Transcript Chromosomal structures and transposable elements Package DNA

Chromosomal structures and transposable
elements
-Package DNA in limited space:
supercoiling
Bacterial chromosome
-Unique DNA molecule
-Naked DNA no histones
Eukaryotic chromosome
-Highly compacted
-Euchromatin and heterochromatin
CHROMATIN
Nucleosomes are DNA wrapped around a
protein octamer made by dimers of
histons H2A, H2B, H3 e H4
HISTONES
H1
are
highly conserved,
small, basic proteins
Linker histone
H2A
H2B
helix
Core histones
variable
H3
H4
conserved
N
Histone acetylation
is a reversible modification
of lysines in the N-termini
of the core histones.
Result:
• reduced binding to DNA
• destabilization of chromatin
Histone octamer assembly
H3-H4
tetramer
Histone
octamer
H2A-H2B
dimer
<
11 nm
>
Histone octamer organizes 145 bp of
DNA
<
6 nm
>
• Each core histone dimer
has 6 DNA binding surfaces
that organize 3 DNA turns;
• The histone octamer
organizes 145 bp of DNA
in 1 3/4 helical turn of DNA:
48 nm of DNA packaged in a disc of 6 x
11nm
30 nm fiber
Chromatin decondensed (beads on a string)
Nuclear - chromosome compaction
+ 2M NaCl
histones
chromatid 1mm
Mitosis
DNA loops
mitotic chromosome
10 mm
radial loop
chrosomosome model
+ 2M NaCl
histones
chromatid
compact size
DNA
length
compaction
nucleus (human)
2 x 23 = 46 chromosomes
92 DNA molecules
10 m ball
12,000 Mbp
4 m DNA
400,000 x
mitotic chromosome
2 chromatids, 1 m thick
2 DNA molecules
10 m long X
2x 130 Mbp
2x 43 mm DNA
10,000 x
DNA domain
anchored DNA loop
1 replicon ?
60 nm x 0.5 m
60 kbp
20 m DNA
35 x
chromatin fiber
approx. 6 nucleosomes per ‘turn’ of 11 nm
30 nm diameter
1200 bp
400 nm DNA
35 x
nucleosome
disk 1 ¾ turn of DNA (146 bp) + linker DNA
6 x 11 nm
200 bp
66 nm DNA
6 - 11 x
Compaction by chromosome scaffold / nuclear matrix
Variation in the chromatin structure
-Polytenic chromosomes in Drosophyla DNA replication non
associated to cell division.
-Sensitivity to Dnasi I correlates with gene activity
Polytenic chromosomes
Dnasi I sensitivity
In chicken erythoblasts,
DNasiI sensitivity
correlates transcription
of globin genes
Chromatin
fibers
11 nm
30 nm
chromatin fiber
+ charged N termini
(bind DNA on neigboring
nucleosomes)
(beads)
highly acetylated
core histones
(especially H3 and H4)
• HIGH level of histone H1
• Reduced level of histone H1
• NO gene transcription
• Gene transcription possible
The structure of the
centromer
-Chromosomes without
centromers are lost in mitosis
Centromers are made of
repetitive sequences
Telomeric structure
Denaturation and renaturation of DNA
Transposable elements
-Mobile DNA elements found in
the genomes of all eukaryotes
-They can cause mutations
Transposition
1. Replicative transposition
Replicative transposition: single strand interruptions, replication,
resolution
2. NON REPLICATIVE transposition
3. RNA intermediates: retrotrasposons
Mutagenic effects
Bacterial transposable elements
Insertion sequences:
- sono semplici elementi trasponibili che portano unicamente le
informazioni necessarie per la trasposizione
Composite transposons:
Intervening DNA
Non composite transposons:
-no Intervening DNA, repetitive sequences
-some phages use transposition to insert their genome into the
bacterial DNA.
Transposable elements in eukaryotes
Negli eucarioti esiste una vasta gamma di elementi trasponibili.
Alcuni ricordano gli elementi trasponibili dei procarioti, con
ripetizioni terminali invertite e un meccanismo di trasposizione
mediante intermedio a DNA. Altri sono retrotrasposoni con
ripetizioni lunghe dirette alle loro estremità e modalità di
trasposizione tramite intermedio a RNA.
Lievito
Barbara McClintock discovered transposable elements
studying varaiagated maize
Ac e Ds transposable elements in maize
Drosophyla transposable elements
Human transposable elements
Distribution of genes and other sequences in the genome
• The majority of transposons are inactive
– Very few are currently active
• The transposons may have played an active role in genome
evolution
•LINEs are one of the most ancient and successful inventions in
eukaryotic genomes. In humans, these transposons are about 6 kb
long, harbour an internal polymerase II promoter and encode two
open reading frames (ORFs). Upon translation, a LINE RNA
assembles with its own encoded proteins and moves to the nucleus,
where an endonuclease activity makes a single-stranded nick and
the reverse transcriptase uses the nicked DNA to prime reverse
transcription from the 3' end of the LINE RNA. Reverse
transcription frequently fails to proceed to the 5' end, resulting in
many truncated, nonfunctional insertions. Indeed, most LINEderived repeats are short, with an average size of 900 bp for all
LINE1 copies, and a median size of 1,070 bp for copies of the
currently active LINE1 element (L1Hs). New insertion sites are
flanked by a small target site duplication of 7–20 bp. The LINE
machinery is believed to be responsible for most reverse
transcription in the genome, including the retrotransposition of the
non-autonomous SINEs [144] and the creation of processed
pseudogenes [145, 146]. Three distantly related LINE families are
found in the human genome: LINE1, LINE2 and LINE3. Only LINE1
is still active.
•SINEs are wildly successful freeloaders on the backs of LINE
elements. They are short (about 100–400 bp), harbour an internal
polymerase III promoter and encode no proteins.These nonautonomous transposons are thought to use the LINE machinery
for transposition. Indeed, most SINEs 'live' by sharing the 3' end
with a resident LINE element [144]. The promoter regions of all
known SINEs are derived from tRNA sequences, with the
exception of a single monophyletic family of SINEs derived from
the signal recognition particle component 7SL. This family, which
also does not share its 3' end with a LINE, includes the only active
SINE in the human genome: the Alu element. By contrast, the
mouse has both tRNA-derived and 7SL-derived SINEs. The human
genome contains three distinct monophyletic families of SINEs:
the active Alu, and the inactive MIR and Ther2/MIR3
•LTR retroposons are flanked by long terminal direct repeats that
contain all of the necessary transcriptional regulatory elements.
The autonomous elements (retrotransposons) contain gag and pol
genes, which encode a protease, reverse transcriptase, RNAse H
and integrase. Exogenous retroviruses seem to have arisen from
endogenous retrotransposons by acquisition of a cellular envelope
gene (env) [147]. Transposition occurs through the retroviral
mechanism with reverse transcription occurring in a cytoplasmic
virus-like particle, primed by a tRNA (in contrast to the nuclear
location and chromosomal priming of LINEs). Although a variety
of LTR retrotransposons exist, only the vertebrate-specific
endogenous retroviruses (ERVs) appear to have been active in the
mammalian genome. Mammalian retroviruses fall into three
classes (I–III), each comprising many families with independent
origins. Most (85%) of the LTR retroposon-derived 'fossils' consist
only of an isolated LTR, with the internal sequence having been
lost by homologous recombination between the flanking LTRs.
•DNA transposons resemble bacterial transposons, having terminal
inverted repeats and encoding a transposase that binds near the
inverted repeats and mediates mobility through a 'cut-and-paste'
mechanism. The human genome contains at least seven major
classes of DNA transposon, which can be subdivided into many
families with independent origins [148] (see RepBase,
http://www.girinst.org/~server/repbase.html DNA transposons
tend to have short life spans within a species. This can be explained
by contrasting the modes of transposition of DNA transposons and
LINE elements. LINE transposition tends to involve only functional
elements, owing to the cis-preference by which LINE proteins
assemble with the RNA from which they were translated. By
contrast, DNA transposons cannot exercise a cis-preference: the
encoded transposase is produced in the cytoplasm and, when it
returns to the nucleus, it cannot distinguish active from inactive
elements. As inactive copies accumulate in the genome,
transposition becomes less efficient. This checks the expansion of
any DNA transposon family and in due course causes it to die out. To
survive, DNA transposons must eventually move by horizontal
transfer to virgin genomes, and there is considerable evidence for
such transfer [149-153].
•Transposable elements employ different strategies to ensure their
evolutionary survival. LINEs and SINEs rely almost exclusively on
vertical transmission within the host genome [154] (but see refs
148, 155).DNA transposons are more promiscuous, requiring
relatively frequent horizontal transfer. LTR retroposons use both
strategies, with some being long-term active residents of the human
genome (such as members of the ERVL family) and others having
only short residence times.