Contents

Review article

1. Scanning probe microscopy for chromosomal research (TAMAYO, J.)
   
   
   
2. The structure of human metaphase chromosomes: Its histological perspective and new horizons by atomic force microscopy (USHIKI, T.)
   
   
   
3. Single-molecule studies of chromatin fibers: A personal report (LEUBA, S. H.)
   
   
   

Original articles

1. Chromatin reconstitution: Development of a salt-dialysis method monitored by nano-technology (HIZUME, K.)
   
   
   
2. Three-dimensional helical coiling structures and band patterns of hydrous metaphase chromosomes observed by low vacuum scanning electron microscopy (INAGA, S.)
   
   
   
3. Imaging of chromosomes at nano-meter scale resolution using scanning near-field optical/atomic force microscopy (OHTANI, T.)
   
   
   
4. Scanning near field optical/atomic force microscopy of bromodeoxyuridine- incorporated human chromosomes (KIMURA, E.)
   
   
   
5. Changes in chromosomal surface structure by different isolation conditions (SONE, T.)
   
   
   
6. Mechanical elongation of the centromere in the barley metaphase chromosome (OTOBE, K.)
   
   
   
7. Integrated microfluidics for chromosome engineering − Preparation, transportation and manipulation (INOUE, T.)
   
   
   
8. Atomic force microscope-based dissection of human metaphase chromosomes and high resolutional imaging by carbon nanotube tip (IWABUCHI, S.)
   
   
   
9. Application of a microchamber array for DNA amplification using a novel dispensing method (MATSUBARA, Y.)

Summary

Title
Scanning Probe Microscopy for Chromosomal Research

Author
Javier TAMAYO and Mervyn MILES

Address
Instituto de Microelectronica de Madrid, IMM (CNM-CSIC), Madrid, Spain; and H. H. Wills Physics Laboratory and IRC for Nanotechnology, University of Bristol, Bristol, United Kingdom.

Summary
The study of chromosome structure with scanning probe microscopy provides a range of information from three-dimensional topographic structures through mechanical properties to optical information, usually fluorescence. For atomic force microscopy studies, the importance of removing cell debris from the chromosome surface has been recognized. Studies in aqueous environments reveal a highy swollen and tough chromosomal structure, but the charge interaction between the probe and specimen needs to be considered if high-resolution images are to be achieved. This charge interaction can be used to extract DNA strands of about 2 kbp from the chromosome. SPM studies of chromosomes may also be of value in the identification of defective chromosomes with either duplications or deletions.


Correspondence: Prof. Mervyn MILES, H. H. Wills Physics Laboratory and IRC for Nanotechnology, University of Bristol, Tyndall Avenue, Bristol, BS8 1TL, U. K.

Title
The Structure of Human Metaphase Chromosomes: Its Histological Perspective and New Horizons by Atomic Force Microscopy

Author
Tatsuo USHIKI, Osamu HOSHI, Kousuke IWAI, Eiji KIMURA and Masatsugu SHIGENO

Address
Division of Microscopic Anatomy and Bio-imaging, Department of Cellular Function, Niigata University Graduate School of Medical and Dental Sciences, Niigata; and Seiko Instruments Inc., Chiba Japan

Summary
Studies on the structure of the human chromosome were reviewed from the histological perspective and discussed in connection with our recent findings obtained mainly by atomic force microscopy (AFM). In this paper, we introduce several hitherto known models of the high-order structure of the metaphase chromosome and discuss the actual structure of chromosomes in relation to such structures as spiral chromatids, chromosome bands, and chromosome scaffolds. In chromosomes treated with Ohnuki・s hypotonic solution, the chromosome arms were elongated and showed a characteristic spiral pattern of chromatid fibers. On the other hand, alternating transverse ridges and grooves were clearly observed on the surface of chromosomes treated with 0.025% trypsin for G-banding, and these ridges and grooves corresponded to the dark and pale bands of G-banded chromosomes. Similar findings were also found in chromosomes treated with quinacrine mastards for Q-banding. Fibers bridging the gap between the sister chromatids were often observed in G/Q-banded chromosomes; these fibers tended to be restricted within the G/Q-positive portions, suggesting the presence of chromatin fibers bridging these regions. Based on these findings in conjunction with previous studies, we outlined the high-order structure of the human chromosome. Recent advances in nanotechnology have provided new AFM techniques for the imaging and handling of materials at nano-scale resolution. Application of these techniques to chromosome research is expected to provide valuable information on the chromosome structure in relation to its function.


Correspondence: Prof. Tatsuo USHIKI, Division of Microscopic Anatomy and Bio-imaging, Department of Cellular Function, Niigata University Graduate School of Medical and Dental Sciences, Asahimachi-dori 1, Niigata 951-8510, Japan
Phone: ＋81-25-227-2057, Fax: ＋81-25-224-1767
E-mail: t-ushiki@med.niigata-u.ac.jp

Title
Single-Molecule Studies of Chromatin Fibers: A Personal Report

Author
Sanford H. LEUBA and Jordanka ZLATANOVA

Address
Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Hillman Cancer Center, Pittsburgh, PA; and Department of Chemistry and Chemical Engineering, Polytechnic University, Brooklyn, NY, USA

Summary
With the advent of single-molecule techniques, macromolecular science has reached new horizons. Nowadays, we can observe, touch, stretch and twist biological macromolecules or their complexes, one-at-a time, in attempts to better understand their mechanical properties and to gain insights into their behavior in the living cell. Chromatin structure and function has been the focus of our research interests for many years. In the past decade, we have added some of the newly emerged single-molecule approaches to the more traditional biochemical and biophysical methods that we have been using throughout the years. This paper briefly summaries our studies on individual chromatin fibers using the atomic force microscope (AFM), optical tweezers, and magnetic tweezers. We believe that our results so far have contributed significantly to our understanding of chromatin, but we also hope that this is only the beginning, and that more exciting times lie ahead.

Correspondence: Dr. Sanford H. LEUBA Department of Cell Biology and Physiology, University of Pittsburgh School of Medicine, Hillman Cancer Center, University of Pittsburgh Cancer Institute Research Pavilion, Pittsburgh, PA 15213, USA
Tel: ＋1-412-623-7788, Fax: ＋1-412-623-4840
E-mail: leuba@pitt.edu

Title
Chromatin Reconstitution: Development of a Salt-dialysis Method Monitored by Nano-technology

Author
Kohji HIZUME, Shige H. YOSHIMURA, Hugo MARUYAMA, Joongbeak KIM, Hitoshi WADA 
and Kunio TAKEYASU

Address
Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto University Graduate School of Biostudies, Kyoto, Japan

Summary
The regulation of DNA replication and transcription is achieved by dynamic structural changes of chromatin in which a series of proteins will acquire accessibility to specific regions of the DNA strand. A combination of biochemistry and nano-technology is essential to address questions regarding the structural basis for such macromolecular mechanisms. In the present study, we established an efficient salt-dialysis method of chromatin reconstitution and employed atomic force microscopy (AFM) as a single-molecule-imaging technique, to monitor the efficiency of the reconstitution. At first, the reconstitution efficiency with short DNA molecules of several kilo-base pairs was low, although the salt dialysis yielded a ・beads-on-a-string・ structure of oligonucleosomes with each nucleosome trapping 158＋/−27 bp DNA. However, the efficiency for nucleosome formation became higher when longer DNA molecules with a super-helical constraint were used. A statistical analysis of the obtained AFM images identified a first-order relationship between the efficiency of the reconstitution and the length of the super-coiled DNA used. A high efficiency of 〜290 bp/nucleosome that is close to the in vivo situation was obtained with a 〜100 kbp template DNA. This enabled the structure-function studies of long chromatin molecules under well-defined conditions.


Correspondence: Prof. Kunio TAKEYASU, Laboratory of Plasma Membrane and Nuclear Signaling, Kyoto Univer-sity Graduate School of Biostudies, Kyoto 606-8502, Japan
Tel & Fax: ＋81-75-753-6852
E-mail: takeyasu@lif.kyoto-u.ac.jp

Title
Three-dimensional Helical Coiling Structures and Band Patterns of Hydrous Metaphase Chromosomes Observed by Low Vacuum Scanning Electron Microscopy

Author
Sumire INAGA, Keiichi TANAKA and Akihiro IINO

Address
Division of Genome Morphology, Department of Functional, Morphological and Regulatory Science, Tottori University Faculty of Medicine, Yonago; and Tanaka SEM Institute, Yonago, Japan

Summary
Helical coiling structures and band patterns of hydrous metaphase chromosomes were documented three-dimensionally by low vacuum scanning electron microscopy (SEM). Fixed or unfixed isolated Chinese hamster metaphase chromosomes were stained with platinum blue (Pt blue) and observed in the backscattered electron mode for low vacuum SEM without any hypotonic treatment or drying processes.
　Fibrous structures were shown both in the fixed and unfixed hydrous chromosomes; helical chromatid coils and their subcoils were clarified especially in the fixed chromosomes having contrasting alternative bands of light and darkness, while the translucent perichromosomal matrix and compact fibrous structures were recognized in the unfixed chromosomes. The helical coils were more clearly represented in a loosened chromatid of metaphase chromosomes. Treatment with a tris-HCl buffer solution and Pt blue staining in a hydrous condition successfully produced banding patterns similar to G-bands on metaphase chromosomes. These banded chromosomes observed by low vacuum SEM were also analyzed stereoscopically by field emission SEM after critical point drying.
　These findings indicate that: 1) native or unfixed chromosomes maintain the compact arrangement of high-order helical structures covered with the perichromosomal matrix; 2) helical coiling appearances of chromatids frequently observed in previous papers might be caused by loosening of the final level of the high-order structure of the metaphase chromosome; and 3) banding patterns might be produced by the rearrangement or reorganization of chromatin fibers at the 30 nm fiber level after the extraction of some chromosomal components including the peri- or intra-chromosomal materials during the banding procedure.


Correspondence: Dr. Sumire INAGA, Division of Genome Morphology, Department of Functional, Morphological and Regulatory Science, Tottori University Faculty of Medicine, Nishi-cho 86, Yonago, 683-8503 Japan
Tel: ＋81-859-34-8002, Fax: ＋81-859-34-8299
E-mail: sumire@grape.med.tottori-u.ac.jp

Title
Imaging of Chromosomes at Nano-Meter Scale Resolution Using Scanning Near-Field Optical/Atomic Force Microscopy

Author
Toshio OHTANI, Motoharu SHICHIRI, Daisuke FUKUSHI, Shigeru SUGIYAMA, Tomoyuki YOSHINO, Toshiro KOBORI, Shoji HAGIWARA and Tatsuo USHIKI

Address
National Food Research Institute, Tsukuba, Ibaraki; and Division of Microscopic Anatomy and Bio-imaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan

Summary
Topographic and fluorescent images of whole barley chromosomes stained with YOYO-1 were observed simultaneously by scanning near-field optical/atomic force microscopy (SNOM/AFM). The chromosome was relatively smooth and flat in the topographic images and no significant difference in height was present between regions of high fluorescent and low fluorescent intensity in the chromosomes. The telomeric region, labeled by fluorescence in situ hybridization (FISH) method, was also observed by SNOM/AFM at high resolution, and fluorescent signals of the telomeric region were clearly defined on the topographic image of chromatin fibers on the chromosome at the nano-meter scale level. Although the telomeric signals were usually visualized as a single fluorescent region at the end of sister chromatids by conventional light microscopy, they were observed separately as two fluorescent regions, less than 100-200 nm distance, using the SNOM/AFM. The SNOM/AFM offers great potential in identifying particular single gene location on chromosomes in the near future.


Correspondence: Dr. Toshio OHTANI, National Food Research Institute, 2-1-12 Kannondai, Tsukuba, Ibaraki 305-8643 Japan 
Phone: ＋81-29-838-8054, Fax: ＋8129-838-7181 
E-mail: ohtani@affrc.go.jp

Title
Scanning Near Field Optical/Atomic Force Microscopy of Bromodeoxyuridine-Incorporated Human Chromosomes

Author
Eiji KIMURA, Jiro HITOMI and Tatsuo USHIKI

Address
Division of Microscopic Anatomy and Bio-imaging, Department of Cellular Function, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

Summary
The present study applied scanning near field optical/atomic force microscopy (SNOM/AFM) to the observation of human chromosomes immunostained with an anti-BrdU antibody after incorporation of BrdU into DNA. Human lymphocytes were cultured in BrdU for 72 h and their chromosomes were prepared with a standard method for light microscopy. After additional fixation with 15% formalin in phosphate buffered saline, the specimens were denatured with 2N HCl with 0.1% Triton-X 100, immunostained with the anti-BrdU antibody, and observed both by fluorescence microscopy and by SNOM/AFM. The preparation technique used in the present study enabled the differential staining of sister chromatids in each chromosome, and sister chromatid exchanges (SCEs) were recognized in some chromosomes of the metaphase spread. Observations of the specimens by SNOM/AFM further provided the simultaneous collection of topographical and fluorescent images of the same portions of BrdU-incorporated chromosomes. The resolution of the fluorescence images by SNOM/AFM was greater than that obtained by fluorescence microscopy. Superimposition of topographical and fluorescent images of the chromosomes is useful for the precise analysis of the fine structure of chromosomes in relation to the SCEs. The application of SNOM/AFM to the BrdU-incorporated chromosomes is thus useful for the analysis of the fine structure of chromosomes in relation to their function.


Correspondence: Dr. Eiji KIMURA, Division of Microscopic Anatomy and Bio-imaging, Department of Cellular Function, Niigata University Graduate School of Medicine and Dental Sciences, Asahimachi-dori 1, Niigata, 951-8510 Japan
Phone: 81-25-227-2062, Fax: 81-25-224-1767
E-mail: eijik@med.niigata-u.ac.jp

Title
Changes in Chromosomal Surface Structure by Different Isolation Conditions

Author
Takefumi SONE, Megumi IWANO, Shouhei KOBAYASHI, Takeshi ISHIHARA, Naoto HORI, Hideaki TAKATA, Tatsuo USHIKI, Susumu UCHIYAMA and Kiichi FUKUI

Address
Department of Biotechnology, Osaka University, Graduate School of Engineering, Suita, Osaka; Graduate School of Biological Science, Nara Advanced Institute of Science and Technology, Ikoma, Nara; Division of Microscopic Anatomy and Bio-imaging, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan

Summary
The human cell cycle was synchronized and the chromosomes were isolated by a centrifugation method using two representative solutions for chromosome isolation (a polyamine buffer, PAB and citric acid solution, CAS) and fixatives. The centrifugation method yielded sufficient amounts of human metaphase chromosomes. Observation of the isolated chromosomes by scanning electron microscopy (SEM) revealed two types of surface structure which have been repeatedly reported to date: the human chromosomes in the PAB were relatively smooth but covered irregularly with scaly structures, while the surface of the chromosomes in the CAS exhibited a dense fibrous structure with a uniform diameter of 50-70 nm. Comparison of proteins extracted from chromosomes isolated with the PAB and CAS clearly indicated the removal of linker histones, H1, from chromosomes isolated with the CAS. These findings imply that the two different images of human chromosomes frequently observed by SEM are due to the removal of peripheral chromosomal materials including linker histones and/or the depletion of linker histones which prevent the surface chromatin fibers from scattering.


Correspondence: Prof. Kiichi FUKUI, Department of Biotechnology, Graduate School of Engineering, Osaka University, 2-1 Yamadaoka, Suita 565-0871, Japan
Tel: ＋81-6-6879-7440, Fax: ＋81-6-6879-7441
E-mail: kfukui@bio.eng.osaka-u.ac.jp

Present address of T. S.: Department of Molecular Biology, Research Institute for Microbial Diseases, Osaka University, 3-1 Yamadaoka, Suita 565-0871, Japan

Title
Mechanical Elongation of the Centromere in the Barley Metaphase Chromosome

Author
Kazunori OTOBE, Motoharu SHICHIRI, Daisuke FUKUSHI, Tomoyuki YOSHINO, Hidenobu NAKAO, Shigeru SUGIYAMA and Toshio OHTANI

Address
Department of Farm Mechanization and Engineering, National Agricultural Research Center, Tsukuba; Food Engineering Division, National Food Research Institute, Tsukuba, Japan

Summary
The present study investigated the mechanical elongation of the centromere in the barley chromosomes by a microneedle manipulation method for the structural analysis of the chromosomes. Chromosomes were extracted from barley root cells, affixed on a cover slip by a standard preparation method, and elongated in either distilled water, phosphate buffered saline (PBS), or 2×sodium saline citrate (SSC). The mechanical property of the chromosome elongation was assessed by the measurement of the force required for the elongation of chromosomes. This assessment has shown that the chromosomes in distilled water were much firmer than those in the PBS or 2×SSC. To confirm the elongation of the centromere, the elongated chromosomes were investigated by fluorescence in situ hybridization with a centromere probe. The fluorescence information indicated that the extent of the loosening of the centromere during elongation differed depending on the buffers used; the centromere elongated in 2×SSC was more loosened than that in the PBS. Atomic force microscopy also revealed the structure of the unpacked centromere after the mechanical elongation, when rows of fibrous structures about 30 to 50 nm thick were clearly observed in the centromere elongated in 2×SSC. The investigation of elongated chromosomes should prove useful for an understanding of the structural analysis of chromosomes.


Correspondence: Dr. Kazunori OTOBE, Department of Farm Mechanization and Engineering, National Agricultural Research Center, 3-1-1, Kannondai, Tsukuba, Ibaraki, 305-8666 Japan
Phone: ＋81-298-38-7389; Fax: ＋81-298-38-8551
E-mail: otobek@affrc.go.jp

Title
Integrated Microfluidics for Chromosome Engineering −Preparation, Transportation and Manipulation

Author
Takahito INOUE, Katsunori TAKAHASHI and Hiroshi YOKOYAMA

Address
Nanotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Japan

Summary
The design and fabrication process of microfluidics for chromosome transportation and manipulation are described. Micro-channels for fluid networks could be built on single-crystal silicon substrates by anisotropic wet etching. After injection of a chromosome-containing buffer solution into the channels by a mechanical pump, chromosome electrophoresis was carried out for its precise transportation. The behavior of chromosomes placed in direct (DC) and alternating (AC) electric fields is explained in detail. The frequency and size dependent amplitude of the chromosome observed in this study showed the existence of inertial hydrodynamic effects in an oscillatory motion. Using a rotating AC electric field, chromosomes could be rotated due to the induced polarization of surface charge and electric double layer of the chromosomes. This study thus shows the possibility of chromosome engineering on microchips, which is expected to enable the development of new devices for chromosome research.


Correspondence: Dr. Takahito INOUE, Nanotechnology Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), 1-1-1 Umezono, Tsukuba, Ibaraki, 305-8568 Japan
Tel: ＋81-298-61-5544, Fax: ＋81-298-61-5400
E-mail: takahito-inoue@aist.go.jp

Title
Atomic Force Microscope-Based Dissection of Human Metaphase Chromosomes and High Resolutional Imaging by Carbon Nanotube Tip

Author
Shinichiro IWABUCHI, Takuya MORI, Kenichi OGAWA, Kaori SATO, Masato SAITO, Yasutaka MORITA, Tatsuo USHIKI and Eiichi TAMIYA

Address
School of Materials Science, Japan Advanced Institute of Science and Technology, Tatsunokuchi, Ishikawa; Division of Microscopic Anatomy and Bio-imaging, Graduate School of Medical and Dental Sciences, Niigata University, Niigata, Japan

Summary
The present study was performed to introduce a novel chromosome dissection method employing atomic force microscopy (AFM) in a dynamic force mode for the chemical or molecular biological analysis of tiny chromosomal fragments. After AFM observation of human chromosomes prepared for light microscopy, a region of interest was dissected by increasing the loading force in a series of single-line scans of the target portion by controlling it with the amplitude reference of the tip in a dynamic force mode. The marker gene of the nucleolar organizing region (NOR) was amplified by our designed primers for 5.8S ribosomal DNA. After the dissection, topographic profiles in the section were then obtained with a carbon nanotube (CNT) probe in ambient condition. These results are discussed in relation to a fundamental technology for chromosomal analysis.


Correspondence: Prof. Eiichi TAMIYA, School of Materials Science, Japan Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa 923-1292, Japan
Phone: ＋81-761-51-1660, Fax: ＋81-761-51-1665
E-mail: tamiya@jaist.ac.jp

Title
Application of a Microchamber Array for DNA Amplification Using a Novel Dispensing Method

Author
Yasutaka MATSUBARA, Masaaki KOBAYASHI, Yasutaka MORITA and Eiichi TAMIYA

Address
The School of Materials Science, Japan Advanced Institute of Science and Technology, Ishikawa, Japan

Summary
We recently developed a microchamber array chip for DNA amplification by adopting semiconductor microfabrication technology; a polymerase chain reaction (PCR) was performed in the microchamber array, and the amplified DNA was detected using a fluorescent dye. In order to manipulate a single cell or sample into each microchamber individually in this system, the chip was directly sealed with a cover glass slip which impeded the retrieval of the products from each chamber. The present study was therefore carried out to improve the system by developing methods for covering the microchambers and introducing the reaction solution. First, we fabricated a microchamber array chip, and the oil layer was coated on the whole chip instead of the cover glass slip. The solution for DNA amplification was introduced into each chamber through an oil layer using a nano-liter dispenser. Following this, the microarray chip was placed onto the thermal cycling system for DNA amplification, and the amplified DNA was subsequently detected by fluorescence microscopy. In this system, the products were easily retrieved using a micromanipulator for further analysis.


Correspondence: Dr. Yasutaka MATSUBARA, The School of Materials Science; Japan
Advanced Institute of Science and Technology, 1-1 Asahidai, Tatsunokuchi, Ishikawa, 923-1292 Japan
Tel: ＋81-761-51-1663, Fax: ＋81-761-51-1665,
E-mail: yasutaka@jaist.ac.jp