G2-population cells in mouse kidney and duodenum and their behavior during the cell division cycle.
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Biomedical subjects
Publications and source records attributed to T Pederson.
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When HeLa cells are fixed with ethanol, extracted with 0.1 N HCl, and incinerated at 540 degrees C, all organic constituents and all ions studied are removed with the exception of iron. The gross outlines of cell structures are preserved and high concentrations of residual ash in interphase nucleoli and mitotic chromosomes suggest that there may be a shift in iron salts during the cell cycle. Experiments with cells isotopically labeled in proteins, nucleic acids, lipids, and polysaccharides indicate that the iron is bound to a polysaccharide. Addition of iron chelating agents to living cells causes a selective inhibition of DNA synthesis. These data suggest that iron may play a crucial role in the mitotic process.
Interphase HeLa cells exposed to solutions that are 1.6 x isotonic manifest a series of morphological transformations, several of which grossly resemble those which occur when untreated cells enter prophase. These include chromosome condensation with preferential localization at the nuclear envelope and nucleolus, ruffling of the nuclear envelope, and polyribosome breakdown. The nucleolus loses its fibrous component and appears diffusely granular. At 2.8 x isotonicity the nuclear envelope is selectively dispersed although other membranes show morphological alterations also. The characteristic transitions of the lysosomes, Golgi complex, and microtubules seen in normal mitosis do not occur during hypertonic treatment. All the changes induced with hypertonic solutions are rapidly reversible, and the nucleus particularly goes through a recovery phase which bears some similarity to that of the telophase nucleus. The prophase-like condensation of the chromatin following exposure of the intact cell to hypertonic medium cannot be reproduced on an ultrastructural level in the isolated nucleus with any known variation in salt concentration, suggesting significant modifications of the nuclear contents during isolation. In addition to these morphological responses, hypertonic solutions also markedly and reversibly depress macromolecular synthesis. The polyribosome disaggregation that results from exposure to hypertonic solutions may be partially prevented by prior exposure to elevated Mg(++) concentrations; this same ion is also partially effective in preventing the polyribosome breakdown which normally occurs as cells enter mitosis.
The cell-free synthesis of histone-like polypeptides has been achieved using a selected class of small polyribosomes as the only particulate fraction. This synthesis is prevented if the deoxyribonucleic acid (DNA) inhibitor, cytosine arabinoside, is added to the cells prior to disruption, and it is not detected when the cytoplasm used is derived from postmitotic (G(1)) cells. When the 100,000 g supernate from pure metaphase populations was compared with that from S phase cells, the cell-free synthesis of histone-like polypeptides in the presence of S phase polyribosomes remained unchanged. These data suggest that, except for the histone messenger RNA-ribosome complex, the cytoplasmic factors requisite for histone synthesis are present throughout the cycle, and that the shut-off of this synthesis is not under translational control.
Interphase HeLa cells manifest a stepwise shutoff of RNA synthesis when the tonicity of the extracellular medium is gradually increased. Synthesis of heterogeneous nuclear RNA is most sensitive and is selectively inhibited at 1.5 times isotonicity (450 milliosmols/liter), while 45S ribosomal RNA synthesis is not affected significantly below 2.0 times isotonicity. Transfer RNA synthesis is least sensitive to increased osmolarity and is not completely inhibited until the electrolyte concentration of the medium is elevated to 2.8 times isotonicity. Although the transcription and methylation of 45S ribosomal precursor is unaffected at 1.5 times isotonicity, there is pronounced impairment of its processing into 32S and 18S RNA. Using a refined cell synchronization technique, we have been able to compare these effects of hypertonicity with the shutoff of RNA synthesis which occurs during the G(2)-prophase interval of the cell division cycle. In this case, as with random cells in hypertonic medium, a selective inhibition of heterogeneous nuclear RNA synthesis and slowed processing of 45S ribosomal RNA were found, whereas synthesis of 45S and transfer RNA continued unabated throughout G(2)-prophase. While it is known that RNA synthesis essentially ceases during metaphase, we have noted that transfer RNA synthesis continues in metaphase at 10-15% of the interphase rate, which is of particular interest in view of the relative resistance of this species to hypertonicity. The close correlation between the patterns of cessation of RNA synthesis at mitosis and during exposure to hypertonic medium supports our earlier contention that alteration of intracellular electrolyte levels provides a useful model for studying the mechanism of mitosis.
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