STM 411 MICROBIAL GENETICS NOTE

Course Outline
• Basic nature and structure of the genetic materials of bacteria and other microorganisms.
• The basic structure of nucleic acids in relation to genetics of microorganisms.
• Basic nature and functions of the informational macromolecules – DNA and RNA.
• The concept of Mutation.
• Modifications that can occur in microorganisms.
• Applications of microbial genetic concepts in biotechnology (genetic engineering)

Introduction

Microbes are very small organisms that cannot be seen without the use of microscope.
Microbes are also known as microorganisms; they are found everywhere including water, air,
rock and soil. microorganisms included bacteria, archaea, yeasts, moulds, viruses, etc.
Genetics is the study of how genes carry information, how they replicate and pass to other
generation, and how they affect the characteristics of an organism.
Microbial genetics is a branch of genetics that deals with transmission of hereditary characters
in microorganisms. Microbial genetics means genetics of microbes such as bacteria, archaea,
viruses such as bacteriophages, and unicellular and multicellular eukaryotes including yeasts,
moulds, algae and protozoa. Microbial genetics has played a unique role in developing the
fields of molecular and cell biology, and also has found applications in medicine, agriculture
and the food and pharmaceutical industries. The study of microbial genetics can be applied to
universal themes and serve as the basis for genetic engineering. Because of the relative
simplicity, microbes are ideally suitable for genetics studies, and have been successful in
providing information on the genetic code and the regulation of genetic activity.

1
 Topic 1:
BASIC NATURE AND STRUCTURE OF GENETIC MATERIALS OF BACTERIA
AND OTHER MICROORGANISMS
Brief Classification of Microorganisms
On the basis of presence of nucleus and membrane-bound organelles, there are two (2) groups
of microorganisms; the prokaryotes and eukaryotes. The prokaryotes include bacteria and
archaea while the eukaryotes include algae, fungi and protozoa.
Prokaryotic Cell Struture
The prokaryotic organisms are characterized by absence of true nucleus. They possess genetic
materials that are not enclosed by nuclear membrane. That is, their genetic material is not
separated from cytoplasmic matrix of the cell. The genetic materials of prokaryotes are
scattered within the cytoplasm, to form an irregular shaped region called nucleoid. Prokaryotes
are generally simple and small in nature, and usually unicellular. Most genetics materials
(DNA) of prokaryotes are found in the nucleoid. The deoxyribonucleic acid (DNA) and non-
histone protein form a complex known a chromatin. The coiled and compacted chromatin form
the chromosome. Prokaryotes also possess ribosome granules within the cytoplasmic matrix.
Likewise, inclusion body, other features such as capsule, flagellum, pili or fimbriae and slime-
layer are possessed by some prokaryotes such as Escherichia coli, Proteus, and Pseudomonas
sp. Plasmid; extrachromosomal DNA is also found in prokaryotes.

2
 A bacterium (Rod-shaped) Structure

Microbial Eukaryotic Cell Structure

The eukaryotes have their genetic materials enclosed within the nuclear membrane to form the
most conspicuous compartment within the cytoplasm. The compartment is known as nucleus.
The genetic materials of eukaryotes are also found in mitochondria; a membrane bound
organelle. Other membrane bound organelles found in eukaryotes include Endoplasmic
Reticulum, Golgi-body, and Chloroplast. In eukaryotic cell, the nuclear material (genetic
material) in folded around the histone proteins. In eukaryotes, there are two (2) or more
chromosomes within the nucleus. The eukaryotes are diploid; that is, they contain a full set of
paired chromosomes.

Eukaryotic cell (Yeast cell) Structure

3
Chromosome
A chromosome is an organised structure of DNA and protein. The DNA and protein complex
is referred to as chromatin. The chromatin is folded or coiled and compacted inorder for it to
be accommodated within the cell or nucleus. A chromosome is a single piece of coiled DNA
and protein complex containing many genes, regulatory elements and other nucleotide
sequences. The proteins that form complex with DNA could be histone or non-histone
proteins.; these proteins help in packaging the DNA and control chromosome functions. The
chromosomes vary widely between different organisms, this depends on the length of the
polynucleotide that makes up the DNA. The chromosomal molecule may be circular or linear,
and composed of 10,000 to 1,000, 000 nucleotides joined together in a very long chain. A single
molecule of DNA within a chromosome may be as long as 8.5 entimeter (cm). For the DNA to
fit within the cell, it must twist and fold into a very complex shape. Each chromosome has a
constriction section called the centromere; divides chromosome into two sections or arms. The
short arm is labelled “p arm” while the long arm is known as “q arm” . The location of the
centromere on each chromosome gives the chromosome its characterisricss shape and can be
used to describe the location of speceific gene. Each half of chromosome is referred to as
chromatid.

Chromosome

Prokaryotic Chromosomes
Prokaryotes usually possess giant chromosomes in their nucleoids. Prokaryotic chromosome
most times consists of a single, circular, double-stranded DNA molecule of 580kbp to 5220kbp.
In prokaryotic chromosome, the DNA and non-histone proteins (nucleoid-associated proteins
[NAPs]) form a complex known as chromatin. Histone proteins are lacking in prokaryotic
chromosomes. Although, most prokaryotes have a single chromosome, the presence of multiple
chromosomes has been found in many bacteria such as Brucella, Leptospira interrogans and
Vibrio cholerae. Most prokaryotic cells have just one chromosome, so they are classified as
haploid cells (1n, without paired chromosome).

4
Moreso, the presence of linear chromosomes has been reported in Borrelia and Streptomyces.
Different prokaryotic species have different size of chromosomes. Genes that are essential for
bacterial growth (often referred to as house-keeping genes) are carried on the chromosomes.
The prokaryotic chromosomes have a single copy of each gene; therefore, they are haploid (1n)
in nature. During cell division, a single copy of each gene or chromosome is transferred to
daughter cell.

Note: When a prokaryote (bacterium, Vibrio cholerae) has two (2) chromosomes, the
chromosomes are unique from one another. That is, they are not a homologous pair, because
they do not contain the same genes in the same location. Prokaryotic DNA also undergoes
supercoiling like eukaryotic DNA but it is not wound around protein first. Supercoiling uses
the application of tension to twist a DNA molecule, so it wraps around itself, creating loop.

Eukaryotic Chromosomes
The eukaryotic cells carry two or more linear chromosomes that are separated from the
cytoplasm matrix by nuclear membrane. Eukaryotic chromosomes consist of a DNA-histone
proteins complex that is organized in a compact manner which permits the large amount of
DNA to be stored in the nucleus of the cell. The eukaryotic cells contain two homologous
(divergent evolutionary copies) of each chromosome. The eukaryotic chromosomes are tightly
coiled DNA around histone proteins. Although chromosomal compaction in eukaryotes
involves the wrapping of DNA around histones to form nucleosome, this is not true in all
eukaryotes. The eukaryotic chromosomes are diploid in nature (2n); they are in pair and have
two copies of each gene. The subunit designation of the chromosome is chromatin, while the
fundamental unit of chromatin in the nucleosome.

The eukaryotic chromosomes differ from the prokaryotic chromosomes in morphology,

chemical composition and molecular structure. Eukaryotic chromosomes contain more genetic
information than prokaryotic and viral chromosomes, therefore, they contain a great amount of
genetic material (DNA molecules). The shape of the eukaryotic chromosome is changeable
from phase to phase in the continuous process of the cell growth and cell division. They are
thin, coiled, elastic, thread-like structures during the interphase and are called chromatin
threads. The morphology of the chromosome is best studied during metaphase and anaphase,
which are the periods of maximal contraction. During these phases, the chromatin threads
become highly coiled and folded to form compact and individual distinct ribbon-shaped
chromosomes.

Virus and Viral Chromosomes

Viruses are microorganisms that are capable of survival but not growth in the absence of a cell
host. Replication of viral genome (genetic material) depends on the metabolic energy and
macromolecules synthetic machinery of the host. There is distinction between viruses
associated with eukaryotes and viruses associated with prokaryotes. The latter is termed
bacteriophages or phages. The bacteriophages constitute the largest of all viral groups. The
nucleic acid of phages is surrounded by a protein coat, considerable variability is found in the
nucleic acid. Many phages contain double-stranded DNA, others contain double-stranded
RNA, single-stranded RNA or single-stranded DNA. Bacteriophages exhibit a wide variety of
morphologies. Many phages contain specialized syringe-like structures (tails) that bind to
receptors on the cell surface and inject the phage nucleic acid into a host cells.

5
The viral chromosomes occur singly in a viral species and chemically may contain either DNA
or RNA. It is a distinguishing feature of viruses from all living organisms which possess both
RNA and DNA. DNA-containing viruses are known as deoxy-viruses and RNA-containing
viruses are ribo-viruses. The DNA containing viral chromosomes may either be either linear
(T2, T3, T4, T5 and most bacteriophages) or circular in shape (most animal viruses and certain
bacteriophages), while the RNA-containing viral chromosomes are made up linear RNA
molecule. Viral chromosomes are tightly packed within the capsid of mature virus particles
(viron) or occur freely inside the host cell. The nucleic acid in virus could be either single-
stranded or double-stranded. The genomes vary in size from 1kb to nearly 30kb.

A T-even Bacteriophage

Extrachromosomal Genetic Elements in Bacteria

In bacteria, there are other genetic elements other than chromosome. The extrachromosomal
genetic elements could be plasmids or phages DNA (bacteriophages DNA). They are non-
essential elements which often determine resistance to antibiotic agents, production of virulent
factors or other factors.

(1) Plasmids: They are small, circular pieces of double-stranded DNA molecules that replicate
independently of the host (bacteria) chromosome. They are found in many bacterial cells.
Plasmid is usually smaller than bacterial chromosome. When plasmids are integrated into the
host bacterial chromosomes, they are referred to as episomes. Plasmids are separated DNA
molecules that contain a replication origin that allows them multiply independently of the host
chromosome. The plasmids consist of genes that are not essential for bacterial survival. The
absence of plasmids does not kill the bacterium, but their presence provides additional benefits
to the bacterial cell. Plasmids vary in size; smallest plasmid is only 846bp long and contains
only one gene. They differ from chromosomes, being small and coding for genes that not
essential for the bacterial survival. Some plasmids carry more than 500 genes. The large
plasmid known carries 1,674 genes. A cell may have different plasmids existing together. The
copy number refers to the number of copies of a plasmid present in a cell. The large plasmids
are present in small numbers (1 to 2) and small plasmids may be found in high copy number

6
(40). Plasmids are capable of transfer to other cells but not all plasmids are transferrable.
Gene(s) carry by plasmids usually give their bacterial hosts a selective advantage over others
that do not carry the plasmid. Single copy of a number of different plasmids may exist within
a bacterial cell, while in some multiple copies of the same plasmid may exist within the cell.
Examples of plasmids are fertility factor (F-factor) or conjugative plasmid, R-plasmid
(antibiotic resistant plasmid), physiological function plasmids, cryptic plasmid and col-plasmid
(colicinogenic plasmid). Plasmids are usually exchanged between bacteria via pili through a
process called conjugation.
Plasmids ae usually ideal structure or molecule for genetic engineering due to their simple
structure, easy for genetic manipulation and transferred back to bacteria, contain genetic
information which can be used by the bacteria, most plasmids are present in high number and
they are not essential for bacterial growth, thus possible to manipulate plasmid DNA without
affecting bacterial growth.
(2) Phage DNA: The phage DNA is also known as bacteriophage DNA. Apart from plasmids,
viral genetic material or molecule (DNA) can be found in bacteria. Some viruses infect
bacteria, these viruses are known as phages or bacteriophages. The phage DNA is usually
incorporated into the bacterial chromosome during transduction. Infection of the bacteria by
phage usually take place during lysogenic cycle.

Advantages of Simple and Haploid Nature of Bacterial Chromosome

Due to simple and haploid nature of bacterial chromosome, majority of the information carried
on the DNA of bacterial chromosome are expressed at all times. Unexpressed information is
just 10% of the total information unlike 90% in eukaryotic chromosome. This unique attribute
makes microorganisms veritable tools in study of genetics. As a result of simple and haploid
nature of bacterial chromosome, rapid cell division is possible, resulting in large population
within a short period or generation time, thus increases the chances of selecting genetically
changed colonies from the larger population.
Microbes as Tools for Genetic Studies:
1. They can be grown on a simple, chemically defined minimal medium.
2. They can be grown and obtained in pure culture.
3. Many different mutants can be easily isolated.
4. Selective technique can be used for their isolation.
5. They exhibit very short generation time under optimum conditions.
6. Easy manipulation of genetic material.

7
 Topic 2:

BASIC STRUCTURE OF NUCLEIC ACIDS IN RELATION TO GENETICS OF

MICROORGANISMS

Nucleic Acids
Nucleic acids are heteropolymers (macromolecules) composed of monomers known as
nucleotides. There are two types of nucleic acids; ribonucleic acid (RNA) and deoxyribonucleic
acid (DNA). Nucleic acids are polynucleotide, which is combination of many molecules of
nucleotides that are linked together by phosphodiester bonds. The DNA carries the genetic blue
print (information) while RNA is the intermediate molecule that converts the blue print into
defined amino acid sequences in protein. The sequence of the monomers (nucleotides)
determines the genetic information carries by deoxyribonucleic acid.

Composition of Nucleotide
The nucleotide is made up of three basic units: nitrogenous base, pentose (5-carbon sugar) and
phosphate molecule. When pentose sugar combines with nitrogenous base, it forms nucleoside.
Addition of phosphate to nucleoside brings about formation of nucleotide. The bases and
pentoses of the common nucleotide are conventionally numbered to facilitate the naming and
identification of compound. The pentoses of nucleotides and nucleoside, the carbon numbers
are given as prime (ʹ) designation to distinguish them from the numbered atoms of the
nitrogenous bases. The base of a nucleotide is joined covalently (at N-1 of pyrimidines or N-9
of purines) with N-β-glycosidic bond to the carbon-1 (1́ʹ or 1-prime) of the pentose, and the
phosphate is esterified to the 5ʹ, that is the phosphate binds carbon-5.

Nitrogenous + Pentose
Base Nucleoside
Sugar
Nitrogenous
Pentose Nucleotide
Base + + Phosphate
Sugar

Pentose Sugar in Nucleotide

The deoxyribonucleotide and ribonucleotide are made up of different type of pentose sugar.
DNA nucleotides contain deoxyribose sugar while RNA on the other hand has ribose sugar.
The two sugars are similar in structure but deoxyribose lacks the hydroxyl group (OH) at
carbon-2 (2ʹ or 2-prime) but hydrogen.

8
 Pentose Sugars (Nucleic acid sugars)

Groups of Nitrogenous Organic Bases in Nucleic Acids

There are five different nitrogenous bases found in nucleic acids. The bases are categorised
into two groups; purines and pyrimidines.

DNA Nitrogenous Bases RNA Nitrogenous Bases

• Adenine (A) • Adenine (A)
• Guanine (G) • Guanine (G)
• Cytosine (C) • Cytosine (C)
• Thymine (T) • Uracil (U)

Purines

Adenine Guanine

Pyrimidines

Cytosine Thymine Uracil

Figure 5: The Nitrogenous Bases

9
Basic Structure of Nucleotide
In nucleic acids, the nitrogenous bases are joined to the pentose sugars by glycosidic bond
between carbon-1 (1ʹ) of the sugar and the nitrogen atom at position 9 of a purine or position 1
of a pyrimidine, to form glycoside. The glycoside could be ribonucleoside or
deoxyribonucleoside, depends on the type of pentose sugar (ribose or deoxyribose). When one
or more phosphate groups are attached to the 5ʹ (carbon-5) of the sugar, the nucleoside is
converted to the corresponding nucleotide. One to three molecules of phosphate can be added
to a nucleoside to form nucleotide such as Adenosine monophosphate (AMP), Adenosine
diphosphate (ADP) and Adenosine triphosphate (ATP). The nucleotide with deoxyribose is
referred to as deoxyribonucleotide while those with ribose are called ribonucleotides.

Formation of Polynucleotide Strand

DNA and RNA are polynucleotide strands that are formed by joining together many varieties
of nucleotide in a repetitive manner. They may vary in length, from a few to many millions of
nucleotides. Each nucleotide is joined to the next one by covalent phosphodiester bond that are
formed when the 3ʹ position of the sugar in a nucleotide is connected with the 5ʹ position of the
sugar in the next nucleotide through a phosphate group. In a nutshell, polynucleotides consist
of nucleotides covalently bonded via phosphate from carbon-3 called the 3ʹ (3-prime) of one
sugar to the carbon-5 (5ʹ) of the adjacent sugar. The phosphate linkage is referred to as
phosphodiester bond because a phosphate molecule connects two sugar molecules by ester
linkage

10
Structure of Deoxyribonucleic Acid
Deoxyribonucleic acid (DNA) is a double stranded extremely long chemical thread, which
made up of two complementary polynucleotide strands that wound together around an
imaginary core to form a double helix with 0.20nm or 20A˚ diameter. Each chromosome
consists of two strands of polynucleotide, each strand contains hundreds of thousands to several
millions’ nucleotide linked by phosphodiester bonds. The strands are associated with each other
by hydrogen bonds that form between the nitrogenous bases in nucleotide of one strand and
those of second strand. DNA thread has polarity; the ends are chemically distinct. The DNA
strands are antiparallel; they lie in opposite direction. The end of the strand carries phosphate
(PO4) group is called 5ʹ end while the end that carries a hydroxyl (OH) group is called 3ʹ end.
The two polynucleotide strands that make up the DNA thread are arranged to lie in double
helix, opposite direction to each other. One strand lies in the 3ʹ→5ʹ direction and the other
strand in 5ʹ→3ʹ direction. With this arrangement, they are said to be antiparallel double helix.

In DNA, adenine (A) pairs with thymine (T) while cytosine (C) pairs guanine (G), thus ensures
the two strands are complementary in base sequences. Whenever G is found in one strand, C
is found in the other strand. Likewise, whenever T is present in one strand, its complementary
strand carries G. For the complementary strands to be held together, hydrogen bonds must exist
between the complementary base pairs. Adenosine and thymine are held together by double
hydrogen bonds (A=T), while cytosine and guanine are bound together by three hydrogen
bonds (G≡C).

11
 (a) (b)

(a) Helical shape and (b) Double stranded DNA

RNA Structure
RNA is a polymer of ribonucleotides linked together by 3′ to 5′ phosphodiester linkage. Most
RNA are single stranded polynucleotides; they are made up of ribose, phosphate and
nitrogenous bases. But a few RNA could be double stranded. The ends of single stranded RNA
can be distinguished by a phosphate (PO4) group at the 5ʹ end and hydroxyl (OH) group at the
3ʹ end. The base composition of RNA is different from that of DNA. Its nitrogenous bases;
adenine, guanine, cytosine and uracil attached to carbon-1 of the ribose sugar. In RNA
complementary base pair is not common since it is single stranded, but when it occurs, adenine
pairs uracil while guanine pairs cytosine. RNA typically folds back upon themselves in regions
where complementary base pairing is possible to form folded structure. This pattern of folding
in RNA is termed secondary structure. There are three primary types of RNA; ribosomal RNA
(rRNA), transfer RNA (tRNA) and message RNA (mRNA).

Major Types of RNA

• Messager RNA (mRNA): It is a single stranded copy of the sequence information in a
gene. The sequence of nucleotides in an mRNA is used as a template to make protein
during translation. Messenger RNA is like a copy of information in DNA that is brought
to the ribosome where the information is translated into a protein. That is, it carries
coded information for the synthesis of specific protein from DNA gene to ribosome for
use. mRNA acts as a template for protein synthesis. It carries information about a
protein sequence. It is coded so that every three nucleotides (a codon) correspond to
one amino acid.
• Transfer RNA (tRNA): It carries the amino acids to the ribosome, where they are
incorporated into protein. tRNAs are short, folded molecules with two distinct ends:
one end of the tRNA attaches to a specific amino acid; the other end has an anticodon,
or group of 3 nitrogenous bases that are complementary to a codon on mRNA. tRNA is

12
 the actual translator. The tRNA anticodon base pairs the codon to decode the type of
amino acid sequence transcribed by mRNA from DNA.
• Ribosomal RNA (rRNA): It is a physical part of a functional ribosome, that carries out
protein synthesis. rRNA is like the protein factories of the cells. While rRNA is rarely
drawn out in diagrams, consider it to be a necessary subunit of a ribosome and that it
acts as an enzyme forming peptide bonds between amino acid. It forms ribosome and
its integral part.

. Differences between DAN and RNA

DNA RNA
• Presence of deoxyribose sugar Ribose sugar is presence
• Always in double-stranded Mostly single-stranded
• Presence of thymine Presence of uracil
• Usually very long Not very long
• Purines and pyrimidines mostly Purines and pyrimidine are not
equal equal
• Bulk of DNA is found within the RNA is mostly found in the
nucleus cytoplasm
• It has small grooves The grooves are large
• It stores, and replicates information. It transcribes and translates genetic
information into protein.

Important of Phosphodiester and Hydrogen Bonds in Polynucleotide Structure

Phosphodiester bonds and hydrogen bonds are covalent bonds that play important roles in
assembling nucleotides together to form a very long chain known as polynucleotides. The size
of polynucleotides depends on the number of phosphodiester bonds that linked the nucleotides
together both in DNA and RNA. The hydrogen bonds between complementary bases are a
fundamental feature of the double helix, contributing to the thermodynamic stability of the
helix and providing the information content and specificity of base pairing. Hydrogen bonds
hold the two complementary strands of DNA together, this is made possible during pairing of
the complementary nitrogenous bases from the two strands. Therefore, in order to separate the
double-stranded DNA, the hydrogen bonds that link the bases together must be broken.
Hydrogen bonding is important for the specificity of base pairing. There are two hydrogen
bonds between A and T, but between G and C, there are three hydrogen bonds.

DNA Replication
DNA replication is a biological process by which existing DNA is used as template for the
synthesis of new DNA strands. It is an extraordinary important process that occurs in all living
organisms, upon which all lives depend. The process involves the following basic steps:
uncoiling of DNA, unzipping of the hydrogen bonds so each strand serves as a template for the
synthesis of a new strand, and synthesis of a new strand whose bases are complementary to
those of the old strand. DNA synthesis is described as semi-conservative type because each
old strand is used as a template for the synthesis of a new strand. So, in every DNA molecule,
one strand is always an old one and other strand is always a new one. During DNA replication,
cellular proofreading and error-checking mechanisms ensure near perfect fidelity of DNA
replication.

13
In cell, prior to cell division, the hydrogen bonds that hold the double-stranded DNA together
are broken and the double helix DNA strands are unwound by DNA helicase (an enzyme).
Unwinding of the double helix begins at a specific location called the origin of replication.
During unwinding, the double helix DNA forms a γ-shaped structure called a replication fork.
The replication fork is created by gradual opening of the two unpaired single strands.
Topoisomerase binds at the region ahead of the replication fork to prevent supercoiling (over-
winding). In order to initiate DNA replication, a very short RNA with few nucleotides of about
10 bases, called RNA primer is required. The RNA primer is synthesized by an enzyme called
Primase or specific RNA polymerase, at the replication fork. A primer provides a point to
which new deoxyribonucleotides may be added by DNA polymerase III. When DNA
replication is initiated, the RNA primer base pairs with the single stranded DNA template,
thereby forms RNA/DNA base hybrid. A free 3ʹ-OH group is supplied by RNA Primer for start
of polymerization. A new deoxyribonucleotide is added at time to the 3ʹ end (3ʹ OH end) of the
RNA primer via phosphodiester bonds. Addition of the new deoxyribonucleotides to the primer
in a continuous manner is facilitated by DNA polymerase III. The DNA polymerase III is an
important enzyme that responsible for the elongation of polydeoxyribonucleotides in
5ʹ→3ʹdirection. DNA polymerase III adds deoxyribonucleotides one by one to the growing
DNA chain.
Since the new strand must be antiparallel to the template, there is important distinction in the
replication of the two DNA strands, due to the fact that replication always proceeds from 5ʹ→3ʹ.
When the 3ʹ to 5ʹ strand is used as template, the new complementary strand, that grows
continuously in 5ʹ→3ʹ direction, towards the replication fork using just one RNA primer is
refers to as leading strand. Moreso, when the complement strand uses the 5ʹ to 3ʹ strand as
template, polydeoxyribonucleotides are synthesized discontinuously away from the replication
fork, as small fragments of DNA (Okazaki fragments); this stand is known lagging strand.
During lagging strand synthesis, the normal 5ˈ→3ˈ is also still followed but several RNA
primers are involved and is synthesized away from the replication fork. Therefore, for lagging
strand to be produced, the RNA primers must be synthesized by primase in multiple times in
order to provide free 3ˈOH group. Finally, the exonuclease, cut off the primers and the Okazaki
fragments are annealed (joined/stitched) together by ligase to form a continuous strand. This is
following by cellular proofreading and error checking.

Replication fork

14
RNA Transcription
Transcription is a process of synthesizing single-stranded RNA; this process involves coping
of genetic information from DNA to RNA using RNA polymerase. During RNA transcription,
only genes section of the DNA is copied. The RNA polymerase, catalyses the formation of
phosphodiester bonds between ribonucleotides. Unlike DNA polymerase, RNA polymerase
initiates new strand of ribonucleotide on its own, without any need for a primer. The mechanism
of RNA synthesis is also much like that of DNA, the ribonucleotides are added to the 3ʹ OH of
the ribose of the proceeding ribonucleotide. During RNA transcription, existing DNA strand is
used as template and only the genes sections are copied. This polymerization process is an
energy driven process. The template for RNA polymerase is the double-stranded DNA
molecule, but only one certain portion of the strand is transcribed for a given gene. The process
is divided into four stages; promoter recognition and identification, initiation, elongation and
transcription termination. In order to initiate RNA synthesis correctly, RNA polymerase must
first recognize the initial sites on the DNA. The important sites are called promoters. Once the
RNA polymerase has bound to the promoter, transcription can process. In this process, the DNA
double-helix at the promoter is opened by the RNA polymerase to form a transcription
bubble. As the RNA polymerase moves, it unwinds the DNA in short segments. This transient
(brief) unwinding, exposes the template strand and allows it to be copied into the RNA
complementary strand. Unlike DNA replication which copies the entire genomes, transcription
involves much smaller unit of DNA, often as little as a single gene. RNA synthesis also
proceeds in a 5ʹ →3ʹ direction, with new ribonucleotide being added to the 3ʹ end of the growing
chain. For example, the sequences of the coding strand, template strand, and RNA transcript
are:
Coding strand: 5' - ATGATCTCGTAA-3'
Template strand: 3'-TACTAGAGCATT-5'
mRNA: 5'-AUGAUC...-3' (the dots indicate where ribonucleotides are still being added to the
RNA strand at its 3' end).

Promoter, RNA coding and terminator regions of DNA

Assigment: Describe extensive protein synthesis.

15
 Topic 3:
NATURE AND FUNCTION OF DNA AND RNA
Genetic Code
Genetic code is a set of triplet nucleotides called the codon; each corresponds to a
specific amino acid or stop signal/stop codon. Codon is a triplet of nucleotide bases that
encodes a specific amino acid or stop signal. The genetic code is written as mRNA
rather DNA, because it is RNA that translates. The RNA is known as Messager RNA
(mRNA). The mRNA carries information (copied from a DNA template) in the form of
a genetic code that directs the synthesis of a particular protein. The message encoded
in a gene takes the form of a series of triplets (codons). There are 64 possible three-
letter combinations of A, C, G and U, 61 correspond to specific amino acids, while the
remaining three represent stop signals (stop codons or nonsense codons), indicating
that reading of the message should cease at that point. The stop codons are UAG, UAA
and UGA (see table below). It is essential that the reading of the message starts at the
correct place, otherwise the reading frame (groups of three nucleotides) may become
disrupted. This would lead to a completely inappropriate sequence of amino acids being
produced. Since there are only 20 amino acids to account for, it means that the genetic
code is degenerate (redundant), that is, a particular amino acid may be coded for by
more than one triplet. Amino acids such as serine and leucine are encoded by as many
as six alternatives each, while tryptophan and methionine are the only amino acids to
have just a single codon (see tabel below).

Characteristics of Genetic Code

a. Specificity: Genetic code is specific or unambiguous in nature. A specific codon is
always encodes for same amino acid or stop sign. For example AUG that codes for
methionine cannot code for another amino acid.
b. Universal: In all living organisms, genetic code is the same, exception to universality
can only be found in mitochondrial codon.
c. Redundant: Genetic code is redundant or degenerate in nature. Although each codon is
corresponds to a specific amino acid, but a particular amino acid may be coded for by
more than one codon, even up to six. Only tryptophan and methionine that have single
codon.
d. Non-Overlapping: The reading of genetic code during protein synthesis does not
involve any overlapping, each nucleotide is specific for a codon. The nucleotide cannot
be shared by adjacent codons.
e. Non-Punctuation: Once the reading commences at a specific codon, there is no
punctuation between the codons. Message is read in continuous sequence of nucleotide
triplet until translation stop codon is reached.
f. Initiator codon: Messages on mRNA are read from a fixed starting point to establish a
reading frame. The triplet codon AUG that specifies methionine serves in some cases
as the nucleotide codon that signals the beginning of coding in nucleotide sequence of
mRNA, that synthesis of a specific polynucleotide chain begins.
g. Non-Sense Codon: There are 64 codons, three of these codons do not code for any
amino acid but stop codon/terminal codon/non-sense codon/stop signal.

16
 h. Polarity: In genetic code, the nucleotide sequence in a always read from a fixed
direction (5ʹ→3ʹ). The first nucleotide in a triplet is at the 5ʹ end of the codon and the
third is at the 3ʹ end, for example 5ʹAUG3ʹ.

Table: Genetic code

Second Position
U C A G

Amino
Amino Amino Amino
Codon Codon Acid Codon Codon
acid Acid acid
U UUU UCU UAU UGU U
Phe Tyr Cys
UUC UCC UAC UGC C
Ser
UUA UCA UAA UGA STOP A
STOP
UUG UCG UAG UGG Trp G

C CUU CCU CAU CGU U

Leu His
CUC CCC CAC CGC C
Pro Arg
CUA CCA CAA CGA A

Third Position
First Position

Gln
CUG CCG CAG CGG G

A AUU ACU AAU AGU U

Asn Ser
AUC Ile ACC AAC AGC C
Thr
AUA ACA AAA AGA A
Lys Arg
AUG Met ACG AAG AGG G

G GUU GCU GAU GGU U

Asp
GUC GCC GAC GGC C
Val Ala Gly
GUA GCA GAA GGA A
Glu
GUG GCG GAG GGG G

Key: Ala = Alanine (A); Arg = Arginine (R); Asn = Asparagine (N); Asp = Aspartic acid
(D); Cys = Cysteine (C); Gln = Glutamine (Q); Glu = Glutamic Acid (E); Gly =
Glycine(G); His = Histidine (H); Ile = Isoleucine(I); Leu = Leucine (L); Lys = Lysine
(K); Met = Methionine (M); Phe = Phenylalanine (P); Ser = Serine (S); The = Threonine
(T); Trp = Tryptophan (W); Tyr = Tyrosine(Y); Val = Valine (V).

3.2 Wobble Concept

In genetic code, although a codon is specific for a particular amino acid, there are 61 possible
sense codons (codons that specific amino acids). In most cases except tryptophan and
methionine, an amino acid has more than one codon, for examples the codons; UCA, UCG,
CCU, CCC, CCA and CCG are specific for Leucine while AAA and AAG code for lysine. A
codon is recognised by specific base-pairing with complementary sequence of three bases
called anticodon, a sequence found in tRNA. If the traditional base-pairing rules are strictly
followed, it means 61 tRNA anticodons would be needed to pair 61 codons on mRNA. In lysine

17
(AAA, AAG), the codons are different at 3rd base but have the same base at positions 1 and 2
(5ʹ→3ʹ). In order to have a single tRNA anticodon for lysine (AAA, AAG), the rules of base-
pairing are relaxed at the third position, so that a base can pair with more than one
complementary base. Therefore there is no need for two (2) different tRNA anticodons
molecules to base pair lysine codons. The first two bases in mRNA codon (5ʹ→3ʹ) base pair
traditionally with the 2nd and 3rd bases of the anticodon in tRNA (3ʹ→5ʹ). The non-traditionally
or irregularly base-pair that occurs between the 3rd base of the codon and the complementary
nucleotide in anticodon is termed Wobble concept or phenomenon

18
 Topic Four:
MUTATION
Concept of Mutation
Mutation is a stable, heritable change in the base sequence or nucleotide sequence of the
genome or DNA of an organism that may be good or bad. All organisms contain specific
sequence of nucleotide base in their genome, a change in this sequence that is stable, and
heritable is known as mutation. Mutation is a permanent change in organism’s genome. When
mutation occurs, this is transferred from generation to generation. Mutation usually occurs in
the gene; a segment of deoxyribonucleic acid (DNA) carries in its nucleotides sequence
information for a physiological or biochemical property. Mutation can arise from the alteration
of single pair of nucleotides and from the addition or deletion of one or two nucleotide pair(s)
in the coding regions of the genome. On the molecular level, a mutation is any change in the
DNA sequence of a gene. Often mutations are deleterious; causing harm to organisms.
However, mutation is one of the ways by which new genes are created, thus, it is sometimes
beneficiary to organisms. Mutation can bring about morphological changes in a microorganism
in numerous ways or different ways. Mutation can also cause death of microorganisms (lethal
mutation). Mutation may or may not have effect on the phenotype (physical manifested
properties) of the organism.

Mutation as it Relates to Microorganisms

In all cells, the genome consists of double-stranded DNA but in viruses, the genome consists
of single or double-stranded DNA or RNA. Any microorganism whose base sequence has been
altered or changed is known as Mutant. An organism that has a natural, non-mutated
characteristics is called the wildtype or prototroph. Prototrophs are the original parents from
which the mutants are derived. The mutant is a strain of any cell or viruses carrying a change
in nucleotide sequence and is definitely different from its parent in genotype (nucleotide
sequence of the genome). Genotype is represented by three small letters and a capital letter e.g.
hisC. The mutant may also produce observable characteristics or properties relatively different
from the parent cell. The process by which mutation takes place in microorganisms is known
as mutagenesis. A mutant different from its parent strain genotype (nucleotide sequence of the
genome), and may also produce observable properties (phenotype) relatively different from
parental cell (strain). In microorganisms, most especially bacteria, as long as the base sequences
of the DNA do not change, the organism will always breed true generation. A change in the
base sequence will alter the information carry by DNA and this is likely producing heritable
change in the structure of at least bacterial protein. Mutation could be spontaneous or induced.
Mutation can affect the whole chromosome and it might be mutations that affect certain genes
(gene mutation).
Causes of Mutation
(1) Tautomeric Shift: Each nitrogenous base exists in two forms, called tautomer. A change
from one tautomer (form) to another is known as tautomeric shift (Tautomerism).
Tautomeric shift occurs naturally/spontaneously during replication (DNA replication).
Erroneous base incorporations do occur during replication, as a result of tautomerism
(tautomeric shift). Nitrogenous bases occur in standard/normal forms (tautomer) and rare
alternative forms/tautomer. The keto (C=O) and amino (C˗NH2) forms are the standard or
normal tautomer while the enol (C˗OH) and imino (C=NH) are the rare/unusual
form(tautomer). Thymine (T) and Guanine (G) occur in keto form as standard/normal form

19
 but in enol form as rare form. Adenine and Cytosine occur in amino form as standard form
and imino form as rare form. These tautomeric shifts are due to rearrangement of hydrogen
atoms that brings about wrong/mispairing of the nitrogenous bases during subsequent
round of replication, one half of the DNA molecules will contain a wrong base pair at that
point. Naturally, Adenine (A) in amino form pairs with Thymine (T) in keto form, whereas
Adenine in its imino form pairs with Cystine (C) and Thymine (T) in enol form pairs with
Guanine (G).
(2) Addition and Deletion of Nucleotide(s): Addition or deletion of one or more nucleotide
base pair(s) could occur spontaneously, during replication. This would cause a change in
the reading frame of the mRNA, resulting in non-functional protein. When the reading
frame of the mRNA is altered, frame-shift mutation occurs. Deletion or addition of
nucleotide(s) can be induced by intercalating agents.
(3) Depurination: Loss of a purine base from a nucleotide causes mutation. Depurination is
a natural phenomenon that occurs when the covalent bond (glycosidic bond) connecting
purine to the carbon-1 atom of deoxyribose sugar is broken and purine is lost. More so, the
loss of a purine base, Adenine (A) or Guanine (G) resulting in apurinic site, which will not
pair normally and may cause a transition mutation after the next round of replication.
During replication, the resulting apurinic site cannot specify a base complementary to the
original purine. So, efficient repair mechanisms remove apurinic site, under certain
conditions, as base can be inserted; this insertion will frequently result in mutation.
(4) Deamination: Loss of amine in nitrogenous base (Cytosine) occurs naturally and can be
induced using mutagen (nitrous acid/HNO2). The deamination of Cytosine yields Uracil.
During replication, the Uracil will pair Adenine (A), resulting in the conversion of a G≡C
pair to an A=T pair (G:C→A:T transition).
(5) Mutagen: Chemical and physical mutagens cause mutation in diverse ways. Mutagens
interact with DNA in a disruptive manner. They physical mutagens (radiations) include X-
ray that causes single-stranded and double-stranded breaks while the UV rays cause
thymine dimers. The chemical mutagens could be in form of nitrogenous base analogues,
intercalating agents, alkylating agents and deaminating/hydroxylating agents.

Types of Mutation
(1) Spontaneous Mutation: Spontaneous mutation is a random change in the DNA due to
errors in replication that occur without known cause. It is a mutation that occurs naturally
without the help of known mutagenic chemicals or radiation. It occurs without the help of
human intervention and it is random in nature. The rate at which it occurs in cells is not
constant. Spontaneous mutation occurs once in every 105 to 1010 replication. Replication
error, DNA repair error and environmental factors are usually responsible spontaneous
mutation. The spontaneous mutation could be due to the following: tautomeric shift, base
substitution, base modification and apurinic site. Spontaneous mutation occurs in the
absence of exogenic agents.
(2) Induced Mutations: They are form of mutations that occur due to deliberate application
of chemical or physical agents, that directly damage the DNA of a cell or alter its chemistry
or interfere with its repair mechanisms. The agents that cause induced mutation are referred
to as mutagens. The mutagens that responsible for induced mutation can be conveniently
classified according to their mechanism of action. Mutagens cause mutation in diverse
ways, these include: dimer formation, mispairing, intercalation, alkylation,

20
 deamination/hydroxylation etc. Induced mutations are caused by agents such as radiation
(X-ray, and ultra-violet ray), and chemicals that include the following types: nitrogenous
base analogue, intercalating agents, and deaminating/hydroxylating agents.
(3) Base Substitution Mutation: It is a gene mutation that occurs when a nitrogenous base at
a particular position is one strand of DNA molecule is substituted with another nitrogenous
base. During replication a new base pair will appear in the daughter molecule generated
from the strand. The base substitution mutation is also known as point mutation. The base
substitution mutation is of two types: transition mutation and transversion mutation.
a) Transition Mutation: This occurs when purine is replaced with a different purine or
alternatively, a pyrimidine is replaced by a different pyrimidine. It could be A for G
(A→G) or G for A (G→A). It could also be when T for C (T→C) or C for T (C→T).

A=T A=T
C≡G C≡G
G≡C Transition Mutation during replication A=T
T=A T=A
G≡C G≡C
A=T A=T
A-T A-T
b) Transversion Mutation: In transversion mutation, purine is replaced or
substituted with pyrimidine or pyrimidine replaced with purine.

A=T A=T
C≡G C≡G
G≡C Transversion Mutation during replication T=A
T=A T=A
G≡C G≡C
A=T A=T
A-T A-T
(2) Frameshift Mutation: This occurs from the insertion or deletion of one or more
nucleotide base pair(s) within the coding region of DNA. The deletion or
insertion/addition could be in form of single nucleotide pair, double pairs, codon or
several genes. Deletion or addition/insertion of one or two nucleotide base pair(s) will
lead to frameshift and wrong coding, since coding always start from 5ʹ tail. Deletion of
a codon will lead to loss amino acid in the polypeptide sequence. Frameshift mutation
changes the readings frame of the genes. There are two types of frameshifts: deletion
mutation and insertion mutation.
a) Deletion Mutation: During deletion mutation, a block of one or more nucleotide
base pair(s) is lost from a DNA molecule

21
 T=A
T=A
A=T
C≡G A=T
C≡G
– G≡C A=T
G≡C
C≡G T=A
C≡G
Deletion G≡C
A=T
T=A
G≡C

b) Insertion Mutation: This occurs when a block of one or more nucleotide base
pair(s) is removed or inserted or added to a DNA molecule.

T=A T=A
C≡G A=T
A=T
G≡C C≡G
A=T +
C≡G G≡C
T=A
Insertion C≡G
G≡C
A=T
T=A
G≡C
(3) Lethal Mutations: Sometimes some mutations affect vital functions and the mutant
(bacterial cell) becomes nonviable. Hence, those mutations that can kill the cell are call
lethal mutations:
(4) Reverse Mutation: It is a type of mutation that occurs in a mutated cell (mutant), that
has to do with previously mutated gene that has reverts the cell back to its initial
wildtypes. Reverse mutation is also known as back mutation. A mutant can mutate back
to being a wildtype, when this happens, the original genotype and phenotype are
restored. Back mutation is very specific, it restores function to a previously damaged
protein by a specific mutation.
(5) Conditional Lethal Mutation: Sometimes a mutation may affect an organism in such
a way that the mutant can survive only in certain environment. For example, a
temperature sensitive mutant can survive at permissive temperature of 35oC but not at
restricted temperature of 30oC.

Mutation Rate
It is the probability of occurrence of a particular mutation per cell division, which is usually
very slime. It is generally express as negative exponential per cell division. Generally,
mutations occur spontaneously in nature at a very low frequency. About one of every ten
thousand (10,000) genes contain an error. If there is one chance in a million that a particular
gene will mutate when the cell divides, the mutation rate for the gene varies between 10 -6 to

22
10-9 per cell division or generation. Mutagens typically increase the mutation rate by a factor
of 10 to 1000 times.
Factors Influencing the Rate of Spontaneous Mutation
(i) Accuracy of the DNA replication mechanism.
(ii) Efficiency of DNA damage repair mechanism.
(iii) The size of the gene; larger genes are prone to mutation.
(iv) Errors made during recombination.

Effects of Substitution Mutations

Base substitution mutations produce different effect on the affected codon(s). The effects could
be in form of silent mutation, missense mutation or nonsense mutation.

(1) Silent Mutation: During base substitution, the base sequence of the DNA is altered
at a point but the altered codon on mRNA still encodes the same amino acid due to
the degenerate or redundancy nature of the genetic code. In silent mutation, the new
codon encodes the same amino acid; there is no change in amino acid sequence. That
is, the substituted base does not change the amino acid.

Mutated DNA
Normal DNA

Peptide

Peptide
mRNA

mRNA

T A T A
A U Met A U Met
C G C G
T A T A
T A Lys T A Lys
T A C G
C G C G
A U Val A U Val
T A T A
Note: T on DNA strand was substituted with C, causing transition mutation which
changed the mRNA codon (AAA) to AAG that encodes for same amino acid (lysine).

(2) Missense Mutation: It is a change in nucleotide sequence as a result of base

substitution at a particular point that changes a codon for one amino acid into a
codon for a different amino acid. Missense mutations usually produce nonfunctional
protein. A simple missense mutation is a change from A to T at point specific site
results in change from glutamic acid to valine.

23
 Mutated DNA
Normal DNA

Peptide

Peptide
mRNA

mRNA
T A T A
A U Met A U Met
C G C G
C G C U
T A Glu A U Val
C G C G
T A T A
T A Lyn T A Lys
T A T A
Note: Thymine (T) on normal DNA template strand was substituted with Adenine (A)
during replication causing transversion mutation that changed the mRNA codon (GAG)
for Glutamic acid to GUG, that encodes for valine.

(3) Nonsense Mutation: Substitution mutation causes conversion of a sense codon to a

nonsense codon or stop codon. A base substitution changes the codon for an amino
acid to any of the three chain terminating codons. This causes premature termination
of translation or protein synthesis, so that an inactive protein is produced.
Mutated DNA
Normal DNA

Peptide

Peptide
mRNA

mRNA

T A T A
A U Met A U Met
C G C G
T A A U
Stop
T A Lys T A
Codon
T A T A
C G C
A U Val A
T A T
Note: Thymine (A) normal DNA template was substituted with Adenine (A) during
replication causing transversion mutation, that changes a sense codon to stop codon
(UAA), therefore causing termination of protein synthesis.

Selection and Detection of Mutants

Almost all characteristics of an organism can be changed due to mutation. Some mutations are
selectable, conferring some types of advantages on the organisms possessing them, whereas
others are non-selectable, even though they may lead to very clear change in the phenotype of

24
an organism. Some mutants are selectable while others are non-selectable. Selectable Mutants
are strains that grow and outgrow and replace their parents e.g. an antibiotic resistant mutant
and can be selected under appropriate environmental condition. Selection is a tool allowing the
isolation of a single mutant form a population containing millions and billions of parental
organisms. Non-Selectable Mutants are usually obtained through screening process e.g.
colour loss in a pigmented organism. The non-selected mutants are referred to as detectable
mutants. Mutants can be isolated from a population based on selection or detection technique.
A selectable mutation confers a clear advantage on the mutant strain under certain
environmental condition, so that the progeny of the mutant is able outgrow and replace the
parent. For example, antibiotic resistant mutant can grow in the presence of antibiotic
concentrations that inhibit or kill the parental cell. Selectable technique is therefore a powerful
genetic tool that allows isolation of a single mutant from a population containing millions of
parent organisms. An example of a non-selectable mutation is a colour loss in a pigmented
organism. Non-pigment cells neither have advantage nor disadvantage over he pigmented
parental cell, when on an agar plate. Non-selectable technique can be used to isolate mutant by
examine large numbers of colonies and look for different one.

Isolation of Mutants
Mutant can be isolated from parent strain using screening techniques e.g. replica plating
technique, and selection techniques e.g. direct method.

(i) Direct Method: This method involves plating cells on a medium that permits the growth
of the mutant but not the parent or wildtype. For example, if penicillin resistant mutant is
to be isolated from a population, the easy way to select out the mutant is by direct plating
of a natural population of cells on a medium containing penicillin. Only a few resistant
cells in the population will form visible colonies.

Direct Plating Technique

(ii) Replica Plating Technique: A nutritional auxotroph can be isolated by replica plating
technique. An auxotroph is a mutant that requires availability of special nutrient for its
growth. An auxotroph may be derived from the wildtype. For example, in order to isolate
lysine auxotroph (lys–) using replica plating, mutants are generated by treating a culture
with a mutagen, The culture containing the wildtype and lysine auxotroph is plated on a
complete medium. (medium contain lysine) and incubated at the required temperature and
time. After colonies have appeared on the plate surface, sterile velveteen is pressed on the
plate surface to pick up that bacterial from each colony. The velveteen is the pressed on

25
 another two plates (complete and minimal medium plates) and the organisms are
transferred to the same position as on the master plate (the first complete medium plate).
The growth on the two replica plates is compared. The colony that grew on the replica
complete medium plate but not on replica minimal medium plate is lysine mutant (lys–).
The lysine auxotroph mutant (lys–) can be isolated from the replica complete medium plate
and culture accordingly.

Replica Plating Technique

Carcinogenicity Test
Ames Test: Many of the known mutagens are carcinogens; they cause cancer in animals. The
potential carcinogenicity of a mutagen can be tested on animal. The procedures for using
animal are timing consuming an expensive. Brue N. Ames in 1975 at University of California
developed a faster and less expensive procedure for the preliminary screening of a potential
carcinogen. This test is called Ames test. The principle of the test is based on back/ reverse
mutation in an auxotroph strain of Salmonella typhimurium TA98. Ames test is a screening test
for detection of carcinogenic property, by testing the ability of chemical agents to induced
bacterial back mutation.
In order to carry out Ames test, a compound whose carcinogenicity is to be tested is mixed with
histidine auxotroph Salmonella typhimurium TA98 strain (his– auxotroph strain). This is
followed mixed with a solution of extract from rat liver cells, and a small or little histidine. The

26
mixture is plated on a minimal medium (histidine deficient medium) and incubated for 2-3 days
at 37oC. After incubation, only colonies that are his+ revertants are able to grow.

Ames test

Mutagens and Their Effects

Chemical Mutagens: The chemical mutagens include base analogues, alkylating agents,
deaminating agents/hydroxylating agents and intercalating agents. They show different effect
on the DNA molecule.

(i) Base Analogues: They are chemicals or compounds that are structurally similar to
normal nitrogenous bases (purines and pyrimidines) but when incorporated into DNA
molecule during replication display faulty or wrong pairing. Once in place, these
compounds typically exhibit base pairing different from the normal base they replace
and cause stable mutation. Examples are 5-BromoUracil and 2-Aminppurine.
• Effect: They induce transition mutation in DNA molecule.
Mechanism: When molecule is exposed to 5-BromoUracil; a thymine analogue, it
replaces Thymine (T) during replication and pair with Adenine (A). After further
subsequent replication, the 5-BU mispairs with Guanine (G). Therefore, causing
TA→CG (transition).
• 2-Aminopurine (2-AP) is an analogue of Adenine (A) which normally pairs with
Thymine (T). 2-AP usually mimic Adenine but pairs with Cytosine instead of Thymine
(T). After a further replication, Cytosine (C) pairs with Guanine, therefore forming a
AT→GC transition mutation.

27
 C≡G C ≡ G
A=T Incorporation of 5-BU A = T Mechanism of 5-BU
T=A 5-BU= A as a mutagen
G≡C G ≡ C
C≡G C ≡ G

Replication 1

C ≡ G C≡G
A = T A=T
5-BU= G + T=A
Insertion of G into Non- mutated DNA
G ≡ C G≡C
New DNA strand
C ≡ G C≡G

Replication 2

C ≡ G C≡G
A = T A=T
5-BU= G + C≡G Mutated DNA
G ≡ C G≡C
C ≡ G C≡G

TA→CG Transition mutation

Replication will
generate more
mutant DNA

28
 Mechanism of 2-Aminopurine (2-AP)
as a mutagen

C≡G C ≡ G
A=T Insertion of 2-AP 2-AP = T
A=T T = A
G≡C G ≡ C
C≡G C ≡ G

Replication 1

C ≡ G C≡G
2-AP = C A=T
T = A + T=A
Insertion of C into New Non- mutated DNA
G ≡ C G≡C
strand C ≡ G C≡G

Replication 2

C ≡ G C≡G
2-AP = C G=C Mutated DNA
T = A + T=A
G ≡ C G≡C
C ≡ G C≡G

AT→GC Transition mutation

Replication will
generate more
mutant DNA

29
 (ii) Alkylating Agents: They are chemicals that alter the structure of nitrogenous base by
adding an alkyl group (e.g. methyl, ethyl etc.) at various position on DNA. Addition of
alkyl group to the nitrogenous base brings about mispairing. They are dangerous
chemicals, not only that, they are highly mutagenic, also may of them are strongly
carcinogenic. They induce changes even in non-replicating DNA. Alkylating agents
include ethylmethanesulphate (EMS), ethylethanesuphate (EES), and
methylnitrosoguadine (MNG) etc.
• Effect: For example, methylnitrosoguadine induces transition mutation GC→AT.
• Mechanism: Methylnitrosoguadine adds methyl group guanine or thymine, therefore
induces GC→AT transition mutation. The modified guanine pairs thymine instead of
cytosine and modified thymine pairs with guanine rather than adenine. The same
mechanism occurs in all alkylating agents.

C≡G C≡G C≡G

Replication 1 T=A
T=A MNG T=A Non- mutated DNA
T=A T=A T=A
G≡C *G≡C G≡C
C≡G C≡G G≡C

C≡G
T=A
T=A
*G≡C
C≡G

Replication 2

C≡G C≡G
T=A A=T
T=A + T=A
*G≡C A=T
C≡G C≡G

Mutated DNA

Further replication
will produce more
mutant DNA

30
 (iii) Deamination/hydroxylating agents: Nitrous) acid (HNO2) is a potent mutagen that
alters the base-pairing affinities of Cytosine and Adenine by replacing an amino
group with an oxygen atom. Transition mutations occur in both directions AT→GC
and GC→AT. Although, guanine is deaminated but its base pairing properties are
unaffected. It affects replicating and non-replicating DNA.
• Effect: Transition mutation AT→GC or GC→AT
• Mechanisms: Nitrous acid deaminates Adenine (A) and Cytosine (C) and
replaces with oxygen atom. The deaminated Adenine (A*) pairs with
Cytosine (C) instead of Thymine (T) while deaminated Cytosine (C*) pairs
Adenine rather than Guanine.

Deamination of Cytosine

C≡G C*≡G Replication 1 C*≡A C≡G

A=T Deamination A =T A=T + A=T Non- mutated DNA
T=A T =A T=A T=A

Replication 2

C*≡A T=A
A=T + A=T Mutated DNA
T=A T=A

Transition Mutation: CG→TA

31
 Deamination of Adenine

C≡G C ≡G
A=T Deamination A*=T
T=A T =A
G≡C G ≡C
C≡G C ≡G

C≡G C≡G
A*=C A=T
T=A Replication 1 T=A Non-Mutated DNA
G≡C G≡C
C≡G C≡G

C ≡G C≡G
A*≡C G≡C
A =T T=A
G≡C Replication 2 G≡C Mutated DNA
C≡G C≡G

More mutant DNA will

be produced during AT→GC (Transition Mutation)
subsequent replication

(iv) Intercalating Agents: They are mutagens that insert themselves between adjacent
nucleotides in a single-stranded DNA. More so, they also insert themselves between
nitrogenous bases. They distort the strand at this site, thereby causing deletion or
addition of nucleotide(s). Examples are acridine dyes (e.g. acridine orange and
ethidium bromide), benzopyrene and proflavine. They are generally called
intercalator. They exert mutagenic effect during replication.
• Effect: They cause nucleotide addition and deletion leading to frameshift or
formation of nonsense codon and stop codon.
• Mechanism: They usually insert themselves in between adjacent
nitrogenous bases in single-stranded DNA. The bound intercalator interferes
with new stranded synthesis, lead to gross deletion of nucleotide.

Physical Agents (Radiation)

X-rays and ultra-violet radiation are the common radiation that cause mutation. X-rays are
ionizing radiation while UV light is a non-ionizing radiation. VU light is not so effective in
inducing mutation like X-ray.

32
 • Effect: X-ray causes physical breakage of DNA strands in the phosphate backbone of
DNA, therefore causing frameshift and deletion. Ultraviolet light causes Thymine-
Thymine dimers.
• Mechanical: In UV light, two adjacent pyrimidines bind together, therefore forming
dimer. That is, binding together of the adjacent pyrimidine in the same strand. The
formation of dimer causes replication error, that is replication of DNA cannot proceed
and gene expression is prevented.
X-ray have sufficient energy to break the sugar-phosphate backbone of DNA, thereby
splitting it into small pieces. If these, pieces are not joined together properly, mutation
due to deletion may occur.

X-rays Mechanism
T=A A=T
T=A – T=A G≡C + A=T
G≡C A=T Deleted
A=T C≡G
C≡G T=A
T=A Frameshift Mutation

Repair of Damaged DNA

Errors might occur in bacteria or other microorganisms during DNA replication and a variety
of mutagen like X-ray and chemicals can alter the nucleotide sequence, therefore
microorganism must be able to repair changes in DNA sequence that might be fatal. All
organisms have developed means to repair their damage done to their DNA, in addition to the
proofreading mechanism by replicator enzymes (DNA polymerase). The repair mechanisms
include mismatch repair, direct repair, and double-stranded break repair. The excision repair
could be in form of base excision repair or nucleotide excision repair.

(i) Mismatch repair: Despite the accuracy of the DNA polymerase action and
continual proofreading, few errors slip through during DNA synthesis, any
remaining mismatch bases and other errors are usually detected and repaired through
a process called mismatch repair. During mismatch repair, shortly after DNA
replication mis-paired bases are removed and replaced.
(ii) Base excision repair: It is a repair mechanism used to detect and remove certain
types of damaged bases, the damaged bases are excised and replaced. The removal
of the bases is catalyzed by a set of enzymes called DNA glycosylases. They
recognized and remove a specific type of modified mase by cleaving the bond that
links the base to the 1-carbon atom of deoxyribose. For example, Uracil produced
by the deamination of Cytosine. In base excision repair, just only the damaged base
that is removed and repaired. Once the base is removed, the gap is filled and sealed
by DNA polymerase and ligase respectively.
(iii) Double-stranded break repair: Sometimes, environmental factor like X-rays can
cause double-stranded breaks. If the breaks are not repair, hundred of genes may be
lost. The break can be repaired either by joining the non-homologous ends or
homologous recombination. In homologous recombination, the broken chromosome

33
 pairs up with it homologous. The damage region is replaced via recombination, using
sequences copies from the homologue. That is, the information from the homologous
chromosome that matches the damaged one is used to repair the break. In non-
homologous, end joining the two broken ends of the chromosome are simply glued
back together.
(iv) Nucleotide excision repair: It is a complex process thar removes bulky DNA
lesions. A set of complex enzymes detect the distorted DNA and additional enzyme
separates the two nucleotide strands at the damaged region. The sugar-phosphate
backbone of the damaged strands it cleaved (cut) on both sides of the damaged. The
damaged portion is removed and the gap is filled in by DNA polymerase and sealed
by DNA ligase.
(v) Direct repair/ chemical reversal: This repair mechanism does not replace the
altered nucleotides or bases but change them back to their original structure. The
best characterized direct repair mechanism is photoreaction of UV induced
pyrimidine dimer. Escherichia coli and eukaryotic cells possess an enzyme called
photolyase, that uses energy captured from light to break the covalent bonds that link
the pyrimidines in a dimer. This is also called light repair or photoreactivation.
Damage caused by alkylating agents can also be repaired by direct repair/ reverser.
Methyl or other alkyl groups added to guanine are removed by enzyme known as
alkyltransferase or methylguanine methylransferase. Therefore, damaged to guanine
by methylnitrosoguadine can be replaced directly.

Types of Mutants
(1) Morphological Mutants: They are microorganisms whose colonial or cellular
morphology or characteristics changed due to mutation. These changes many include
loss of ability to produce pigments, spores, capsule, flagella or genetic make-up.
Morphology mutants can also be bacterial cells that have lost their attachment sites for
bacteriophages (bacterial viruses) and are therefore resistant to the activities of phage
(bacteriophages).
(2) Biochemical Mutants: They are mutants formed as a result of a change or changes in
the biochemistry of the cell; that is, the mutant cannot grow on minimal medium and
therefore requires nutrient supplement. Such mutants are called Auxotroph. Whereas,
the wildtype microbial strains which can grow on minimal medium are called
Prototrophs. Auxotroph is formed by a forward mutation on a prototroph (wildtype
organism).

Phenotypic and Genotypic Changes

(i) Phenotypical change: It is a reversible change in the sum total of the observable
characteristics of an organism, resulting from interaction of the environment with the
genotype. Phenotypic change usually affects all the cells in the population, without
having any effect on the genes. Phenotypic change is usually morphological change
(morphological in nature) during which cells are probably responding to changes in
temperature or type of nutrient in their immediate environment.
(ii) Genotypic change: This is an irreversible, rare event among microorganisms such that
only a few cells in a population have a chance in the sum total of their genetics

34
 constituent. Genotypic change is also referred to a mutation, as long as it is not lethal
and the altered gene(s) or mutant gene(s) will breed from generation to generation. It is
usually non-reversible.

Recombination/Genetic Recombination
Genetic recombination is a process through which a new DNA molecule is formed by
combining genetic materials from two different organisms or different regions within the same
chromosome. Genetic recombination brings a lot changes to the genetic make-up of an
organism unlike mutation that causes same change in genome. Recombination contributes to
population diversity to produce beneficial change, since it intends not to destroy gene function.
This process occurs naturally in both prokaryotes and eukaryotes, and can be carried out in the
laboratory.

In eukaryotes, it is a process by which genes are exchanged between two DNA molecules to
form new combination of genes on the chromosomes. It involves cross-over. In eukaryotes,
recombination occurs through meiosis (sexual reproduction), this process creates diversity in
offspring but parents remain unchanged. During recombination, the eukaryotes undergo It is
vertical gene transfer process, that is, genes are passed from parents to offspring. More so,
recombination in eukaryotes is reciprocal.

In prokaryotes, genetic recombination occurs via gene transfer within the chromosome or
between two cells. It is a one-way process by which a piece of genetic material (exogenote) is
donated to the chromosome of a recipient cell and integrated into its endogenote. Prokaryotes
undergo recombination through horizontal gene transfer via conjugation, transformation and
transduction; theses processes require donor cell that gives portion of its DNA to the recipient
cell. The recipient cell that receives the donor’s DNA (exogenote) and incorporates it into its
chromosome is called recombinant cell. If the recombinant cell shows new character due to
incorporation of the new DNA, the cell has been transformed (Transformed cell). Generation
of recombinant cell is usually very low, only few cells in a population are capable of
transferring or receiving and incorporating of genetic material.

Types of Recombination
(i) Homologous or General Recombination: This form of recombination involves exchange
of genetic material between complementary strands of two homologous DNA molecules
(identical or similar molecules or sequences). It is a reciprocal exchange of genetic
materials between two homologous DNA molecules. Homologous recombination occurs
anywhere/place on the chromosome and it results in DNA strands breakage and reunion,
leading to cross-over. Double helix of two DNA molecules breaks and the two ends join
to their opposite partner to re-unite to form double helix. The site of exchange can occur
anywhere in the homologous nucleotide sequence. When a strand of one DNA molecule
becomes base paired to the second strand to yield heteroduplex, just between two double
helices. Eukaryotic cells carryout homologous recombination.
(ii) Non-homologous Recombination: The non-homologous recombination on the other hand
occurs between DNA molecules that are not necessarily similar. It occurs in regions where
no large-scale sequence similarity is apparent, e.g. translocation between different
chromosomes or deletions that removed several genes along a chromosome. For example,

35
 during bacterial transformation (a non-reciprocal gene transfer), a piece of genetic material
is inserted into the chromosome through the incorporation of a single strand to form a
stretch of heteroduplex DNA.
(iii) Site-specific Recombination: It is a type of recombination in which DNA molecules are
rearranged by breaking and joining the strands at specific points. Site-specific
recombination involves two shorts DNA (12-24bp) sequences (sites) which may be within
the same molecule or in different molecules. Dissimilar parental materials are involved in
site-specific recombination. The process requires. A specialized enzymatic machinery
basically one enzyme or enzymatic system for each particular site. Integration of virus
genomes into bacterial chromosome is a good example of site-specific type of
recombination, which happens during transduction. Bacteriophages genome is injected
into a bacterial chromosome when the bacterium is infected with the phage (bacterial
virus). The virus genetic material is not homologous with the bacterial chromosome, hence
the enzymes that are involved are specific for that particular virus and bacterium.
(iv) Transposition Recombination: Transposition is a highly specialized form of recombination
in which a discrete DNA segment called transposable element or transposon moves from
one location to another, either on the same chromosome or different chromosomes.
Transposition can also promote other kinds of DNA rearrangements such as deletion,
inversion and replicon fusion. Such rearrangements of DNA can have profound effects. It
can result in stable acquisition of new genetic information. The acquisition of novel DNA
segment or other transposon-mediated DNA rearrangement can also have considerable
impact on the host genetic information. The transposons (mobile DNA segments) serve as
agents of genetic change and their actions contribute substantially to genetic diversity.
Element integration, deletion, and inversion can all result in alterations in the host DNA,
which can lead to subsequent change in gene expression. Transposons (mobile DNA
segments) also often encode properties/genes such as antibiotic resistant, degradation of
organic compounds, exotoxin, change in colony morphology, pigmentation, pili, viral and
virulent factor genes. The transposon is also referred to as jumping gene. The movement
of the gene is mediated by transposase (enzyme). Since they can be transferred between
unrelated organisms, they create genetic diversity essential for the evolution and survival
of organisms. The transposase mediates specific recognition of the transposable element
and also executes DNA strand breakage and joining reaction which underlie element
translocation. Transposons occur in both prokaryotes and eukaryotes. Transposition in
bacteria is of two forms or types; non-replicating (cut and paste) transposition and
replication transposition.
(a) Non-Replication Transposition: In non-replication transposition (cut-and-paste
transposition), the transposon is excised from the donor DNA and inserted into the
target DNA to form a simple inversion. The role of replication in this reaction is limited
to repair of the new joints between the transposon and the target DNA. It is also results
in a gapped donor DNA which may be processed in a variety of ways.
(b) Replication Transposition: In replication transposition, the transposon is directly
copied by DNA replication during element translocation to generate a structure called
a cointegrate. The cointegrate contains two copies of the transposon joining the donor
and target backbone. The transposon is is excised form the donor backbone and
inserted into the target DNA to yield a simple inversion. Another product of this
reaction is a gapped donor backbone.

36
Methods of Genetic Material Transfer in Bacteria
Bacteria are haploid organisms; they do not undergo sexual reproduction to accompany genetic
recombination process. Recombination takes place in bacteria by following horizontal gene
transfer. Genes or genetic materials are not transferred by inheritance from parents to offspring
(vertical gene transfer) but rather genes are transferred from one independent mature organism
to another. Genetic material or gene can be transferred horizontally to bacteria through the
following methods: transformation, conjugation and transduction.

(i) Transformation: It is a process by which free DNA fragment from one bacterium is
incorporated into a recipient bacterium/cell and brings about genetic change or
incorporate it to its chromosome in inheritable form. Transformation may occur either
in nature or in the laboratory. The natural process of this genetic material transfer
appears to play no role in disease. In laboratory conditions, DNA may be extracted from
one type of bacterium and introduced into genetically different bacterium. The cell
walls of bacteria in vitro are made more permeable for DNA uptake by treating with
Calcium Chloride.
In natural transformation, the DNA comes from donor bacterium. The process is
random in nature; any portion of a genome can be transferred between bacteria. When
bacteria are lysed, they released considerable amount of DNA into the surrounding
environment. These fragments may be relatively large and contain several genes. When
a fragment makes contact with a competent cell (cell that is able to take up DNA and
transformed), it binds to the cell and be taken inside. For effective transformation to
take place, four stage are involved: preparation of the transforming DNA, formation of
a competent recipient cell, uptake of transferred DNA by recipient cell and integration.

37
(ii) Conjugation: It is process of transferring genetic material (DNA) from the donor
bacterium to the recipient bacterium during mating between the two cells. Conjugation
usually occurs between two closely related species, most especially Gram-positive
bacteria. Conjugation mechanism is plasmid mediated and direct cell-to-cell contact is
involved. The donor cell possesses conjugative plasmid called fertility (F) plasmid or
fertility factor or sex plasmid. The recipient bacterium/cell lacks conjugative plasmid.
The F-plasmid controls the mating process of the bacteria. Pilus is the most important
protein that forms conjugative tube/bridge between the cells (mating cells).
Mating Between F+ and F– Cells/Bacterium
Mating between donor cell (male bacterium) carrying the F-factor (F+) and the recipient
female bacterium (bacterium lacking F-factor/ F– cells) begins with cell-to-cell contact.
The F– cell/bacterium attaches itself to receptor on the surface of the F– bacterium/cell
with the aid of pilus. A conjugation bridge is then formed between the two cells. This
is followed by enzymatic cleavage of the F-factor Double-stranded DNA into separate
two strands. A strand of the F-factor DNA is transferred into F– cell through the
conjugation bridge. Synthesis of the complementary strand to form a double-stranded
F-factor (plasmid-factor) in both the donor cell (F+ cell) and recipient cell (/ F– cell)
completes the process of conjugation. The recipient cell becomes F+ cell that is capable
of transmitting the plasmid (F-factor) to other F– cells (female cells).

38
39
Conjugation Between High-frequency Recombination Bacterium/Cell and F–bacterium/Cell
High-frequency recombinant cell (Hfr) is a strain of F+ cell that has fertility (F) plasmid
integrated into its chromosome. The F-factor contains sequences which enable it to get
integrated into the host bacteria chromosome. The F-factor is an episome. Hence, the Hfr
strains have the capacity to transfer chromosome to another cell. The whole chromosome can
be transferred if integrated with F-plasmid. When a cell is in the Hfr state, the integrated F-
factor is replicated as of the chromosome and becomes inheritable by the progeny. The self-
transfer function is not repressed in the Hfr state, lit still directs the formation of pili on the cell
surface, so that Hfr cell can conjugate and transfer DNA.

Mating between Hfr cell and F– cell begins with cell-to-cell contact. A conjugative tube or
bridge is then formed between the Hfr cell and F– cell. During mating between the two cells,
single strand of Hfr DNA that enters the recipient cell (F– cell), contains a part of the F-factor
at one end, followed by the bacteria chromosome and then part of the F-factor (F-plasmid).
Full F-plasmid is not transferred with the bacterial chromosome to the recipient cell (F– cell),
that is, part of the F-factor is left in the Hf cell. During mating between the Hfr cell and F– cell,
part of F-factor segment is first transferred through the conjugal tube, followed by the entire
chromosome, as the remaining segment of the F-plasmid containing the tra region is
transferred, a cut occurs. Since the recombinant produces after mating between Hfr cell and F–
cell fails to inherit the entire set of F-plasmid genes, therefore the recipient remains F– cell.

(iii) Transduction: The transfer of a portion of DNA from a bacterium to another that is
mediated by phage/bacteriophage (virus that infects bacteria) is known as transduction.
This only happen when the lytic cycle of the bacteriophage is followed by lysogenic cycle.
During replication of virus within a bacterial cell, a piece of the bacterial DNA may be
incorporated into the phage genome. Such phage particle is defective. During subsequent
infection of another bacterium, the phage injects its DNA into the new bacterium which
neither produce new phages nor be destroyed. Non-pathogenic bacteria can become
pathogenic, for example when genes encode diphtheria toxin, botulinum toxin, cholera
toxin, and erythrogenic toxin can be transferred from one bacterium to another by
transduction.

40
Bacterial Plasmids
Plasmids are small, circular, double-stranded extrachromosomal piece of genetic material
(DNA), that can replicate and segregate autonomously in bacterial cells. Plasmid can maintain
itself in the cytoplasm of a bacterium for many generations. It contains genes that are not
essential but often beneficial to the bacterium, for example resistance gene. Some plasmids can
exist either independently or integrated into the host chromosome, such plasmids are called
Episomes. All bacterial plasmids that contain genetic information for self-transfer to another
by conjugation are known as conjugative plasmids.

41
Main Types of Bacterial Plasmids
(i) Fertility Plasmids: The fertility plasmids (F-plasmids) contain transfer genes that allow
genes to be transferred from one bacterium to another through conjugation. F-plasmids
that are inserted into chromosomal DNA are known as episomes. Bacteria that have the
F-plasmid are known as F+ cells (F+ bacteria), and bacteria without it are F– cells (F–
bacteria). When F+ bacterium conjugates with F– bacterium, two F+ bacteria result. They
can only be one F-plasmid in each bacterium.

(ii) Resistance Plasmids: The R plasmids contain genes that help a bacterial cell defend
against environmental factors such as poisons or antibiotics. Some resistance plasmids
can transfer themselves through conjugation. When this happens, a strain of bacteria
can become resistant to antibiotics. Recently, the type bacterium that causes the
sexually transmitted infection gonorrhea has become so resistant to a class of antibiotics
called quinolones that a new class of antibiotics, called cephalosporins, has started to
be recommended by the World Health Organization instead. The bacteria may even
become resistant to these antibiotics within new few years. Also overuse of antibiotics
to treat other infections, like urinary tract infections, may lead to the proliferation of
drug-resistant strains.

(iii)Virulence Plasmids: These plasmids determine the pathogenicity of bacteria. When a

virulence plasmid is inside a bacterium, it turns that bacterium into a pathogen, which
is an agent of disease. Bacteria that cause disease can be easily spread and replicated
among affected individuals. The bacterium Escherichia coli (E. coli) has several
virulence plasmids. E. coli is found naturally in the human gut and in other animals, but
certain strains of E. coli can cause severe diarrhea and vomiting. Salmonella enterica
is another bacterium that contains virulence plasmids.

(iv) Degradative Plasmids: Degradative plasmids help the host bacterium to digest
compounds that are not commonly found in nature, such as camphor, xylene, toluene,
and salicylic acid. These plasmids contain genes for special enzymes that break down
specific compounds. Degradative plasmids are conjugative.

(v) Col Plasmids: The Col plasmids contain genes that make bacteriocins (also known as
colicins), which are proteins that kill other bacteria and thus defend the host bacterium.
Bacteriocins are found in many types of bacteria including E. coli, which gets them
from the plasmid ColE1.

Plasmid Curing
Plasmids are usually not stable; they can easily be lost. The loss of plasmid is called curing.
Curing can occur spontaneously or be induced by treatments that inhibit plasmid replication
but not cell replication. Exposure to physical and chemical agents cause curing of plasmids.
The chemical agents include acridine orange, acriflavine, ethidium bromide and sodium
dodecyl sulphate, while the physical agents include super-optimum temperatures, pH or
extreme environmental conditions. Curing is usually carried out in order to determine where a
plasmid encodes a particular genetic information, for example, antibiotic resistance. The
efficiency of curing varies widely, depending on the plasmid and the particular bacterial host
carry it.

42
 Topic six:

MICROBIAL GENETICS IN BIOTECHNOLOGY

Biotechnology and Genetic Engineering

Over the years the uses of microorganism, plants and even animals have gone beyond the initial
known applications. In order solve some of the challenges that confront man, biotechnology
has played very vital roles. Biotechnology is the application of biological techniques and
genetic engineering to produce desired products. The knowledge of genetic engineering is
applied to microorganisms and there derivatives (enzymes) to produce useful products or for
the purpose other than their original intension. The genetic make-up (genome) of an organism
can be deliberately manipulated by changing the nucleotide sequence for a desire purpose, this
is termed genetic engineering. This is achieved by using recombinant DNA technology.
Recombinant DNA technology provide methods by which DNA molecules are cut, modified
and joined together by means of enzymes, so as to provide recombinant DNA molecules.
Specified DNA fragments can be isolated and amplified, and their genes can be expressed at
high levels. The nucleotide specificity required for cleavage by restriction enzymes allows
fragments containing genes or parts of genes to be incorporated into plasmids (vectors) that
can in turn be used to transform bacterial cells. In genetic engineering, recombinant process is
carried out in vitro rather than in vivo (inside the cell).

Major Steps in Genetic Engineering

i. Construction of the recombinant DNA molecules.

ii. Introduction of the recombinant DNA into a host organism.
iii. Screening and selection of those host cells that contain the recombinant DNA
molecules.
iv. Expression of the genes of interest.

Construction of Recombinant Molecules

For recombinant DNA molecules to be constructed, DNA fragment carrying desired genes must
first be generated. The desired genes can be isolated from the organism possessing them using
physical fragmentation method or enzymatic approach. Physical fragmentation involves
disruption of cells, nuclease inhibition and denaturation of nucleoprotein complex and
purification of DNA from the macromolecules. Using enzymatic approach, DAN fragment can
be obtained by cutting linear DNA molecules using restriction enzymes (endonuclease).

Restriction Endonuclease
Restriction endonucleases (restriction enzymes) are the enzymes that recognise specific base
sequences within DNA and cleave (cut) the DNA. They are found in a wide variety of
procaryotes (bacterial species), but very rare in eucaryotes. In vivo, restriction enzymes protect
procaryotes from hostile foreign DNA such as viral genome. In vitro, they are essential in DNA
manipulation and their discovery gave birth to the field of genetic engineering. The fragment

43
generated by restriction enzymes are called restriction fragment, and the act of cutting is often
referred to as digestion.
Restriction enzymes are named based on the abbreviation of the organisms from which they
were isolated. The first letter of the generic name, followed by the first two letters of the species
name, form the basic name of the enzyme. For example, Eco from Escherichia coli and Hin
from Haemophilus influenzae. When the enzyme is present in a specific strain of an organism,
the name may be followed by a strain designation. For example, Eco R from Escherichia coli
RY. The restriction enzyme can cut the recognised sequence in either of the following two
ways:

(i) Restriction enzymes can cut the recognised sequence at different position on the two
DNA strands that is asymmetrically, to generate fragments which are single stranded,
with protruding 5ʹ or 3ʹ terminal. This is usually referred to as sticky or staggered cuts
or ends.

44
 (ii) The enzyme can also cut through both DNA strands at the same point right at the line
of symmetry to produce fragment. This is usually referred to as blunt ends or cuts.

Vectors
Vectors are specialized DNA molecules which are used as vehicles to transport or carry
exogenous DNA or foreign genetic material into the host cell. A vector is capable of self-
replication and stable integration inside the host cell. In the host cell, the transferred exogenous
DNA can be further replicated and/or expressed. A vector is capable of self-replication and
stable integration inside the host cell. The vector itself is a DNA sequence that consists of an
insert (transgene) and a larger sequence that serves as the backbone of the vector. It is a self-
replicating DNA molecule using in gene cloning. The sequence to be cloned is inserted into the
vector and replicate along with it. The insert is usually gene (genetic material) of interest.
Vectors are capable of self-replication in the host cell, so that the inserted DNA fragment
(insert) will also replicate along with the vector and clones (copies) of DNA can be produced.

Types of Vectors

(i) Plasmids: They are simple vectors. They are extrachromosomal, simple, circular
double-stranded DNA, found in Gram-positive and Gram-negative bacteria.

45
 (ii) Phages: They are viruses that infect bacteria. The most common used phages is
Lambda (λ); a naturally occurring double-stranded DNA that infects Escherichia coli.
It is a large vector capable of carry up to 25kb of foreign DNA.
(iii) Cosmids: They are hybrids between plasmid and phage vector. They are large capacity
vector that is capable of carry up to 45kb of foreign DNA.
(iv) Yeast Artificial Chromosome: They are hybrid of plasmid and yeast DNA sequence.
They are relatively small (25kb) vectors that can carry large foreign DNA of several
hundreds of kilobases (kb).

Importance of Genetic Engineering

(i) Agricultural Application: With increase in world population and decrease in amount of
land, transgenic plants and animals are being used to solve the problem of food shortage.
In addition to food productivity, transgenic plants and animals are also aimed in improving
food quality. Genetic engineering brings about development of drought resistant crops.
Likewise, transgenic plants or crops are also resistant to frost, herbicides and pesticides.
More so, transgenic plants are more resistant to plant viruses and pests, they are more
nutritious with short period of reaching harvest time. Example of genetically engineered
animal is Lamb Dolly that was produced in 1997. The livestock produced in this way
usually have improved feed efficiency, fertility, milk output, disease resistance as well as
better meat quality.
(ii) Environmental Application: Genetically engineered microorganisms have found their
application in cleaning up of polluted environment and highly toxic mining waste. Some
organisms are also specifically engineered to degrade recalcitrant compounds.
(iii) Applications in Medicine: Clinically important proteins such as human insulin, interferon
and tissue plasminogen activator that dissolved blood clots can now be produced in large
quantity using genetically engineered microorganisms. For example, gene for human
insulin can now be cloned in Escherichia coli to produce large quantity of insulin.
Genetically engineered microorganisms are also being used in the production of vaccines.
Likewise, genetically engineered microorganisms also have important role to play in the
pharmaceutical industry in the nearest future.
(iv) Gene therapy application: Genetic engineering has also found application in area of
genetic disease. Gene therapy is a technique for replacing a faulty gene with a normal one
in individuals with fatal or extremely debilitating genetic diseases. The inherent benefit of
this therapy is to permanently cure the physiological dysfunction by repairing the genetic
defect.

46