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DNA synthesis in cytokinin-autotrophic tobacco cells

Effect of bromodeoxyuridine, fluorodeoxyuridine, and kinetin

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Abstract

DNA isolated from various Nicotiana tabacum cell types, differing in their degree of hormone autotrophy and incubated in the presence of bromodeoxyuridine (BrdUrd), was analyzed by isopycnic CsCl gradient centrifugation. All cell types incorporate BrdUrd into DNA in such a way that hybrid DNA is formed with 60–80% of thymine (Thy) residues replaced by bromouracil (BrUra) in the newly synthesized strand. This DNA is not replicated further under ordinary culture conditions. Whereas in “normal” hormone-dependent cells this state is final and cells necrotize, in tumor (cytokinin-auxin autotrophic) and cytokinin-autotrophic cells a mechanism is induced leading to the reduction of BrUra content in DNA. As a result a decrease in the buoyant density (in CsCl) of BrUra DNA can be observed. In the case of cytokinin-autotrophic cells supplemented with kinetin, the buoyant density of the whole DNA decreases gradually to the value of that of unsubstituted DNA, but specific radioactivities of different DNA fractions reflect the retention of the pyrimidine ring of BrUra in DNA. This is interpreted as debromination of DNA in situ. The process can be inhibited by fluorodeoxyuridine (FdUrd) and deoxycytidine (dCyd). Moreover, FdUrd (but not dCyd) allows replication of hybrid DNA in tumor cells in such a way that HH DNA with all Thy residues replaced by BrUra is formed. For cytokinin-autotrophic cells FdUrd and kinetin are required. In hormone-dependent cells replication of hybrid DNA cannot be induced under any conditions. Most of these conclusions complement our previous findings that BrdUrd tolerance in hormone-autotrophic tobacco cells in hormone controlled. It is postulated that a modulation of thymidylate synthetase specificity is one factor affecting the level of BrUra substitution in DNA. The possibility of cytokinins being involved in the control of DNA synthesis is discussed.

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Abbreviations

BrdUrd:

5-bromo-2′-deoxyuridine

BrUra:

5-bromouracil

dCyd:

2′-deoxycytidine

FdUrd:

5-fluoro-2′-deoxyuridine

dThd:

thymidine

Thy:

thymine

EDTA:

Na2-ethylenedia-minotetraacetate

IAA:

idole-3-acetic acid (auxin)

SDS:

Na-dodecylsulphate

LL, HL, HH DNA:

“light-light” (unsubstituted), “heavy-light” (unifilarly BrUra substituted), “heavy-heavy” (bifilarly BrUra substituted) DNAs, respectively

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Authors and Affiliations

  1. Institute of Biophysics, Czechoslovak Academy of Sciences, 612 65 Brno, 135, Královopolská, Czechoslovakia

    Milan Bezděk & Boris Vyskot

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  1. Milan Bezděk
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  2. Boris Vyskot
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Bezděk, M., Vyskot, B. DNA synthesis in cytokinin-autotrophic tobacco cells. Planta 152, 215–224 (1981). https://doi.org/10.1007/BF00385147

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  • DOI: https://doi.org/10.1007/BF00385147

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