and that RPA is, on its own, unable to perform this auxiliary function.

The Arabidopsis SDG4 contributes to the regulation of pollen tube growth by methylation of histone H3 lysines 4 and 36 in mature pollen.

Plant SET domain proteins are known to be involved in the epigenetic control of gene expression during plant development. Here, we report that the Arabidopsis SET domain protein, SDG4, contributes to the epigenetic regulation of pollen tube growth, thus affecting fertilization. Using an SDG4-GFP fusion construct, the chromosomal localization of SDG4 was established in tobacco BY-2 cells. In Arabidopsis, sdg4 knockout showed reproductive defects. Tissue-specific expression analyses indicated that SDG4 is the major ASH1-related gene expressed in the pollen. Immunological analyses demonstrated that SDG4 was involved in the methylation of histone H3 in the inflorescence and pollen grains. The significant reduction in the amount of methylated histone H3 K4 and K36 in sdg4 pollen vegetative nuclei resulted in suppression of pollen tube growth. Our results indicate that SDG4 is capable of modulating the expression of genes that function in the growth of pollen tube by methylation of specific lysine residues of the histone H3 in the vegetative nuclei.

Characterization of a splicing variant of plant Aurora kinase.

Aurora kinases play a key role in chromosome segregation and cytokinesis. In plants, three Aurora kinases (AtAUR1-AtAUR3) have been identified in Arabidopsis thaliana. Here, we report an AtAUR2 splicing variant (AtAUR2S), which lacks the fourth exon encoding a part of the kinase domain of AtAUR2. AtAUR2S was shown to have lost its kinase activity to phosphorylate histone H3 at Ser10; however, it maintained its ability to bind to histone H3. The localization pattern of AtAUR2S was the same as that of AtAUR2. The findings suggest that AtAUR2S affects cell division by competing with AtAUR2.

Aurora kinase is required for chromosome segregation in tobacco BY-2 cells.

Post-translational modifications of core histone tails play crucial roles in chromatin structure and function. Although phosphorylation of Ser10 and Ser28 (H3S10ph and H3S28ph) of histone H3 is ubiquitous among eukaryotes, the phosphorylation mechanism during the cell cycle remains unclear. In the present study, H3S10ph and H3S28ph in tobacco BY-2 cells were observed in the pericentromeric regions during mitosis. Moreover, the Aurora kinase inhibitor Hesperadin inhibited the kinase activity of Arabidopsis thaliana Aurora kinase 3 (AtAUR3) in phosphorylating both Ser10 and Ser28 of histone H3 in vitro. Consistently, Hesperadin inhibited both H3S10ph and H3S28ph during mitosis in BY-2 cells. These results indicate that plant Aurora kinases phosphorylate not only Ser10, but also Ser28 of histone H3 in vivo. Hesperadin treatment increased the ratio of metaphase cells, while the ratio of anaphase/telophase cells decreased, although the mitotic index was not affected in Hesperadin-treated cells. These results suggest that Hesperadin induces delayed transition from metaphase to anaphase, and early exit from mitosis after chromosome segregation. In addition, micronuclei were observed frequently and lagging chromosomes, caused by the delay and failure of sister chromatid separation, were observed at anaphase and telophase in Hesperadin-treated BY-2 cells. The data obtained here suggest that plant Aurora kinases and H3S10ph/H3S28ph may have a role in chromosome segregation and metaphase/anaphase transition.

Generation of monoclonal antibodies against chromosomal antigens that have a high sequence similarity between human and mouse.

We raised monoclonal antibodies by immunizing mice with total chromosome proteins extracted from isolated human metaphase chromosomes. The indirect immunofluorescence screening of hybridoma cell lines provided 15 monoclonal antibodies against the chromosomal antigens. The antigen proteins of the mAbs were identified by immunoblotting as core histones or by immunoprecipitation followed by a peptide mass fingerprinting method as nuclear mitotic apparatus protein, heterogeneous nuclear ribonucleoprotein A2/B1, ribosomal protein S4, linker histone and beta-actin. During mitosis, localizations of these proteins on chromosomes were clearly observed using the obtained antibodies. These results indicate that the current strategy is effective for obtaining monoclonal antibodies useful for immunoblotting and/or immunofluorescent staining of human proteins, using the antigens with high homology to mouse proteins.

Characterization and dynamic analysis of Arabidopsis condensin subunits, AtCAP-H and AtCAP-H2.

Condensin complexes are thought to play essential roles in mitotic chromosome assembly and segregation in eukaryotes. To date, two condensin complexes (condensin I and II) have been identified. Both complexes contain two structural maintenance of chromosome (SMC) subunits and three non-SMC subunits. In plants, little is known about the localization and function of all the condensin subunits. Here, we report the analyses on the localization of a non-SMC subunit of Arabidopsis condensin I and II, AtCAP-H, and AtCAP-H2, respectively. Our study indicated that localization of AtCAP-H and AtCAP-H2 is dynamically changed through the mitotic cell cycle using GFP-tagged AtCAP-H and AtCAP-H2 in tobacco cultured cells. They are localized at mitotic chromosomes from prometaphase to telophase. However, their localization in interphase is quite different. AtCAP-H was mainly found in the cytoplasm whereas AtCAP-H2 was mainly found in a nucleolus. It is revealed using GFP-tagged deletion mutant s of AtCAP-H that the kleisin-gamma middle domain (GM domain) is a unique domain only in AtCAP-H, responsible for chromosomal localization. We propose that the GM domain of CAP-H is essential for its chromosomal localization at mitosis and thus proper function of CAP-H. Differences in localization of AtCAP-H and AtCAP-H2 at interphase also suggest their functional differentiation.

Crystallization and preliminary X-ray crystallographic analysis of a conserved domain in plants and prokaryotes from Pyrococcus horikoshii OT3.

A plant- and prokaryote-conserved domain (PPC) has previously been found in AT-hook motif nuclear localized protein 1 (AHL1) localized in the nuclear matrix of Arabidopsis thaliana (AtAHL1). AtAHL1 has a DNA-binding function. Mutation analyses of AtAHL1 has previously revealed that the hydrophobic region of the PPC domain is essential for its nuclear localization. In this study, the PPC of the hyperthermophilic archaebacterium Pyrococcus horikoshii (PhPPC) was crystallized using the hanging-drop vapour-diffusion method. The crystals belonged to the hexagonal space group P6(3)22, with unit-cell parameters a = b = 53.69, c = 159.2 A. Data were obtained at 100 K, with diffraction being observed to a resolution of 1.7 A. A complete data set from crystals of the SeMet-substituted protein was also obtained.

An upper limit of the ratio of DNA volume to nuclear volume exists in plants.

The variations in nuclear DNA content from 2 x 10(2) to 2.5 x 10(5) Mbp are reported in higher plants. The major finding so far is that the genome size of plant species differs by three orders of magnitude, which are more variable than the other organisms. Investigations pertaining to the manner in which DNA is packaged in the nucleus provide us with basic information on the made of DNA existence in the plant nucleus. However, the fundamentals on nuclear DNA content and nuclear size, which underlie and enable the flexible containment of such large differences in nuclear DNA content, remain unknown. We analyzed the nuclear volumes of plants with 2C value DNA contents ranging from 3.2 x 10(2) to 1.0 x 10(5) Mbp. As a result, we obtained a constant ratio between the DNA volume and nuclear volume, which does not exceed 3%. Furthermore, we also demonstrate that the nuclear Rabl model of chromatin organisation is not a common 3-D structure, even in plants with large nuclear DNA contents. The existence of an upper limit of DNA volume ratio would present a basal parameter for the future insight into the nuclear organisation in higher plants.

Identification of a novel plant MAR DNA binding protein localized on chromosomal surfaces.

We identified a novel nucleoplasm localized protein in Arabidopsis called AT-hook motif nuclear localized protein 1 (AHL1), which was isolated by visual screening of transformants using random GFP::cDNA fusions. AHL1 contains an AT-hook motif and unknown conserved PPC (plants and prokaryotes conserved) domain that includes a hydrophobic region. Approximately 30 paralogues were identified in the Arabidopsis genome. Proteins with PPC-like domains are found in Bacteria, Archaea and the plant kingdom, but in Bacteria and Archaea the PPC containing proteins of do not have an AT-hook motif. Thus, the PPC domain is evolutionary conserved and has a new function such as AT-rich DNA binding. AHL1 was mainly localized in the nucleoplasm, but little in the nucleolus and heterochromatic region, and was concentrated in the boundary region between euchromatin and heterochromatin. Biochemically, AHL1 was also found in the nuclear matrix fraction. In the M phase, AHL1 was localized on the chromosomal surface. The AT-hook motif was essential for matrix attachment region (MAR) binding, and the hydrophobic region of the PPC was indispensable for nuclear localization. Our results suggest that AHL1 is a novel plant MAR binding protein, which is related to the positioning of chromatin fibers in the nucleus by the presence of an AT-hook motif and PPC domain. In addition, AHL1 is located on the surface of chromosomes during mitosis.