Reconstitution plasmids

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... 

Further modifications using the same strain of ODC S. cerevisiae reconstituted a bacterial/plant polyamine synthesis pathway in yeast [41], The ODC strain was transformed with plasmids encoding arginine decarboxylase and ag-matine ureohydrolase, which conferred polyamine-independent growth on the recombinant microbe. A similar construction could be used to screen for inhibitors of the homologous enzymes from Apicomplexan protozoa, which synthesize poly amines through this pathway [42]. 

Efforts to test hypothesis two by biochemical reconstitution of a functional desaturase complex in vitro proved technically difficult and failed to provide a reproducible assay for functional expression of cloned lepidopteran desaturase sequences. In contrast, an in vivo expression system consisting of the yeast olel mutant and YEpOLEX plasmid confirmed hypothesis two and provided a technically facile and robust assay for determining the functional identities of many moth desaturase-encoding cDNAs. Particularly desirable features of this... 

In cloning, the plasmid vector is incubated with a restriction endonuclease that cuts open the plasmid DNA. Exposure of the open DNA to a new DNA fragment plus a ligase reconstitutes the plasmid DNA with a new nucleotide sequence. The resulting recombinant DNA is inserted into bacterial cells, which then multiply. Each cell can contain 50-100 copies of the recombinant plasmid and can duplicate every 20-30 minutes. 

Recent reports on facilitation of protein folding by molecular chaperones 46-48) provide another possibility the difference between these two pathways could be a consequence of lack of certain cellular components in the in vitro reaction. It was observed that the products of gr< -bearing plasmid were able to rescue some temperature sensitive P22 tailspike mutants in Salmonella at the restrictive temperature (39 C), though very weakly 49), On the other hand, high yield of the native protein was also reported in the refolding (reconstitution) of the acid urea denatured tailspike polypeptide chains without the addition of cellular factors at low temperatures (10°C) (57). This in vitro result indicates that auxiliary factors inside the cell are not absolutely required for the folding of this protein, at least under these experimental conditions. However, this does not rule out that the cellular factors could play a role under in vivo conditions. 

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