Insertional mutagenesis

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In molecular biology, insertional mutagenesis is the creation of mutations in DNA by the addition of one or more base pairs. Such insertional mutations can occur naturally, mediated by viruses or transposons, or can be artificially created for research purposes in the lab.

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This is a technique used to study the function of genes. A transposon such as the P element of Drosophila melanogaster 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 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 by using the polymerase chain reaction 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. Historically, lentiviral vectors included strong viral promoters which had a side effect of insertional mutagenesis, nuclear DNA mutations that effect the function of a gene. [1] These strong viral promotors were shown to be the main cause of cancer formation. [1] As a result, viral promotors have been replaced by cellular promotors and regulatory sequences. [1] 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 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 a 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, 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 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, 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 is an example of a virus that causes disease by insertional mutagenesis. Newly hatched chicks infected with the Avian leukosis virus will begin to form tumours that will begin to appear in their bursa of Fabricius (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. [2]

Insertional inactivation

Insertional inactivation is a technique used in recombinant DNA engineering where a plasmid (such as pBR322) [3] is used to disable the expression of a gene. [4]

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 the 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 the expression of an interrupted gene at the site of the insertion and then restart the expression of a second truncated gene. This method has been used to identify oncogenes. [5] [6]

See also

Related Research Articles

<span class="mw-page-title-main">Mutation</span> Alteration in the nucleotide sequence of a genome

In biology, a mutation is an alteration in the nucleic acid sequence of the genome of an organism, virus, or extrachromosomal DNA. Viral genomes contain either DNA or RNA. Mutations result from errors during DNA or viral replication, mitosis, or meiosis or other types of damage to DNA, which then may undergo error-prone repair, cause an error during other forms of repair, or cause an error during replication. Mutations may also result from insertion or deletion of segments of DNA due to mobile genetic elements.

<span class="mw-page-title-main">Retrovirus</span> Family of viruses

A retrovirus is a type of virus that inserts a DNA copy of its RNA genome into the DNA of a host cell that it invades, thus changing the genome of that cell. After invading a host cell's cytoplasm, the virus uses its own reverse transcriptase enzyme to produce DNA from its RNA genome, the reverse of the usual pattern, thus retro (backwards). The new DNA is then incorporated into the host cell genome by an integrase enzyme, at which point the retroviral DNA is referred to as a provirus. The host cell then treats the viral DNA as part of its own genome, transcribing and translating the viral genes along with the cell's own genes, producing the proteins required to assemble new copies of the virus. Many retroviruses cause serious diseases in humans, other mammals, and birds.

<span class="mw-page-title-main">Transposable element</span> Semiparasitic DNA sequence

A transposable element 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. Transposition often results in duplication of the same genetic material. In the human genome, L1 and Alu elements are two examples. Barbara McClintock's discovery of them earned her a Nobel Prize in 1983. Its importance in personalized medicine is becoming increasingly relevant, as well as gaining more attention in data analytics given the difficulty of analysis in very high dimensional spaces.

<span class="mw-page-title-main">Molecular genetics</span> Scientific study of genes at the molecular level

Molecular genetics is a sub-field of biology that addresses how differences in the structures or expression of DNA molecules manifests as variation among organisms. Molecular genetics often applies an "investigative approach" to determine the structure and/or function of genes in an organism's genome using genetic screens. The field of study is based on the merging of several sub-fields in biology: classical Mendelian inheritance, cellular biology, molecular biology, biochemistry, and biotechnology. Researchers search for mutations in a gene or induce mutations in a gene to link a gene sequence to a specific phenotype. Molecular genetics is a powerful methodology for linking mutations to genetic conditions that may aid the search for treatments/cures for various genetics diseases.

<span class="mw-page-title-main">Transduction (genetics)</span> Transfer process in genetics

Transduction is the process by which foreign DNA is introduced into a cell by a virus or viral vector. An example is the viral transfer of DNA from one bacterium to another and hence an example of horizontal gene transfer. Transduction does not require physical contact between the cell donating the DNA and the cell receiving the DNA, and it is DNase resistant. Transduction is a common tool used by molecular biologists to stably introduce a foreign gene into a host cell's genome.

Forward genetics is a molecular genetics approach of determining the genetic basis responsible for a phenotype. Forward genetics provides an unbiased approach because it relies heavily on identifying the genes or genetic factors that cause a particular phenotype or trait of interest.

P elements are transposable elements that were discovered in Drosophila as the causative agents of genetic traits called hybrid dysgenesis. The transposon is responsible for the P trait of the P element and it is found only in wild flies. They are also found in many other eukaryotes.

Viral vectors are tools commonly used by molecular biologists to deliver genetic material into cells. This process can be performed inside a living organism or in cell culture. Viruses have evolved specialized molecular mechanisms to efficiently transport their genomes inside the cells they infect. Delivery of genes or other genetic material by a vector is termed transduction and the infected cells are described as transduced. Molecular biologists first harnessed this machinery in the 1970s. Paul Berg used a modified SV40 virus containing DNA from the bacteriophage λ to infect monkey kidney cells maintained in culture.

<span class="mw-page-title-main">Mobile genetic elements</span> DNA sequence whose position in the genome is variable

Mobile genetic elements (MGEs) sometimes called selfish genetic elements are a type of genetic material that can move around within a genome, or that can be transferred from one species or replicon to another. MGEs are found in all organisms. In humans, approximately 50% of the genome is thought to be MGEs. MGEs play a distinct role in evolution. Gene duplication events can also happen through the mechanism of MGEs. MGEs can also cause mutations in protein coding regions, which alters the protein functions. These mechanisms can also rearrange genes in the host genome generating variation. These mechanism can increase fitness by gaining new or additional functions. An example of MGEs in evolutionary context are that virulence factors and antibiotic resistance genes of MGEs can be transported to share genetic code with neighboring bacteria. However, MGEs can also decrease fitness by introducing disease-causing alleles or mutations. The set of MGEs in an organism is called a mobilome, which is composed of a large number of plasmids, transposons and viruses.

In molecular cloning, a vector is any particle used as a vehicle to artificially carry a foreign nucleic sequence – usually DNA – into another cell, where it can be replicated and/or expressed. A vector containing foreign DNA is termed recombinant DNA. The four major types of vectors are plasmids, viral vectors, cosmids, and artificial chromosomes. Of these, the most commonly used vectors are plasmids. Common to all engineered vectors are an origin of replication, a multicloning site, and a selectable marker.

Transposon 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. Transposon mutagenesis is much more effective than chemical mutagenesis, with a higher mutation frequency and a lower chance of killing the organism. Other advantages include being able to induce single hit mutations, being able to incorporate selectable markers in strain construction, and being able to recover genes after mutagenesis. Disadvantages include the low frequency of transposition in living systems, and the inaccuracy of most transposition systems.

<span class="mw-page-title-main">Knockout rat</span> Type of genetically engineered rat

A knockout rat is a genetically engineered rat with a single gene turned off through a targeted mutation used for academic and pharmaceutical research. Knockout rats can mimic human diseases and are important tools for studying gene function and for drug discovery and development. The production of knockout rats was not economically or technically feasible until 2008.

Gene therapy using lentiviral vectors was being explored in early stage trials as of 2009.

Transposons are semi-parasitic DNA sequences which can replicate and spread through the host's genome. They can be harnessed as a genetic tool for analysis of gene and protein function. The use of transposons is well-developed in Drosophila and in Thale cress and bacteria such as Escherichia coli.

The Sleeping Beauty transposon system is a synthetic DNA transposon designed 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. It is a Tc1/mariner-type system, with the transposase resurrected from multiple inactive fish sequences.

<span class="mw-page-title-main">Genetic engineering techniques</span> Methods used to change the DNA of organisms

Genetic engineering techniques allow the modification of animal and plant genomes. Techniques have been devised to insert, delete, and modify DNA at multiple levels, ranging from a specific base pair in a specific gene to entire genes. There are a number of steps that are followed before a genetically modified organism (GMO) is created. Genetic engineers must first choose what gene they wish to insert, modify, or delete. The gene must then be isolated and incorporated, along with other genetic elements, into a suitable vector. This vector is then used to insert the gene into the host genome, creating a transgenic or edited organism.

<span class="mw-page-title-main">Reverse genetics</span> Method in molecular genetics

Reverse genetics is a method in molecular genetics that is used to help understand the function(s) of a gene by analysing the phenotypic effects caused by genetically engineering specific nucleic acid sequences within the gene. The process proceeds in the opposite direction to forward genetic screens of classical genetics. While forward genetics seeks to find the genetic basis of a phenotype or trait, reverse genetics seeks to find what phenotypes are controlled by particular genetic sequences.

<span class="mw-page-title-main">Lentiviral vector in gene therapy</span>

Lentiviral vectors in gene therapy is a method by which genes can be inserted, modified, or deleted in organisms using lentiviruses.

<span class="mw-page-title-main">Mutagenesis (molecular biology technique)</span>

In molecular biology, mutagenesis is an important laboratory technique whereby DNA mutations are deliberately engineered to produce libraries of mutant genes, proteins, strains of bacteria, or other genetically modified organisms. The various constituents of a gene, as well as its regulatory elements and its gene products, may be mutated so that the functioning of a genetic locus, process, or product can be examined in detail. The mutation may produce mutant proteins with interesting properties or enhanced or novel functions that may be of commercial use. Mutant strains may also be produced that have practical application or allow the molecular basis of a particular cell function to be investigated.

<span class="mw-page-title-main">Conservative transposition</span>

Transposition is the process by which a specific genetic sequence, known as a transposon, is moved from one location of the genome to another. Simple, or conservative transposition, is a non-replicative mode of transposition. That is, in conservative transposition the transposon is completely removed from the genome and reintegrated into a new, non-homologous locus, the same genetic sequence is conserved throughout the entire process. The site in which the transposon is reintegrated into the genome is called the target site. A target site can be in the same chromosome as the transposon or within a different chromosome. Conservative transposition uses the "cut-and-paste" mechanism driven by the catalytic activity of the enzyme transposase. Transposase acts like DNA scissors; it is an enzyme that cuts through double-stranded DNA to remove the transposon, then transfers and pastes it into a target site.

References

  1. 1 2 3 Poletti, Valentina; Mavilio, Fulvio (2021). "Designing Lentiviral Vectors for Gene Therapy of Genetic Diseases". Viruses. 13 (8): 5. doi: 10.3390/v13081526 . ISSN   1999-4915. PMC   8402868 . PMID   34452394.{{cite journal}}: CS1 maint: date and year (link)
  2. ↑ "Retroviruses". Archived from the original on May 4, 2015.
  3. ↑ "Recombinant DNA". Archived from the original on 2012-08-05. Retrieved 2010-04-11.
  4. ↑ "Insertional inactivation". Archived from the original on 2009-02-18. Retrieved 2010-04-11.
  5. ↑ Carlson CM, Largaespada DA (July 2005). "Insertional mutagenesis in mice: new perspectives and tools". Nat. Rev. Genet. 6 (7): 568–80. doi:10.1038/nrg1638. PMID   15995698. S2CID   3194633.
  6. ↑ Ivics Z, Izsvák Z (2004). "Transposable elements for transgenesis and insertional mutagenesis in vertebrates: a contemporary review of experimental strategies". Mobile Genetic Elements. Methods Mol. Biol. Vol. 260. pp. 255–76. doi:10.1385/1-59259-755-6:255. ISBN   1-59259-755-6. PMID   15020812.
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