TITLE
A 34x208 Base Pair Repeat Array Containing the 601 Nucleosome Positioning Sequence
CASE NUMBER
C12555
ABSTRACT
Previous published work by Widom and colleagues showed that nucleosomes can be tightly positioned on DNA by the so-called "601" nucleosome positioning sequence. We have generated a vector containing 34 repeats of the 601 nucleosome positioning sequence in a 208 base pair repeat. The array can be excised from the vector with restriction enzyme digestion as a single fragment, or used to produce large quantities of smaller mononucleosome-sized fragments containing the 601 sequence at a central or end position on the DNA.
Description
“601” DNA sequence is a strong nucleosome positioning sequence, meaning that when DNA fragments containing this sequence are reconstituted with histone proteins, the histones preferentially occupy this DNA sequence. The array of 601 repeats described here can serve two basic purposes. First, digestion of the array with either EcoRI or EcoRV restriction enzymes will produce 208 base pair DNA fragments, with the 601 positioning sequence at a defined location on the DNA fragments. Whereas such fragments can also be generated with PCR, the incorporation of many identical repeats into a plasmid allows the production of large quantities of DNA (milligrams) that would otherwise be practically unattainable. These DNA fragments can be used for producing large quantities of nucleosomes for biochemical or biophysical experiments.
A second use of this 601 repeat sequence is that the entire 34-repeat can be used as a single fragment, which when reconstituted with histones can produce an evenly spaced nucleosome array. Such a nucleosome array is considered a chromatin fiber, and the defined, regular spacing of 601 sequences (and therefore nucleosomes) can allow study of different factors, posttranslational modifications, and buffer conditions that may influence condensation/collapse of the chromatin fiber. Chromatin fiber condensation is generally believed to be important for gene regulation, and the biophysical properties of chromatin fibers and how their organization is affected by other factors is a current area of interest.
Features
Novel aspects include the precise spacing between 601 repeats (63 base pairs) and the ability to use two different restriction enzymes to generate two products: a “centered” nucleosome with 28 bp on one side and 35 bp on the other, or an “end-positioned” nucleosome with 2 bp on one side and 61 bp on the other.
ASSOCIATED PUBLICATIONS
■ PMC3561990 ■
TECHNOLOGY CLASSIFICATION
Primary Category: Discovery/Research Tools
Primary Subcategory: Research Reagent
