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Molecular Biology BSc First Year > BMS11005 -Introductory Developmental, Stem Cell and Regenerative Biology > Flashcards

BMS11005 -Introductory Developmental, Stem Cell and Regenerative Biology Flashcards

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1
Q

What are the advantages of using zebrafish (Danis rerio) to study DevSteR?

A

-vertebrate model
-produce large numbers of embryos
-relatively transparent (allows individual cells to be tracked)
-fertilisation is external

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2
Q

What are the disadvantages of using zebrafish (Danis rerio) to study DevSteR?

A

-have a complex genome with gene duplication
-have a high variation (not inbred)

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3
Q

What useful techniques can be used with zebrafish (Danis rerio)?

A

-mutagenesis/genetics
-cell transplantation
-transgenes

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4
Q

What are the advantages of using mice (Mus musculus) to study DevSteR?

A

-mammal (similar to humans)
-have a rapid generation time (8 weeks)
-have a low variation (inbred strains)

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5
Q

What are the disadvantages of using mice (Mus musculus) to study DevSteR?

A

-internal embryos (poor access)
-produce small batches of embryos
-expensive (to house and maintain)
-ethical issues

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6
Q

What useful techniques can be used with mice (Mus musculus)?

A

-embryonic stem cells for gene knockout

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7
Q

What are the advantages of using African clawed frog (Xenopus laelvis/tropicalis) to study DevSteR?

A

-external fertilisation
-produce large batches of embryos
-embryos and cells are large

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8
Q

What are the disadvantages of using African clawed frog (Xenopus laelvis/tropicalis) to study DevSteR?

A

-low generation time
-yolky embryo (aka not transparent so can’t easily study individual cells)

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9
Q

What useful techniques can be used with African clawed frog (Xenopus laelvis/tropicalis)?

A

-injections
-tissue transplantations
-tissue culture
-transgenesis (introducing foreign genes)

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10
Q

What are the advantages of using chicks (Gallus gallus domesticus) to study DevSteR?

A

-large embryos
-tetrapod (allows limb development to be studied)

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11
Q

What are the disadvantages of using chicks (Gallus gallus domesticus) to study DevSteR?

A

-not accessible early (in hen)
-limited genetics

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12
Q

What useful techniques can be used with chicks (Gallus gallus domesticus)?

A

-tissue transplantation
-transcient genetic manipulation

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13
Q

What is cell fate mapping?

A

-marking a cell in an embryo and observing it to see which cells it gives rise to as its descendants
-can be plotted in a cell fate map

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14
Q

What does it mean when a cell is committed/determined to its fate?

A

it will develop according to its fate whichever region they are in
-if a cell is not yet determined, its fate will change according to the region
-determination can be shown by transplantation experiments
-more likely to be determined at later stages in development
-even when cells aren’t determined, they may be specified

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15
Q

What does it mean when a cell is competent?

A

it is capable of receiving a signal (receptor present)

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16
Q

What does it mean when a cell is specified?

A

when isolated and cultured in a medium away from the embryo, it will still develop according to its normal fate

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17
Q

What is RNA in situ hybridisation?

A

a method of gene expression analysis where cells are stained blue/purple if they express RNA for a particular gene

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18
Q

What is antibody staining?

A

a method of gene expression analysis where gene specific antibodies are used to detect where proteins are expressed

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19
Q

What is a sea urchin phyletically?

A

an echindoderm (sister group to chorodates, which vertebrates are)
-part of the deuterostomes

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20
Q

What are the advantages of using sea urchins to study DevSteR?

A

-produce large numbers of embryos
-easily bred in lab
-can easily be used for experimental manipulation

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21
Q

What two ideas of how development occurs were come up with from sea urchin experiments?

A

1) Mosaic development -egg nucleus contains determinants which specify different fates to difference cells by specific segregation
2) Regulative development -cells communicate whilst they develop, causing differences to be generated
-or both!

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22
Q

How are genetic models used to investigate development?

A

genes within a genome are altered to study its impact on development
-genes control development by controlling where and when proteins are synthesised

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23
Q

What are the ideal characteristics of an organism for genetic analysis?

A

-small (so lots can be kept)
-large batches of embryos
-short generation time
-easy to breed
-easy scoring of phenotypes
-genome has been sequenced

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24
Q

Why is C.elegans an ideal genetic model?

A

v. convenient bc…
-transparent
-can be frozen
-develops within 72hrs

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25
Q

Why is C.elegans described as having a stereotypical cleavage pattern?

A

-each worm has same pattern
-first cleavage is asymmetric

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26
Q

What is apoptosis?

A

programmed cell death
-highly controlled process
-essential for proper development and homeostasis

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27
Q

Which organism was apoptosis originally understood in?

A

C.elegans
(apoptosis is very stereotypical in C.elegans development)

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28
Q

What is apoptosis essential for?

A

-proper development (formation of reproductive organs, digestive system maturing, removing skin between digits)
-homeostasis (maintaining a constant number of cells, removing damaged cells)

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29
Q

What is RNA interference (RNAi)?

A

a mechanism to control flow of genetic information
-identical mRNAs degraded by biochemical process triggered by double-stranded RNA

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30
Q

Which organism was RNA interference discovered in?

A

C.elegans

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31
Q

How does RNA interference happen?

A

-cells take up double-stranded RNA, which is recognised and cut up into siRNAs (short interfering RNAs) by the Dicer enzyme
-siRNAs are loaded into RISC (RNA-induced silencing complex), which uses the siRNA sequence to find complementary RNAs
-when mRNAs complementary bind to siRNA sequence, RNAases are activated and degrade it

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32
Q

What are siRNAs?

A

short interfering RNAs
small double-stranded RNAs involved in the RNA interference process, generated by Dicer cutting up longer double-stranded RNAs

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33
Q

What is RISC?

A

RNA-induced silencing complex
ribonucleoprotein complex which uses siRNA or miRNA to find complementary mRNA which activates RNAases to degrade the siRNA or miRNA

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34
Q

What are flies?

A

protostomes

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35
Q

What is forward genetics?

A

genetic analysis where a mutant is identified based on its unusual phenotype and then experiments are done to identify the genes behind it

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36
Q

What is reverse genetics?

A

genetic analysis where a known gene/amino acid sequence is used to determine the gene’s function

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37
Q

What did mutant screening of Drosophila lead to?

A

-basic understanding of how genes control development of body plan
-identifying new genes and biological signalling pathways
-confirmed genetics

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38
Q

What happens in the life cycle of a fly?

A

-sperm and egg nuclei fuse
-nuclear division occurs, creating syncytium, and the nuclei migrate to periphery of cytoplasm
-pole cells develop at edge
-embryo cellularises, forming cellular blastoderm
-gasturlation occurs where cells become specified to their fate (mesoderm, ectoderm or endoderm)
-segmentation occurs
-further development occurs, where first instar hatches second instar, which hatches third instar pupa
-metamorphosis occurs, where different imaginal discs in larvae form different parts of adult fly

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39
Q

What fate are mesoderm cells specified to (in gastrulation) in Drosophila?

A

muscle

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40
Q

What fate are ectoderm cells specified to (in gastrulation) in Drosophila?

A

epidermis
nervous system

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41
Q

What fate are endoderm cells specified to (in gastrulation) in Drosophila?

A

gut

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42
Q

What regions does the Drosophila embryo become divided into along the antero-posterior axis?

A

-head
-thorax
-abdomen

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43
Q

What regions does the Drosophila embryo become divided into along the dorsal-ventricular axis?

A

-mesoderm
-dorsal
-amnioserosa
-ventral

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44
Q

What are gap genes?

A

zygotic genes which code for transcription factors in early Drosophila embryo development that subdivide the embryo into regions along the antero-posterior axis
-create combinatory code, which defines regions in embryo
-responsible for striped pair-rule genes being expressed
-first zygotic genes activated
-determines spatial expression of Hox genes

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45
Q

What are pair-rule genes?

A

genes in Drosophila that are involved in determining parasegments (developmental units which later give rise to segments in adult Drosophila)
-expressed in transverse stripes in the blastoderm so that each pair-rule gene is expressed in alternate parasegments

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46
Q

What are parasegments?

A

developmental units arranged along the body of a developing Drosophila embryo which give rise to the segments of larva and adult Drosophila

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47
Q

What is the role of segmentation genes?

A

cause patterning within each segment of the embryo
-cause the shift from pair-rule patterning into segmental patterning -ending up with 14 stripes of engrailed expression

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48
Q

What is the role of homeotic selector genes?

A

determine segment identity

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49
Q

When does patterning occur in the embryo?

A

when the embryo is cellularised
-cell signalling and transcription factors are both required

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50
Q

Which signalling proteins are needed for anterior/posterior patterning once the Drosophila embryo has become cellularised?

A

Wingless (Wg)
Hedgehog (Hh)
Engrailed (En)
-Wg expression is maintained by Hh
-Hh and En expression is maintained by Wg (under influence of pair-rule genes)

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51
Q

What can defects in Wingless and Hedgehog lead to?

A

Colon cancer and Basal cell cancer

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52
Q

What classes of maternal genes are involved in setting up the antero-posterior axis in the Drosophila embryo?

A

-bicoid
-nanos
-torso

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53
Q

What region is affected by a bicoid mutant in a Drosophila embryo?

A

anterior

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54
Q

What region is affected by a nanos mutant in a Drosophila embryo?

A

posterior

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55
Q

What region is affected by a torso mutant in a Drosophila embryo?

A

terminal

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56
Q

What is a morphogen?

A

a molecule present in a concentration gradient that specifies the fate of the cells present along its concentration gradient
-involved in patterning
-can define more than one cell’s fate

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57
Q

Why is bicoid an unusual morphogen?

A

can only function as a morphogen because Drosophila egg is a syncytium (single cell with many nuclei)

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58
Q

What is bicoid?

A

-transcription factor (switches on other genes)
-morphogen -it forms a H+ gradient across A/P axis

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59
Q

What is the role of nanos?

A

prevent translation of Hunchback

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60
Q

Which genes are involved in posterior patterning in Drosophila embryos?

A

nanos and caudal

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61
Q

Which genes are involved in anterior patterning in Drosophila embryos?

A

bicoid

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62
Q

Which genes are involved in terminal patterning in Drosophila embryos?

A

torso

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63
Q

What is the ligand for the torso receptor?

A

trunk protein
-protease (present at poles of egg) is required for its release

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64
Q

Where is the torso signal activated?

A

tip of egg
-even though torso receptors and trunk protein are both everywhere in the egg, torso signal is only activated at tip because protease is only present at poles
-protease is required to release trunk protein (ligand)

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65
Q

What is the cell signalling involved in terminal patterning of the A/P axis in Drosophila embryos?

A

Torso receptor and Trunk ligand

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66
Q

What is the cell signalling involved in dorsal-ventricular patterning in Drosophila embryos?

A

Toll receptor and Spatzle ligand

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67
Q

Where is the toll signal activated?

A

on ventral side
-Pipe enzyme is localised to ventral side, where is activates Spatzle ligand
- resulting in Dorsal protein undergoing nuclear localisation on ventral side

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68
Q

How does the polarisation of the oocyte occur in Drosophila embryos?

A

-signals from egg chamber induces stalk cells
-oocyte adheres to stalk cells, causing oocyte to be positioned at end
-if stalk signals to follicle cells (unpaired-Jak cell signalling) coincides with Gurken signal from oocyte, they become posterior follicle cells (if the signals don’t coincide, they become posterior follicle cells)

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69
Q

What is the role of Dorsal (protein)?

A

controls patterning along the dorsal-ventral axis

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70
Q

How is Dorsal distributed in the Drosophila egg?

A

uniformly
-initially only in cytoplasm, enters cytoplasm due to signals from activated Toll proteins in ventral region
-high conc in ventral nuclei (where Spatzle is active) -conc decreases in dorsal direction as Toll signal becomes weaker
-little/no Dorsal in dorsal nuclei

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71
Q

What are Hox genes?

A

homeobox-containing genes that are present in all animals involved in antero-posterior patterning (specify segment identity)
-often clustered on chromosomes in one or more gene clusters known as gene complexes
-combinations of expressions of diff Hox genes characterizes diff regions/structures along axis (determine identity of imaginal discs)
-order of Hox genes on genome reflects spatial and timing of expression (3’ expressed first and most anteriorly, 5’ expressed last and most posteriorly)

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72
Q

How were Hox genes discovered?

A

through homeotic mutations in Drosophila where one structure replaced another (eg. organs replaced by wings)

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73
Q

How many Hox complexes are there in Drosophila?

A

1

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74
Q

How many Hox complexes are there in vertebrates?

A

4

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75
Q

What is neurogenesis?

A

formation of neurones

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76
Q

Where do neurones form?

A

in neuroectoderm
-some cells in neuroectoderm do remain ectodermal

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77
Q

What levels of nuclear Dorsal protein are there when neurogenic region is established?

A

low

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78
Q

How are neuronal cells selected from a proneural cluster?

A

via lateral inhibition
-cells in proneural cluster compete by producing delta ligands
-delta bind to Notch receptors on the other cells in the proneural cluster, which activated the Notch receptors
-Notch signal downregulates Achaete/Scute
-the small difference in Achaete/Scute expression becomes amplified
-high Achaete/Scute expression in one of the cells in the cluster activates its neural genes, meaning this cell is selected to become the neuroblast (while the other cell reverts back to epidermal fate

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79
Q

Which genes set up the proneural cluster?

A

Achaete and Scute

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80
Q

What is a neuroblast?

A

embryonic cell that gives rise to neural tissue (neurons and glia)

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81
Q

What is a proneural cluster?

A

cluster of equivalent cells in the neuroectoderm where one cell will be selected and become a neuroblast

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82
Q

Which signal does the proneural cluster activate?

A

Notch/Delta

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83
Q

Which gene codes for the localised protein complex involved in Drosophila neuroblasts’ memory of their apical-basal polarity?

A

Bazooka

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84
Q

Which gene codes for the localised protein complex involved in mammal neuroblasts’ memory of their apical-basal polarity?

A

Par3

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85
Q

What does the localised protein complex (coded for by Bazooka/Par3) do in neuroblasts once they have been selected for?

A

-orientates mitotic spindle (to determine the plane of division in mitosis)
-directs localisation of proteins and RNA molecules to opposite sides of the cell

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86
Q

What cells are produced by the mitosis of a selected neuroblast?

A

-a stem cell (continues to undergo further asymmetric divisions)
-ganglion mother cell (which undergoes one more division to produce a neurone or a glia)

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87
Q

What does the ganglion mother complex (produced by the asymmetric division of a neuroblast) divide into?

A

-neuronal cell
-glial cell (cell which provides physical and chemical support to neurones eg. Schwann cells)

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88
Q

What determines which is the stem cell and which is the ganglion mother cell out of the cells produced by the mitosis of a selected neuroblast?

A

the orientation of the spindle (and localisation of the determinants)

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89
Q

Do cells with a high level of Par3 have a neuronal fate?

A

no

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90
Q

Do cells with a low level of Par3 have a neuronal fate?

A

yes

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91
Q

Which two developmental mechanisms are involved in neuronal development?

A

-lateral inhibition (using Delta-Notch ligand-receptor system to select one cell from a proneural cluster)
-asymmetric cell division (Bazooka/Par3/Pins ensure that the daughter cells produced take on different fates to either become a stem cell or a neurone/glia)

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92
Q

Do all vertebrates develop in the same way?

A

v. diff in early stages (fertilisation)
diff in gastrulation
similar during head and tail formation (pharyngula stage)
diff in final stages

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93
Q

What is the Pharyngula?

A

highly segmented stage in vertebrate embryo development where embryos of all vertebrates look v similar with repeated structures known as somites (all along body axis, form vertebrate structures) and pharyngula pouches (form facial region)

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94
Q

What are the similarities between vertebrate embryos in the Pharyngula stage thought to be due to?

A

evolutionarily conserved bottle necks during development

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95
Q

What happens in the idealised early development of embryos?

A

-oocyte and sperm fuse during fertilisation, forming a zygote (diploid)
-cell divides (cleavage!) forming blastomeres (individual cells)
-blastula stage (blastoderm = outer layer, blastocoel = fluid-filled space inside)

-in this early development, embryo size doesn’t increase but number of cells does

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96
Q

What is the structure of the blastula stage of early embryo development?

A

outer layer = blastoderm
inner fluid-filled space = blastocoel

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97
Q

Nfinish =

(final number of cells in early vertebrate embryo development)

A

Nstart x 2^(tf)
when N = no. cells
t = time
f = frequency of divisions
in early embryo development

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98
Q

How does egg activation occur after fertilisation in vertebrates?

A

-after fertilisation, a wave of free Ca2+ move across egg, allowing development to continue
-Ca2+ act on proteins controlling the cell cycle, which initiates cell division (cleavage of egg)
-oscillations in Ca2+ levels synchronise cell divisions (bc all cells get signal at same time)

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99
Q

How is the cell cycle different in early vertebrate embryos?

A

-only synthesis and mitosis phases (no growth phases)
-maternal stores (of proteins and RNA) provide building blocks for DNA synthesis and growth (no transcription needed)

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100
Q

What is zygotic genome activation?

A

the initiation of gene activation after fertilisation
-as maternal RNA levels decrease, zygotic genome activation occurs, causing the embryo to produce its own (zygotic) RNAs -transcription occurs in the embryo
-cell cycle becomes asynchronous

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101
Q

What happens in gastrulation in vertebrates?

A

the formation of 3 germ layers: mesoderm, endoderm, ectoderm
-cells move to inside to form endoderm and mesoderm
-cells remaining on surface form ectoderm
-establishes body axis (A/P and D/V)

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102
Q

What tissues does the ectoderm germ layer give rise to in vertebrates?

A

-neurones
-glia
-epidermis
-pigment cells

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103
Q

What tissues does the mesoderm germ layer give rise to in vertebrates?

A

-muscle
-cartilage/bone
-dermis
-kidney
-heart
-blood

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104
Q

What tissues does the endoderm germ layer give rise to in vertebrates?

A

-gut
-lungs
-associated organs

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105
Q

What are the first two tissue types formed in vertebrates?

A

epithelium and mesenchyme

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106
Q

What is the mobility of epithelium like?

A

can migrate but moves as a sheet/cluster

107
Q

What is the mobility of mesenchyme like?

A

move easily

108
Q

What is the structure of epithelium?

A

cuboidal cells in rows
-basal side, attached to basal membrane
-junctional complexes between cells

109
Q

What is the structure of mesenchyme tissue?

A

amorphous cells (have no defined shape) in a matrix

110
Q

What do cells do in vertebrate embryos?

A

-condensation
-change from epithelium to mesenchyme or vice versa
-involution
-invagination

111
Q

What forces drive cell and tissue rearrangements in vertebrate embryos?

A

-changes in cell shape (cytoskeletal rearrangements)
-changes in expression of cell surface proteins (cell adhesion molecules)
-cell migration
-localised cell proliferation
-cell death
=>morphogenesis

112
Q

What is morphogenesis?

A

processes involved in bringing about changes in form in the developing embryo
-dependent on cellular movement/motility and cell-cell adhesion
-occurs by mechanisms such as folding of cell sheets, cell death, cell migration

113
Q

What is somitogenesis?

A

the formation of somites
-from anterior to posterior

114
Q

How are somites formed?/What happens in somitogenesis?

A

-mesenchyme cells gather dorsally after gastrulation has ended
-cells on outside of somite epithelise
-somites disassemble and revert to mesenchyme cells

115
Q

What mesodermal progenitor cells are in vertebrate somites?

A

-dermomyotome
-sclerotome

116
Q

What are progenitor cells?

A

descendants of stem cells which further differentiate into specialised cell types

117
Q

What do dermomyotomes further specialise into?

A

dermis and skeletal muscle

118
Q

What do sclerotome further specialise into?

A

vertebrae and ribs

119
Q

How does the neural tube form in vertebrate embryos?/What happens in neurulation?

A

-neural plate (in ectoderm) invaginates (two neural folds create a neural furrow w/a medial hinge point)
-notochord (skeletal rod) forms and grows next to medial hinge region
-surface ectoderm forms outside of dorsal-lateral hinge points
-when folds reach, they fuse, forming the neural tube inside
-neural tube develops into brain and spinal cord

120
Q

What is neurulation?

A

process where the brain and spinal cord are formed from ectodermal neural plate in vertebrate embryos

121
Q

What are the first signs of limbs in a vertebrate embryo?

A

limb buds

122
Q

What are the distinct regions in a limb bud?

A

in ectoderm
-apical ectodermal ridge (AER) (forms in epithelium)
in mesoderm
-progress zone (mesenchyme cells below AER which divides rapidly to drive growth of limb)
-differentiating tissues (cells behind progress zone)

123
Q

What is the apical ectodermal ridge?

A

a thickened region of the ectoderm at the distal end of a developing limb bud, which is essential for limb bud outgrowth and patterning along proximo-distal axis of limb

124
Q

What is the progress zone?

A

mesodermal area at the tip of developing limb bud where cells acquire positional values

125
Q

What does the removal of the apical ectodermal ridge in a developing limb bud do to the limb?

A

truncates the limb (limb develops shorter than it should be)
∴progress zone requires something (FGFs) secreted by AER

126
Q

What signal molecules are secreted by the apical ectodermal ridge to drive the progress zone in a developing limb bud?

A

Fibroblast growth factors (FGFs)
-genes fgf4 and fgf8

127
Q

How are FGFs investigated?

A

gene knockouts in mice
-crossed fgf4 mutation (causing smaller limb) and fgf8 mutation (has no limb defect) together
-this causes no limb growth
∴fgf signalling is required for limb development

128
Q

What is cell ablation?

A

selectively destroying or removing cells in an organism
-biotechnological tool

129
Q

What experimental evidence proves FGF8 is responsible for driving the growth of limb buds?

A

beads soaked in fgf8 act as a substitute for apical ectodermal ridge when it’s been removed
-higher levels of fgf8, the more of the limb grows (dose dependent)

130
Q

What experimental evidence proves FGF8 is sufficient for limb development?

A

ectopic expression of fgf8 in flank leads to ectopic limbs (extra limbs)

131
Q

What is the zone of polarised activity (ZPA)?

A

area at posterior of chick/mice limb buds which produces a signal which specifies its position along the anterior-posterior axis
-important in patterning

132
Q

What is the role of Sonic hedgehog (Shh)?

A

anterior-posterior patterning -required for posterior fates and polarises the limb
-gene expression correlates to ZPA
-forms a gradient which acts as a morphogen

133
Q

What is the terminally differentiated state?

A

final fate of a cell

134
Q

What are totipotent cells?

A

cells which can differentiate into all cell types in body

135
Q

What are pluripotent cells?

A

cells which can differentiate into most cells

136
Q

What are multipotent cells?

A

cells which can differentiate into multiple cell types

137
Q

What are bipotent cells?

A

cells which can differentiate into two cell types

138
Q

What are unipotent cells?

A

cells which can differentiate into only one cell type

139
Q

What are used to track different stages of differentiation?

A

markers

140
Q

What do transcription factors control?

A

-how much protein is made (high/low levels)
-which proteins are made

141
Q

What is the transcriptome?

A

all the genes transcribed in a specific cell type
-diff in diff cell types
-reflects total no. genes expressed in cell at any given time

142
Q

What is the proteome?

A

the proteins in a cell at any given time

143
Q

Where do DNA binding proteins bind?

A

to DNA backbone, reaching into major groove to form specific bonds

144
Q

What is a binding site?

A

section of DNA with a sequenced recognised and bound by DNA binding proteins
-aka cis acting elements
-found in and around a gene

145
Q

What is an enhancer?

A

a binding site for transcriptional activators
-work on any gene (promiscuous -useful for research)

146
Q

What is a silencer?

A

a binding site for transcriptional repressors

147
Q

What are transcription factors regulated by?

A

-extrinsic signals
-intrinsic factors (inherited)
-regulatory binding sites
-accessibility of chromatin (tightly/loosely wound)

148
Q

What is myoD?

A

transcription factor expressed in muscle precursors and muscle cells which controls the expression of genes involved in muscle differentiation
-sufficient but not required for muscle differentiation

149
Q

What muscle-specific proteins are there?

A

-myosin II
-muscle-specific actin
-tropomyosin
-muscle specific enzymes

150
Q

What experiment can be done to prove MyoD is sufficient for muscle differentiation?

A

introduce foreign DNA to fibroblasts and transfect them with acitivated myoD gene
result: fibroblasts differentiate into muscle
∴ myoD is sufficient

151
Q

What experiment can be done to prove MyoD is not required for muscle differentiation?

A

mice lacking myoD function have normal skeletal muscle
∴ myoD is not necessary

152
Q

What genes are involved in muscle differentiation?

A

mrf4
myoD
myf5
pax3

153
Q

Where does most muscle differentiate from in embryos?

A

somites

154
Q

Where does most muscle differentiate from in adults?

A

satellite stem cells

155
Q

Where are blood cells made in mammalian embryos?

A

yolk sac
later on liver

156
Q

Where are blood cells made in mammalian adults?

A

bone marrow

157
Q

What are mutations?

A

changes in DNA base sequence
-naturally occuring or induced in labs

158
Q

How are mutations randomly induced in labs?

A

-radiation eg. UV light, x-rays, radioactivity
-chemical eg. base modifiers, intercalating agents

159
Q

How are targeted mutations induced in labs?

A

-CRISPR
-gene knock-out

160
Q

How do changes in regulatory sequences in DNA affect gene function?

A

affect transcription

161
Q

How do changes in non-coding sequences of transcript affect gene function?

A

affect RNA splicing, stability or translation

162
Q

How do changes in coding sequence affect gene function?

A

affect protein folding or create premature stop codon, causing a truncated (shortened) protein

163
Q

How do mutations affect protein function?

A

-loss of function (non-functioning protein)
-altered function
-reduction in function
-gain in function (over expression of protein)

164
Q

What is an amorphic phenotype?

A

loss of function (protein becomes non-functioning)
-usually recessive -bc most genes are haplosufficient in diploids
-typically non-sense or deletion mutations

165
Q

What is an antimorphic phenotype?

A

altered function (partially active) or for homozygous, completely inactive
-aka dominant negative
-competitive inhibitors
-mutation affects 1 domain of protein
-dominant

166
Q

What is a hypomorphic phenotype?

A

reduction in function
-usually recessive
-typically missense mutations

167
Q

What is a hypermorphic phenotype?

A

over expression of a protein (function gain)
-dominant

168
Q

Are amorphic phenotypes usually dominant or recessive?

A

recessive

169
Q

Are antimorphic phenotypes usually dominant or recessive?

A

dominant

170
Q

Are hypomorphic phenotypes usually dominant or recessive?

A

recessive

171
Q

Are hypermorphic phenotypes usually dominant or recessive?

A

dominant

172
Q

How is a GFP transgenic line generated?

A

-genetical engineering to add GFP to last exon of DNA (via gene fusion) or replace the gene (via reporter construct)
-reintroduce GFP into animal

173
Q

What are the uses of a GFP transgenic line?

A

-follow gene expression or cell behaviour in vitro
-follow subcellular localisation of a protein

174
Q

What stem cells are present in embryo?

A

embryonic stem cells

175
Q

What stem cells are present in mammalian adults?

A

-mesenchymal stem cells
-blood stem cells
-satellite stem cells
-germ cells

176
Q

What do mesenchymal stem cells differentiate into?

A

bone cells (osteoblasts)
cartilage cells (chondrocytes)
fat cells (adicytes)

177
Q

What do satellite stem cells differentiate into?

A

muscle

178
Q

What do germ cells differentiate into?

A

oocyte
sperm

179
Q

Why do adults need stem cells?

A

to replenish cells
-diff cells have diff lifespans (based on their use) so need to be replenished at diff rates

180
Q

How are stem cell populations maintained?

A

-stem cells undergo three types of division to produce 2 stem cells, 1 stem cell and 1 differentiating cell or 2 differentiating cells
-division rates are balanced to keep stem cell population constant

181
Q

What are the different layers of the epidermis?

A

-basal layer
-spinous layer
-granular layer
-stratum corneum

keratinocytes make up these layers -stem cells which renew the keratinocytes are found in the basal layer

182
Q

What cells make up the epidermis?

A

keratinocytes

183
Q

How is the epidermis (in skin) continuously renewed?

A

-keratinocytes (cells) move upwards through skin (dead cells on outer layer)
-cells produce diff keratins as they move towards surface
-stem cells which renew the keratinocytes are found in the basal layer

184
Q

How do signals regulate the basal stem cell niche?

A

-Wnt signals (from dermis) inhibit differentiation and activate stem cell maintenance
-integrins aid the adhesion of cells to basal layer, which activates stem cell maintenance -Notch signals inhibit integrins but notch can be inhibited by Egf

185
Q

How are bulge cells (surrounding end of hair follicle) involved in renewing the epidermis?

A

after injury there is a greater need for cells so bulge cells can provide replacement cells

186
Q

What is junctional epidermolysis bullosa (JEB)?

A

genetic disease where adhesion between the dermis and epidermis is impaired due to mutations in adhesion genes eg. LAMB3
-treatments developed include culturing skin epidermis

187
Q

What cells are in the small intestine?

A

-enterocyte (absorption)
-goblet cells (secrete mucus)
-enteroendocrine cells (secrete peptide hormones)
-paneth cells (secrete antimicrobial peptides)
-stem cells
-submucosa cells (help maintain stem cells)

188
Q

How is the gut continuously replenished?

A

-cells continuously move up

189
Q

Which genes are markers for stem cells?

A

Bim1 (expressed in slow dividing stem cells)
Lgr5 (expressed in fast dividing stem cells)

190
Q

How do we know that cells expressing Lgr5 are stem cells?

A

by lineage tracing
-transgene controlled by Lgr5 promotor and marker is beta galactosidase (blue) -expressed in cell permanently
-marker also expressed in cell’s offspring permanently

191
Q

How do signals maintain intestinal stem cells?

A

-Wnt activates stem cell maintenance
-BMP inhibits stem cell maintenance -BMP itself is inhibited by Noggin (which in turn, activates stem cell maintenance)

192
Q

How can pluripotency be assayed/investigated?

A

-expression of epiblast markers
-chimeras (mix of embryonic stem cells with normal embryonic cells)
-teratomers (tumours containing differentiated tissues)

193
Q

How can mouse embryonic stem cells be made in the lab?

A

-Yamanaka factors (eg.Oct3/4, Sox2, Klf4, c-Myc) applied to differentiated cells
-induced pluripotent stem cells (iPS cells) produced

future potential to make stem cells from a patient’s own cells

194
Q

What experiment shows terminal differentiation can be reversed?

A

nuclear transfer

195
Q

What can be concluded from the fact damage can induce cells reprogramming?

A

-gene expression in nuclei from terminally differentiated cells can be changed under special circumstances
-cytoplasmic factors can control gene expression
-tissue loss can be sensed, resulting in regeneration

196
Q

What is a clone?

A

a group of genetically identical cells that share a common ancestry (derived from same cell)

197
Q

What is done in somatic cell nuclear transfer?

A

-nucleus is separated from donor’s somatic cell
-an egg cell is enucleated (nucleus removed)
-nucleus from the donor is fused with the enucleated egg cell using an electric shock
-the fused cell divides and forms an embryo
-the embryo is transferred to a surrogate mother and develops and a clone is born

198
Q

How can stem cells be used in regenerative medicine?

A

-stem cells from patient can be differentiated in vitro into cell type needed for repair
-these can then be transplanted directly into tissue or added to a scaffold to be transplanted
alternatively… in vivo
-surrounding, healthy tissue can be stimulated by drugs so that they differentiate

199
Q

How are stem cell therapies based on iPS cells carried out?

A

-patient’s cells are given Yamanaka factors, inducing them to become pluripotent to produce self-renewing iPS cells
-iPS cells differentiate and can be transplanted back into patient as corrected cells
-alternatively, iPS cells can be used as disease models for compound screening and drug discovery

200
Q

What are the advantages of stem cell therapies based on iPS cells?

A

-can correct genetic defects (eg. junctional epidermolysis bullosa)
-can replace simple tissues/cell types (eg. bladder, retinal cells)
-can test how cells respond to different pharmaceuticals (personalised medicine)

201
Q

What are the limitations of stem cell therapies based on iPS cells?

A

-genetic changes can be introduced when culturing cells
-transplantations
-organogenesis (making organs) is very complex -tissue engineering scaffolds have limited success

202
Q

What are organoids?

A

small, 3D tissue cultures derived from stem cells cultured in special media which resemble organs or tissues
-used to study regenerative medicine eg. testing potential cures

203
Q

What was Wilson’s experiment using sponges as organoids?

A

-he dissociated sponges
-put the dissociated sponges though a fine sieve
-cells reorganised themselves into intact sponges
∴ cell signalling and adhesion molecules involved

204
Q

Why does cell growth need to be regulated?

A

to maintain correct proportions and drive morphogenesis (eg. limb development)

205
Q

What are the different ways tissue growth occurs?

A

-cell proliferation (cell divisions)
-cell hypotrophy (cell enlargement)
-growth by accretion (secretion of extracellular matrix by cells to cause tissue size to increase)

206
Q

How does tissue growth occur by accretion?

A

cells secrete extracellular matrix, causing tissue to increase in size

207
Q

What are the stages in the cell cycle?

A

Interphase
-G1
-Synthesis
-G2
Mitotic phase
-mitosis
-cytokinesis

208
Q

What is the start checkpoint (during G1) promoted by?

A

FGF

209
Q

What is the start checkpoint (during G1) inhibited by?

A

Rb

210
Q

What is checked in the start checkpoint (during G1)?

A

if environment is favourable

211
Q

What is checked in the G2/M checkpoint?

A

if DNA is replicated and if environment is favourable

212
Q

What is checked in the metaphase-to-anaphase checkpoint?

A

if chromatids are attached to spindle

213
Q

What do mutations in checkpoint genes lead to?

A

excess proliferation, leading to tumour formation

214
Q

How is body size regulated?

A

by hormones -growth hormone (GH) produced by pituitary gland
-in -ve feedback loops

215
Q

How is organ size regulated?

A

by the organ itself using TOR and Hippo pathways

216
Q

What does TOR do?

A

-promote cell growth, which maintains cell size and in effect maintains organ/body size homeostasis

217
Q

What does Hippo do?

A

-inhibit cell proliferation to maintain cell number
-promote cell death to maintain cell number
in effect maintains organ/body size homeostasis

218
Q

What do mutations in Hippo cause?

A

organhypertrophy (increase in organ volume due to cells enlarging)

219
Q

What do mutations in myostatin cause?

A

increased muscle mass
-promotes Rb (which itself inhibits myoblasts from proliferating)
-inhibits myoD (which would promote muscle differentiation)

220
Q

How does the skeleton initially form in vertebrate embryos?

A

as a cartilage template

221
Q

What is ossification?

A

the formation of bone from cartilage template in embryos
-starts in diaphysis (central part of bone)

222
Q

What drives postnatal growth (growth in childhood and adolescence) in long bones (eg. fibula)?

A

growth plate -proliferating chondrocytes (cartilage cells) which increase in size and undergo apoptosis and ossification

223
Q

What sex specific differences in growth are there?

A

-growth spurt occurs earlier in girls than boys
-females have smaller ring finger and larger index finger (boys opposite way around)

224
Q

What is regeneration?

A

an organism’s ability to restore structures in form and function

225
Q

Why are aquatic organisms typically used as models for regeneration?

A

-better at regeneration than terrestrial vertebrates
-can regenerate limbs, eyes, spinal cords, hearts
-simple aquatic organisms (eg. starfish) can undergo whole body regeneration

226
Q

What are the ways tissue regeneration can occur?

A

-morphallaxis
-epimorphosis

227
Q

What is morphallaxis?

A

regeneration which occurs by repatterning currently existing tissues without any growth
-regenerated organism/organ is much smaller
eg. in hydra

228
Q

What is epimorphosis?

A

regeneration which occurs by regrowth
-regenerated organism/organ is same size as original
eg. salamander limb regeneration

229
Q

What act as early wound signals?

A

-ATP (released by damaged cells into extracellular space)
-calcium (intracellular levels increase)
-hydrogen peroxide (released)

230
Q

What happens in the wound response?

A

-cytoskeletal changes occur to close wound (if small, cells on edge of wound form “purse strings” to close wound)
-immune cells are recruited to the wound site
-regeneration or scar formation (depending on organism) is initiated

231
Q

What is fibrosis?

A

scarring
permanent change to tissue caused when fibroblasts secrete high levels of extracellular matrix

232
Q

What happens when a salamander’s limb is amputated?

A

-early wound signals are released (ATP, Ca2+, H2O2)
-wound closes via cytoskeletal changes and movement of epithelium
-wound epithelium secretes signals to induce dedifferentiation
-blastema cells proliferate and regrowth begins

233
Q

How can planaria (flatworms) regenerate?

A

-small fragments can regenerate to form smaller flatworms via morphallaxis
-head region can regenerate via epimorphosis
-some neoblasts (planaria stem cells) are pluripotent, some are lineage restricted

234
Q

How can hydra regenerate?

A

-via morphallaxis so interstitial cells (hydra stem cells) not needed for regeneration

235
Q

How can a zebrafish heart regenerate?

A

-clotting
-wound activates epicardium (cells surrounding heart)
-different signals (retionic acid, hedgehog) are produced
-cardiomyocytes dedifferentiate and proliferate at wound site
-vascularisation (growth of blood vessels into tissue) occurs and cardiomyocytes are activated

236
Q

What are the set of genes used in development also used in?

A

regeneration

237
Q

What do phylogenetic trees show?

A

relationships between organisms
-originally based upon morphological (structural) similarities -now using molecular sequence data

238
Q

How can we tell if proteins are similar?

A

comparison using computer programmes eg. Blast protein alignment

239
Q

How have extra copies of genes arised?

A

gene duplications as errors in mitosis
-improper segregation of chromosomes
-local duplications

240
Q

What are paralogs?

A

new genes which have arised in the genome as a result of gene duplications

241
Q

How can extra copies of genes (from gene duplications) change?

A

-expression pattern during development
-protein structure (large change- domain swapping, small change- point mutations)

242
Q

Why are changes in expression patterns of genes thought to play an important role in evolution?

A

regulatory elements can change
-relatively easy to add/delete transcription factors binding sites by rearrangements/insertions/deletions

243
Q

What happens as a result of experimentally changing gene expression?

A

ectopic organs
-caused by master regulatory genes
-organs often functional ->evolutionary robustness

244
Q

What order do stages of early vertebrate embryo development occur in?

A

-cleavage
-gastrulation
-neurulation
-organogenesis

245
Q

What does chimeric mean?

A

organism contains tissues of different genetic origins -due to mutations, grafting, fusion of early embryos

246
Q

How many parasegments are formed during segmentation in Drosophila?

A

14

-3 mouth
-3 thorax
-8 abdomen

247
Q

What is the hierarchy of gene activity in Drosophila development?

A

-maternal genes
-gap genes
-pair-rule genes
-segment polarity genes

248
Q

What is the common mutation in gap genes?

A

segments A2 to A6 missing

249
Q

What is the notochord?

A

rod-like cellular structure in vertebrate embryos found below the neural tube and derived from the mesoderm
-eventually cells become part of the vertebral column

250
Q

What is ingression?

A

movement of cells from outside to inside of the embryo during gasturlation

251
Q

What is epiboly?

A

flattening and spreading of epithelial cells to increase the surface they cover
-extension of ectoderm during gastrulation to cover the whole of the embryo

252
Q

What are somites?

A

mesodermal block which segment from the mesoderm on either side of the notochord that give rise to trunk/limb muscles, vertebral column/ribs and the dermis

253
Q

What is convergent extension?

A

process where a sheet of cells changes shape by converging (extending in one direction and narrowing) perpendicular to extension by intercalation of cells (cells exchanging positions)
-used in the elongation of the antero-posterior body axis during vertebrate gastrulation, elongation of the notochord, lengthening and closure of the neural tube, germ-band extension in Drosophila, etc

254
Q

What Yamanaka factors are used to generate iPS cells?

A

-Oct3/4
-Sox2
-Klf4
-c-Myc

255
Q

What is the BMP signalling pathway involved in?

A

apoptosis

256
Q

What are imaginal discs?

A

small sacs of epithelium in Drosophila which during metamorphosis give rise to adult stuctures, such as wings, legs, antennae

257
Q

What signalling pathway signals for the growth of neuronal growth tubes?

A

semaphorsis and Eph ligands

258
Q

Which stage of the cell cycle are cells which rarely divide (eg. red blood cells, neurons, keratinocytes) in?

A

G0

259
Q

What are osteoblasts?

A

bone-forming cells derived from stem cells in bone marrow
-have no lineage relationship with chondrocytes

260
Q

What signalling molecules stimulate growth?

A

IGF-1 and IGF-2

261
Q

What can inactivation or overexpression of Hox genes lead to?

A

-localized abnormalities
-homeotic transformations of one ‘segment’ of the axis into another
(indicates that these genes are crucial in specifying regional identity

262
Q

How is the spherical shape of a blastula/blastocyst maintained?

A

-flow of fluid into interior
-hydrostatic pressure

263
Q

What is the role of mrf?

A

muscle determination

264
Q

What is the role of myogenin?

A

muscle maturation

Molecular Biology BSc First Year (10 decks)

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