LINEs insert themselves into regions of the eukaryotic genome that are rich in bases AT. At AT regions LINE uses its nuclease to cut one strand of the eukaryotic double-stranded DNA. The adenine-rich sequence in LINE transcript base pairs with the cut strand to flag where the LINE will be inserted with hydroxyl groups. Reverse transcriptase recognises these hydroxyl groups to synthesise LINE retrotransposon where the DNA is cut. Like with LTR retrotransposons, this new inserted LINE contains eukaryotic genome information so it can be copied and pasted into other genomic regions easily. The information sequences are longer and more variable than those in LTR retrotransposons.


Most LINE copies have variable length at the start because reverse transcription usually stops before DNA synthesis is complete. In some cases this causes RNA polymerase II promoter to be lost so LINEs cannot transpose further. [ 15 ]


LINE-1 (L1) retrotransposons make up a significant portion of the human genome, with an estimated 500,000 copies per genome. Genes encoding for human LINE1 usually have their transcription inhibited by methyl groups binding to its DNA carried out by PIWI proteins and enzymes DNA methyltransferases. L1 retrotransposition can disrupt the nature of genes transcribed by pasting themselves inside or near genes which could in turn lead to human disease. LINE1s can only retrotranspose in some cases to form different chromosome structures contributing to differences in genetics between individuals. [ 17 ]  There is an estimate of 80–100 active L1s in the reference genome of the Human Genome Project, and an even smaller number of L1s within those active L1s retrotranspose often. L1 insertions have been associated with  tumorigenesis  by activating cancer-related genes oncogenes and diminishing tumor suppressor genes.


Each human LINE1 contains two regions from which gene products can be encoded. The first coding region contains a leucine zipper protein involved in protein-protein interactions and a protein that binds to the terminus of nucleic acids. The second coding region has a purine/pyrimidine nuclease, reverse transcriptase and protein rich in amino acids cysteines and histidines. The end of the human LINE1, as with other retrotransposons is adenine-rich. [ 18 ] [ 19 ] [ 20 ]


Human L1 actively retrotransposes in the human genome. A recent study identified 1,708 somatic L1 retrotransposition events, especially in colorectal epithelial cells. These events occur from early embryogenesis and retrotransposition rate is substantially increased during colorectal tumourigenesis. [ 21 ]


SINEs are much shorter (300bp) than LINEs. [ 22 ]  They share similarity with genes transcribed by RNA polymerase II, the enzyme that transcribes genes into mRNA transcripts, and the initiation sequence of RNA polymerase III, the enzyme that transcribes genes into ribosomal RNA, tRNA and other small RNA molecules. [ 23 ]  SINEs such as mammalian MIR elements have tRNA gene at the start and adenine-rich at the end like in LINEs.


SINEs do not encode a functional reverse transcriptase protein and rely on other mobile transposons, especially  LINEs . [ 24 ]  SINEs exploit LINE transposition components despite LINE-binding proteins prefer binding to LINE RNA. SINEs cannot transpose by themselves because they cannot encode SINE transcripts. They usually consist of parts derived from tRNA and LINEs. The tRNA portion contains an RNA polymerase III promoter which the same kind of enzyme as RNA polymerase II. This makes sure the LINE copies would be transcribed into RNA for further transposition. The LINE component remains so LINE-binding proteins can recognise the LINE part of the SINE.


Alu s  are the most common SINE in primates. They are approximately 350 base pairs long, do not encode proteins and can be recognized by the  restriction enzyme   AluI  (hence the name). Their distribution may be important in some genetic diseases and cancers. Copy and pasting Alu RNA requires the Alu's adenine-rich end and the rest of the sequence bound to a signal. The signal-bound Alu can then associate with ribosomes. LINE RNA associates on the same ribosomes as the Alu. Binding to the same ribosome allows Alus of SINEs to interact with LINE. This simultaneous translation of Alu element and LINE allows SINE copy and pasting.


SVA elements are present at lower levels than SINES and LINEs in humans. The starts of SVA and Alu elements are similar, followed by repeats and an end similar to endogenous retrovirus. LINEs bind to sites flanking SVA elements to transpose them. SVA are one of the youngest transposons in great apes genome and among the most active and polymorphic in the human population. SVA was created by a fusion between an Alu element, a VNTR (variable number tandem repeat), and an LTR fragment. [ 25 ]


Retrotransposons ensure they are not lost by chance by occurring only in cell genetics that can be passed on from one generation to the next from parent gametes. However, LINEs can transpose into the human embryo cells that eventually develop into the nervous system, raising the question whether this LINE retrotransposition affects brain function. LINE retrotransposition is also a feature of several cancers, but it is unclear whether retrotransposition itself causes cancer instead of just a symptom. Uncontrolled retrotransposition is bad for both the host organism and retrotransposons themselves so they have to be regulated. Retrotransposons are regulated by  RNA interference . RNA interference is carried out by a bunch of short  non-coding RNAs . The short non-coding RNA interacts with protein Argonaute to degrade retrotransposon transcripts and change their DNA histone structure to reduce their transcription.


LTR retrotransposons came about later than non-LTR retrotransposons, possibly from an ancestral non-LTR retrotransposon acquiring an integrase from a DNA transposon. Retroviruses gained additional properties to their virus envelopes by taking the relevant genes from other viruses using the power of LTR retrotransposon.


Due to their retrotransposition mechanism, retrotransposons amplify in number quickly, composing 40% of the human genome. The insertion rates for LINE1, Alu and SVA elements are 1/200 – 1/20, 1/20 and 1/900 respectively. The LINE1 insertion rates have varied a lot over the past 35 million years, so they indicate points in genome evolution.


Notably a large number of 100 kilobases in the maize genome show variety due to the presence or absence of retrotransposons. However since maize is unusual genetically as compared to other plants it cannot be used to predict retrotransposition in other plants.


Mutations caused by retrotransposons include:


Transposable elements  are pieces of genetic material that are capable of splicing themselves into a host  genome  and then self propagating throughout the genome, much like a  virus .  Retrotransposons  are a subset of transposable elements that use an RNA intermediate and reverse transcribe themselves into the genome. Retrotransposon proliferation may lead to  insertional mutagenesis , disrupt the process of  DNA repair , or cause errors during  chromosomal crossover , and so it is advantageous for an organism to possess the means to suppress or "silence" retrotransposon activity.


Piwi-interacting RNA  (piRNA) and endogenous short interfering RNA (esiRNA) are small  RNA  fragments that partner with cleaving proteins to act as guides to a retrotransposon. [ 1 ]  piRNA and  PIWI  protein complexes are a common defense mechanism against retrotransposons in the  germline —sperm or egg cells—cells that are extremely vulnerable to retrotransposon induced mutations. [ 2 ]  Once retrotransposon RNA has been encountered, the piRNA/ PIWI protein complex cleaves the RNA, rendering it unable to reverse transcribe. EsiRNA and its protein complex may also function to promote  heterochromatin  creation near retrotransposon sites, silencing the DNA by making it difficult for  DNA polymerases  to access it. Although it is unclear how piRNAs and esiRNAs are produced, it is thought that retrotransposon cleaving by PIWI may produce new piRNA, in the so called  ping pong theory . Production of piRNA in both the male and female germline is known to be regulated by the  nuage , a protein-RNA hybrid also sometimes called the intermitochondrial cement, the absence of which causes infertility in males but not females. [ 3 ]  This may imply that retrotransposon induced  mutations may be more prevalent or more devastating in the male germline. A  microRNA —another type of small RNA—from strawberries has been shown to post transcriptionally silence retrotransposon function by blocking the binding site for proteins involved in  reverse transcription . [ 4 ]


Research indicates both PIWI proteins and esiRNAs may also play a role in silencing retrotransposons through  DNA methylation . [ 5 ] [ 6 ]  esiRNA's methylation of LINE-1 retrotransposon is one process that can be disrupted in  cancer  cells, leading to the development of the high mutation rates required to develop a tumor. [ 7 ]


EsiRNA and its protein complex may also function to promote the creation of  heterochromatin , protein around which DNA is very tightly coiled, near retrotransposon sites, silencing the DNA by making it difficult for transcription related proteins to access it. [ 8 ]  Likewise, pi-RNA mediated DNA methylation also contributes to the formation of heterochromatin around the methylated site. [ 9 ]  Methylation and heterochromatin formation allow PIWI and piRNAs to continue to repress retrotransposon even when PIWI's ability to cleave retrotransposons is disrupted (Darricarrere et al 2013).


Though the mechanism is as of yet unclear, RNA-dependent RNA  polymerase  RrpC has been shown to post-transcriptionally silence retrotransposons in Dictyostelium. [ 10 ]   Cryptic unstable transcripts  (CUTs), another type of noncoding RNA, are also involved in  histone  methylation and deacetylation, both of which form heterochromatin (Berretta et al. 2008).  Centromere binding protein B , a DNA binding factor, has also been indicated to play a role in counteracting retrotransposon-induced instability during  DNA replication . [ 11 ]


Retrozymes  are a family of  retrotransposons  first discovered in the genomes of  plants [ 1 ]  but now also known in genomes of  animals . [ 2 ]  Retrozymes contain a  hammerhead ribozyme  (HHR) in their sequences (and so the name  retrozyme  is a combination of  retrotransposon  and  hammerhead ribozyme ), although they do not possess any coding regions. Retrozymes are nonautonomous retroelements, and so borrow  proteins  from other elements to move into new regions of a  genome . Retrozymes are actively transcribed into covalently closed circular RNAs (circRNAs or cccRNAs) and are detected in both polarities, which may indicate the use of  rolling circle replication  in their lifecycle. [ 3 ]


The genomic structure of a retrozyme in plants involves a central  non-coding region  that may stretch about 300–600nt flanked by  long terminal repeats  about 300–400nt containing the HHR motif. They also have two sequences (a  primer binding site  (PBS) complementary to the tRNA-Met sequence and a poly-purine tract (PPT)) needed to prime DNA synthesis during mobilization. The most distinguishing feature of the retrozyme compared with other elements of plant genomes are the hammerhead ribozyme. Otherwise, they resemble other known features of plant genomes such as terminal-repeat retrotransposons in miniature (TRIMs) and small LTR retrotransposons (SMARTs). The PBS, PPT, and the HHR motif are the only parts of the retrozyme sequences which shows conservation and  homology . [ 4 ]  Currently, it is thought retrozymes evolved from a large retrotransposon family known across many  eukaryotes  as the Penelope-like elements (PLEs). Retrozymes share a number of peculiar features with PLEs, including a type I HHR, occurrence as tandem copies, and co-existence in all analyzed metazoans to date. [ 2 ] [ 4 ]


Retrozymes are presently known to reach sequence sizes as small as 170nt and as big as 1,116nt. Smaller retrozymes are typically found in  invertebrates , such as a 300nt retrozyme in the genome of the  Mediterranean mussel  ( Mytilus galloprovincialis ). The largest known retrozyme is 1,116nt in length, discovered in the genome of a strain of  Jatropha curcas . [ 5 ]


Presently, the only database for retrozymes and similar elements is ViroidDB, which currently contains sequences of 73 retrozymes taken from the  National Center for Biotechnology Information  nucleotide database. [ 6 ]  Sequences of retrozymes in particular were initially directly and separately found and downloaded from  GenBank , as retrozymes currently have no taxonomic classification. [ 6 ]  Some methods have been developed to study retrozymes in the laboratory. [ 7 ]


Retrozymes differentially accumulate in different tissues of plants. Furthermore, the domesticated equivalents of some species of plants contain substantially fewer copies of retrozymes, indicating that  domestication  applies a  negative selection pressure  on retrozyme sequences. Another interesting trait of retrozymes in plants is their active transcription, even though the majority of  retrotransposons  are inactive. [ 1 ]


The smallest known retrozymes are those found in  invertebrates , where they can range from 170–400nt. They appear to be expressed in, at the least, most cell types. Just as with plants, retrozymes in animals are also expressed at high levels in both  somatic cells  and  germ cells . While retrozymes have been found in both linear and circularized forms, levels of circularized retrozymes have been seen much more abundantly  in vivo  and the linear forms may be a product of self-cleavage by the HHR motif during replication or a result of spontaneous breakage during purification. [ 2 ]


Animal retrozymes have several differences with plant retrozymes. Different proteins circularize and reversibly transcribe plant and animal retrozymes during the replication cycle. Animal retrozymes lack all the characteristic long-terminal repeats, PBSs, and PPTs known in plant retrozymes. And while plant retrozymes only have one or two copies of the HHR motif, animal retrozymes may have many such copies. Animal retrozymes also have smaller tandem repeats that are often flanked by target side duplications (TSDs). TSDs in animals are typically 8–12bp, slightly larger than the 4bp TSDs found in plants. [ 8 ]


The retrozyme sequence is first transcribed by a  polymerase  in the host. The product is an oligomeric RNA sequence which is a single transcript containing multiple copies of the retrozyme sequence. The hammerhead ribozyme motif then autocatalytically performs self-cleavage to separate the oligomeric transcript into several monomeric transcripts, each containing only one copy of the retrozyme sequence. This copy is an intermediate of the replication cycle, containing the opposite polarity of the original sequence with a 5'-hydroxyl and a 2'-3'-cyclic phosphate ends. A  ligase  protein in the host may then circularize this intermediate into a stable, circular RNA molecule. In plants, this ligase is a chloroplast tRNA ligase. Dependence on chloroplast tRNA ligase for circularization is also seen in the  Avsunviroidae  family of  viroids . In animals, the ligase is an RtcB tRNA ligase.  Reverse transcriptase  activity is required from a different  retrotransposon  to generate a corresponding  complementary DNA  of the retrozyme RNA, and the polarity of this cDNA corresponds to the polarity of the original sequence. Plant and animal retrozymes rely on different retrotransposons to produce a cDNA copy of their RNA molecule. In plants, LTR retrotransposons of the Gypsy family are used. Although it is not clear which type of retrotransposons are relied on in animals, these could be classes such as LINEs or PLEs. After the DNA copy has been produced, the retrozyme sequence has the opportunity to re-insert itself into a genomic loci. [ 2 ]


Retrozymes possess close similarities to types of  mobile genetic elements  (MGE), especially  viroids ,  satellite RNAs  (satRNAs), and  Ribozyviria  (a recently described  realm  of  viruses [ 9 ] ). For one, the hammerhead ribozyme (HHR) motif is found in all these elements. These elements also replicate through  rolling circle replication , where the HHR motif plays the  autocatalytic  role of cleaving the circular RNA molecule at a conserved site. Furthermore, all these elements depend on a host polymerase to transcribe their sequence and a ligase to recircularize them into a circular RNA molecule. Retrozymes form branched conformations, as do some satRNAs and  Avsunviroidae  (one of the two classes of viroids). [ 3 ]


Due to their simplicity, many have suggested that viroids originated and are remnants of the  RNA world . [ 10 ] [ 11 ] [ 12 ]  Other suggestions include that viroids derive from other viruses, having degenerated in size and lost any protein-coding genes. Several challenges have been raised to these suggestions. The limited range of viroids and satellite RNAs in  flowering plants  (with none discovered in  bacteria  and  archaea ) indicates that their origins post-date the emergence of  eukaryotes . [ 11 ]  The recent discovery and advances related to retrozymes have led to the current hypothesis that retrozymes were the source of the origins of viroids and satRNAs. [ 4 ]  The relationship with ribozyviruses is less straight forward. Ribozyviruses are more complex than retrozymes, viroids, and satellites. They are the only viroid-like element to harbour a protein-coding gene. This gene codes for a  capsid  which undergoes  post-translational modifications  to give rise to different forms which together perform a variety of functions in the host, enabling their lifecycle. Furthermore, ribozyviruses are only found narrowly in animal lineages whereas both viroids and satellite RNAs are only known to be infectious in plants. The narrow spread of ribozyviruses in animals, combined with strong evidence for the origins of viroids in plants, suggests that ribozyviruses are the more recent class of MGEs. Ribozyviruses may have emerged from viroids and then transferred into animals through  horizontal gene transfer , at some point acquiring a protein-coding gene. Alternatively, because retrozymes are known in both plants and animals, retrozymes may have independently given rise to ribozyviruses in animal lineages. [ 3 ]  It is unclear if viroids and other viroid-like elements emerged from retrozymes once or several times, and while they are unlikely to trace back to RNA world, some still stress their importance as minimal replicators close to the theoretical lower limit of replicator size. [ 3 ]


The  RK2 Plasmid   is a broad-host-range  plasmid  belonging to the incP incompatibility group [ 1 ]  It is notable for its ability to replicate in a wide variety of  single-celled organisms , which makes it suitable as a  genetic engineering  tool. [ 2 ]  It is capable of transfer, replication, and maintenance in most  genera  of  Gram-negative  bacteria. RK2 may sometimes be referred to as pRK2, which is also the name of another, unrelated plasmid. [ 3 ] [ 4 ] [ 5 ]  Other names for RK2 include R18, R68, RP1, and RP4. These were all separate isolates, and later found to be identical plasmids. [ 6 ]
The IncP-1 plasmid group (IncP plasmids in  Escherichia coli ) of which RK2 is a part has been described as "highly potent, self-transmissible, selfish DNA molecules with a complicated regulatory circuit" [ 7 ]


RK2 was first isolated in connection with an outbreak of  antibiotic-resistant   Pseudomonas aeruginosa  and  Klebsiella aerogenes  in  Birmingham  in 1969, as one of a family of plasmids implicated in transfer of  Ampicillin  resistance between bacterial strains. [ 8 ]  Plasmids in the IncP-1 subgroup has been isolated from wastewater, agricultural soil, and hospitals. [ 9 ]


RK2 is approximately 60  kbp  long and contains  genes  for  replication , maintenance,  conjugation  and  antibiotic resistance . The resistance genes confer resistance to the antibiotics kanamycin, ampicillin and tetracycline. [ 8 ]  In addition, RK2 contains a set of potentially lethal (to the cell) genes, called  kil  genes, and a set of complementary  transcriptional repressor  genes, called  kor  (short for "kil-override") genes, which inactivate the  kil  genes. The  kil  and  kor  genes together are suspected to play a role in the broad host range of RK2. [ 10 ]


The essential replication system in RK2 consists of an origin of replication,  oriV , and a gene,  trfA , whose gene product, the TrfA protein, binds to and activates  oriV . [ 11 ] [ 12 ]  In  Escherichia coli , replication proceeds unidirectionally from  oriV  after activation by TrfA. [ 13 ]  In E. coli, multiple plasmid copies appear to cluster together, creating a few multiplasmid clusters in each cell. [ 14 ] [ 15 ]  The copy number of RK2 is about 4-7 per cell in  E. coli  and 3 in  P. aeruginosa . [ 16 ]


Several minimal derivatives of RK2 have been prepared. In these plasmids most of the genes have been removed, leaving only genes essential for replication and one or more  selectable markers . One such "mini-replicon" is the plasmid PFF1, which is 5873 basepairs long.


PFF1 consists of an  origin of replication , oriV, an  origin of transfer , oriT, a gene coding for plasmid replication proteins, trfA, and two  antibiotic resistance  genes,  bla  and  cat , which confer resistance to  Ampicillin  and  Chloramphenicol , respectively. Minimal plasmids such as PFF1 are useful for studying the basic mechanisms of plasmid replication and copy number regulation, as there are less superfluous genetic elements which might affect the processes being studied. Several mutants of PFF1 which affect the copy number of the plasmid have been identified. Two such mutants, PFF1cop254D and PFF1cop271C, increase the copy number of PFF1 in  E. coli  from approximately 39-40 to about 501 and 113 plasmids per cell, respectively. [ 17 ]  An increase in copy number is useful for genetic engineering applications to increase the production yield of  recombinant protein . [ 18 ]


SaPI s ( Staphylococcus aureus    pathogenicity islands ) are a family of ~15 kb  mobile genetic elements  resident in the  genomes  of the vast majority of  S. aureus  strains. [ 1 ] [ 2 ]    Much like  bacteriophages , SaPIs can be transferred to uninfected cells and integrate into the host chromosome.  Unlike the bacterial viruses, however, integrated SaPIs are mobilized by host infection with "helper" bacteriophages (specific SaPIs may require specific helper bacteriophages for mobilization, though Staphylococcus phage 80alpha appears to mobilize all known SaPIs). SaPIs are used by the host bacteria to co-opt the phage reproduction cycle for their own  genetic transduction  and also inhibit phage reproduction in the process. [ 2 ]


SaPIs can infect many strains that are resistant to phages and can readily be transferred to  Listeria monocytogenes  even though staphylococcal phages cannot grow in Listeria cells. [ 3 ]   SaPIs occur infrequently in other staphylococcal species, but SaPI-like elements are common and widespread in other  Gram-positive cocci . [ 4 ]


SaPI DNA is maintained in a  prophage -like state under the control of a master repressor and is induced to excise and replicate by helper phages encoding proteins that counter the SaPI  repressor . [ 5 ]  One set of SaPIs is induced  by other co-resident SaPIs which must first be induced by helper phages, setting up a 3-way cascade [ 6 ]   For most SaPIs, following replication, concatemeric SaPI DNA is packaged by the  headful  mechanism, utilizing a small  terminase  subunit (TerS) encoded by the SaPI [ 7 ]  in phage-like particles that are released in numbers approaching 10 9 /ml by phage-induced lysis. [ 8 ]  A subset of SaPIs have acquired a  cos  site  and use the phage’s TerS for packaging by the  cos   mechanism. [ 9 ] ' The released SaPI particles infect other staphylococcal cells, injecting their DNA which integrates into a specific chromosomal attachment site [ 10 ]  of which there are 5 in  S. aureus . [ 10 ]  SaPIs are ancestrally related to  Siphoviridae  and possess phage-related genes enabling their integration/excision, replication, and packaging. [ 2 ]  They lack structural phage proteins and lysis proteins and utilize those of the helper phage, which they modify to form small  capsids  commensurate with their genomes.


Their evolutionary divergence from an ancestral prophage has involved not only the loss of structural and lytic proteins, but also the development (or acquisition) of several different means of partially blocking reproduction of their helper phages. [ 11 ] [ 12 ]  These phage interference functions are advantageous both to the host cell, as they (partially) block predation by exogenous phages, and to the SaPI, as they enable it to keep pace with helper phage reproduction. Unlike the  CRISPRs , which simply destroy infecting phages and thus totally block phage-mediated  horizontal gene transfer  (HGT), the SaPIs  are major agents of phage–dependent HGT, as they only partially block phage reproduction and also package chromosomal fragments as well as their own genomes. [ 13 ]


SaPIs were discovered on the basis of their carriage of the gene for toxic shock syndrome toxin-1 and they are uniquely responsible for staphylococcal toxic shock. They also carry other superantigen toxins as well as other virulence factors among which is a set of genes enabling their host strains to  coagulate  farm animal  blood plasma . These genes encode different  alleles  of a coagulase, the  von Willebrand factor -binding protein, which has a role in determining the animal host specificity of  S. aureus . [ 14 ]  The toxin and other accessory genes are expressed by the integrated and repressed SaPI genomes, as are the toxin genes of converting prophages.


One of the original SaPIs, SaPI2, has been converted to a non-antibiotic therapeutic agent for the treatment of staphylococcal and listerial infections. [ 15 ]  This conversion involved the removal of all toxin and other  virulence genes , and of the capsid morphogenesis genes, and the addition, by cloning, of antibacterial genes including those encoding  CRISPR/cas9  with spacers targeting conserved chromosomal genes. These cause a lethal double-stranded DNA cleavage in the target  protospacer . Alternative cargos include CRISPR/dcas9 with spacers targeting and inhibiting genes that regulate bacterial virulence, or the gene for  lysostaphin , a powerful staphylolytic enzyme. [ 16 ]  The production of these transformed SaPI2s, known as antibacterial drones (ABDs), is greatly enhanced by deleting the helper prophage’s t erS  gene so that only ABD DNA is packaged. [ 7 ]  The ABDs cure experimental staph infections in mice and are being further developed for clinical use. To preclude the development of resistance, ABDs for potential clinical use will always contain at least two different antibacterial modules that act by different mechanisms.


SCC mec , or  staphylococcal cassette chromosome  mec , is a  mobile genetic element  of  Staphylococcus   bacterial  species. This genetic sequence includes the  mecA  gene coding for resistance to the  antibiotic   methicillin  and is the only known way for  Staphylococcus  strains to spread the gene in the wild by  horizontal gene transfer . [ 1 ]  SCC mec  is a 21 to 60 kb long genetic element that confers broad-spectrum β-lactam resistance to MRSA. [ 2 ]  Moreover, additional genetic elements like Tn 554 , pT181, and pUB110 can be found in SCC mec,  which have the capability to render resistance to various non-β-lactam drugs. [ 3 ]


Not all SCC mec  elements are identical (in fact, SCC elements without the  mecA  gene do exist. [ 4 ] ) As of December 2021,  SCCmec  elements have been classified into fourteen types (I through XIV). [ 5 ]  One region is the  mec  complex including the  mecA  gene.  The other is the  ccr  gene complex including genes coding for  recombinases . [ 6 ]


The  mec  complex is divided further into five types (I through V) based on the arrangement of regulatory genetic features such as  mecR1 , an  inducer . [ 7 ]  The  mec  gene complex in SCC mec , comprising  mec  gene, its regulators ( mecR1 ,  mecI ), and insertion sequences (IS), is categorized into five classes (A to E). Class A includes  mecA , full  mecR1 ,  mecI , and IS 431 . Class B has IS 1272 ,  mecA , partial  mecR1 , and IS 431 . Class C, with two versions (C1, C2), contains  mecA , partial  mecR1 , IS 431 , differing in IS 431  orientation. Class D includes IS 431 ,  mecA , partial  mecR1 ; Class E consists of  blaZ ,  mecC ,  mecR1 ,  mecI . [ 7 ] [ 8 ] [ 9 ]


The  ccr  and  mec  gene complexes in SCC mec  are connected by joining (J) regions, considered non-essential but capable of carrying extra antimicrobial resistance determinants. [ 10 ] [ 11 ]  These are categorized as J1, J2, and J3, based on their SCC mec  positions. J1, also known as the L-C region, lies between the right chromosomal junction and upstream of the  ccr  gene. J2, previously the C-M region, is situated between the  ccr  and  mec  gene complexes. J3 (formerly the I-R region) is found downstream of the mec gene complex, extending to the left chromosomal junction. [ 12 ]


The SCC mec  found in  methicillin-resistant  Staphylococcus aureus  likely originated in  coagulase -negative staphylococcal species and was acquired by  S. aureus . [ 13 ]


Staphylococcal strains isolated from pig farms were found to carry several different types of SCC mec , suggesting that they may serve as a reservoir of these elements. [ 14 ]


Short interspersed nuclear elements  ( SINEs ) are non-autonomous,  non-coding   transposable elements (TEs)  that are about 100 to 700  base pairs  in length. [ 1 ]  They are a class of  retrotransposons , DNA elements that amplify themselves throughout  eukaryotic   genomes , often through  RNA  intermediates. SINEs compose about 13% of the  mammalian  genome. [ 2 ]


The internal regions of SINEs originate from  tRNA  and remain highly conserved, suggesting positive pressure to preserve structure and function of SINEs. [ 3 ]   While SINEs are present in many species of vertebrates and invertebrates, SINEs are often lineage specific, making them useful markers of  divergent evolution  between species. Copy number variation and  mutations  in the SINE sequence make it possible to construct  phylogenies  based on differences in SINEs between species. SINEs are also implicated in certain types of genetic disease in humans and other  eukaryotes .


In essence, short interspersed nuclear elements are genetic parasites which have evolved very early in the history of eukaryotes to utilize protein machinery within the organism as well as to co-opt the machinery from similarly parasitic genomic elements. The simplicity of these elements make them remarkably successful at persisting and amplifying (through retrotransposition)  within the genomes of eukaryotes. These "parasites" which have become ubiquitous in genomes can be very deleterious to organisms as discussed below. However, eukaryotes have been able to integrate short-interspersed nuclear elements into different signaling, metabolic and regulatory pathways and SINEs have become a great source of genetic variability. They seem to play a particularly important role in the regulation of  gene expression  and the creation of  RNA genes . This regulation extends to  chromatin  re-organization and the regulation of genomic architecture. The different lineages, mutations, and activities among eukaryotes make short-interspersed nuclear elements a useful tool in phylogenetic analysis.


SINEs are classified as non-LTR  retrotransposons  because they do not contain  long terminal repeats (LTRs) . [ 4 ]  There are three types of SINEs common to vertebrates and invertebrates: CORE-SINEs, V-SINEs, and AmnSINEs. [ 3 ]  SINEs have 50-500 base pair internal regions which contain a tRNA-derived segment with A and B boxes that serve as an internal promoter for  RNA polymerase III . [ 5 ] [ 3 ]


SINEs are characterized by their different modules, which are essentially a sectioning of their sequence. SINEs can, but do not necessarily have to possess a head, a body, and a tail. The head is at the  5' end  of short-interspersed nuclear elements and is evolutionarily derived from an RNA synthesized by RNA Polymerase III such as ribosomal RNAs and tRNAs; the 5' head is indicative of which endogenous element that SINE was derived from and was able to parasitically utilize its transcriptional machinery. [ 1 ]  For example, the 5' of the  Alu SINE  is derived from  7SL RNA , a sequence transcribed by RNA Polymerase III, giving rise to the RNA element of SRP, an abundant ribonucleoprotein. [ 6 ]  The body of SINEs possess an unknown origin but often share much homology with a corresponding  LINE  which thus allows SINEs to parasitically co-opt  endonucleases  coded by LINEs (which recognize certain sequence motifs). Lastly, the  3′ tail  of SINEs is composed of short simple repeats of varying lengths; these simple repeats are sites where two (or more) short-interspersed nuclear elements can combine to form a dimeric SINE. [ 7 ]  Short-interspersed nuclear elements which only possess a head and tail are called simple SINEs whereas short-interspersed nuclear elements which also possess a body or are a combination of two or more SINEs are complex SINEs. [ 1 ]


Short-interspersed nuclear elements are transcribed by  RNA polymerase III  which is known to transcribe  ribosomal RNA  and  tRNA , two types of RNA vital to  ribosomal  assembly and  mRNA translation . [ 8 ]  SINEs, like tRNAs and many small-nuclear RNAs possess an internal promoter and thus are transcribed differently than most protein-coding genes. [ 1 ]   In other words, short-interspersed nuclear elements have their key promoter elements within the transcribed region itself. Though transcribed by RNA polymerase III, SINEs and other genes possessing internal promoters, recruit different transcriptional machinery and factors than genes possessing upstream promoters. [ 9 ]


Changes in chromosome structure influence  gene expression  primarily by affecting the accessibility of genes to transcriptional machinery. The chromosome has a very complex and hierarchical system of organizing the genome. This system of organization, which includes  histones ,  methyl  groups,  acetyl  groups, and a variety of proteins and RNAs allows different domains within a chromosome to be accessible to polymerases,  transcription factors , and other associated proteins to different degrees. [ 10 ]   Furthermore, the shape and density of certain areas of a chromosome can affect the shape and density of neighboring (or even distant regions) on the chromosome through interaction facilitated by different proteins and elements. Non-coding RNAs  such as short-interspersed nuclear elements, which have been known to associate with and contribute to chromatin structure, can thus play huge role in regulating gene expression. [ 11 ]   Short-interspersed-nuclear-elements similarly can be involved in gene regulation by modifying genomic architecture.


In fact Usmanova et al. 2008 suggested that short-interspersed nuclear elements can serve as direct signals in  chromatin  rearrangement and structure.  The paper examined the global distribution of SINEs in mouse and human chromosomes and determined that this distribution was very similar to genomic distributions of genes and  CpG motifs . [ 12 ]  The distribution of SINEs to genes was significantly more similar than that of other non-coding genetic elements and even differed significantly from the distribution of long-interspersed nuclear elements. [ 12 ]  This suggested that the SINE distribution was not a mere accident caused by LINE-mediated retrotransposition but rather that SINEs possessed a role in gene-regulation. Furthermore, SINEs frequently contain motifs for  YY1  polycomb proteins. [ 12 ]  YY1 is a zinc-finger protein that acts as a transcriptional repressor for a wide-variety of genes essential for development and signaling. [ 13 ]   Polycomb protein YY1 is believed to mediate the activity of histone deacetylases and histone acetyltransferases to facilitate chromatin re-organization; this is often to facilitate the formation of  heterochromatin  (gene-silencing state). [ 14 ]  Thus, the analysis suggests that short-interspersed nuclear elements can function as a ‘signal-booster' in the polycomb-dependent silencing of gene-sets through chromatin re-organization. [ 12 ]   In essence, it is the cumulative effect of many types of interactions that leads to the difference between  euchromatin , which is not tightly packed and generally more accessible to transcriptional machinery, and  heterochromatin , which is tightly packed and generally not accessible to transcriptional machinery; SINEs seem to play an evolutionary role in this process.


In addition to directly affecting chromatin structure, there are a number of ways in which SINEs can potentially regulate gene expression. For example, long non-coding RNA can directly interact with transcriptional repressors and activators, attenuating or modifying their function. [ 15 ]   This type of regulation can occur in different ways: the RNA transcript can directly bind to the transcription factor as a co-regulator; also, the RNA can regulate and modify the ability of co-regulators to associate with the transcription factor. [ 15 ]   For example, Evf-2, a certain long non-coding RNA, has been known to function as a co-activator for certain homeobox transcription factors which are critical to nervous system development and organization. [ 16 ]  Furthermore, RNA transcripts can interfere with the functionality of the transcriptional complex by interacting or associating with RNA polymerases during the transcription or loading processes. [ 15 ]  Moreover, non-coding RNAs like SINEs can bind or interact directly with the DNA duplex coding the gene and thus prevent its transcription. [ 15 ]


Also, many non-coding RNAs are distributed near protein-coding genes, often in the reverse direction. This is especially true for short-interspersed nuclear elements as seen in Usmanova et al. These non-coding RNAs, which lie adjacent to or overlap gene-sets provide a mechanism by which transcription factors and machinery can be recruited to increase or repress the transcription of local genes. The particular example of SINEs potentially recruiting the YY1  polycomb  transcriptional repressor is discussed above. [ 12 ]   Alternatively, it also provides a mechanism by which local gene expression can be curtailed and regulated because the transcriptional complexes can hinder or prevent nearby genes from being transcribed. There is research to suggest that this phenomenon is particularly seen in the gene-regulation of pluripotent cells. [ 17 ]


In conclusion, non-coding RNAs such as SINEs are capable of affecting gene expression on a multitude of different levels and in different ways. Short-interspersed nuclear elements are believed to be deeply integrated into a complex regulatory network capable of fine-tuning gene expression across the eukaryotic genome.


The RNA transcribed from the short-interspersed nuclear element does not code for any protein product but is nonetheless  reverse-transcribed  and inserted back into an alternate region in the genome. For this reason, short interspersed nuclear elements are believed to have co-evolved with  long interspersed nuclear element  (LINEs), as LINEs do in fact encode protein products which enable them to be reverse- transcribed and integrated back into the genome. [ 4 ]   SINEs are  believed to have co-opted the proteins coded by LINEs which are contained in 2 reading frames.  Open reading frame  1 (ORF 1)  encodes a protein which binds to RNA and acts as a chaperone to facilitate and maintain the LINE protein-RNA complex structure. [ 18 ]  Open reading frame 2 (ORF 2) codes a protein which possesses both endonuclease and reverse transcriptase activities. [ 19 ]  This enables the LINE mRNA to be reverse-transcribed into DNA and integrated into the genome based on the sequence-motifs recognized by the protein's endonuclease domain.


LINE-1 (L1) is transcribed and retrotransposed most frequently in the  germ-line  and during early development; as a result SINEs move around the genome most during these periods. SINE transcription is down-regulated by transcription factors in  somatic cells  after early development, though stress can cause up-regulation of normally silent SINEs. [ 20 ]  SINEs can be transferred between individuals or species via  horizontal transfer  through a  viral vector . [ 21 ]


SINEs are known to share sequence homology with LINES which gives a basis by which the LINE machinery can reverse transcribe and integrate SINE transcripts. [ 22 ]  Alternately, some SINEs are believed to use a much more complex system of integrating back into the genome; this system involves the use random double-stranded DNA breaks (rather than the endonuclease coded by related long-interspersed nuclear elements creating an insertion-site). [ 22 ]  These DNA breaks are utilized to prime reverse transcriptase, ultimately integrating the SINE transcript back into the genome. [ 22 ]  SINEs nonetheless depend on enzymes coded by other DNA elements and are thus known as non-autonomous retrotransposons as they depend on the machinery of LINEs, which are known as autonomous retrotransposons. [ 23 ]


The theory that short-interspersed nuclear elements have evolved to utilize the retrotransposon machinery of long-interspersed nuclear elements is supported by studies which examine the presence and distribution of LINEs and SINEs in taxa of different species. [ 24 ]  For example, LINEs and SINEs in rodents and primates show very strong homology at the insertion-site motif. [ 24 ]  Such evidence is a basis for the proposed mechanism in which integration of the SINE transcript can be co-opted with LINE-coded protein products. This is specifically demonstrated by a detailed analysis of over 20 rodent species profiled LINEs and SINEs, mainly L1s and B1s respectively; these are families of LINEs and SINEs found at high frequencies in rodents along with other mammals. [ 24 ]  The study sought to provide phylogenetic clarity within the context of LINE and SINE activity.


The study arrived at a candidate taxa believed to be the first instance of L1 LINE extinction; it expectedly discovered that there was no evidence to suggest that B1 SINE activity occurred in species which did not have L1 LINE activity. [ 24 ]  Also, the study suggested that B1 short-interspersed nuclear element silencing in fact occurred before L1 long-interspersed nuclear element extinction; this is due to the fact that B1 SINEs are silenced in the genus most-closely related to the genus which does not contain active L1 LINEs (though the genus with B1 SINE silencing still contains active L1 LINEs). [ 24 ]   Another genus was also found which similarly contained active L1 long-interspersed nuclear elements but did not contain B1 short-interspersed nuclear elements; the opposite scenario, in which active B1 SINEs were present in a genus which did not possess active L1 LINEs was not found. [ 24 ]  This result was expected and strongly supports the theory that SINEs have evolved to co-opt the RNA-binding proteins, endonucleases, and reverse-transcriptases coded by LINEs. In taxa which do not actively transcribe and translate long-interspersed nuclear elements protein-products, SINEs do not have the theoretical foundation by which to retrotranspose within the genome. The results obtained in Rinehart et al. are thus very supportive of the current model of SINE retrotransposition.


Insertion of a SINE upstream of a coding region may result in  exon shuffling  or changes to the regulatory region of the gene. Insertion of a SINE into the coding sequence of a gene can have deleterious effects and unregulated transposition can cause  genetic disease . The transposition and recombination of SINEs and other active nuclear elements is thought to be one of the major contributions of genetic diversity between lineages during speciation. [ 21 ]


Short-interspersed nuclear elements are believed to have  parasitic  origins in eukaryotic genomes. These SINEs have mutated and replicated themselves a large number of times on an evolutionary time-scale and thus form many different lineages. Their early evolutionary origin has caused them to be ubiquitous in many eukaryotic lineages.


Alu elements , short-interspersed nuclear element of about 300 nucleotides, are the most common SINE in humans, with >1,000,000 copies throughout the genome, which is over 10 percent of the total genome; this is not uncommon among other species. [ 25 ]  Alu element copy number differences can be used to distinguish between and construct phylogenies of primate species. [ 21 ]  Canines differ primarily in their abundance of SINEC_Cf repeats throughout the genome, rather than other gene or allele level mutations. These dog-specific SINEs may code for a splice acceptor site, altering the sequences that appear as exons or introns in each species. [ 26 ]


Apart from mammals, SINEs can reach high copy numbers in a range of species, including nonbony vertebrates (elephant shark) and some fish species (coelacanths). [ 27 ]  In plants, SINEs are often restricted to closely related species and have emerged, decayed, and vanished frequently during evolution. [ 28 ]  Nevertheless, some SINE families such as the Au-SINEs [ 29 ]  and the Angio-SINEs [ 30 ]  are unusually widespread across many often unrelated plant species.


There are >50 human diseases associated with SINEs. [ 20 ]  When inserted near or within the exon, SINEs can cause improper splicing, become coding regions, or change the  reading frame , often leading to disease phenotypes in humans and other animals. [ 26 ]  Insertion of Alu elements in the human genome is associated with  breast cancer ,  colon cancer ,  leukemia ,  hemophilia ,  Dent's disease ,  cystic fibrosis ,  neurofibromatosis , and many others. [ 4 ]


The role of short-interspersed nuclear elements in gene regulation within cells has been supported by multiple studies. One such study examined the correlation between a certain family of SINEs with  microRNAs  (in  zebrafish ). [ 31 ]  The specific family of SINEs being examined was the Anamnia V-SINEs; this family of short interspersed nuclear elements is often found in the untranslated region of the 3' end of many genes and is present in vertebrate genomes. [ 31 ]   The study involved a computational analysis in which the genomic distribution and activity of the Anamnia V-SINEs in  Danio rerio  zebrafish was examined; furthermore, these V-SINEs potential to generate novel microRNA loci was analyzed. [ 31 ]   It was found that genes which were predicted to possess V-SINEs were targeted by microRNAs with significantly higher hybridization E-values (relative to other areas in the genome). [ 31 ]   The genes that had high hybridization E-values were genes particularly involved in metabolic and signaling pathways. [ 31 ]  Almost all miRNAs identified to have a strong ability to hybridize to putative V-SINE sequence motifs in genes have been identified (in mammals) to have regulatory roles. [ 31 ]  These results which establish a correlation between short-interspersed nuclear elements and different regulatory microRNAs strongly suggest that V-SINEs have a significant role in attenuating responses to different signals and stimuli related to metabolism, proliferation and differentiation. Many other studies must be undertaken to establish the validity and extent of short-interspersed nuclear element retrotransposons' role in regulatory gene-expression networks. In conclusion, though not much is known about the role and mechanism by which SINEs generate miRNA gene loci  it is generally understood that SINEs have played a significant evolutionary role in the creation of "RNA-genes", this is also touched upon above in SINEs and pseudogenes.


With such evidence suggesting that short-interspersed nuclear elements have been evolutionary sources for microRNA loci generation it is important to further discuss the potential relationships between the two as well as the mechanism by which the microRNA regulates RNA degradation and more broadly, gene expression. A microRNA is a non-coding RNA generally 22 nucleotides in length. [ 32 ]  This non-protein coding oligonucleotide is usually transcribed from a longer nuclear DNA sequence by RNA polymerase II which is also responsible for the transcription of most mRNAs and snRNAs in eukaryotes. [ 33 ]   However, some research suggests that some microRNAs that possess upstream short-interspersed nuclear elements are transcribed by RNA polymerase III which is widely implicated in ribosomal RNA and tRNA, two transcripts vital to mRNA translation. [ 34 ]   This provides an alternate mechanism by which short-interspersed nuclear elements could be interacting with or mediating gene-regulatory networks involving microRNAs.


The genomic regions producing miRNA can be independent RNA-genes often being anti-sense to neighboring protein-coding genes, or can be found within the introns of protein-coding genes. [ 35 ]  The co-localization of microRNA and protein-coding genes provides a mechanistic foundation by which microRNA regulates gene-expression. Furthermore, Scarpato et al.  reveals (as discussed above) that genes predicted to possess short-interspersed nuclear elements (SINEs) through sequence analysis were targeted and hybridized by microRNAs significantly greater than other genes. [ 31 ]  This provides an evolutionarily path by which the parasitic SINEs were co-opted and utilized to form RNA-genes (such as microRNAs) which have evolved to play a role in complex gene-regulatory networks.


The microRNAs are transcribed as part of longer RNA strands of generally about 80 nucleotides which through complementary base-pairing are able to form hairpin loop structures [ 36 ]  These structures are recognized and processed in the nucleus by the nuclear protein DiGeorge Syndrome Critical Region 8 (DGCR8) which recruits and associates with the Drosha protein. [ 37 ]   This complex is responsible for cleaving some of the hair-pin structures from the pre-microRNA which is transported to the cytoplasm. The pre-miRNA is processed by the protein DICER into a double stranded 22 nucleotide. [ 38 ]   Thereafter, one of the strands is incorporated into a multi-protein  RNA-induced silencing complex  (RISC). [ 39 ]  Among these proteins are proteins from the Argonaute family which are critical to the complex's ability to interact with and repress the translation of the target mRNA. [ 40 ]


Understanding the different ways in which microRNA regulates gene-expression, including mRNA-translation and degradation is key to understanding the potential evolutionary role of SINEs in gene-regulation and in the generation of microRNA loci. This, in addition to SINEs' direct role in regulatory networks (as discussed in SINEs as long non-coding RNAs) is crucial to beginning to understand the relationship between SINEs and certain diseases. Multiple studies have suggested that increased SINE activity is correlated with certain gene-expression profiles and post-transcription regulation of certain genes. [ 41 ] [ 42 ] [ 43 ]   In fact, Peterson et al. 2013 demonstrated that high SINE RNA expression correlates with post-transcriptional downregulation of  BRCA1 , a tumor suppressor implicated in multiple forms of cancer, namely breast cancer. [ 43 ]  Furthermore, studies have established a strong correlation between transcriptional mobilization of SINEs and certain cancers and conditions such as hypoxia; this can be due to the genomic instability caused by SINE activity as well as more direct-downstream effects. [ 42 ]   SINEs have also been implicated in countless other diseases. In essence, short-interspersed nuclear elements have become deeply integrated in countless regulatory, metabolic and signaling pathways and thus play an inevitable role in causing disease. Much is still to be known about these genomic parasites but it is clear they play a significant role within eukaryotic organisms.


The activity of SINEs however has genetic vestiges which do not seem to play a significant role, positive or negative, and manifest themselves in the genome as  pseudogenes .  SINEs however should not be mistaken as RNA pseudogenes. [ 1 ]  In general, pseudogenes are generated when processed mRNAs of protein-coding genes are reverse-transcribed and incorporated back into the genome (RNA pseudogenes are reverse transcribed RNA genes). [ 44 ]  Pseudogenes are generally functionless as they descend from processed RNAs independent of their evolutionary-context which includes introns and different regulatory elements which enable transcription and processing. These pseudogenes, though non-functional may in some cases still possess promoters, CpG islands, and other features which enable transcription; they thus can still be transcribed and may possess a role in the regulation of gene expression (like SINEs and other non-coding elements). [ 44 ]   Pseudogenes thus differ from SINEs in that they are derived from transcribed- functional RNA whereas SINEs are DNA elements which retrotranspose by co-opting RNA genes transcriptional machinery. However, there are studies which suggest  that retro-transposable elements such as short-interspersed nuclear elements are not only capable of copying themselves in alternate regions in the genome but are also able to do so for random genes too. [ 45 ] [ 46 ]   Thus SINEs can be playing a vital role in the generation of pseudogenes, which themselves are known to be involved in regulatory networks. This is perhaps another means by which SINEs have been able to influence and contribute to gene-regulation.


A  simple transposon  also called "conservative  transposon " is an  insertion sequence  (IS element) that contains its own coding  transposase  between the short, inverted,  repeated sequences  that flank (present) its gene coding region. Transposase is responsible for excision and transfer while resolvase is responsible for resolution of the transfer. [ 1 ]


Simple transposition is also called cut-and-paste transposition because the element is cut out of its original site and pasted into a new one. [ 2 ]


Sister chromatid exchange  ( SCE ) is the exchange of genetic material between two identical  sister chromatids .


It was first discovered by using the  Giemsa staining  method on one  chromatid  belonging to the sister chromatid complex before  anaphase  in  mitosis . The staining revealed that few segments were passed to the sister chromatid which were not dyed.
The Giemsa staining was able to stain due to the presence of  bromodeoxyuridine  analogous base which was introduced to the desired chromatid.


The reason for the (SCE) is not known but it is required and used as a  mutagenic  testing of many products. Four to five sister chromatid exchanges per chromosome pair, per mitosis is in the normal distribution, while 14–100 exchanges is not normal and presents a danger to the organism. SCE is elevated in pathologies including  Bloom syndrome , having recombination rates ~10–100 times above normal, depending on cell type. [ 1 ] [ 2 ]  Frequent SCEs may also be related to formation of  tumors .


Sister chromatid exchange has also been observed more frequently in  B51 (+)  Behçet's disease . [ 3 ]


Mitotic  recombination in the budding yeast  Saccharomyces cerevisiae  is primarily a result of  DNA repair  processes responding to spontaneous or induced damages that occur during vegetative growth. [ 4 ] } (Also reviewed in Bernstein and Bernstein, pp 220–221 [ 5 ] ).  In order for yeast cells to repair damage by  homologous recombination , there must be present, in the same nucleus, a second DNA molecule containing sequence homology with the region to be repaired.  In a  diploid  cell in  G1 phase  of the cell cycle, such a molecule is present in the form of the homologous chromosome.  However, in the  G2 phase  of the cell cycle (following DNA replication), a second homologous DNA molecule is also present: the  sister chromatid .  Evidence indicates that, due to the special nearby relationship they share, sister chromatids are not only preferred over distant homologous chromatids as substrates for recombinational repair, but have the capacity to repair more DNA damage than do homologs. [ 6 ]


The  genomes  of  diploid  organisms in natural populations are highly polymorphic for  insertions  and  deletions .  During  meiosis  double-strand breaks (DSBs) that form within such polymorphic regions must be repaired by inter- sister chromatid  exchange, rather than by inter-homolog exchange.  A molecular-level study of recombination during budding yeast meiosis has shown that recombination events initiated by DSBs in regions that lack corresponding sequences in the non-sister homolog are efficiently repaired by inter-sister chromatid recombination. [ 7 ]   This recombination occurs with the same timing as inter-homolog recombination, but with reduced (2- to 3-fold) yields of  Holliday junction  joint molecules.  This study, and comparable evidence from other organisms (e.g. Peacock [ 8 ] ), indicates that inter-sister recombination occurs frequently during meiosis, and up to one-third of all recombination events occur between sister chromatids, although mainly by a pathway that does not involve Holliday junction intermediates. [ 7 ]


During  oogenesis  in the nematode  Caenorhabditis elegans  the sister chromatid, or even the same  DNA  molecule, can serve as a  meiotic  repair template for both crossover and non- crossover recombination . [ 9 ]  Non-crossover events are the most frequent  recombination  outcome.  For DNA double strand breaks induced throughout meiotic prophase I, the sister or intra-chromatid substrate is available as a recombinational repair partner. [ 9 ]


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  Sleeping Beauty  transposon system is composed of a  Sleeping Beauty  (SB)  transposase  and a  transposon  that was designed in 1997 to insert specific sequences of  DNA  into genomes of vertebrate animals. DNA transposons translocate from one DNA site to another in a simple, cut-and-paste manner (Fig. 1).  Transposition is a precise process in which a defined DNA segment is excised from one DNA molecule and moved to another site in the same or different DNA molecule or  genome . [ 1 ]


As do all other  Tc1/mariner -type transposases, SB transposase inserts a transposon into a TA dinucleotide  base pair  in a recipient DNA sequence. [ 2 ]  The insertion site can be elsewhere in the same DNA molecule, or in another DNA molecule (or chromosome). In mammalian genomes, including humans, there are approximately 200 million TA sites. The TA insertion site is duplicated in the process of transposon integration. This duplication of the TA sequence is a hallmark of transposition and used to ascertain the mechanism in some experiments. However, a recent study indicated that SB also integrates into non-TA dinucleotides at a low frequency. [ 3 ]  The transposase can be encoded either within the transposon (e.g., the putative transposon shown in Fig. 2) or the transposase can be supplied by another source, in which case the transposon becomes a non-autonomous element. Non-autonomous transposons (e.g., Fig. 1) are most useful as genetic tools because after insertion they cannot independently continue to excise and re-insert. All of the DNA transposons identified in the human genome and other mammalian genomes are non-autonomous because even though they contain transposase genes, the genes are non-functional and unable to generate a transposase that can mobilize the transposon.


This resurrected transposase gene was named " Sleeping Beauty (SB) " because it was brought back to activity from a long evolutionary sleep. [ 4 ]   The SB transposon system is synthetic in that the SB transposase was re-constructed from extinct (fossil) transposase sequences belonging to the Tc1/mariner class of transposons [ 5 ] [ 6 ]  found in the genomes of salmonid fish. [ 7 ]  As in humans, where about 20,000 inactivated Tc1/mariner-type transposons comprise almost 3% of the  human genome , [ 8 ] [ 9 ]  the transposase genes found in fish have been inactive for more than 10 million years due to accumulated mutations. The reconstruction of SB transposase was based on the concept that there was a primordial Tc1-like transposon that was the ancestor to the sequences found in fish genomes. Although there were many sequences that looked like Tc-1 transposons in all the fish genomes studied, the transposon sequences were all inactive due to mutations. By assuming that the variations in sequences were due to independent mutations that accumulated in the different transposons, a putative ancestral transposon (Fig. 2) was postulated. [ 10 ]


The construction for the transposase began by fusing portions of two inactive transposon sequences from  Atlantic salmon  ( Salmo salar ) and one inactive transposon sequence from  rainbow trout  ( Oncorhynchus mykiss ) and then repairing small deficits in the functional domains of the transposase  enzyme  (Fig. 3). Each  amino acid  in the first completed transposase, called SB10, was determined by a “majority-rule  consensus  sequence” based on 12 partial genes found in eight fish species. The first steps (1->3 in Fig. 3) were to restore a complete protein by filling in gaps in the sequence and reversing termination codons that would keep the putative 360-amino acid polypeptide from being synthesized. The next step (4 in Fig. 3) was to reverse mutations in the  nuclear localization signal  (NLS) that is required to import the transposase enzyme from the  cytoplasm  where it is made to the  nucleus  where it acts. The amino-terminus of the transposase, which contains the DNA-binding motifs for recognition of the direct repeats (DRs), was restored in steps 5->8. The last two steps restored the catalytic domain, which features conserved  aspartic acid  ( D ) and  glutamic acid  ( E ) amino acids with specific spacing that are found in  integrases  and  recombinases . [ 11 ]  The result was SB10, which contains all of the motifs required for function. [ 4 ]


SB10 transposase has been improved over the decade since its construction by increasing the consensus with a greater number of extinct Tc1 transposon sequences and testing various combinations of changes. [ 12 ] [ 13 ] [ 14 ] [ 15 ] [ 16 ] [ 17 ]  Further work has shown that the DNA-binding domain consists of two paired sequences, which are homologous to sequence motifs found in certain transcription factors. The paired subdomains in SB transposase were designated PAI and RED. The PAI subdomain plays a dominant role in recognition of the DR sequences in the transposon. The RED subdomain overlaps with the nuclear localization signal, but its function remains unclear. [ 18 ]  The most recent version of SB transposase, SB100X, has about 100 times the activity of SB10 as determined by transposition assays of antibiotic-resistance genes conducted in tissue cultured human HeLa cells. [ 16 ]  The International Society for Molecular and Cell Biology and Biotechnology Protocols and Research (ISMCBBPR) named SB100X the molecule of the year for 2009 for recognition of the potential it has in future genome engineering. [ 19 ]


The transposon recognized by SB transposase was named T because it was isolated from the genome of another salmond fish,  Tanichthys albonubes . The transposon consists of a genetic sequence of interest that is flanked by  inverted repeats  (IRs) that themselves contain short direct repeats (DR) (tandem arrowheads IR-DR in Figs. 1 and 2). T had the closest IR/DR sequence to the consensus sequence for the extinct Tc-1 like transposons in fish. The consensus transposon has IRs of 231 base pairs.  The innermost DRs are 29 base pairs long whereas the outermost DRs are 31 base pairs long. The difference in length is critical for maximal transposition rates. [ 20 ]  The original T transposon component of the SB transposon system has been improved with minor changes to conform to the consensus of many related extinct and active transposons. [ 20 ] [ 21 ]


Over the past decade, SB transposons have been developed as non-viral vectors for introduction of genes into genomes of vertebrate animals and for  gene therapy . The genetic cargo can be an  expression cassette —a  transgene  and associated elements that confer  transcriptional regulation  for expression at a desired level in specific tissue(s). An alternative use of SB transposons is to discover functions of genes, especially those that cause  cancer , [ 22 ] [ 23 ]  by delivering DNA sequences that maximally disrupt expression of genes close to the insertion site. This process is referred to as  insertional mutagenesis  or  transposon mutagenesis . When a gene is inactivated by insertion of a transposon (or other mechanism), that gene is “knocked out”.  Knockout mice  and  knockout rats  have been made with the SB system. [ 24 ] [ 25 ]   Figure 4 illustrates these two uses of SB transposons.


For either gene delivery or gene disruption, SB transposons combine the advantages of viruses and naked DNA. Viruses have been evolutionarily selected based on their abilities to infect and replicate in new host cells. Simultaneously, cells have evolved major molecular defense mechanisms to protect themselves against viral infections. For some applications of genome engineering such as some forms of gene therapy, [ 26 ] [ 27 ] [ 28 ]  avoiding the use of viruses is also important for social and regulatory reasons. The use of non-viral vectors avoids many, but not all, of the defenses that cells employ against vectors.


Plasmids , the circular DNAs shown in Fig. 1, are generally used for non-viral gene delivery. However, there are two major problems with most methods for delivering DNA to cellular chromosomes using plasmids, the most common form of non-viral gene delivery. First, expression of transgenes from plasmids is brief due to lack of integration and due to cellular responses that turn off expression. Second, uptake of plasmid molecules into cells is difficult and inefficient. The Sleeping Beauty Transposon System was engineered to overcome the first problem. DNA transposons precisely insert defined DNA sequences (Fig. 1) almost randomly into host genomes thereby increasing the longevity of gene expression (even through multiple generations). Moreover, transposition avoids the formation of multiple, tandem integrations, which often results in switching off expression of the transgene. Currently, insertion of transgenes into chromosomes using plasmids is much less efficient than using viruses. However, by using powerful promoters to regulate expression of a transgene, delivery of transposons to a few cells can provide useful levels of secreted gene products for an entire animal. [ 29 ] [ 30 ]


Arguably the most exciting potential application of  Sleeping Beauty  transposons will be for human gene therapy. The widespread human application of gene therapy in first-world nations as well as countries with developing economies can be envisioned if the costs of the vector system are affordable. Because the SB system is composed solely of DNA, the costs of production and delivery are considerably reduced compared to viral vectors. The first clinical trials using SB transposons in genetically modified T cells will test the efficacy of this form of gene therapy in patients at risk of death from advanced malignancies. [ 31 ]


A  transposable element  ( TE ), also    transposon , or  jumping gene , is a type of  mobile genetic element , 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 . [ 1 ] [ 2 ]


Transposition often results in duplication of the same genetic material. The discovery of mobile genetic elements earned  Barbara McClintock  a  Nobel Prize  in 1983. [ 3 ]  Further research into transposons has potential for use in  gene therapy , and the finding of new drug targets in  personalized medicine . The vast number of variables in the transposon makes  data analytics  difficult but combined with other sequencing technologies significant advances may be made in the understanding and treatment of disease. [ 4 ]


Transposable elements make up about half of the genome in a  eukaryotic cell , accounting for much of human  genetic diversity . [ 4 ]   Although TEs are  selfish genetic elements , many are important in genome function and evolution. [ 5 ]  Transposons are also very useful to researchers as a means to alter DNA inside a living organism.


There are at least two classes of TEs: Class I TEs or  retrotransposons  generally function via  reverse transcription , while Class II TEs or  DNA transposons  encode the protein  transposase , which they require for insertion and excision, and some of these TEs also encode other proteins. [ 6 ]


Barbara McClintock  discovered the first TEs in  maize  ( Zea mays ) at the  Cold Spring Harbor Laboratory  in New York. McClintock was experimenting with maize plants that had broken chromosomes. [ 7 ]


In the winter of 1944–1945, McClintock planted corn kernels that were self-pollinated, meaning that the silk ( style ) of the flower received pollen from its own  anther . [ 7 ]  These kernels came from a long line of plants that had been self-pollinated, causing broken arms on the end of their ninth chromosomes. [ 7 ]  As the maize plants began to grow, McClintock noted unusual color patterns on the leaves. [ 7 ]  For example, one leaf had two albino patches of almost identical size, located side by side on the leaf. [ 7 ]  McClintock hypothesized that during cell division certain cells lost genetic material, while others gained what they had lost. [ 8 ]  However, when comparing the chromosomes of the current generation of plants with the parent generation, she found certain parts of the chromosome had switched position. [ 8 ]  This refuted the popular genetic theory of the time that genes were fixed in their position on a chromosome. McClintock found that genes could not only move but they could also be turned on or off due to certain environmental conditions or during different stages of cell development. [ 8 ]


McClintock also showed that gene mutations could be reversed. [ 9 ]  She presented her report on her findings in 1951, and published an article on her discoveries in  Genetics  in November 1953 entitled "Induction of Instability at Selected Loci in Maize". [ 10 ]


At the 1951 Cold Spring Harbor Symposium where she first publicized her findings, her talk was met with silence. [ 11 ]  Her work was largely dismissed and ignored until the late 1960s–1970s when, after TEs were found in bacteria, it was rediscovered. [ 12 ]  She was awarded a  Nobel Prize in Physiology or Medicine  in 1983 for her discovery of TEs, more than thirty years after her initial research. [ 13 ]


Transposable elements represent one of several types of  mobile genetic elements . TEs are assigned to one of two classes according to their mechanism of transposition, which can be described as either  copy and paste  (Class I TEs) or  cut and paste  (Class II TEs). [ 14 ]


Class I TEs are copied in two stages: first, they are  transcribed  from DNA to  RNA , and the RNA produced is then  reverse transcribed  to DNA. This  copied DNA  is then inserted back into the genome at a new position. The reverse transcription step is catalyzed by a  reverse transcriptase , which is often encoded by the TE itself. The characteristics of retrotransposons are similar to  retroviruses , such as  HIV .


Despite the potential negative effects of retrotransposons, like inserting itself into the middle of a necessary DNA sequence, which can render important genes unusable, they are still essential to keep a species'  ribosomal DNA  intact over the generations, preventing infertility. [ 15 ]


Retrotransposons are commonly grouped into three main orders:


Retroviruses can also be considered TEs. For example, after the conversion of retroviral RNA into DNA inside a host cell, the newly produced retroviral DNA is integrated into the  genome  of the host cell. These integrated DNAs are termed  proviruses . The provirus is a specialized form of  eukaryotic  retrotransposon, which can produce RNA intermediates that may leave the host cell and infect other cells. The transposition cycle of retroviruses has similarities to that of  prokaryotic  TEs, suggesting a distant relationship between the two.


The cut-and-paste transposition mechanism of class II TEs does not involve an RNA intermediate. The transpositions are catalyzed by several  transposase  enzymes. Some transposases non-specifically bind to any target site in DNA, whereas others bind to specific target sequences. The transposase makes a staggered cut at the target site producing  sticky ends , cuts out the DNA transposon and ligates it into the target site. A  DNA polymerase  fills in the resulting gaps from the sticky ends 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 TE  excision  by  transposase ).


Cut-and-paste TEs may be duplicated if their transposition takes place during  S phase  of the  cell cycle , when a donor site has already been replicated but a target site has not yet been replicated. [ citation needed ]  Such duplications at the target site can result in  gene duplication , which plays an important role in genomic  evolution . [ 17 ] : 284


Not all DNA transposons transpose through the cut-and-paste mechanism. In some cases, a  replicative transposition  is observed in which a transposon replicates itself to a new target site (e.g.  helitron ).


Class II TEs comprise less than 2% of the human genome, making the rest Class I. [ 18 ]


Transposition can be classified as either "autonomous" or "non-autonomous" in both Class I and Class II TEs. Autonomous TEs can move by themselves, whereas non-autonomous TEs require the presence of another TE to move. This is often because dependent TEs lack transposase (for Class II) or reverse transcriptase (for Class I).


Activator element ( Ac ) is an example of an autonomous TE, and dissociation elements ( Ds ) is an example of a non-autonomous TE. Without  Ac,   Ds  is not able to transpose.


Some researchers also identify a third class of transposable elements, [ 19 ]  which has been described as "a grab-bag consisting of transposons that don't clearly fit into the other two categories". [ 20 ]  Examples of such TEs are the Foldback (FB) elements of  Drosophila melanogaster , the TU elements of  Strongylocentrotus purpuratus , and  Miniature Inverted-repeat Transposable Elements . [ 21 ] [ 22 ]


Approximately 64% of the maize genome is made up of TEs, [ 23 ] [ 24 ]  as is 44% of the human genome, [ 25 ]  and almost half of  murine  genomes. [ 26 ]


New discoveries of transposable elements have shown the exact distribution of TEs with respect to their transcription start sites (TSSs) and enhancers. A recent study found that a promoter contains 25% of regions that harbor TEs. It is known that older TEs are not found in TSS locations because TEs frequency starts as a function once there is a distance from the TSS. A possible theory for this is that TEs might interfere with the transcription pausing or the first-intro splicing. [ 27 ]  Also as mentioned before, the presence of TEs closed by the TSS locations is correlated to their evolutionary age (number of different mutations that TEs can develop during the time).


Transposons have coexisted with eukaryotes for thousands of years and through their coexistence have become integrated in many organisms' genomes. Colloquially known as 'jumping genes', transposons can move within and between genomes allowing for this integration.


While there are many positive effects of transposons in their host eukaryotic genomes, [ further explanation needed ]  there are some instances of mutagenic effects that TEs have on genomes leading to disease and malignant genetic alterations. [ 41 ]


TEs are  mutagens  and due to the contribution to the formation of new cis-regulatory DNA elements that are connected to many transcription factors that are found in living cells; TEs can undergo many evolutionary mutations and alterations. These are often the causes of genetic disease, and gives the potential lethal effects of ectopic expression. [ 27 ]


TEs can damage the genome of their host cell in different ways: [ 41 ]


TEs use a number of different mechanisms to cause genetic instability and disease in their host genomes.


Diseases often caused by TEs include


One study estimated the rate of transposition of a particular retrotransposon, the  Ty1  element in  Saccharomyces cerevisiae . Using several assumptions, the rate of successful transposition event per single Ty1 element came out to be about once every few months to once every few years. [ 49 ]  Some TEs contain  heat-shock like  promoters and their rate of transposition increases if the cell is subjected to stress, [ 50 ]  thus increasing the mutation rate under these conditions, which might be beneficial to the cell.


Cells defend against the proliferation of TEs in a number of ways. These include  piRNAs  and  siRNAs , [ 51 ]  which  silence  TEs after they have been transcribed.


If organisms are mostly composed of TEs, one might assume that disease caused by misplaced TEs is very common, but in most cases TEs are silenced through  epigenetic  mechanisms like  DNA methylation , chromatin remodeling and piRNA, such that little to no phenotypic effects nor movements of TEs occur as in some wild-type plant TEs. Certain mutated plants have been found to have defects in methylation-related enzymes (methyl transferase) which cause the transcription of TEs, thus affecting the phenotype. [ 6 ] [ 52 ]


One hypothesis suggests that only approximately 100 LINE1 related sequences are active, despite their sequences making up 17% of the human genome. In human cells, silencing of LINE1 sequences is triggered by an  RNA interference  (RNAi) mechanism. Surprisingly, the RNAi sequences are derived from the 5′ untranslated region (UTR) of the LINE1, a long terminal which repeats itself. Supposedly, the 5′ LINE1 UTR that codes for the sense promoter for LINE1 transcription also encodes the antisense promoter for the  miRNA  that becomes the substrate for siRNA production. Inhibition of the RNAi silencing mechanism in this region showed an increase in LINE1 transcription. [ 6 ] [ 53 ]


TEs are found in almost all life forms, and the scientific community is still exploring their evolution and their effect on genome evolution. It is unclear whether TEs originated in the  last universal common ancestor , arose independently multiple times, or arose once and then spread to other kingdoms by  horizontal gene transfer . [ 54 ]  While some TEs confer benefits on their hosts, most are regarded as  selfish DNA   parasites . In this way, they are similar to  viruses . Various viruses and TEs also share features in their genome structures and biochemical abilities, leading to speculation that they share a common ancestor. [ 55 ]


Because excessive TE activity can damage  exons , many organisms have acquired mechanisms to inhibit their activity. Bacteria may undergo high rates of  gene deletion  as part of a mechanism to remove TEs and viruses from their genomes, while  eukaryotic  organisms typically use  RNA interference  to inhibit TE activity. Nevertheless, some TEs generate large families often associated with  speciation  events. [ 56 ]  Evolution often deactivates DNA transposons, leaving them as  introns  (inactive gene sequences). In vertebrate animal cells, nearly all 100,000+ DNA transposons per genome have genes that encode inactive transposase polypeptides. [ 57 ]  The first synthetic transposon designed for use in vertebrate (including human) cells, the  Sleeping Beauty transposon system , is a Tc1/mariner-like transposon. Its dead ("fossil") versions are spread widely in the salmonid genome and a functional version was engineered by comparing those versions. [ 58 ]  Human Tc1-like transposons are divided into Hsmar1 and Hsmar2 subfamilies. Although both types are inactive, one copy of Hsmar1 found in the  SETMAR  gene is under selection as it provides DNA-binding for the histone-modifying protein. [ 59 ]  Many other human genes are similarly derived from transposons. [ 60 ]  Hsmar2 has been reconstructed multiple times from the fossil sequences. [ 61 ]


The frequency and location of TE integrations influence genomic structure and evolution and affect gene and protein regulatory networks during development and in differentiated cell types. [ 62 ]  Large quantities of TEs within genomes may still present evolutionary advantages, however.  Interspersed repeats  within genomes are created by transposition events accumulating over evolutionary time. Because interspersed repeats block  gene conversion , they protect novel gene sequences from being overwritten by similar gene sequences and thereby facilitate the development of new genes. TEs may also have been co-opted by the  vertebrate immune system  as a means of producing antibody diversity. The  V(D)J recombination  system operates by a mechanism similar to that of some TEs. TEs also serve to generate repeating sequences that can form  dsRNA  to act as a substrate for the action of  ADAR  in RNA editing. [ 63 ]


TEs can contain many types of genes, including those conferring antibiotic resistance and the ability to transpose to conjugative plasmids. Some TEs also contain  integrons , genetic elements that can capture and express genes from other sources. These contain  integrase , which can integrate  gene cassettes . There are over 40 antibiotic resistance genes identified on cassettes, as well as virulence genes.


Transposons do not always excise their elements precisely, sometimes removing the adjacent base pairs; this phenomenon is called  exon shuffling . Shuffling two unrelated exons can create a novel gene product or, more likely, an intron. [ 64 ]


Some non-autonomous DNA TEs found in plants can capture coding DNA from genes and shuffle them across the genome. [ 65 ]  This process can duplicate genes in the genome (a phenomenon called transduplication), and can contribute to generate novel genes by exon shuffling. [ 66 ]


There is a hypothesis that states that TEs might provide a ready source of DNA that could be co-opted by the cell to help regulate gene expression. Research showed that many diverse modes of TEs co-evolution along with some transcription factors targeting TE-associated genomic elements and chromatin are evolving from TE sequences. Most of the time, these particular modes do not follow the simple model of TEs and regulating host gene expression. [ 27 ]


Transposable elements can be harnessed in laboratory and research settings to study genomes of organisms and even engineer genetic sequences. The use of transposable elements can be split into two categories: for genetic engineering and as a genetic tool.


In addition to the qualities mentioned for Genetic engineering, a Genetic tool also:-


De novo  repeat identification is an initial scan of sequence data that seeks to find the repetitive regions of the genome, and to classify these repeats. Many computer programs exist to perform  de novo  repeat identification, all operating under the same general principles. [ 68 ]  As short tandem repeats are generally 1–6 base pairs in length and are often consecutive, their identification is relatively simple. [ 67 ]  Dispersed repetitive elements, on the other hand, are more challenging to identify, due to the fact that they are longer and have often acquired mutations. However, it is important to identify these repeats as they are often found to be transposable elements (TEs). [ 68 ]


De novo  identification of transposons involves three steps: 1) find all repeats within the genome, 2) build a  consensus  of each family of sequences, and 3) classify these repeats.  There are three groups of algorithms for the first step. One group is referred to as the  k-mer  approach, where a k-mer is a sequence of length k. In this approach, the genome is scanned for overrepresented k-mers; that is, k-mers that occur more often than is likely based on probability alone. The length k is determined by the type of transposon being searched for. The k-mer approach also allows mismatches, the number of which is determined by the analyst. Some k-mer approach programs use the k-mer as a base, and extend both ends of each repeated k-mer until there is no more similarity between them, indicating the ends of the repeats. [ 68 ]  Another group of algorithms employs a method called sequence self-comparison. Sequence self-comparison programs use databases such as  AB-BLAST  to conduct an initial  sequence alignment . As these programs find groups of elements that partially overlap, they are useful for finding highly diverged transposons, or transposons with only a small region copied into other parts of the genome. [ 69 ]  Another group of algorithms follows the periodicity approach. These algorithms perform a  Fourier transformation  on the sequence data, identifying periodicities, regions that are repeated periodically, and are able to use peaks in the resultant spectrum to find candidate repetitive elements. This method works best for tandem repeats, but can be used for dispersed repeats as well. However, it is a slow process, making it an unlikely choice for genome-scale analysis. [ 68 ]


The second step of  de novo  repeat identification involves building a consensus of each family of sequences. A  consensus sequence  is a sequence that is created based on the repeats that comprise a TE family. A base pair in a consensus is the one that occurred most often in the sequences being compared to make the consensus. For example, in a family of 50 repeats where 42 have a T base pair in the same position, the consensus sequence would have a T at this position as well, as the base pair is representative of the family as a whole at that particular position, and is most likely the base pair found in the family's ancestor at that position. [ 68 ]  Once a consensus sequence has been made for each family, it is then possible to move on to further analysis, such as TE classification and genome masking in order to quantify the overall TE content of the genome.


Transposable elements have been recognized as good candidates for stimulating gene adaptation, through their ability to regulate the expression levels of nearby genes. [ 70 ]  Combined with their "mobility", transposable elements can be relocated adjacent to their targeted genes, and control the expression levels of the gene, dependent upon the circumstances.


The study conducted in 2008, "High Rate of Recent Transposable Element–Induced Adaptation in Drosophila melanogaster", used  D. melanogaster  that had recently migrated from Africa to other parts of the world, as a basis for studying adaptations caused by transposable elements. Although most of the TEs were located on introns, the experiment showed a significant difference in gene expressions between the population in Africa and other parts of the world. The four TEs that caused the selective sweep were more prevalent in  D. melanogaster  from temperate climates, leading the researchers to conclude that the selective pressures of the climate prompted genetic adaptation. [ 71 ]  From this experiment, it has been confirmed that adaptive TEs are prevalent in nature, by enabling organisms to adapt gene expression as a result of new selective pressures.


However, not all effects of adaptive TEs are beneficial to the population. In the research conducted in 2009, "A Recent Adaptive Transposable Element Insertion Near Highly Conserved Developmental Loci in Drosophila melanogaster", a TE, inserted between Jheh 2 and Jheh 3, revealed a downgrade in the expression level of both of the genes. Downregulation of such genes has caused  Drosophila  to exhibit extended developmental time and reduced egg to adult viability. Although this adaptation was observed in high frequency in all non-African populations, it was not fixed in any of them. [ 72 ]  This is not hard to believe, since it is logical for a population to favor higher egg to adult viability, therefore trying to purge the trait caused by this specific TE adaptation.


At the same time, there have been several reports showing the advantageous adaptation caused by TEs. In the research done with silkworms, "An Adaptive Transposable Element insertion in the Regulatory Region of the EO Gene in the Domesticated Silkworm", a TE insertion was observed in the cis-regulatory region of the EO gene, which regulates molting hormone 20E, and enhanced expression was recorded. While populations without the TE insert are often unable to effectively regulate hormone 20E under starvation conditions, those with the insert had a more stable development, which resulted in higher developmental uniformity. [ 74 ]


These three experiments all demonstrated different ways in which TE insertions can be advantageous or disadvantageous, through means of regulating the expression level of adjacent genes. The field of adaptive TE research is still under development and more findings can be expected in the future.


Recent studies have confirmed that TEs can contribute to the generation of transcription factors. However, how this process of contribution can have an impact on the participation of genome control networks. TEs are more common in many regions of the DNA and it makes up 45% of total human DNA. Also, TEs contributed to 16% of transcription factor binding sites. A larger number of motifs are also found in non-TE-derived DNA, and the number is larger than TE-derived DNA. All these factors correlate to the direct participation of TEs in many ways of gene control networks. [ 27 ]


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 . [ 1 ]  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 (which coded for color) allowed other genes for pigment to be expressed. [ 2 ]  Transposition in maize causes changes in color; however, in other organisms, such as bacteria, it can cause  antibiotic resistance . [ 2 ]  Transposition is also important in creating genetic diversity within species and generating adaptability to changing living conditions. [ 3 ]


Transposases are classified under  EC number  EC 2.7.7. Genes encoding transposases are widespread in the genomes of most organisms and are the most abundant genes known. [ 4 ]  During the course of human evolution, as much as 40% of the human genome has moved around via methods such as transposition of transposons. [ 2 ]


Transposase (Tnp) Tn5 is a member of the  RNase  superfamily of proteins which includes retroviral  integrases . Tn5 can be found in  Shewanella  and  Escherichia  bacteria. [ 5 ]  The transposon codes for antibiotic resistance to  kanamycin  and other aminoglycoside antibiotics. [ 3 ] [ 6 ]


Tn5 and other transposases are notably inactive. Because DNA transposition events are inherently mutagenic, the low activity of transposases is necessary to reduce the risk of causing a fatal mutation in the host, and thus eliminating the  transposable element . One of the reasons Tn5 is so unreactive is because the N- and C-termini are located in relatively close proximity to one another and tend to inhibit each other. This was elucidated by the characterization of several mutations which resulted in hyperactive forms of transposases. One such mutation, L372P, is a mutation of amino acid 372 in the Tn5 transposase. This amino acid is generally a leucine residue in the middle of an alpha helix. When this leucine is replaced with a proline residue the alpha helix is broken, introducing a conformational change to the C-terminal domain, separating it from the N-terminal domain enough to promote higher activity of the protein. [ 3 ]  The transposition of a transposon often needs only three pieces: the transposon, the transposase enzyme, and the target DNA for the insertion of the transposon. [ 3 ]  This is the case with Tn5, which uses a cut-and-paste mechanism for moving around transposons. [ 3 ]


Tn5 and most other transposases contain a DDE motif, which is the active site that catalyzes the movement of the transposon. Aspartate-97, aspartate-188, and glutamate-326 make up the active site, which is a triad of acidic residues. [ 7 ]  The DDE motif is said to coordinate divalent metal ions, most often magnesium and manganese, which are important in the catalytic reaction. [ 7 ]  Because transposase is incredibly inactive, the DDE region is mutated so that the transposase becomes hyperactive and catalyzes the movement of the transposon. [ 7 ]  The glutamate is transformed into an aspartate and the two aspartates into glutamates. [ 7 ]  Through this mutation, the study of Tn5 becomes possible, but some steps in the catalytic process are lost as a result. [ 3 ]


There are several steps which catalyze the movement of the transposon, including Tnp binding, synapsis (the creation of a synaptic complex), cleavage, target capture, and strand transfer. Transposase then binds to the DNA strand and creates a clamp over the transposon end of the DNA and inserts into the active site. Once the transposase binds to the transposon, it produces a synaptic complex in which two transposases are bound in a cis/trans relationship with the transposon. [ 3 ]


In cleavage, the magnesium ions activate oxygen from water molecules and expose them to nucleophilic attack. [ 6 ]  This allows the water molecules to nick the 3' strands on both ends and create a hairpin formation, which separates the transposon from the donor DNA. [ 3 ]  Next, the transposase moves the transposon to a suitable location. Not much is known about the target capture, although there is a sequence bias which has not yet been determined. [ 3 ]  After target capture, the transposase attacks the target DNA nine base pairs apart, resulting in the integration of the transposon into the target DNA. [ 3 ]


As mentioned before, due to the mutations of the DDE, some steps of the process are lost—for example, when this experiment is performed  in vitro , and SDS heat treatment denatures the transposase. However, it is still uncertain what happens to the transposase  in vivo . [ 3 ]


The study of transposase Tn5 is of general importance because of its similarities to  HIV -1 and other retroviral diseases. By studying Tn5, much can also be discovered about other transposases and their activities. [ 3 ]


Tn5 is utilized in genome sequencing by using the Tn5 to append sequencing adaptors and fragment the DNA in a single enzymatic reaction in 2010, [ 8 ]  reducing the time and input requirements over traditional  next-generation sequencing  library preparation. The Tn5-based strategy can simplify the library preparation protocol significantly and can even can be incorporated into the direct colony-PCR for large numbers of bacterial isolates with no obvious coverage bias. [ 8 ]  The main disadvantages are less control of fragmented size compared to enzymatic fragmentation and mechanical fragmentation, and a bias toward high G-C content. [ 8 ]  This means of library preparation is also used in the  ATAC-seq  technique.


The Sleeping Beauty (SB) transposase is the recombinase that drives the  Sleeping Beauty transposon system . [ 9 ]   SB transposase belongs to the DD[E/D] family of transposases, which in turn belong to a large superfamily of polynucleotidyl transferases that includes RNase H, RuvC Holliday resolvase, RAG proteins, and retroviral integrases. [ 10 ] [ 11 ]  The SB system is used primarily in vertebrate animals for gene transfer, [ 12 ]  including gene therapy, [ 13 ] [ 14 ]  and gene discovery. [ 15 ] [ 16 ]   The engineered SB100X is an enzyme that directs the high levels of transposon integration. [ 17 ] [ 18 ]