Evidence has been obtained which indicates that disulphide bond crosslinks contribute to the morphological integrity of isolated lampbrush chromosomes (both chromomeres and lateral loops) and nucleoli. It is suggested that the progressive formation of these bonds in vitro by aerial oxidation may provide the basis for the previously recognized time-dependent hardening or ‘denaturing’ of these structures.
Manually isolated germinal vesicle nuclei have been massed and fractionated by low-speed centrifugation into nucleoplasm and chromatin. Phase-contrast microscopy demonstrates the chromatin to consist of nucleoli, lampbrush chromosomes and nuclear membranes.
Urea gel electrophoresis has been employed to resolve the reduced and S-carboxymethylated proteins of whole nuclei into some 12 components, negatively charged at pH 8. The nucleoplasm alone gives an essentially similar pattern, but with the distinct depletion of one component and slight depletion of another. Both of these components are much enriched in the chromatin pellet where they predominate over all other proteins.
The total chromatin has been subfractionated by microdissection, taking advantage of the differential attachment of nucleoli to the nuclear membrane at different stages of oogenesis. It is concluded that the nuclear membrane per se does not contribute to the major chromatin proteins. The two major polypeptides are components of the nucleoli. Preparations of isolated lampbrush chromosomes have not, to date, provided sufficient material to give a distinctive electropherogram; only one faint band, a major component of whole nuclei, was apparent.
Sodium dodecyl sulphate gel electrophoresis has resolved some 25 components in whole nuclei, and again demonstrates the enrichment of the two major species in the total chromatin fraction. The apparent molecular weights of these two species are 43 kilodaltons and 110 kilodaltons. Approximately 20 minor species are also present in the chromatin and are obviously good candidates as components of the nucleolar and chromosomal structures. Histones, at most, make only a minor contribution to the overall chromatin protein population.