The Cell Nucleus

Question 1

What are the functional elements of a chromosome?

Answer 1

A single molecule of DNA
Linear (in eukaryotes) 
Contains genes
Structural elements:
Telomeres: protect chromosome ends
Centromere: needed during cell division
Origins of replication

Question 2

What do centromeres do?

Answer 2

Locks sister chromatids together
Attachment site for chromosomes to the mitotic spindle via a protein structure called the kinetochore
Mega-bases of repetitive DNA, major component is the alpha satellite DNA in humans
All chromosomes have different satellite DNA configurations

Question 3

What do telomeres do?

Answer 3

The end of the chromosomes is made up of a tandem repeat (TTAGGG in human)
With each round of replication, we lose a little bit of the end of our chromosome- the end replication problem
These repeats are lost as the cell divides
The Hayflick limit is the number of times a cell can divide before it dies

Question 4

How does the telomeres end replication problem happen?

Answer 4

Happens on the lagging strand
Whenever a primer is removed and you fill in the gaps the only place where this can’t be done is at the end of the chromosomes

Question 5

How is the telomeres end replication problem solved?

Answer 5

Telomerase, an RNA-dependent DNA polymerase that adds telomeric DNA to telomeres
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 chromosomal DNA has been restored
Note the gap where the primer for DNA replication has been removed

Question 6

What are the origins of replication?

Answer 6

DNA synthesis begins at the replication origins
Bacteria have a single origin of replication
Eukaryotic chromosomes are large so multiple origins must fire simultaneously for replication to be completed within a reasonable timescale
Origins are clustered in replication units

Question 7

What is a G banded metaphase spread?

Answer 7

If you take a blood sample, culture it and add a cell cycle blocking agent so that you get a higher proportion of the cells during metaphase- when a chromosomes are most condensed
Then adds the cells to a hypotonic solution which would swell the cells up before dropping them on a slide
When dropped on a slide, they burst open and release the chromosomes

Question 8

How does one identify chromosomes?

Answer 8

G banding- chromosomes partially digested and stained with Giemsa
G dark- gene poor, tends to be heterochromatin rich
G light- gene rich, tends to be euchromatin rich

Question 9

What are the three types of chromosomes?

Answer 9

Chromosomes have two arms- p (above the centromere) and q (below it)
Metacentric- the arms are divided evenly
Submetacentric- the p arm is shorter than the q arm
Acrocentric- there is barely a p arm at all

Question 10

What is FISH? What is spectral karyotyping?

Answer 10

FISH- Fluorescent In Situ Hybridisation
Use a chromosome paint to colour in an entire chromosome here with a yellow fluorescent tag in order to spot their individual space during G1
Spectral karyotyping is where all the chromosomes are a different colour
This way you can see the spatial organisation and changes of the chromosomes within the nucleus

Question 11

What are chromosome territories?

Answer 11

(Chromosome arms and bands are distinct and mutually exclusive)
Chromosomes form non-overlapping domains in the interphase nucleus

Question 12

What is the spatial location of different chromosomes?

Answer 12

Gene poor- periphery of the nucleus preference
Gene rich- interior of the nucleus preference
Genes can have preferential locations at the surface of the chromosome territory and can dynamically loop out in response to transcriptional activation

Question 13

What happens with the MHC gene cluster?

Answer 13

MHC gene cluster with all these genes was most often found at the surface of a chromosome territory
When you added interferon to these cells the MHC gene cluster has gone and is well away from the chromosome territory it has projected itself out

Question 14

List the nuclear compartment and their functions

Answer 14

Chromosome territories- store DNA and control access to DNA
Replication factories- nascent DNA production
Transcription factories- nascent RNA production
Spliceosome- irregular domains contain splicing factors
Nucleoli- ribosome biogenesis
PML nuclear bodies- possible nuclear depot
Plus Cajal bodies, Gems paraspeckles, OPT domains, DNA repair factories, Ikaros, PcG bodies

Question 15

What is the nucleolus?

Answer 15

A self-organising nuclear compartment
The nucleolus is the largest substructure in the nucleus
Function is ribosome subunit production
The nucleolus forms around the Nuclear Organising Regions (NORs)
NORs are the location of the rRNA genes

Question 16

How does ribosome subunit production occur in the nucleolus?

Answer 16

RNA Pol I transcribes precursor rRNA
Processed to 18S, 5.8S and 28S- endo and exonuclease cleavage
5S is transcribed in the nucleoplasm by RNA Pol III and transported to the nucleolus
rRNA folded and associate with 79 ribosomal proteins to assemble the 40S and 60S ribosomal subunits (eukaryotic)
Subunits transported to the cytoplasm

Question 17

What are the three zones that make up the nucleolus?

Answer 17

Fibrillar centre- ribosomal RNA genes
Dense fibrillar component- rRNA transcripts
Granular component- processing and assembly

Question 18

Where are the rRNA genes?

Answer 18

There are 200 rRNA gene copies per haploid genome
The genes are located in tandem copies on the acrocentric chromosomes- 13, 14, 15, 21 and 22
The cell has multiple copies of the rRNA genes
This is because the rRNA molecule is not translated into the protein, the rRNA molecule transcribed is the final product and the cell requires many ribosomes

Question 19

What is nucleolus proteomic analysis?

Answer 19

Mass spectrometry organelle proteomics
700 endogenous nucleolar proteins
Role in processing of endogenous nuclear siRNA (small interfering)
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 some telomeres*

Question 20

What are splicing speckles/ spliceosomes?

Answer 20

Composed of splicing factors and other mRNA processing factors needed for splicing to occur
20-50 per cell
146 known proteins localise to this
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 21

What are the two experiments carried out on splicing speckles and what conclusions came of them?

Answer 21

Change the internal conditions to the cell and observe effects on the compartment
1. Inhibit transcription so (i.e. no splicing in the cell)- speckles round up and become larger
This supports speckles as a storage/ assembly compartment, not direct sites of splicing
2. 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 splicing factors being utilised with shuttling between the speckles and the transcription site
(More recent research suggests speckles may play a role in regulating access to splicing factors in the cell)

Question 22

What is the replication factories hypothesis?

Answer 22

Multiple replicons (DNA replicated from a single origin) ust operate in parallel to ensure all genomic DNA is replicated through S phase
The number of foci is smaller than the number of replicons, leading to the concept of a replication factory
Factories all contain the enzymes and other factors required to produce two new DNA strands
Super resolution microscopy can resolve single replication foci in S phase- 5583 per cell nucleus
The predicted number of replicons is 5149 firing at the same time
This challenges the conventional interpretation of nuclear foci as replication factories; disproving the existence of replication factories

Question 23

What is the transcription factories hypothesis?

Answer 23

Transcription can be visualised by pulse labelled with BrUTP to detect nascent RNA or by immunofluorescence to active elongating RNA Pol II
Transcription factories are sites where multiple active RNA polymerases are concentrated
It is proposed that genes pass through the factory as they are transcribed
Proposed that genes from the same or different chromosomes may associate at the same factory
Through super-resolution microscopy they found:
Spatial organisation of RNA Pol II-mediated transcription
Transcription foci consist of only one RNA Pol II molecule
No clustering, no factories
Transcription machinery assembling at each site of transcription

Question 24

What are nuclear pores and what do they do?

Answer 24

Communication between the nucleoplasm and the cytoplasm happens via the nuclear pore
The nuclear envelope contains about 3000-4000 nuclear pore complexes
Movement through the nuclear pore is strictly controlled and only small water soluble molecules can diffuse freely through them
Larger molecules to be actively transported
Nuclear exports, e.g. ribosomal subunits and mRNA require a nuclear export signal
Nuclear imports, e.g. histones, DNA/RNA polymerases etc., require a nuclear localisation sequence