The Cell Nucleus Flashcards
What is the functional compartmentalisation?
How the nucleus contains no internal membranes but subnuclear compartments exists anyways
What are the functional elements of a chromosome?
A single molecule of DNA
Linear (in eukaryotes) and double stranded
Contains genes
What are the structural elements of a chromosome
Telomeres (protecting chromosome ends)
Centromere (needed during cell division)
Replication origins
What are the functions of a centromere?
- Locks sister chromatids together
- Attachment site for chromosomes to the mitotic spindle via a protein structure called the kinetochore
- made up of megabuses of repetitive DNA, (majority of which is the alpha satellite DNA in humans)
- all chromosomes have different satellite DNA configurations.
How many base pairs is alpha satellite DNA made up of
171
What is a hierarchy of repeats?
When you have one unit that is repeated, then the repeats get repeated etc etc etc
What is a telomere made up of?
Tandem repeat TTAGGG
What is the end replication problem?
With each round of replication we loose a bit of the end of the replication
What is the hayflick limit?
The number of times a cell can divide before it dies
What type of loop is a telomere? (Double or single strand)
Single stranded
Why does the telomere shorten after each round of replication?
Removal of the RNA primer on the lagging strand
What is telomerase?
An RNA dependant DNA polymerase that adds telomeric DNA to telomeres
How does telomerease work?
Uses an RNA template that follows the TTAGGG sequence, which is able to bind to the DNA which means the original length of the chromosomes is restored
How do you get a G banded metaphase spread?
- take a blood sample
- culture cells
- add a cell cycle blocking agent to get lots of the cells in metaphase (the chromosomes are most condensed here)
- add cells to hypertonic solution (swells cells)
- drop them onto the slide so the cells burst open
- end up with a karyotype
What are the 3 identifying features of chromosomes?
- size
- banding pattern
- centromere position
What is G bonding
When chromosomes are partially digested and stained with giemsa
What does G dark mean?
Gene poor- lots of heterochromatin
What does G light mean?
Gene rich- lots of euchromatin
How are chromosomes organised in the interphase nucleus?
Heterochromatin on the outside and euchromatin on the inside
What does FISH stand for?
Fluorescent in situe hybridisation
What does FISH do?
Allows decondensed chromosomes to be visualised
Give the steps of FISH
- stain metaphase chromosomes red by propidium iodide
- as the cells enter G1 the chromosomes begin to decondense
- when it is fully in G1, the whole nucleus fluoresces red and a chromosome pend is used to colour in entire chromosomes
What are the benefits of FISH?
Means you are able to see where specific chromosomes are in relation to one another and the cell in 3D
What does spectral karyotyping allow?
You to see what happens to the chromosomes in interphase as it is coloured
What happens to the arms and the bands of chromosomes in interphase?
They stay very distinct and mutually exclusive
Where do gene poor/ gene rich chromosomes prefer to be?
Gene poor like to be on the outside (like heterochromatin) and gene rich like to be in the inside (like euchromatin)
Where is an MHC (Major histocompatibility complex) gene cluster found in a territory?
At the surface
What happens to the genes when interferon is added?
They loop out in response to transcriptional activation
What is the largest substructure in the nucleus and what does it do?
Nucleolous is the site of ribosomal subunit production
How are ribosomal subunits made?
- RNA poll transcribes precursor rRNA
- processed to 18s, 5.8s and 28s- endo and Exonuclease cleavage
- 5s is transcribed in the nucleoplasm by RNA Poll2 and transported to the nucleolus
- rRNA is folded and associated with 79 ribosomal proteins to assemble the 40 and 60s ribosomal subunits
- subunits transported to the cytoplasm
What are the three distinct zones of the nucleolus (seen by electron microscopy)
Fibrillar centre- ribosomal RNA genes
Dense fibril or component - processing and assembly
Granular component - processing and assembly
Where does the nucleolus form around?
The Nuclear organising regions (NORS) location of the rRNA genes
What are NORS the location of?
rRNA genes
How many rRNA gene copies per haploid genome and where are they located?
200- in tandem copies on the Acrocentric chromosomes
Which chromosomes are acrocentric?
13, 14, 15, 21, 22
What is a tandem copy of genes?
Copies that follow on from each other along the length of the chromosome
When would a single mRNA molecule be translated many time to give amplification of the final product?
on Single copy genes
Give the features of a splicing speckle
20-50 per cell
146 known proteins
Composed of splicing factors and other mRNA processing factors
Variable size and shape used as a model system to study nuclear organisation
What is nucleolus proteomics analysis?
Mass spectrometry organelle proteomics
Give some other functions of the nucleolus (not subunit production)
- role in processing of endogenous nuclear siRNAs
- assembly of the six proteins and RNA molecules making up the signal recognition particles
- biogenesis of other classes of RNPs such as the spliceosome small nuclear snRNP and telomerase
Do slicking speckles contain DNA?
No, but they are associated with active transcription sites
What happens when you inhibit transcription in speckles and what does this show?
The speckles round up and become larger, which shows they support speckles as a storage/assembly compartment not a direct site of splicing
What happens when you add more intron genes to cells and what does this show?
Splicing factors redistribute to transcription sites and the speckles get smaller. This shows that the speckles are a reservoir of splicing factors and the spicing factors are shuttled between the speckles and transcription sites
What defines nuclear body size/shape and pool in the nucleoplasm?
The continuous association and dissociation of components
What does more recent research suggest about the speckles?
They may play a role in regulating access to splicing factors
What is the predicted number of replicons firing at the same time and what does this mean?
5149- this challenges conventional interpretation of nuclear replication foci as replication factories
How can transcription factories be visualised
Pulse labelled with BrUTP to detect nascent RNA or by immunofluorescence to active elongating RNA poll1
What is pulse labelling?
Incorporating a tag into the RNA thats being transcribed so the mRNA takes it
What do we think transcription factories do?
- sites where multiple active RNA polymerases are concentrated
- termed transcription factories and proposed that genes pass through the factory as they are transcribed
- genes from the same or different chromosomes may associate at the same factory
How has super-resolution microscopy disproved the transcription factory theories?
- spacial organisation of RNA Poll1 mediated transcription
- transcription foci consist of only one RNA poll2 molecule
- no clustering, no factories
- transcription machinery assembling at the site of transcription
How many nuclear pore complexes does a typical mammalian cell contain?
3000-4000
What is the nuclear envelope made up of?
Two lipid bilayers
Are nuclear pores an open space?
No, more like a gateway- only small water soluble molecules can diffuse freely through the pore
What do nuclear exports (give some examples) need to get through the nuclear pore?
Ribosomal subunits and mRNA need a nuclear export signal
What do nuclear imports (give some examples) need to get through the nuclear pore?
Histones, DNA/RNA polymerases and other nuclear proteins need a nuclear localisation sequence
