The cell nucleus

• Cell Nucleus


  The part of a cell that contains the genetic material and controls the activities of the cell
• Functions of the Nucleus

  • Stores and maintains the cell's DNA
  • DNA replication
  • Transcription to make the various types of RNA, one of which is the messenger RNA to make proteins
  • Ribosomal biogenesis — ribosomes need to be transported into the cytoplasm so they can be used for protein translation
  • Controls communication between the nucleoplasm and the cytoplasm — controls via the nuclear membrane what goes in and out of the nucleus

  
• Functional Compartmentalisation of the Nucleus

  • Subnuclear compartments exist despite the absence of internal membranes in the nucleus
  • Whenever there's a requirement for multiple enzymes and proteins to come together to perform a function in the nucleus, such as transcription, replication or making ribosome subunits, they somehow come together to perform their functions in the absence of any membranes
  • Functions are compartmentalised in certain parts of the nucleus, which is thought be very self-organising
• Functional Elements of a Chromosome

  • Stores the DNA
  • Chromosomes are linear (in eukaryotes), double-stranded molecules of DNA and has a structure called a telomere at each end to protect the chromosome ends
  • Contains genes and has multiple origins of replication along the length of the chromosome, which are required to initiate DNA replication during the S-phase
  • Centromeres are needed during cell division

  
• Centromeres


  • Lock the replicated sister chromatids together after S-phase of the cell cycle and during G2 of the cell cycle
  • Attach the chromosomes to the mitotic spindle during cell division via a protein structure called the kinetochore
  • Made up of megabases of repetitive DNA, a piece of DNA with a particular sequence that's repeated over and over again

  
• Telomeres


  • Found at the ends of a chromosome and are made up of a tandem repeat, which is a 6 BP repeat TTAGGG in humans)
  • This is repeated over and over again at the ends of the chromosomes
  • There's a single-stranded region at the end of the telomere that loops around to form a loop that protects the ends of the chromosome

  
• Telomere End Replication Problem

  1. The 5' to 3' strand is the DNA strand being copied

  2. In the leading strand, the DNA can be replicated all the way to the end of the chromosome

  3. The lagging strand, however, will go outward from a replication bubble; the primer (in yellow) will attach and an Okazaki fragment forms

  4. When the Okazaki fragments are removed with the primer removal and there are gaps where the primer was, the only place this can't be done is at the end of the chromosome

  5. As DNA is replicated repeatedly, the lagging strand becomes shorter and shorter

  
• Telomerase


  • An enzyme that can fix the telomere end replication problem
  • It's an RNA-dependent DNA polymerase that adds telomeric DNA to telomeres
  • It has an RNA template that is complementary to the ends of the chromosome, and binds to the chromosome
  • The RNA sequence acts as a template for DNA and the enzyme adds the telomeric sequence to the 3' end of the chromosome

  
• Origins of Replication


  • Bacteria have a single origin of replication as their genes aren't large, so single replication origin still allows them to replicate their entire genome in a reasonable time frame
  • Eukaryotic chromosomes are large (and DNA replication is also slower as it has higher fidelity and is more accurate) so multiple origins must fire simultaneously for replication to be completed within a reasonable time frame
  • Origins are clustered in replication units
• Different organisms have different numbers of chromosomes in their cells: each human cell has 46 chromosomes or 23 pairs of chromosomes
• 1. banded Metaphase Spread


  • A staining pattern method where a blood sample is taken from an individual, cells are cultured and a cell cycle-blocking agent is added so a higher proportion of the cells are blocked during cell division in metaphase of the cell cycle
  • Cells are then added to a hypotonic solution, which would have the effect of swelling the cells up before they're dropped onto a slide. Because they're now swollen, they'll burst open and release the chromosomes

  
• Karyotyping


  • After carrying out a G-banded metaphase spread, a karyotype is created
  • The G-banding gives a characteristic G-banding pattern that's specific for each of the chromosomes in our cells
  • There are 23 pairs of chromosomes in human cells: 22 1 to 22 pairs are autosome chromosomes (any chromosome not considered a sex chromosome) and 1 pair of sex chromosomes (XX for females or XY for males)

  
• Identifying Chromosomes


  • Size — chromosome 1 is the largest while chromosome 21 and 22 are the smallest
  • Banding pattern — each chromosome has a distinct banding pattern
  • Centromere position — where the p and q arms are in relation to the centromere (metacentric, submetacentric, acrocentric)

  
• Electron Microscope


  • Information on the locations of the chromosomes under an electron microscope can't be discerned except for the heterochromatin (at the periphery) and euchromatin (in the interior)
  • Beyond that, the microscope doesn't allow for the identification of individual chromosomes, so it's hard to tell where they're located in the nucleus
• FISH (fluorescence in situ hybridisation)


  • This technique allows the decondensed chromosomes in the interphase nucleus to be visualised
  • Chromosome paints can be used to colour entire chromosomes, making it easier to see where individual chromosomes are located in the 3D space of the nucleus
• Spectral Karyotyping


  If multiple chromosome paints are used on all of the different chromosomes in the cell, spectral karyotyping can be carried out, where any rearrangements that have occurred can be seen clearly
• Chromosome Territories


  • Chromosomes form non-overlapping domains in the interphase nucleus
• Gene poor chromosomes are most likely found on the periphery of the interphase nucleus while gene rich chromosomes are most likely found in the interior

  
• Chromosome Territories


  • Spectral Karyotyping allows for the fluorescent dissection of what was happening to the chromosome arms in interphase of the cell cycle
  • It also allows for the examination of the ends of the chromosomes

  
• Chromosome arms and bands are distinct and mutually exclusive
• Chromosomes form non-overlapping domains in the interphase nucleus
• Gene poor chromosomes


  Most likely found on the periphery of the interphase nucleus
• Gene rich chromosomes


  Most likely found in the interior of the interphase nucleus
• There are different functions within the nuclear volume with more genes and transcription in the interior compared to the exterior
• Genes can have preferential locations at the surface of the chromosome territory and can dynamically loop out in response to transcriptional activation
• Nuclear Compartments and Their Functions

  • Chromosome territories: store DNA and control access to DNA
  • Replication factories: nascent DNA production
  • Transcription factories: nascent RNA production
  • Spliceosome: irregular domains containing splicing factors
  • Nucleoli: ribosome biogenesis
  • PML nuclear bodies: possible nuclear depot

  
• Nucleolus


  The largest substructure in the nucleus, typically forms one or two large domains within the nuclear volume, and is the site of ribosomal subunit production

  
• Ribosomal RNA (rRNA) production in the nucleolus

  1. Transcription of rRNA genes by RNA polymerase

  2. Endo and exonuclease cleavage/cutting to form 18S, 5.8S and 28S rRNA molecules

  3. Folding and association with 79 ribosomal proteins to assemble the 40S (small subunit) and 60S (large subunit) ribosomal subunits

  4. Export of subunits through the nuclear pore before coming together to form the functional ribosome in the cytoplasm

  
• Nucleolar Organising Regions (NORs)

  The location of the rRNA genes, where the nucleolus forms as the cell exits cell division and enters G1 phase

  
• There are 200 rRNA gene copies per haploid genome, located in tandem copies on the acrocentric chromosomes 13, 14, 15, 21 and 22
• Having multiple copies of rRNA genes enables the cell to make many ribosomes within a reasonable time
• Nucleolus Proteomic Analysis


  Examination of the proteins within the nucleolus, which has suggested it has many functions beyond just ribosomal RNA subunit production

  
• Additional functions of the nucleolus

  • Processing of endogenous nuclear siRNAs
  • Assembly of the Signal Recognition Particle (SRP)
  • Biogenesis of other classes of RNPs such as spliceosomes and telomerase

  
• Splicing Speckles (aka Spliceosomes, SC35)


  Variable in number, composed of splicing factors and other mRNA processing factors needed for splicing, and used as a model system to study nuclear organisation
• Splicing speckles do not contain DNA and are not sites of transcription, but are associated with/close to highly active transcription sites
• Inhibiting transcription


  Leads to splicing speckles rounding up and becoming larger
• Adding more intron containing genes to a cell


  Leads to splicing factors redistributing to transcription sites, and the speckles getting smaller
• Splicing speckles are thought of as a model of self assembly through transient macromolecular interactions, with continuous association and disassociation of components defining their size/shape and the pool in the nucleoplasm

  
• More recent research suggests splicing speckles may play a role in regulating access to splicing factors in the cell and in turn, controlling and regulating gene expression in the nucleus
• Replication Factories Hypothesis


  Chromosomes have multiple replicons that must operate in parallel, and cluster together to access the enzymes and factors needed for DNA replication