Chromatin nuclei isolation

Figure 5. (Continued) different panels (e and f for HeLa, j and k for chicken erythrocyte, and o and p for yeast). A section profile obtained along X-Y line shows a typical granular structure in die nucleus (e, j, o), and the peak-to-peak distance between the granular structure was distributed from 60 nm to 120 nm (e). The diickness of the chromatin fibers released out of die nucleus varied possibly due to the assembly of diinner fibers (f, k, p). A section profile for the spread fibers was obtained along X-Y line (f, k, p). Isolated HeLa cell nucleus was treated widi (r, s) or without (q) RNase. The treatment releases SOnmfiber from the nucleus. The histogram of die fiber width is shown in an inset of (s). Bars, 250 nm. (See Colour Plate 2.)...

Recent progress in confocal micro Raman spectroscopy has made it possible to investigate chromosomes and whole single living cells (Puppels et al., 1990, 1991). A spatial resolution of less than 1 )im was obtained. Different Raman spectra have been recorded of the cytoplasm and the cell nucleus. The spectrum of the nucleus consists of lines attributed to DNA and protein vibrations, strongly ressembling the spectra of isolated chromatin. The search for left-handed Z form DNA in metaphase chromosomes is in progress. 

In eukaryotic organisms the genetic processes of DNA replication and RNA synthesis are isolated by the nuclear envelope from translation which occurs on the ribosomes in the cytoplasm. In the nucleus, DNA forms a compact complex with various proteins, known as chromatin. Packing of chromatin into the nucleus is achieved through several orders of folding as illustrated schematically in Fig. 1. 

This process has not yet been studied at all, and the mechanism of the movement of macromolecules from chromosomes to the cytoplasm is quite unclear. The only useful information is based on electron microscopic observations. One can observe particles presumably containing D-RNA, such as Beerman granules or perichromatin granules, interchromatin granules, and helices in different parts of the nucleus. They may be bound to chromatin threads or lie freely in the nuclear sap or even be in contact with the nuclear membrane. These pictures may reflect the movement of the particles from chromatin to the nuclear membrane (Beerman and Bahr, 1954 Stevens and Swift, 1966, Monneron and Bernhard, 1969). As all these types of particles are of ribonucleoprotein nature and have many properties similar to those of isolated nuclear D-RNP, it is possible to suggest that D-RNA moves to the nuclear membrane in a complex with informofers. 

In the nucleus the estrogens bind to chromatin from which they can be dissociated in the form of a 5S hormone-receptor complex. While the cytosol receptor is found in uteri of animals untreated with estrogens, pretreatment with estrogens is needed for the appearance of a 5 S complex in the nucleus. Moreover, when isolated nuclei are incubated with estradiol, the 5 S complex will appear only if cytosol is present in the incubation mixture. These experiments and others... 

The interaction of retinol-CRBP with die nucleus has been further studied (Liau etal., 1981). Using CRBP labeled with tritium by reductive methylation, it was found that CRBP delivers retinol to the specific binding sites within the nucleus without itself remaining bound. The outer nuclear envelope is apparendy not involved because Triton X-lOO-treated nuclei retained 70% of the retinol binding sites found in intact nuclei. Isolated liver chromatin also had the same... 

It was demonstrated also that the acidic proteins stimulate synthesis in isolated chromatin fractions and increase the transcriptional activity of the DNA-histone complex (Frenster, 1965 Teng et al., 1970). These proteins promote the transcription of free DNA (Allfrey et al., 1972), and they determine transcriptional specificity. The accumulation of acidic nucleus proteins with a molecular weight of20,000-40,000 occurs during puff formation in Drosophila salivary gland chromosomes (Berendes, 1972). An important role in this process is played by phosphoproteins which demonstrate a high level of tissue specificity (Allfrey et al., 1972 Rickwood et al., 1972 Fig. 60). 

Isolation of Sperm Heads and/or Cell Nucleus Fraction or Chromatin from Fish Testes during Spermatogenesis... 

The main subcellular PA-binding sites are probably (I) RNA, both ribosomal and transfer, (2) DNA, both nuclear and oiganellar, (3) cell wall, and (4) membranes (Bachrach, 1970). It is well known that PAs, like Mg, bind to ribosomes and facilitate the association of ribosomal subunits (see reviews in Tabor and Tabor, 1976,1984). Polyamines can bind to specific sites on tRNA molecules (Pochon and Cohen, 1972 Sakai and Cohen, 1973). In plants, bound putrescine, spermidine, and spermine have been found in ribosomes (Cocucci and Bagni, 1968), as well as in complexes with rRNA and tRNA (Bagni et ai, 1973 Serafini-Fracassini et ai, 1984). On the other hand, numerous studies have shown the interaction and stabilization of DNA by PAs as well as their binding to this macromolecule (see reviews in Bachrach, 1970 Cohen and McCormick, 1979 Tabor and Tabor, 1984). In plants the information is scarce, but the occurrence of spermidine and spermine in chromatin isolated from com has been reported. Most of the spermine is probably chromatin bound in vivo (Hirasawa and Suzuki, 1985). However, the localization of PAs in the nucleus, in vivo, still remains somewhat uncertain (Tabor and Tabor, 1984). 

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