Fractionation of histone

ACS Symposium Series American Chemical Society Washington, DC, 1980. 

Incorporation of ribonucleotides, magnesium or cobalt are to be preferred because these make fewer mistakes. 

Now let us consider the case of essential metal Ions that, nevertheless, produce deleterious effects in the wrong concentration. Magnesium Ions are required for protein synthesis, yet Mg Ions in too high concentration lead to errors, as is illustrated by the studies of Szer and Ochoa (16) on the incorporation of phenylalanine and leucine in a ribosomal preparation using poly(U) as the messenger RNA. UUC as well as the UUU codon in poly(U) code for phenylalanine, so that incorporation of the latter represents correct translation. UUA and UUG code for leucine, so that leucine incorporation in this system is incorrect. At low Mg2+ concentration only phenylalanine is in fact incorporated. Phenylalanine incorporation is maximal at lOmM Mg as the Mg concentration is increased, however, leucine also becomes incorporated and its maximal incorporation is at 20mM Mg2+ ( ). 

Metal Ions can produce a large variety of other effects on nucleic acids that could be deleterious if they occur during genetic information transfer. Metal ions can bring about the degradation of RNA (17, 18, 19), changes in the specificity of enzymes that act on DNA (20), changes in the conformation of polynucleotides and nucleic acid - protein complexes (21). 

It is also known that cellular metal ion concentrations change with age. An illustration of such age changes in human lens nuclei is given in Table I (22). We hypothesize that these changing concentrations of metal ions that are essential to genetic information transfer, yet can alter it, can affect information transfer and therefore contribute to the changes that are associated with the aging process. 

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Histone methylation is another posttranslational modification which involves a transfer of a methyl group from the methyl donor S-adenosyl methionine (SAM) to lysine or arginine residues (Fig. 1). In sharp contrast with histone acetylation, this modification occurs particularly in histones H3 and H4 with a remarkable specificity (Kouzarides, 2002 Shilatifard, 2006) (Fig. 1, Table 2). Another feature of histone methylation is that a large fraction of histones in mature chromatin is... 

A small fraction of histone H2A undergoes phosphorylation and dephosphorylation continuously,43 but HI and H3 are phosphorylated and dephosphor-ylated at specific stages of the cell cycle. Phosphorylation of HI has been thought essential for "condensation" of chromatin,44 45 the folding into the tightly packed chromosome structures. However, more recent experiments point to the N terminus of histone H2B as the required site of phosphorylation for chro-... 

In order to assess the nuclear nonhistone proteins being ADP-ribosylated in association with DNA repair we have isolated the endogenous conjugates formed in DMS-treated cells by applying aminophenyl boronic acid chromatography to 0.2 M sulfuric acid precipitates of isolated nuclei [1]. With hepatoma cells, this procedure provided rou y 80% of the newly-formed conjugates in a highly purified form. The isolated fraction of histone-depleted (ADP-ribosyl) proteins comprised about 1% of total nuclear... 

Some investigations supposed that the mechanism of action of various fractions of histones can differ, i.e., the lysine-rich histone I (fl) inhibits transcription when it is associated with DNA the arginine-rich histone III (f3) inhibits RNA-polymerase activity when the two are associated (Spelsberg et al., 1969). 

Hie use of polyacrylamide gel instead of starch for fractionation of histones enabled an even finer fractionation of total histone and its principal fractions to be carried out (Driedger... 

The use of other methods of fractionation of histones (car-boxymethylcellulose, Amberlite IRC-50, DEAE-cellulose) has also... 

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. 

Specifically, when histones are dissociated from DNA in 2 M NaCl, and H3 H4 tetramer and H2A-H2B dimer may be identified after fractionation of the histones at pH 5.0 and cross-linking with dimethylsu-berimidate (Komberg and Thomas, 1974). The inference was drawn that these complexes exist as such in the intact nucleosome. Furthermore, since both the un-cross-linked H3 H4 tetramer, and the uncross-linked H2A-H2B dimer are stable complexes, it has proved possible to characterize their physical properties in solution. Some of these results are summarized here. 

Infrared and circular dichroism (CD) measurements (Moss et al., 1976b) are both consistent with a sizable fraction of the tetramer being in the a-helical configuration, —29% a-helix with negligible j3 structure. This is rather similar to the 25% a-helix and —0% /3 structure obtained for the tetramer prepared from acid-extracted histones (D Anna and Isenberg, 1974b). 

Upon increase in salt concentration to 2 M, histone octamers were obtained (Thomas and Butler, 1978). The octamer could be assembled from acid-extracted as well as from salt-extracted histones (Thomas and Butler, 1978). In a concentrated solution of the four core histones (prepared by acid extraction) at an ionic strength higher than 2 M NaCl (minimum 10 mg/ml histone concentration), there is a small fraction of assembled fibrous structures which can be observed in the electron microscope (Sperling and Bustin, 1976 Wachtel and Sperling, 1979). These fibers (see Fig. 3d) are 60 A in diameter and have a 330 A axial repeat, and were shown to be composed of the four core histones in an equimolar ratio (Wachtel and Sperling, 1979). The percentage of fibers in the solution of the four core histones is promoted by increase in histone and salt concentrations. 

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). 

Another group of non-histone proteins have been identified as essential components for the formation of the condensed chromosome (Table 1). Topoisomerase II (topo II) localizes in the scaffold/matrix fraction of the interphase nuclear (Berrios et al., 1985) and the mitotic chromosome (Maeshima and Laemmli, 2003) (see section 3.1). Topo II forms a ring-shaped homodimer (Berger et al, 1996 Nettikadan et al, 1998) and catalyzes the decatenation and relaxation of DNA double strand (Wang, 2002). In fission yeast, chromosomes cannot be condensed without functional topo II (Uemura et al, 1987). In addition, in in vitro experiment, mitotic extracts containing topo II induce chromatin condensation in the isolated nuclei from HeLa and chicken erythrocyte cells (Adachi et al., 1991). 

Van Lint C, Emiliani S, Ott M, Verdin E (1996a) Transcriptional activation and chromatin remodeling of the HIV-1 promoter in response to histone acetylation. EMBO J 15 1112-1120 Van Lint C, Emiliani S, Verdin E (1996b) The expression of a small fraction of cellular genes is changed in response to histone hyperacetylation. Gene Expr 5 245-53 Van Lint C, Amelia CA, Emiliani S, John M, Jie T, Verdin E (1997) Transcription factor binding sites downstream of the human immunodeficiency virus type 1 transcription start site are important for vims infectivity. J Virol 71 6113-6127... 

The proteins known today as linker or HI histones were initially described as the abundant lysine-rich nuclear proteins that could be separated by chromatography on ion exchange resin from other major basic nuclear proteins known today as core histones (for review see Refs. [1,2]). During gel electrophoresis of histones the HI fraction migrated as the slowest and most heterogeneous band. Upon the discovery of nucleosomal organization of chromatin in the mid 1970s it turned out that linker histones are not involved in the assembly of the nucleosomal protein core, but bind to DNA between nucleosomes (hence their name). 

Van Lint C, Emiliani S, Verdin E. (1996) The expression of a small fraction of cellular genes is changed in response to histone hyperacetylation. Gene Expression 5 245-253. 

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