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Chapter 9
Chromosomes
Jocelyn E. Krebs
Figure 09.CO: Microphotograph of living cells undergoing mitosis. Individual
chromosomes lined up at the metaphase plate or separating are visible in several cells.
© Dimarion/ShutterStock, Inc.
9.1 Introduction
nucleoid – The structure in a prokaryotic cell that contains the
genome.
The DNA is bound to proteins and is not enclosed by a
membrane.
chromatin – The state of nuclear DNA and its associated proteins.
• chromosome – A discrete unit of the genome carrying many
genes. Each consists of a very long molecule of duplex DNA
and an approximately equal mass of proteins.
– It is visible as a morphological entity only during cell
division.
– Tight packing (5-10 fold than chromatin)
Density of DNA—10 mg/ml (bacteria), 100 mg/ml
(eukaryotic), 500 mg/ml (T4 head)
Figure 09.01: The length of nucleic acid is much greater than the dimensions of the
surrounding compartment.
9.2 Viral Genomes Are Packaged into Their
Coats
• Packing mechanism
•
•
1) protein shells are assembled around nucleic acid
2) capsid forms DNA packing
• capsid – The external protein coat of a virus particle.
• The length of DNA that can be incorporated into a virus is
limited by the structure of the headshell.
• Nucleic acid within the headshell is extremely condensed.
Figure 09.02: A helical path for TMV
RNA is created by the stacking of
protein subunits in the virion.
TMV virus packing (capsid protein-RNA
associated)
1. Duplex hairpin in RNA (nucleation center)
2. Capsid proteins assembled the RNA
bidirections up to RNA end
Figure 09.02: A helical path for TMV RNA is created by the stacking of protein subunits
in the virion.
• Filamentous RNA
viruses condense the
RNA genome as they
assemble the headshell
around it.
• nucleation center – A
duplex hairpin in TMV
(tobacco mosaic virus)
in which assembly of
coat protein with RNA is
initiated.
9.2 Viral Genomes Are Packaged into Their
Coats-Lambda Phage
Head structure DNA packing
Head shell core
Two step: translocation and condensation
Translocation ATP-dependent mechanism
Rolling circle model
Figure 09.03: Maturation of phage
lambda passes through several stages.
Top photo reproduced from Cue, D. and Feiss, M., Proc. Natl. Acad. Sci.
USA 90 (1993): 9290-9294. Copyright 1993 National Academy of Science,
USA. Photo courtesy of Michael G. Feiss, University of Iowa. Bottom
photo courtesy of Robert Duda, University of Pittsburgh.
Figure 09.04: Terminase protein binds to specific sites on a multimer of virus genomes
generated by rolling circle replication.
• Spherical DNA viruses insert the
DNA into a preassembled protein
shell.
• terminase – An enzyme that
cleaves multimers of a viral
genome and then uses hydrolysis
of ATP to provide the energy to
translocate the DNA into an
empty viral capsid starting with
the cleaved end.
• Cos site
9.3 The Bacterial Genome Is a Supercoiled
Nucleoid
• The bacterial nucleoid is ~80% DNA by mass and can be unfolded by
agents that act on RNA or protein.
•  protein and RNA are essential for genome structure maintaining
• The proteins that are responsible for condensing the DNA have not been
identified.
Genomes are organized in definite bodies
(compact clump; occupied 1/3 volume)
Figure 09.05: E. coli bacterium, colored transmission electron micrograph (TEM).
© Dr. Klaus Boller/Photo Researchers, Inc.
DNA intercalating agent; Et-Br
Generating positive supercoil in closed
circular DNA
Open DNA, tension is reduced by rotation
E.coli, negative supercoiled , domain
Figure 09.06: The nucleoid spills out of a lysed E. coli cell in the
form of loops of a fiber.
© G. Murti/Photo Researchers, Inc.
• The nucleoid has ~400
independent
negatively supercoiled
domains.
• 400 domain/10kb
• The average density
of supercoiling is ~1
supercoil/100bp.
Figure 09.07: The bacterial genome consists of a large number of loops of duplex DNA
(in the form of a fiber), each of which is secured at the base to form an independent
structural domain.
Figure 09.08: An unconstrained supercoil in the DNA path creates tension, but no
tension is transmitted along DNA when a supercoil is constrained by protein binding.
9.4 Eukaryotic DNA Has Loops and Domains
Attached to a Scaffold
• DNA of interphase chromatin is
negatively supercoiled into
independent domains of ~85 kb.
• Metaphase chromosomes have a
protein scaffold to which the loops
of supercoiled DNA are attached.
Figure 09.09: Histone-depleted
chromosomes consist of a protein
scaffold to which loops of DNA are
anchored.
• DNA is attached to the nuclear matrix at specific sequences
called MARs or SARs.
• The MARs are A-T-rich but do not have any specific consensus
sequence.
• metaphase ( or mitotic) scaffold – A proteinaceous structure
in the shape of a sister chromatid pair, generated when
chromosomes are depleted of histones.
9.5 Chromatin Is
Divided into
Euchromatin and
Heterochromatin
Figure 09.10: The sister chromatids of a mitotic pair each consist of a fiber (~30 nm in
diameter) compactly folded into the chromosome.
© Biophoto Associates/Photo Researchers, Inc.
Figure 09.B01
Adapted from an illustration by Darryl Leja, National
Human Genome Research Institute (www.genome.gov).
Figure 09.B02A: Spectral karyotyping (SKY), an application of chromosome painting.
Photo courtesy of Johannes Wienberg, LudwigMaximilians-University, and Thomas Ried, National
Institutes of Health.
Figure 09.B02B:
Photo courtesy of Johannes Wienberg, LudwigMaximilians-University, and Thomas Ried, National
Institutes of Health.
9.5 Chromatin Is Divided into Euchromatin and
Heterochromatin
• Individual chromosomes can be seen
only during mitosis.
• During interphase, the general mass of
chromatin is in the form of euchromatin,
which is slightly less tightly packed than
mitotic chromosomes.
• Regions of heterochromatin remain
densely packed throughout interphase.
Figure 09.11: A thin section through a
nucleus stained with Feulgen shows • chromocenter – An aggregate of
heterochromatin as compact regions
heterochromatin from different
clustered near the nucleolus and
chromosomes.
nuclear membrane.
• Constitutive and facultative
heterochromatin
Photo courtesy of Edmond Puvion, Centre National
de la Recherche Scientifique
Common features of heterochromatin
1.
2.
3.
4.
5.
Permanently condensed
Late replication and low recombination
Multi-repeated sequence and very low or absent transcription
Gene is rare
Translocation into this region induce silencing (exception; ribosomal DNA)
For active transcription, location of euchromatin is necessary but not sufficient
9.6 Chromosomes Have Banding Patterns
• Certain staining techniques cause
the chromosomes to have the
appearance of a series of
striations, which are called Gbands.
• The bands are lower in G-C
content than the interbands.
• Genes are concentrated in the GC-rich interbands.
Figure 09.13: The human X
chromosome can be divided into
distinct regions by its banding pattern.
Figure 09.12: G-banding generates a characteristic lateral series of bands in each
member of the chromosome set.
Photo courtesy of Lisa Shaffer, Washington State University-Spokane
Figure 09.13: The human X chromosome can be divided into distinct regions by its
banding pattern.
Chromosome 12
Contains over 1600 genes
Contains over 130 million base pairs, of which over 95% have been determined
See the diseases associated with chromosome 12 in the MapViewer.
Phenylketonuria
Phenylketonuria (PKU) is an inherited error of metabolism caused by a
deficiency in the enzyme phenylalanine hydroxylase. Loss of this enzyme
results in mental retardation, organ damage, unusual posture and can, in
cases of maternal PKU, severely compromise pregnancy.
Classical PKU is an autosomal recessive disorder, caused by mutations in
both alleles of the gene for phenylalanine hydroxylase (PAH), found on
chromosome 12. In the body, phenylalanine hydroxylase converts the amino
acid phenylalanine to tyrosine, another amino acid. Mutations in both copies of
the gene for PAH means that the enzyme is inactive or is less efficient, and the
concentration of phenylalanine in the body can build up to toxic levels. In some
cases, mutations in PAH will result in a phenotypically mild form of PKU called
hyperphenylalanemia. Both diseases are the result of a variety of mutations in
the PAH locus; in those cases where a patient is heterozygous for two
mutations of PAH (ie each copy of the gene has a different mutation), the
milder mutation will predominate.
A form of PKU has been discovered in mice, and these model organisms are
helping us to better understand the disease, and find treatments against it.
With careful dietary supervision, children born with PKU can lead normal lives,
and mothers who have the disease can produce healthy children
Chromosome 15
Contains approximately 1200 genes
Contains approximately 100 million base pairs, of which over 80% have been determined
See the diseases associated with chromosome 15 in the MapViewer.
Prader-Willi syndrome
Prader-Willi syndrome (PWS) is an uncommon inherited disorder characterized by mental retardation, decreased muscle tone,
short stature, emotional lability and an insatiable appetite which can lead to life-threatening obesity. The syndrome was first
described in 1956 by Drs. Prader, Labhart, and Willi.
PWS is caused by the absence of segment 11-13 on the long arm of the paternally derived chromosome 15. In 70-80% of
PWS cases, the region is missing due to a deletion. Certain genes in this region are normally suppressed on the maternal
chromosome, so, for normal development to occur, they must be expressed on the paternal chromosome. When these
paternally derived genes are absent or disrupted, the PWS phenotype results. When this same segment is missing from the
maternally derived chromosome 15, a completely different disease, Angelman syndrome, arises. This pattern of inheritance
when expression of a gene depends on whether it is inherited from the mother or the father is called genomic imprinting.
The mechanism of imprinting is uncertain, but, it may involve DNA methylation.
Genes found in the PWS chromosomal region code for the small ribonucleoprotein N (SNRPN). SNRPN is involved in mRNA
processing, an intermediate step between DNA transcripton and protein formation. A mouse model of PWS has been
developed with a large deletion which includes the SNRPN region and the PWS 'imprinting centre' (IC) and shows a
phenotype similar to infants with PWS. These and other molecular biology techniques may lead to a better understanding of
PWS and the mechanisms of genomic imprinting
Angelman syndrome
Angelman syndrome (AS) is an uncommon neurogenetic disorder
characterized by mental retardation, abnormal gait, speech
impairment, seizures, and an inappropriate happy demeanor that
includes frequent laughing, smiling, and excitability. The
uncoordinated gait and laughter have caused some people to refer
to this disorder as the "happy puppet" syndrome.
The genetic basis of AS is very complex, but the majority of cases
are due to a deletion of segment 15q11 q13 on the maternally
derived chromosome 15. When this same region is missing from
the paternally derived chromosome, an entirely different disorder,
Prader Willi syndrome, results. This phenomenon when the
expression of genetic material depends on whether it has been
inherited from the mother or the father is termed genomic
imprinting.
The ubiquitin ligase gene (UBE3A) is found in the AS chromosomal
region. It codes for an enzyme that is a key part of a cellular protein
degradation system. AS is thought to occur when mutations in
UBE3A disrupt protein break down during brain development.
In a mouse model of AS, affected animals had much less
maternally inherited UBE3A than their unaffected litter mates.
However, this difference in UBE3A levels was only found in the
hippocampus and the cerebellum, and not all of the brain. This
animal model and other molecular techniques are helping us learn
more about the disparate maternal and paternal expression of the
UBE3A gene
9.7 Polytene Chromosomes Form Bands
That Expand at Sites of Gene Expression
Figure 09.14: The polytene
chromosomes of D. melanogaster
form an alternating series of
bands and interbands.
Photo courtesy of José Bonner, Indiana University
• chromomeres – Densely staining
granules visible in chromosomes
under certain conditions, especially
early in meiosis, when a
chromosome may appear to consist
of a series of chromomeres.
• polytene chromosomes –
Chromosomes that are generated by
successive replications of a
chromosome set without separation
of the replicas.
Some specialized eukaryotic
cells increase cell volume via
endomitosis, where DNA
synthesis is repeated without
cell division and a normal
chromosome develops into a
giant polytene chromosome
results
Figure 09.14: The polytene chromosomes of D. melanogaster
form an alternating series of bands and interbands.
Photo courtesy of José Bonner, Indiana University
in situ hybridization – Hybridization performed
by denaturing the DNA of cells squashed on a
microscope slide so that reaction is possible
with an added single-stranded RNA or DNA;
the added preparation is radioactively labeled
and its hybridization is followed by
autoradiography.
Figure 09.15: A magnified view of bands 87A and 87C shows their hybridization in situ
with labeled RNA
extracted from heat-shocked cells.
Photo courtesy of José Bonner, Indiana University
•
Bands that are sites of gene expression on
polytene chromosomes expand to give
“puffs.”
Figure 09.16: Displayed is a small segment of chromosome 3 before (top) and after
(bottom) heat shock. Chromosomes are stained for DNA (blue) and for Pol II (green).
Photo courtesy of Victor G. Corces, Emory University
9.8 The Eukaryotic Chromosome Is a
Segregation Device
•
•
A eukaryotic chromosome is held on the mitotic spindle by the attachment of microtubules
to the kinetochore that forms in its centromeric region.
microtubule organizing center (MTOC) – A region from which microtubules emanate.
– In animal cells the centrosome is the major microtubule organizing center.
centromere – A constricted region of a chromosome that includes the site of attachment (the
kinetochore) to the mitotic or meiotic spindle.
It may consist of unique DNA sequences or highly repetitive sequences and proteins not
found anywhere else in the chromosome.
acentric fragment – A fragment of a chromosome (generated by breakage) that lacks a
centromere and is lost at cell division.
Figure 09.17: Chromosomes are pulled to the poles via microtubules that attach at the
centromeres.
Figure 09.18: The centromere is identified by a DNA sequence that binds specific
proteins.
Adapted from Y. Datal et al., Proc. Natl. Acad. Sci. USA
104 (2007): 15974–15981.
9.9 Regional Centromeres Contain a
Centromeric Histone H3 Variant and Repetitive
DNA
• Centromeres are
characterized by a
centromere-specific histone
H3 variant and often contain
heterochromatin that is rich
in satellite DNA sequences.
• The function of the
repetitive DNA is not known.
• Satellite DNA-171 Bp repeated
Figure 09.19: A model of the overall structure of a regional centromere.
9.10 Point Centromeres in S. cerevisiae Contain
Short, Essential Protein-Binding DNA Sequences
• CEN elements are identified in S. cerevisiae by the ability to
allow a plasmid to segregate accurately at mitosis.
• CEN elements consist of the short, conserved sequences CDE-I
and CDE-III that flank the A-T-rich region CDE-II.
Figure 09.20: Three conserved regions can be identified by the sequence homologies
between yeast CEN elements.
9.10 Point Centromeres in S. cerevisiae Contain
Short, Essential Protein-Binding DNA Sequences
Figure 09.21: The DNA at CDE-II is
wound around a protein aggregate
including Cse4p, CDE-III is bound to
CBF3 and CDE-I is bound to CBF1.
• A specialized protein complex
containing the histone variant Cse4
is formed at CDE-II.
• The CBF3 protein complex that
binds to CDE-III is essential for
centromeric function.
• The proteins that bind CEN serve as
an assembly platform for the
kinetochore and provide the
connection to microtubules.
9.11 Telomeres Have Simple Repeating
Sequences That Seal the Chromosome Ends
• The telomere is required for the stability of the chromosome
end.
• A telomere consists of a simple repeat where a C+A-rich
strand has the sequence C>1(A/T)1–4.
Figure 09.22: A typical telomere has a simple repeating structure with a G-T-rich strand
that extends beyond the C-A-rich strand.
Figure 10.11 The Biology of Cancer (© Garland Science 2007)
Figure 10.13a The Biology of Cancer (© Garland Science 2007)
Figure 09.23: The crystal structure of a short repeating sequence from the human
telomere forms three stakced G quartets.
Figure 09.24: Colored transmission electron micrograph (TEM) of a telomere.
© Dr. Gopal Murti/Photo Researchers, Inc.
Figure 10.17a The Biology of Cancer (© Garland Science 2007)
• The protein TRF2 catalyzes a
reaction in which the 3′ repeating
unit of the G+T-rich strand forms a
loop by displacing its homolog in
an upstream region of the
telomere.
Figure 09.25: The 3' single-stranded end of the telomere (TTAGGG)n displaces the
homologous repeats from duplex DNA to form a t-loop. The reaction is catalyzed by
TRF2.
• Telomerase uses the 3′-OH of the
G+T telomeric strand to prime
synthesis of tandem TTGGGG
repeats.
• The RNA component of telomerase
has a sequence that pairs with the
C+A-rich repeats.
• One of the protein subunits is a
reverse transcriptase that uses the
RNA as template to synthesis the
G+T-rich sequence.
Figure 09.27: Telomerase positions itself by base pairing between the RNA template
and the protruding single-stranded DNA primer.
Figure 10.18 The Biology of Cancer (© Garland Science 2007)
Figure 10.19 The Biology of Cancer (© Garland Science 2007)
9.11 Telomeres Have Simple Repeating
Sequences That Seal the Chromosome Ends
• Telomerase is expressed in actively dividing cells and is not
expressed in differentiated cells.
• Loss of telomeres results in senescence.
• Escape from senescence can occur if telomerase is reactivated,
or via unequal homologous recombination to restore
telomeres.
9.11 Telomeres Have Simple Repeating
Sequences That Seal the Chromosome Ends
Figure 09.28: Mutation in telomerase causes telomeres to shorten in
each cell division. Eventual loss of the telomere causes chromosome
breaks and rearrangements.
Figure 10.20 The Biology of Cancer (© Garland Science 2007)
Figure 10.21a The Biology of Cancer (© Garland Science 2007)
Figure 10.31 The Biology of Cancer (© Garland Science 2007)
Dyskerin-hTR
X-linked dyskeratosis congenia
Figure 10.32a The Biology of Cancer (© Garland Science 2007)
Figure 09.22: A typical telomere has a simple repeating structure with a G-T-rich
strand that extends beyond the C-A-rich strand.
Figure 09.FTR01A: FISH, Chromosome Painting, and Spectral Karyotyping
Figure 09.FTR01B: FISH, Chromosome Painting, and Spectral Karyotyping
1. Senescence
Progeroid disorders; prematured senescence
Human progeroid diseases
1.
2.
3.
4.
Werner syndrome
Hutchinson-Gilford Syndrome
Ataxia-Telangiactia
Cockayne syndrome
HGS
Trigger of senescence
1. Oxidative stress
2. IGF
3. Energy balance
4. replication
Cockayne syndrome
Recent publications- ageing is hot issue
Cell, 2006- Sirt homologue SIRT6 is also linked to aging
Science 2006, Lamin A (HGS) is