Transcript w + gene is silenced in some cells

PowerPoint to accompany
Genetics: From Genes to
Genomes
Fourth Edition
Leland H. Hartwell, Leroy Hood,
Michael L. Goldberg, Ann E. Reynolds,
and Lee M. Silver
Prepared by Mary A. Bedell
University of Georgia
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PART
IV
How Genes Travel on Chromosomes
CHAPTER
The Eukaryotic
Chromosome
CHAPTER OUTLINE
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
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12.1
12.2
12.3
12.4
Chromosomal DNA and Proteins
Chromosome Structure and Compaction
Chromosomal Packaging and Function
Replication and Segregation of Chromosomes
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Chromosomal DNA and proteins
Chromosomes have a versatile, modular structure for
packaging DNA that supports flexibility of form and function
Chromatin is the generic term for any complex of DNA and
protein found in a nucleus of a cell
Chromosomes are the separate pieces of chromatin that
behave as a unit during cell division
Chromatin is ~ 1/3 DNA, 1/3 histones, 1/3 nonhistone
proteins
DNA interaction with histones and nonhistone proteins
produces sufficient level of compaction to fit into a cell
nucleus
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Histone proteins
Histones – small, positively-charged, and highly conserved
• Bind to and neutralize negatively charged DNA
• Make up half of all chromatin protein by weight
• Five types - H1, H2A, H2B, H3, and H4
• Core histones (H2A, H2B, H3, and H4) make up the
nucleosome
Posttranslational modifications of histones H3 and H4
• Methylation and acetylation of histone tails
• Affect chromatin structure and gene expression in
specific chromosomal regions
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Nonhistone proteins
Hundreds of other proteins that make up chromatin and are
not histones
200 – 200,000 molecules of each kind of nonhistone protein
Large variety of functions
• Structural role – chromosome scaffold (see Figure
12.2)
• Chromosome replication – e.g. DNA polymerases
• Chromosome segregation – e.g. kinetochore proteins
(see Figure 12.3)
• Active in transcription – largest group
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Different levels of chromosome compaction
Table 12.1
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The nucleosome is the fundamental unit of
chromosomal packaging
DNA wraps twice around nucleosome core octamer (Figure
12.5) and forms 100 Å fiber
• Results in 7-fold compaction of DNA
Spacing and structure of nucleosomes affect genetic
function
• Determines whether DNA between nucleosomes is
accessible for proteins that initiate transcription,
replication and further compaction
• Arrangement along chromatin is highly defined and
transmitted from parent to daughter cells during DNA
replication
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Nucleosomes look like "beads on a string"
in the electron microscope
Diameter of DNA helix (string)
is 20 Å
Diameter of nucleosome core
(bead) is 100 Å
Fig. 12.4
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The nucleosome core is an octamer
of two each of histones H2A, H2B, H3, and H4
160 bp of DNA wraps twice
around a nucleosome core
Fig. 12.5
40 bp of linker DNA connects
adjacent nucleosomes
Histone H1 associates with
linker DNA as it enters and
leaves the nucleosome core
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X-ray crystallography of a nucleosome
DNA bends sharply at
several places as it wraps
around the core histone
octamer
Base sequence dictates
preferred nucleosome
positions along the DNA
Fig. 12.6
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Models of higher-order packaging
100 Å fiber is compacted into 300 Å fiber by supercoiling
• Results in an additional 6-fold compaction of DNA
Fig. 12.7a
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The radial loop-scaffold model for
higher levels of compaction
Several nonhistone proteins (NHPs) bind to chromatin every
60-100 kb and tether the 300 Å fiber into structural loops
Other NHPs gather several loops together into daisylike
rosettes
Fig. 12.7b
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The radial loop-scaffold model for
higher levels of compaction (cont)
Condensins may further condense chromosomes into a
compact bundle for mitosis
Fig. 12.7c
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The karyotype of a human female
examined by high-resolution G-banding
Metaphase chromosomes
stained with Giemsa have
alternating bands of light
and dark staining
Each band is contains
many DNA loops and
ranges from 1 to 10 Mb in
length
Banding patterns on each
chromosome are highly
reproducible
Fig. 12.9
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Locations of genes in relation
to chromosomal bands
Short arm = p arm
Fig. 12.10
Long arm = q arm
Within each arm, light and
dark bands are numbered
consecutively
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Comparison of
chromosomes from
humans and great apes
Banding patterns in some
chromosomes (i.e. Chr 1) are
nearly identical
The metacentric Chr 2 in
humans was formed by fusion
of two acrocentric
chromosomes in great apes
Chromosome 1
Fig. 12.11
Acrocentric
chromosomes
of great apes
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Chromosomal packaging and function
Heterochromatin – highly condensed, usually inactive
transcriptionally
•Darkly stained regions of chromosomes
•Constitutive – condensed in all cells [e.g. most of the Y
chromosome and all pericentromeric regions (see Fig
12.12)]
•Facultative – condensed in only some cells and relaxed in
other cells (e.g. position effect variegation, X chromosome
in female mammals)
Euchromatin – relaxed, usually active transcriptionally
•Lightly stained regions of chromosomes
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Position-effect variegation (PEV) in Drosophila
White+ (w+) gene is normally located in euchromatin
Chromosomal inversion can result in w+ gene being located
adjacent to heterochromatin
w+ gene is
expressed in all
cells (red pigment)
Fig. 12.13a
w+ gene is silenced
in some cells (no
pigment) but is
expressed in other
cells (red pigment)
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PEV in
Drosophila (cont)
Gene silencing can be
caused by spreading of
heterochromatin into
nearby genes
Spreading can occur
over > 1000 kb of
chromatin
Heterochromatin
spreads further in some
cells than in others
Fig. 12.13b
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Identification of molecules involved in
heterochromatin formation
Genetic screens in Drosophila were used to identify
mutations that enhance or suppress the extent of PEV
• Mutation in enhancer of PEV results in more cells
having gene inactivation
 Encodes protein that localizes to heterochromatin
• Mutation in suppressor of PEV results in fewer cells
having gene inactivation
 Encodes protein that adds methyl group to lysines on
histone H3, signal for chromatin condensation
Barriers – specific sites on DNA that block the spread of
heterochromatin
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X-chromosome inactivation in female
mammals occurs through heterochromatin
formation
Example of facultative heterochromatin
Dosage compensation in mammals so that X-linked genes
in XX and XY individuals are expressed at same level
Random inactivation of all except one X chromosome in XX
Barr bodies – darkly stained heterochromatin masses
observed in somatic cells at interphase
• XX person has one Barr body
• XXX person has two Barr bodies
• XXY person has one Barr body
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X chromosome mosaicism
In very early embryo, both X chromosomes are active
In humans, random X-inactivation occurs ~ 2 weeks after
fertilization
• Some cells have maternal X inactivated, other cells
have paternal X inactivated
• All cell descendants have the same inactive X
Adult females are mosaic at X-linked genes
• In females heterozygous for X-linked mutation:
 Some cells have wild-type allele inactivated
 Some cells have mutant allele inactivated
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X-inactivation is initiated by expression
of the Xist gene
Xist, X inactivation specific transcript
• One of the few genes expressed on the inactive X but
is not expressed on the active X
Xist RNA is a large, non-coding, cis-acting regulatory RNA
• Binds to the X-chromosome that it was expressed from
• Initiates histone modifications (methylation,
deacetylation) that result in heterochromatin formation
Deletion of the Xist gene abolishes X inactivation
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Transcription is controlled by chromatin
structure and nucleosome position
Spacing and structure of nucleosomes affect transcription
Three major mechanisms can regulate chromatin patterns
1.Histone modifications – addition of methyl or acetyl
groups
2.Remodeling complexes can alter nucleosome patterns
• Change accessibility of promoter sequences
 Assays for DNase hypersensitive sites (Figure 12.14)
• Remove or reposition promoter-blocking nucleosomes
3.Histone variants can cause different nucleosomal
structures, e.g. CENP-A at centromeres
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Assay for chromosomal regions
that lack nucleosomes
DNase hypersensitive sites - promoters of transcribed
genes are more susceptible to nuclease digestion than
promoters of non-transcribed genes
Fig. 12.14
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Origins of replication in eukaryotes
Rate of DNA synthesis in human cells ~ 50 nt/sec
Most mammalian cells have ~ 10,000 origins
 It would take 800 hours to replicate the human genome if
there was only one origin of replication!
Many origins are active at the same time (see Fig. 12.15)
Accessible regions of DNA that are devoid of nucleosomes
Replication unit (replicon) – DNA running both ways from
one origin to the endpoints
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Origins of replication in yeast are
"autonomously replicating sequences" (ARSs)
ARSs permit replication of plasmids in yeast cells
AT-rich consensus sequence found in all ARS elements,
flanked by sequences that promote replication initiation
ARS1 (below) is the first ARS to be characterized
Fig. 12.16
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Telomeres are "caps" that protect the ends
of eukaryotic chromosomes
Telomeres consist of specific
repetitive sequences and
don't contain genes
 Species-specific sequences
 e.g. TTAGGG in humans,
TTGGGG in Tetrahymena
 250-1500 repeats with
variable number between
different cell types
Prevent chromosome
fusions and maintain
integrity of chromosomal
ends
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Fig. 12.17
28
Replication at the ends
of chromosomes
After removal of the last RNA
primer, DNA polymerase
cannot replicate some of the
sequences at the 5' end
• DNA synthesis occurs
only in 5'-to-3' direction
Without a special
mechanism, DNA would be
lost from every new DNA
strand at each cell cycle
Fig. 12.18
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Telomerase is a ribonucleoprotein that
extends telomeres
Telomerase RNA is
complementary to telomere
repeat sequences
Serves as template for
addition of new DNA repeat
sequences to telomere
Additional rounds of
telomere elongation occur
after telomeres translocate
to newly-synthesized end
Fig. 12.19
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Telomerase activity and cell proliferation
In yeast that has deletion of telomerase, telomeres shorten
by 3 bp per generation
• Eventually the chromosomes break and the cells die
In humans, the levels of telomerase and cellular life-span
varies between different types of cells
• Most somatic cells have low expression of telomerase
 Telomeres shorten slightly at each cell division
 Senescence after < 50 generations in culture
• Germ cells, stem cells, and tumor cells have high
expression of telomerase
 At each generation, telomere length is maintained
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Chromosome duplication includes
reproduction of chromatin structure
Chromatin fiber unwinds before DNA synthesis occurs
Synthesis of histones and their transport into nucleus must
be tightly coordinated with DNA synthesis
Newly-synthesized DNA must associate with either
preexisting histones or with newly-synthesized histones
• After DNA replication, nucleosomal DNA must produce
the same level of compaction as before replication
• In differentiating cells, a slightly different chromatin
condensation pattern can appear after replication
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Segregation of condensed chromosomes
depends on centromeres
During anaphase of mitosis and meiosis II, sister
chromatids must segregate to different daughter cells
During anaphase of meiosis I, sister chromatids do not
separate and homologous chromosomes segregate to
different daughter cells
Centromeres have two functions:
• Hold sister chromatids together (through action of
cohesin)
• Attachment sites for chromosome segregation
machinery (through formation of kinetochore)
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Characteristics of centromeres
Appear as constrictions in chromosomes
Can be in middle of chromosome (metacentric) or near one
end of the chromosome (acrocentric)
Consist of satellite DNAs, which are repetitive, noncoding
sequences
• Tandem repeats of 5 – 300 bp long, can extend over
megabases of DNA
• Have different chromatin structure and higher-order
packaging than other chromosomal regions
• Predominant human satellite DNA is a-satellite
 171 bp repeat, present in > 1 Mb block of tandem repeats
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Action of cohesin during mitosis
Cohesin is a protein complex that holds sister chromatids
during metaphase
At anaphase, cohesin is enzymatically cleaved and sister
chromatids are released from each other
Fig. 12.20a
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Action of cohesin during meiosis I
At anaphase I, cohesin along chromosome arms is
enzymatically cleaved but cohesin at centromeres is not
cleaved
Shugoshin protects centromeric cohesin from degradation
Fig. 12.20b
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Action of cohesin during meiosis II
After entry into metaphase II, shugoshin is removed and
centromeric cohesin is degraded
Fig. 12.20c
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Structure of centromeres in
higher organisms
Centromeres hold sister chromatids together and contain
information for construction of a kinetochore
Cohesin binds sister chromatids together at the centromere
Fig. 12.21
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Structure and DNA sequence
organization of yeast centromeres
Fig. 12.22
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Histone variants at centromeres
Specialized chromatin in central core of centromere marks
this region for attachment of kinetochore proteins
Central core of each centromere:
• Composed of unique chromatin that does not
recombine and is not transcribed
• Surrounded by regions of heterochromatin
interspersed with euchromatin
Histone variant CENP-A is present in central core of all
eukaryotes examined
• Differs from histone H3 in N-terminal region
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