Transposable Elements

TRANSPOSABLE ELEMENTS The traditional view of the DNA of a cell has been that of a fixed sequence of bases that is only subject to occasional changes by means of mutation or by recombination when cells exchange genetic material. There has therefore been a tendency to think of all cells in a pure bacterial culture as having an identical genetic make-up. It is now known that this is far from the truth. The genetic material is much more fluid than that and is subject to a range of larger-scale alterations in its structure, including insertions, transpositions, inversions and deletions. Some of these variations are readily reversible and generate high levels of genetic diversity The world of transposable elements is rich with diversity. Many different types of elements have been identified in assortment of organisms. They form prominent parts of the genome and clearly have roles in modifying the structure of chromosomes and in modulating the expressions of genes Transposons are segments of DNA that can move around to different positions in the genome of a single cell. In the process, they may cause mutations, increase (or decrease) the amount of DNA in the genome. These mobile segments of DNA are sometimes called "jumping genes". Transposons cannot exist independent of a replicon (because they don't have an ori of their own) Transposition Transposase must be able to: 1) Recognize the inverted repeats and 2) Recognize the target sequence (or a partial match of the target sequence) Target sequence is 5-9 bp on the recipient DNA molecule Conservative transposition Requires Transposase 1) Transposase cuts transposon out of donor DNA (blunt cut) 2) Transposase makes a staggered cut at the target sequence 3) Transposase ligates transposon into the target 4) Gaps are filled in with DNA pol I and ligase Results: Target DNA has been duplicated in process Usually destroys donor DNA Replicative transposition Requires transposase, resolvase and an IRS (internal resolution site) recognized by resolvase 1) Transposase makes nicks at ends of transposon and at ends of target 2) Transposase ligates the donor and target together such that they're linked together via single strands of the transposon 3) Gaps are filled in with host DNA pol I and ligase (transposon is replicated) 4) Resolvase catalyzes recombination between the 2 IRSs Results: Transposon does not leave the donor DNA so donor is not destroyed Transposon is replicated so 2 copies of the transposon are produced Depending upon how they transpose; There are three distinct types: Class I Transposons consisting only of DNA that moves directly from place to place. Class II Transposons; also known as Miniature Inverted-repeats Transposable Elements or MITEs. Class III Retrotransposons that first transcribe the DNA into RNA and then use reverse transcriptase to make a DNA copy of the RNA to insert in a new location.

Class I Transposons They usually move by a mechanism analogous to cut and paste where the element is physically cut out to transpose it to a new position. The new position is situated any where in the cells genome using the transposase enzyme which is encoded by the element itself. This mechanism also called cut and paste mechanism. Different types of Transposase work in different ways. Some can bind to any part of the DNA molecule, and the target site can therefore be anywhere, while others bind to specific sequences Transposase binds to: 1) Both ends of the transposon, which consist of inverted repeats; that is, identical sequences reading in opposite directions. 2) A sequence of DNA that makes up the target site. Some transposases require a specific sequence as their target site; other can insert the transposon anywhere in the genome. Transposase makes a staggered cut at the target site producing sticky ends produced by some restriction enzymes cuts out the transposon and ligates it into the target site.The gaps are filled in by Watson-Crick base pairing by DNA polymerase and DNA ligase closes the sugar-phosphate backbone. This results in target site duplication and the insertion sites of DNA transposons may be identified by short direct repeats (a staggered cut in the target DNA filled by DNA polymerase) followed by inverted repeats (which are important for the transposon excision by transposase). Often transposons lose their gene for transposase; but as long as somewhere in the cell there is a transposon that can synthesize the enzyme, their inverted repeats are recognized and they, too, can be moved to a new location. These transposons undergo conservative transposition The transposons which only move by cut and paste may duplicate themselves if the transposition happens during S phase of the cell cycle when the "donor" site has already been replicated, but the "target" site has not. Class II Transposons Replication of transposable elements is involved in these elements transposition by Transposase which interacts between the element and the potential insertion site. During the interaction the element is replicated and one copy of it is inserted in the new site. The other copy remains in the original site. This mechanism is also called replicative transposition Transposase makes a staggered cut at the target site producing sticky ends produced by some restriction enzymes ligates it into the target site.The gaps are filled in by Watson-Crick base pairing by DNA polymerase and DNA ligase closes the sugar-phosphate backbone. This results in target site duplication and the insertion sites of DNA transposons may be identified by short direct repeats (a staggered cut in the target DNA filled by DNA polymerase) followed by inverted repeats (which are important for the transposon excision by transposase). These transposons undergo replicative transposition Class III: Retrotransposons Retrotransposons work by copying themselves and pasting copies back into the genome in multiple places. Initially retrotransposons copy themselves to RNA (transcription) but, in addition to being transcribed, the RNA is copied into DNA by a reverse transcriptase (often coded by the transposon itself) and inserted back into the genome. Retrotransposons behave very similarly to retroviruses, such as HIV, giving a clue to the evolutionary origins of such viruses. Like DNA transposons, retrotransposons generate direct repeats at their new sites of insertion. In fact, it is the presence of these direct repeats that often is the clue that the intervening stretch of DNA arrived there by retrotransposition. About 40% of the entire human genome consists of retrotransposons There are three main classes of retrotransposons: Retrovirus like elements-Viral: encode reverse transcriptase (to reverse transcribe RNA into DNA), have long terminal repeats (LTRs), similar to retroviruses Retroposons 1) LINEs: encode reverse transcriptase, lack LTRs, transcribed by RNA polymerase II 2) SINEs:Nonviral superfamily: do not code for reverse transcriptase, transcribed by RNA polymerase III . Many retrotransposons have long terminal repeats (LTRs) at their ends that may contain over 1000 base pairs in each.

Transposable elements in Bacteria

` Though eukaryotic transposons were first identified, bacterial transposons were studied at the molecular level.They are basically of 3 types 1). IS elements which are simple and contains a gene coding for transposition 2). Composite transposons which have extra genes can code for products unrelated to transposition 3). Tn 3 elements which are slightly more complex than composite transposons

IS Elements An insertion sequence (also known as an IS, an insertion sequence element, or an IS element) is a short DNA sequence that acts as a simple transposable element. They were first discovered in certain Lac- mutations of E.Coli which had high rate to revert back to wild type caused due to presence or absence of transposons near the Lac gene. Insertion sequences have two major characteristics: they are small relative to other transposable elements (generally around 700 to 2500 bp in length) and only code for proteins implicated in the transposition activity. These proteins are usually the Transposase which catalyses the enzymatic reaction allowing the IS to move by attaching to ends of the element and cutting the Dna strand, and also one regulatory protein which either stimulates or inhibits the transposition activity. There are many IS elements known. They differ in size and other details, but the overall structure of most such elements is similar. One example IS1 is 768 bases long but many other IS elements are longer (usually 1300–1500 bases). The central region of an IS element codes for Transposase protein. At the ends of the insertion sequence are almost perfect inverted terminal repeat (IR) sequences, which in IS1 consist of 23 nucleotides. A minority of elements, such as IS900 from Mycobacterium paratuberculosis do not have inverted terminal repeat ends. An inverted repeat of a DNA sequence does NOT mean that the sequence on an individual strand is repeated backwards, but that the sequence from left to right on the ‘top’ strand is repeated from right to left on the ‘bottom’ strand so that reading either copy of the IR in the 5’ to 3’ direction will result in the same sequence of bases. Since DNA sequences are often presented as just one of the two strands, an inverted repeat of the sequence CAT will appear as ATG. They range between 9 – 40 bp. Mutation occurring here renders the element immobile. In addition to the inverted repeats, inspection of a DNA region containing an insertion sequence usually shows a further short sequence that is duplicated – but this sequence is repeated in the same orientation and is therefore referred to as a direct repeat (DR). This is not part of the IS, but arises from duplication of the DNA at the insertion site and therefore different copies of IS1 will have different target sequence repeats depending on the point of insertion hence it is also called target site duplication. The range varies between 2-13 bp. Transposition of IS1 generates rather long direct repeats (9 base pairs). The presence of these direct repeats is linked to the mechanism of transposition . The well-known IS911 (1250 bp) is flanked by two 36bp inverted repeat extremities and the coding region has two genes partially overlapping orfA and orfAB, coding the transposase (OrfAB) and a regulatory protein (OrfA). A particular insertion sequence may be named according to the form ISn, where n is a number (e.g. IS1, IS2, IS3, IS10, IS50, IS911, etc.); this is not the only naming scheme used, however. Although insertion sequences are usually discussed in the context of prokaryotic genomes, certain eukaryotic DNA sequences belonging to the family of Tc1/mariner transposable elements may be considered to be insertion sequences Insertion sequences have been identified in most bacterial genera, although the presence and the number of copies of any one element often vary from strain to strain. A typical laboratory strain of E. coli for example might contain six copies of IS1 as well as a number of copies of other insertion sequences. In addition to occurring autonomously, insertion sequences may also occur as parts of composite transposons

Plasmid may also contain IS elements E.g. F plasmid which has IS2 ,IS3.hence if plasmid and the circular genome cointegrates due to homology of the IS sequences. when they loop out Hfr strains can be produced. Hence IS elements help in transfer or exchange of genes.

Composite transposons They are denoted by the symbol Tn and are created when 2 IS sequences are inserted beside each other. The region between them can be transported along with the inserted IS. Hence the ISs have made an immobile gene mobile. These genes are nothing related to transposition they mostly code for resistance to a certain antibiotic These composite transposons consist of two copies of an insertion sequence on either side of a set of resistance genes. For example the tetracycline resistance transposonTn10, which is about 9300 bp in length, consists of a central region carrying the resistance determinants flanked by two copies of the IS10 insertion sequence in opposite orientations IS10 itself is about 1300 bp long with 23-bp inverted repeat ends and contains a Transposase gene. Composite transposons may have their flanking IS regions in inverted orientation or as direct repeats. For example Tn10 and Tn5 both have inverted repeats of an IS (IS10 and IS50 respectively) at their ends, while Tn9 has direct repeats of IS1. The transposition behaviour of such composite elements can be quite complex; the insertion sequences themselves may transpose independently or transposition of the entire region may occur. Furthermore, recombination between the IS elements can occur, leading to deletion or inversion of the region separating them .Even more complex arrangements can occur. For example, Tn4 appears to be related to Tn21 but contains a complete copy of Tn3 within it. The ampicillin resistance gene of Tn4 can thus be transposed as part of the complete Tn4 transposon or by transposition of the Tn3 element. Thus several layers of transposons can occur, nested within one another. In Tn5 the IS50R is capable of producing Transposase but IS50Lcannot due to deletion of a nucleotide pair.Tn5 when carried by non lytic bacteriophage the frequency of transposition is reduced if the virus already carries a copy on Tn5.Sinc the resident Tn5 inhibits the entry of incoming Tn5 by synthesis of a repressor

Tn 3 elements These are larger than IS elements but are different from composite transposons due to absence of IS element flanks. instead have terminal inverted repeats. The structure of Tn3; it consists of about 4957 base pairs and has a short (38 bp) inverted repeat sequence at each end. It is therefore analogous to an insertion sequence, the distinction being that a transposon carries an identifiable genetic marker – in this case the ampicillin

resistance gene (bla, β-lactamase). Tn3 codes for two other proteins as well: a Transposase (TnpA), and TnpR , resolvase a bi functional protein that acts as a repressor and is also responsible for one stage of transposition known as resolution. There is a short direct repeat at either end of the transposon (five base pairs in the case of Tn3). Transposition of Tn3 occurs in 2 stages. Firstly the transposase fuses 2 circular genomes one with Tn3 and the other without it. Resulting structure is called Cointegrate during this process Tn3 replicates hence one copy of Tn3 is rendered to each genome which are oriented in the same direction. Secondly Resolvase bring about recombination between the 2 Tn3 elements in the Cointegrate at the res (resolution site) resulting in two circular genomes each with a copy of Tn3.Resolvase also prevents the expression on the two genes by binding at the res site interfering with the transcription of the genes creating a chronic shortage of the products hence Tn3 remains to be Immobile. These also come under the category replicative transposons

Transposons in Eukaryotes There are many different types of transposons in Eukaryotes. Some are abundant in the genome others are rare. These elements have inverted repeats and direct repeats and also encode transposase for the mobility of the transposase

Maize - Ac/Ds System Barbara McClintock was the first scientist to predict that transposable elements, mobile pieces of the genetic material (DNA), were present in eukaryotic genomes The first transposons were discovered in maize (Zea mays), by Barbara McClintock in 1948, for which she was awarded a Nobel Prize in 1983. .She performed her work on corn and specifically followed seed color phenotypes.. McClintock discovered transposable elements by analyzing genetic stocks of corn that were phenotypically unstable. In particular, she was analyzing genes that control the color of the aleurone layer of the endosperm by using genetic markers. Remember that this tissue is triploid (3n). The genes that she was following were located on the short arm of chromosome 9 of corn and were involved in the development of the color of the seed. The genetic map of this region and the allelic designations follow. ___________________________________ C Bz Ds CI = dominant allele that prevents color from being expressed in the aleurone layer- Inhibitor C = recessive allele that leads to color development in the aleurone layer Bz = dominant allele that produces purple aleurone color bz = recessive allele that produces a dark brown to purple-brown aleurone color ds = a genetic location where chromosome breakage occurs

Homozygous stocks were created and CC bzbz -- females (without ds, denoted by the dash) were mated with CICI BzBz dsds males. The aleurone layer of the endosperm would thus have the genotype CICC Bzbzbz --ds. Because of the presence of the inhibitor allele, the aleurone layer was expected to be colorless. For many of the kernels this was the case but a few kernels had dark brown colored sectors on an otherwise colorless background. McClintock concluded that in some manner the CI and Bz alleles were lost because chromosome breakage had occurred at the Ds locus. This breakage apparently did not occur during gamete formation, but had occurred after fertilization and during the development of the seed. This breakage and loss of genes occurred in a single cell, but all cells that developed from mitotic division of that cell did not contain the inhibitor gene, so the color expression was controlled by the bz allele in those cells.

Female gametes: __________________________________ C bz Male gametes: __________________________________ CI Bz ds The following is the expected chromosomal composition of triploid endosperm. Because of the dominant CI allele the endosperm should be colorless without breakage ____________________________ (from female) C bz ____________________________ (from female) C bz ___________________________ (from male) CI Bz ds But if breakage at ds occurred, then the genotype of the endosperm would be: ____________________________ (from female) C bz ____________________________ (from female) C bz ….……………………… _______ (from male) and any cells with this genotype would be dark brown in color. Breakage at Ds had been established by McClintock prior to performing these experiments. The designation Ds was short for dissociation or a locus were breakage of chromosomes occurred. But after crossing with a number of different genetic stocks, she realized that Ds alone could not induce the breakage. A second factor, Ac, short for activator, was also necessary since its transposase causes the breakage of the chromosome. Ds can be activate dif Ac transposase is present anywhere in the genome (Thus, some genetic stocks contained Ac whereas other stocks did not contain that locus.) This system is called a two-element system and historically has been called the Ac/Ds system. Additional genetic stocks were analyzed by McClintock and she determined that in the presence of Ac, Ds could move locations as well as cause breakages. She was able to isolate a corn line where Ds had moved into the normal Bz allele and caused a mutation in that gene. But as was mentioned this only occurred when Ac was present. Furthermore, when this new line was used and Ac was present, the Ds element was shown to move out of the Bz locus and reversion to the original phenotype was detected. This mutated allele was designated bzm1. But in the absence of Ac, bzm1 was a stable allele. Another unstable Bz allele was found that contained an Ac insertion and was designated bzm2. One difference between this allele and bzm1 was its higher rate of transposition and reversion back to the original phenotype .Ac/Ds are also called controlling elements since there insertion into various position renders the nearby gene altered or inactive. Multiple copies of these are present in the genome . So the conclusions drawn from these experiments and observations are: 1) Ds requires some factor provided by Ac to move, whereas Ac is independent 2) Because of their relationship, Ac is termed an autonomous element and Ds a non- autonomous element.

3) Because both Ac and Ds can move, they are called transposable genetic elements. About 50% of the total genome of maize consists of transposons. The Ac/Ds system McClintock described are class II transposons. Ac is 4563 bp in length contain essential 11-bp inverted repeats at the ends encodes a 3.5 kb mRNA that is translated into a 92 kDa transposase protein 101 N-terminal amino acids not required for transposition about 200 bp on each end of element necessary for transposition transposase binds to the hexamer AAACGG 8-bp direct repeats of target DNA are generated footprints (residual DNA sequences) are often left behind after the element is excised Ds are truncated versions of Ac; these can lack a transposase or the inverted repeat. Ds is structurally heterogeneous and have similar IRs but the internal sequence varies. Some Ds are derived from Ac by loss of certain internal sequences. Other Ds contain non-Ac DNA between their IRs. Other Ds types are Aberrant Ds, piggyback arranged Ds where one Ds element is inserted into an other but in the inverted orientation hence also called double Ds elements

Maize - Spm / Dspm system The Suppressor-mutator (Spm) family of maize transposable elements consists of autonomous Sprn elements and nonautonomous defective Spm (dSpm) elements. It was discovered by Barbara McClintock. One characteristic of this family is that the insertion of dSpm elements into a structural gene often permits some level of structural gene expression in the absence of Spm activity, but the structural gene expression is suppressed in Trans by Spm activity. The Spm’s sub terminal repetitive regions (SRRs) contain several iterations of a 12-bp repeat motif. It had been proposed that binding of an Spm-encoded protein to these repeat motifs blocks structural gene transcriptional read through, thus suppressing gene expression. The bz-m13 allele of the bronze I locus contains a 2.24-kb dSpm insertion in the second exon of a Bz allele. In the absence of Spm activity, bz-m13 displays substantial Bz expression, and this expression is fully suppressed by Spm. Four intra-dSpm deletion derivatives are described in which this Bz expression is only partially suppressed by Spm. Each of these derivatives retains at least 12 SRR repeat motifs. Thus the presence of these repeat motifs is not sufficient to guarantee complete suppression by Spm. Some other property such as secondary structure or element size plays a role. Spm element is 8287 bp long with 13 bp IR and creates a 3 bp target site duplication. Dspm are smaller than the spm’s because they are derived from the spm element due to loss of internal sequences hence the complete function of spm is not expressed in Dspm. Dspm requires presence of Spm for their mobility since only Spm can code for transposase

Drosophila – P elements A P element is a transposon that is present in the fruit fly Drosophila melanogaster and is used widely for mutagenesis and the creation of genetically modified flies used for genetic research. The P element gives rise to a phenotype known as hybrid dysgenesis. P elements seem to have first appeared in the species only in the middle of the twentieth century. Over the last fifty years, they have spread through every wild population, so that only older laboratory stocks lack them. It was identified by geneticists Margaret and James Kidwell in 1977 Hybrid dysgenic mutant’s white gene was compared with that of recessive white gene and found that the former had a small element inserted. These were the P elements Characteristics

The P element is a class II transposon, which means that its movement within the genome is made possible by a transposase. The complete element is 2907 bp and is autonomous because it encodes a functional transposase; nonautonomous P elements which lack a functional transposase gene due to mutation also exist. Non-autonomous P elements can still move within the genome if there are autonomous elements to produce transposase. The P element can be identified by its terminal 31-bp inverted repeats, 11-bp subterminal inverted repeat and the 8 bp direct repeats its movement into and out of DNA sequence produces. Several lines of evidence show that P elements transpose non replicatively and without an RNA intermediate donor element is excised and reinserted into a recipient site creating a direct duplication of 8 bp at the site of insertion P element insertions have been found at thousands of genomic positions, but not all sites are equally likely to be hit. The mechanism of insertion site selection is not known, but several generalizations can be made: (i) Euchromatic sites are hit more often than the heterochromatin (ii) Some euchromatic loci are much more susceptible to P mutagenesis than others. For example, the singed gene is hit at frequencies approaching 10-2 whereas the vestigial gene has a rate of less than 10-6 .Despite this variability, there is no evidence that any loci are immune from P element mutagenesis given a sufficiently large sample size; (iii) Within genes there is a preference for insertion in the non coding upstream sequences (iv) Target sites with close matches to the consensus octamer GGCCAGAC are more likely to receive P element insertions (v) P elements tend to insert into or near other P elements, with a particular preference for base pairs 19-26 of the target P element (vi) Some P elements have been observed to jump preferentially to sites closely linked to the donor site The P element transposase is an 87 kiloDalton protein encoded by autonomous P elements .It binds to subterminal regions at both ends of the element and represses transcription. GTP is also bound by the transposase, and is required for transposition in vitro . Some natural populations contain a few P elements, but others contain up to 50 copies. Interestingly, strains captured before 1950 do not contain any P elements. These are called "empty" strains. These strains may represent the primitive condition and P elements may have invaded these natural populations. Close relatives of the lab species D. melanaogaster have maintained the empty condition, but more distantly-related strains like D.willostoni acquired the element. It has been suggested that the element entered the species by piggy-backing on viruses that naturally infect Drosophila Hybrid dysgenesis When P elements are mobilized they produce a syndrome of traits known collectively as hybrid dysgenesis Hybrid dysgenesis refers to the high rate of mutation in germ line cells of Drosophila strains resulting from a cross of males with autonomous P elements (P Strain/P cytotype) and females that lack P elements (M Strain/M cytotype). The hybrid dysgenesis syndrome is marked by temperature-dependent sterility, elevated mutation rates, and increased chromosome rearrangement and recombination. The hybrid dysgenesis phenotype is affected by the transposition of P elements within the germ-line cells of offspring of P strain males with M strain females. These two kinds of strains are called "P" and "M" because they contribute paternally and maternally respectively to hybrid dysgenesis. Transposition only occurs in germ-line cells, because a splicing event needed to make transposase mRNA does not occur in somatic cells. Hybrid dysgenesis manifests when crossing P strain males with M strain females and not when crossing P strain females (females with autonomous P elements) with M strain males. The eggs of P strain females contain high amounts of a repressor protein that prevents transcription of the transposase gene. The eggs of M strain mothers, which do not contain the repressor protein, allow for transposition of P elements from the sperm of fathers. The dysgenic traits can be explained largely by genomic changes due to P element transposition and excision in developing germ cells. The sterility is due to loss of germ cells early in development It is more pronounced in females, where there are fewer germ cells to spare than in males, and at temperatures above 25°C. The mutations come about through several mechanisms, but are primarily P insertions into genes and imprecise excision of P elements near genes Chromosome rearrangements usually result from breakage at the sites of two or more P element insertions, followed

by rejoining of the chromosome segments in a different order). P-induced recombination occurs preferentially in the genetic intervals containing mobile P elements, and usually within 2 kb of the insertion site P element movement within the fly is a maternally inherited trait. Movement is suppressed in flies with the P cytotype, but is permissible in the M cytotype. Furthermore, P-cytotype flies contain P elements, whereas M-cytotype elements do not. When movement does occur it happens in the germ line, that is those cells which produce the gametes. This movement of P elements creates many different changes in phenotype, but few are fatal. The progeny are described as being dysgenic that refers to biological deficiencies that are a result of the P element movement. Movement is suppressed in somatic cells where the effects would be more damaging this is because at the level of RNA splicing metabolic block occurs hence one of the introns remains in the transcript and is prevented from translation of the transposase gene by a stop codon that prematurely terminates translation. This condition of germ line abnormalities which generates mutations, chromosomal breakage and sterility is called P-M hybrid dysgenesis and is mediated by P element movement.

It is currently thought that P elements encode a transposase as well as a repressor of the transposase activity. The expression of the repressor protein is also thought to be a maternally-expressed trait. P-cytotype flies contain large numbers of P elements. Thus, the females would contain high levels of the repressor protein. Therefore, in a cross between a P-cytotype female and a M- cytotype male the high level of repressor expressed by the female would prevent P element movement and hybrid dysgenesis. Conversely, a M-cytotype female does not express the repressor and when they are mated with a P-cytotype female, P element movement can occur and the progeny would be dysgenic. P elements are repressed not only within P strains, but also in the hybrids of P(female) x M(male) crosses. They are not repressed in hybrids from the reciprocal cross, suggesting that repressor products produced in the P strain germline can be inherited maternally. Interestingly, this effect goes beyond simple maternal inheritance. Progeny from the cross MP(female) x P(male) show more P mobility than those from the cross PM(female) x PM(male) where "MP" and "PM" represent the two reciprocal F1 hybrids with the female component shown first. Thus, the repressed state, called the P cytotype, is jointly determined by chromosomal and maternal components. One explanation proposed for this unusual inheritance was that the repressor-making P elements in the MP hybrids are more likely to be excised than those of the PM hybrids However, that explanation was ruled out by the finding that a single repressor-producing P element was sufficient for this mode of inheritance An alternative model involving differential splicing of the same intron that is involved in tissue-specific regulation, provides an adequate explanation for available data..

Yeast – Ty elements This element has several features that are unique, and it appears to resemble a primitive retrovirus. A retrovirus is a RNA virus that, after being uncoated in the host cell, converts its RNA to a DNA copy by the enzyme reverse transcriptase. This enzyme is encoded by the retroviral pol gene. The DNA copy of the retrovirus is inserted into the eukaryotic genome, and it remains there as a provirus until it is excised and undergoes transcription to produce new viral particles. Features of TY elements • about 35 copies in the haploid yeast gene • They about 5900 bp long and are bounded at each end by δ sequence

• • • • •

δ sequence has 340 bp sequence and has orientation in the same direction; these are called long terminal repeats or LTRs They are flanked by 5 bp direct repeats Target sit duplication are rich in A:T bp They resemble eukaryotic retroviruses; because they lack some of the retroviral functions they are considered to be primitive retroviruses; because of their similarities they are called retrotransposons transposition involves an RNA intermediate that is generated by transcription of the TY element; a reverse transcriptase (encoded by the TyB gene of the element) makes a DNA copy of the element which is then inserted into a new site in the yeast genome

Ty Elements in Yeast has 4 genes: 1) gag for a virion internal protein, 2) int for integrase, 3) env (may not be present) for virion envelope protein, and 4) pol for reverse transcriptase Transposition of Ty elements: 1) Transcription to produce ssRNA 2) Translation to produce reverse transcriptase (RT) and integrase 3) RT produces a RNA/DNA hybrid from the ssRNA 4) RT degrades the RNA in the hybrid, leaving ssDNA 5) RT makes another strand of DNA using the ssDNA as the template 6) Integrase inserts dsDNA into chromosome at a different sites

Retrotransposons Retroviruses (HIV-1) HIV-1 — the cause of AIDS — and other human retroviruses (e.g., HTLV-1, the human T-cell leukemia virus) behave like retrotransposons. The RNA genome of HIV-1 contains a gene for reverse transcriptase and one for integrase. The integrase serves the same function as the transposases of DNA transposons. The DNA copies can be inserted anywhere in the genome. Molecules of both enzymes are incorporated in the virus particle. Retroviruses like elements They are found in many different organisms including yeast plants and animals. Despite difference in size and nucleotide sequence, they all have same basic structure. A central coding region flanked by long terminal repeats (LTR) which are oriented in the same direction. Each LTR is bounded by short inverted repeats. The coding region contains a small number of genes usually two. One is homologous to gag (encodes structural protein of virus capsule) the other is homologous to pol(encodes reverse transcriptase/ integrase protein).Retrovirus have a third gene env (encodes a protein component of the virus envelope)

Retroposons These are also called non LTR transposons. They are large widely distributed transposons. They move through an RNA molecule and then reverse transcribed into DNA. They create target site duplication but do not have inverted or direct repeats as integral parts of their termini. They are distinguished by a homogenous sequence of A:T base pairs ant one end. This sequence is derived from the poly(A) tail that is added near the 3’ of the retroposon RNA during its maturation. Integrated retroposons therefore exihibit a vestige of their origin as reverse transcripts of polyadenylated RNAs 1.LINEs (Long interspersed elements) The human genome contains some 850,000 LINEs (representing some 21% of the genome). Most of these belong to a family called LINE-1 (L1). These L1 elements are DNA sequences that range in length from a few hundred to as many as 9,000 base pairs. Only about 50 L1 elements are functional "genes"; that is, can be transcribed and translated. The functional L1 elements are about 6,500 bp in length and encode three proteins, including an endonuclease that cuts DNA and a reverse transcriptase that makes a DNA copy of an RNA transcript. L1 activity proceeds as follows: RNA polymerase II transcribes the L1 DNA into RNA. The RNA is translated by ribosomes in the cytoplasm into the proteins. The proteins and RNA join together and reenter the nucleus. The endonuclease cuts a strand of "target" DNA, often in the intron of a gene. The reverse transcriptase copies the L1 RNA into L1 DNA which is inserted into the target DNA forming a new L1 element there. Through this copy-paste mechanism, the number of LINEs can increase in the genome. The diversity of LINEs between individual human genomes makes them useful markers for DNA "fingerprinting". Variation in the length of L1 elements: Transcription of active L1 elements sometimes continues downstream into additional DNA producing a longer transposed element. Reverse transcription of L1 RNA often concludes prematurely and produces a shortened transposed element. While these elements are not functional, they may play a role in regulating the efficiency of transcription of the gene in which they reside . Occasionally, L1 activity makes and inserts a copy of a cellular mRNA (thus a natural cDNA). Lacking introns as well as the necessary control elements like promoters, these genes are not expressed. They represent one category of pseudogene. 2.SINEs (Short interspersed elements) SINEs are short DNA sequences (100–400 base pairs) that represent reverse-transcribed RNA molecules originally transcribed by RNA polymerase III; that is, molecules of tRNA, 5S rRNA, and some other small nuclear RNAs. The most abundant SINEs are the Alu elements. There are over one million copies in the human genome (representing about 11% of the total DNA). Alu elements consist of a sequence of 300 base pairs containing a site that is recognized by the restriction enzyme AluI. They appear to be reverse transcripts of 7S RNA, part of the signal recognition particle. Most SINEs do not encode any functional molecules and depend on the machinery of active L1 elements to be transposed; that is, copied and pasted in new locations. Miniature Inverted-repeat Transposable Elements (MITEs) The recent completion of the genome sequence of rice and C. elegans has revealed that their genomes contain thousands of copies of a recurring motif consisting of almost identical sequences of about 400 base pairs flanked by characteristic inverted repeats of about 15 base pairs such as 5' GGCCAGTCACAATGG..~400 nt..CCATTGTGACTGGCC 3' 3' CCGGTCAGTGTTACC..~400 nt..GGTAACACTGACCGG 5' MITEs are too small to encode any protein. Just how they are copied and moved to new locations is still uncertain. Probably larger transposons that do encode the necessary enzyme and recognize the same inverted repeatsare responsible.

There are over 100,000 MITEs in the rice genome (representing some 6% of the total genome). Some of the mutations found in certain strains of rice are caused by insertion of a MITE in the gene. MITEs have also been found in the genome of humans, Xenopus, and apples.