Phosphate ions in dividing root-tip cells: a combined trapping and squash method. Implications in nuclear tran-scription

TANDLER, CJ; RIOS, H

Biotechnic & Histochemistry

Elsevier

Año: 2001 vol. 76 p. 239 - 245

Inorganic phosphate ions (Pi) were first identified in the nuclei of eukaryotic cells by an ion-trapping method. Pi were later reported to stimulate the chain elongation reac-tion in RNA synthesis by isolated subnuclear structures. We examined the pattern of Pi distri-bution in Zea mays root-tip dividing cells. Unfixed and paraformaldehyde or glutaraldheyde vapour-fixed tissues were immersed into 0.15M lead acetate-2%glutaraldehyde ? 0.1M caco-dylate buffer pH 6.8-7.0 for 4 h to capture Pi as the insoluble orthophosphate lead hydroxyap-atite. The excess of lead ions was removed with 0.4 M sodium citrate (4h) and then permea-bilized in 25% ammonia for 2 h. The refringent precipitates was stained overnight with 0.5% potassium sulfide, washed with distilled water and squashed in a drop of 60% glycerine. Pi showed cyclic accumulation in the cytoplasm during mitosis and surrounded all chromosomes at metaphase-anaphase. The beginning of partition between dividing cells had a high Pi con-centration at sites where plasma membrane and cell walls were formed. The small daughter cells and those in G1 phase showed Pi concentrated to the nucleolus and lower levels else-where in the nucleus. Later on, the larger nuclei, revealed greater amounts of Pi in relation to condensed chromatin. In Xenopus laevis oocytes, Pi concentrated to the nucleus, mainly to the active central core of the multiple nucleoli. These results, taken together with previous da-ta, strongly suggested a compartmentation in the intact cell. A correlation may exist between the rate of transcription and the Pi concentration in distinct functional domains within the nucleus.