A circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA. Plasmids are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells.
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plas·mid (plăz'mĭd) ![]() |
A circular, double-stranded unit of DNA that replicates within a cell independently of the chromosomal DNA. Plasmids are most often found in bacteria and are used in recombinant DNA research to transfer genes between cells.
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| Sci-Tech Encyclopedia: Plasmid |
A circular extrachromosomal genetic element that is ubiquitous in prokaryotes and has also been identified in a number of eukaryotes. In general, bacterial plasmids can be classified into two groups on the basis of the number of genes and functions they carry. The larger plasmids are deoxyribonucleic acid (DNA) molecules of around 100 kilobase (kb) pairs, which is sufficient to code for approximately 100 genes. There is usually a small number of copies of these plasmids per host chromosome, so that their replication must be precisely coordinated with the cell division cycle. The plasmids in the second group are smaller in size, about 6–10 kb. These plasmids may harbor 6–10 genes and are usually present in multiple copies (10–20 per chromosome). See also Gene.
Plasmids have been identified in a large number of bacterial genera. Some bacterial species harbor plasmids with no known functions (cryptic plasmids) which have been identified as small circular molecules present in the bacterial DNA. The host range of a particular plasmid is usually limited to closely related genera. Some plasmids, however, are much more promiscuous and have a much broader host range.
The functions specified by different bacterial plasmids are usually quite specialized in nature. Moreover, they are not essential for cell growth since the host bacteria are viable without a plasmid when the cells are cultured under conditions that do not select for plasmid-specified gene products. Plasmids thus introduce specialized functions to host cells which provide versatility and adaptability for growth and survival. Plasmids which confer antibiotic resistance (R plasmids) have been extensively characterized because of their medical importance. Plasmids have played a seminal role in the spectacular advances in the area of genetic engineering. Individual genes can be inserted into specific sites on plasmids in cell cultures and the recombinant plasmid thus formed introduced into a living cell by the process of bacterial transformation. See also Genetic engineering.
| Dental Dictionary: plasmid |
Any type of intracellular inclusion considered to have a genetic function.
| Genetics Encyclopedia: Plasmid |
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.
Replication
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.
Bibliography
Alberts, Bruce, et al. Molecular Biology of the Cell, 4th ed. New York: Garland Science,2002.
Bloom, Mark V., Greg A. Freyer, and David A. Micklos. Laboratory DNA Science: An Introduction to Recombinant DNA Techniques and Methods of Genome Analysis. Menlo Park, CA: Addison-Wesley, 1996.
Alcamo, I. Edward. DNA Technology: The Awesome Skill, 2nd ed. Burlington, MA: Harcourt Press, 2000.
Lodish, Harvey, et al. Molecular Cell Biology, 4th ed. New York: W. H. Freeman,1999.
—Linnea Fletcher
| Britannica Concise Encyclopedia: plasmid |
For more information on plasmid, visit Britannica.com.
| Science Dictionary: plasmid |
A circular bacterial DNA, sometimes used as a vector for gene insertion or genetic engineering. Plasmids are often the site of genes that code for resistance to antibiotics.
| Veterinary Dictionary: plasmid |
An extrachromosomal self-replicating genetic element of a cell. In bacteria, plasmids are circular DNA molecules that reproduce themselves and are thus conserved, apart from the chromosome, through successive cell divisions; they include the F factor and R factor.
| Wikipedia: Plasmid |
A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA.[1] In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e.g., the 2-micrometre-ring in Saccharomyces cerevisiae).
Plasmid size varies from 1 to over 1,000 kilobase pairs (kbp).[2][3][4] The number of identical plasmids within a single cell can range anywhere from one to even thousands under some circumstances. Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer.
The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952.[5]
Plasmids are considered transferable genetic elements, or "replicons", capable of autonomous replication within a suitable host. Plasmids can be found in all three major domains, Archea, Bacteria and Eukarya.[1] Similar to viruses, plasmids are not considered a form of "life" as it is currently defined.[6] Unlike viruses, plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host. Plasmid host-to-host transfer requires direct, mechanical transfer by "conjugation" or changes in host gene expression allowing the intentional uptake of the genetic element by "transformation".[1] Microbial transformation with plasmid DNA is neither parasitic nor symbiotic in nature, since each implies the presence of an independent species living in a commensal or detrimental state with the host organism. Rather, plasmids provide a mechanism for horizontal gene transfer within a population of microbes and typically provide a selective advantage under a given environmental state. Plasmids may carry genes that provide resistance to naturally occurring antibiotics in a competitive environmental niche, or alternatively the proteins produced may act as toxins under similar circumstances. Plasmids also can provide bacteria with an ability to fix elemental nitrogen or to degrade calcitrant organic compounds which provide an advantage under conditions of nutrient deprivation.[1]
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Plasmid classification is very important to biological scientists, a plasmid classification system is available from the website: http://www.computationalmedicalbiology.org/plasclas.aspx. [7]
Plasmids used in genetic engineering are called vectors. Plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to multiply (make many copies of) or express particular genes.[2] Many plasmids are commercially available for such uses. The gene to be replicated is inserted into copies of a plasmid containing genes that make cells resistant to particular antibiotics and a multiple cloning site (MCS, or polylinker), which is a short region containing several commonly used restriction sites allowing the easy insertion of DNA fragments at this location. Next, the plasmids are inserted into bacteria by a process called transformation. Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive , since the plasmid makes them resistant. In particular, the protecting genes are expressed (used to make a protein) and the expressed protein breaks down the antibiotics. In this way the antibiotics act as a filter to select only the modified bacteria. Now these bacteria can be grown in large amounts, harvested and lysed (often using the alkaline lysis method) to isolate the plasmid of interest.
Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacteria produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing a gene or the protein it then codes for, for example, insulin or even antibiotics.
However, a plasmid can only contain inserts of about 1-10 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used.
The success of gene therapy depends on the efficient insertion of therapeutic genes at the appropriate chromosomal target sites within the human genome, without causing cell injury, oncogenic mutations (cancer) or an immune response. Plasmid vectors could be used for this purpose. Zinc finger nucleases (ZFNs) offer a way to cause a site-specific double strand break to the DNA genome and cause homologous recombination. This makes targeted gene correction a viable option in human cells. Plasmids encoding ZFN could be used to deliver a therapeutic gene to a pre-selected chromosomal site. This approach to gene therapy could be less problematic to the alternative viral-based delivery of therapeutic genes.[8]
One way of grouping plasmids is by their ability to transfer to other bacteria. Conjugative plasmids contain so-called tra-genes, which perform the complex process of conjugation, the transfer of plasmids to another bacterium (Fig. 4). Non-conjugative plasmids are incapable of initiating conjugation, hence they can only be transferred with the assistance of conjugative plasmids, by 'accident'. An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can 'parasitize' a conjugative plasmid, transferring at high frequency only in its presence. Plasmids are now being used to manipulate DNA and may possibly be a tool for curing many diseases.
It is possible for plasmids of different types to coexist in a single cell. Several different plasmids have been found in E. coli. But related plasmids are often incompatible, in the sense that only one of them survives in the cell line, due to the regulation of vital plasmid functions. Therefore, plasmids can be assigned into compatibility groups.
Another way to classify plasmids is by function. There are five main classes:
Plasmids can belong to more than one of these functional groups.
Plasmids that exist only as one or a few copies in each bacterium are, upon cell division, in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems which attempt to actively distribute a copy to both daughter cells.
Some plasmids include an addiction system or "postsegregational killing system (PSK)", such as the hok/sok (host killing/suppressor of killing) system of plasmid R1 in Escherichia coli.[9] They produce both a long-lived poison and a short-lived antidote. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell.
Other types of plasmids, often related to yeast cloning vectors include:
For further information see: http://dbb.urmc.rochester.edu/labs/sherman_f/yeast/Cont.html
As alluded to above, plasmids are often used to purify a specific sequence, since they can easily be purified away from the rest of the genome. For their use as vectors, and for molecular cloning, plasmids often need to be isolated.
There are several methods to isolate plasmid DNA from bacteria, the archetypes of which are the miniprep and the maxiprep/bulkprep.[2] The former can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by restriction digest and for some cloning techniques.
In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. Essentially this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several micrograms) of very pure plasmid DNA.
In recent times many commercial kits have been created to perform plasmid extraction at various scales, purity and levels of automation. Commercial services can prepare plasmid DNA at quoted prices below $300/mg in milligram quantities and $15/mg in gram quantities (early 2007[update]).
Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:
The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continually increasing yet different rates. Therefore the resolution of a gel decreases with increased voltage.
At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length. Large linear fragments (over 20kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules 'reptate', with the bulk of the molecule following the leading end through the gel matrix. Restriction digests are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after gel electrophoresis. It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments.
Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.
The use of plasmids as a technique in molecular biology is supported by bioinformatics software. These programmes record the DNA sequence of plasmid vectors, help to predict cut sites of restriction enzymes, and to plan manipulations. Examples of software packages that handle plasmid maps are Lasergene, GeneConstructionKit, and Vector NTI.
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