Introductory Genetics BIOL2104

Lecture 2: DNA and Chromosome Structure

Reading: Chapters 10 and 12
Next Lecture: Chapter 11
Watson-Crick DNA model of B-DNA
Seen under aqueous, low-salt conditions

Alternative forms of DNA observed under different conditions

• A-DNA Slightly more compact than B-DNA

Prevalent under high-salt or dehydration conditions

• B-DNA

• C-DNA
• D-DNA
• E-DNA
• P-DNA

• Z-DNA Left-handed double helix

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RNA
• Sugar ribose replaces deoxyribose of DNA
• Nitrogenous base uracil replaces thymine of DNA
• Most are single-stranded (ss)

Three classes of cellular RNAs (function during gene expression)

Ribosomal RNA (rRNA) [80% of total RNA]

Structural components of ribosomes for protein synthesis

Transfer RNA (tRNA) [15% of total RNA]

Carry amino acids for protein synthesis

Messenger RNA (mRNA) [< 5% of total RNA]

Template for protein synthesis
Carry genetic information from gene to ribosome
In recent years a number of additional RNA products with regulatory function have been
characterized:
Small nuclear RNA (snRNA)-important in pre-mRNA splicing
Small nucleolar RNA (snoRNA) – modification of other RNA, especially rRNA, tRNA and snRNA
Small interfering RNA (siRNA)
microRNA (miRNA) Small noncoding RNAs that are primarily involved in
regulation of gene expression-mostly silencing
Piwi interacting RNA (piRNA)
Long non coding (lncRNA) - regulation of gene expression and genome organization
Telomerase RNA Component (TERC)
Bacteria—clustered regularly interspaced short palindromic repeats (CRISPR) RNA (crRNA)
Discovered in bacteria, CRISPR technique mimics the immune system of the bacteria where it was found
Analytical techniques useful during DNA and RNA investigation
• Absorption of UV light
• Denaturation and renaturation of nucleic acids
• Molecular hybridization
Nucleic acids
• Absorb UV light strongly at 254–260 nm due to interaction between UV light and ring
systems of the bases

Left Image- Textbook

Right - Probably more accurate

Can use absorbance at 260 nm to quantitate the

amount of DNA in a sample
Double Stranded DNA, UV
absorbance of 1.0

In a 1 cm cuvette, 50 ug/ml sample has an A260 of 1.0

You have purified DNA from a culture of bacteria and your DNA is dissolved in 1 mL of water.
From this you pipette 50 uL into a tube and make the volume up to 1 mL with water. You
measure the UV absorbance spectrum in a 1 cm cuvette.

Extract and purify DNA

Sample: 1ml DNA in water
- 50 ug/ml sample has an A260 of 1.0
- 0.04 x 50 ug/ml = 2ug/ml
- Dilution Factor is 20
- 2 ug/ml x 20 = 40 ug/ml

ANSWER: 40 ug

How many ug of DNA are in sample?

DNA absorbs UV at 260 nm
Protein absorbs UV at 280 nm
and 230 nm

How might you determine purity of

your DNA sample?
 Pure DNA

DNA absorbs UV at 260 nm

Protein absorbs UV at 280 nm
and 230 nm

How might you determine purity of

your DNA sample?
 Is DNA in your sample pure?
Yes you can use the sample, its alot more than 1.8 (slightly less than 2.0)
DNA can denature due to heat or stress
Hyperchromic shift
Increase in UV absorption of heated
DNA in solution
Denaturation used to determine
melting temperature (Tm)

denaturation
DNA can denature due to heat or stress
Hyperchromic shift
Increase in UV absorption of heated
DNA in solution
Denaturation used to determine
melting temperature (Tm)

denaturation

renaturation (slowly)
DNA can denature due to heat or stress
Hyperchromic shift
Increase in UV absorption of heated
DNA in solution
Denaturation used to determine
melting temperature (Tm)

• which curve would you expect to have the

highest G:C content?
G-C base pairs are 3 hyrogen bonds, instead of only 2 hydrogen bonds

• Why? and are harder to pull apart

The green curve has more G:C content
Molecular hybridization
• Denaturation and renaturation of
nucleic acids are the basis for
molecular hybridization T T TGCGC
AAACGCG

• Example: Single strands of nucleic

acids combine in duplex structures,
• If DNA is isolated from two
distinct sources, double-
stranded hybrids will form

• This is the basis for many

procedures that are central to
modern molecular genetics
techniques
Fluorescent in situ hybridization (FISH)
• Uses fluorescent probes to monitor
hybridization
• Probes are short polynucleotides
(oligonucleotides) with a specific
sequence that will hybridize ONLY
with specific chromosomal areas
• Mitotic cells fixed to slides and
subjected to hybridization
• ssDNA is added and hybridization is
monitored

hybridization of fluorescent probe

Probably a probe to a centremeric region
Probs are usually DNA sequences, because we can synthesize the DNA in a machine
I’m going to design an oligonucleotide to hybridize to a piece of DNA
-what factors will influence the Tm of the oligonucleotide and whether it is an effective probe?
1. Higher G:C content: Higher content means higher Tm

2. The Length: longer sequences have higher Tm

3. The sequence of the oligonucleotide: nearest neighbour effects

4. Whether there are any mistakes, anything not totally complimentary lower the Tm

5. Whether the end are complementary, would mean it has competition by itself

3’ 5’
5’ 3’
DNA Topology
DNA forms topological domains
-circular forms
-segments of DNA in which the ends are fixed
-for example, fixed on a membrane or
on a protein scaffold
Supercoiled DNA
-DNA is not totally relaxed (contain exactly
10.4 base pairs per helical turn)
-is normally slightly underwound
-is wrapped around proteins (nucleosomes)
-is being opened up (replication and
transcription)

Supercoiling occurs when DNA duplex is

underwound or overwound wrt its relaxed
negative
state supercoils
DNA gyrase is a type II
topoisomerase that introduces
negative supercoils to counteract
the accumulating positive
supercoils and therefore relieves
topological stress
Supercoiling facilitates compaction of DNA

Supercoiled DNA
Closed-circular molecules are more
compact and sediment more rapidly than
linear forms
 Of course, within the cell, the
DNA is not naked but is tightly
bound with different proteins in
the form of chromatin.

In bacteria, nucleoid associated

proteins (NAPs) (such as H-NS,
and HU) create domains, and
maintain the topological
organization of the chromosome,
which is essential for function.

In addition, DNA is compacted by

supercoiling

M. Macvanin, S. Adhya / Biochimica et Biophysica Acta 1819 (2012) 830–835

Dillon and Dorman, Nature Reviews Microbiology 8, 185–195 (2010)
Metaphase of mitosis

interphase
 Chromosomes are not randomly
distributed around the nucleus
but appear to occupy
chromosome territories

This has enormous implications

for regulation of gene expression,
replication, and repair

https://cellbiology.med.unsw.edu.au/cellbiology/index.php/Cell_Nucleus
 Chromosomes are not randomly
distributed around the nucleus
but appear to occupy
chromosome territories

This has enormous implications

for regulation of gene expression,
replication, and repair

Geyer et al, Current Opinion in Cell Biology 2011, 23:354–359

 Chromosomes Following DNA replication,
each chromosome contains
two identical sister chromatids

Telomeres

S phase

maternal paternal

During G1, each chromosome of a

diploid pair contains one chromatid
 karyotype
Metaphase preparation

Genome: Genetic information in haploid set

Centromeres
• Constricted regions on
chromosomes
• function in chromosome segregation
during cell division
• Location of centromere establishes
appearance of chromosome
• Metacentric
• Submetacentric
• Acrocentric
• Telocentric
 Mouse chromosomes hybridized with a probe that binds to centromeres
Centromeres
• Constricted regions on chromosomes
• function in chromosome segregation
during cell division
• Location of centromere establishes
appearance of chromosome
• Metacentric
• Submetacentric
• Acrocentric
• Telocentric

What can we say about mouse chromosomes?

All telocentric, all located right at the end on the chromosomes
Only about 2% of the human genome contains genes that encode proteins

Over 50 % of the human genome is repetitive DNA

Are repeated many times within eukaryotic chromosomes
Several categories of repetitive DNA
Satellite DNA
• Highly repetitive and consists of short repeated sequences
• Makes up variable proportion of total DNA (~5% in humans)
• Found in heterochromatic centromeric regions of chromosomes
• In humans can be over 1 Mbp and consist of 170 bp repeats

Centromeres
• Separation of homologs during mitosis and
meiosis depends on centromeres
• Are the primary constrictions along
eukaryotic chromosomes

Kinetochore proteins
• Bind to centromere region and interact with
microtubules during cell division
 Moderately (middle) repetitive DNA
Tandem repeats
• Minisatellites, Variable number tandem repeats (VNTRs)
• 15-100 bp per repeat Occur in long arrays
dispersed throughout the
• Microsatellites (short tandem repeats, STRs) genome
• 2-5 bp per repeat

……..
Highly polymorphic (any one individual will have arrays that differ in the number of repeats)

• rDNA: tandem arrays of rRNA genes (43kb repeated about 70 times)

• In humans there are five rDNA clusters; on chromosomes 13, 14, 15, 21 and 22
Interspersed retrotransposons
Short interspersed elements (SINES) – ~13 % of the human
genome
• Less than 500 bp in length
• Present in > 1.5 M copies dispersed throughout the
genome
• e.g Alu sequences

Long interspersed elements (LINES) - ~21 % of the human

genome
• >5000 bp in length
• Present in almost 1 M copies and dispersed
throughout the genome
• E.g L1 sequences

SINES and LINES appear to have evolved through

retrotransposition, a process during which the transposable Important in cancers, and have a structural role for the genome.
Allows the genome to be folded and pack in major way.
gene is copied into an RNA intermediate, reverse transcribed
into DNA, and inserted into the genome of its host at a new
location.
C-banding
banding occurs at the centromeres
G-banding
Nomenclature for
human chromosome
banding
• Based on G-banding
• dark bands are A:T rich,
relatively gene poor
• light bands are G:C rich

Xq21.33
bands, sub-bands, sub-
sub-bands et. can be
observed at higher
resolution
 Unstructured histone tails are
not packed into folded histone
domains within nucleosome
• can be modified by chromatin
remodeling proteins
Acetylation
Methylation
Phosphorylation

Annunziato, A. (2008) DNA Packaging: Nucleosomes and Chromatin. Nature Education 1(1):26
 Chromatin remodeling
• To accommodate DNA-protein interactions, chromatin structure must change
• To allow replication and gene expression, chromatin must
• relax compact structure
• expose regions of DNA to regulatory proteins
Chromatin Acetylation DNA
remodeling Methylation Methylation modifications
modifications Phosphorylation

Euchromatin
• Uncoiled and transcriptionally active
Heterochromatin
• Condensed areas are mostly inactive (lacks genes or contains repressed genes)
• Replicates later in S phase than euchromatin
• Telomere maintains chromosome integrity
• Centromere involved in chromosome movement
 tails are rich in lysine residues (which can be
acetylated and methylated) and serine
residues (which can be phosphorylated)

These modifications are added/removed by

a range of enzymes

For example, Acetylation is carried out by

Enzyme histone acetyltransferase (HAT)
Addition of acetyl group to positively
charged amino group on side chain
(lysine) changes net charge of protein by
neutralizing positive charge

Phillips, T. & Shaw, K. (2008) Chromatin Remodeling in Eukaryotes. Nature Education 1(1):209
Nucleic acid electrophoresis
• Separates DNA and RNA fragments by size
• Smaller fragments migrate through gel at faster rate than large fragments
Agarose or polyacrylamide gel
• Porous matrix restricts migration of larger molecules more than it restricts smaller ones

Why does DNA migrate in the gel?

Why does DNA migrate in the gel?
Chromosome Banding Differentiates Regions along the Mitotic Chromosome
Used by cytogeneticists to identify chromosomes and assemble karyotypes
Can identify location of specific genes (before human genome project)

Most common patterns are C-banding and G-banding

C-banding
Mitotic chromosomes have a characteristic banding pattern
Only centromeres (heterochromatin) take up stain

G-banding
Differential staining along length of each chromosome
Digestion of mitotic chromosome by enzyme

Banding based on different types of stains are also used (e.g. R-banding, Q-banding)
C-banding
banding occurs at the centromeres
G-banding
Nomenclature for
human chromosome
banding
• Based on G-banding
• dark bands are A:T rich,
relatively gene poor
• light bands are G:C rich

Xq21.33
bands, sub-bands, sub-
sub-bands et. can be
observed at higher
resolution
 There are a couple of atypical examples of eukaryotic chromosomes
1) Polytene chromosomes
• Found in some tissues of flies, and also in
some protozoans and plants
• DNA of paired homologs undergoes many
rounds of replication without strand
separation or cytoplasmic division. Can
have 1000-5000 DNA strands in parallel
 There are a couple of atypical examples of eukaryotic chromosomes
1) Polytene chromosomes
• Found in some tissues of flies, and also in
some protozoans and plants
• DNA of paired homologs undergoes many
rounds of replication without strand
separation or cytoplasmic division. Can
have 1000-5000 DNA strands in parallel
• Puff regions. Bulges where DNA has
uncoiled that are visible manifestations of
high level gene activity (transcription that
produces RNA)
2) Lampbrush chromosomes
• Found in most vertebrate oocytes and
spermatocytes of some insects
• Easily isolated from oocytes in diplotene stage
of prophase I of meiosis
• Large with extensive DNA looping that are sites
of gene activity