DNA and

Chromosomes

Mayurika Lahiri
BIO313
2019
 The Concept of a Gene as a Unit of Inheritance
•  Life depends on the ability of cells to store, retrieve and translate the
genetic instructions required to make and maintain an organism. This
hereditary information is passed on from a cell to its daughter cells at cell
division and from one generation of an organism to the next through
reproductive cells. These instructions are stored within every living cell as its
genes.

Gregor Johann Mendel [1822-1884]

Mendel’s experiments with garden peas led to his discovery of
the basic principles of genetics
16th century monastery at Brno where Gregor Mendel lived and worked. Brno, Czech Republic
 Discovery of DNA
Mendel’s studies with pea plants concluded that hereditary factors determine
an organism’s traits.
•  But what are these factors?
•  How did these molecules store hereditary information?

The answer to these questions began to emerge during an epidemic of

pneumonia in London in 1920s when British microbiologist, Frederick Griffith
studied Streptococcus pneumoniae
Griffith’s Experiments [1928]
 Avery, McLeod and
McCarty

In early 1940s, American researcher

Oswald Avery and his colleagues set out
to test whether the transforming agent in
Griffith’s experiment was protein, RNA or
DNA.
- Protease to destroy protein in heat –
killed cells
- RNase to destroy RNA
- DNase to destroy DNA

Cells missing DNA were not able to transform

CONCLUSION: DNA is responsible for

transformation in bacteria
 Hershey-Chase Experiment
The hereditary information
injected into the bacteria that
specified the new generation
of viruses was DNA and not
protein

"This protein probably has no function in the growth of intracellular phage. The DNA
has some function. Further chemical inferences should not be drawn from the
experiments presented" [Hershey, A. D., & Chase, M. Independent functions of viral protein and nucleic
acid in growth of bacteriophage. Journal of General Physiology 36, 39–56 (1952)]
 What is the Structure of DNA?
The Chemical Nature of Nucleic Acids

Friedrich Miescher, a German chemist discovered DNA in

1869.
He extracted a white substance from the nuclei of human
cells and fish sperm. Since the constituent of nitrogen and
phosphorus in the substance was different from that in any
other known constituent of cells, Miescher was convinced
that he had discovered a new biological substance which
he called nuclein since it seemed to specifically associate
with the nucleus.

Because Miescher’s nuclein was slightly acidic, it came to

be called nucleic acid

Miescher’s laboratory in
Tubingen
 What is the Structure of DNA?
DNA is a polymer

P A Levene, a biochemist determined the basic

structure of nucleic acids in 1920.
DNA contains three main components
- phosphate groups
- five-carbon sugars, and
- nitrogen-containing bases called purines
(adenine, guanine) and pyrimidines (thymine and
cytosine)

Levene concluded correctly that DNA and RNA

molecules are made of repeating units of the three
components. Each unit consisting of a sugar
attached to a phosphate group and a base, called a
nucleotide
 The Structure and Function of DNA
The Three-Dimensional Structure of DNA

Rosalind Franklin X-ray diffraction photograph of James Watson and Francis

DNA fibers made in 1953 by Crick deduced the structure of
Rosalind Franklin in the DNA in 1953 from Chargaff’s
laboratory of Maurice Wilkins rules and Franklin’s diffraction
studies
The Three-Dimensional Structure of DNA
The structure of DNA provides a mechanism for heredity
In eukaryotes, DNA is enclosed in a cell nucleus
 Eukaryotic DNA is packaged into a set of Chromosomes
Complete set of human chromosomes visualized following “chromosome painting”
or spectral karyotyping
 Banding Pattern of Human Chromosomes

Ideogram showing Geimsa stain; observed under light microscope

 Chromosome Banding of Mitotic
Chromosomes

•  C-banding
–  Giemsa stain only stains centromeric regions

•  G-banding
–  Protease treat the chromosome with Geimsa dye and is thought
to result from interactions of both DNA and protein with the
thiazine and eosin of the stain (Bickmore, Encyclopedia of Life Sciences, 2001)
–  Provides a unique series of bands along each chromosome
–  Provided uniform nomenclature for human chromosomes/
locations in 1971
C-Banding
G-Banding
Chromosome Banding Patterns and Nomenclature
sub-
region band band
telomere = pter (terminus)

3 p22.3

2 2 p22.2
2
Centromere divides chromosomes into a short arm (p; petit)
1 p22.1 and a long arm (q)
SHORT ARM = p (petit) Centromere is defined by the first band on the short arm (p10)
p21
1 and the first band on the long arm (q10)

4 p11.4 Te l o m e r e s a r e p r e s e n t a t t h e t e r m i n i ( p t e r a n d q t e r )
3 p11.3
1 1
2 p11.2 Each chromosome arm is divided into regions based on landmarks
1 p10 centromere = p10+ q10 (consistent and distinct morphologic area of a chromosome)
1 q10
2 q12 The regions immediately adjacent to the centromere are designated as “1”
1 (p1 and q1)
3 q13
Region numbers increase distally to the centromere

Regions are divided into bands and bands into sub-bands

1 q21
LONG ARM = q Xp22.3 = X chromosome, short arm region 2, band 2, sub-band 3
2 q22
Band is read as X q two-two point three, not X q twenty-two point three
3 q23
2 4 q24

5 q25

6 q26
7 q27

8 q28
telomere = qter (terminus)

X Chromosome Band Pattern

metacentric submetacentric acrocentric telocentric

Centromere Locations
Chromosome Anomalies
21-trisomy [Down’s Syndrome]
Chromosome Anomalies
 Chromosomes contain long strings of genes
The arrangement of genes in the genome of S. cerevisae
Light red shading: transcribed genes

Two closely related species of deer

with very different chromosome
numbers
The two species contain a similar number of
genes
The organization of genes on a human chromosome
Table 4-1 Molecular Biology of the Cell (© Garland Science 2008)
 Representation of the nucleotide sequence content
of the completely sequenced human genome
REPEATED SEQUENCES ARE A FEATURE OF EULARYTOIC DNA

LINE: Long Interspersed Element (moderately repetitive DNA); eg. L1 repeats

SINE: Short Interspersed Element (highly repetitive DNA); eg. Alu repeats
Figure 4-17 Molecular Biology of the Cell (© Garland Science 2008)
 Genome comparisons reveal
evolutionarily conserved DNA
sequences

conserved synteny:
large blocks of the
genome containing
genes in the same order

Figure 4-18 Molecular Biology of the Cell (© Garland Science 2008)

 Chromosomes exist in different states throughout
the life of the Cell

Figure 4-19 Molecular Biology of the Cell (© Garland Science 2008)

 Three DNA sequences required to produce a
eukaryotic chromosome that can be replicated and
then segregated at mitosis
 Three DNA sequences required to produce a
eukaryotic chromosome that can be replicated and
then segregated at mitosis
 Eukaryotic DNA is packaged into a set of Chromosomes
If the double helices comprising of all 46 chromosomes in a human cell could be
laid end-to-end, it would reach ~ 2m. But nucleus is only 6µm in diameter
- Therefore packaging of the DNA is necessary and is done by specialized proteins
that bind to and fold DNA, generating a series of coils and loops.
-  Proteins bound to DNA are subject to change during the life of the cell.
-  These changes affect the degree of chromatin compaction
 Nucleosome are a basic unit of eukaryotic
chromosome structure
Proteins that bind to DNA are of two categories: histones and non-histone
chromosomal proteins

Complex of both classes of protein with nuclear DNA is known as

chromatin

Histones bring about chromosome packing to form nucleosome, a protein-

DNA complex, discovered in 1974.

Beads on a string
 Nucleosome

Each individual nucleosome core particle

consists of a complex of eight histone
proteins – 2 molecules each of histones
H2A, H2B, H3 and H4 along with double-
stranded DNA of 147 nucleotide pairs
long. The histone octamer forms a
protein core around which the double-
stranded DNA makes 1.7 turns in a left-
handed coil

Each nucleosome core particle is

separated from the next region by a
region of linker DNA

Histones are among the most highly

conserved eukaryotic proteins
 Vary in length between 20 to 100 bp,
depending on species and cell type Diameter of the
nucleosome

Overall structure of connected nucleosomes resembles “beads on a string”

–  This structure shortens the DNA length about seven-fold

Copyright ©The McGraw-Hill Companies, Inc. Permission required for reproduction or display
Play a role in the organization
and compaction of the
chromosome
 Beads on a String

Overall structure of connected nucleosomes resembles “beads on a string”

Shortens DNA length ~ seven-fold
Nucleosomes Join to
Form 30 nm Fiber
Nucleosomes associate to form
more compact structure - the 30
nm fiber

Histone H1 plays a role in this

compaction via its interaction with
linker DNA
 Compaction level
in euchromatin

During interphase
most chromosomal Compaction level
regions are in heterochromatin
euchromatic
 Nuclear Matrix Association
Nuclear matrix composed of two parts
–  Nuclear lamina
–  Internal matrix proteins
•  10 nm fiber and associated proteins
 DNA Loops on Nuclear Matrix
The third mechanism of DNA compaction involves the formation of radial loop domains

Matrix-attachment
regions
25,000 to
or 200,000 bp

Scaffold-attachment
regions (SARs)

MARs are anchored to

the nuclear matrix,
thus creating radial
loops
 A model for the organization of an interphase
chromosome

Figure 4-57 Molecular Biology of the Cell (© Garland Science 2008)

 Chromosomes are folded into large loops of chromatin
Lampbrush chromosomes
in an amphibian oocyte

Figure 4-54 Molecular Biology of the Cell (© Garland Science 2008)

 A model for the structure of a lampbrush chromosome

Figure 4-55 Molecular Biology of the Cell (© Garland Science 2008)

Polytene chromosomes are uniquely useful for
visualizing chromatin structures

Figure 4-58 Molecular Biology of the Cell (© Garland Science 2008)

 Micrographs of polytene chromosomes from
Drosophila salivary glands
~3700 bands and
3700 interbands

Figure 4-59 Molecular Biology of the Cell (© Garland Science 2008)

 Chromosome loops decondense when the genes
within them are expressed
RNA synthesis in polytene chromosome puffs

3H-uridine

labeling

Figure 4-62 Molecular Biology of the Cell (© Garland Science 2008)

Levels of DNA
Condensation

•  DNA
•  11 nm fiber
•  30 nm fiber
•  300 nm fiber
•  700 nm fiber
Nucleosome are a basic unit of eukaryotic chromosome structure
 Further Compaction of the Chromosome
The attachment of radial loops to the nuclear matrix is important in two ways
•  It plays a role in gene regulation
•  It serves to organize the chromosomes within the nucleus

Each chromosome in the nucleus is located in a discrete and non-overlapping

chromosome territory

Chromosome Territories, Nuclear Architecture and Gene

Regulation in Mammalian Cells
Thomas Cremer and Christoph Crèmer
Nature Reviews Genetics vol. 2, no. 4, pp. 292-301 (2001)
Advances in microscopy and fluorescence imaging have revealed a multitude of sub-
structures in the nucleus collectively referred to as nuclear bodies.

Spector D L. J Cell Sci (2001) 114: 2891-2893

Gene density plot of human chromosomes
Chromosomes are non-randomly positioned in the
interphase nucleus

Cremer (2002) Nature Rev. Genet.

Chromosome positioning is conserved in evolution

Tanabe (2002) PNAS

Nuclear organization of active gene loci in the nucleus

Chubb & Bickmore (2003) Cell 112:403-406

A schematic of gene loci and its association with
nuclear neighborhoods
Visualizing all 46 chromosome territories in the
interphase nucleus by Fluorescence in situ
hybridization (FISH)

Bolzer et al., (2005) PLoS Biology

 Figure 1. 24-Color 3D FISH Representation and Classification of Chromosomes in a Human G0
Fibroblast Nucleus

Bolzer A, Kreth G, Solovei I, Koehler D, et al. (2005) Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei
and Prometaphase Rosettes. PLoS Biol 3(5): e157. doi:10.1371/journal.pbio.0030157
http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030157
 Localization of Alu Sequences in Nuclei of
Fibroblasts and Lymphocytes

Alu: green
TO-PRO-3: red

Bolzer A, Kreth G, Solovei I, Koehler D, et al. (2005) Three-Dimensional Maps of All Chromosomes in Human Male Fibroblast Nuclei
and Prometaphase Rosettes. PLoS Biol 3(5): e157. doi:10.1371/journal.pbio.0030157
http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0030157
 THE REGULATION OF
CHROMATIN STRUCTURE
Structure of Nucleosome Core Particle Reveals How
DNA is Packaged

“handshake” interaction
The Assembly of a Histone Octamer on DNA
Bending of DNA in a Nucleosome
 Chromatin Remodeling

•  Chromatin structure is dynamic

•  Induced change in chromatin structure
–  Replication, gene expression
•  Histone modification
–  Acetylation by histone acetyltransferases (HATs)
–  Deacetylation by histone acetyl complex (HDACs)
–  Methylation by methyl transferases (HMTs)
–  Demethylation by histone demethylases
–  Phosphorylation by kinases
•  DNA modifications
–  Methylation of cytosine (5-methyl C) in CpG islands
Nucleosomes have dynamic structure and are frequently subjected to
changes catalyzed by chromatin-remodeling complexes
Kinetics show that the DNA from an isolated nucleosome unwraps from each
end at the rate of about 4 times per second, remaining exposed for 10-50
milliseconds before the partially unwrapped nucleosome recloses. Thus, most
of the DNA in an isolated nucleosome is in principle available for binding other
proteins
 Nucleosomes have a dynamic structure and are frequently
subjected to changes catalyzed by ATP-dependent chromatin
remodeling complexes
Nucleosome removal and histone exchange catalyzed by ATP-
dependent chromatin remodeling complexes
Nucleosome removal and histone exchange catalyzed by ATP-
dependent chromatin remodeling complexes
 Structure of Histone Variants

Histones variants are synthesized throughout interphase; inserted into

already-formed chromatin catalyzed by ATP-dependent chromatin
remodeling complexes
 Histone modifying enzymes - Outcome of
gene expression

Music is encoded in the form of notations on sheet music, that define the
notes to be played and the tempo at which they are played.
A guitarist translates the sheet music into audible music by spatial and
temporal control of the guitar strings.
In a like manner, gene transcription is spatially and temporally controlled
by epigenetic modifications, catalyzed by specific enzymes.
 Histone modifying enzymes - Outcome of
gene expression

Each of the core

histones has an N-
terminal amino acid
tail which extends
out from the histone
core

Schematic of the nucleosome, illustrating the types of post-

translational modifications that can occur on the histone tails and the
enzymes responsible for these modification reactions.
The covalent modification of core histone tails
 The covalent modifications and the histone variants act in
concert to produce a “Histone Code” that helps to determine
biological function
The covalent modification of core histone tails
A complex of Code-reader and Code-writer proteins can spread specific
chromatin modifications for long distances along a chromosome
Some mechanisms of barrier action
Some mechanisms of barrier action
Some mechanisms of barrier action
A model for the structure of a simple centromere
The organization and function of the chromatin that
forms human centromere
 A model for the direct inheritance of centromeric
heterochromatin

Figure 4-51 Molecular Biology of the Cell (© Garland Science 2008)

 A model for the direct inheritance of centromeric
heterochromatin

Figure 4-51 Molecular Biology of the Cell (© Garland Science 2008)

How packaging of DNA in chromatin can be inherited
during chromosome replication
 Heterochromatin
•  1928, staining differences in nuclei lead to terms euchromatin and
heterochromatin
•  Heterochromatin
–  Dark staining
–  Genetically inactive
•  Few genes, those present repressed
•  Replicates late in S phase
•  Centromeres and telomeres are heterochromatic
•  Portion of Y and inactivated X chromosomes
•  Position effect when genes are translocated to location adjacent
to heterochromatin
Position effect variegation
 Types of Heterochromatin

•  Constitutive heterochromatin
–  Always heterochromatic
–  Permanently inactive with regard to transcription
•  Facultative heterochromatin
–  Regions that can interconvert between euchromatin and
heterochromatin
–  Example: Barr body
Barr body (named after discoverer Murray Barr) is the
inactive X chromosome in a female somatic cell
Barr Bodies: heterochromatinized X-chromosomes
In mammals, males are heterogametic (XY) and females homogametic
(XX). Dosage compensation is achieved by inactivation at random of one of the
two X chromosomes. The heterochromatized X chromosome appears as a
darkly-staining bodies attached to the nuclear membrane. The phenomenon was
first described by Dr Murray L. Barr, a Canadian cytogeneticist, and
heterochromatin bodies are now called Barr Bodies. [The other dark bodies
within the nucleus are nucleoli, which represent repetitive rDNA genes].
 Chromosome Organization
Telomere

•  Genes located between centromere &

Origin of replication
Origin of replication
Kinetochore proteins
Centromere
Origin of replication
•  Non-gene sequences
Origin of replication
Genes
Repetitive sequences
Telomere
telomeres
–  hundreds to thousands of genes
–  lower eukaryotes (i.e. yeast)
•  Genes are relatively small
•  Very few introns
–  higher eukaryotes (i.e. mammals)
•  Genes are long
•  Have many introns
–  Repetitive DNA
•  Telomere
•  Centromere
•  Satellite
 Eukaryotic Gene Structure

Start Codon Stop Codon

Cis- ATG TAA, TAG, TGA
Regulatory
Elements
Exon1 Exon2 Exon3
Promoter/
Enhancer