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Transformation (genetics) | Wikipedia audio article

October 13, 2019

In molecular biology, transformation is the
genetic alteration of a cell resulting from the direct uptake and incorporation of exogenous
genetic material from its surroundings through the cell membrane(s). For transformation to take place, the recipient
bacteria must be in a state of competence, which might occur in nature as a time-limited
response to environmental conditions such as starvation and cell density, and may also
be induced in a laboratory.Transformation is one of three processes for horizontal gene
transfer, in which exogenous genetic material passes from one bacterium to another, the
other two being conjugation (transfer of genetic material between two bacterial cells in direct
contact) and transduction (injection of foreign DNA by a bacteriophage virus into the host
bacterium). In transformation, the genetic material passes
through the intervening medium, and uptake is completely dependent on the recipient bacterium.As
of 2014 about 80 species of bacteria were known to be capable of transformation, about
evenly divided between Gram-positive and Gram-negative bacteria; the number might be an overestimate
since several of the reports are supported by single papers.”Transformation” may also
be used to describe the insertion of new genetic material into nonbacterial cells, including
animal and plant cells; however, because “transformation” has a special meaning in relation to animal
cells, indicating progression to a cancerous state, the process is usually called “transfection”.==History==
Transformation in bacteria was first demonstrated in 1928 by the British bacteriologist Frederick
Griffith. Griffith was interested in determining whether
injections of heat-killed bacteria could be used to vaccinate mice against pneumonia. However, he discovered that a non-virulent
strain of Streptococcus pneumoniae could be made virulent after being exposed to heat-killed
virulent strains. Griffith hypothesized that some “transforming
principle” from the heat-killed strain was responsible for making the harmless strain
virulent. In 1944 this “transforming principle” was
identified as being genetic by Oswald Avery, Colin MacLeod, and Maclyn McCarty. They isolated DNA from a virulent strain of
S. pneumoniae and using just this DNA were able to make a harmless strain virulent. They called this uptake and incorporation
of DNA by bacteria “transformation” (See Avery-MacLeod-McCarty experiment) The results of Avery et al.’s
experiments were at first skeptically received by the scientific community and it was not
until the development of genetic markers and the discovery of other methods of genetic
transfer (conjugation in 1947 and transduction in 1953) by Joshua Lederberg that Avery’s
experiments were accepted.It was originally thought that Escherichia coli, a commonly
used laboratory organism, was refractory to transformation. However, in 1970, Morton Mandel and Akiko
Higa showed that E. coli may be induced to take up DNA from bacteriophage λ without
the use of helper phage after treatment with calcium chloride solution. Two years later in 1972, Stanley Norman Cohen,
Annie Chang and Leslie Hsu showed that CaCl2 treatment is also effective for transformation
of plasmid DNA. The method of transformation by Mandel and
Higa was later improved upon by Douglas Hanahan. The discovery of artificially induced competence
in E. coli created an efficient and convenient procedure for transforming bacteria which
allows for simpler molecular cloning methods in biotechnology and research, and it is now
a routinely used laboratory procedure. Transformation using electroporation was developed
in the late 1980s, increasing the efficiency of in-vitro transformation and increasing
the number of bacterial strains that could be transformed. Transformation of animal and plant cells was
also investigated with the first transgenic mouse being created by injecting a gene for
a rat growth hormone into a mouse embryo in 1982. In 1907 a bacterium that caused plant tumors,
Agrobacterium tumefaciens, was discovered and in the early 1970s the tumor-inducing
agent was found to be a DNA plasmid called the Ti plasmid. By removing the genes in the plasmid that
caused the tumor and adding in novel genes, researchers were able to infect plants with
A. tumefaciens and let the bacteria insert their chosen DNA into the genomes of the plants. Not all plant cells are susceptible to infection
by A. tumefaciens, so other methods were developed, including electroporation and micro-injection. Particle bombardment was made possible with
the invention of the Biolistic Particle Delivery System (gene gun) by John Sanford in the 1980s.==Definitions==
Transformation is one of three forms of horizontal gene transfer that occur in nature among bacteria,
in which DNA encoding for a trait passes from one bacterium to another and is integrated
into the recipient genome by homologous recombination; the other two are transduction, carried out
by means of a bacteriophage, and conjugation, in which a gene is passed through direct contact
between bacteria. In transformation, the genetic material passes
through the intervening medium, and uptake is completely dependent on the recipient bacterium.Competence
refers to a temporary state of being able to take up exogenous DNA from the environment;
it may be induced in a laboratory.It appears to be an ancient process inherited from a
common prokaryotic ancestor that is a beneficial adaptation for promoting recombinational repair
of DNA damage, especially damage acquired under stressful conditions. Natural genetic transformation appears to
be an adaptation for repair of DNA damage that also generates genetic diversity.Transformation
has been studied in medically important Gram-negative bacteria species such as Helicobacter pylori,
Legionella pneumophila, Neisseria meningitidis, Neisseria gonorrhoeae, Haemophilus influenzae
and Vibrio cholerae. It has also been studied in Gram-negative
species found in soil such as Pseudomonas stutzeri, Acinetobacter baylyi, and Gram-negative
plant pathogens such as Ralstonia solanacearum and Xylella fastidiosa. Transformation among Gram-positive bacteria
has been studied in medically important species such as Streptococcus pneumoniae, Streptococcus
mutans, Staphylococcus aureus and Streptococcus sanguinis and in Gram-positive soil bacterium
Bacillus subtilis. It has also been reported in at least 30 species
of Proteobacteria distributed in the classes alpha, beta, gamma and epsilon. The best studied Proteobacteria with respect
to transformation are the medically important human pathogens Neisseria gonorrhoeae (class
beta), Haemophilus influenzae (class gamma) and Helicobacter pylori (class epsilon)”Transformation”
may also be used to describe the insertion of new genetic material into nonbacterial
cells, including animal and plant cells; however, because “transformation” has a special meaning
in relation to animal cells, indicating progression to a cancerous state, the process is usually
called “transfection”.==Natural competence and transformation==As of 2014 about 80 species of bacteria were
known to be capable of transformation, about evenly divided between Gram-positive and Gram-negative
bacteria; the number might be an overestimate since several of the reports are supported
by single papers.Naturally competent bacteria carry sets of genes that provide the protein
machinery to bring DNA across the cell membrane(s). The transport of the exogenous DNA into the
cells may require proteins that are involved in the assembly of type IV pili and type II
secretion system, as well as DNA translocase complex at the cytoplasmic membrane.Due to
the differences in structure of the cell envelope between Gram-positive and Gram-negative bacteria,
there are some differences in the mechanisms of DNA uptake in these cells, however most
of them share common features that involve related proteins. The DNA first binds to the surface of the
competent cells on a DNA receptor, and passes through the cytoplasmic membrane via DNA translocase. Only single-stranded DNA may pass through,
the other strand being degraded by nucleases in the process. The translocated single-stranded DNA may then
be integrated into the bacterial chromosomes by a RecA-dependent process. In Gram-negative cells, due to the presence
of an extra membrane, the DNA requires the presence of a channel formed by secretins
on the outer membrane. Pilin may be required for competence, but
its role is uncertain. The uptake of DNA is generally non-sequence
specific, although in some species the presence of specific DNA uptake sequences may facilitate
efficient DNA uptake.===Natural transformation===
Natural transformation is a bacterial adaptation for DNA transfer that depends on the expression
of numerous bacterial genes whose products appear to be responsible for this process. In general, transformation is a complex, energy-requiring
developmental process. In order for a bacterium to bind, take up
and recombine exogenous DNA into its chromosome, it must become competent, that is, enter a
special physiological state. Competence development in Bacillus subtilis
requires expression of about 40 genes. The DNA integrated into the host chromosome
is usually (but with rare exceptions) derived from another bacterium of the same species,
and is thus homologous to the resident chromosome. In B. subtilis the length of the transferred
DNA is greater than 1271 kb (more than 1 million bases). The length transferred is likely double stranded
DNA and is often more than a third of the total chromosome length of 4215 kb. It appears that about 7-9% of the recipient
cells take up an entire chromosome.The capacity for natural transformation appears to occur
in a number of prokaryotes, and thus far 67 prokaryotic species (in seven different phyla)
are known to undergo this process.Competence for transformation is typically induced by
high cell density and/or nutritional limitation, conditions associated with the stationary
phase of bacterial growth. Transformation in Haemophilus influenzae occurs
most efficiently at the end of exponential growth as bacterial growth approaches stationary
phase. Transformation in Streptococcus mutans, as
well as in many other streptococci, occurs at high cell density and is associated with
biofilm formation. Competence in B. subtilis is induced toward
the end of logarithmic growth, especially under conditions of amino acid limitation. Similarly, in Micrococcus luteus (a representative
of the less well studied Actinobacteria phylum), competence develops during the mid-late exponential
growth phase and is also triggered by amino acids starvation.By releasing intact host
and plasmid DNA, certain bacteriophages are thought to contribute to transformation.===Transformation, as an adaptation for DNA
repair===Competence is specifically induced by DNA
damaging conditions. For instance, transformation is induced in
Streptococcus pneumoniae by the DNA damaging agents mitomycin C (a DNA cross-linking agent)
and fluoroquinolone (a topoisomerase inhibitor that causes double-strand breaks). In B. subtilis, transformation is increased
by UV light, a DNA damaging agent. In Helicobacter pylori, ciprofloxacin, which
interacts with DNA gyrase and introduces double-strand breaks, induces expression of competence genes,
thus enhancing the frequency of transformation Using Legionella pneumophila, Charpentier
et al. tested 64 toxic molecules to determine which of these induce competence. Of these, only six, all DNA damaging agents,
caused strong induction. These DNA damaging agents were mitomycin C
(which causes DNA inter-strand crosslinks), norfloxacin, ofloxacin and nalidixic acid
(inhibitors of DNA gyrase that cause double-strand breaks), bicyclomycin (causes single- and
double-strand breaks), and hydroxyurea (induces DNA base oxidation). UV light also induced competence in L. pneumophila. Charpentier et al. suggested that competence
for transformation probably evolved as a DNA damage response. Logarithmically growing bacteria differ from
stationary phase bacteria with respect to the number of genome copies present in the
cell, and this has implications for the capability to carry out an important DNA repair process. During logarithmic growth, two or more copies
of any particular region of the chromosome may be present in a bacterial cell, as cell
division is not precisely matched with chromosome replication. The process of homologous recombinational
repair (HRR) is a key DNA repair process that is especially effective for repairing double-strand
damages, such as double-strand breaks. This process depends on a second homologous
chromosome in addition to the damaged chromosome. During logarithmic growth, a DNA damage in
one chromosome may be repaired by HRR using sequence information from the other homologous
chromosome. Once cells approach stationary phase, however,
they typically have just one copy of the chromosome, and HRR requires input of homologous template
from outside the cell by transformation.To test whether the adaptive function of transformation
is repair of DNA damages, a series of experiments were carried out using B. subtilis irradiated
by UV light as the damaging agent (reviewed by Michod et al. and Bernstein et al.) The results of these experiments indicated
that transforming DNA acts to repair potentially lethal DNA damages introduced by UV light
in the recipient DNA. The particular process responsible for repair
was likely HRR. Transformation in bacteria can be viewed as
a primitive sexual process, since it involves interaction of homologous DNA from two individuals
to form recombinant DNA that is passed on to succeeding generations. Bacterial transformation in prokaryotes may
have been the ancestral process that gave rise to meiotic sexual reproduction in eukaryotes
(see Evolution of sexual reproduction; Meiosis.)==
Methods and mechanisms of transformation in laboratory=====Bacterial===
Artificial competence can be induced in laboratory procedures that involve making the cell passively
permeable to DNA by exposing it to conditions that do not normally occur in nature. Typically the cells are incubated in a solution
containing divalent cations (often calcium chloride) under cold conditions, before being
exposed to a heat pulse (heat shock). Calcium chloride partially disrupts the cell
membrane, which allows the recombinant DNA to enter the host cell. Cells that are able to take up the DNA are
called competent cells. It has been found that growth of Gram-negative
bacteria in 20 mM Mg reduces the number of protein-to-lipopolysaccharide bonds by increasing
the ratio of ionic to covalent bonds, which increases membrane fluidity, facilitating
transformation. The role of lipopolysaccharides here are verified
from the observation that shorter O-side chains are more effectively transformed – perhaps
because of improved DNA accessibility. The surface of bacteria such as E. coli is
negatively charged due to phospholipids and lipopolysaccharides on its cell surface, and
the DNA is also negatively charged. One function of the divalent cation therefore
would be to shield the charges by coordinating the phosphate groups and other negative charges,
thereby allowing a DNA molecule to adhere to the cell surface. DNA entry into E. coli cells is through channels
known as zones of adhesion or Bayer’s junction, with a typical cell carrying as many as 400
such zones. Their role was established when cobalamine
(which also uses these channels) was found to competitively inhibit DNA uptake. Another type of channel implicated in DNA
uptake consists of poly (HB):poly P:Ca. In this poly (HB) is envisioned to wrap around
DNA (itself a polyphosphate), and is carried in a shield formed by Ca ions.It is suggested
that exposing the cells to divalent cations in cold condition may also change or weaken
the cell surface structure, making it more permeable to DNA. The heat-pulse is thought to create a thermal
imbalance across the cell membrane, which forces the DNA to enter the cells through
either cell pores or the damaged cell wall. Electroporation is another method of promoting
competence. In this method the cells are briefly shocked
with an electric field of 10-20 kV/cm, which is thought to create holes in the cell membrane
through which the plasmid DNA may enter. After the electric shock, the holes are rapidly
closed by the cell’s membrane-repair mechanisms.===Yeast===
Most species of yeast, including Saccharomyces cerevisiae, may be transformed by exogenous
DNA in the environment. Several methods have been developed to facilitate
this transformation at high frequency in the lab. Yeast cells may be treated with enzymes to
degrade their cell walls, yielding spheroplasts. These cells are very fragile but take up foreign
DNA at a high rate. Exposing intact yeast cells to alkali cations
such as those of caesium or lithium allows the cells to take up plasmid DNA. Later protocols adapted this transformation
method, using lithium acetate, polyethylene glycol, and single-stranded DNA. In these protocols, the single-stranded DNA
preferentially binds to the yeast cell wall, preventing plasmid DNA from doing so and leaving
it available for transformation. Electroporation: Formation of transient holes
in the cell membranes using electric shock; this allows DNA to enter as described above
for bacteria. Enzymatic digestion or agitation with glass
beads may also be used to transform yeast cells.Efficiency – Different yeast genera
and species take up foreign DNA with different efficiencies. Also, most transformation protocols have been
developed for baker’s yeast, S. cerevisiae, and thus may not be optimal for other species. Even within one species, different strains
have different transformation efficiencies, sometimes different by three orders of magnitude. For instance, when S. cerevisiae strains were
transformed with 10 ug of plasmid YEp13, the strain DKD-5D-H yielded between 550 and 3115
colonies while strain OS1 yielded fewer than five colonies.===Plants===
A number of methods are available to transfer DNA into plant cells. Some vector-mediated methods are: Agrobacterium-mediated transformation is the
easiest and most simple plant transformation. Plant tissue (often leaves) are cut into small
pieces, e.g. 10x10mm, and soaked for ten minutes in a fluid containing suspended Agrobacterium. The bacteria will attach to many of the plant
cells exposed by the cut. The plant cells secrete wound-related phenolic
compounds which in turn act to upregulate the virulence operon of the Agrobacterium. The virulence operon includes many genes that
encode for proteins that are part of a Type IV secretion system that exports from the
bacterium proteins and DNA (delineated by specific recognition motifs called border
sequences and excised as a single strand from the virulence plasmid) into the plant cell
through a structure called a pilus. The transferred DNA (called T-DNA) is piloted
to the plant cell nucleus by nuclear localization signals present in the Agrobacterium protein
VirD2, which is covalently attached to the end of the T-DNA at the Right border (RB). Exactly how the T-DNA is integrated into the
host plant genomic DNA is an active area of plant biology research. Assuming that a selection marker (such as
an antibiotic resistance gene) was included in the T-DNA, the transformed plant tissue
can be cultured on selective media to produce shoots. The shoots are then transferred to a different
medium to promote root formation. Once roots begin to grow from the transgenic
shoot, the plants can be transferred to soil to complete a normal life cycle (make seeds). The seeds from this first plant (called the
T1, for first transgenic generation) can be planted on a selective (containing an antibiotic),
or if an herbicide resistance gene was used, could alternatively be planted in soil, then
later treated with herbicide to kill wildtype segregants. Some plants species, such as Arabidopsis thaliana
can be transformed by dipping the flowers or whole plant, into a suspension of Agrobacterium
tumefaciens, typically strain C58 (C=Cherry, 58=1958, the year in which this particular
strain of A. tumefaciens was isolated from a cherry tree in an orchard at Cornell University
in Ithaca, New York). Though many plants remain recalcitrant to
transformation by this method, research is ongoing that continues to add to the list
the species that have been successfully modified in this manner. Viral transformation (transduction): Package
the desired genetic material into a suitable plant virus and allow this modified virus
to infect the plant. If the genetic material is DNA, it can recombine
with the chromosomes to produce transformant cells. However, genomes of most plant viruses consist
of single stranded RNA which replicates in the cytoplasm of infected cell. For such genomes this method is a form of
transfection and not a real transformation, since the inserted genes never reach the nucleus
of the cell and do not integrate into the host genome. The progeny of the infected plants is virus-free
and also free of the inserted gene.Some vector-less methods include: Gene gun: Also referred to as particle bombardment,
microprojectile bombardment, or biolistics. Particles of gold or tungsten are coated with
DNA and then shot into young plant cells or plant embryos. Some genetic material will stay in the cells
and transform them. This method also allows transformation of
plant plastids. The transformation efficiency is lower than
in Agrobacterium-mediated transformation, but most plants can be transformed with this
method. Electroporation: Formation of transient holes
in cell membranes using electric pulses of high field strength; this allows DNA to enter
as described above for bacteria.===Fungi===
There are some methods to produce transgenic fungi most of them being analogous to those
used for plants. However, fungi have to be treated differently
due to some of their microscopic and biochemical traits: A major issue is the dikaryotic state that
parts of some fungi are in; dikaryotic cells contain two haploid nuclei, one of each parent
fungus. If only one of these gets transformed, which
is the rule, the percentage of transformed nuclei decreases after each sporulation. Fungal cell walls are quite thick hindering
DNA uptake so (partial) removal is often required; complete degradation, which is sometimes necessary,
yields protoplasts. Mycelial fungi consist of filamentous hyphae,
which are, if at all, separated by internal cell walls interrupted by pores big enough
to enable nutrients and organelles, sometimes even nuclei, to travel through each hypha. As a result, individual cells usually cannot
be separated. This is problematic as neighbouring transformed
cells may render untransformed ones immune to selection treatments, e.g. by delivering
nutrients or proteins for antibiotic resistance. Additionally, growth (and thereby mitosis)
of these fungi exclusively occurs at the tip of their hyphae which can also deliver issues.As
stated earlier, an array of methods used for plant transformation do also work in fungi: Agrobacterium is not only capable of infecting
plants but also fungi, however, unlike plants, fungi do not secrete the phenolic compounds
necessary to triggger Agrobacterium so that they have to be added e.g. in the form of
acetosyringone. Thanks to development of an expression system
for small RNAs in fungi the introduction of a CRISPR/CAS9-system in fungal cells became
possible. In 2016 the USDA declared that it will not
regulate a white button mushroom strain edited with CRISPR/CAS9 to prevent fruit body browning
causing a broad discussion about placing CRISPR/CAS9-edited crops on the market. Physical methods like electroporation, biolistics
(“gene gun”), sonoporation that uses cavitation of gas bubbles produced by ultrasound to penetrate
the cell membrane, etc. are also applicable to fungi.===Animals===
Introduction of DNA into animal cells is usually called transfection, and is discussed in the
corresponding article.==Practical aspects of transformation in
molecular biology==The discovery of artificially induced competence
in bacteria allow bacteria such as Escherichia coli to be used as a convenient host for the
manipulation of DNA as well as expressing proteins. Typically plasmids are used for transformation
in E. coli. In order to be stably maintained in the cell,
a plasmid DNA molecule must contain an origin of replication, which allows it to be replicated
in the cell independently of the replication of the cell’s own chromosome. The efficiency with which a competent culture
can take up exogenous DNA and express its genes is known as transformation efficiency
and is measured in colony forming unit (cfu) per μg DNA used. A transformation efficiency of 1×108 cfu/μg
for a small plasmid like pUC19 is roughly equivalent to 1 in 2000 molecules of the plasmid
used being transformed. In calcium chloride transformation, the cells
are prepared by chilling cells in the presence of Ca2+ (in CaCl2 solution), making the cell
become permeable to plasmid DNA. The cells are incubated on ice with the DNA,
and then briefly heat-shocked (e.g., at 42 °C for 30–120 seconds). This method works very well for circular plasmid
DNA. Non-commercial preparations should normally
give 106 to 107 transformants per microgram of plasmid; a poor preparation will be about
104/μg or less, but a good preparation of competent cells can give up to ~108 colonies
per microgram of plasmid. Protocols, however, exist for making supercompetent
cells that may yield a transformation efficiency of over 109. The chemical method, however, usually does
not work well for linear DNA, such as fragments of chromosomal DNA, probably because the cell’s
native exonuclease enzymes rapidly degrade linear DNA. In contrast, cells that are naturally competent
are usually transformed more efficiently with linear DNA than with plasmid DNA. The transformation efficiency using the CaCl2
method decreases with plasmid size, and electroporation therefore may be a more effective method for
the uptake of large plasmid DNA. Cells used in electroporation should be prepared
first by washing in cold double-distilled water to remove charged particles that may
create sparks during the electroporation process.===Selection and screening in plasmid transformation
===Because transformation usually produces a
mixture of relatively few transformed cells and an abundance of non-transformed cells,
a method is necessary to select for the cells that have acquired the plasmid. The plasmid therefore requires a selectable
marker such that those cells without the plasmid may be killed or have their growth arrested. Antibiotic resistance is the most commonly
used marker for prokaryotes. The transforming plasmid contains a gene that
confers resistance to an antibiotic that the bacteria are otherwise sensitive to. The mixture of treated cells is cultured on
media that contain the antibiotic so that only transformed cells are able to grow. Another method of selection is the use of
certain auxotrophic markers that can compensate for an inability to metabolise certain amino
acids, nucleotides, or sugars. This method requires the use of suitably mutated
strains that are deficient in the synthesis or utility of a particular biomolecule, and
the transformed cells are cultured in a medium that allows only cells containing the plasmid
to grow. In a cloning experiment, a gene may be inserted
into a plasmid used for transformation. However, in such experiment, not all the plasmids
may contain a successfully inserted gene. Additional techniques may therefore be employed
further to screen for transformed cells that contain plasmid with the insert. Reporter genes can be used as markers, such
as the lacZ gene which codes for β-galactosidase used in blue-white screening. This method of screening relies on the principle
of α-complementation, where a fragment of the lacZ gene (lacZα) in the plasmid can
complement another mutant lacZ gene (lacZΔM15) in the cell. Both genes by themselves produce non-functional
peptides, however, when expressed together, as when a plasmid containing lacZ-α is transformed
into a lacZΔM15 cells, they form a functional β-galactosidase. The presence of an active β-galactosidase
may be detected when cells are grown in plates containing X-gal, forming characteristic blue
colonies. However, the multiple cloning site, where
a gene of interest may be ligated into the plasmid vector, is located within the lacZα
gene. Successful ligation therefore disrupts the
lacZα gene, and no functional β-galactosidase can form, resulting in white colonies. Cells containing successfully ligated insert
can then be easily identified by its white coloration from the unsuccessful blue ones. Other commonly used reporter genes are green
fluorescent protein (GFP), which produces cells that glow green under blue light, and
the enzyme luciferase, which catalyzes a reaction with luciferin to emit light. The recombinant DNA may also be detected using
other methods such as nucleic acid hybridization with radioactive RNA probe, while cells that
expressed the desired protein from the plasmid may also be detected using immunological methods

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