Molekularbiologie. Menu of Organisatorisches Kurze Wiederholung aus der Grundvorlesung
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1 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 1 Molekularbiologie Menu of Organisatorisches Kurze Wiederholung aus der Grundvorlesung DNA: More than just... the protein coding information carrier ---- Structure of Genes and Chromosomes Organisation of Genomes Nuclear Organisation News from Stockholm Summary
2 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 2 Molekularbiologie (Lern)ziele: Wiederholung und Vertiefung der Grundvorlesungsinhalte DNA (Matthias Mayer, ZMBH) DNA im Zellkern (Elmar Schiebel, ZMBH) Einführung weiterer Inhalte und Beispiele Kennenlernen experimenteller Strategien und Systeme Klausur bestehen!
3 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 3 Molekularbiologie Dozenten: Termine: Oliver Gruß Victor Sourjik Claus Bartram Bernd Bukau Günter Kramer Frauke Melchior Georg Stöcklin Christof Niehrs ZMBH ZMBH Institut für Humangenetik ZMBH ZMBH ZMBH DKFZ DKFZ s. Nächste Folien Raum: ZMBH 512 ZMBH 001 (tba)
4 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 4
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7 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 7 1. DNA: More than just... the protein coding information carrier
8 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 8 Oswald Avery,
9 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 9 Avery experiment, 1944 Enzymatic digestion of different substance classes Figure 4-2 Molecular Biology of the Cell ( Garland Science 2008)
10 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 10 3D model of the DNA Double Helix Antiparallel strands explain base pairing and chemically different ends Figure 4-5 Molecular Biology of the Cell ( Garland Science 2008)
11 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 11 The 3D-organisation of the DNA immediately explains the mechanism of replication: One strand is used for the generation of a complementary strand (semiconservative mechanism) Figure 4-8 Molecular Biology of the Cell ( Garland Science 2008)
12 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 12 The Human Genome Project (HGP) endeavored to map the human genome down to the nucleotide (or base pair) level and to identify all the genes present in it. 1. The HGP was formally founded in 1990 by the United States Department of Energy and the U.S. National Institutes of Health, and was expected to take 15 years. Costs: $3 billion an identical quest was initiated separately with private venture capital by a company called Celera Genomics (founded by Craig Venter). Costs: $300 million of private research funding). 3. The completion of the human DNA sequence in the spring of 2003 coincided with the 50th anniversary of Watson and Crick's description of the fundamental structure of DNA.
13 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 13 Other genome projects: C57BL/6J Mouse Sequencing Sus scrofa Xenopus tropicalis Danio rerio Sequencing Project
14 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 14 Table 4-1 Molecular Biology of the Cell ( Garland Science 2008)
15 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 15 Today: Deep sequencing technology can generate 500 million (5 x 10 8!!) bp in a normal 10 hour run on a single sequencing unit. This means you can analyse your genome in less than a week. Details from Victor Sourjik
16 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 16 Sequence Your Genome for Only $5,000 Mountain View, CA (OBBeC) - Complete Genomics, a third-generation human genome sequencing company, has announced its launch as the world's first provider of large-scale human genome sequencing services. Starting next spring the company will be offering a complete human genome sequencing services to pharmaceut and biotechnology companies as well as other medical researchers that were previously priced out of the marke By offering a sequencing service instead of following the traditional instrument sales model, Complete Genomics relieving its customers of operational, computational and capital purchase burdens, allowing them to focus their resources on scientific discovery. "We will be the first company to sequence complete human genomes for less than $1,000 in material costs," said Dr. Clifford Reid, chairman, president and chief executive officer of Complete Genomics. "This breakthrough materials cost, combined with our low per-genome instrument, labour and overhead costs, will allow us to offer complete human genomes for just $5,000 in Q " "This $5,000 price point, combined with the scale of our sequencing centre, will dramatically increase the availability and affordability of human genome sequencing. For the first time, our customers can conduct systematic studies of the genetic basis of disease and drug response. Our sequencing services will be one of the core enablers of the impending revolution in personalized medicine," Reid added.
17 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 17 A complete Neandertal mitochondrial genome sequence determined by high-throughput sequencing Cell Aug 8;134(3): Svante Pääbo and colleagues A complete mitochondrial (mt) genome sequence was reconstructed from a 38,000 year-old Neandertal individual with 8341 mtdna sequences identified among 4.8 Gb of DNA generated from approximately 0.3 g of bone. Analysis of the assembled sequence unequivocally establishes that the Neandertal mtdna falls outside the variation of extant human mtdnas, and allows an estimate of the divergence date between the two mtdna lineages of 660,000 +/- 140,000 years. Of the 13 proteins encoded in the mtdna, subunit 2 of cytochrome c oxidase of the mitochondrial electron transport chain has experienced the largest number of amino acid substitutions in human ancestors since the separation from Neandertals. There is evidence that purifying selection in the Neandertal mtdna was reduced compared with other primate lineages, suggesting that the effective population size of Neandertals was small.
18 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite Organisation of Genes and Chromosomes
19 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 19
20 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 20 Gene: The entire DNA - including exons, introns and non-coding transcriptional control regions -, that are essential for the production of a functional protein or RNA-molecule. What Are Pseudogenes? Pseudogenes are genomic DNA sequences similar to normal genes but are not expressed into functional proteins; they are regarded as defunct relatives of functional genes. Open Reading Frame (ORF): Open Reading Frame is a stretch of DNA of at least 100 codons starting with an ATG start codon and ending with a stop codon (TGA/TAA/TAG). An ORFs has the potential to encode a protein.
21 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 21
22 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 22 Only a small subset of the genomic DNA represents protein coding sequences (ca. 1.5%). In human Chr. 22, the short (p) arm is e.g. heterochromatin, which comes in short, repetitive sequence Elements. Figure 4-15 Molecular Biology of the Cell ( Garland Science 2008)
23 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 23 A good 20% of DNA is assigned to protein coding genes, only 1.5% in exonic sequences. More than 50% is found in different kinds of repeats. Short/Long Interspersed elements (S/LINEs) Figure 4-17 Molecular Biology of the Cell ( Garland Science 2008)
24 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 24
25 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 25 The Nuclear compartment harbours the DNA to enable efficiently high concentrations of Enzymes with DNA-related functions and interference with/by cytoplasmic functions Figure 4-9 Molecular Biology of the Cell ( Garland Science 2008)
26 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 26 Of the ca. 3.2 x 10 9 bp of the human genome, a marginal but essential part (ca. 1.7 x 10 4 ) are encoded on mitochondrial DNA. Replication of the mitochondrial DNA works via a separate ( bacterial ) replication system. The Mitochondrial Genome Mitochondrial DNA in most verterates (incl. Humans) is inherited maternally. Due to the production of ROS in mitochondria, mtdna is often damaged and mutated. Many human diseases occur as a consequence of mtdna mutations.
27 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 27 DNA organisation in Interphase and Mitosis: Mitosis includes Nuclear Envelope Breakdown in Higher Eukaryotes (Open Mitosis) Figure 4-1 Molecular Biology of the Cell ( Garland Science 2008)
28 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 28 Faithful segregation of sister chromatids after replication is essential for the maintenance of genome stability. Chromosomes are highly condensed in metaphase in order to be separated by the mitotic spindle. The cell cycle control machinery ensures correct timing of events and their proper execution. Figure 4-19 Molecular Biology of the Cell ( Garland Science 2008)
29 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 29 Chromosomes need basic elements in order to be stably inherited. Replication starts at replications origins, while spindle microtubules attach on kinetochores assembled on centromeric DNA. The special telomere structure ensures homeostasis of chromosome length. Figure 4-21 Molecular Biology of the Cell ( Garland Science 2008)
30 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 30
31 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 31 Figure 4-73 Molecular Biology of the Cell ( Garland Science 2008) Figure 4-74 Molecular Biology of the Cell ( Garland Science 2008)
32 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 32 DNA condensation in Mitosis and Interphase Figure 4-20 Molecular Biology of the Cell ( Garland Science 2008)
33 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 33 Figure 4-71 Molecular Biology of the Cell ( Garland Science 2008) Figure 4-70 Molecular Biology of the Cell ( Garland Science 2008) Figure 4-72 Molecular Biology of the Cell ( Garland Science 2008)
34 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 34 Chromosome painting: Hybridising with fluorescently labeled DNA probes reveals 2x23 human (condensed) chromosomes in mitosis Figure 4-10 Molecular Biology of the Cell ( Garland Science 2008)
35 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 35 Giemsa staining of human chromosomes in early mitosis shows a characteristic Pattern. Genes encoding for Ribosomal RNAs Figure 4-11 Molecular Biology of the Cell ( Garland Science 2008)
36 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 36 Chromosome Translocation: Pathological Translocation from human Chromosome 4 to human Chromosome 12 t (4;12) is associated with Ataxia. Patterns of translocations upon primate evolution can be used to decipher chromosome development. Figure 4-12 Molecular Biology of the Cell ( Garland Science 2008) Figure 4-18 Molecular Biology of the Cell ( Garland Science 2008)
37 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 37
38 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite nm-fiber and DNA organised in nucleosomes. Figure 4-22 Molecular Biology of the Cell ( Garland Science 2008)
39 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 39 Schemes of the primary structures of Histones. They contain a conserved histone fold, which accounts for dimerisation ( handshake ). The N-terminal tail region is often heavily modified to modulate DNA packaging and to recruit additional Chromatin proteins. Figure 4-25 Molecular Biology of the Cell ( Garland Science 2008)
40 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 40 High-Resolution Structure of Nucleosomes Figure 4-24 Molecular Biology of the Cell ( Garland Science 2008)
41 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 41 Assembly of a nucleosome: Two dimers of H3-H4 form a tetramer, which is incorporated into DNA by the help of Histone Chaperones. The H3-H4 tetramer then further recruits tow dimers of H2A/H2B. Figure 4-26 Molecular Biology of the Cell ( Garland Science 2008)
42 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 42 Nucleosome Dynaimcs: The structure of nucleosomes was long considered as a rigid, rather non-flexible packaging. Figure 4-28 Molecular Biology of the Cell ( Garland Science 2008) We now know that they are in fact highly dynamic and can unwrap and dewrap several times per second.
43 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 43 Chromatin Remodeling Complexes: These DNA binding complexes use the energy of ATP to actively move histone octamers in their position on the DNA. Figure 4-29 Molecular Biology of the Cell ( Garland Science 2008)
44 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 44 Exchange of Nucleosomes: Chromatin remodeling complexes cooperate with Histone chaperones to replace Histone dimers or even complete octamers from nucleosomes. Histone chaperones are also required for the deposit of new octamers into chromatin upon replication. Figure 4-30 Molecular Biology of the Cell ( Garland Science 2008)
45 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 45 Incorporation of Nucleosomes: Replication of DNA requires also the duplication of Histones... Figure 5-38a Molecular Biology of the Cell ( Garland Science 2008)
46 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 46 Figure 5-38b Molecular Biology of the Cell ( Garland Science 2008)... and respective Histone chaperones to dispose the new Histones into the nucleosomes.
47 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 47 The Histone code: The combinations of modifications (in the N-terminals tails) of core Histones Details from Victor Sourjik Figure 4-44a Molecular Biology of the Cell ( Garland Science 2008)
48 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 48 Inheritance of Epigenetic Marks Figure 5-39 Molecular Biology of the Cell ( Garland Science 2008) Details from Victor Sourjik
49 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 49 Figure 4-61 Molecular Biology of the Cell ( Garland Science 2008)
50 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 50 Superstructure of the 30nm fiber: Zic-Zac-Model deduced from the x-ray structure of the nucleosome tetramer, or more complicated structures deduced Figure 4-31 Molecular Biology of the Cell ( Garland Science 2008) from oligomers after cryo-em.
51 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 51 Figure 4-33a Molecular Biology of the Cell ( Garland Science 2008) Interactions between nucleosomes are mediated by tail-tail-interactions (histone code!)... Figure 4-33b Molecular Biology of the Cell ( Garland Science 2008)
52 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite and the linker Histone H1. Figure 4-34 Molecular Biology of the Cell ( Garland Science 2008) Histone H1 bends the linker DNA and thus promotes a more compact chromatin structure.
53 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 53 Lampbrush-Chromosomes: Oocytes arrest in Meiosis I before chromosome hypercondensation with highly active gene expression. Figure 4-54 Molecular Biology of the Cell ( Garland Science 2008)
54 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 54 Joseph Gall, Carnegie Intitute Oogenesis: Stages of Xenopus laevis oocytes Stage: Classification according to Dumont (1972)
55 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 55 Figure 4-55 Molecular Biology of the Cell ( Garland Science 2008)
56 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 56 Chromatin conformation capture, a method for detecting proteinmediated loop structures of chromatin: Next neighbouring sequences in 3D are crosslinked, free DNA is digested and re-ligated. The residual DNA can be sequenced. Figure 4-56 Molecular Biology of the Cell ( Garland Science 2008)
57 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 57 Different activities cooperate to loop out regions for active gene expression. These loops are organised by a protein-rich scaffold, which contains e.g. Topoisomerase II and other enzymes. Figure 4-57 Molecular Biology of the Cell ( Garland Science 2008)
58 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 58 Salivery Gland Polytene Chromosomes: Homologous chromosomes of polyploid chromosomes (2 10!) perfectly align to each other and therefore reveal details of chromosome organisation upon microscopy analysis. Figure 4-58 Molecular Biology of the Cell ( Garland Science 2008)
59 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 59 Salivery Gland Polytene Chromosomes: Transcriptionally active regions ( puffs ) loose their characteristic banding pattern, i.e. they are unfolded and Figure 4-59 Molecular Biology of the Cell ( Garland Science 2008) Figure 4-62 Molecular Biology of the Cell ( Garland Science 2008)
60 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite Organisation of Genomes
61 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 61 Gene duplication enabled the evolution pf different globin genes with specialised and separate functions in different tissues or during development. Figure 4-86 Molecular Biology of the Cell ( Garland Science 2008) Figure 4-87 Molecular Biology of the Cell ( Garland Science 2008)
62 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 62 Gene duplication of sequences coding For protein domains enabled the evolution of proteins with repetitive structure, like e.g the immunoglobulin fold. Figure 4-88 Molecular Biology of the Cell ( Garland Science 2008)
63 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 63 Individual Variations in the human population can include frequent gene duplications to different copy numbers of the same gene, or rather rare differences in the number of repetitive elements. Figure 4-89 Molecular Biology of the Cell ( Garland Science 2008) Figure 4-90 Molecular Biology of the Cell ( Garland Science 2008)
64 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 64 Figure 4-81 Molecular Biology of the Cell ( Garland Science 2008) Fugu Rubripes The puffer fish minimised the size of introns in its sequences. Shown is the Huntingtin gene of humans and Fubu. Figure 4-82 Molecular Biology of the Cell ( Garland Science 2008)
65 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 65 Conservation of the Leptin gene Figure 4-75 Molecular Biology of the Cell ( Garland Science 2008) Phylogenetic trees: The distance of two species (in % nucleotide changes)is half the distance to the last common ancestor. Figure 4-76 Molecular Biology of the Cell ( Garland Science 2008)
66 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 66 Figure 4-78 Molecular Biology of the Cell ( Garland Science 2008) Figure 4-77 Molecular Biology of the Cell ( Garland Science 2008)
67 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 67 Interspecies alignment The CFTR gene was aligned to detect multispecies conserved sequences, which show highest homology in the consensus of all species. Figure 4-83 Molecular Biology of the Cell ( Garland Science 2008)
68 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 68 Figure 4-84 Molecular Biology of the Cell ( Garland Science 2008)
69 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite Nuclear Organisation
70 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 70 Typical examples of subnuclear compartments: Nucleoli and Cajal (Coiled) bodies. Figure 4-67 Molecular Biology of the Cell ( Garland Science 2008)
71 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 71 Figure 4-68 Molecular Biology of the Cell ( Garland Science 2008)
72 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 72 Figure 4-69 Molecular Biology of the Cell ( Garland Science 2008) ifrap detects dynamics of different Cajal body components. Coilin and SMN (not shown) have A different residence time than Sm proteins, which are substrates of Cajal bodies. (from M. Dundr et al., JCB, 2004)
73 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 73 Gene rich regions (here: Alu interspersed repeats) are stained in green, while red indicates the absence of those repeats. Regions rich in genes are therefore centered within the nucleus. Figure 4-64 Molecular Biology of the Cell ( Garland Science 2008)
74 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 74 A FISH experiment was used to detect terretories of human chromosomes. The multi-colour detection allowed a 3D-reconstruction of the terretories. Figure 4-63 Molecular Biology of the Cell ( Garland Science 2008)
75 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 75 FISH was used to detect rearrangements of regions, which relocate upon activation of transcription. Shown is a region of chromosome 11, which contains 47 different genes. Figure 4-65 Molecular Biology of the Cell ( Garland Science 2008)
76 Zyklusvorlesung Molekularbiologie WS 2009/10 Oliver Gruß, Seite 76 Figure 4-66 Molecular Biology of the Cell ( Garland Science 2008)
77 The Human Genome Project (HGP) endeavored to map the human genome down to the nucleotide (or base pair) level and to identify all the genes present in it. 1. DNA: More than just... the protein coding information carrier
78 2. Organisation of Genes and Chromosomes
79 3. Organisation of Genomes
80 4. Nuclear Organisation
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