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Chromosomes and DNA Packaging

Chromosomes and DNA Packaging. Source: From teaching resources of Bloomsburg University.

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Chromosomes and DNA Packaging

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  1. Chromosomes and DNA Packaging Source: From teaching resources of Bloomsburg University SBL201

  2. Nobody can pack as nature has packed: DNA CondensationIf you were to take one molecule of DNA from a human cell and stretch it out to its full length, it would be approximately two meters long. So it is truly incredible that such an enormously long molecule can be compressed into the microscopic space of the nucleus of a cell. Unpacking of DNA as a function of time (femto seconds) and space (1014 cells) is a far far bigger challenge

  3. The Problem • Human genome (in diploid cells) = 6 x 109bp • 6 x 109bp X 0.34 nm/bp = 2.04 x 109 nm = 2 m/cell • Very thin (2.0 nm), extremely fragile • Diameter of nucleus = 5-10 mm • DNA must be packaged to protect it, but must still be accessible to allow gene expression and cellular responsiveness SBL201

  4. Solution: Chromosomes • Single DNA Molecule and associated proteins • Karyotype • Chromatin vs. Chromosomes SBL201

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  7. HISTONES • Main packaging proteins • 5 classes: H1, H2A, H2B, H3, H4. • Rich in Lysine and Arginine SBL201

  8. HISTONES • NOTE: if histones from different species are added to any eukaryotic DNA sample, chromatin is reconstituted. Implication? • Very highly conserved in eukaryotes in both • Structure • Function • Protection • Must allow gene activity SBL201

  9. VARIABILITY in HISTONES • Fairly uniform, some variability Highly Conserved: Core Histones H3, H4, can assemble DNA into particles Moderate variations amongst tissues and species: Core Histones H2A, H2B, More peripheral Varies markedly among tissues and species: H Variable, mostly in non-replicating cells: H10 Very Variable, only in transcriptionally inactive cells of some species: H5 • Why variable if only function is packaging? SBL201

  10. Histones—Degrees of Conservation • H4---Only 2 variations ever discovered • H3—also highly conserved • H2A, H2B---Some variation between tissues and species • H1-like histones • H1—Varies markedly between tissues and species • H1º--Variable, mostly present in non-replicating cells • H5---Extremely variable SBL201

  11. Fig. 9 Orders of chromatin structure from naked DNA to chromatin to fully condensed chromosomes... SBL201

  12. Beads on a String—10 nm Fiber SBL201

  13. 10 nm filament; nucleosomes protein purification H1 H3 H2A histones (= 1g per g DNA) DNA H2B H4 • Basic (arg, lys); • + charges bind to - phosphates on DNA SBL201

  14. 10 nm Fiber • A string of nucleosomes is seen under EM as a 10 nm fiber SBL201

  15. 30 nm Fiber • 30 nm fiber is coil of nucleosomes with 6/turn SBL201

  16. The 30 nm Fiber(Compacts DNA 7X more) SBL201

  17. Different forms of chromatin show differential gene activity. heterochromatin euchromatin SBL201

  18. Other DNA Associated Proteins • Gene expression in a dynamic environment • DNA associated non-histones: • Palindromes • -helices • Symmetrical dimers • Interaction between R-groups and specific bases • Role of basic amino acids SBL201

  19. back SBL201

  20. back Phosphorylation (Ser and Thr) SBL201

  21. Histidine: N-phosphorylation • How will this affect His’s ability to participate in weak interactions? SBL201

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  23. E H Euchromatin (E) vs Heterochromatin (H) DNase Being more condensed (tightly packed), heterochromatin is resistant to DNase digestion. SBL201

  24. Hypothesis: Transcriptionally active DNA (i.e., an active gene) is in euchromatin. nascent transcripts E H Can test with DNase I, which should preferentially digest active genes in a cell... SBL201

  25. An Experiment: denature DNA, “spot” on filter make 2 of these... Increasing time of digestion Fig. 12 chick red blood cells chick RBC nuclei chromatin extraction total chromatin digest with DNase I for different times… partially digested chromatin; extract undigested DNA SBL201

  26. Increasing time of digestion globin gene probe ovalbumin gene probe DNA from digested RBC chromatin • Radiolabel, denature cloned globin and other gene DNA to use as *probe. • Allow *probe DNA to hybridizeto complementary sequences on filters… expose, develop autoradio- graph... SBL201 Fig. 13

  27. Conclusions: • Compared to globin gene DNA, ovalbumin gene DNA in RBC chromatin resists DNase digestion. • Globin gene DNA in RBC chromatin must be more accessible to DNase (less condensed, more euchromatic) than ovalbumin DNA. SBL201

  28. Variations In Histones • How can cells introduce changes in protein structure and thus protein function? • Mutations • Post transcriptional modifications—ex alternate splicing • Post translational modifications • Acetylation • Methylation • Ser-Thr O-phosphorylation • His N-phosphorylation • NOTE: These processes are dynamic. They give the cell another means to regulate gene expression SBL201

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