Perdy is NOT Bae

Question 1

(1) Form of DNA used in most of cells

Answer 1

B form (2 chains are in opposite orientation)

Question 2

(1) 2 ways torsional stress is accommodated in supercoiling

Answer 2

1) Formation of superhelices

2) Altering number of base pairs per turn of helix

Question 3

(1) What topoisomerase type 1 does?

Answer 3

Breaks one strand of DNA, pass other strand through the gap and seal the break (Linking number changed by +-1)
-Removes one -ve supercoil
(Maintains supercoiling DNA with DNA gyrase)

Question 4

(1) What topoisomerase type 2 does?

Answer 4

Breaks both strands of the DNA, pass another part of the helix through the gap (change linking number by +-2)

Question 5

(1) Function of DNA gyrase?

Answer 5

Creates -ve supercoils (using ATP), opens up strands

essential for DNA replication

Question 6

(1) Prophages definition

Answer 6

Sequenced bacterial genomes harbour phage-like elements, implicated in pathogenesis

Question 7

(1) Genomic islands definition

Answer 7

Horizontally acquired genomic regions that may have mutated to mask modes of transmission and integration.

Question 8

(1) Main replicative enzyme

Answer 8

DNA Polymerase III

Question 9

(1) Role of Polymerase I in DNA replication

Answer 9

Role in removal of RNA primers from Okazaki fragments

Question 10

(1) Outline process of DNA replication

Answer 10

1) DNA Pol III synthesises new DNA and Okazaki fragment
2) DNA Pol III stops when it reaches the RNA primer
3) DNA Pol I continues synthesis
4) DNA ligase links the 2 DNA fragments

Question 11

(1) What is a replicon

Answer 11

Basic unit of replication (a DNA molecule or sequence which has functional origin of replication)

Question 12

(1) 3 main features of OriC replication origin

Answer 12

• GATC present 14x
• 13-nucleotide motifs
• 9-nucleotide motifs (DnaA binding sites)

Question 13

(1) How is initiation of replication controlled?

Answer 13

• Dam methylase methylates adenine residues in GATC seqeuences
• All 14 GATCs in oriC are fully methylated in initiation
• Newly synthesised strands NOT methylated but old strand is, to differ the strands.

Question 14

(1) How does termination of circular chromosomes work?

Answer 14

• The 2 replication forks from OriC move bi-directionally away and fuse within a region diametrically opposed to OriC.
• DNA terminators are polar and only arrest forks in one direction
• Termination is achieved when the forks meet in the correct termination point, enabled by the stalling by ter regions.

Question 15

(1) Why are replication forks arrested in one direction?

Answer 15

• The ter site must bind a specific terminator protein Tus (terminus utilisation substance) in E.coli
• Fork arrest results from inhibition of helicase-mediated unwinding of DNA duplex at apex of fork

Question 16

(1) Definition of recombination

Answer 16

Breaking and rejoining of DNA molecules in new combinations

Question 17

(1) 6 steps of homologous recombination

Answer 17

1) Alignment
2) Cleavage: One strand of each duplex is cleaved (RecBCD endonuclease at chi sites)
3) Invasion: Holliday junction formed (via RecA) (not identical molecules, heteroduplex formed)
4) Branch migration: Increase heteroduplex region (via RuvAB)
5) Isomerisation: Strands of HJ cross and uncross
6) Resolution: 2 crossed strands of HJ cleaved by RuvC
OUTCOME:
-2 duplex molecules with region of heteroduplex
OR 2 recombinant duplex molecules with a region of heteroduplex

Question 18

(1) 5 components of RecBCD enzyme

Answer 18

1) ssDNA exonuclease (5’ to 3’ and vice versa)
2) ssDDNA endonuclease
3) dsDNA exonuclease
4) DNA-dependent ATPase
5) DNA helicase

Question 19

(1) 2 activity alterations to RecBCD when it encounters a chi site

Answer 19

1) 3’ to 5’ exonuclease activity INHIBITED
2) 5’ to 3’ exonuclease activity STIMULATED
(Helicase activity unaffected)

Question 20

(2) Examples of single base changes (exert no effect on replication, but are mutations that are inherited)`

Answer 20

• Replication errors via keto-enol tautomerisation
• Deamination of C to U
• Incorporation of U instead of T in repli
• Chemical mod of bases

Question 21

(2) Examples of structural distortions (impeding transcription/repli)

Answer 21

• Single strand breaks
• Cov mod of bases (alkylation)
• Removal of base
• Inter/intrastrand cov bonds

Question 22

(2) Def Direct repair and example

Answer 22

Reversal/simple removal of damage

e.g. Photolyase reparing UV intrastrand pyrimidine dimers

Question 23

(2) Def Mismatch repair and example

Answer 23

Detection and repair of mismatched bases

e.g. Uracil DNA glycosidase, removes U and adds T

Question 24

(2) Def Excision repair

Answer 24

Recognition of damage followed by excision of a patch of DNA and replacement by undamaged DNA. (Types: Very short patch (bp), short patch (20bp), long patch (1500-10,000 bp)

Question 25

(2) Def Tolerance systems and example

Answer 25

Allow DNA replication to proceed through damaged regions of DNA (e.g. Translesion synthesis polymerases able to synthesise DNA past damaged bases)

Question 26

(2) Retreval systems and example

Answer 26

Recombinational processes to repair damaged DNA | (e.g. Daughter strand gap repair, allows replication after damage and relies on other systems to do it) Lazy sod

Question 27

(2) Explain the mut system

Answer 27

1) MutS recognises mismatches and binds 2) MutL stabilises complex 3) DAM methylase methylates GATC seq on old strand 4) MutS-MutL complex activates MutH that finds methyl group and nicks new strand opposite meth group 5) MutU unwinds DNA from nick (towards mismatch) 6) DNA Pol 1 degrades and replaces unwound DNA 7) DNA ligase seals single strand break.

Question 28

(2) Def of SOS response

Answer 28

If there is severe DNA damage, a large number of unlinked genes are activated to save the cell. -LexA repress SOS operons -RecA induces SOS response (by inactivating LexA) SOS response allows cell to survive massive DNA damage by allowing DNA repli but expense of DNA repli fidelity

Question 29

(2) Def transposable genetic elements

Answer 29

-Specific DNA sequences that are able to transfer copies of themselves to other parts of DNA.

Question 30

(2) The 7 classes of of transposable elements in bacteria

Answer 30

1) Insertion sequences 2) Composite transposons 3) TN3 family transposons (e.g. drug resistance) alos known as replicative transposition 4) Transposable phages 5) TN7 transposons 6) Inversion sequences 7) Gram +ve transposons

Question 31

(2) Explain phase variation in Salmonella spp

Answer 31

- Can express 2 different flagellum - Culture of phase 1 cells will produce phase 2 at low frequency - Phase variation determined by orientation of hin region - Hin genes codes an invertase that catalyses inversion

Question 32

(2) 4 stages of transcription

Answer 32

1) Template recognition: Binding of RNA polymerase to DNA template at promoter 2) Initiation: Formation of first 9 phosdiester bond and release sigma-factor 3) Elongation: Addition of nucleotides of growing RNA. 4) Termination: Recognition of a signal that E should cease followed by dissociation of enzyme, RNA and DNA. (Rho-dependent or independent)

Question 33

(2) Promoter sequence features

Answer 33

1) -10 seq TATAAT | 2) -35 seq TTGACA

Question 34

(2) What are UP and DOWN mutations

Answer 34

UP mutations make promoter more similar to consensus | DOWN mutations make less similar consensus.

Question 35

(3) Thy inducer?

Answer 35

Activate activators or inactivate repressors

Question 36

(3) Thy Corepressors?

Answer 36

Activate repressors or inactivate activators

Question 37

(3) What is diauxic growth?

Answer 37

When glucose is used first, and alternative carbon source is used after

Question 38

(3) 3 structural genes of the lac operon

Answer 38

1) lacZ: B-galactosidase (Cleaves B-galactosides to monosaccarides 2) lacY: B-galactosidase (Cytoplasmic membrane protein that facilitates into cell) 3) lacA: B-galactosidase transacetylase (Detoxify toxic B-galactosides by acetylation)

Question 39

(3) 3 alternative regions of lac operon

Answer 39

1) Promoter: lacP at which RNA Pol binds 2) Operator: lacO where lac repressor binds 3) CAP site: cAMP acceptor proteins (CAP) and catabolite respression protein binds here.

Question 40

(3) 2 ways lac operon is ACTIVATED BRO

Answer 40

1) Inactivation of lac repressor (lactose to allolactose and this thing binds to it) 2) CAP activation (absence of glucose) by cAMP (by adenyl cyclase)

Question 41

(3) How Jacob and Monod found the lac operon model

Answer 41

- Used merodipliod (partially diploid) E.coli by inserting lac operon genes into F' plasmid - Some genes encoded for diffusable products that regulated gene expression on BOTH DNA molecules - Regulatory proteins can affect both DNA strands at site distant to their genes. TRANS-acting elements

Question 42

(3) WAT is Trans-acting elements

Answer 42

Gene product diffuses to affect both molecules

Question 43

(3) WAT is Cis-acting elements

Answer 43

Mutated lacO has no gene product, only affects DNA molecule its on. -No repression occurs on chromosome operon

Question 44

(3) WAT is gene complementation

Answer 44

Both operons are repressed BUT f' plasmid functional lacY | -Chromosome's functional lacYZ complement mutations so phenotype is normal

Question 45

(3) HOW TO CLONE

Answer 45

1) Plasmid transform E.coli 2) Selectable marker to select for clones of vector (e.g. antibiotic resistance 3) Bacteria grown 4) Use a screenable marker (e.g. x-galactose blue thingy)

Question 46

(3) Enzyme to convert RNA to cDNA

Answer 46

Reverse Transcriptase

Question 47

(3) Why phages good vectors?

Answer 47

1) Good at producing large numbers and incorporating DNA into chromosome 2) Can replace non-essential genes (doesn't affect lytic cycle) with cloned DNA

Question 48

(3) 2 Types of lambda vectors

Answer 48

Insertion vectors: Non-essential DNA removed already and DNA can be cloned into this site Replacement vectors: Non-essential is replaced with non-coding stuffer DNA that can be replaced by DNA to be cloned

Question 49

(3) Thy Fosmids?

Answer 49

Large plasmids based on F plasmid with cos-sites, lacZ and a T7 promoter added

Question 50

(3) Ti plasmid uses?

Answer 50

Used to transfect plant cells | -Only specific protion of Ti plasmid is transferred into the plant cell and then integrated into plant genome

Question 51

(3) How to clone large fragments?

Answer 51

USe BACs/YACs: Bacterial/Yeast artificial chromosomes

Question 52

(3) 3 areas of a chromosome

Answer 52

Telomere Centromere Origin of replication

Question 53

(3) Example of expression controlled by non coding RNA molecules

Answer 53

IF IRON IS FREELY AVAILABLE 1) Protein FUR attaches to free iron, all-iron requiring proteins are made IF IRON IS LIMITED 1) FUR stops repressing ryhB 2) RhyB ncRNA binds to mRNA of non-essential iron proteins which degrades 3) Need of iron of cell falls 4) Self regulates as iron concentration rises ryhB repression reoccurs

Question 54

(4) 2 Types of microbial community

Answer 54

Positive interactions: Toxic product of one organism may be substrate for another Negative interactions: Antibiotic production that targets other microbes or competition for substrates

Question 55

(4) Process of Quorum sensing

Answer 55

1) Cells excrete a signal molecule (autoinducer) which allow them to sense population size 2) Once autoinducer reaches a threshold then cells respond (expression/function) EXAMPLE Staphylococcus Aureus can form biofilms on wounds but can switch to invasive phenotype driven by quorum sensing (via Agr system)

Question 56

(4) Process of accessory regulator regulatory sysyem

Answer 56

Arg is 2 component reg system based around operon with 2 promoter Agr D = autoinducing peptide (AIP) Agr B = Transmembrane protein, secretes AIP Agr C = AIP receptor, binds AIP then phos Agr Agr A = Response protein, activates P3 producing RNAIII Low cell density: Low AIP conc High cell density (biofilm forms): AIP con rises

Question 57

(4) What are biofilms?

Answer 57

Structured clusters of cells, enclosed in self-produced polymer matrix and attached to surface (allows pathogens survival in environment and in hosts)

Question 58

(4) Common characteristics of biofilms

Answer 58

- Cells enclosed in polymer matrix of exopolysaccharides, proteins, nucleic acid - Formation initiated by extracellular signals in environment - Biofilm protects bacteria against the host immune response

Question 59

(4) 5 stages of biofilms

Answer 59

1) Initial attachment: flagella, type I pili 2) Irreversible attachment: LPS, Type IV pili 3) Maturation I: microcolonies, repress flagella 4) Maturation II: Quorum sensing 5) Dispersion: release planktonic cells

Question 60

(4) 2 examples of genes in biofilms

Answer 60

1) Pseudomonas aeruginosa (twitching motility essential for maturation into microcolonies = express type IV pili) 2) Vibrio parahaemolyticus (Switch between 2 flagella systems is important in maturation = interference with flagella rotation by sruface induces 2ndary flagella system linked to swarming motility)