2. Genome, Transcriptome and Proteome
Genome: The complete DNA sequence of an organism.
Transcriptome: The complete set of genes expressed
under particular conditions. It is defined in terms of the
set of RNA molecules present. It includes non-coding
RNAs and mRNAs.
Proteome: The complete set of proteins coded by the
whole genome or produced in any particular cell or
tissue.
3. Genome Organization
• There is no correlation between genome size and genetic
complexity.
• The total amount of DNA in the haploid genome is the
characteristic of each living species known as its C-value.
Drosophila – 4 pairs of chromosomes
Arabidopsis thaliana – 5 pairs of chromosomes
Yeast – 16 pairs of chromosomes
• Eukaryotic genomes are always linear
and contained in chromosomes.
• Chromosome number is unrelated to
genome size and function.
4. Packaging of DNA into chromosomes
• Chromosomes are much
shorter than DNA molecules.
• Nuclear DNA is associated
with DNA binding proteins
called histones.
• Histones are basic proteins
(having positive charge) and
rich in lysine and arginine.
• Chromatin – DNA-histone
complex.
5. • Nucleosome organization was discovered by means of nuclease protection
experiments in 1973-74.
• Nuclease will cut DNA at positions not protected by histone.
After nuclease treatment:
Under non-limiting conditions, ~146
bp fragments were obtained.
Under limiting conditions, ~200 bp
fragments were obtained.
Nuclease Protection Assays
6.
7. Nucleosome: DNA + core octamer
Chromatosome: DNA + core octamer + H1 + linker DNA
8.
9. Nucleosome
• Electron micrograph of chromatin was obtained in 1974 which
demonstrated “beads on a string” structure.
• Nucleosome consists of 8 histones (2 each of H2A, H2B, H3, H4)
forming a barrel shaped core octamer with DNA wound twice
around the outside.
• 140-150 bp DNA is associated with each nucleosome particle.
• 50-70 bp linker DNA separates each nucleosome.
• So, total repeat length is 190-220 bp.
• Linker histone - H1 is attached to the nucleosome to form the
chromatosome.
• 30 nm fiber – Nucleosomes are further packaged in a condensed
form to form the 30 nm fiber.
10. Complexity of
eukaryotic genomes
• Eukaryotic genomes are larger and
much more complex than prokaryotic
genomes.
• Among eukaryotes, the genome size
is not related to genetic complexity.
For e.g., the genomes of Salamanders
and Lilies have 10 times more DNA
as humans, but are not as complex as
humans.
• This is because eukaryotic genomes
contain large amounts of non-coding
sequences along with coding
sequences.
11. Repetitive DNA sequences
• Eukaryotic genomes consist of unique and repetitive
regions.
• Repetitive DNA does not code for proteins and are present
in hundreds and thousands of copies per genome.
• Repeat sequences were first demonstrated by Roy Britten
and David Kohne by studying the rates of reassociation
of denatured DNA.
• The complexity of eukaryotic genomes can thus be studied
by denaturation and renaturation analysis of DNA.
13. Denaturation
• When DNA is heated, it separates into single strands. This is called
Melting or Denaturation.
• Melting temperature (Tm) – The temperature at which 50% of the
DNA is melted or denatured.
14. Denaturation
• Tm increases if %GC of the DNA is high.
• When DNA becomes single stranded, its
absorbance at 260 nm increases. This is
called hyperchromic effect.
Hydrogen bonding between bases in DNA
Absorption spectrum of DNA
Hyperchromic effect
15. Renaturation
• When DNA is cooled, single strands re-associate (anneal). This is
called renaturation or re-association or annealing.
• For this, it is important that correct base pairs collide with each
other.
• This is difficult if the DNA is long and complex.
• Annealing generally occurs 25°C below Tm.
Single
strands
16. Renaturation
• Rate of renaturation gives an idea about the complexity of DNA.
• When DNA renatures, there is a decrease in A260. This is known as
hypochromic effect.
• The degree of renaturation after a given time depends on:
1. C0 – concentration of double stranded DNA prior to denaturation
2. T – duration of renaturation in seconds.
17. Renaturation
• C0t curve – extent of renaturation plotted against log C0t.
• In C0t curve, some part of DNA renatures quickly – this means
these sequences are present at a higher concentration. This part
of the curve represents repeat DNA.
• Some of the DNA renatures slowly. This is the unique part of
DNA.
18. C0t curve of Eukaryotes v/s Prokaryotes
• When denatured E. coli genome is allowed to renature, all of the DNA
re-associates at the same rate.
• However, in case of mammalian genome, ~40% of the DNA
fragments re-associate rapidly (repeated sequences) and ~60% of
DNA fragments re-associate slowly (non-repeated or unique
sequences).
19. Other features of Eukaryotic Nuclear
Genomes
• Genes are not arranged evenly.
• In Arabidopsis thaliana, the gene density is 1-38 genes per 100 kb,
whereas, in humans, the gene density is 0 to 64 genes per 100 kb.
Frequency of repeated DNA in
different genomes
Organism % repeat DNA in
the genome
Human 46%
Mouse 38%
Arabidopsis 14%
Yeast 2.4%
Fruit fly 2%
Caenorhabditis
elegans
<1%
