What are plasmid relationships and applications?
The structure of plasmids is usually circular, although linear forms do exist. Their size ranges from a few thousand base pairs to hundreds of thousands of base pairs. They are found primarily in bacteria but have also been found in fungi, plants, and even humans.
In its commensal relationship with its host, the plasmid can be thought of as a molecular parasite whose primary function is to maintain itself within its host and to spread itself as widely as possible to other hosts. The majority of genes that are present on a plasmid will be dedicated to this function. Researchers have discovered that despite the great diversity of plasmids, most of them have similar genes, dedicated to this function. This relative simplicity of plasmids makes them ideal models of gene function, as well as useful tools for molecular biology. Genes of interest can be placed on a plasmid, which can easily be moved in and out of cells. Using plasmids isolated from the wild, molecular biologists have designed many varieties of artificial plasmids, which have greatly facilitated research in molecular biology.
To survive and propagate, a plasmid must be able to copy itself, or replicate. The genes that direct this process are known as the replication genes. These genes do not carry out all the functions of replication, but instead coopt the host’s replication machinery to replicate the plasmid. Replication allows the plasmid to propagate by creating copies of itself that can be passed to each daughter cell when the host divides. In this manner, the plasmid propagates along with the host.
A second function of the replication genes is to control the copy number of the plasmid. The number of copies of a plasmid that exist inside a host can vary considerably. Plasmids can exist at a very low copy number (one or two copies per cell) or at a higher copy number, with dozens of copies per cell. Adjusting the copy number is an important consideration for a plasmid. Plasmid replication is an expensive process that consumes energy and resources of the host cell. A plasmid with a high copy number can place a significant energy drain on its host cell. In environments where the nutrient supply is low, a plasmid-bearing cell may not be able to compete successfully with other, non-plasmid-containing cells. Wild plasmids often exist at a low copy number, or create a high copy number for only a brief period of time.
Because the presence of a plasmid is expensive in terms of energy, a cell harboring a plasmid will grow more slowly than a similar cell with no plasmid. This can cause a problem for a plasmid if it fails to partition properly during its host’s division. If the plasmid does not partition properly, then one of the host’s daughter cells will not contain a plasmid. Since this cell does not have to spend energy replicating a plasmid, it will gain an ability to grow faster, as will all of its offspring. In such a situation, the population of non-plasmid-containing cells could outgrow the population of plasmid-containing cells and use up all the nutrients in the environment. To avoid this problem, plasmids have evolved strategies to prevent improper partitioning. One strategy is for the plasmid to contain partitioning genes. Partitioning genes encode proteins that actively partition plasmids into each daughter cell during the cell division of the host. Active partitioning greatly reduces the errors in partitioning that might occur if partitioning were left to chance.
A second strategy that plasmids use to prevent partitioning errors is the plasmid addition system. In this strategy, genes on the plasmid direct the production of both a toxin and an antidote. The antidote protein is very unstable and degrades quickly, but the toxin is quite stable. As long as the plasmid is present, the cytoplasm of the cell will be full of toxin and antidote. Should a daughter cell fail to receive a plasmid during division, the residual antidote and toxin present in the cytoplasm from the mother cell will begin to degrade, since there is no longer a plasmid present to direct the synthesis of either toxin or antidote. Since the antidote is very unstable, it will degrade first, leaving only toxin, which will kill the cell.
Propagation of plasmids can occur through the spread of plasmids from parent cells to their offspring (referred to as vertical transfer), but propagation can also occur between two different cells (referred to as horizontal transfer). Many plasmids are able to transfer themselves from one host to another through the process of conjugation. Conjugal plasmids contain a collection of genes that direct the host cell that contains them to attach to other cells and transfer a copy of the plasmid. In this manner, the plasmid can spread itself to other hosts and is not limited to spreading itself only to the descendants of the original host cell.
One of the first plasmids to be identified was discovered because of its ability to conjugate. This plasmid, known as the F plasmid, or F factor, is a plasmid found in the bacterium Escherichia coli . Cells harboring the F plasmid are designated F+ cells and can transfer their plasmid to other E. coli cells that do not contain the F plasmid (called F- cells).
Conjugal plasmids can be very specific and transfer only between closely related members of the same species (such as the F plasmid), or they can be very promiscuous and allow transfer between unrelated species. An extreme example of cross-species transfer is the Ti plasmid of the bacterial species Agrobacterium tumefaciens. The Ti plasmid is capable of transferring part of itself from A. tumefaciens into the cells of dicotyledonous plants. Plant cells that receive parts of the Ti plasmid are induced to grow and form a tumorlike structure, called a gall, that provides a hospitable environment for A. tumefaciens.
In most commensal relationships, there is an exchange of benefits between the two partners. The same is true for plasmids and their hosts. In many cases, plasmids provide their host cells with a collection of genes that enhance the ability of the host cell to survive. Enhancements include the ability to metabolize a wider range of materials for food and the ability to survive in hostile environments. One particular hostile environment in which plasmids can provide the ability to survive is the human body. A number of pathogenic microorganisms gain their ability to inhabit the human body, and thus cause disease, from genes contained on plasmids. An example of this is Bacillus anthracis, the agent that causes anthrax. Many of the genes that allow this organism to cause disease are contained on one of two plasmids, called pXO1 and pXO2. Yersinia pestis, the causative agent of bubonic plague, also gains its disease-causing ability from plasmids.
Another example of plasmids conferring on their hosts the ability to survive in a hostile environment is antibiotic resistance. Plasmids known as R factors contain genes that make their bacterial hosts resistant to antibiotics. These R factors are usually conjugal plasmids, so they can move easily from cell to cell. Because the antibiotic resistance genes they carry are usually parts of transposons, they can readily copy themselves from one piece of DNA to another. Two different R factors that happened to be together in one cell could exchange copies of each other’s antibiotic resistance genes. A number of R factors exist that contain multiple antibiotic resistance genes. Such plasmids can result in the formation of “multidrug resistant” (MDR) strains of pathogenic bacteria, which are difficult to treat. There is much evidence to suggest that the widespread use of antibiotics has contributed to the development of MDR pathogens, which are emerging as an important health concern.
Through conjugation, plasmids can transfer genetic information from one species of bacterial cell to another. During its stay in a particular host, a plasmid may acquire some of the chromosomal genes of the host, which it then carries to a new host by conjugation. These genes can then be transferred from the plasmid to the chromosome of the new host. If the new host and the old host are different species, this gene transfer can result in the introduction of new genes, and thus new traits, into a cell. Bacteria, being asexual, produce daughter cells that are genetically identical to their parent. The existence of conjugal plasmids, which allow for the transfer of genes between bacterial species, may represent an important mechanism by which bacteria generate diversity and create new species.
The identification of restriction endonuclease sites within plasmids allowed scientists to manipulate the organization and makeup of these molecules. Researchers were now able to insert genes of interest into these restriction sites within the plasmids and have the recombined plasmid vector taken up by bacteria through the process of transformation. Transcription of the inserted gene by the host bacterium is dependent on an upstream promoter that is active in that bacterial strain. The use of genetic engineering and the creation of artificial plasmid vectors have revolutionized basic research and have led to the creation of modern industrial microbiology. Because bacteria containing the vector are able to express proteins encoded by the inserted gene(s) at a high level, mass quantities of desired proteins can be produced on an industrial scale. Vectors have been used to express medically important proteins such as insulin, human growth hormone, and human factor IX, a blood-clotting factor. Vectors have also been used to express proteins within eukaryotic cells. Additional DNA elements are necessary for the optimal production of proteins in these cells. Viral or mammalian promoters recognized by host RNA polymerases, as well as enhancer sequences, allow proteins to be expressed from the vector. Poly-adenylation sequences are added downstream of the inserted gene for proper messenger RNA (mRNA) expression.
The ability to express proteins within mammalian cells has enabled the development of DNA vaccines in which antigenic proteins are expressed from plasmids. These plasmids are taken up by cells following introduction into the animal or human subject by injection or alternate means of inoculation. Expression of these proteins in the cell then allows for an immunological response by the vaccine. Expression of cytokine or other immunomodulatory molecules from the same plasmid has been explored as a means of enhancing the immune response to the coexpressed antigenic protein using DNA vaccines. Plasmid DNA itself can sometimes have immunostimulatory effects due to the presence of CpG dinucleotides. Clinical trials using DNA vaccines targeting cancer have demonstrated the immunogenicity of these vaccines in humans. Veterinary applications of DNA vaccines have been approved for use.
Plasmids have also been used as delivery vehicles for the expression of double-stranded RNAs (dsRNAs) in order to suppress specific mRNAs by RNA interference (RNAi). Because of the abbreviated length (about 21 nucleotides) and need for a distinction termination point of these dsRNAs, RNA polymerase III promoters and the accompanying termination signals have often been included on the plasmids to express these transcripts within cells. Delivery of the RNAi expression plasmids to the desired location within the body remains a challenge for the development of this technology.
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