MBMB 451A Section One - Fall 2007

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Higher Ordered Structure

Hierarchies of organization
	A.  Winding of DNA into bead like particles
		1.  10 nm fiber - does not require H1 histone under low ionic conditions
		2.  Packing ratio (length of DNA/length of unit) is ~6
		3.  Part of euchromatin, heterochromatin, and chromosomes - invariant
	B.  Coiling of series of beads into a helical array
		1.  30 nm fiber requires H1 and greater ionic conditions
		2.  packing ratio of ~40
		3.  basic component of both mitotic chromosome and interphase chromatin
		4.  easily interconverted into 10 nm structure by changing salt concentration
	C.  Packing of 30 nm fiber
		packing ratio of > or = 1000 in euchromatin and < or = 10,000 for heterchromatin
	D.  Nucleosome assembly - chaperone
		1.  Nucleoplasmin - acidic protein
		2.  Topoisomerase I
	E.  Nuclear scaffold and nuclear matrix figures 26.6 and 26.7 (Genes VI)
	F.  Domains on chromatin 
		1.  LCR locus control region
		2.  MAR matrix attachment regions
		3.  delimiter or scs (specialized chromatin structures) - 
			prevent effects from spreading beyound the functional unit

Phasing of Nucleosomes
	A.  Definition of nucleosome phasing - the binding of nucleosomes to a specific region of DNA 
		rather than the more normal random binding of nucleosomes to DNA
		1.  Translational positioning figure 27.30 (Genes VI)
		2.  Rotational positioning figure 27.31 (Genes VI)
	B.  Nucleosome phasing occurs because of
		1.  binding is influenced by sequence specific topology
		2.  the first nucleosome in a region is preferentially assembled at a particular site because
			a.  region excluded by prior protein-DNA complex or other higher order structure
			b.  followed by sequential  assembly of nucleosomes
			c.  phasing would be expected to be well maintained close to boundary, 
				but not farther away (variability in linker)
			d.  example is the central region of Tetrahymena rDNA

Nucleosomes - Transcribed Genes
	A.  Some perturbation must occur in euchromatin to allow transcription to occur
		1.  Entry of RNA polymerase and transcription factors
		2.  Transcription bubble formation
		3.  Passage of polymerase and transcription bubble
	B.  Visualizing transcription and chromatin in vivo
		1.  Indirect labeling
			example of heat shock activated transcription in Drosophila and the URA3 gene 
				figure 27.37 (Genes VI)
			need to keep in mind the fraction of genes expressed vs total genes
		2.  Electron Microscopy Analysis
			a.  rDNA complexes - classic picture
			b.  SV-40 minichromosomes - detect presence of nucleosomes and RNA tail on same 
				minichromosome, also can detect gap where no nucleosomes are associated
		3.  Follow extent of protein modification of histone proteins - according to phase in cell cycle
			a.  acetylation
			b.  methylation
			c.  phosphorlyation
		4.  Chromatin immunoprecipitation or CHIP assay
			will discuss in lecture
		5.  DNase sensitivity
			a.  hypersensitive sites - typically 100X more sensitive than the rest of the chromatin.
				use indirect labeling to detect; example is the SV40 gap region in the minichromosome
				next to the major late promoter
			b.  more extensive DNase digestion - helps define large domains
				look for loss of selected sequence after digestion figure 27.41 (Genes VI) 

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Nucleic Acids  |  Methods-Nucleic Acids: Part A and Part B  and Molecular Cloning | DNA Supercoiling  |  Nucleosome | Transcription: Part A and Part B  | Chromatin Transcription  |  Regulation of Transcription  |  Course Material

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Last updated on August 30, 2007 .