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MIMS > The Genome in Health and Disease > Flashcards

The Genome in Health and Disease Flashcards

1
Q

what are carcinomas?

A

malignant tumours of epithelial cells

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2
Q

what are sarcomas?

A

tumours derived from the mesenchymal layer

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3
Q

what are the 6 hallmarks of cancer?

A

self-sufficiency in growth signals, insensitivity to anti-growth signals, evading apoptosis, limitless replicative potential, sustained angiogenesis, tissue invasion and metastasis

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4
Q

what are the extra updated hallmarks of cancer?

A

avoiding immune destruction, tumour-promoting inflammation, genome instability and mutation, deregulating cellular energetics

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5
Q

how many bases and genes does the human genome comprise?

A

3 x 10^9 bases, around 20000 genes

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6
Q

how close must a cell be to a blood vessel to avoid hypoxia?

A

about 10 cells distance

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7
Q

what is hypoxia?

A

inadequate oxygen diffusion to a cell

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8
Q

what is cachexia?

A

body wasting caused by cancer

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9
Q

who first isolated DNA?

A

Miescher

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10
Q

what are the 3 functions of DNA?

A

maintenance of the information encoded in the genome, propagation of the genetic information via cell division, reading the genetic information

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11
Q

what is the 5 carbon sugar used in DNA?

A

deoxyribose

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12
Q

which position has the extra-cyclic C atom in DNA?

A

5

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13
Q

which carbon position does not have a hydroxyl group attached in DNA?

A

2

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14
Q

which bases are purines?

A

adenosine and guanine

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15
Q

which bases are pyrimidines?

A

thymine and cytosine

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16
Q

what is a nucleoside?

A

a base and a ribose sugar

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17
Q

what is a nucleotide?

A

a base, a ribose and phosphate

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18
Q

which carbon are bases attached to in DNA?

A

the 1’ carbon

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19
Q

which carbon is the phosphate group attached to in DNA?

A

the 5’ carbon

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20
Q

which atoms do phosphodiester bonds form between in DNA?

A

the 3’ C of one ribose, the phosphate and the 5’ C of the ribose in the next nucleotide

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21
Q

what is the charge of a DNA or RNA strand at neutral pH?

A

negative

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22
Q

which is more prone to degradation in alkaline conditions, DNA or RNA?

A

RNA

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23
Q

what do riboswitches control?

A

gene expression

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24
Q

what does ribozyme catalyse?

A

peptide bond synthesis

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25
Q

are the DNA strands in the double helix left or right-handed?

A

right handed

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26
Q

are the DNA strands in the double helix parallel or anti-parallel?

A

anti-parallel

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27
Q

what is the charge of the phosphates in DNA?

A

negative

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28
Q

how many H bonds form between A and T?

A

2

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29
Q

how many H bonds form between G and C?

A

3

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30
Q

what is the angle between the plane of bases and the vertical axis of the DNA helix?

A

almost perpendicular

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31
Q

how many base pairs are present in one turn in B-DNA?

A

10.5

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32
Q

what is the rise per base pair in B-DNA?

A

3.4A

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33
Q

what is the width of the helix in B-DNA?

A

around 20A

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34
Q

what is the rise per helical turn called?

A

pitch

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35
Q

what is the rise per helical turn in B-DNA?

A

10.5 x 3.4A

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36
Q

what allows DNA to form sequence specific interactions with DNA binding proteins?

A

exposed functional groups on the edge of the aromatic bases

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37
Q

what is the most common structure of DNA in chromosomal DNA?

A

the B form

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38
Q

what form does double stranded RNA adopt?

A

the A-form

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39
Q

which is wider, the A form or the B form?

A

the A form

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40
Q

which has a more compressed, narrower major groove, the A form or the B form?

A

the A form

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41
Q

what type of DNA tends to adopt an A like conformation?

A

GC-rich

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42
Q

which has a wider minor groove, the A form or B form?

A

the A form

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43
Q

what is different about the Z form to the B form?

A

left-handed, phosphate backbone follows zigzag trajectory

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44
Q

what role does the Z form seem to have?

A

role in modulating gene expression

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45
Q

what causes local changes in the shape of B form DNA?

A

local nucleotide composition

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46
Q

what is the diameter of a human cell nucleus and how long is the DNA housed within it?

A

nucleus 5 microns diameter, DNA 2 metres long

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47
Q

what mechanism does DNA use to compact into the nucleus?

A

supercoiling

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48
Q

which direction is the twisting in positive supercoiling?

A

right-handed

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49
Q

what does overwound supercoiling cause?

A

the DNA helix to distort and ‘knot’

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50
Q

which direction is the twisting in negative supercoiling?

A

left-handed

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51
Q

what does underwound supercoiling cause?

A

DNA knotted into negative supercoils

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52
Q

what resists supercoiling in DNA?

A

ends of DNA fixed to proteinaceous scaffold, long DNA tails have high viscosity

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53
Q

how is supercoiling achieved in the cell DNA?

A

by a small degree of underwinding which reduces the number of turns of the double helix

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54
Q

what does underwinding of DNA facilitate?

A

compaction (important for packing DNA into cell) and strand separation (important for DNA metabolism)

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55
Q

what are two cellular processes which generate supercoiling?

A

mRNA transcription and DNA replication

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56
Q

what is the name of the enzymes that are involved in supercoiling?

A

topoisomerases

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57
Q

how do topoisomerases work?

A

by cleaving and rejoining the DNA strands

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58
Q

what is the nucleoid?

A

a ‘rosette’ model of DNA orgainsation with around 500 loops emanating from a proteinaceous scaffold-like core

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59
Q

what is chromatin?

A

the structure formed by chromosomal DNA bound to proteins such as histones and other DNA packaging factors

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60
Q

what is the basic unit of chromatin in eukaryotic cells?

A

the nucleosome

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61
Q

what is DNA wound around in the nucleosome?

A

a protein ‘barrel’ made up of 8 histone proteins (a histone octamer)

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62
Q

what are the histones present in each nucleosome?

A

2 copies of H2A, H2B, H3 and H4

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63
Q

how many nucleotides of DNA are present in a nucleosome?

A

147

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64
Q

how many left-handed turns does the DNA make around the histone octamer in the nucleosome core particle?

A

1.7

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65
Q

what property helps histones bind to the phosphate groups in DNA?

A

histones are positively charged, phosphate groups are negatively charged

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66
Q

what part of the histones in the NCP particle are susceptible to chemical modification by cellular enzymes?

A

the amino terminal tails which project beyond the DNA gyres

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67
Q

what does H1 bind in the NCP?

A

nucleosomal DNA at the entry and exit positions

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68
Q

what is H1 also known as and why?

A

linker histone as it binds the linker DNA between nucleosomes

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69
Q

what is the most common form of chromatin?

A

the 10-nm fibre

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70
Q

what gives the 10nm chromatin fibre its beads-on-a-string appearance?

A

a linear array of NCPs separated by linker DNA

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71
Q

what is the nucleosome repeat length in chromatin?

A

around 200 nucleotides

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72
Q

what causes the 10nm fibre to transition into the 30nm fibre?

A

specific buffer conditions of low salt and the presence of divalent metal ions

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73
Q

what are the characteristics of the 30nm chromatin fibre?

A

compact and regular

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74
Q

what does the large majority of chromatin in the cell nucleus fold into?

A

local heterogenous clusters or globules of nucleosomes without long-range regularity

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75
Q

what does chromatin compaction regulate?

A

transcription

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76
Q

what is euchromatin?

A

the more open form of chromatin - transcriptionally active

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77
Q

what is the transcriptionally active form of chromatin?

A

euchromatin

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78
Q

what is heterochromatin?

A

the more condensed form of chromatin, transcriptionally inactive

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79
Q

what is the transcriptionally inactive form of chromatin?

A

heterochromatin

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80
Q

what is the DNA loop?

A

a large region of DNA that is anchored at its base to a proteinaceous chromosomal scaffold and is spacially and transcriptionally segregated from rest of genome

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81
Q

what are the consequences of the DNA loop on gene reglation?

A

bring enhancer and promoter regions together

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82
Q

what is the role of the DNA loop in DNA recombination?

A

involved in recombination in maturation of immunoglobulin genes

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83
Q

what does SMC stand for?

A

structural maintenance of chromosome

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84
Q

what are the SMC proteins?

A

cohesin and condensin

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85
Q

how are SMC proteins typically arranged?

A

split ATPase domain separated by a long helical region that folds in on itself at hinge position to reconstitute a globular ATPase domain

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86
Q

what forms the characteristic V-shaped appearance of SMC proteins?

A

2 SMC proteins coming together by hinge dimerisation

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87
Q

what links the head domains in cohesin and condensin?

A

a non-SMC subunit

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88
Q

what does the head domains being linked by a non-SMC subunit mean for cohesin and condensin?

A

they have a topological ring structure that can trap 1 or more DNA molecules in the ring

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89
Q

what determines the size of the DNA loop in chromosomal DNA?

A

adjacent binding sites of CTCF

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90
Q

what is CTCF?

A

a DNA-binding protein that recognises specific sequence motifs and recruits cohesin

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91
Q

what does cohesin connect?

A

physically distant sites on DNA

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92
Q

what is the loop-extrusion model?

A

condensin extrudes loops of DNA. condensin molecules approach each other. chromosome loops around the longitudinal axis form threadlike structures, chromatin packs into a metaphase chromosome

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93
Q

when does cohesin compact chromosomes?

A

at interphase

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94
Q

when does condensin compact chromosomes?

A

in mitosis

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95
Q

what are the constituent units of chromosomal DNA at the megabase scale?

A

DNA loops

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96
Q

what is the megabase scale?

A

10e6

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97
Q

what does TAD stand for?

A

topologically associated domain

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98
Q

what is a TAD?

A

several DNA loops folded together

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99
Q

what is the chromosome territory?

A

the unique volume occupied by each chromosome in the nucleus

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100
Q

what can each chromosome territory be divided into transcriptionally?

A

2 compartments, A and B, that are respectively transcriptionally active (predominantly euchromatin) and inactive (mainly heterochromatin)

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101
Q

where does the heterochromatin compartment usually sit in the chromosome territory?

A

on the outside

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102
Q

where does the euchromatin compartment usually sit in the chromosome territory?

A

in the centre

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103
Q

what can the likelihood of translocations be determined by?

A

spatial proximity

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104
Q

what is Burkitt’s lymphoma characterised by?

A

a translocation between the MYC gene and 1 of 3 immunoglobulin gene variants located on different chromosomes

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105
Q

what is the most common translocation in Burkitt’s lymphoma and why?

A

MYC:IGH as IGH is spatially the closest immunoglobulin to Myc

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106
Q

what can modify chromatin status?

A

binding of H1 causing compaction, post-translational modifications of histone tails, chromatin remodellers

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107
Q

what are chromatin remodellers?

A

multi-subunit protein complexes with ATPase activity that can alter the position of nucleosomes

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108
Q

can histone modifications be inheritable?

A

yes

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109
Q

what is the effect of lysine acetylation?

A

reduces the overall positive charge of the histones, so reduces ability to interact with DNA, reduces chromatin compaction

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110
Q

what do chromatin ‘writer’ enzymes do?

A

add chemical signals

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111
Q

what do chromatin ‘eraser’ enzymes do?

A

remove chemical signals

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112
Q

what doe chromatin ‘reader’ proteins do?

A

recognise each unique set of chromatin modifications and trigger a transcriptional response

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113
Q

what doe nucleosome remodellers do?

A

use the energy from ATP hydrolysis to shift histones and alter local chromatin structure

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114
Q

what do nucleosome remodellers contain?

A

a DNA translocation motor and ‘reader’ subunits for targeting the remodeller to a specific chromatin site

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115
Q

when does DNA replication take place?

A

S phase

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116
Q

how many base pairs does each cell contain?

A

3 billion

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117
Q

what type of replication is DNA replication?

A

semi-conservative

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118
Q

what would dispersive replication produce?

A

strands with fragments of parental and daughter DNA in a mosaic

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119
Q

what is the origin of replication?

A

the specific sequence at which replication initiates in prokaryotes

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120
Q

how long is the origin of replication in E.coli?

A

around 250bp

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121
Q

what is the origin of replication known as in E.coli?

A

OriC

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122
Q

what binds to the origin of replication to begin the process of replication?

A

DnaA, an initiator protein

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123
Q

what does DnaA bind to?

A

DnaA boxes in the origin of replication

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124
Q

what are DnaA boxes?

A

a tandem repeat of DNA sequences

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125
Q

what causes local melting of the double helix in DNA replication?

A

DNA unwinding element (DUE)

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126
Q

what is DnaB?

A

a DNA helicase that uses the energy from ATP hydrolysis to unwind dsDNA

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127
Q

what are some features shared by all replicative helicases?

A

hexameric ring proteins, ATP-driven molecular motors

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128
Q

what loads the DnaB helicase onto the unwound origin DNA?

A

the protein loader DnaC

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129
Q

what does DnaC cause?

A

transient opening of the helicase ring and its subsequent closure around the DNA strand- steric exclusion- 1 DNA strand threaded through the ring, peeling off the 2nd strand

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130
Q

what stabilises the exposed ssDNA in DNA replication?

A

interaction with the single-stranded DNA-binding protein

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131
Q

what does SSB stand for?

A

single stranded DNA-binding protein

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132
Q

what are the properties of SSB protein?

A

binds to ssDNA with high affinity and no sequence specificity

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133
Q

how does SSB protein work?

A

coats the DNA strand to prevent reannealing and protect it from possible nuclease degradation

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134
Q

in what direction does DNA replication proceed after initiation?

A

bidirectionally from origin creating replication bubble

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135
Q

what are replication forks?

A

the Y-shaped structures that move away from the initiation point in DNA replication

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136
Q

by means of what process and enzyme does DNA synthesis take place?

A

nucleotide polymerisation by DNA polymerase

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137
Q

what is needed for nucleotide polymerisation?

A

a primer annealed to template with free 3’ end and deoxynucleotide triphosphates

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138
Q

what direction does nucleotide polymerisation take place in?

A

5’ to 3’ direction

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139
Q

how many DNA polymerases does E.coli have?

A

5

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140
Q

what is the role of DNA polymerase I in E. coli?

A

roles in nick-translation during Okazaki fragment processing

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141
Q

what is the main replicative enzyme in E. coli?

A

DNA polymerase III

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142
Q

how many DNA polymerase have been found in humans?

A

at least 15

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143
Q

how many DNA polymerases perform the bulk of DNA synthesis in DNA replication in humans?

A

3

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144
Q

which DNA polymerases perform the bulk of DNA synthesis during DNA replication in humans?

A

alpha, delta, epsilon

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145
Q

what are the roles of DNA polymerases not involved in DNA synthesis in DNA replication in humans?

A

nucleotide polymerisation in DNA repair in presence of damage or gaps in DNA

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146
Q

what do DNA polymerases rely on to start DNA synthesis?

A

specialised polymerase called primase

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147
Q

what does primase synthetise?

A

a short RNA primer on the template DNA that is extended by DNA polymerase

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148
Q

what is primase called in bacteria?

A

DnaG

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149
Q

what is primase strictly speaking?

A

a DNA dependent RNA polymerase

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150
Q

how long is the RNA primer primase synthesises?

A

5-15 nucleotides

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151
Q

is primase more important in leading or lagging strand synthesis?

A

Lagging

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152
Q

in which strand does nucleotide polymerisation proceed in the same direction as the advancing fork?

A

the leading strand

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153
Q

how is the lagging strand replicated?

A

in short segments called Okazaki fragments

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154
Q

how long are Okazaki fragments in bacteria?

A

about 1000 bases

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155
Q

how long are Okazaki fragments in eukaryotes?

A

about 100-200 bases

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156
Q

from what end does DNA pol I hydrolyses RNA and DNA?

A

the 5’ end

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157
Q

what replaces the RNA primer with DNA in the lagging strand?

A

DNA pol I

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158
Q

what seals the Okazaki fragments together?

A

DNA ligase

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159
Q

how many bases does the leading strand DNA polymerase keep going for?

A

about 2.5 million

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160
Q

what is processivity?

A

the ability of a polymerase to polymerise nucleotide without stopping

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161
Q

what ‘sliding clamp’ protein does DNA pol II rely on?

A

the β-clamp

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162
Q

what does the β-clamp provide DNA pol II?

A

a topological link to the DNA template ensuring that the polymerase remains attached to the template

163
Q

what is the difference between the action of the β-clamp in leading and lagging strand synthesis?

A

continuous attachment in leading, in lagging repeatedly binds and releases

164
Q

how many copies of DNA polymerase does evidence suggest act together to synthesise leading and lagging strand DNA at the replication fork?

165
Q

what do the 2 DNA polymerases interact with to form a replisome?

A

other replication factors such as helicase and primase

166
Q

what is a replisome?

A

a replication machine (all of the replication factors acting together)

167
Q

what does the replisome do?

A

couples leading and lagging strand synthesis with replication fork progression

168
Q

what happens in the trombone model?

A

the lagging strand template is primed then bent back around to engage with DNA pol III forming a loop of increasing size that is periodically released allowing novel priming event to occur

169
Q

what are the steps of DNA replication?

A

origin recognition; helicase recruitment; DNA melting; priming; elongation; association of polymerase with sliding clamp

170
Q

what is the origin recognition protein in bacteria?

171
Q

what is the origin recognition protein in eukaryotes?

172
Q

what is the helicase loading protein in bacteria?

173
Q

what are the helicase loading proteins in eukaryotes?

A

Cdc6 and Cdt1

174
Q

what is the replicative helicase in bacteria?

175
Q

what is the replicative helicase in eukaryotes?

A

MCM2-7 helicase

176
Q

what is the ss binding protein in bacteria?

177
Q

what is the ss binding protein in eukaryotes

178
Q

what is the primase in bacteria?

179
Q

what are the primases in eukaryotes?

A

PriS, PriL, heterodimer

180
Q

what is the main replicative DNA polymerase in bacteria?

A

DNA Pol III

181
Q

what is the main replicative DNA polymerase in eukaryotes?

A

DNA Pol α, δ, ε

182
Q

what is the sliding clamp in bacteria?

183
Q

what is the sliding clamp in eukaryotes?

184
Q

what is the clamp loader in bacteria?

A

γ Complex

185
Q

what is the clamp loader in eukaryotes?

186
Q

what is the DNA ligase in bacteria dependent on?

187
Q

what is the DNA ligase in eukaryotes dependent on?

188
Q

what is the primer remover in bacteria?

189
Q

what is the primer remover in eukaryotes?

190
Q

which eukaryotes have ARSs?

A

simple, monocellular eukaryotes such as budding yeast

191
Q

what are Autonomously Replicating Sequences (ARSs)?

A

origins of replication of defined sequence in simple monocellular eukaryotes

192
Q

what is an additional consequence of having multiple origins of replication in eukaryote cells?

A

cells develop temporal programme of origin activation (origin firing) with early and late firing origins

193
Q

which regions of the genome are usually replicated last in eukaryotes?

A

heterochromatic regions

194
Q

what is eukaryotic DNA replication tightly coupled in eukaryotes?

A

the cell cycle

195
Q

what is aneuploidy?

A

an unequal number of chromosomes

196
Q

how is DNA replication coupling to the cell cycle achieved in eukaryotes?

A

by separating the process of origin licensing in G1

197
Q

what does MCM2-7 stand for?

A

mini chromosome maintenance 2-7

198
Q

what loads inactive MCM2-7 onto the DNA?

A

the Origin Recognition Complex (ORC)

199
Q

when is MCM2-7 activated?

A

at the beginning of S-phase

200
Q

where is the replisome assembled in eukaryotes?

A

on the MCM2-7 helicase

201
Q

what controlling steps does origin firing require?

A

recruitment of co-activator proteins Cdc45 and GINS to the MCM2-7 helicase and CDK phosphorylation activity

202
Q

what is replication primase a subunit of in eukaryotic replication?

A

DNA polymerase α

203
Q

what extends the RNA primer synthetised by primase with dNTPs in eukaryotic replication?

A

DNA Pol α

204
Q

what ribonuclease removes the RNA primer in eukaryotes?

205
Q

what does RNaseH do?

A

digests RNA that is base paired to DNA in eukaryotes

206
Q

what cuts off the flap structure created by the replicative DNA polymerase displacing the residual primer in eukaryotes?

A

the Flap Endonuclease Fen1

207
Q

what seals the DNA nick produce by Fen1?

A

DNA ligase I

208
Q

what coordinates the actions of Fen1 and DNA ligase I?

A

the eukaryotic sliding clamp PCNA

209
Q

what are topoisomerases?

A

enzymes that act to release topological tension in DNA molecules and untangle molecules

210
Q

what are the classes of topoisomerase?

A

type I and type II

211
Q

what do type I topoisomerases do?

A

remove positive supercoiling in front of the advancing replication fork. they nick a single strand of DNA and swivel or pass it around the other strand before resealing the nick

212
Q

what do type II enzymes do?

A

completely cut both strands of one duplex and allow another duplex to pass through the gap which is then resealed

213
Q

what attachment do topoisomerase form with DNA?

A

a covalent protein-DNA attachment

214
Q

what is the purpose of the covalent attachment of topoisomerases to DNA?

A

preserve energy of the chemical bond and ensures inadvertent release of broken DNA molecules doesn’t occur

215
Q

what is the genome of an organism?

A

the complete set of genetic material for that organism

216
Q

what doesn’t genome usually refer to in eukaryotes?

A

mitochondrial DNA

217
Q

what is the most commonly used technique for sequencing DNA?

A

chain termination method

218
Q

what does the chain termination method of DNA sequencing rely on?

A

DNA polymerase can incorporate 2’,3’-dideoxynucleotides (ddNTPs) into growing DNA chains

219
Q

who created the chain termination method?

220
Q

why do ddNTPs terminate the DNA chain?

A

don’t have a 3’ OH group

221
Q

how many reactions were set up in the original chain termination technique?

222
Q

what did the 4 reactions in the original chain termination technique contain?

A

dATP, dCTP, dGTP, dTTP, radiolabel, small amounts of either ddATP, ddCTP, ddGTP or ddTTP

223
Q

what is used to visualise the DNA bands in the original chain termination technique?

A

gel electrophoresis and X-ray film to visualise the DNA bands

224
Q

what has been used to enhance the chain termination technique?

A

using ddNTPs conjugated with specifically-coloured fluorescent markers, using fine capillaries to separate the DNA fragments rather than large gels

225
Q

what sequencing method did the Human Genome project use?

A

chain termination method

226
Q

what is the more modern technique of DNA sequencing?

A

next generation sequencing (NGS)

227
Q

what does NGS rely on?

A

massively parallel sequencing whereby millions of short sequence reads are obtained simultaneously and then assembled by computer into chromosomes and genomes

228
Q

which will have a higher melting temperature, A:T rich or G:C rich sequences?

229
Q

why do G:C rich sequences have a higher melting temperature than A:T rich ones?

A

because of the different number of H bonds holding together the base pairs

230
Q

what is the C-value paradox?

A

there is a lack of correlation between genome size and organismal complexity

231
Q

how much of the human genome is non-coding DNA without a clearly defined function?

233
Q

what is the telomere sequence in humans?

234
Q

what adds the telomere sequences to DNA?

A

telomerase

235
Q

what are the components of telomerase?

A

protein and RNA component

236
Q

what does the RNA component of telomerase function as?

A

template for DNA synthesis

237
Q

what are reverse transcriptases?

A

a class of enzyme that use RNA to make DNA

238
Q

how does the action of telomerase counteract the lagging-strand problem?

A

by adding long, repetitive stretches of single-stranded DNA to the end of the chromosome

239
Q

what is the T loop of DNA?

A

a loop that closes off the end of the chromosome

240
Q

what is quadruplex DNA?

A

when 4 GGG triplets come together to form 3 stacked planar G quartets held together by Hoogsteen H bonds

241
Q

what happens to the length of telomeres when adult cells divide?

A

they get shorter as they don’t have enough telomerase

242
Q

what is the Hayflick limit?

A

when telomeres get to a short enough length that cells read it as a signal to stop dividing

243
Q

what can extend the life span of cells at the Hayflick limit?

A

re-introduction of telomerase into cells

244
Q

what are mobile genetic elements?

A

regions of DNA that can move around and insert in other parts of the genome, and thus cause mutations

245
Q

what are the 2 types of mobile genetic elements known?

A

transposons and retrotransposons

246
Q

what enzyme moves DNA transposons around the genome?

A

transposase

247
Q

what occurs in movement of retrotransposons?

A

DNA is transcribed to RNA, RNA then reverse transcribed to DNA which is inserted back into different part of genome

248
Q

how can transposons propagate antibiotic resistance?

A

in bacteria many transposons carry antibiotic resistance genes, so can propagate antibiotic resistance within and between strains and species

249
Q

how much of the human genome is composed of mobile genetic elements or their remnants?

250
Q

how may transposons be beneficial on an evolutionary timescale?

A

facilitate shuffling of coding sequences or moving genes so they’re under control of a new promoter

251
Q

what are the recombinases that mediate antibody diversity regulation related to?

A

transposon-encoded enzymes

252
Q

what is DNA melting?

A

denaturation of the double helix leading to separation of the 2 strands of DNA

253
Q

what factors affect DNA melting and re-annealing?

A

temperature, length of DNA, base composition, ionic composition of solvent

254
Q

what is the process of re-annealing of DNA known as?

A

DNA hybridisation

255
Q

what techniques is DNA hybridisation the backbone of? (5)

A

PCR, genotyping by in situ hybridisation, Southern blotting, FISH, DNA microarrays

256
Q

what does FISH stand for?

A

fluorescence in situ hybridisation of DNA

257
Q

what does PCR stand for?

A

polymerase chain reaction

258
Q

what gives DNA and RNA strong UV light absorption?

A

aromatic bases

259
Q

what is the peak absorbance of DNA?

260
Q

why is UV absorbance of bases lower in the double helix?

A

base stacking (hypochromicity effect)

261
Q

what is the hypochromicity effect?

A

UV absorbance of bases is lower in the double helix due to base stacking

262
Q

how does the hypochromicity effect allow for following the melting/re-annealing process?

A

base stacking in the double helix reduces the absorption compared to the denatured state

263
Q

what is the restriction-modification system?

A

anti-viral defence mechanism in bacteria that uses enzymes to digest viral DNA

264
Q

what type of enzymes does the restriction-modification system use?

A

DNA endonucleases

265
Q

how do DNA endonucleases distinguish between bacterial and viral DNA?

A

they recognise specific DNA sequences which may be absent from the bacterial genome + are specific for either methylated or non-methylated DNA

266
Q

which bases can methylation occur at?

267
Q

what performs DNA methylation in bacteria?

A

an SNA methyltransferase enzyme

268
Q

where do bacteria methyltransferases methylate adenine?

A

within restriction sequences

269
Q

what is the most prevalent position of adenine methylation in viral DNA by bacterial methyltransferases?

A

at position N6 of adenine

270
Q

what are the most widely used type of restriction enzymes?

271
Q

do type II restriction enzymes typically form homo or heterodimers?

A

homodimers

272
Q

how long are the sequences recognised by type II restriction enzymes?

273
Q

what types of ends does EcoRI cleavage produce?

A

sticky ends

274
Q

what sort of ends does EcoRV cleavage produce?

A

blunt ends

275
Q

what are RFLPs?

A

restriction fragment length polymorphisms

276
Q

what is RFLP?

A

the study of genetic differences between individuals

277
Q

what is recombinant DNA?

A

DNA molecules that are the result of laboratory manipulation

278
Q

what properties allow DNA to be separated by gel electrophoresis?

A

negatively charged, chemically relatively stable in solution

279
Q

what sorts of gel can be used in gel electrophoresis?

A

agarose, polyacrylamide

280
Q

which electrode does DNA run towards in gel electrophoresis?

A

the positive electrode

281
Q

how can DNA be visualised after separation by gel electrophoresis?

A

dyes that bind dsDNA and fluoresce under UV light

282
Q

what is the most common dye used to visualise DNA separated by gel electrophoresis?

A

ethidium bromide

283
Q

what is the Southern blot technique?

A

transferring separated DNA from gel to a filter then analysing by hybridisation to check for specific sequences

284
Q

what is the blot method used for RNA?

A

Northern blot

285
Q

what is the blot method used for DNA?

A

Southern blot

286
Q

what is the blot method used for proteins?

A

Western blot

287
Q

what is the most common way to make lots of copies of a piece of DNA?

A

cutting and pasting into a bacterial plasmid

288
Q

how many origins of replication does a plasmid have?

289
Q

what are plasmids?

A

circular molecules of extrachromosomal DNA in bacteria

290
Q

what are bacteriophage?

A

viruses that infect bacteria

291
Q

what are more specialised systems for manipulating larger DNA fragments than plasmids or bacteriophage?

A

yeast and bacterial artificial chromosomes

292
Q

what is the downside to using plasmids or bacteriophage to amplify DNA?

A

limit to length of the piece of DNA, several thousand base pairs

293
Q

what is the polymerase chain reaction?

A

a method for making large amounts of a defined region of DNA in a test tube without having to use bacteria

294
Q

what information about the DNA is needed for the PCR?

A

DNA sequence you want to amplify or the DNA flanking the region of interest

295
Q

what do the primers provide in PCR?

A

a free 3’ end of DNA that DNA polymerase can extend from

296
Q

what is the first step of PCR?

A

over 90 degrees C to separate the dsDNA (melting)

297
Q

what is the second step of PCR?

A

55-60 degrees C to allow primers to hybridise to their target sequence (annealing)

298
Q

what is the third step of PCR?

A

72 degrees C to allow the DNA polymerase to make the DNA copy (extension)

299
Q

what components are required for PCR amplification of a chosen template DNA?

A

short primers (oligonucleotides); stock of nucleotides (dNTPs); a buffer solution; a DNA polymerase

300
Q

which is present in a large molar excess in PCR, primers or template?

301
Q

what specific DNA polymerase is able to withstand heating and cooling in PCR?

A

Taq polymerase

302
Q

what is Friedrich’s ataxia?

A

a genetic disorder causing progressive degeneration of the nervous system with loss of controlled movement

303
Q

what is Friedrich’s ataxia caused by?

A

a defect in the FXN gene which codes for Frataxin protein

304
Q

what is the defect in the FXN gene that causes Friedrich’s ataxia?

A

abnormally high (>100) number of copies of the GAA repeat in the FXN gene

305
Q

what does CRISPR stand for?

A

clustered regularly-interspersed short palindromic repeats

306
Q

what are CRISPRs?

A

short viral DNA sequences present in the microbial genome

307
Q

what do CRISPRs do?

A

they are transcribed into RNA and used as guides to direct the Cas9 nuclease to cleave the DNA of the invading virus during an infection

308
Q

what does Cas protein stand for?

A

CRISPR-associated

309
Q

what is Cas protein?

A

a nuclease that cleaves the DNA sequence targeted by the guide RNA

310
Q

what is the CRISPR-Cas system?

A

1) the viral DNA is integrated at the CRISPR locus; 2) RNA is transcribed from the CRISPR locus; 3) RNA guides the CAS nuclease to the invading DNA; 4) the CAS nuclease degrades the viral DNA

311
Q

what is the first approved genetic treatment based on CRISPR for?

A

patients suffering from sickle-cell anaemia and beta-thalassemia

312
Q

what does the CRISPR therapy for sickle-cell anaemia and beta-thalassemia do?

A

reactivates fetal haemoglobin in patients with defective adult haemoglobin

313
Q

what is the error rate for DNA replication?

A

1 error in 10^9 nucleotides

314
Q

what do mutations in the ‘proof-reading’ exonuclease domain of Pol epsilon cause?

A

a ‘hyper mutation’ phenotype that drives cancer formation

315
Q

what are the types of replication error?

A

base mismatches, nucleotide misincorporation by error-prone TLS polymerases, deletions, insertions

316
Q

what are TLS polymerases?

A

translesion synthesis polymerases

317
Q

what is translesion DNA synthesis?

A

a mechanism to traverse damaged sites (roadblocks) on the DNA template

318
Q

what is the advantage of translesion DNA synthesis?

A

DNA replication doesn’t stop

319
Q

what is the disadvantage of translesion DNA synthesis?

A

increased mutagenesis due to lack of proof-reading and poor nucleotide selectivity

320
Q

what is the mechanism of translesion DNA synthesis?

A

they have larger active sites that can accommodate bulkier modified bases

321
Q

what sort of condition is Huntington’s disease?

A

autosomal dominant genetic condition

322
Q

what causes Huntington’s disease?

A

mutations in huntingtin gene on chromosome 4

323
Q

what is the mutation that causes Huntington’s?

A

an expansion of a repeated stretch of CAGs in the protein coding part of the gene

324
Q

how many repeats of CAGs are needed to cause symptomatic Huntington’s?

A

> 40 repeats

325
Q

what does the mechanism of triplet expansion depend on?

A

the propensity of (CNG)n to depart from B-form DNA

326
Q

what are endogenous causes of DNA damage? (not examples)

A

causes within organisms

327
Q

what are some examples of endogenous causes of DNA damage?

A

replication errors, by-products of metabolism, chemical instability

328
Q

what are exogenous causes of DNA damage? (not examples)

A

environmental causes of DNA damage

329
Q

what are examples of exogenous causes of DNA damage?

A

UV light, ionising radiation, genotoxic chemicals

330
Q

how many damage events occur per mammalian cell per day?

A

around 30000

331
Q

what does the Ames test assess?

A

the mutagenic potential of a chemical compound

332
Q

what is the Ames test used for?

A

screening new chemicals

333
Q

what do we assume mutagenicity in bacteria leads to in humans?

A

toxicity (carcinogenicity)

334
Q

what are the pathways of DNA repair?

A

base excision repair, nucleotide excision repair, mismatch mediated repair

335
Q

what does BER stand for?

A

base excision repair

336
Q

what does NER stand for?

A

nucleotide excision repair

337
Q

what does MMR stand for?

A

mismatch mediated repair

338
Q

what sort of DNA do BER, NER and MMR repair damage to?

A

single stranded

339
Q

what sort of damage does BER repair?

A

single-base damage

340
Q

what sort of damage does NER repair?

A

bulky lesions

341
Q

what sort of damage does MMR repair?

A

base mismatch

342
Q

what pathways repair damage to dsDNA?

A

homologous recombination and non-homologous end joining

343
Q

what does HR stand for in DNA repair?

A

homologous recombination

344
Q

what does NHEJ stand for?

A

non-homologous end joining

345
Q

what does CPD stand for?

A

cyclo-butane pyrimidine dimers

346
Q

what do photolyase enzymes in bacteria do?

A

directly reverse DNA damage by absorbing light and using its energy to split the pyrimidine dimer

347
Q

what is a transversion mutation?

A

purine to pyrimidine base

348
Q

what is a transition mutation?

A

pyrimidine to purine base

349
Q

what does cleavage of a sugar-base bond of a damaged base leave?

A

an abasic site

350
Q

what is extra-helical recognition?

A

specific recognition of different types of base damage by specific DNA glycolyases

351
Q

what are the first and second steps of BER?

A

excision of the damaged base; APE1 nuclease cuts DNA strand

352
Q

what are the 2 pathways of BER called?

A

short patch and long patch

353
Q

what is the base that is methylated in eukaryotic DNA?

354
Q

where does DNA methylation occur predominantly in eukaryotic DNA?

A

at the CpG dinucleotide

355
Q

what catalyses DNA methylation in eukaryotes?

A

DNA-methyltransferase (DNMT)

356
Q

where are CpG nucleotides enriched?

A

at CpG islands (CGIs)

357
Q

what are CGIs?

A

several hundred bp long regions containing a high number of CpGs

358
Q

where are CGIs found?

A

at promoter regions

359
Q

what enzyme catalyses the reverse of DNA methylation in eukaryotes?

A

ten eleven translocase (TET)

360
Q

what is genomic imprinting?

A

when genes are expressed depending on the parent of origin

361
Q

what does genomic imprinting depend on?

A

DNA methylation which shuts down transcription in 1 parental chromosome

362
Q

what is the major type of damage repaired by Nucleotide Excision Repair?

A

DNA lesions caused by UV light

363
Q

what sort of lesions does NER repair?

A

bulky lesions that cause local distortions in the double helix

364
Q

what is the difference in NER from BER?

A

NER has no specific recognition of the lesion type

365
Q

what are the 2 types of NER?

A

global genomic NER and transcription coupled NER

366
Q

what is the difference between the 2 types of NER?

A

the mechanism of lesion recognition

367
Q

what causes Xeroderma pigmentosum?

A

mutations in a number of NER genes

368
Q

what does MMR correct?

A

base mismatches missed by replicative DNA polymerase proof-reading

369
Q

what is MMR usually coupled to?

A

DNA replication, to aid mismatched strand identification

370
Q

what do mutations in MMR genes increase the risk of?

A

colorectal cancer

371
Q

why are double strand breaks the most dangerous type of damage?

A

generates free ends that can lead to GCRs, unrepaired DSBs can cause cell death or genomic instability

372
Q

what are GCRs?

A

gross chromosomal rearrangements

373
Q

does NHEJ have a high or low fidelity?

374
Q

does HR have a high or low fidelity?

375
Q

what phase of the cell cycle is HR repair restricted to?

A

the S phase, when a template (sister chromatid) is available

376
Q

what protein binds DNA ends in NHEJ?

A

Ku protein

377
Q

what does the Ku protein do?

A

recruits DNA-PKcs (DNA-dependent protein kinase)

378
Q

what nuclease ‘cleans up’ the ends in NHEJ?

379
Q

what ligates the edited ends in NHEJ?

A

DNA ligase IV, Xrcc4, XLF

380
Q

what initiate 5’ end resection in HR repair?

A

MRN and CtIP

381
Q

what binds the 3’-overhangs in HR repair?

382
Q

which is more complicated, NHEJ or HR?

383
Q

what is combinatorial joining of gene segments the combination of?

A

combinatorial segment assembly, error-prone NHEJ joining, somatic hypermutation, terminal transferase activity

384
Q

what pathway is required for antibody generation?

A

the NHEJ pathway

385
Q

what sort of mutant is a SCID mouse?

A

a DNA-PKcs mutant

386
Q

what is ataxia telangiectasia mutated?

A

a kinase involved in detecting DSB DNA damage

387
Q

what does ATM stand for? (protein kinase)

A

ataxia telangiectasia mutated

388
Q

what is BRCA2?

A

the breast cancer susceptibility protein

389
Q

what is BRCA2 a chaperone of?

A

RAD51 activity

390
Q

how does BRCA2 work?

A

BRCA2 binds RAD51 and transports it to the site of damage, then displaces RPA and loads RAD51 on the 3’-overhangs

391
Q

what does synthetic lethality do?

A

exploits DNA repair deficiencies of cancer cells

392
Q

what is lncRNA?

A

long non-coding RNA

393
Q

what are tandem repeats?

A

short nucleotide stretches in head-to-tail arrangement

394
Q

what is miRNA?

395
Q

what are interspersed repeats?

A

mobile genetic elements that can move around the genome (transposons or Alu repeats)

396
Q

what percentage of the human genome do segmental duplications represent?

397
Q

what are 2 major regions of heterochromatin in eukaryotic cells?

A

the telomeres and centromeres

398
Q

where are the telomeres?

A

the ends of linear chromosomes

399
Q

what are the centromeres?

A

region of attachment of the 2 sister chromatids, forms structure that is bound by microtubules in mitosis

400
Q

what determines the length of the 2 arms of the chromosome?

A

centromere position

401
Q

what is the shorter arm of the chromosome called?

402
Q

what is the longer arm of the chromosome called?

403
Q

what are the large tandem arrays of repeats in centromeres known as?

A

satellite DNA

404
Q

how long is satellite DNA?

A

171 bp long

MIMS (15 decks)

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