WRITTEN REPORT:

HOW CELLULAR INFORMATION IS ALTERED

A. Mutation: B. Selecting Desirable Mutants: C. Natural Mechanism
for Gene Transfer and Rearrangement: D. Genetically Engineered Cells
ChE 444
Introduction to Biotechnology

Submitted to:
Engr. Luomar Jake Cabatas
Faculty of College of Engineering Education

Submitted by:
Quenne A. Belocura

Room/Time
BE 208
2:00-3:30 PM

Date
May 16, 2019
HOW CELLULAR INFORMATION IS ALTERED
8.1 Mutation
An alteration in the genetic material (the genome) of a cell of a living organism or of
a virus that is more or less permanent and that can be transmitted to the cell’s or the virus’s
descendants. Mutation in the DNA of a body cell of a multicellular organism (somatic
mutation) may be transmitted to descendant cells by DNA replication and hence result in a
sector or patch of cells having abnormal function, an example being cancer. Mutations in egg
or sperm cells (germinal mutations) may result in an individual offspring all of whose cells
carry the mutation, which often confers some serious malfunction, as in the case of a human
genetic disease such as cystic fibrosis. Mutations result either from accidents during the
normal chemical transactions of DNA, often during replication, or from exposure to high-
energy electromagnetic radiation (e.g., ultraviolet light or X-rays) or particle radiation or to
highly reactive chemicals in the environment. Because mutations are random changes, they
are expected to be mostly deleterious, but some may be beneficial in certain environments. In
general, mutation is the main source of genetic variation, which is the raw material
for evolution by natural selection.
The genome is composed of one to several long molecules of DNA, and mutation can
occur potentially anywhere on these molecules at any time. The most serious changes take
place in the functional units of DNA, the genes. A mutated form of a gene is called
a mutant allele. A gene is typically composed of a regulatory region, which is responsible for
turning the gene’s transcription on and off at the appropriate times during development, and a
coding region, which carries the genetic code for the structure of a functional molecule,
generally a protein. A protein is a chain of usually several hundred amino acids. Cells make
20 common amino acids, and it is the unique number and sequence of these that give a
protein its specific function. Each amino acid is encoded by a unique sequence, or codon, of
three of the four possible base pairs in the DNA (A–T, T–A, G–C, and C–G, the individual
letters referring to the four nitrogenous bases adenine, thymine, guanine, and cytosine).
Hence, a mutation that changes DNA sequence can change amino acid sequence and in this
way potentially reduce or inactivate a protein’s function. A change in the DNA sequence of a
gene’s regulatory region can adversely affect the timing and availability of the gene’s protein
and also lead to serious cellular malfunction. On the other hand, many mutations are silent,
showing no obvious effect at the functional level. Some silent mutations are in the DNA
between genes, or they are of a type that results in no significant amino acid changes.
 Changes within genes are called point mutations. The simplest kinds are changes to
single base pairs, called base-pair substitutions. Many of these substitutes an incorrect amino
acid in the corresponding position in the encoded protein, and of these a large proportion
result in altered protein function.

8.1.2. Selecting for Desirable Mutants

Mutants can serve as powerful tools to better understand cell physiology; they are also
valuable as industrial organisms because mutation can be used to alter metabolic regulation
and cause overproduction of a desired compound. Methods to induce mutations and then
select for mutants are important tools for catalyst development in bioprocessing.

The selection of a mutant with desirable properties is no easy task. Mutations are
classified as selectable and unselectable. A selectable mutation confers upon the mutant an
advantage for growth or survival under a specific set of environmental conditions; thus, the
mutant can grow and the wild type will die. An unselectable mutant requires a cell-by-cell
examination to find a mutant with the desired characteristics (e.g., green pigment). Even with
mutagens, the frequency of mutation is sufficiently low to make prohibitive a brute-force
screening effort for most unselectable mutants.

Selection can be direct or indirect. An example of direct selection would be to find a

mutant resistant to an antibiotic or toxic compound. Indirect selection is used for isolating
mutants that are deficient in their capacity to produce a necessary growth factor (e.g., an
amino acid or a vitamin). Auxotrophic mutants would not grow on such a simple medium
unless it were supplemented with the growth factor that the cell could no longer make (e.g., a
lysine auxotroph has lost the capacity to make lysine, so lysine must be added to the glucose
and salts to enable the cell to grow). The wild-type cell that needs no supplements to a
minimal medium is called a prototroph.

A method that facilitates selection greatly is called replica plating. A master plate using
lysine-supplemented medium will grow both the auxotroph and wild-type cells. Once
colonies are well formed on the master plate, an imprint is made on sterile velveteen. 

Another class of mutants is called conditional mutants. Mutations that would normally be

lethal to the cell could not be detected by methods we have described so far. 
8.2 Natural Mechanisms for Gene Transfer and Rearrangement
Bacteria can gain and express wholly different biochemical capabilities (e.g., the
ability to degrade an antibiotic or detoxify a hazardous chemical in their environment)
literally overnight. These alterations cannot be explained through inheritance and small
evolutionary changes in the chromosome. Rather, they arise from gene transfer from one
organism to another and/or large rearrangements in chromosomal DNA. 

In this section, we discuss genetic recombination, gene transfer, and genetic rearrangements
—all mechanisms that can be exploited to genetically engineer cells

8.2.1. Genetic Recombination

Genetic recombination is a process that brings genetic elements from two different
genomes into one unit, resulting in new genotypes in the absence of mutations. Genetic
recombination in prokaryotes is a rare event but sufficiently frequent to be important
ecologically as well as industrially. The three main mechanisms for gene transfer
are transformation, transduction, and conjugation. Transformation is a process in which free
DNA is taken up by a cell. Transduction is a process in which DNA is transferred by a
bacteriophage, and conjugation is DNA transfer between intact cells that are in direct contact
with one another.

8.2.2. Transformation
Transformation is the genetic alteration of a cell resulting from the direct uptake and
incorporation of exogenous genetic material (e.g., DNA) from its surroundings through the
cell membrane(s). Transformation is a naturally occurring process in some bacteria, but it
cannot be performed by all genera of bacteria. Even within transformable genera, only certain
strains are transformable (competent). Competent cells have a much higher capacity for
binding DNA to the cell surface than do noncompetent cells. Competency can depend on the
physiological state of the cell (current and previous growth conditions). Even in a competent
population, not all cells are transformable. Typically, about 0.1% to 1.0% are transformable.

8.2.3. Transduction
Transduction is the process by which DNA is transferred from one bacterium to
another by a vector, which is usually a virus, and certainly plays an important role in nature.
In the most common type of transduction,  generalized transduction, infection of a recipient
cell results in fragmentation of the bacterial DNA into 100 or so pieces. One of these
fragments can become packaged randomly into a phage particle. The altered phage particle
then injects bacterial DNA into another cell, where it can recombine with that cell’s DNA.
With generalized transduction, any bacterial gene may be transferred.

Another method of transfer, which is far more specific with respect to the genes that
are transferred, is specialized transduction. Here the phage incorporates into specific sites in
the chromosome, and the frequency of transduction of a gene is related to its distance away
from the site of incorporation. 

8.2.4. Episomes and Conjugation

A third type of gene transfer involves another genetic element. This element is called
an episome. It is a DNA molecule that may exist either integrated into the chromosome or
separate from it. When it exists separately from the chromosome (i.e., extrachromosomally),
it is essentially a plasmid. A well-known episome is the F or fertility factor. Such factors are
responsible for the process known as conjugation.

Most experiments with conjugation are done with the F factor, which is present in low
copy number. Direct cell-to-cell contact is required. This DNA molecule encodes at least 13
genes involved in its self-transfer from one cell to another.

Conjugation, transduction, and transformation all represent forms of gene transfer

from one cell to another. However, gene transfer can occur within a cell.

8.2.5. Transposons: Internal Gene Transfer

Previously, we discussed the presence of IS elements on the chromosome. A closely
related phenomenon is a transposon, which refers to a gene or genes that have the ability to
“jump” from one piece of DNA to another or to another position on the original piece of
DNA. The transposon integrates itself into the new position independently of any homology
with the recipient piece of DNA. Transposons differ from IS elements in that they code for
proteins. Transposons appear to arise when a gene becomes bounded on both sides by
insertion sequences. Many of the transposons encode antibiotic resistance.

Transposons are important because they can induce mutations when they insert into
the middle of a gene, they can bring once-separate genes together, and in combination with
plasmid- or viral-mediated gene transfer, they can mediate the movement of genes between
unrelated bacteria (e.g., multiple antibiotic resistances on newly formed plasmids).
Transposon mutagenesis can be a very powerful tool in altering cellular properties usually by
insertional inactivation of chromosomal genes.
8.3. Genetically Engineered Cells
Our description of DNA replication, mutation, and selection and the natural
mechanisms for gene transfer covered all the tools necessary to genetically engineer a cell.

Genetic engineering is a set of tools and not a scientific discipline. Although difficult
to define precisely, it involves the manipulation of DNA outside the cell to create artificial
genes or novel combinations of genes with predesigned control elements. 

8.3.1. Basic Elements of Genetic Engineering

The strategy makes use of recombinant DNA techniques, the ability to isolate genes
from one organism and recombine the isolated gene with other DNA that can be propagated
in a similar or unrelated host. Most of our discussion is drawn from approaches for
genetically engineering bacteria.

8.3.1.1. Obtaining the gene of interest

The first step is obtaining the gene of interest. A simple, brute-force approach
is shotgun cloning. Here the DNA from the donor organism is cut into fragments
using restriction enzymes. If an efficient screening procedure is available, large numbers of
host cells with random fragments of DNA can be screened for those exhibiting a property
related to the desired gene.

Most often, such a shotgun approach is very inefficient. More specific approaches
use hybridization. A probe can be synthesized chemically to be complementary to a portion
of the gene. The probe is usually much shorter than the gene but sufficiently long that it is
unlikely for other genes to have the same complementary DNA sequence. The construction of
the probe requires some knowledge of either the nucleotide sequence of the desired gene or a
partial amino acid sequence for the desired gene. Since the genetic code is degenerate, the
deduction of the actual nucleotide sequence is ambiguous. This ambiguity requires that a
variety of probes be generated. Hybridization reactions require the donor DNA to be both
fragmented and converted into single strands that can react with the single-stranded probes

8.3.1.2. Inserting the gene into DNA

Once the desired gene is isolated or made, it can be inserted into a small piece of
carrier DNA called a vector. Typically, vectors are plasmids, although temperate viruses can
be used. The process for preparing the donor DNA and vector for recombination and the
actual joining of the DNA segments usually requires special enzymes. We discussed these
enzymes in our previous consideration of DNA replication and genetic recombination. A
wide variety of restriction enzymes exist that will cut DNA at a different prespecified site.

8.3.2. Genetic Engineering of Higher Organisms

The direct genetic engineering of higher organisms can be a great deal more difficult
than for bacteria because of a lack of good effective techniques to introduce foreign DNA and
lack of an understanding of host cell genetics. Be aware of the techniques being developed to
work with some of these host systems. The introduction of foreign DNA into higher
organisms is usually termed transfection.

Some plants are subject to infection with the bacterium Agrobacterium

tumefaciens. A. tumefaciens contains a plasmid that contains a section known as T-DNA.
This T-DNA can integrate into the plant chromosome. If genes are inserted into the T-DNA
region, they can be incorporated ultimately in the plant chromosome. Unfortunately, most
cereal plants are not readily susceptible to Agrobacteriuminfection.

The biolistic process for obtaining plant transformation is to coat small (1-μm

diameter) projectiles (e.g., of tungsten) and shoot them into cells at high velocity. Results
with this approach have been remarkably successful, and this technique is fairly general.

Another fairly general approach is electroporation, which involves a brief high

voltage electric discharge that renders cells permeable to DNA. Electroporation can be used
with animal, plant, fungal, and bacterial systems. The formation of protoplasts can enhance
transfection but is not essential. A protoplast is a cell in which the outer cell envelope has
been removed so that only the cytoplasmic membrane remains.

8.3.3. Genome Engineering

Genome engineering, or genome editing, refers to the collection of strategies and
techniques developed in recent years for the targeted, specific modification of the genetic
information—or genome—of living organisms. Genome engineering has the potential to
impact a wide range of applications, particularly in the areas of human health, agricultural
and industrial biotechnology, and research tool development. Examples include the
inactivation or modification of a specific gene for understanding its function, the correction
of a gene carrying a harmful mutation, the production of therapeutic proteins, the elimination
of persistent viral sequences, and the development of new generations of genetically modified
crops.
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