Insertional mutagenesis
Encyclopedia
Insertional mutagenesis is mutagenesis
of DNA by the insertion of one or more bases
.
Insertional mutations can occur naturally, mediated by virus or transposon, or can be artificially created for research purposes in the lab.
. A transposon
, such as the Drosophilla Melanogaster P-element, is allowed to integrate at random locations in the genome
of the organism being studied. Mutants generated by this method are then screened for any unusual phenotypes. If such a phenotype is found then it can be assumed that the insertion has caused the gene relating to that phenotype to be inactivated. Because the sequence of the transposon is known, the gene can be identified, either by sequencing the whole genome and searching for the sequence, or using the polymerase chain reaction
to amplify specifically that gene.
It is important to note that not all DNA insertions will lead to a noticeable mutation. In fact, most will not. However, it is a common enough occurrence in viral DNA insertions that biologists researching gene therapy
will avoid using viruses that integrate their DNA in the host genome when it is not necessary to do so, opting instead for viruses that transiently express their DNA (leave their DNA free-floating within the cell, rather than integrate it into the genome of the host). For those viruses that do integrate their DNA into that of the host, the severity of any ensuing mutation depends entirely on the location within the host's genome wherein the viral DNA is inserted. If the DNA is inserted into the middle of an essential gene the effects on the cell will be drastic. Additionally, insertion into the promoter region of a gene can cause equally drastic effects. For instance, if the viral DNA is inserted into a repressor
, the gene corresponding to that promoter may be over expressed - leading to an overabundance of its product and altered cellular activity. If the DNA is inserted into an enhancer region, the gene may be under-expressed - leading to relative absence of its product, which can significantly interrupt the activity of the cell.
Alteration of different genes will have varying effects on the cell. Not all mutations will significantly effect the proliferation of the cell. However, if the insertion occurs in an essential gene or a gene that is involved in cellular replication or programmed cell death
, the insertion may compromise the viability of the cell or even cause the cell to replicate interminably - leading to the formation of a tumor, which may become cancerous.
Below is an example of a significant change in cell activity due to insertion of a viral gene into a portion of the hosts genome that controls replication.
Virus insertional mutagenesis is only possible with a replication competent virus. The virus inserts a gene (known as a viral onocogene) normally near the cellular myc (c-myc)gene. The c-myc gene is normally turned off in the cell, however when it is turned on it is able to push the cell into the G1 phase
of the cell cycle
and cause the cell to begin replication which allows the viral gene to be replicated. After many replications where the viral gene stays latent tumours begin to grow. These tumours are normally derived from one mutated/ transformed cell (clonal in origin). Avian leukosis virus is an example of a virus that causes a disease by insertional mutagenesis. Newly hatched chicks infected with Avian leukosis virus will begin to form tumours begin to appear in their bursa of fabricus (like the human thymus). This viral gene insertion is also known as a promoter insertion as it drives the expression of the c-myc gene. There is an example of an insertional mutagenesis event caused by a retrotransposon
in the human genome where it causes Fukuyama-type muscular dystrophy http://www.microbiologybytes.com/virology/Retroviruses.html.
engineering where a plasmid (such as pBR322
) is used to disable expression of a gene.
The inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence. Any future products from the inactivated gene will not work because of the extra codes added to it. An example is the use of pBR322, which has genes that respectively encode polypeptides that confer resistance to ampicillin and tetracyclin antibiotics. Hence, when a genetic region is interrupted by integration of pBR322, the gene function is lost but new gene function (resistance to specific antibiotics) is gained.
An alternative strategy for insertional mutagenesis has been used in vertebrate animals to find genes that cause cancer. In this case a transposon, e.g. Sleeping Beauty
, is designed to interrupt a gene in such a way that it causes maximal genetic havoc. Specifically, the transposon contains signals to truncate expression of an interrupted gene at the site of the insertion and then restart expression of a second truncated gene. This method has been used to identify oncogenes
Mutagenesis
Mutagenesis is a process by which the genetic information of an organism is changed in a stable manner, resulting in a mutation. It may occur spontaneously in nature, or as a result of exposure to mutagens. It can also be achieved experimentally using laboratory procedures...
of DNA by the insertion of one or more bases
Base pair
In molecular biology and genetics, the linking between two nitrogenous bases on opposite complementary DNA or certain types of RNA strands that are connected via hydrogen bonds is called a base pair...
.
Insertional mutations can occur naturally, mediated by virus or transposon, or can be artificially created for research purposes in the lab.
Signature tagged mutagenesis
This is a technique used to study the function of genesGênes
Gênes is the name of a département of the First French Empire in present Italy, named after the city of Genoa. It was formed in 1805, when Napoleon Bonaparte occupied the Republic of Genoa. Its capital was Genoa, and it was divided in the arrondissements of Genoa, Bobbio, Novi Ligure, Tortona and...
. A transposon
Transposon
Transposable elements are sequences of DNA that can move or transpose themselves to new positions within the genome of a single cell. The mechanism of transposition can be either "copy and paste" or "cut and paste". Transposition can create phenotypically significant mutations and alter the cell's...
, such as the Drosophilla Melanogaster P-element, is allowed to integrate at random locations in the genome
Genome
In modern molecular biology and genetics, the genome is the entirety of an organism's hereditary information. It is encoded either in DNA or, for many types of virus, in RNA. The genome includes both the genes and the non-coding sequences of the DNA/RNA....
of the organism being studied. Mutants generated by this method are then screened for any unusual phenotypes. If such a phenotype is found then it can be assumed that the insertion has caused the gene relating to that phenotype to be inactivated. Because the sequence of the transposon is known, the gene can be identified, either by sequencing the whole genome and searching for the sequence, or using the polymerase chain reaction
Polymerase chain reaction
The polymerase chain reaction is a scientific technique in molecular biology to amplify a single or a few copies of a piece of DNA across several orders of magnitude, generating thousands to millions of copies of a particular DNA sequence....
to amplify specifically that gene.
Virus insertional mutagenesis
As mentioned in the introduction, insertional mutagenesis refers to mutation of an organism caused by the insertion of additional DNA bases into the organism's preexisting DNA. Because many viruses (not all of them) integrate their own genome into the genome of their host cells in order to replicate, mutagenesis caused by viral infections is a fairly common occurrence. Not all integrating viruses cause insertional mutagenesis, however.It is important to note that not all DNA insertions will lead to a noticeable mutation. In fact, most will not. However, it is a common enough occurrence in viral DNA insertions that biologists researching gene therapy
Gene therapy
Gene therapy is the insertion, alteration, or removal of genes within an individual's cells and biological tissues to treat disease. It is a technique for correcting defective genes that are responsible for disease development...
will avoid using viruses that integrate their DNA in the host genome when it is not necessary to do so, opting instead for viruses that transiently express their DNA (leave their DNA free-floating within the cell, rather than integrate it into the genome of the host). For those viruses that do integrate their DNA into that of the host, the severity of any ensuing mutation depends entirely on the location within the host's genome wherein the viral DNA is inserted. If the DNA is inserted into the middle of an essential gene the effects on the cell will be drastic. Additionally, insertion into the promoter region of a gene can cause equally drastic effects. For instance, if the viral DNA is inserted into a repressor
Repressor
In molecular genetics, a repressor is a DNA-binding protein that regulates the expression of one or more genes by binding to the operator and blocking the attachment of RNA polymerase to the promoter, thus preventing transcription of the genes. This blocking of expression is called...
, the gene corresponding to that promoter may be over expressed - leading to an overabundance of its product and altered cellular activity. If the DNA is inserted into an enhancer region, the gene may be under-expressed - leading to relative absence of its product, which can significantly interrupt the activity of the cell.
Alteration of different genes will have varying effects on the cell. Not all mutations will significantly effect the proliferation of the cell. However, if the insertion occurs in an essential gene or a gene that is involved in cellular replication or programmed cell death
Programmed cell death
Programmed cell-death is death of a cell in any form, mediated by an intracellular program. PCD is carried out in a regulated process which generally confers advantage during an organism's life-cycle...
, the insertion may compromise the viability of the cell or even cause the cell to replicate interminably - leading to the formation of a tumor, which may become cancerous.
Below is an example of a significant change in cell activity due to insertion of a viral gene into a portion of the hosts genome that controls replication.
Virus insertional mutagenesis is only possible with a replication competent virus. The virus inserts a gene (known as a viral onocogene) normally near the cellular myc (c-myc)gene. The c-myc gene is normally turned off in the cell, however when it is turned on it is able to push the cell into the G1 phase
G1 phase
The G1 phase is a period in the cell cycle during interphase, before the S phase. For many cells, this phase is the major period of cell growth during its lifespan. During this stage new organelles are being synthesized, so the cell requires both structural proteins and enzymes, resulting in great...
of the cell cycle
Cell cycle
The cell cycle, or cell-division cycle, is the series of events that takes place in a cell leading to its division and duplication . In cells without a nucleus , the cell cycle occurs via a process termed binary fission...
and cause the cell to begin replication which allows the viral gene to be replicated. After many replications where the viral gene stays latent tumours begin to grow. These tumours are normally derived from one mutated/ transformed cell (clonal in origin). Avian leukosis virus is an example of a virus that causes a disease by insertional mutagenesis. Newly hatched chicks infected with Avian leukosis virus will begin to form tumours begin to appear in their bursa of fabricus (like the human thymus). This viral gene insertion is also known as a promoter insertion as it drives the expression of the c-myc gene. There is an example of an insertional mutagenesis event caused by a retrotransposon
Retrotransposon
Retrotransposons are genetic elements that can amplify themselves in a genome and are ubiquitous components of the DNA of many eukaryotic organisms. They are a subclass of transposon. They are particularly abundant in plants, where they are often a principal component of nuclear DNA...
in the human genome where it causes Fukuyama-type muscular dystrophy http://www.microbiologybytes.com/virology/Retroviruses.html.
Insertional inactivation
Insertional inactivation is a technique used in recombinant DNARecombinant DNA
Recombinant DNA molecules are DNA sequences that result from the use of laboratory methods to bring together genetic material from multiple sources, creating sequences that would not otherwise be found in biological organisms...
engineering where a plasmid (such as pBR322
PBR322
pBR322 is a plasmid and was the first widely-used E. coli cloning vectors. Created in 1977, it was named after its Mexican creators, p standing for plasmid, and BR for Bolivar and Rodriguez....
) is used to disable expression of a gene.
The inactivation of a gene by inserting a fragment of DNA into the middle of its coding sequence. Any future products from the inactivated gene will not work because of the extra codes added to it. An example is the use of pBR322, which has genes that respectively encode polypeptides that confer resistance to ampicillin and tetracyclin antibiotics. Hence, when a genetic region is interrupted by integration of pBR322, the gene function is lost but new gene function (resistance to specific antibiotics) is gained.
An alternative strategy for insertional mutagenesis has been used in vertebrate animals to find genes that cause cancer. In this case a transposon, e.g. Sleeping Beauty
Sleeping Beauty transposon system
The Sleeping Beauty transposon system is a synthetic DNA transposon that was constructed to introduce precisely defined DNA sequences into the chromosomes of vertebrate animals for the purposes of introducing new traits and to discover new genes and their functions.-Mechanism of Action:The Sleeping...
, is designed to interrupt a gene in such a way that it causes maximal genetic havoc. Specifically, the transposon contains signals to truncate expression of an interrupted gene at the site of the insertion and then restart expression of a second truncated gene. This method has been used to identify oncogenes
See Also
- MutationMutationIn molecular biology and genetics, mutations are changes in a genomic sequence: the DNA sequence of a cell's genome or the DNA or RNA sequence of a virus. They can be defined as sudden and spontaneous changes in the cell. Mutations are caused by radiation, viruses, transposons and mutagenic...
- Directed mutagenesisDirected mutagenesisDirected mutagenesis, also known as directed mutation, is a hypothesis proposing that organisms can respond to environmental stresses through directing mutations to certain genes or areas of the genome....
- PCR mutagenesisPCR mutagenesisPCR mutagenesis is a method for generating site-directed mutagenesis. This method can generate mutations without the need for subcloning into M13-based bacteriophage vectors and for ssDNA rescue...
- Signature tagged mutagenesisSignature tagged mutagenesisSignature-tagged mutagenesis is a genetic technique used to study gene function. Recent advances in genome sequencing have allowed us to catalogue a large variety of organisms genomes, but the function of the genes they contain is still largely unknown...
- Site-directed mutagenesisSite-directed mutagenesisSite-directed mutagenesis, also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, is a molecular biology technique in which a mutation is created at a defined site in a DNA molecule. In general, this form of mutagenesis requires that the wild type gene sequence be known...
- Transposon mutagenesisTransposon MutagenesisTransposon mutagenesis, or transposition mutagenesis, is a biological process that allows genes to be transferred to a host organism's chromosome, interrupting or modifying the function of an extant gene on the chromosome and causing mutation.-History:...