Transposons as a genetic tool

Last updated May 09, 2021

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 (in which P elements are most commonly used) and in Thale cress ( Arabidopsis thaliana ) and bacteria such as Escherichia coli (E. coli ).^[1]^[2]

Currently transposons can be used in genetic research and recombinant genetic engineering for insertional mutagenesis. Insertional mutagenesis is when transposons function as vectors to help remove and integrate genetic sequences. Given their relatively simple design and inherent ability to move DNA sequences, transposons are highly compatible at transducing genetic material, making them ideal genetic tools.

Signature-Tagging Mutagenesis

Signature-tagging mutagenesis (also known as STM) is a technique focused on using transposable element insertion to determine the phenotype of a locus in an organism's genome. While genetic sequencing techniques can determine the genotype of a genome, they cannot determine the function or phenotypic expression of gene sequences.^[3]^[4] STM can bypass this issue by mutating a locus, causing it form a new phenotype; by comparing the observed phenotypic expressions of the mutated and unaltered locus, one can deduce the phenotypic expression of the locus.

In STM, specially tagged transposons are inserted into an organism, such as a bacterium, and randomly integrated into the host genome. In theory, the modified mutant organism should express the altered gene, thus altering the phenotype. If a new phenotype is observed, the genome is sequenced and searched for tagged transposons.^[3] If the site of transposon integration is found, then the locus may be responsible for expressing the phenotypes.^[5]^[6]

There have been many studies conducted transposon based STM, most notably with the P elements ^[4] in Drosophila. P elements are transposons originally described in Drosophila melanogaster genome capable of being artificially synthesized or spread to other Drosophila species through horizontal transfer.^[4] In experimental trials, artificially created P elements and transposase genes are inserted into the genomes of Drosophila embryos. Subsequently, embryos that exhibit mutations have their genomes sequenced and compared, thus revealing the loci that have been affected by insertion and the roles of the loci.,^[4]^[6]

Insertional Inactivation

Insertional inactivation focuses on suppressing the expression of a gene by disrupting its sequence with an insertion. When additional nucleotides are inserted near or into a locus, the locus can suffer a frameshift mutation that could prevent it from being properly expressed into polypeptide chain. Transposon-based Insertional inactivation is considered for medical research from suppression of antibiotic resistance in bacteria to the treatment of genetic diseases.^[7] In the treatment of genetic diseases, the insertion of a transposon into deleterious gene locus of organism's genome would misalign the locus sequence, truncating any harmful proteins formed and rendering them non-functional. Alternatively insertional inactivation could be used to suppress genes that express antibiotic-resistance in bacteria.,^[7]^[8]

Sleeping Beauty

While transposons have been used successfully in plants and invertebrate subjects through insertional mutagenesis and insertional activation, the usage of transposons in vertebrates has been limited due to a lack of transposons specific to vertebrates. Nearly all transposons compatible to and present within vertebrate genomes are inactive and are often relegated to "junk" DNA.^[6] However it is possible to identify dormant transposons and artificially recreate them as active agents.^[6] Genetic researchers Zsuzsanna Izsvák and Zoltán Ivics discovered a fish transposon sequence that, despite being dormant for 15 million years, could be resurrected as a vector for introducing foreign genes into the vertebrate genomes, including those of humans. This transposon, called Sleeping Beauty was described in 1997, and could be artificially reactivated into a functioning transposon.^[6]

Sleeping Beauty can also be viable in gene therapy procedures by helping introduce beneficial transgenes into host genomes. Belcher et al. tested this notion by using Sleeping Beauty transposons to help insert sequences into mice with sickle cell anemia so they can produce the enzymes need to counteract their anemia.^[6] Belcher et al. began their experiment by constructing a genetic sequence consisting of the Hmox-1 transposable element and transposase from Sleeping Beauty. This sequence was then added inserted into a plasmid and introduced into the cells of the mice. The transposase from Sleeping Beauty helped insert the Hmox-1 transposon into the mice genome, allowing the production of enzyme heme oxygenase-1 (HO-1). The mice that receive the insertion showed a fivefold increase in the expression of HO-1, which in turn reduced blood vessel blockage from sickle-cell anemia. The publication of the experiment in 2010 showed that transposons can be useful in gene therapy.^[6]

P Elements as a tool (Drosophila)

Naturally occurring P elements contain:

coding sequence for the enzyme transposase;
recognition sequences for transposase action.

Transposase is an enzyme which regulates and catalyzes the excision of a P element from the host DNA, cutting at two recognition sites, and then reinserts the P element randomly. It is the random-insertion process, that can interfere with existing genes, or carry an additional gene, that can be used as a process for genetic research.

To use this process as a useful and controllable genetic tool, the two parts of the P element must be separated to prevent uncontrolled transposition. The normal genetic tools are therefore:

DNA coding for transposase (or occasionally simply transposase) with no transposase recognition sequences so it cannot insert; and
a "P Plasmid".

P Plasmids always contain:

a Drosophila reporter gene, often a red-eye marker (the product of the white gene);
transposase recognition sequences;

and may contain:

a gene of interest;
an E. coli reporter gene (often some kind of antibiotic resistance); and
origin of replication and other associated plasmid 'housekeeping' sequences.

Methods of usage (Drosophila)

(Forward genetics methods) There are two main ways to utilise these tools: Fly Transformation, and Insertional Mutagenesis, each described below.

Fly Transformation

(hoping for insertion in non-coding regions)

Microinject the posterior end of an early-stage (pre-cellularization) embryo with coding for transposase and a plasmid with the reporter gene, gene of interest and transposase recognition sequences.
Random transposition occurs, inserting the gene of interest and reporter gene.
Grow flies and cross to remove genetic variation between the cells of the organism. (Only some of the cells of the organism will have been transformed. By breeding only the genotype of the gametes is passed on, removing this variation).
Look for flies expressing the reporter gene. These carry the inserted gene of interest, so can be investigated to determine the phenotype due to the gene of interest.

It is important to note that the inserted gene may have damaged the function of one of the host's genes. Several lines of flies are required so comparison can take place and ensure that no additional genes have been knocked out.

Insertional Mutagenesis

(hoping for insertion in coding region)

Microinject the embryo with coding for transposase and a plasmid with the reporter gene and transposase recognition sequences (and often the E. coli reporter gene and origin of replication, etc.).
Random transposition occurs, inserting the reporter gene randomly. The insertion tends to occur near actively transcribed genes, as this is where the chromatin structure is loosest, so the DNA most accessible.
Grow flies and cross to remove genetic variation between the cells of the organism (see above).
Look for flies expressing the reporter gene. These have experienced a successful transposition, so can be investigated to determine the phenotype due to mutation of existing genes.

Possible mutations:

Insertion in a translated region => hybrid protein/truncated protein. Usually causes loss of protein function, although more complex effects are seen.
Insertion in an intron => altered splicing pattern/splicing failure. Usually results in protein truncation or the production of inactive mis-spliced products, although more complex effects are common.
Insertion in 5' (the sequence that will become the mRNA 5' UTR) untranslated region => truncation of transcript. Usually results in failure of the mRNA to contain a 5' cap, leading to less efficient translation.
Insertion in promoter => reduction/complete loss of expression. Always results in greatly reduced protein production levels. The most useful type of insertion for analysis due to the simplicity of the situation.
Insertion between promoter and upstream enhancers => loss of enhancer function/hijack of enhancer function for reporter gene.† Generally reduces the level of protein specificity to cell type, although complex effects are often seen.

† Enhancer trapping

The hijack of an enhancer from another gene allows the analysis of the function of that enhancer. This, especially if the reporter gene is for a fluorescent protein, can be used to help map expression of the mutated gene through the organism, and is a very powerful tool.

Other usage of P Elements (Drosophila)

(Reverse genetics method)

Secondary mobilisation

If there is an old P element near the gene of interest (with a broken transposase) you can remobilise by microinjection of the embryo with coding for transposase or transposase itself. The P element will often transpose within a few kilobases of the original location, hopefully affecting your gene of interest as for 'Insertional Mutagenisis'.

Analysis of Mutagenesis Products (Drosophila)

Once the function of the mutated protein has been determined it is possible to sequence/purify/clone the regions flanking the insertion by the following methods:

Inverse PCR

Isolate the fly genome.
Undergo a light digest (using an enzyme [enzyme 1] known NOT to cut in the reporter gene), giving fragments of a few kilobases, a few with the insertion and its flanking DNA.
Self ligate the digest (low DNA concentration to ensure self ligation) giving a selection of circular DNA fragments, a few with the insertion and its flanking DNA.
Cut the plasmids at some point in the reporter gene (with an enzyme [enzyme 2] known to cut very rarely in genomic DNA, but is known to in the reporter gene).
Using primers for the reporter gene sections, the DNA can be amplified for sequencing.

The process of cutting, self ligation and re cutting allows the amplification of the flanking regions of DNA without knowing the sequence. The point at which the ligation occurred can be seen by identifying the cut site of [enzyme 1].

Plasmid Rescue (E. coli Transformation)

Isolate the fly genome.
Undergo a light digest (using an enzyme [enzyme 1] known to cut in the boundary between the reporter gene and the E. coli reporter gene and plasmid sequences), giving fragments of a few kilobases, a few with the E. coli reporter, the plasmid sequences and its flanking DNA.
Self ligate the digest (low DNA concentration to ensure self ligation) giving a selection of circular DNA fragments, a few with the E. coli reporter, the plasmid sequences and its flanking DNA.
Insert the plasmids into E. coli cells (e.g. by electroporation).
Screen plasmids for the E. coli reporter gene. Only successful inserts of plasmids with the plasmid 'housekeeping' sequences will express this gene.

7. The gene can be cloned for further analysis.

Transposable Element Application Other organisms

The genomes of other organisms can be analysed in a similar way, although with different transposable elements. The recent discovery of the 'mariner transposon' (from the reconstruction of the original sequence from many 'dead' versions in the human genome) has allowed many new experiments, mariner has well conserved homologues across a wide range of species and is a very versatile tool.

Related Research Articles

A transposable element is a DNA sequence 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. Barbara McClintock's discovery of them earned her a Nobel Prize in 1983.

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.

Transposase is an enzyme that binds to the end of a transposon and catalyses its movement to another part of the genome by a cut and paste mechanism or a replicative transposition mechanism. The word "transposase" was first coined by the individuals who cloned the enzyme required for transposition of the Tn3 transposon. The existence of transposons was postulated in the late 1940s by Barbara McClintock, who was studying the inheritance of maize, but the actual molecular basis for transposition was described by later groups. McClintock discovered that pieces of the chromosomes changed their position, jumping from one chromosome to another. The repositioning of these transposons allowed other genes for pigment to be expressed. Transposition in maize causes changes in color; however, in other organisms, such as bacteria, it can cause antibiotic resistance. Transposition is also important in creating genetic diversity within species and adaptability to changing living conditions. During the course of human evolution, as much as 40% of the human genome has moved around via methods such as transposition of transposons.

Forward genetics is a molecular genetics approach of determining the genetic basis responsible for a phenotype. Forward genetics methods begin with the identification of a phenotype, and finds or creates model organisms that display the characteristic being studied.

Genetics, a discipline of biology, is the science of heredity and variation in living organisms.

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.

A transgene is a gene that has been transferred naturally, or by any of a number of genetic engineering techniques from one organism to another. The introduction of a transgene, in a process known as transgenesis, has the potential to change the phenotype of an organism. Transgene describes a segment of DNA containing a gene sequence that has been isolated from one organism and is introduced into a different organism. This non-native segment of DNA may either retain the ability to produce RNA or protein in the transgenic organism or alter the normal function of the transgenic organism's genetic code. In general, the DNA is incorporated into the organism's germ line. For example, in higher vertebrates this can be accomplished by injecting the foreign DNA into the nucleus of a fertilized ovum. This technique is routinely used to introduce human disease genes or other genes of interest into strains of laboratory mice to study the function or pathology involved with that particular gene.

Tn10 is a transposable element, which is a sequence of DNA that is capable of mediating its own movement from one position in the DNA of the host organism to another. There are a number of different transposition mechanisms in nature, but Tn10 uses the non-replicative cut-and-paste mechanism. The transposase protein recognizes the ends of the element and cuts it from the original locus. The protein-DNA complex then diffuses away from the donor site until random collisions brings it in contact with a new target site, where it is integrated. To accomplish this reaction the 50 kDa transposase protein must break four DNA strands to free the transposon from the donor site, and perform two strand exchange reactions to integrate the element at the target site. This leaves two strands unjoined at the target site, but the host DNA repair proteins take care of this. The target site selection is essentially random, but there is a preference for the sequence 5'-GCTNAGC-3'. The 6-9 base pairs that flank the sequence also influence selection of the insertion site.

In molecular biology, 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 or transposons, or can be artificially created for research purposes in the lab.

In the fields of bioinformatics and computational biology, Genome survey sequences (GSS) are nucleotide sequences similar to expressed sequence tags (ESTs) that the only difference is that most of them are genomic in origin, rather than mRNA.

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.

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.

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.

The PiggyBac (PB) transposon is a mobile genetic element that efficiently transposes between vectors and chromosomes via a "cut and paste" mechanism. During transposition, the PB transposase recognizes transposon-specific inverted terminal repeat sequences (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and integrates them into TTAA chromosomal sites. The powerful activity of the PiggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes. The TTAA-specific transposon piggyBac is rapidly becoming a highly useful transposon for genetic engineering of a wide variety of species, particularly insects. They were discovered in 1989 by Malcolm Fraser at the University of Notre Dame.

Genetic engineering can be accomplished using multiple techniques. 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. The ability to genetically engineer organisms is built on years of research and discovery on how genes function and how we can manipulate them. Important advances included the discovery of restriction enzymes and DNA ligases and the development of polymerase chain reaction and sequencing.

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.

Ac/Ds transposable controlling elements was the first transposable element system recognized in maize. The Ac Activator element is autonomous, whereas the Ds Dissociation element requires an Activator element to transpose. Ac was initially discovered as enabling a Ds element to break chromosomes. Both Ac and Ds can also insert into genes, causing mutants that may revert to normal on excision of the element. The phenotypic consequence of Ac/Ds transposable element includes mosaic colors in kernels and leaves in maize.

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.

DNA transposons are DNA sequences, sometimes referred to "jumping genes", that can move and integrate to different locations within the genome. They are class II transposable elements (TEs) that move through a DNA intermediate, as opposed to class I TEs, retrotransposons, that move through an RNA intermediate. DNA transposons can move in the DNA of an organism via a single-or double-stranded DNA intermediate. DNA transposons have been found in both prokaryotic and eukaryotic organisms. They can make up a significant portion of an organism's genome, particularly in eukaryotes. In prokaryotes, TE's can facilitate the horizontal transfer of antibiotic resistance or other genes associated with virulence. After replicating and propagating in a host, all transposon copies become inactivated and are lost unless the transposon passes to a genome by starting a new life cycle with horizontal transfer. It is important to note that DNA transposons do not randomly insert themselves into the genome, but rather show preference for specific sites.

Tc1/mariner is a class and superfamily of interspersed repeats DNA transposons. The elements of this class are found in all animals, including humans. They can also be found in protists and bacteria.

References

This article incorporates material from the Citizendium article "Transposons as a genetic tool", which is licensed under the Creative Commons Attribution-ShareAlike 3.0 Unported License but not under the GFDL.

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