Citizendia

Figure 1: Illustration of a bacterium with plasmids enclosed showing chromosomal DNA and plasmids.
Figure 1: Illustration of a bacterium with plasmids enclosed showing chromosomal DNA and plasmids.

A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA. Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known A chromosome is an organized structure of DNA and Protein that is found in cells. [1] In many cases, it is circular and double-stranded. Plasmids usually occur naturally in bacteria, but are sometimes found in eukaryotic organisms (e. The Bacteria ( singular: bacterium) are a large group of unicellular Microorganisms Typically a few Micrometres in length bacteria have Animals Plants fungi, and Protists are eukaryotes (juːˈkærɪɒt or -oʊt Organisms whose cells are organized into complex g. , the 2-micrometre-ring in Saccharomyces cerevisiae). Saccharomyces cerevisiae is a Species of Budding Yeast. It is perhaps the most useful Yeast owing to its use since ancient times

Plasmid size varies from 1 to over 200 kilobase pairs (kbp). In Molecular biology, two Nucleotides on opposite complementary DNA or RNA strands that are connected via Hydrogen bonds are called [2] The number of identical plasmids within a single cell can be zero, one, or even thousands under some circumstances. The cell is the structural and functional unit of all known living Organisms It is the smallest unit of an organism that is classified as living and is often called Plasmids can be considered to be part of the mobilome, since they are often associated with conjugation, a mechanism of horizontal gene transfer. The mobilome is the total of all Mobile genetic elements in a Genome. Bacterial conjugation is the transfer of genetic material between bacteria through direct cell-to-cell contact

The term plasmid was first introduced by the American molecular biologist Joshua Lederberg in 1952. The United States of America —commonly referred to as the Molecular biology is the study of Biology at a molecular level Joshua Lederberg ( May 23, 1925 &ndash February 2, 2008) was an American molecular biologist known for his work in [3]

Plasmids can be considered to be independent life-forms similar to viruses, since both are capable of autonomous replication in suitable (host) environments. However the plasmid-host relationship tends to be more symbiotic than parasitic (although this can also occur for viruses, for example with Endoviruses) since plasmids can endow their hosts with useful packages of DNA to assist mutual survival in times of severe stress. For example, plasmids can convey antibiotic resistance to host bacteria, who may then survive along with their life-saving guests who are carried along into future host generations.

Contents

Vectors

There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as in the top instance; whereas episomes, the lower example, integrate into the host chromosome.
There are two types of plasmid integration into a host bacteria: Non-integrating plasmids replicate as in the top instance; whereas episomes, the lower example, integrate into the host chromosome. A plasmid is an extra-chromosomal DNA molecule separate from the chromosomal DNA which is capable of replicating independently of the chromosomal DNA A chromosome is an organized structure of DNA and Protein that is found in cells.
Further information: Vector (molecular biology)

Plasmids used in genetic engineering are called vectors. In Molecular biology, a vector is any vehicle used to transfer foreign genetic material into another cell Genetic engineering, Recombinant DNA technology, genetic modification/manipulation (GM and gene splicing are terms that apply to the direct In Molecular biology, a vector is any vehicle used to transfer foreign genetic material into another cell 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. Protein expression is a subcomponent of Gene expression. It consists of the stages after DNA has been translated into Amino acid chains which are ultimately folded [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. A multiple cloning site ( MCS) also called a polylinker, is a short segment of DNA which contains many (usually 20+ Restriction sites - a standard Restriction sites, or restriction recognition sites, are specific sequences of Nucleotides that are recognized by Restriction enzymes The sites are generally Next, the plasmids are inserted into bacteria by a process called transformation. In Molecular biology, transformation is the genetic alteration of a cell resulting from the uptake genomic incorporation and expression of foreign Then, the bacteria are exposed to the particular antibiotics. Only bacteria which take up copies of the plasmid survive the antibiotic, 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. Alkaline lysis is a method used in Molecular biology to break cells open to isolate Plasmid DNA or other cell components such as Proteins Bacteria

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. Insulin is a Hormone with intensive effects on both metabolism and several other body systems (eg vascular compliance In modern usage an antibiotic is a Chemotherapeutic agent with activity against Microorganisms such as Bacteria, fungi or Protozoa

However, a plasmid can only contain inserts of about 1-10 kbp. In Molecular biology, two Nucleotides on opposite complementary DNA or RNA strands that are connected via Hydrogen bonds are called To clone longer lengths of DNA, lambda phage with lysogeny genes deleted, cosmids, bacterial artificial chromosomes or yeast artificial chromosomes could be used. Enterobacteria phage λ ( Lambda phage) is a temperate bacteriophage that infects Escherichia coli A cosmid, first described by Collins and Hohn in 1978 is a type of hybrid Plasmid (often used as a Cloning vector) that contains cos sequences, DNA sequences A bacterial artificial chromosome (BAC is a DNA construct, based on a fertility Plasmid (or F-plasmid) used for transforming and Cloning A yeast artificial chromosome (short YAC) is a vector used to clone large DNA fragments (larger than 100 kb and up to 3000 kb)

Gene Therapy

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 or an immune response. Gene therapy is the insertion of Genes into an individual's cells and tissues to treat a Disease, and Hereditary diseases in which a History See also History of genetics The existence of genes was first suggested by Gregor Mendel (1822-1884 who in the 1860s studied inheritance A chromosome is an organized structure of DNA and Protein that is found in cells. In classical genetics the genome of a Diploid Organism including Eukarya refers to a full set of chromosomes or genes in a Gamete, thereby Oncogenesis is the process of Malignant transformation leading to the formation of a tumor ( Tumorigenesis) An immune system is a collection of mechanisms within an Organism that protects against Disease by identifying and killing Pathogens and Tumor 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. Zinc finger nucleases ( ZFN s are protein chimera comprised of a Zinc finger -based DNA-binding domain and a DNA-cleavage domain Homologous recombination is a type of Genetic recombination, a process of physical rearrangement occurring between two strands of DNA. 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. [4]

Types

Figure 4: Overview of Bacterial conjugation
Figure 4: Overview of Bacterial conjugation

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. Bacterial conjugation is the transfer of genetic material between bacteria through direct cell-to-cell contact 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. Seven 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. Cell division is a process by which a cell, called the parent cell divides into two or more cells called daughter cells. 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. The host killing/suppressor of killing system, also known as hok/sok system, in Molecular biology, is a postsegregational killing system of the Plasmid [5] They produce both a long-lived poison and a short-lived antidote. In the context of Biology, poisons are substances that can cause damage, Illness, or Death to Organisms usually by An antidote or counterdose is a substance which can counteract a form of Poisoning. 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.

Plasmid DNA extraction

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. Cloning in Biology is the process of producing populations of genetically-identical individuals that occurs in nature when organisms such as Bacteria, Insects

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. A restriction digest is a procedure used in Molecular biology to prepare DNA for analysis or other processing

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). Year 2007 ( MMVII) was a Common year starting on Monday of the Gregorian calendar in the 21st century.

Conformations

Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during electrophoresis. Electrophoresis is the most well-known electrokinetic phenomenon. 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. A restriction digest is a procedure used in Molecular biology to prepare DNA for analysis or other processing 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.

Simulation of plasmids

The use of plasmids as a technique in molecular biology is supported by bioinformatics software. Bioinformatics is the application of information technology to the field of molecular biology These programmes record the DNA sequence of plasmid vectors, help to predict cut sites of restriction enzymes, and to plan manipulations. Deoxyribonucleic acid ( DNA) is a Nucleic acid that contains the genetic instructions used in the development and functioning of all known A restriction enzyme (or restriction Endonuclease) is an Enzyme that cuts double-stranded DNA at specific recognition Nucleotide Examples of software packages that handle plasmid maps are Lasergene, GeneConstructionKit, and Vector NTI. Vector NTI is a Bioinformatics software package It is available for free to academic governmental and non-profit researchers

See also

References

  1. ^ Lipps G (editor). A bacterial artificial chromosome (BAC is a DNA construct, based on a fertility Plasmid (or F-plasmid) used for transforming and Cloning Triparental mating is a form of Bacterial conjugation where a conjugative plasmid present in one bacterial strain assists the transfer of a mobilizable plasmid present in a second Transposons are sequences of DNA that can move around to different positions within the Genome of a single cell, a process called transposition This article is about a biological infectious particle for other uses see Phage (disambiguation. A provirus is a Virus genome that has integrated itself into the DNA of a host cell. Segrosomes are Protein complexes that ensure accurate segregation (partitioning of Plasmids or Chromosomes during bacterial cell division. (2008). Plasmids: Current Research and Future Trends. Caister Academic Press. ISBN 978-1-904455-35-6.  
  2. ^ a b c Russell, David W. ; Sambrook, Joseph (2001). Molecular cloning: a laboratory manual. Cold Spring Harbor, N. Y: Cold Spring Harbor Laboratory.  
  3. ^ LEDERBERG J (1952). "Cell genetics and hereditary symbiosis". Physiol. Rev. 32 (4): 403-30.  
  4. ^ Kandavelou K; Chandrasegaran S (2008). "Plasmids for Gene Therapy", Plasmids: Current Research and Future Trends. Caister Academic Press. ISBN 978-1-904455-35-6.  
  5. ^ Gerdes K, Rasmussen PB, Molin S (1986). "Unique type of plasmid maintenance function: postsegregational killing of plasmid-free cells". Proc. Natl. Acad. Sci. U. S. A. 83 (10): 3116. doi:10.1073/pnas.83.10.3116. A digital object identifier ( DOI) is a permanent identifier given to an Electronic document.  

External links

Episomes

Dictionary

plasmid

-noun

  1. (cytology) A circle of double-stranded DNA that is separate from the chromosomes, and which is found in bacteria and protozoa.
© 2009 citizendia.org; parts available under the terms of GNU Free Documentation License, from http://en.wikipedia.org
 Dapyx Software network: MP3 Explorer | Ebook Manager | Zenithic