TBL 2 DNA Flashcards
(164 cards)
1
Q
DNA stored in the _______ is always ______ before the cell undergoes mitosis.
A
stored in the nucleus; replicated
2
Q
_________ replication suggests that the parental DNA strands separate and serve as a template strand for replication, and do not re-anneal with each other. All daughter molecules contain ___________.
A
Semi-conservative replication;
all daughter molecules contain 1 parental strand and 1 daughter newly-synthesised strand
3
Q
DNA replication always proceeds in the __’ to __; direction.
A
5’ to 3’
parental strand is read in 3’ to 5’ direction
4
Q
DNA replication requires a ______ because DNA Polymerase cannot initiate DNA synthesis on their own, they can only add deoxyribonucleotides to the 3’ end of an existing chain.
A
Primers (usually RNA)
5
Q
There are 5 classes of DNA polymerases (a, B, y, d, E). DNA polymerase __ is the main DNA polymerase for DNA replication.
A
DNA Polymerase delta
6
Q
The bond between 2 nucleotides is a _________ bond, catalysed by DNA polymerase.
A
phosphodiester bond (between the 5’ phosphate group of the incoming dNTP and the 3’ OH group of the elongating strand)
7
Q
Hydrolysis of ________ releases pyrophosphate and a large amount of energy needed to drive DNA replication reaction.
A
Hydrolysis of triphosphate on the incoming dNTP
8
Q
Dideoxycytidine/zalcitabine and Azidothymidine/zidovudine are nucleoside analogues, which are used as inhibitors to _________ and therefore a HIV antiviral.
These analogues do not provide a free 3’ OH group for DNA P to extend and thus terminates chain replication.
A
Reverse Transcriptase
9
Q
_________ are used as a drugs which has similar structure to DNA nucleosides, and therefore can be added to stop DNA replication.
A
Nucleoside analogues
10
Q
Acyclovir (nucleoside analogue) is an antiviral drug used for ________, ______.
A
herpes simplex infections, chicken pox and shingles
11
Q
Cytarabine/cytosine arabinose (nucleoside analogue) is a chemotherapy medication for various ________.
A
leukaemias
12
Q
DNA replication begins at one or more specific _____ sites on the DNA molecule known as _________.
A
A-T rich sites; origins of replication
13
Q
In prokaryotes, how many oriC are there?
A
single unique origin
replication proceeds bidirectionally from oriC simultaneously
14
Q
In _________, there are multiple origins of replication for the long, linear chromosome.
A
eukaryotes
15
Q
______ are a family of proteins which bind to one strand of DNA and use ____ as an energy source to break the hydrogen bonds holding the two strands together.
A
Helicases; ATP
16
Q
____________ temporarily stabilises the unwound single-stranded portion of the DNA molecule.
This is cooperative binding, and is to prevent the ssDNA from reannealing to each other.
It also prevents ssDNA from being _______ by exonucleases.
A
Single-strand binding proteins (SSB proteins); degraded
17
Q
_________ are proteins which cleave a strand of the helix to create a transient single-stranded nick, and allow free rotation around the intact strand to relieve strain on the DNA molecule, then resealing the broken strand.
A
Topoisomerases
18
Q
_______ (specialised RNA polymerase) will synthesise a short RNA primer using the template strand to provide a free 3’ OH end which DNA P can extend.
A
DNA Primase
19
Q
The RNA primer is transient, and will later be removed by _______ with _____ exonuclease activity and replace with a DNA version.
A
DNA Polymerase; with 5’ to 3’ exonuclease activity
20
Q
As the templates for the two new daughter strands are _________, the replication fork is asymmetrical. Both daughter strands are only synthesised in the 5’ to 3’ direction, so there are two types of strands - _______ and _______.
A
antiparallel; leading and lagging
21
Q
The ______ strand is synthesised continuously as a single polymer towards the replication fork; whereas the _______ strand is synthesised discontinuously in a series of _________, each synthesised (towards/against) the direction of the replication fork.
A
Leading strand;
Lagging strand; Okazaki fragments; each synthesised AGAINST the overall direction of the replication fork
22
Q
______ catalyses the formation of phosphodiester bonds between the 3’ end of the new Okazaki fragment (replaced with dNTPs) and the 5’ end of the growing DNA strand with ATP hydrolysis.
A
DNA ligase
23
Q
Which of the following requires ATP?
- DNA ligase
- DNA Polymerase
- DNA primase
A
Answer: ALL
24
Q
_______ of the template for the (leading/lagging strand) allows both daughter strands to be synthesised in a coordinated manner.
A
Looping of the template for the LAGGING strand
=> allows both the DNA P to be in the same orientation
25
DNA P has a _______ mechanism to ensure no mistakes are made in the process.
proofreading mechanism
26
Another DNA repair mechanism is _______ repair, where deformities in the double helical structure due to incorrect base-pairing are recognised after replication is complete.
Enzymes determine the template and non-template strand and excise the wrongly incorporated base then replacing it with the correct nucleotide.
mismatch repair
27
All cells in the human body contains the same genetic material (all the genes), but serve different functions due to differences in ___________.
gene expression
28
The complete DNA sequence of an organism is known as its ______.
genome
29
The human genome is arranged into ___ chromosomes. __ autosomal pairs, 1 pair of sex chromosomes.
46 chromosomes; 22 autosomal pairs and 1 pair of sex chromosomes
30
_______ genes are genes which are being constitutively expressed by all cells, as they are needed for normal cell function.
Housekeeping genes
31
DNA replication and transcription occurs in the ______; whereas translation occurs in the _______.
DNA replication and transcription: nucleus
Translation: cytoplasm
32
The template DNA strand in transcription is known as the _______ strand. It is read from the _______ direction and is complementary to the mRNA produced.
antisense strand; read in the 3' to 5' direction
33
The non-template strand in transcription is known as the ______ strand, and it has the same sequence as mRNA, except that the T has been replaced by U.
sense strand
34
The promoter contains the _______ (5'-TATAAA-3' on the non-template strand) and ___________.
TATA box and transcription start site
35
The TATA box is a binding site for _____ (general transcription factor) which facilitates the binding of RNA P.
TFIID
36
Is the promoter transcribed?
No, except for the transcription start site.
37
The terminator sequence is found at the end of the gene, coding for a polyadenylation signal sequence: 5' _______ 3' in the pre-mRNA.
5'-AAUAAA-3'
38
Transcription factor binding sites lie (upstream/downstream) of the promoter sequence and control the rate of transcription via transcription factor binding.
upstream
39
There are 3 types of RNA P.
RNA P 1: Transcribes ____ genes
RNA P 2**: Transcribes genes encoding proteins into ____
RNA P 3:
Transcribes _____ and 5S RNA genes
RNA P 1: Transcribes rRNA genes
RNA P 2**: Transcribes genes encoding proteins into mRNA
RNA P 3:
Transcribes tRNA and 5S RNA genes
40
_________ are required for RNA P to bind to its promoter and initiate transcription, achieving a basal rate.
General/basal transcription factors
41
Transcription factors can assess the DNA base pairs of the promoter and TF binding sites via the _______ and _______ of DNA, but the main point is through the _______.
major and minor grooves; mainly through major grooves
42
TFIID contains the _________ and TBP accessory factors (TAFs), and upon binding to the _______, it partially unwinds the DNA helix and widens the minor groove to allow extensive contact with DNA.
TATA binding protein (TBP); binds to the TATA box
43
After TFIID binds to TATA box, TFII__ and TFII__ binds, followed by the binding of TFII__, TFII__, TFII__ and TFII__.
1) TFIID
2) TFIIA and TFIIB
3) TFIIE, TFIIF, TFIIH and TFIIJ
44
Binding of TFII__ promotes further unwinding of the DNA helix to facilitate RNA synthesis, with kinase activity phosphorylating _____ residues on RNA P II to activate it for transcription.
TFIIH
- promotes further unwinding of helix
- activates RNA P by phosphorylating serine residues
45
RNA P _____ the unwound DNA helix behind it, dissociating the growing RNA chain from the template.
reanneals
46
The transcribed terminator encodes a polyadenylation signal sequence in the RNA, and transcription continues until ___ to ___ bp downstream of the signal sequence.
Proteins bind to cut and free the pre-mRNA from RNA P II.
10 to 35 bp
47
Histone acetylation will (reduce/increase) rate of transcription.
increase rate of transcription
=> Acetyl groups are negatively-charged. Acetylation will neutralise positive charge of histones, causing histones to bind less tightly to DNA => more accessible for transcription factors,
48
Histone deacetylation will (reduce/increase) rate of transcription.
reduce rate of transcription
49
Protein ____ is an inhibitor of transcription factor NFKB, which upregulates cytokine genes in the nucleus to promote inflammation.
How does aspirin, an anti-inflammatory drug, work?
IKB
Aspirin will inhibit the breakdown of IKB, which will bind to the TF NFKB, such that it remains in the cytoplasm and is unable to initiate the transcription of the cytokine genes, minimising inflammation.
50
Oestrogen receptors are ________ which regulate oestrogen regulated genes.
Over 70% of breast cancers overexpress the oestrogen receptor, resulting in uncontrollable tumor growth and proliferation.
transcription factors
51
Anti-oestrogen drugs like _______ act as progenitors of highly competitive inhibitors of the oestrogen receptor to repress its activity and prevent overactivation of genes controlling cell cycle progression.
Tamoxifen
52
Prokaryotes only have one type of RNA P.
| The transcription factor (known as ____ factor) binds to the ________ to initiate transcription.
sigma factor binds to the Pribnow box to initiate transcription
53
RNA processing occurs in the _____.
nucleus
54
_____ are protein-coding sequences which form part of the mature mRNA; whereas _______ are nucleotides inserted between exons which are non-coding and thus edited out of the mature mRNA.
Exons; introns
55
5' end of pre-mRNA is modified by the addition of a cap structure consisting of ___________.
a methylated guanine nucleotide/methylguanosine triphosphate
56
How is the 5' methylated guanosine cap added?
It is formed by the hydrolysis of terminal triphosphate of mRNA to a diphosphate, which then reacts with the 5' phosphate of GTP to form a 5'-5' phosphate linkage.
This is further modified by methylation at the N7 position in the purine ring to form a 7-methylguanylate cap.
57
3 functions of 5' cap:
1. Protects mRNA from _______ by hydrolytic enzymes like nucleases.
2. Defines the 5' end of the mRNA, which serves to recruit the _______ for translation initiation.
3. Distinguishes mRNA from other types of RNA.
1. degradation
| 2. binding to the 40S ribosomal subunit for translation initiation
58
Viruses like ________ can interfere with the recognition of the cap during translation, thus blocking the formation of the translation initiation complex and preventing vital protein synthesis.
polioviruses
59
After the pre-mRNA has been cleaved at a site 11-30 bp downstream of the polyadenylation signal sequence, most 3' ends are modified by addition of a series of about 200 ______ nucleotides catalysed by the ___________.
adenine nucleotides, catalysed by poly(A)-polymerase
60
Function of the 3' polyA tail:
1. Protects mRNA from _______ by nucleases
2. Required to facilitate export of mRNA out of nucleus via _______.
1. degradation
| 2. nuclear pores
61
Introns start with the splice donor site containing the sequence 5'___3' and end with a splice acceptor site containing the sequence 5'___3'.
Splice donor site: 5'-GU-3'
Splice acceptor site: 5'-AG-3'
62
__________ are large complexes comprising of several subunits called snRNPS which contain small nuclear RNAs and sets of proteins.
Spliceosomes
63
RNA Splicing:
First protein is ___, which binds to the splice donor site of intron.
After which, the binding of ___, ___, ___ and ___ complete the formation of the spliceosome, which cleaves the splice donor sequence at the 5' end.
1. U1 to splice donor site (5' GU 3')
2. U2, U4, U5 and U6 bind
=> spliceosome
3. Cleaves splice donor sequence
64
An ______ residue in the intron is used as a branch point in the intermediate step, binding to the __' end of the intron.
(2' OH group) adenine residue within the intron; binding to the 5' end of the intron (G)
65
Phosphodiester bond between G at the end of the intron (i.e. the splice acceptor site) and the next exon is cleaved, and the intron is removed as a ______-like structure.
lariat-like structure
66
After the introns are removed, simultaneous ______ of the exposed adjacent exon sequences occurs.
simultaneous ligation
67
________ is a group of inherited autosomal recessive disorders in which there is an imbalance in the relative amount of globin chains.
Thalassaemia
68
In B-thalassaemia, there is a relative deficiency of ___-globin chains.
Deficiency of B-globin chains
69
Several types of B-thalassaemia is due to ________ mutations in the B-globin genes, resulting in improper mRNA splicing and thus production of non-functional B-globin chains.
splice site mutations
70
Patient with B-thalassaemia presents with severe anemia and hepatosplenomegaly. There is iron overload (__________) due to chronic anaemia, causing hepatic fibrosis and cirrhosis, darkening of skin.
haemosiderosis
71
The genetic code is made of ___ codons, which code for ___ amino acids.
64 codons; 20 amino acids
72
The genetic code is _________, meaning a single amino acid may be coded by more than one different codon.
degenerate
73
Third base position in a codon is a _____ position as base pairing at the 3rd base is not so specific.
wobble position
74
The start codon is ____, which codes for amino acid _______.
AUG - methionine
75
The stop codons are: ____, ____, ____. They do not code for any amino acids.
UAA, UGA, UAG
76
The mRNA template is read by the ribosome in the ____ direction.
5' to 3' direction
| i.e. the ribosome binds to the 5' end of the mRNA and looks for the start codon AUG
77
tRNA molecules have an anticodon loop that will bind to specific mRNA codon via ___________.
complementary base pairing
78
The 3' end of the tRNA (____) is the ________ attachment site that binds to an amino acid via an _____ bond.
3' end - CCA stem;
| amino acid attachment site; via an ester bond
79
The tertiary structure of the tRNA refers to the actual twisting and folding of the secondary structure into a compact 3D ______ structure maintained by hydrogen bonds.
L-shaped structure
80
The 3D structure of tRNA is recognised by the enzyme ____________, which catalyses the formation of an ester linkage between the CCA stem and specific amino acid's carboxyl group --> forms _________.
enzyme amino-acyl tRNA synthetase; forming aminoacyl tRNA
81
The binding interactions of the tRNA with the third mRNA codon base is weaker (wobble) and tolerates other bases in that position.
-
82
The active site of amino acyl-tRNA synthetase is complementary to the 3D conformation of the _________ and the __________.
1) specific amino acid
| 2) specific anticodon of the tRNA
83
Ribosomes are large ribonucleoprotein complexes composed of ________ and _______.
ribosomal proteins and ribosomal RNA (rRNA)
84
Each eukaryotic ribosome consists of two subunits:
Large (60S) and small (40S) ribosomal subunits
85
The large ribosomal subunit contains 3 binding sites for tRNA.
1) A site (holds the incoming tRNA carrying the next AA to be added)
2) P site (holds the tRNA carrying the growing polypeptide chain)
3) E site
86
The small ribosomal subunit contains the binding site for _______.
mRNA (5' end)
87
rRNA in the large ribosomal subunit has _________ (enzyme) activity and catalyses the formation of peptide bonds between the amino acids.
peptidyl transferase activity
88
Pre-initiation complex for translation involves ________ binding to the (small/large) ribosomal subunit, positioning the tRNA-met to the __ site.
eukaryotic initiation factors (eIF-2) binding to the small subunit of the ribosome, positioning tRNA-met to the P site
89
eIF-__ and eIF-__ bind to the 5' cap of mRNA, and are recognised by the pre-initiation complex, leading to the 40S subunit of ribosome binding to the mRNA.
eIF-4E and eIF-4G
90
Binding of the 60S large ribosomal unit occurs with the dissociation of ____ with the hydrolysis of ____, leading to conformational changes.
dissociation of eIFs with the hydrolysis of GTP to GDP --> conformational change occurs
91
The translocation of tRNA occurs along the ribosome, guided by the elongation factors with the hydrolysis of ___ to promote the movement of ribosome.
hydrolysis of GTP promotes movement of ribosome along the mRNA
92
Termination of translation: When the in frame stop codons (___, ___ and ___) reach the A site of the ribosome, a protein ________ binds directly to the stop codon at the A site. (There are no tRNAs with anticodons complementary to the stop codons.)
UAA, UAG and UGA
Protein release factor
93
Bound ribosomes on the rough ER are responsible for synthesising ________ and ______ proteins.
secretory and transmembrane proteins
94
Polypeptides of proteins destined for secretion (i.e. the endomembrane system) are marked by a __________ (first 20-24 amino acids rich in hydrophobic amino acids), targeting them to the ER.
signal sequence/peptide
95
Marking polypeptides for secretion:
The signal peptide near the N-terminus of the polypeptide is recognised as it emerges from the ribosome by a ____________ (protein-RNA complex).
Signal recognition particle (SRP)
96
The ___________ is an adaptor which brings the ribosome to the rER membrane by binding to it, causing translation to resume. The growing polypeptide chain is synthesised into the lumen of the rER.
Signal recognition particle (SRP)
97
(Secreted/transmembrane) proteins are translocated fully into the rER lumen during translation.
These proteins are completely contained within the vesicle and directed towards the GA or other organelles.
Secreted
98
(Secreted/transmembrane) proteins have an additional hydrophobic sequence which holds them in the ER membrane.
These proteins are anchored to the vesicle membrane such that when the vesicle fuses with another membrane, the protein is embedded within the membrane.
Transmembrane
99
Once the secretory and transmembrane proteins are translated, the enzyme _________ cleaves the signal sequence on the N-terminus of the polypeptide, and the protein folds into its native conformation.
signal peptidase
100
__________ (antibiotic) inhibits initiation of translation by binding to the _________ of the prokaryotic ribosome. This interferes with the binding of fMet-tRNA to the 30S subunit during initiation.
Streptomycin; binds to the 16S rRNA of the 30S small ribosomal subunit
101
_________ inhibits aminoacyl-tRNA binding by binding to the 30S ribosomal subunit.
Tetracycline
102
________ inhibits translocation of the ribosomal complex by binding to the 50S ribosomal subunit.`
Erythromycin (E for Eukaryotic factors which help with translocation)
103
Chloramphenicol inhibits ___________ in the 50S large ribosomal subunit, preventing polypeptide elongation.
Peptidyl transferase
104
_________ terminates elongation prematurely, acting as an analogue of the 3' end of aminoacyl-tRNA.
Puromycin (P for premature termination)
105
In glycosylation of polypeptides, pre-assembled carbohydrate chains are N-linked to asparagine (Asn) of the _________ sequence by (enzyme).
This stage occurs in the ______ (organelle), as glycan on the lipid-linked precursor exist within the ____ lumen.
Asn-X-Ser/Thr sequence (where X is any AA except for proline)
By glycosyl transferases
Occurs in the rER; rER lumen
106
Glycan addition occurs in the ________, whereas glycan processing occurs at the ________. (where carbohydrate chains are processed by trimming followed by extension)
ER; golgi apparatus
107
Prenylation/acylation of polypeptides refer to addition of _____ groups.
lipid groups
108
Insulin (protein hormone) biosynthesis:
1. _______ bond formation from the oxidation of two cysteine residue side chains. (preproinsulin ---> proinsulin)
2. Proteolytic cleavage (i.e. removing a sequence of amino acids):
Proinsulin is cut _____ after the disulfide bonds are formed, and ________ is removed from the middle of the protein in a secretory vesicle.
=>Active insulin consisting of two polypeptide chains.
1. Disulphide bond formation
| 2. Proteolytic cleavage - Proinsulin is cut twice and the connecting peptide C chain is removed.
109
________ marks intracellular proteins for proteolysis (degradation) by the proteasome.
Ubiquitin
110
Endocrine: __________ produced in the hypothalamus acts on the kidneys.
Vasopressin
111
Endocrine:
| _______ produced by the adrenal glands act on the trachea.
Adrenaline
112
_______ effect is when the hormone acts on an adjacent cell.
Paracrine
113
Paracrine:
| Insulin produced by the B-cells in the islets of Langerhans acting on _________ to inhibit glucagon secretion.
neighbouring alpha cells
114
Paracrine:
| ________ and ______ produced by the endothelial cells in blood vessels, causing vasoconstriction.
Endothelin-1 and nitric oxide
115
Autocrine:
Activated T-cells secrete _____, which binds to the receptor on the same cell, signalling the cell to enter the cell cycle and inducing cell proliferation and differentiation.
IL-2
116
Cells can communicate via signalling between __________ via physical contact.
E.g. TCR on T-cells binding to MHC class II complexes on APC.
membrane-attached proteins (MAPs)
117
Intracellular receptors will receive signals from _________ ligands.
hydrophobic ligands
| that can cross the membrane
118
________ receptors are membrane receptors which allow specific ions to pass through when the receptor changes its conformation due to a ligand binding.
Ionotropic receptors (ion channel receptors)
119
Acetylcholine is a ligand to the sodium ion channel receptor __________ on skeletal muscles.
Nicotinic acetylcholine
120
_______ is a general anaethesia that is administered to compete with acetylcholine for binding sites on the nicotinic acetylcholine receptors, leading to reduced muscle contraction.
Atracurium
121
Structure of GPCRs: 7 transmembrane _______
7 transmembrane a-helices
122
G-protein has __ subunits.
| Active G-protein will dissociate into two active components:
3 subunits
2 active components: a and By subunits
123
Internal GTPase activity of the __ subunit of G protein will cause the hydrolysis of GTP back to GDP.
alpha subunit
124
___ protein-linked receptor: B1 adrenergic receptor
Ligand:
Pharmocology:
Gs protein-linked receptor
Ligand: Adrenaline, noradrenaline
Pharmocology: Beta blockers
125
G_ protein activates adenylyl cyclase.
| G_ protein inhibits adenylyl cyclase.
Gs protein
| Gi protein
126
________ catalyses the synthesis of cAMP (second messenger).
cAMP then activates ________, which phosphorylates other proteins and may lead to different effects depending on the linked G protein.
Gi/Gs protein --> Adenylyl cyclase ---> cAMP ---> Protein Kinase A (PKA) --> phosphorylates other proteins
127
Gq proteins activate _______, which cleaves PIP2 into ___ and IP3.
IP3 binds to IP3-gated _______ channel in the ER membrane, causing ___ ions to diffuse out of the ER and into the cytoplasm.
Gq proteins --> Phospholipase C (PLC) --> cleaves PIP2 into DAG and IP3 (second messengers)
IP3 binds to gated Ca2+ channels, causing Ca2+ ions to diffuse out of the ER into the cytoplasm and activate calcium-sensitive proteins to produce a cellular response.
128
Dopamine is a ligand that binds to ___________ receptor, which is G_ protein linked.
Receptor: D2 dopaminergic GPCR
Gi protein linked
129
Glucocorticoid receptor is an _________ receptor. Its ligand is ______.
Glucocorticoid receptor - intracellular
Ligand: Cortisol, corticosterone
130
Thyroid hormone receptor is an _________ receptor. Its ligands are:
Thyroid hormone receptor - intracellular
Ligands: T4 (thyroxine) and T3 (triiodothyronine)
131
Carcinogens can cause DNA damage in the following ways:
1.
2. (Physically link two DNA bases)
3. (Chemical/oxidate changes around the bases leading to non-recognition of DNA)
4. (Covalently link two DNA molecules via G/A)
1. Double and single strand breaks
2. Base dimer
3. Base hydroxylations and abasic sites
4. DNA adducts and alkylation
132
___________ are common environmental pollutants formed from the combustion of fossil fuels or tobacco with complex, aromatic multi-benzene ring structure.
Polycyclic Aromatic Hydrocarbons (PAH)
E.g. Benzo[a]pyrene (B[a]p)
133
PAH:
Benzo[a]pyrene (B[a]p) undergoes a two-step ________ process which activates it.
Enzyme 1: ________ oxidises B[a]p to an epoxide, a strained molecule which is highly reactive.
Enzyme 2:
________ splits the functional group of the epoxide into two hydroxyl groups. [detoxification pathway protective mechanism]
The resulting dihydrodiol becomes a substrate for _____, which oxidises it to an epoxide with a dihydrodiol --> extremely reactive and electrophilic.
Epoxidation;
Enzyme 1: P450 enzyme;
Enzyme 2:
Epoxide hydrolase
Resulting dihydrodiol becomes a substrate for P450, forming an extremely reactive epoxide.
134
DNA is nucleopholic/electrophilic.
nucleophilic (due to the large source of electrons)
135
PAH:
__________ is a potent human liver carcinogen.
It is a substrate of P450, which oxidises it to form an epoxide.
This will cause it to be highly reactive/electrophilic, reacting with DNA bases (particularly ______) to form DNA conjugates/adducts.
Aflatoxin-B1;
| reacts with DNA bases to form guanine-alfatoxin B1 conjugate (adduct)
136
PAH:
___________ is a potent human bladder carcinogen.
It is a substrate for P450 (CYP1A2), which converts it to a _________. (DNA-damaging)
2-napthylamine; converted to N-hydroxy derivative (DNA-damaging)
137
Body's mechanism of coping with mutagens:
Phase I: Addition of ___________ to generate an intermediate (may be DNA-damaging)
Phase II:
Conjugation of phase I functional groups; generating _________ metabolites that can be safely excreted from the body.
Phase I: Addition of functional groups to generate intermediate
Phase II:
Conjugation of functional groups which may generate polar, harmless metabolites
138
2-napthylamine Phase II enzyme system involves __________ adding sugar molecules to the amines, detoxifying the molecule --> non-toxic, polar and excretable in urine.
However, as urine pH is (acidic/alkaline), this breaks up the molecule (losing the sugar), generating a (positively/negatively)-charged nitrenium ion that is highly reactive in the urine stored in bladder.
=> attack bladder _______ cells, causing mutations and tumors.
2-napthylamine:
Phase II - Glucuronyl Transferase adding sugar molecules
```
urine pH acidic
=> breaks up molecule
=> positively-charged nitrenium ion
=> highly reactive in the urine
=> attack the bladder epithelial cells
```
139
Solar (UV) radiation results in the formation of __________ between the adjacent thymine bases in DNA.
formation of pyrimidine (thymine) dimers
| => no longer recognised as thymine molecules, leading to mutations
140
_________ radiation generates free radicals in cells.
Oxygen free radicals possess unpaired, single electrons and are highly (nucleophilic/electrophilic), targeting nucleophilic DNA molecules.
This may result in __________, base removal and base modifications.
Ionising radiation
Free radicals are highly electrophilic.
May result in double/single strand breaks, base removal and base modifications
141
____ protein deals with cellular stresses by initiating a series of events to fix the problem.
p53 protein
| DNA repair
142
There are 5 types of DNA repair mechanisms:
1. Direct reversal
2. Base excision repair
3. Nucleotide excision repair
4. Mismatch repair
5. Recombinational repair
143
Direct reversal DNA damage repair:
_______ splits cyclobutene pyrimidine dimers.
__________ and alkyltransferases remove alkyl groups from bases.
1) Photolyase
| 2) Methyltransferases
144
Base excision repair occurs mainly for ________ damage.
1. DNA _______ and endonucleases excise the bases.
2. __________ fills the gap.
3. _____ completes the repair.
apurinic/apyrmidinic damage
1. DNA glycosylases and endonucleases excise the bases.
2. Repair polymerases (e.g. Pol b) fills the gap.
3. DNA ligase completes repair.
145
For base excision repair, DNA glycosylase will only remove the affected __________.
Endonuclease then cleaves the backbone to allow for the addition of the new nucleotide, before reforming the backbone by DNA ligase.
```
nitrogenous base
(not the entire nucleotide --> sugar-phosphate backbone left intact)
```
146
Nucleotide excision repair occurs mainly for _______.
bulky DNA adducts (i.e. two DNA molecules covalently link together via G or A)
147
Nucleotide excision repair:
_________ proteins assemble at the damage site, excising a stretch of nucleotides on either side of the damage.
Xeroderma pigmentosum (XP) proteins
=> Deficiency of XP proteins severely predisposes individuals to skin cancer.
148
________ repair looks for mismatched base pairs in DNA due to incorrect complementary base pairing.
Mismatch repair
149
_________ repair is repairing double-stranded DNA damage by recombination with a homologous intact duplex (usually sister chromosome)
Recombinational repair
150
Cell-based: In vivo/in vitro
Cell-free: In vivo/in vitro
Cell-based: In vivo
Cell-free: In vitro
151
In-vivo (cell-based) DNA cloning first involves the in vitro construction of _________.
(1) Cut target DNA and replicon with the same ________ enzyme, so that the two ends of the DNA are compatible.
(2) Mix and join DNA fragments with DNA ligase.
(3) Transform recombinant DNA molecules into host cells. (e.g. bacteria)
(4) Selective propagation on agar plate
recombinant DNA molecules/plasmids
(1) Cut using restriction endonucleases
(2) Mix using DNA ligase
152
Restriction sites are usually 4-8bp _______ sequences.
palindromic = same sequence on both strands when read from 5' to 3' direction.
153
Restriction endonucleases produce either _____ ends or ______ ends.
```
Blunt ends (straight cuts with no nucleotide overhang)
or sticky ends (staggered cuts with nucleotide overhangs which make annealing of DNA fragments more efficient)
```
154
Transformation of recombinant DNA molecules into host bacterial cells is done through ________ or _________.
heat shock or electroporation
155
What is the function of selecting colonies after bacterial transformation?
Colonies that are ultimately selected for are the transformed bacterial cells with recombinant plasmids containing the target DNA fragment.
156
Selection of colonies
1) Positive selection: Selection for transformed bacteria which __________
2) Negative selection: Selection for transformed bacteria which _________.
1) Positive selection: Selection for bacteria which have taken up plasmid
2) Negative selection: Selection for bacteria which have taken up recombinant plasmid
157
Recombinant bacterial colonies (should/should not) survive on amp+ tet+ agar plates.
Should survive on amp+
Should not survive on tet+ (as the insertion of DNA fragment into a plasmid results in disruption of the tet gene sequence)
158
DNA is negatively-charged, so it moves towards the (cathode/anode) during gel electrophoresis when electrical force is applied.
Anode - Positive electrode
159
__________ is a method for detecting specific nucleic acid sequences in which homologous single-stranded DNA or RNA molecules combine to form double-stranded molecules.
Nucleic acid hybridisation
160
In nucleic acid hybridisation, target DNA is immobilised on a solid support - _________ or _____ membrane.
nylon or nitrocellulose membrane
161
__________ refers to the power to distinguish related sequences during hybridisation between the probe and target DNA for nucleic acid hybridisation.
Hybridisation stringency
162
```
Hybridisation strigency (increases/decreases) with increasing temperature, and
(increases/decreases) with increasing Na+ concentration.
```
- Increases with increasing temperature
- Decreases with increasing Na+ concentration
(i. e. more stringent = high temperature, low salt concentration)
163
_____ is an in-vitro method to selectively amplify a specific region of target DNA.
Polymerase Chain Reaction (PCR)
[in vitro - cell free]
164
_________ is a collection of microscopic DNA spots representing single genes, robotically arrayed on a solid surface.
DNA microarray
