Histones, electrophoresis

Lindner, H., M. Wurm, A. Dirschhnayer, B. Sarg, and W. Helliger, Application of high-performance capillary electrophoresis to the analysis of HI histones. Electrophoresis, 14,480-485, 1993. 

Fig. 4. Electrophoresis of the proteins of nuclear D-RNP s in polyacrylamide gel. A, B. Total protein of particles C, D. Fraction I from CM-cellulose E, F. Fraction II from CM-cellulose, A, C, E. Without treatment with mercaptoethanol B, D, F. After treatment with mercaptoethanol G, Ribosomal proteins. H. Histones. Electrophoresis was performed at pH 4.5 (From Samarina et al., 1968a. J. Molec. Biol., 33 251-263 Krichevskaya and Georgiev, 1969. Biochim. Biophys. Acta, 194 619-621.)...

The nucleus contains a large number of proteins other than histones. These so-called nonhistone proteins may or may not be tightly associated with the chromosomes. For example, the nucleus contains enzymes associated with the synthesis of RNA and DNA these are nonhistone proteins, but they are not part of the structure of chromosomes. One group of nonhistone proteins are the high mobility group (HMG) proteins, named for their rapid movement on polyacryl-amide gel electrophoresis. The HMG proteins, but not histone HI, are associated with the chromatin that is most active in RNA synthesis. 

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). 

Figure 20.35 Mechanisms by which external or internal stress leads to cell damage resulting in apoptosis. The stress leads to activation of initiator proteolytic enzymes (caspases) that initiate activation of effector caspases. These enzymes cause proteolytic damage to the cytoskeleton, plasma membrane and DNA. The activation of DNAases in the nucleus results in cleavage of DNA chains between histones that produces a specific pattern of DNA damage which, upon electrophoresis, gives a specific pattern of DNA fragments. The major endproduct of apoptosis are the apoptolic bodies which are removed by the phagocytes.

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). 

Fig. 10. A. Acetic acid-urea-triton-X-100 polyacrylamide gel electrophoresis [15] of the histones used to reconstitute 208-12 nucleosome arrays consisting of recombinant H2A.Z (lane 2) or recombinant H2A.1 (lane 3). Lanes 1 and 4 respectively are chicken erythrocyte and calf thymus histones used as markers [42]. B. Ionic strength (NaCl concentration) dependence of the average sedimentation coelRcient (s2o,w) of reconstituted 208-12 nucleosome arrays containing either H2A.1 (O) or H2A.Z ( ) [42]. The dotted line represents the behavior of a 208-12 complex reconstituted with chicken erythrocyte histones [406]. [Reproduced from Abbott D.W. et al. (2001) I. Biol. Chem. 276, 41945-41949, with permission from The American Society for Biochemistry and Molecular Biology.]...

Analyses of the reeonstituted eomplexes by quantitative agarose gel electrophoresis [404,405] and analytical ultracentrifugation [266,406] in the presence of MgCl2 showed that the arrays were able to fold in a way that is almost indistinguishable from complexes reconstituted with major histones (see Fig. 14A-B). Despite this, it was found that histone H2A ubiquitination affects the MgCl2 solubility of the reconstituted complexes (see Fig. 14C) suggesting that this modification may play a role in enhancing the intermolecular associations between chromatin fibers [221]. 

A further improvement of the more traditional slab gel analysis is the use of high-performance capillary electrophoresis (HPCE), which combines the separation power of high-performance liquid chromatography (HPLC) with the selectivity and speed of conventional gel electrophoresis. However, as HPCE separations are often performed using fused silica capillaries the positively charged histone molecules... 

Aguilar, C., Hofte, A.J., Tjaden, U.R. and van der Greef, J. (2001) Analysis of histones by on-line capillary zone electrophoresis-electrospray ionisation mass spectrometry. Journal of Chromatography. A, 926, 57-67. 

The sequencing gel electrophoresis of the DNA fragment irradiated in the nucleosome core particle NCP (DNA bound to the histones) show a regular alternation of regions (of around 10.4 base pairs, which is also the number of base pairs on one helical... 

Figure 4 Footprint of histones on the DNA of irradiated core nucleosome. A) Nucleosome core particle structure extracted from lAOl entry of PDB. The 146 base pairs DNA fragment Is In green and yellow, and the histone octamer is in blue and magenta tones. B) Experimental relative probabilities of FSB deduced from the radioactive profile (electrophoresis ) and relative probability of attack to deoxyriboses leading to FSB (calculated with RADACK). C) The Fourier transform (power) of the probabilities showing that probability variation has a period of 10.4 base pairs both for the experlmen t and for the calculated values.

Northern blotting to characterize RNA species in various cell types. The RNA is separated by gel electrophoresis in a denaturing formaldehyde agarose gel, transferred to a nitrocellulose filter, and hybridized with a gene-specific cDNA probe (gene A cDNA). Note that a second probe is also used to detect the presence of RNA molecules, such as histone RNA, that can be used to control for loading differences. 

Analysis of histones is frequently carried out in the presence of detergents which may bind to them. Binding of the nonionic detergent Triton X-100 to histones from calf thymus can be analysed by gel chromatography. The binding of Triton to histones was abolished if 8 M urea was present in 0.01 M HCl or 0.9 M acetic acid [110]. Subsequently [111], the effects of the cationic detergent cetyltrimethyl ammonium bromide (CTAB), and the anionic detergent sodium dodecyl sulphate (SDS), in GPC and electrophoresis were compared. The results showed that, in the presence of CTAB, the values of the retardation and distribution coefficients were linear in respect to the molecular mass of histones, while in the presence of SDS, deviations from this type were observed. 

HMC proteins, high mobility group proteins, a family of chromatin proteins first described during the 1970s. The name is related to their high mobility in polyacrylamide gel electrophoresis. According to this behavior and other properties, they resemble histones. The highly conserved HMG proteins contain 25% basic and 30% acidic amino acids. The major members of this family constitute two pairs of... 

You have isolated and separated a number of proteins from a cell lysate by 2D gel electrophoresis. You then estimate the molecular weight and pi of these proteins. The properties of a number of your proteins, labeled A-E, are presented in the table below. Which one(s) could be histones ... 

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