Protoplasma

, Volume 173, Issue 3–4, pp 123–132 | Cite as

Quantification of nuclear DNA and G-C content in marine macroalgae by flow cytometry of isolated nuclei

  • Y. Le Gall
  • S. Brown
  • Dominique Marie
  • Meftaha Mejjad
  • B. Kloareg
Article

Summary

The amounts of nuclear DNA in ten species of seaweeds belonging to the Rhodophyceae, Phaeophyceae, and Chlorophyceae were determined by flow cytometric analysis of nuclei isolated from protoplasts. Genome size was determined from the fluorescence of the nuclei stained with ethidium bromide. The size of the nuclear genome ranged from 0.13 pg per cell in the 1 C population ofUlva rigida to 3.40 pg per cell in the 2 C population ofSphacelaria sp. GC% analysis was based on staining with either Hoechst 33342 or mithramycin A, two fluorochromes specific for the bases A-T and G-C, respectively. Two models were used for the estimation of the proportion of guanine plus cytosine in the nuclear genome. The first one was based on the linear relationships mithramycin A fluorescence/G-C content and ethidium bromide fluorescence/total DNA content. The second model, based on the curvilinear relationships Hoechst 33342 fluorescence/A-T content and mithramycin A fluorescence/G-C content, resulted in comparatively more homogenous and consistent data and appears more accurate. Comparison with previous reports from other methods for the physical investigation of nuclear genomes shows that flow cytometry of nuclei isolated from protoplasts is an accurate, convenient and robust technique to assay for genome sizes and base pair composition in marine macroalgae.

Keywords

Flow cytometry Genome size G-C composition Protoplasts Seaweeds 

Abbreviations

A-T

nucleic bases adenine and thymine

CRBC

chicken red blood cell

FALS

forward-angle light scatter

G-C

nucleic bases guanine and cytosine

SEIM

sorbitol enzymatic incubation medium

SWIM

sea water incubation medium

Tm

thermal denaturation temperature of DNA

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Copyright information

© Springer-Verlag 1993

Authors and Affiliations

  • Y. Le Gall
    • 1
  • S. Brown
    • 2
  • Dominique Marie
    • 2
  • Meftaha Mejjad
    • 3
  • B. Kloareg
    • 1
  1. 1.Centre d'Etudes Océanographiques et de Biologie MarineCNRS (UPR 4601) and Université Paris VI, Station BiologiqueRoscoff
  2. 2.Service de CytométrieISV, CNRSGif-sur-Yvette
  3. 3.Laboratoire de Morphogénèse Végétale ExpérimentaleCNRS, URA 115, Université Paris XIOrsay

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