Transposons: Definition and Types (With Diagram)

Transposons: Definition and Types!

Definition of Transposons:

Presence of transposable elements was first predicted by Barbara McClintock in maize (corn) in late 1940s. After several careful studies, she found that certain genetic elements were moving from one site to an entirely different site in the chromosome. She called this phenomenon of changing sites of genetic elements as transposition and those genetic elements were called by her as controlling elements.

These controlling elements were later on called as transposable elements by Alexander Brink. In late 1960s this phenomenon was also discovered in bacteria.

Consequently, the molecular biologists called them as Transposons. A transposon may be defined as: “a DNA sequence that is able to move or insert itself at a new location in the genome.” The phenomenon of movement of a transposon to a new site in the genome is referred to as transposition.

Transposons are found to encode a special protein named as transposase which catalyses the process of transposition. Transposons are particular to different groups of organisms. They constitute a fairly accountable fraction of genome of organisms like fungi, bacteria, plants, animals and humans. Transposons have had a major impact on changing or altering the genetic composition of organisms.

Transposons or transposable genetic elements are often referred to as ‘mobile genetic elements’ also. They can be categorized on different bases like their mode of transposition or on the basis of the organisms in which they are present.

Types of Transposons:

Different transposons may change their sites by following different transposition mechanisms.

On the basis of their transposition mechanism, transposons may be categorized into following types:

(i) Cut-and-Paste Transposons:

They transpose by excision (cutting) of the transposable sequence from one position in the genome and its insertion (pasting) to another position within the genome (Fig. 1).

The cut-and-paste transposition involves two transposase subunits. Each transposase submit binds to the specific sequences at the two ends of transposon. These subunits of transposase protein then come together and lead to the excision of transposon.

This excised ‘transposon-Transposase Complex’ then gets integrated to the target recipient site. In this manner, the transposon is cut from one site and then pasted on other site by a mechanism mediated by transposase protein (Fig. 2).

Examples of cut-and-paste type of transposons are IS-elements, P-elements in maize, hobo-elements in Drosophila etc.

(ii) Replicative Transposons:

They transpose by a mechanism which involves replication of transposable sequence and this copy of DNA, so formed, is inserted into the target site while the donor site remains unchanged (Fig. 3). Thus, in this type of transposition, there is a gain of one copy of transposon and both-the donor and the recipient DNA molecule are having one-one transposable sequence each, after transposition.

Tn3-elements found in bacteria are good examples of such type of transposons.

(iii) Retro Elements:

Their transposition is accomplished through a process which involves the synthesis of DNA by reverse transcription (i.e. RNA DNA) by using elements RNA as the template (Fig. 4). This type of transposition involves an RNA intermediate, the transposable DNA is transcribed to produce an RNA molecule.

This RNA is then used as a template for producing a complementary DNA by the activity of enzyme reverse transcriptase. This single stranded DNA copy so formed, is then made double stranded and then inserted into the target DNA site. The transposable elements which require reverse transcriptase tor their movement are called retro transposons.

The Retro elements may be viral or non-viral. Out of these two, the non-viral retro elements are important and may further be classified as:

(A) Retrovirus like elements:

They carry long terminal repeats (LTR). Examples are copia, gypsy elements in Drosophila.

Retroposons:

LTR are absent. Examples are LINEs and SINEs in humans.

Transposable Elements in Prokaryotes:

Although the presence of transposons was predicted in eukaryotes but first observation at molecular level was done in bacteria, which is a prokaryote.

Bacterial transposable elements are of the following types:

(a) Insertion Sequences or IS Elements:

They are the transposable sequences which can insert at different sites in the bacterial chromosomes.

IS-elements contain ITRs (Inverted Terminal Repeats), these were first observed in E.coli. IS elements are relatively short usually not exceeding 2500 bp. The ITRs present at the ends of IS-elements are an important feature which enables their mobility. The ITRs present in the IS-elements of E.coli usually range between 18-40 bp.

The term ‘Inverted Terminal Repeat’ (ITR) implies that the sequence at 5 end of one strand is identical to the sequence at 5′ end of the other strand but they run in inverse opposite direction (Fig. 5). In Exoli chromosome, a number of copies of several IS-elements like IS1, IS2, IS3, IS4 and IS5 are present.

(b) Prokaryotic Transposon Element:

These are also called composite transposons and are shown by the symbol Tn. It is made up of two IS elements, one present at each end of a DNA sequence which contains genes whose functions are not related to the transposition process. These transposons have been found to have inverted repeats at the ends. The length of these inverted repeats ranges from a few nucleotides to about 1500 bp.

It can be said that these are the large transposons which are formed by capturing of an immobile DNA sequence within two insertion sequences thus enabling it to move. Examples of such transposons include the members of Tn series like Tn1, Tn5, Tn9, Tn10, etc.

Transposable Elements in Eukaryotes:

(a) Transposons in Maize:

Different types of transposons present in maize are described below:

Ac-Ds system:

This system of transposable elements in maize was analysed and given by Barbara Mc. Clintock. Here Ac stands for Activator and Ds for Dissociation. Barbara found that Ds and Ac genes were sometimes mobile and moved to different chromosomal locations thus resulting in different kernel phenotypes.

Ds element is activated by Ac and on activation it serves as the site provider for breakage in chromosome. Ac can move autonomously while Ds can move only in the presence of Ac (Fig. 6). The transposition involving this Ac-Ds system produces altered kernel phenotypes.

Other transposable elements of maize are:

i. spm (suppressor mutator) system,

ii. dt (dotted) system,

iii. Mu (Mutator) system, etc.

(b) Transposons in Drosophila:

A number of transposable elements are found in Drosophila which are of different types and account for a quite high fraction of Drosophila genome.

Some of these transposons are given below:

P-elements:

These were discovered during the study of ‘hybrid-dysgenesis’ which is a sterility causing condition. They are 2.9 kb long and contain 31 bp long inverted terminal repeats High rate of P-element transposition causes hybrid dysgenesis. P-elements encode transposase enzyme which helps in their transposition. These are also useful as vectors for introducing foreign genes into Drosophila.

Copia-elements:

Their transposition causes mutations for eye-colour in Drosophila. They are of size approximately 5-8 kb with direct terminal repeat (DTR) of about 276 bp at each end. Within each of this direct repeats is present short inverted repeat (IR) of about 17 bp length. About 10-80 copia- elements are present in cell-genome (Fig. 7).

FB Elements:

These are the fold back elements present in Drosophila genome. These have ability to fold back to form a stem and loop structure due to the presence of long inverted terminal repeats. Their transposition results into a changed expression by causing mutation by insertion or by affecting the normal gene expression.

Other important types of transposable elements found in Drosophila are:

i. I elements,

ii. Mariner elements,

iii. Gypsy elements,

iv. Hobo elements, etc.

(c) Transposons in Humans:

Transposons in humans are in the form of repetitive DNA which consists of sequences that are interspersed within the entire human genome. These sequences are transposable and can move to different locations within the genome.

These are of following two types:

(1) SINEs (Short Interspersed Elements):

They are ~ 300 bp long and may be present about 5 lakh times in human genome. Alu sequences are the best characterized SINEs in humans.

These are termed as ‘Alu’ elements because they contain specific nucleotide sequences which are cleaved by the restriction enzyme named Alul. Alu elements contain Direct Terminal Repeats (DTR) of 7-20 bp length. These DTRs help them in the insertion process during transposition.

(2) LINEs (Long Interspersed Elements):

They are ~ 6400 bp long and are present about 1 lakh times in the human genome. Most prominent example is LI sequence. These transposable elements are some of the most abundant and common families of moderately repeated sequences in human DNA.

Significance of Transposable Elements:

1. Transposons may change the structural and functional characteristics of genome by changing their position in the genome.

2. Transposable elements cause mutation by insertion, deletion, etc.

3. Transposons make positive contribution in evolution as they have tremendous impact on the alteration of genetic organisation of organisms.

4. They are useful as cloning vectors also, in gene cloning. For example, P-elements are frequently used as vector for introducing transgenes into Drosophila.

5. Transposons may also be used as genetic markers while mapping the genomes.

6. Transposon-mediated gene tagging is done for searching and isolation of a particular gene.

Techniques of Gene Mapping:

A gene map is the detailed schematic representation of the positions of genes or sequences of interest in a chromosome. It may also provide details of relative distances between these genes. A gene map may also be referred to as a genome map or molecular map. It attempts to describe the structural and functional organization of genome of an organism.

Gene mapping has attained much attention during the last two decades not only in case of plants but also in animals. These maps provide immense benefit in improvement of commercially important plants or animals. Gene mapping of human genome is the prime goal of Human Genome Project (HGP). It will surely help to solve the genetic disorders in humans.

Gene maps also aid for assessment of genetic diversity and taxonomic classification of organisms by comparison. Most common technique of development of a gene map is by using the molecular markers. For functioning of molecular markers, the basis is provided by polymorphism.

Polymorphism:

Genome of the organism contains a number of polymorphisms (literal meaning being many forms). These polymorphisms are actually the positions in genome where the nucleotide sequence is not the same in every member of a population. These variable sites may be used as the DNA markers (or molecular marker) for genome mapping.

The detection of polymorphism can be done by either of the following ways:

(i) Biochemically:

This detection can be done on account of the differences in structure and composition of the proteins encoded by the polymorphic sites of genomes.

(ii) Morphologically:

It can be done by visual examination, without involving any specialized biochemical or molecular technique.

(iii) Molecular Detection:

This is done at the DNA level i.e., the DNA sequences having the differences can be detected. The polymorphism occurring at the molecular level i.e., in DNA sequence, can be used efficiently as the molecular/DNA marker and hence is important for preparing gene maps.

Molecular Markers:

These may also be termed as the DNA markers. They represent primary method of development of gene maps. Molecular markers, actually display the variability at DNA level. Molecular markers may be described as the DNA sequences which reveal variations at DNA level which can be easily detected and monitored in the subsequent generations.

On the basis of principles and methods employed for the development and use of the molecular markers, they may be classified as the hybridization-based markers and PCR-based markers.

A good molecular marker must be polymorphic and should be evenly distributed within the genome. A molecular marker should be easy and quick to be detected. As stated earlier also, major use of molecular marker is in the development of gene maps.

Different important DNA markers which are utilized for gene mapping are given below:

(i) RFLP (Restriction Fragment Length Polymorphism):

It is a hybridization based molecular marker. The principle of RFLP marker is the restriction digestion of pure DNA sample by restriction endonuclease enzymes. It represents the polymorphism as single base changes.

(ii) RAPD (Random Amplified Polymorphic DNA):

It is a PCR (Polymerase Chain Reaction) based technique. Basic principle involved in the functioning of RAPD is the DNA amplification using PCR. RAPD is a quicker technique.

(iii) AFLP (Amplified Fragment Length Polymorphism):

It is a combination of features of both RAPD and RFLP. The basic principle of working of AFLP is the PCR amplification of fragments of genome produced by applying restriction enzymes. AFLP is a faster and less laborious technique.

(iv) VNTR (Variable Number of Tandem Repeats):

These are also called as mini-satellites. Polymorphism in VNTRs is associated with the number of repeat units at a given position in a chromosome in different individuals,

(v) STRs (Short Tandem Repeats):

These are also termed as microsatellites. They show polymorphism due to the variation in the number of repeats. These are ideal markers to develop a high resolution molecular map. The molecular markers are employed for construction of genomic maps in plants as well as in animals and microorganisms also. However many other techniques as in-situ hybridisation etc., are also utilized for mapping.

Types of Gene Maps:

On the basis of strategies followed for the preparation of gene maps, there are following types of gene maps:

Genetic Map:

It is obtained by genetic studies using mendelian principles like crossing over, linkage, etc. Such maps are not considered to be very accurate. They only provide information about the position of concerned genes (i.e., on which chromosome they are present) and also give an idea, roughly, about the relative distance between the concerned genes.

Linkage Map is the term which is given especially for those genetic maps in which the relative distances between genetic markers or concerned genes is measured in terms of recombination frequencies between them. The unit of distance in a linkage map is centiMorgan (cM) or map-unit. One cM is defined as that distance which allows 1% recombination between the genes.

During the preparation of linkage maps, recombination frequencies between the genes are studied. The data based on such recombination frequency is processed to measure the distance between genes.

In the recent years, a number of advanced computer software have been introduced which help in fast and accurate processing of data on recombination frequency and thus construct the linkage maps of genomes. Some such computer software’s are—LINKAGE, MAPMAKER, CRI-MAP, etc.

One main drawback of genetic map is that sometimes, the distances between genes in a genetic map do not correspond to the actual distance between them on the chromosome. Many times there may remain gaps on the genetic maps.

This happens basically because the recombination frequencies (which are used as the measure of distance between genes) obviously get affected by environmental conditions and nature and position of mutants used for study.

Recently different techniques have been devised for genetic mapping. These involve the use of DNA sequences which are not genes but display variations in a population. Such sequences are called as the molecular markers.

Most important molecular markers used for genetic mapping are:

(a) RFLP: These polymorphic markers have been used for genetic mapping in a number of crop plants along with mice, human beings, Drosophila, etc. also. RFLP genetic maps have been so far developed successfully in wheat, rice, rye, barley, tomato, potato, etc.

(b) RAPD: Genetic maps are successfully prepared by using PCR-based technique named RAPD. These markers provide more convenient and quick method of genetic mapping than RFLPs. However, less information is served by RAPD genetic map.

(c) Genetic maps have been successfully constructed by using the mini-satellites (also called variable number of tandem repeats, VNTR) and microsatellites (i.e., short tandom repeats, STR) also.

(d) For production of genetic maps, the AFLPs, may also be used. Technique of AFLP has the features of both RFLP and RAPD and it is more advantageous and faster technique for constructing genetic maps.

Cytogenetic Map:

They may also be called as cytological maps or chromosome maps. When genes are assigned to specific chromosome arms and their distances from the centromere are also shown, then the map is known as a cytogenetic map and the technique is called the cytogenetic mapping.

A cytogenetic map makes possible to locate concerned genes, not only on a specific chromosome but also on the specific regions of this chromosome. Cytogenetic mapping is generally better used in case of those organisms which have larger microscopically observable chromosomes.

There are different techniques which may be employed for construction of cytogenetic maps. Some of them are given below:

(a) FISH (Fluorescence In-situ Hybridization):

The technique of in-situ hybridization involves the detection and location of desired sequence of DNA directly inside the cell. When in situ hybridization involves labelling with fluorescent molecules, then, it is called as fluorescent in-situ hybridization (FISH). By using FISH, the genes can be located on the chromosome within the cell in correct order and thus provide a cytogenetic map.

(b) RFLP:

The cytogenetic maps can also be prepared by using molecular markers like RFLP. In this approach, RFLP locus is located on the specific regions of particular chromosome.

(c) Use of somatic cell Hybrids:

The somatic cell hybrids having chromosomes of known identity have been used successfully for preparation of cytogenetic maps preferably in humans. For example, the man-mouse somatic cell hybrids have resulted in mapping of genes on human chromosome to more specific regions.

Physical Map:

These maps represent the correct order of genes on chromosome and they are based on the physical distances between genes or between a gene and the centromere.

In a physical map, distance is never given in centiMorgan, instead, it is given in terms of number of base pairs between the genes. Physical maps are considered as more accurate than genetic maps. They ultimately result in obtaining the entire sequencing of whole genome and that too with the knowledge of physical distances between genes.

Restriction Mapping:

It is a type of physical mapping because in it, distances are given in terms of base pairs. It is a successful technique for mapping the prokaryotic genome. Preparation of restriction maps involves the use of restriction endonuclease enzymes which cleave the DNA at specific sites. To prepare a restriction map, one or more restriction endonuclease enzymes are used to cleave DNA at different sites (Fig. 9).

As a result, DNA fragments of varying lengths are obtained. The sample having such DNA fragments of different size are then subjected to a technique called gel electrophoresis for separation. Consequently, a series of bands is obtained on the gel where position of bands depends on its size.

This gel with different bands is then calibrated with the help of DNA fragments of known lengths, to obtain the sites of cleavage. These sites of cleavage are then identified and mapped together to produce a complete restriction map.

Different techniques for preparing physical maps are:

(a) ISH (in situ Hybridisation) Technique:

It can be used for physical mapping, this has been successfully utilized for obtaining physical maps in rye, wheat and barley. An advanced technique FISH (Fluorescence in-situ hybridisation) can also be employed for physical mapping.

(b) Physical mapping can also be performed by using chromosomal aberrations like duplication, deletion and translocation.

(c) Physical maps can also be developed by using a mapping reagent. The mapping reagent is actually a collection of DNA fragments spanning a complete chromosome or the entire genome.

(d) The technique of chromosome walking is also helpful for developing physical maps. However, this technique is not of much use in case of higher eukaryotes due to the presence of highly repeated sequences.

(e) Using YAC (Yeast Artificial Chromosome) has also proved to be of great use for physical mapping of Drosophila and human beings.

Significance of Gene Maps:

1. Gene maps play an important role in the researches related to plant as well as animal biotechnology.

2. Genetic and physical maps are important for the Human Genome Project (HGP).

3. Gene maps aid the genetists to study phylogenetic relationships and evolutionary patterns of organisms.

4. These are helpful for characterisation of genetic resources and estimation of genetic diversity.

5. Gene maps have enormous utility in crop improvement programmes.

6. They may provide an extra aid for solving the problems related to a number of harmful genetic disorders in human beings.

Chromosome Walking:

Chromosome walking is an important aspect of cytogenetics:

It is a method for analysing long stretches of DNA. By using this technique, large regions of chromosome of about 1000 kb length can be easily characterized. In contrast, the conventional cloning methods or PFGE (Pulse Field Gel Electrophoresis) etc., can characterize only about 100 kb long segments of chromosome.

Chromosome walking is done on the DNA fragment containing the gene of interest:

The process starts with a known gene present near the gene of interest on the DNA fragment. For chromosome walking, clones of interest are derived from the genomic library.

During chromosome walking, the end-piece of a cloned DNA fragment is sub cloned and is used as a probe to recover another overlapping clone from the genomic library. Restriction mapping of such overlapping sequences may be used to construct the original sequence of DNA stretch under study.

Probes:

These are the labelled DNA or RNA molecules which are used to identify target genes or molecules.

Sub cloning:

The cloning of a clone is called sub cloning.

Steps in Chromosome Walking:

i. First clone of interest is selected from the genomic library after identifying by a probe.

ii. A small fragment from one end of this clone is sub cloned.

iii. This sub cloned fragment is now used as a probe and is hybridized with other clone from the genomic library.

iv. Now, the second clone hybridized with the sub clone of the first clone is identified due to the presence of overlapping region.

v. End piece of the second clone is then sub cloned and used for hybridization with another clone from library.

vi. Again, the third clone hybridized with the sub clone of second clone is also identified due to the presence of overlapping region.

vii. This process of sub cloning and probing the genomic library is repeated to recover overlapping clones until the gene of interest is reached.

viii. Restriction maps of the overlapping clones may be constructed so as to get the entire sequence of original DNA stretch (Fig. 10).

Applications of Chromosome Walking:

1. This technique is used successfully for the characterization of large regions of chromosomes.

2. Chromosome walking is applied for the identification of specific genes.

3. It can be used for the isolation of specific genes also.

4. Chromosome walking is a technique which is frequently used for the preparation of genome maps specially the physical maps.

5. It is of great importance for the identification of genetic disorders in human like cystic fibrosis, muscular dystrophy, etc.

Limitations of Chromosome Walking:

1. This technique is time-consuming.

2. It is a laborious technique.

3. It requires the construction of a genomic library.

4. It is usually difficult to perform chromosome walking in complex eukaryotic genomes like those of human beings because they carry highly repetitive sequences.