Molecular biology semester 2 Flashcards
(646 cards)
1
Q
What is the definition of an organelle?
A
Any discrete intracellular space specialised for a specific function. They can be membrane bound or not membrane bound.
2
Q
After synthesis where are organelles transported to?
A
The ER.
3
Q
Peroxisomes, endosomes and lysosomes contain a membrane, true or false?
A
True. They are membrane bound.
4
Q
Are microtubules, chromosomes and microfillament classed as organelles?
A
Yes. They are all non membrane bound organelles.
5
Q
Where are most proteins synthesised?
A
The cytoplasm.
6
Q
What is targeting?
A
When newly synthesised proteins are delivered to a particular membrane of an organelle from the cytosol.
7
Q
What is translocation?
A
When an organelle is transported across the membrane.
8
Q
What is sorting?
A
When an organelle is transported from one membrane bound compartment to another.
9
Q
Although sorting normally involves transportation between compartments, where can it happen in the same compartment?
A
The ER.
10
Q
Who came up with the signal sequence hypothesis?
A
Gunther Blobel.
11
Q
<p>What is a signal sequence?</p>
A
<p>A relatively short amino acid sequence that directs a protein to a specific location within a cell.</p>
12
Q
Does translocation require an energy source?
A
Yes.
13
Q
Protein folding usually occurs in the cytosol, apart from when?
A
When channels are too narrow for the folds protein to fit through.
14
Q
What organelle was used to establish the signal sequence hypothesis?
A
The ER.
15
Q
In the ER what is translocation coupled to?
A
Translation.
16
Q
When cells where radioactively labelled and homogenised the radioactive labels were contained showing coupling of translation and translocation at the ER. What happened when a detergent was also added?
A
The radioactivity had not been contained meaning it was not imported.
17
Q
What did Ceaser Milstein discover in regards to the length of proteins translocated?
A
He showed that proteins made in vitro were longer than the ones recovered from the ER. This showed that the signal sequence was cleaved.
18
Q
Ceaser Milstein showed that proteins made in vitro could still be imported into the ER. True or false?
A
False. He showed that fully synthesised proteins could not enter the ER.
19
Q
<p>What is a major piece of evidence for co translational import regarding ribosomes?</p>
A
<p>They (and newly synthesised proteins) can be found associated at the ER around the point of translation.</p>
20
Q
Can protein important to the ER happen in vitro when microsomes are not present?
A
No.
21
Q
What end of the signal peptide is synthesised first, hence also enters the ER first?
A
The N terminus.
22
Q
The signal peptide has a core of __________ which are often proceeded by _________ .
A
6-12 hydrophobic amino acids
Several positive amino acids.
23
Q
What is the SRP?
A
The recognises the signal sequence by acting as a receptor. It is a recognition particle and is not found on the membrane.
24
Q
What subunit in SRP recognises the signal sequence?
A
P54.
25
When the SRP binds to the signal does translation stop?
Yes.
26
Once the SRP is bound to the signal where can the ribosome complex dock and how?
The ribosome complex can dock to the ER membrane due to complementation between the attached SRP recognition particle and the SRP receptor on the ER membrane.
27
What stabilises SRP binding to it's receptor on the ER membrane?
GTP.
28
What is the membrane bound SRP associator associated with?
The channel in the ER.
29
Does the GTP hydrolyse allowing release of SRP before or after translation has been completed?
Before. Once the pore has open and the nascent transcript is through the pore it does not need to be present and can be used again.
30
What is the driving force of translocation?
Translation.
31
Is the signal sequence cleaved before or after the pore has closed?
Before.
32
What releases the SRP from it's receptor?
Hydrolysis of GTP. This happens as soon as the nascent polypeptide is in the channel as it is no longer needed.
33
What percentage of proteins need to be localised?
50%
34
What end of a transcript is inserted into a type one channel?
NH3+ end.
35
What causes translation to stop in a type one channel?
The stop anchor sequence as it prevents further translation.
36
What stops the stop anchor sequence in type one channels?
Hydrophobic residues.
37
Once the nascent polypeptide has been stopped by the stop anchor sequence in type one channels what way does the pore open to allow translation to continue?
Sideways.
38
Once translation has resumed in type one channels is translocation also resumed?
No.
39
What is the only channel type to have an internal stop anchor sequence?
Type 1.
40
What way is the N Terminus orientated in type 2 channels?
Towards the cytosol.
41
How does the signal anchor sequence insert into a type 2 membrane?
Spontaneously. It is recognised by the SRP receptor and delivered to the translocon.
42
Why is it believed that the N terminus is orientated to the cytosol in type 2 channels?
As there is a positively charged N terminal on the signal anchor sequence.
43
Is there a signal sequence in type 3 channels?
No as can never be imported co translationally.
44
Where is the positive end pointing in a type 3 channel?
Towards the cystol.
45
For a protein to be imported post translationally what state does it need to be in?
Unfolded. This is done by chaperons.
46
Why is ATP needed to insert a protein post-translationally?
As translocation is no longer acting as the driving force.
47
What is HSP70 and what is it's role in the translocation of protons?
It is a heat shock protein. It binds to the unfolded polypeptide in the lumen to stop it diffusing back out.
48
What does HSP70 recognise in order to bind to the unfolded polypeptide?
Hydrophobic residues which would not normally be exposed.
49
What leads to the conformational change of HSP70 allowing it to bind, close and refold the protein?
ATP hydrolysis.
50
What causes HSP70 to open?
Bound ADP present after ATP hydrolysis.
51
How long is the mitochondrial signal sequence?
20-25 amino acids long.
52
What does the mitochondrial signal sequence consist of?
An amphiphilic helix.
53
Can post translational import occur in the ER, mitochondria or both?
Both.
54
Does post translational import in the mitochondria require ATP?
Yes.
55
What has to be close together for post translational import to occur in the mitochondria?
The membranes of the mitochondria.
56
What blocks the import of chimeric DHFR?
MTX drug.
57
Chimeric DHFR can not enter the lumen when the drug MTX is present. What can bind to DHFR so this can be visualised?
An antibody attached to gold particles.
58
How many signal pathways target proteins to sub mitochondrial compartments?
Multiple.
59
What does the peroxisomal targeting signal contain at the carboxyl terminus?
Leucine, serine and lysine.
60
What can some proteins form in cytoplasm?
Oligomers. This allows the assembly of prosethic groups.
61
What is Hyperoxaluria type 1?
A heredity kidney stone disease.
62
Accumulation of what causes Hyperoxaluria?
Calcium oxulate.
63
What do parents lack in Hyperoxaluria type 1?
AGT- alanine/glyoxylate amino transferase.
64
What does a mutation of proline- Leucine cause?
The generation of a amphiphilic helix at the N terminus causing a MTC.
65
What point mutation is needed for someone to have Hyperoxaluria type 1?
GLY170ARG
66
If someone only has a proline-leucine mutation will the signal be targeted to the peroxisome or the mitochondria?
The peroxisome. To be targeted at the mitochondria you also need the GLY170ARG mutation .
67
What does GLY170ARG delay?
Dimerisation of the protein meaning it can enter the mitochondria.
68
What does a class A secretion mutation result in?
Accumulation in the cystol.
69
What does a class B secretion mutation result in?
Accumulation in the RER.
70
What does a class C secretion mutation result in?
The accumulation of ER Golgi transport vesicles.
71
What does a class D secretion mutation result in?
Accumulation in the Golgi.
72
What does a class E secretion mutation result in?
Accumulation of secretory vesicles.
73
What defective function causes a class A secretory mutation?
Transport into the ER is defective.
74
What defective function causes a class B secretory mutation?
Vesicle budding from the RER is defective.
75
What defective function causes a class C secretory mutation?
Transport of Golgi vesicles is defected.
76
What defective function causes a class D secretory mutation?
A defect in Golgi secretory vesicle transport.
77
What defective function causes a class E secretory mutation?
A defect in transporting secretory vesicles to the cell surface.
78
What allows you to see which part of the secretory pathway is blocked?
Double mutants.
79
Video microscopy showed that the temperature sensitive mutant of the VSV-G protein could stay in the ER at what temperature and why?
40 degrees, as it would have be unfolded.
80
When coat proteins bind to the receptors what do they do?
They oligomerise with themselves.
81
What do coat proteins do to allow cargo molecule into the vesicle?
They recruit receptors.
82
What do transporting terminates on vesicles allow?
Targeting.
83
Are sorting signals long or short peptide signals?
Short.
84
Can signal sequences be modified with tags?
Yes.
85
What does oligomerisation of coat proteins cause the membrane to do?
Bend.
86
Why can't proteins leave vesicles once they have bound?
There are receptors in the vesicle.
87
What is coat assembly under control of?
GTPase.
88
What does sar1-GDP bind to?
SEC12.
89
When GDP exchanges for GTP what anchors to the ER membrane?
SER1.
90
What does SER1 bound to GTP drive?
Polymerisation of soluble coat factors.
91
What do coat proteins recognise?
Sorting signals in cargo receptors.
92
What does polymerisation of soluble coat factors, caused by SER1 eventually cause?
Budding.
93
What initiates uncoating?
GTP hydrolysis.
94
Does uncoating happen before or after budding?
After. It happens before docking and fusion of the vesicle to the membrane.
95
What are snare proteins required for?
Specificity and mechanism for fusion.
96
What two types of snare proteins are there?
Vesicle (V) and target membrane (T).
97
Is the complex formed between T and V snares tight or loose?
Very tight.
98
Where Rab-GTP found?
On the vesicle.
99
What type of specificity does the rab have to it's effector?
Dual specificity.
100
What type of complex does the snare protein form?
A four helical bundle.
101
What is disassembly of the snare complex dependant on?
ATP.
102
When GTP hydrolyses what does rab do?
It dissociates.
103
What assembles the snare complex?
NSF and alpha SNAP.
104
Where is the rab effector found?
The target membrane.
105
Apart from unfolding snares what else does NFS have to do to them?
Unfold them as the complex is very tight.
106
What is the forward pathway also called?
The anterograde pathway.
107
How many subunits of clathrin assemble a coat protein?
3.
108
Are different coats and signals are needed to transport snares back?
Yes.
109
What happens to the proteins when they enter the Golgi?
Glycosalation.
110
In retrograde transport what is found at the COOH terminus?
KDEL to bind the specific receptor to the Golgi stack to help incorporation into a retrograde vesicle.
111
What two models show transport through the Golgi?
Cisternal maturation model
| Stationary cisternae model
112
What is the cis/early Golgi form from in the Cisternal model?
Multiple ER vesicles.
113
When the middle Golgi looses it's enzymes what are these replaced by in the Cisternal model?
Enzymes from the late Golgi.
114
Are proteins transported in the in the Cisternal model?
No, the organelles change.
115
What happens at the end Cisternal model?
The network breaks up and is transported back to the plasma membrane.
116
Does the stationary cisternal model work for the retrograde transport or the anterograde pathway?
Retrograde transport.
117
What model for Golgi transport is favoured?
The cisternal maturation model.
118
How does transport happen between stacks in the stationary cisternal model?
By vesicles.
119
What happens to the enzymes in the stationary cisternal model?
They are retained in that compartment or retrieved from a later compartment.
120
What is the default pathway when there is no specific signal?
Constitutive secretory pathway.
121
How many secretory pathways are involved in trafficking from the trans golgi network?
2 secretory, 1 to the lysosomes and 1 to the endosomes.
122
What can late endosomes change to?
Lysosomes.
123
For late endosomes to change to lysosomes what specific modification is needed. Is this recognised be an enzyme by the transfer of what?
Phosphorylated glcNac moving to the carbon 6 atom to make mannose 6 phosphate.
124
In transport to the lysosomes what is added to the cis golgi initially?
Man6P.
125
After being added to the cis golgi, where is mannose 6 phosphate then added too?
The trans golgi.
126
Where is the mannose 6 phosphate incorporated into the clathrin coated protein?
The trans golgi.
127
After budding the coat dissembles. What causes this?
A very low pH.
128
Does the coated or uncoated vesicle fuse with the late endosome?
Uncoated.
129
When the late endosome fuses with the lysosomes what two things need to happen?
The receptor needs to be released and dephosphorylation needs to occur.
130
Receptors are released from the late endosome and they are recycled. However this is not a very efficient process meaning some of the receptors end up where?
The plasma membrane.
131
What occasionally happens to the phosphorylated lysosomal proteins?
They occasionally secreted but are picked up again by endocytosis which then take them back to the endosomes.
132
What sort of deformations are caused by I cell disease?
Skeletal, psychomotor and mental.
133
What do lysosomes contain large exclusions of in someone who suffers from I cell disease?
Glycolipids and glycoaminoglycans.
134
How many acid hydrolyses does someone with I cell disease lack?
8.
135
What can not form probably in I cell disease?
Mannose 6 phosphate.
136
What is macropinocytosis an example of?
Endocytosis.
137
Apart from being clathrin mediated, what other molecule can mediate endocytosis?
Caveolin.
138
What can clathrin coat adaptors bind?
AD1 and AD2.
139
Does clathrin bind directly to the cargo?
No.
140
Why does dynamin form an extensive spiral structure?
It allows it to pinch of the vesicle.
141
A mutation in what causes Familial Hypercholestrolaemia?
A mutation in LDL receptor.
142
Apart from atheromas plaque, what else can Familial Hypercholestrolaemia cause?
Xanthomas- fatty acid deposition in skin and tendons.
143
What 5 processes are blocked in someone who has Familial Hypercholestrolaemia?
1. Null alleles.
2. Signal for endocytosis meaning internalisation can not happen.
3. Recycling of alleles.
4. Binding of defective alleles.
5. Transport if deficient alleles.
144
What does an amplification protein cause?
A normal protein to be expressed at the wrong time.
145
What causes a chimeric protein with an altered functionto form?
When a gene fuses with another gene, possibly as a result of a chromosome breaking then rearranging.
146
What do epigenetic modifications cause?
Gene silencing.
147
What cancer is common in Japan?
Stomach cancer. Although breast cancer is rare.
148
Deamination of cytosine into what is an example of spontaneous DNA damage?
Uridine.
149
What can breakage of bond between the purines and deoxyribose result in?
Random base insertions.
150
Deamination methyl cytosine into what is an example of spontaneous DNA damage?
Thymidine.
151
What type of compounds can cause bladder cancer when they are inhaled?
Rubber.
152
When iodine 131 enters the body where does it concentrate itself?
The thyroid gland.
153
When DNA is damaged on one side and is repaired it can result in what forming?
A thymidine a diner, held together my covalent cross links. This can stop normal base pairing and block DNAP resulting in DNA not being correctly repaired.
154
Why are double stranded DNA breaks not easily repairable?
There is no template. This can cause recombination and inactivation of essential genes.
155
What is the role of p53?
It surveys the DNA for damage and allows repairs to any damage found.
156
How can thymidine dimers be repaired?
The whole stretch of DNA is removed, resynthesis occurs and the opposite strand is used as a template.
157
What type of DNA damage can be directly removed without breaking the phosphate backbone?
O-6 methyl guanine.
158
What can p53 delay?
DNA replication.
159
If p53 detects damage that is to severe to fix what happens?
Apoptosis.
160
What type of signals generate growth factors?
Mitogenic growth signals.
161
There are four major families of growth factors. Two are found in all cell cultures, what are they?
EGF (Epidermal growth factor) and FGF ( Fibroblast growth factor).
162
What does the extracelluar matrix support?
Epithelial cells so they connect with their neighbours and the basement.
163
What three things can occur to make the cell autonomous to growth signals?
1. More GF can be produced.
2. Receptors to GF can have more activity.
3. Through the modulation of posh ways less GF can be needed to bring about a response.
164
Cancers can not produce their own growth signal. True or false?
False. Many cancers can.
165
Cells that produce growth factors are not stimulated by it. What type of mechanism does this cause?
Feedback.
166
The Glioblast PDGF factors are over expressed and in a different isoform to that of a normal cell. What type of cancer does this cause?
Brain cancer.
167
When receptors become activated by growth factors what do they become?
Tyrosine kinases.
168
Receptors are activated as tyrosine kinases. What process does this allow to happen on tyrosine residues?
Autophosphorylation.
169
Receptors can also be serine kinases as well as tyrosine kinases. Tyrosine kinases can autophosphroylate, can this happen in serine kinases?
No. However they can phosphorylate other proteins.
Tyrosine residues are also able to do this.
170
What can phosphorylated proteins activate to modify gene expression?
Transcription factors.
171
What are EGF-R and Erb-B examples of?
Receptors.
172
What receptors are unregulated in stomach, brain and breast tumours?
EGF-R (receptor for EGF) and Erb-B (receptor for heregulin).
173
Overexpression of what receptor can result in ligand independent signalling?
HER2/neu
174
Over expression of HER2/neu causes what type of cancer?
Stomach and brain.
175
What is an intigrin?
An extracelluar matrix receptor. These anchor cells to the extracelluar matrix.
176
What happens if intergrins fail to bin to the extracelluar matrix?
Motility is impaired and apoptosis may occur.
177
What point mutation makes receptors always active?
Val-Gln point mutation.
178
Receptors can become always active by being structurally altered. This is causes by extracelluar matrix proteins being stimulates causing intergrins to relax and allowing extra space to divide. What domain to the receptors then lack?
Cytoplasmic.
179
When receptors are structurally altered what do they become?
Ligand dependant.
180
When GF receptors are activated and pro growth intigrins are present, what pathway is often activated?
SOS-Ras-Raf-MAP kinase pathway.
181
What percentage of tumours have a mutated RAS protein?
25%
182
When Ras is mutated can mitogenic signals be produced without upstream activation of the pathway?
Yes.
183
What normally binds to Ras to produce a conformational change?
GTP.
184
Mutated Ras looses GTP activity. This means GTP is permanently associated with Ras. What does this result in?
Ras becomes permanently active.
185
Ras can also interact with p53. This cause growth signals to automatically stimulate what?
Survival signals- protecting the cell from apoptosis.
186
What is NF1?
Another factor that is able to mediate a live or die response, it is similar to Ras.
187
Cells need to respond to antipoliferative signals. What do these signals do?
Stop growth.
188
What do antipoliferative signals depend on?
Soluble or immobilised inhibitors on nearby cells. When these inhibitors activate the signals circuits are formed.
189
What methods can block proliferation?
Cells can differentiate or become quiescent.
190
Can terminally differentiated cells or quiescent cells re enter the cell cycle?
Quiescent as these cells are in G0 phase. Terminally differentiated cells have had a change in morphology and can not do this.
191
What is TCFB an example off?
An antigrowth factor.
192
Is TCFB more or less active in tumours?
Less active.
193
TCFB is an antigrowth factor that causes a mutation in what?
The TCFB receptor.
194
When the TCFB receptor is mutated, what proteins are produced and what do they do?
p15, p21 and p27. They are proteins which inhibit the cell cycle. These also inhibit the formation of the CDK/cyclin complex.
195
What do SMADS produce?
Antigrowth signals.
196
Mutations in what cause less antigrowth signals in tumours?
SMADS.
197
TCFB is an antigrowth factor which causes mutations in its receptor which in turn produces proteins that can inhibit the cell cycle. It can also supress another gene which regulates G1 machinery. What gene is this?
cymc gene.
198
TCFB is less active in tumours. This is caused by single point mutations which can also do what?
Cause a loss of p15 and mutations in the smad and rb protein.
199
A decreased sensitivity to p15 can be due to a mutation in what?
CDK14.
200
What does rb stand for?
Retinoblastoma protein.
201
Rb binds _____ preventing it from acting as a transcription factor thus reducing gene expression in the G1-S stage of the cell cycle.
E2F.
202
How is E2F released from rb?
Rb becomes phosphorylated by cyclin dependant kinases. This causes the release of E2F allowing it to act as a transcription factor, allowing progression through the cell cycle.
203
When is the activity of CDK2, CDK4 and Cyclin D increased?
When the cell cycle is active. CDK2 helps rb maintain its phosphorylated state.
204
E2F can act as a transcription factor on two major classes. What does this result in?
1. Products essential for DNA synthesis become present.
eg thymide kinase and dihydrotolarate reductase.
2. The phosphorylation state of rb is maintained.
205
There is a 70% reduction in rb activity in which type of cancer?
Cervical cancer caused by the HPV virus.
206
The HPV virus can cause cervical cancer partially by reducing rb activity. It also produces a protein which can bind to p53. What viral protein is this?
E6.
207
When E6 is bound to p53 what is promoted?
Apoptosis. This also degrades p53.
208
The HPV virus can produce the E6 protein which can inactive p53. It can also produce the E7 protein. What does this do?
E7 can bind to rb preventing it from binding to E2F.
209
What can cell adhesion molecules produce?
Antigrowth signals.
210
Probably acting through rb, what can cancer cells turn of the expression of?
Cell adhesion molecules meaning less antigrowth signals are produced.
211
The antigrowth signals directed to rb and the cell cycle are _______ in most human tumours.
Blocked.
212
What can turning of the expression of antigrowth signals promote the expression off?
Progrowth signals.
213
What does the c-myc gene encode for?
The transcription factor myc.
214
What can some tumour cells avoid?
Terminal differentiation.
215
When myc is bound to max is the cell dividing or has it differentiated?
It is still growing/ dividing. Overproduction of myc happens in tumour cells to avoid terminal differentiation.
216
When max is bound to mad is the cell dividing or has it differentiated ?
The cell has differentiated.
217
Overexpression of c-myc gene favours the myc max complex. Does this have any effect on the mad max complex?
Yes. It means the mad max complex is formed less as less max is available. This means less cells terminally differentiate.
218
Apoptosis and necrosis are the same thing. True or False?
False. Necrosis is the process which occurs when the cell is poisoned.
219
What happens in apoptosis?
1. The nuclear envelope breaks down.
| 2. The DNA forms fragments.
220
How long does apoptosis take?
30-120 minutes.
221
What two things initiate the apoptosis cascade?
Death factors and surface receptors.
222
FAS and TNFA are receptors. What do they bind?
Death factors.
223
TNF and FASL are examples of what?
Death factors.
224
Signals for apoptosis are generated inside and outside of the cell. Do FAS and TNFA pick up all these signals?
No they are only triggered by outside signals.
225
Some proteins promote apoptosis. Are there also proteins within the cell that prevent apoptosis?
Yes.
226
Apart form the binding of death factors via outside signals what else can trigger apoptosis?
1. p53
2. DNA damage.
3. Lack of oncre gene action (oncre genes promote growth).
4. Cytochome C present in the cystol (when it should be present in the mitochondria.)
227
What is IGF1 an example of?
A survival factor.
228
What is BCL2?
It is regulatory protein which can promote or prevent apoptosis.
229
What regulates cytochrome C release?
BCL2.
230
What does p53 upregulated when it comes into contact with damaged DNA.
Bax.
231
Cytc and death receptors are both involved in different cascades to cause apoptosis. Which one of the two triggers capase 9 and other capases?
Cytc triggers capase 9 with in turn triggers other capases to cause cell death
Death factors trigger capase 8 which directly causes the final breakdown of the cells.
232
Some tumours are hormone dependant. What happens when the hormones are removed from these tumours?
They undergo massive apoptosis.
233
Apoptosis is switched of by oncre gene over expression. True or False?
False. This would normally trigger apoptosis allowing the constant removal of mutant cells. For a cancer to develop it has to avoid the apoptotic machinery.
234
What percentage of B cell lymphomas had a mutation in the myc oncre gene?
50%. These also had a mutated BCL2 gene.
235
How can overexpression of BCL2 be forced meaning the rate of apoptosis is lowered.
Adding survival factors such as IGF1.
236
Inactivation of what will cause tumour cells to become larger?
p53.
237
It is possible for a cell to abolish the FAS death signal. What decoy is used to allow this to happen?
A non signalling decoy receptor for a FAS ligand is upregulated.
238
How many times can a cell divide before it dies?
60-70 times.
239
If rb and P53 are disabled the cell will keep on growing. Before a cell with limitless replication poteinal arises, what happens to the rest of the cells in the colony?
There is a mass crisis of cell deaths where only 1 cell in 10^7 divide.
240
What does the massive crisis of cell death to cells with limitless replication explain in regards to tumour size?
Why the tumour can not exceed the number of body cells. This is because of the widespread apoptosis present. The size of the tumour hence greatly underestimates the number of divisions required to make the tumour.
241
What may be a barrier to cancer development, in regards to cell number?
The generational limit of normal somatic cells. It made no sense that tumour cells were immortal.
242
Telomeres contain 250-1500 copies of which sequence?
TTAGGG.
243
After each cell division how much DNA is lost from the end of every chromosome?
50-100 bp of telomeric DNA.
244
Where does telomere maintenance occur most?
In malignant cells.
245
What does terlomerase add to the ends of the telomeric DNA?
Hexanucleotide repeats.
246
What does keeping the telomeres above the length of the critical threshold mean?
That the cell has the potential for unlimited multiplication.
247
What does 'angiogenesis' translate to?
The birth of new blood vessels.
248
Angiogenesis is normally established in early development apart from when?
After surgery/ injury. It is why scars are red.
249
How close do cells in a tissue need to be to a blood vessel?
100um.
250
A what size will a cancer cell recruit signals to recruit surrounding tissue and vascular cells causing the formation of a new blood vessel.
2mm.
251
What are the two types of angiogenic signals?
Positive and negative.
252
What three growth factors are involved in positive angiogenesis?
VEGF, FGF1 and FCF2.
253
What is involved in negative angiogenesis?
Thrombosin 1.
This is also able to bind to the transmembrane receptor CD36 on epithelial cells.
254
Activation of what gene may upregulate VEGF?
The ras oncre gene.
255
What postivley regulates thrombosin 1?
p53.
256
How do tumours activate angiogenesis?
By up regulating inducers and down regulating inhibitors.
257
Where does the stage of tissue invasion and metastasis come in tumour development?
It is one of the final stages.
258
Cancer cells need to be anchored to the basement membrane and to other cells. What happens if they become unanchored?
A similar process to apoptosis.
259
What two things hold the tissue cells in place?
Adhesion molecules and intergrins.
260
Catenins link cadherins to the internal cytoskeleton. What is the role of cadherins?
They span the membrane and lock onto cadherin molecules on adjacent cells.
B catenin is an example of a transcription factor.
261
Intergins bind to firbronectin and this in turn binds collagen to what?
The basel lamina.
Epithelial cells also use collagen to bind to the basel lamina.
262
What are the only cells that can migrate out of the blood?
White blood cells.
263
Localised spread to the lymph nodes is normally a sign of what?
Poor prognosis.
Ie not a very good diagnosis.
264
Where does prostrate cancer normally spread to?
The bone.
265
Where does prostate cancer almost never spread to?
The lung and the brain.
266
Occular melanoma only ever really spreads to where?
The liver.
267
Where does stomach cancer often spread to?
The ovary.
268
Is metasis random?
No. However a process called shredding is.
269
When a cancer spreads and infects a new organ, do the cells of the second organ become cancerous?
No. They just contain the cancerous cells from the first organ.
270
For a cancer cell to spread what connections need to be broken?
The connections to the neighbouring cells and the ECM (epitheal cell membrane.)
271
Is it more common for their to be a mutation in the e. cadherin gene or for there just to be a decrease in amount of e.cadherin?
It is more common for there to be a decreased amount of e.cadherin. Mutations in the gene are relatively rare.
272
There is often a decreased amount of e. cadherin in a cancer cell. How are the levels of catenins effected?
Catenins are often absent or non functioning.
273
Do cancer cells produce more or less intergins?
Less. The intergins are often also a different type.
274
The intergins produced in a cancer cell about to metastasis can be in fewer number and a different type. The new type contains a different protein which allows the cell to do what?
It allows the cell to migrate through the walls of the blood vessels and through connective tissue.
This is different in different cancer cells. There are 46 families of the protein.
275
How do cells about to metastasis have a different structure to other cancer cells?
They are often elongated into a star shape
276
Metastatic cells have to become mobile. What needs to be reconfigured to allow this to happen?
The cytoskeleton.
277
Cancer cells elongated into a star shape resemble cells generally only seen when?
In early development.
278
What can cancer cells sometimes secrete to degrade surrounding cells, connective tissue and the basel membrane?
Proteases.
279
Serine proteases are plasminogen activators. What does this lead to the conversion off?
Plasminogen to plasmin.
280
What is cathepsin B and example of?
A cysteine protease.
281
Where is cathepsin B often expressed in the cell and is this universal?
At the front of the cell/ the direction in which the cell is traveling, It is not even universal in cell type.
282
What often converts matrix metalloproteinases (MMPS)to an active form?
Plasmin.
283
What are matrix metalloproteinases dependant on?
Zinc.
284
Urokinase is a type of what?
Serine protease.
285
Why is metastasis not a very efficient process?
Tumour cells are often relatively large compared to blood vessels and often the tumour is detected as foreign by the immune system.
286
What are MMP's capable of degrading?
Many types of extracellular matrix proteins.
287
Are MMP's found in all cancers?
No.
288
What secretes MMP's?
Catherpsin B or a urokinase activator.
289
How do cancer cells often travel in the blood?
They surround themselves in platlets and travel in clusters.
290
Why do cancer cells surround themselves in platlets when they travel through the blood?
It helps mask the cancer from immune surveillance.
291
Carbohydrates on the cancer cells bind to a specific receptor on the endotheial cells. What is this receptor called?
A selectin.
292
Are the carbohydrates attached to the surface of cancer cells always the same?
No. Specific carbohydrates are recognised by specific selectins.
293
What other cells use carbohydrate-selectin interactions to identify certain tissues?
White blood cells.
294
Selectins can be used to explain what?
Why secondary tumours form in specific tissues in the body.
295
When the cancer cell contacts a surface with the specific select what happens?
New bonds, mediated by integrin's form between the cell.
296
What does the cancer cell use to migrate back through the blood vessel wall to the secondary site?
Proteases.
297
Does the cancer cell use the same set of proteases to enter the blood as it does to leave?
It is not known.
298
What do the proteases degrade when they are secreted by the cancerous cells in order for them to leave the blood?
Connective tissue matrix.
299
Will adult cells divide without a signal?
No.
300
What are the 6 established treatments available to treat cancer?
1. Surgery.
2. Radiotherapy.
3. Chemotherapy.
4. Endocrine therapy.
5. Immunotherapy.
6. Biological (targeted) therapy.
301
It costs £10 million to bring a new drug to market. True or false?
False. It costs approximately £100 million.
302
Which two used cancer treatments only have limited success?
Immunotherapy and biological therapy.
303
What type of cancer therapy involves using small peptide chemicals to bind to the receptors?
Biological (targeted) therapy.
304
Biological therapy can involve the use of antibodies to target specific receptors. Will this form of treatment be used on its own to treat cancer?
No.
305
What two methods of treatment are best for localised cancer?
Radiotherapy and surgery.
306
What does systemic treatment involve?
Prolongation of the patients life.
307
Drug treatment is used with all cancers. True or false?
False. Drug treatment will only work on certain types of cancers.
308
Leukaemia can be treated through drugs and surgery. True or false?
False it can not be treated by surgery, it can however be cured by drugs.
309
Cis platin can be used to treat ovarian cancer. What type of cancer can it not treat?
Colon.
310
What age range is screened to help detect female breast cancer?
55-60.
People will be screened at a younger age if they have a family history of breast cancer.
311
What is the aim of adjuvant therapy?
To eradicate residual microscopic cancer after surgery.
312
What does neoadjuvant therapy involve?
Shrinking the cancer to a size which allows it to be removed surgically.
313
What to cytotoxic drugs inhibit?
Proliferation. Cell death is induced by apoptosis.
314
How do cytotoxic drugs damage the DNA?
They can cause strand breakage or cross linking. Certain drugs can also act as alkylating agents.
315
What are nitrogen mustards and ethylenimines also known as?
Alkylating agents.
316
What do alkylating agents do?
They transfer an alky group to the N7 position of guanine during cell division.
317
Apart from DNA damage, what can cytotoxic drugs also do in the cell?
Act as synthetic analogues of normal metabolites which compete for metabolism.
318
Methotrexate is used to treat leukaemia. It works by interfering with the production of what?
Tetrahydrofolic acid. It does this by blocking toxic acid reducatase.
319
What does 5-Flurouracil block?
Enzymes that produce materials for DNA and RNA synthesis. It does this by binding to thymidylate synthase.
320
Arabinosides inhibit DNA synthesis. Why do they produce less side effects than other drugs?
They are not active until they are in the cell.
321
How do anthrcyclins and related compounds work in cancer treatments?
Antibiotics are produced by microorganisms which interfere with DNA and RNA synthesis.
322
What do tropisomerase inhibitor's ultimately prevent?
DNA replication.
323
What chemicals are classed as spindle poisons?
Plant alkaloids and taxoids.
324
What do spindle poisons to do the cell?
Inhibit microtubule assembly which causes cell cycle arrest in mitosis.
325
Platnium drugs do what to the DNA?
Cause it to crosslink.
10% of the worlds platinum is now used in cis platin.
326
What does recombinant DNA refer too?
DNA that has been generated by joining and amplifying DNA from different biological sources.
327
What three processes allow DNA to be inserted into host cells?
Transformation, transduction, transfection.
328
Placing plasmids into microorganisms is called ________.
Transformation.
329
Placing a phage into a bacteria is called _______.
Transduction.
330
Placing a plasmid into eukaryotic cells is called _______.
Transfection.
331
Temperate phages such as lambda can exist as a latent _______ during lysogenic growth or go through the ______ _____.
Prophage
Lytic cycle.
332
What represses the expression of phage genes during lysogenic growth?
The cI repressor.
333
Where does the cI repressor bind to when it is inhibiting gene expression of the phage in lysogenic growth?
The OL and OR operators. These are found at the PL and PR promoters.
334
How is cI degraded when the cell is stressed?
Proteolytically.
335
What genes are immediately transcribed by OL and OR when cI is degraded?
N and cro.
336
N is an early gene which is encoded for straight away when cI is degraded. What is N?
An antiterminator allowing expression further along the phage genome.
337
Cro is an early gene transcribed when cI is degraded. what is cro?
A transcription repressor which blocks the expression of cI.
338
When cI levels decrease and cro and N levels increase what is phage irreversibly committed too?
The lytic pathway.
339
cI and cro are both what?
Transciption repressors.
340
Does the OL/PL region of the gene express N or cro?
N.
341
Does the OR/PR region of the gene express N or cro?
cro.
342
How long are cos sites?
12 nucleotides.
343
What happens to the cos sites once the lytic cycle has been entered?
They anneal.
344
Where are cos sites found?
At the terminal ends of the linear phage DNA.
345
what does the att site in the phage genome allow?
It allows integration into the hosts DNA.
346
How is bacterial DNA protected against phage DNA?
It has its own specific methylation pattern allowing it to recognise foreign DNA. Sometimes phages can adopt this pattern.
347
What are Dam and Dcm?
E.coli methylases.
348
What does Dam allow the production of?
m6A.
349
What does Dcm allow the production of?
m5C.
350
What does each E.coli daughter cell receive during semi conservative DNA replication?
One methylated strand and one new non methylated strand.
351
What is used as the methionine donor to methylate hemimethylated DNA?
S-adenosyl-methionine.
352
What do type one restriction/modfication systems involve?
Complexes contain both nuclease and methylase activity.
353
What do type 1 restriction/modification systems do to non methlayed DNA?
They cause random double strand breaks by hydrolysing the DNA.
354
Are type 1 or type 2 restriction systems used in molecular biology?
Type 2.
355
What do type 2 restriction/modification systems contain?
Separate nuclease and methylate activity.
356
Where do type 2 restriction systems cut?
Specific palindromic sequences in the DNA.
357
Do the enzymes involved in the type 2 recognition systems bind specifically or non specifically at first?
Non specfifcally. They they 'slide' or 'hop' to the specific site. When cut a 5' phosphate and a 3' hydroxyl group is generated.
358
What molecule is essential for cleavage?
Mg2+.
359
How long are the palindromic sequences recognised by type 2 restriction enzymes?
4-8 nucleotides long.
360
Do the cleavage sites normally lie within or beside the palindromic sequences?
Within.
361
Distinct fragments cut with the same enzyme will have single stranded overhangs with complementary _____ _____.
Sticky ends.
362
What type of ends are generated when the enzyme Sma1 is used?
Blunt.
363
What enzyme covalently links the cos sites in the linear lambda phage?
DNA ligase.
364
What is the DNA ligase commonly used in molecular biology from?
Bacteriophage T4.
365
Would you treat the DNA with polymerases, phosphatases and kinases before or after treatment with ligases?
Before.
366
DNA polymerases and polynucleotide kinases are examples of what?
Modular proteins.
367
What type of enzyme helps to join blunt ends to help prevent reannealing?
Phosphatases
368
DNA ligase was purified from ecoli using liner lambda DNA in an assay. This involved centrifugation under what conditions?
Alkaline.
369
Do all phages encode their own ligases?
No.
370
Liner lambda DNA is circulised after transduction. Is the ligase from the phage or the host cell?
The host cell.
371
Can DNA ligase join blunt ends as well as overhangs?
Yes.
372
The ligase uses three steps to anneal the DNA. What is the first step?
An adenylate group is added to a lysine residue.
373
The ligase uses three steps to anneal DNA. What is the second step?
The adenylate group is transferred to the 5' phosphate group.
374
The ligase uses three steps to anneal the DNA. What is the third step?
A phosphodiester bond forms.
375
Step one of ligation involves the attachment of a adenylate group to a lysine residue. What brings the adenylate group to the DNA ligase?
NAD or ATP.
376
In the third step of ligation the activated 3' hydroxyl group attacks the 5' phosphate group to form a phosphodiester bond. True or false?
False. The activated 5' phosphate group attacks the 3' hydroxyl group to form a phosphoester bond.
377
If the ends of a fragment are non cohesive they can still be ligated together. What two processes allow this to happen?
Filling in overhangs by an a polymerase or degrading an overhang with an exonuclease.
378
Why is it sometimes useful to convert sticky ends into blunt ends?
Allows you to ligate fragments from different enzymes and allows the removal of restriction sites.
379
Apart from polymerase activity, what other activity does DNA polymerase 1 have?
Exonuclease activity, in both directions.
380
Why is the klenow fragment ideal for blunt end cloning?
The domain for 5-3 exonuclease activity is not present meaning it only has 3-5 exonuclease activity and polymerase activity. This means the overhang will not be cleaved.
381
What protease will degraded DNA polymerase 1 leaving the klenow fragment?
Subtilisin.
382
What does klenow do in blunt end cloning?
Fill in overhangs.
383
What do most restriction enzymes leave?
A 5' overhang.
384
Can you add the missing nucleotides to a 3' overhang.
No. To generate blunt ends it has to be removed.
385
What do alkaline phosphates remove phosphate groups from?
polynucleotides and proteins.
386
Why are alkaline phosphatases often used in cloning?
They prevent self ligation of linearised plasmids with cohesive ends by removing the terminal phosphate. This causes nicked ends.
387
What do alkaline phosphatases depend on?
Mg2+ and Zn2+.
388
What is polynucleotide kinase an example off?
A DNA repair enzyme.
389
What is nicked DNA?
DNA where the phosphate backbone of one strand is broken.
390
What does nicked DNA contain?
A 5 hydroxyl group and a 3' phosphate group.
391
What does polynucleotide kinase convert nicked DNA too?
To DNA with a 5' phosphate and a 3' hydroxyl nick that can be sealed with DNA ligase.
392
Polynucleotide kinase has a phosphatase activity. What does this allow?
For the 3' phosphate group to be removed from the nicked DNA.
393
The kinase activity of phosphatase kinase allows for the addition of a phosphate group to the 5' end of the nicked DNA. Where does this phospahate group come from?
The gamma phosphate of ATP.
394
Apart from converting nicked DNA into DNA that can be ligated, what else is polynucleotide kinase used for?
To radiolabel oligonucleotides for the use of hybridisation probes.
395
What is reverse transcriptase used to produce?
cDNA from RNA.
396
Reverse transcriptase is error prone. True or false?
True. This allows diversity in viruses such as HIV.
397
What do reterovirus and retrotransposons both encode?
Reverse transcriptase.
398
What is the most commonly used cloning vector?
Plasmids.
399
What is the insert size for a phage cloning vector?
15kb.
400
What is the insert size of a cosmid cloning vector ?
40kb.
A cosmid is also a derivative of a phage.
401
What is M13 f1ori an example off?
A phagemid.
402
What cloning vector has the largest insert size?
An artificial chromosome.
403
What is a polylinker also known as?
A multiple cloning site.
404
What three things must a plasmid contain to be used as a cloning vector?
1. Origin of replication.
2. Selectable marker.
3. Multiple cloning site.
405
Can plasmids autonomously replicate in bacterial cells?
Yes.
406
What must E.coli strains used in molecular biology be deficient in?
Restriction/modification systems and nonspecific endonuclease activity.
407
What E.coli strains are used to propagate
recA- mutants. These can not undergo homologous recombination.
408
Most plasmids used in molecular biology replicate by what mechanism?
'theta mechanism'
This is the same mechanism found in E.coli chromosomes.
409
The replication of origin influences the number of plasmid cells. True or false?
True.
410
What influences the number of plasmid cells?
How many origins of replication are present and regulatory proteins/RNA which control the rate of replication initiation.
411
Can plasmids also use the rolling circle model of replication?
Yes.
412
How does the rolling circle model of DNA replication work in plasmids?
The DNA is nicked at the 3' end and is extended with DNA polymerase 2. This displaces the nicked strand. The whole thing is then cleaved to ligated to give double stranded circular DNA.
413
There are two ways in which an E.coli cell can be made 'transformation competent'. What are these?
1. Treatment with high concentrations of divalent cations.
| 2. The DNA can be introduced by electroporation.
414
What cations are often used to make the E.coli cell 'transformation competent'?
Ca2+, Mg2+, Rb2+.
415
What can increase the efficiency of transformation?
Heatshock at 42 degrees.
416
Larger plasmids are transformed more efficiently. True or false?
False. Smaller plasmids are transformed more efficiently.
417
What two ways are normally used to detect transformed plasmids?
Expression of drug resistance genes or prototrophic markers.
418
What does ampicillin inhibit?
D,D-transpeptidase.
419
Ampicillin inhibits D,D- transpeptidase. What is the purpose of D, D-transpeptidase within the cell?
It cross links cell wall peptidoglycan.
420
What are chloramphenicol and tetracycline?
Ribosome inhibitors. This stops the translation of new proteins.
N.B if resistance to these antibiotics is used as a selective marker it has to be ensured that the plasmid is able to translate the gene required for resistance first, before protein production is blocked after exposure to the drug.
421
What do most drug resistance genes do?
Modify the antibiotic (eg B-lactamase and CAT) or secrete the drug (tetA).
422
What causes Chloramphenicol resistance?
Modification of the enzyme Chloramphenocial acyltransferase (CAT).
423
Blue- white selection is a form of what?
Alpha complementation.
424
What vector is used in Blue-White selection?
A vector which encodes for the N-terminal domain of B-galactosidase.
425
In Blue-White complementation an E.coli strain is used which has a mutant copy of lacz. What does this mutation result in?
Only the C terminal of B-galactosidase is encoded for.
426
In the Blue-White complementation test what is the colour do cells which have taken up the plasmid?
Blue.
427
In Blue-White complementation why do cells which have taken up the plasmid turn blue?
They express a functional B-galactosidase which is able to hydrolyse the colourless X-gal substrate into a blue product which in turn gives rise to blue colonies.
428
Have any white cells in the complementation test taken up the plasmid?
Some may have done but it may have been incorporated into a polylinker region meaning the coding of the N terminal is disrupted and no B-galactosidase is produced.
429
What non functional allele is present in the E.coli in the Blue-White complementation test?
∆M15.
430
What happens to the E.coli cell when it is lysed in NAOH and SDS?
The cell contents are solubilised and the DNA dentures.
431
What is the lysate neutralised by in the alkaline lysis mehtod?
Potassium acetate.
432
What happens after the lysate is neutralised by potassium acetate in the alkaline lysis method?
Genomic DNA and protein precipitate and the plasmid DNA refolds and stays in the solution.
433
Which two methods allow the plasmid to be recovered from the supernatant in the alkaline lysis method?
1. Ethanol precipitation.
| 2. Affinity chromatography using silica.
434
Why is the plasmid denatured less than the bacteria's genomic DNA in the alkaline lysis method?
It is smaller and the DNA is supercoiled.
435
To allow the purification of plasmids cells are grown under selection and resuspended in a solution containing _____. This blocks DNase and RNase activity.
EDTA.
436
What are DNA libraries?
Collections of recombinant DNA molecules that allow the isolation of specific genes/ complete transcription units from a particular organism, cell line or tissue source. Genes from organisms with small genomes can be readily cloned from libraries generated from recombinant DNA.
437
What do cDNA libraries allow?
They allow you to isolate ORFs from organisms with larger genomes. This allows functional gene analysis.
438
cDNA libraries allow you to construct a genomic map. True or False?
False. Genomic DNA libraries allow you to construct a genomic map.
439
Are genomic DNA libraries practical in higher organisms?
No.
440
MRNA can be isolated by looking for what?
A specific protein encoding gene. The MRNA can then be turned into DNA.
441
How can you generate a DNA library?
By cloning a collection of DNA fragments into a vector.
442
Ideally how many genome equivilents will a DNA library contain?
Several.
443
What is a genome equivalent?
The number of transformants that are theoretically required to cover the complete genome.
1 genome equivalent is calculated by dividing the genome size by the average insert length.
444
Does the 'vector only background' want to be increased or reduced when making a genomic DNA library?
Reduced.
445
DNA cut with _____ and ______ have complementary 5' overhangs.
Sau3A and BamH1.
446
Partial digestion of genomic DNA with Sau3A generates overlapping fragments that can be directly cloned into what vector to make a genomic DNA library?
BAMH1.
447
Instead of genomic DNA libraries what is it more practical to make instead for eukaryotes?
CDNA libraries.
448
Sau31 cuts every how many nucleotides?
256 nucleotides.
449
During a partial digest should you or should you not use a restriction enzyme that cuts every restriction site?
No. This way it does not matter if the restriction site is within your gene of interest.
450
What percentage of the total amount of RNA in a cell is mRNA?
5%.
451
How do you enrich mRNA from the total RNA?
Oligo(dt) affinity chromatography.
452
Why is poly(A) and RNA eluted in the absence of salt?
As it weakens ionic interactions between base pairs.
453
What is used to select mRNA from rRNA and tRNA?
Poly(A) tail.
454
What is mRNA converted to to make a CDNA library?
single stranded cDNA, then double stranded cDNA.
455
What does first strand synthesis refer to?
Making a single strand DNA molecule from RNA.
456
What is often used as a primer in first strand synthesis?
Oglio(dt).
457
What does 'cap trapping' ensure?
That full length cDNAs are generated.
458
Due to stalling incomplete cDNA products are formed. Why are incomplete cDNAs digested by RNAseI?
As they still have the cap. (RNA is degraded)
459
Does cap trapping occur after a single stranded cDNA or a double stranded cDNA is produced?
After a single stranded cDNA is produced.
460
What happens after the cap trapping enzymes have degraded the RNA?
The cDNA is extended by homopolymer tailing
| with terminal deoxynucleotidyltransferase. PCR then amplifies the product.
461
What does homopolymer tailing involve?
The addition of a series of G residues to the 3' end of the tail using deoxynucleotidyltransferase.
462
After homopolymer tailing PCR makes the cDNA double stranded. What primers does it use to do this?
A forward primer with a 3'C tract and a reverse primer with a 3'T tract.
463
What is normally added to the end of DNA to mediate cloning?
Linkers. These are artificial sequences that contain specific restriction sites.
464
What is the DNA treated with before it is cloned?
Corresponding methylases such as EcoRI methylases to methylate any restriction sites within the cloned DNA. This destroys the restriction sites.
465
Are linkers added before or after methylation?
After.
466
What three methods can you use to screen gene libraries?
1. Hybridisation techniques.
2. Immunological techniques.
3. Complementation.
467
Why would you screen a gene library?
To find a DNA sequence you didn't know.
468
What two methods allow you to make a hybridisation probe?
1. Chemical synthesis.
| 2. Generation of a radiolabeled cDNA from a DNA fragment.
469
The N terminal sequencing of purified proteins or the use of mass spec normally provides enough data for what?
To synthesise a short oligonucleotide which can act as a gene specific probe.
470
What is generated to cover all possible variant nucleic acid sequences that encode a given polypeptide sequence?
A pool of degenerate oglionucleotides.
471
What does random primed labelling of a DNA fragment involve?
Annealing short primers of random nucleotides and the extending with radiolaballed nucletiodes, by using either poly1 or the klenow fragment. To do this the double strand is initially denatured.
472
For hybridisation assays what must the DNA from transformants be bound to?
The membrane plate (In the filter replica method).
473
Once the transformants are replicated on the agar plates onto the membrane what happens to the cells?
They are lysed.
474
In filter replica once the cells are lysed the membrane is washed and hybridised with what?
The radiolaballed gene specific probe.
475
How is the correct clone finally picked from the plate in the filter replica method?
The colony pattern is compared with the autoradiographic signal.
476
How are the cells lysed in filer replica?
With Naoh. They are then washed and baked.
477
What do cDNA library inserts lack?
A promoter.
478
What must cDNA sequences be expressed in so polypeptides can be detected?
In an insert expression vector.
479
What does an expression vector contain?
Multiple coding sites downstream of an inducible promoter. This is often the lac promoter/operator. This means that when the library is switched on the cloned DNA sequence will be expressed.
480
For immunological screening what is the library constructed using?
An expression plasmid.
481
Does expression screening involve filter replica?
Yes.
482
What is often added to the medium to induce expression when immunological DNA libraries are being screened?
IPTG.
483
In immunological screening, once IPTG is added what is done to the cell?
They are lysed to release the protein inside of them. These proteins can they bind to the filter replica membrane.
484
Is the secondary or primary antibody radiolaballed?
The secondary antibody.
485
What method of screening oinvloves the use of antiobodies?
Immunological screening.
486
What can be cloned from genomic libraries with genetic complementation?
Recessive mutants with a clear growth phenotype.
487
What type of vector is used in the screening method of genetic complementation?
A shuttle vector.
488
How many systems can a shuttle vector be maintained in.
E.coli and an additional system.
489
Anaylasis of what organism often uses shuttle vectors?
Yeast.
490
Why is genetic complemtation often used as the screening procedure in yeast?
Because it is a haploid organims with a small genome.
491
What is the ars gene used for in the complementation screening of yeast?
It is the replication origin in yeast.
492
Apart from the yeasts origin of replication what else is needed for complementation screening in yeast?
A centromic region (CEN) and a selectable marker, eg the URA3 gene.
493
Why is the bla gene required in a yeast shuttle vector?
It allows maintenance and selection in E.coli. A n ecoli multiple cloning site and origin of replication is also required.
494
What does the URA3 gene encode for?
Uracil biosynthesis. Often used as a selectable marker.
495
In complementation screening of yeast what will cells grow without if they have taken up the plasmid?
Uracil.
496
cDNA libraries lack replicatory systems. True or false?
True.
497
When making a yeast genomic library what is used to partially digest the genomic DNA?
Sau1.
498
When making a yeast genomic library what is the shuttle vector linearised with?
BamH1.
499
When making a yeast genomic library why is the shuttle vector linearised by BamH1 and theDNAgenomic library is partially digested by Sau1.
Because they have complementary 5' overhangs.
500
When making a yeast genomic library what do you have to make sure the digest genomic DNA is before using DNA ligase and ATP to insert it into the plasmid?
The correct size.
501
What autotrophic mutants are used in order to screen a yeast genomic DNA library?
ura3- mutants. These mutants can not synthesis uracil.
502
When screening a yeast genomic DNA library what are the plasmids first grown on?
A medium lacking uracil at a permissive temperature.
503
When screening a yeast genomic DNA library what are the transfomants secondarily grown under?
A non permissive temperature. Mutants will only growth that contain a functional copy of the gene mutant in the temperature sensitive strain.
504
What phage is often used to clone large fragments of DNA
Phage lambda.
505
The genome size of phage lambda is 48kb. It can be packaged with ____kb to ____kb of DNA.
37 and 53.
506
What to phage lambda cloning vectors are used to clone large fragments of DNA?
International vectors and substitution vectors.
507
What is the size of an insert limited to in lambda insertion vectors?
12kb. This is large enough for most full lengh cDNA>
508
Can insertion vectors be packaged without containing an insert?
Yes.
509
What two reasons caused phage lambda to be developed into a cloning vector?
1. Not all its gene products are required for lytic growth.
| 2. Mutations where isolated with single restriction sites. You can engineer these to have other restriction sites.
510
What unique restriction site is found in lambdagt11?
A unique EcoR1 restriction site at the 3' end of the 3' end of the lacz gene. When something is inserted into this site the lacz gene lacks funciton and Blue-White screening can be used.
511
What can cDNAs in lambdag11 be expressed as to allow lambdag11 to also act as an expression vector allowing clones to be screened with anti-lacZ antibodies?
Fusion proteins.
512
Is southern blotting used for DNA, RNA or proteins?
DNA.
513
What method is used to determine the following...
1. Is the gene duplicated or amplified?
2. Has the gene undergone rearrangement?
3. Does the gene have related homologous genes?
4. The conformation of insertions and deletions.
Southern Blotting.
514
How are specific sequences detected in the use of southern blotting?
Through hybridisation with a specific probes.
515
What three steps are involved in southern blotting?
1. DNA is degraded by restriction enzymes.
2. The DNA is then resolved through gels and then transferred through a membrane.
3. Specific sequences can then be identified with a hybridisation probes.
516
What are zoo blots ?
The process of southern blotting used on different animals. It helps detect homologous genes. It also helps identify mutants.
517
Does northern blotting use DNA, RNA or proteins?
RNA.
518
What is resolved through gels and transferred to a blot in the northern blotting technique?
Total RNA or poly(A)+ RNA.
519
What three things can northern blots show?
1. Lengths of RNA transcripts.
2. The size/ abundance of RNA transcripts from a specific gene.
3. Tissue specific expression.
520
What can FISH in regards to RNA?
It can show spatial expression patterns. This helps show where RNA is found during development.
521
Is western blotting used to look at DNA, RNA or proteins?
Proteins.
522
What are proteins resolved by in Northern blotting?
SDS Phage.
523
Once proteins are transferred to a blot in the western blotting method, what is the protein then incubated with?
A primary antibody and the secondary antibody. When proteins are expression as fusion proteins they can be detected by these antibodies.
524
What are microarrays used for?
To look at the relative expression of genes on a genome wide scale.
525
What are microarrays also known as?
Gene chips.
526
What are 2D arrays of gene specific probes hybridised and pooled with during the formation of a microarray?
Florescently labelled cDNA molecules.
527
What two sample types are used in a microarray?
A test and a control sample.
528
What does transcriptional profiling describe?
The expression of RNA on a genome wide scale.
529
Microarrays reveal changes in gene expression. What 4 things can cause these changes?
1. Changes in growth conditions.
2. Changes in developmental stage.
3. An altered genotype.
4. An infection.
530
What does cluster analysis identify?
A set of genes whose expression varies in a similar manner.
531
What type of primers are used to make cDNA?
OgliodT primers.
532
What is meant by the term 'competitively hybridised'
When cDNA molecules are labelled with different colours so you can compare the amount of expression of both.
533
What do coordinated regulated genes define?
Regulons.
534
What do mammalian shuttle vectors typically contain?
The SV40 virus origin and promoter of replication. It also has polyadenylation elements from viral or highly expressed genes.
535
What do transient infections involve?
Expression from plasmids that have not segregated correctly so are lost during growth.
536
What does stable transfections involve?
Integrating DNA into the genome.
537
Expression fusion proteins that include an epitope tag enable what?
Immunoflorescence- detecting of the protein within the cell.
538
Where are epitope tags derived from?
Naturally fluorescent proteins from jellyfish (green) or naturally fluorescent proteins from reef coral (red).
539
What to the left and right arms of the phage genome contain?
All the genes required for lytic growth.
540
How much of the lambda genome is required for lytic growth?
23kb.
541
What is the central stuffer region of substitution vectors flanked with?
EcoR1 sites.
542
Can substitution vectors be used to generate genomic DNA libraries?
Yes as they allow cloning of approximately 25kb of DNA.
543
What is replaced in substitution vectors to allow genomic DNA libraries to be made?
The stuffer sequences.
544
What are cosmids?
Plasmids containing a phage cos site.
545
What are cleaved cosmids ligated with?
Genomic DNA fragments of up to 45 kb in length.
546
What is used to package ligated DNA into cosmids?
A helper phage. Provides needed proteins but not expressed themselves.
547
Do cosmid transformants grow as colonies or plagues?
Colonies.
548
Can phage particles regulate E.coli cells?
No, but it can effect them.
549
What does chromosome walking identify?
Ordered arrays of overlapping lambda clones.
550
Chromosome walking can identify overlapping lambda clones. What is the region at the end of the identify clone used for?
It is used to generate new hybridization probes. This can then identify adjacent and overlapping clones.
551
Chromosome walking helps generate hybridization probes. What can this ultimately generate?
A minimal ordered array.
552
What are yeast artificial chromosomes?
Plasmids that can be digested to give two fragments each with a telomere sequences (ie. two artificial chromosomes are produced.)
553
YACS allowing the cloning of large fragments at high efficiency. true or false.
False. It can produce large fragments but at low efficiency.
554
What is produced after ligation of the fragments produced by a YAc and genomic DNA?
Linear DNA molecules with an ARS origin of replication, centromeric elements and telomeres. This allows them to function as a chromosome in yeast.
555
What are bacterial artificial chromosomes based on?
F plasmid.
556
What size fragment can be cloned into a bacterial artificial chromosome?
700kb.
557
What genome does the M13 bacteriophage have?
A single-stranded (+) circular DNA genome.
558
What form does M13 exist in in E.coli?
Double stranded (+/-) replicative form. this can be replicated like a plasmid.
559
What can you do M13 in E.coli?
It can be modified to contain unique restriction sites.
560
When the M13 vector is used to form libraries why can all sequences be sequenced in parallel?
As all the fragments are the same size.
561
Can M13 be isolated as a ssDNA from phage particles?
Yes.
562
What is known as shotgun sequencing?
When a large number of randomly selected clones are sequenced in parallel using universal primers.
563
What does primer walking involve?
Reiterative sequencing of reactions that extend and read from the previous round.
564
What is contig?
When a combined sequence is made by sticking together overlapping sequences.
565
Minimal order arrays and analysing random clones in parallel can do what?
Sequence a whole genome.
566
How many people are expected to get cancer?
1/3.
567
How many people are expected to die from cancer?
1/5.
568
Retinoblastoma and Wilms tumour (neuroblastoma) are most common in what age group?
Children.
569
Cancer is a group of diseases characterised by what?
Uncontrolled cell growth and the spread of abnormal cells.
570
What age does breast cancer peak at?
80.
571
Where do all cancers develop?
In tissues.
572
What are the epithelial cells anchored too?
The basement membrane.
573
When is metastis possible?
Once the tumour has broken through the membrane.
574
How many mutations do you need to develop a tumour?
5-8.
575
Once cells have the initial mutation they expand in colony size. Where does the second mutation need to be?
At a critical locus. This allows a second clonal expansion.
576
Why is screening for prostrate cancer not always necessary?
It is very slow to develop and does not always kill. By a certain age most men will have prostrate cancer.
577
Can any cell differentiate into a tumour or only cancer stem cells?
It is not known completely but it is thought that both types can form tumours.
578
Where can benign tumours grow?
In any tissue.
579
Do benign tumours histologically resemble the tissue or origin?
Yes.
580
What type of tumour is easily treatable?
Benign tumours.
581
Benign tumours can produce wart like outgrowths. What else do they normal cause the over production off?
A particular hormone.
582
Where do in situ tumours develop?
The epithelium.
583
What size are in situ tumours?
Normally very small. They often contain abnormal chromosomes.
584
Do benign tumours or in situ tumours not resemble their tissue of origin?
In situ tumours have an altered histological appearance.
585
Do in situ tumours invade the basement membrane and supporting mesenchyme?
No they do not.
586
Why is an in situ tumour in the bladder?
Bladder will loose flexibility and therefore will bleed. Blood in the urine allows it to be easily detected.
587
Cancer mutations are somatic. Can mutations in the germline also cause cancer?
They can increase the risk of cancer developing.
588
Does a cancer cell need to do all 6 features to be cancerous?
Not necessarily, if it has less it will be less aggressive.
589
Does the rate of cancer development vary between tumours?
Yes.
590
Where can tumours spread too?
The blood and the lymph underneath (the mesenchyme).
591
How are primary tumours often treated?
With chemotherapy and radiotherapy.
592
90% of cancer patients that die die from what?
From metacyosis.
593
Colon cancer often travels to the liver. Where does it go next?
The lungs.
594
What do the tissues where cancer cells develop have to contain?
Proliferating cells.
595
What is responsible for most cancers?
Exogenous agents.
596
What type of cells do not differentiate meaning cancer rarely develops there?
Muscle cells and nerve cells.
597
What are the two major classes of genes that mutate to cause cancer?
Proto-oncogenes and tumour suppressor genes.
598
What are proto-oncogenes involved in?
Growth promotion.
599
What do tumour suppressor genes restrain?
Cell growth.
600
What organism is taq isolated from?
Thermus aquaticus.
601
What organism is Pfu isolated from?
Pyrococcus furiosus.
602
Does taq or Pfu have a high fidelity?
Pfu.
603
In 30 cycles of PCR how many copies of the DNA will be made?
10^9.
604
What can be added to the DNA being amplified via the primers?
Restriction sites. This means that the product can then be used in a restriction digest and used in cloning. The restriction sites are normally added to the 5' ends.
605
PCR allows genes to be directly cloned from cDNA and DNA without the need of what?
A gene library.
606
What is quantitative PCR also known as?
Real time PCR.
607
Real time PCR allows you to see how much DNA is being made. Fluorescent dyes are often use which bind to the double stranded DNA product to allow this to happen. What is the name of the common green dye used?
SYBR.
608
Through real time PCR it is possible to see that abundant DNA and rare DNA will eventually both make the same maximum amount of product. Why is this the case?
The primers do eventually run out.
609
What can PCR be coupled to?
A reverse transcription reaction of RNA allowing genes to be cloned from a specific transcript. To do this an Ogliodt primer is used.
610
What would be generated through the use of imperfect primers in PCR?
Specific mutants.
611
In site directed mutagenesis how can you isolate the original plasmid from the mutant PCR copy? What specific enzyme is needed?
Through the use of the Dpnl enzyme. This cuts methylated DNA sites, which are not present in the PCR product meaning only the template gets degraded.
612
is taq used in site-directed mutagenesis?
No DNAP is used due to its proof reading ability.
613
Why is taq used in random mutagenesis and not in site directed mutagenesis?
Because in random mutagenesis you want to generate as many mutations as possible and taq is error prone which allows this. In site- directed mutagenesis the error is found in the primer and you want that primer to be amplified correctly to allow the correct mutant to be made. The proofreading of DNAP allows this.
614
Apart from through the use of taq, what 4 other processes allow errors to be made in random mutagenesis?
1. Excess of one nucleotide to generate a bias pool.
2. Use of Mn2+.
3. Use of chemical mutagens.
4. Use of mutated strains.
615
Why is Mn2+ often used in random mutagenesis?
Mn2+ competes with Mg2+ decreasing the polymerases fidelity.
616
In chemical mutagenesis what chemicals are used and why?
Bisulphite and nitrous acid.
| These increase deamination, through changing C-U and A-I.
617
What is XL-Ired and why is it used in random mutagenesus?
It is a mutated version of E.coli which has multiple deficient repair mechanisms meaning it allows more error to be introduced to the DNA.
618
PCR fragments transformed into yeast can be targeted efficiently to specific sites through _________ ________ by using primers with appropriate _______ ________ at their _____ end.
Homologous recombination, homologous sequences, 5'.
619
Homologous recombination in yeast involves cassettes. What is meant by the term cassettes?
The gene being introduced into the genome. This is often a gene for antibiotic resistance.
620
_______sequences are amplified using PCR with primers with a 3' end complementary to the _____ and a 5' end complementary to the ______ ______.
Cassette, cassette, target gene.
621
What type of PCR os the gene KANMX4 often involved in?
Homologous recombination PCR- it is a antibiotic resistance gene.
622
How much synteny is there between mice and humans?
90%.
623
What percentage of homologous genes are there between mice and humans?
At least 99%.
624
What type of integration creates transgenic mice?
Non homologous integration.
625
In the production of transgenic mice where is the DNA microinjected into?
The pronucleus.
626
What two things are often expressed in transgenic mice?
Foreign genes and the overexpression of mice genes.
627
What do knock out mice lack?
The ability to produce a specific gene product.
628
What process happens in the mouse embryonic stem cell to disrupt the gene in knock out mice?
Homologous recombination.
629
When the cells with disrupted genes are implanted into a normal blastocyst what is produced?
Chimeric mice.
630
What dominant phenotypic marker is often used in the production of knock out mice?
White fur colour.
631
What type of mouse is created when a wild type is crossed with a chimeric mouse?
A mouse heterozygous to the mutation which is also white in colour.
632
What needs to be crossed to created a white mouse homozygous to the genetic disorder?
Two heterozygous white mice.
633
What two things can not be studied through the use of knock out mice?
Essential genes and systemically expressed genes.
634
How are essential genes and systemically expressed genes studied in mice?
A gene is flanked with loxP sites.
635
What can Cre recombinase recognise?
LoxP sites.
636
What do recombinases do?
Promote recombination between homologous sequences.
637
Once cre recombinase has caused recombination of loxp sites what happens?
The gene is excised.
638
Mice strains are generated that express cre recombinase from promoters of genes that are only expressed in _ _____ ______ ______ or after ______ ________.
A tissue specific manner, after drug treatment.
639
What are floxed mice crossed with to isolate strains that only produce cre recombinase in a tissue specific or drug responsive manner?
Transgenic mice.
640
What is synaptic plasticity?
The ability to generate new neuronal circuits in response to stimuli.
641
What is the NMDA receptor also known as?
The N-methyl-D-aspartate receptor.
642
What type of receptor is the NMDA receptor?
Glutamate.
643
What ligands can the NMDA receptor respond too?
Glycine and glutamate.
644
Where does gene inactivation occur which prevents the NMDA receptor from learning?
Hippocampus.
645
What happens when NMDA is over expressed?
The mice are more intelligent.
646
What happens when NMDA is not expressed through the formation of knock out mice?
The mice have a decreased learning ability.
