The challenge of DNA packaging1. Largest human chromosome: ~3 x 108 bp3 x 108 bp x 3.4 Å/bp x 1 m/1010 Å = 10 x 10-2 m =10 cm!2. A typical cell = 10 µm = 10 x 10-6 m3. Therefore the DNA must be compacted ~104-foldMany levels of sequential packagingThe double helixWrapping around nucleosomesEuchromatin: genes can beexpressed“Loops” of 30-nm fibers seen atinterphaseHeterochromatin: genes aresilenced, DNA is replicatedlater in S phaseMitosis: chromosomes need to becondensed and separated tosegregate to different cellsNucleosome arrays: beads on a stringEM: electron micrographAtomic-resolution structure of a nucleosomeDNA is white; proteins are colored. DNA makes two loopsarounda central protein scaffold with peptide ‘tails’ sticking out. [Luger et al., 1997, Nature 389:251]Packaging unit first evident in nuclease digest1. Isolate nuclei2. Partial digest with nonspecificendonuclease3. Run agarose gelResult: Ladder of multiples of 150-200 bpConclusion: Nucleosome is protecting theDNA in units of 150-200 bp.Proteins = histones H1, H2A, H2B, H3 & H44. Treat with even more nuclease5. Run acrylamide gelResult: Ladder of multiples of ~10 bp.Conclusion: Rougly B-form DNA is wrappedaround the outside of the nucleosome.The histone foldRibbondrawingSimple. Conserved.Adopted by all 4 “core” histones(H2A, H2B, H3 and H4).H3-H4 tetramer binds two H2A-H2B dimersto form the histone octamerRibbondrawings“Top” view“Side” view147 bp of DNA wrappedalmost twice around 8core histonesCarolyn Luger and Tim RichmondHistone “tails” stick outfrom the nucleosomePost-translational chemical modification of thetails marks some regions as euchromatin andothers as heterochromatin. These modificationpatterns comprise the “histone code”.Examples of the histone codeAc - acetyl, Me - methyl, P - phosphorylReplication distributes marked nucleosomes toboth daughter strands but not random DNA:some epigenetic inheritance of chromatin stateMany levels of sequential packagingThe double helixWrapping around nucleosomesEuchromatin: genes can beexpressed“Loops” of 30-nm fibers seen atinterphaseHeterochromatin: genes aresilenced, DNA is replicatedlater in S phaseMitosis: chromosomes need to becondensed and separated tosegregate to different cellsA higher-order organization ofnucleosomes: the 30 nm fiberElectron micrographA partiallydecondensed humanchromosome, withloops of DNA anchoredin a central scaffold.Haploid genome DNA content in variousorganismsGenome size increases out of proportion with gene numberEvolution of new genesGene families arise by duplication and divergence“Pseudogenes” arise by mRNA reverse transcriptionand integration in to the genome, probably byretroviral RTsNew types of gene may arise by “exon shuffling”Exon shuffling in eukaryotesMechanism 1: Recombination between homologousinterspersed repeats in the introns of separategenes would produce a new combination of exons.Exon shuffling in eukaryotesMechanism 2: Exon transposition(a) Exon hopping by getting a ride between the outerinverted repeats of two nearby transposons.Exon shuffling in eukaryotesMechanism 2: Exon transposition(b) Reverse transcription of a non-LTR LINE genomic RNAextending into the 3’ exon of gene 1 can gives gene 2 anew 3’ exon upon integration.About half the genes in any genome encodeproteins of unknown
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