Nucleus and ChromosomesChapter 8
Learning Objectives
(1) A typical nonmitotic nucleus includes several major
components.
(2) Nuclear envelope and nuclear pore complex.
(3) The components of chromatin and packaging of
chromosome.
(4) Nucleolus.
1,The nucleus,Nuclear envelope and NPC
A,Structure,Double-membrane nuclear
envelope surrounds the nucleus
B,The nuclear envelope consists consists of
two membranes by a perinuclear space.
?The inner surface of the nuclear envelope is
lined by the nuclear lamina
?The nuclear lamina supports the nuclear envelope,
Gives shape and stability of nuclear envelope;
Provides a structure link between chromatin and
nuclear envelope
?The nuclear lamina is composed of lamins.
?The integrity of the nuclear lamina is regulated by
phosphorylation and dephosphorylation.
Breakdown and reformation of nuclear envelope during mitosis
?The phosphorylation of the lamins triggers the
disassembly of the nuclear lamina,which in turn cause the
nuclear envelope to break up into vesicles,
Dephosphorylation of the lamins helps reverse the process,
?Because the new nuclear envelope is so closely applied to
the surface of the chromosomes,it excludes all of the
proteins in the cell except those bound to the chromosomes.
So nuclear localization signals are not cleaved off after
transport into the nucleus.
?Old nuclear envelope reforms new nuclear envelope,
C,Nuclear pore complex (NPC)
Cytoplasmic
Ring
Nuclear
Ring
D,Molecules enter and exit the nucleus
through nuclear pore complex
Bidirectional traffic
?Passive transport—passively diffuse
?3000-4000 NPC/cell(mammalian);
To import about 106 histone/3 mins.(DNA-sythesizing
cell) = 100 histone/ min/NPC
?Each NPC contains one or more open aqueous
channels,9 nm in diameter and 15 nm long
?The effective size of these channels has been
determined by injecting various sizes of colloidal gold
particles and examined by electron microscopy.
<10 nm in diameter
<60kd globular protein Able to enter the nucleus
? Active transport
?Transport of large proteins into nucleus needs nuclear
localization signal (NLS)
?NLS are present only in nuclear proteins
A typical NLS contains several consecutive
basic amino acids (the positively charged Aa,
Lys,Arg)
Normal pyruvate kinase,
in cytosol
Chimeric pyruvate kinase
containing SV40 NLS,in nucleus
Wild-type,T-antigen
in nucleus
Mutant-type,T-antigen
in cytosol
?Nuclear import and export
?Nuclear import receptors bind NLS and
Nucleoporins
?The Ran GTPase drives directional transport
through NPC
The compartmentalization of
Ran-GDP and Ran-GTP.
?A model for how GTP hydrolysis by Ran provides
directionality for nuclear transport
?Nuclear export works like nuclear import,
but in reverse
hnRNP proteins contain a nuclear-export
signal (NES)
Reference:
Cell 92,327,1998
2,Eukaryotes package DNA in
Chromatin and chromosomes
?Chromosomes exist in different states
throughout the life of a cell
Chromatin,(Interphase)
Fibers,10-30nm in diameter,
Dispersed through the nucleus.
DNA+Proteins+RNA.
Chromosomes,(M phase)
Cell division,these fibers condense
and fold into larger,compact structure
A,The complexity of eukaryotic genomes
?Genome,One copy of all the genetic
information of an organism,
?Genome size generally increases with
an organism’s complexity.
If each nucleotide pair is 1 mm,then the
human genome would extend 3200 km,
far enough to stretch across the center
of Africa,the site of our human origins.
Species Genome size
SV40 5 × 103bp
E.coli 4.6× 106bp
Yeast 2× 107bp
Fruit fly 2× 108bp
Human 3× 109bp
Some amphibian and
plants have larger
genome size than
human.
?DNA sequencing is being applied to whole
genomes as well as to genes
Early,
E.coli,6 years to complete its sequence.
If do as so,
Almost 6000 years to sequence the,
Each DNA molecule that forms a linear chromosome.
?Protein-coding sequences (Nonrepeated)
~25-50% of the protein-coding genes are solitary genes.
Others belong to gene family,which encode proteins
with similar but nonidentical amino acid sequences.
?-like globin gene family:
Contains 5 functional genes
?Tandemly repeated genes encode rRNAs,
tRNAs,and Histones (Moderately repeated)
Tandemly repeated genes encode identical or
nearly identical proteins or functional RNAs.
These genes are needed to meet the great
cellular demand for their transcripts.
5S rRNA and pre-rRNA genes,More than 100
copy/Genome.
?Tandemly repeated DNA,Simple-sequence,
Highly repeated,Noncoding
Satellite DNA:
Centromeres,Telomeres (105~107bp)
Microsatellite DNA:
102~105bp at each site
Minisatellite DNA:
10~100bp at each site
DNA fingerprinting depends on differences in
length of minisatellite or microsatellite DNA
Unequal exchange
during meiosis.
?Interspersed moderately repeated DNA
Interspersed repeated DNA makes up 25-40%
of most mammalian genomes.
Alu family,
~300bp long/unit,accounting for about
10% of the human DNA
B,DNA packaging
? Nucleosomes are the basic unit of
chromatin structure
?Each human cell contains about 2 m of DNA
within nucleus if stretched end-to-end,yet the
nucleus of a human cell itself is only about 6 um
in diameter,
Compaction ratio=nearly 10000-fold.
(Chromosome 22,DNA 1.5cm?2um)
?Evidence:
(1)Electron micrographs of chromatin fibers
Isolated from interphase nucleus,30nm thick
Chromatin unpacked,show the unclesome
?Evidence:
(2)Nuclease digestion (Rat liver chromatin)
Gel electrophoresis
after removal of
chromatin proteins
Analyzed by electron
microscopy
The basic repeat unit,
containing an average
of 200bp of DNA
associated with a
protein particle,is the
nucleosome
?Evidence:
(3)X-ray diffraction studies
3-D of nucleosome Nature 389:251,1997
?Structural organization of the
nucleosome
? A histone octamer forms the nucleosome core
?Histone octamer:
(H2A-H2B)-(H3-H4)-(H3-H4)-(H2A-H2B)
?Where is the histone H1?
H1 molecules are associated with the linker
region,146+15~50bp linker DNA
200bpDNA:
Linker DNA:15-50bp
Nucleosomal DNA:146bp to wrap
1.65 times around the histone core.
Structure of nucleosome
Digested briefly:
H1+Octamer+200
bpDNA
Digested longer:
Octamer+146bp
H1 is released
Linker Core particle
?Histones:
The most abundant proteins associated with
eukaryotic DNA
Rich in positively charged basic amino acids,which
interact with the negtively charged phosphate
groups in DNA
The amino acid sequences of histones H2A,H2B,
H3,H4 are remarkably similar among distantly
species
Covalent modification of histone tails,Ac,P and/or Met…..
?Localization of nucleosome is affected
by two factors:
Sequence-specific
DNA binding proteins
DNA bending
C,Nucleosomes are packed together to
form chromatin fibers and chromosomes
?Scaffold radial loop structure model
Nonhistone proteins provide a structural
scaffold for long chromatin loops
?2nm DNA –11nm nucleosome – 30nm
fiber – Loop– Metaphase chromosome
Evidence for Scaffold Radial Loop Model
Loop
D,Euchromatin and Heterochromatin
?Euchromatin
? The possibility of transcription;
? The types of chromosomal structure—
30-nm fibers and looped domains;
? Light-staining,less condensed;
? Pulse labeling with 3H-uridine,
Transcriptional activity
?Heterochromatin:
? Dark-staining,condensed chromatin;
? No transcriptional activity; There are position
effects;
? in a typical mammalian cell,approximately 10%
of the genome is packaged into heterochromatins
forming CEN and TEL
? Divided into two classes:
Constitutive & facultative
Compacted state
at all time,
Centromere
Inactivated at
certain phase
of life
Example of facultative heterochromatin:
Random inactivation of X chromosome in different
cells during early embryonic development creates a
mosaic of tissue patches.
Barr body in a woman’s cell A calico cat,a mosaic patches
Male
Or?
female
? The position effects
of hetrochromatin
A,yeast;
B,Fluit fly.
3,Chromosome number,size,and shape
at metaphase are species specific
Chromatids
Karyotype
Binding
Human mitotic chromosomes and karyotype
A,Main structures of chromosome
? Centromere & Kinetochore
Centromere,Highly repeated DNA+Kinetochore
?The Centromere and Kinetochore,serve as a site
for the attachment of spindle microtubules during
mitosis and meiosis
The structure of a human CEN The plasticity of human CEN
formation (MBC pp228)
Human chromosome No.14
secondary constriction
nucleolar organizing region,NOR
satellite
telomere
centromere
A typical mitotic chromosome
at metaphase
? Three functional elements are required
for replication and stable inheritance of
chromosomes
?Autonomously replicating sequences (ARS)
ARS act as
an origin of
replication
?Centromeres
Transfected
Leu- cell by
Plasmid with
sequence
Progeny of
transfected
cell
Growth
without
Leu
Mitotic
segregation Conclusion
?Telomeres:
Functions
For the complete replication of chromosome
Protect the chromosomes from nuclease influence
Prevent the ends of chromosomes from fusing
Tetrahymena
FISH (Human Probe ),
TTAGGG
Anti-RAP1 antibody:
Yeast cell
?Telomere,telomerase and cellular
aging,cancer cell
?Telomerase is found in germ cells,not in somatic cells.
?The telomere length of adult is shorter than that of
younger.
?Telomere shortening is thought to activate a suicide
program,
So,telomere shortening plays a key role in protecting
the body from cancer,
90% of human tumors contain an active telomerase.
?Yeast Artificial Chromosome (YAC)
? Giant chromosomes
?Polytene chromosomes
Endomitosis
Chromocenter
Bands and interbands
Luxury
genes
House-keeping
genes
?Puffs in plolytene chromosome,Gene activity
Puffs,transcriptionally
active chromatin
Labeled with anti-
RNA polymerase II
antibody
If labeled with 3H-U,
then…
?Lampbrush chromosomes
In growing
amphibian oocyte
Stay in meiosis I
Structure
Functions:
Transcript into
RNA,storage
4,Nucleolus
A,Structure
Fibrillar centers
Dense fibrillar component
Granular component
B,Functions of nucleolus,Ribosomal Biogenesis
? Ribosomal Biogenesis:
pre-rRNA synthesis Process Assembly
FC,DFC DFC.GC Cytosol
Directional process
? Characteristics of RNA transcription
NORs in human
chromosomes:
13\14\15\21\22
Code for (in eukaryotes):
18s,28s,5.8s rRNA
?rRNA genes are tandemly arrayed in genome,
?Christmas-Like tree when transcripted
?Synthesis and processing of rRNA precursor
General structure of eukaryotic pre-rRNA transcription units
pre-rRNA transcription units
?Cleavage of pre-rRNA as RNP particles
Mammalia
?Synthesis and processing of 5s rRNA
?5s rRNAs are encoded by a large number genes
(Human,2000)
?5s rRNA gene are located outside the nucleolus.
?5s rRNAs are transcibed by RNA poly III.
?The 3’ end of 5s rRNA is removed during
processing.
? The assembly
of ribosomes
? The nucleolus disappears during mitosis
Nucleolar fusion
of human
fibroblast in
cultrue
Learning Objectives
(1) A typical nonmitotic nucleus includes several major
components.
(2) Nuclear envelope and nuclear pore complex.
(3) The components of chromatin and packaging of
chromosome.
(4) Nucleolus.
1,The nucleus,Nuclear envelope and NPC
A,Structure,Double-membrane nuclear
envelope surrounds the nucleus
B,The nuclear envelope consists consists of
two membranes by a perinuclear space.
?The inner surface of the nuclear envelope is
lined by the nuclear lamina
?The nuclear lamina supports the nuclear envelope,
Gives shape and stability of nuclear envelope;
Provides a structure link between chromatin and
nuclear envelope
?The nuclear lamina is composed of lamins.
?The integrity of the nuclear lamina is regulated by
phosphorylation and dephosphorylation.
Breakdown and reformation of nuclear envelope during mitosis
?The phosphorylation of the lamins triggers the
disassembly of the nuclear lamina,which in turn cause the
nuclear envelope to break up into vesicles,
Dephosphorylation of the lamins helps reverse the process,
?Because the new nuclear envelope is so closely applied to
the surface of the chromosomes,it excludes all of the
proteins in the cell except those bound to the chromosomes.
So nuclear localization signals are not cleaved off after
transport into the nucleus.
?Old nuclear envelope reforms new nuclear envelope,
C,Nuclear pore complex (NPC)
Cytoplasmic
Ring
Nuclear
Ring
D,Molecules enter and exit the nucleus
through nuclear pore complex
Bidirectional traffic
?Passive transport—passively diffuse
?3000-4000 NPC/cell(mammalian);
To import about 106 histone/3 mins.(DNA-sythesizing
cell) = 100 histone/ min/NPC
?Each NPC contains one or more open aqueous
channels,9 nm in diameter and 15 nm long
?The effective size of these channels has been
determined by injecting various sizes of colloidal gold
particles and examined by electron microscopy.
<10 nm in diameter
<60kd globular protein Able to enter the nucleus
? Active transport
?Transport of large proteins into nucleus needs nuclear
localization signal (NLS)
?NLS are present only in nuclear proteins
A typical NLS contains several consecutive
basic amino acids (the positively charged Aa,
Lys,Arg)
Normal pyruvate kinase,
in cytosol
Chimeric pyruvate kinase
containing SV40 NLS,in nucleus
Wild-type,T-antigen
in nucleus
Mutant-type,T-antigen
in cytosol
?Nuclear import and export
?Nuclear import receptors bind NLS and
Nucleoporins
?The Ran GTPase drives directional transport
through NPC
The compartmentalization of
Ran-GDP and Ran-GTP.
?A model for how GTP hydrolysis by Ran provides
directionality for nuclear transport
?Nuclear export works like nuclear import,
but in reverse
hnRNP proteins contain a nuclear-export
signal (NES)
Reference:
Cell 92,327,1998
2,Eukaryotes package DNA in
Chromatin and chromosomes
?Chromosomes exist in different states
throughout the life of a cell
Chromatin,(Interphase)
Fibers,10-30nm in diameter,
Dispersed through the nucleus.
DNA+Proteins+RNA.
Chromosomes,(M phase)
Cell division,these fibers condense
and fold into larger,compact structure
A,The complexity of eukaryotic genomes
?Genome,One copy of all the genetic
information of an organism,
?Genome size generally increases with
an organism’s complexity.
If each nucleotide pair is 1 mm,then the
human genome would extend 3200 km,
far enough to stretch across the center
of Africa,the site of our human origins.
Species Genome size
SV40 5 × 103bp
E.coli 4.6× 106bp
Yeast 2× 107bp
Fruit fly 2× 108bp
Human 3× 109bp
Some amphibian and
plants have larger
genome size than
human.
?DNA sequencing is being applied to whole
genomes as well as to genes
Early,
E.coli,6 years to complete its sequence.
If do as so,
Almost 6000 years to sequence the,
Each DNA molecule that forms a linear chromosome.
?Protein-coding sequences (Nonrepeated)
~25-50% of the protein-coding genes are solitary genes.
Others belong to gene family,which encode proteins
with similar but nonidentical amino acid sequences.
?-like globin gene family:
Contains 5 functional genes
?Tandemly repeated genes encode rRNAs,
tRNAs,and Histones (Moderately repeated)
Tandemly repeated genes encode identical or
nearly identical proteins or functional RNAs.
These genes are needed to meet the great
cellular demand for their transcripts.
5S rRNA and pre-rRNA genes,More than 100
copy/Genome.
?Tandemly repeated DNA,Simple-sequence,
Highly repeated,Noncoding
Satellite DNA:
Centromeres,Telomeres (105~107bp)
Microsatellite DNA:
102~105bp at each site
Minisatellite DNA:
10~100bp at each site
DNA fingerprinting depends on differences in
length of minisatellite or microsatellite DNA
Unequal exchange
during meiosis.
?Interspersed moderately repeated DNA
Interspersed repeated DNA makes up 25-40%
of most mammalian genomes.
Alu family,
~300bp long/unit,accounting for about
10% of the human DNA
B,DNA packaging
? Nucleosomes are the basic unit of
chromatin structure
?Each human cell contains about 2 m of DNA
within nucleus if stretched end-to-end,yet the
nucleus of a human cell itself is only about 6 um
in diameter,
Compaction ratio=nearly 10000-fold.
(Chromosome 22,DNA 1.5cm?2um)
?Evidence:
(1)Electron micrographs of chromatin fibers
Isolated from interphase nucleus,30nm thick
Chromatin unpacked,show the unclesome
?Evidence:
(2)Nuclease digestion (Rat liver chromatin)
Gel electrophoresis
after removal of
chromatin proteins
Analyzed by electron
microscopy
The basic repeat unit,
containing an average
of 200bp of DNA
associated with a
protein particle,is the
nucleosome
?Evidence:
(3)X-ray diffraction studies
3-D of nucleosome Nature 389:251,1997
?Structural organization of the
nucleosome
? A histone octamer forms the nucleosome core
?Histone octamer:
(H2A-H2B)-(H3-H4)-(H3-H4)-(H2A-H2B)
?Where is the histone H1?
H1 molecules are associated with the linker
region,146+15~50bp linker DNA
200bpDNA:
Linker DNA:15-50bp
Nucleosomal DNA:146bp to wrap
1.65 times around the histone core.
Structure of nucleosome
Digested briefly:
H1+Octamer+200
bpDNA
Digested longer:
Octamer+146bp
H1 is released
Linker Core particle
?Histones:
The most abundant proteins associated with
eukaryotic DNA
Rich in positively charged basic amino acids,which
interact with the negtively charged phosphate
groups in DNA
The amino acid sequences of histones H2A,H2B,
H3,H4 are remarkably similar among distantly
species
Covalent modification of histone tails,Ac,P and/or Met…..
?Localization of nucleosome is affected
by two factors:
Sequence-specific
DNA binding proteins
DNA bending
C,Nucleosomes are packed together to
form chromatin fibers and chromosomes
?Scaffold radial loop structure model
Nonhistone proteins provide a structural
scaffold for long chromatin loops
?2nm DNA –11nm nucleosome – 30nm
fiber – Loop– Metaphase chromosome
Evidence for Scaffold Radial Loop Model
Loop
D,Euchromatin and Heterochromatin
?Euchromatin
? The possibility of transcription;
? The types of chromosomal structure—
30-nm fibers and looped domains;
? Light-staining,less condensed;
? Pulse labeling with 3H-uridine,
Transcriptional activity
?Heterochromatin:
? Dark-staining,condensed chromatin;
? No transcriptional activity; There are position
effects;
? in a typical mammalian cell,approximately 10%
of the genome is packaged into heterochromatins
forming CEN and TEL
? Divided into two classes:
Constitutive & facultative
Compacted state
at all time,
Centromere
Inactivated at
certain phase
of life
Example of facultative heterochromatin:
Random inactivation of X chromosome in different
cells during early embryonic development creates a
mosaic of tissue patches.
Barr body in a woman’s cell A calico cat,a mosaic patches
Male
Or?
female
? The position effects
of hetrochromatin
A,yeast;
B,Fluit fly.
3,Chromosome number,size,and shape
at metaphase are species specific
Chromatids
Karyotype
Binding
Human mitotic chromosomes and karyotype
A,Main structures of chromosome
? Centromere & Kinetochore
Centromere,Highly repeated DNA+Kinetochore
?The Centromere and Kinetochore,serve as a site
for the attachment of spindle microtubules during
mitosis and meiosis
The structure of a human CEN The plasticity of human CEN
formation (MBC pp228)
Human chromosome No.14
secondary constriction
nucleolar organizing region,NOR
satellite
telomere
centromere
A typical mitotic chromosome
at metaphase
? Three functional elements are required
for replication and stable inheritance of
chromosomes
?Autonomously replicating sequences (ARS)
ARS act as
an origin of
replication
?Centromeres
Transfected
Leu- cell by
Plasmid with
sequence
Progeny of
transfected
cell
Growth
without
Leu
Mitotic
segregation Conclusion
?Telomeres:
Functions
For the complete replication of chromosome
Protect the chromosomes from nuclease influence
Prevent the ends of chromosomes from fusing
Tetrahymena
FISH (Human Probe ),
TTAGGG
Anti-RAP1 antibody:
Yeast cell
?Telomere,telomerase and cellular
aging,cancer cell
?Telomerase is found in germ cells,not in somatic cells.
?The telomere length of adult is shorter than that of
younger.
?Telomere shortening is thought to activate a suicide
program,
So,telomere shortening plays a key role in protecting
the body from cancer,
90% of human tumors contain an active telomerase.
?Yeast Artificial Chromosome (YAC)
? Giant chromosomes
?Polytene chromosomes
Endomitosis
Chromocenter
Bands and interbands
Luxury
genes
House-keeping
genes
?Puffs in plolytene chromosome,Gene activity
Puffs,transcriptionally
active chromatin
Labeled with anti-
RNA polymerase II
antibody
If labeled with 3H-U,
then…
?Lampbrush chromosomes
In growing
amphibian oocyte
Stay in meiosis I
Structure
Functions:
Transcript into
RNA,storage
4,Nucleolus
A,Structure
Fibrillar centers
Dense fibrillar component
Granular component
B,Functions of nucleolus,Ribosomal Biogenesis
? Ribosomal Biogenesis:
pre-rRNA synthesis Process Assembly
FC,DFC DFC.GC Cytosol
Directional process
? Characteristics of RNA transcription
NORs in human
chromosomes:
13\14\15\21\22
Code for (in eukaryotes):
18s,28s,5.8s rRNA
?rRNA genes are tandemly arrayed in genome,
?Christmas-Like tree when transcripted
?Synthesis and processing of rRNA precursor
General structure of eukaryotic pre-rRNA transcription units
pre-rRNA transcription units
?Cleavage of pre-rRNA as RNP particles
Mammalia
?Synthesis and processing of 5s rRNA
?5s rRNAs are encoded by a large number genes
(Human,2000)
?5s rRNA gene are located outside the nucleolus.
?5s rRNAs are transcibed by RNA poly III.
?The 3’ end of 5s rRNA is removed during
processing.
? The assembly
of ribosomes
? The nucleolus disappears during mitosis
Nucleolar fusion
of human
fibroblast in
cultrue
