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Biomedical subjects

Kiichi Fukui

Publications and source records attributed to Kiichi Fukui.

At least 37 records · Page 2Linked to original sources
PubMed2005

Fluorescent labeling of plant chromosomes in suspension by FISH.

By optimizing the concentration and time of treatment with hydroxyurea (HU), a DNA synthesis inhibitor, and trifluralin, a microtubule inhibitor, a highly effective (over 60%) cell cycle synchronization method for rye and barley meristem cells was developed. Chromosome suspensions containing highly purified and morphologically intact rye and barley chromosomes were prepared from the meristems of their root tips by homogenization. Digoxigenin-labeled 5S rDNA was used as a probe in FISH for the rye chromosomes in the suspension, and biotin-labeled 17S rDNA and centromeric DNA were used in FISH for the rye and barley chromosome suspensions, respectively. Bright signals were detected at the specific regions of interest on the chromosomes. The results indicate that the method developed in this study is useful for selection and sorting of chromosomes that are not distinguishable by other means, using specific fluorescent labeling by FISH of the chromosomes in suspension.

Chromosomes, Plant↗
PubMed2004

Hydrophobic core around tyrosine for human endothelin-1 investigated by photochemically induced dynamic nuclear polarization nuclear magnetic resonance and matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

Human endothelin-1 (ET-1) is a potent cardiovascular bioactive peptide. Its activity is based on the C-terminal residues, e.g., Trp 21 in particular. Recently, we reported an NMR solution structure of ET-1, which has a C-terminal hydrophobic core around Tyr 13. This C-terminal conformation does not agree with a previously reported X-ray crystal structure. To clarify the discrepancy, we performed photo-CIDNP NMR in combination with MALDI-TOF MS. The photo-CIDNP results revealed that the Tyr 13 aromatic ring is concealed in a hydrophobic interaction. MALDI-TOF MS experiments showed this is an intramolecular interaction in monomeric form, which is also supported by sedimentation analysis and two-dimensional NMR cross-peak line shapes. Thus, we confirmed the intramolecular hydrophobic core around Tyr 13 in aqueous solution, which agrees with the solution structure. The C-terminal conformational discrepancy between the solution and crystal was caused by the intermolecular hydrogen bond between Tyr 13 of one molecule and Asp 8 of the other in a dimer-like formation of crystalline ET-1. On the other hand, we indicated that endothelin-3, another isoform of the endothelin, has an apparent self-association equilibrium under the same condition in which three tyrosines participate.

Amino Acid Sequence↗
PubMed2004

Synthesis of long Poly(dG).Poly(dC) DNA using enzymatic reaction.

Non-defect Poly(dG).Poly(dC) of 500 bp (170 nm) has been synthesized by using enzymatic reactions and was characterized by its UV spectrum, showing that conjugated pi-electrons between base pairs are spread over the DNA molecule suggesting the absence of structural defects.

DNA↗
PubMed2004

Obtaining transgenic plants using the bio-active beads method.

Several methods of transformation are currently available for delivering exogenous DNA into animal and plant cells. In this study, a novel and efficient transformation system for DNA delivery/expression with a capacity to transport DNA of high molecular weight was developed. This system can overcome the shortcomings of traditional transformation methods such as Agrobacterium-mediated transformation, particle bombardment, and the electroporation method. The method developed in this study uses calcium alginate micro beads to immobilize DNA molecules in combination with polyethylene glycol treatment. In addition, it is simple and low-cost, and requires limited equipment. Using this method, we have successfully transformed tobacco plants, screening by kanamycin resistance. The transformed genes in the transformants were confirmed by PCR and Southern hybridization.

Alginates↗
PubMed2004

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.

Amino Acid Sequence↗
PubMed2004

Cell culture in a closed nano-space.

The minimum size of a closed nano-space in which cells can survive was determined using 4-nl nanowells. One or two cells could divide in the nanowell. Our results suggest that the cell division activity in the nano-space is determined by the conflict between intercellular effects and consumption of substrates.

Journal Article↗
PubMed2004

Development of new dosimetry using extended DNA fibers.

We applied fluorescent microscopy to monitor the damage of DNA upon exposure to gamma radiation. Our developed dosimetry demonstrated that the number of breaks in DNA is proportional to the dose of the irradiation but is not dependent on dose rate of the irradiation and the GC content of DNA.

Journal Article↗
PubMed2004

A novel transfection method for mammalian cells using calcium alginate microbeads.

The direct transfer of genetic materials into mammalian cells is an indispensable technique. We have developed calcium alginate (CA) microbeads which can deliver plasmid DNAs and yeast artificial chromosomes into plant and yeast cells. In this paper, we demonstrate the effective transfection of mammalian cells by CA microbeads immobilizing plasmid DNAs. The transfection was performed using the pEGFP-C1 plasmid containing the cytomegalovirus (CMV) promoter and enhanced green fluorescent protein (EGFP) gene. The transient expression of EGFP was observed 24 h after transfection. The expression efficiency was maximum when the concentration of sodium alginate was 1% and the amount of plasmid DNA was increased to 100 microg. The expression efficiency of our method using CA microbeads is 2-10 times higher than that of the polyethylene glycol (PEG) method. Our results suggest that the CA microbead mediated transfection of mammalian cells effectively delivers genetic materials into mammalian suspension cells.

Journal Article↗
PubMed2004

Flow karyotypes and chromosomal DNA contents of genus Triticum species and rye (Secale cereale).

The flow cytometry and chromosome imaging method were jointly used for analyzing genome content and chromosomal DNA content of hexaploid wheat (AABBDD), hexaploid triticale (AABBRR), tetraploid wheat (AABB), and AA, BB, DD genome donors and RR genome rye. Their genome sizes were 34.4 pg, 40.9 pg, 26.2 pg, 12.1 pg, 13.7 pg, 10.5 pg, and 16.9 pg, respectively. The 2C nuclear DNA content of BB genome donor with 13.7 pg was the highest value among the other genome donors, AA or DD. The genome content of tetraploid wheat, unlike hexaploid wheat or hexaploid triticale, was larger than the sum of the genomes of AA and BB genome donors. The DNA content of each chromosome ranged from 1.22 pg in DD genome donor to 2.61 pg in rye. Each chromosome peak was divided into three to four groups. Only one chromosome was included in the highest chromosomal DNA peak in hexaploid wheat, tetraploid wheat, DD genome donor and rye but two chromosomes in AA, BB genome donors, and hexaploid triticale. Correlation between 2C nuclear DNA content and chromosome density volume was the highest value compared with the other chromosomal parameters of chromosome area, or chromosome length.

Chromosomes, Plant↗
PubMed2003

Repeated chromosome splitting targeted to delta sequences in Saccharomyces cerevisiae.

We have previously developed a chromosome-splitting technique based on homologous recombination in Saccharomyces cerevisiae. To facilitate chromosome splitting at multiple sites, we focused on the delta sequences that are distributed in more than 200 copies throughout the yeast genome. We constructed a new chromosome-splitting vector harboring the YFLWdelta4 sequence and the hisG-URA3-hisG cassette, and transformed yeast cells with this vector. The karyotype analysis of transformants showed that chromosomes XIV, III, and IV, or other chromosomes are split. After the excision of the URA3 gene, the transformant with split chromosome IV was subsequently transformed with the same vector. Karyotype analysis revealed that further splitting occurred at chromosome X, the split chromosome IV, or other chromosomes. These results indicate that delta sequences are efficient target sites for repeated chromosome splitting at multiple sites with a single vector.

Journal Article↗
PubMed2003

Centromere-specific acetylation of histone H4 in barley detected through three-dimensional microscopy.

Histone acetylation affects chromatin conformation and transcriptional activity. However, the structural role of histone acetylation at specific chromosomal regions, such as the centromere, is poorly understood. In this study, histone H4 acetylation and its localization in barley interphase nuclei are revealed by three-dimensional microscopy. The centromeres form a ring-like allocation near the nuclear membrane in barley. Immunofluorescence studies on non-fixed, interphase nuclei treatment revealed ring-like distribution of the highly acetylated histone H4, located near the nuclear membrane at one pole of the nucleus. This fluorescent structure was similar to the centromere cluster and referred to as hyperacetylated region (HAR). The distribution pattern of the acetylated histone H4 was similar to each of the K5, K8, K12 and K16 lysine residues, although H4 acetylated at K5, K8 and K12 residues was found in almost all nuclei, whereas H4 acetylated at K16 was weakly observed in only half of the nuclei. Each HAR consists of two strongly acetylated cores and a halo-like, less acetylated surrounding area. Fluorescence signals from centromere-specific repetitive sequences of barley, detected through three-dimensional fluorescence in situ hybridization (3D-FISH), co-localized with the HAR corresponding to the K5 residue acetylation, but the signals did not completely overlap each other. These findings indicate that histone acetylation specifically occurring at the centromeres likely have certain structural roles for the centromere.

Acetylation↗
PubMed2003

Development of a quantitative pachytene chromosome map in Oryza sativa by imaging methods.

A higher GC content region of an Oryza sativa chromosome can be specifically visualized by double staining with propidium iodide (PI) and 4, 6-diamidino-2-phenylindole (DAPI). This procedure allows identification of chromosome 9 from the other rice chromosomes at the pachytene stage. Using rice chromosome 9 as a model, an imaging method to construct a pachytene chromosomal map was developed by quantifying the fluorescence profile (FP) of each chromomere. The pachytene map of chromosome 9 consists of twenty-two chromomeres including four chromomeres within the nucleolar organizing region (NOR) and satellite region. The pachytene map was compared with the corresponding somatic prometaphase map and the linkage map. The differences among the three maps indicate that each map depicts specific biological information, which is difficult to be substituted by the other maps.

Chromosome Mapping↗
PubMed2003

Transformation of yeast using calcium alginate microbeads with surface-immobilized chromosomal DNA.

Yeast artificial chromosomes (YACs) are useful cloning vectors that have the capacity to carry large DNA inserts. The largest barrier to using such large DNA molecules in transformation experiments has been their physical instability in solution. We developed a new method of transforming yeast using chromosome-sized DNA. The method uses calcium alginate microbeads to immobilize high-density yeast chromosomal DNA. Chromosomal DNA immobilized on microbeads is physically stabilized compared with naked chromosomal DNA. The microbead-mediated transformation performed well, not only with respect to the transformation frequency with large DNA molecules (> 100 kb) but also in successful transformation using split chromosome DNA that exceeded 450 kb.

Adsorption↗
PubMed2002

Variability of chromosomal DNA contents in maize (Zea mays L.) inbred and hybrid lines.

The flow karyotypes of different maize (Zea mays L.) inbred and hybrid lines were analyzed. The accumulation and isolation of large quantities of high-quality metaphase chromosomes from root tips was achieved from many kinds of maize lines. The chromosome suspensions were prepared by a simple slicing method from synchronized maize root tips and analyzed by flow cytometry. Variations of experimental flow karyotypes were detected among inbred and hybrid lines in terms of the positions and/or the numbers of chromosome peaks. The 2C DNA amount among eight inbred lines ranged from 5.09 to 5.52 pg. The selection of appropriate maize lines is critical for sorting specific single chromosome types. At least five different chromosome types can be discriminated and sorted from five maize lines. The variability of DNA content in maize chromosome 1 was 9.1%, ranging from 0.685 to 0.747 pg. Differences were detected in the DNA content of homologous chromosome 1 of hybrid lines.

Algorithms↗
PubMed2002

Trends in site-number change of rDNA loci during polyploid evolution in Sanguisorba (Rosaceae).

To elucidate the evolutionary dynamics of rDNA site number in polyploid plants, we determined 5S and 18S-5.8S-26S rDNA sites for ten species of Sanguisorba (2n=14, 28, 56) and a single species of each of three outgroup genera, Agrimonia (2n=28), Rosa (2n=14), and Rubus (2n=14) by the fluorescence in situ hybridization (FISH) method. We also estimated phylogenetic relationships among these species using matK chloroplast DNA (cpDNA) sequences, and reconstructed the evolutionary history of rDNA site number based on the maximum parsimony method. The 2n=14 and 2n=28 plants of all genera except Rosa carried two 5S rDNA sites, whereas Rosa and 2n=56 plants carried four sites. The 2n=14 plants had two 18S-5.8S-26S rDNA sites, whereas Sanguisorba annua and 2n=28 plants had four or six sites. Phylogenetic analysis showed that polyploidization from 2n=14 to 2n=28 has occurred once or three times in Sanguisorba and Agrimonia. The 5S rDNA sites duplicated during each ancestral polyploidization were evidently lost after each polyploidization. However, the duplicated 18S-5.8S-26S rDNA sites were all conserved after each polyploidization. Thus, the duplicated 5S rDNA sites tend to have been eliminated, whereas those of 18S-5.8S-26S rDNA tend to have been conserved in Sanguisorba. In the most parsimonious hypothesis, 2n=14 in S. annua is a secondary, putatively dysploid state, reduced from 2n=28.

Biological Evolution↗
PubMed2002

Cell cycle-dependent and lysine residue-specific dynamic changes of histone H4 acetylation in barley.

Histone acetylation affects chromatin conformation and regulates various cellular functions, such as transcription and cell cycle progression. Although mitosis dependent transcriptional silencing and large-scale chromatin structural changes are well established, acetylation of histone H4 during the mitosis is poorly understood in plants. Here, the dynamics of acetylation of histone H4 in defined genome regions has been examined in the fixed barley cells throughout the mitosis by three-dimensional microscopy. Patterns of strong acetylation of the two lysine residues K5 and K16 of histone H4 in the barley genomes were found to be different. In interphase nuclei, H4 acetylated at K 16 was associated with the gene-rich, telomere-associated hemispheres, whereas K5 acetylation was detected in centromeric regions where the heterochromatin is distributed. Regions of strong K5 acetylation changed dynamically as the cell cycle proceeded. At prometaphase, centromeric acetylation at K5 decreased suddenly, with accompanying rapid increases of acetylation in the nucleolar organizing regions (NORs). Reverse changes occurred at telophase. On the other hand, the strongly acetylated regions of the K16 showed changes compatible with transcriptional activities and chromosome condensation throughout the cell cycle. Telomeric acetylation at K16 was detected throughout the cell cycle, although it was reduced at metaphase which corresponds to the most condensed stage of the chromosomes. It is concluded that dynamic changes in H4 acetylation occur in a lysine residue-, stage-, and region-specific manner and that they correlate with changes in the chromosome structure through the cell cycle.

Acetylation↗