Cell nucleus session 2

Nucleus
DR . SUMAN MUKHERJEE
M.D.S. (1ST YEAR )
V.S.D.C.H.
2

Contents (Session 1)
Introduction
Timeline of Nucleus
Evolution of Nucleus
Sizing and Shaping of
Nucleus:
Significance & Mechanism
Nucleus Structures
Nucleus bodies & Domains
Chromosome Territories
Chromatin
Eu-chromatin & Hetero-
chromatin
Nuclear Transport
Cell Nucleus & Aging
Nuclear bodies : Disease
Relevance
Conclusions.
3

Contents (session 2)
 Function of Nucleus
 DNA
 Structure of DNA
 Semiconservative method of DNA
replication
 DNA Packing
 RNA
 The human genome project
 Gene therapy
 DNA Mutations
 DNA Repair
 DNA Cloning
 DNA data storage
 Conclusion
 Bibliography
4

Introduction
• The nucleus is center control of all eukaryotic cell.
• The nucleus contains the genetic information that
deﬁnes the appearance and behavior of all
eukaryotic organisms.
5

Nucleus Function
Gene Expression
 Gene expression is dictated by DNA
sequences within gene promoters that
determine how the RNA synthetic
machinery, RNA polymerase, is
positioned on the gene.
6

• In contrast to prokaryotes, which have a single
RNA polymerase, the synthesis of RNA in
eukaryotic cells is carried out by three different
RNA polymerase complexes.
• In most mammalian cells, RNA polymerase II
(RNA Pol II) is the major activity, transcribing
all protein-coding genes to generate patterns of
gene expression that determine the cell type
(humans have about 250 distinct cell types).
7

DNA Replication
• It is self-evident that, if cells are to maintain
their genetic integrity, the proliferation
process must be tightly controlled so that
cell division is only possible once the
duplication of DNA is complete.
• This process of DNA replication is clearly
central to the activities of cell proliferation,
and is the target for a wide variety of cellular
controls and checkpoints (quality controls)
8

The Cell Division
Cycle
 Multicellular organisms must continuously
replace cells that are damaged or have otherwise
fulﬁlled their natural purpose.
 Cell replacement operates through a
proliferative ‘‘cell cycle’’ during which mitogenic
cues (growth factors) from the local
environment activate a complex series of events
that, ultimately will lead each parental or mother
cell to divide into two genetically identical
daughters.
9

Nucleic Acids
 A complex organic substance present in living cells, especially DNA or RNA, whose
molecules consist of many nucleotides linked in a long chain.
 They are biopolymers, or small biomolecules, essential to all known forms of life.
 They are composed of nucleotides, which are monomers made of three components:
a 5-carbon sugar, a phosphate group and a nitrogenous base.
10

RNA is
present in the
cytoplasm and
in particularly
high conc. in
the nucleolus
of the nucleus.
DNA on the
other hand, is
found mainly
in the
chromosomes.
11

DNA
(deoxyribonucleic acid)
• DNA, a self-replicating material which
is present in nearly all living organisms
as the main constituent of
chromosomes.
• It is the carrier of genetic information.
The shape of a strand of DNA,
photographed with an electron
microscope
13

Structure of DNA
Each strand of DNA contains
 A Sugar Phosphate Backbone
 Four Base Chemicals (Attached in
pairs)
• Adenine pairs with Thymine
• Guanine pairs with Cytosine
14

Semi Replication Structure of DNA
• Semiconservative replication describes the
mechanism by which DNA is replicated in
all known cells.
• Conservative replication would leave the
two original template DNA strands
together in a double helix and would
produce a copy composed of two new
strands containing all of the new DNA
base pairs.
15

Mode of DNA replication: Meselson-Stahl experiment
A key historical experiment that demonstrated the semi-conservative
mechanism of DNA replication.
16

Watson & Crick model of DNA
 In 1953, J.D. Watson (an American
biologist) and F.H.C. Crick (a British
Physicist) proposed the three-dimensional
model of physiological DNA (i. e B-DNA)
on the basis of X-ray diffraction data of
DNA obtained by Franklin and Wilkins.
 Watson, Crick and Wilkins got Nobel Prize
in medicine in 1962.
 Term DNA was given by Zaccharis.
17

 The sides of the ladder are made up of
alternate molecule of phosphate and
deoxyribose.
 The bases make up the rungs of the ladder are
attracted by a weak chemical bonds called
hydrogen bonds.
 The DNA double helix & antiparallel, which
means that the 5’ end of one strand is paired
with the 3’ end of its complementary strand &
vice-versa.
18

 5’--------------->3’
 3’<---------------5’
 2 hydrogen bonds connect T to A; 3
hydrogen bonds connect G to C
 In the DNA, each strand acts as a
template for the synthesis of the
opposite strands during replication
process
19

 In each single strand of DNA :
• Spiral has a pitch of 3.4nm/turn
• Within single turn, 10 base pairs
are seen
• Thus, adjacent bases are separated
by 0.34nm
• Diameter and Width of helix is
1.9 to 2.0nm
20

DNA
TERTIARY
STRUCTURE
• DNA double helix structure coils
around histones
• Nucleosomes(146 nucleotides)
21

Different forms of DNA helix
A DNA
B DNA
C DNA
Z DNA
22

MITOCHONDRIAL DNA
• In addition to nuclear DNA, the
several thousand mitochondria of each
cell possess their own circular double
stranded DNA, mitochondrial DNA
or mtDNA.
• The mitochondrial DNA genome is
very compact containing little
repetitive DNA, and codes for 37
genes.
24

Biological Importance of DNA:
 Hereditary material.
 Autocatalytic role DNA.
 Hetero catalytic role.
 DNA controls cellular metabolism, growth, and differentiation.
 Variations : DNA undergoes recombination its meiosis and occasional mutation
(changes in nucleotide sequences) which creates variations in population and ultimately
contributes to evolution.
 DNA finger printing (-DNA typing or profiling)
 Recombinant DNA technology (Genetic engineering)
25

• The central dogma of molecular biology explains the flow of genetic information,
from DNA to RNA, to make a functional product, a protein.
• The central dogma suggests that DNA contains the information needed to make all
of our proteins, and that RNA is a messenger that carries this information to the
ribosomes.
• The ribosomes serve as factories in the cell where the information is ‘translated’ from
a code into the functional product.
27

• The process by which the DNA instructions are converted into the functional
product is called gene expression.
• Gene expression has two key stages - transcription and translation.
• In transcription, the information in the DNA of every cell is converted into small,
portable RNA messages.
• During translation, these messages travel from where the DNA is in the cell nucleus
to the ribosomes where they are ‘read’ to make specific proteins.
28

• DNA replication, through the action of the enzyme DNA polymerase, takes place
at multiple points known as origins of replication, forming bifurcated Y-shaped
structures known as replication forks.
• The synthesis of both complementary antiparallel DNA strands occurs in the 5'to
3' direction.
• One strand, known as the leading strand, is synthesized as a continuous process.
32

• One of these is called the leading strand, and it
is replicated continuously in the 3' to 5'
direction.
• The other strand is the lagging strand, and it is
replicated discontinuously in short sections.
• These sections are called Okazaki fragments, and
they are short lengths of DNA.
34

Each of us has enough DNA to reach from here to the sun
and back, more than 300 times. How is all of that DNA
packaged so tightly into chromosomes and squeezed into a
tiny nucleus?
It is estimated that the human body contains about 50 trillion cells—which works out
to 100 trillion meters of DNA per human. Now, consider the fact that the Sun is 150
billion meters from Earth. This means that each of us has enough DNA to go from
here to the Sun and back more than 300 times, or around Earth's equator 2.5 million
times! How is this possible?
37

• The answer to this question lies in the fact that certain proteins compact
chromosomal DNA into the microscopic space of
the eukaryotic nucleus.
• These proteins are called histones, and the resulting DNA-protein
complex is called chromatin.
38

• Histones are a family of small,
positively charged proteins termed
H1, H2A, H2B, H3, and H4 (Van
Holde, 1988).
• DNA is negatively charged, due to the
phosphate groups in its phosphate-
sugar backbone, so histones bind with
DNA very tightly.
39

Chromosomes are composed of
DNA tightly-wound around
histones.
Chromosomal DNA is packaged inside
microscopic nuclei with the help of
histones. These are positively-charged
proteins that strongly adhere to negatively-
charged DNA and form complexes called
nucleosomes. Each nuclesome is composed
of DNA wound 1.65 times around eight
histone proteins. Nucleosomes fold up to
form a 30 nm chromatin fiber, which
forms loops averaging 300 nm in length.
The 300 nm fibers are compressed and
folded to produce a 250 nm-wide fiber,
which is tightly coiled into the chromatid of
a chromosome. 40

RNA (Ribonucleic acid)
• RNA is a polymer of ribonucleotides
linked together by 3’-5’ phosphodiester
linkage
• The main job of RNA is to transfer the
genetic code need for the creation of
proteins from the nucleus to the
ribosome.
• This process prevents the DNA from
having to leave the nucleus. This keeps
the DNA and genetic code protected
from damage.
Although single stranded, RNA is not always
linear. It has the ability to fold into complex
three dimensional shapes and form hairpin
loops.
41

Seeing RNA at the nanoscale Strand Of RNA On DNA Fragment.
43

TRIPLET CODONS
• The triplet of nucleotide bases in the
mRNA that codes for a particular amino
acid is called a codon.
• Termination of translation of the mRNA
is signalled by the presence of one of the
three stop or termination codons.
44

Human Genome Project
• The Human Genome Project (HGP) was the
international, collaborative research program
whose goal was the complete mapping and
understanding of all the genes of human beings.
• All the genes together are known as "genome."
Integral pieces of our cellular
machinery have been altered by
coming into contact with viruses.
45

• The HGP has revealed that there are probably about 20,500 human genes.
• The completed human sequence can now identify their locations.
• This ultimate product of the HGP has given the world a resource of detailed
information about the structure, organization and function of the complete set of
human genes.
• This information can be thought of as the basic set of inheritable "instructions" for
the development and function of a human being.
46

Gene Therapy
• Gene therapy is an experimental technique that uses genes to treat or prevent disease.
In the future, this technique may allow doctors to treat a disorder by inserting a gene
into a patient’s cells instead of using drugs or surgery.
• Researchers are testing several approaches to gene therapy, including:
• Replacing a mutated gene that causes disease with a healthy copy of the gene.
• Inactivating, or “knocking out,” a mutated gene that is functioning improperly.
• Introducing a new gene into the body to help fight a disease.
47

• Although gene therapy is a promising treatment option for a number of diseases
(including inherited disorders, some types of cancer, and certain viral infections), the
technique remains risky and is still under study to make sure that it will be safe and
effective.
• Gene therapy is currently being tested only for diseases that have no other cures.
48

• The treatment, which was first tested in humans in 1990, can be performed inside or
outside of the body.
• When it’s done inside the body, doctors may inject the virus carrying the gene in
question directly into the part of the body that has defective cells.
49

• This is useful when only certain populations of
cells need to be “fixed.”
• For example, researchers are using it to try to
treat Parkinson's disease, because only part of the
brain must be targeted.
• This approach is also being used to treat eye
diseases and hemophilia, an inherited disease that
leads to a high risk for excess bleeding, even from
minor cuts.
50

• Out-of-the-body gene therapy has already been used to treat severe combined
immunodeficiency—also referred to as SCID or boy-in-the-bubble syndrome—
where patients are unable to fight infection and die in childhood.
• In this type of gene therapy, scientists use retroviruses, of which HIV is an
example.
• These agents are extremely good at inserting their genes into the DNA of host
cells.
• More than 30 patients have been treated for SCID, and more than 90 percent of
those children have been cured of their disorder—an improvement over the 50
percent chance of recovery offered by bone marrow transplants.
51

Timing is everything when it comes
to treating this rare genetic disorder
that causes complete
immunodeficiency
52

• A risk involved with retroviruses is that
they may stitch their gene anywhere into
DNA, disrupting other genes and causing
leukemia.
• Unfortunately, five of the 30 children
treated for SCID have experienced this
complication; four of those five, however,
have beaten the cancer.
• Researchers are now designing delivery
systems that will carry a much lower risk
of causing this condition.
53

DNA Mutation
• DNA mutations are permanent changes in
the DNA sequence of a gene.
• Mutations range in their severity.
• Some damage the way a cell or whole
organism functions, or even cause lethality,
while others have no effect.
• Mutations also range in the amount of
DNA altered. They can involve from a
single nucleotide up to large segments of
chromosomes.
54

• Inherited: parents that have
mutations can pass them to their
offspring.
• Germ line mutations: are present in
every cell of an individual, including
the egg or sperm used in the
production of offspring.
55

• De novo (new) mutations: occur by chance in one or a few eggs or sperm, or
just after fertilization, and are NOT present in every cell of a parent. These
explain situations where a child has a genetic disorder that is unseen in the
family history.
• Acquired: environmental agents, called mutagens, can alter DNA. An example
of a common mutagen are the UV wavelengths in sunlight associated with
skin cancer (see image). Acquired mutations are typically not passed to
offspring but can be if they alter DNA sequences in egg or sperm.
56

Types of mutation
• Insertion/Duplication/Deletion: the addition or subtraction of nucleotides
from DNA sequence. They can be as small as single nucleotides or large
enough to visualize on a chromosome and involve tens to hundreds of
thousands of nucleotides.
• Point Mutation: the change in one nucleotide for another. For example, an “A”
becomes a “T”.
57

• Translocation: the movement of a segment of DNA from one chromosome to
another.
• Inversion: the 180° flip of a DNA segment so that that it is reversed compared
to the original structure.
• Ultimately whether or not a particular mutation causes a detrimental effect is
due to the location of the mutation within a gene (or genes) as well as the
significance of that gene’s function.
58

DNA Repair
• DNA repair is a collection of processes by which a cell identifies and corrects
damage to the DNA molecules that encode its genome.
60

Examples of DNA damage induced
by radiation and chemicals
Direct repair of thymine dimers
UV-induced thymine dimers can be repaired
by photoreactivation, in which energy from
visible light is used to split the bonds forming
the cyclobutane ring. 61

Nucleotide-excision repair of thymine
dimers
Damaged DNA is recognized and then
cleaved on both sides of a thymine
dimer by 3′ and 5′ nucleases. Unwinding
by a helicase results in excision of an
oligonucleotide containing the damaged
bases. The resulting gap is then filled by
DNA polymerase and sealed by ligase.
62

Mismatch repair in mammalian cells
Mismatch repair in mammalian cells is
similar to E. coli, except that the
newly replicated strand is
distinguished from the parental strand
because it contains strand breaks.
MutS and MutL bind to the
mismatched base and direct excision
of the DNA between the strand
break and the mismatch.
63

Postreplication repair
The presence of a thymine dimer blocks
replication, but DNA polymerase can bypass
the lesion and reinitiate replication at a new
site downstream of the dimer. The result is a
gap opposite the dimer in the newly
synthesized DNA strand. In recombinational
repair, this gap is filled by recombination with
the undamaged parental strand. Although
this leaves a gap in the previously intact
parental strand, the gap can be filled by the
actions of polymerase and ligase, using the
intact daughter strand as a template. Two
intact DNA molecules are thus formed, and
the remaining thymine dimer eventually can
be removed by excision repair.
64

DNA Cloning
• DNA cloning is the process of making multiple,
identical copies of a particular piece of DNA.
• DNA cloning is the starting point for
many genetic engineering approaches to
biotechnology research.
• Large amounts of DNA are needed for genetic
engineering. Multiple copies of a piece of DNA
can be made either by using polymerase chain
reaction (PCR) or by cloning DNA in cells.
65

• The term ‘cloning’ is also used to
describe other laboratory processes:
• Reproductive cloning is the process
of making a genetically identical copy
of an organism.
• Therapeutic cloning is the process of
making multiple copies of a cell to
treat a disease.
66

DNA cloning is used to create a large number
of copies of a gene or other piece of DNA.
The cloned DNA can be used to :
• Work out the function of the gene
• Investigate a gene’s characteristics (size,
expression, tissue distribution)
• Look at how mutations may affect a gene’s
function
• Make large concentrations of the protein
coded for by the gene
67

Cloned
monkeys
Zhong Zhong
and Hua Hua
are seen at
the non-
human
primate
facility
68

DNA Data Storage
“The libraries of the future will be made of DNA.”
• Various scientists have begun to explore the
possibility of using DNA to store information,
called Nuclear Acid Memory (NAM).
• This would involve the data being “translated”
into the letters GATC, the base nucleic acids of
DNA. DNA strands would then be created which
could be translated back into the “original” by
being sequenced. Researchers recently stored
archival-quality versions of music by Miles Davis
and Deep Purple and also of a short GIF in DNA
form. 69

• DNA is durable and increasingly easy to
produce and read. It will keep for thousands
of years in the right storage conditions. DNA
might be stored anywhere that is dark, dry,
cold, and arguably would not take up a great
deal of room
• It remains to be seen whether future storage
and writing will be as easy to access, and who
will be in control of humanity’s information
and memory in the coming decades and
centuries.
Tens of years from now, 100 years from now,
1,000 years from now [people] will be able to
take that speck of DNA and read it back on
a machine that reads DNA.
70

conclusion
• DNA is very important for life.
• It can replicate well, which means that the
next generation will retain the
characteristics of the parents.
• It is capable of change, which means that it
provides for variation and was crucial for
evolution to occur.
• It also codes for proteins that help express
genes and traits of the organism.
71

Bibliography …
• How does gene therapy work? Arthur Nienhuis scientific american.
• The Cell: A Molecular Approach.
72

Different parts of chromosome
1. Pellicle and Matrix
2. Chromatids, Chromonema and
Chromomeres
3. Centromeres
4. Secondary Constriction
5. Satellite
6. Telomere.
74

Structure of chromosome
contains
• Sister chromatids
• Centromere
• DNA
• telomeres
75

karyolymph
• karyolymph kar·y·o·lymph (kār'ē-ə-lĭmf') n.
The colorless gel or liquid component of
the cell nucleus in which stainable elements
are suspended, now known to be
euchromatin. Also called nuclear
hyaloplasm .
76

karyohexis
• Karyorrhexis (from Greek κάρυον
karyon, "kernel, seed or nucleus", and
ῥῆξις rhexis, "bursting") is the
destructive fragmentation of the nucleus
of a dying cell whereby its chromatin is
distributed irregularly throughout the
cytoplasm.
77

Nucleus
DR . SUMAN MUKHERJEE
M.D.S. (1ST YEAR )
V.S.D.C.H.
78

Cell nucleus session 2

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