Lecture 2
DNA quaternary and eukaryotic
genome structure
Course design
Different levels of complexity
DNA
Primary
structure
DNA
2ndary
structure
DNA
3tiary
structure
DNA
4nary structure
cell
Individual/
families
Groups
DNA structure: Revision.
Primary structure
Secondary structure
Tertiary structure
Linear sequence of dNTPs
Interaction between
dNTPs, H bonds
B-DNA
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
Histones
H1
H2A
H2B
H3
H4
1.7 turns of DNA
around the octamer.
Lys-rich
slightly lys rich
slightly lys rich
Arg-rich
Arg-rich
DNA length reduced
to 1/3 of its original
length.
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
Scanning electron microscopy
 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
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
Middle repetitive DNA:
Euchromatic
Minisatellites
Tandem repeats Microsatellites
Multiple copy genes
Interspersed repeats
SINES
LINES
Cytogenetic
definition
DNA elements
Repetition
Position etc
Heterochromatin
Satellite DNA
Highly repeated
(105 106 copies)
Telomers
centromers
Euchromatin
Genes (introns,
exons), gene
families etc
Single copy
Dispersed, central
Euchromatin
Histones and other
multicopy genes
Middle repetitive
DNA (TANDEM)
(100s)
dispersed
Euchromatin
rRNA coding genes
Middle repetitive
DNA (TANDEM)
(100s)
Specific location in
chromosomes
Euchromatin
Minisatellites
Middle repetitive
DNA (TANDEM)
dispersed
Euchromatin
Mobile elements
(SINES, LINES)
Middle repetitive
DNA (DISPERSED)
Dispersed
Euchromatin
microsatellites
Middle-low
repetition
Introns, intergenic
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
keeps chromatids together
site of kinetochore formation
In situ hybridization, radioactive probe for
mouse satellite DNA
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)