In this article we will discuss about the transposable elements in the eukaryotic organisms.
Transposons have been discovered in eukaryotic organisms also, e.g., controlling elements in maize, Tam1 elements in Antirrhinum majus, Ty elements in Yeast and FB elements in Drosophila. These elements can be divided into two main classes.
1. This class includes the transposable elements that are similar to those found in bacteria. These elements contain inverted repeats at their ends and generate short direct repeats of the target DNA at the sites of their insertion. These elements are always located in the host genome and cannot survive outside the genome. Controlling elements in maize and P elements in Drosophila belong to this class of transposable elements.
2. Retroposons (Retro transposons). Retroposons are DNA elements formed by the reverse transcription of retroviruses. This class of retroviruses and other sequences are transposed via RNA. Transposition of retroposons occurs through RNA intermediates.
Controlling Elements in Maize:
In 1940’s Barbara McClintock discovered changes in maize genome during somatic cell division. The changes were genetically controlled aberrations, such as, deficiencies, duplications, inversions, translocations and ring chromosomes. These changes were found to be caused by a genetic system named Dissociation-Activator (Ds-Ac) system.
McClintock termed these genetic elements Ds and Ac as controlling elements in 1956. Since then, several systems of controlling elements have been discovered in maize. These elements are classified into two groups: autonomous and non-autonomous.
1. Autonomous elements:
The controlling elements which have the ability of their own excision and transposition are called autonomous elements, e.g., Activator (Ac), Suppressor mutator (Spm) and Enhancer (En).
2. Non-autonomous elements:
These elements do not have the ability of transposition. Non-autonomous elements have originated from autonomous elements through the loss of transacting functions which are required for transposition. A single type of autonomous element and different non-autonomous elements derived from it form a family.
Deletions of different lengths and different regions from an autonomous element give rise to different types of non-autonomous elements. Such elements change their position in response to an autonomous element of the same family present in the genome.
Non-autonomous element is activated in trans by its related autonomous element. Examples of non-autonomous elements are Dissociation (Ds), defective suppressor mutator (dSpm), and Inhibitor (I).
Dissociation-Activator (DS-Ac) System:
The main features of the Ds-Ac system in maize are as follows:
(i) An Ac element can exist in a number of states similar to other genes, and it controls the activity and time of action of the Ds element.
(ii) Ac and Ds, both exhibit inter-chromosomal as well as intra-chromosomal movements (transposition). The movement occurs through excision of these elements from one site and their insertion at a new site.
(iii) Ds element is unstable in the presence of the Ac element in the same nucleus. When both the elements are present, loss (deletion) of a part of the chromosome 9 occurs if the chromosome 9 carries the Ds element. The deletion is caused by breakage of the chromosome at the site of Ds.
(iv) The genes lying adjacent to the Ds become inactivated.
(v) The number of Ac elements present in a genome has a negative relationship with the time of Ds action during the development. Therefore, the presence of Ac in a greater number delays the transposition of Ds during the development. This can be well explained in the maize endosperm which is triploid.
In maize endosperm, the number of Ac element may vary from 0 to 6. The dominant allel I at the C locus on chromosome 9 inhibits colour formation in the aleurones of kernels so that the kernels having the I allele are colourless. In the presence of recessive allele i, colour develops normally in the aleurone. Therefore, an ii secondary nucleus fertilized by a pollen carrying i allele will produce colouredaleurone iii.
But an ii secondary nucleus fertilized by a pollen carrying the dominant allele I will produce colourless endosperm Iii.
If both, Ac and Ds are present in the above I pollen, and Ds occupies a place within or near the C locus, coloured spots would be observed in many kernels. The coloured spots develop due to the transposition of the Ds element from the allele I during the stages of seed development which permits the c locus to produce aleurone colour.
An increase in the number of Ac elements delays the dissociation of Ds. Thus variegation pattern in the kernel will differ according to the number of Ac. (Table 5.3)
Organization of Ac and Ds elements:
Activator (Ac):
Activator (Ac) element is 4563 bp long and is autonomous in action. It has 11 bp inverted repeats at its both ends (Fig. 5.11). The target site for Ac insertion is 8 bp long; this target sequence is duplicated during the insertion as direct repeats. The Ac element has 5 exons (Fig. 5.11); transcription produces and mRNA of 35 00 bases which has a coding sequence for 807 codons. This element has two open reading frames.
Dissociation (Ds) element:
These elements are non-autonomous and are produced through interstitial deletions in the Ac element (Fig. 5.11). Based on the length and the region of deletion, Ds elements are grouped into several types as, Ds1, Ds2, Ds6, Ds9 Ds 2dl, and Ds2d2 etc.
All the Ds elements contain the 11 bp inverted repeats at their ends. The Ds1 element represents an extreme case in that it has a large interstitial deletion so that only the terminal 11bp inverted repeats are retained.
Ds6 element possesses 1000 bp from each end of the Ac, the rest portion being deleted. Ds9 on the other hand, represents a very short deletion of about 194 bp. Further changes may also occur in the non-autonomous elements leaving them incapable of transposition, i.e., they become permanently stabilized.
Autonomous elements may also be subject to changes. During the different developmental periods of an individual these elements may undergo cycles of active and inactive phases; the phase changes are brought about by methylation of their DNA. A methylation in the target sequence 
of an element leads to a reversible inactivation of the element.
Effects of transposition of Ds:
Transposition of Ds causes breakage in the chromosome at the site from which the Ds element moves out. The mechanism of transposition is non-replicative. Following breakage, the acentric chromosome fragment is lost.
If the chromosome carrying Ds has dominant alleles, e.g. A, B, C and its homologue carries the recessive alleles a, b, c, the transposition of Ds will lead to breakage and loss of the fragment carrying the dominant alleles (Fig. 5 12.). In the progeny cells as a result only the recessive alleles a, b, c will be expressed.
Alternatively, the broken ends of the two sister chromatids may join together as they are produced through chromosome replication. The acentric fragment, as a result, will form a U- shaped structure which would be lost. The centric fragment, on the other hand, will form a dicentric chromatid bridge during anaphase.
As a result of the tension created due to the centromere movement, the chromatid bridge will break at some point between them producing two dissimilar chromatids. In the next cycle, the broken chromatid ends will again fuse during chromosome replication.
This will, as earlier lead to the formation of dicentric chromatid bridges in both the daughter cells. Thus a chromatid-fusion-bridge-breakage cycle is generated (Fig. 5.13). In such a condition cells contain duplication for one or more genes along with deficiency for some other genes.
Duplication and deletion:
When the transposons are located on both the homologues but at different positions, pairing and recombination between them leads to the production of one deficient chromosome and one chromosome with duplication (Fig. 5.14).
Translocations caused by transposons:
Transposons located on non-homologous chromosomes can pair, and crossing over between them produces reciprocal translocations (Fig. 5.15).
Suppressor-Mutator (Spm) Elements:
Spm is nearly similar to the En (enhancer). It is a larger transposon than Ac and contains inverted repeats of 13bp at its ends. Its promoter is situated at the left and is responsible for the transcription of 8300bp of DNA.
Spm is composed of two genes, tnpA and tnpB. The tnpA is a split gene containing 11 exons, the first intron being very long (Fig. 5.11). After splicing, a 2500 base mRNA is obtained which is translated into a protein of 621 amino acids. The first intron of the tnpA gene possesses two additional reading frames ORF l and ORF 2; both the reading frames are jointly called the tnpB gene. The proteins coded by tnpA and tnpB are required for several functions related to transposition. A deletion in the ORF 1 and ORF 2 regions produce defective Spm elements (dSpm).
Transposition of the Spm into a gene completely inhibits the expression of the gene. But when dSpm insertion occurs within a gene the expression of the gene is reduced; such a gene is called dSpm- suppressive gene. Insertion of dSpm in the vicinity of a gene does not inhibit or reduce its expression but, on the other hand, enhances its expression. Therefore, such a gene is called “dSpm- dependent gene”.
P Elements in Drosophila Melanogaster:
In Drosophila, certain strains when mated together produce hybrid dysgenesis (mutations, chromosome aberrations, distorted segregation at meiosis and sterility). In such crosses, the F, flies have normal somatic tissues but, their gonads do not develop.
A transposable genetic element known as “P element” or “P factor” has been found to be responsible for this condition. On the basis of the presence or the absence of the P factors, the flies are divided into two types.
I. P-type (Paternal Contributing):
Strains containing P elements in their chromosomes are designated as P-type. The number of P elements varies from 30 to 40 in the genome.
P elements occupy several different positions on the chromosome. These factors are present on chromosomes as inactive component in the P-strain.
2. M-Type (Maternal Contributing):
M-strains do not contain P-elements in their chromosomes. Crosses involving P-Type male flies and M-type female flies produce F, flies showing hybrids dysgenesis. But when the female flies are P-type, the hybrids are normal irrespective of the male being P-type or M-type. (Fig. 5.16.).
Organisation of P elements:
There are different types of the P element. The full size elements are about 3000 bp long and contain inverted repeats of 31 bp at their ends. Transposition of the P elements generates direct repeats of 8 bp on both sides of the site where transposition has taken place.
Interstitial deletions in P element produce different P elements of smaller sizes. Some of the small P element possess the gene coding for transposase while others do not have the complete gene. The latter type of P elements (which have an incomplete transposase gene) are activated by the transposase enzyme produced by another normal P element. The full size P element has 4 open reading frames designated as ORFO, ORF1, ORF2 and ORF3. The processing of the primary RNA transcript occurs by separate mechanisms in somatic and germinal tissues.
1. Processing in somatic tissues:
In somatic tissues, splicing occurs producing the mRNA which contains only three open reading frames, namely, ORFO, ORF1 and ORF2; the intron 3 is not spliced. Somatic cells contain a protein that binds to the intron 3, thus inhibiting the removal of this intron. This mRNA produces a 66,000 Dalton protein which functions as a repressor for transposition so that somatic tissues are not affected by P element.
2. Processing in the germ line:
In the germ line, the intron 3 binding protein is absent and therefore, all the introns are removed during processing of the primary transcript. The four reading frames ORFO, ORF1, ORF2 and ORF3 are joined together to produce a large mRNA. This mRNA produces the 87,000 Dalton protein (the enzyme transposase) which leads to transposition.
Transposition occurs by the no-replicative mechanisms similar to that of Tn10 transposon. The enzyme transposase binds to 10 bp sequence adjacent to the 31 bp inverted repeats at ends of P. Transposition leaves a break at the original site of P and it produces gene mutation at the new (insertion) site of P. Both the events, therefore, generate adverse effects on the individual.
The P line contains P-cytotype, while the M line contains M-cytotype. When chromosomes bearing P factors come into the M-cytotype, transposition occurs leading to hybrid dysgenesis. However, when chromosomes carrying the P elements come into the P cytoplasm, there is no transposition. This can be explained as follows (Fig. 5.16).
A repressor protein called 66,000 Dalton protein is present in the egg cytoplasm of flies containing P elements. However, the repressor protein is absent from the egg cytoplasm of M- females. When a P-male is crossed to an M-female, in the F1 their P elements present in the paternal chromosome undergo transposition due to the absence of the repressor; this produces hybrid dysgenesis. But in crosses involving P-females, the transposition of P elements is prevented by the 66,000 Dalton repressor protein present in the egg cytoplasm; this yields normal fertile hybrids.
Retroposons (Retro-transposons):
Retroposons are transposable genetic elements which are mobilized through an RNA form. The DNA element is transcribed into RNA and then the RNA is copied by the enzyme reverse transcriptase into DNA which is inserted at a new site into the host genome. Retroposons include processed pseudo genes, small RNA pseudo genes (SnRNA) and Alu family in primates and rodents.
Some of the eukaryotic transposons are related to retroviral proviruses and mobilize through RNA intermediates. Retroposons differ from retroviruses in the sense that they do not pass through an independent infectious form. However, they do use the reverse transcription process to produce DNA. In order to understand retroposons, it is desirable to study the life cycle of a retrovirus.
Retroviruses:
They are infectious viruses containing single-stranded RNA (+ strand) which infect eukaryotic cells. Through reverse transcription, the viral RNA produces a complementary DNA (-) strand. The enzyme reverse transcriptase has exonuclease activity (RNAase H) by which it degrades the RNA strand from the RNA-DNA hybrid so produced.
The same enzyme also synthesizes, by its polymerase activity, the complementary DNA strand (+ strand) to (-) strand.
This double-stranded DNA moves to the nucleus of the cell where one or more copies of it become integrated into the host genome; the enzyme involved in the insertion is integrase. This viral DNA genome integrated into eukaryotic host chromosome is called a provirus or retroposon.
It remains as a endogenous provirus in the germ line. In other cells, the pro-viral DNA is transcribed to produce RNAs which function as (i) viral genome and (ii) mRNA to produce proteins that are structural components of the retrovirus.
In every viral particle, 2 copies of RNAs are packaged, making it a diploid virion. When two different retroviruses infect a single cell, the new viral particle may contain one chromosome from each of the two viruses; thus some virions may be heterozygous.
Organization of retroviral RNA and pro-viral DNA (Retroposon):
The retroviral RNA has direct repeats (R) varying from 10 to 80 nucleotides at its both ends (Fig. 5.17). An 80-100 base long unique region (U5) lies next to the R segment at the 5′-end. Similarly, at the 3′-end, left to the R segment, there occurs a unique region (U3) containing 170- 1260 nucleotides.
The coding region of the virus contains the genes gag (2000 bases), pol (2900 bases) and env (1800 bases). The reverse-transcribed DNA has a long terminal repeat (LTR) that s composed of the sequences “U3-R-U5” at both the ends (Fig. 5.17).
Integration:
Integration of DNA into the host chromosome occurs through the linear form of DNA. Integration events are similar to those of transposable elements. The enzyme integrase makes staggered cuts at the target site which may be 4-6 bp in length. Direct repeats of the target DNA is produced during the integration.
During this process, the U3 sequence loses 2 bp from left end and the U5 sequence loses 2 bp from the right end. A single cell receives up to 10 copies of a provirus.
Expression of viral genes:
The coding region of retroviruses consists of 3-4 genes, such as, gag pol, env sequences. Transcription of provirus produces the genomic RNA from which env mRNA is obtained by splicing (Fig. 5.17). Translation of the genomic RNA yields Gag, Gag-Pol and Env poly-proteins. Specific proteases cleave the poly-proteins into individual proteins through processing.
After about 8 hour of infection, the poly-proteins together with viral genomic RNA begin to assemble under the plasma membrane. They attract the envelope proteins already present in the membrane. Nucleocapsid is formed by protein cleavages.
A segment of the host cell membrane is pinched off (like budding) and viral particle is released. During the process of infection, the viral particle becomes fused with the plasma membrane as a result of which, the RNA is released into the cell.
Transducing viral particles:
A retrovirus may carry genes from its host cell. This occurs when a deletion in the provirus (retroposon) occurs, thus fusing the viral and host genes. As shown in the Figure 5.18, the deletion of a part of the pro-viral DNA causes the transcription of DNA containing both, pro-viral DNA and the host DNA to produce a “fused mRNA.”
After splicing of the host mRNA the fused mRNA becomes shorter. In some cases, the c-onc gene of the host may be transcribed and fused with the viral mRNA. But this RNA is defective and cannot produce new virus. If the cell contains some normal provirus, it acts as a helper.
Some of the viral particles produced in such a way will contain one fused (defective) and one normal viral genome. Recombination between the two RNAs will produce an RNA genome that contains LTR along with the viral genes and host genes.
The c-onc gene is called v-onc gene when present in the viral genome. The properties of the host cell are drastically changed when it is infected by such a viral particle; it becomes a cancerous cell.
Retroposon-like Elements in Eukaryotes:
Retroposons or retro-transposon like elements are found in different eukaryotic organisms, such as, yeast, Drosophila, and mammals including human. These elements are classified into the following two groups.
I. Viral super family:
The retroposons that code for reverse transcriptase and integrase, and possess the ability of transposition belong to this family. They have long terminal repeats. Many retroposons also contain introns. They generate direct repeats of 4-6 bases in the target DNA. Examples of such elements are: Ty elements in yeast, copia in Drosophila and LINES LI in mammals.
II. Non-viral super family:
The retroposons belonging to this family do not code for proteins that have role in transposition. They are believed to have originated from RNA sequences through the process of reverse transcription; they do not contain either terminal repeats or introns. They generate direct repeats of target DNA containing 7-21 bp. Examples are SINES B1/Alu family in mammals, processed pseudo genes, transcript of RNA polymerase II.
Ty Elements in Yeast (Saccharomyces Cerevisiae):
Ty (Transposon yeast) elements are of divergent types and made a family of dispersed repetitive sequences on yeast genome. These elements are 63000 bases long and are grouped into two main classes, Ty1 and Ty917. A typical yeast genome contains about 30 copies of Ty] type and about 6 copies of Ty917 type elements. They have direct repeats of 350 bp at each end; these repeats are called delta (8).
A Ty element has two open reading frames TyA and TyB. The TyA protein represents the TyA reading frame, while the TyB protein represents the joint TyA and TyB regions. The TyA region codes for DNA-binding proteins, while TyB codes for reverse transcriptase, protease and integrase (like retrovirus). Ty elements are mobilized through an RNA intermediate and transposition is controlled by its genes. The element behaves like a retrovirus which has lost the coding region for the viral envelope.
Copia Elements in Drosophila:
The term copia denotes a large number of closely related sequences in Drosophila. The number of these retroposons per genome varies from 20 to 60. The copia element is 5146 bp long with terminal direct repeats of 276 bp and terminal inverted repeats of 13 bp.
It generates direct repeats of 5 bp in the target DNA at the site of insertion. Copia elements are dispersed and take different locations in different strains of Drosophila.
Sometimes copia elements are found as circular molecules of 5000 bp and 4700 bp in length. They contain a single reading frame of 4227 bp which shows homologous relationship with gag and pol sequences of retrovirus but the env sequence is absent. Therefore, copia cannot produce a virus particle.
Retroposon-Like Elements in Mammals:
In mammals, a large part of the repetitive DNA consists of retroposons. These are two main groups of these elements, called LINES and SINES. The LINES sequences are also called LI. They are long interspersed sequences dispersed in the genome.
The average length of LINES is 6.5 kb. At their end, they contain sequences rich in adenine (A) nucleotides. LINES sequences are derived from the transcripts of RNA polymerase II. The number of copies of LINES ranges from 20,000 to 50,000 per mammalian genome.
Short interspersed sequences in mammalian genomes are called SINES. They are derived from the transcripts of RNA polymerase III. These elements do not posses a coding region, and are about 300 bp in length. Probably they originated from a transposition event like retroviruses and the RNA was copied into DNA by reverse transcriptase.
The SINES family includes the “Alu- family”. Alu family is a set of dispersed, related sequences (about 300 bp long) formed in human genome. Individual sequences have Alu cleavage sites at each end. There are about 300,000 (3 x 105) Alu sequences dispersed in the haploid human genome. The Alu sequences are flanked ‘ by short direct repeats indicating their resemblance to transposons.