Principles of Cell Biology (BIOL2060)

Department of Biology
Memorial University of Newfoundland

DNA, Chromosomes & the Nucleus

In 1952, Watson and Crick proposed that DNA is a double helix which is known to have alternative forms.

The Genome

The organization of total sum of genetic information (or genome) of an organism is in the form of double-stranded DNA, except that viruses may have single-stranded DNA, single-stranded RNA or double-stranded RNA genomes.
In many viruses and prokaryotes, the genome is a single linear or circular molecule.
In eukaryotes, the nuclear genome consists of linear chromosomes (usually as a diploid set) and the mitochondrial and chloroplast (in plants) genomes are small circular DNA molecules.
The DNA is converted from relaxed to supercoiled by DNA specific enzymes.
In general, genome size increases with organism's complexity.

Through the analysis of DNA renaturation studies, the large sizes of eukaryotic genomes reveal large amounts of repeated DNA.
Heating sheared DNA in solution produces single-stranded DNA.
Lowering the temperature allows the DNA to reanneal (the higher the concentration the faster the process of reannealing) which can be followed by optical density readings (single- versus double-stranded).
For a set concentration of DNA, viral genomes (small) anneal faster than bacterial genomes (far larger than viral genomes, smaller than eukaryotes).
Eukaryotic genomes undergo a complex pattern of reannealing which reveals a large amount of repeated DNA sequences (fast annealing) and unique, nonrepeated DNA (slow annealing).
For example, calf DNA has ~40% repeated and ~60% non-repeated DNA.

The repeated DNA is present in two categories, 1) tandemly repeated DNA and 2) interspersed repeated DNA.
Tandemly repeated DNA (10-15% of mammalian genomes) is made up of rows of many copies of the same sequence.
The repeated unit ranges from 1 to 2000 basepairs (bps) in length.
Often the repeat is less than 10 bps and is referred to as simple-sequence repeated DNA or satellite DNA (due to centrifugation "satellite" bands).
These may provide special physical properties to some stretches of the chromosome.
Centromeres and telomeres are rich in simple-sequence repeated DNA.
At a given site, the amount of simple-sequence repeated DNA may vary greatly.
In DNA minisatellites, the satellites may vary between 100 and 100,000 bps in length.
DNA fingerprinting is used to distinguish individuals by analyzing microsatellites (repeats of 1 to 4 bps) which often differ by 10 to 100 bps.
A number of human diseases are caused by having triplet repeat amplification such as in Huntington's Disease where 11-34 repeats of CAG in the Huntington's Disease gene is normal but ~50 to 100 results in the disease.
Interspersed repeated DNA make up 25 to 40% of most mammalian genomes that are hundreds to thousands of bps long.
Many interspersed repeated DNA sequences are transposable elements.

Eukaryotic Genome Packaging

The cell must handle amounts of DNA that are many times longer than the cells they are in.
DNA packaging must be very efficient while still allowing for DNA replication and transcription to occur.
Prokaryotes package DNA in bacterial chromosomes and plasmids.
The bacterial chromosome is localized in the nucleoid region of the cell (no nucleus) and is looped into negative coils.
The loops are 50,000 to 100,000 bps in length (similar to eukaryotic chromosomes) which are held in place by RNA and small basic (histone-like) proteins.
Plasmids, small negatively supercoiled circular DNA molecules, carry usually non-essential genes (often drug resistance).

Eukaryotes package DNA in the nucleus into chromatin and chromosomes.
Chromatin fibres are 10 to 30 nm in diameter which condense into much more compact (packaged) structure for cell division.
The histones, a group of positive charged (lysine and arginine rich) proteins, which stabilize DNA (negatively charged).
Chromatin contains equal amounts of H2A, H2B, H3 and H4 and ~ one-half the amount of H1 plus numerous nonhistone proteins.
Histones provide the basis for the nucleosome, the basic unit of chromatin structure, as seen as "beads-on-a-string" structures on electron micrographs.
The nucleosome core is comprised of a histone octomer [(H2A-H2B)X2,(H3-H4)X2].
The DNA double helix is wrapped around (~1.7 times) the histone octomer.
With nuclease digestion, 146 bps of DNA are tightly associated with the nucleosome but ~200 bps of DNA in total are associated with the nucleosome.
The difference is the linker DNA.
H1 is associated with the linker DNA between nucleosome cores.

Nucleosomes are packed to form chromatin fibres and chromosomes
The nucleosome is only the first level of packaging nuclear DNA.
The "beads on a string" fibres are 10-nm in diameter.
The next level of organization forms the 30-nm chromatin fibre.
Looped (active?) domains (50 to 100 kilobases) of the 30-nm fibre are attached to the non-histone chromosomal scaffold.
Heterochromatin is more compacted and mostly transcriptionally inactive.
The most compact level of chromatin is, of course, the microscopically visible chromosomes (chromatids).

The mitochondria and chloroplasts also have a DNA genome (or chromosome).
These resemble procaryotic genomes (likely due to the endosymbiotic origin of these organelles) but are much smaller.
The mitochondrial genome varies in size among eukaryotes (mammals =16.5 kb & 37 genes, yeast and plants are greater than 5X this).
Chloroplasts are ~120 kb and have ~120 genes.

The Nucleus

A double-membrane nuclear envelope surrounds the nucleus.
The inner and outer nuclear membranes (7-8 nm thick) are surrounded by the periplasmic space of 20-40 nm thickness.
The inner nuclear membrane is supported by the nuclear lamina fibres.
The outer membrane is continuous with the endoplasmic reticulum and ribosomes often on the cytosolic side.
The periplasmic space is continuous with the ER's lumen.
Nuclear pores are channels that pass through both nuclear membranes that join the cytosol with the nucleoplasm.
The inner and outer membranes fuse to form a channel that is lined with the nuclear pore complex (diameter 120 nm).
A mammalian nucleus may have 3000-4000 nuclear pores.
Nuclear pores are site of molecular entry and exit both the passive transport of small molecules and the active transport of large proteins and RNA.
Proteins that are required in the nucleus contain nuclear localization sequences (NLS) that target them through the nuclear pores.
The NLS-containing proteins bind importin (a cytosolic receptor protein) which binds the protein to the nuclear pore.
The nuclear pore transporter then passes the protein into the nucleoplasm.

Ribosome Synthesis
The nucleolus is the intranuclear site where rRNA is made.
Ribosomal proteins are imported into the nucleus and ribosomal subunits are made in the nucleolus.
The ribosomal subunits are then exported through the nuclear pores to the cytoplasm.

Notes prepared from Becker's World of the Cell, 9th edition
Hardin & Bertoni, 2015
Figures copyright of Pearson Education Inc.
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