GODFREY OKOYE UNIVERSITY, ENUGU.

MICROBIOLOGY DEPARTMENT
BTG 343 - MOLECULAR GENETICS FIRST SEMESTER NOTES

INSTRUCTOR: Prof. Emmanuel A. Eze

COURSE OUTLINE
 Introduction and Definition of Concepts
 Nature of the Genetic Material
 The Significance of DNA Structure
 DNA Replication
 Applications of the DNA Code: Transcription and Translation
 Protein Synthesis
 Changes in Genetic Information
 Genetic Transfers
 Transformation
 Conjugation
 Transduction
 Mutation

INTRODUCTION:

Genetics is the study inheritance of biological characteristics by living things. It is the science of heredity
and its main focus include the transmission of biological properties (traits) from parent to offspring, the
expression and variation of those traits, the structure and function of the genetic material, and how this
material changes or evolves. Molecular genetics deals with the biochemistry of gene function.

The Nature of the Genetic Material:

Deoxyribonucleic acid or DNA is the central molecule of genetics. The basic unit of DNA structure is a
nucleotide, composed of phosphate, deoxyribose sugar, and a nitrogen base. Each deoxyribose sugar
bonds covalently in a repeating pattern with two phosphates. One of the bonds is to the number 5´ (“five
prime”) carbon on deoxyribose, and the other is to the 3´ carbon, which specifies the order and direction
of each strand. This formation results in an elongate strand with a sugar-phosphate backbone. The
nitrogen bases, purines and pyrimidines, attach by covalent bonds at the 19 position of the sugar. They
span the center of the molecule and pair with appropriate complementary bases from the other strand,
thereby forming a double-stranded helix. The paired bases are so aligned as to be joined by hydrogen
bonds. Such weak bonds are easily broken, allowing the molecule to be “unzipped” into its
complementary strands. This feature is of great importance in gaining access to the information encoded
in the nitrogen base sequence. Pairing of purines and pyrimidines is not random; it is dictated by the
formation of hydrogen bonds between certain bases. Thus, in DNA, the purine adenine (A) pairs with the
pyrimidine thymine (T), and the purine guanine (G) pairs with the pyrimidine cytosine (C). Adenine
forms two hydrogen bonds with thymine, and cytosine forms three hydrogen bonds with guanine. This
difference influences a number of DNA functions. Research indicates that the bases are attracted to each
other in this pattern because each has a complementary three-dimensional shape that matches its pair.
Although the base-pairing partners generally do not vary, the sequence of base pairs along the DNA
molecule can assume any order, resulting in an infinite number of possible nucleotide sequences. The two
strands are not oriented in the same direction. One side of the helix runs in the opposite direction of the
other, in what is called an antiparallel arrangement. The order of the bond between the carbon on
deoxyribose and the phosphates is used to keep track of the direction of the two sides of the helix. Thus,
one helix runs from the 5´ to 3´ direction, and the other runs from the 3´ to 5´ direction. This
characteristic is a significant factor in DNA synthesis and translation.
It is the sequence of bases along the length of DNA that constitutes its “language.” For this language to be
preserved for hundreds of generations, the codes it contains will need to be duplicated with high fidelity.
This process of duplication is called DNA replication.

The Levels of Structure and Function of the Genome:

The genome is the sum total of genetic material carried within a cell. Although most of the genome exists
in the form of chromosomes, genetic material can appear in extrachromosomal sites as well. For example,
bacteria and some fungi contain tiny extra pieces of DNA called plasmids, and the mitochondria and
chloroplasts of eukaryotes are equipped with their own functional chromosomes. Genomes of cells are
composed exclusively of DNA, but viruses contain either DNA or RNA as the principal genetic material.
Although the specific genome of an individual organism is unique, the general pattern of nucleic acid
structure and function is similar among all organisms. In general, a chromosome is a discrete cellular
structure composed of a neatly packaged DNA molecule. The chromosomes of eukaryotes and bacterial
cells differ in several respects. The structure of eukaryotic chromosomes consists of a DNA molecule
tightly wound around histone proteins, whereas a bacterial chromosome is condensed and secured into a
packet by means of a different type of protein. Eukaryotic chromosomes are located in the nucleus; they
vary in number from a few to hundreds; they can occur in pairs (diploid) or singles (haploid); and they are
linear in format. In contrast, most bacteria have a single, circular chromosome, although some have
multiple chromosomes and a few have linear chromosomes.

All chromosomes contain a series of basic informational “packets” called genes. A gene can be defined
from more than one perspective. In classical genetics, the term refers to the fundamental unit of heredity
responsible for a given trait in an organism. In the molecular and biochemical sense, it is a portion of the
chromosome that provides information for a given cell function. More specifically still, it is a specific
segment of DNA that contains the necessary codes to make a protein or RNA molecule.

Genes fall into three basic categories: structural genes that code for proteins, genes that code for RNA,
and regulatory genes that control gene expression. The sum of all of these types of genes constitutes an
organism’s distinctive genetic makeup, or genotype. The expression of the genotype creates traits (certain
structures or functions) referred to as the phenotype. For example, a person inherits a combination of
genes (genotype) that gives a certain eye color or height (phenotype), a bacterium inherits genes that
direct the formation of a flagellum or the ability to metabolize a certain substrate, and a virus has genes
that dictate its capsid structure. All organisms contain more genes in their genotypes than are being seen
as a phenotype at any given time. In other words, the phenotype can change depending on which genes
are “turned on” or expressed.

The Significance of DNA Structure: The arrangement of nitrogen bases in DNA has two essential
effects.

1. Maintenance of the genetic codes during reproduction. The constancy of base-pairing guarantees
that the codes will be retained during cell growth and division. When the two strands are separated, each
one provides a template (pattern or model) for the replication (exact copying) of a new molecule.
Because the sequence of one strand automatically gives the sequence of its partner, the codes can be
duplicated with fidelity.
2. Providing variety. The order of bases along the length of the DNA strand provides the information
needed to produce RNA and protein molecules, which in turn are responsible for the phenotype of the
cell. Changing the identity of the bases or their order in the DNA molecule can have a dramatic effect on
the phenotype of the organism.

DNA REPLICATION:

DNA replication requires a careful orchestration of the actions of 30 different enzymes which separate the
strands of the existing DNA molecule, copy its template, and produce two complete daughter molecules.
The key steps include:

1. Uncoiling the parent DNA molecule, beginning at a predetermined point of origin;

2. Unzipping the hydrogen bonds between the base pairs, thus separating the two strands and exposing the
nucleotide sequence of each strand (which is normally buried in the center of the helix) to serve as
templates; and

3. Synthesizing two new strands by attachment of the correct complementary nucleotides to each single-
stranded template. A critical feature of DNA replication is that each daughter molecule will be identical to
the parent in composition, but neither one is completely new; the strand that serves as a template is an
original parental DNA strand. The preservation of the parent molecule in this way, termed
semiconservative replication, helps explain the reliability and fidelity of replication.

The Replication Process: All chromosomes have a specific origin of replication site that serves as the
place where replication will be initiated. It is recognized by a short sequence rich in adenine and thymine
that are held together by only two hydrogen bonds rather than three. Because the origin of replication is
AT-rich, less energy is required to separate the two strands than would be required if the origin were rich
in guanine and cytosine. The process of synthesizing a new daughter strand of DNA using the parental
strand as a template is carried out by a giant enzyme complex that brings in the primary replication
enzyme—DNA polymerase III— along with numerous accessory enzymes (see table). The DNA
polymerase III is responsible for deciphering and duplicating DNA codes. DNA polymerase cannot add
nucleotides to a DNA strand without a preexisting unbound nucleotide. Before replication can begin, a
piece of RNA called a primer is inserted at the origin of replication to serve as the starting point for bases
to be added. The polymerase can add nucleotides only in the direction of 5´ to 3´, which means that
synthesis of the two template strands will be somewhat different. Prior to the start of replication, enzymes
called helicases (unzipping enzymes) bind to the DNA at the origin. These enzymes untwist the helix and
break the hydrogen bonds holding the two strands together, resulting in two separate strands, each of
which will be used as a template for the synthesis of a new strand.

Some Enzymes Involved in DNA Replication and Their Functions

Enzyme Function
Helicase Unzipping the DNA helix
Primase Synthesizing an RNA prime
Adding bases to the new DNA chain; proofreading the chain for
DNA polymerase III
mistakes
DNA polymerase I Removing RNA primer, closing gaps and repairing mismatches
Ligase Final binding of nicks or cuts in DNA during synthesis and repair
Gyrase Supercoiling

Replication begins when an RNA primer is synthesized by a primase at the origin of replication. DNA
polymerase III must have this short strand of RNA to serve as a starting point for adding nucleotides.
Because the bacterial DNA molecule is circular, opening of the circle forms two replication forks, each
containing its own set of replication enzymes. Each fork contains two active DNA polymerases along
with several other proteins and enzymes whose main functions are to stabilize the polymerase and provide
a means of removing improperly incorporated nucleotides. The enzyme complex encircles the DNA near
the replication fork and synthesizes new DNA using the parent strand as a template. As synthesis
proceeds, the forks continually open up to expose the template for replication. Because DNA polymerase
III can only synthesize new DNA in the 5´ to 3´ direction, just one strand, called the leading strand, can
be synthesized continuously. One enzyme does this as it moves along the template strand toward the
replication fork. The other strand, which is oriented 3´ to 5´ with respect to the polymerase, is termed the
lagging strand. Because this strand cannot be synthesized continuously, an RNA primer is placed ahead
of the polymerase to initiate synthesis. It then adds nucleotides a few at a time in the direction away from
the fork (5´ to 3´). As the fork opens up a bit, the next segment is synthesized backward near the point of
the previous segment. In this way, the two new strands can be synthesized simultaneously. This manner
of synthesis produces short fragments of DNA (100 to 1,000 bases long) called Okazaki fragments on
the side of the lagging strand.
The lagging strand will be completed when the enzyme, DNA polymerase I, removes the RNA primers
from the Okazaki fragments and a ligase follows behind and inserts the correct DNA nucleotides into the
open spaces. The final result should is to the leading strand. The addition of nucleotides proceeds at an
astonishing pace, estimated in some bacteria to be 750 bases per second at each fork! As replication
proceeds, one newly synthesized strand loops down. When the forks have gone full circle, a termination
site shuts replication down. The two circular daughter molecules remain connected briefly but are nicked
and separated by a helicase. Like any language, DNA is occasionally “misspelled” when an incorrect base
is added to the growing chain. Studies have shown that such mistakes are made once in approximately
108 to 109 bases, but most of these are corrected. If not corrected, they are referred to as mutations and
can lead to serious cell dysfunction. Because continued cellular integrity is very dependent on accurate
replication, cells have evolved their own proofreading function for DNA. DNA polymerase III, the
enzyme that elongates the molecule, can also detect incorrect, unmatching bases; excise them; and replace
them with the correct base. DNA polymerase I is involved in proofreading the molecule and repairing
damaged DNA.

Figure: DNA Replication

Applications of the DNA Code: Transcription and Translation – Protein Synthesis

Gene expression involves two separate but interrelated processes - transcription and translation.
A. Transcription: Transcription is the process of synthesizing RNA from a DNA template. The
DNA acts as a template for the transcription of RNA by RNA polymerase for subsequent protein
production within the cell. RNA polymerase attaches itself to the beginning of a gene on
DNA and synthesizes mRNA, using one of the strands in DNA as a template. This process is
known as transcription. The bases in mRNA will be complementary to one strand of DNA since DNA
acts as a template for synthesis of mRNA.

B. Translation: Translation is the process of decoding the information carried on the mRNA to
synthesize the specified protein.

Process of translation: The process of translation requires three major components—mRNA, ribosomes,
and tRNAs, in addition to various accessory proteins.

Messenger RNA (mRNA): The mRNA is a temporary copy of genetic information. It carries the coded
information for making specific proteins from DNA to ribosomes, where proteins are synthesized.
Ribosomes: Serve as the sites of translation, and their structure facilitates the joining of one amino acid
to another

Transfer RNA (tRNA): The tRNA molecule contains a triplet at one end and amino acid at the other end.
The ribosome moves along the mRNA until the entire mRNA molecule has been translated into
corresponding sequences of amino acids. Finally, the sequence of amino acids in the resulting polypeptide
chain determines the configuration into which the polypeptide chain folds itself, which in many cases
determines the enzymatic properties of the completed protein.

Central dogma of molecular biology: The flow of information from DNA to RNA to protein is often
referred to as the Genetic dogma or central dogma of molecular biology (DNA→RNA→ polypeptide).

PROTEIN SYNTHESIS:
A protein consists of one or more polypeptides; a polypeptide being a chain of amino acids covalently
linked by peptide bonds (- CO. NH-). For any given polypeptide, the nature, number and sequence of
amino acids are dictated by a particular sequence of bases in a DNA strand. This sequence of basis
conforms to a gene and the message a genetic code. During protein synthesis, a gene is first transcribed.
The transcript (RNA) of the gene which carries the message from DNA is the mRNA. Along the length of
the mRNA molecule, groups of three consecutive bases (each) encode a particular amino acid. Each of
these three – base groups is called a codon, hence the sequence of codons in mRNA encodes the sequence
of amino acids in a polypeptide.

The synthesis of a polypeptide on mRNA (the translation process) takes place on a ribosome. The first
codon to be translated (initiator codon) is commonly AUG which aligns with ribosomal ‘P’ site initiating
the process of protein synthesis. P site is the peptidyl or donor site. The first step of protein synthesis is
amino acid activation – a process in which amino acids are attached to transfer RNA molecules. This
activation is catalyzed by aminoacyl – tRNA synthetase. The acceptor or amino acid stem holds the
activated amino acid on the 3 1end of the tRNA. The 31end of all tRNAs has the same -C-C-A sequence;
the amino acid is attached to the terminal adenylic acid.

The actual process of protein synthesis takes place on the ribosomes that serve as workbenches, with
mRNA acting as the blueprint. This process may be divided into three stages: Initiation; Elongation and
Termination.

Initiation begins with a specially modified aminoacyl – tRNA, N-formylmethionyl-tRNA (fMet- tRNA).
This initiator fMet- tRNA binds to the free 30S (70S ribosome subunit) subunit first. This process
involves the participation of protein initiation factors 1,2 and 3. Next, mRNA attaches to the 30S subunit.
Finally, the 50S subunit binds to the 30S subunit- mRNA forming an active ribosome – mRNA complex.
The fMet – tRNA is positioned at the P site.

Elongation involves amino acid addition to a growing polypeptide chain in an elongation cycle composed
of three phases:

(a). Aminoacyl – tRNA binding

(b). Transpeptidation reaction in which peptide grows by one amino acid and is transferred to ‘A’ site.
The enzyme which catalyzes transpeptidation is peptidyl transferase.

(c). Translocation in which the ribosome moves along the mRNA by one codon in the 5 1 -to-31
direction.

Termination. Protein synthesis stops when the ribosome reaches one of the three special nonsense or stop
codons – UAA, UAG, and UGA. Three release factors – RF-1, RF-2 and RF-3 aid the ribosome in
recognizing these codons. After the ribosome has stopped, peptidyl transferase hydrolyzes the peptide
free from its tRNA and the empty tRNA is released. Next the ribosome dissociates from its mRNA and
separates into 30S and 50S subunits. IF-3 binds to the 30S subunit and prevents it from reassociation with
the 50S subunit until proper stage in initiation is again reached. Thus, ribosomal subunits associate during
protein synthesis and separate afterwards.

CHANGES IN GENETIC INFORMATION

GENETIC TRANSFERS

In addition to mutation, genetic information in a bacterium can change as a result of genes acquired
through transfers. There are three fundamentally distinct mechanisms by which transfers can occur

Transformation – in which a cell takes up isolated DNA molecule from the surrounding medium.

Conjugation – which involves the direct transfer of DNA from one cell to another.

Transduction – in which the transfer is mediated by bacterial viruses (bacteriophages)

TRANSFORMATION: Transformation plays a key role as the principal method of introducing DNA
into bacteria after in vitro genetic manipulation. There are basically four types of transformation – Natural
Competence, Induced Competence, Protoplast Transformation and Electroporation. Competence is the
ability of a cell to be transformed.

Natural Competence – This generally occurs at a specific stage of growth, most commonly in the late
log phase, just as the cells are entering stationary phase. The development of competence is associated
with nutrient depletion and accumulation of specific secreted products i.e. competence factors.
Concentration of competence factors is dependent on cell concentration; thus, competence develops at
high cell density. Following the development of competence, double stranded DNA binds to receptors on
the cell surface but only one of the strands enters the cell. Generally, transformation occurs with related
DNA because the incoming DNA has to combine with the host chromosome in order to be replicated and
inherited.

Induced Competence – In many organisms e.g. E. coli, competent cells do not appear to occur naturally.
For these organisms, competence is induced artificially. This is achieved, for example, by washing the
cells repeatedly with cold calcium chloride solution. After this induction, the competent cells are mixed
with the DNA solution and subjected to a heat shock treatment, such as heating them at 42 oC for 1-2
minutes and then transferring them back onto ice. They are then diluted into broth and incubated at 37oC
to allow expression of the newly acquired DNA before plating out onto an appropriate medium.

Protoplast Transformation – Enzymatic removal of the cell wall of a bacterium in the presence of an
osmotic stabilizer such as sucrose generates protoplast in which large areas of the cytoplasmic membrane
are exposed. Addition of DNA solution together with polyethylene glycol (PEG) causes the cells to take
up the DNA. The PEG is the removed by centrifugation and the cells are allowed to regenerate on an
osmotically stabilized medium.

Electroporation – In this procedure, bacterial cells are mixed with plasmid DNA and are subjected to a
brief pulse of high voltage electricity. This causes transient tiny holes in the cell membrane through which
the DNA enters the cell. The transformants can then be selected, for example, on the basis of the
antibiotic resistance conferred by the plasmid.

CONJUGATION: Conjugation is the direct transmission of DNA from one bacterial cell to another. In
most cases, this involves the transfer of plasmid DNA, although with some organisms, chromosomal
transfer can also be demonstrated. As with other modes of gene transfer in bacteria, there is a one-way
transfer of a copy of part of the DNA from one parent (donor) to the other (recipient) in conjugation. The
DNA recipient is called a transconjugant. In majority of the cases, the occurrence of conjugation is
dependent on the presence in the donor strain of a plasmid that carries the genetic information required
for promoting DNA transfer.

Mechanism of conjugation varies from case to another but generally the donor cell carries pili (long thin
appendages on the cell surface) which make contact with receptors on the surface of the recipient cell,
thus establishing the formation of a mating pair. The pili then contract to bring the cells into intimate
contact. Donors which bear pili are designated F+ cells while recipients (which lack the F plasmid) are F-
cells. On mixing F+ and F- cells, the tips of the pili bind to the surface of F- cells to bring the cells into
contact as earlier stated.

The process of transfer of plasmid DNA from the donor to the recipient is initiated by a protein that
makes a single – stranded break (nick) at a specific site in the DNA known as the origin of transfer (oriT).
A plasmid encoded helicase unwinds the plasmid DNA and the single nicked strand is transferred to the
recipient starting with the 51end generated by the nick. Concurrently, the free 3 1end of the nicked strand is
extended to replace the DNA transferred by a process known as rolling cycle. DNA synthesis in the
recipient converts the transferred single strand into a double stranded molecule.

TRANSDUCTION: Transduction involves phage-mediated transfer of genetic material. The key step is
the packaging of DNA into the empty phage heads. This process is normally highly specific for phage
DNA. Not all bacteriophages are capable of carrying out transduction. The basic requirements of an
effective transducing phage are that infection should result in an appropriate level of degradation of the
chromosomal DNA to form suitable sized fragments at the right time for packaging, and that the
specificity of the packaging process should be comparatively low.

Generalized Transduction – In this, any gene has an equal chance of being transduced from one cell to
another. In the recipient cell (transductant), the transduced DNA may:

Be degraded by restriction endonucleases.

Undergo recombination with the chromosome or plasmid so that some donor genes can be stably
inherited (complete transduction).

Persist in a stable but non-replicating state (abortive transduction).

Donor genes with functional promoters (whether in a complete or abortive transduction) may be
expressed in the transductant. If two or more genes are transduced together (co-transduction), it is seen as
evidence of closeness on the chromosome.

Specialized or Restricted Transduction – This can be brought about only by a temperate phage in which
there is a phase of integration with the host’s chromosome. Temperate phages are able to establish a
stable state known as lysogeny in which expression of phage genes and replication of phage is repressed.
In some cases, the prophage is inserted into the bacterial DNA and replicates as part of the chromosome.
Integration and excision of the phage DNA occur via a site-specific recombination. When lysogeny
breaks down and the phage enters the lytic cycle, it is excised from the chromosome by recombination
between sequences at each end of the integrated prophage. If this recombination event happens in the
wrong place, an adjacent region of bacterial DNA is incorporated into the phage DNA. The progeny
phage produced will then all contain this bacterial gene which will therefore be transduced at a very high
frequency once the transducing phage has been isolated. However, the DNA transferred is limited to a
very small region of the chromosome. The phenomenon is therefore known as specialized or restricted
transduction.

MUTATION

In addition to gene transfers as above, genetic information in a bacterium can change as a result of
mutation.

Mutation is a random, undirected, heritable change or variation caused by an alteration in the nucleotide
sequence at some point of the DNA of the cell. It may be due to addition, deletion or substitution of one
or more bases. The molecular basis of mutation is that during DNA replication, an ‘error’ creeps in while
the progeny strands are copied.

Mutation is a natural event, taking place all the time at its particular frequency in all the dividing
forms of life. Particular mutations occur at fairly constant rates, normally between once per 10 4 and
once per 1010 cell divisions. Though mutations take place all the time, most mutants go unrecognized as
the mutation may involve minor function or it may be lethal. Mutation is best appreciated when it
involves a function which can be readily observed. Mutants can be detected by selecting or testing for an
altered phenotype. For example, an E. coli mutant that loses its ability to ferment lactose can be readily
detected on MacConkey agar but is unrecognizable on nutrient agar.

Lethal mutation: Some mutations involve vital functions, and such mutants are nonviable (lethal
mutation). An important type of lethal mutation is conditional mutation.

Conditional mutation: A conditional lethal mutant may be able to live under certain permissive
conditions but not under other or nonpermissive conditions. The most common type of conditional
mutant is the temperature sensitive (ts) mutant, which is able to live at the permissive temperature
(say, 35°C), but not at the restrictive temperature (say, 39°C).

Recognition of mutation: Mutation can be best recognized when it involves a function which can
be readily observed by experimental methods like alteration in colonial morphology, pigmentation,
alteration in cell surface antigens, sensitivity to bacteriophages or bacteriocins, loss of ability to
produce capsule or flagella, loss of virulence and change in biochemical characters.

Types of Mutation: Mutations can be divided conveniently into:

A. Spontaneous mutation: Many mutations occur spontaneously in nature in the absence of any
mutation-causing agents.

B. Induced mutation: The frequency of mutation is greatly enhanced by exposure of cells to several
agents (mutagens) which may be physical or chemical.

C. Point mutations: As the name suggests, point mutations affect just one point (base pair) in a gene.
Such mutations may be a change to or substitution of a different base pair. Alternatively, a point mutation
can result in the deletion or addition of a base pair. It is, in general, reversible and is of two classes:
1. Base pair substitution: This comprises those mutants in which a single base pair (nucleotide) has been
substituted for another pair, and can be subdivided into transition (one purine is replaced by another
purine or a pyrimidine is replaced by another pyrimidine) and transversion (substitution of a purine for a
pyrimidine and vice versa in base pairing).

Normal sequence: The Fat Cat Ate The Rat.

Substitution: The Fat Can Ate The Rat.

Depending on the placement of the substituted base, when the mRNA is translated this may cause no
change (silent mutation), lead to the insertion of the wrong amino acid (missense mutation) or generate
a stop codon (nonsense mutation), prematurely terminating the polypeptide.

Missense mutation: If the triplet code is altered so as to specify an amino acid different from that
normally located at a particular position in the protein. This is called missense mutation.

Nonsense mutation: Deletion of a nucleotide within a gene may cause premature polypeptide chain
termination by generating a nonsense codon (UAG, UAA or UGA). This is known as nonsense mutation.

2. Base pair deletion or insertion:

Frameshift mutations: If the number of bases inserted or deleted is not a multiple of three, there
will be shift in the reading frame, i.e. frameshift mutations. This shifts the normal ‘reading frame’ of
the coded message forming newest of triplet codon. The coded message is read correctly up to the point
of addition or deletion, but the subsequent codons will specify the incorrect amino acids.

Survival Advantage of Mutation: When mutation confers a survival advantage, it is of vital importance.
For example, if a streptomycin resistant mutant of the tubercle bacillus develops in a patient under
treatment with the drug, it multiplies selectively and ultimately replaces the original drug sensitive
population of bacteria.
Mutagens
1. Physical agents: (i) UV rays; (ii) Ionizing radiation, e.g. X-rays; (iii) Visible light; (iv) Heat

2. Chemical agents: (i) Alkylating agents; (ii) Acridine dyes; (iii) 5-Bromouracil; (iv) 2-aminopurine; (v)
Nitrous acid.