RAPID COMMUNICATION |
Correspondence to: U.C. Lavania, Cytogenetics Division, Central Institute of Medicinal and Aromatic Plants, Lucknow 226 015, India. E-mail: lavania@cimap.res.in
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Summary |
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The conventional protocol for isolation of cell wall free nuclei for release of DNA fibers for plants involves mechanical removal of the cell wall and separation of debris by sieve filtration. The mechanical grinding pressure applied during the process leaves only the more tolerant G1 nuclei intact, and all other states of active nuclei that may be present in the target tissues (e.g., leaf) are simply crushed/disrupted during the isolation process. Here we describe an alternative enzymatic protocol for isolation of nuclei from root tip tissue. Cell wall free nuclei at a given stage of cell cycle, free of any cell debris, could be realized in suspension that are fit for preparation of extended fibers suitable for fiber FISH applications. The protocol utilizes selective harvest of active nuclei from root tip tissue in liquid suspension under the influence of cell wall-degrading enzymes, and provides opportunities to target cell cycle-specific nuclei from interphase through division phase for the release of extended DNA fibers. Availability of cell cycle-specific fibers may have added value in transcriptional analysis, DNA:RNA hybridization, visualization of DNA replication and replication forks, and improved FISH efficiency. (J Histochem Cytochem 51:12491253, 2003)
Key Words: fiber FISH, nuclear isolation, extended DNA fibers, mitotic fibers, chromosomal fibers
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Introduction |
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Fluorescence in situ hybridization (FISH), allows distinct positioning of unique and repetitive DNA sequences for the top-down chromosomal approach to complement bottom-up DNA marker and clone-based analysis for chromosome structure and landmarks for looking at genes, their clustering, and their orientation. This is greatly facilitating the development of physical and genetic maps (
Lately, in any mapping strategy that implement a bottom-up approach, the application of FISH to extended DNA fibers (
In DNA fiber mapping, the DNA molecules are released from interphase cell nuclei, stretched to some extent, and then immobilized on a solid support. Such DNA bound to solid support, called "DNA fibers," may consist of a single thread of DNA molecules in some experiments or bundles of chromatin fibers in others. Because the released DNA fibers become more accessible to probes and detection reagents, the efficiency of FISH is greatly enhanced, allowing the detection of DNA targets as small as a few hundred base pairs (
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Materials and Methods |
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Plant Materials
Fast-growing roots obtained from germinating seeds of Triticum aestivum L. var. Chinese Spring, bulbs of Allium cepa L., Allium sativum L., runners of Chlorophytum comosum L., and slips of Cymbopogon winterianus Sprengel were used as the test systems, representing moderately hard, soft and hard roots, and medium, large, and small size chromosomes/nuclei.
Preparation of Cell Wall Free Nuclei
Fast-growing roots about 1 cm long were excised and fixed in acetic acid:ethanol (1:3) for 5 min and then transferred to 1 x enzyme buffer pH 4.6 (prepared by mixing 40 ml of 10 mM citric acid + 60 ml of 10 mM trisodium citrate) in a small Petri dish. Depending on the thickness of roots, about 30 roots of Triticum aestivum (in which the roots are thin) or 10 roots of Allium (in which roots are relatively thicker in diameter) were taken out, their root caps removed, and placed in an Eppendorf tube containing 50 µl of enzyme solution [i.e., mixture of 3% (v/v) pectinase from Aspergillus niger and 2% (w/v) cellulase, i.e., 1.8% cellulase from Aspergillus niger and 0.2% "Onozuka" RS cellulase] with root tip side immersed in enzyme solution, and incubated at 37C for 2050 min. For harder roots such as those of wheat the incubation time could be 50 min, and for Allium just 20 min are sufficient. The enzymatic incubation time must be adjusted in such a way that the root tip zone gets just soft but not so soft that the tips lose their identity on being taken out. The incubated bundle of the roots is taken out and placed on a solid surface of a glass slide with a drop of enzyme buffer. The loose cells in the root tip meristematic zone are squeezed out by applying gentle pressure with the help of a needle or forceps. The nuclear suspension thus obtained is collected in an Eppendorf tube and the nuclei sedimented by gentle centrifugation. Again, 50 µl of enzyme solution is added to the nuclear sediment, pipette-mixed, and re-incubated for 46 hr at 37C. One should check intermittently the situation of the nuclei to see whether the cell wall has been completely dissolved. This can be easily done by taking out just 1 µl of nuclei suspended in enzyme solution, followed by staining in aceto-carmine and observation under a microscope. Once the optimal stage for the nuclei is achieved, i.e., completely free of cell wall, the enzymatic incubation is terminated by repeated washing in 1 x PBS and sedimentation of nuclei by pulses of mild centrifugation at 20003000 rpm. This leads to a nuclear suspension in 1 x PBS providing 50100 µl of nuclear suspension in PBS of desired nuclear density suitable for release of DNA chromatin fibers.
Monitoring the Cell Cycle Stage
Depending on the requirements, the fast-growing roots can be subjected to suitable prefixation. Normally, any fast-growing root would contain heterogeneous groups of nuclei covering all stages of the cell cycle. However, to enhance the frequency of a particular stage, the growing roots may be chilled for 24 hr at 4C, followed by growth at 25C for 2024 hr. This treatment brings about a sort of cell synchronization in the growing roots. Depending on the duration of the cell cycle for a given species, e.g., for wheat, 2324 hr would have a high frequency at metaphase and less than this at other stages of interphase. To obtain mitotic nuclei with scattered chromosomes, it is desirable for the roots to be chilled at 0C overnight in ice-cold water before fixation.
Preparation of Fibers
It is very important for the nuclear density in the nuclear suspension to be optimal, i.e., the nuclei do not overlap or collide when applied onto the slide. There should be an available internuclear space between the nuclei equal to the size of at least one nucleus or a little more, and most of the nuclei in suspension should be free of cell wall and other debris. Apply two drops of 1 µl each consecutively (to facilitate proper spread and uniform distribution) of the nuclear suspension on a clean glass slide towards one end and leave to air-dry for about 10 min. Then apply 10 µl of STE buffer [STE-1, i.e., 0.5% (w/v) SDS, 50 mM EDTA and 100 mM Tris, pH 7.0, or STE-2 i.e., STE-1 buffer containing 5 mM EDTA instead], leave for 5 min, and then gently tilt the slide at 45° to let buffer solution flow downward. The hydrodynamic kinetics developed with downward flowing liquid facilitates stretching of the DNA fibers. Air-dry the slides for 15 min and fix the chromatin/DNA fibers on the slides in freshly prepared acetic-ethanol (1:3) for 3 min, air-dry, and store in a moisture-free slide box for subsequent use for fiber FISH application. About 10 slides can be prepared at a time, and a sample from the batch can be tested for suitability of fiber spreads by staining with DAPI for observation under a microscope with UV excitation.
Probe Labeling and Hybridization
As probes for in situ hybridization (ISH), pTa71 plasmid DNA with a wheat 45S rDNA, an onion BAC clone with centromeric repeat sequences (
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Results |
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The procedure detailed here allows isolation of mitotically active nuclei in suspension from fast-growing roots of diverse systems encompassing germinating seeds, bulbous tissues, and vegetative shoots representing thin/thick, soft/hard root tips. Isolated nuclei free of cell wall and cytoplasm could be obtained in suspension enriched with active nuclei at a given stage of the cell cycle.
The nuclear suspension obtained comprises nuclei representing all stages of the cell cycle, not just G1-phase as is the case with the nuclear isolation protocol in vogue for plants (
The nuclear isolation procedure is quite rapid and simple, and the nuclei could be stored in PBS buffer at 4C for use over a long period. The nuclear suspension stored for 45 days had no problem with the release of DNA fibers and for the fiber FISH response. It might be possible to release fibers from all nuclear stages, from interphase, prophase, and other division phases including metaphase, all of which respond suitably to fiber FISH. Both STE-1 and STE-2 lysis buffers were effective in the release of DNA/chromatin fibers, but STE-1 was more effective for more compact nuclei and metaphase chromosomes, e.g. wheat, and STE-2 was sufficient for material with softer tissues and more relaxed chromatin, e.g., Allium. Representative figures depicting released fibers from interphase and metaphase nuclei are shown in Fig 1A and Fig 1B and for fiber FISH in Fig 1C and Fig 1D.
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Discussion |
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Chromosome Compactness and Release of DNA Fibers
To ensure accurate segregation of genetic information during mitosis and meiosis, the DNA is systematically compacted through a fundamental process of chromosomal condensation. For each chromosome, this means packing of about 4 cm DNA into a rod 10 µm long and 1 µm in diameter (
However, a differential gradient of DNA condensation for unique and repetitive DNA sequences also affects resolution of FISH signals. Resolution of FISH is distinct in the heterochromatin regions compared to euchromatin. With a microscopic resolution limit of 0.1 µm, FISH can resolve 1.2 mb in heterochromatin and 120 kb in euchromatin at pachytene, and approximately 1 kb on extended DNA fibers in heterochromatin, which is far better than the estimated 45 mB for mitotic metaphase chromosomes in plants, e.g., potato (
Cell Cycle-specific DNA Fibers and FISH Resolution
The existing protocol for the release of DNA fibers for fiber FISH applications in plants makes use of cell wall free nuclei obtained from fresh leaves/plant tissues after mechanical crushing followed by nuclear filtration (
Implications of Fiber FISH Using Cell Cycle-specific Fibers
In addition to several applications of fiber FISH, such as chromatin and EST mapping, positional cloning and transgene analysis, and a host of others highlighted by
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Acknowledgments |
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Supported by a JSPS invitational fellowship to UCL and by DBT, New Delhi.
Received for publication April 4, 2003; accepted June 25, 2003.
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