Lecture 2

DNA quaternary and eukaryotic

genome structure
 Course design
Different levels of complexity

DNA DNA DNA DNA

Primary 2ndary 3tiary 4nary structure
structure structure structure

cell
Individual/
families

Groups
 DNA structure: Revision.

Primary structure Linear sequence of dNTPs

Secondary structure Interaction between

dNTPs, H bonds

B-DNA
Tertiary structure A-DNA
Z-DNA
Source: http://palaeos.com/eukarya/glossary/glossaryP.htm

Quaternary structure Highest level of organization

DNA interacts with other
molecules
Absent in viruses, bacteria.
 Quaternary DNA structure
• Eukaryotic DNA is compacted in a structure of DNA + proteins.
• Chromosome condensation: length contraction at ~ 10 000 times.
• Visible after cell division, chromatin is visible: partial de-condensation.

Evidence:

1) Electron microscopy allowed to see stringed

structures: nucleosomes ( “beads on a string”)

2) X-ray diffraction (1984)

Beads on a string 30nm fibre
Our body has enough DNA to reach from earth to the sun and
back 300 times
 1st level of packaging:
the nucleosome

Nucleosome:
11 nm diameter
DNA: 2 nm diameter
1.7 turns of DNA
around the octamer.
Histones
DNA length reduced
H1 Lys-rich
to 1/3 of its original
H2A slightly lys rich
length.
H2B slightly lys rich
H3 Arg-rich
H4 Arg-rich
DNA is further compacted

30nm fibre
chromatin
 Protruding histone tails

Nucleosome:
2X 4 histones = octamer

Histone tails are sites for chemical

modification

Higher resolution of X-ray diffraction,

from 7 Å (1984) to 2.8 Å (1997), and 1.9 Å (2003).
 Chromatin remodeling

• Change in structure of the protein-DNA structure

• Chromatin remodeling must occur to allow the

DNA to be accessed by DNA binding proteins

• To allow replication and gene expression,

chromatin must relax its compact structure and
expose regions of DNA to regulatory proteins
 Chromatin remodelling
Chromosomes achieve maximum condensation in cell division

Histones tails provide targets for

chemical modification:
• acetylation of lysine: acetyl is transferred to NH2
neutralizes (+) in Lys
gene activation
HAT (Histone acetyltransferases)
H4 is underacetylated in mammal inactive X-chromosome
(Barr body).

• methylation: added to Arg AND Lys - gene activation by methyltransferases.

• phosphorylation: introduces (-) charge to the proteins by kinases.

PO4H2- group is added to OH- in Ser and His in H3 – [cell cycle]

Polar uncharged
Positive charge
Chromatin and the structure of the
eukaryotic genome

Relative levels of condensation and

decondensation of chromatin provided an initial
clue to the differential levels of genetic activity
 DNA Is Organized into Chromatin
in Eukaryotes
Light microscopy !!!
• Polytene chromosomes
– in various tissues in the larvae of some flies
and several species of protozoans and plants.
– Can be seen in interphase cells.
– have distinctive banding patterns
(chromomeres) for chromosome and species.
– represent paired homologs (somatic cells)
– are composed of large numbers of identical
DNA strands.
– the DNA of the paired homologs of polytene
chromosomes undergoes many rounds of
replication without strand separation or
cytoplasmic division.
– Chromosomes have 1,000–5,000 DNA strands
in precise parallel alignment with each other.
 Polytene chromosomes Light microscopy !!!

band

interband

• Polytene chromosomes have puff regions where the DNA has uncoiled
that are visible manifestations of a high level of gene activity
(transcription that produces RNA)

• The cell has many copies of each gene, it can transcribe at a much
higher rate than with only two copies in diploid cells.
 Lampbrush chromosomes
Light microscopy
Meiotic chromosomes  Lampbrush chromosomes are large
and have extensive DNA looping
 They are found in most vertebrate
oocytes as well as spermatocytes
 They are found in the diplotene
stage of prophase I of meiosis
 Lampbrush loops are similar to
puffs in polytene chromosomes
and are sites of gene activity

Scanning electron microscopy

Cytogenetics: branch of genetics science that
studies chromosome morphology and behavior:

The light microscopy visualization of chromosomes

allowed to defined two types of chromatin:

• Heterochromatin: tightly packed DNA

inactive genes
centromeric + telomeric satellites

• Euchromatin: loosely packed DNA,

active section of the genome (~92%)
 States of chromosome condensation:
euchromatin and heterochromatin
(1928)

Heterochromatin: condensed
DNA-proteins after cell division
(interphase).

Euchromatin: DNA in active

transcription

Heterochromatin is unique to eukaryotes.

 Heterochromatin:
Constitutive vs Facultative

Constitutive Heterochromatin :
• associated with structural functions.
• REPETITIVE

Centromere: involved in chromosome movement in cell division

Telomeres: maintenance of chromosomal physical integrity

Other examples: Barr body (mammal females),

most of Y-chromosome,
 Facultative heterochromatin

• Facultative heterochromatin is the result of

genes that are silenced through a mechanism
such as histone deacetylation

• non-repetitive
Chromosome banding
Techniques that allow to characterize / identify
regions within chromosomes due to their different
staining properties

•C-banding: heat + Giemsa

•G-banding: trypsine + Giemsa
•R-banding : Heat+Phosphatase + Giemsa, GC regions (reverse of G)
• NOR (Nucleolar Organization Region): silver nitrate , rRNA genes
• restriction enzymes – based staining: satellite DNA
• fluorescent staining : e.g DAPI, pericentromeric breaking points

Interesting link: http://geneticssuite.net/node/25

 Modern staining techniques:
fluorophores

Different dyes allows to detect GC-rich , AT-rich and ribosomal transcriptional

activity (NOR regions)

Applications: medicine, evolution and characterization of biological species

DAPI (blue): 4′,6-diamidino-2-phenylindole, is a fluorescent stain that binds strongly

to adenine–thymine rich regions in DNA
 Cytogenetics
Light microscopy
C-banding (heat + Giemsa dye), early ‘70s
allows to locate centromeres.

Giesma dye: Binds to regions of DNA where there are high amounts of
adenine-thymine bonding
 Cytogenetics
G-banding (trypsin + Giemsa dye), early ‘70s
allows to locate heterochromatin.

Human
chromosomes

Light microscopy
 Karyotype:
number and morphology of chromosomes of a species

In Diploid organisms, chromosomes

occur in pairs : HOMOLOGOUS

Metaphasic chromosomes:
2 chromatides each.
Chromosome banding application

In 1971, G-banding was adopted to

define nomenclature for regions in
human chromosomes.
This nomenclature is still
maintained today.

Further reading

http://www.nature.com/scitable/topicpage/ch
romosome-mapping-idiograms-302
 This nomenclature is maintained nowadays:
Genbank website, human Y chromosome
 Genome composition

Highly repetitive DNA: satellite DNA, telomeres

Heterochromatic

Minisatellites
Middle repetitive DNA: Tandem repeats Microsatellites
Euchromatic Multiple copy genes

SINES
Interspersed repeats
LINES
Cytogenetic DNA elements Repetition Position etc
definition
Heterochromatin Satellite DNA Highly repeated Telomers
(105 106 copies) centromers
Euchromatin Genes (introns, Single copy Dispersed, central
exons), gene
families etc
Euchromatin Histones and other Middle repetitive dispersed
multicopy genes DNA (TANDEM)
(100s)
Euchromatin rRNA coding genes Middle repetitive Specific location in
DNA (TANDEM) chromosomes
(100s)
Euchromatin Minisatellites Middle repetitive dispersed
DNA (TANDEM)
Euchromatin Mobile elements Middle repetitive Dispersed
(SINES, LINES) DNA (DISPERSED)
Euchromatin microsatellites Middle-low Introns, intergenic
repetition
The human genome

The Human Genome was sequenced in 2001

• Haploid set : 3 billion bp long

3 x 109 bases long

• ~20 000 – 25 000 genes

• ~ 1.5 % of the genomes codes for proteins
and enzymes
• Rest = non-coding, regulatory DNA
sequences, LINEs, SINEs, introns, etc.
 In perspective..
If the genome was a text document , at 50 lines per
page and 80 characters OR spaces per line,

the genome would be = 750 000 pages long

@ 200 pages / volume = 3750 volumes

Coding regions would occupy …56.25 volumes

Heterochromatin

Euchromatin
 Highly repetitive DNA
5% of Human Genome

Concentrated in pericentromeric
and telomeric regions
100s-1000s bp repeated in tandem
 Highly repetitive DNA
Centromere = primary constriction
In situ hybridization, radioactive probe for
mouse satellite DNA
keeps chromatids together
site of kinetochore formation

FISH with a human alphoid "pan-centromeric" probe. All

centromeres light up red

Source:
http://www.chrombios.com/cms/website.php?id=/en/index/infogallery
/pagesrep/gal_rep2.htm&sid=ok5vgn9h0fsk58b00bqqa12mc7
 Highly repetitive DNA
Telomeres
• stability of chromosomes

• chromosome tips: hexamer TTAGGG repeats is conserved in

evolution, present in all vertebrates.

• This DNA is transcribed, TERRA (Telomere repeat containing

RNA), integral part of the telomere.

• highly repetitive DNA is found adjacent to these telomeric

repeats
 FISH: fluorescent in situ hybridization
Repetitive DNA Sequences
Human a satellite, vertebrate
telomeric and rDNA sequences, in a
three color FISH experiment

Human metaphase chromosomes

hybridized with a telomeric repeat
(TTAGGG)n labeled in red,
a pan-centromeric probe labelled in
yellow and a probe for the rRNA genes
at the NORs labeled in green.

Source
http://www.chrombios.com/cms/website.php?id=/en/index/infogallery/pagesrep/gal_rep7.htm&sid=ok5vgn9h0fsk58b00bqqa12mc7
 Middle repetitive DNA:
multiple copies (coding / non coding)
arranged in tandem or dispersed through the genome
Transposons: mobile elements
SINES: (Short Interspersed Elements)
located between and within genes
Alu seqs in primates (~ 300 bp), 5% human genome
SINES: 13 % human genome
some Alu elements are transcribed, uncertain function

LINEs: (Long Interspersed Elements)

retrotransposons
21% of human genome
Transposon-like elements are very common in
eukaryotes genomes
Figure from Trends in Genetics, 2005, 21:8-11.
A whole lotta jumpin' goin' on: new transposon tools for
vertebrate functional genomics
 Middle Repetitive DNA
Histone coding genes

http://www.eplantscience.com/botanical_biotechnology_biology_chemistry/genetics/multigene_familie
s_in_eukaryotes/multigene_families_with_identical_genes.php
 Histone genes
Multiple copies of histone genes. The five major histone proteins, namely H1;
H2A, H2B, H3, H4 are involved in the packing of DNA into nucleosomes, the
chromatin subunits. When DNA is duplicated during S phase of the cell cycle,
large quantities of these histone proteins are needed. To meet this demand, for
each of the histone genes, there are present (i) 10-20 copies in birds and
mammals and (ii) 600-800 copies in sea urchin and newt (amphibians). A higher
number in amphibians suggests a response to their need for rapid cell division.
The five genes for five histones form a basic unit that is repeated, although
different genes within a repeat unit may differ in orientation (Fig. 44.5). These
genes (coding sequences) in a repeat unit are highly conserved, but the spacer
region differs in different organisms.

The histone genes differ from most other eukaryotic genes

in having their transcripts devoid of introns and poly A
tails.
 Middle repetitive DNA

rRNA encoding genes

Current Opinion in Cell Biology 2010, 22:351–356

Humans have 200 rRNA gene copies per haploid

genome, spread out in small clusters on five different
chromosomes (chromosomes 13, 14, 15, 21, 22)

Source:
http://www.rzuser.uni-heidelberg.de/~bu6/Introduction11.html
 Non coding Middle repetitive DNA

Minisatellites or
VNTRs (Variable Number of Tandem Repeats)

In humans:
Tandem repeats 10-100 bp
Interspersed in euchromatin
Stretches 1-20 kb

Individual variation in repeat numbers,

Mendelian inheritance
The beginning of Forensic Genetics
Prof Alec Jeffreys, Leicester University, UK.
First forensic case resolved, based on DNA
evidence, 1985.
Non Coding Middle Repetitive DNA

Microsatellites.
• 2-6 bp repeat motifs
• Interspersed in euchromatin, located in intergenic or intronic
regions.

•High allelic variation between individuals

• Mendelian inheritance
• Markers of choice for forensic applications
Microsatellites or STRs (Short Tandem Repeats)

Alleged sister

Corpse
 Questions:
1. How is DNA compactly packed in the nucleus?
2. What is chromatin remodeling? What control mechanisms allow for localized
“unpacking” ?
3. Why does it happen? Why does the cell need unpacked DNA?
4. What is a karyotype?
5. How is the genome composed ? What types of DNA sequences can be found?
6. What is the proportion of coding vs non-coding DNA in eukaryotes genomes?
7. What categories of repetitive DNA are there in eukaryotes genomes?
8. What is their function , if any..?
9. What method of visualization of DNA sequences at the cytological level can you
mention ? (at least 3)
10.What model organisms were utilized in the development of techniques and the
development of knowledge on the topics shown in this class? (answers are not
necessarily in this presentation, bibliographic search is advisable)