Linker DNA preparation

The nucleosome core particle is a relatively stable and homogenous structure that is easily prepared, and as such has formed the basis for numerous studies into chromatin structure and function. However, several recent studies have suggested that what is true for the nucleosome core may not always be true for nucleosome arrays, nor even for nucleosomes containing linker DNA. For example, the core histone tails preferentially interact with linker DNA when is it present, whereas they are constrained to bind intranucleosomal DNA in core particles [46 8]. Consequently, the activities of proteins that require access to the tails or the DNA may be affected, and it has been shown that both DNA ligase and P/CAF are less active on nucleosome core particles than other chromatin substrates [49,50]. Similar concerns apply to the interaction of HMGN proteins with nucleosome core particles, and results from studies of these complexes must be considered in the wider context of how these proteins may interact with nucleosome arrays. 

Fig. 4. Images of unfixed and unstained chromatin in a frozen and hydrated state. All samples shown contain linker histone H5. (A) Soluble chromatin prepared from chicken erythrocyte nuclei. Arrow indicates a nucleosome with a linker histone stem conformation. (B-E) Chromatin reconstituted onto an array of the 5S rDNA nucleosome positioning sequence. En face views (B-D) of nucleosomes show the linker DNA entering and exiting the nucleosome tangentially, before interacting and remaining associated for 3-5 nm before separating (arrows). An edge-on view (E) shows the two gyres of DNA (arrow heads) and the apposed linker DNA (arrow) (from Ref. [30]). Scale bar 20 nm (A) and 10 nm (B-E).

While structural transitions at the level of nucleosomes are still outside the scope of current models, applying a constant force to both ends of the liber allows to simulate the low end of the force-extension curve. First results from our own work (Aumann, Caudron, Wedemann, and Langowski, manuscript in preparation) are shown in Fig. 5. In this particular example, a nucleosome repeat of 200 bp and a linker DNA length of 11 bp was used. 

Where the linker DNA sequence was modified for better codon usage in E. coli. The linker was originally prepared by complementarity oligonudeotide synthesis to generate the sequence ... 

Estimate the quantities of VH-linker and linker-VL DNA prepared by the jumping PCR reactions (Protocol 7) using a dot spot technique." Adjust the concentrations of VH-linker and linker-VL PCR products to 5 ng/jcl. 

To perform the DNA assembly step, a mixture of all the 3-kb sets, plasmid, and linker DNAs will be cotransformed in yeast (Fig. lb). For this, the following four samples, including controls, have to be prepared ... 

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