|previous page||PLNT3140 Introductory Cytogenetics
Lecture 9, part 4 of 4
Note: These treatments are done using isolated nuclei,
not naked DNA
|In the autoradiogram, we see that even at the highest DNAseI concentration, the 4.6kb fragment is relatively insensitive to digestion in nuclei from a cell line that does not express the globin gene. In the erythroblasts, however, specific degredation of this sequence can be seen to occur even at the lowest concentration. The interpretation of this is that the globin gene is in a more open chromatin configuration, which provides greater access to DNAseI. This phenomenon is referred to as general nuclease sensitivity, because the whole gene appears to be digested. However, more detailed studies of certain genes can detect specific sites within transcriptionally active genes that are hypersensitive to nuclease digestion.||
From Lodish et al. Molecualr Cell Biology
|M is a marker lane, with a mixture of DNAs generated by digesting this gene with either BamH1, Xba1, Pst1 or Alu1, and then mixing all of these DNAs together.Now, when the DNAse-ed DNA is digested with Hind3, the +1 to +210 probe will only detect fragments upstream from the Hind3 site. If we do a limiting digestion with DNAseI, in any given fragment, only one of several possible hypersensitive sites will be digested in a given molecule. Thus, we get a "ladder", in which each rung represents a different cut site. Note that there are two constitutive hypersensitive sites, even in uninduced (aerobic) nuclei, whereas in induced nuclei, we see 8 distinct hypersensitive sites.||
from Anna-Lisa Paul, Vimla Vasil, Indra K. Vasil, and Robert J. Ferl. Constitutive and anaerobically induced DNase-I-hypersensitive sites in the 5′ region of the maize Adh1 gene. PNAS 84:799-803
Mol. Biol. of the
Cell, 2nd Ed., p410, Fig. 8-37]
Electron micrograph shows nucleosome structure on both sides of an RNA poymerase II transcription complex.
CROSS-LINKING OF SV40 MINI-CHROMOSOMES
[Darnell, Molecular Cell Biology, 2nd ed.,p335 Fig. 9-22]
Nucleosomes seem to protect
little bubbles of DNA in transcribing genes from treatment
with DNA crosslinking reagents (eg. 6-methyl psoralen). The
bubbles can be shown to be roughly 200bp apart. Only the
inter-nucleosome DNA is crosslinked. The long "tail" on the
circle is the growing mRNA transcript.
Chromatin remodeling: insights and intrigue from single-molecule studies
Bradley R Cairns. Nature Structural & Molecular Biology 14, 989 - 996 (2007) Published online: 5 November 2007
The precise details of chromatin remodeling are still under investigation. However, the figure below illustrates one model for chromatin remodeling. In (d), D and Tr domains of the ocatmer "walk" along the double helix by rotating around the Hinge domain. Thus, while the DNA is more accessible to the solvent, and hence, more sensitive to nucleases, the nucleosome never completely dissociates from the DNA helix.
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