Plasmid

Plasmids are small, generally circular, autonomously replicating, double stranded DNA molecules that may be present in bacteria and archaea in, but not to the bacterial chromosome ( nucleus equivalent) are so present extrachromosomally ( Fig. 1). Rarely enter plasmids and eukaryotes (eg, 2 -micron ring in baker's yeast ). Their size is from 1 to 1000 kbp.

Construction

Plasmids can contain many different genes. Mediating these genes, for example, antibiotic resistance, may result from a selective advantage for the host bacterium. Each plasmid contains at least one sequence ( shortly ORI Sheet origin of replication ) is used as a replication origin. The origin of replication is compatible with the strain of bacteria, the plasmid can be replicated independently of the chromosomal DNA ( Fig. 2). Plasmids are self-replicating thus ( replicons ) and are inherited. Depending on the nature of the replication origin are present in a bacterial cell a few ( low-copy ) or very many copies (high -copy).

Episomes are in bacterial plasmids that can integrate into the chromosomal DNA of the host organism, such as the F- plasmid ( Fig. 3). There they can remain for a long time, be it with every cell division of the host with replicates and can even become an integral part of its DNA. However, you may also like other plasmids exist independently.

In eukaryotes occasionally occurring viral episomes as cccDNA of the hepatitis B virus or episomes of the herpes viruses, and adenoviruses do not integrate into the genome of the host, but in addition are present in the cell nucleus. An exception are the episomes of poxviruses, which occur in the cytosol. Viral episomes are mechanisms for immune evasion in persistent infection with pathogens.

Classification

There are two main groups of plasmids, conjugated and non- conjugated. Conjugative plasmids contain so -called tra - gene conjugation, the parasexual exchange of plasmids between two bacteria that can trigger ( Fig. 4). Non - conjugative plasmids not have this feature, but it can be transmitted along with conjugating plasmids during conjugation.

Different types of plasmids can coexist in the same cell and there are, in Escherichia coli, for example, up to seven. If two plasmids incompatible with each other, is one of them destroyed. Depending on the ability to co-exist in the same cell, so they can be divided into Inkompatiblitätsgruppen.

The classification of plasmids can be done by their function. There are five main classes:

• Fertility (F- ) plasmids ( see F plasmid) containing only tra genes. Their sole function is the initiation of conjugation.
• Resistance ( R ) plasmids, the genes for resistance to antibiotics or toxins contained.
• Col include plasmids, the genes for colicins (proteins that are toxic to other bacteria ) encoding.
• Degradation plasmids that allow the removal of unusual compounds such as toluene or salicylic acid.
• Virulence plasmids that make a bacterium into a pathogen.

A special type of plasmids represent the so-called Ti plasmids (tumor inducing); they are often a component of certain bacteria ( Agrobacterium tumefaciens or Agrobacterium rhizogenes ) and are transferred from these in plants. There they cause the only known cancer in plants.

Use

Plasmids are important tools of molecular biology, genetics, biochemistry and other biological and medical areas. You will be called vectors and used to amplify genes or express. The specific adaptation of a vector is called the vector design.

Many of the plasmids used for these purposes are commercially available. They are derived from the naturally occurring plasmids and still have their structural components such as the origin of replication ( origin of replication, ORI). They have also been changed so that they can be easily used for cloning (they contain for a so-called MCS ( multiple cloning site ) which recognition sequences containing a variety of restriction enzymes ): The field to be reproduced gene is inserted into plasmids which a have gene with antibiotic resistance. Then, these plasmids are introduced into bacteria that grow in a culture medium treated with the appropriate antibiotic. There are thus survival of the bacteria that have taken up the plasmid containing the information for the antibiotic resistance. Bacteria that have not received the plasmid die off by the antibiotic. Thus, the antibiotic acts as a selection marker, which can only survive the bacteria with the resistance plasmid. Usually a different gene ( reporter gene) will be interrupted by the insertion of the gene into the plasmid, which then can no longer be expressed. This lack of property can be used for screening, because only those bacteria that have taken up a plasmid with the desired gene, this property is no longer possess. Bring to genes in plasmids, which ensure that the introduced genes are expressed in a large amount ( expression ), one can obtain the corresponding gene products in large quantities. This procedure allows the production of recombinant insulin today (that is identical to human insulin insulin), which had to be used to complex isolated from the pancreas of pigs.

If a segment of DNA into an organism ( bacteria, plant, animal, human, etc. ) are introduced, plasmids can also be used as carriers. In animals and humans, this process is really established only in cells in cell culture, since there are easily accessible cells as a monolayer. Depending on the cell type needed for the transfer either precipitated DNA ( muscle cells by endocytosis ) or in liposomes packaged DNA. There are other methods such as electroporation, which may further increase the transfection efficiency.

Visualization

Plasmids can be obtained by suitable processes in the electron microscope to visualize, for example, by staining with uranium atoms ( BAC spreading). The ring structure of the plasmid in the form of a spiral around itself has this DNA topological consequences. The plasmids can be present in three different forms shape. The supercoiled form of the native conformation of the plasmid. Since the DNA double helix is wound around itself and can not wrest in a closed plasmid produces a torsional stress, which bends the plasmid in the space around themselves. The same effect can be observed in phone lines, so the fixed wind around its own axis. In the open-chain form one of the two DNA strands is broken in one place, so that the open string is free to rotate about the fixed itself; thereby relaxes the torsional strain, the plasmid lies open before. In the linear DNA, both strands are broken, the circuit structure is removed.