Жасушаның сыртқы ДНҚ арқылы генетикалық өзгерісі

Genetic alteration of a cell by uptake of genetic material from the environment

In molecular biology and genetics, 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 bacterium 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. In transformation, the genetic material passes through the intervening medium, and uptake is completely dependent on the recipient bacterium.

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 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 1897 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.

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. 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. It has also been reported in at least 30 species of Pseudomonadota distributed in several different classes. The best studied Pseudomonadota with respect to transformation are the medically important human pathogens Neisseria gonorrhoeae, Haemophilus influenzae, and Helicobacter pylori. 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 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. 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. 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 Actinomycetota 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.

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. 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. 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. 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.

Introduction of DNA into animal cells is usually called transfection, and is discussed in the corresponding article.

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 (in link=calcium chlorideCaCl2 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 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.

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.