In
molecular biology
Molecular biology is the branch of biology that seeks to understand the molecular basis of biological activity in and between cells, including biomolecular synthesis, modification, mechanisms, and interactions. The study of chemical and physi ...
, insertional mutagenesis is the creation of
mutations of DNA by addition of one or more
base pairs. Such insertional mutations can occur naturally, mediated by
viruses
A virus is a submicroscopic infectious agent that replicates only inside the living cells of an organism. Viruses infect all life forms, from animals and plants to microorganisms, including bacteria and archaea.
Since Dmitri Ivanovsky's ...
or
transposons
A transposable element (TE, transposon, or jumping gene) is a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Tran ...
, or can be artificially created for research purposes in the lab.
Signature tagged mutagenesis
This is a technique used to study the function of
genes. A
transposon
A transposable element (TE, transposon, or jumping gene) is a nucleic acid sequence in DNA that can change its position within a genome, sometimes creating or reversing mutations and altering the cell's genetic identity and genome size. Tra ...
such as the
P element of ''
Drosophila melanogaster
''Drosophila melanogaster'' is a species of fly (the taxonomic order Diptera) in the family Drosophilidae. The species is often referred to as the fruit fly or lesser fruit fly, or less commonly the " vinegar fly" or "pomace fly". Starting with ...
'' is allowed to integrate at random locations in the
genome
In the fields of molecular biology and genetics, a genome is all the genetic information of an organism. It consists of nucleotide sequences of DNA (or RNA in RNA viruses). The nuclear genome includes protein-coding genes and non-coding g ...
of the organism being studied. Mutants generated by this method are then screened for any unusual
phenotype
In genetics, the phenotype () is the set of observable characteristics or traits of an organism. The term covers the organism's morphology or physical form and structure, its developmental processes, its biochemical and physiological pr ...
s. If such a phenotype is found then it can be assumed that the insertion has caused the gene relating to the usual 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
The polymerase chain reaction (PCR) is a method widely used to rapidly make millions to billions of copies (complete or partial) of a specific DNA sample, allowing scientists to take a very small sample of DNA and amplify it (or a part of it) ...
to amplify specifically that gene.
Virus insertional mutagenesis
Because many viruses integrate their own genomes into the genomes 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.
Some DNA insertions will lead to no noticeable mutation. In recent gene therapy trials, the
lentiviral
''Lentivirus'' is a genus of retroviruses that cause chronic and deadly diseases characterized by long incubation periods, in humans and other mammalian species. The genus includes the human immunodeficiency virus (HIV), which causes AIDS. Lent ...
vectors used to insert therapeutic DNA showed no tendency to disrupt gene function or promote
oncogenic
Carcinogenesis, also called oncogenesis or tumorigenesis, is the formation of a cancer, whereby normal cells are transformed into cancer cells. The process is characterized by changes at the cellular, genetic, and epigenetic levels and abno ...
development. Because of these advances, it is now considered safe to use such integrating vectors for gene therapy. An advantage is that the lentiviral vectors integrate the DNA permanently, whereas other, non-integrating, viruses' effect is transient. For those viruses such as
gammaretroviruses that tend to integrate their DNA in genetically unfavorable locations, 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 a gene's
promoter region
In genetics, a promoter is a sequence of DNA to which proteins bind to initiate transcription of a single RNA transcript from the DNA downstream of the promoter. The RNA transcript may encode a protein (mRNA), or can have a function in and of ...
can have equally drastic effects. Likewise, if the viral DNA is inserted into a
repressor, the promoter's corresponding gene may be over-expressed – leading to an overabundance of its product and altered cellular activity. If the DNA is inserted into a gene's
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 affect 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 (PCD; sometimes referred to as cellular suicide) is the death of a cell (biology), cell as a result of events inside of a cell, such as apoptosis or autophagy. PCD is carried out in a biological process, which usually confers ...
, 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.
Insertional mutagenesis is possible whether the virus is of the self-inactivating types commonly used in gene therapy or competent to replicate. The virus inserts a gene (known as a viral oncogene) normally near the cellular myc (c-myc)gene. The
c-myc
''Myc'' is a family of regulator genes and proto-oncogenes that code for transcription factors. The ''Myc'' family consists of three related human genes: ''c-myc'' ( MYC), ''l-myc'' ( MYCL), and ''n-myc'' ( MYCN). ''c-myc'' (also sometimes re ...
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
The G1 phase, gap 1 phase, or growth 1 phase, is the first of four phases of the cell cycle that takes place in eukaryotic cell division. In this part of interphase, the cell synthesizes mRNA and proteins in preparation for subsequent steps lead ...
of the
cell cycle
The cell cycle, or cell-division cycle, is the series of events that take place in a cell that cause it to divide into two daughter cells. These events include the duplication of its DNA (DNA replication) and some of its organelles, and sub ...
and cause the cell to begin replication, causing unchecked cell proliferation while allowing 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
Avian sarcoma leukosis virus (ASLV) is an endogenous retrovirus that infects and can lead to cancer in chickens; experimentally it can infect other species of birds and mammals. ASLV replicates in chicken embryo fibroblasts, the cells that contr ...
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 that will begin to appear in their
bursa of Fabricius In birds, the bursa of Fabricius (Latin: ''bursa cloacalis'' or ''bursa fabricii'') is the site of hematopoiesis. It is a specialized organ that, as first demonstrated by Bruce Glick and later by Max Dale Cooper and Robert Good, is necessary fo ...
(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
Retrotransposons (also called Class I transposable elements or transposons via RNA intermediates) are a type of genetic component that copy and paste themselves into different genomic locations (transposon) by converting RNA back into DNA through ...
in the human genome where it causes Fukuyama-type muscular dystrophy.
Insertional inactivation
Insertional inactivation is a technique used in
recombinant DNA
Recombinant DNA (rDNA) molecules are DNA molecules formed by laboratory methods of genetic recombination (such as molecular cloning) that bring together genetic material from multiple sources, creating sequences that would not otherwise be fo ...
engineering where a plasmid (such as
pBR322
pBR322 is a plasmid and was one of the first widely used '' E. coli'' cloning vectors. Created in 1977 in the laboratory of Herbert Boyer at the University of California, San Francisco, it was named after Francisco Bolivar Zapata, the postdocto ...
) 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'' (french: La belle au bois dormant, or ''The Beauty in the Sleeping Forest''; german: Dornröschen, or ''Little Briar Rose''), also titled in English as ''The Sleeping Beauty in the Woods'', is a fairy tale about a princess cu ...
, 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
An oncogene is a gene that has the potential to cause cancer. In tumor cells, these genes are often mutated, or expressed at high levels. .
See also
References
External links
*
Diagram at gene-technology-online.com
{{DEFAULTSORT:Insertional Mutagenesis
Genetics
Mutagenesis