NATIONAL PHARMACEUTICAL UNIVERSITY

DEPARTMENT OF VETERINARY MEDICINE AND

PHARMACY
Lecture on Biology and
genetics principles
specialty 226 Pharmacy

The Nucleus
Hereditary material organization
Lecturer: associate professor Department
of Veterinary medicine and pharmacy
Ph.D. Dotsenko Roman Valeryevich
 LECTURE PLAN
• Introduction
• History Note
• Function
• Structural Component
• Chromatin
• Nucleolus
• DNA
• RNA
• Mutation
• Genetic Engineering
 Recommended literature
Maloshtan L.M., Filiptsova O.V. 2011. Biology and genetics principles,
Publisher house of NUPh, Kharkiv, pp. 4-18,56-68.

https://www.britannica.com/list/6-cell-organelles

Craig, Nancy (2014). Molecular Biology, Principles of Genome Function.

ISBN 978-0-19-965857-2.

Darwin, Charles (1859). On the Origin of Species, John Murray.

"biology, n". Oxford English Dictionary online version. Oxford University

Press. September 2011. Retrieved 1 November 2011.

Bruce Alberts: Molecular.Biology.Of.The.Cell.5th.Ed

Arthur C. Guyton; John E. Hall. Text book of Medical Physiology. Tenth

edition
 The Cell

The cell (from Latin cello, meaning "small room") is the basic structural,
functional and biological unit of all known living organisms.
Each living cell carries out the tasks of taking food, transforming food
into energy, getting rid of wastes, and reproducing.

Every living thing - from the tiniest bacterium to the largest whale - is
made of one or more cells.
 Discovery Of Cells

Robert Hooke (1665):

Observed a thin slice of cork (dead plant cells) with a
microscope.
He described what he observed as "little boxes" (cells).
The idea that all living things are made of cells
was put forward in about 1840 and in 1855 came
'Cell Theory' - i.e. 'cells only come from other cells'
- contradicting the earlier theory of 'Spontaneous Generation'

Principles Of Cell Theory

1. All living things are composed of one or more cells.
2. Cells are organisms' basic units of structure and function.
3. Cells come only from existing cells.
The Nucleus is a large, often rounded organelle. Most animal and plant
cells have a nucleus, which contains a copy of the DNA of the organism (a
notable exception would be mammalian red blood cells, which lack a
nucleus).

Chemically coded on the DNA are the instructions to produce every

protein an organism needs to make new cells, digest foods, produce
necessary chemicals, move, and all other cell-level life functions.

The exact sequences are copied inside the nucleus by molecules of

messenger RNA (mRNA), which pass out of the nucleus to ribosomes for
production.

Ribosomes are able to read the sequence of ingredients on the mRNA

and attach amino acids together to form the new protein.
 Introduction
The nucleus is a cellular organelle that is found in eukaryotes, but
not in prokaryotes.
The nucleus contains the genetic information that defines the
appearance and behavior of all eukaryotic organisms.
The genetic material—DNA—is packaged as a DNA—protein
complex, known as chromatin, into units of manageable size,
termed chromosomes.

At the other, it is viewed as a largely disordered, membrane bound bag of

DNA and other molecules, in which all "structures" are no more than
transient complexes that form and disperse as a result of Transcription,
Replication, & RNA Processing activities in various regions of the
genome.
 “The Immortal Molecule within our body is DNA and as such could
represent the reductionist Answer to the old Question....
“Where Were You Before Your Grandmother Was Born?"

The continuity and capability of a DNA sequence to cycle on through subsequent

human generations and to adapt and evolve into a complex Human
system, which is able to build Skyscrapers and travel in Space, represents a
spectacular feat of Molecular Management”.

Donald S. Coffey Commentary In “Nature

Medicine" Vol. 4 No. 8, 882 - 885 (1998)
 Function

The Nucleus (House of Genetic Material) contains a Blueprint for all cell structures
and activities encoded in the DNA of the chromosomes.

It contains the molecular machinery to replicate its DNA and to synthesize RNA.

* Pre-rRNA Processing
* Ribosomal RNA (rRNA) Transcription
* Production of Ribosome Subunit & Exports them into Cytoplasm for Assembly.

Interactions and translocation of a large number of proteins and RNAs are conducted
and coordinated. Because functional ribosomes do not occur in the nucleus, no
proteins are produced here.

Macromolecular transfer between the nuclear and cytoplasmic compartments is

regulated.
NUCLEAR ENVELOPE
СHROMATIN
NUCLEOLUS
NUCLEAR MATRIX
(NUCLEOPLASM)
 Nuclear Envelope
First description : M. Watson , 1955

Nuclear Envelope (NE) that surrounds the nucleus

is made of two membranes separated by the
Perinuclear Space (PS).

The Outer membrane (Cytoplasmic Surface) bound to it and is continuous with the
Rough Endoplasmic Reticulum (RER).

The Inner membrane (Nuclear Surface) is associated with a Nuclear Lamina (NL) for
the attachment of Chromatin.

The two membranes fuse at many places to form Nuclear Pores (NP).

It controls movement of molecules between Nucleus & Cytoplasm

 Nuclear Lamina
The Nuclear lamina is formed of filaments proteins, the Lamins, which assemble as a
lattice adjacent to the inner nuclear membrane.

When the nuclear envelope disperses during early prophase of cell division, at least
some Lamin proteins remain attached to the membrane fragments and reassembly
of the nuclear lamina immediately after cell division facilitates re-formation of the
nuclear envelopes of the two new nuclei.

The nuclear lamina also contains Binding sites

for Chromatin, helping to organize this material
in the nucleus.

Nucto*
\90nnm
 Nuclear Pore Complex
A nuclear pore complex (NPC) is membrane-bound channels and a made of
transmembrane proteins (Nucleoporins) which form an octagonal annulus or ring,
with filaments extending into both the cytoplasm and the nucleus.

The Nucleus of a typical mammalian cell contains 3000-4000 pore complexes. Vary
in number according to the cell's function and stage of development.

Regulates most Bidirectional Transport between the nucleus and the cytoplasm.

The nuclear envelope is impermeable to all sizes of ions and the exchange of
substances between the nucleus and the cytoplasm occur only through the nuclear
pores.

Ions and small molecules pass through the nuclear pore by passive diffusion.
 Passage of Substances Through
Nuclear Pore

The molecules that pass from the cytoplasm into the nucleus are mainly:

Ions
Nucleotides for DNA synthesis;
Proteins for binding to DNA

The molecules that pass from the nucleus into the cytoplasm are m-RNA and r-RNA
bound to protein as RNA-protein complexes.

Protein molecules require a special mechanism to pass through the nuclear pores.
 Chromatin

Nucléosome (U-body) : Basic Structural unit of Chromatin

Core of Histone Octamers.
Two Loops of DNA -146 Base Pairs

Composition

Coiled Strands of DNA & Proteins:

Histone Proteins (80%) -Basic Proteins only in the Nucleus H2A,

H2B, H3, H4 - Nucleosome Core
H1-Keeping in place the wrapped DNA, Binds to the "Linker DNA"
Non-Histone Proteins (20%) - Acid Proteins Nuclear protein matrix
 Chromatin
Chromatin, in non-dividing nuclei, is in fact the Chromosomes in a different degree of
uncoiling.
According to the degree of chromosome condensation, two types of
chromatin, (Heterochromatin & Euchromatin) can be distinguished with both
the light and electron microscopes.

Heterochromatin
(Condensed) - 90% Gr. heteros, other, + chroma, color

Heterochromatin is electron dense, appears as coarse granules in the electron

microscope and as basophilic clumps in the light microscope.
• Marginal chromatin - Nuclearmembrane
• Chromocenters - nucleoplasm
• Nucleolar-associated chromatin
• Constitutive heterochromatin - Inactive, around the chromosome
centromere.
• Facultative heterochromatin - Sex chromatin
 Chromatin

Euchromatin

( Extended) -10% Gr. eu, good, true + chroma, color

A lightly packed form of chromatin (DNA, RNA and Protein)

Comprises the most active portion of the Genome - Replication and Transcription

Euchromatin is the less coiled portion of the chromosomes, visible as a

finely dispersed granular material in the electron microscope and as lightly
stained basophilic areas in the light microscope.
The proportion of heterochromatin to euchromatin accounts for the light-to-dark
appearance of nuclei in tissue sections as seen in light and electron microscopes.

Chromatin is composed mainly of coiled strands of DNA bound to basic proteins

called histones and to various non-histone proteins.
 Chromatin
The basic structural unit of chromatin is the Nucleosome which consists of

* A core of four types of Histones: two copies each of histones H2A, H2B, H3, and
H4, around which are wrapped 146 DNA Base Pairs.
* An additional 48-base pair segment forms a link between adjacent
nucleosomes, and another type of histone (H1 or H5) is bound to this DNA.
 Chromatin
This organization of chromatin has been referred to as "Beads-on-a-String."

Non-Histone Proteins are also associated with chromatin, but their arrangement is
less well understood.

Medical Application
The intensity of nuclear staining of the chromatin is frequently used to distinguish
and identify different tissues and cell types in the light microscope.
The presence of a prominent nucleolus indicates that the cell is actively synthesizing
protein. A prominent nucleolus is present in protein-secreting cells such as those of
the pancreas, plasma cells, developing blood cell precursors.
 Chromosome

Denomination: W. von Waldeyer, 1888

Definition : a very long DNA molecule (that contains many genes) and associated proteins,
carrying portions of the hereditary information of an organism.

Total number in humans - 46 (2n) (23 homologous pairs)

22 pairs of Autosomes

1 pair of Gonosomes (sex chromosomes) - X and Y Size:

length- 0.1 to 30 pm (average 3-8pm)
(51 million to 245 million base pairs)

Thickness - 0.5-2 pm
Single DNA molecule-1.7-8.5 cm
Total length-1.7 m
 Chromosomal Aberrations

A chromosomal aberration is a major change in DNA that can be observed at the

level of the chromosome.
There are four types of aberrations: Inversions, Translocations, Duplications, and
Deletions.
An Inversion occurs when a chromosome is broken and a piece becomes reattached
to its original chromosome but in reverse order—it has been cut out and flipped
around.
A Translocation occurs when one broken segment of DNA becomes integrated into a
different chromosome.
Duplications occur when a portion of a chromosome is replicated and attached to
the original section in sequence.

Deletion aberrations result when a broken piece becomes lost or is destroyed before
it can be reattached.
 Nucleolus

The nucleolus is a generally spherical, highly basophilic structure present in

the nuclei of cells active in protein synthesis.

Fibrillar Centre - Pale-stained region:

• RNA polymerase I

Fibrillar part, pars fibrosa:

• Nucleolonema
• Newly synthesizes rRNA
Granular part, pars granulosa:
• Ribonucleoprotein particles

It is the site of rRNA Transcription and Processing, & of Ribosome Assembly.

 Transcription & Processing of rRNA

The nucleolus, which is not surrounded by a membrane, is organized around the

chromosomal regions that contain the genes for the 5.8Sf 18S, & 28S rRNAs.
Eukaryotic ribosomes contain Four types of RNA, designated the 5S, 5.8S, 18S, and
28S rRNAs.
The 5.8S, 18S, and 28S rRNAs are transcribed as a single unit within the nucleolus by
RNA Polymerase I, yielding a 45S ribosomal precursor.
Transcription of the 5S rRNA, which is also found in the 60S ribosomal subunit, takes
place outside of the nucleolus and is catalysed by RNA Polymerase III.
The 45S pre-rRNA is processed to the 18S rRNA of the 40S (Small) ribosomal subunit
and to the 5.8S and 28S rRNAs of the 60S (Large) ribosomal subunit.
The final steps of maturation follow the export of preribosomal particles to the
Cytoplasm, yielding the 40S and 60S ribosomal subunits.

The Smaller Ribosomal Subunit (40S), which contains only the 18S rRNA, matures
more rapidly than the Larger Subunit (60S), which contains 28S, 5.8S, and 5S rRNAs.
 Ribosome Assembly
The formation of ribosomes involves the assembly of the ribosomal precursor RNA
with both Ribosomal Proteins and 5S rRNA.
The genes that encode Ribosomal Proteins are transcribed outside of the nucleolus
by RNA polymerases II, yielding mRNAs that are translated on cytoplasmic
ribosomes.
Ribosomal Proteins are imported to the nucleolus from the cytoplasm and begin to
assemble on pre-rRNA to form preribosomal particles prior to its cleavage.
The genes for 5S rRNA are also transcribed outside of the nucleolus, in this case by
RNA polymerase III, 5S rRNAs similarly are assembled into preribosomal particles
within the nucleolus.
Ribosomal proteins and the 5S rRNA are incorporated into preribosomal particles as
cleavage of the pre-rRNA proceeds.
 Nuclear Matrix (Nucleoplasm)

A highly Dynamic structure

Composition - Amorphous
* Nucleoskeleton -- Proteins+ RNA
* Nuclear Lamina
* Numerous enzymes
* Metabolites
* Ions
* Crystalline inclusions
* Viruses and
* Other inclusions
 Barr Body
In 1949, Murray Barr and Ewart Bertram found that staining cat cells with Feulgen, a
stain that binds to DNA, often resulted in the appearance of a "drumstick".
They identified a highly condensed structure in the interphase nuclei of somatic cells
in female cats that was not found in male cats.
This structure became known as the Barr body. In 1960, Susumu Ohno correctly
proposed that the Barr body is a highly condensed X chromosome.
 Barr Body
Medical Application
S Diagnostics in Endocrinology
S Forensic Medicine Practice
S Study of Inherited Chromosome Anomalies - Klinefelter's & Turner Syndromes.
S Disclosure of the Genetic sex - In Hermaphroditism & Pseudohermaphroditism

Number of X Number of
Chromosome Phenotype Composition Chromosomes Barr Bodies

Normal female XX
2 1
Normal male XY
1 0

Turner syndrome XO 1 0
(female)

Triple X syndrome XXX 3 2

(female)

Klinefelter syndrome XXY 2 1

(male)
 DNA
DNA: A Nucleic Acid that contains the Genetic Information (Genes)

Chemical composition: Long Polymers of Nucleotides

Phosphate Group
Deoxyribose - Five-Carbon Sugar
Nitrogenous Bases
Purine - Adenine and Guanine
Pyrimidine - Thymine and Cytosine

Structure
Arranged in the form of a Double Helix, Two anti-parallel strands
It looks like a Twisted Ladder: The sides of the ladder are composed of nucleotides

The strands of the ladder are bonds between the bases where Adenine only forms a
bond with Thymine, and Guanine with Cytosine.
Nucleotides are joined together by a phosphodiester linkage between 5 1 and 31
carbon atoms to form nucleic acids.
The linear sequence of nucleotides in a nucleic acid chain is commonly abbreviated
by a one-letter code, A-G-C-T-T-A-C-A, with the 5' end of the chain at the left.

The relationship between Genetic information carried in DNA and proteins

 The Elucidation of DNA Structure

The basic chemical composition of nucleic acids was elucidated in the 1920s through the efforts of
P. A.Levene.

Despite his major contributions to nucleic acid chemistry, Levene mistakenly believed that DNA was
a very small molecule, probably only four nucleotides long, composed of equal amounts of the four
different nucleotides arranged in a fixed sequence.

Further advances in our understanding of DNA structure awaited the development of significant
new analytical techniques in chemistry. One development was the invention of paper
chromatography by Archer Martin and Richard Synge between 1941 and 1944.

By 1948 the chemist Erwin Chargaff had begun using paper chromatography to analyse the base
composition of DNA from a number of species. He soon found that the base composition of DNA
from genetic material did indeed vary among species just as he expected.

Furthermore, the total amount of purines always equalled the total amount of pyrimidines; and the
adenine/thymine and guanine/ cytosine ratios were always 1. These findings, known as Chargaff's
rules, were a key to the understanding of DNA structure.
Chargaff’s rules
Another turning point in research on DNA structure was reached in 1951 when Rosalind Franklin
arrived at King's College, London, and joined Maurice Wilkins in his efforts to prepare highly
oriented DNA fibers and study them by X-ray crystallography.

By the winter of 1952-1953, Franklin had obtained an excellent X-ray diffraction photograph of
DNA.

"While the biological properties of

deoxypentose nucleic acid suggest a
molecular structure containing
great complexity, X-ray diffraction
studies described here... show the
basic molecular configuration has
great simplicity. "
Rosalind Fraaklin
The same year that Franklin began work at King's College, the American biologist James Watson went
to Cambridge University and met Francis Crick. Although Crick was a physicist, he was very interested
in the structure and function of DNA, and the two soon began to work on its structure.

Their attempts were unsuccessful until Franklin's data provided them with the necessary clues. Her
photograph of fibrous DNA contained a crossing pattern of dark spots, which showed that the
molecule was Helical. The dark regions at the top and bottom of the photograph showed that the
purine and pyrimidine bases were stacked on top of each other and separated by 0.34 nm. Franklin
had already concluded that the phosphate groups lay to the outside of the cylinder.

It now seems certain that the amino

acid sequence of any protein is
determined by the sequence of
bases in some region of a particular
nucleic acid molecule.
(Francis Crick)
Finally, the X-ray data and her determination of the density of DNA indicated that the helix
contained two strands, not three or more as some had proposed.

Without actually doing any experiments themselves, Watson and Crick constructed their model by
combining Chargaff's rules on base composition with Franklin's X-ray data and their predictions
about how genetic material should behave.

By building models, they found that a smooth, two-stranded helix of constant diameter could be
constructed only when an adenine hydrogen bonded with thymine and when a guanine bonded
with cytosine in the centre of the helix.

They immediately realized that the double helical structure provided a mechanism by which
genetic material might be Replicated. The two parental strands could unwind and direct the
synthesis of complementary strands, thus forming two new identical DNA molecules.

Watson, Crick, and Wilkins received the Nobel Prize in 1962 for their discoveries.

Franklin could not be considered for the prize because she had died of cancer in 1958 at the age
of thirty-seven.
"It Has Not Escaped Our Notice That The Specific Pairing
We Have Postulated Immediately Suggests A Possible
Copying Mechanism For The Genetic
Material."

James Watson and Francis Crick Proposing

the Double Helical Structure of DNA
(Nature 171:737-738 (1953)
DNA has four properties that enable it to function as genetic material. It can...

■ Store Information that determines the characteristics of cells and organisms;

■ Use this information to Direct the Synthesis of structural and regulatory proteins
essential to the operation of the cell or organism;
■ Mutate, or chemically change, and transmit these changes to future
generations; and
■ Replicate by directing the manufacture of copies of itself.

Each strand of the DNA double helix contains a sequence of nucleotides that is
exactly complementary to the nucleotide sequence of its partner strand. Each strand
can therefore act as a template for the synthesis of a new complementary strand.
 Central Dogma
The Central Dogma of Molecular Biology is an explanation of the flow of genetic information within
a biological system. It was first stated by Francis Crick in 1958 and re-stated in a Nature paper
published in 1970.

DNA contains the complete genetic information that defines the structure and function of an
organism. Proteins are formed using the genetic code of the DNA. Three different processes are
responsible for the inheritance of genetic information and for its conversion from one form to
another.

Replication : A double stranded nucleic acid is duplicated to give identical copies. This process
propagates the genetic information.

Transcription : A DNA segment that constitutes a gene is read and transcribed into a single
stranded sequence of RNA. The RNA moves from the nucleus into the cytoplasm.

Translation : The RNA sequence is translated into a sequence of amino acids as the Protein is
formed. During translation, the ribosome reads three bases (Codon) at a time from the RNA and
translates them into one amino acid.
 Central Dogma
structural proteins

(transcription) (translation) proteins carrier

enzymatic/hormonal
 Replication
Before a cell divides, its DNA must replicate so that each daughter cell receives the
same set of genetic instructions. Clues to the self-replication mechanism came from
Watson and Crick's report on DNA's chemical structure. The paper ends with the
tantalizing statement,

"It has not escaped our notice that the specific pairing we have postulated
immediately suggests a possible copying mechanism for the genetic material."

They envisioned DNA unwinding, exposing unpaired bases that would attract their
complements, and neatly knitting two double helices from one. This route to
replication is called Semiconservative because each DNA double helix conserves half
of the original molecule

Bidirectional Replication.
 The copying of genetic information by DNA replication. In this process, the two
strands of a DNA double helix are pulled apart, and each serves as a template for
synthesis of a new complementary strand.
 Replication
Each strand of the DNA double helix contains a sequence of nucleotides that is exactly
complementary to the nucleotide sequence of its partner strand. Each strand can therefore act as a
template for the synthesis of a new complementary strand.

DNA Synthesis Begins at Replication Origins

The process of DNA replication is begun by initiator proteins that bind to the DNA and pry the two
strands apart, breaking the hydrogen bonds between the bases. The positions at which the DNA is
first opened are called Replication Origin. They usually marked by a particular sequence of
nucleotides.
 New DNA Synthesis Occurs at Replication Forks
During the DNA replication it is possible to see Y-shaped junctions in the DNA, called REPLICATION
FORKS. At these forks, the replication machine is moving along the DNA, opening up the two strands
of double helix and using each strand as a template to make new daughter strand. Two replication
forks are formed starting from each replication origin, and they move away from the origin in both
directions, unzipping DNA as they go. Enzymes called Helicases unwind and hold apart replicating
DNA

The Replication Fork Is Asymmetrical

The 5'-to-3' direction of the DNA polymerization mechanism poses a problem at replication fork
One new DNA is being made on a template that runs in one direction (3'to 5'), whereas the other
new strand is being made on a template that runs in the opposite direction (5' to 3').
 DNA Polymerase
DNA Polymerase, however, can catalyse the growth of the DNA chain in only one direction: it can
add subunits only to the 3'end of the chain. As a result, a new DNA chain can be synthesized only in
5' to 3' direction.

The DNA strand whose 5' end must grow is made DISCONTINUOUSLY, in successive separate small
pieces. These small pieces are called Okazaki fragments.
Helicase unwinds double helix.

Primase adds short RNA primer to template strand.

Binding proteins stabilize each strand.

DNA polymerase binds nucleotides to form new strands.

Ligase joins Okazaki fragments and seals nicks in sugar phosphate backbone.
DNA replication is incredibly accurate; after proofreading, DNA polymerase
incorrectly incorporates only about 1 in a billion nucleotides.
Other repair enzymes help ensure the accuracy of DNA replication by cutting out and
replacing incorrect nucleotides.
Nevertheless, mistakes occasionally remain. The result is a Mutation, which is any
change in a cell's DNA sequence.
 RNA
We now turn our attention to RNA, which differs from DNA in three respects.

First, the backbone of RNA contains Ribose rather than 2-deoxyribose. That is, ribose
has a hydroxyl group at the 2nd position.
Second, RNA contains Uracil in place of thymine. Uracil has the same single-ringed
structure as thymine, except that it lacks the 5-methyl group.
Third, RNA is usually found as a Single Polynucleotide Chain.

Nitrogenous base O
Except for the case of certain viruses, RNA is not the genetic material and does not
need to be capable of serving as a template for its own replication.
As a consequence of being a single strand, RNA can fold back on itself to form short
stretches of double helix between regions that are complementary to each other.
 RNA
RNA allows a greater range of base pairing than does DNA. Thus, as well as A:U and
C:G pairing, U can also pair with G.

This capacity to form a non-Watson-Crick base pair adds to the propensity of RNA to
form double helical segments.

Freed of the constraint of forming long range regular helices, RNA can form complex
tertiary structures, which are often based on unconventional interactions between
bases and between bases and the sugar-phosphate backbone.

The main role 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, so it stays safe.
Without RNA, proteins could never be made.
TYPES OF RNA

Type of RNA Description

A linear molecule that carries the gene’s information
mRNA from the cell's nucleus to the cytoplasm

An adaptor molecule that aids in the pairing of

amino acids to the correct codons of the
tRNA
mRNA

rRNA Important part of ribosomal molecular structure

and the ribosome’s ability to carry out
translation
 RNA Polymerase
RNA polymerase I catalyses the transcription of all rRNA genes except 5S.

These rRNA genes are organized into a single transcriptional unit and are transcribed
into a continuous transcript. This precursor is then processed into three rRNAs: 18S,
5.8S, and 28S.
The transcription of rRNA genes takes place in a specialized structure of the nucleus
called the nucleolus, where the transcribed rRNAs are combined with proteins to
form ribosomes.
RNA polymerase II is responsible for the transcription of all mRNAs. Many Pol II
transcripts exist transiently as single strand precursor RNAs (pre-RNAs) that are
further processed to generate mature RNAs.
For example, precursor mRNAs (pre-mRNAs) are extensively processed before exiting
into the cytoplasm through the nuclear pore for protein translation.
RNA polymerase III transcribes small non-coding RNAs, including tRNAs, 5S rRNA, U6
snRNA, SRP RNA, and other stable short RNAs such as ribonucléase P-RNA.
 Transcription & Processing of rRNA
Eukaryotic transcription occurs within the nucleus where DNA is packaged into
nucleosomes and higher order chromatin structures.
Transcription is the process of copying genetic information stored in a DNA strand
into a transportable complementary strand of RNA.
Transcription takes place in the nucleus of the cell and proceeds in basic three
sequential stages: Initiation, Elongation, and Termination.
The transcriptional machinery that catalyses this complex reaction has at its core
three multi-subunit RNA polymerases.
Transcription is divided into

❖ Pre-initiation
❖ Initiation
❖ Promoter clearance
❖ Elongation and
❖ Termination
Transcription & Processing of rRNA
 Reverse Transcription

Reverse Transcription is widely used in the laboratory to convert RNA to DNA for use in molecular
cloning, RNA sequencing, polymerase chain reaction (PCR), or genome analysis.
 Transfer RNA (tRNA)
A Transfer RNA is an adaptor molecule composed of RNA, typically 73 to 94 nucleotides in length,
that serves as the physical link between the nucleotide sequence of nucleic acids and the amino
acid sequence of proteins.

It does this by carrying an amino acid to the protein synthetic machinery of a cell (ribosome) as
directed by a three-nucleotide sequence (codon) in a messenger RNA (mRNA).
 Translation

Reading Frames and Their Importance. The place at which DNA sequence reading begins
determines the way nucleotides are grouped together in clusters of three (outlined with
brackets), and this specifies the mRNA codons and the peptide product. In the example, a
change in the reading frame by one nucleotide yields a quite different mRNA and final
peptide.
 Mutation

Any change in the nucleotide sequence of DNA—the genetic information—is a

mutation.
Some mutations result in changes in the proteins produced and some do not. As
stated earlier, as a genetic material, DNA must be able to change, or mutate.
Different types of changes can occur and these changes have a potential impact on
protein synthesis.
Mutagenic agents are substances or conditions that can cause the sequence of DNA
to change. Agents known to cause damage to DNA are certain viruses (e.g.,
papillomavirus), weak or "fragile" spots in the DNA, radiation (X rays or UV light),
and chemicals found in foods and other products such as nicotine in tobacco.
 Forward Mutation

1. Single Nucleotide Base Pair Substitution (At DNA Level)

Transition:
purine --> purine or pyrimidine --> pyrimidine
A-> G or G-> A T—> C or C-> T

Transversion:
purine -> pyrimidine or vice versa

A—> T, C; G “>T,C; T-->A, G; C-->A,G

Causes......(At Protein Level)

* Silent Mutation
* Neutral or Splicing Mutation
* Missense Mutation
* Nonsense Mutation.
Genetic Engineering & Biotechnology
Biotechnology includes the use of a method of splicing genes from one organism into
another, resulting in a new form of DNA called recombinant DNA.
Organisms with these genetic changes are referred to as genetically modified (GM),
or transgenic organisms.
These organisms or their offspring have been engineered so that they contain
genes from at least one unrelated organism, which could be a virus, a bacterium, a
fungus, a plant, or an animal.
 Gene cloning

Gene cloning is accomplished by using enzymes that are naturally involved in the
DNA replication process and other enzymes that are naturally produced by bacteria.
When genes are spliced from different organisms into host cells, the host cell
replicates these new "foreign" genes and synthesizes proteins encoded by them.
Gene splicing begins with the laboratory isolation of DNA from an organism that
contains the desired gene—for example, from human cells that contain the gene for
the manufacture of insulin.
If the gene is short enough and its base sequence is known, it can be synthesized in
the laboratory from separate nucleotides.
If the gene is too long and complex, it is cut from the chromosome with enzymes
called restriction endonucleases.
 Polymerase Chain Reaction (PCR)

The polymerase chain reaction (PCR) is a biochemical technology in molecular

biology to amplify a single or a few copies of a piece of DNA across several orders of
magnitude, generating thousands to millions of copies of a particular DNA sequence.
Developed in 1983 by Kary Mullis, PCR is now a common and often indispensable
technique used in medical and biological research labs for a variety of applications.
These include......
❖ DNA cloning for Sequencing, DNA-based Phylogeny, or Functional Analysis of
Genes;
❖ The diagnosis of Hereditary Diseases;
❖ The identification of Genetic Fingerprints (used in forensic sciences and
paternity testing)
❖ The detection and Diagnosis of Infectious Diseases.
In 1993, Mullis was awarded the Nobel Prize in Chemistry along with Michael Smith
for his work on PCR.
Thank you for attention