The objective of this project was to investigate the chromatin structure of the mutant Chinese hamster ovary cell line, xrs, the project consisted of two parts by; a radiobiological study, and a protein chemistry study and to specifically ask, whether or not the increased susceptibility of the x family of mutants compared to wt could be attributed to modifications to structure of chromatin or are the modifications a pleiotropic effect of the mutated gene. The radiobiological study used the change in DNA radiosensitivity with progressive removal of DNA bound proteins as a probe to investigate the chromatin conformation of CHO KI and xrs. The DNA alkaline unwinding assay, showed that there was a difference in sensitivity to ionising radiation (IR)-induced damage between xrs-5 and CHO KI after treatment with various concentrations of salt (0.25-0.75 M NaCl) and y-rays (0.25-0.75 Gy), 0.75M NaCl and 0.75 Gy gave maximal effects. The sensitivity difference between mutant and wt was less obvious with NaCl treatment at 0.75 Gy y-irradiation but nonetheless apparent. This radiosensitivity could be attributed to structural modifications in the mutant cell lines. These structural modifications are represented graphically in two algebraic forms i.e. a quadratic (y=mx2) and a quartic sine (y= ax4 + bx3 + cx4 + dx+ C) in CHO KI and xrs-5 respectively. The decrease in unwinding in hypertonic solutions was thought to be attributable to either ongoing fast ssb repair, a faster unwinding population, or, loss of DNA supercoiling due to high salt and irradiation, or negative interaction between one unwound DNA population and a adjacent break points leading to saturation. Partial reversion of .xrs-5 did not alter the unwinding profile markedly. As deduced from the observation that despite the partial reversion the mutant was consistently more susceptible to IR-induced DNA damage compared to wt. The difference in DNA radiosensitivity in this assay of mutant compared to wt may not necessarily reflect the radiosensitivity of the xrs cells. The protein chemistry study showed that there was a difference observed between protein extracts of mutant xrs-6 and wt, with respect to histone-related proteins. Specifically, a species of 31 kDa was detectable in extracts of wt but not xrs-6 mutant cells. The possible identity of this polypeptide and the role of candidate proteins in chromatin structure and radiosensitivity is discussed.

The ultrastructure of the nucleus pulposus was studied with conventional staining methods. The multinucleated cell masses seen in toluidine blue stained thick sections could be resolved into a large number of closely packed cells at the ultrastructural level. Numerous desmosomes were observed between adjacent cells; a second type of intercellular junction was identified as a gap junction after extracellular tracer treatment. The possible functions of these junctions were discussed, the notochordal cell cytoplasmic organelles include centrioles, mitochondria, rough endoplasmic reticulum, Golgi apparatus, cytofilaments, glycogen and lysosomes. Large vesicles, a characteristic component of notochordal cells contain material which from its staining reaction was judged to be proteoglycan. Large numbers of pinocytotic vesicles were seen in the vicinity of extracellular bands of electron-dense material applied to the cells surface. Degenerating cells were occasionally seen in the young nucleus pulposus but were seen more frequently with advancing age. The matrix contains metachromatic material which is seen after lead citrate and uranyl acetate staining cis branched and unbranched beaded filaments. From its staining reaction the material was considered to represent proteoglycan. Isolated collagen fibres were occasionally seen. Banded fibres consisting of alternate dark and light bands, periodicity 84 nm were found in the matrix. From their staining reaction these structures appear to consist of longitudinal protein filaments with transverse bands of fibrillar matrix proteoglycan. The possible relationship of this material to collagen was discussed. With a view to selective staining and further identification of the matrix components the theory of the critical electrolyte concentration (C.E.C.) technique was outlined. The most promising organic cations containing a heavy metal for the ultrastructural application of this technique were chelates of haematein with heavy metals. Chelate formation is accompanied by a decrease in pH and in some cases a spectral change. The characteristic absorption spectrum of indium haematein allowed the optimum molar ratio to be ascertained. Preliminary experiments of polyanions spotted on filter papers, unfixed collagen and thin sections of pancreas indicated a progressive loss of staining of sulphate, carboxyl and phosphate groups with increasing salt molarity. Unosmicated thin sections of nucleus pulposus showed loss of staining of vesicle material followed by matrix material as the salt molarity was increased; nuclear staining was largely unaffected. This staining pattern was discussed in terms of the C.E.C. theory. Although iron, lead and uranium haematein chelates also produced staining consistent with localisation of tissue polyanions each had disadvantages which did not commend their use in the C.E.C. approach to Selective staining.