Transposons

Since McClintock’s discovery, three basic types of transposons have been identified. They are: class I transposons or retrotransposons, class II Transposons: DNA Transposons, and class III transposons: miniature inverted-repeat transposable elements (MITEs).

Class I Transposons: Retrotransposons

Class I transposons are also known as retrotransposons because the DNA segment is first copied into an RNA, then reverse transcribed back to DNA by the enzyme reverse transcriptase. They are replicative since they make copies of themselves before they move. They have a similar structure to retroviruses, like HIV, and follow a similar replication mechanism. Class I transposons do not encode a transposase enzyme.  

There are 2 types of class I transposons: one with long terminal repeats (LTRs) and the other without them (non-LTRs). LTR retrotransposons contain gag and pol genes, where pol codes for the reverse transcriptase and integrase enzymes required for transposition. On the contrary, non-LTR retrotransposons have 2 open reading frames (ORFs), which often end with a poly(A) tail. ORF2 encodes endonuclease and reverse transcriptase.

Class II Transposons: DNA Transposons

Class II transposons are also known as DNA transposons, as unlike the class I enzymes, they do not involve RNA intermediate for movement. Most class II transposons follow a replicative cut-and-paste process. They initially excise themselves from one location and get inserted into another site. Class II transposons have LTRs on both ends.

They are found in both eukaryotic and prokaryotic genomes. Their organization is simple, containing a structural gene, which codes for the transposase enzyme responsible for the mobility of Class II elements, flanked by short terminal inverted repeats (TIRs).

During transposition, TIRs are the binding sites for the transposase. After binding, it promotes excision by making double-strand breaks at each element’s end. Following this excision, the transposase promotes double-strand breaks at the insertion site and directs the component’s insertion in the new position.

In prokaryotes, the transposable element is known as the insertion sequence or IS. When two identical IS sequences flank specific genes, it is called a composite transposon. These genes code for antibiotic resistance or virulence, providing an advantage to the host bacterium. In non-composite transposons, the transposition and non-transposition genes are clustered and flanked by terminal IR sequences.

Class III Transposons: Miniature Inverted-Repeat Transposable Elements (MITEs)

These transposons are characterized by their short lengths, about 400 to 600 base pairs, and 5 base pairs stretch at each element’s end in the inverted form. MITEs are too small to encode proteins. Thousands of these transposons are found in the genomes of Oryza sativa, Caenorhabditis elegans, and other organisms, including humans.

Transposons can be autonomous and non-autonomous.

Autonomous transposons can move by themselves. The activator element (Ac) is an example of an autonomous transposon. In contrast, non-autonomous transposons require the help of other transposons for their movement as they lack enzymes reverse transcriptase (for Class I) or transposase (for Class II). Dissociation elements (Ds) are an example of a non-autonomous.

In the 1940s, transposons were discovered by Barbara McClintock on Zea mays. Transposons in maize were found to cause gene mutations like insertions, deletions, and translocations to occur.

The P-elements in Drosophila are Class II transposons that typically remain repressed. However, when male flies containing P elements mate with females that lack P elements, transposase becomes active, causing multiple mutations in their offspring. P elements appeared in Drosophila melanogaster almost 50 years ago. Since then, they have spread to every population of the species.

Transposons in bacteria sometimes contain antibiotic-resistance genes, along with the transposase gene. When the transposon is inserted in a plasmid vector, it leaves the original host cell and moves to another one. The mechanism of transposition in bacteria is a copy-paste process. It is how the antibiotic-resistance genes spread.  

Although there are many different transposons, DNA transposons are most commonly used in the laboratory. Some common types of transposon systems used in research are described below:

Sleeping Beauty

Sleeping Beauty is a synthetic transposable element from inactivated Tc1/mariner transposons found in fish. Its preferred target site for integration is TA dinucleotides, leaving a CAG DNA footprint from its terminal sequences at the site of excision after cleavage by the transposase.

It has a cargo capacity of more than 100 kilobase pairs, although integration efficiency decreases with cargo size. Sleeping Beauty has almost a random integration profile in mammalian genomes. It is found in vertebrates and inserted in humans at rates similar to retroviral vectors.

piggyBac

piggyBac was identified in the cabbage looper moth. The target site of piggyBac is TTAA, and unlike other transposons, it does not leave behind a footprint after excision. It also moves DNA over 100 kilobase pairs in size and is active in vitro and in vivo in yeasts, plants, insects, and mammalian cells, including humans.

They are biased to integrate at transcription start sites, CpG islands, and DNase I hypersensitivity sites.

Tol2

It is the first active DNA transposon in the vertebrate (found in the Japanese medaka fish). The insertion of Tol2 into the fish’s tyrosinase gene caused albinism. The gene has a weak consensus sequence for its integration site compared to the other two transposons. Like piggyBac, these transposons also prefer integrating at transcription start sites, CpG islands, and DNase I hypersensitivity sites.

Mutagenesis Screenings

Transposons are potent tools for mutagenesis studies, where researchers deliberately induce mutations to study gene function. By inserting transposons into specific locations in the genome, scientists can disrupt the normal operation of genes and observe the resulting phenotypic changes. This approach helps uncover the roles of different genes and their contributions to various biological processes.

Generating Transgenic Animals

Transposons play a pivotal role in the creation of transgenic animals. Scientists use transposons to deliver and integrate desired genes into the germline cells of animals, ensuring that the introduced genetic material is passed on to the next generations. This application is particularly crucial for studying gene function, disease modeling, and the development of animals with desirable traits for agriculture.

Gene Transfer in Plant

Transposons are employed as powerful tools in plant genetic engineering. Researchers use transposons to transfer and express genes in plant genomes, enabling the development of genetically modified (GM) crops with improved traits such as resistance to pests, diseases, or environmental stress. The ability of transposons to integrate efficiently into plant genomes facilitates the creation of stable and heritable genetic modifications.

RNA Guided Transposons Insertion

Advances in genetic engineering caused the development of RNA-guided transposon insertion systems, leveraging the principles of RNA interference (RNAi) and the precision of the CRISPR-Cas system. This innovative approach allows researchers to guide transposons to specific genomic locations by designing complementary RNA sequences. The combination of CRISPR technology and transposons enhances the precision and control over gene insertion, reducing off-target effects and providing a more sophisticated method for genome editing.

Understanding Genome Structure and Function

The study of transposons has deepened our understanding of genome structure and function. Transposons can influence the regulation of nearby genes by affecting chromatin structure and gene expression. Investigating transposon dynamics provides valuable information about the intricate mechanisms that govern the organization and regulation of genetic material.

Forensic Applications

Transposons have been used in forensic genetics to analyze DNA samples. The unique patterns of transposon insertion in individual genomes can serve as genetic markers, aiding in the identification of individuals. This application has proven helpful in forensic investigations and paternity testing.

Enhancing Precision in Gene Editing

Using transposons with modern gene-editing technologies like CRISPR-Cas provides a versatile platform for precise gene editing. Transposons are engineered to carry specific DNA sequences that encode therapeutic genes. By integrating these transposons at exact genomic locations using CRISPR technology, scientists can target and modify genes with unprecedented accuracy, opening up new possibilities for therapeutic interventions in human health.

Studying Gene Function and Regulation

Transposons aid in functional genomics studies by enabling the controlled disruption or modification of specific genes. This approach is invaluable for understanding the roles of individual genes and their contributions to various biological processes. Researchers can uncover gene function and regulation mechanisms using transposons to manipulate gene expression.

Creating Disease Models

Transposons are instrumental in the generation of animal models for human diseases. By introducing transposons carrying disease-associated genes or mutations into the genomes of animals, scientists can mimic pathological conditions observed in humans. These models are crucial for studying disease mechanisms, testing potential therapeutic interventions, and advancing our understanding of complex genetic disorders.