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CHAPTER 8
NUCLEUS AND
CHROMOSOME
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Nucleus is the most important organelle in cell. In mammalian cells,
excepting RBC, all cells else are the nucleus contained cells. In prokaryotic
cells, there is no membrane to package the nucleic acid substance, so, we call
this nucleic substance enriched area as “Nucleoid”.
The major structures of nucleus include: ① nuclear envelope. ②
nucleolus. ③ nuclear matrix. ④ chromatin. ⑤ nuclear lamina.
The major functions of nucleus: ① inheritance: maintain the genetic
continuity of generation by the replication of DNA chromatin and the
proliferation of cell. ② development: regulate the cell differentiation by the
regulation of spatiotemporal sequence of gene expression.
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Structure of nucleus:
(n: nucleolus; N:
euchromatin)
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I. Nuclear envelope (Nuclear membrane)
Nuclear envelope is the lipid bilayer that packages the nucleus. Nuclear envelope
separates the DNA from cell plasma and forms a stable inner environment to: ① protect
the DNA from damage, ② separate the replication of DNA from the translation of RNA
spatiotemporally, ③ the chromatin is anchored on to the nuclear envelope, that is
beneficial to be despiraled, replicated, condensed, and distributed into new nuclei
equally, ④ the pores on the envelope are the channels for the substance exchange.
Nuclear envelope is bilayer membrane:
Nuclear envelope is composed of inner nuclear membrane, outer nuclear
membrane, and perinuclear space. There are nuclear pores on the membrane that are
linked with plasma.
Ribosome is attached to the plasma side of outer nuclear membrane, and the
ribosome is linked with ER. The perinuclear space is linked with ER space. The
intermediate filament (10nm) is attached to the outer nuclear membrane, so, the
locations of nucleus and ER are not movable because of the intermediate filament. The
unmovable locations are convenient to the co-function of nucleus and ER.
The nuclear lamina (meshwork filament proteins) attached to the inner side of the
inner nuclear membrane can stabilize nuclear membrane shape.
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The photo of nuclear envelope by TEM
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Structure of nuclear envelope
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The functions of nuclear lamina:
1．Keeps nuclear shape no changed: If you use the high
concentration salt solution, detergent or nuclease to move away the nuclear
substance, the remaining (nuclear lamina) still presents a nuclear shape. In
addition, the nuclear lamina links nuclear skeleton meshwork and
intermediate filament together to form a continued meshwork for nucleus.
2．Is associated with the assembly of chromatin and nucleus: The
shape of nuclear lamina can be changed during the cell proliferation
phases. In the G1 phase, nuclear lamina can present the anchoring sites for
heterochromatin on the inner side of inner nuclear membrane. At the
ending of the G1 phase, the nuclear lamina will be phosphorylated and the
nuclear envelope will disappear. The B type of nuclear lamina will combine
to the residual vesicles of nuclear membrane, and A type of lamina will be
dissolved in plasma. In the later of M phase, All types of lamina will be
dephosphorylated and assembled again to form nuclear lamina and mediate
the nuclear envelope construction.
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Structure of nuclear lamina
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The nuclear pores are the channels for the substance
transportation:
Nuclear proteins are synthesized in plasma, then will be imported into nucleus
by the pores. The RNAs and the ribosome subunits synthesized in nucleus will be
exported into plasma by the pores also. In addition, it is indicated by a injection
experiment that small molecules can enter the nucleus by diffusion from the pores.
Nuclear pores are composed of 50 different nucleoporins at least, and we call
these pore structure as nuclear pore complex (NPC). Usually, a mammalian nucleus
contains 3,000 nuclear pores. The more activities a cell takes, the more nuclear pores
the cell contains. For example, a frog ovum can contain 37.7X106 nuclear pores, but
a matured cell contains 150~300 nuclear pores only.
The structures of nuclear pore include ① cytoplasmic ring located on the cell
plasma part of the pore complex contains 8 filaments extending into plasma. ②
nuclear ring located on the nuclear plasma part of the pore complex extending 8
filaments also. ③ transporter located in center of the pore as a plug particle. ④
Spoke located on the edge of the pore as the spines.
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The nuclear pore structures on the cell plasma side after an extraction
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The nuclear pore structures on the nuclear plasma side after an extraction
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A model
structure of
the nuclear
pore
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The transportation by nuclear pore is associated with signal transduction:
1982, R. Laskey identified a signal sequence on the C terminal of
nucleoplasmin enriched in nucleus, and the signal can lead protein to enter
nucleus. This signal sequence was named as nuclear localization signal (NLS).
The firstly identified NLS is the T antigen of SV40. This antigen is synthesized
in cell plasma, and transported into nucleus quickly. Its NLS is pro-pro-lyslys-lys-Arg-Lys-val.
NLS is composed of 4 – 8 amino acids containing Pro, Lys and Arg.
NLS is not specific to target protein and will not be cleaved by protease.
Karyopherin is a protein family that is associated with the selective
transportation by the pore, and it is a receptor family actually. The imporin of
them imports proteins into nucleus from cell plasma and the exportin of them
exports the proteins on an opposite direction.
Ran is another protein involved in the transportation by the pore
complex. Ran is a G protein that regulates the assembly and disassembly of
the complex of the protein transported and the receptor used. Ran-GTP
concentration is much higher in the nucleus than in cell plasma.
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Gold labeled
nucleoplasmin
is passing
through the
nuclear pore
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Nuclear plasma protein (nucleoplasmin) is transported by the following
steps: ① The protein combines to the α / β dimer of the receptor (imporin). ②
The complex of the protein transported and the receptor used combines to the
filaments located on the NPC cytoplasmic ring. ③ The filaments curve to the
nuclear center, the transporter structure will be changed to form a hydrophilic
channel, and the protein passes through the channel. ④ The complex of the
protein transported and the receptor used combines to Ran-GTP, the complex is
disassembled and releases out the protein transported. ⑤ The imporin β
combined with Ran-GTP will be exported out of the nucleus, the GTP combined
with Ran will be hydrolyzed in cell plasma, and the Ran-GDP will go back to
nucleus to be transformed to Ran-GTP again. ⑥ The imporin α will be
transported back to cell plasma with the help from exportin.
We know a little about how the macromolecules are transported to cell
plasma from nucleus. In most of cases, the RNA in nucleus is combined with
protein to form an RNP complex, then, transported into cell plasma. There is
nuclear exportation signal (NES) on the protein of RNP complex that can
combine to the intracellular receptor, exportin, to form the complex of RNPexportin-Ran-GTP. In the cell plasma, this complex will be disassembled and
release out the Ran-GTP, RNA, Ran-GDP, exportin, and RNP protein.
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The nucleoplasmin is transported into nucleus
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II. Chromosome
Chromatin was named by W. Flemming in 1879.
Chromosome was named by Waldeyer in 1888.
Chromatin and chromosome are same substance with different shape
presentation in different cell cycle phases.
The chemical components of chromatin:
Chromatin is composed of DNA, histone, nonhistone protein, and some
RNA at ratio about 1:1:(1-1.5):0.05.
DNA:
DNA is the carrier of genetic information. DNA sequences can be sorted
as 3 types: nonrepeated fraction, moderately repeated fraction (101-105),
and highly repeated fraction (>105). DNA forms: B-DNA, Z-DNA, and A-DNA.
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DNA forms (Red color shows the couple backbones)
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Chromosome DNA contains three basic sequences: ① autonomously
replicating DNA sequence (ARS). ARS is the starting site of DNA replication. In
yeast genome, there are 200-400 ARSs included, and most of them contain a AT
enriched 11bp sequence called as ARS consensus sequence (ACS). ②
centromere DNA sequence (CEN) composed of a lot of repeated sequences. ③
telomere DNA sequence (TEL). TEL is similar in different bio organisms, and
composed of 5 – 10bp repeated sequences. Human TEL repeated sequence is
TTAGGG.
In 1983, A. W. Murray et al constructed yeast artificial chromosome
(YAC) contains ARS, CEN, TEL and exogenous DNA with the length of 55kb.
YAC is very useful to transgenic technology and construction of cDNA library
because the length of insert to YAC can be much longer than that to plasmid.
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Three basic sequences of chromosome
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Histone:
Histone is positively charged and contains arginine and lycine. Histone is
alkaline protein.
Histones can be sorted as two types:
1. Highly conserved core histone including H2A, H2B, H3, and H4.
2. Non conserved linker histone including H1 only.
The core histone is highly conserved, especially the H4 is. For example, 2
of 102 amino acids of the H4 of cattle and pea are different, but cattle has been
evoluted 300 million years earlier than pea. The reasons for that may be as the
follows:
1. Most of the amino acids of core histone interact with DNA or other
histones, so, any change of them will cause the fatal mutation.
2. In all bio organisms, the DNA phosphodiester skeleton that interacts with
histone is same.
The core histone head part makes DNA winded round the histone center by
the electronic force between arginine residue and phosphodiester skeleton. By
the described as above, nucleosome can be formed. The tail part of core histone
containing a lot of arginine and lysine residues. The tail part is the site to be
modified after translation.
H1 is easy to be mutated, and it is species specific and tissue specific.
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Nonhistone protein:
Nonhistone protein is the protein that binds to the specific DNA sequence of
chromosome, so, we call it as sequence specific DNA binding protein.
The features of nonhistone protein are as the follows: ① Nonhistone protein
is negatively charged and acidic protein that contains a large number of aspartic
acids and glutamic acids. ② Nonhistone protein can be synthesized during the
whole cell cycle, but histone protein is synthesized during the S phase only. ③
Nonhistone protein can recognize the specific DNA sequence.
The functions of nonhistone are as the follows: ① Help DNA molecules to
be pleated and form different structure domains that are beneficial to DNA
replication and gene transcription. ② Help to start DNA replication reaction. ③
Regulate transcription and gene expression.
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From DNA to chromosome:
There are 23 pairs of chromosomes in a human nucleus. If you open and
extend the DNA molecule in each chromosome, it will be 5cm long. If you link all
DNA molecules in a nucleus together, it will be 1.7 – 2.0 m long. But, the
diameter of nucleus is shorter than 10μm. That is why I told you the genome
information is packaged into the space of a cell nucleus —— thousands of times
smaller than the dot on this i. The primary structure formed by the powerful
compaction is called as nucleosome.
Nucleosome:
If the chromatin is treated by a nonspecific nuclease, the DNA fragments
around 200bp can be obtained in most of cases. If you treat the null DNA with
that enzyme, you will obtain the randomly degenerated fragments of DNA.
Based on this experiment, R. Kornberg figured out the model of nucleosome.
Nucleosome is a beaded structure composed of core particles and linker
DNA. We can describe the structure as the follows: ① Each nucleosome
includes about 200bp DNA, one histone core, and an H1. ② The octameric
histone core is composed of 8 molecules from H2A, H2B, H3, and H4 by two
molecules from each. ③ DNA molecule winds the core particle with a left hand
helix and 80bp for each circle. 1.75 circles for each structure. ④ Adjacent core
particles are linked by a 60bp linker DNA.
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Structures of nucleosome
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Chromatin DNA filament:
The DNA is compacted to be shortened by 7 folds and forms the DNA
filament in 11nm diameter when it was transformed to the beaded nucleosome
chain.
Chromatin DNA exists in another style by that the beaded nucleosome
chain is condensed by 6 folds. Under electron microscope, we can see the
chromatin DNA filament in 30nm diameter that is formed by the overlapped
helix structure of the beaded nucleosome chain.
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The DNA filaments in 30nm and 11nm diameter
(A) Is composed of (B)
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For the advanced package of the chromosome, we keep detail unknown
so far. Probably, it is the serial overlapped or pleated like the follows:
From DNA to Chromosome:
DNA
11nm filament (beaded nucleosome chain)
30nm filament
pleat as loop chain
bind to the sites on nuclear skeleton where is AT
enriched
assembly of chromosome
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Assembly of chromosome
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Heterochromatin and euchromatin:
In the inter phase (G1 and G2) of cell cycle, the chromatin in the nucleus
can be sorted as heterochromatin and euchromatin.
Euchromatin is the DNA regions where the transcription is very active.
Euchromatin looks like loose loop and bright staining under electron
microscope. Euchromatin is easy to be cleaved by nuclease at some
hypersensitive sites.
Heterochromatin is condensed in G phase without any transcription, so, it
was named as inactive chromatin. Heterochromatin is the genetic lazy regions,
and replicated lately, condensed early, that is called as heteropyknosis.
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Heterochromatin
Heterochromatin
(dark staining)
and euchromatin
(bright staining)
Euchromatin
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Constitutive
heterochromatin is
heteropyknosed
chromatin in
each type of
cell and
located in
centromere
region.
The Fig shows
you the
Constitutive
heterochromatin
displayed by
fluorescence
hybridization in
situ.
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Facultative heterochromatin
is heterochromatin appeared
in some special cell type or
developing stage. The X
chromosome of female
mammalians is the facultative
heterochromatin. Usually,
female mammalian cell
contains double X
chromosomes, and one of
them is heterochromatin
called barr body. When a
human embryo is developed
after 16 days, one X
chromosome will be
transformed as barr body with
dark staining. So, we can
identify the sex of a human
embryo by checking the barr
body of the embryo cells in
the amniotic fluid.
The barr body like a drumstick in a white cell
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The structure of chromosome:
In the M phase of cell cycle, chromatin will be transformed as chromosomes
by the powerful condensation. Chromosomes are stick shape with different length.
The metaphase chromosome is the best stage to observe and number them
because the morphology of chromosome is stable at this time.
The number of chromosome is same in the same type of cells from different
individuals of one species. The chromosomes of sex cells are haploid, we mark it
as n. The chromosomes of other cells are diploid, we mark it as 2n. The
chromosomes of some cells of some species are polyploid, such as, 4n, 6n, and
8n.
The different cells from same individual can be different chromosome types.
For example, body cells of rat are 2n, but its liver cells can be 4n, 8n, and 16n.
The chromosome number of human endometrial cell is variable from 2n =17 - 2n
=103, that is not euploidy.
The chromosome number can be different in different species cells. For
examples, human 2n = 46, chimpanzee 2n = 48, fruit fly 2n = 8, wheat 2n = 42,
rice 2n = 24, onion 2n=16.
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The terms used to the structure of chromosome:
1．Chromatid: Metaphase chromosome is composed of two chromatids with a
junction at the centromere site. Each chromatid is formed by the overlapped and
pleated DNA double strands. When the cell is dividing the chromatids can be
separated into two new cells.
2．Chromonema: In the S or G phase cells, each chromonema indicates a
chromatid.
3．Chromomere: Chromomere is the linear beaded particles chain DNA. The
chromomere of heterochromatin is bigger than that of euchromatin.
4．Primary constriction: It is a bright stained hang ditch on the metaphase
chromosome where the centromere is located, so, it can be called as centromere
region. Each chromosome has one localized centromere. The chromosome from
some species has centromere function every where. We call this chromosome as
holocentromere chromosome. For examples, ascarid (round worm) and other
nemas, butterfly. The chromosomes can be sorted by the location of centromere
as following: ① metacentric chromosome. ② submetacentric chromosome. ③
subtelocentric chromosome. ④ telocentric chromosome.
5．Secondary constriction: Excepting primary constriction, the second ditch is
called as secondary constriction. The location of secondary constriction is
unmovable by that we can identify chromosome.
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The terms of chromosome structure
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6．Nucleolar organizing regions (NORs): They are the areas where the genes
for ribosome RNA are located. They can synthesize the 28S, 18S, and 5.8S rRNA
for ribosome. NORs can exist in secondary constriction.
7．Satellite: It is a ball part located at the terminal of chromosome, and linked to
the main part of chromosome by secondary constriction. The satellite located at
terminal of chromosome is called as terminal satellite, and located between two
secondary constrictions is called as intermediate satellite.
8．Telomere: It is the specialized part located at the terminal of chromosome. The
function of telomere is maintenance of the stability of chromosome. Telomere is
composed of the highly repeated fractions, and it is so conserved that it is similar
between the totally different life beings. The component of human telomere is
TTAGGG. Telomere is associated with aging. After each replication of telomere
DNA, the telomere will be shortened by 50 – 100bp. The replication of telomere is
droved by telomerase that has reverse transcriptase activity. This enzyme lacks in
normal cells, so, telomere will become short with the cell proliferation. So, cell will
be aging during this action.
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The nucleolus will be formed in the center of nucleolar organizing regions
The telomeres
displayed using
fluorescence
hybridization in situ.
The sequences of
telomere
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The structure of
centromere:
Centromere and
kinetochore are different
concepts, the former
means the special region
by that the chromatids of
metaphase chromosome
are linked together, and
the later means the outer
surface structure located
on the primary constriction
that is linked to spindle
fibers. Centromere
contains 3 domains:
kinetochore domain,
central domain, paring
domain.
Three domains of centromere
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Kinetochore
domain:
Kinetochore domain
is composed of outer
plate, inner plate,
interzone, and
fibrous corona. The
inner plate combines
to the
heterochromatin of
central domain, outer
plate combines to
filaments of spindle
fibers. Their motor
proteins located on
the fibrous corona to
supply energy to the
chromosomes
separation.
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Central domain: It is located below the centromere and contains the
heterochromatin composed of highly repeated α satellite DNA.
Paring domain: It is located in centromere by that the chromatids of metaphase
chromosome are linked together. There are two types of proteins in this domain:
inner centromere protein (INCENP), and chromatid linking protein (CLIP).
By using anti-centromere antibodies (ACA), INCENP or CENP (centromere protein)
can be identified and sorted as the follows:
Types
Functions
CENP-A
Specific histone to centromere
CENP-B
Binds to satellite DNA in central domain
CENP-C
Binds to kinetochore
CENP-D
Binds to kinetochore
CENP-E
Drive motor protein
CENP-F
Binds to kinetochore
INCENP-A
Link partner chromatid
INCENP-B
Link partner chromatid
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Karyotype and bands display:
The bands display technology of chromosome was developed in 1960s to
1970s, and it brought the chromosome researches to a new and fast developing
stage. The result data about chromosome bands is the very useful background
research for modern genome research programs, gene molecular research
programs, and genetic research programs.
Karyotype is the total features of the chromosomes in M phase. It
includes the number, size, and shape of chromosome. If the paired
chromosomes are arranged by shape and size, a figure will be obtained, and we
call it as karyogram. Karyogram is of characters of species.
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Karyogram of Platypleura kaempferi
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The chromosome banding technology is very important to genetics
research, species classification, and others. This technology includes the cell
and chromosome treatments by physical and chemical methods, chromosome
staining and bands display.
Bands display technologies can be sorted as two types: 1. The bands
distribute on entire chromosome, such as G, Q, and R banding technologies. 2.
The bands distribute in localized region of chromosome, such as C, Cd, T, and
N banding technologies.
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Human G Karyotype. G bands display the regions where AT is enriched
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Human Q Karyotype. Q bands display same thing of G bands
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Human C Karyotype. C bands display the centromere heterochromatin
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Special chromosomes:
Polytene chromosome:
It was identified in some insect saliva cells in 1881.
Polytene chromosome: ① 1,000 – 2,000 folds huger than others. It is the
reason that chromosomes are replicated without separation. ② Polytene. Each
polytene chromosome is composed by 500 – 4,000 helix opened chromosomes.
③ Cell junction and homologous chromosomes combination. ④ Striation. ⑤ In
some life stage of insects, some bands of polytene chromosome become loosed
and form puff and Balbiani ring. Puff can be labeled by H3-TdR, that means puff
is the region where the gene transcription is very active.
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The chromosomes from saliva cells
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Lamp-brush chromosome:
Lamp-brush chromosome was identified in fishes firstly. There are lateral loops
on the chromosome like lamp brush. It is composed of two homologous
chromosomes. Lateral loops are the region with RNA active transcription.
B chromosome:
In 1928, Randolph, a scientist, call normal chromosomes as A chromosomes, and
call abnormal chromosomes existed in many animals and plants as B chromosome.
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III. Nucleolus
Nucleolus may be visible in G phase nucleus. They are spherical and 1 – 2 for
each cell usually. The number and size of nucleolus are depended on the cell type
and function. The more proteins synthesis and the faster proliferation the cell
takes, the more and bigger nucleoli the cell has. Nucleolus disappears before the
cell division, and appears in the end of division. The major functions of nucleolus
are rRNA transcription and ribosome assembly.
Structure of nucleolus:
No any membrane packages nucleolus area. There are three special areas
can be identified under electron microscope: ① fibrillar centers (FC) that are
surrounded by dense fibers, and low electric density. FC contains RNA
polymerase and rDNA that is naked molecule. ② dense fibrillar component (DFC)
that is a loop or half loop to surround FC. Transcription is carried out in the border
region of FC and DFC. ③ granular component (GC) composed of 15-20nm
particles that are the RNPs in different manufactured steps. RNP means the RNA
combined with protein.
Nucleolus chromatins can be sorted as two types: heterochromatin and
euchromatin. The nucleolus heterochromatin is always located around the
nucleolus, so we call them as nucleolus peripheral chromatin. The nucleolus
euchromatin is located in nucleolus, and nucleolus organizing region in that the
rDNA is located.
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Structure of nucleolus
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IV. Ribosome
Ribosome is the manufacturing shop to synthesize proteins. There are about
20,000 ribosomes in an actively growing bacterium. Ribosome proteins are 10% of
total proteins of cell, and its RNA is 80% of cell total RNA.
Structure of ribosome:
The ratios of protein and RNA to ribosome components are 40% and 60%.
The ribosome subunits are composed of the combination of the protein and RNA.
The catalytic activities needed by the translation are presented by ribosome
protein, rRNA and other helper factors.
The ribosomes can be sorted as two types. 70S ribosome exists in bacteria,
mitochondrion, and chloroplast. 80S ribosome exists in the plasma of eukaryotic
cells.
Ribosome is composed of a large subunit and a small subunit. The both
subunits will be combined together when the ribosome synthesizes protein with
mRNA as template. After the translation, the ribosome will be separated as two
parts again. When a protein is translated on an mRNA, many ribosomes can bind
to the mRNA to synthesize the protein. We call these ribosomes for one protein
synthesis as polyribosome. The longer mRNA is used, the more ribosomes are
combined. The polyribosome enhances the efficiency of protein synthesis.
Prokaryotic 5S rRNA and eukaryotic 5.8S rRNA are very conserved for their
structures, so, they can be used to research the bio-evolution.
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Assembly of ribosome:
The DNA fragment encoding rRNA is called as rRNA gene. There are about
200 copies of this gene in a human cell. rDNA contains no histone core, so, it is a
naked DNA.
To transcript rRNA, the RNA polymerase moves ahead along the DNA
molecule. The synthesized rRNA molecules extend out their molecules from the
complex of polymerase and DNA, and form a featherlike structure under
microscope.
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rRNA transcription
The filaments are the
new synthesized 45S
rRNA that combines to
protein to form RNP
complex. The
methylated 45S rRNA
can be cleaved as the
two parts by RNase:
18S rRNA and 32S
rRNA，the latter is
cleaved as 28S rRNA
and 5.8S rRNA. The
synthesized 5S rRNA
will be transported into
nucleolus to join the
assembly of the large
subunits of ribosome.
There is a 60bp non-transcription DNA
fragment between adjacent rRNA genes
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Assembly of ribosome
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Model of a
ribosome
mRNA
Synthesized protein
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http://www.wadsworth.org
V. Nuclear matrix
Nuclear matrix is called as nucleoskeleton that is a meshwork in eukaryotic
cells, that is what I told you before. Because nuclear matrix is associated with
DNA replication, RNA transcription and modification, chromosome assembly, and
virus replication, nuclear matrix is now paid more attentions to.
Components of nuclear matrix:
① Non-histone filaments at ratio of 96%. The nucleoskeleton contains three
scaffold proteins: SC Ⅰ, SCⅡ, and SC Ⅲ.
② A little RNA and DNA: The RNA is important to maintain the skeleton
structure. The DNA is called as matrix /scaffold associated region (MAR or SAR)
where the AT is enriched to form the heterochromatin binding sites.
③ A little phospholipids (1.6%) and sugars (0.9%).
Nuclear skeleton – nuclear lamina – inter filaments – pore complex is a
meshwork system with very good stability.
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The function of nuclear skeleton:
1. Present the scaffolds for DNA replication. DNA can be anchored on to the
scaffold with a replication loop. The enzymes needed by DNA replication are
located on the skeleton, such as DNA polymerase α, DNA primerase, DNA
topoisomerase II.
2. Is the place where gene can be transcripted and modified. There are RNA
polymerase binding sites on the skeleton. New synthesized RNA is
combined to the skeleton for further modification.
3. Is associated with the assembly of chromosome. The nuclear skeleton may
be same thing to chromosome skeleton. 30nm chromatin fibers are
combined to nuclear skeleton to form loops that will be packaged further in M
phase to be assembled as chromosome.
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Chromatin bound on nuclear skeleton or chromosome skeleton
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