Purification Of Nuclei Research Articles

The proteins of polytene chromosomes of Drosophila hydei.

It is reported that chromatin can be prepared from highly purified polytene nuclei from the salivary glands of third instar larvae of Drosophila hydei; such chromatin differs from that of diploid nuclei mainly by deficiencies in certain nonhistone chromosomal proteins. It is suggested that these proteins are important components of constitutive heterochromatin, which is severely underrepresented in polytene chromosomes. Chromosome morphology, including the pattern of induced puffs, is maintained throughout the mass isolation of glands and sucrose gradient purification of nuclei, as indicated by studies on temperature-shocked and control larvae. No significant alteration in the chromosomal proteins following puff induction by heat shock could be detected on analysis of the isolated protein fractions by disc gel electrophoresis. More sensitive techniques must be developed to study the apparent rearrangement or accumulation of protein at puff sites, and to elucidate the role of this protein in gene activation.

Further studies on testosterone 5 -reduction in rat ventral prostate.

The increase in weight of the rat ventral prostate after testosterone administration to castrated rats was shown to be accompanied with concomitant elevations of both the rate of testosterone 5α-reduction and that of the incorporation of choline-3H into phospholipids in the glands. Time course study revealed that the increase in rate of 5α-reduction and of choline-3H incorporation became evident by 24 hrs after testosterone administration. The increase in rate of 5α-reduction and of choline-3H incorporation into phospholipids was observed in both microsomal and crude nuclear fractions from castrated rats 24 hrs after testosterone administration. The rate of 5α-reduction and DNA content exhibited the similar recovery after the purification of nuclei, however the radioactivity of choline-3H in the crude prostatic nuclei was lost during the procedure for purification of the nuclei. Therefore, it was concluded that the increase in rate of 5areduction and choline-3H incorporation into phospholipids took place simultaneously after the administration of testosterone, however sites of increased testosterone 5α-reductase did not seem to be located on the newly synthetized membranes.5α-Androst-l-ene-17β-ol-3-one, known to be a potent androgen, evoked a similar increase in DNA content, the rate of 5α-reduction and DNA polymerase activity after testosterone administration to ventral prostate of castrated rats. This suggested that the increase in testosterone 5α-reduction observed did not seem to be a substrate-induced change.

Uptake of oestradiol by the rabbit hypothalamus. Specificity of binding by nuclei in vitro.

The binding of [6,7-(3)H]oestradiol-17beta to subcellular fractions of the hypothalamus and the cerebellum of the rabbit was studied in vitro. Uptake of steroid was higher in hypothalamic nuclei than in cerebellar nuclei. Lower binding was observed in other fractions of both tissues. After dialysis of the fractions, hypothalamic nuclei retained a high percentage of oestradiol whereas cerebellar nuclei lost most of the bound steroid. Supernatant fractions of both tissues retained a significant proportion of label after dialysis and after gel filtration on Sephadex G-200. No specific binding was observed in these fractions when subjected to sucrose-density-gradient centrifugation. Purification of nuclei followed by incubation with labelled oestradiol in the absence of the supernatant fraction resulted in loss of binding of steroid by hypothalamic nuclei. Incubation of the purified hypothalamic nuclei with supernatant fraction maintained the binding specificity of hormone retention.

The isolation of cell nuclei from rat brain.

A method for preparing highly pure cell nuclei from adult rat brain, using both differential and isopycnic centrifugation in sucrose media, is described. The morphology of these preparations was examined by both phase contrast and electron microscopy. The isolated nuclei retained many aspects of their in situ morphology; in particular, the nuclear envelope was double layered and interrupted by pore-like discontinuities, and the nucleoli consisted of irregular masses of densely packed granules. Analyses of these nuclear preparations for cytochrome oxidase and cholinesterase activity, as well as RNA/DNA ratio, indicated minimal contamination with mitochondria and microsomes. Problems involving the homogenization technique, choice of ionic conditions in the homogenization medium, and choice of optimal density of the sucrose solution used for the final purification of nuclei are discussed. Results of application of the technique to isolation of adult rat liver nuclei are also reported.