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Chromosome Structure

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The DNA and protein complex found in eukaryotic chromosomes is called chromatin ... Region of sister chromatid cohesion. Constitutive heterochromatin ... – PowerPoint PPT presentation

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Title: Chromosome Structure


1
  • Chromsomes
  • Chromosome structure
  • Chromatin structure
  • Chromosome variations
  • The new cytogenetics

2
Prokaryotic chromosomes
  • Circular double helix
  • Complexed with protein in a structure termed the
    nucleoid
  • Attached to plasma membrane

3
Eukaryotic Chromosomes
  • Located in the nucleus
  • Each chromosome consists of a single molecule of
    DNA and its associated proteins
  • The DNA and protein complex found in eukaryotic
    chromosomes is called chromatin
  • 1/3 DNA and 2/3 protein
  • Complex interactions between proteins and nucleic
    acids in the chromosomes regulate gene and
    chromosomal function

4
(No Transcript)
5
Some evidence that chromosomes contain a single
DNA molecule
  • Pulsed-field gel electrophoresis - separation of
    chromosomes
  • Analysis of the complete nucleotide sequence of
    many genomes now
  • In situ hybridization (below)

6
Karyotype The representation of entire
metaphase chromosomes in a cell, arranged in
order of size and other characteristics
7
  • Ideogram
  • Diagramatic representation
  • of a karyotype
  • Individual chromsomes are recognized by
  • -arm lengths
  • p, short
  • q, long
  • -centromere position
  • metacentric
  • sub-metacentric
  • acrocentric
  • telocentric
  • -staining (banding) patterns

From Miller Therman (2001) Human Chromosomes,
Springer
8
(No Transcript)
9
Chromsome banding
  • Q (quinicrine) G (Giemsa) banding
    preferentially stain AT rich regions
  • R (reverse banding) preferentially stains GC-rich
    regions
  • C-banding (denaturation staining)
    preferentially stains constitutive
    heterochromatin, found in the centromere regions
    and distal Yq

10
C-banded karyotype of XY cell
From Miller Therman (2001) Human Chromosomes,
Springer
11
Constitutive heterochromatin
  • Tandem, highly repeated short sequences of DNA
  • Non-coding and non-expressing
  • Buoyant density discrete from the bulk of the
    genome (satellite DNA )
  • C-banding
  • Late replicating
  • Maintains a highly compacted organization
  • Never transcribed

12
Facultative heterochromatin
  • All types of sequences
  • C-banding negative
  • Late replicating
  • Condensed conformation
  • Not transcribed
  • Includes genes silenced in specific cell types
    and/or at specific times in development
  • e.g. inactivated X chromosomes

13
Euchromatin
  • Actively expressed sequences
  • More open conformation

14
Fluorescence in situ hybridization (FISH)
a The basic elements of fluorescence in situ
hybridization are a DNA probe and a target
sequence. b Before hybridization, the DNA probe
is labelled by various means such as NICK
TRANSLATION, RANDOM-PRIMED LABELLING and PCR. Two
labelling strategies are commonly used indirect
labelling (left panel) and direct labelling
(right panel). For indirect labelling, probes are
labelled with modified nucleotides that contain a
HAPTEN, whereas direct labelling uses the
incorporation of nucleotides that have been
directly modified to contain a fluorophore. c
The labelled probe and the target DNA are
denatured to yield ssDNA. d They are then
combined, which allows the annealing of
complementary DNA sequences. e If the probe has
been labelled indirectly, an extra step is
required for visualization of the non-fluorescent
hapten that uses an enzymatic or immunological
detection system. Whereas FISH is faster with
directly labelled probes, indirect labelling
offers the advantage of signal amplification by
using several layers of antibodies, and might
therefore produce a signal that is brighter
compared with background levels. Finally, the
signals are evaluated by fluorescence microscopy
(not shown). From Speicher Carter (2005)
Nature Rev Genet 6782
15
Fluorescence in situ hybridization (FISH)
From Speicher Carter (2005) Nature Rev Genet
6782
a Painting probes stain entire chromosomes. b
Regional painting probes can be generated by
chromosome microdissection c Centromeric-repeat
probes are available for almost all human
chromosomes. d Large-insert clones are
available for most genomic regions. Subtelomeric
probes, which are often used to screen for
cryptic translocations that are not usually
visible in conventional chromosome-banding
analyses, are shown in this example. e Special
probe sets can be designed to facilitate
diagnosis of known structural rearrangements. In
this example, the probe set includes a
breakpoint-spanning probe (red) and two
breakpoint-flanking probes (green and blue). f
Genomic DNA is used as the probe in comparative
genomic hybridization (CGH) to establish copy
number. An analysis of chromosome 8 is shown as
an example. Simultaneous visualization of both
test DNA (green region) and normal reference DNA
(red region) fluorochromes shows balanced regions
in orange (equal amounts of green and red
fluorochromes). g For high-resolution analysis,
DNA fibres can be used as the target for probe
hybridization. The simultaneous hybridization of
two different probes is shown, labelled green and
red. h Microarrays can be used as targets for
hybridization to provide resolutions down to the
single-nucleotide level. A BAC array is shown, to
which test DNA and reference DNA are hybridized.
Individual clones show different colours after
hybridization depending on whether the
corresponding DNA in the test sample is lost (red
on the array), gained (green on the array) or
neither (yellow on the array).
16
Fluorescence in situ hybridization (FISH) probes
on metaphase chromosomes
17
Chromosome-specific paints for FISH
18
Fluorescence in situ hybridization (FISH)
metaphase chromosome painting
19
  • Chromosome maintenance
  • Origins of replication
  • Telomeres
  • Centromeres

20
Origins of replication
  • Multiple origins
  • -every 100 kb on average in humans
  • Heterochromatin is late replicating
  • Replication times correspond to banding patterns
  • Each band replicated independently

From Miller Therman (2001) Human Chromosomes,
Springer
21
Telomeres
  • End structures of linear chromosomes
  • Serve to replicate chromosome ends
  • Serve to stabilize chromosome ends (i.e. prevent
    non-homologous end joining, NHEJ)
  • G-rich tandem repeats
  • - TTAGG, insects
  • - TTAGGG, vertebrates
  • - TTTAGGG, plants
  • Length is under genetic and developmental control
  • - e.g. 2-5 kb in Arabidopsis, 60-160 kb in
    Tobacco, 15 kb in humans
  • Sequence and proteins conserved across taxa,
    mammals to plants

22
FISH with a telomere-specific probe
23
Telomeres telomerase in the replication of
linear chromosome ends
24
Telomerase
  • Reverse transcriptase RNA primer
  • Repeating cycles of parental strand extension
  • - build template for lagging strand replication
  • - build up the number of telomeres
  • Abundant in mammalian embryos, stem cells and
    cancer cells
  • Absent in mammalian somatic cells
  • - telomeres shorten with each cell division
  • - cells cease division and begin senescence
  • Abundant in rapidly dividing and germ-line cells
    of plants
  • Absent in vegetative tissues of plants

25
Centromeres
  • Primary constriction
  • Kinetochore - spindle fiber attachment
  • Region of sister chromatid cohesion
  • Constitutive heterochromatin
  • Repeat sequences - CENs - 5 to 170 bp
  • e.g. human alphoid satellite repeat
  • No universal centromere repeat, but the same
    repeat can be found in more than one centromere
    of a species or between species
  • Centromere repeats can change rapidly in
    evolution via mutation, new elements, recruitment
    of other genomic repeats
  • Specific associated proteins
  • e.g. Centromere-specific histone HE (CenH3)

26
A model of centromere structure
27
Chromatin structure
Compacts DNA 10,000 X
From Miller Therman (2001) Human Chromosomes,
Springer
28
Chromatin structure
  • 11 nm fiber
  • Nucleosomes
  • 147 bp DNA wound on histone core
  • - Histones H3, H4, H2A, H2B (2 each)
  • Internucleosomal spacer
  • - 60 bp linker DNA
  • 30 nm fiber
  • Histone H1 (linker) binds and compacts
    nucleosomes
  • Exact structure is controversial
  • - Solenoid single helix coiling of 11 nm fiber
  • - Zig-zag stacking of nucleosomes then coiling
    double helix of 11 nm fiber

From Woodcock (2006) Curr Opin Struct Biol 16213
29
Chromatin structure
  • 300 nm fiber
  • Loops of 30 nm fibers
  • Attached to protein scaffold
  • Attachment points correspond to boundary
    elements, isolating regions of differential gene
    expression
  • Metaphase chromatin
  • Coiling of the 300 nm fiber

30
Chromatin structure histone modifications
  • Post-translational modifications on histone
    proteins
  • Establish global chromatin structure
  • -heterochromatin vs euchromatin
  • Regulate DNA-based functions
  • - Transcription
  • - Replication, recombination repair
  • Complex interactions
  • - Not really a simple histone code
  • - The truth is likely to be that any given
    modification has the potential to activate or
    repress under different conditions.

From Kouzarides (2007) Cell 128693
31
Chromatin structure histone modifications
Post-translational modifications on histone
proteins alter chromatin structure and,
consequently, chromatin function
Table 1. Different Classes of Modifications
Identified on Histones
Overview of different classes of modification
identified on histones. The functions that have
been associated with each modification are shown.
Each modification is discussed in detail in the
text under the heading of the function it
regulates. From Kouzarides (2007) Cell 128693
32
Chromatin structure histone modifications
Post-translational modifications on histone
proteins alter chromatin structure and,
consequently, chromatin function
Figure 1. Recruitment of Proteins to Histones (A)
Domains used for the recognition of methylated
lysines, acetylated lysines, or phosphorylated
serines. (B) Proteins found that associate
preferentially with modified versions of histone
H3 and histone H4. From Kouzarides (2007) Cell
128693
33
Chromatin structure histone modifications
  • Post-translational modifications on histone
    proteins
  • The truth is likely to be that any given
    modification has the potential to activate or
    repress under different conditions.
  • Histone acetylation
  • - generally associated with activation of
    transcription
  • Histone de-acetylation
  • - generally associated with repression of
    transcription
  • - Histone de-acetylase targeted to methylated CpG
    islands

Kouzarides (2007) Cell 128693
34
Chromatin structure histone modifications
  • Post-translational modifications on histone
    proteins
  • The truth is likely to be that any given
    modification has the potential to activate or
    repress under different conditions.
  • Lysine methyation associated with activation of
    transcription H3K4, H3K36, H3K79
  • Lysine methyation associated with repression of
    transcription H3K9, H3K27, H4K20

Kouzarides (2007) Cell 128693
35
Chromatin structure functional consequences of
histone modifications
Figure 3. Functional Consequences of Histone
Modifications (A) Gene-expression changes are
brought about by the recruitment of the NURF
complex, which contains a component BRTF
recognizing H3K4me and a component-remodeling
chromatin. (B) The Crb2 protein of fission yeast
is recruited to DNA-repair foci during a
DNA-repair response. Crb2 is partly tethered
there by association with methylated H4 and
phosphorylated H2A. (C) The HBO1
acetyltransferase is an ING5-associated factor
and is therefore tethered to sites of replication
via methylated H3K4. HBO1 also binds to the MCM
proteins found at replication sites. Evidence
exists that HBO1 augments the formation of the
preinitiation complex and is required for DNA
replication. From Kouzarides (2007) Cell 128693
36
Nuclear architecture Chromosome territories
aAll the chromosome territories that make up the
human genome can be visualized simultaneously in
intact interphase nuclei, each in a different
colour. a A red, green and blue image of the 24
labelled chromosomes (122, X and Y) was produced
from deconvoluted mid-plane nuclear sections from
a three-dimensional stack by superposition of the
7 colour channels. b As in 24-colour
KARYOTYPING, each chromosome can be identified by
using a combination labelling scheme in which
each chromosome is labelled with a different set
of fluorochromes. In this way, each chromosome
territory can be automatically classified using
appropriate software, which assigns the
corresponding chromosome number to a territory.
If a stack of these images is collected
throughout the nucleus, a simultaneous
three-dimensional reconstruction of all
chromosome territories is possible. Some of the
dark regions represent unstained nucleoli. For
further details see Ref. 90. From Speicher
Carter (2005) Nature Rev Genet 6782
37
Nuclear architecture Chromosome territories
  • Nonrandom chromosome positioning
  • Gene rich chromosomes toward center
  • Gene poor chromosomes toward periphery
  • Centromeres are not the determining factor
  • Chromosomes with adjacent positions more likely
    to interact cytolologically

38
Nuclear architecture consequences of chromosome
territories
Figure 3. Functional Consequences of Global
Chromatin Organization (A and B) Spatial
clustering of genes on distinct chromosomes
facilitates their expression by (A) association
with shared transcription and processing sites or
(B) physical interactions with regulatory
elements on separate chromosomes. (C) The
physical proximity of chromosomes contributes to
the probability of chromosomal translocations.
From Misteli (2007) Cell 128787
39
Nuclear architecture Nuclear factories
Figure 1. Compartmentalization of Nuclear
Processes Transcription, replication, and DNA
repair are compartmentalized. (A) Transcription
sites visualized by incorporation of bromo-UTP,
(B) replication sites visualized by incorporation
of bromo-dUTP, and (C) repair sites visualized by
accumulation of repair factor 53BP1 at a
double-strand break (DSB) are shown. In all
cases, components are dynamically recruited from
the nucleoplasm as single subunits or small
preassembled subcomplexes.
(A) is reprinted with permission from Elbi et
al., 2002, (B) is courtesy of Rong Wu and David
Gilbert at Florida State University, and (C) is
courtesy of Evi Soutoglou from the National
Cancer Institute, NIH. From Misteli (2007) Cell
128787
40
Model of functional nuclear architecture
Figure 3. Structural features that support the
chromosome-territoryinterchromatin-compartment
(CTIC) model are shown. These features are drawn
roughly to scale on an optical section taken from
the nucleus of a living HeLa cell. Although
experimental evidence is available to support
these features, the overall model of functional
nuclear architecture is speculative (see text).
From Cremer Cremer (2001) Nature Rev Genet
2292
41
Model of functional nuclear architecture
Figure 3. Structural features that support the
chromosome-territoryinterchromatin-compartment
(CTIC) model are shown. These features are drawn
roughly to scale on an optical section taken from
the nucleus of a living HeLa cell. Although
experimental evidence is available to support
these features, the overall model of functional
nuclear architecture is speculative (see text). a
CTs have complex folded surfaces. Inset
topological model of gene regulation23. A giant
chromatin loop with several active genes (red)
expands from the CT surface into the IC space. b
CTs contain separate arm domains for the short
(p) and long chromosome arms (q), and a
centromeric domain (asterisks). Inset
topological model of gene regulation78, 79. Top,
actively transcribed genes (white) are located on
a chromatin loop that is remote from centromeric
heterochromatin. Bottom, recruitment of the same
genes (black) to the centromeric heterochromatin
leads to their silencing. c CTs have variable
chromatin density (dark brown, high density
light yellow, low density). Loose chromatin
expands into the IC, whereas the most dense
chromatin is remote from the IC. d CT showing
early-replicating chromatin domains (green) and
mid-to-late-replicating chromatin domains (red).
Each domain comprises 1 Mb. Gene-poor chromatin
(red), is preferentially located at the nuclear
periphery and in close contact with the nuclear
lamina (yellow), as well as with infoldings of
the lamina and around the nucleolus (nu).
Gene-rich chromatin (green) is located between
the gene-poor compartments. e Higher-order
chromatin structures built up from a hierarchy of
chromatin fibres88. Inset this topological view
of gene regulation27, 68 indicates that active
genes (white dots) are at the surface of
convoluted chromatin fibres. Silenced genes
(black dots) may be located towards the interior
of the chromatin structure. f The CTIC model
predicts that the IC (green) contains complexes
(orange dots) and larger non-chromatin domains
(aggregations of orange dots) for transcription,
splicing, DNA replication and repair. g CT with
1-Mb chromatin domains (red) and IC (green)
expanding between these domains. Inset the
topological relationships between the IC, and
active and inactive genes72. The finest branches
of the IC end between 100-kb chromatin domains.
Top active genes (white dots) are located at the
surface of these domains, whereas silenced genes
(black dots) are located in the interior. Bottom
alternatively, closed 100-kb chromatin domains
with silenced genes are transformed into an open
configuration before transcriptional activation.
From Cremer Cremer (2001) Nature Rev Genet
2292
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