253 genetics Flashcards
(520 cards)
1
Q
what did fred griffith do in 1928
A
transforming principle
material isolated from heat-killed virulent bacteria could transform non-virulent bacteria into a virulent form
used streptococcus pneumoniae
2
Q
what did oswald avery,Colin Macleod, Maclyn McCarty discover in 1944
A
they idenitified griffiths transforming principle as DNA
by transforming activity destroyed when nucleic acids treated with deoxyribonuclease but not ribonuclease
3
Q
what did Alfred Hershey and Martha Chase discover in 1952
A
labelled bacteriophage T2 with either 35S or 32P
only the 32P etected in infected bacteria and in phage progeny 35S in phage ‘ghosts’ fail to enter bacteria
4
Q
whats the name for DNA and RNA polymers
A
polynucleotides
5
Q
which way are nucleic acids directional
A
write 5’ to 3’
5
Q
how are nucleotides joined
A
phosphodiester bond between 3’ OH of one sugar and the phosphate attached to the 5’ hydroxyl of next sugar
6
Q
whats a tautomer
A
a molecule in which a proton has migrated to a different place
7
Q
how many base pairs does human genome have
A
3 Gbp = 3 000 000 000 base pairs
8
Q
how many rare tautomers does human genome have
A
approx 100 000 (0.0033%) are rare tautomers
9
Q
do tautomers have implications for the accuracy of DNA replication ?
A
yes
so can provide genetic variation
10
Q
whats a nucleoside
A
base plus sugar
named for bases e.g. adenosine (if sugar is ribose) and deoxyadenosine (if deoxyribose)
11
Q
what do you get if you add a phosphate to a nucleoside
A
a nucelotide
12
Q
how are bases joined to a sugar
A
glycosidic bond between the c1’ of the sugar and the n1 of a pyrimidine or n9 of a purine
13
Q
do nucleotides have additonal biological functions such as energy storage (ATP) and molecular transport
A
yes
14
Q
chargaffs rules
A
Amount of purine base = Amount of pyrimidine bases: [A]+[G]=[C]+[T]
Amount of guanine = Amount of cytosine: [G]=[C]
Amount of adenine = Amount of thymine: [A]=[T]
15
Q
dimensions of a-helix
A
34 Å (3.4 nm) and approx. 10 bp/turn
3.4 Å (0.34 nm) rise per bp
Helix diameter 20 Å (2 nm)
16
Q
who proposed structure of base pairs
A
watson and crick
17
Q
B-DNA structure
A
-2 complementary strands are antiparallel and wind around each other in a right handed double helix (clockwise)
-hydrophillic sugar phosphate backbone on outside
-van der waals interaactions between bases (stability)
- contribution of base stacking to stability varies with sequence (neighbouring bases)
- helix forms a major groove and a minor groove which governs interactions with other molecules
18
Q
diameter of B-DNA helix
A
around 2nm
19
Q
how many base pairs in one complete turn of helix B-DNA
A
10.5 base pairs
20
Q
how far apart are base pairs in B-DNA
A
0.34nm apart
21
Q
how long is one full turn of B-DNA helix
A
3.57nm
22
Q
major groove of DNA helix
A
-Each BP presents diff chem info
-info diff based on diff sequence combos
- rich in chemical info
- Different DNA sequences have different combinations of hydrogen bond acceptors (A), hydrogen bond donors (D), and methyl groups (M) available (CH3)
23
Q
minor groove base pair stuff
A
T-A and AT base pairs and G-C and C-G base pairs present the same chemical groups
cant be distinguished
24
what conformation of DNA is most predominant in cells
B-DNA
other less seen conformations are A-DNA (low humidity), Z-DNA (alternating pyrimidine/purines
25
diameter of A-DNA helix
2.6nm
26
how many base pairs per turn in A-DNA
11
27
the grooves are uneven in A-DNA
true or false
false
the grooves are even sized
28
how is A-DNA conformation induced
DNA binding proteins
29
whats the diameter of Z-DNA helix
1.8nm
30
is A-DNA right or left handed helix
right
31
is Z-DNA left or right handed helix
left
32
how many bp are there per turn in Z-DNA
12
33
how is Z-DNA conformation induced
by methylation of cytosine, torsional stress, high salt conc
34
non-B DNA structures formed in genomic repetitive sequences and they general seq. requirements
- cruciform - inverted repeats
- slipped (hairpin) structure- direct repeats
-quadruplex- oligo tracts
35
does closed circular DNA isolated from cells appear intertwined and tangled
yes
36
supercoils cannot form in constrained linear DNA
true or false
false
37
how do you generate supercoiling in DNA
-circular molecule is cut and held at one end while the other is twisted
linear DNA unwound by 2 turns and then circle closed again
- the 2 ends are reattached the DNA twists to restore the preferred number of bases per turn
this causes the DNA to wrap around itself in a coiled structure
38
what does LK stand for
linking number
39
equation to find the LK
to quantify supercoiling
LK= Tw + Wr
Tw= number of turns in a DNA fragment
Wr= writhe is the number of supercoils and can be positive or negative
40
topoisomerase
introduce or remove supercoils from DNA in an energy-requiring process by temporarily breaking DNA and twisting it
41
key features of Watson and cricks model (B-DNA)
Two polynucleotide chains wound around each other in a right-handed helix
Two chains are antiparallel
Sugar-phosphate backbones on outside, bases on inside
Bases of two strands held by H-bonds – 3 between G-C base pairs, 2 between A-T base pairs
Base stacking contributes significantly to helix stability – sequence dependent
Bases 0.34 nm apart, one full turn (10.5 bp) 3.6 nm
Helix diameter 2 nm
Because of base H-bonding, the opposite sugar-phosphate backbones not equally spaced – major and minor groove
42
genome definition
sequence of nucleotides in DNA that comprises the genetic makeup of an organism
43
how is genome size always presented
the total amount of DNA contained within 1 copy of a single genome
44
how are viral genomes diverese
can be DNA or RNA
size variations
single or double stranded
circular or linear genomes
viral genes may overlap
45
bacteriophage (upside down) y genome size and structure
48502bp
dsDNA
linear when virion
circular when infect
46
do single cell eukarytoes generally have more genes than multicellular organisms
no
they normally have fewer genes
exceptions to this
47
is there a correlation between genome size, gene number and organism complexity
no not a simple correlation
48
C-value paradox
genome size doesnt consistently correlate with organism complexity
similar organisms can show a large range in genome size
genomes often have transposable elements- peices of DNA that copy themselves leading to an increase in genome size
49
intergenic regions
used to be deemed 'junk' DNA
transposable elements
junk DNA includes coding for RNA species that are not translated (pseudo genes)
50
gene definition
region that controls a discrete hereditary characteristic, usually a specific product like a protein
51
how many copies/genomes doesa unique genome have
1 to a few
52
how many copies/genomes does moderatly repetitive genomes have
few to 10^5
53
how many copies/genomes does highly repetitive genome have
10^5 to 10^7
54
prokaryotes have mostly moderately repetitive sequence DNA
true or false
false
have mostly unique DNA
55
eukaryotes have a mix of unique and repetitive sequences
true or false
true
56
how much of human genome code , codes for proteins
2%
57
do bacteria generally have a single chromosome
yes
58
what bends bacterial DNA to facilitate packaging and supercoiling
bacterial nucleoid associated proteins (NAPS)and integration host factor
59
how is DNA organised in bacterial chromosomes
around 400 independent supercoiled looped domains each if around 10kb
60
does topoisomerases generate supercoils
yes
61
are most eukaryotes diploid or haploid
diploid
produce haploid gametes
62
how long is DNA per human cell
around 2 meters
63
diameter of human nucleus
around 10um
64
what AAs are histones rich in
lysine and arginine
as they counteract negatice charge on phosphate group on DNA
these charges stabilise DNA-histone interactions
65
how is a nucleosome formed
when DNA wraps around a histone complex in a left handed manner
66
histone octamer core histones
H2A
H2B
H3
H4
histone complex is at the center is the histone octamer
67
nucleosome assembly detail
two H3-H4 dimers associate with DNA
two H2A-H2B dimers then associate to form octamer
146bp of DNA wraps around octamer 1.75 in left handed direction supercoiling
68
does removing histone octamer leave DNA postive or negative supercoiled
negatively supercoiled DNA left
this negative strand supercoiling makes strand separation easier
which is required for replication/transcription
69
structure of nucleosome core
N-terminal tail extending outwards between DNA coils
tails are up to 25 AA (undefined strucutre)
tails interact with other nucleosomes to further compact DNA
tails can be chem modified - important for chromatin structure and function
70
where are other variations (not the core 4) histones befound
at special chromatin locations
71
what is CENP-A a variant of
variant of histone H3 thats incorporated into nucleosomes at centromeres
essential for centrosome formation and function
72
whats the least compact visualized chromatin called
beads on a string appearance
the 10nm fiber
73
what can the 10nm chromatin fiber be folded into
the 30nm fiber
a regular arrangment that brings nucleosomes together
DNA is compacted around 50fold in 30nm fibre
74
which histone is needed for teh formation of 30nm fiber
H1
75
how does H1facilitate formation of the 30nm fibre
H1 is linker histone that binds the linker DNA in between successive nucleosomes helping compaction
the core histone tails are involved in this process but its not fully understood how
76
is chromatin further compacted in metaphase ?
yes
- 30nm chromatin fiber folded into looped domains via anchoring to central non-histone proteins chromosome scaffold
- chromosome-level condensation acheived throguh packaging of 10nm fibre - without the 30nm fibre intermediate
77
euchromatin stains how
chromtin here relatively decondensed and stains lightly actively transcribed genes
78
heterochromatin stains how
chromatin is more compacted and stains more darkly
less active transcription
79
what locations are chromosomes rich in hetercochromatin
telomeres- ends of chromosomes
DNA near centromers
regions with highly repetitive sequences
80
what does chromatic structure affect
transcription
DNA replication
recombination
chromosome transmissio
81
how does chromatin structure affect transcription
change of gene from euchromatic to heterchroamtic can prevent transcription
82
how does chromatin structure affect DNA replication
rearrangements that place an origin of replication into heterochromatin result in late replciation
83
how does chromatin structure affect recombination
in which DNA is broken and joined to a different DNA molecule is decreased in heterchromatin regions - protects genome from rearrangments
84
how does chromatin structure affect chromsome transmission
special histone CENP-A is needed to form a functional centromere, which is needed for proper chromosome separation
85
Modification of histones affects chromatin structure and function
Dynamic changes in chromatin are key to regulation of gene activity
Amino acid side chains in protruding N-terminal histone tails modified
Side chains in globular regions of histones can also be modified
Combination of specific modifications influences function of DNA
Histone modifications are reversible – specialized enzymes add and remove the chemical groups
Euchromatin is more acetylated than heterochromatin
86
what do histone acetyltransferases add to side chains
add acetyl groups to lysine side chains
87
what do histone deacetylases remove
acetyl groups
88
what do hsitone methyltransferases add to side chains
add methyl groups to lysine and arginine side chains
89
what do histone demethylases remove
methyl groups
90
how are phospahtes added and removed from chromatin
added by kinases
removed by phosphatases
91
how is ubiquitin added and removed from DNA
added by chain of enzymatic reactions
removed by a deubiquitinase (DUB)
92
elements required for chromosome functions
-origins of replication (ori)
- in bacteria ori determines distribution of replicated chromosomes to the daughter cells, ter specifies replication termination
-in eukarytoes centromeres direct chromosome segregation
-telomers stabilize ends of chromsomes
93
centromere
all eukaryotic chromosomes
normly 1 per chromosome
in heterochromatic region
inherited epigenetically- marked by H3 variant - CENP-A
bind specific proteins to form a kinetochore
94
kinetochores attach what
attach to microtubules from opposite spindle poles
allows spindle to separate sister chromatids
95
what does it mean by semi conservative dna
each strand of the parental double helix acts as a template for synthesis of new daughter DNA strand
96
what does semi conservative replication ensure
that each daughter double helix is identical to the parent
and each daughter cell will recieve identical DNA molecules
97
where does DNA synthesis occur
replication forks
98
which way along DNA does DNA synthesis occur
bidirectionally in 5' to 3' direction on both strands
99
what are the 3 phases of chromosome replication
initiation
elongation
termination
100
do helicase bind near ori
yes
then move along and unwind DNA
101
DNA primase binding location in chrmosome replication
DNA primase is aprimer that bidns to ori
synthesises short peice of RNA
102
initiation of replication
origin of replication is recognised by initator proteins that open up helix and recruit helicases
DNA helicase unwinds to expose sinlge strand
DNA synthesis needs primer. DNA polymerase can only add nucleotides to an existing 3' end
103
what is a DNA primer
a short RNA strand synthesised by primase
104
elongation of replication in chromosomes
sliding clamp recruited after RNA primer is synthesised
DNA polymerase assocaites with DNA via the sliding clamp
each base in the parental DNA is read by the DNA polymerase and complimentary bases added to the growing strand in a 5 to 3 direction
105
is DNA synthesis continuous or discontinous and where
continuous of leading strand
discontinuous on lagging strand
106
termination of replication of chromosomes
termination occurs when
-DNA polymerase encounters DNA that has been replicated
- 2 diff forks meet
- fork reaches end of linear chromosome
RNA primers removed and replaced with DNA
DNA ligase connects adjacent strands
107
what does DNA polymerase catalyse
the addition of new nucleotide to the 3' OH of the last nucleotide of the growing strand
108
is DNA antiparallel
yes
109
which hand does DNA polymerase resemble
right hand
palm = catalytic site for nucleotide addition and forms a cleft where elongating dsDNA fits
fingers= ssDNA tmeplate wrap through finger domains which helps position the incoming nucleotides
thumb= holds elongating dsDNA and maintains contact with single strand template necessary for processive synthesis
110
whats the main DNA polymerase in bacteria
DNA polymerase III
leading and lagging strand synthesis
111
whats the main DNA polymerases in eukaryotes
DNA polymerase δ in lagging stand
DNA polymerase ε in leading strand
112
animal cell mtDNA structure
compact
normally codes for 13 proteins
3 rRNAs
22tRNAs
113
is animal cell mtDNA packaged into chromatin
no
its anchored to mitochondrial inner membrane
114
do mitochondrialgenes follow rules to mendelian inheritance in mammals
no
decribedas non-mendelian
115
who are mitochondria inherited from and examples of inherited disease
maternal inheritance
lebers hereditary optic neuropathy
kearns-sayre syndrome
116
how mant proteins do chloroplasts code for
50-100
as well as the rRNAsand tRNAs
117
mechanism of catalysis by DNA polymerase
activesite links 5' p of incoming nucleotide to 3' OH of growing DNA= phosphodiester bond
nucelophilic attack by 3' OH on a-p of incoming dNTP releasing 2 phosphates pyrophosphate
hydrolysis of released pyrophosphate provides energy
118
is DNA polymerase specific
yes the specificity promotes faithful DNA replication
119
how is DNA polymerase specific
active site selective for bp
correct nucleotide fits precisely in the active site only when base-paired with template strand
mismatches have diff shapes so dont fit well
no energy rewuired for proof reading
base selectivity ensures error rate of <1 in 100 000
120
whats the error rate in DNA polymerase base pairing
<1 in 100 000
121
does DNA polymerase require energy to proof read DNA
no
122
DNA polymerisation active site has increased affinity for 3’OH when incorrect nt present
true or false
false
it has reduced affinity
123
Proofreading exonuclease active site has increased affinity for 3’OH when incorrect nt present.
true or false
true
124
is energy required to remove the incorrect nucleotide
yes
125
do all organisms have multiple DNA polymerases with specialised functions
yes
126
DNA helicases protein shape
hexameric ring proteins
127
does different polarity of helicase suggest independent or dependent evolution
independent
128
which way does DNA helicase unwind strands in bacteria and eukaryotes
lagging- 5--> 3 in bacteria
leading 3-->5 in eukaryotic
129
is bacteria homo or hetero hexameric
bacteria= homo-hexamer
eukaryotes= heter-hexameric
130
what subunits is eukaryote hetero-hexameric MCM made up of
MCM2-7
131
how is unwound DNA strands kept open and protected from nucleases
single-stranded binding proteins bind to ssDNA
ss DNA-binding protein SSB in bacteria
replication protein RPA in eukaryotes
132
what do topoisomerases do
assist helicases by removing supercoils from DNA
positive supercoils made by exposed ssDNA impede DNA progress
topoisomerases release the overwound DNA by transiently breaking DNA and allowing supercoils to relax
133
types of topoisomerases
done to reduce tension in supercoiling DNA
type 1- cuts 1 strand
type 2- cuts 2 strands
134
DNA gyrase
topoisomerase
type II
introduces negative supercoils and maintains bacterial DNA in negative supercoiled state
in bacteria only
maintains negative supercoiled state
negative supercoiling allows for easier strand separatio
135
type IB topoisomerases
cut1 strand and cleave and rotate
cut and become transiently covalently attached to 3' end
free end swivels and releases supercoils
DNA ends rejoined
rely on tension in DNA to drive relaxation supercoils
136
type 1A topoisomerase
cut 1 strand and cleave and pass
cut and become transiently covalently attached to 5' end
ends rejoined
relax supercoils ahead of the replication fork in both bacteria and eukaryotes
137
type II topoisomerase
cut both strands and cleave and pass
become transiently covalently attached to the 5’ ends of the cleaved DNA.
activity requires ATP hydrolysis.
relax positive supercoils ahead of the replication fork
138
what are origins (ori)
regions where the dsDNA is separated and replication is intiated
139
what binds to origins
initiator proteins
and recruit helicases to unwind the DNA
all initiator proteins are AAA+ ATPases
140
how many oris do bacteria have per chromosome
1
141
how many oris to eukaryotes have per chromosome
many as they initiate replication at many origins
142
what DNA binds at oriC
DnaA proteins bind to DnaA boxes which are found on oriC
143
what happens when DnaA binds to oriC
initiator protein binds to ATP and DnaA self associates into a helical multisubunit complex which binds to initiator binding site which is adjacent to AT-rich region
its this AT rich region that is unwound as a result of DNA wrapping around spiral DnaA complex and bending the DNA
144
what does DNA binding at oriC recruit
DnaB helicase
145
where does DnaC assemble
onto the DnaB helicase and binds to DnaA
146
what does DnaC do
loads the DnaB helicase onto the DNA
147
How does initiation occur at bacterial origins (oriC)?
DnaA binds to DnaA boxes at oriC to unwind DNA.
DnaC loads DnaB helicase onto ssDNA.
SSB proteins stabilize unwound DNA.
148
How are origins licensed in eukaryotes?
ORC binds to origins in G1 phase.
Cdc6 and Cdt1 load MCM2-7 helicase.
Licensing occurs in G1, activation in S phase by DDK and CDK phosphorylation.
149
What is the role of DNA primase?
Synthesizes short RNA primers for DNA polymerase.
Leading strand: Single primer required.
Lagging strand: Multiple primers for Okazaki fragments.
150
Which enzymes synthesize primers in bacteria and eukaryotes?
Bacteria: DnaG primase (10-30 bases).
Eukaryotes: RNA primase + DNA polymerase α (RNA-DNA hybrid primers).
151
What is the function of sliding clamps?
Enhance DNA polymerase processivity by tethering it to DNA.
Bacteria: β-protein (homodimer).
Eukaryotes: PCNA (homotrimer).
152
How are sliding clamps loaded onto DNA?
Clamp loaders (e.g., RFC in eukaryotes) open the clamp.
ATP binding facilitates loading onto the primer-template junction.
153
How is leading and lagging strand synthesis coordinated in bacteria and eukaryotes?
Bacteria: Replisome with Tau proteins links polymerases to helicase.
Eukaryotes: Ctf4 links CMG helicase, polymerase ε, and primase.
154
How are Okazaki fragments processed in bacteria?
DNA polymerase I removes RNA primers and fills gaps with DNA.
DNA ligase seals remaining nicks.
155
How are Okazaki fragments processed in eukaryotes?
DNA polymerase δ displaces primers, creating flaps.
Fen1 cleaves flaps.
DNA ligase seals nicks.
156
How does termination occur in bacterial DNA replication?
Ter sites and Tus proteins stall replication forks in one direction.
DNA polymerase I and ligase complete replication.
157
What enzymes unlink replicated circular chromosomes?
type II topoisomerases separate fully replicated molecules.
Type IA topoisomerases handle incompletely replicated molecules.
158
What role does DnaA play in bacterial DNA unwinding?
DnaA binds to DnaA boxes at the origin.
DNA wraps around DnaA, causing bending and local unwinding at AT-rich regions.
Recruits DnaB helicase via DnaC loader.
159
How is DNA unwound in eukaryotic replication origins?
ORC binds to DNA in late M/early G1 phase.
Cdc6 and Cdt1 load MCM2-7 helicase as a head-to-head dimer.
Activation in S phase involves DDK and CDK phosphorylation, forming the CMG complex.
160
What is the CMG helicase, and how is it activated?
CMG = Cdc45-MCM-GINS complex.
Formed when MCM2-7 helicase recruits Cdc45 and GINS in S phase.
Transitions from encircling dsDNA to ssDNA during unwinding.
161
Why do DNA polymerases require RNA primers?
DNA polymerases cannot synthesize DNA de novo.
RNA primers provide a 3’OH group for elongation by DNA polymerases.
162
What are the components of the eukaryotic primase-polymerase complex?
Primase subunits: Synthesizes short RNA primers (~10 nucleotides).
polymerase α: Extends primers with a short DNA stretch (iDNA).
163
How does the sliding clamp ensure processivity of DNA polymerase?
Encircles DNA and tethers DNA polymerase to the template.
Prevents dissociation, allowing continuous DNA synthesis.
164
Describe the ATP-dependent mechanism of clamp loading
Clamp loader binds ATP, increasing affinity for the clamp.
Opens the clamp and positions it at the primer-template junction.
ATP hydrolysis closes the clamp and releases the loader.
165
What is polymerase switching, and why is it important?
Primase-polymerase α complex initiates synthesis.
Sliding clamp recruits DNA polymerase δ or ε for processive replication.
Ensures replicative polymerases take over elongation efficiently.
166
What is the role of Ctf4 in eukaryotic replication fork coordination?
Couples CMG helicase to DNA polymerase ε (leading strand) and polymerase α-primase complex.
Acts as a multisubunit hub for replication fork stability
167
How are Okazaki fragments joined in bacteria?
DNA polymerase III stops at the next RNA primer.
DNA polymerase I removes primers and fills gaps.
DNA ligase seals nicks.
168
How is the RNA flap from Okazaki fragments removed in eukaryotes?
DNA polymerase δ displaces primers, creating flaps.
Flap endonuclease (Fen1) cleaves the flap.
DNA ligase seals the remaining nick.
169
What are Ter sites, and how do they work
Ter sites (e.g., TerC) are replication fork traps.
Tus proteins bind Ter sites and block forks in one direction.
Ensure proper termination as forks meet.
170
What is the role of topoisomerases in circular chromosome replication?
Type II topoisomerases unlink interlinked daughter DNA (catenated molecules).
Type IA topoisomerases resolve incomplete replication products.
171
How is lagging strand synthesis coordinated with the leading strand?
Lagging strand forms loops to synchronize polymerase movement with leading strand synthesis.
Tau proteins (bacteria) or Ctf4 (eukaryotes) stabilize coordination.
172
What is the function of DNA ligase in replication?
Seals "nicks" in the DNA backbone after primer removal or flap cleavage.
Ensures continuous and intact DNA strands.
173
What triggers activation of eukaryotic origins during replication?
In S phase, DDK phosphorylates MCM2-7.
CDK phosphorylates Sld2 and Sld3, recruiting GINS and forming the active CMG helicase complex.
174
What structural differences exist between bacterial and eukaryotic sliding clamps?
Bacteria: β-protein (homodimer with three similar domains).
Eukaryotes: PCNA (homotrimer with two similar domains per subunit).
175
What are the components of clamp loaders in bacteria and eukaryotes?
Bacteria: γ-complex (3 γ or τ subunits, 1 δ, and 1 δ’).
Eukaryotes: Replication Factor C (RFC) with 5 different subunits
176
What is the pre-replicative complex in eukaryotic cells?
Composed of ORC, Cdc6, Cdt1, and MCM2-7 helicase.
Loaded at origins during G1 but inactive until S phase
177
What role does ATP play in the clamp loading process?
ATP binding increases clamp loader affinity for the sliding clamp.
ATP hydrolysis closes the clamp around DNA and releases the loader.
178
Why is the lagging strand looped during replication?
Allows lagging strand polymerase to move in the same direction as the replication fork.
Ensures coordination with the leading strand.
179
What ensures that polymerase switching occurs at the right time and place?
Sliding clamps recruit replicative polymerases (DNA polymerase δ or ε).
Primase-polymerase α complex dissociates after primer synthesis.
180
What happens if Fen1 does not properly cleave the RNA flap?
Unremoved flaps can cause replication errors or strand instability.
Backup endonucleases may step in, but inefficiency can lead to genomic instability.
181
Which polymerases synthesize the leading strand in bacteria and eukaryotes?
Bacteria: DNA polymerase III.
Eukaryotes: DNA polymerase ε.
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182
Which polymerases synthesize the lagging strand in bacteria and eukaryotes?
Bacteria: DNA polymerase III.
Eukaryotes: DNA polymerase δ.
183
What are Okazaki fragments, and why are they important?
Short, discontinuous DNA fragments synthesized on the lagging strand.
Allow replication of the strand opposite to the fork movement.
184
Why are topoisomerases critical at the end of replication?
Resolve supercoiling and unlink catenated circular chromosomes.
Prevents mechanical stress on DNA molecules.
185
How does the Tus-Ter system prevent bidirectional fork collisions?
Tus proteins block forks only when approaching from one direction.
Ensures that replication forks terminate efficiently without overlap.
186
Why is replication more complex in eukaryotes than in bacteria?
Larger genome with multiple origins.
Presence of chromatin and histone modifications.
Requirement to coordinate replication with the cell cycle.
187
When does replication occur in the cell cycle, and how is it regulated?
Occurs during the S phase.
Regulated by DDK and CDK to ensure proper activation of helicases and polymerases.
188
What is the difference between the pre-replicative and active replication complex?
Pre-replicative Complex: Inactive, includes ORC and MCM2-7 helicase (G1 phase).
Active Complex: CMG helicase, loaded with polymerases and associated factors (S phase).
189
What is the role of SSB proteins in replication?
Stabilize single-stranded DNA (ssDNA) to prevent secondary structures.
Protect ssDNA from nuclease degradation.
190
How long are RNA primers in bacteria and eukaryotes?
Bacteria: ~10-30 nucleotides (DnaG primase).
Eukaryotes: ~10 nucleotides of RNA + short DNA extension (polymerase α).
191
Name three key proteins or complexes present at the replication fork.
Helicase (DnaB in bacteria, CMG in eukaryotes).
Sliding clamp (β-clamp or PCNA).
DNA polymerases (III in bacteria, δ/ε in eukaryotes).
192
How does the termination zone ensure replication completion in bacteria?
Multiple Ter sites trap forks in both directions.
Tus ensures that forks meet and terminate without over-replication.
193
does termination occur at multiple sites in eukaryotes
yes
194
termination of DNA replicaiton in eukaryotes
replication forks converge- CMG complexes move past each other on leading strand and CMG transitions to encircling dsDNA
DNA polymerase δ, Fen1 and DNA ligase I are recruited to complete processing.
MCM7 is ubiquitinated and protein p97 unloads the CMG complex.
Double-stranded DNA remains entwined, and this is resolved by a topoisomerase II.
195
longer wavelength lower the energy
true or false
true
196
what is UVC and UVB absorbed by
all UVC absorbed by atmospheric o2
most UVB absorbed by ozone layer
197
which types of UV are important to human health
UVA
UVB
198
how much UVB reaches teh earths surface
10%
199
what absorbance does DNA absorb UV radiation at
peak absorbance is 254nm
200
what happens when dna absorbs uv
becomes photoexcited and formation of intrastrand cross-linked pyrimidine dimers
not accommodated in active site of dna replication polymerases - replicated by low-fidelity TLs polymerases - leads to mismatches and distortion/mutation
201
ionising radiation examples
x-ray
y-ray
202
wavelength of ionisng radiation
short wavelength
high energy radiation
203
where does ionising radiation come from
natural- e.g. aberdeen has a bunch of radiation gas related to granite
therapeutic
diagnostic
occupational
204
what leads to damage to dna by ionising radiation
35%- direct interaction of radiation energy
65%- indirectly by reative oxigen species formed by ionisation of cellular water- water covers dna leads to OH radicals etc whcih damages bases and breaking strands
205
whats the ames test used for
assaying mutagenicity
wont grow on agar with histidine as cant synthesise it.
some chemicals are converted to mutagens by liver enzymes, potential mutagens are treated with a mixture of liver enzymes prior to addition
206
whys ames test a well used test
cheaper
easier
ethical than testing on animals
207
2 main headings of DNA repair
direct reversal of DNA damage
excision of damages DNA
208
2 examples of direct reversal of DNA damage
-repair of O^6 alkylguanine
-enzymatic photoreactivation
209
3 examples of excision pathways of damages DNA
-Mismatch repair (MMR)
-Base excision repair (BER)
-Nucleotide excision repair (NER)
210
What are the two major physical agents that cause DNA damage?
UV radiation and ionizing radiation.
211
What are the two general categories of DNA repair mechanisms?
Damage excision-based repair and direct reversal of damage.
212
What type of DNA damage does UV radiation cause?
Formation of intrastrand cross-linked pyrimidine dimers.
213
Why are pyrimidine dimers harmful to DNA replication?
They create a kink in the DNA helix, preventing replication by high-fidelity polymerases
214
What are the two types of DNA damage caused by ionizing radiation?
Direct interaction with DNA (35%) and indirect damage via reactive oxygen species (65%)
215
What is the most lethal type of DNA damage caused by ionizing radiation?
Double-strand breaks (DSBs).
216
What is the Ames test used for?
Detecting mutagenic potential of chemicals.
217
Why are liver enzymes added to the Ames test?
some chemicals require metabolic activation to become mutagenic.
218
What enzyme repairs O6-alkylguanine damage?
O6-Methylguanine-DNA Methyltransferase.
219
How does photolyase repair pyrimidine dimers?
it absorbs blue light and transfers an electron to break the dimer bond.
220
What are the three main types of excision repair mechanisms?
mismatch repair (MMR), base excision repair (BER), and nucleotide excision repair (NER).
221
What does the mismatch repair system correct?
Errors made by DNA polymerase during replication.
222
How does the system distinguish between the correct and incorrect strand?
The newly synthesized strand is unmethylated.
223
What enzyme removes the damaged base in BER?
DNA glycosylase.
224
What enzyme cuts the DNA backbone at the abasic site? in BER
AP endonuclease
225
What type of DNA damage does NER repair?
Bulky lesions that distort the DNA helix.
226
What genetic disorder is caused by defects in NER?
Xeroderma Pigmentosum (XP).
227
Which UV rays reach the Earth's surface?
10% of UVB and most UVA rays.
228
What type of DNA polymerases replicate UV-induced damage?
ow-fidelity translesion synthesis (TLS) polymerases.
229
What are two major types of DNA damage caused by ionizing radiation?
Oxidative damage to bases and strand breaks (single-strand and double-strand).
230
What kind of mutations does the Ames test detect
Frameshift and point mutations.
231
What does O6-Methylguanine-DNA Methyltransferase (MGMT) do?
transfers the methyl group from damaged guanine to itself, permanently inactivating the enzyme.
232
233
Why is MGMT considered a "suicide enzyme"?
Because it is inactivated after repairing a single lesion.
234
What are the two types of photolyases, and what do they repair?
CPD photolyase (repairs cyclobutane pyrimidine dimers) and 6-4 photolyase (repairs 6-4 photoproducts).
235
What proteins recognize and repair mismatches in bacteria?
MutS, MutL, and MutH.
236
how does MutH recognize the newly synthesized DNA strand?
It is not yet methylated.
237
What enzyme unwinds the DNA around the mismatch?
UvrD helicase.
238
What type of exonuclease is used if MutH nicks the DNA 5’ to the mismatch?
A 5’-3’ exonuclease (RecJ or exonuclease VII).
239
What type of exonuclease is used if MutH nicks the DNA 3’ to the mismatch?
A 3’-5’ exonuclease (Exonuclease I).
240
what enzyme resynthesizes the DNA after mismatch removal?
DNA polymerase III.
241
What types of DNA damage does BER repair?
Small, non-helix-distorting lesions (e.g., oxidized bases, uracil incorporation, alkylation damage).
242
What enzyme removes the damaged base in BER?
A DNA glycosylase specific for the type of damage.
243
What is an abasic site?
A site in DNA where the base has been removed, leaving just the sugar-phosphate backbone.
244
What enzyme recognizes and cleaves abasic sites?
AP endonuclease.
245
What enzyme fills the gap after AP endonuclease action?
DNA polymerase.
246
What enzyme seals the repaired strand? in BER
DNA ligase
247
What types of DNA damage does NER repair?
Bulky DNA adducts, pyrimidine dimers, and chemically modified bases.
248
What are the key NER proteins in E. coli?
UvrA, UvrB, UvrC, and UvrD.
249
What is the role of UvrA in NER?
it recognizes DNA damage and recruits UvrB.
250
What is the role of UvrB in NER?
It binds the damaged site and separates the DNA strands.
251
What is the role of UvrC in NER?
it makes two cuts around the damaged DNA.
252
What is the role of UvrD in NER?
It acts as a helicase to remove the damaged strand.
253
What enzyme fills in the gap after damage removal in NER?
DNA polymerase.
254
What are the symptoms of Xeroderma Pigmentosum (XP)?
Extreme sensitivity to sunlight and a high predisposition to skin cancer.
255
What other disorder is linked to NER defects?
Cockayne Syndrome.
256
how many bp in e.coli genome
4.6mbp
4600000bp
257
what does non essential extrachromosomal dna in bacteria do
confer advantages to host cell
258
e.coli genome
1essential circular ds dna chromosome
4.6mbp
mainly unique sequence
259
what are the 3 distinct types of plasmids in bacterial genomes
sex plasmids
r plasmids
col plasmids
260
examples of a sex plasmid for bacteria
f (fertility) plasmids of e.coli
kinda large 100kbp
selfmobile
stringent replicaiton
Approx 35% of sequence encode functions permitting transfer between individual bacteria
Remaining sequence contains four insertion sequence elements (1 x IS2, 2 x IS3, 1 x IS1000 aka gd)
261
what does episome mean
may exist as a free circular plasmid or be integrated into chromosome
F plasmid is one
262
how do sex plasmids mediate transfer of bacterial genes
conjugation
263
size of r plasmids
relatively large
30-100kbp
vary in size
264
what do r plasmids do
encode resistance to one ormoreantimicrobial drugs, heavy metals toxins
265
how do r plasmids spread
through environment and between unrelated bacterial species
poses a threat to treatment of infectious disease
266
what does the r in r plasmids stand for
resistance
267
size of col plasmids
small plasmids
mostley under 25kbp
268
what do col plasmids encode
biological factors such as colicin
dont encode functions permitting transfer between individual bacteria
269
why might col plasmids be transferred
if f or r plasmids present in same cell encoding functions required for contact and transfer
270
are col plasmids used by scientists
col plasmids have been extensively manipulated by molecular biologists to generate useful vectors for DNA cloning. e.g. pGEM3zf derived from colE1 plasmid
271
when was f plasmid mediated conjugation in e.coli discouvered
1946
by lederberg and tatum
272
f plasmid mediated conjugation experiment
Mixed two auxotrophic strains of bacteria (A & B)
Observed some prototrophic colonies when mixture plated on minimal media
Colonies resulted from genetic exchange between original strains
U-tube expt confirmed physical contact between strains required
273
is bacterial conjugation uni or bi directional
unidirectional
bacterial mating
274
what happens in bacterial conjugation
F+ cell contact F- cell - initial connection by long tubular F-pilus
F+ pilus contracts forming cytoplasmic bridge- sharing cytoplasm and transfers genetic material
F plasmid carries Transfer genes for contact and mobilisation functions - encodes pilin protein to build pilus
275
mechanism of F plasmid transfer
initiated by a nick in DNA at ORiT
5' end of ssDNA transferred to recipient
rolling circle replication forms ssDNA from F plasmid
DNA synthesis in F- recipient restores second strand
276
whats Hfr
high frequency of recombination
occurs through crossing over and recombination
F plasmid can be integrated in either orientation according to orientation of recombining sequences
F plasmid integrated into chromosome via recombination between insertion sequences on F plasmid and chromosome
F plasmid integration is reversible
277
who discovered Hfr strains
Hayes and Cavalli-Sforza
278
Hfr transfer of bacterial genes
F-plasmid integrated into chromosome
encodes transfer functions
integrated F plasmid oriT nicked
F factor initiates transfer to recipient
bacterial chromosome follows
transferred chromosomal DNA recombines with recipient chromosome
279
can Hfr strains conjugate withF- strains
yes
280
is Hfr conjugation species specific
no
this allows horizontal gene transfer
281
what is success of promiscuous conjugation dependent on
DNA homology- need recombination to integrate transferred fragments
282
can Hfr conjugation be used to map bacterial genes
yup
jacob and wollman discouvered or something
283
how to determine if genes have been transferred from Hfr to F-
Mix bacteria and then at various times after mating commences withdraw samples, break mating cells apart and plate bacteria on selective media to determine which genes have been transferred from Hfr to F-
284
What is the mechanism of F plasmid transfer in bacterial conjugation?
The F+ cell establishes contact with an F- cell via a pilus. The F plasmid's tra genes encode the transfer functions, and DNA transfer starts at OriT (origin of transfer). Rolling circle replication ensures that a single-stranded copy of the plasmid is transferred, and the recipient synthesizes the complementary strand.
285
How does conjugation with an Hfr strain differ from standard F+ conjugation?
In Hfr x F- matings, DNA transfer starts at OriT within the F plasmid and continues into the bacterial chromosome. The entire F factor is rarely transferred because the conjugation bridge is fragile, leading to only partial chromosome transfer.
286
What was the significance of the Jacob and Wollman interrupted mating experiment?
It helped determine the order of genes on the E. coli chromosome. By stopping conjugation at different times and checking which genes had transferred, they showed that the E. coli genome is circular.
287
How do F' plasmids form, and what is their significance?
F' plasmids arise when the F plasmid imprecisely excises from the chromosome, taking bacterial genes with it. This enables sexduction, where the F' plasmid transfers these genes to a recipient, creating a partial diploid (merodiploid).
288
How does generalized transduction occur?
A bacteriophage accidentally packages bacterial DNA instead of its own. When this phage infects a new bacterium, it injects bacterial genes, which may recombine with the recipient’s genome.
289
How can transduction be used for gene mapping?
Genes closer together on the bacterial chromosome are more likely to be co-transduced (transferred together in the same phage particle). The frequency of cotransduction helps determine gene order and distance.
290
What did the Jacob and Wollman interrupted mating experiment reveal?
Goal: Determine the order of bacterial genes on the E. coli chromosome.
Method:
Mixed Hfr (thr⁺ leu⁺) donor with F- (thr⁻ leu⁻) recipient.
Allowed conjugation to proceed for specific time intervals.
Used a blender to disrupt mating pairs.
Plated bacteria on selective media to determine which genes had transferred.
Findings:
Gene transfer follows a linear sequence.
The E. coli chromosome is circular.
The time taken for genes to transfer reflects gene distance from OriT.
291
What is an F' plasmid, and how does it form?
Sometimes, the F plasmid excises imprecisely from the chromosome, carrying adjacent chromosomal genes with it.
This modified plasmid is now an F' plasmid (e.g., F’lac if it carries the lac gene).
F' plasmids can transfer chromosomal genes to recipients, creating merodiploids (partial diploids).
292
How is cotransduction frequency used to map bacterial genes?
Genes closer together are more likely to be packaged into the same phage particle.
The higher the cotransduction frequency, the closer two genes are on the chromosome.
This allows mapping of genes relative to each other.
293
what happens in lysogenic cycle
the phage dna becomes incorporated into the bacterial chromosome becomes prophage
replicates and a population of bacteria now infected
294
what life cycle does bacteriophage upside down y adopt
can adopt both lytic and lysogenic cycles
In both the linear bacteriophage chromosome is first circularized in bacteria by annealing of complementary ends followed by ligation
295
What are the two main pathways for repairing DNA double-strand breaks (DSBs)?
Non-homologous end joining (NHEJ) and homology-directed repair (HDR).
296
What are the four steps of homology-directed repair?
1) Presynapsis: Generation of single-stranded DNA (ssDNA).
2) Synapsis: Pairing of ssDNA with an intact homologous duplex to form a heteroduplex.
3) Postsynapsis: DNA synthesis using homologous DNA as a template.
4) Resolution: Separation of two DNA duplexes.
297
What role does the RecBCD complex play in homology-directed repair?
It generates single-stranded tails at DSBs by helicase and nuclease activity, with modulation by Chi sequences.
298
What is a Holliday junction, and how is it resolved?
A Holliday junction is a four-armed DNA structure formed during homologous recombination. It is resolved by cleavage, which can result in crossover or non-crossover products.
299
What are plasmids, and how do they differ from the bacterial chromosome?
Plasmids are small, circular, extrachromosomal DNA molecules in bacteria that are non-essential but may provide advantages (e.g., antibiotic resistance).
300
How does the F plasmid enable bacterial conjugation?
The F plasmid contains tra genes that encode the pilus and mobilization functions. DNA transfer is initiated at the oriT site.
301
does trasncription need a primer to begin
no
as soon as promotor sequence binds to the RNA polymerase transcription begins
302
which direction is rna transcribed in
5' to 3'
303
what happens once the transcription has unwound past promotor sequence
complex undergoes a conformational change
stabilising its interaction with DNA
the rna polymerase elongates the rna transcript further
happens till stop codon where the rna is then released from the template
304
what are the core subunits of a prokaryotic RNA polymerase
core enzyme- 5 polypeptides, 2 copies of a subunit and 1 b,b' and w
sigma factor- recognsies promotor region and allows rna pol to bind
holoenzyme - core enzyme + sigma factor
305
What are plasmids, and how do they differ from the bacterial chromosome?
Plasmids are small, circular, extrachromosomal DNA molecules in bacteria that are non-essential but may provide advantages (e.g., antibiotic resistance).
306
How does the F plasmid enable bacterial conjugation?
The F plasmid contains tra genes that encode the pilus and mobilization functions. DNA transfer is initiated at the oriT site.
307
What is an Hfr strain, and how is it formed?
An Hfr strain arises when the F plasmid integrates into the bacterial chromosome via recombination between insertion sequences (IS).
308
What is generalized transduction, and which bacteriophage mediates it?
Generalized transduction occurs when bacteriophage P1 packages fragments of bacterial DNA and transfers them to a new host. Any part of the bacterial genome can be transferred.
309
How does specialized transduction differ from generalized transduction?
Specialized transduction is mediated by temperate bacteriophages like lambda and only transfers genes near the phage integration site.
310
What are the two possible life cycles of lambda phage in bacteria?
Lytic pathway: The phage replicates and lyses the host cell.
Lysogenic pathway: The phage integrates into the bacterial genome as a prophage.
311
What are transposable elements, and how do they move within the genome?
Transposable elements (transposons) are DNA sequences that move within the genome via "cut-and-paste" or "copy-and-paste" mechanisms.
312
What are the three main types of transposons in bacteria?
1) Insertion sequences (IS).
2) Composite transposons (e.g., Tn10).
3) Non-composite transposons (e.g., Tn3).
313
What is the difference between "cut-and-paste" and "nick-and-paste" transposition?
"Cut-and-paste" involves excision and insertion of the transposon, while "nick-and-paste" forms a cointegrate, eventually resolved into separate molecules.
314
What are the two major pathways for repairing DNA double-strand breaks?
Non-homologous end joining (NHEJ) and homology-directed repair (HDR).
315
Which repair pathway is predominant in non-dividing cells?
Non-homologous end joining (NHEJ).
316
What does homology-directed repair (HDR) require that NHEJ does not?
A homologous DNA template.
317
In which cell cycle phases does HDR occur most frequently?
Late S-phase and G2.
318
What is the main risk of using homologous chromosomes as a template in HDR?
Loss of heterozygosity.
319
What are the four main steps of HDR?
Presynapsis, Synapsis, Postsynapsis, and Resolution.
320
What occurs during presynapsis?
Generation of single-stranded DNA (ssDNA) at the break site
321
Which proteins are involved in the presynapsis step?
Helicases and nucleases such as the RecBCD complex.
322
What is the purpose of synapsis in HDR?
To allow the ssDNA to invade a homologous duplex and form a D-loop.
323
How does the D-loop contribute to repair?
It stabilizes strand invasion and initiates DNA synthesis.
324
What is the main function of RecA in HDR?
It facilitates homologous strand invasion and exchange.
325
What eukaryotic protein is functionally similar to RecA?
Rad51
326
What complex generates single-stranded tails at DSBs?
RecBCD complex.
327
What role does the Chi sequence play in RecBCD activity?
It modulates RecBCD activity, reducing nuclease degradation on the 3’ end.
328
What is the function of RuvAB?
It facilitates branch migration of Holliday junctions.
329
Q: How do BRCA1 and BRCA2 contribute to HDR?
They help load Rad51 onto ssDNA, ensuring efficient repair.
330
What is the D-loop, and why is it important?
A displaced loop of DNA that stabilizes strand invasion during HDR.
331
What promotes the formation of the D-loop?
RecA in bacteria and Rad51 in eukaryotes.
332
What is the function of the heteroduplex in HDR?
It stabilizes the interaction between damaged and template DNA strands.
333
How is new DNA synthesized during HDR?
DNA polymerase extends the invading 3’ strand using the homologous template.
334
What happens if the invading strand is not properly stabilized in HDR?
The repair process may fail, leading to mutations or chromosomal instability.
335
What is a Holliday junction?
A four-stranded DNA structure formed during homologous recombination.
336
How are Holliday junctions resolved?
They are cleaved by enzymes like RuvC in bacteria or GEN1 in eukaryotes.
337
What determines whether recombination results in crossover or non-crossover products?
The orientation of the Holliday junction resolution.
338
What is the function of the RuvAB complex?
It promotes branch migration of Holliday junctions
339
What is the role of RuvC in HDR?
It cleaves Holliday junctions to complete recombination.
340
What is the SDSA pathway in HDR?
synthesis-dependent strand annealing
A mechanism where the invading strand is extended but does not form Holliday junctions.
341
Why does SDSA not result in crossover?
The newly synthesized strand is displaced and annealed back to its original strand.
342
What is an advantage of SDSA over the Holliday junction pathway?
It avoids chromosomal crossover, preventing unwanted genetic exchange
343
Which organisms primarily use SDSA for DSB repair?
Eukaryotic somatic cells
344
How does SDSA differ from the classical HDR pathway?
It does not involve double Holliday junction formation.
345
How does homologous recombination contribute to genetic diversity?
It facilitates crossover between homologous chromosomes during meiosis.
346
What initiates recombination in meiosis?
The enzyme Spo11 creates programmed DSBs.
347
How does meiotic recombination ensure accurate chromosome segregation?
It creates physical links between homologous chromosomes.
348
What is the role of the synaptonemal complex?
It aligns homologous chromosomes and facilitates recombination.
349
What happens if recombination fails during meiosis?
It can lead to aneuploidy, resulting in conditions like Down syndrome.
350
What is gene conversion?
A non-reciprocal transfer of genetic information during HDR.
351
When does gene conversion occur?
When HDR uses a homologous chromosome as a template instead of a sister chromatid.
352
How does gene conversion lead to loss of heterozygosity?
It replaces one allele with another from the homologous chromosome.
353
Why is loss of heterozygosity significant in cancer?
It can inactivate tumor suppressor genes like BRCA1.
354
What type of DNA repair event is most likely to cause gene conversion?
HDR using a homologous chromosome instead of a sister chromatid.
355
How does defective HDR contribute to cancer?
Cells accumulate mutations, leading to genomic instability.
356
Why are BRCA1/2-deficient tumors sensitive to PARP inhibitors?
They rely on error-prone repair pathways, making them vulnerable to DNA damage.
357
What is synthetic lethality in the context of HDR-deficient cells?
When blocking an alternative repair pathway leads to cell death.
358
How is HDR used in genome editing?
It allows precise gene insertion using CRISPR-Cas9 and donor DNA.
359
What is the role of homologous recombination in bacterial gene transfer?
It allows integration of foreign DNA from transformation, conjugation, or transduction.
360
Summarize the key steps of HDR in five sentences.
1) A DSB is resected to generate ssDNA.
2) The ssDNA invades a homologous duplex, forming a D-loop.
3) DNA synthesis extends the invading strand.
4) Resolution of recombination intermediates occurs.
5) The DNA structure is restored, ensuring accurate repair.
361
What are plasmids?
Small, circular, extrachromosomal DNA molecules that replicate independently in bacteria.
362
Are plasmids essential for bacterial survival?
No, but they can provide advantages such as antibiotic resistance and virulence factors.
363
What are the three main types of plasmids?
F (fertility) plasmids, R (resistance) plasmids, and col (colicin) plasmids.
364
What is the function of an F plasmid?
It enables bacterial conjugation by carrying genes necessary for pilus formation and DNA transfer.
365
What is the function of an R plasmid?
It provides resistance to antibiotics, allowing bacteria to survive antimicrobial treatment.
366
What are col plasmids?
Plasmids that encode colicins, which are proteins toxic to competing bacteria
367
What does the term “self-mobilizable” mean in relation to plasmids?
A plasmid that can transfer itself between bacterial cells via conjugation.
368
What is an episome?
A plasmid that can integrate into the bacterial chromosome
369
What determines the copy number of a plasmid in a bacterial cell?
The plasmid’s origin of replication and regulatory mechanisms controlling replication
370
How do plasmids contribute to bacterial evolution?
They enable horizontal gene transfer, spreading beneficial traits such as antibiotic resistance.
371
What is bacterial conjugation?
A process where genetic material is transferred from one bacterium to another through direct contact.
372
What is the F pilus, and what is its function?
A filamentous structure encoded by the F plasmid, used to connect donor and recipient bacteria during conjugation.
373
What is the difference between F+ and F- bacterial cells?
F+ cells contain the F plasmid and can initiate conjugation, while F- cells lack it.
374
What are tra genes, and where are they located?
Genes on the F plasmid are responsible for pilus formation and DNA transfer.
375
What is the oriT site, and why is it important?
The origin of transfer on the F plasmid where DNA nicking occurs to initiate transfer.
376
What is rolling circle replication, and how does it function in conjugation?
A mechanism of DNA replication where the leading strand is continuously synthesized while the lagging strand is displaced and transferred.
377
What happens to the recipient bacterium after conjugation?
If the entire F plasmid is transferred, the recipient becomes F+ and can act as a donor.
378
Why is conjugation considered a form of horizontal gene transfer?
It allows the direct exchange of genetic material between bacteria, independent of cell division.
379
Can conjugation occur between different bacterial species?
Yes, some plasmids can transfer between distantly related bacteria, facilitating interspecies gene exchange.
380
What is the ecological significance of conjugation?
It spreads genetic traits such as antibiotic resistance, affecting microbial evolution and human health.
381
What is an Hfr strain?
A bacterium where the F plasmid has integrated into the chromosome, enabling chromosomal gene transfer.
382
How does an Hfr strain arise
Through recombination between insertion sequences (IS) on the F plasmid and bacterial chromosome.
383
How does gene transfer in an Hfr × F- mating differ from an F+ × F- mating?
Hfr strains transfer chromosomal genes, while F+ strains transfer only the F plasmid.
384
Why does the entire chromosome rarely transfer during Hfr conjugation?
The mating bridge is fragile and often breaks before complete transfer.
385
What happens if the F plasmid excises imprecisely from an Hfr strain?
It may carry adjacent chromosomal genes, forming an F’ (F prime) plasmid.
386
What is the significance of F’ plasmids?
They allow partial diploids (merodiploids), useful in genetic analysis.
387
How was conjugation used to map the E. coli chromosome?
By interrupting mating at different times and determining the order of transferred genes
388
What was the key finding from Jacob and Wollman’s interrupted mating experiments?
That the bacterial chromosome is circular.
389
How does recombination integrate transferred chromosomal DNA into the recipient?
Through homologous recombination.
390
What determines the order of gene transfer in an Hfr strain?
The location and orientation of F plasmid integration.
391
What is transduction?
Gene transfer mediated by bacteriophages.
392
What is the difference between generalized and specialized transduction?
Generalized transduction transfers any bacterial gene, while specialized transduction transfers only genes near the phage integration site.
393
Which bacteriophage is commonly associated with generalized transduction?
Bacteriophage P1.
394
How does a phage package bacterial DNA during generalized transduction?
A packaging error results in fragments of bacterial DNA being enclosed in the phage capsid.
395
What happens when a transducing phage infects a new bacterial cell?
The bacterial DNA it carries can be recombined into the recipient’s genome.
396
Why is generalized transduction useful in bacterial genetics?
It enables precise mapping of bacterial genes based on cotransduction frequency.
397
What is cotransduction?
The simultaneous transfer of two closely linked genes in one transducing particle
398
How can cotransduction frequency help determine gene order?
Genes that are more frequently cotransduced are located closer together.
399
What is the size limit for DNA fragments transferred by generalized transduction?
Approximately 100 kilobase pairs (kbp), the size of the P1 genome.
400
Why do transducing phages produce non-lytic infections?
They lack phage genes required for replication and lysis.
401
What are the three main methods of horizontal gene transfer in bacteria?
Transformation, conjugation, and transduction.
402
How does bacterial conjugation differ from transduction?
Conjugation requires cell-to-cell contact, while transduction is mediated by phages
403
Why is conjugation an important mechanism for antibiotic resistance spread?
It allows rapid transfer of resistance genes between bacteria.
404
How is transduction used in bacterial genome engineering?
It enables precise introduction of genetic modifications using phage-mediated gene transfer.
405
Summarize conjugation and transduction in five key points.
(1) Plasmids enable conjugation; (2) F+ and Hfr strains transfer DNA; (3) Generalized transduction transfers random genes; (4) Cotransduction maps gene order; (5) Both processes drive bacterial evolution.
406
What is transduction?
A process where bacteriophages transfer genetic material between bacteria.
407
What are the two types of transduction?
Generalized transduction and specialized transduction.
408
What type of bacteriophage mediates specialized transduction?
Temperate bacteriophages, such as lambda (λ) phage.
409
What are the two life cycles of a temperate bacteriophage?
The lytic cycle and the lysogenic cycle.
410
What happens during the lysogenic cycle?
The phage genome integrates into the bacterial chromosome as a prophage.
411
What is the integration site for lambda (λ) phage in E. coli?
The attB site on the bacterial chromosome.
412
What is the attP site?
The attachment site on the phage genome for site-specific recombination.
the integration site for lambda (λ) phage in E. coli?
413
What enzyme mediates integration of lambda phage into the bacterial genome?
Integrase.
414
What triggers excision of a prophage from the bacterial genome?
Environmental stress, such as DNA damage (e.g., UV radiation).
415
What is aberrant excision in specialized transduction?
When the prophage excises incorrectly, carrying bacterial genes with it.
416
What genes are typically transferred by lambda phage during specialized transduction?
Only genes adjacent to the attB integration site (e.g., gal and bio genes in E. coli).
417
Why are defective phages produced in specialized transduction?
The phage genome is missing some essential genes due to aberrant excision.
418
How does a defective transducing phage infect recipient cells?
It delivers bacterial DNA but cannot enter the lytic cycle.
419
What are transposable elements?
DNA sequences that can move within the genome.
420
What is another term for transposable elements?
Jumping genes
421
Who discovered transposons, and in what organism?
Barbara McClintock in maize.
422
Do transposons exist in all organisms?
Yes, they are found in bacteria, plants, and animals.
423
How do transposons contribute to genetic variation?
By inserting into new genomic locations, they can disrupt or regulate genes.
424
What are the three main types of transposable elements?
DNA-only transposons, long terminal repeat (LTR) retrotransposons, and non-LTR retrotransposons.
425
What is the only type of transposon found in bacteria?
DNA-only transposons.
426
What are insertion sequences (IS elements)?
The simplest transposons, containing only a transposase gene flanked by inverted repeats
427
What is a composite transposon
A transposon with two IS elements flanking additional genes, such as antibiotic resistance genes.
428
What is a non-composite transposon?
A transposon that does not rely on IS elements for movement but has its own transposition machinery.
429
What are the two mechanisms of transposition?
Cut-and-paste transposition and replicative transposition.
430
How does cut-and-paste transposition work?
The transposon is excised from one location and inserted into another
431
What enzyme is required for cut-and-paste transposition?
Transposase
432
How does replicative transposition work?
The transposon is copied and inserted at a new site, leaving the original in place.
433
What is a target site duplication?
A short sequence of duplicated DNA flanking a transposon after insertion.
434
How do transposons contribute to antibiotic resistance?
They carry resistance genes that can integrate into plasmids or chromosomes.
435
What is an example of an antibiotic resistance transposon?
Tn3, which carries the β-lactamase gene for ampicillin resistance.
436
How do transposons spread resistance genes between bacteria?
Through horizontal gene transfer mechanisms like conjugation and transduction.
437
Why is transposon-mediated resistance a concern in medicine??
It enables rapid bacterial adaptation to antibiotics, leading to multidrug-resistant strains.
438
Can transposons move between bacterial chromosomes and plasmids
Yes, allowing the transfer of resistance genes within and between bacterial species.a
439
How are transposons used in genetic engineering?
They serve as tools for mutagenesis and gene delivery in research.
440
What is the Sleeping Beauty transposon system?
A synthetic transposon system used in gene therapy and genome modification.
441
What are transposon-based mutagenesis studies?
Experiments using transposons to disrupt genes and study their functions.
442
Why are transposons useful in biotechnology?
They enable stable gene integration without using viral vectors.
443
Can transposons be used in cancer therapy?
Yes, engineered transposons are being tested for gene therapy in certain cancers.
444
Summarize the key points of specialized transduction and transposition.
(1) Specialized transduction transfers genes near the phage integration site.
(2) Transposons are mobile DNA elements that impact genetic diversity.
(3) Transposons move by cut-and-paste or replicative mechanisms.
(4) Antibiotic resistance can spread via transposons.
(5) Transposons have applications in genetic engineering and medicine.
445
what is transcription
The process of synthesizing RNA from a DNA template.
446
What is the central dogma of molecular biology?
DNA → RNA → Protein.
447
What enzyme is responsible for transcription?
RNA polymerase
448
In which direction is RNA synthesized
5 to 3
449
How does transcription differ from DNA replication?
How does transcription differ from DNA replication?
450
What are the three main stages of transcription?
Initiation, elongation, and termination.
451
What is the function of a promoter?
t is a DNA sequence that signals RNA polymerase where to start transcription.
452
What is abortive initiation in transcription?
When RNA polymerase repeatedly synthesizes short RNA fragments before producing full-length RNA.
453
What is the transcription bubble?
A region of unwound DNA where transcription occurs.
454
How does RNA polymerase stabilize itself after initiation?
It undergoes a conformational change that strengthens its interaction with DNA.
455
cancer overview
heterogeneous group of around 200 disease
uncontrolled cell growth
cell spreading - metastise
can be undifferentiated or dedifferentiate
cell signalling responses dysregulated
cancer formation from carcinogenesis
456
examples of cell signalling responses dysregulated by cancers
contact inhibition
apoptosis
proliferation
inhibition signalling
457
metastasis
spread of cancer cells to distant sites
458
carcinoma orgin
epithelial origin
about 80-90% human cancers
459
sarcoma origin
in supportive and connective tissues like bones, tendons, cartilage, muscle, fat
460
lymphoma origin
blood
lymph tumours thatdevelop from lymphocytes
461
leukaemia origin
begin in bone marrow and result in high numbers of abnormal blood cells
462
brain cancer origin
blastoma
brain
spinal cord cancers
463
whats the correlation between risk of cancer and total stem cell divisions
directly proportional
as more divisions = more risk
e.g. colon cells divide more than in bone cancers
464
dysplasia
cells change form
loose regulation and contact inhibition etc
465
hyperplasia
cell divides more rapidly than normal
466
in situ cancer
cells stay in 1 place
benign
localised overgrowth
467
malignant tumour
cells invade neighbouring tissues
enter blood and lymph
metastasise form at distant site
468
why are tumours monoclonal
all arise from a single starting cell
doesnt mean all tumour cells are identical
469
driver mutations
tumour has about 5-8
a mutation that directly/indirectly confers a selective growth advantage
early driver mutation give cells a growth advantage normally
470
passenger mutations
hundreds in typical tumour
no in/direct effect on selective growth advantage of the cell
471
what are positive regulators in cancer susceptibility gene
classical oncogenes
telomerase
anti-apoptosis genes
promote cell proliferation
472
examples of negative cancer regulators
classical tumour suppressor genes
indirectly acting tumour suppressor genes
apoptic genes
473
what happens in s phase of cell cycle
DNA synthesis
creating 2 identical sister chromatids
474
G2 phase of cell cycle
A gap phase allowing growth and preparation for separation of the sister chromatids. The mitotic spindle begins to form
475
M-phase of cell cylce
Includes mitosis (nuclear envelope breaks down, chromosomes are attached to spindle and sister chromatids separated to opposite sides of the cell) and cytokinesis (the cell divides to create two daughter cells ‘born’ in G1)
476
G1 phase in cell cycle
growth before chromosome duplication
477
what are the checkpoints in cell cycle
restriction point- is envi favourable for division?
S phase checkpoint
G2/M transition- is DNA replicated? is envi favourable for division?
Metaphase to anaphase transition- are chromo attached to spindle
478
what is a checkpoint pathway
interanl quality control mechanism that halt the cell cycle when things go wrong
479
what does each cyclin-Cdk complex activate in cell cycle
events in cell cycle phase
cyclin-Cdk in next cycle phase
over 400 cyclin-Cdk substrate with roles in dna replication, protein synthesis, chromosome condenstion, mitosos
480
how do cyclin-Cdks regulate protein degradation
phosphorylate cell cycle regulators mkaingthem substrates for SCF complex (ubiquitin ligase)
phoshorylating some ubiquitin ligases, activating the (anaphase promoting complex or cyclosome, APC/C)
481
what is the progression of cell cycle dependent on
activation of cell cycle phase specific cdk activity
elimination of proteins from previous stages, by degradation by ubiquitin mediated proteolysis machinery
482
the structure of a checkpoint pathway
have sensors transducers and effectors
483
how does a checkpoint pathway work e.g. DNA DSB
Damage-specific sensors bind to the damaged DNA (RPA/MRN complex – L8)
Sensors activate transducers, which launch the damage response (ATR/ATM – L8)
Transducers activate effectors, which perform checkpoint functions e.g DNA repair proteins, proteins that arrest cell cycle
Prolonged arrest leads to apoptosis in many multicellular eukaryotes.
484
what distinguishing biological characteristics do cancer cells acquire during their evolution (10)
-. Self-sufficiency in growth signalling
- Insensitive to signals suppressing growth
- . Activating invasion and metastasis
-Enabling replicative immortality
-Inducing angiogenesis
-Ability to avoid apoptosis
-. Deregulating cellular energetics
-Avoiding immune destruction
-Genome instability and mutation
-Tumour promoting inflammation
485
how many genes have casual contributions to cancer
729
not a single gene that causes cancer
486
oncogene summary
normal role is proto-oncogene
when genetic change leads to increase in activity of protein an oncogene is made
they are genetically dominant- single allele is sufficient to make sig contribution to develop of cancer
487
what purpose of proto-oncogene
genes that allows the cell cycle to be induced or for the cell to go through the cycle
function in growth signalling pathways that promote cell proliferation or inhibit apoptosis.
488
Six major functional classes of cellular oncogenes that work in cell growth signalling or apoptosis
Secreted growth factors/mitogens
Growth factor/mitogen receptors
Signal transduction component
Transcription factors
Cell cycle regulators/drivers
Cell death inhibitors
489
how do oncogene drive increased cell proliferation
Six major functional classes of cellular oncogenes that work in cell growth signalling or apoptosis
490
what are the 3 ways proto-oncogenes turn to oncogoenes
translocation(gene moved to new locus)
gene amplification(multuple copies)
point mutation
491
epidermal growth factor and oncogenes
mutation in EGFR promotes proliferation by constitutive actiavtion of pathway from growth factor receptor (without binding to receptor)
Gene encoding epidermal growth factor receptor (EGFR) mutated in 40-50 % brain tumours, 20 % breast cancers, 15-30% ovarian cancers
EGFR mutations include:
amplification – more receptors – lower threshold for capture of mitogen
deletion – results in truncated receptor that lacks extracellular domain – triggers intracellular signalling in absence of mitogen
492
when Ras protein is bound to GDP is it active or inactive
inactive
493
how is Ras activated
when mitogen binds
GDP swapped for GTP
activates signalling by raf kinase
transient
ras intrinsic GTPAse hydrolyses GTP to GDP
494
Ras protein as an oncogene
Ras intrinsic GTPase activity hydrolyses GTP to GDP
30% cancers have Ras mutations
Mutations at codons 12, 13, 61 compromise GTPase activity – signalling pathway becomes constitutively active.
Sustained signalling – in absence of mitogen
495
what does Myc protein do
in nucleus
stimulates cell growth and division
496
does oncogene mutation of Myc lead to increased or decreased expression of myc
increased
497
where is myc found in normal and oncogenes
Myc in centrosomes only in normal cells
Throughout the cell in oncogene- hypertranscription
498
how is Myc oncogene made
point mutation- that stabilises Myc (rapid turnover)
translocation- results in increased gene expression. e.g Burkitt’s lymphoma - translocation brings MYC gene under the control of sequences that normally drive the expression of antibody genes in B lymphocytes – mutant B cells proliferate and form a tumour
499
where is cyclin D amplified in
wide range of cancers including 35% oesophageal carcinomas, 15 % bladder cancers, 15 % breast cancers
500
what does overecpression of cyclin D promote
unscheduled entry into S phase
501
where is CDK4 amplified
in some tumours including 12 % gliomas, 11 % sarcomas
502
what does inappropriate expression of G1-Cdk drive
of G1-Cdk drives progression through start in the absence of mitogen stimulation
503
where is unregulated phosphorylation of Rb from
amplification of CCND1(cyclin D) or Cdk4
In G0 and early G1, transcription activator E2F is bound to and inhibited by Rb protein
To enter cell cycle cyclin D associates with Cdk4 or Cdk6 – G1 Cdk
G1 Cdk phosphorylates Rb - releases E2F
E2F activates transcription of genes required for G1/S transition
504
what are the characteristic changes of apoptosis
Cells shrink and condense
Cytoskeleton collapses
Nuclear envelope disassembles
Nuclear chromatin condenses and breaks into fragments.
Cell surface chemically altered – ‘eat me’ signal – engulfed by neighbouring cell or macrophage
505
is apoptosis a nice and neat way of getting rid of cells
yes
506
the 2 ways of triggering apoptosis
extrinsic – triggered by extracellular signal proteins binding to cell surface death receptors
intrinsic – depends on the release into the cytosol of mitochondrial proteins that are normally in the intermembrane space
507
what does MDM2 regulate
Tumour suppressor gene activity
508
what does MDM2 regulate
levels of p53 which is required fro damage checkpoint activation
509
where is MDM2 amplification found
19/28 tumour types – common in tumours of adipose tissue (42%), soft tissue sarcomas (20%)
510
what does MDM2 mutation prevent
accumulation of p53if cant accumulate the cells are released freely through cell cycle
511
how does MDM2 work
In absence of DNA damage MDM2 ubiquitinates lysines in the p53 C-terminal domain, targeting it for degradation
Any remaining p53 is exported from the nucleus
In presence of DNA damage MDM2 and p53 are phosphorylated, disrupting interaction, allowing p53 accumulation
512
whats the hayflick limit
Normal cells in culture cease to divide after 50-70 doublings – senescent
513
do most somatic cells havelow or high levels of telomerase
low levels
they get shorter with successive division
514
what do oncogenes do totelomerase expression
reactive the expression of telomerase so the cell is no longer senescent
so immortalised
515
where is telomerase activity detected
in all major cancer types
516
what is the family of proteins that regulate the intrinsic pathway of apoptosis
Bcl2
517
how does the pro-apoptotic protein BH123 work
increased Bax due to p53 activation results in Bax homodimers and induction of apoptosis
518
how does the anti-apoptotic protein Bcl2 work
proto-oncogenes
increased levels of activated Bcl2 brought about through eg oncogenes activation block apoptosis
cells immortalized
divide and grow indefinetly
519
