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Volledige uitgebreide samenvatting van moleculaire biologie (ik haalde 19/20)

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deze samenvatting is gebaseerd op de slides van professor Aleyde Vandeneynde. voor het vak moleculaire biologie, onderdeel van Biochemie en moleculaire biologie in het nieuwe curriculum van de eerste bachelor in geneeskunde

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  • June 12, 2024
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DEEL 2: MOLECULAIRE BIOLOGIE




Eerste Bachelor Geneeskunde
KU LEUVEN

,INHOUDSOPGAVE

16.1 de chemische structuur van het genetische materiaal ................................................................................ 7
16.1.1 DNA als drager van genetisch materiaal ................................................................................................................... 7
16.1.2 Empirisch onderzoek van naar DNA naar de drager ............................................................................................... 8
Onderzoek 1: Griffith – Ontdekking van een transformerend agens ........................................................................ 8
Onderzoek 2: Avery, Macleod en McCarty – Tonen DNA als transformerend agens ............................................ 8
Onderzoek 3: Hersey Chase experiment – Ontdekking van DNA als het genetisch materiaal van een
bacteriofaag .......................................................................................................................................................................... 9
Onderzoek 4: Hersey Chase experiment – RNA is het genetisch materiaal van tabaksmozaïekvirus ................ 9
Samenvatting van de conclusies van (verder) onderzoek naar genetische dragers: ............................................ 10
16.1.3 uitzondering: retrovirussen hebben RNA als drager van het erfelijk materiaal ................................................ 10
Werking en structuur van retrovirussen .......................................................................................................................... 10
Levenscyclus van een typisch retrovirus .......................................................................................................................... 11
16.1.4 Uitzondering 2: Tumorvirussen hebben rNA als genoom ..................................................................................... 11

16.2 DNA structuur........................................................................................................................................... 12
16.2.1 basensamenstelling van DNA .................................................................................................................................... 12
Conclusie van dit onderzoek: Regels van Chagraff .................................................................................................... 12
16.2.2 dubbele-helix structuur van DNA ............................................................................................................................. 13
Ontstaan van de major en de minor groove ................................................................................................................ 13
16.2.3 drie structuurvarianten van DNA .............................................................................................................................. 14
16.2.4 Ruimtelijke structuur van DNA .................................................................................................................................. 14
Topoisomerasen verwijderen supercoils in DNA: .......................................................................................................... 15
Denaturatie en renaturatie: afbraak en heropbouw van de ruimtelijke structuren............................................... 15

16.3 compactering van DNA ............................................................................................................................ 16
16.3.1 compactering van bacterieel DNA ........................................................................................................................... 16
Eigenschappen van bacterieel DNA: .............................................................................................................................. 16
Compactering van bacterieel DNA: ............................................................................................................................... 16
16.3.2 compactering van het eukaryotisch DNA ................................................................................................................ 17
Stap 1: vorming van de Nucleosomen............................................................................................................................ 17
Stap 2: vorming van chromatine ..................................................................................................................................... 17
Stap 3: vorming van euchromatine ................................................................................................................................. 18
Stap 4: vorming van heterochromatine en chromosomen ........................................................................................... 18
16.3.3 regulering van de compactering van chromatine .................................................................................................. 18
Post-translationele modificaties van histonen die een direct effect hebben op de compactering van
chromatine: .......................................................................................................................................................................... 18
Post-translationele modificaties van histonen die indirect effect hebben op de compactering van chromatine:
............................................................................................................................................................................................... 19

16.4 de samenstelling van het humaan genoom............................................................................................. 19
16.4.1 de samenstelling van het nucleair genoom.............................................................................................................. 19
Transposons: ........................................................................................................................................................................ 19
16.4.2 de samenstelling van het mitochondriaal genoom ................................................................................................. 20

16.5 de celkern ................................................................................................................................................. 21
16.5.1 structurele organisatie van de celkern ..................................................................................................................... 21

, Human connections: Progeria ........................................................................................................................................... 21
16.5.2 transport doorheen de kernporiën............................................................................................................................ 22
Actieve nucleaire import: .................................................................................................................................................. 22
Actieve nucleaire export ................................................................................................................................................... 23
16.5.3 De Nucleolus ................................................................................................................................................................ 23

17.1 DNA replicatie .......................................................................................................................................... 24
17.2 mechanisme DNA-synthese tijdens de S-fase ............................................................................................................. 25
Meselson en Stahl experiment: ........................................................................................................................................ 25
17.3 het verloop van de S-fase bij bacteriën ...................................................................................................................... 26
Stap 1: initiatie van de replicatie ................................................................................................................................... 26
Stap 2: de fase van de DNA-synthese .......................................................................................................................... 26
Stap 3: proeflezing door DNA-polymerase ................................................................................................................. 28
Overzicht van de DNA replicatie in bacteriën: ............................................................................................................ 28
17.4 het verloop van de S-fase bij eukaryoten .................................................................................................................. 29
Stap 1: initiatie van de replicatie bij eukaryoten........................................................................................................ 29
Verdere verloop replicatie bij de eukaryoten ............................................................................................................. 29
Het probleem van de replicatie van de chromosoomuiteinden van telomeren bij eukaryoten: ......................... 29
17.5 key technique: polymerasekettingreactie (PCR)......................................................................................................... 30

17.2 DNA schade en DNA foutenherstel .......................................................................................................... 31
17.2.1 oorzaken van mutaties ............................................................................................................................................... 31
Replicatiefouten ten gevolge van tautomeren:............................................................................................................. 31
Replicatiefouten in het repetitief DNA: .......................................................................................................................... 31
Replicatiefouten ten gevolge van spontane depurineringen en deamineringen: .................................................. 32
Replicatiefouten ten gevolge van spontane oxidatie van basen door reactieve zuurstof soorten (ROS) ........ 32
Mutaties ten gevolge van de chemische mutagenen ................................................................................................... 32
Mutaties ten gevolge van stralingen .............................................................................................................................. 32
17.2.2 DNA herstelmechanismen ........................................................................................................................................... 33
Fotolyase: ............................................................................................................................................................................. 33
Base excision repair (BER): ............................................................................................................................................... 33
Nucleotide excision repair (NER) ..................................................................................................................................... 33
Mismatch repair .................................................................................................................................................................. 34
Translesie synthese (geen volwaardig foutenherstel) .................................................................................................. 34
Herstel van een dubbelstrengige DNA breuk............................................................................................................... 34
Apoptose .............................................................................................................................................................................. 36
17.2.3 key technique: crispr/cas9 genoom bewerking ...................................................................................................... 36

17.3 Homologe recombinatie en mobiele genetische elementen ..................................................................... 37
17.3.1 homologe recombinatie ............................................................................................................................................. 37
17.3.2 mobiele genetische elementen ................................................................................................................................... 38
DNA-transposons ................................................................................................................................................................ 38
Retrotransposons:................................................................................................................................................................ 39

18.1 de richting van de genetische informatiestroom ...................................................................................... 40
18.1.1 genetische informatiestroom volgens het centrale dogma.................................................................................... 40
18.1.2 uitzonderingen op het centrale dogma.................................................................................................................... 40
Toepassing: positief enkelstrengig RNA virus: SARS-CoV-2 ...................................................................................... 41
Toepassing: telomerase ..................................................................................................................................................... 41

,18.2 het mechanisme van de transcriptie ........................................................................................................ 42
18.2.1 Transcriptie bij de prokaryoten................................................................................................................................. 42
Transcriptionele unit bij de prokaryoten: ....................................................................................................................... 42
Katalyserende functie van RNA-polymerase ................................................................................................................ 43
Promotorsequentie bij prokaryoten ................................................................................................................................ 43
Effectieve verloop van de transcriptie bij prokaryoten: ............................................................................................ 44
18.2.2 transcriptie bij de eukaryoten ................................................................................................................................... 45
Toepassing: Amanita Phalloïdes poisoning.................................................................................................................... 46
Promotoren voor eukaryoten ........................................................................................................................................... 46
Mechanisme van transcriptie bij eukaryoten................................................................................................................. 46
18.2.3 Key technique: hunting for DNA-protein interacties .............................................................................................. 47

18.3 RNA processing en turnover .................................................................................................................... 48
18.3.1 rRna synthese ............................................................................................................................................................... 48
Eukaryotische rRNA synthese: .......................................................................................................................................... 49
Het mechanisme van de pre rRNA processing ............................................................................................................ 49
18.3.2 tRNA-synthese ............................................................................................................................................................. 50
18.3.3 mRNA synthese ............................................................................................................................................................ 51
5’ capping ........................................................................................................................................................................... 51
3’ polyadenylering ............................................................................................................................................................ 51
Pre-mRNA splicing .............................................................................................................................................................. 52
RNA-editing ......................................................................................................................................................................... 53
Toepassing 2: trypsanosoma ............................................................................................................................................ 54
Toepassing 3: APOBEC3G ............................................................................................................................................... 54

19.1 de genetische code ................................................................................................................................... 55
19.1.1 experiment van Beadle en tatum .............................................................................................................................. 55
19.1.2 experiment van pauling, ingram en yanofsky ........................................................................................................ 55
Pauling experiment: ........................................................................................................................................................... 55
Ingram experiment: ............................................................................................................................................................ 56
19.1.3 experiment van crick en brenner .............................................................................................................................. 56
19.1.4 experiment van nirenberg, matthie en khorana ..................................................................................................... 58
19.1.5 besluit genetische code .............................................................................................................................................. 58

19.2 de hoofdrolspelers van de translatie ....................................................................................................... 59
19.2.1 ribosomen .................................................................................................................................................................... 59
19.2.2 tRNA ............................................................................................................................................................................. 59
19.2.3 aminoacyl-tRNA synthetase ....................................................................................................................................... 61
19.2.4 mRNA ........................................................................................................................................................................... 62
19.2.5 proteïnefactoren ......................................................................................................................................................... 62

19.3 mechanisme van de translatie ................................................................................................................. 62
19.3.1 initiatie.......................................................................................................................................................................... 63
Initiatiefase bij de prokaryoten: ..................................................................................................................................... 63
Initiatiefase bij de eukaryoten:........................................................................................................................................ 64
19.3.2 elongatie ...................................................................................................................................................................... 65
19.3.3 terminatie van de translatie ....................................................................................................................................... 67

, 19.3.4 samenvatting van de volledige translatie................................................................................................................ 68
19.3.5 Co- en post-translationele processen ....................................................................................................................... 69
Moleculaire chaperones .................................................................................................................................................... 69

19.4 Mutatie en translatie ................................................................................................................................ 70
19.4.1 mutaties waarbij één nucleotide is veranderd ......................................................................................................... 70
Voorbeelden van hoe de cel omgaat met nonsense/nonstop mutaties in mRNA’s ................................................ 70
19.4.2 mutaties waarbij één of meerder nucleotiden zijn toegevoegd (insertie) of verwijderd (deletie) .................. 71
19.4.3 mutaties waarbij grote stukken DNA veranderen van plaats ............................................................................... 71
19.4.4 key technique – protein localization using fluorescent fusion proteins ............................................................... 71

19.5 post-translationele processing ................................................................................................................. 71
Voorbeelden: ...................................................................................................................................................................... 71

20.1 regeling van de genexpressie in bacteriën .............................................................................................. 73
20.1.1 Katabole paden: lac operon ..................................................................................................................................... 74
Positieve regulatie van Lac operond door CAP-cAMP: .............................................................................................. 75
20.1.2 anabole paden ............................................................................................................................................................ 76
20.1.3 sigmafactoren ............................................................................................................................................................. 76
20.1.4 regeling na de initiatie van transcriptie................................................................................................................... 76
Mechanisme van de leadersequentie: ............................................................................................................................ 77
20.1.5 ribo-schakelaars (riboswitches) ................................................................................................................................ 78
20.1.6 toepassing van de regulatie van de genexpressie in bacteriën: CRISPR ............................................................ 78

20.2 regeling van de genexpressie in eukaryoten: eerste niveau.................................................................... 79
Uitzondering op het genomic equivalence principe in specifieke celtypes............................................................. 80
De (de)condensatie graad van het chromatine is verschillend tussen de celtypen: .............................................. 80
20.1.1 conclusie van de genexpressie op genoom niveau ................................................................................................ 81
Vormen van epigenetische veranderingen .................................................................................................................... 81

20.3 regeling van de genexpressie in eukaryoten: tweede niveau ................................................................. 83
20.3.1 algemene regulerende trancriptiefactoren (TF) ..................................................................................................... 83
Het werkingsmechanisme van de enhancers: ................................................................................................................ 84
20.3.2 structuur van DNA-bindingsdomeinen van regulerende TF................................................................................... 85
20.3.3 gecoördineerde genexpressie door binding van een TF op een DNA respons element ................................... 86
DNA respons elementen voor hormoon receptoren: .................................................................................................... 86
DNA respons elementen voor CREB: ............................................................................................................................... 86
DNA respons elementen voor STAT: ............................................................................................................................... 87
DNA respons elementen voor heat-shock transcriptie factoren:................................................................................ 87
DNA respons elementen voor homeotische transcriptiefactoren: .............................................................................. 88

20.4 regeling van de genexpressie in eukaryoten: 3de , 4de en 5de niveau ...................................................... 88
20.4.1 controle van de RNA processing en het nucleair export ....................................................................................... 88
20.4.2 translationele controle ................................................................................................................................................ 89
20.4.3 Key technique: Gene knockdown via RNAi zie slides............................................................................................. 92
20.4.4 post-translationele controle ....................................................................................................................................... 92

,24.1 overzicht van de celcyclus ....................................................................................................................... 94
24.1.1 key technique: meting van milioenen cellen tegelijk .............................................................................................. 95

24.2 kern en celdeling ...................................................................................................................................... 95
24.2.1 profase ......................................................................................................................................................................... 96
24.2.2 prometafase ................................................................................................................................................................ 96
Microtubili-kinetochoor-interactie: ................................................................................................................................... 97
Herkenning van de prometafase .................................................................................................................................... 97
24.2.3 metafase ...................................................................................................................................................................... 98
24.2.4 anafase ........................................................................................................................................................................ 98
Werking van de verkorting en verlenging van de microtubili: ................................................................................. 98
24.2.5 telofase ........................................................................................................................................................................ 99
24.2.6 cytokinese..................................................................................................................................................................... 99

24.3 regulatie van de celcyclus ...................................................................................................................... 100
24.3.1 Progressie van de celcyclus: 3 sleutel transitiepunten.......................................................................................... 100
24.3.2 Celfusie-experimenten, celcyclus mutanten in gist en biochemische experimenten .......................................... 100
24.3.3 cycline-afhankelijk eiwitkinasen (cdks) .................................................................................................................. 101
Functie van de Cdk’s tijdens de G1/S transitie .......................................................................................................... 102
Functie van de Cdk’s tijdens de G2/M transitie ........................................................................................................ 102
24.3.4 Anafase promoting complex (APC)....................................................................................................................... 102
24.3.5 Checkpoint (controlepunten) pathways.................................................................................................................. 103
Checkpunt van de S-fase ................................................................................................................................................ 103
DNA-replicatie controlepunt........................................................................................................................................... 103
Het mid-mitose controlepunt ........................................................................................................................................... 104
Het DNA-schade controlepunt:....................................................................................................................................... 104
24.3.6 samenvatting van de regulering van de celcyclus ................................................................................................ 104

24.4 groeifactoren en celproliferatie .............................................................................................................. 105
24.4.1 regeling van de celcyclus door groeifactoren ................................................................................................. 105
24.4.2 regeling van de celcyclus door inhibitorische groeifactoren ......................................................................... 106

24.5 apoptosis................................................................................................................................................ 107
24.5.1 het mechanisme van de apoptose ........................................................................................................................... 107

25.1 inleiding tot het mechanisme van het sexuele reproductie .................................................................... 109
25.1.1 genetische samenstelling tijdens verschillende levensfasen ................................................................................. 109

25.2 meiose .................................................................................................................................................... 111
25.2.1 meiose 1 ..................................................................................................................................................................... 111
Profase 1: .......................................................................................................................................................................... 111
Metafase 1: ....................................................................................................................................................................... 112
Anafase 1: ......................................................................................................................................................................... 112
Telofase 1: ......................................................................................................................................................................... 113
Cytokinese: ........................................................................................................................................................................ 113
Meiose 2: ................................................................................................................................................................................ 113

,25.3 meiose Vs mitose ................................................................................................................................... 113

25.4 fouten in de meiose ............................................................................................................................... 114
25.4.1 niet-disjunctie ............................................................................................................................................................. 114

25.5 differentiatie van de gameten ................................................................................................................ 115
25.5.1 spermatogenese ........................................................................................................................................................ 115
25.5.2 oögenese .................................................................................................................................................................... 115

25.6 genetische variabiliteit ........................................................................................................................... 116
25.6.1 experimenten van mendel ........................................................................................................................................ 116
25.6.2 experimenten van de morgan ................................................................................................................................. 117
25.6.3 Homologe recombinatie en crossing-over............................................................................................................. 117
25.6.4 conclusie: herkomst van de genetische variabiliteit.............................................................................................. 118

25.7 genetische recombinatie in bacteriën en virussen ................................................................................. 118
25.7.1 Genetische recombinatie in bacteriofagen: ........................................................................................................... 118
25.7.2 genetische recombinatie in bacteriën ..................................................................................................................... 118

, HOOFDSTUK 16: DNA EN
CHROMOSOMEN
16.1 DE CHEMISCHE STRUCTUUR VAN HET GENETISCHE MATERIAAL

16.1.1 DNA ALS DRAGER VAN GENETISCH MATERIAAL

Onderstaande foto geeft de genetische informatiestroom binnen een cel en tussen generaties van cellen weer:

Hier is dus zichtbaar dat een aanwezige
DNA streng alle informatie zal bevatten om
processen in de cel te laten voorkomen. De
DNA streng wordt namelijk afgeschreven
via de transcriptie in een RNA streng die
dan de kern zal verlaten om in de ribosomen
de aanmaak van eiwitten mogelijk te
maken. Deze eiwitten zullen allerlei
functionele rollen opnemen in de cel.

Hieruit is dus duidelijk dat DNA de
algemene drager is van het “genetisch
materiaal” die de cel nodig heeft om een
bepaalde functie te kunnen uitvoeren.
Onderstaande foto geeft dan weer hoe de
informatiestroom van genetisch materiaal
tussen cellen gebeurt.

De aanwezige hoeveelheid erfelijk materiaal is dus in de kern geconcentreert onder de vorm van DNA, die zal
onveranderlijk doorgegeven moeten worden naar de dochtercel om deze naar behoren te laten functioneren (zie
uitleg over de informatiestroom in de cel) Om deze doorgave te verwezelijken zal er een verdubbeling van het
erfelijk materiaal plaatsvinden en zal er vervolgens een splitsing in twee cellen voortbrengen.

De inwendige informatiestroom die hierboven wordt uitgelegd verloopt volgens onderstaand schema en zal in de
hieropvolgende hoofdstukken in detail aan bod komen.

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