Extrachromosomal DNA - Lecture notes 1 PDF

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Extrachromosomal DNA Extrachromosomal DNA is any DNA that is found outside of the nucleus of a cell. It is also referred to as extranuclear DNA or cytoplasmic DNA. Most DNA in an individual genome is found in chromosomes but DNA found outside of the nucleus also serves important biological functions. In prokaryotes, nonviral extrachromosomal DNA is primarily found in plasmids whereas in eukaryotes extrachromosomal DNA is primarily found in organelles. Mitochondrial DNA is a main source of this extrachromosomal DNA in eukaryotes. Plasmid

Illustration of a bacterium showing chromosomal DNA and plasmids. A plasmid is a small DNA molecule within a cell that is physically separated from a chromosomal DNA and can replicate independently. They are most commonly found in bacteria as small circular, double-stranded DNA molecules; however, plasmids are sometimes present in archaea and eukaryotic organisms. In nature, plasmids often carry genes that may benefit the survival of the organism, for example antibiotic resistance. While the chromosomes are big and contain all the essential genetic information for living under normal conditions, plasmids usually are very small and contain only additional genes that may be useful to the organism under certain situations or particular conditions. Artificial plasmids are widely used as vectors in molecular cloning, serving to drive the replication of recombinant DNA sequences within host organisms. Plasmids are considered replicons, a unit of DNA capable of replicating autonomously within a suitable host. However, plasmids, like viruses, are not generally classified as life. Plasmids can be transmitted from one bacterium to another (even of another species) via three main mechanisms: transformation, transduction, and conjugation.

Properties and characteristics

There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as with the top instance, whereas episomes, the lower example, can integrate into the host chromosome. The American molecular biologist Joshua Lederberg first introduced the term plasmid in 1952 originally to describe any bacterial genetic material that exists in an extrachromosomal state for at least part of its replication cycle In order for plasmids to replicate independently within a cell, they must possess a stretch of DNA that can act as an origin of replication. The self-replicating unit, in this case the plasmid, is called a replicon. A typical bacterial replicon may consist of a number of elements, such as the gene for plasmid-specific replication initiation protein (Rep), repeating units called iterons, DnaA boxes, and an adjacent AT-rich region. Smaller plasmids make use of the host replicative enzymes to make copies of themselves, while larger plasmids may carry genes specific for the replication of those plasmids. A few types of plasmids can also insert into the host chromosome, and these integrative plasmids are sometimes referred to as episomes in prokaryotes.

Classifications and types

Overview of bacterial conjugation

Electron micrograph of a DNA fiber bundle, presumably of a single bacterial chromosome loop.

Electron micrograph of a bacterial DNA plasmid (chromosome fragment). Plasmids may be classified in a number of ways. Plasmids can be broadly classified into conjugative plasmids and non-conjugative plasmids. Conjugative plasmids contain a set of transfer or tra genes which promote sexual conjugation between different cells. In the complex process of conjugation, plasmid may be transferred from one bacterium to another via sex pili encoded by some of the tra genes (see figure). Non-conjugative plasmids are incapable of initiating conjugation, hence they can be transferred only with the assistance of conjugative plasmids. 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 can also be classified into incompatibility groups. A microbe can harbour different types of plasmids; however, different plasmids can only exist in a single bacterial cell if they are compatible. If two plasmids are not compatible, one or the other will be rapidly lost from the cell. Different plasmids may therefore be assigned to different incompatibility groups depending on whether they can coexist together. Incompatible plasmids (belonging to the same incompatibility group) normally share the same replication or partition mechanisms and can thus not be kept together in a single cell. Another way to classify plasmids is by function. There are five main classes: 

Fertility F-plasmids, which contain tra genes. They are capable of conjugation and result in the expression of sex pili.

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Resistance (R) plasmids, which contain genes that provide resistance against antibiotics or poisons. Historically known as R-factors, before the nature of plasmids was understood.

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Col plasmids, which contain genes that code for bacteriocins, proteins that can kill other bacteria.

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Degradative plasmids, which e.g. toluene and salicylic acid.

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Virulence plasmids, which turn the bacterium into a pathogen.

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Vectors Artificially constructed plasmids may be used as vectors in genetic engineering. These plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to clone and amplify (make many copies of) or express particular genes. A wide variety of plasmids are commercially available for such uses. The gene to be replicated is normally inserted into a plasmid that typically contains a number of features for their use. These include a gene that confers resistance to particular antibiotics (ampicillin is most frequently used for bacterial strains), an origin of replication to allow the bacterial cells to replicate the plasmid DNA, and a suitable site for cloning.

A schematic representation of the pBR322 plasmid, one of the first plasmids to be used widely as a cloning vector. Shown on the plasmid diagram are the genes encoded (amp and tet for ampicillin and tetracycline resistance respectively), its origin of replication (ori), and various restriction sites (indicated in blue).

Cloning Plasmids are the most-commonly used bacterial cloning vectors. These cloning vectors contain a site that allows DNA fragments to be inserted, for example a multiple cloning site or polylinker which has several commonly used restriction sites to which DNA fragments may be ligated. After the gene of interest is inserted, the plasmids are introduced into bacteria by a process called transformation. These plasmids contain a selectable marker, usually an antibiotic resistance gene, which confer on the bacteria an ability to survive and proliferate in a selective growth medium containing the particular antibiotics. The cells after transformation are exposed to the selective media, and only cells containing the plasmid may survive. In this way, the antibiotics act as a filter to select only the bacteria containing the plasmid DNA. The vector may also contain other marker genes or reporter genes to facilitate selection of plasmid with cloned insert. Bacteria containing the plasmid can then be grown in large amounts, harvested, and the plasmid of interest may then be isolated using various methods of plasmid preparation. A plasmid cloning vector is typically used to clone DNA fragments of up to 15 kbp. To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes, or yeast artificial chromosomes are used. Protein production 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 bacterium 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 the protein the gene codes for, for example, insulin. Gene therapy Plasmid may also be used for gene transfer into human cells as potential treatment in gene therapy so that it may express the protein that is lacking in the cells. Some strategies of gene therapy require the insertion of therapeutic genes at pre-selected chromosomal target sites within the human genome. Plasmid vectors are one of many approaches that 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. Plasmids encoding ZFN could help deliver a therapeutic gene to a specific site so that cell damage, cancer-causing mutations, or an immune response is avoided. Disease models Plasmids were historically used to genetically engineer the embryonic stem cells of rats in order to create rat genetic disease models. The limited efficiency of plasmid-based techniques precluded their use in the creation of more accurate human cell models. However, developments in Adenoassociated virus recombination techniques, and Zinc finger nucleases, have enabled the creation of a new generation of isogenic human disease models....