cell nucleus

Question 1

what is meant by functional compartmentilization of the nucleus

Answer 1

• Subnuclear compartments exist despite the absence of internal membranes in a nucleus. Whenever there is a requirement for multiple enzymes and proteins to come together to perform a function in the nucleus, they are able to do so in the absence of any membrane

Question 2

describe the functional elements of the chromosome and its key features.

Answer 2

Function : store cells dna
Chromosomes features :
- A single molecule of DNA
- Linear and double stranded ( in eukaryotea)
- Contain genes
- Telomeres : protect chromosome ends
- Centromere : needed during cell division
- Multiple Origins of replication along the chromosome- required to initiate DNA replication during the S phase. This is because chromosomes are very long and if
they only had a single origin, it would be too slow to replicate it.
Eg if you were making a mammalian artificial chromosome for the purposes of gene therapy, then these are the three functional elements required to make that chromosome.

Question 3

what are the functions of a centromere

Answer 3

• Locks sister chromatids together after s phase of the cell cycle and during G2.
- Attachment site for chromosomes to the mitotic spindle via a protein structure called the kinetochore during cell divison.

Question 4

what are centromeres made out out?

Answer 4

• Centromeres are made out of Megabases of repetitive DNA, major component of centromeres is the alpha satellite DNA in humans
• All chromosomes have different satellite DNA configurations
• alpha satelite dna is made out 171 bp monomers which are repeated. forming a hierchary of repeats until it forms megabases of dna. this is called a homogenous higher order alpha satellite array.

Question 5

what are telomeres ?

Answer 5

they are found at the double stranded ends of chromosomes and is made up of a tandem repeat (TTAGGG in human)
- In telomeres, there is a single stranded region at the end of the telomere, which loops around to form a loop and protect the ends of chromosomes.

Question 6

what is the telomere end replication problem

Answer 6

During DNA replication, the RNA primers are replaced and the gaps are filled. this cannot occur at the end of the chromosome, therefore there is a gap at the end of the lagging strand. With each round of replication the lagging strand shortens for a certain kB of DNA leading to loss of telomeric repeats, reaching hayflicks limit

Question 7

which dna polymerase helps to solve the telomere end replication problem

Answer 7

Telomerase – an RNA-dependent DNA polymerase that adds telomeric DNA to telomeres

Question 8

explain how we solve the telomere end replication problem

Answer 8

• Unless we were able to solve the hayflicks problem oour germ cells and stem cells would not be able to infinitely replicate our DNA and we start losing genes from the end of our chromosomes. So we wouldn’t be a successful organism. So we have solbutions
• Removal of the RNA primer leads to the shortening of the chromosome after each round of replication. Chromosome shortening eventually leads to cell death.
• an rna sequence in telomerase acts as a template for DNA. this enzyme adds the telomeric sequence to the 3’ end of the chromosome.
• the original length of the chromosome is retained

Question 9

why do eukaryotes have multiple origins of replication?

Answer 9

• Bacteria have a single origin of replication
• Eukaryotic chromosomes are large (and DNA replication is also slower) so multiple origins must fire simultaneously for replication to be completed within a reasonable timescale

Question 10

origins are clustered in ?

Answer 10

replication units

Question 11

How can we visualise and identify chromosomes in a cell

Answer 11

g-banding producing a g banded metaphase spread

Question 12

explain the gbanding method

Answer 12

Take a blood sample
Culture cells
Add cell cycle blocking agent so higher portion of cells blocked during cell division in metaphase, when the chromosomes are most condensed and can be visualised.
Add cells to a hypotonic solution 0 swells the cells before youadd to the glass slide. Ebcasue theyre swollen when you drop it into a glass slide they burst and release the chromosome. This is called a metaphase spread. This results with a karyotype
This is because the g banding gives a characteristic g banding pattern which is specific to each chromosome.

Question 13

how do we identify chromosomes

Answer 13

1. Size- chromosomes differ in length. Chromosome 1 being longest and y
   shortest.
2. Banding patter- Gene rich (euchromatin rich) sections are G light and gene
   poor (heterochromatin rich) sections are G dark. Gene rich and gene poor
   sections will differ in different chromosomes, resulting in different patterns
   of light and dark bands.
3. Centromere position- centromere may be metacentric (in the centre/two
   arms equal), sub metacentric (off-centre) or acrocentric (to the very end of
   the chromosome- short p arm contains mostly repetitive DNA).

Question 14

what can we tell about Chromosome organisation in the interphase nucleus using EM ?

Answer 14

we cant discern any information about the location of the chromosomes, except that heterochromatin is in the periphery and euchromatin in the anterior

Question 15

what can we tell about Chromosome organisation in the interphase nucleus using fish

Answer 15

• This technique FISH allows decondensed chromosomes in interphase to be visualised, and allowed us to visualise the cell in 3D
• In FISH , you use a chromosome paint, this allows youto colour an entire
  chromosome
  – Allowed us to observe that chromosomes don’t overlap with one another and form domains, chromosome arms are separated from one another, the locations of genes within chromosome territories.

Question 16

what is spectral karyotyping

Answer 16

each chromosome is differentially labelled by different coloured flouresecent tags. This allows spectral karytyping. Easier to see any rearrangements that have taken place.
- allowed us to observe chromosome arms, bands, territories, spatial location of gene rich vs gene poor chromosomes, and that Genes can have preferential locations at the surface of the chromosome territory and can dynamically loop out in response to transcriptional activation

Question 17

explain what spectral karyotyping visualises about chromosome arms

Answer 17

• Spectral karyotyping allowed us to see what was happening in the chromosome arms in interphase.
• no intermingling between the p and q arms as the cell goes form metaphase to interphase. the arms were separate and distinct.
• also able to visualise telomeric ends and they retained their identity.
• the chromosome arms are and bands are distinct and mutually exclusive.

Question 18

explain how we visualised chromosome territories.

Answer 18

• using spectral karyotyping
• By observing interphase nuclei, we were able to observe how chromosomes are distributed in interphase.
• Chromosomes form non-overlapping domains in the interphase nucleus. Ie they have their own space in the nucleus

Question 19

explain the spatial location of gene rich vs gene poor

Answer 19

• visualised by spectral karyotyping

– Spectral karyotyping : gene poor chromosome 18 ( coloured orange) tended to be found at the periphery of the interphase nuclueus. Its also where the heterochromatin is also most likely to be found . they compared this to the gene rich chromosome 19 ( coloured 19) and was more likely to be found in the interior of the nucleus.

• there was different functions within the nuclear volume, with more genes and transcription found in the interior .

-Gene poor: periphery of the nucleus
Gene rich: interior of the nucleus

Question 20

explain Genes can have preferential locations at the surface of the chromosome territory and can dynamically loop out in response to transcriptional activation

Answer 20

• visualised by spectral karyotyping

red: MHC gene cluster
Green: chromosome 6
1) MHC gene clusters were tend to be found on the surface of the chromosome territory
2) When interferon is added,– you can dynamically loop out in response to transcriptional activation. The gene cluster has projected away from the chromosome territory

Question 21

list the nucleur compartments and their functions

Answer 21

• Distinct sections with a high concentration of specific proteins.
• E.g. nucleoli territory- ribosome biogenesis,

chromosome territory- DNA storage,

nascent RNA (newly transcribed) territory-transcription factories, splicosomes

territory- irregular domains containing splicing factors,

PML nuclear bodies territory-
possible nuclear depot.

Question 22

what is the function of the nucleolus

Answer 22

it is the largest substrucutre in the nucleus and its function is ribosome subunit production.

Question 23

outline the steps in ribosome production

Answer 23

1. the fibular centre, this is where the ribosomal RNA GENES are located and is where transcription is taking place with RNA polymerase.
2. RNA PolI transcribes precursor rRNA
3. Processed to 18S, 5.8S and 28S - endo and exonuclease cleavage
4. 5S is transcribed in the nucleoplasm by RNA PolIII and transported to the nucleolus
5. rRNA folded and associate with 79 ribosomal proteins to assemble the 40S and 60S ribosomal subunits (eukaryotic)
6. Subunits transported to the cytoplasm

Question 24

what are the zones of the nucleolous and how can they be observed ?

Answer 24

EM,

1. Fibular centre : ribosomal RNA genes
2. Dense fibular component : rRNA transcription
3. Granular component. : processing and assembly

Question 25

what are NORs

Answer 25

- The nucleolus forms around the nucleolar organising regions (NORS) - NORS are the location of the rRNA genes - Initially there are 10 NOR regions for each of the locations of the ribosomal rna genes, they coalesce as the cell enters interphase until there is one large nucleolus.

Question 26

why do we have so many copies of ribosomal rrna genes

Answer 26

- compare to single copy gene – a single mRNA molecule can be translated many times to give amplification of the final protein product - the ribosomal RNA molecule is not translated into protein, the ribosomal RNA molecule transcribed is the final product and the cell requires many ribosomes, then having multiple copies of rRNA enables it to make these in a reasonable time.

Question 27

what method has been used to analyse the nucleolus ? and what are some of the functions that have been postulated via this anaylsis.

Answer 27

nucleolus proteonomic analysis - 700 endogenous nucleolar proteins - Role in processing of endogenous nuclear siRNAs (small interfering RNAs) - Assembly of the six proteins and RNA molecule making up the Signal Recognition Particle (SRP) - Biogenesis of other classes of RNPs such as the spliceosomes small nuclear (sn)RNPs and telomerase

Question 28

describe splicing speckles

Answer 28

- aka splicesome • 20-50 per cell • 146 known proteins localise ( due to splicing is a huge macromolecule and requires proteins ) • Composed of splicing factors and other mRNA processing factors • Variable size and shape • Used as a model system to study nuclear organisation • Do not contain DNA • Not a site of transcription • But are associated with highly active transcription sites

Question 29

outline the investigations on the functions of splicing specles

Answer 29

• Inhibit transcription so (ie.e no splicing in the cell) – speckles round up and become larger Support speckles as a storage/assembly compartment. Not a direct sites of splicing • Add more intron containing genes to a cell (i.e. increase splicing) – splicing factors redistribute to transcription sites, speckles get smaller Supports speckles as a reservoir of splicing factors with slicing factors being utilised with shuttling between the speckles and the transcription site

Question 30

Using Splicing Speckles as a model system what can we conclude about nuclear compartment organisation?

Answer 30

* A model of self assembly through transient macromolecular interactions * Continuous association and disassociation of components define nuclear body size/shapes and the pool in the nucleoplasm

Question 31

explain what has been visualised in dna replication

Answer 31

• Different patterns of replication foci have been observed in S phase by many researchers • No high quality 3D analysis due to limited resolution of optical microscopy - Visualies replication by incorporating a molecule into a piece of dna whenever its being replicated, tagging it fluoriesenetly visualising it. - Different parts of the genome get replicated at diff times in the s phase - Euchromatic tends to get replicated first, and heterochromatin gets replicated later in the s phase.

Question 32

what is the replication factor hypothesis and how was it disproved

Answer 32

* Multiple replicons (DNA replicated from a single origin) must operate in parallel to ensure all genomic DNA is replicated during S phase * The number of foci is smaller than the number of replicons, leading to the concept of a replication factory * Factories contain all the enzymes and other factors required to produce two new DNA strands Visualisation of replication foci by super resolution microscopy • Can resolve single repliation foci in S phase: 5583 per cell nucleus • Predicted number of replicons: 5149 firing at the same time Challenges the conventional interpretation of nuclear replication foci as replication factories - Therefore you can visualise more foci - So if you have a genome to be replicated, all factors go to that place and all the enzymes go to the particular genome location to replication. So there are no nuclear .compartments for replication. Everything assembles at the site its occurring.

Question 33

outline the methods on visualising transcription .

Answer 33

• Using confocal microscopy Transcription can be visualised in the cell nucleus, estimates from a few hundred to many thousand (variability from both cell type and thresholding) Can be visualised by pulse labelling with BrUTP to detect nascent RNA or by immunofluorescence to active elongating RNA PolII Transcription factories proposed :

Question 34

what is the transcription factories hypothesis and how was it disproved ?

Answer 34

* Sites where multiple active RNA polymerases are concentrated * Termed transcription factories and proposed that genes pass through the factory as they are transcribed (large arrows) * Proposed that genes from the same or different chromosomes may associate at the same factory Hwoever super resolution microscopy disproved this theory : Spatial organisation of RNA PolII mediated transcription • Transcription foci consist of only one RNA POLII molecule • No clustering, no factories • Transcription machinery assembling at each site of transcription

Question 35

describe the nucleur envelope

Answer 35

- The nucleus is surrounded by a nuclear envelope, which is 2 lipid bilayers, and is cotinous with er. - It has nuclear pores which allows communication between nucleoplasm and cytoplasm. - Movement through the nuclear pore is strictly controlled and only small water soluble molecules can diffuse freely through the pore - Larger molecules must be actively transported through the nuclear pore and require specific tags to get in and out and m - These tags are called nuclear export singals and nuclear localisation sequences.

Question 36

what does nucleur export require

Answer 36

proteins require a nucleur export signal e.g ribosomal subunits and mrna

Question 37

what do nucleur imports require

Answer 37

proteins require a nuclear localisation sequence E.G histones, dna/rna , polymerases and other nucleus proteins.

Question 38

What is the nuclear material stored as in the nucleoplasm?

Answer 38

chromatin