Histone fractionation

Ruiz-Carrillo, A., and Allfrey, V.G. (1973) A method for the purification of histone fraction F3 by affinity chromatography. Arch. Biochem. Biophys. 154, 185-191. 

The use of urea triton gel electrophoresis (Zweidler and Cohen, 1972 Alfageme et al., 1974) has permitted the further resolution of histone fractions and the identification of histone variants. Microcomponents or variants of histone HI have been known for a long time (for references, see Cole, 1977). Recently, it has been shown that microcomponents or variants of histones H2B, H2A, and H3 occur in many systems (Marzluff et al., 1972 Laine et al., 1976 Garrard, 1976 Blankstein and Levy, 1976 Zweidler, 1976 Franklin and Zweidler, 1977). Furthermore, in sea urchin embryo the synthesis of new histone variants has been correlated with development (Cohen et al., 1975 Newrock et al., 1978 Von Holt et al., 1979). 

It has been reported that the plasmid vector is unable to translocate to the nucleus unless complexed in the cytoplasm with nuclear proteins possessing NLS. NLS are short karyophilic peptides on proteins that bind to specific transporter molecules in the cytoplasm, mediating their passage through the pore complexes to the nucleus. Examples of these peptides will be given later in this section. DNA can also be presented to cells in culture as a complex with polycations such as polylysine, or basic proteins such as protamine, total histones or specific histone fractions (110), cationized albumin, and others. These molecules increase the transfection efficiency. In addition histone HI is identified as transfection-enhancing protein in cell culture (111). 

Shepherd, G. R., Hardin, J. M. and Noland, B. J. (1971) Methylation of lysine residues of histone fractions in synchronized mammalian cells. Arch. Biochem. Biophys. 143 1-5. 

Bradbury et al. (1967) have studied partial nucleoproteins produced by removal of histone fractions from nucleohistone. Figure 12.6 shows a polarized infrared spectrum of one of the nucleoproteins oriented by shearing. The spectrum is that of a film deuterated by D2O vapor. The bands of the in-plane vibrations of cytosine and guanine moieties are highly polarized when the electric vector is placed perpendicular to the fiber axis. The 1452 cm absorption is that of the amide IF band (Miyazawa et al., 1956 Miyazawa, 1962) for the readily deuterated fraction of the partial nucleoprotein. As seen in Fig. 12.6, the 1452 cm band is polarized considerably in the direction parallel to the fiber axis, giving evidence that the extended polypeptide chain is situated so that its axis lies between the angle of the groove in the DNA double helix and the axis of the DNA helices (Bradbury et al., 1967). 

It is unlikely that protein synthesis is required solely for the initiation of DNA synthesis at specific chromosomal sites during the S phase in order that replication continue. The inhibition of cellular protein synthesis would affect many metabolic functions, causing gradual cessation of cell activity. Closely associated with DNA replication is the synthesis of chromosomal proteins necessary for the formation of new chromosomes. Different histone fractions, for example, show differences in their degree of dependence on DNA synthesis (Sagopal and Bonner, 1969). The converse may also hold continued DNA synthesis may be dependent on the availability of specific proteins, possibly for the incorporation of newly synthesized DNA into new chromosomal structure. 

In work with the bovine thymus synthetase, Tanaka et al. 212) demonstrated that the enzyme was completely dependent on histone when Mg + was omitted from the assay histone HI was ADP-ribosylated under these reaction conditions. Maximum stimulation and ADP-ribosylation occurred when the ratio of DNA to histone HI was 1 to 10 on a weight basis stimulation was lost when the amount of DNA was increased to 50% of histone HI. All other histone fractions were efiective in stimulating the reaction but none was as active as histone HI. Kawaichi et al. 109) and Ueda and co-workers 222) also observed synthesis of poly(ADP-ribosyl) histone using an apparently homogeneous preparation of rat liver poly( ADP-ribose) synthetase. As opposed to the requirement for a large excess of histone over DNA used by Tanaka et al. 212) to demonstrate modification of Hi, a ratio of DNA to histone of 1 1 (on a weight basis) appeared to be optimal. The amount of ADP-ribose incorporated into histone Hi increased linearly as the DNA to histone Hi ratio was fixed at unity and their concentrations increased from 25 to 150 jug/ml. The ADP-ribose incorporated into histone HI represented, however, only about 50% of the total poly ADP-ribosylation the remainder was polymer associated with the synthetase itself. In these studies 212), Mg + was present at a concentration of 10 mAf. All histone subfractions were tested as acceptor proteins Hi was best, followed by H2B H2A, H3, and H4 were poor acceptors. This order of effectiveness is nearly identical to that found in experiments with intact nuclei (2,28,30, 64, 76,102,103,149,162,164,178-180, 200, 215, 229). 

These brief considerations of the various histone fractionation methods indicate that different types of histones are obtained, depending upon the method used. Sometimes a histone isolated by one method has an amino acid composition homologous to a histone isolated by another procedure. Often a procedure will yield a type of histone that cannot be obtained by other procedures. Whether the different fractions are representative of native histones or whether they are the result of degradation remains to be established. 

An energy-dependent histone phosphorylation was discovered in calf and rat thymus nuclei [68, 69], A histone phosphokinase has been purified from liver, and some of its properties have been studied. The kinase phosphorylates only the serine residues of histones or protamines. It is inactive on other proteins and therefore different from the typical phosphopro-tein kinase. In vitro the enzyme phosphorylates all histone fractions, but FI appears to be a preferred substrate. 

Small amounts of 5 N-methyl lysine have been found in some histone fractions (Ilaa, Ila, IIIo, and IV). Surprisingly, methyl lysine is absent in the lysine-rich fractions la and Ib, suggesting that methylation is specific. The methyl group can be donated to lysine by methionine. This was established in experiments utilizing C-labeled (methyl) methionine as a precursor. 

Histones and DNA, Perhaps one of the most intriguing observations with respect to the role of histones in the control of genetic transfer stems from the experiments in which DNA-dependent RNA synthesis and DNA synthesis are measured in the absence and presence of various histone fractions. 

Therefore, various histone fractions have different specializations in chromatin structure. The phosphorylation of HI histone determines the molecular level of chromatin organization the phosphorylation of H2a histone determines the heterochromatization pf chromatin and the superphosphorylation of H1 histone, and normal phosphorylation of H3 histone affects the microscopic level of organization (Curley et al., 1978). 

Mauritzen and Stedman (1959, 1960) found slight but definite tissue differences in the amino acid composition of nuclear histones isolated from different organs. These differences concerned only some amino acids (aspartic and glutamic acids, alanine, leucine, isoleucine, and valine). They postulate that histones may be tissue-specific proteins. The relative quantities of the same histone fractions also differ appreciably from one tissue to another. 

Tissue differences in the distribution of histone fractions have also been discovered by chromatography on carbox5methyl-cellulose (Davis and Busch, 1959 Starbuckand Busch, 1960). Of later work in this direction, we need mention only the very detailed investigation made by Driedger and co-workers (1963). Using electrophoresis on polyacrylamide gels, these workers showed... 

In another investigation (Agrell and Christensson, 1965), also devoted to changes in the composition of histones in organs of the chick during embryogenesis, it was clearly shown that the ratio between the histone fractions varied with the phases of development (considerably in the erythrocytes, brain, and liver, and slightly in the heart and eyes). 

This difference in inhibitory functions between the histone fractions was also confirmed by other experiments. These showed that different histone fractions differ in their ability to protect DNA from thermal denaturation, and also that the level of the inhibitory properties of histones relative to DNA-dependent RNA synthesis bore a direct relationship to the level of DNA stabilization against thermal denaturation. 

The principal fact to which Butler directs attention is the absence of sharply defined tissue differences in the composition of the histone fractions, for he considers that if they possess repressor functions such differences should be present. He also emphasizes the small number of electrophoretically homogeneous... 

These experiments thus confirm the possibility that the mechanism of cortisone action is through its link with chromosomal proteins. Further data on the mechanism of the RNA-stimulating action of hydrocortisone were obtained by Sluyser (1966) who showed that this hormone is bound with histones, selectively, in fact, with the histone fraction 3 characterized by a low lysine content. 

On the other hand, slight tissue differences in the ratio between fractions of jS-histones isolated from chicken erythrocytes, liver, and spleen were observed by Bellair and Mauritzen (1967). Gutierrez and Hnilica (1967) studied phosphorylation of various histone fractions in rat tissues (by determining incorporation of -labeled phosphates into histones) and observed definite fraction and tissue specificity in histone phosphorylation activity. 

Observations revealing chaises in fee pattern of histone fractions obtained by electrophoresis on polyacrylamide gel were made during the study of a morphogenetic process taking place in plants, the formation of lily pollen (Sheridan and Stern, 1967). 

Gurley, L. R., Walters, R. A., and Tobey, R. A., 1973b, The metabolism of histone fractions. VI. Differences in the phosphorylation of histone fractions during the cell cycle. Arch. Biochem. Biophys. 154 212. 

Desai L, Adams R, Pothier L et al (1972) Immunofluorescent labeling of chromosomes with antisera to histones and histone fractions. Exp Cell Res 70 468-471... 

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