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30 Cards in this Set
- Front
- Back
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Nuclear reprogramming can change the differentiation state of a cell. What are the 4 different ways of reprogramming? |
- by nuclear transfer into egg - by nuclear transfer into oocyte - by overexpression of transcription factors (induced pluripotency) - reprogramming by cell fusion |
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Muscle differentiation exemplifies important concepts: |
- transdifferentiation: reprogramming by cell fusion and by overexpression of transcription factors - master transcription factors initiate a programme of differentiation; the relationship between cell division and cell differentiation; criteria of proof that a gene product is involved in a bio process |
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The nucleus contain genetic information that directs cellular development. Give experimental evidence: |
- remove nucleus from foot of Acetabularia cell and cut off cap; Cells can regenerate a cap up to 70 days
- same as exp 1 but in the presence of ribonuclease; cap regeneration is abolished - replace nucleus in sp 1 with nucleaus from sp 2 and cut off cap - initially cap grows back the same as sp1 but then with time starts looking more like sp2 |
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What is a heterokaryon? |
- a multinucleated cell that contains genetically different nuclei; |
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Specialised cells have a memory of their developmental history. |
- the differentiated state is normally stably maintained, with tissue-specific genes activated and expression of other genes repressed - e.g. single committed muscle cell continues to express muscle-specific genes even when surrounded by non-muscle cells |
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A stable relationship exists between nucleus and cytoplasm |
- nucleus dictates cytoplasmic syntheses - cytoplasmically-derived factors regulate nuclear activity including transcription |
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How does the relationship between nucleus and cytoplasm manifest itself in differentiated cells? |
- in characteristic patterns of gene expression for specific cell types - maintained by epigenetic modifications and modulated only by progress through the cell cycle and external signals such as growth factors and hormones |
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Somatic cell nuclear transfer demonstrated that the differentiated state can be reversed |
- genomic equivalence - injected donor nucleus - produce a normal tadpole = no loss of genetic information during differentiation - nuclear reprogramming - somatic nucleus behaves like a zygote nucleus and directs normal development, indicating that the egg has reversed the differentiated state of the somatic nucleus |
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What are the methods of nuclear reprogramming? |
- de-differentiation - a differentiated cell reverting to a state of increased developmental plasticity. It reprogrammed to a state of totipotency, the cell can give rise to a new adult - transdifferentiation - a cell switching from one differentiated cell type to another |
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What are the requirements for successful reprogramming? |
- a change in gene expression profile (new lineage-specific genes turned on, and previous lineage-specific genes turned off) - resetting the epigenetic mechanisms that maintain stable gene expression (DNA methylation, histone modifications) |
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1) Somatic cell nuclear reprogramming (SCNT) |
- nuclear transfer into eggs (meiotic metaphase II oocytes) - mimics natural fertilisation - factors in the egg reprogram the somatic nucleus they same way they would reprogram the sperm pronucleus - the somatic cell undergoes extensive DNA replication and division to generate a new org |
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What happens if you use adult donor nuclei for SCNT? |
- success rate declines to <1-2% - the nuclei suffers major DNA damage and chromosome loss upon being forced to undergo rapid DNA replication - resetting epigenetic marks - incomplete = persistent memory of gene expression pattern of differentiated state (strongly expressed genes may fail to be switched off and vice versa) |
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2) Direct transcriptional reprogramming by nuclear transfer into meiotic prophase I oocytes |
- oocytes like eggs contain factors needed to activate the sperm nucleus - same factors can reprogram somatic nuclei - use of oocytes enables analysis of somatic cell nuclear reprogramming without damaging effects of enforced rapid DNA replication and cell division - no new cell types are generated |
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What model organism oocyte is ideal for studying mechanisms of nuclear reprogramming? |
- one oocyte can reprogram hundreds of injected mouse nuclei
- injected oocyte can be cultured for a month - oocyte can be manipulated to alter the concentration of candidate reprogramming factors |
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The somatic nucleus undergoes nucleosome remodelling |
- the linker histone (B4 in amphibians or H1) is incorporated into the transplanted nuclei as well because its necessary for reactivationg the transcription of pluripotency genes required for early development |
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What are the factors that enhance reprogramming efficiency? |
- chromatin remodelling - histone modification - histone variants - DNA modification - RNA expression (miRNA) |
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3) induced pluripotency: reprogramming by overexpresson of transcription factors |
- transfection of Oct4, Sox2, Klf4 and Myc silences somatic gene expression and generates induced pluripotent stem cells (iPSCs) which can differentiate into any cell type |
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4) reprogramming by cell fusion |
- the nucleus of 1 donor cell is induced to express genes charact. of the other donor cell - in heterokaryons the nuclei remain separate within the shared cytoplasm (division and nuclear fusion generates synkaryons) - nuclei from more specialised cells are more resistant to reprogramming than less specialised cells |
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Proof that a gene product performs a particular function in normal development: |
- expressed at right cells at right time and in bio active form - necessary for that function to take place (test by knockout) - sufficient for that function to take place (test by gain of function experiments) |
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Experimental evidence - MyoD |
- MyoD isolated, transfected into fibroblasts, keratinocytes, hepatocytes converts them into muscle cells - evidence that a single gene can act as a master switch initiating a complex programme of differentiation |
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How are Myogenic Regulatory Factors (MRFs) identified? |
- on the basis of their ability to impose a muscle-specific programme of gene expression - expression of a single MRF converts fibroblasts into committed myoblasts |
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What are MRFs? |
- bHLH transcription factors - basic helix-loop helix - basic region binds DNA |
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What happens when different muscle genes are knockout |
- MyoD - skeletal muscle still present - myf5 - skeletal muscle still present - MyoD and Myf5 - some skeletal muscle - MyoD, Myf5 and MRF4 - no skeletal muscle ( MyoD and Myf5 compensate for loss of the other) |
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What activates MyoD, Myf5 and MRF4? |
- external signals - Wnt, shh - they activate myogenin - responsible for myoblast formation |
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MyoD initiates a temporal cascade of gene activity |
- MyoD directly activates transcription of some muscle-specific genes, e.g. creatine phosphokinase - MyoD binds an enhancer upstream of MyoD (+ve feedback) - MyoD activates genes whose products act as cofactors for MyoD binding to a later group of enhancers, activating further muscle specific genes |
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Proliferating myoblasts express |
- MyoD and Myf5 - but do not differentiate |
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What happens to proliferating myoblasts in the presence of growth factors? |
- Cdk4 phosphorylates MyoD and Myf5, hastening their degradation - Cyclin-cdk inactivates Rb - pRB cannot bind E2F - E2F is free to promote expression of S-specific genes i.e. promotes cell proliferation - FGF stimulates expression of inhibitory proteins |
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Which transcription factor blocks differentiation in proliferationg myoblasts? |
- HLH Id - heterodimerises with MRF or their binding partners |
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What does growth factor withdrawal trigger? |
- cell cycle arrest - allows MRF-driven differentiation |
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What regulates the balance between proliferation and differentiation? |
- MRFs |
