Transposon mutagenesis

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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. [1] 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. [2] Disadvantages include the low frequency of transposition in living systems, and the inaccuracy of most transposition systems.

Contents

History

Transposon mutagenesis was first studied by Barbara McClintock in the mid-20th century during her Nobel Prize-winning work with corn. McClintock received her BSc in 1923 from Cornell’s College of Agriculture. By 1927 she had her PhD in botany, and she immediately began working on the topic of maize chromosomes. [3] In the early 1940s, McClintock was studying the progeny of self-pollinated maize plants which resulted from crosses having a broken chromosome 9. These plants were missing their telomeres. This research prompted the first discovery of a transposable element, [4] and from there transposon mutagenesis have been exploited as a biological tool.

Dynamics

In the case of bacteria, transposition mutagenesis is usually accomplished by way of a plasmid from which a transposon is extracted and inserted into the host chromosome. This usually requires a set of enzymes including transposase to be translated. The transposase can be expressed either on a separate plasmid, or on the plasmid containing the gene to be integrated. Alternatively, an injection of transposase mRNA into the host cell can induce translation and expression. [5] Early transposon mutagenesis experiments relied on bacteriophages and conjugative bacterial plasmids for the insertion of sequences. These were very non-specific, and made it difficult to incorporate specific genes. A newer technique called shuttle mutagenesis uses specific cloned genes from the host species to incorporate genetic elements. [2] Another effective approach is to deliver transposons through viral capsids. This facilitates integration into the chromosome and long-term transgene expression. [5]

Tn5 transposon system

Structure of the Tn5 transposon, with insertion sequence flanking a cassette of genes, in this case drug resistance genes. Tn5 gene diagram.png
Structure of the Tn5 transposon, with insertion sequence flanking a cassette of genes, in this case drug resistance genes.

The Tn5 transposon system is a model system for the study of transposition and for the application of transposon mutagenesis. Tn5 is a bacterial composite transposon in which genes (the original system containing antibiotic resistance genes) are flanked by two nearly identical insertion sequences, named IS50R and IS50L corresponding to the right and left sides of the transposon respectively. [6] The IS50R sequence codes for two proteins, Tnp and Inh. These two proteins are identical in sequence, save for the fact that Inh is lacking the 55 N-terminal amino acids. Tnp codes for a transposase for the entire system, and Inh encodes an inhibitor of transposase. The DNA-binding domain of Tnp resides in the 55 N-terminal amino acids, and so these residues are essential for function. [6] The IS50R and IS50L sequences are both flanked by 19-base pair elements on the inside and outside ends of the transposon, labelled IE and OE respectively. Mutation of these regions results in an inability of transposase genes to bind to the sequences. The binding interactions between transposase and these sequences is very complicated, and is affected by DNA methylation and other epigenetic marks. [6] In addition, other proteins seem to be able to bind with and affect the transposition of the IS50 elements, such as DnaA.

The most likely pathway of Tn 5 transposition is the common pathway for all transposon systems. It begins with Tnp binding the OE and IE sequences of each IS50 sequence. The two ends are brought together, and through oligomerization of DNA, the sequence is cut out of the chromosome. After introducing 9-base pair 5' ends in target DNA, the transposon and its incorporated genes are inserted into the target DNA, duplicating the regions on either end of the transposon. [6] Genes of interest can be genetically engineered into the transposon system between the IS50 sequences. By placing the transposon under the control of a host promoter, the genes will be expressed. Incorporated genes usually include, in addition to the gene of interest, a selectable marker to identify transformants, a eukaryotic promoter/terminator (if expressing in a eukaryote), and 3' UTR sequences to separate genes in a polycistronic stretch of sequence.

Sleeping Beauty transposon system

The Sleeping Beauty transposon system (SBTS) is the first successful non-viral vector for incorporation of a gene cassette into a vertebrate genome. Up until the development of this system, the major problems with non-viral gene therapy have been the intracellular breakdown of the transgene due to it being recognized as Prokaryotes and the inefficient delivery of the transgene into organ systems. The SBTS revolutionized these issues by combining the advantages of viruses and naked DNA. It consists of a transposon containing the cassette of genes to be expressed, as well as its own transposase enzyme. By transposing the cassette directly into the genome of the organism from the plasmid, sustained expression of the transgene can be attained. [2] This can be further refined by enhancing the transposon sequences and the transposase enzymes used. SB100X is a hyperactive mammalian transposase which is roughly 100x more efficient than the typical first-generation transposase. Incorporation of this enzyme into the cassette results in even more sustained transgene expression (over one year). Additionally, transgenesis frequencies can be as high as 45% when using pronuclear injection into mouse zygotes. [7]

Sleeping Beauty Transposon System. Transposase enzyme can be expressed in cis or in trans to the gene cassette. SBTS.png
Sleeping Beauty Transposon System. Transposase enzyme can be expressed in cis or in trans to the gene cassette.

The mechanism of the SBTS is similar to the Tn5 transposon system, however the enzyme and gene sequences are eukaryotic in nature as opposed to prokaryotic. The system's tranposase can act in trans as well as in cis, allowing a diverse collection of transposon structures. The transposon itself is flanked by inverted repeat sequences, which are each repeated twice in a direct fashion, designated IR/DR sequences. The internal region consists of the gene or sequence to be transposed, and could also contain the transposase gene. Alternatively, the transposase can be encoded on a separate plasmid or injected in its protein form. Yet another approach is to incorporate both the transposon and the transposase genes into a viral vector, which can target a cell or tissue of choice. The transposase protein is extremely specific in the sequences that it binds, and is able to discern its IR/DR sequences from a similar sequence by three base pairs. Once the enzyme is bound to both ends of the transposon, the IR/DR sequences are brought together and held by the transposase in a Synaptic Complex Formation (SCF). The formation of the SCF is a checkpoint ensuring proper cleavage. HMGB1 is a non-histone protein from the host which is associated with eukaryotic chromatin. It enhances the preferential binding of the transposase to the IR/DR sequences and is likely essential for SCF complex formation/stability. Transposase cleaves the DNA at the target sites, generating 3' overhangs. The enzyme then targets TA dinucleotides in the host genome as target sites for integration. The same enzymatic catalytic site which cleaved the DNA is responsible for integrating the DNA into the genome, duplicating the region of the genome in the process. Although transposase is specific for TA dinucleotides, the high frequency of these pairs in the genome indicates that the transposon undergoes fairly random integration. [8]

Practical applications

As a result of the capacity of transposon mutagenesis to incorporate genes into most areas of target chromosomes, there are a number of functions associated with the process.

Specific examples

Mycobacterium tuberculosis virulence gene cluster identification

In 1999, the virulence genes associated with Mycobacterium tuberculosis were identified through transposon mutagenesis-mediated gene knockout. A plasmid named pCG113 containing kanamycin resistance genes and the IS1096 insertion sequence was engineered to contain variable 80-base pair tags. The plasmids were then transformed into M. tuberculosis cells by electroporation. Colonies were plated on kanamycin to select for resistant cells. Colonies that underwent random transposition events were identified by Bam HI digestion and Southern blotting using an internal IS1096 DNA probe. Colonies were screened for attenuated multiplication to identify colonies with mutations in candidate virulence genes. Mutations leading to an attenuated phenotype were mapped by amplification of adjacent regions to the IS1096 sequences and compared with the published M. tuberculosis genome. In this instance transposon mutagenesis identified 13 pathogenic loci in the M. tuberculosis genome which were not previously associated with disease. [10] This is essential information in understanding the infectious cycle of the bacterium.

PiggyBac (PB) transposon mutagenesis for cancer gene discovery

The PiggyBac (PB) transposon from the cabbage looper moth Trichoplusiani was engineered to be highly active in mammalian cells, and is capable of genome-wide mutagenesis. Transposons contained both PB and Sleeping Beauty inverted repeats, in order to be recognized by both transposases and increase the frequency of transposition. In addition, the transposon contained promoter and enhancer elements, a splice donor and acceptors to allow gain- or loss-of-function mutations depending on the transposon's orientation, and bidirectional polyadenylation signals. The transposons were transformed into mouse cells in vitro and mutants containing tumours were analyzed. The mechanism of the mutation leading to tumour formation determined if the gene was classified as an oncogene or a tumour-suppressor gene. Oncogenes tended to be characterized by insertions in regions leading to overexpression of a gene, whereas tumour-suppressor genes were classified as such based on loss-of-function mutations. Since the mouse is a model organism for the study of human physiology and disease, this research will help lead to an increased understanding of cancer-causing genes and potential therapeutic targets. [9]

See also

Related Research Articles

<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.

Site-directed mutagenesis is a molecular biology method that is used to make specific and intentional mutating changes to the DNA sequence of a gene and any gene products. Also called site-specific mutagenesis or oligonucleotide-directed mutagenesis, it is used for investigating the structure and biological activity of DNA, RNA, and protein molecules, and for protein engineering.

<span class="mw-page-title-main">Library (biology)</span>

In molecular biology, a library is a collection of DNA fragments that is stored and propagated in a population of micro-organisms through the process of molecular cloning. There are different types of DNA libraries, including cDNA libraries, genomic libraries and randomized mutant libraries. DNA library technology is a mainstay of current molecular biology, genetic engineering, and protein engineering, and the applications of these libraries depend on the source of the original DNA fragments. There are differences in the cloning vectors and techniques used in library preparation, but in general each DNA fragment is uniquely inserted into a cloning vector and the pool of recombinant DNA molecules is then transferred into a population of bacteria or yeast such that each organism contains on average one construct. As the population of organisms is grown in culture, the DNA molecules contained within them are copied and propagated.

<span class="mw-page-title-main">Transfer DNA</span>

The transfer DNA is the transferred DNA of the tumor-inducing (Ti) plasmid of some species of bacteria such as Agrobacterium tumefaciens and Agrobacterium rhizogenes . The T-DNA is transferred from bacterium into the host plant's nuclear DNA genome. The capability of this specialized tumor-inducing (Ti) plasmid is attributed to two essential regions required for DNA transfer to the host cell. The T-DNA is bordered by 25-base-pair repeats on each end. Transfer is initiated at the right border and terminated at the left border and requires the vir genes of the Ti plasmid.

A transposase is any of a class of enzymes capable of binding to the end of a transposon and catalysing its movement to another part of a genome, typically by a cut-and-paste mechanism or a replicative mechanism, in a process known as transposition. 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 some segments of chromosomes changed their position, jumping between different loci or 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 generating adaptability to changing living conditions.

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.

<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.

The Tn3 transposon is a 4957 base pair mobile genetic element, found in prokaryotes. It encodes three proteins:

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.

<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.

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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.

<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.

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.

<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.

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.

References

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  9. 1 2 Rad, Roland; Rad, Lena; Wang, Wei; Cadinanos, Juan; Vassiliou, George; Rice, Stephen; Campos, Lia S.; Yusa, Kosuke; Banerjee, Ruby (2010-11-19). "PiggyBac Transposon Mutagenesis: A Tool for Cancer Gene Discovery in Mice". Science. 330 (6007): 1104–1107. Bibcode:2010Sci...330.1104R. doi:10.1126/science.1193004. ISSN   0036-8075. PMC   3719098 . PMID   20947725.
  10. ↑ Camacho, Luis Reinaldo; Ensergueix, Danielle; Perez, Esther; Gicquel, Brigitte; Guilhot, Christophe (1999-10-01). "Identification of a virulence gene cluster of Mycobacterium tuberculosis by signature-tagged transposon mutagenesis". Molecular Microbiology. 34 (2): 257–267. doi:10.1046/j.1365-2958.1999.01593.x. ISSN   1365-2958. PMID   10564470. S2CID   21119819.
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