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Journal of Histochemistry and Cytochemistry, Vol. 51, 1249-1253, October 2003, Copyright © 2003, The Histochemical Society, Inc.

RAPID COMMUNICATION

Extended Chromatin and DNA Fibers from Active Plant Nuclei for High-resolution FISH

U.C. Lavaniaa, M. Yamamotob, and Y. Mukaic
a Cytogenetics Division, Central Institute of Medicinal and Aromatic Plants, Lucknow, India
b Kansai Women's College, Kashiwara, Osaka, Japan
c Division of Natural Sciences, Osaka Kyoiku University, Kashiwara, Osaka, Japan

Correspondence to: U.C. Lavania, Cytogenetics Division, Central Institute of Medicinal and Aromatic Plants, Lucknow 226 015, India. E-mail: lavania@cimap.res.in

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 51:1249–1253, 2003)

Key Words: fiber FISH, nuclear isolation, extended DNA fibers, mitotic fibers, chromosomal fibers


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U. C. Lavania, S. Basu, S. Srivastava, Y. Mukai, and S. Lavania
In Situ Chromosomal Localization of rDNA Sites in "Safed Musli" Chlorophytum Ker-Gawl and Their Physical Measurement by Fiber FISH
J. Hered., March 1, 2005; 96(2): 155 - 160.
[Abstract] [Full Text] [PDF]



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