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Nucleic acid Basics

Hybridization. Diagnostic tools. Nucleic acid Basics. PCR. Electrophoresis. DNA-Protein interactions. Chromatin. Gene expression. Chromosome Structure: Essential elements. Origins of DNA replication: - required for the start of DNA replication - many origins per chromosome.

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Nucleic acid Basics

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  1. Hybridization Diagnostic tools Nucleic acid Basics PCR Electrophoresis DNA-Protein interactions Chromatin Gene expression

  2. Chromosome Structure:Essential elements Origins of DNA replication: - required for the start of DNA replication - many origins per chromosome Telomeres: -located at each end of each chromosome -required for completon of DNA replication Centromeres: - involved in mitotic spindle formation - required for segregation of sister chromatids to daughter cells Chromosome 9 during mitosis

  3. Chromosome Structure:Genes • Genes make up a small portion of the genome. A gene contains: • Transcriptional conrol elements • Introns • Coding regions in the form of exons • Translational control elements (if translated)

  4. Chromosome Structure:Intergenic Regons • Most of the genome is intergenic DNA • Transcriptionally silent • Intergenic loci can be examined by molecular tools: polymorphic markers such as STRs. • Important in forensics. • Important for finding disease genes. • STRs are also present in some introns (and in some cases, exons).

  5. Chromatin:The nucleosome Histone Octamer: (2 each of histones 2a, 2b, 3 and 4) Double stranded DNA (146 bp) wrapped around histone octamer Histone termini are extended Chemical modification of the histones (primarily at the NH3 termini) leads to altered nucleosome-nucleosome interactions. These alterations are important forthe regulation of gene expression.

  6. Chromatin:10nm filament Nucleosomes are spaced along the DNA - There are about 50 bp of DNA between each nucleosome. - When extended, this structure looks like “beads-on-a-string” when viewed by electron microscopy. Nucleosomes 50 bp of DNA between nucleosomes

  7. Chromatin:30nm filament The 10nm filament can be further condensed, to a 30nm filament. Important effectors: Histone H1addition DNA methylation Histone deacetylation One possible structure (other structures have been proposed)

  8. Folding of DNA into chromatin Horn & Peterson Science 297:1824-27

  9. Packing of chromatin & transcriptional activity Less compact More active Highly compact inactive Horn & Peterson Science 297:1824-27

  10. Chromatin Domains • A particular Chromatin structure can extend over a large domain. • Domains are separated by DNA sequences referred to as insulators • Domains are loops of about 50 kb, anchored to the nuclear substructure • In mitotic chromosomes, the loops are anchored to a protein core

  11. An insulator-protein complex Gaszner & Felsenfeld (2006) Nat Rev Genet 7:703-713

  12. Functional states of Chromatin • Repressed chromatin • Contains DNA that will never be transcribed in a particular cell line. • Seen histologically as heterochromatin • Inactive & Potentially active chromatin • Contains DNA that is not transcribed, but may be in the future. • Seen histologically as euchromatin • Active chromatin • Contains DNA that is being actively transcribed. • Seen histologically as euchromatin

  13. Important regulators of chromatin structure - Histone modification acetylation methylation phosphorylation other modifications - Chromatin remodeling complexes - Non-histone proteins - DNA modification methylation of C at CpG (Lecture topics) (Lecture topic)

  14. Histone ModificationAcetylation • Histone acetylation leads to a loose chromatin structure. • Transcription factors that stimulate transcription of genes bind histone acetylases. Decondensed chromatin Condensed chromatin Ac Transcription Ac Ac Histone acetylase Activated transcription factor Histone acetylation

  15. Histone ModificationAcetylation • Transcription factors that repress transcription of genes bind histone deacetlyases. Decondensed chromatin Condensed chromatin No Transcription Ac Ac Ac Ac Ac Ac Ac Ac Activated repressor Histone deacetylase Ac

  16. Histone ModificationMethylation • Methylation of some histone lysines condenses chromatin. Examples: • Histone H3 lysine 9 • Histone H3 lysine 27 • Histone H4 lysine 20 • Methylation of some histone lysines decondenses chromatin. Examples: • Histone H3 lysine 4 • Histone H3 lysine 36 • Histone H3 lysine 79 (Lecture topic) (Lecture topic)

  17. Methylation of histone H3 lysine 27 (H3K27) Histone Modification Maintaining the undifferentiated state of embryonic stem cells. Genes required for differentiation Genes with methylated H3K27 (not transcribed) Genes required for proliferation Genes with unmethylated H3K27 (are transcribed) Lee et al (2006) Cell 125: 301-313

  18. Histone ModificationMethylation in Embryonic Stem Cells Bernstein et al (2006) Cell 125:315-326 • Histone H3 methylation patterns: H3K27me (condensed chromatin; repressed transcription) H3K27me/H3K4me (condensed chromatin; minimal transcription) H3K4me (decondensed chromatin; active transcription) K27 - lysine 27 K4 - lysine 4

  19. Histone ModificationMethylation in Embryonic Development Bernstein et al (2006) Cell 125:315-326 Typical methylation pattern of key regulatory genes in undifferentiated embryonic stem cells: H3K27me/H3K4me Differentiation H3K27me H3K4me Methylation pattern of genes that are repressed Methylathion pattern of genes that are expressed

  20. Epigenetic inheritance • Regions of chromatin can be silenced (become permanently transcriptionally inactive). • The pattern of silencing can be maintained and passed to daughter cells, thereby defining a differentiated cell type. • Example: Methylation of histone H3 lysine 27. • Example: Methylation of DNA at CpG

  21. Histone ModificationMaintaining the methylation state(a hypothesis) Trojer & Reinberg (2006) 125:213-217 H3K27 methylase PC binds to histone H3 amino tails

  22. DNA Methylation In mammalian cells, DNA methylation is associated with chromatin inactivity. DNA is methylated on the 5 position of C at some CG sequences. Nucleotide: Sequence: ...CG... ...C5mG...

  23. DNA Methylation and Epigenetic Inheritance A specific pattern of DNA methylation can be transferred to daughter cells: ...CmG ... ...G Cm... + ...CmG ... ...G Cm... ...CmG ... ...G C ... + ...C G ... ...G Cm... DNA replication CmG specific methylation ...CmG ... ...G Cm... Parent cell Newly replicated DNA Daughter cells (both strands (one strand (both strands methylated) methylated) methylated)

  24. 5-methyl-C and MutationsCaused by Deamination If dC is deaminated, dU is the product. dU is rapidly removed by DNA repair enzymes. If d5-methylC is deaminated, T is the product. It is not rapidly removed.

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