Further studies of DNA-nucleoprotein gels and residual protein of isolated cell nuclei.

Abstract 1. 1. Data are presented which provide further tests of a previously proposed model for the fundamental structure of chromosomes at the molecular level. 2. 2. Observations concerning the effects on the gel-forming capabilities of the residues left after extracting with various reagents the DNA-residual protein complexes isolated from rat liver or calf thymus nuclei are presented and discussed. These results are consistent with the postulate that the formation of viscoelastic gels is dependent on linear continuity between tracts of DNA and tracts of residual protein. 3. 3. Observations are presented concerning the use of formaldehyde to investigate the possibility of damage to DNA caused by extracting DNA-nucleoprotein with 0.1 N HCl in 8 M urea. DNA appears to survive acid denaturation because of the presence of the portion of the residual protein which, together with the DNA, remains insoluble in the acid urea. It is inferred that protection might be conferred on the DNA by the insoluble residual protein if the latter, which appears to be covalently bonded to the DNA, keeps the strands in register after hydrogen bond rupture brought about by acid, thus facilitating renaturation. 4. 4. It was found that DNA-nucleoprotein materials which had been frozen failed to produce the gels customarily observed on raising the pH to 8–9. Pure DNA dissolved in water and frozen could be subsequently recovered in fibrous form, indicating that freezing damages the protein component of DNA-nucleoprotein rather than the DNA. 5. 5. Data are presented which show that Bailey's reagent and Cleland's reagent for the disruption of disulfide bridges destroy the viscoelastic properties of DNA-nucleoprotein gels and render soluble at pH values above neutrality the residual protein of cell nuclei. These observations on disulfide bond behavior appear to leave little doubt that intact disulfide bridges play a crucial role in maintaining the structure of residual protein, DNA-nucleoprotein gels, and by inference, chromosomes. 6. 6. The effects on the residual protein component caused by extracting with acid urea were studied by performing amino acid analysis on whole residual protein, the soluble extract, and the insoluble residue; by performing gel filtration studies on these materials; by examining the gelability of the acid urea insoluble residues, and by analysis for deoxyribonucleotides in the acid urea soluble extracts. The evidence indicates that some degradation of the residual protein component results but the bulk of the DNA appears to remain firmly bound to the acid urea insoluble component of the residual protein. 7. 7. Polarographic data showing the concentrations of sulfhydryl and disulfide sulfur in whole residual protein, the acid urea soluble extract, and the insoluble residue are presented. 8. 8. Paper electrophoresis of solubilized residual protein performed under conditions which answer a possible objection to previous data still indicate the presence of two types of peptide chains in the residual protein. 9. 9. Gel filtration studies performed on solubilized residual protein indicate that the components of this material are probably greater than 400,000 in molecular weight.