Jimma Univesity College of Agriculture

and Veterinary Medicine

School of Veterinary Medicine

Department Of Biomedical Sciences

Molecular Biology (BISC 103)

For Week End AH Year I

Instructor:
Prof. Yosef Deneke (DVM, MVSc,PhD)
Professor in Microbial Biotechnology and Daignostic
Immunology
  Introduction Molecular Biology

Chapter 1. Introduction to Molecular Biology

 Molecular biology grew out of the discipline of biology, genetics and

biochemistry.

 Biology: originated from a Greek word : Bios, means “life” and logos “
knowledge”- is a branch of science that studies about life ( living
things). It examines the structure, function , growth, origin, evolution
and distribution of living things both in the past and present.
The four principles of Biology:

1) Cell Theory: All living things are made of at least one cell. The basic
unit of functions in all organisms (i.e. it is the fundamental unit of life).
- Cells arise from other cells through cell division, and in multicellular
organisms, every cell in the organism's body is produced from a
single cell in a fertilized egg.
- Furthermore, the cell is considered to be the basic part of the
pathological processes of an organism.

2) Evolution: Through natural selection and genetic drift, a population's

inherited traits change from generation to generation.
 Con’d

• Charles Darwin theorized that species and breeds developed through

the processes of natural selection, as well as by artificial selection or
selective breeding.

• Genetic drift was embraced as an additional mechanism of evolutionary

development in the modern synthesis theory.

• The evolutionary history of a species –which describes the characteristics

of various species from which it descended together with genealogical
relationship to every other species is called it's phylogeny.
 Cont’d

3) Gene Theory: A living organism's traits are encoded in their DNA, the
fundamental components of genes.

Traits are passed on from one generation to the next by way of these
genes.

All information flow from genes to the phenotype , the observable physical
or biochemical characteristics of the organism.

 Genes are the primary unit of inheritance.

 But physiological adaptation to an organism's environment can not be

inherited by its offsprings.
 Cont’d

The total complement of genes in an organism or cell is known as its

genome which is stored on one or more chromosomes.

• A chromosome is a single, long DNA strand on which thousands of

genes, depending on the organism, are encoded.

• When a gene is active, the DNA code is transcribed into an RNA copy
of the gene‘s information. A ribosome then translates the RNA into a
structural protein or catalytic protein.
4) Homeostasis:

 The physiological process that allows an organism to maintain its

internal environment notwithstanding its external environment.

 Homeostasis is the ability of an open system to regulate its internal

environment to maintain a stable condition by means of multiple
dynamic equilibrium adjustments controlled by interrelated regulation
mechanisms.

 All living organisms, weather unicellular or multi-cellular, exhibits

homeostasis.
E.G.,

- Maintenance of stable internal acidity (pH) at the cellular level

- Maintenance of internal body temperature by warm-blooded animals

(at the level of organism).

• A central concept in science and scientific method is that all

evidence must be empirical or empirically based, that is, dependent
on facts or consequences that are observable by the senses.
 The topics studied in molecular biology also overlap directly with
genetics , cell- and developmental biology and biochemistry.

Cell Biology: studies the physiological properties of cells, as well as their

behavior, interactions and environment. This is done both on microscopic
and molecular level.

Developmental Biology: studies the process by which organisms grow

and develop.

- Originating in embryology, modern developmental biology studies the

genetic control of cell growth, differentiation and “ morphogenesis”, which
is the process that gives rise to tissue, organs and body system
(anatomy).
Model organism for developmental biology

☞The round worm Caenorhabditis elegans

The fruit fly Drosophila melanogaster

The zebra fish Brachyodanio rerio

The mouse Mus musculus

The weed Arbidopsis thaliana

Genetics:

is the science of genes, heredity and the variation of organisms.

 It involves the study of cells, individuals , their offspring and the population
within which organisms live.

 The gene is the contained within each DNA (deoxyribonucleic acid).

 Chemically it is a linear array of nucleotides-which are the chemical building

blocks of DNA and RNA (ribonucleic acid).

 It is an informational storage unit capable of undergoing replication and

expression.
Biochemistry:

 interfaces with biology and chemistry and is concerned with the

chemical processes that take place within living cells.

 It studies the chemical substances (such as organic molecules like

protein, fat, and carbohydrates) and vital processes occurring in
living organisms.
1.1.What is Molecular Biology?

Definitions:

Molecular biology is the study of life at a molecular level. It chiefly

concerns itself with understanding the interaction between the various
systems of cells including the interrelation of DNA, RNA and protein
biosynthesis and learning how these interactions are regulated.

 is the study of molecular underpinnings of the process of replication,

transcription, translation , structure and function of the genetic material
(Nucleic acids) at the molecular level.

 studies macromolecules and the macromolecular mechanism found in

living things, such as the molecular nature of the gene and its
mechanisms of gene replication, mutation and expression.
 The Central Dogma of Molecular Biology:

 The genetic material (DNA) is transcribed into

RNA and then translated into protein.

DNA  RNA  Protein

The attributes of living things are:

1) The major characteristic of living things is that they are highly organized
and complicated.

• E.g., Human Body  Parts (systems)  Organs  Tissues  Cells 

Supra molecular assemblies  Macromolecules  Nucleic Acids 
 Proteins
Small Molecules - Elements like C,H,N,O,S.

- Small molecules of life – like amino acids, nucleic acids, sugars, lipids-
give rise to macromolecules.

- Informational molecules such as nucleic acids and proteins are giving

precise information about growth and reproduction.
2) Living things are able to extract, transform and use energy from the
environment in the process of metabolism and synthesis of proteins.

3) Each component (Part ) of a living system has its own function

(purpose)

 Ribosome – Synthesis of protein

Mitochondria – Storage place for energy ( the power House of the

cell)
4) Growth – An increase in size and mass because of metabolic
activities with the help of enzymes.
- Enzymes are proteins, which catalyze a chemical reaction in the
body. Proteins are made up of amino acids.
So we can say, molecular biology involves the study of :

Storage of Information by Nucleic acids

Transmission of the information - Nucleic acid replication

Utilization of hereditary information – to investigate how the process

of transcription and translation are going on.
  Must know Terms in Molecular Biology:

- Biomolecule: A minute particles of matter from a living or once

living thing.

- Chromosomes: Thread-like structures in the nucleus of the cells.

Chromosomes are made of genes. Genes are made of DNA.

- DNA: Means Deoxyribo Nucleic acids. DNA is found in the nucleus

and is the material that stores genetic information. DNA controls
inherited traits such as appearance and eye color.

- Gene: A short length of a chromosome which controls the

characteristic of an organism.
- Genome: The total amount of the genetic information for a given
species such as human or yeast.
-Genetic engineering: Altering genetic material by combining
fragments of DNA from different organisms.

- Heredity: Science of passing traits from parents to young.

-Macromolecules: Large molecules made up of many small organic molecules that

are often referred to as monomers; e.g., carbohydrates, lipids, proteins, and nucleic
acids. They are polymers of monomers.

-Messenger RNA (mRNA): “ Blue Print” for protein synthesis that is transcribed from
one strand of the DNA (gene) and which is translated at the ribosome into a
polypeptide sequence.

-Meiosis: Cell division in which the chromosomes replicate, followed by two

nuclear divisions. Each of the resulting gametes in animals, spores in
plants, receive a haploid set of chromosome.

- Reduction division by which ploidy, the number of set of homologous

chromosomes is reduced in the formation of haploid cells that become
gametes (gametophytes in plants).
 Cont’d

-Mitosis: The division of the cell's nucleus and nuclear material of a cells,
consists of four stages: Prophase. Metaphase, Anaphase, and Telophase.
As Cell Xeroxing, mitosis occurs only in eukaryotes. The DNA of the cell is
replicated during the four phases of mitosis.

-Genomics: The study of genes and their function. Recent advances in

genomics help us in understanding of the molecular mechanism of disease,
including the complex interplay of genetic and environmental factors.

-Nucleic acid: Polymers made of nucleotides that carry genetic information.

- Replication: Duplication of DNA prior to cell division.

-Ribonucleic acid (RNA): Nucleic acid that differs from DNA in having
ribose in place of deoxyribose and having uracil in place of thymine.

-Ribosomes: The cell's machinery in making protein.

 Cont’d

-Base: A key component of DNA and RNA molecules. Four

different bases are found in DNA: adenine (A), cytosine (C), guanine
(G) and thymine (T). Also known as nitrogenous bases.
A base, a phosphate molecule and a sugar joined together constitute
a nucleotide.

- Base pairs: Two nucleotide bases on different strands of the

nucleic acid molecule that bond together. The bases can pair in only
one way: adenine (A) with thymine (T) in DNA or with uracil(U) in
RNA and guanine with cytosine.
1.2. Goals and concepts of Molecular Biology

• The ultimate goal of molecular biology is ambitious: to understand the

five fundamental behavior patterns (growth, division, specialization,
movement, and interaction) in terms of the various molecules that are
responsible for them.

•That is, molecular biology wants to generate a complete description of the

structure, function, and interrelationships of the cell's macromolecules,
and thereby to understand why living cells behave the way they do.
 In conclusion the goals of Molecular Biology are:

 Sequence and compare full genome of organism

 Identify the genes and determine the foundation of the protein they
encode

 Understand the genetic diseases

 Understand evolution and evolutionary history

 Understand proteins, which means predict the folding from the

amino acids sequence, and characterize the function of the protein
from this folding.

 Synthesize proteins, which means create an amino acid sequence,

such that the desired dose for a prescribed function.
Concepts of Molecular Biology:

The key concepts in molecular biology are: mechanisms,

information, and the gene. The heart of the discipline of molecular
biology consists of understanding the “ mechanism” of
information exchange within cells.

 Mechanism: In molecular biological mechanisms, types of

entities include macromolecules ( Proteins, Nucleic acids, DNA and
RNA) and sub-cellular structure, such as ribosomal particles
(composed of RNA and Proteins).

• Types of activities include geometric-mechanical activities, as lock

and key of an enzyme and its substrate, and chemical bonding
activities, as formation of strong covalent bond and weak hydrogen
bonds.
 Information: The term “information” is used ubiquitously by molecular
biologists. Gene as linear DNA sequences of bases are said to carry information
for the production of proteins.

- Transcription from DNA to messenger RNA and Translation from

RNA to protein. The protein folds into three-dimensional structure;
protein folding is the only part of this mechanism not yet well
understood.

- During DNA replication and subsequent inheritance, it may be said that

what is passed from one generation to the next is the linear ordering
of bases along the complementary DNA strands.

- The exact sequence of the base is the code which carries the genetic
information. It is important not to confuse the genetic code with the
genetic information.
 Cont’d

 The genetic code refers to the relationship between three

bases of DNA, called a “codon” and one amino acid.

 The genetic information refers to the linear sequence of codons

along the DNA, which (in the simplest case) are transcribed to
mRNA and translated to linear order of the amino acids in a
protein.
 Summary of concepts of Molecular Biology:

 The “Biopolymer “ concept: Polymers of small molecules such as

amino acids (proteins) or nucleotides (Nucleic acids) are extra ordinarily
versatile, and hence have evolved in complexity to generate the diverse
functional repertories of living cells.

 The “Shape and Size” concept: The specific function of a molecule

best suited to perform is largely dictated by its size and shape. Size and
shape set distinct limits on the number and type of inheritance and
relationships in which macromolecules can participate.

E.g. - Subunits of proteins- Antibodies (Immunoglobulins -

proteins of the Immune system- Interact with a foreign molecule
(antigen) forming Antigen-Antibody complex. Subunit of the
Immunoglobulins are L- light chain and H- heavy chain.
 The “Gene” concept: Genes contain all the information for the
synthesis and functioning of cellular components.

 The “RNA processing” concept: Eukaryotic mRNAs are extensively

processed( edited) prior to functioning as templates for protein
synthesis.

 The “Regulation” concept: A multitude of regulatory mechanisms

function in cells to insure the supply of various macromolecules matches
up with the cell's changing needs.

 The “ Simple Genome” concept: Viral genomes are extra ordinarily

simple, in parts because viruses borrow functions from the host cells.
The “ Level of sophistication” concept: Prokaryotes such as viruses and
bacteria, under heavy natural selection pressure, have evolved more
highly streamlined mechanism for controlling gene expression than
higher organisms (e.g., human).

 The “ Instability-Flexibility” concept: The inherent instability of most

proteins and RNAs insures that living cells are in a constant state of flux,
and therefore amenable to rapid change.

 The “ Changing Genome” concept: the sequence or arrangement of

nucleotides in genes is constantly evolving, either by mutation, by
occasional recombination events or from the action of transposable
elements.
1.3. Historical Background of Molecular Biology

Origins:- The field of molecular biology arose from the convergence of

work by geneticists, Physicists, and structural chemists on a
common problem:
the structure and function of the gene.

Early 1920th - Genetics was guided by Mendel's low of segregation

(two alleles of a gene separate, i.e. segregate, during the formation
of the germ cells so that each has one but not the other) and

- law of independent assortment ( genes in different linkage groups

assort independently in the formation of germ cells), the actual
mechanisms of gene reproduction, mutation and expression
remained unknown.
 Molecular Biology Time line:-

1856 - 1863 : Gregor Mendel -conducted his famous pea experiment

concerning gene segregation.

1859 : Charles Darwin - Published on the origin species; modern theory of

evolution is identified with Darwin

1866 : Gregor Mendel - Published a research paper in the inheritance of

seven different traits in the garden pea (establishing the basic principles of
heredity) - dominance and recessivness, equal segregation, independent
assortment.
Mendel's conclusions - Parents contribute particles, genetic units, to the
offspring = genes.
Phenotype - comes from the same Greek root as Phenomenon meaning
appearance - e.g. yellow seed, white flower.
Gregor Mendel
Cont’d
Genes can exist in different forms called - alleles.

E.g. The pea can have either yellow or green seeds.

i.e. - One allele of the gene for seed color gives rise to yellow seeds, the
other to green.
• One allele can be dominant over the other recessive, allele.
• The term filial comes from the Latin: filus, meaning son; filia,
meaning daughter. Therefore the filial generation (F1) contains
the offsprings (sons and daughters) of the original parents.

The second filial generation (F2) is the offsprings of the F1

individuals.
1869: Friedrich Miescher- Discovered DNA : first called "nuclein"
- as an acid containing substance found in cell nuclei.

• In treating wounded solder's from the Franco-Prussian war in

Tuebingen (Germany) he obtained a ready supply of white blood cells
from the Pus on the solder's bandages.

• When he digested the pus with pepsin and HCI and extracted the digest
with ether, he obtained a preparation of cell nuclei the first sub cellular
fractionation.

• Then by treating the nuclei with alkali, he obtained a precipitate which

he called nuclein and which on account of its acidic properties was later
called nucleic acid.
 Cont’d
1875: O. Hertwig - showed that nucleus is required for fertilization and cell
division, and hence contained information for those processes.

1889: Altman - Discovered and purified the acidic nature of the nuclein and
gave the name nucleic acid.

1891: Kossel- Found the chemical composition of sugar in nucleic acid.

1900: Hugo de Vries, Carl Correns, Erich von Tschermak- Seysenegg -

in dependently rediscovered Mendel's work. (The principles of heridity)

1901 : Ascoli - Identification of Uracil

1902 : Waltor Sutton and Theodor Boveri- Realized that genes are
located on the chromosomes. Since chromosomes contained nucleic acid
and protein this led to the search to determine which was the true genetic
material.
- Proposed the chromosome theory of inheritance
 1908: Leven and Jacob - Showed that the sugar in nucleic acids was
Deoxy-ribose.

1909: W.Johannsen - Introduced the word "gene“

1910: Thomas Hunt Morgern - Chromosomes theory of Inheritance

(Genes on chromosomes) - Proposed that genetic linkage was the result of
the genes involved and being on the same chromosome.

1928: Fredrick Griffth - Discovered the genetic transformative of a

bacterium and called the agent responsible "the transforming principle"

1941: George Beadle, & E.L. Tatum - Proposed the one gene - one
enzyme hypothesis

1944: Oswald Avery, Colin Macleod & Maclyn Macarty - Showed that
Grifth's transforming principle was DNA - as the material genes are made
of.
1952 : Alfred Hershey& Martha Chase- in a Blender experiment
showed that the genetic material of bacteriophage T2 is DNA.

1953: James Watson, Francis Crick, Rosalind Franklin, Maurice

Wilkins - Discovered the double helical structure of the DNA

• Rosalind Franklin }
• Maurice wilkins } worked on X-ray diffraction pattern of DNA fibers.

1957: Heinz Fraenkel - Conrat, B.Singer- Showed that the genetic

material in Tobacco mosaic virus was RNA.

1958: Matthew Messlson & Franklin Stahl - Demonstrated the semi-

conservative replication of DNA.
 Cont’d

1959: Arthur kornberg - Discovered DNA Polymerase I from Nuclei -

Synthesis of DNA in vitro (in a test tube)

Severo Ochoa - Synthesis of RNA using RNA polymerase.

RNA. Polymerase


Nucleoside Diphosphoester  RNA

1960: Francis Crick - Proposed the central dogma of molecular Biology:

DNA  RNA  Protein

1961: Sydney Brenner, Francois Jacob, Matthew Meselesen

Discovered messenger RNA (mRNA).
• Francois Jacob Sydney Brenner Matthew Meselsen
1961 - Francis Jacob & Jacques Monod - Put forward the operon model

for the regulation of gene expression

1962 : Arber and Co-worker - Discovered Restriction endonuclease-Type I

(RE - Type I).

1964: Khorana and Co-worker -Chemical synthesis of DNA

1966: Marshall Nirenberg & Gobind khorana - worked out the complete
genetic code.

1970: Hamilton smith - Discovered Restriction Enzymes that cut DNA at a

specific sites (RE - Type II), Which made cutting and Pasting DNA easy, thus
facilitating DNA cloning.

1972: Paul Berg - Made the first Recombinant DNA in vitro

1973: Herb Boyer and Stanley Cohen - First use a plasmid to clone DNA
Cont’d

1975: Edward M. Southern - Developed a method for transferring

DNA fragments separated in a gel to a filler, preserving the relative
positioning of the fragments. This is one of the most valuable
techniques for identifying cloned genes.

1977: Walter Gilbert and Fredrick Sanger- Worked out methods to

determine the sequence of bases in DNA

1985: Kary Mullis and others - Develop the Polymerase Chain Reaction
(PCR), a technique for amplification of selected DNA segment without
cloning.
 Cont’d

1990 : James Watson and many others - launched the Human

Genomic Project to map and sequence the complete genomes of a
number genetically important organisms, including humans.

1997: Ian Wilmut and colleagues - Cloned a sheep (Dolly) from an

adult sheep udder cell.

2000 : Alian Fischer and colleagues - First successful gene therapy

trial - on two patients with severe combined immuno deficiency
( "bubble boy" syndrome)
2001: Human Genome project: announced the completion of a "working
Draft" - DNA sequence of the entire human genome

2004: Human genome sequence finished - Analysis indicated that only

20,000 - 25000 protein coding genes.

2007 : Controversies continue over human and animal cloning, research

on stem cells, and genetic modification of crops.
1.4 Rapid Progress in Molecular Biology :

• Molecular biology was established in the 1930s, the term was

coined by Waren Weaver in 1938 however.

• Many of the most significant advances in molecular biology have

come about through the application of genetic analysis to molecular
phenomena.

• Direct experimentation using the model systems and laboratory

methods has proven to be a powerful driving forces in molecular
biology.
 Cont’d

Since the late 1950s and early1960s- Characterization, isolation, and

manipulation of the molecular components of cells and organisms.

 In the 1970s- Description of restriction enzymes and cloning.

 In the 1980s - mid 1990s -genome-scale sequencing, interface of

molecular biology and computer science in bioinformatics and
computational biology.
 As early of 2000s until present time- molecular biology blossomed
as a field
Important Laboratory Methods-for Rapid Development of Molecular
Biology

Ultra Centrifuge- in the 1920 used for estimation of size and shape
of molecules in centrifugal field

Gel Electrophoresis:- Gel electrophoresis is one of the principal

tools of molecular biology.

- The basic principle is that DNA ,RNA , and proteins can be separated
using electric field.
- In agarose gel electrophoresis, DNA and RNA can be separated
based on size by running the DNA through an agarose gel. Proteins
can be separated on size using an SDS-PAGE gel or by size and their
electric charge.
 Cont’d

 Southern Blotting:- named after its inventor biologist Edwin Southern,

the southern blot is a method for probing for the presence of a specific
DNA sequence within a DNA sample before or after restriction enzyme
digestion,
- which are separated by gel electrophoresis and then transferred to a
membrane blotting via capillary action.

 The membrane can be probed using a DNA probe labeled using a

complement of the sequence of interest.
Western Blotting:
 Antibodies to most of proteins can be created by injecting small amount of
the protein into an animal such as mouse, rabbit, sheep, or donkey
(polyclonal antibodies) or produced in cell culture (monoclonal antibodies).
These antibodies can be used for a variety of analytical and preparative
techniques.

 In Western blotting, proteins are first separated by size, in A thin gel

sandwiched between two glass plates in a technique known as SDS-
PAGE ( Sodium dodecyl sulphate polyacrylamide gel electrophoresis).
 The protein in the gel are then transferred to a nitrocellulose, nylon, or
other support membrane.
 The membrane can then be probed with solutions of antibodies.
Antibodies that specifically bind to the protein of interest can then be
visualized by colored products, chemiluminescence or autoradiography.
Northern blotting:-

The Northern blot is used to study the expression patterns a specific type of
RNA molecule as relative comparison among a set of different sample of
RNA.

- It is essentially a combination of denaturing RNA gel electrophoresis and a

blot.

- In this process RNA is separated based on size and is then transferred to

a membrane that is then probed with a labeled complement of a sequence
of interest.
- The result may be visualized by chemiluminescence or autoradiography.

- The procedure is commonly used to study when and how much gene
expression is occurring by measuring how much of that RNA is present in
different samples.
- It is one of the most basic tools for determining at what time, and under
what conditions, certain genes are expressed in living tissues .
 Cont’d
Recombinant DNA technology:- used for the genetic engineering of
novel genes.

Expression cloning:- The most basic technique to protein function is

expression cloning.
- In this technique, DNA coding for a portion of interest is cloned
( using PCR and/or restriction enzymes ) into a plasmid ( known as expression
cloning vector).

- This plasmid may have special promoter elements to drive production of the
portion of interest, and may also have antibiotic resistance makers to help
follow the plasmid.
- The plasmid can be inserted into either bacterial or animal cells.
- Introducing DNA into bacterial cells is called transformation, and can be
completed with several methods, including electroporation, microinjection,
passive uptake and conjugation.

- Introducing DNA into eukaryotic cell, such as animal cells, is called transfection.
The Polymerase Chain Reaction (PCR):-

 The polymerase chain reaction is an extremely versatile technique for

copying DNA. In brief PCR allows a single DNA sequence to be
copied ( million times ) or altered in predetermined ways. For example
PCR can be used :
– To introduce restriction enzyme site or mutate
( change) particular bases of DNA
– To determine whether a particular DNA fragment is found in a
cDNA library.

Varieties of PCR
-Reverse- transcriptase PCR ( RT. PCR ) for amplification of RNA
-Real -TIME PCR ( qPCR ) – for quantitative measurement of DNA or
RNA molecules.
DNA-arrays:-

 A DNA array is a collection of spots attached to a solid support

such as a microscope slide, each spot contains one or more DNA
oligonucleotides.

 Arrays make it possible to put down a large number of very small

( 100 micrometer diameter ) spots on a single slide.

 If each spot has a DNA molecule that is complementary to a

single gene ( similar to Southern blotting ) one can analyze the
expression of every gene in an organization in a single
experiment.
 E.g. Baker’s yeast- Saccharomyces cerevisiae

– Contains about 7000 genes- with a micro array one can

measure quantitatively how each gene is expressed and how
that expression changes for example with a change in
temperature.

Materials used for micro array fabrication

– Silicon Chip-most common

– Microscope slides-with spot of 100 µm diameter
– Custom arrays & arrays with larger spots on porous
membranes ( Micro arrays)
– Other array type e.g. Antibody array- used to determine what
proteins or bacteria are present in a blood samples.