Chromosome biology lecture 1

Question 1

DNA replication is

Answer 1

• Accurate + precise copying of DNA of genome
• Happens 1 per cell division cycle
• Each daughter cell inherits identical DNA
• Evidence

Question 2

Stages in DNA replication

Answer 2

• Initiation (origin recognised + opened)
• Elongation (DNA synthesised)
• Termination (stops polymerase)

Question 3

Organisation of chromosome

Answer 3

• Chromosomes = scaffolded by proteinaceous matrix

- 2nm diameter duplex DNA → wrapped around histone octamer → 30nm fibre → loops that condense in metaphase

Question 4

Chromosome banding

Answer 4

• Different points of chromosome replicated at different points in S phase
• Stain w/ Giemsa dye, gives G band (region w/o actively transcribed gene
• Oligomycin gives R bands that are GC rich

Question 5

Gene-rich DNA replicated early

Answer 5

• R bands = G rich, early
• G bands = gene poor, replicate later
• Micro-array analysis of replication origin

Question 6

Spatial localisation of replication in 3D

Answer 6

• Label cells w/ different times, take serial selections using 3D FISH
• Green (early-replicating) localised more to centre of nucleus
• Relate to chromatin in TAD (Repressed TAG = towards periphery)

Question 7

Comparison of prokaryotic + eukaryotic DNA replication

Answer 7

• Prokaryotes = 4.5x10^6 nt, single circular chromosome, 1 bi-directional origin
• Eukaryotes = 3x10^9 nt, many linear chromosomes, 2x10^4-5 origins

Question 8

Evidence

Answer 8

1. Pulse-labelled cells
2. EM = origins seems as open ‘bubbles’
3. confocal microscopy (synchronise or arrest cells)

Question 9

Mapping origins of bi-directional replication

Answer 9

Shotgun cloning

• Extract genome, cut w/ RE, clone fragment into vector, see if grow on medium w/o His
• If grow, sequence acts as autonomously replicating sequence, supports replication of plasmid

Question 10

Mapping origins of bi-directional replication

E.g. S cerevisiae

Answer 10

• Consensus in ARS consensus in ACS box
• Recognised by ORC
• B domain = 3’ to T rich strand of ACS
• Str2 histone deacetylase silences some origins in yeast- epigenetic control
• In addition to AT rich seq, also have ORB + DUE

Question 11

Does the origin act in vivo?

Answer 11

• 2D gel mapping
• Yeast genome, cleave w/ RE, run down well, rotate 90o, apply ↑ V, transfer to nitrocellulose, probe against region of genome of interest, hybridise
• Bubble arch = if have origin
• If have passive replication by replication fork outside, give y arch
• If replication origin is to one side, start off with bubble arch then get y arch
• But messy

Question 12

Replication of eukaryotic DNA viruses

Answer 12

• like SV40
• Has small ds genome, requires viral protein for recognition of origin to let virus replicate independent of host
• Region that binds Tag (recognition origin protein)

Question 13

Simple origin

Answer 13

• Origin consists of core origin (CORE) that binds initiator flanked by DUE + auxiliary sequence that binds TF
• AT rich