The Genome in Health and Disease Flashcards
1
Q
what are carcinomas?
A
malignant tumours of epithelial cells
2
Q
what are sarcomas?
A
tumours derived from the mesenchymal layer
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
4
Q
what are the extra updated hallmarks of cancer?
A
avoiding immune destruction, tumour-promoting inflammation, genome instability and mutation, deregulating cellular energetics
5
Q
how many bases and genes does the human genome comprise?
A
3 x 10^9 bases, around 20000 genes
6
Q
how close must a cell be to a blood vessel to avoid hypoxia?
A
about 10 cells distance
7
Q
what is hypoxia?
A
inadequate oxygen diffusion to a cell
8
Q
what is cachexia?
A
body wasting caused by cancer
9
Q
who first isolated DNA?
A
Miescher
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
11
Q
what is the 5 carbon sugar used in DNA?
A
deoxyribose
12
Q
which position has the extra-cyclic C atom in DNA?
A
5
13
Q
which carbon position does not have a hydroxyl group attached in DNA?
A
2
14
Q
which bases are purines?
A
adenosine and guanine
15
Q
which bases are pyrimidines?
A
thymine and cytosine
16
Q
what is a nucleoside?
A
a base and a ribose sugar
17
Q
what is a nucleotide?
A
a base, a ribose and phosphate
18
Q
which carbon are bases attached to in DNA?
A
the 1’ carbon
19
Q
which carbon is the phosphate group attached to in DNA?
A
the 5’ carbon
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
21
Q
what is the charge of a DNA or RNA strand at neutral pH?
A
negative
22
Q
which is more prone to degradation in alkaline conditions, DNA or RNA?
A
RNA
23
Q
what do riboswitches control?
A
gene expression
24
Q
what does ribozyme catalyse?
A
peptide bond synthesis
25
are the DNA strands in the double helix left or right-handed?
right handed
26
are the DNA strands in the double helix parallel or anti-parallel?
anti-parallel
27
what is the charge of the phosphates in DNA?
negative
28
how many H bonds form between A and T?
2
29
how many H bonds form between G and C?
3
30
what is the angle between the plane of bases and the vertical axis of the DNA helix?
almost perpendicular
31
how many base pairs are present in one turn in B-DNA?
10.5
32
what is the rise per base pair in B-DNA?
3.4A
33
what is the width of the helix in B-DNA?
around 20A
34
what is the rise per helical turn called?
pitch
35
what is the rise per helical turn in B-DNA?
10.5 x 3.4A
36
what allows DNA to form sequence specific interactions with DNA binding proteins?
exposed functional groups on the edge of the aromatic bases
37
what is the most common structure of DNA in chromosomal DNA?
the B form
38
what form does double stranded RNA adopt?
the A-form
39
which is wider, the A form or the B form?
the A form
40
which has a more compressed, narrower major groove, the A form or the B form?
the A form
41
what type of DNA tends to adopt an A like conformation?
GC-rich
42
which has a wider minor groove, the A form or B form?
the A form
43
what is different about the Z form to the B form?
left-handed, phosphate backbone follows zigzag trajectory
44
what role does the Z form seem to have?
role in modulating gene expression
45
what causes local changes in the shape of B form DNA?
local nucleotide composition
46
what is the diameter of a human cell nucleus and how long is the DNA housed within it?
nucleus 5 microns diameter, DNA 2 metres long
47
what mechanism does DNA use to compact into the nucleus?
supercoiling
48
which direction is the twisting in positive supercoiling?
right-handed
49
what does overwound supercoiling cause?
the DNA helix to distort and 'knot'
50
which direction is the twisting in negative supercoiling?
left-handed
51
what does underwound supercoiling cause?
DNA knotted into negative supercoils
52
what resists supercoiling in DNA?
ends of DNA fixed to proteinaceous scaffold, long DNA tails have high viscosity
53
how is supercoiling achieved in the cell DNA?
by a small degree of underwinding which reduces the number of turns of the double helix
54
what does underwinding of DNA facilitate?
compaction (important for packing DNA into cell) and strand separation (important for DNA metabolism)
55
what are two cellular processes which generate supercoiling?
mRNA transcription and DNA replication
56
what is the name of the enzymes that are involved in supercoiling?
topoisomerases
57
how do topoisomerases work?
by cleaving and rejoining the DNA strands
58
what is the nucleoid?
a 'rosette' model of DNA orgainsation with around 500 loops emanating from a proteinaceous scaffold-like core
59
what is chromatin?
the structure formed by chromosomal DNA bound to proteins such as histones and other DNA packaging factors
60
what is the basic unit of chromatin in eukaryotic cells?
the nucleosome
61
what is DNA wound around in the nucleosome?
a protein 'barrel' made up of 8 histone proteins (a histone octamer)
62
what are the histones present in each nucleosome?
2 copies of H2A, H2B, H3 and H4
63
how many nucleotides of DNA are present in a nucleosome?
147
64
how many left-handed turns does the DNA make around the histone octamer in the nucleosome core particle?
1.7
65
what property helps histones bind to the phosphate groups in DNA?
histones are positively charged, phosphate groups are negatively charged
66
what part of the histones in the NCP particle are susceptible to chemical modification by cellular enzymes?
the amino terminal tails which project beyond the DNA gyres
67
what does H1 bind in the NCP?
nucleosomal DNA at the entry and exit positions
68
what is H1 also known as and why?
linker histone as it binds the linker DNA between nucleosomes
69
what is the most common form of chromatin?
the 10-nm fibre
70
what gives the 10nm chromatin fibre its beads-on-a-string appearance?
a linear array of NCPs separated by linker DNA
71
what is the nucleosome repeat length in chromatin?
around 200 nucleotides
72
what causes the 10nm fibre to transition into the 30nm fibre?
specific buffer conditions of low salt and the presence of divalent metal ions
73
what are the characteristics of the 30nm chromatin fibre?
compact and regular
74
what does the large majority of chromatin in the cell nucleus fold into?
local heterogenous clusters or globules of nucleosomes without long-range regularity
75
what does chromatin compaction regulate?
transcription
76
what is euchromatin?
the more open form of chromatin - transcriptionally active
77
what is the transcriptionally active form of chromatin?
euchromatin
78
what is heterochromatin?
the more condensed form of chromatin, transcriptionally inactive
79
what is the transcriptionally inactive form of chromatin?
heterochromatin
80
what is the DNA loop?
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
81
what are the consequences of the DNA loop on gene reglation?
bring enhancer and promoter regions together
82
what is the role of the DNA loop in DNA recombination?
involved in recombination in maturation of immunoglobulin genes
83
what does SMC stand for?
structural maintenance of chromosome
84
what are the SMC proteins?
cohesin and condensin
85
how are SMC proteins typically arranged?
split ATPase domain separated by a long helical region that folds in on itself at hinge position to reconstitute a globular ATPase domain
86
what forms the characteristic V-shaped appearance of SMC proteins?
2 SMC proteins coming together by hinge dimerisation
87
what links the head domains in cohesin and condensin?
a non-SMC subunit
88
what does the head domains being linked by a non-SMC subunit mean for cohesin and condensin?
they have a topological ring structure that can trap 1 or more DNA molecules in the ring
89
what determines the size of the DNA loop in chromosomal DNA?
adjacent binding sites of CTCF
90
what is CTCF?
a DNA-binding protein that recognises specific sequence motifs and recruits cohesin
91
what does cohesin connect?
physically distant sites on DNA
92
what is the loop-extrusion model?
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
93
when does cohesin compact chromosomes?
at interphase
94
when does condensin compact chromosomes?
in mitosis
95
what are the constituent units of chromosomal DNA at the megabase scale?
DNA loops
96
what is the megabase scale?
10e6
97
what does TAD stand for?
topologically associated domain
98
what is a TAD?
several DNA loops folded together
99
what is the chromosome territory?
the unique volume occupied by each chromosome in the nucleus
100
what can each chromosome territory be divided into transcriptionally?
2 compartments, A and B, that are respectively transcriptionally active (predominantly euchromatin) and inactive (mainly heterochromatin)
101
where does the heterochromatin compartment usually sit in the chromosome territory?
on the outside
102
where does the euchromatin compartment usually sit in the chromosome territory?
in the centre
103
what can the likelihood of translocations be determined by?
spatial proximity
104
what is Burkitt's lymphoma characterised by?
a translocation between the MYC gene and 1 of 3 immunoglobulin gene variants located on different chromosomes
105
what is the most common translocation in Burkitt's lymphoma and why?
MYC:IGH as IGH is spatially the closest immunoglobulin to Myc
106
what can modify chromatin status?
binding of H1 causing compaction, post-translational modifications of histone tails, chromatin remodellers
107
what are chromatin remodellers?
multi-subunit protein complexes with ATPase activity that can alter the position of nucleosomes
108
can histone modifications be inheritable?
yes
109
what is the effect of lysine acetylation?
reduces the overall positive charge of the histones, so reduces ability to interact with DNA, reduces chromatin compaction
110
what do chromatin 'writer' enzymes do?
add chemical signals
111
what do chromatin 'eraser' enzymes do?
remove chemical signals
112
what doe chromatin 'reader' proteins do?
recognise each unique set of chromatin modifications and trigger a transcriptional response
113
what doe nucleosome remodellers do?
use the energy from ATP hydrolysis to shift histones and alter local chromatin structure
114
what do nucleosome remodellers contain?
a DNA translocation motor and 'reader' subunits for targeting the remodeller to a specific chromatin site
115
when does DNA replication take place?
S phase
116
how many base pairs does each cell contain?
3 billion
117
what type of replication is DNA replication?
semi-conservative
118
what would dispersive replication produce?
strands with fragments of parental and daughter DNA in a mosaic
119
what is the origin of replication?
the specific sequence at which replication initiates in prokaryotes
120
how long is the origin of replication in E.coli?
around 250bp
121
what is the origin of replication known as in E.coli?
OriC
122
what binds to the origin of replication to begin the process of replication?
DnaA, an initiator protein
123
what does DnaA bind to?
DnaA boxes in the origin of replication
124
what are DnaA boxes?
a tandem repeat of DNA sequences
125
what causes local melting of the double helix in DNA replication?
DNA unwinding element (DUE)
126
what is DnaB?
a DNA helicase that uses the energy from ATP hydrolysis to unwind dsDNA
127
what are some features shared by all replicative helicases?
hexameric ring proteins, ATP-driven molecular motors
128
what loads the DnaB helicase onto the unwound origin DNA?
the protein loader DnaC
129
what does DnaC cause?
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
130
what stabilises the exposed ssDNA in DNA replication?
interaction with the single-stranded DNA-binding protein
131
what does SSB stand for?
single stranded DNA-binding protein
132
what are the properties of SSB protein?
binds to ssDNA with high affinity and no sequence specificity
133
how does SSB protein work?
coats the DNA strand to prevent reannealing and protect it from possible nuclease degradation
134
in what direction does DNA replication proceed after initiation?
bidirectionally from origin creating replication bubble
135
what are replication forks?
the Y-shaped structures that move away from the initiation point in DNA replication
136
by means of what process and enzyme does DNA synthesis take place?
nucleotide polymerisation by DNA polymerase
137
what is needed for nucleotide polymerisation?
a primer annealed to template with free 3' end and deoxynucleotide triphosphates
138
what direction does nucleotide polymerisation take place in?
5' to 3' direction
139
how many DNA polymerases does E.coli have?
5
140
what is the role of DNA polymerase I in E. coli?
roles in nick-translation during Okazaki fragment processing
141
what is the main replicative enzyme in E. coli?
DNA polymerase III
142
how many DNA polymerase have been found in humans?
at least 15
143
how many DNA polymerases perform the bulk of DNA synthesis in DNA replication in humans?
3
144
which DNA polymerases perform the bulk of DNA synthesis during DNA replication in humans?
alpha, delta, epsilon
145
what are the roles of DNA polymerases not involved in DNA synthesis in DNA replication in humans?
nucleotide polymerisation in DNA repair in presence of damage or gaps in DNA
146
what do DNA polymerases rely on to start DNA synthesis?
specialised polymerase called primase
147
what does primase synthetise?
a short RNA primer on the template DNA that is extended by DNA polymerase
148
what is primase called in bacteria?
DnaG
149
what is primase strictly speaking?
a DNA dependent RNA polymerase
150
how long is the RNA primer primase synthesises?
5-15 nucleotides
151
is primase more important in leading or lagging strand synthesis?
Lagging
152
in which strand does nucleotide polymerisation proceed in the same direction as the advancing fork?
the leading strand
153
how is the lagging strand replicated?
in short segments called Okazaki fragments
154
how long are Okazaki fragments in bacteria?
about 1000 bases
155
how long are Okazaki fragments in eukaryotes?
about 100-200 bases
156
from what end does DNA pol I hydrolyses RNA and DNA?
the 5' end
157
what replaces the RNA primer with DNA in the lagging strand?
DNA pol I
158
what seals the Okazaki fragments together?
DNA ligase
159
how many bases does the leading strand DNA polymerase keep going for?
about 2.5 million
160
what is processivity?
the ability of a polymerase to polymerise nucleotide without stopping
161
what 'sliding clamp' protein does DNA pol II rely on?
the β-clamp
162
what does the β-clamp provide DNA pol II?
a topological link to the DNA template ensuring that the polymerase remains attached to the template
163
what is the difference between the action of the β-clamp in leading and lagging strand synthesis?
continuous attachment in leading, in lagging repeatedly binds and releases
164
how many copies of DNA polymerase does evidence suggest act together to synthesise leading and lagging strand DNA at the replication fork?
2
165
what do the 2 DNA polymerases interact with to form a replisome?
other replication factors such as helicase and primase
166
what is a replisome?
a replication machine (all of the replication factors acting together)
167
what does the replisome do?
couples leading and lagging strand synthesis with replication fork progression
168
what happens in the trombone model?
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
what are the steps of DNA replication?
origin recognition; helicase recruitment; DNA melting; priming; elongation; association of polymerase with sliding clamp
170
what is the origin recognition protein in bacteria?
DnaA
171
what is the origin recognition protein in eukaryotes?
ORC1-6
172
what is the helicase loading protein in bacteria?
DnaC
173
what are the helicase loading proteins in eukaryotes?
Cdc6 and Cdt1
174
what is the replicative helicase in bacteria?
DnaB
175
what is the replicative helicase in eukaryotes?
MCM2-7 helicase
176
what is the ss binding protein in bacteria?
SSB
177
what is the ss binding protein in eukaryotes
RPA
178
what is the primase in bacteria?
DnaG
179
what are the primases in eukaryotes?
PriS, PriL, heterodimer
180
what is the main replicative DNA polymerase in bacteria?
DNA Pol III
181
what is the main replicative DNA polymerase in eukaryotes?
DNA Pol α, δ, ε
182
what is the sliding clamp in bacteria?
β-clamp
183
what is the sliding clamp in eukaryotes?
PCNA
184
what is the clamp loader in bacteria?
γ Complex
185
what is the clamp loader in eukaryotes?
RFC
186
what is the DNA ligase in bacteria dependent on?
NAD
187
what is the DNA ligase in eukaryotes dependent on?
ATP
188
what is the primer remover in bacteria?
DNA Pol I
189
what is the primer remover in eukaryotes?
FEN1
190
which eukaryotes have ARSs?
simple, monocellular eukaryotes such as budding yeast
191
what are Autonomously Replicating Sequences (ARSs)?
origins of replication of defined sequence in simple monocellular eukaryotes
192
what is an additional consequence of having multiple origins of replication in eukaryote cells?
cells develop temporal programme of origin activation (origin firing) with early and late firing origins
193
which regions of the genome are usually replicated last in eukaryotes?
heterochromatic regions
194
what is eukaryotic DNA replication tightly coupled in eukaryotes?
the cell cycle
195
what is aneuploidy?
an unequal number of chromosomes
196
how is DNA replication coupling to the cell cycle achieved in eukaryotes?
by separating the process of origin licensing in G1
197
what does MCM2-7 stand for?
mini chromosome maintenance 2-7
198
what loads inactive MCM2-7 onto the DNA?
the Origin Recognition Complex (ORC)
199
when is MCM2-7 activated?
at the beginning of S-phase
200
where is the replisome assembled in eukaryotes?
on the MCM2-7 helicase
201
what controlling steps does origin firing require?
recruitment of co-activator proteins Cdc45 and GINS to the MCM2-7 helicase and CDK phosphorylation activity
202
what is replication primase a subunit of in eukaryotic replication?
DNA polymerase α
203
what extends the RNA primer synthetised by primase with dNTPs in eukaryotic replication?
DNA Pol α
204
what ribonuclease removes the RNA primer in eukaryotes?
RNaseH
205
what does RNaseH do?
digests RNA that is base paired to DNA in eukaryotes
206
what cuts off the flap structure created by the replicative DNA polymerase displacing the residual primer in eukaryotes?
the Flap Endonuclease Fen1
207
what seals the DNA nick produce by Fen1?
DNA ligase I
208
what coordinates the actions of Fen1 and DNA ligase I?
the eukaryotic sliding clamp PCNA
209
what are topoisomerases?
enzymes that act to release topological tension in DNA molecules and untangle molecules
210
what are the classes of topoisomerase?
type I and type II
211
what do type I topoisomerases do?
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
what do type II enzymes do?
completely cut both strands of one duplex and allow another duplex to pass through the gap which is then resealed
213
what attachment do topoisomerase form with DNA?
a covalent protein-DNA attachment
214
what is the purpose of the covalent attachment of topoisomerases to DNA?
preserve energy of the chemical bond and ensures inadvertent release of broken DNA molecules doesn't occur
215
what is the genome of an organism?
the complete set of genetic material for that organism
216
what doesn't genome usually refer to in eukaryotes?
mitochondrial DNA
217
what is the most commonly used technique for sequencing DNA?
chain termination method
218
what does the chain termination method of DNA sequencing rely on?
DNA polymerase can incorporate 2',3'-dideoxynucleotides (ddNTPs) into growing DNA chains
219
who created the chain termination method?
Sanger
220
why do ddNTPs terminate the DNA chain?
don't have a 3' OH group
221
how many reactions were set up in the original chain termination technique?
4
222
what did the 4 reactions in the original chain termination technique contain?
dATP, dCTP, dGTP, dTTP, radiolabel, small amounts of either ddATP, ddCTP, ddGTP or ddTTP
223
what is used to visualise the DNA bands in the original chain termination technique?
gel electrophoresis and X-ray film to visualise the DNA bands
224
what has been used to enhance the chain termination technique?
using ddNTPs conjugated with specifically-coloured fluorescent markers, using fine capillaries to separate the DNA fragments rather than large gels
225
what sequencing method did the Human Genome project use?
chain termination method
226
what is the more modern technique of DNA sequencing?
next generation sequencing (NGS)
227
what does NGS rely on?
massively parallel sequencing whereby millions of short sequence reads are obtained simultaneously and then assembled by computer into chromosomes and genomes
228
which will have a higher melting temperature, A:T rich or G:C rich sequences?
G:C rich
229
why do G:C rich sequences have a higher melting temperature than A:T rich ones?
because of the different number of H bonds holding together the base pairs
230
what is the C-value paradox?
there is a lack of correlation between genome size and organismal complexity
231
how much of the human genome is non-coding DNA without a clearly defined function?
98.5%
232
233
what is the telomere sequence in humans?
TTAGGG
234
what adds the telomere sequences to DNA?
telomerase
235
what are the components of telomerase?
protein and RNA component
236
what does the RNA component of telomerase function as?
template for DNA synthesis
237
what are reverse transcriptases?
a class of enzyme that use RNA to make DNA
238
how does the action of telomerase counteract the lagging-strand problem?
by adding long, repetitive stretches of single-stranded DNA to the end of the chromosome
239
what is the T loop of DNA?
a loop that closes off the end of the chromosome
240
what is quadruplex DNA?
when 4 GGG triplets come together to form 3 stacked planar G quartets held together by Hoogsteen H bonds
241
what happens to the length of telomeres when adult cells divide?
they get shorter as they don't have enough telomerase
242
what is the Hayflick limit?
when telomeres get to a short enough length that cells read it as a signal to stop dividing
243
what can extend the life span of cells at the Hayflick limit?
re-introduction of telomerase into cells
244
what are mobile genetic elements?
regions of DNA that can move around and insert in other parts of the genome, and thus cause mutations
245
what are the 2 types of mobile genetic elements known?
transposons and retrotransposons
246
what enzyme moves DNA transposons around the genome?
transposase
247
what occurs in movement of retrotransposons?
DNA is transcribed to RNA, RNA then reverse transcribed to DNA which is inserted back into different part of genome
248
how can transposons propagate antibiotic resistance?
in bacteria many transposons carry antibiotic resistance genes, so can propagate antibiotic resistance within and between strains and species
249
how much of the human genome is composed of mobile genetic elements or their remnants?
45%
250
how may transposons be beneficial on an evolutionary timescale?
facilitate shuffling of coding sequences or moving genes so they're under control of a new promoter
251
what are the recombinases that mediate antibody diversity regulation related to?
transposon-encoded enzymes
252
what is DNA melting?
denaturation of the double helix leading to separation of the 2 strands of DNA
253
what factors affect DNA melting and re-annealing?
temperature, length of DNA, base composition, ionic composition of solvent
254
what is the process of re-annealing of DNA known as?
DNA hybridisation
255
what techniques is DNA hybridisation the backbone of? (5)
PCR, genotyping by in situ hybridisation, Southern blotting, FISH, DNA microarrays
256
what does FISH stand for?
fluorescence in situ hybridisation of DNA
257
what does PCR stand for?
polymerase chain reaction
258
what gives DNA and RNA strong UV light absorption?
aromatic bases
259
what is the peak absorbance of DNA?
260nm
260
why is UV absorbance of bases lower in the double helix?
base stacking (hypochromicity effect)
261
what is the hypochromicity effect?
UV absorbance of bases is lower in the double helix due to base stacking
262
how does the hypochromicity effect allow for following the melting/re-annealing process?
base stacking in the double helix reduces the absorption compared to the denatured state
263
what is the restriction-modification system?
anti-viral defence mechanism in bacteria that uses enzymes to digest viral DNA
264
what type of enzymes does the restriction-modification system use?
DNA endonucleases
265
how do DNA endonucleases distinguish between bacterial and viral DNA?
they recognise specific DNA sequences which may be absent from the bacterial genome + are specific for either methylated or non-methylated DNA
266
which bases can methylation occur at?
A or C
267
what performs DNA methylation in bacteria?
an SNA methyltransferase enzyme
268
where do bacteria methyltransferases methylate adenine?
within restriction sequences
269
what is the most prevalent position of adenine methylation in viral DNA by bacterial methyltransferases?
at position N6 of adenine
270
what are the most widely used type of restriction enzymes?
Type II
271
do type II restriction enzymes typically form homo or heterodimers?
homodimers
272
how long are the sequences recognised by type II restriction enzymes?
4-8nt
273
what types of ends does EcoRI cleavage produce?
sticky ends
274
what sort of ends does EcoRV cleavage produce?
blunt ends
275
what are RFLPs?
restriction fragment length polymorphisms
276
what is RFLP?
the study of genetic differences between individuals
277
what is recombinant DNA?
DNA molecules that are the result of laboratory manipulation
278
what properties allow DNA to be separated by gel electrophoresis?
negatively charged, chemically relatively stable in solution
279
what sorts of gel can be used in gel electrophoresis?
agarose, polyacrylamide
280
which electrode does DNA run towards in gel electrophoresis?
the positive electrode
281
how can DNA be visualised after separation by gel electrophoresis?
dyes that bind dsDNA and fluoresce under UV light
282
what is the most common dye used to visualise DNA separated by gel electrophoresis?
ethidium bromide
283
what is the Southern blot technique?
transferring separated DNA from gel to a filter then analysing by hybridisation to check for specific sequences
284
what is the blot method used for RNA?
Northern blot
285
what is the blot method used for DNA?
Southern blot
286
what is the blot method used for proteins?
Western blot
287
what is the most common way to make lots of copies of a piece of DNA?
cutting and pasting into a bacterial plasmid
288
how many origins of replication does a plasmid have?
1
289
what are plasmids?
circular molecules of extrachromosomal DNA in bacteria
290
what are bacteriophage?
viruses that infect bacteria
291
what are more specialised systems for manipulating larger DNA fragments than plasmids or bacteriophage?
yeast and bacterial artificial chromosomes
292
what is the downside to using plasmids or bacteriophage to amplify DNA?
limit to length of the piece of DNA, several thousand base pairs
293
what is the polymerase chain reaction?
a method for making large amounts of a defined region of DNA in a test tube without having to use bacteria
294
what information about the DNA is needed for the PCR?
DNA sequence you want to amplify or the DNA flanking the region of interest
295
what do the primers provide in PCR?
a free 3' end of DNA that DNA polymerase can extend from
296
what is the first step of PCR?
over 90 degrees C to separate the dsDNA (melting)
297
what is the second step of PCR?
55-60 degrees C to allow primers to hybridise to their target sequence (annealing)
298
what is the third step of PCR?
72 degrees C to allow the DNA polymerase to make the DNA copy (extension)
299
what components are required for PCR amplification of a chosen template DNA?
short primers (oligonucleotides); stock of nucleotides (dNTPs); a buffer solution; a DNA polymerase
300
which is present in a large molar excess in PCR, primers or template?
primers
301
what specific DNA polymerase is able to withstand heating and cooling in PCR?
Taq polymerase
302
what is Friedrich's ataxia?
a genetic disorder causing progressive degeneration of the nervous system with loss of controlled movement
303
what is Friedrich's ataxia caused by?
a defect in the FXN gene which codes for Frataxin protein
304
what is the defect in the FXN gene that causes Friedrich's ataxia?
abnormally high (>100) number of copies of the GAA repeat in the FXN gene
305
what does CRISPR stand for?
clustered regularly-interspersed short palindromic repeats
306
what are CRISPRs?
short viral DNA sequences present in the microbial genome
307
what do CRISPRs do?
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
what does Cas protein stand for?
CRISPR-associated
309
what is Cas protein?
a nuclease that cleaves the DNA sequence targeted by the guide RNA
310
what is the CRISPR-Cas system?
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
what is the first approved genetic treatment based on CRISPR for?
patients suffering from sickle-cell anaemia and beta-thalassemia
312
what does the CRISPR therapy for sickle-cell anaemia and beta-thalassemia do?
reactivates fetal haemoglobin in patients with defective adult haemoglobin
313
what is the error rate for DNA replication?
1 error in 10^9 nucleotides
314
what do mutations in the 'proof-reading' exonuclease domain of Pol epsilon cause?
a 'hyper mutation' phenotype that drives cancer formation
315
what are the types of replication error?
base mismatches, nucleotide misincorporation by error-prone TLS polymerases, deletions, insertions
316
what are TLS polymerases?
translesion synthesis polymerases
317
what is translesion DNA synthesis?
a mechanism to traverse damaged sites (roadblocks) on the DNA template
318
what is the advantage of translesion DNA synthesis?
DNA replication doesn't stop
319
what is the disadvantage of translesion DNA synthesis?
increased mutagenesis due to lack of proof-reading and poor nucleotide selectivity
320
what is the mechanism of translesion DNA synthesis?
they have larger active sites that can accommodate bulkier modified bases
321
what sort of condition is Huntington's disease?
autosomal dominant genetic condition
322
what causes Huntington's disease?
mutations in huntingtin gene on chromosome 4
323
what is the mutation that causes Huntington's?
an expansion of a repeated stretch of CAGs in the protein coding part of the gene
324
how many repeats of CAGs are needed to cause symptomatic Huntington's?
>40 repeats
325
what does the mechanism of triplet expansion depend on?
the propensity of (CNG)n to depart from B-form DNA
326
what are endogenous causes of DNA damage? (not examples)
causes within organisms
327
what are some examples of endogenous causes of DNA damage?
replication errors, by-products of metabolism, chemical instability
328
what are exogenous causes of DNA damage? (not examples)
environmental causes of DNA damage
329
what are examples of exogenous causes of DNA damage?
UV light, ionising radiation, genotoxic chemicals
330
how many damage events occur per mammalian cell per day?
around 30000
331
what does the Ames test assess?
the mutagenic potential of a chemical compound
332
what is the Ames test used for?
screening new chemicals
333
what do we assume mutagenicity in bacteria leads to in humans?
toxicity (carcinogenicity)
334
what are the pathways of DNA repair?
base excision repair, nucleotide excision repair, mismatch mediated repair
335
what does BER stand for?
base excision repair
336
what does NER stand for?
nucleotide excision repair
337
what does MMR stand for?
mismatch mediated repair
338
what sort of DNA do BER, NER and MMR repair damage to?
single stranded
339
what sort of damage does BER repair?
single-base damage
340
what sort of damage does NER repair?
bulky lesions
341
what sort of damage does MMR repair?
base mismatch
342
what pathways repair damage to dsDNA?
homologous recombination and non-homologous end joining
343
what does HR stand for in DNA repair?
homologous recombination
344
what does NHEJ stand for?
non-homologous end joining
345
what does CPD stand for?
cyclo-butane pyrimidine dimers
346
what do photolyase enzymes in bacteria do?
directly reverse DNA damage by absorbing light and using its energy to split the pyrimidine dimer
347
what is a transversion mutation?
purine to pyrimidine base
348
what is a transition mutation?
pyrimidine to purine base
349
what does cleavage of a sugar-base bond of a damaged base leave?
an abasic site
350
what is extra-helical recognition?
specific recognition of different types of base damage by specific DNA glycolyases
351
what are the first and second steps of BER?
excision of the damaged base; APE1 nuclease cuts DNA strand
352
what are the 2 pathways of BER called?
short patch and long patch
353
what is the base that is methylated in eukaryotic DNA?
cytosine
354
where does DNA methylation occur predominantly in eukaryotic DNA?
at the CpG dinucleotide
355
what catalyses DNA methylation in eukaryotes?
DNA-methyltransferase (DNMT)
356
where are CpG nucleotides enriched?
at CpG islands (CGIs)
357
what are CGIs?
several hundred bp long regions containing a high number of CpGs
358
where are CGIs found?
at promoter regions
359
what enzyme catalyses the reverse of DNA methylation in eukaryotes?
ten eleven translocase (TET)
360
what is genomic imprinting?
when genes are expressed depending on the parent of origin
361
what does genomic imprinting depend on?
DNA methylation which shuts down transcription in 1 parental chromosome
362
what is the major type of damage repaired by Nucleotide Excision Repair?
DNA lesions caused by UV light
363
what sort of lesions does NER repair?
bulky lesions that cause local distortions in the double helix
364
what is the difference in NER from BER?
NER has no specific recognition of the lesion type
365
what are the 2 types of NER?
global genomic NER and transcription coupled NER
366
what is the difference between the 2 types of NER?
the mechanism of lesion recognition
367
what causes Xeroderma pigmentosum?
mutations in a number of NER genes
368
what does MMR correct?
base mismatches missed by replicative DNA polymerase proof-reading
369
what is MMR usually coupled to?
DNA replication, to aid mismatched strand identification
370
what do mutations in MMR genes increase the risk of?
colorectal cancer
371
why are double strand breaks the most dangerous type of damage?
generates free ends that can lead to GCRs, unrepaired DSBs can cause cell death or genomic instability
372
what are GCRs?
gross chromosomal rearrangements
373
does NHEJ have a high or low fidelity?
low
374
does HR have a high or low fidelity?
high
375
what phase of the cell cycle is HR repair restricted to?
the S phase, when a template (sister chromatid) is available
376
what protein binds DNA ends in NHEJ?
Ku protein
377
what does the Ku protein do?
recruits DNA-PKcs (DNA-dependent protein kinase)
378
what nuclease 'cleans up' the ends in NHEJ?
Artemis
379
what ligates the edited ends in NHEJ?
DNA ligase IV, Xrcc4, XLF
380
what initiate 5' end resection in HR repair?
MRN and CtIP
381
what binds the 3'-overhangs in HR repair?
RPA
382
which is more complicated, NHEJ or HR?
HR
383
what is combinatorial joining of gene segments the combination of?
combinatorial segment assembly, error-prone NHEJ joining, somatic hypermutation, terminal transferase activity
384
what pathway is required for antibody generation?
the NHEJ pathway
385
what sort of mutant is a SCID mouse?
a DNA-PKcs mutant
386
what is ataxia telangiectasia mutated?
a kinase involved in detecting DSB DNA damage
387
what does ATM stand for? (protein kinase)
ataxia telangiectasia mutated
388
what is BRCA2?
the breast cancer susceptibility protein
389
what is BRCA2 a chaperone of?
RAD51 activity
390
how does BRCA2 work?
BRCA2 binds RAD51 and transports it to the site of damage, then displaces RPA and loads RAD51 on the 3'-overhangs
391
what does synthetic lethality do?
exploits DNA repair deficiencies of cancer cells
392
what is lncRNA?
long non-coding RNA
393
what are tandem repeats?
short nucleotide stretches in head-to-tail arrangement
394
what is miRNA?
microRNA
395
what are interspersed repeats?
mobile genetic elements that can move around the genome (transposons or Alu repeats)
396
what percentage of the human genome do segmental duplications represent?
around 5%
397
what are 2 major regions of heterochromatin in eukaryotic cells?
the telomeres and centromeres
398
where are the telomeres?
the ends of linear chromosomes
399
what are the centromeres?
region of attachment of the 2 sister chromatids, forms structure that is bound by microtubules in mitosis
400
what determines the length of the 2 arms of the chromosome?
centromere position
401
what is the shorter arm of the chromosome called?
p
402
what is the longer arm of the chromosome called?
q
403
what are the large tandem arrays of repeats in centromeres known as?
satellite DNA
404
how long is satellite DNA?
171 bp long
