Plasmids are naturally occurring, stable genetic elements found in bacteria, fungi, and even in the mitochondria of some plants. They may be composed of DNA or RNA, double-stranded or single-stranded, linear or circular. Plasmids almost always exist and replicate independently of the chromosome of the cell in which they are found.
Types of Plasmids
Plasmids are not usually required by their host cell for its survival. Instead, they carry genes that confer a selective advantage on their host, such as resistance to heavy metals or resistance to naturally made antibiotics carried by other organisms. Alternatively, they may produce antibiotics (toxins) that help the host to compete for food or space. For instance, antibiotic resistance genes produced by a plasmid will allow its host bacteria to grow even in the presence of competing bacteria or fungi that produce these antibiotics.
Plasmids are subgrouped into five main types based on phenotypic function. R plasmids carry genes encoding resistance to antibiotics. Col plasmids confer on their host the ability to produce antibacterial polypeptides called bacteriocins that are often lethal to closely related or other bacteria. The col proteins of E. coli are encoded by plasmids such as ColE1. F plasmids contain the F or fertility system required for conjugation (the transfer of genetic information between two cells). These are also known as episomes because, under some circumstances, they can integrate into the host chromosome and thereby promote the transfer of chromosomal DNA between bacterial cells. Degradative or catabolic plasmids allow a host bacterium to metabolize normally undegradable or difficult compounds such as various pesticides. Finally, virulence plasmids confer pathogenicity on a host organism by the production of toxins or other virulence factors.
One common feature of all plasmids is a specific sequence of nucleotides termed an origin of replication (ori). This sequence, together with other regulatory sequences, is referred to as a replicon. The replicon allows a plasmid to replicate within a host cell independently of the host cell's own replication cycle. If the plasmid makes many copies of itself per cell, it is termed a "relaxed" plasmid. If it maintains itself in fewer numbers within the cell it is termed a "stringent" plasmid. Two different plasmids can coexist in the same cell only if they share the same replication elements. If they do not, they will be unable to be propagated stably in the same cell line, and are termed incompatible.
In nature, plasmid inheritance can occur through a variety of mechanisms. During conjugation between two bacterial strains, plasmids can be transferred along with the bacterial DNA, and this activity is controlled by a set of transfer (tra ) genes that are located on the plasmid and not on the bacterial chromosome. The proteins produced by these transfer genes bind to the DNA at the ori site to form a DNA-protein complex known as a relaxosome. This complex makes a nick, or break, in one of the two strands of the double-stranded plasmid DNA molecule. The place where this break occurs is called the "nic" site, and the nicked DNA is said to be "relaxed" because the DNA unwinds as a result of the nick in one of the strands. The single-stranded DNA that is generated by the nick is thought to be unwound and transferred through the pilus, or mating bridge, that connects the two bacteria entering the recipient bacteria. The other strand is left in the donor bacteria. It acts as a template for the synthesis of a new complementary DNA strand forming a double-stranded plasmid DNA molecule.
Some nonconjugative plasmids can also be transferred into bacteria by means of a process called mobilization, as long as they carry the necessary (mob ) genes. Others are taken up by bacterial cells during the process known as transformation. Finally, plasmids that exist in a host cell that undergoes fission (cell division) are simply divided between the resultant two daughter cells.
Use in Research and Technology
Because of their ability to move genes from cell to cell, plasmids have become versatile tools for both research and biotechnology. In the laboratory, researchers use plasmids to carry marker genes, allowing them to trace the plasmid's inheritance across host cells. Transferred or "cloned" genes are used to produce a variety of important medical, agricultural, or environmental products that can be economically used by humans.
Researchers have also engineered plasmids to be extremely efficient cloning vectors . To be used in this way, the plasmid must contain at least one origin of replication, a multiple cloning site (called a polylinker) where a variety of restriction enzymes can cut so that foreign DNA can be inserted, a selectable genetic marker, and transcription and translation signals recognized by the host cell, so that the expression of a cloned gene can be easily identified.
The foreign DNA is often inserted in such a way that the expression of the foreign gene is tied to the expression of a marker gene. For example, one of the most popular methods to show that a foreign DNA has been inserted and expressed in the host is by the insertional inactivation of the lac Z gene. In this case, the foreign DNA is inserted in the middle of the lac Z gene so that the gene becomes defective and the enzyme it codes for no longer works. The damaged enzyme therefore cannot cleave the artificial substrate Xgal to produce a blue color or blue colony, as it normally would, and white colonies of bacteria are produced. Therefore, the white colonies indicate that artificial DNA has been successfully cloned or recombined into the plasmid in the lac Z gene, whereas nonrecombinant colonies are blue. The white colonies can thus be easily isolated for further expansion and experimentation.
Under certain circumstances, recombinant DNA experiments using plasmids are considered to be hazardous, and the ease with which plasmids are acquired by bacteria has led them to be classed as biohazards. They are therefore subject to guidelines and may require registration and approval. A publication produced by the National Institutes of Health, titled Guidelines for Research Involving Recombinant DNA Molecules, is the definitive reference for recombinant DNA research in the United States and should be consulted when considering research, particularly biomedical research, involving plasmids.
see also Antibiotic Resistance; Cloning Genes; Conjugation; Inheritance, Extranuclear; Marker Systems; Restriction Enzymes; Transformation.
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