Genes, Chromosomes Flashcards
1
Q
What are ddATP nucleotides and what is their role in Sanger Sequencing?
A
ddATPs are terminator nucleotides that halt chain extension; markers on these nucleotides allow for generation of a sequence.
2
Q
How does the structure of ddATP nucleotides differ from dATP?
A
ddATP doesn’t have an OH group attached to the deoxyribose ring, preventing further chain extension.
3
Q
What are Sanger’s two greatest accolades?
A
2 nobel prizes:
1958 - structure of proteins and insulin
1977 - sequencing nucleic acid (sanger sequencing)
4
Q
How is sanger sequencing performed in a singular reaction?
A
Utilising fluorescently labelled ddNTPs, allows for the process to occur in a single capillary tube; these are then separated by length during electrophoresis.
5
Q
Give an example of a device that can electronically read DNA sequences by Sanger Sequencing:
A
ABI 3730 sequencer -> can sequence up to 800bp from a single reaction
6
Q
How long is the human genome?
A
3 billion bp across 24 chromosomes.
7
Q
What was the human genome project’s approach to sequencing the genome?
A
Extract the human genome DNA anonymously -> fragment the DNA via sonication -> select by size (via gel electrophoresis) -> 100-200kb fragments were cloned in BACs -> E.coli then used to amplify DNA.
8
Q
What are BACs (Human genome project)
A
Bacterial Artificial Chromosomes (pretty much plasmids)
9
Q
What is the loop of BAC clone by clone method? (human genome project)
A
Start with a genetic map marker -> perform PCR on known BAC library -> Identify BAC clone with genetic marker ->[ PCR using primers based on seq. from ened of BAC insert -> add to BAC library -> identify the clone containing the end seq ->] repeat.
10
Q
Summarise how BAC clone by clone method worked? (Human genome project)
A
The genome is slowly identified by the overlapping sequence of clones, which span the difference between markers on the genetic map.
11
Q
What is a genetic map?
A
A genetic map shows the relative location of markers on a genome.
12
Q
Shotgun Approach:
Celera
A
40-100Kb DNA fragment from BAC clone -> broken into 5-10kb fragments -> inserted into vectors -> 800bp chunks sequenced from either end -> generates pairs of sequences that can be pieced together computationally.
13
Q
What had shotgun sequencing been used for before the human genome project?
A
The sequencing of Haemophilus Influenzae genome.
14
Q
Why didn’t the human genome use shotgun sequencing?
A
Government funders are risk adverse.
Assembly and closing gaps was easier using clone by clone.
15
Q
What was the name of the private company that competed with human genome project?
A
Celera
16
Q
When was the human genome published?
A
February 15th 2001
17
Q
What strategy is commonly used in modern sequencing?
A
Shotgun Strategy.
18
Q
Original idea/method of Sanger sequencing:
A
To produce many fragments of DNA of varying sizes by performing PCR with free nucleotides and a specific ddATP. gel electrophoresis is then performed to identify the positions of the base attached to the ddATP.; this is repeated four times each time with different base.
19
Q
What machine uses massively parallel sequencing?
A
Illumina’s HiSeq.
20
Q
How many reads can be produced from one HiSeq run?
A
8 billion sequence reads -> each with a max length of 125bp.
21
Q
What type of amplification does illumina sequencing employ to amplify the template molecule?
A
Bridge amplification (illumina occurs on surface rather than in solution)
22
Q
Why does illumina need to amplify the template molecule:
A
The optical sensors aren’t sensitive enough to detect a signal from a single molecule.
23
Q
What are the keystages of illumina?
A
-Genomic DNA extraction
-DNA fragmentation
and size selection
-Library preparation (addition of adapter molecules)
-Cluster generation (bridge amplification)
-Sequencing by synthesis
-Data analysis
24
Q
What size fragments are used in illumina sequencing?
A
500bp
25
What is attached to DNA fragments in illumina sequencing?
adaptor sequences on the 5' and 3' end. This allows for the hybridisation of primer sequences (base pairing) -> allowing for amplification and formation of new strand.
26
How does bridge amplification work?
The template strand binds to one of the fragment strands on their adaptor region. A polymerase creates a complement of the hybridised fragment, the molecule is denatured and the template is washed away. The strand folds over and binds to a complimentary adaptor seq. A polymerase forms a complement. The molecule denatures however both strands are fixed to the surface and-so can repeat the process with other adaptor strands.
27
How do clusters form in illumina sequencing?
The template sequence fragments attach to the flow cell surface at random positions, undergo bridge amplification, forming clusters.
28
How can separate clusters be formed from one flow cell?
By controlling the concentration of template fragments across the cell.
29
What method underpins the approach of illumina sequencing?
Shot gun sequencing -> many random clusters simultaneously sequences and analysed to build sequence,
30
What approach does illumina use to build-up a DNA sequence?
USes a sequencing-by-synthesis approach.
31
What is a sequencing-by-synthesis approach?
Uses fluorescently labelled reversible terminator nucleotides which can then be cleaved and further extended. Extension process can be controlled chemically.
32
What enzyme breaks down DNA strands, strand specifically?
Endonuclease
33
In which direction does sequencing occur in illumina sequencing?
Common for it to occur in both directions
34
How far apart are reads sequenced from the same fragment in illumina sequencing?
500bp apart.
35
What is purpose of adding a 3rd primer in illumina sequencing?
For index sequencing:
Allows for different samples to be distinguished. -> maximising the data generated by the machine.
36
What generation of sequencing is illumina?
Next gen Illumina
37
What is an example of next gen sequencing?
Illumina
38
How many primers are needed for bridge amplification?
2 primers
39
What are the advantages of Second Gen Seq?
-Massively Parallel
-Much cheaper to sequence per base
"built-in" shotgun sequencing without cloning step
40
What are the disadvantages of Second Gen Seq?
-Library Prep can be expensive and slow
-Amplification of DNA fragments is required which can introduce bias against CG sequences
-Read lengths are short and smaller than sanger
41
What are the advantages of third gen seq?
- Single molecule sequencing (no amplification) -> no bias
-Real time sequences (generated during run)
-Ultra-long read of up to 50kb (PacBio) or >2Mb(nanopore)
-Can identify base modifications
42
Disadvantages of third Gen seq:
-Fewer reads per run. (more expensive per base)
-Individual reads have high error rate - (Caused by no redundancy because it's a single molecule.)
43
How can the high error rate of third gen sequencing be circumvented?
By performing multiple reads to retrieve a consensus read.
44
What are the key features of PacBio seq?
-Sequence-by-synthesis
-Fluorescently labelled nucleotides
-Single molecule at a time
-No pause required between base incorporations
-Zero-mode waveguides
-Individual reads have high error rates.
45
How are the high error rates of PacBio Seq compensated?
Circular consensus seq.
46
What is the role of zero-mode waveguides?
To allow for the detection of fluorescence at a single molecule level.
47
What is a key distinction between marker nucleotides used in third gen vs second and first gen?
The nucleotides aren't terminator nucleotides, and instead the fluorescent label is cleaved.
48
Why don't most the free nucleotides fluoresce in PacBio seq?
The illuminated region is very small surrounding the template fragment, and therefore there's only small amounts of background noise from some diffusing into the illuminated space.
49
What is the structure of a PacBio SMRT chip?
The sequences on the surface of 150,000 ZMW wells, each well has an immobilised polymerase.
50
What is the process of sequencing in PacBio SMRT chips?
Replicate template DNA molecule, incorporates fluorescent nucleotide into single DNA strand -> label is cleaved as the polymerase moves along the sequence -> sequence extension.
51
What is the process of circular consensus seq?
-Double stranded DNA -> SMRT-bell adaptors added -> creates closed loop of DNA -> Primer used to hybridise with adaptor seq -> polymerase replicates the fragment. -> at junction the polymerase displace the second strand, allowing for replication of the lower strand -> this process loops -> the subreads can then be sequences and a consensus sequence can made.
52
What are the components of Oxford nanopore?
-An artificial electrically insulating membrane
-Motor Protein (polymerase or helicase)
-CsgG pore protein
53
What is the error rate of Oxford Nanopore?
~1%
54
What can Oxford nanopore sequence?
DNA, RNA, and proteins.
55
How does oxford nanopore work?
As each nucleotide/component crosses the membrane through the CsgG pore the shift in electric current is used to deduce its identity sequentially.
56
What happens to the motor protein in oxford nanopore once sequencing has finished?
the motor protein will fall off.
57
Why are repetitive regions an issue for shotgun sequencing?
If they're large relative to the size of fragment or reads they can be difficult to locate.
58
What does software do if it can't form a continuous sequence due to repeat regions?
It will form contigs/draft sequences.
59
What is the process of resequencing?
Mapping to a reference genome, taking sequence reads, working out where they derive from the reference, differences can be compared.
60
What is meant by functional genomics?
Methods to understand genome function by mapping regions of the genome to reference.
61
What are examples of functional genomic methods?
RNAseq + Chip Seq.
62
Genomics:
The structure of the genome.
63
Transcriptomics:
Which genes are when + how much are they expressed.
64
How can the genomics of a biological sample be investigated?
Either the generation of a consensus seq. via DNA sequencing or Resequencing from a reference genome
65
Steps of Northern Blotting:
RNA extraction from sample -> electrophoresis (separate by size) -> northern blotting (Transfer of RNA to Membrane) -> tag RNA using radiolabelled probes -> visualise under x-ray
66
What is RT-qPCR?
Reverse Transcriptase quantitative PCR is a method to investigate Transcriptomics.
67
What is the process of RT-qPCR?
One gene at a time: RNA reverse transcribed into cDNA -> undergoes PCR amplification -> relative amounts of fluorescent primers will give information on relative expression of different regions at different times.
68
How are microarrays used for transcriptomics?
Spots loaded with different sequences -> fluorescently tagged mRNA nucleotides bind to complementary bases in samples -> relative fluorescence of each sample gives level of expression.
69
What are technical replicates?
Same procedure with same sample -> variation caused by equipment
70
What are biological replicates?
Independent experiments with cells from different cultures.
71
Transcriptomic experiment output equation:
Log2(full change)
72
What is the transcriptomic experiment output?
A measurement of the effect size of variation.
73
How can RNA seq. be used for transcriptomics?
RNA is fragmented -> reverse transcribed into cDNA -> sequenced. The reads are then mapped and genes that match more of the reads than other are more expressed.
73
Limitations of MicroArrays:
-Low resolution
-Exact sequence present unknown
-No info on sequences not covered by probes
-Limit to how much RNA can hybridise to a particular spot -> limits ability to distinguish expression at higher levels.
74
RNA seq vs Micro Arrays: Transcriptomics
- Both well developed with minimal technical variation
- RNA-seq has larger more dynamic range + can distinguish levels of expression more clearly
-MicroArrays only give info on preselected region
-RNA-seq is genome wide
-RNA-seq allows us to detect differences from the ref genome
75
Alternative splicing:
1 pre-mRNA -> multiple alternate spliced forms -> multiple protein isoforms
76
What is DNA methylation?
A method of modifiying/regulating gene expression wherein a methyl group is added to the 5 carbon of the cytosine base -> this down regulates gene exrpession.
77
Examples of DNA methylation:
-When genes are silences - e.g. X inactivation
-Imprinting: distinguishing maternal and parental alleles.
-In bacteria to distinguish between "self" DNA and foreign DNA.
78
What is a CpG site?
A site where a C is connected to G by a phosphate group rather than base paring.
79
What is the role of CpG islands in gene supression?
CpG islands are sometimes within promoter regions. methylation of cytosine groups will cause the gene to be repressed, however this will then spontaneously deaminate into thymine, permanently silencing the gene.
80
What is Bisulphite Conversion a method used for?
To investigate the methylation of DNA,
81
How does Bisulphite Conversion work?
DNA is treated with sodium bisulphites -> incubated -> unmethylated cytosine will convert into uracil and-so remaining cytosine bases are methylated.
82
What is RRBS?
Reduced Representation Bisulphite Sequencing.
83
What is A-tailing?
An enzymatic method used to add a non-templated nucleotide to the 3' blunt end of a double stranded DNA molecule.
84
Steps of RRBS (Reduced Representation Bisulphite Seq.)
Purify genomic DNA -> restriction enzyme digest (with CpG recognition site) -> A-tailing -> adapter ligation -> gel electrophoresis (size selection for smaller fragments)-> bisulphite conversion -> PCR amplification -> sequence.
85
How can a methylated base be identified by PacBio seq?
They will be a longer pause between two signals.
86
What is Chromatin Immunoprecipitation?
A method used to isolate DNA bound by specific proteins.
87
In Chromatin Immunoprecipitation, how is the DNA crosslinked to protein>
Protein-DNA crosslinks are formed using formaldehyde.
88
Steps of ChIP (Chromatin Immunoprecipitation)
DNA and protein treated with formaldehyde -> chromatin sheared by sonication or endonuclease (get rid of protein unbound DNA) -> use of exonuclease allows for DNA to be trimmed to the binding site -> Antibody specific to the protein of interest is bound to a membrane/matrix and used to purify and immunoprecipitate the protein and its bound DNA.
89
How can the DNA purified by ChIP be analysed?
The DNA can be purified and separated from the bound protein and then either hybridised to a microarray, or fluorescently tagged
90
What is ChIP-seq?
A method using Illumina sequencing to characterise the binding sites of a protein.
91
What is the process of ChIP-seq?
Purify DNA from bound protein -> Digest DNA upstream of the protein using 5' to 3' exonucleases. -> map sequences to reference -> both strands will have a small over hang/overlap on reference map -> this is the region the protein binds.
92
What is Chromosomal Conformation Capture?
A method of observing long range chromosomal interactions. (E.g. sequences/components very far apart in the sequence but close in proximity)
93
Give an example of a long range chromosomal interaction:
Enhancers -> similar to promoters they promote the expression of a specific gene -> however unlike promoters they are very far away in the sequence, but close by physical proximity.
94
What are the different types of Chromosomal Conformation Capture?
3C, 4C, 5C, and Hi-C
95
3C Chromosomal Conformation Capture: Outcome
3C assesses the presence of interactions between known sequences.
96
How does Chromosomal Conformation Capture work?
It looks at cross-links between "remote regions" of DNA -> the DNA is digested using a restriction digest (fragmented) -> the ends of the fragment are ligated to form closed "chimeric DNA" -> the cross-link is removed -> either purified and amplified by PCR or sequenced.
97
4C Chromosomal Conformation Capture: Outcome
Allows for the assessment of one known sequence with multiple others.
98
5C Chromosomal Conformation Capture: Outcome
Allows for many different interactions within one region to be observed.
99
Hi-C Chromosomal Conformation Capture: Outcome
Allows for the observation of every long-range interaction within the genome
100
3C Chromosomal Conformation Capture: How does it work
PCR with primers specific to both interacting regions
101
4C Chromosomal Conformation Capture: How does it work
Digestion with 4bp by restriction enzyme -> ligate to form circular DNA -> amplify by PCR with primers from one region-> Microarray
102
5C Chromosomal Conformation Capture: How does it work
Design lots of primers for many potential interacting partners -> ligation and PCR -> Microarray/sequening
103
Hi-C Chromosomal Conformation Capture: How does it work
Biotin is used to label junction -> This complex is sheared -> ligation -> travertine recognises the biotin and purifies the DNA -> adaptors are ligated + amplification _> sequencing
104
5C Chromosomal Conformation Capture: Primers
Each primer has a universal tail, meaning once they hybridise they can be amplified.
105
What is ENCODE:
The Encyclopaedia of DNA elements.
106
What were the techniques employed by ENCODE to find functional genes?
-5C Chromosomal Conformation Capture -> ChIA-PET
-DNase-seq and FAIRE-seq
-WGBS (whole genome bisulfide seq.)
-PCR
-RNA-seq
-CLIP-seq + RIP-seq
107
Role of CLIP-seq and RIP-seq in ENCODE:
TO identify proteins crosslinked to RNA
108
What was the role of DNase-seq and FAIRE-seq in ENCODE?
To look for open chromatin
109
DNase-seq: How does it work?
Exploits that when DNA is unwound it's accessible for degradation
110
FAIRE-seq: How does it work?
Crosslinks histones to DNA and identifies regions that are too tightly wound to be bound to proteins
111
Evolutionary Conservation as an indication of function:
-Histograms can be used to compare relation between organisms.
-Exons are more highly conserved between species -> due to mutations in exons likely being negative for viability
112
What are transposons?
DNA sequences that can more from one genetic element to another, containing genes additional to those require for transposition.
113
What is TraDIS?
Transposon Directed Insertion-site Sequencing
114
Structure of a transposon:
Short DNA sequence with a transposase gene flanked by two regions of inverted repeats.
115
What is the role of the inverted repeats on a transposon?
They are recognised by transposase and allow for the transposon to then move and integrate elsewhere.
116
How does TraDIS operate?
Millions of bacterial mutants are produced by inserting an engineered transposon into the cell -> genes are disrupted when a transposon inserts into them -> sequence reads are taken of each mutant -> reads are mapped to reference genome -> regions of no noise are essential.
117
What would a change in TraDIS results between environments suggest?
That specific genes are more/less essential to the fitness of the bacterium dependent on the host/environment.
118
What are the advantages of using microbes in genetics?
-Simple to maintain
-Reproduce Rapidly
-Spontaneous mutations will occur in large populations
-Easy to select
-Haploid -> phenotype of mutation is immediate
-Small genome size
119
What are known as conditional lethal mutations?
Mis-sense mutations that cause changes to protein structure/function in specific conditions (e.g. high temperature or pH)
120
What is an alternative name for a point mutation?
A single nucleotide polymorphism.
121
What causes frame shift?
Insertion or deletion of a single base pair, causing changes in the bases read by the ribosome, altering all codons downstream of the mutation.
122
What are potential causes of spontaneous mutations in microbes?
-Deamination by water
-EM radiation
-Analogue Bases
- Base Modifying Chemicals
-Transposons
-Recombination
-Lambda Red
-Intercalator insertion
123
Error Proof DNA repair:
There are many repair systems, each recognising a specific type of damage.
124
Hemi-methylated DNA:
DNA that is methylated on the parent strand, but not the newly synthesised strand.
125
What does the MUT system do?
The MUT system repairs mis-matched regions that don't base pair on Hemi-methylated DNA.
126
How does the MUT system work?
Mut S binds to the region where base pairing does not occur -> this recruits MutL and MutH -> MutL brings its domains together to form a loop with the damaged DNA -> MutH acts as an endonuclease and creates a nick -> UvrD unwinds the two strands -> an exonuclease will then cleave the erroneous DNA -> DNA polymerase 1 then ligates this gap
127
What is a thymine dimer?
Where two adjacent thymine bases on the same DNA strand base pair with eachother.
128
What does Nucleotide Excision Repair Do?
Repairs thymine dimers induced by UV damage.
129
How does Nucleotide Excision Repair work?
UvrA and UvrB recognise the thymine dimer -> they bind to it and bend the DNA -> This is then recognised by UvrC (endonuclease) -> this creates a nick either side of the dimer -> helicase then unwinds the DNA and erroneous portion is removed -> DNA polymerase 1 fills and ligates the gap.
130
What does base excision repair fix?
Portions of DNA where the base has been damaged.
131
How does base excision repair function?
Base excision repair -> DNA glycosylate removes the damaged base leaving an "AP" site-> Ap endonuclease creates nick by AP site -> UvrD helicase unwinds the DNA -> DNA Pol1 fills in the base -> ligase then ligates the strands together.
132
What does recombinational repair fix?
Gaps in strands formed during strand replication. e.g. caused by replication of strand with thymine dimer.
133
Slip-strand mis-pairing(slippage):
Tends to occur in sets of three bases in large repeat sequences -> the strand will slip back, causing the addition of an amino acid.
134
What is the term given to the bacterial use of slippage in control of gene expression?
Phase shift.
135
How does recombinational repair work?
RecA aligns homologous regions in the DNA -> finds region homologous to the gap -> strand invasion occur -> sister strand loses region of strand to fill in gap -> DNA polymerase 1 will fill in the gap of the sister strand.
136
What does the SOS repair system fix?
Used when there is extensive DNA damage across the chromosome.
137
How does the SOS repair system work?
Once activated, SulA (stops cell div), UmuDC - (encodes DNA Pol V), and UrvA (NER pathway) are expressed.
138
How is the SOS system regulated?
The system is kept of when the genome is healthy, by using a LexA repressor. When DNA is damaged ssDNA builds up -> this is recognised by RecA which will then repress LexA -> activating the SOS sytsem.
139
What is DNA Pol V not used ordinarily despite it's fast speed?
It has a high error rate.
140
What is mutation frequency?
The number of mutants within a population.
141
What is the mutation rate?
The estimation of the probability of a mutation occurring per cell division.
142
What is the difference between bacterial selection and screening?
Selection uses growth media that will only grow the mutant whereas screening both the wild type and mutant can grow however can be phenotypically distinguished.
143
Site-specific Recombination:
The phage DNA has an att site (attP) -> integrase helps the DNA integrate at the attB on the chromosome.
144
Homologous Recombination:
When two pieces of DNA with identical sequence recombine.
145
When does Homologous recombination occur?
When linear of plasmid DNA is unable to replicate but has homology with the chromosome between 500-1000Bp.
146
What is horizontal gene transfer?
Mechanism whereby genes can be spread to different cells and species -> occurs when a piece of DNA is taken into a cell.
147
What are the two functions of Recombination?
Horizontal Gene Transfer and Recombinational Repair.
148
What is the Chi-site?
A nucleotide sequence which stimulates the Rec-BCD pathway in homologous recombination.
149
How does strand invasion in homologous recombination occur?
RecB, RecC, and RecD enter at the end of the Donor DNA fragment and unwind it -> this occurs until the chi site -> the DNA is nicked and further unwound -> forms an ssDNA arm. RecA binds to the ssDNA and aligns it to a region of homology on the chromosome. Strand invasion occurs.
150
What structure forms during strand invasion during homologous recombination?
A D-loop (single strand crossover)
151
What occurs after strand invasion during homologous recombination?
RuvA and RuvB assemble at the single strand crossover and pull the donor and recipient strands in opposite directions (branch migration), an endonuclease cleave the end of the D-loop and the strands are ligated together. This leaves one crossover known as the "Holliday Junction"
152
How many crossovers are present in a D loop (single strand crossover?)
2 crossovers.
153
What are the two possible outcomes of cleaving the Holliday junction?
There will either be two identical strands of DNA or the two strands will vary with one being sandwiched by donor DNA and one sandwiched by recipient DNA.
154
What happens in homologous recombination is the donor DNA is plasmid DNA?
A co-integrate will form -> the two will merge as a singular product.
155
What occurs in homologous recombination if the donor DNA is linear?
A 2nd crossover event must occur to maintain the circularity of the bacterial genome. Some of the DNA from the linear will have been incorporated into the chromosome. However the leftover linear molecule is degraded by nucleases.
156
Summarise homologous recombination:
New DNA fragment introduced -> will be digested by nucleases unless it shares region of homology with host chromosome -> Linear DNA processed by RecBCD -> RecA binds to the ssDNA strand and aligns it to region of homology on chromosome -> Intermolecular recombination by Ruv proteins leads to formation of D-loop -> crossover is cleaved, followed by Holliday junction.
157
What enzyme cleaves the Holliday junction?
RuvC
158
What are the 3 primary components of lambda red?
Exo, Beta, and Gam.
159
Lambda Red: Exo
Exo is a 5' to 3' exonuclease that degrades the 5' ends of linear DNA to form ssDNA.
160
Lambda Red: Beta
Beta binds to ssDNA at 3' end and promotes annealing to complementary DNA. (similar to RecA)
161
Lambda Red: Gam
Binds to the host RecBCD system to inhibit its activity.
162
What is Lambda?
A bacteriophage that has its own recombination system.
163
How do we use homologous recombination to make knockout genes in labs:
Host Chromosome has 3 neighbouring genes A,B, and C. To replace B with b -> construct PCR product with 1000 base pairs homologous to Genes A and C, in between b gene with kanamycin resistant antibiotic cassette -> double recombination event occurs -> gene B will be replaced and linear bi-product is digested. -> can select for mutant using kanamycin plates.
164
Why is it beneficial to co-opt the lambda red system?
Lambda Red is a much better and faster method of gene knockout in the lab.
165
How are protein components of the lambda red system introduced into badcteria?
A plasmid delivery system is used.
Specifically plasmid PKD46 -> which has a temp sensitive origin of replication.
166
How do PKD46 operate in lambda red gene knockout?
It operates as a plasmid delivery system. The plasmid has a temperature sensitive origin of replication. At normal growth temperature the proteins are expressed (at 42'C they aren't). Each protein gene is contained within the same operon controlled by the pBAD promoter (activated by AraC TF detecting arabinose).
167
Why is the temperature sensitive nature of PKD46 important in lambda red gene knockout?
The plasmid can be controlled to prevent unwanted expression. -> once expression is halted the plasmid will be broken down by nucleases.
168
How long does the lambda red gene knockout take for E.coli?
One week.
169
How can the lambda red gene knock out be improved?
By limiting the exposure of the cell to labra red activities and include sacB based counter-selection.
170
What is sacB counterselection:
sacB is a gene from Bacillus Subtylus -> uses sucrose to make levans -> toxic to -ve bacteria .
171
What is counterselection?
Genetic engineering techniques used to eliminate specific gene fragment's containing selectable markers.
172
Example of Lambda Red more efficient method:
-Lambda red now also includes mega nuclease SceI and has a SceI cleavage site (cassettehas two cleavage sites)-> cleavage causes formation of linear donor DNA -> increases the amount of homologous DNA to the genome + sucrose to ensure leftover plasmid stops functioning -> select using kanamycin to select for successful mutants.
173
What are the two groups of transposable elements?
Insertion Sequences + Transposons
174
What are insertion sequences?
DNA sequences that can travel around the DNA but only code for their transposition.
175
Transposable Element Characteristics:
-Infrequently move (10^-7 per generation)
-Don't require homology to DNA seq.
-Utilise Transposase
-Ubiquitous
176
In which type of organisms does Transposon Mutagenesis function?
Bacteria because they are genetically tractable.
177
Structure of insertion sequences:
1-2kb, contains DNA encoding for the transposase gene flanked by terminal inverted repeats.
178
What are the two types of transposons:
Complex Transposons and Composite Transposons.
179
Composite transposon:
-Interior inverted repeats
-Additional genes flanked by degenerated inverted repeats insertion sequences on either end of the transposon.
-Unable to replicate
180
Complex transposon:
-encodes for transposase, resolvase, and more.
-All flanked by degenerated inverted repeats.
-Replicative
181
Replicative Transposition:
Transposable element is copied, with one remaining within original site.
182
Non-Replicative Transposition:
Transposable element jumps between sites.
183
What is the process of non-replicative transposition?
Transposase aligns the inverted repeats by folding the DNA -> cleaves phosphodiester bond on both strands at both side of the IS element-> 3'-OH' attack produces hairpin structure and host carrier DNA is ejected and repaired. -> hairpins are re-nicked and 3'-OH attack recipient DNA -> transposasome move to target DNA seq -> insertion.
184
What kind of cut is made to DNA by transposase?
A staggered cut to allow for ligation.
185
What is a transposasome?
The complex of transposon and transposase.
186
How do transposons change the genome even if they leave a site again?
Every jump creates duplicated regions created by transposase's staggered cuts.
187
What are polar effects:
A change to the genome that causes downstream effects to other genes (e.g. a transposon inserting in the first gene of an operon)
188
What is the effect of a transposon inserting into the gene on its function?
They will inactivate a gene, by causing a frame shift/ null mutation.
189
What is TMDH?
Transposon-mediated differential hybridisation.
190
What is TMDH a precursor of?
TraDIS
191
What is the specific about the modified transposon used in TMDH?
Instead of inverted repeats the transposons have a T7 promoter on the 5' end and SP6 promoter on the 3' end.
192
What was the goal of TMDH?
To generate a library of mutants
193
Process of TMDH:
Insert modified transposon (Tn) into Bacterium -> culture -> extract genomic DNA and fragment by restriction endonuclease. -> transcription in vivo using a polymerase for each promoter region on the Tn and marked dNTPs-> generate run-offs -> hybridise to whole genome microarray.
194
Does the lambda red system also use the chi site?
No
195
What is RNAP?
RNA polymerase/
196
Where does RNAP bind to a gene?
Upstream of the gene's promoter region.
197
Transcription Fusions:
A transcript is engineered with the promoter of a test gene and the translational elements of a reporter/marker gene to observe changes to the expression of the test gene. -> forms two proteins
198
Translational Fusions:
The promoter and translational elements of the test gene are used to drive the expression of the reporter -> therefore allows for the determination of gene expression in different environments. -> forms one chimeric protein
199
lacZ: function in lab
Encodes Beta-galatcosidase -> can observed using colourimetric assay
200
cat: function in lab
Encodes chloramphenicol acetyltransferase -> will be resistant to chloramphenicol (screening)
201
lux: function in lab
Encodes luciferase -> can be quantitatively measured -> needs O2 to function
202
gfp: function in lab
encodes green fluorescent protein -> allows for cell imaging of protein expression and localisation
203
What does a difference in the expression between transcriptional and translational fusion confirm?
That post-translational transformations are occuring.
204
What are single copy reporter fusions used for?
A method to investigate the transcriptional and translational expression of gene.
205
What is FACS?
Fluorescence Activated Cell Sorting -> to separate clones that are already activated outside the host -> isolate not-activating genes.
206
GFP promoter trap: what is this method kind of similar to?
TraDIS combined with innoculation.
207
GFP promoter trap: Process
Digest bacterial DNA and clone into promoter-less GFP plasmids -> isolate unactivated clones by FACs -> innocculated into host-> bacteria is recovered and isolate activated clones by FACs -> this indicates which genes are activated in vivo.
208
What are the 3 bacterial defence strategies against infection?
- Surface Receptor modification (prevent phage recognition)
-Restriction digest of foreign DNA (breakdown Phage DNA)
-CRIPSR-Cas (acts as memory immunity system)
209
How many bacterial species utilise the CRISPR-Cas system?
Half of bacterial species.
210
What is the CRISPR-Cas system?
A small interfering RNA system in which bacteria and archaea that survive bacteriophage attack capture a piece of the attacker's DNA to counteract future attack.
211
What does CRISPR stand for?
Clustered Regularly Inter-Spaced Short Palindromic Repeats
212
What are the 3 key features of the CRISPR system?
Adaption, Expression, Interference.
213
Structure of CRISPR loci?
-Short area on the genome with direct repeat sequences (~30bp) separated by spaces of phage DNA (20-72bp)
-Adjacent to these clusters are protein encoding genes that synthesis nuclease enzymes.
214
What is the most common cas protein used in lab genetics?
cas9 derived from Streptococcus pyogenes
215
What is the Adaption step of CRISPR-cas:
The insertion of new spacers into the CRISPR locus.
216
What is the expression of CRISPR-Cas?
Transcription of the repeat spacer array to express cas proteins
217
What is the Interference step of CRISPR cas?
The detections and degradation of foreign mobile genetic elements by CRISPR RNA and cas proteins.
218
CRISPR-cas DEPTH: Adpation
cas1,2, and csn2 form a complex and scan the phage DNA for PAM site -> initiates the insertion of small sequence into the chromosome
219
What is a PAM site:
Sequence searched for by cas1,2, and csn2 during the adaption step of CRISPR-cas.
220
CRISPR-cas DEPTH: Expression
Upon re-entry of a phage's DNA -> crRNA is transcribed from the repeat-spacer array -> tracrRNA and cas9 are also transcribed. -> RNase III will then cleave each repeat and space into different complexes each can recognise different phage DNA sequences.
221
What is the function of tracrRNA in CRISPR-cas?
tracrRNA has a role in the expression step, wherein it will bind to cas9 nuclease and base pair with repeat sequences between phage spacers, localising the nuclease to the crRNA.
222
CRISPR-cas DEPTH: Interference
Nuclease will bind near PAM site and form an R-loop -> both strands of incoming DNA then cleaved using the nuclease's two nuclease domains (RuvC and HNH) -> results in a double stranded break -> causing the phage DNA to "Die".
223
What are the improvements made to the CRISPR system when co-opting the mechanism to modify genomes?
tracr RNA is fused to guide RNA -> therefore only one piece of RNA needs expressing (they are linked by a linker RNA loop.
224
How many methods of CRISPR-cas9 are there to modify the genome?
3 methods.
225
What are the 3 possible uses for cas9 in gene modification?
-Cas9 can introduce blunt breaks to the DNA -> can then be filled either by homologous recombination or non homologous end joining
-Cas9 creates a nick -> forms staggered breaks -> more specific than method 1 -> as binding occurs twice.
-Cas9 acts as a DNA binding protein to down or upregulate transcription when bound to activator or repressor domains.
226
What modifications are made to cas9 for its use as a nickase or DNA binding protein in gene modification?
-For cas9 to become a nickase it loses one of its nuclease domains
-For cas9 to become a DNA binding protein it loses both of its nuclease domains.
227
What are some issues associated with CRISPR-Cas9 gene modification?
The spacer RNA can base pair with different sites in the human genome -> can cause off-target effects that can lead to death (circumvented by cas9 nickase)
228
In which CRISPR systems in cas9 found?
type 2 CRISPR systems.
229
What is a phagemid? (and what does it contain in CRISPR-cas gene modification?)
A large plasmid with genes that encode for phage particles and the genes of CRISPR-cas system -> encapsulated in capsule specific to receptors on target cell.
230
What are the 3 elements required for a phagemid delivery system to work?
crRNA (target the gene), tracr RNA (binds to the repeat beside spacer to localise cas9 nuclease) and the cas9 gene (will induce a double stranded sequence break)
231
What is and Origin of Replication? (Ori)
An AT-rich sequence that initiates the replication of an entire plasmids DNA sequence.
232
What are replication bubbles?
Structure/region formed when double helix H-bonds break between nucleotide -> where bidirectional replication occurs.
233
What is the function of the origin recognition protein complex (ORC)?
To make sure DNA replication is linked to the cell cycle and controlled.
234
What is an ARS?
Autonomously replicating sequence
235
What attracts the ORC and helicase enzymes to the bacterial genome to initiate replication?
The ORC binds to an 11bp autonomous consensus sequence -> attracts helicase enzymes to the upstream AT rich region.
236
How many origins of replication are on a eukaryotic genome?
Multiple, which are activated during S-phase
237
What are characteristics of Metazoan sequences?
-Poorly conserved between animals
-Link to transcriptional activity
-Some used often and some used randomly
-No clear consensus sequence
238
What are OGREs?
Origin G-rich repeated elements
239
What are the features of OGREs?
-G-rich octamers found at many origins
-forms G-quadruplex structures upstream of initiation sites
-Important in regulation
-Deletion reduces origin activity
-nucleosome free -> remove nucleosome from initiation sites.
240
How can the evolutionary relation between different organisms be compared?
-Isolate nascent DNA seq -> synthesise with BrdU -> isolate modified DNA seq -> Isolate DNA associated with an RNA primer -> extract total DNA -> isolate small DNA fragments by gel electrophoresis -> treat with lambda exonuclease to degrade broken DNA ->Okazaki fragments with RNA primer then amplified by PCR and sequenced. -> compare to libraries.
241
What is nascent DNA?
Newly replicated DNA.
242
What produces Okazaki fragments?
By the lagging of DNA replication
243
What is the mitotic spindle?
A cytoskeletal structure, made of most microtubules, which associates with kinetochores on the sister microtubules.
244
What is the kinetochore?
An elaborate kinetochore protein complex that recognises centromeric chromatin and attaches the centromere to the microtubules, allowing for the segregation of sister chromosomes/chromatids.
e.g. Ncd80
245
What are nucleosomes?
Complex where DNA is wrapped around 8 histone proteins -> which can be modified by methylation and acetylation.
246
What is CENP-A?
a histone that binds to alphoid satellite DNA of AT-rich sequences. each repeat is 171~bp -> marks centrosomes.
247
What are telomeres?
Highly conserved DNA sequence at the end of chromosomes consisting of a number of repeats and a 3' overhang.
248
What is the Hayflick limit?
Differentiated human cells can only undergo mitosis a certain number of times before they stop dividing -> occurs when telomere length drops between certain threshold -> triggering senescence;
249
What is senescence ?
The loss of a cells ability to grow and divide -> mechanism to protect genome and reduce cancer risk.
250
Problems of telomere length?
- 3' overhang is caused by lack of adjacent okazaki fragments.
-DNA polymerase always build DNA 5' to 3'.
251
What are the two components of the telomerase enzyme?
Protein component TERT and Telomerase RNA component TERC.
252
What is the role of TERC in telomerase?
TERC provides a template for the reverse transcriptase enzymatic activity
253
What is the role of TERT in telomerase?
TERT acts as Telomeric Reverse Transcriptase.
254
Mechanism of telomerase:
TERC base-pairs with the 3' overhang -> elongation of overhang using TERC template -> telomerase translocates further out along the 3' -> RNA removed and synthesis of the second strand by DNA polymerase occurs, using overhang as template.
255
In which cells are telomerases active?
-Single-celled species (always)
-Stem cells and germline cells
256
What can speed up the shortening of telomeres?
Oxidative stress (build-up of single-stranded breaks)
257
What condition is associated with elevated telomerase activity:
Cancers~ -> 90% of cancer cells have elevated telomerae activity -> allowing for continuous proliferation -> often caused by mutation in the promoter of telomerase coding gene.
258
Why is the telomerase gene useful in the treatment of cancer?
Most cancers have uncontrolled cell proliferation due to elevated activity of the telomerase gene -> therefore it's used as a drug target to interfere with its expression and reduce the growth and proliferation of cancer cells.
259
Immortalised Cell Lines:
Some cells can be cultured infinitely -> e.g. most cell lines are derived from cancer cells (e.g. HeLa cells)
260
Telomere-binding proteins: Role
Form Shelterin complex -> responsible for the protection of chromosome ends and promote the formation of the t-loop.
261
What is the C-value paradox?
The complexity of an organisms doesn't correspond to genome size. -> massive variation in sizes between similar living organisms.
261
What is a T loop?
A special tertiary structure in the telomeres where the telomere DNA loops to protect the 3' DNA overhang, conserving it for telomerase activity.
261
What are 3 of the telomere-binding proteins in humans?
TRF1, TRF2, and POT1.
262
What is meant by humans having a low gene density?
They have a very small proportion of protein-coding sequences (exons) compared to non-coding (intron) regions.
263
What are microsatellites?
Repeated sequences of 1-13bp that repeat for less than 150 repeats.
264
What are minisatelittes?
Repeated sequences off 14-100bp that form tandem arrays of 1-5kb
265
How much of the human genome is formed by satellite sequences?
6%
266
What is the DNA sequence of the centromere?
There's a homogenous higher order alpha satellite array (200-5000kb), each(1-3kb) with a slightly divergent sequence of Alphoid satellite DNA sequences (171bp).
267
What disease can be caused by the presence of a specific repeat sequence?
Huntington Disease is caused by CAG repeats within a certain gene sequence.
268
What is DNA finger printing?
Use of polymorphism in minisatellite length between individual to identify them -> used for paternity, forensics, and genetic mapping.
269
What is the process of DNA finger printing?
Extract DNA -> digest with restriction digest -> separate fragment on an agarose gel -> southern blot using minisatellite as probe-> observe characteristics of bands for each individual
270
What can mistake in replication can cause elongation of repeat sequences?
Replication slippage.
271
What is the potential effect on satellite regions if the chromatids misalign during metaphase?
One gamete will have more copies and the other will have fewer.
272
DNA transposon mechanism:
Transposase is transcribed and translated by host machinery -> transposase binds to inverted repeats at the end of the transposon sequence -> enzyme cuts the DNA to remove the transposon from its original genomic location -> creates break at the target site to allow for insertion of the transposon into new location.
273
What kind of cut does transposase make to the target DNA?
Staggered cuts (of around 5bp) -> the blunt transposon then inserts and the gaps are ligated.
274
What are LTR regions in LTR retrotransposons?
Long Terminal Repeats-> These palindromic regions flank the internal coding region -> contain binding sites for enzymes and regulatory elements
275
LTR retrotransposon Mechanism: "copy and paste"
Generation of RNA molecule and protein products by host machinery -> Reverse transcriptase forms cDNA from mRNA template -> RNAseh degrades the template -> dsDNA molecule formed -> integrase guides dsDNA to insertion site -> insertion
276
What does LINE stand for?
Long Intersperse element.
277
What is ORF1?
RNA-binding protein
278
What is ORF2?
Reverse transcriptase and DNA endonuclease.
279
What key proteins are involved in the LINE transposon mechanism?
ORF1, ORF2, poly A,
280
LINE transposon mechanism:
Transposon is transcribed and translated -> ORF1 binds to LINE RNA< ORF2 binds LINE polyA and binds to polyT DNA seq. -> RNA transported to the nucleus -> ORF2 nicks DNA -> Reverse transcription of LINE RNA -> polyA fills in gaps using host DNA template ->ligation
281
What are the two mechanisms by which transposition of transposons occur?
Cut and paste vs copy and paste
282
Autonomous transposons:
Has its own transposase gene and therefore can activate its own transposition
283
Nonautonomous transposons:
Need to use transposase enzyme coded for by other transposons.
284
Examples of system with both autonomous and nonautonomous transposons.
Ac/Ds system, and LINEs and SINEs
285
What is a SINE?
Short interspersed element -> nonautonomous transposon and requires enzymes from LINEs to function.
286
What do SINEs contain which allow them to bind to ORF1 and ORF2?
AT-rich sequences.
287
Why do RNA transposons increase in overall number?
Due to their copy-paste mechanism -> leaves sequence in original site and in new insertion site.
288
How do DNA transposons increase in number?
If a transposon has already been replicated but then transposes from one fork further back in the sequence -> one daughter chromosome will have one copy and the other will have two copies at the original and new location.
289
Why aren't transpositions very impactful (short term):
They don't occur regularly due to many cellular defence systems -> will only be passed down if occur in the germline genome.
290
P elements: What is it an example of?
P elements are an example of a transposon control mechanism. P+ drosophila (wild type) -> low mutation rate if P+ egg
If P- egg the fly transposes at very high rates -> high mutation and loss of fertility (unviable)
291
What are the 3 mechanisms of gene duplication?
-Replication Slippage
-Unequal crossing over
-Retro Transposition of an mRNA.
292
What are the two outcomes of Exon Shuffling?
An exon codes for a new
293
What is exon shuffling?
Exon shuffling is the recombining of existing gene parts to create new genes with altered functions. Playing a key role in evolution.
294
What is a pseudogene?
A sequence derived from a functional gene however is not capable of transcribing for a functional protein.
295
How can pseudogenes be formed by Retro-transposition?
The mRNA might be spliced before being reverse transcribed -> altering the sequence -> no longer functional as a coding sequence.
296
What occurs in intender/correct exon shuffling?
Exons of different genes both flanked by the same transposon are swapped over when recombination occurs in the transposon sequence.
297
What are two ways Exon shuffling can occur by mistakes in transposition?
-An exon between two DNA transposons might be excised from the genome and inserted at a new location. (caused by transposase misreading and moving the entire length as one transposon)
-A LINE transposon might use polyA signal of neighbouring gene instead of its own -> both the LINE and exon will transpose.
298
What are orthologous genes?
The same gene in two different species; e.g alpha tubulin is shared between multiple species.
299
Paralogous genes:
Two copies of a gene within the same species. e.g. alpha and beta tubulin (evolved by duplication and then subfunctionalisation)
300
Neofunctionalisation:
One copy of a duplicated gene gains a new function
301
Subfunctionalisation:
Each copy of a duplicated gene specialises to allow for adaptation for different conditions -> allowing flexibility
302
What are the 3 different fates of a duplicated gene?
-Degradation of the copy
-Neofunctionalisation
-Subfunctionalisation
303
What is a tandem repeat?
A sequence of two or more DNA bases that are repeated adjacently.
304
What causes the degradation of gene copies within species over time?
Because of redundancy there is less selection pressure on the genes; Therefore mutations can accumulate on a copy without affecting viability -> accumulate leading to loss of function.
305
What causes the formation of conventional pseudogenes?
The degradation of duplicated gene copies.
306
What does each branch on a phylogenetic tree correspond to?
A different duplication event.
307
Why is gene expression control important?
To allow for adaption to conditions, specialisation, sensitive control of reactions, +loss of regulated controls often leads to defects and cancers.
308
TATA box:
Nucleotide sequence important in the targeting of polymerase to the transcription site.
309
What are CpG islands?
Clusters of dinucleotide sequence CG -> responsive to methylation and-so can be modulated to downregulate expression of downstream sequence.
310
What are enhancers?
similar to promoters they promote the expression of a specific gene -> however unlike promoters they are very far away in the sequence, but close by physical proximity.
311
What are silencer elements?
Regions recognised by factors that inhibit transcription.
312
What is UAS?
An upstream activating sequence
313
What is URS?
An upstream repressor sequence.
314
What do all yeast genes contain?
A UAS or URS and a TATA box promoter element.
315
What is the process of a Primer Extension Assay?
Reverse transcriptase enzyme extend DNA primers which then anneal to RNA to produce a single-stranded complimentary cDNA strand. -> lengths of the cDNA products can be measured by PAGE -> positions of the 5' end of the RNA can then be inferred.
316
What is PAGE?
Polyacrylamide gel electrophoresis.
317
What method of assay can be used to define regulatory regions within promoters?
Deletion analysis using a reporter genes assay.
318
Reporter Gene Assay:
DNA fragment containing promoter region of gene upstream -> fusions(transcriptional or translational)-> expression observed
319
Luciferase (lux):
Marker protein/gene that fluoresces orange.
320
What does the Linker Scanning Mutagenesis of promoters method do?
Assays short regions within the promoter for their role/requirement in promoter regions (when compared to a reporter gene)
321
What is the process of Linker Scanning Mutagenesis?
Chosen promoter region is restricted and randomised and linker sequences are inserted to generate a series of constructs to be assayed -> constructs are mapped and constructs not supporting expression will mean the mutagenised linker has interrupted a key promoter sequence in that loci.
322
Enhancer Trapping:
Transposons with reporter gene is inserted randomly across genome of eukaryote -> cells with upregulation are selected -> DNA extracted cloned and sequenced -> this is the mapped to identify regions in the genome that are in proximity to enhancers.
323
What are Specific Transcription Factors (sTFs)?
sTFs are factors that have a modular domain structure that mediate changes in RNA polymerase activity by combinatorial control.
324
How many RNA polymerase enzymes are used by prokaryotes?
1 which is directly targeted by proteins
325
What are gTFs?
General transcription factors which directly bind to polymerases to modulate transcription.
326
How are gTFs formed?
The hierarchal assembly of substituent protein groups.
327
What complex forms upon binding of polymerase to a promoter?
The mediator complex.
328
What is the mediator complex?
A large protein complex that assembles on the RNA polymerase upon its binding to the promoters -> it mediates signals from transcription factors to up/down regulate transcription.
329
How is the chromatin structure altered to modulate transcription?
Protein factors are recruited to the chromatin structure to alter its packing -> increasing proteins increases packing which makes gene less accessible for transcription.
330
How are functionally linked genes co-ordinated by sTFs?
sTFs can be organised into families of monocistronic transcription factors, each family specific to promoters of genes with related functions (regulons).
331
What are regulons?
Families of genes coordinately regulated at the level of transcription by sTFs.
332
What is cDNA?
Copy DNA or Complimentary DNA e.g. cDNA can be produced from the reverse transcription of mRNA.
333
Yeast GaL Gene Switch: Function
Allows for yeast to grow in both medium containing glucose or galactose -> by expressing enzymes required for the breakdown of galactose when in its presence. -> to ensure resources and energy aren't wasted by their synthesis.
334
What is the name of the pathway in which galactose is broken down?
The Leloir Pathway
335
Yeast GaL Gene Switch: GaL 1 Function
GaL1 is a kinase that phosphorylates Galactose into Gal-Pi
336
Yeast GaL Gene Switch: GaL7 Function
Converts Gal-Pi into Glc-Pi
337
Yeast GaL Gene Switch: GaL10 Function
Restores UDP-Gal into UDP-Glc
338
Yeast GaL Gene Switch: GaL2 Function
Permease required for Galactose to enter the cell
339
Yeast GaL Gene Switch: GaL3 Function
Galactose binding protein which will bind to Gal80 when bound to Galactose
340
Yeast GaL Gene Switch: GaL80 Function
Regulatory Protein which will bind to GaL3 when galactose levels are high in the cytoplasm, but when levels are low will enter the nucleus and repress Gal4 and promotion at the UAS.
341
Yeast GaL Gene Switch: GaL4 Function
GaL4 is an sTF that binds to the GAL gene UAS -> sti,ulating the activation and expression of Gal responsive genes.
342
Yeast GaL Gene Switch: Where does Gal80 bind to decrease GAL gene expression?
The activation domain of GaL4.
343
Yeast GaL Gene Switch: What region of the DNA is targeted for switching?
The UAS (Upstream activation sequence)
344
What is meant by transcription factors being modular?
Different parts of sTFs can be dissembled and each domain can be separated and have distinct functions.
345
Yeast GaL Gene Switch: How can you use this system to investigate the modular nature of transcription factors?
The Switch Gene acts as the reporter gene, and you use this reporter gene to assay many different mutations of Gal4 sTF's encoding gene to see if they continue to bind to the DNA and activate lacZ.
346
What domains are typically in sTFs?
A DNA-binding domain and an activation/repression domain -> separated by flexible regions.
347
What is the function of a Yeast Two-hybrid assay?
To analyse protein-protein interactions
348
Give a very brief summary of a yeast two-hybrid assay:
A distinct activation domain and DNA binding domains of Gal4 are fused to separate fixed bait and variable prey proteins. Expression of reporter gene reflects successful reconstitution of Gal4 by the proteins' interaction.
349
Why can the DNA binding domain and activation of Gal4 be split?
Because Gal4 is a transcription factor and therefore modular.
350
What are the reporter genes that can be used in a Two-hybrid assay?
HIS3, ADE2, and LacZ
351
Two-Hybrid Assay: ADE2
Yeast with ade2 mutants develop red pigments, ADE2 expression suppresses this phenotype.
352
Two-Hybrid Assay: HIS3
HIS3 expression used to complement auxotrophic allele _. increasing concentration of inhibitor 3-AT -> allows for selection of clones that express higher levels of HIS3.
353
Substrate is used to detect/observe lacZ expression?
The chromogenic substrate X-gal -> will express a chromophore blue/green colour if expression is positive.
354
What is the benefit to cell systems of using a combination of enhancers and repressors?
The combination of both allows for a wide range of intermediary levels of expression -> allowing for more sensitive control.
355
What is meant by the combinatorial control of sTFs?
sTFs often operate as homo- or hetero- dimer combinations. Operating as a heterodimer effects the gene targeted and the sTFs effect. e.g. some heterodimers recognise the same DNA sequence, however are responsive to different conditions -> allowing for the activation of gene required in a range of conditions but not all.
356
What is the homeodomain structure in sTFs?
A structure within some sTFs similar to the helix-turn-helix motif in prokaryotic DNA-binding proteins -> function as dimers with each subunit having a recognition helix that will interact with the opposite side of a helical turn in DNA -> allowing the fold to slot into a major groove.
357
What does the order of hox genes along the chromosome correlate to?
Their spatial expression pattern along the axis of the developing embryo.
358
What is the name of the directional axis on bilateral organisms?
The anterior-posterior body axis,
359
Why are Homeobox genes readily identified?
They have a high level of nucleotide sequence conservation within the homeodomain box.
360
Leucine Zipper Domains: Structure
Coiled-coil dimers formed by extended amphipathic alpha-helices. Contain a leucine residue at every 7th position along the helix, forming 3-D structure along the hydrophobic surface.
361
What are Basic Leucine Zipper Proteins (bZIP)?
Family of transcription factors that comprise of a leucine zipper to allow dimerisation and a region rich in basic residues to allow DNA binding.
362
Why do nucleic binding proteins tend to have basic properties?
To allow the binding to phosphates.
363
Zinc-Finger Domains:
Most common DNA-binding domain.
-> Zinc finger domains are folded around central zinc ion through contacts with either two cysteines and two histidines or 4 cysteines. Amino acids on recognition helix make contact with nucleotides.
364
What's the difference between function of C4 and C2H2 zinc fingers?
C2H2 finger domains have predictable binding specificity allowing for proteins to be designed that recognise specific sequences. C4 domains are found in nuclear receptors -> class of sTFs that bind to small lipid-soluble hormones.
365
RNA Recognition Motif (RRM) interaction with RNA:
Hydrophobic stacking of nucleotide bases and two conserved aromatic residues within the beta sheet.
Other interactions include hydrogen bonding between basic residues and the phosphodiester backbone.
366
RNA-binding proteins:
Proteins containing multiple RNA-binding domains with their interaction with RNA being dependent upon these synergistic individual interactions.
367
What is the RNA recognition motif?
Common eukaryotic domain which interacts with and attaches to RNA.
368
Band Shift Assays: Function
Detect nucleic acid binding activity, purify nucleic acid binding protein, or determine the specificity and affinity of a given interaction.
369
Band Shift Assay: Process
Labelled DNA or RNA is mixed with cell extract or purified protein and resolved by gel electrophoresis -> different shifts caused by different proteins -> protein can be identified by incubating the reaction mix with a protein-specific antibody -> upon binding reduces mobility -> "supershift"
370
What are the two regulatory pathways of sTF activity?
Either:
Extracellular signal binds to receptor -> signal transduction pathway activates an sTF -> modulates activity of gene.
or small lipid-soluble hormone passes through the plasma membrane -> binds to the sTF -> modulates activity of gene
371
What kind of change occurs to activate an sTF?
The sTFs activation domain is structurally altered to allow for interactions with coactivators/repressors -> allows for function to be carried out.
372
Example of sTF binding to coactivator:
cAMP response element binding protein (CREB) interacts with its coactivator CREB binding protein (CBP) upon phosphorylation by protein kinase A.
373
Nucleosome Structure:
Octameric structure -> 2 copies of 4 different histone proteins (H2A, H2B, H3, H4).
Moveable along the DNA and have regular spacing between nucleosomes.
Occupy 10 Bp (one helical turn)
374
What structure in the nucleosome allows for interactions between histones and other proteins?
Core histones have flexible N-terminal regions known as "tails" that mediate these interactions.
375
Chromosome Painting:
A fluorescence in situ hybridisation (FISH)-based technique that visualises individual chromosomes within the cell nucleus.
376
What is a chromosome territory?
A restricted sub-region of a cell nucleus in which a specific chromosome is restricted to.
377
What movements within the nucleus observed by chromosome painting are associated with transcription activation?
The movement of specific genes away from the chromosomal territory into regions that are accessible to transcription machinery.
378
What are the sites in which genes move to for transcription activation?
Transcription Factories.
379
What are topologically associated domains?
Spatially restricted regions for the chromosome that undergo coordinated gene expression and replication.
380
What establish TAD (Topologically associated domain) boundaries?
CTCF and cohesin -> gene looping
381
Lamina associated domains:
Contain transcriptionally silent DNA -> serve function to organise chromosomes.
382
What are epigenetic markers?
Structures that influence gene expression and chromatin structure.
383
What component of histone proteins is often post-translationally modified?
Their N-terminus and C-terminus tails. More specifically lysine side chains (e.g. for acetylation, methylation, and ubiquitylation)
384
What enzyme group is responsible for the addition of acetyl group to lysine groups of Histone tails?
Histone acetyltransferases.
(Neutralise positive charge)
385
What enzyme is responsible for reversing the acetylation of lysine residues on histone tails?
Histone deacetylase complexes.
386
What enzyme group is responsible for the addition of methyl groups to lysine residues on histone tails?
Histone methyltransferases.
387
What enzyme is responsible for reversing the methylation of lysine residues on histone tails?
Lysine-specific demthylases
388
What is the impact of the methylation of lysine residues on histone tails?
Prevents the acetylation of the tail, protecting the positive charge.
389
Heterochromatin:
-Densely packaged chromatin -> darker staining -> found nearer periphery of nucleus -> transcriptionally inactive.
390
Constitutive Heterochromatin:
Includes repetitive sequences -> including telomeres, centromeres, and satellite DNA -> stably inherited through cell division and has very little transcriptional activity.
391
Facultative Heterochromatin:
Is readily able to decondense and change between states.
392
What specific histone markers are associated with actively transcribed DNA?
H3K9Ac and H3K4me
393
What specific histone markers are associated with heterochromatin?
H3K9me and H3K27
394
Chromatin Remodelling Complexes:
Slide DNA sequences into nucleosomes using ATP-dependent helicase activity -> sliding the nucleosome along the DNA
395
How are chromatin remodelling complexes recruited to the DNA?
CRMs are recruited to promoter regions through interactions with sTF factors.
396
How do activators direct histone acetylation? example
Histone acetyltransferase (HAT) complex with SAGA coactivator -> SAGA is recruited to UAS element through interactions with transcriptional activators e.g. Gcn4 and Gal4. -> Acetylation leads to nucleosomes loosening providing the recognition sites for proteins with bromo-domains.
397
What are the 3 examples of gene activation through acetylation of histones?
HAT and SAGA,
TFIID, and CRMa
398
TFIID: What is it?
A key transcriptional factor recruited to hyper-acetylated regions of DNA -> allows recruitment of coactivator which allows for acetylation of neighbouring regions -> positive feedback.
399
Chromodomain:
A domain associated with the remodelling and manipulation of chromatin.
400
What is a Chromo Shadow Domain?
A protein-protein interaction domain which can form multiple interactions with other Chromo Shadow Domains -. pulling nucleosomes together.
401
What is the role of Heterochromatin protein 1(HP1)?
HP1 binds to specifically methylated histone tails -> draws together adjacent nucleosomes carrying H3M9me mark
402
What are gene insulators?
DNA protein complexes that function to block the spread of heterochromatin into actively transcribed genes, and to block the cross-activation or repression of enhancer or silencer elements.
403
What are boundary elements?
Structures and complexes that bind to specific regions of DNA to prevent heterochromatin spreading. (e.g. insulator elements)
404
Nuclease Sensitivity Assay:
Chromatin is digested with micrococcal nuclease enzymes -> DNA fragments with one or more nucleosomes can be resolved through agarose gels -. hybridisation with gene-specific probes reveals whether gene has high or low nucleosome density. -> ChIP techniques (identify modifications) -> isolate cross-linked DNA seq. -> analysis.
| Function is to identify which regions are hetero/euchromatin
405
What assay is used to investigate the chromatin structure of specific genes?
Nuclease Sensitivity Assays -> using ChIP techniques
406
What does it mean if processes are coupled?
Processes occur simultaneously and are linked to one another.
407
What is a CTD?
C-terminal domain
408
Why is the CTD region of RNA polymerase differentially phosphorylated?
The CTD's amino acids are capable of being phosphorylated, and due to the dynamic nature of phosphorylation the pattern of this will change dependent on the RNA sequence.
409
What repeated structure makes up the RNA polymerase CTD?
The heptapeptide YSPTSPS is repeated.
410
What are the two forms of serine present on the CTD?
Serine 2 (more prevalent on 3' end)
Serine 5 (more prevalent on 5' end)
411
What is the function of the CTD during RNA processing?
Capping modification has affinity for CTD when ser5 is phosphorylated -> meaning that capping will occur during transcription.
412
What is the role of the RNA polymerase CTD?
To allow for the coordination of processing factors using their affinities for different phosphorylated residues.
413
What is heteronuclear RNA (hnRNA)?
Rapidly degraded newly synthesised RNA -> only a small fraction of which become mRNA.
414
What are the two reactions involved in pre-mRNA splicing?
Transesterification and Spliceosome assembly
415
Tranesterification: (pre-mRNA splicing)
Two steps -> step 1 an ester linkage is formed between the 2' hydroxyl group of the branchpoint adenosine and the phosphate group at the 3' end of the 5' exon, while the phosphoester linkage is lost. -> Step 2 an esterlinkage is formed between the hydroxyl group on the 5' exon and the phosphoester on the 3' exon. -> this is then degraded by debranching enzyme and exoribonucleases.
416
What is the function of the first step of transesterification? (pre-mRNA splicing)
The generation of a free 5' exon and a 3' exon-lariat intermediate
417
What is the spliceosome?
A large dynamic RNP complex that carries out nuclear pre-mRNA splicing
418
What are "snurps"?
Small nuclear RNP complexes
419
What are the components of a spliceosome?
Snurps (U1, U2, U4, U5, and U6), and protein complexes (e.g the PRP complex)
420
What is the mechanism by which the spliceosome assembles on the mRNA called?
The spliceosome assembly cycle.
421
What is the the process of the spliceosome assembly cycle?
U1 binds to 5' splice site -> U2 binds to branchpoint -> combination forms A complex. U4/U6 complex is formed by bp between them -> complex associates with U5 -> forms tri-snurp -> recruited by spliceosome. -> Major rearrangement driven by RNA helicases to form B complex -> U6 bp with U2 and interacts with 5' splice site -> displacing U1 and U4 -> Prp19 recruited by U4 -> complex disassembles once RNA helicase is absent
422
What occur throughout the spliceosome assembly cycle to ensure the fidelity of pre-mRNA splicing?
Checkpoints.
423
Kinetic Proofreading model:
ATP hydrolysis by Prp16 provides a molecular clock that selects correct substrates during productive splicing -> ensure fidelity.
424
In Vitro Splicing Assays: Process
Radiolabelled substrates containing a single intron -> Changing levels of substrate intermediates and products can be followed as a function of time -> by depleting nuclear extracts of specific proteins using antibodies -> the function of proteins can be tested.
425
What forms of nucleic acids show decreased mobility through gels?
Circular forms of nucleic acid.
426
What is spliceosome assembly dependent on?
Splicing factors.
427
What is the role of U2 (U2AF) in the spliceosome assembly cycle?
U2 is an auxillary factor that binds the polypyrimidine tract and aids U2 snRNP binding to the branchpoint.
428
What is splicing by intron definition?
(Occurs for short introns mRNAs containing a small number of introns)The 5' and 3' splice sites are recognised and the spliceosome is recruited through the factors that bridge U1 and U2 snRNPS.
429
What is splicing by exon definition?
(Occurs in large pre-mRNAs that contain long introns and short exons) Complexes are assembled across exons that link the U1 and U2 snRNPs.
430
What are SR proteins?
Serine/arginine rich proteins that mediate both protein/RNA and protein/protein interactions (recognise splicing factors)
431
What complexes are assembled by SR proteins?
cross-exon recognition complexes -> connect the U2 and U1 snRNPs at the 5' and 3' ends.
432
What is a splicing method that only occurs in pre-mRNA with very large intron sequences (50kb <)?
Recursive splicing -> the intron is removed in a stepwise manner -> by regenerating a functional 5' splice site at the spliced junction.
433
What is alternative splicing?
Alternative splicing patterns exist for the same gene -> this can be used to form protein isoforms or regulate the expression of the protein post translation.
434
What governs the choice of splice site during pre-mRNA splicing?
Alternative splicing factors -> either activate or suppress splicing at either site -> bind to RNA sequences close to the 5' or 3' splice site.
435
How many genetically inherited disease reflect mutations affecting alternative splicing:
1/3, 15% of which are cause b y defects in exon definition,
436
Example of Genetic Disease that results from mutations affecting alternative splicing:
Spinal Muscular Atrophy
437
Spinal Muscular Atrophy:
Recessive genetic disease characterised by a loss of function of motor neuron proteins caused by a point mutation in SMN1 into SMN2 within exon 7 -> decreases synthesis of SMN protein by 80%.
438
What is Sxl (sex lethal)?
a splice site suppressor that reinforces its own expression in female flies by binding to intronic silencing elements at the 3' end of inron 2 of the sxl transcript -> causes exclusion of "poison exon".
Also acts as a translational repressor.
439
What is Tra? (Drosophila)
A splice site activator that is expressed specifically in female-flies. The activator together with tra2 promotes the inclusion of exon 4 (doublesex) transcript -> causes expression of sex specific isoforms of Dsx -> controls set of genes that differentiate male and female flies.
440
Trans-splicing:
The spliceosome mediates splicing between one RNA containing a 5' splice site and another transcript containing the branch point at the 3' splice site.
441
What is the purpose of the at-ac spliceosome/
Allows for the splicing of sequences without the conserved GU and AG sequences at the splice sites.
442
What complexes perforate the nuclear membrane, allowing for the directional movements of protein and RNA?
Nuclear Pore Complexes (NPCs)
443
Nucleoporins:
A family of around 30 proteins which are the main components of Nuclear Pore Complexes (NPCs)
444
What proteins line the interior of an NPC?
FG-repeat nucleoporins -> form a hydrophobic gel
445
What is the key function of FG-repeat nucleoporins?
They interact with proteins and RNA complexes allowing them to translocated by facilitated diffusion.
446
What are FG-repeat nucleoporins?
Nucleoporins which have repeats of the FG dipeptide -> form a hydrophobic plug within the pore -> makes the pore size selective.
447
In what state do proteins and complexes translocated through NPCs?
A folded state.
448
What is the name of proteins that mediate nuclear transport through nuclear pores?
Karyopherins (importins or exportins)
449
What is the function of GEF proteins?
A guanine exchange factor -> Ran-GDP converts into RanGTP in the nucleus
450
IN vivo nuclear protein import assay: Function
To observe whether or not transport across a nuclear membrane of a specific protein will take place.
451
IN vivo nuclear protein import assay:
Cells resuspended in digitonin -> to permeabilise the cell membrane -> ruptures the cell surface membrane but doesn't interfere with the nuclear membrane -> cytoplasmic material can then be extracted -> plasma membrane re-sealed -> marked protein/molecule injected into cell -> observe.
452
What is the function of Ran in nuclear transport?
Ran is a GTPase that associates with importin or exportin to aid transport.
453
What is the function of Ran-GAP?
Converts RanGTP -> RanGDP in the cytoplasm.
454
To what state of Ran do exportins associate with?
RanGTP
455
To what state of Ran do importins associate with?
RanGDP
456
What interactions of Importins are mutually exclusive?
Association to Ran and association to cargo.
457
What is the key difference in mechanism between importins and exportins?
Importins release cargo upon association to RanGTP, whereas Exportins take-up cargo upon association to RanGTP.
458
What are NLS and NES?
Nuclear localisation signals and nuclear export signals -> short polypeptide sequences that mediate the transport of nuclear proteins into and out of the nucleus respectively.
459
What molecules are transported across the nuclear membrane independently of Ran-GTP cycle?
mRNAs
460
What transporter protein mediates the transport of mRNA molecules across the nuclear membrane?
Nuclear export factor 1 (NXF1)
461
What can be done to block the export of mRNA from the nucleus into the cytoplasm?
The genetic depletion of NXF1
462
What is the exon junction complex?
A protein complex which forms on a pre-mRNA strand at the junction of two exons joined together by splicing.
463
What complex allows for NXF1 to interact with mRNA?
The ref component of the exon junction complex -> ensures spliced mRNA is exported.
464
How does the exon junction complex allow for the interaction of NXF1 and mRNA?
EJC has a Ref component -> interacts with NXF1 -> releases RNA from EJC -> allows for NXF1 to bind to RNA. -> forms NXF1/mRNA complex
465
How is mRNA released from NXF1 in the cytoplasm?
RNA helicase Dbp5 disassembles the NXF1/mRNP complex -> mRNP is extensively remodelled -> nuclear associated proteins removed and cytoplasmic factors bind -> promote mRNA translation.
466
Where are Dbp5 helicases located?
Dbp5 is localised to the cytoplasmic fibrils of nuclear pore complexes.
467
Linker DNA:
DNA sequence between nucleosome
468
How can Gene expression be spatially regulated by mRNA?
mRNA localisation
469
mRNA localisation: Summary
Once mRNA have been exported from the nucleus into the cytoplasm -> can localise to specific sites within the cell/system -> site will be close to ribosomes to allow for translation
470
What are the mechanisms by which mRNA localisation occurs?:
-Random Diffusion + anchoring
-Active Transport -> mRNA are transported by actin-mediated transport
-Selective degradation -> caused by factors blocking translation or promoting degradation
471
What are zip code binding proteins?
Proteins that bind to zip code sequences (localisation signals) within the 3' UTR of mRNAs -> guide to localisation site
472
Bromo-domains:
Sites on CRMs that positively induce transcription by interacting with acetylated nucleosomes
473
Example of genes regulated by mRNA localisation: Drosophila
Maternal effect genes -> bicoid (bcd) and nanos (nos) -> dictate the polarity of the developing genome
474
What are zip codes?
nucleotide sequences found in the 3' UTR region that dictate the localisation of mRNA transcripts
475
mRNA localisation in Saccharomyces cerevisiae: ASH1
Ash1 is a transcriptional repressor which has a specific localisation during cell division -> inhibits expression of the HO gene in the daughter cell to make the daughter cell inherit the parent's mating type.
476
mRNA localisation in Saccharomyces cerevisiae: What components localise ASH1 into the daughter cell?
The zipcode binding protein She2, adaptor protein She3, and motorprotein Myo4.
477
What are the two Saccharomyces cerevisiae mating types?
Mat A and Mat Alpha
478
What is PolyA binding protein:
Binds to PolyA tail
479
What proteins make up the cap binding complex?
eIF4E, eIF4A, eIF4G
480
eIF4E: Function
Recognises the cap structure of mRNA
481
eIF4A: Function
Acts as a helicase that helps remove structures within the 5' leader sequence , allowing the ribosomal subunit to move through a scan the initiation codon
482
eIF4G: Function
Interacts with the PAB proteins bound to the PolyA tail -> makes complex form a circular structure -> increasing efficiency of translation.
483
What is a method of translational control that can be performed by modification to the cap binding complex?
Breaking the circularise structure of the cap binding complex by disrupting the interactions between the eIF4G and PABP.
484
What are two methods by which the circularised structure of a cap binding complex can be broken?
The phosphorylation of eIF4E binding proteins
485
eIF4E Binding proteins:
Have a particular domain mimicking the domain of eIF4G that binds to 4E -> therefore competes with 4G -> dampening the interaction and linearising the structure -> disrupting initiation and down regulating translation.
486
What is the mTOR complex?
An important hub within the signal transduction system of the cell -> promotes growth and cell division -> stimulates protein kinase pathways to increase translation. + inhibits 4EBP (inhibitor)
487
How is mTOR regulated?
mTOR is controlled by signals that upregulate it in the presence of growth promoting nutrients and downregulate at low energy/infection.
488
What drug targets the mTOR complex?
Rapamycin -> growth inhibitor that inhibits mTOR.
489
How can the translation initiation be globally regulated without altering the cap binding complex?
Effecting the pool of active eIF2 transcription factor.
490
eIF2-alpha:
Brings tRNA to ribosome to initiate translation -> acts as GTPase -> regenerate using GEF post initiation -> if phosphorylate it'll be stuck bound to GEF -> down regulating translation.
491
Autoregulation of transcription in bacteria:
if the pool of free ribosomal protein becomes too high, they'll bind to the 5' ladder of the mRNA -> preventing the small ribosomal subunit from bine shine-dalgarno sequence.
492
What is the shine-dalgarno sequence?
Sequence upstream
of start site in the ribosomal binding site that upregulates expression.
493
Ferratin mRNA in homeostasis:
Has stem loop structure within 5' leader seq. -> iron regulatory proteins (IRPs) bind to it -> blocks scanning mechanism -> prevents translation. In high Fe levels -> IRPs bind to Fe to form complex -> don't bind to mRNA loop -> translation occurs.
494
X chromosome inactivation in Mammalian Females:
Single X chromosome stochastically chosen -> silenced by tightly winding into heterochromatin -> formr Barr body
495
What is dosage compensation:
A system in which equivalent expression levels within cells of males and females that have unequal numbers of sex chromosomes. -> ensures transcriptional output of males and females is equal.
496
Dosage compensation in Flies:
Sxl only in females -> inhibits Msl-2 -> dampens male characteristics. -> in males there's no sxl and-so exon 1 will koin exon 2 -> Msl2 will be expressed.
497
What enzymes degrade mRNA polyA tails?
Deadenylases.
498
What is the lifetime of an mRNA molecules?
10min - 9 hours
499
At what length of PolyA tail mRNA quickly degraded and depleted?
10 residues
500
What is the m7G cap?
It's the 5' methylation cap.
501
What enzyme removes the 5' methyl cap after deadenylation of the polyA tail?
A decapping enzyme consisting of a Dcp1 and Dcp2 subunits.
502
What bond is broken by decapping enzymes?
5'-5' triphosphate linkage between the cap and the m7g cap structure -> releasing m7GDP.
503
Maternal effect genes:
Genes inherited from the mother (e.g. P+) -> from the cytoplasm environment.
504
What enzymes degrade adenylated transcripts:
3'-5' exoribonucleases e.g. Xrn1
505
What set of factors regulate mRNA turnover?
A/U-rich element binding sites.
506
A/U-rich element binding proteins: function
Recognise A/U-rich elements within the 3' UTR of mRNAs -> increase or decrease the stability of the mRNA.
507
A/U-rich element binding proteins: examples
HuR -> Increases mRNA stability
AUF1 -> Decreases mRNA stability.
508
LSD:
Lysine specific demethylase -> loosen chromatin
509
What is the benefit of using more unstable mRNA?
More unstable mRNA is much more responsive to changes in transcription -> will reach steady state level far more rapidly once transcriptional rate is changed.
510
MicroRNAs:
-> short RNA sequences generated from larger endogenous precursor RNAs -> processed by Drosha and Dicer -> incorporated into RNA-induced silencing complexes.
->Direct post-transcriptional regulation.
511
What is the function of RNA-induced silencing complexes?
accelerate mRNA turnover and translational repression by irregular base-pairing and recruit deadenylases complexes
512
What causes the inhibition of Sxl?
Inclusion of exon 3 -> early stop codon -> prevents.
513
Why are mRNAs with early stop codons rapidly degraded?
They are rapidly degraded by nonsense-mediated decay (NMD)
514
Nonsense-mediated decay (NMD): Why does it initiate?
Absence of interaction between the ribosome and the mRNP 3'UTR domain
515
Why aren't truncated proteins not often expressed?
They will be degraded due to not folding into a stable or by eukaryotic system that eradicate mRNAs with nonsense mutations.
516
Why could functional mRNA transcripts be subjected to NMD?
If an mRNA transcript has an unusual 3' UTR structure.
517
What is used to form genome crosslinks during Chromosomal Conformation Capture?
Formaldehyde
