1 Transposons DNA transposons: leave their site of insertion (nonreplicative transpostion) or duplicate during transposition (replicative transpositions RNA (Retro) transposons: stay at the site of insertion, integrate a new copy
2 DNA-Transposable elements typically carry inverted repeats at their ends and generate a short target site duplication upon insertion
3 Excision-insertion typically leads to a duplication of the insertion site During replicative transposition, the TE is copied to a new site Some excision-insertion events are highly conservative (e.g. Lambda)
4 Replicative versus non-replicative transposition Both mechansims can use a common intermediate. Depending on the resolution of this crossover structure either type of transposition can occurr. The type of transposition is typical for a particular type of TE
5 Transposition can lead to DNA rearrangements Insertion of multiple copies of a transposon can lead to loss of DNA at the site of excision, or DNA rearrangement. Imprecise excision can induce mutations Loss Inversion
6 DNA transposons typically encode their own transposase can have deletions in their transposase can carry other genes between their LTRs can be activated by a transposase provided in trans frequently prefer to jump locally excision can be precise or unprecise transposition frequently leads to target site duplication after excision the duplicated target site leads to a toeprint of the transposon
7 Organisation des Mais Transposons AC Kunze et al., 1997
8 Ac hat einen offenen Leserahmen (ORF); Ds Elemente sind meist ähnlich wie Ac, aber mit internen Deletionen oder mit Fremd-DNA. Lewin, 2000
9 Kunze et al., 1997 Die Transposasemoleküle an den Ac/Ds Enden machen je einen Endonuklease- Schnitt, der jedoch in den beiden Strängen um 8 Basen versetzt ist. ( Target-site duplication = TSD )
10 The maize Ac-Ds System Activation-Dissociation system Discovered by B. McClintock in the 1940s The dissociator induces frequent brakes at chromosome 9 but only in the presence of the Ac (activator)
11 Descendants of the Ac-DS strain show a variety of kernel phenotypes These can be explained by local hopping and excision Clonal (mitotic) expansion of a particular phenotyp leads to spotted appearance Ac: is an autonomous element carrying its own transposase Ds: is a non-autonomous element that depends on Ac s transposase
12 TEs can give rise to a variety of different phenotypes
14 Drosphila P-elements (DNA transposons) show hybrid dysgenesis no repressor repressor element transposes in germ-line of F1 no transposition in germ-line of F1
15 A repressor prevents transposition Since oocytes contribute to the nucleo and cytoplasm a female with P cytotype produces the repressor protein that prevents P-element hopping The reciprocal cross leads to P activation, accumulation of mutations and sterility Exon 1-4 : Repressor Exon 2-4: Transposase
17 The use of transposable elements in mutagenesis The helper plasmid has a marker to allow selection for its loss (to prevent additional hoppings in subsequent generations). Cosegregation of the ry phenotype with a particular mutation allows mapping of the gene responsible for the mutation. Identification of flanking sequences occur via inverse PCR cut with R-enzyme Ligate, PCR amplify clone
18 The life-cycle of a retrovirus
19 Retrotransposons and retroviruses are structurally and functionally related Env: envelope (unique to viruses) Gag: Required for RNA processing (in some REs) Pol: Reverse transcripase (in all REs, but not always active) also encodes integrase
20 The life cycle of a Ty RE Splicing of an inserted intron proves that transposition occurs through an RNA intermediate
21 Retroviral replication requires two consecutive priming events
23 Integration/transposition reaction target site duplication
25 Non-LTR retroelements (LINES) require priming by a nick in DNA The nicking and RTAse activities are encoded by the RE The gene products of LINES can promote activation of SINES
26 read through
27 The human genome is full of TE and RE but: the majority of elements is inactive Insertion or RE in the human HGO gene (homogenisate 1,2, dioxygenase)
28 HeT-A and TART, two non-ltr TEs are located at the end of Drosophila chromosomes. (Drosophila lacks Telomerase) Priming by the chromosome end allows reverse transcription and elongation of telomeric sequences
29 Nukleolus und Ribosomen Biogenese
30 5S RNA Gene Satelliten DNA im Heterochromatin rrna Gene Telomer Repeats Transposase Transposon Non-LTR Retrotransposons (non viral) LTR Retrotransposons (viral type)
32 Ribosomale Gene sind tandemartig organisiert
33 Ribosomale Transcripte sind polycistronisch und werden processiert Pol I transcribes rrna Pol III transcribes 5s rrna, trna and some snrnas
34 small nucleolar RNAs (snornas) guide base conversions in the nucleolus the conversions are essential for rrna processing. C/D box snornas guide methylation, H/ACA box RNAs guide pseudouridinylation
35 Im Nukleolus werden ribosomale Untereinheite n assembliert
37 Meiotische Zellteilung Reduziert das diploide Genom auf einen haploiden Chromosomensatz Durchmischt das maternale und paternale Genom durch Rekombination
38 To allow pairing and recombination meiotic prophase is extended and can be separated into different stages Leptotän Zygotän Pachytän Diplotän Diakinese Metaphase I (früh) Anaphase I (spät) Telophase I /Interkinese Metaphase II Anaphase II Tetrade Paarung Rekombination Kondensation Segregation
40 Diakinese Chiasmata in den Bivalenten gut sichtbar
41 Komplette Chromosomen aus je 2 Chromatiden wandern an die Pole der Zelle Anaphase I
42 (Telophase I) Interkinese (Prophase II)
43 Anaphase II Schwestercentromere trennen sich
45 Mitose Körperzelle Meiose R! I. S! II. Körperzellen Geschlechtszellen oder Beginn der haploiden Generation S!
46 Salamander (Oedipina) Schistocerca
47 Interchromosomale (intergenomische) Rekombination Intrachromosomale Rekombination Crossing-over I II III IV V I II III IV V Diploider Satz aus 2 elterlichen Sätzen Diploider Satz aus 2 elterlichen Sätzen Meiose Meiose Haploide Gameten I II 3 IV III 4 V In der Meiose erfolgen fast immer interchromosomale und intrachromosomale Rekombination gemeinsam
48 h a H A Crossing Over Rekombination zwischen den Loci Haarfarbe und Augenfarbe
49 h h H H a A a A >25% <25% <25% >25%
50 Formen der Segregation ablesbar von den linearen Asci von Neurospora kein C.O. zwischen Centromer und Marker C.O. Reduktionale Segregation Äquationale Segregation 4 : 4 2 : 2 : 2 : 2
51 Perkins & Raju 1995
52 Recombination can have crossover and non-crossover outcomes D-loop formation double holyday junction
53 DHJs can lead to gene conversion taken from Lodish et al. Molecular Cell Biology.W.H.Freeman, New York, 2001 Figure 12-31
54 Gene conversion PART II taken from Lodish et al. Molecular Cell Biology.W.H.Freeman, New York, 2001 Figure 12-31
55 Observation of gene conversion in Neurospora
56 Double strand breaks are induced by Spo11, a Topo II-like (ancient) protein
58 Processing of double strand breaks requires strand resection, strand invasion, and the DNA repair protein Rad51
59 Properties of RecA/Rad51 RecA coats ss DNA invades homologous sequences in duplex DNA displaces the non-complimentary strand of a homologous duplex in an ATP-dependent manner releases newly formed duplex is greatly stimulated by SSB -which may help undo DNA secondary structures Performs these events in three stages: 1) presynapsis, 2) synapsis, 3) post-synapsis
60 Structure and Model of mechanism of RuvAB function
61 RuvC binds and resolves Holliday junctions RuvC binds to junctions like RuvA Binds to RuvB but not RuvA Two active sites (30Å apart) Square planar surface match junctions Displays sequence preference (WTT*S) Associates with RuvAB complex to form a resolvasome
62 The Synaptonemal Complex (SC) a proteineous structure formed during zygotene and pachytene (essential for homolog chromosome alignment, pairing, and synapsis and for meiotic recombination-depending on the organism)
65 The SC might be required for crossover interference (regulated the number of COs per bivalent)
66 Chiasmata halten homologe Chromosomen verbunden
67 In der ersten meiotischen Teilung wird genetisches Material zwischen homologen Chromosomen ausgetauscht In der ersten meiotischen Teilung wird Cohesion distal von Chiasmata gelöst In der zweiten meiotischen Teilung wird Cohesion in Zentromernähe gelöst Sgo1 (Shugoshin) rekrutiert eine Phosphatase zum Zentromer und verhindert die vorzeitige Trennung der Zentromere Der Ansatzpunkt der Spindel am Kinetochor (Zentromer) wird zwischen Meiose 1 & 2 umstrukturiert
68 Die weibliche Meiose arretiert für viele Jahre in Säugern
69 Der Vorzeitige Verlust von Cohesion führt zur vorzeitigen Trennung homologer Chromosomen und deren zufälliger Aufteilung auf Tochterzellen
70 Die Verankerung der Spindel an homologen Chromosomen erfordert Monopolin
71 Ursachen von Trisomien
72 Geringe Rekombinationsfrequenz und Rekombination nahe der Chromosomenenden führen zu zufälliger Chromosomensegregation Kleine Chromosomen tendieren zur aberranten Segregation
73 Entstehung von Polkörperchen
74 Das Produkt der männlichen Meiose sind 4 Spermien Spermatogonium Spermatogonium Primäre Spermatozyte Sekundäre Spermatozyte Spermatiden Spermien
75 Das Produkt der weiblichen Meiose sind eine Eizelle und drei Polkörperchen Primäre Oocyte Sekundäre Oocyte Polkörperchen Eizelle 3 Polkörperchen
76 Die zweite meiotische Teilung wird durch die Befruchtung eingeleitet Spermium Polkörperchen Primäre Oozyte Sekundäre Oozyte Eizelle
77 Eizelle und Polkörperchen erhalten unterschiedliches genetisches Material Eizelle Polkörperchen
78 Forward versus reverse genetics
79 Forward genetics From phenotype to genotype Mapping of genes relies on the identification of mutant phenotypes
80 Chromosome Mapping in Eukaryotes Copyright 2006 Pearson Prentice Hall, Inc.
81 Cosegregation: Genes Linked on the Same Chromosome Segregate Together complete linkage
82 The Linkage Ratio complete linkage: in the absence of recombination In theory there are as many linkage groups as there are chromosomes in an organism
83 (instead of 9:3:3:1 observed in independent assortments completely linked alleles behave like single loci in back-crosses
84 But: Recombination can lead to a new allele combination on single chromosomes
85 The larger the distance between two loci the higher the probability of a crossover 4 COs between B and C 1 CO between A and B
86 Crossing Over Serves as the Basis of Determining the Distance between Genes during Chromosome Mapping Morgan and Crossing Over
87 Genes on the same chromsome rarely show complete linkage: determining the genetic distance of two genes y= yellow body w= white eye m= miniature wings
88 Sturtevant (student of Thomas Morgan) and mapping assumed that recombination between different loci is additive introduced the term map unit (one map unit = 1% recombination between two genes) 1 map unit = 1 centimorgan Advantage of Drosophila for mapping: No recombination in male meiosis
89 Single Crossovers 50% Recombination occurs on non-sister chromatids even if recombination occurs at 100% frequency between two chromatids recombination frequency is at 50% (50 centimorgans)
90 The distance of 50 cm resembles random segregation Figure 5-6 Copyright 2006 Pearson Prentice Hall, Inc.
91 Multiple Exchanges (double crossovers) since each individual crossover occurs (almost) independent of a neighboring crossover, probabilities of double crossovers multiply
92 Three-Point Mapping in Drosophila allows the creation of detailed chromosome maps
93 3-factor crosses allow the determination of gene order Single cross overs indicate the distance of two genes but does not allow to determine their order. Since double cross overs are least likely, the correct order of genes can be determined Figure 5-9 Copyright 2006 Pearson Prentice Hall, Inc.
94 A Mapping Problem in Maize mapping becomes more complex when the arrangement of alleles is not known
95 1) The noncrossover situation indicates the arrangement of genes in the paternal gametes 2) The double-crossover situation allows mapping of the central gene Figure 5-10b Copyright 2006 Pearson Prentice Hall, Inc.
96 Three possible sequences of alleles Only one fits the observation Figure 5-11 Copyright 2006 Pearson Prentice Hall, Inc.
97 Interference Affects the Recovery of Multiple Exchanges The presence of a chiasma frequently suppresses a second chiasma in its vicinity. This type of positive interference decays over distance. However, the distribution of chiasmata is therefore not completely random.
98 As the Distance between Two Genes Increases, Mapping Experiments Become More Inaccurate The occurence of double-cos leads to an inaccurate calculation of gene distance
99 The occurence of interference and double crossovers leads to a discrepancy between hypothetical and observed map units Figure 5-13 Copyright 2006 Pearson Prentice Hall, Inc.
100 Drosophila Genes Have Been Extensively Mapped
101 Modern genetic analysis involves the use of single nucleotide polymorphisms (snp) Individuals carrying a snp are crossed to individuals lacking a snp The F1 is heterozygous with respect to the mutation and the snp cosegregation of a phenotype (homozygous) with snps (homozygous) is analyzed by sequencing or restriction cleavage in the F2 Once a region of interest has been narrowed down, the gene(s) can be isolated and sequenced to verify the mutation Alternatively, complementation with a wild type sequence can be performed (rescue of phenotype by exogenous sequence added in trans)
103 Genetic screens are performed in model organisms Requirements: Mutability (Chemicals or transposons -give a genomic tag) Rapid generation cycle and easy to hold in the laboratory Small genome (diploid or haploid) Genetic analyis (phenotypic markers) (Sequenced genomes) Transformable
104 Reverse genetics from gene to phenotype can be done when genome information is given identify gene of interest by bioinformatic screen (homology, functional domain) inhibit gene function observe and study phenotype
105 Mechanisms to inhibit gene function gene knock out: destroy or replace endogenous sequence (by homologous recombination) By RNA interference
106 A strategy to for gene knock outs in the yeast Figure Copyright 2006 Pearson Prentice Hall, Inc.
107 A strategy to create a gene knock out in the mouse Figure Copyright 2006 Pearson Prentice Hall, Inc.
108 Figure 21-18a Copyright 2006 Pearson Prentice Hall, Inc.
109 Figure 21-18b Copyright 2006 Pearson Prentice Hall, Inc.
110 RNA interference (RNAi) Presumably evolved as an antiviral mechanism (to prevent RNA viruses from replicating) and to inhibit the activity of retroelements works in almost all organisms destruction of mrna by RNAi translational inhibition by mirnas occurs endogenously to regulate gene activity
111 Different types of RNAs can be administered RNA oligos synthesized in vitro long RNA synthesized in vitro (activates PKR pathway) short haripins synthesized in vitro or in vivo
112 Different expression systems can be used to synthesize hairpin RNAs in vivo
113 Genome wide reverse genetic screens have been performed in S. cerevisiae (gene knock out) C. elegans (RNAi) Drosophila (in progress) Problems in reverse genetic screens: Phenotype is not obvious since only a number of conditions are screened for Phenotype is leaky (RNAi) since not all RNAi works with different efficiency in different tissues Complete loss of function might be lethal (weak mutant alleles might give more insight into gene functions. ie function is impaired but not missing)
114 25.2 The Hardy Weinberg Law Describes the Relationship between Allele Frequencies and Genotype Frequencies in an Ideal Population an ideal population is infinitely large, mating is random, no evolutionary forces (migration, selection, mutation ) apply
115 Figure 25-1 Copyright 2006 Pearson Prentice Hall, Inc.
116 Allele frequencies can be calculated by: p 2 + 2pq +q 2 Figure 25-2 Copyright 2006 Pearson Prentice Hall, Inc.
117 25.3 The Hardy Weinberg Law Can Be Applied to (a receptor) Human Populations
118 Figure 25-3 Copyright 2006 Pearson Prentice Hall, Inc.
119 Observed allele frequencies fit the expected allele frequencies, i.e. no selection applies Table 25-2 Copyright 2006 Pearson Prentice Hall, Inc.
120 25.3 The Hardy Weinberg Law Can Be Applied to Human Populations Testing for Hardy Weinberg Equilibrium For instance, in a situation involving three alleles (p + q + r = 1), the frequencies of the genotypes are given by (p + q + r) 2 = p 2 + q 2 + r 2 + 2pq + 2pr + 2qr = 1. Blood groups AB0 (0 is recessive) A is AA and A0 B is BB and B0 AB is AB and BA 0 is 00
121 By knowing the recessive allele frequency, all others can be calculated p 2 + 2pr + r 2 = (p+r) 2 = 0.79 p+r = 0.89 p= = 0.38
122 Genotype frequency versus allele frequency Heterozygosity increases rapidly Figure 25-5 Copyright 2006 Pearson Prentice Hall, Inc.
123 Selection against a homozygous recessive allele rapidly reduces the frequency of the allele but never approaches 0 e.g cystic fibrosis is present at 4% (heterozygous) in the human population Figure 25-6 Copyright 2006 Pearson Prentice Hall, Inc.