DNA Packaging in Prokaryotes and Eukaryotes

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Genetic Material

Genetic material in eukaryotes and prokaryotes is dsDNA.

Genetic material in prokaryotes can be found in the nuleoid

Genetic material in eukaryotes is found in the chloroplasts, mitochondria and nucleus

DNA is acidic, very long and relatively fragile.

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Prokaryotic and eukaryotic genomes

DNA is the genetic material in both prokaryotes and eukaryotes however the arrangement of genetic material between the two are different:

  • nucleus or nucleoid
  • linear or circular DNA molecules
  • diploid or haploid life cycle
  • transcription/translation, separation (or not) of these sterps by the nuclear membrane.
  • packaging of DNA
  • arrangement of genes within the genome
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Packaging of DNA genomes

Packaging involes:

  • Association with proteins to stabilise the acidic nature of the DNA and to aid in packaging.
  • Histones:
    • in eukaryotes
    • small basic proteins
    • makes up 50% of the proteins in chromosomes.
  • Histone-like proteins
    • in prokaryotes
    • small basic proteins
    • not related to histones in terms of their sequence

In prokaryotes supercoiling is important for compression of the DNA in the nucleoid.

Twisting DNA in the direction to unwind the double helix will generate negative (right handed) superhelical DNA.

Twisting DNA in the same direction as the double helix generates positive superhelical DNA.

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Supercoiling in E.coli

in vivo: in regions of twisted DNA +ve superhelices.

  • regions of disrupted H bonds = bubbles
  • strand separation
  • replication/recombination/transcription require ssDNA rather than dsDNA

Enzymes which control supercoiling:

  • topoisomerases - cccDNA => supercoiled DNA (I), supercoiled DNA => cccDNA (II)
  • ligases - open circular DNA => cccDNA
  • endonucleases - cccDNA => open circular DNA, supercoiled DNA => open circular DNA
  • gyrases - cccDNA => supercoiled DNA
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Prokaryote Genomes

Nucleoid:

  • DNA + proteins associated with the DNA
  • polymerases, repressors, DNA binding/packaging proteins etc.
  • DNA is being replicated continuously during normal growth

DNA binding proteins - histone-like proteins:

package DNA into the nucleoid, stabilise DNA and contrain the supercoiling...

  • Protein HU
    • small basic dimeric protein
    • most abundant
    • DNA wraps around it
    • not sequence specific
  • Protein H-NS
    • monomeric neutral protein
    • not sequence specific
    • prefers 'bent' DNA
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Bacteria and Plasmids

Bacteria may also harbour small independent DNA molecules known as plasmids

Plasmids:

  • carry genes not found on the main DNA molecule
  • maintain an independent existence
  • size varies: 1-250kb - circular dsDNA
  • small plasmids use the cell's own replication machinery
  • larger plasmids may carry genes for their own replication 
  • episomes are plasmids that can insert themselves into the cell's genome, or exist as a circular DNA molecule.
  • have at least one origin of replication 
  • classified according to the gene that they carry - 5 types
  • used by molecular biologists as vectors i.e. to carry DNA from one cell to another
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Plasmid Types

Fertility, F plasmids - direct conjugation between different bacteria 

Resistance, R plasmids - confer antibiotic resistance to the host, e.g. chloramphenicol, penicillin etc. May confer resistance to more than one antibiotic.

Virulence plasmids - confer pathogenicity on the host bacterium 

Col plasmids - carry genes coding for colicins (anti-bacterial proteins) 

Degradative plasmids - allows the host to degrade unusual molecules

Scientific use:

  • Used as vectors
  • Cut at a specific site
  • Ligate in a piece of foreign DNA
  • Host bacteria transformed
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More complex vectors

  • colour markers/metabolic markers
    • rather than antibiotic resistance
  • expression vectors
    • allows the gene that is now carried on the plasmid to be transceribed and then translated
  • insertion cassettes
    • increases the number of single site restriction enzyme recognition sequences and to allow the DNA being inserted to be places in one reading frame rather than another.
  • plasmids designed for mammalian and yeast cells as we as bacterial and plant cells
  • plasmids designed to allow the inserted DNA to be sequenced


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Eukaryotic Genomes

H.sapiens -

  • 3.3 x 10^9 bp, linear dsDNA found in the nucleus, arranged as 23 chromosomes (haploid) each containing one linear DNA molecule.
  • Chromosomes are only visible during mitosis and meiosis when the DNA is at its most compressed
  • DNA is always packaged in the form of chromatin,

Chromatin-

  • highly ordered DNA-protein-RNA complex
  • serves to package and stabilise DNA
  • organises the DNA
  • alters level of packaging during the cell cycle
  • 50% of proteins in chromatin are histones:-
    • small basic proteins
    • bind tightly to DNA
    • five types - H1, H2A, H2B, H3, and H4.
    • highloy conserved
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Basic unit of chromatin

Kornberg (1975)

  • Core histone octamer + DNA wrapped round it
    • DNA wraps round the nucleosome core 1.8 times 
    • = 146 bp.
    • Histones: 2 x [H2A, H2B, H3, H4]
  • Histone H1 sits on top of the DNA/histone core partide
    • this histone is not as well conserved as the other 4 core histones

The arrangement of DNA around nucleosome is highly regular and increases the packing 1 x (naked DNA) to 6-7x.

The DNA between the nuecloeosomes is termed Linker DNA

  • 10-100 bp depending on species - usual range in mammals of 20-80, an average of 50-55 bp
  • this gives a nucleosome repeat of ~200 bp
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Further packaging

Further packaging is necessary, in vivo:

  • 30nm solenoid
  • 6 nucleosomes per turn
  • H1 on the inner face of the solenoid
  • increases packing ration to 40x

This is still not approaching the levels of condensation seen during mitosis. To achieve these, the solenoid must be looped and attached to the cnetral protein core which forms the chromosome scaffold.


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Model for the chromosome

Levels of condensation:

naked DNA - 10 bp per turn, 1x, no level of condensation

beads on a string - nucleosome, 6-7x, DNA being transcribed or replicated

solenoid - 30nm, 40x, normal state for most of the interphase nucleus

loops/chromosome at metaphase - interactions with the nuclear matrix + chromosome scaffold, 1.2 x 10^4x, needs non-histone proteins.

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Over the Cell Cycle

Chromosomes are at their most condensed at metaphase and then relax during interpahse. All DNA is replicated, therefore all DNA must be at the lowest level of packaging at least once in S phase to allow the replication enzymes to access.

  • S phase of interphase:
    • replication of DNA
    • must be accessible to the replication enzymes
  • G1, G2 phase
    • most of the genome is packaged as 30nm fibre
  • Mitosis
    • entire genome is highly condensed
    • no transcription or DNA replication seen
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Different regions of the genome

At interphase, it can be seen that chromatin is more dense in areas of the nucleus than others.

The degree of packaging varies between different parts of the genome:

  • Euchromatin
    • loosely packaged
    • transcribable
  • Heterochromatin
    • tightly packaged
    • not transcribed
      • e.g. Barr body
      • inactive X chromosomes of female mammals


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