Chapter 19
Nucleosomes
19.1 Introduction
19.2 The nucleosome is the subunit of all chromatin
19.3 DNA is coiled in arrays of nucleosomes
19.4 Nucleosomes have a common structure
19.5 DNA structure varies on the nucleosomal surface
19.6 Supercoiling and the periodicity of DNA
19.7 The path of nucleosomes in the chromatin fiber
19.8 Organization of the histone octamer
19.9 Histones are modified
19.10 Reproduction of chromatin requires assembly of nucleosomes
19.11 Do nucleosomes lie at specific positions?
19.12 Are transcribed genes organized in nucleosomes?
19.13 Histone octamers are displaced by transcription
19.14 DNAase hypersensitive sites change chromatin structure
19.15 Domains define regions that contain active genes
19.16 Heterochromatin propagates from a nucleation event
19.17 Heterochromatin depends on interactions with histones
19.18 X chromosomes undergo global changes
19.19 Chromosome condensation is caused by condensins
19.20 Methylation is perpetuated by a maintenance methylase
19.21 Methylation is responsible for imprinting
19.22 Epigenetic effects can be inherited
19.23 Yeast prions show unusual inheritance
19.24 Prions cause diseases in mammals
Histones are conserved DNA-binding
proteins of eukaryotes that form the
nucleosome,the basic subunit of chromatin.
Nucleosome is the basic structural subunit of
chromatin,consisting of ~200 bp of DNA
and an octamer of histone proteins.
19.1 Introduction
Figure 18.9 The sister
chromatids of a mitotic pair each
consist of a fiber (~30 nm in
diameter) compactly folded into
the chromosome,Photograph
kindly provided by E,J,DuPraw.
19.1 Introduction
Micrococcal nuclease is an
endonuclease that cleaves DNA; in
chromatin,DNA is cleaved
preferentially between nucleosomes.
19.2 The nucleosome is the
subunit of all chromatin
Figure 19.1 Chromatin
spilling out of lysed nuclei
consists of a compactly
organized series of particles,
The bar is 100 nm,
Photograph kindly provided
by Pierre Chambon.
19.2 The nucleosome
is the subunit of all
chromatin
Figure 19.2
Individual
nucleosomes are
released by
digestion of
chromatin with
micrococcal
nuclease,The bar is
100 nm,Photograph
kindly provided by
Pierre Chambon.
19.2 The nucleosome is the
subunit of all chromatin
Figure 19.3 The
nucleosome consists of
approximately equal
masses of DNA and
histones (including H1),
The predicted mass of the
nucleosome is 262 kD.
19.2 The nucleosome is the
subunit of all chromatin
Figure 19.4 The
nucleosome may be
a cylinder with
DNA organized
into two turns
around the surface.
19.2 The nucleosome is the
subunit of all chromatin
Figure 19.5 The
two turns of DNA
on the nucleosome
lie close together.
19.2 The
nucleosome
is the subunit
of all
chromatin
Figure 19.6
Sequences
on the DNA
that lie on
different
turns around
the
nucleosome
may be close
together.
19.2 The nucleosome is the
subunit of all chromatin
Core DNA is the 146 bp of DNA contained on a core
particle.
Core particle is a digestion product of the nucleosome
that retains the histone octamer and has 146 bp of
DNA; its structure appears similar to that of the
nucleosome itself.
Linker DNA is all DNA contained on a nucleosome in
excess of the 146 bp core DNA.
19.3 DNA is coiled in arrays of nucleosomes
Figure 19.7 Micrococcal nuclease
digests chromatin in nuclei into a
multimeric series of DNA bands
that can be separated by gel
electrophoresis,Photograph kindly
provided by Markus Noll.
19.3 DNA is coiled in
arrays of nucleosomes
Figure 19.8 Each multimer of
nucleosomes contains the
appropriate number of unit
lengths of DNA,Photograph
kindly provided by John Finch.
19.3 DNA is coiled in
arrays of nucleosomes
Figure 19.9 Micrococcal
nuclease reduces the length
of nucleosome monomers in
discrete steps,Photograph
kindly provided by Roger
Kornberg.
19.3 DNA is coiled in arrays of nucleosomes
Figure 19.10 Microccocal nuclease initially cleaves between
nucleosomes,Mononucleosomes typically have ~200 bp DNA,
End-trimming reduces the length of DNA first to ~165 bp,and
then generates core particles with 146 bp.
19.3 DNA is coiled in arrays of nucleosomes
Figure 19.4 The
nucleosome may be
a cylinder with
DNA organized
into two turns
around the surface.
19.3 DNA is coiled in arrays of nucleosomes
Figure 19.11 Nicks in double-stranded DNA are revealed by
fragments when the DNA is denatured to give single strands,If
the DNA is labeled at (say) 5 ends,only the 5 fragments
are visible by autoradiography,The size of the fragment
identifies the distance of the nick from the labeled end,
19.4 DNA structure varies
on the nucleosomal surface
Figure 2.4 When
restriction fragments are
identified by their
possession of a labeled
end,each fragment
directly shows the
distance of a cutting site
from the end,
Successive fragments
increase in length by the
distance between
adjacent restriction sites.
19.4 DNA structure varies
on the nucleosomal surface
Figure 19.12 Sites for nicking lie at
regular intervals along core DNA,as
seen in a DNAase I digest of nuclei,
Photograph kindly provided by
Leonard Lutter.
19.4 DNA structure varies
on the nucleosomal surface
Figure 19.13 Two
numbering schemes
divide core particle
DNA into 10 bp
segments,Sites may
be numbered S1 to
S13 from one end; or
taking S7 to identify
coordinate 0 of the
dyad symmetry,they
may be numbered -7
to +7.
19.4 DNA structure varies
on the nucleosomal surface
Figure 19.4 The
nucleosome may
be a cylinder with
DNA organized
into two turns
around the
surface.
19.4 DNA structure varies
on the nucleosomal surface
Figure 19.14 The most exposed positions on DNA recur
with a periodicity that reflects the structure of the double
helix,(For clarity,sites are shown for only one strand.)
19.4 DNA structure varies
on the nucleosomal surface
Figure 19.15 High resolution analysis
shows that each site for DNAase I
consists of several adjacent susceptible
phosphodiester bonds as seen in this
example of sites S4 and S5 analyzed in
end-labeled core particles,Photograph
kindly provided by Leonard Lutter.
19.4 DNA structure varies
on the nucleosomal surface
Linking number paradox describes the
discrepancy between the existence of -2
supercoils in the path of DNA on the nucleosome
compared with the measurement of -1 supercoil
released when histones are removed.
Minichromosome of SV40 or polyoma is the
nucleosomal form of the viral circular DNA.
19.5 Supercoiling and the periodicity of DNA
Figure 19.16 The supercoils
of the SV40 minichromosome
can be relaxed to generate a
circular structure,whose loss
of histones then generates
supercoils in the free DNA.
19.5 Supercoiling and the periodicity of DNA
Figure 19.4
The
nucleosome
may be a
cylinder with
DNA
organized
into two turns
around the
surface.
19.5 Supercoiling and the periodicity of DNA
Figure 19.17 The 10
nm fiber in partially
unwound state can be
seen to consist of a
string of nucleosomes,
Photograph kindly
provided by Barbara
Hamkalo.
19.6 The path of
nucleosomes in
the chromatin
fiber
Figure 19.18 The 10
nm fiber is a
continuous strong of
nucleosomes.
19.6 The path of nucleosomes in the chromatin fiber
Figure 19.19 The 30 nm
fiber has a coiled structure,
Photograph kindly provided
by Barbara Hamkalo.
19.6 The path of
nucleosomes in the
chromatin fiber
Figure 19.20 The
30 nm fiber may
have a helical coil
of 6 nucleosomes
per turn,organized
radially.
19.6 The path of nucleosomes in the chromatin fiber
Figure 19.21 In a
symmetrical model
for the nucleosome,
the H32- H42
tetramer provides a
kernel for the shape,
One H2A-H2B
dimer can be seen
in the top view; the
other is underneath.
19.7 Organization of the histone octamer
Figure 19.22 The crystal structure of the
histone core octamer is represented in a
space-filling model with the H32-H42
tetramer shown in white and the H2A-H2B
dimers shown in blue,Only one of the
H2A-H2B dimers is visible in the top view,
because the other is hidden underneath,The
potential path of the DNA is shown in the
top view as a narrow tube (one quarter the
diameter of DNA),and in the side view by
the parallel lines in a 20? wide bundle,
Photographs kindly provided by Evangelos
Moudrianakis.
19.7 Organization of the histone octamer
Figure 19.4
The
nucleosome
may be a
cylinder with
DNA
organized into
two turns
around the
surface.
19.7 Organization of the histone octamer
Figure 19.23 Histone positions
in a top view show H3-H4 and
H2A-H2B pairs in a half
nucleosome; the symmetrical
organization can be seen in the
superimposition of both
halves.
19.7 Organization of
the histone octamer
Figure 19.24 The
globular bodies of the
histones are localized
in the histone octamer
of the core particle,
but the locations of
the N-terminal tails,
which carry the sites
for modification,are
not known,and could
be more flexible.
19.7 Organization of the histone octamer
Figure 19.25 Acetylation of
lysine or phosphorylation of
serine reduces the overall
positive charge of a protein.
19.7 Organization
of the histone
octamer
Figure 19.26
Replicated
DNA is
immediately
incorporated
into
nucleosomes,
Photograph
kindly
provided by S,
MacKnight.
19.8 Reproduction of chromatin
requires assembly of nucleosomes
Figure 19.27 In vitro,DNA can
either interact directly with an
intact (crosslinked) histone
octamer or can assemble with
the H32-H42 tetramer,after
which two H2A-H2B dimers
are added.
19.8 Reproduction of
chromatin requires
assembly of nucleosomes
Figure 19.28 If histone octamers
were conserved,old and new
octamers would band at
different densities when
replication of heavy octamers
occurs in light amino acids (part
1); but actually the octamers
band diffusely between heavy
and light densities,suggesting
disassembly and reassembly
(part 2).
19.8 Reproduction of chromatin
requires assembly of nucleosomes
Figure 19.29 Nucleosome
positioning places restriction
sites at unique positions relative
to the linker sites cleaved by
micrococcal nuclease.
19.9 Do nucleosomes lie
at specific positions?
Figure 19.30 In the absence of
nucleosome positioning,a
restriction site lies at all possible
locations in different copies of the
genome,Fragments of all possible
sizes are produced when a
restriction enzyme cuts at a target
site (red) and micrococcal
nuclease cuts at the junctions
between nucleosomes (green).
19.9 Do nucleosomes lie at specific positions?
Figure 19.31 Translational positioning describes the linear position of
DNA relative to the histone octamer,Displacement of the DNA by 10 bp
changes the sequences that are in the more exposed linker regions,but
does not alter which face of DNA is protected by the histone surface and
which is exposed to the exterior,DNA is really coiled around the
nucleosomes,and is shown in linear form only for convenience.
19.9 Do
nucleosomes
lie at specific
positions?
Figure 19.32 Rotational positioning
describes the exposure of DNA on
the surface of the nucleosome,Any
movement that differs from the
helical repeat (~10.2 bp/turn)
displaces DNA with reference to
the histone surface,Nucleotides on
the inside are more protected
against nucleases than nucleotides
on the outside.
19.9 Do nucleosomes lie
at specific positions?
Figure 19.33 The extended
axis of an rDNA
transcription unit alternates
with the only slightly less
extended non-transcribed
spacer,Photograph kindly
provided by Charles Laird.
19.10 Are transcribed genes organized in nucleosomes?
Figure 19.34
An SV40
minichromo
some can be
transcribed,
Photograph
kindly
provided by
Pierre
Chambon.
19.10 Are transcribed genes organized in nucleosomes?
Figure 19.35
RNA
polymerase is
comparable in
size to the
nucleosome and
might encounter
difficulties in
following the
DNA around the
histone octamer.
19.10 Are transcribed genes organized in nucleosomes?
Figure 19.36 A protocol to
test the effect of
transcription on
nucleosomes shows that
the histone octamer is
displaced from DNA and
rebinds at a new position.
19.10 Are transcribed
genes organized in
nucleosomes?
Figure 19.37 RNA polymerase
displaces DNA from the histone
octamer as it advances,The DNA
loops back and attaches (to
polymerase or to the octamer) to
form a closed loop,As the
polymerase proceeds,it generates
positive supercoiling ahead,This
displaces the octamer,which
keeps contact with DNA and/or
polymerase,and is inserted behind
the RNA polymerase.
19.10 Are transcribed
genes organized in
nucleosomes?
Figure 19.38 The URA3 gene
has translationally positioned
nucleosomes before
transcription,When
transcription is induced,
nucleosome positions are
randomized,When transcription
is repressed,the nucleosomes
resume their particular positions,
Photograph kindly provided by
Fritz Thoma.
19.10 Are transcribed genes organized in nucleosomes?
Figure 19.39 Indirect end-labeling
identifies the distance of a DNAase
hypersensitive site from a restriction
cleavage site,The existence of a
particular cutting site for DNAase I
generates a discrete fragment,whose
size indicates the distance of the
DNAase I hypersensitive site from
the restriction site.
19.11 DNAase hypersensitive
sites change chromatin
structure
Figure 19.40 The
SV40
minichromosome
has a nucleosome
gap,Photograph
kindly provided by
Moshe Yaniv.
19.11 DNAase hypersensitive sites
change chromatin structure
Figure 19.41 The
SV40 gap includes
hypersensitive
sites,sensitive
regions,and a
protected region
of DNA,The
hypersensitive site
of a chicken b-
globin gene
comprises a region
that is susceptible
to several
nucleases.
19.11 DNAase hypersensitive sites
change chromatin structure
Domain of a chromosome may refer either
to a discrete structural entity defined as a
region within which supercoiling is
independent of other domains; or to an
extensive region including an expressed
gene that has heightened sensitivity to
degradation by the enzyme DNAase I.
19.12 Domains define regions
that contain active genes
Figure 19.42 Sensitivity to
DNAase I can be measured by
determining the rate of
disappearance of the material
hybridizing with a particular probe.
19.12 Domains define regions
that contain active genes
Figure 19.43 In adult erythroid cells,the adult
b-globin gene is highly sensitive to DNAase I
digestion,the embryonic b-globin gene is
partially sensitive (probably due tIn adult
erythroid cells,the adult b-globin gene is
highly sensitive to DNAase I digestion,the
embryonic b-globin gene is partially sensitive
(probably due to spreading effects),but
ovalbumin is not sensitive,Data kindly
provided by Harold Weintraub.
19.12 Domains define regions
that contain active genes
Epigenetic changes influence the
phenotype without altering the genotype,
They consist of changes in the properties
of a cell that are inherited but that do not
represent a change in genetic information.
19.13 Heterochromatin depends
on interactions with histones
Figure 19.44 Position effect
variegation in eye color results
when the white gene is integrated
near heterochromatin,Cells in
which white is inactive give
patches of white eye,while cells in
which white is active give red
paPosition effect variegation in eye
color results when the white gene is
integrated near heterochromatin,
Cells in which white is inactive
give patches of white eye,while
cells in which white is active give
red patches,The severity of the
effect is determined by the
closeness of the integrated gene to
heterochromatin,Photograph
kindly provided by Steve Henikoff.
19.13 Heterochromatin depends
on interactions with histones
Figure 19.45 Extension
of heterochromatin
inactivates genes,The
probability that a gene
will be inactivated
depends on its distance
from the
heterochromatin region.
19.13 Heterochromatin depends
on interactions with histones
Figure 19.46 Formation of
heterochromatin is initiated
when RAP1 binds to DNA,
SIR3/4 bind to RAP1 and also to
histones H3/H4,The complex
polymerizes along chromatin
and may connect telomeres to
the nuclear matrix.
19.13 Heterochromatin
depends on interactions
with histones
Constitutive heterochromatin describes the inert state of permanently
nonexpressed sequences,usually satellite DNA.
Dosage compensation describes mechanisms employed to compensate
for the discrepancy between the presence of two X chromosomes in
one sex but only one X chromosome in the other sex.
Facultative heterochromatin describes the inert state of sequences that
also exist in active copies-for example,one mammalian X
chromosome in females.
Single X hypothesis describes the inactivation of one X chromosome
in female mammals.
19.14 Global changes in X chromosomes
Figure 19.47
Different
means of
dosage
compensation
are used to
equalize X
chromosome
expression in
male and
female.
19.14 Global changes in X chromosomes
Figure 19.48 X-linked
variegation is caused by the
random inactivation of one
X chromosome in each
precursor cell,Cells in
which the + allele is on the
active chromosome have
wild phenotype; but cells in
which the - allele is on the
active chromosome have
mutant phenotype.
19.14 Global changes
in X chromosomes
Figure 19.49 X-
inactivation involves
stabilization of XIST
RNA,which coats the
inactive chromosome.
19.14 Global
changes in X
chromosomes
Imprinting describes a change in a gene
that occurs during passage through the
sperm or egg with the result that the
paternal and maternal alleles have
different properties in the very early
embryo,May be caused by methylation
of DNA.
19.15 Methylation is responsible for imprinting
Figure 19.50 The state
of methylated sites
could be perpetuated
by an enzyme that
recognizes only
hemimethylated sites
as substrates.
19.15 Methylation is
responsible for imprinting
Figure 19.51 The state of
methylation is controlled
by three enzymes.
19.15 Methylation
is responsible for
imprinting
Figure 19.52 The parental
alleles of Igf2 are
differentially methylated
in the early embryo,but
the patterns of
methylation are reset
when gametes are formed
by the adult.
19.15 Methylation
is responsible for
imprinting
Prion is a proteinaceous infectious agent,
which behaves as an inheritable trait,
although it contains no nucleic acid,
Examples are PrPSc,the agent of scrapie
in sheep and bovine spongiform
encephalopathy,and Psi,which confers an
inherited state in yeast.
19.16 Epigenetic effects can be inherited
Figure 19.50 The state of
methylated sites could be
perpetuated by an enzyme
that recognizes only
hemimethylated sites as
substrates.
19.16 Epigenetic
effects can be
inherited
Figure 19.53 What
happens to protein
complexes on
chromatin during
replication?
19.16 Epigenetic
effects can be
inherited
Figure 19.45
Extension of
heterochromatin
inactivates
genes,The
probability that
a gene will be
inactivated
depends on its
distance from
the
heterochromatin
region.
19.16 Epigenetic effects can be inherited
Figure 19.54 Acetylated
cores are conserved and
distributed at random to
the daughter chromatin
fibers at replication,Each
daughter fiber has a
mixture of old (acetylated)
cores and new
(unacetylated) cores.
19.16 Epigenetic effects can be inherited
Figure 19.55 The
state of the Sup35
protein determines
whether termination
of translation occurs.
19.17 Yeast prions show
unusual inheritance
Figure 19.56 Newly
synthesized Sup35
protein is converted into
the [PSI+] state by the
presence of pre-existing
[PSI+] protein.
19.17 Yeast prions show
unusual inheritance
Figure 19.57 Purified
protein can convert
the[psi-] state of yeast
to [PSI+].
19.17 Yeast prions show
unusual inheritance
Scrapie is a infective
agent made of protein.
19.18 Prions cause diseases in mammals
Figure 19.58
A PrpSc
protein can
only infect
an animal
that has the
same type of
endogenous
PrPC protein.
19.18 Prions cause diseases in mammals
1,All eukaryotic chromatin consists of nucleosomes.
2,The path of DNA around the histone octamer creates
3,Nucleosomes are organized into a fiber of 30 nm diameter
which has 6 nucleosomes per turn and a packing ratio of 40,
4,RNA polymerase displaces histone octamers during
transcription,
5,Two types of changes in sensitivity to nucleases are
associated with gene activity,
6,Formation of heterochromatin may be initiated at certain
sites and then propagated for a distance that is not precisely
determined.
19.19 Summary
7,Inactive chromatin at yeast telomeres and silent mating
type loci appears to have a common cause,and involves the
interaction of certain proteins with the N-terminal tails of
histones H3 and H4.
8,Inactivation of one X chromosome in female (eutherian)
mammals occurs at random,
9,Methylation of DNA is inherited epigenetically.
10,Prions are proteinaceous infectious agents that are
responsible for the disease ofscrapie in sheep and for related
diseases in man.
19.19 Summary
Nucleosomes
19.1 Introduction
19.2 The nucleosome is the subunit of all chromatin
19.3 DNA is coiled in arrays of nucleosomes
19.4 Nucleosomes have a common structure
19.5 DNA structure varies on the nucleosomal surface
19.6 Supercoiling and the periodicity of DNA
19.7 The path of nucleosomes in the chromatin fiber
19.8 Organization of the histone octamer
19.9 Histones are modified
19.10 Reproduction of chromatin requires assembly of nucleosomes
19.11 Do nucleosomes lie at specific positions?
19.12 Are transcribed genes organized in nucleosomes?
19.13 Histone octamers are displaced by transcription
19.14 DNAase hypersensitive sites change chromatin structure
19.15 Domains define regions that contain active genes
19.16 Heterochromatin propagates from a nucleation event
19.17 Heterochromatin depends on interactions with histones
19.18 X chromosomes undergo global changes
19.19 Chromosome condensation is caused by condensins
19.20 Methylation is perpetuated by a maintenance methylase
19.21 Methylation is responsible for imprinting
19.22 Epigenetic effects can be inherited
19.23 Yeast prions show unusual inheritance
19.24 Prions cause diseases in mammals
Histones are conserved DNA-binding
proteins of eukaryotes that form the
nucleosome,the basic subunit of chromatin.
Nucleosome is the basic structural subunit of
chromatin,consisting of ~200 bp of DNA
and an octamer of histone proteins.
19.1 Introduction
Figure 18.9 The sister
chromatids of a mitotic pair each
consist of a fiber (~30 nm in
diameter) compactly folded into
the chromosome,Photograph
kindly provided by E,J,DuPraw.
19.1 Introduction
Micrococcal nuclease is an
endonuclease that cleaves DNA; in
chromatin,DNA is cleaved
preferentially between nucleosomes.
19.2 The nucleosome is the
subunit of all chromatin
Figure 19.1 Chromatin
spilling out of lysed nuclei
consists of a compactly
organized series of particles,
The bar is 100 nm,
Photograph kindly provided
by Pierre Chambon.
19.2 The nucleosome
is the subunit of all
chromatin
Figure 19.2
Individual
nucleosomes are
released by
digestion of
chromatin with
micrococcal
nuclease,The bar is
100 nm,Photograph
kindly provided by
Pierre Chambon.
19.2 The nucleosome is the
subunit of all chromatin
Figure 19.3 The
nucleosome consists of
approximately equal
masses of DNA and
histones (including H1),
The predicted mass of the
nucleosome is 262 kD.
19.2 The nucleosome is the
subunit of all chromatin
Figure 19.4 The
nucleosome may be
a cylinder with
DNA organized
into two turns
around the surface.
19.2 The nucleosome is the
subunit of all chromatin
Figure 19.5 The
two turns of DNA
on the nucleosome
lie close together.
19.2 The
nucleosome
is the subunit
of all
chromatin
Figure 19.6
Sequences
on the DNA
that lie on
different
turns around
the
nucleosome
may be close
together.
19.2 The nucleosome is the
subunit of all chromatin
Core DNA is the 146 bp of DNA contained on a core
particle.
Core particle is a digestion product of the nucleosome
that retains the histone octamer and has 146 bp of
DNA; its structure appears similar to that of the
nucleosome itself.
Linker DNA is all DNA contained on a nucleosome in
excess of the 146 bp core DNA.
19.3 DNA is coiled in arrays of nucleosomes
Figure 19.7 Micrococcal nuclease
digests chromatin in nuclei into a
multimeric series of DNA bands
that can be separated by gel
electrophoresis,Photograph kindly
provided by Markus Noll.
19.3 DNA is coiled in
arrays of nucleosomes
Figure 19.8 Each multimer of
nucleosomes contains the
appropriate number of unit
lengths of DNA,Photograph
kindly provided by John Finch.
19.3 DNA is coiled in
arrays of nucleosomes
Figure 19.9 Micrococcal
nuclease reduces the length
of nucleosome monomers in
discrete steps,Photograph
kindly provided by Roger
Kornberg.
19.3 DNA is coiled in arrays of nucleosomes
Figure 19.10 Microccocal nuclease initially cleaves between
nucleosomes,Mononucleosomes typically have ~200 bp DNA,
End-trimming reduces the length of DNA first to ~165 bp,and
then generates core particles with 146 bp.
19.3 DNA is coiled in arrays of nucleosomes
Figure 19.4 The
nucleosome may be
a cylinder with
DNA organized
into two turns
around the surface.
19.3 DNA is coiled in arrays of nucleosomes
Figure 19.11 Nicks in double-stranded DNA are revealed by
fragments when the DNA is denatured to give single strands,If
the DNA is labeled at (say) 5 ends,only the 5 fragments
are visible by autoradiography,The size of the fragment
identifies the distance of the nick from the labeled end,
19.4 DNA structure varies
on the nucleosomal surface
Figure 2.4 When
restriction fragments are
identified by their
possession of a labeled
end,each fragment
directly shows the
distance of a cutting site
from the end,
Successive fragments
increase in length by the
distance between
adjacent restriction sites.
19.4 DNA structure varies
on the nucleosomal surface
Figure 19.12 Sites for nicking lie at
regular intervals along core DNA,as
seen in a DNAase I digest of nuclei,
Photograph kindly provided by
Leonard Lutter.
19.4 DNA structure varies
on the nucleosomal surface
Figure 19.13 Two
numbering schemes
divide core particle
DNA into 10 bp
segments,Sites may
be numbered S1 to
S13 from one end; or
taking S7 to identify
coordinate 0 of the
dyad symmetry,they
may be numbered -7
to +7.
19.4 DNA structure varies
on the nucleosomal surface
Figure 19.4 The
nucleosome may
be a cylinder with
DNA organized
into two turns
around the
surface.
19.4 DNA structure varies
on the nucleosomal surface
Figure 19.14 The most exposed positions on DNA recur
with a periodicity that reflects the structure of the double
helix,(For clarity,sites are shown for only one strand.)
19.4 DNA structure varies
on the nucleosomal surface
Figure 19.15 High resolution analysis
shows that each site for DNAase I
consists of several adjacent susceptible
phosphodiester bonds as seen in this
example of sites S4 and S5 analyzed in
end-labeled core particles,Photograph
kindly provided by Leonard Lutter.
19.4 DNA structure varies
on the nucleosomal surface
Linking number paradox describes the
discrepancy between the existence of -2
supercoils in the path of DNA on the nucleosome
compared with the measurement of -1 supercoil
released when histones are removed.
Minichromosome of SV40 or polyoma is the
nucleosomal form of the viral circular DNA.
19.5 Supercoiling and the periodicity of DNA
Figure 19.16 The supercoils
of the SV40 minichromosome
can be relaxed to generate a
circular structure,whose loss
of histones then generates
supercoils in the free DNA.
19.5 Supercoiling and the periodicity of DNA
Figure 19.4
The
nucleosome
may be a
cylinder with
DNA
organized
into two turns
around the
surface.
19.5 Supercoiling and the periodicity of DNA
Figure 19.17 The 10
nm fiber in partially
unwound state can be
seen to consist of a
string of nucleosomes,
Photograph kindly
provided by Barbara
Hamkalo.
19.6 The path of
nucleosomes in
the chromatin
fiber
Figure 19.18 The 10
nm fiber is a
continuous strong of
nucleosomes.
19.6 The path of nucleosomes in the chromatin fiber
Figure 19.19 The 30 nm
fiber has a coiled structure,
Photograph kindly provided
by Barbara Hamkalo.
19.6 The path of
nucleosomes in the
chromatin fiber
Figure 19.20 The
30 nm fiber may
have a helical coil
of 6 nucleosomes
per turn,organized
radially.
19.6 The path of nucleosomes in the chromatin fiber
Figure 19.21 In a
symmetrical model
for the nucleosome,
the H32- H42
tetramer provides a
kernel for the shape,
One H2A-H2B
dimer can be seen
in the top view; the
other is underneath.
19.7 Organization of the histone octamer
Figure 19.22 The crystal structure of the
histone core octamer is represented in a
space-filling model with the H32-H42
tetramer shown in white and the H2A-H2B
dimers shown in blue,Only one of the
H2A-H2B dimers is visible in the top view,
because the other is hidden underneath,The
potential path of the DNA is shown in the
top view as a narrow tube (one quarter the
diameter of DNA),and in the side view by
the parallel lines in a 20? wide bundle,
Photographs kindly provided by Evangelos
Moudrianakis.
19.7 Organization of the histone octamer
Figure 19.4
The
nucleosome
may be a
cylinder with
DNA
organized into
two turns
around the
surface.
19.7 Organization of the histone octamer
Figure 19.23 Histone positions
in a top view show H3-H4 and
H2A-H2B pairs in a half
nucleosome; the symmetrical
organization can be seen in the
superimposition of both
halves.
19.7 Organization of
the histone octamer
Figure 19.24 The
globular bodies of the
histones are localized
in the histone octamer
of the core particle,
but the locations of
the N-terminal tails,
which carry the sites
for modification,are
not known,and could
be more flexible.
19.7 Organization of the histone octamer
Figure 19.25 Acetylation of
lysine or phosphorylation of
serine reduces the overall
positive charge of a protein.
19.7 Organization
of the histone
octamer
Figure 19.26
Replicated
DNA is
immediately
incorporated
into
nucleosomes,
Photograph
kindly
provided by S,
MacKnight.
19.8 Reproduction of chromatin
requires assembly of nucleosomes
Figure 19.27 In vitro,DNA can
either interact directly with an
intact (crosslinked) histone
octamer or can assemble with
the H32-H42 tetramer,after
which two H2A-H2B dimers
are added.
19.8 Reproduction of
chromatin requires
assembly of nucleosomes
Figure 19.28 If histone octamers
were conserved,old and new
octamers would band at
different densities when
replication of heavy octamers
occurs in light amino acids (part
1); but actually the octamers
band diffusely between heavy
and light densities,suggesting
disassembly and reassembly
(part 2).
19.8 Reproduction of chromatin
requires assembly of nucleosomes
Figure 19.29 Nucleosome
positioning places restriction
sites at unique positions relative
to the linker sites cleaved by
micrococcal nuclease.
19.9 Do nucleosomes lie
at specific positions?
Figure 19.30 In the absence of
nucleosome positioning,a
restriction site lies at all possible
locations in different copies of the
genome,Fragments of all possible
sizes are produced when a
restriction enzyme cuts at a target
site (red) and micrococcal
nuclease cuts at the junctions
between nucleosomes (green).
19.9 Do nucleosomes lie at specific positions?
Figure 19.31 Translational positioning describes the linear position of
DNA relative to the histone octamer,Displacement of the DNA by 10 bp
changes the sequences that are in the more exposed linker regions,but
does not alter which face of DNA is protected by the histone surface and
which is exposed to the exterior,DNA is really coiled around the
nucleosomes,and is shown in linear form only for convenience.
19.9 Do
nucleosomes
lie at specific
positions?
Figure 19.32 Rotational positioning
describes the exposure of DNA on
the surface of the nucleosome,Any
movement that differs from the
helical repeat (~10.2 bp/turn)
displaces DNA with reference to
the histone surface,Nucleotides on
the inside are more protected
against nucleases than nucleotides
on the outside.
19.9 Do nucleosomes lie
at specific positions?
Figure 19.33 The extended
axis of an rDNA
transcription unit alternates
with the only slightly less
extended non-transcribed
spacer,Photograph kindly
provided by Charles Laird.
19.10 Are transcribed genes organized in nucleosomes?
Figure 19.34
An SV40
minichromo
some can be
transcribed,
Photograph
kindly
provided by
Pierre
Chambon.
19.10 Are transcribed genes organized in nucleosomes?
Figure 19.35
RNA
polymerase is
comparable in
size to the
nucleosome and
might encounter
difficulties in
following the
DNA around the
histone octamer.
19.10 Are transcribed genes organized in nucleosomes?
Figure 19.36 A protocol to
test the effect of
transcription on
nucleosomes shows that
the histone octamer is
displaced from DNA and
rebinds at a new position.
19.10 Are transcribed
genes organized in
nucleosomes?
Figure 19.37 RNA polymerase
displaces DNA from the histone
octamer as it advances,The DNA
loops back and attaches (to
polymerase or to the octamer) to
form a closed loop,As the
polymerase proceeds,it generates
positive supercoiling ahead,This
displaces the octamer,which
keeps contact with DNA and/or
polymerase,and is inserted behind
the RNA polymerase.
19.10 Are transcribed
genes organized in
nucleosomes?
Figure 19.38 The URA3 gene
has translationally positioned
nucleosomes before
transcription,When
transcription is induced,
nucleosome positions are
randomized,When transcription
is repressed,the nucleosomes
resume their particular positions,
Photograph kindly provided by
Fritz Thoma.
19.10 Are transcribed genes organized in nucleosomes?
Figure 19.39 Indirect end-labeling
identifies the distance of a DNAase
hypersensitive site from a restriction
cleavage site,The existence of a
particular cutting site for DNAase I
generates a discrete fragment,whose
size indicates the distance of the
DNAase I hypersensitive site from
the restriction site.
19.11 DNAase hypersensitive
sites change chromatin
structure
Figure 19.40 The
SV40
minichromosome
has a nucleosome
gap,Photograph
kindly provided by
Moshe Yaniv.
19.11 DNAase hypersensitive sites
change chromatin structure
Figure 19.41 The
SV40 gap includes
hypersensitive
sites,sensitive
regions,and a
protected region
of DNA,The
hypersensitive site
of a chicken b-
globin gene
comprises a region
that is susceptible
to several
nucleases.
19.11 DNAase hypersensitive sites
change chromatin structure
Domain of a chromosome may refer either
to a discrete structural entity defined as a
region within which supercoiling is
independent of other domains; or to an
extensive region including an expressed
gene that has heightened sensitivity to
degradation by the enzyme DNAase I.
19.12 Domains define regions
that contain active genes
Figure 19.42 Sensitivity to
DNAase I can be measured by
determining the rate of
disappearance of the material
hybridizing with a particular probe.
19.12 Domains define regions
that contain active genes
Figure 19.43 In adult erythroid cells,the adult
b-globin gene is highly sensitive to DNAase I
digestion,the embryonic b-globin gene is
partially sensitive (probably due tIn adult
erythroid cells,the adult b-globin gene is
highly sensitive to DNAase I digestion,the
embryonic b-globin gene is partially sensitive
(probably due to spreading effects),but
ovalbumin is not sensitive,Data kindly
provided by Harold Weintraub.
19.12 Domains define regions
that contain active genes
Epigenetic changes influence the
phenotype without altering the genotype,
They consist of changes in the properties
of a cell that are inherited but that do not
represent a change in genetic information.
19.13 Heterochromatin depends
on interactions with histones
Figure 19.44 Position effect
variegation in eye color results
when the white gene is integrated
near heterochromatin,Cells in
which white is inactive give
patches of white eye,while cells in
which white is active give red
paPosition effect variegation in eye
color results when the white gene is
integrated near heterochromatin,
Cells in which white is inactive
give patches of white eye,while
cells in which white is active give
red patches,The severity of the
effect is determined by the
closeness of the integrated gene to
heterochromatin,Photograph
kindly provided by Steve Henikoff.
19.13 Heterochromatin depends
on interactions with histones
Figure 19.45 Extension
of heterochromatin
inactivates genes,The
probability that a gene
will be inactivated
depends on its distance
from the
heterochromatin region.
19.13 Heterochromatin depends
on interactions with histones
Figure 19.46 Formation of
heterochromatin is initiated
when RAP1 binds to DNA,
SIR3/4 bind to RAP1 and also to
histones H3/H4,The complex
polymerizes along chromatin
and may connect telomeres to
the nuclear matrix.
19.13 Heterochromatin
depends on interactions
with histones
Constitutive heterochromatin describes the inert state of permanently
nonexpressed sequences,usually satellite DNA.
Dosage compensation describes mechanisms employed to compensate
for the discrepancy between the presence of two X chromosomes in
one sex but only one X chromosome in the other sex.
Facultative heterochromatin describes the inert state of sequences that
also exist in active copies-for example,one mammalian X
chromosome in females.
Single X hypothesis describes the inactivation of one X chromosome
in female mammals.
19.14 Global changes in X chromosomes
Figure 19.47
Different
means of
dosage
compensation
are used to
equalize X
chromosome
expression in
male and
female.
19.14 Global changes in X chromosomes
Figure 19.48 X-linked
variegation is caused by the
random inactivation of one
X chromosome in each
precursor cell,Cells in
which the + allele is on the
active chromosome have
wild phenotype; but cells in
which the - allele is on the
active chromosome have
mutant phenotype.
19.14 Global changes
in X chromosomes
Figure 19.49 X-
inactivation involves
stabilization of XIST
RNA,which coats the
inactive chromosome.
19.14 Global
changes in X
chromosomes
Imprinting describes a change in a gene
that occurs during passage through the
sperm or egg with the result that the
paternal and maternal alleles have
different properties in the very early
embryo,May be caused by methylation
of DNA.
19.15 Methylation is responsible for imprinting
Figure 19.50 The state
of methylated sites
could be perpetuated
by an enzyme that
recognizes only
hemimethylated sites
as substrates.
19.15 Methylation is
responsible for imprinting
Figure 19.51 The state of
methylation is controlled
by three enzymes.
19.15 Methylation
is responsible for
imprinting
Figure 19.52 The parental
alleles of Igf2 are
differentially methylated
in the early embryo,but
the patterns of
methylation are reset
when gametes are formed
by the adult.
19.15 Methylation
is responsible for
imprinting
Prion is a proteinaceous infectious agent,
which behaves as an inheritable trait,
although it contains no nucleic acid,
Examples are PrPSc,the agent of scrapie
in sheep and bovine spongiform
encephalopathy,and Psi,which confers an
inherited state in yeast.
19.16 Epigenetic effects can be inherited
Figure 19.50 The state of
methylated sites could be
perpetuated by an enzyme
that recognizes only
hemimethylated sites as
substrates.
19.16 Epigenetic
effects can be
inherited
Figure 19.53 What
happens to protein
complexes on
chromatin during
replication?
19.16 Epigenetic
effects can be
inherited
Figure 19.45
Extension of
heterochromatin
inactivates
genes,The
probability that
a gene will be
inactivated
depends on its
distance from
the
heterochromatin
region.
19.16 Epigenetic effects can be inherited
Figure 19.54 Acetylated
cores are conserved and
distributed at random to
the daughter chromatin
fibers at replication,Each
daughter fiber has a
mixture of old (acetylated)
cores and new
(unacetylated) cores.
19.16 Epigenetic effects can be inherited
Figure 19.55 The
state of the Sup35
protein determines
whether termination
of translation occurs.
19.17 Yeast prions show
unusual inheritance
Figure 19.56 Newly
synthesized Sup35
protein is converted into
the [PSI+] state by the
presence of pre-existing
[PSI+] protein.
19.17 Yeast prions show
unusual inheritance
Figure 19.57 Purified
protein can convert
the[psi-] state of yeast
to [PSI+].
19.17 Yeast prions show
unusual inheritance
Scrapie is a infective
agent made of protein.
19.18 Prions cause diseases in mammals
Figure 19.58
A PrpSc
protein can
only infect
an animal
that has the
same type of
endogenous
PrPC protein.
19.18 Prions cause diseases in mammals
1,All eukaryotic chromatin consists of nucleosomes.
2,The path of DNA around the histone octamer creates
3,Nucleosomes are organized into a fiber of 30 nm diameter
which has 6 nucleosomes per turn and a packing ratio of 40,
4,RNA polymerase displaces histone octamers during
transcription,
5,Two types of changes in sensitivity to nucleases are
associated with gene activity,
6,Formation of heterochromatin may be initiated at certain
sites and then propagated for a distance that is not precisely
determined.
19.19 Summary
7,Inactive chromatin at yeast telomeres and silent mating
type loci appears to have a common cause,and involves the
interaction of certain proteins with the N-terminal tails of
histones H3 and H4.
8,Inactivation of one X chromosome in female (eutherian)
mammals occurs at random,
9,Methylation of DNA is inherited epigenetically.
10,Prions are proteinaceous infectious agents that are
responsible for the disease ofscrapie in sheep and for related
diseases in man.
19.19 Summary
