Nucleosomal plasmid

The effect of the superhelical strain of the DNA template on the nucleosome structure can be investigated from the in vitro chromatin reconstitution system (for the detail of in vitro chromatin reconstitution, see sections 2.1 and 2.3). Interestingly, the efficiency of the reconstitution becomes higher as the lengths of the DNA used are longer (Hizume et al, 2004) (Fig. 3a-c). In the 3 kb reconstituted chromatin, one nucleosome could be formed in every 826 bp DNA on average, while in the 106 kb chromatin fibers, one nucleosome can be formed in every 260 bp of DNA. The chromatin reconstituted on the any length of linearized plasmid, the efficiency of the reconstitution becomes one nucleosome per 800 bp DNA. The treatment of the... 

Figure 3. The stability of the nucleosome is affected by the length and the superhelicity of DNA. (a-b) The chromatin fibers were reconstituted from the purified plasmids and the histone octamers by a salt-dialysis method and observed under AFM. The 3 kb (a) or 106 kb (e) supercoiled circular plasmid was used as a template, (c) Relationship between the plasmid length and the frequency of nucleosome formation in the reconstitution process. The nucleosome frequency is represented as the number of base pairs per nucleosome and plotted against the length of the template DNA in supercoiled (filled circle) and linear (open circle) forms, (d) AFM image of the chromatin fiber reconstituted on the topoisomerase 1-treated plasmid, (e) Chromatin fiber reconstituted with Drosophila embryo extract. The chromatin fiber was reconstituted from plasmid DNA of 10kband the embryo extract of Drosophila, and was observed by AFM...

Figure 4. In vitro reconstituted 30 nm chromatin fiber. Dynamic structural changes in the chromatin fiber in the absence (top) or presence (bottom) of linker histone HI with different NaCl concentration were observed by AFM. Nucleosomes were reconstituted on the 106 kb plasmid and then fixed in the buffer containing 50 mM (top left) or 100 mM NaCl (top right). Nucleosomes were well-spread in 50 mM NaCl but attached each other and partially aggregated in 100 mM NaCl. After the addition of histone HI, the thicker fibers were formed. The width of the fibers is 20nm in 50mM NaCl (bottom left) or 30 nm in lOOmM NaCl (bottom right)...

In the absence of conclusive data on the role of a positive supercoiling wave, static positive supercoiling elicited by nucleosome reconstitution on relaxed or slightly positively-supercoiled plasmids [51] or by ethidium bromide intercalation in the loop of mononucleosomes on DNA minicircles [52] did not succeed either in releasing dimers. Moreover, circular dichroism, histone chemical modi-flcation and H3-thiol accessibility failed to detect an even slight alteration in the structure of such torsionally-stressed nucleosomes [51]. The reason was later found to lie in the ability of nucleosome entry/exit DNAs to form a positive crossing [52]. 

The contribution of individual core histone N-tails to transcriptional regulation has been recently addressed in a reconstitution study [42]. Varied combinations of recombinant and mutant cores histones have been assembled on circular plasmids, and the resultant nucleosomal arrays were tested for transcriptional... 

Typically, 1.5 pg plasmid DNA is incubated with embryo extract (3 mg protein) containing 30 mM creatine phosphate, 3 mM MgCl2,3 mM ATP (pH 8.0) 0.1 pg/ ml creatine phosphokinase (type 1, Sigma), 1 mM DTT, and EX buffer to a total volume of 200 /u.1. The conductivity of the assembly reaction mix is equivalent to 65 mM KCl. The reaction is carried out for up to 6 hr at 26°C. Preblastoderm embryo extracts contain a maternal stockpile of core histones that are utilized in the assembly reaction. The linker histone, HI, on the other hand, is absent from the early embryo and, hence, from the reconstituted chromatin. However, exogenous HI can be incorporated into chromatin during the assembly reaction, where it increases the nucleosome repeat length from 180 to 197 bp DNA (Becker and Wu, 1992). 

Fig. 4b shows the complex of pBR322 plasmid DNA with DOGS, dioctadecylamidoglycylspermine. It is expected that the DDNA/DOGS complex is composed of an aggregate of DOGS wrapped by DNA stands, resulting in the formation of a spool or a nucleosome-like structure [16]. 

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