Updated version of III-A, 1 & 2. Revised 02/15/2011 03:18 PM
III. Structure of Chromatin -- Histones & Nucleosomes
A. How are proteins and DNA arranged? How does all that DNA fit into a tiny cell? The evidence.
→ resistant core of around 145 BP. Implies part of 200 BP repeat is relatively protected in/on core of "bead"; rest goes between beads and is more exposed.
1. Appearance of chromatin in EM -- -- low salt ("beads on a string" appearance) vs physiological salt (30 nm fiber). See Becker fig. 18-17 or Alberts (Or go to the PubMed Bookshelf at http://www.ncbi.nlm.nih.gov/books/ and use the search term box. ) Shows DNA and protein arranged in repeating structure of 'beads on a string'.
2. Treatment with DNase
a. Specificity of this DNase. Enzyme used here is microccocal nuclease; cuts DNA in areas that are most exposed or least protected. Where it cuts depends on local chromatin structure, not on the base sequence of the DNA. (Restriction enzymes are specific for certain sequences; this enzyme is not.)
b. Procedure: Treat chromatin (not DNA, but DNA with associated proteins!) with microccocal nuclease; then remove protein and isolate the DNA, then run DNA on a gel.
(1). If you use a little nuclease: A little digestion with nuclease → 200 Base Pair (BP) "ladder" = sequences of multiples of 200 BP on gel. Implies repeating structure ("bead") with about 200 BP per repeat; DNA exposed (so easily cut) about once per 200 BP. (See Becker fig. 18-18 & 18-19.)
(2). If you use a lot of nuclease: More treatment with nuclease
(1). DNA is in Nucleosomes: Chromatin = DNA-protein complex with a repeating structure. One nucleosome = one repeat unit = 200 BP + associated proteins.
(2). Nucleosome core: About 145 PB of 200 BP repeat is relatively protected in/on core of "bead" -- rest is a 'linker' that goes between beads and is more exposed.
(3). Linker: Linker DNA has one site every 200 BP that is relatively unprotected and readily cut by micrococcal nuclease.