Eukaryotic genome organisation and cell cycle Flashcards
Tell me the following about eukaryotic DNA…
- Number of base pairs
- Length of base pair
- Length of haploid genomic DNA stretched out
- Human genome has 3 billion base pairs (3 x 109 bp)
- Each base pair is 0.34 nm long (0.34 x 10-9m/bp)
- Length of haploid genomic DNA stretched out =
- (3 x 109) x (0.34 x 10-9m) = 1 m
How much DNA is found in a human chromosome?
19 to 73 mm (22 pairs plue XX or XY)
But it’s condensed to fit into cell nucleus (5 to 10 um in diameter)
How can nucleosomes be seperated?
By incubation with DNase enzymes
What are the histone proteins found in a nucleosome?
The four core histones have what protruding from the nucleosome?
The four core histones have N-terminal tails protruding from the nucleosome
Whats the role of the H1 histone?
It helps with packing the chromatin into further fibres
How can histone N-terminal tails be modified?
By phosphorylation, acetylation methylation
What are nucleosomes inportant for?
What happens if they are packed too tightly?
This is important for regulation of gene expression and condensing chromosomes during mitosis
if packed too tightly, no other factors can “interrogate” the chromosome and transcription is silenced
What happens in the interphase step if the cell cycle?
Gene expression
Genome amplification
What happens in the M phase of the cell cycle?
Mitosis
segregation of chromosomes (cytokinesis)
Whats Chromatin?
What is it used for?
The complex of histones, non-histone proteins and nuclear DNA. Chromatin allows for the DNA compaction and is involved in the regulation of all DNA activites
What kind of proteins are non-histone proteins?
Non-histone proteins are usually structural or regulatory proteins which effect chromatin structure and usually effect enzymatic activity within chromatin
What are nucleosomes?
The basic structural unit of chromatin
What makes up the histone octamer?
The Dimers H3-H4 and H2A-H2B are repeated twice which makes up the histone octamer
What do each of the core histones (from the octamer) possess?
Two functional domains; amino-terminal tail and histone fold (not only histones which have this domain, found in some transcription factors.
What do histone folds interact with?
Histone folds interact with each other, allowing formation of dimers (via the ‘handshake’ interaction)
How many bp from the dsDNA winds around the histone core?
How many turns is this?
147 bp
It forms slightly less than two turns
How does the linker histone, H1 work?
H1 bind both to DNA and the nucleosome core
They can change the path of DNA that exits the nucleosome. Hence, it affects the linker DNA accessibility, organisation of higher order chromatin fibre and chromatin compaction
What do Hooks on the histone represent?
Places where DNA interacts directly with histones
Whats the name of the model that explains the chromatin structure?
The ‘Zig-Zag’ model
During interphase, how is chromatin arranged?
What helps to form these structures?
What two components were identified so far?
in loops
Architectural proteins are involved in the formation of these loops
The two components identified so far are; Protein complex called cohesin and a protein called CTCF
What does cohesin and CTCF form?
Chromatin loops
The size of the chromatin loops may change, what mechanism do chromatin loops grow via?
The loop extrusion mechanism
during the loop extrusion mechanism, what are the chromatin fibres help together by?
What is the size of the loop regulated by?
Cohesin and the size of the loop is regulated by CTCF
IS loop extrusion a dynamic process?
yes, which requires ATP
What does loop extrusion help to do?
Position specific DNA sequences in areas of chromatin that are transcriptionally either active or inactive
When do loops stop growing?
When CTCF comes into close proximity with cohesin
All structurally regulated
How are the loops of chromatin further organised into higher order organisation of chromatin?
Further loops can form from pre-existing loops (TADs)
Further hierarchical organisation of interphase chromatin: groups of chromatin loops form Topologically Associating domain (TADs)
Whats are TADs?
TADs are grouped into compartments; compartments may be transcriptionally active (type A) or inactive (type B)
compartments belong to individual chromosomal territories, which are occupied by single chromosomes decondensed after mitosis
How can chromosomal territories be visualised?
using fluorescent staining
A technique to paint chromosomes using multi-colour FISH (spectral karyotyping) helps to visualise entire chromosomes
Tell me some features of chromosomes territories?
- some chromosomes localize toward the periphery, often touching the nuclear membrane, whereas others are located toward the center of the nucleus
- recurrent clusters of chromosomes. For example, in mouse lymphocytes, chromosome 12 often sits next to chromosome 14, which in turn is adjacent to chromosome 15, thereby forming a triplet cluster
- there are large areas of chromosomal identity between different species that have been maintained throughout evolution
- patterns of chromosome arrangement are specific to both cell type and tissue type
- chromosome territories can reposition in disease
Conclusions…
- In eukaryotic chromatin DNA is wrapped around histone octamers (nucleosomes)
- During interphase chromatin fibres are arranged in loops. Cohesins and CTCF proteins define boundaries of most of these loops.
- Looping of chromatin fibres has a function in chromatic compaction and in the regulation of gene expression.
- Chromatin loops are organised in Topologically associating Domains (TADs)
- TADs are organised into compartments, which can be transcriptionally active or inactive
- Compartments form Chromosome Territories within interphase nuclei
- Position of a gene within these structures affects the activity of that gene.
- Genome organization depends on an organism, cell type (tissue), stage of development, cell cycle phase, current physiological status (e.g. stimuli from environment, stress) and is disturbed in many pathological states.
Label this mitotic chromosomes
In a metaphase chromosome, how much DNA does an individual chromatid contain?
one
What are condensins and what are the similar to?
Condensin are multiprotein complexes; in their structure they are similar to cohesins
What are the important events of the cell cycle?
What are the phases of the cell cycle ?
Is the length of the cell cycle the same in all species?
Why may it vary?
no
- Cell cycle timing and structure varies in different cells and organisms
- Adapt cell cycle to needs (length and presence of individual stages of cell cycle)
Where on the cell cycle are the three transition checkpoints?
What occurs in each transition?
- Start – major transition at the entry into the cell cycle in mid to late G1; progression past this point is prevented if cell growth is insufficient, DNA is damaged or other preparations are not complete. Cells, instead of arresting, enter a prolonged nondividing state, if the conditions to pass this checkpoint are not met.
- G2/M checkpoint – regulatory transition where entry into M phase can be controlled by various factors, such as DNA damage or the completion of DNA replication
- Metaphase-to-Anaphase transition – transition during M phase where the initiation of the sister chromatid separation can be blocked, if chromosomes are not properly attached to microtubules of the mitotic spindle
When cyclin forms a complex with Cdk, what happens?
The protein kinase is activated to trigger specific cell-cycle events.
Without cyclin, Cdk is inactive
Oscillations in Cdk activity during the cell cycle are primarily due to what?
They are primarily due to changes in the amounts of cyclins. Different types of cyclins are produced at different cell-cycle phases. This results in the periodic formation of distinct cyclin-Cdk complexes that trigger different cell-cycle events
Where are the cyclin-Cdk complexes in the cell-cycle control system?
The enzyme CAKs, is shown in three states. What are these states?
(A) In the inactive state, without cyclin bound, the active site is blocked by a region of the protein called the T-loop (red)
(B) The binding of cyclin causes the T-loop to move out of the active site, resulting in partial activation of the Cdk2
(C) Phosphorylation of Cdk2 (by CAK) at a threonine residue in the T-loop further activates the enzyme by changing the shape of the T-loop, improving the ability of the enzyme to bind its protein substrates
What does the Dephosphorylation by the phosphatase Cdc25 leads to?
the reactivation
Why do cells use CKIs?
Primarily to help govern the activites of G1/S and S-Cdks early in the cell cycle
How can these mechanisms regulate cell cycle?
What happens after DNA damage?
→ Regulation of the cell cycle involves signal transduction via multi-step signalling pathways.
→ Cell-cycle control depends also on transcriptional regulation.
