Also known as  retrotransposons , these employ a strategy of self-copying via RNA transcriptase and subsequently inserting themselves into a new site within the host genome. The presence or absence of transcriptase (the enzyme that allows for self-copying) within the coding of the transposon defines class I elements as autonomous or non-autonomous. [ 2 ]  Class I transposons can take the form of:


Retrotransposons have been discovered to be the predominant form of transpositional element in plants with large genomes, such as  maize  and  wheat , potentially indicating the rapid success of this class of transposon in the creation of  hybrids , such as wheat, and peppermint and, in the distant past, maize. Plant hybridization often creates polyploids, with double, triple, quadruple or more the number of chromosomes present in the parent generation. Polyploid hybrids seem to be particularly susceptible to genetic intrusion by retrotransposons, as supported by a study in sunflower hybridization, which showed that the hybridized flowers possessed genomes that were about 50% larger than that of their parents, with the majority of this increase linked to the amplification of a single retrotransposon class. [ 3 ]


Also known as  DNA transposons , these employ a strategy by which the transposon is excised from its position via  transposase , and re-integrated elsewhere in the genome. [ 2 ]  These can be identified by the following:


Those DNA transposons lacking the coding necessary to synthesize transposase function non-autonomously, likely piggy-backing off of the machinery generated by neighboring transposons of the same class. An example of this would be MITEs, miniature inverted-repeat transposable elements, which, while having both TIRs and TSDs, cannot produce transposase. These are particularly prevalent in plants and are thought to be derived from deletions in the more autonomous DNA transposons. Similarly, these types of transposons can become non-autonomous by capturing or replicating pieces of host DNA. [ 3 ]


Another variety of transposons, discovered in 2001, can also potentially capture host DNA.  Helitrons  are thought to replicate via a "rolling circle", in which transposase links the helitron to two distinct regions of the genome at once, using a  helicase , ligase, and nuclease in the process to unravel the strands involved, replicate the helitron, and subsequently ligate the replicated material into the new site. During this process, it is thought that the helitrons often encode for the surrounding DNA and integrate this into their own material. Non-autonomous helitrons may lack a  transposase , a helicase, a ligase, or a nuclease. All are thought to be necessary for this complex process of transposition. [ 3 ]


Due to their invasive nature, and their potentially disruptive production of non-coding RNAs ( ncRNAs ), most transposons are dangerous to plants and  metazoans  alike. Given the lack of distinction between germ-line and somatic cells in the plant kingdom, this is doubly so, since alterations to the genetic and  epigenetic code  will be more easily inherited. [ 4 ] [ 3 ]  While transposable elements may affect any number of different cell types in an animal, be a skin cell, a liver cell, a brain cell, these changes are not heritable, due to the fact that an animal inherits only a parent's gametic genetic code. In plants, however, there is no such distinction; a flower develops from a meristem, which is a form of somatic cell, and which will pass down to the flower, and thus to the offspring, any genetic or epigenetic alteration. Since each meristem will have developed differently, each different flower from each meristem of the same plant will potentially possess different modifications. In contrast to animals, however, plants do not undergo  chromatin remodelling  between generations, making the maintenance and inheritance of silencing an entirely different process. [ 4 ] [ 5 ] [ 3 ]  There are distinct and identifiable mechanisms for the maintenance of transposon inactivation in plants but, unfortunately, there is significantly less information on the initiation of these inactivation mechanisms. [ 3 ]


Though the effects of transposition can sometimes manifest phenotypically, and indeed, this effect led to  their discovery , transposons can be difficult for the cellular machinery to detect. Many TEs contain stretches of genuine coding DNA, copied from the host, and there is no distinct structure, code, or identifying characteristic of any kind that would allow a cell to recognize the full range of transposable elements with accuracy. Even besides coding for functional proteins or RNAs, some transposons, like class II elements, contain code copied from the nearby strand, allowing them to blend in. This fact suggests that transposons are recognized by hosts more by their effect than their structure. Thus, cell machinery, as detailed in the next section, exists that is capable of detecting transcripts that are atypical of host genomes, such as:


and, more specifically:


Silencing of transposon transcripts can vary in the completeness of silencing as well as in the duration of alteration. Plants employ a number of methods, which range from the elimination of transcripts to complete epigenetic silencing. In general, these can be sorted into two 'strategies':


In general, the initiation of transposon silencing has yet to be fully explained. For example, there have been recorded examples of spontaneous silencing in maize, which carries a high number of transposons (~85% of the genome), though the mechanism by which this occurs is unknown. [ 3 ]  While it is known that heritable methylation occurs, must occur with frequency, and must be initiated, triggered by some distinct factor, the only known example of this is in the case of Mu killer (Muk). This gene in maize silences MuDR, a class II autonomous transposable element. Muk encodes a natural inverted derivative of the transposase coding sequence in MuDR, which, when transcribed, forms a dsRNA that is subsequently cut into siRNA, which renders MUDR incapable of 'cutting and pasting' itself by way of RNAi interference of the transposase. Muk also engages in RNai-directed methylation to create a stable and heritable suppression. [ 2 ] [ 6 ]


Though transposable elements were discovered due in large part to their deleterious effects, epigenetic research has shown that they may be, in some cases, beneficial to the host organism. [ 3 ] (1,5) This research indicates that the distinction between those two aspects, mutualist and parasite, may be harder to accurately describe than was once thought. [ 3 ]


The primary mutualistic interaction between transposon and host organism is in the formation of epialleles. True to the name, an epiallele is a kind of epigenetic mutant of a certain allelic type that produces distinct morphological differences from the wild type. [ 3 ]  The predominant research into this subject has been conducted on Arabidopsis thaliana, which has the dual disadvantages of being both TE-poor and an overly genetically stable organism. [ 4 ] [ 5 ] [ 3 ]  The manner of formation of epialleles is somewhat unclear, but it is thought to be due to the fact that some transposable elements, in stealing pieces of genetic code from their host organism, blend in so well as to confuse the host cellular machinery into thinking that its own genes are the transposons, which leads to epigenetic silencing of certain alleles, forming an epiallele. Some examples of this are:


There is also evidence to suggest that transposons play a more general role than was previously thought in the formation of miRNAs as well as in the silencing of centromeres. [ 7 ] [ 2 ]


Though the majority of information on transposons is in relation to their parasitic effect, it is sometimes unclear as to how exactly they hurt the host organism. To clarify, there are several ways in which a negative effect can be produced by transposable elements. [ 3 ] [ 4 ] [ 5 ] [ 2 ] [ 6 ]


Any one of these can have an extreme or minimal effect, depending on what systems the mutation affects. For example, if a transposon were to interrupt the coding for the enzyme which allows for seeds to digest the nourishing endosperm, then the seed would fail to propagate at all, meaning that the mutation was, in essence, fatal. As a counter-example, a transposon could be inserted into a non-coding region (likely the remnant of a now inactive transposon) and have no effect.


Very little is known about the initiation of epigenetic silencing of transposable elements and there are many unclear aspects of how transposons are regulated in plant genomes. Future research into this field will possibly change our conceptions of transposons and their role in eukaryote development.


A  genomic island  ( GI ) is part of a  genome  that has evidence of  horizontal origins . [ 1 ]  The term is usually used in  microbiology , especially with regard to  bacteria . A GI can code for many functions, can be involved in  symbiosis  or  pathogenesis , and may help an organism's adaptation. Many sub-classes of GIs exist that are based on the function that they confer. [ 2 ]  For example, a GI associated with pathogenesis is often called a  pathogenicity island  (PAIs), while GIs that contain many  antibiotic resistant genes  are referred to as antibiotic resistance islands. The same GI can occur in distantly related species as a result of various types of  horizontal gene transfer  (transformation, conjugation, transduction). This can be determined by base composition analysis, as well as  phylogeny  estimations.


Various genomic island predictions programs have been developed. These tools can be broadly grouped into sequence based methods and  comparative genomics /phylogeny based methods.


Sequence based methods depend on the naturally occurring variation that exists between the genome sequence composition of different species. Genomic regions that show abnormal sequence composition (such as nucleotide bias or codon bias) suggests that these regions may have been horizontally transferred. Two major problems with these methods are that false predictions can occur due to natural variation in the genome (sometimes due to highly expressed genes) and that horizontally transferred DNA will ameliorate (change to the host genome) over time; therefore, limiting predictions to only recently acquired GIs.


Comparative genomics  based methods try to identify regions that show signs that they have been horizontally transferred using information from several related species. For example, a genomic region that is present in one species, but is not present in several other related species suggests that the region may have been horizontally transferred. The alternative explanations are (i) that the region was present in the common ancestor but has been lost in all the other species being compared, or (ii) that the region was absent in the common ancestor but was acquired through mutation and selection in the species in which it is still found. The argument for multiple deletions of the region would be strengthened if there is evidence from outgroups that the region was present in the common ancestor, or if the phylogeny implies relatively few actual deletion events would be required. The argument for acquisition via mutation would be strengthened if the species with the region is known to have diverged substantially from the other species, or if the region in question is small. The plausibility of either (i) or (ii) would be modified if taxon sampling omitted many extinct species that may have possessed the region, and particularly if extinction was correlated with the presence of the region.


One example of a method that integrates several of the most accurate GI prediction methods is IslandViewer. [ 3 ]


In  bacteria , many  type III  and  type IV secretion systems  are located on genomic islands. These "islands" are characterised by their large size(>10  Kb ), their frequent association with  tRNA-encoding genes  and a different  G+C content  compared with the rest of the genome. Many genomic islands are flanked by  repeat structures  and carry fragments of other  mobile elements  such as  phages  and  plasmids . Some genomic islands, including those adjacent to  integrative and conjugative elements  (ICEs), can excise themselves spontaneously from the chromosome and can be transferred to other suitable recipients. [ 4 ]  While excision is dependent on the ICE machinery present, integration is attributed to integrases present on the genomic islands.


Gypsy  (GyDB) is a  wiki -style database of  mobile genetic elements . [ 1 ] [ 2 ] [ 3 ]


hAT transposons  are a superfamily of  DNA transposons , or Class II  transposable elements , that are common in the  genomes  of plants, animals, and fungi. [ 1 ] [ 2 ] [ 3 ]


Superfamilies are identified by shared  DNA sequence  and ability to respond to the same  transposase . [ 1 ]  Common features of hAT transposons include a size of 2.5-5  kilobases  with short  terminal inverted repeats  and short flanking target site duplications generated during the transposition process. [ 3 ]


The hAT superfamily's name derives from three of its members: the  hobo  element from  Drosophila melanogaster , the  Activator  or  Ac  element from  Zea mays , and the  Tam3  element from  Antirrhinum majus . [ 4 ]  The superfamily has been divided based on  bioinformatics  analysis into at least two clusters defined by their  phylogenetic  relationships: the  Ac  family and the  Buster  family. [ 1 ]  More recently, a third group called  Tip  has been described. [ 3 ]


The hAT transposon superfamily includes the first transposon discovered,  Ac  from  Zea mays  ( maize ), first reported by  Barbara McClintock . [ 1 ] [ 5 ]  McClintock was awarded the  Nobel Prize in Physiology or Medicine  in 1983 for this discovery. [ 6 ]  The family also includes a subgroup known as  space invaders  or  SPIN  elements, which have very high  copy numbers  in some genomes and which are among the most efficient known transposons. Although no extant active example is known, laboratory-generated  consensus sequences  of active SPIN elements are able to generate high  copy numbers  when introduced to cells from a wide range of species. [ 1 ] [ 7 ]


hAT transposons are widely distributed across  eukaryotic   genomes , but are not active in all organisms. Inactive hAT transposon sequences are present in  mammal  genomes, including the  human genome ; [ 1 ]  they are among the transposon families believed to have been present in the ancestral  vertebrate  genome. [ 8 ]   Among mammals, the genome of the little brown bat  Myotis lucifugus  is notable for its relatively high and recently acquired number of inactive hAT transposons. [ 1 ]


The distribution of  SPIN  elements is patchy and does not relate well to known phylogenetic relationships, prompting suggestions that these elements may have spread through  horizontal gene transfer . [ 1 ] [ 7 ]


Transposons are said to be  exapted  or "domesticated" when they have acquired functional roles in the host genome. Several sequences evolutionarily related to the hAT family have been exapted in diverse organisms, including  Homo sapiens . [ 1 ]  An example is the  ZBED  gene family, which encode a group of  zinc finger -containing regulatory proteins. [ 9 ]


Insertion element  (also known as an  IS , an  insertion sequence element , or an  IS element ) is a short  DNA  sequence that acts as a simple  transposable element . Insertion sequences have two major characteristics: they are small relative to other transposable elements (generally around 700 to 2500  bp  in length) and only code for proteins implicated in the  transposition  activity (they are thus different from other  transposons , which also carry accessory genes such as  antibiotic resistance  genes).


A particular insertion sequence may be named according to the form IS n , where  n  is a  number  (e.g. IS 1 , IS 2 , IS 3 , IS 10 , IS 50 , IS 911 , IS 26  etc.); this is not the only naming scheme used, however.


These proteins are usually the  transposase  which catalyses the enzymatic reaction allowing the IS to move, and also one regulatory protein which either stimulates or inhibits the transposition activity. The coding region in an insertion sequence is usually flanked by  inverted repeats . For example, the well-known IS 911  (1250 bp) is flanked by two 36bp inverted repeat extremities and the coding region has two genes partially overlapping  orfA  and  orfAB , coding the transposase (OrfAB) and a regulatory protein (OrfA).


In addition to occurring autonomously, insertion sequences may also occur as parts of  composite transposons . In a composite transposon, two insertion sequences flank one or more accessory genes, such as an antibiotic resistance gene (e.g.  Tn10 , Tn 5 ). Nevertheless, there exist another sort of transposons, called unit transposons, that do not carry insertion sequences at their extremities (e.g. Tn 7 ).


A complex transposon does not rely on flanking insertion sequences for resolvase. The resolvase is part of the tns genome and cuts at flanking inverted repeats.


Although insertion sequences are usually discussed in the context of  prokaryotic   genomes , certain  eukaryotic  DNA sequences belonging to the family of Tc1/ mariner  transposable elements may be considered to be insertion sequences. [ 1 ]


Transposition frequency of IS elements is dependent of multiple parameters, including culture growth phase, medium composition, oxygen tension, growth scale, and structural conformation of target sites (e.g.: curvature, presence of certain motifs, DNA composition). [ 2 ]  Recombination between genomic IS sites can enable bacteria to adapt to new environments, making IS elements an important mechanism for evolution in bacteria. [ 3 ]


Interspersed repetitive DNA  is found in all eukaryotic  genomes . They differ from  tandem repeat  DNA in that rather than the repeat sequences coming right after one another, they are dispersed throughout the genome and nonadjacent. The sequence that repeats can vary depending on the type of organism, and many other factors. Certain classes of interspersed repeat sequences propagate themselves by RNA mediated  transposition ; they have been called  retrotransposons , and they constitute 25–40% of most mammalian genomes. Some types of interspersed repetitive DNA elements allow new genes to evolve by uncoupling similar DNA sequences from  gene conversion  during  meiosis . [ 1 ]


Gene conversion  acts on DNA  sequence homology  as its substrate. There is no requirement that the sequence homologies lie at the  allelic  positions on their respective chromosomes or even that the homologies lie on different chromosomes. Gene conversion events can occur between different members of a  gene family  situated on the same chromosome. [ 2 ]  When this happens, it is called  intra chromosomal gene conversion as distinguished from  inter chromosomal gene conversion. The effect of homogenizing DNA sequences is the same.


Repetitive sequences play the role of uncoupling the gene conversion network, thereby allowing new genes to evolve. The shorter  Alu  or  SINE  repetitive DNA are specialized for uncoupling intrachromosomal gene conversion while the longer  LINE  repetitive DNA are specialized for uncoupling interchromosomal gene conversion. In both cases, the interspersed repeats block gene conversion by inserting regions of non-homology within otherwise similar DNA sequences. The homogenizing forces linking DNA sequences are thereby broken and the DNA sequences are free to evolve independently. This leads to the creation of new genes and new species during  evolution . [ 3 ]  By breaking the links that would otherwise overwrite novel DNA sequence variations, interspersed repeats catalyse evolution, allowing the new genes and new species to develop.


DNA sequences are linked together in a gene pool by gene conversion events. Insertion of an interspersed DNA element breaks this linkage, allowing independent evolution of a new gene. The interspersed repeat is an  isolating mechanism  enabling new genes to evolve without interference from the progenitor gene. Because insertion of an interspersed repeat is a saltatory event the evolution of the new gene will also be saltatory. Because  speciation  ultimately depends on the creation of new genes, this naturally causes  punctuated equilibria . Interspersed repeats are thus responsible for punctuated evolution and  rapid modes of evolution .


Islander  is a database of  integrative islands  in  prokaryotic  genomes. [ 1 ]


L1Base  is a database of functional annotations and predictions of active  LINE1  elements. [ 1 ] [ 2 ]


LINE1  (an abbreviation of  Long interspersed nuclear element-1 , also known as  L1  and  LINE-1 ) is a family of related class I  transposable elements  in the  DNA  of many groups of  eukaryotes , including animals and plants, classified with the  long interspersed nuclear elements  (LINEs). [ 1 ]  L1 transposons are most ubiquitous in mammals, where they make up a significant fraction of the total genome length, [ 1 ] [ 2 ]  for example they comprise approximately 17% of the  human genome . [ 3 ]  These active L1s can interrupt the genome through insertions, deletions, rearrangements, and  copy number variations . [ 4 ]  L1 activity has contributed to the instability and evolution of genomes and is tightly regulated in the germline by  DNA methylation ,  histone modifications , and  piRNA . [ 5 ]  L1s can further impact genome variation through mispairing and  unequal crossing over  during meiosis due to its repetitive DNA sequences. [ 4 ]


L1 gene products are also required by many non-autonomous  Alu  and SVA  SINE  retrotransposons.  Mutations  induced by L1 and its non-autonomous counterparts have been found to cause a variety of heritable and somatic diseases. [ 6 ] [ 7 ]


In 2011, human L1 was reportedly discovered in the genome of the  gonorrhea  bacteria, evidently having arrived there by  horizontal gene transfer . [ 8 ] [ 9 ]


A typical L1 element is approximately 6,000  base pairs  (bp) long and consists of two non-overlapping  open reading frames  (ORFs) which are flanked by  untranslated regions  (UTRs) and target site duplications. In humans, ORF2 is thought to be translated by an unconventional termination/reinitiation mechanism, [ 10 ]  while mouse L1s contain an  internal ribosome entry site  (IRES) upstream of each ORF. [ 11 ]


The 5' UTRs of mouse L1s contain a variable number of GC-rich  tandemly repeated  monomers of around 200 bp, followed by a short non-monomeric region. Human 5’ UTRs are ~900 bp in length and do not contain repeated motifs. All families of human L1s harbor in their most 5’ extremity a binding motif for the transcription factor  YY1 . [ 12 ]  Younger families also have two binding sites for  SOX -family transcription factors, and both YY1 and SOX sites were shown to be required for human L1 transcription initiation and activation. [ 13 ] [ 14 ]  Both mouse and human 5’ UTRs also contain a weak  antisense  promoter of unknown function. [ 15 ] [ 16 ]


The first  ORF  of L1 encodes a 500-amino acid, 40- kDa  protein that lacks homology with any protein of known function. In vertebrates, it contains a conserved  C-terminus  domain and a highly variable coiled-coil  N-terminus  that mediates the formation of ORF1 trimeric complexes. ORF1 trimers have RNA-binding and nucleic acid chaperone activity that are necessary for retrotransposition. [ 17 ]


The second  ORF  of L1 encodes a protein that has  endonuclease  and  reverse transcriptase  activity. The encoded protein has a molecular weight of 150  kDa . The structure of the ORF2 protein was solved in 2023. Its protein core contains three domains of unknown functions, termed "tower/EN-linker" and "wrist/RNA-binding domain" that bind Alu RNA's polyA tail and C-terminal domain that binds Alu RNA stem loop.


The  nicking  and  reverse transcriptase  activities of L1 ORF2p are boosted by  single-stranded DNA  structures likely present on the active  replication forks . Unlike viral RTs, L1 ORF2p can be primed by RNA, including RNA hairpin primers produced by the Alu element.


As with other transposable elements, the host organism keeps a heavy check on LINE1 to prevent it from becoming overly active. In the primitive eukaryote  Entamoeba histolytica , ORF2 is massively expressed in  antisense , resulting in no detectable amounts of its protein product. [ 18 ]


L1 activity has been observed in numerous types of  cancers , with particularly extensive insertions found in colorectal and lung cancers. [ 19 ]  It is currently unclear if these insertions are causes or secondary effects of cancer progression. However, at least two cases have found somatic L1 insertions causative of cancer by disrupting the coding sequences of genes  APC  and  PTEN  in colon and  endometrial  cancer, respectively. [ 4 ]


Quantification of L1 copy number by  qPCR  or L1 methylation levels with  bisulfite sequencing  are used as diagnostic biomarkers in some types of cancers. L1 hypomethylation of colon tumor samples is correlated with cancer stage progression. [ 20 ] [ 21 ]  Furthermore, less invasive blood assays for L1 copy number or methylation levels are indicative of breast or bladder cancer progression and may serve as methods for early detection. [ 22 ] [ 23 ]


Higher L1 copy numbers have been observed in the human  brain  compared to other organs. [ 24 ] [ 25 ]  Studies of animal models and human cell lines have shown that L1s become active in neural progenitor cells (NPCs), and that experimental deregulation of or overexpression of L1 increases somatic  mosaicism . This phenomenon is negatively regulated by  Sox2 , which is downregulated in NPCs, and by  MeCP2  and methylation of the L1 5' UTR. [ 26 ]  Human cell lines modeling the neurological disorder  Rett syndrome , which carry MeCP2 mutations, exhibit increased L1 transposition, suggesting a link between L1 activity and neurological disorders. [ 27 ] [ 26 ]  Current studies are aimed at investigating the potential roles of L1 activity in various neuropsychiatric disorders including  schizophrenia ,  autism spectrum disorders ,  epilepsy ,  bipolar disorder ,  Tourette syndrome , and drug  addiction . [ 28 ]  L1s are also highly expressed in octopus brain, suggesting a convergent mechanism in complex cognition. [ 29 ]


Increased RNA levels of  Alu , which requires L1 proteins, are associated with a form of age-related  macular degeneration , a neurological disorder of the  eyes . [ 30 ]


The naturally occurring mouse retinal degeneration model rd7 is caused by an L1 insertion in the  Nr2e3  gene. [ 31 ]


In 2021, a study proposed that L1 elements may be responsible for potential  endogenisation  of the  SARS-CoV-2  genome in  Huh7  mutant cancer cells, [ 32 ]  which would possibly explain why some patients test PCR positive for SARS-CoV-2 even after clearance of the virus. These results however have been criticized as "mechanistically plausible but likely very rare", [ 33 ]  misleading and infrequent [ 34 ]  or artefactual. [ 35 ]


Long interspersed nuclear elements  ( LINEs ) [ 1 ]  (also known as  long interspersed nucleotide elements [ 2 ]  or  long interspersed elements [ 3 ] ) are a group of non-LTR ( long terminal repeat )  retrotransposons  that are widespread in the genome of many  eukaryotes . [ 4 ] [ 5 ]  LINEs contain an internal  Pol II  promoter to initiate  transcription  into  mRNA , and encode one or two proteins, ORF1 and ORF2. [ 6 ]  The functional domains present within ORF1 vary greatly among LINEs, but often exhibit RNA/DNA binding activity. ORF2 is essential to successful retrotransposition, and encodes a protein with both  reverse transcriptase  and  endonuclease  activity. [ 7 ]


LINEs are the most abundant  transposable element  within the  human genome , [ 8 ]  with approximately 20.7% of the sequences identified as being derived from LINEs. The only active lineage of LINE found within humans belongs to the  LINE-1  class, and is referred to as L1Hs. [ 9 ]  The human genome contains an estimated 100,000 truncated and 4,000 full-length LINE-1 elements. [ 10 ]  Due to the accumulation of random mutations, the sequence of many LINEs has degenerated to the extent that they are no longer transcribed or translated. Comparisons of LINE DNA sequences can be used to date transposon insertions in the genome.


The first description of an approximately 6.4 kb long LINE-derived sequence was published by J. Adams  et al.  in 1980. [ 11 ]


Based on structural features and the phylogeny of the essential protein ORF2p, LINEs can be separated into six main groups, referred to as R2, RanI, L1, RTE, I and Jockey. These groups can further be subdivided into at least 28 clades. [ 12 ]


In plant genomes, so far only LINEs of the L1 and RTE clade have been reported. [ 13 ] [ 14 ] [ 15 ]  Whereas L1 elements diversify into several subclades, RTE-type LINEs are highly conserved, often constituting a single family. [ 16 ] [ 17 ]


In fungi, Tad, L1, CRE, Deceiver and Inkcap-like elements have been identified, [ 18 ]  with Tad-like elements appearing exclusively in fungal genomes. [ 19 ]


All LINEs encode a least one protein, ORF2, which contains an RT and an endonuclease (EN) domain, either an N-terminal  APE  or a C-terminal  RLE  or rarely both. A  ribonuclease H  domain is occasionally present. Except for the evolutionary ancient R2 and RTE superfamilies, LINEs usually encode for another protein named ORF1, which may contain an  Gag-knuckle , a  L1-like RRM  ( InterPro :  IPR035300 ), and/or an esterase. LINE elements are relatively rare compared to  LTR-retrotransposons  in plants, fungi or insects, but are dominant in vertebrates and especially in mammals, where they represent around 20% of the genome. [ 12 ] : fig. 1


The  LINE-1/L1 -element is one of the elements that are still active in the human genome today. It is found in all therian  mammals [ 20 ] [ 21 ]  except  megabats . [ 22 ]


Remnants of L2 and L3 elements are found in the human genome. [ 23 ]  It is estimated that L2 and L3 elements were active ~200-300 million years ago. Due to the age of L2 elements found within therian genomes, they lack flanking target site duplications. [ 24 ]  The L2 (and L3) elements are in the same group as the CR1 clade, Jockey. [ 25 ]


In the first human genome draft the fraction of LINE elements of the human genome was given as 21% and their copy number as 850,000. Of these,  L1 , L2 and L3 elements made up 516,000, 315,000 and 37,000 copies, respectively. The non-autonomous  SINE  elements which depend on  L1  elements for their proliferation make up 13% of the human genome and have a copy number of around 1.5 million. [ 23 ]  They probably originated from the RTE family of LINEs. [ 26 ]  Recent estimates show the typical human genome contains on average 100 L1 elements with potential for mobilization, however there is a fair amount of variation and some individuals may contain a larger number of active L1 elements, making these individuals more prone to L1-induced mutagenesis. [ 27 ]


Increased  L1  copy numbers have also been found in the brains of people with schizophrenia, indicating that LINE elements may play a role in some neuronal diseases. [ 28 ]


LINE elements propagate by a so-called target primed reverse transcription mechanism (TPRT), which was first described for the  R2  element from the silkworm  Bombyx mori .


ORF2 (and ORF1 when present) proteins primarily associate in cis with their encoding  mRNA , forming a  ribonucleoprotein  (RNP) complex, likely composed of two ORF2s and an unknown number of ORF1 trimers. [ 29 ]  The complex is transported back into the  nucleus , where the ORF2 endonuclease domain opens the DNA (at TTAAAA hexanucleotide motifs in mammals [ 30 ] ). Thus, a 3'OH group is freed for the reverse transcriptase to prime reverse transcription of the LINE RNA transcript. Following the reverse transcription the target strand is cleaved and the newly created  cDNA  is integrated [ 31 ]


New insertions create short target site duplications (TSDs), and the majority of new inserts are severely 5’-truncated (average insert size of 900bp in humans) and often inverted (Szak et al., 2002). Because they lack their 5’UTR, most of new inserts are non functional.


It has been shown that host cells regulate L1 retrotransposition activity, for example through epigenetic silencing.
For example, the  RNA interference (RNAi)  mechanism of  small interfering RNAs  derived from  L1  sequences can cause suppression of  L1  retrotransposition. [ 32 ]


In plant genomes, epigenetic modification of LINEs can lead to expression changes of nearby genes and even to phenotypic changes: In the oil palm genome, methylation of a Karma-type LINE underlies the somaclonal, 'mantled' variant of this plant, responsible for drastic yield loss. [ 33 ]


Human  APOBEC3C  mediated restriction of LINE-1 elements were reported and it is due to the interaction between A3C with the ORF1p that affects the reverse transcriptase activity. [ 34 ]


A historic example of L1-conferred disease is Haemophilia A, which is caused by  insertional mutagenesis . [ 35 ]  There are nearly 100 examples of known diseases caused by retroelement insertions, including some types of cancer and neurological disorders. [ 36 ]  Correlation between  L1  mobilization and oncogenesis has been reported for epithelial cell cancer ( carcinoma ). [ 37 ]  Hypomethylation of LINES is associated with chromosomal instability and altered gene expression [ 38 ]  and is found in various cancer cell types in various tissues types. [ 39 ] [ 38 ]  Hypomethylation of a specific L1 located in the MET onco gene is associated with bladder cancer tumorogenesis, [ 40 ]   Shift work sleep disorder [ 41 ]  is associated with increased cancer risk because light exposure at night reduces  melatonin , a hormone that has been shown to reduce L1-induced  genome instability . [ 42 ]


LTR retrotransposons  are class I  transposable elements  (TEs) characterized by the presence of  long terminal repeats  (LTRs) directly flanking an internal coding region. As retrotransposons, they mobilize through  reverse transcription  of their  mRNA  and integration of the newly created  cDNA  into another genomic location. Their mechanism of retrotransposition is shared with  retroviruses , with the difference that the rate of horizontal transfer in LTR-retrotransposons is much lower than the vertical transfer by passing active TE insertions to the progeny. LTR retrotransposons that form  virus-like particles  are classified under  Ortervirales .


Their size ranges from a few hundred base pairs to 30 kb, the largest species reported to date are members of the Burro retrotransposon family in  Schmidtea mediterranea . [ 1 ]


In plant genomes, LTR retrotransposons are the major repetitive sequence class constituting more than 75% of the maize genome. [ 2 ]  LTR retrotransposons make up about 8% of the human genome and approximately 10% of the mouse genome. [ 3 ]


LTR retrotransposons have  direct   long terminal repeats  that range from ~100 bp to over 5 kb in size. LTR retrotransposons are further sub-classified into the Ty1- copia -like ( Pseudoviridae ), Ty3-like ( Metaviridae , formally referred to as Gypsy-like, a name that is being considered for retirement [ 4 ] ), and BEL-Pao-like ( Belpaoviridae ) groups based on both their degree of sequence similarity and the order of encoded gene products. Ty1- copia  and Ty3-Metaviridae groups of retrotransposons are commonly found in high copy number (up to a few million copies per  haploid   nucleus ) in animals, fungi, protista, and plants genomes. BEL-Pao like elements have so far only been found in animals. [ 5 ] [ 6 ]


All functional LTR-retrotransposons encode a minimum of two genes,  gag  and  pol , that are sufficient for their replication.  Gag  encodes a polyprotein with a capsid and a nucleocapsid domain. [ 7 ]  Gag proteins form virus-like particles in the cytoplasm inside which reverse-transcription occurs. The  Pol  gene produces three proteins: a  protease  (PR), a  reverse transcriptase  endowed with an RT (reverse-transcriptase) and an  RNAse H  domains, and an  integrase  (IN). [ 8 ]


Typically, LTR-retrotransposon mRNAs are produced by the host  RNA pol II  acting on a  promoter  located in their 5’ LTR. The Gag and Pol genes are encoded in the same mRNA. Depending on the host species, two different strategies can be used to express the two polyproteins: a fusion into a single  open reading frame  (ORF) that is then cleaved or the introduction of a frameshift between the two ORFs. [ 9 ]  Occasional ribosomal frameshifting allows the production of both proteins, while ensuring that much more Gag protein is produced to form virus-like particles.


Reverse transcription  usually initiates at a short sequence located immediately downstream of the 5’-LTR and termed the  primer binding site  (PBS). Specific host  tRNAs  bind to the PBS and act as primers for reverse-transcription, which occurs in a complex and multi-step process, ultimately producing a double- stranded  cDNA  molecule. The cDNA is finally integrated into a new location, creating short TSDs (Target Site Duplications)  [ 10 ]  and adding a new copy in the host genome


Ty1- copia  retrotransposons are abundant in species ranging from single-cell  algae  to  bryophytes ,  gymnosperms , and  angiosperms . They encode four protein domains in the following order:  protease ,  integrase ,  reverse transcriptase , and  ribonuclease H .


At least two classification systems exist for the subdivision of Ty1- copia  retrotransposons into five lineages: [ 11 ] [ 12 ]   Sireviruses /Maximus, Oryco/Ivana, Retrofit/Ale, TORK (subdivided in Angela/Sto, TAR/Fourf, GMR/Tork), and Bianca.


Sireviruses /Maximus retrotransposons contain an additional putative envelope gene. This lineage is named for the founder element SIRE1 in the  Glycine max  genome, [ 13 ]  and was later described in many species such as  Zea mays , [ 14 ]   Arabidopsis thaliana , [ 15 ]   Beta vulgaris , [ 16 ]  and  Pinus pinaster . [ 17 ]  Plant  Sireviruses  of many sequenced plant genomes are summarized at the MASIVEdb  Sirevirus  database. [ 18 ]


Ty3-retrotransposons are widely distributed in the plant kingdom, including both  gymnosperms  and  angiosperms . They encode at least four protein domains in the order:  protease ,  reverse transcriptase ,  ribonuclease H , and  integrase . Based on structure, presence/absence of specific protein domains, and conserved protein sequence motifs, they can be subdivided into several lineages:


Errantiviruses  contain an additional defective envelope ORF with similarities to the retroviral envelope gene. First described as Athila-elements in  Arabidopsis thaliana , [ 19 ] [ 20 ]  they have been later identified in many species, such as  Glycine max [ 21 ]  and  Beta vulgaris . [ 22 ]


Chromoviruses  contain an additional chromodomain ( chr omatin  o rganization  mo difier domain) at the C-terminus of their integrase protein. [ 23 ] [ 24 ]  They are widespread in plants and fungi, probably retaining protein domains during evolution of these two kingdoms. [ 25 ]  It is thought that the chromodomain directs retrotransposon integration to specific target sites. [ 26 ]  According to sequence and structure of the chromodomain, chromoviruses are subdivided into the four clades CRM, Tekay, Reina and Galadriel. Chromoviruses from each clade show distinctive integration patterns, e.g. into centromeres or into the rRNA genes. [ 27 ] [ 28 ]


Ogre-elements are gigantic Ty3-retrotransposons reaching lengths up to 25 kb. [ 29 ]   Ogre elements have been first described in  Pisum sativum . [ 30 ]


Metaviruses  describe conventional Ty3- gypsy  retrotransposons that do not contain additional domains or ORFs.


The Sushi family of Ty3 long terminal repeat retrotransposons were first identified in teleost fish and Sushi-like neogenes were subsequently identified in mammals. [ 31 ]  Mammalian retrotransposon-derived transcripts (MARTs) cannot transpose but have retained open reading frames, demonstrate high levels of evolutionary conservation and are subject to selective pressures, which suggests some have become neofunctionalized genes with new cellular functions. [ 31 ]  Retrotransposon gag-like-3 (RTL3/ZCCHC5/MART3) is one of eleven Sushi-like neogenes identified in the human genome. [ 31 ]


The BEL/pao family is found in animals. [ 32 ]


Although  retroviruses  are often classified separately, they share many features with LTR retrotransposons. A major difference with Ty1- copia  and Ty3- gypsy  retrotransposons is that retroviruses have an envelope protein (ENV). A retrovirus can be transformed into an LTR retrotransposon through inactivation or deletion of the domains that enable extracellular mobility. If such a retrovirus infects and subsequently inserts itself in the genome in germ line cells, it may become transmitted vertically and become an  Endogenous Retrovirus . [ 6 ]


Some LTR retrotransposons lack all of their coding domains. Due to their short size, they are referred to as terminal repeat retrotransposons in miniature (TRIMs). [ 33 ] [ 34 ]  Nevertheless, TRIMs can be able to retrotranspose, as they may rely on the coding domains of autonomous Ty1- copia  or Ty3- gypsy  retrotransposons. Among the TRIMs, the Cassandra family plays an exceptional role, as the family is unusually wide-spread among higher plants. [ 35 ]  In contrast to all other characterized TRIMs, Cassandra elements harbor a 5S rRNA promoter in their LTR sequence. [ 36 ]  Due to their short overall length and the relatively high contribution of the flanking LTRs, TRIMs are prone to re-arrangements by recombination. [ 37 ]


Mammalian-wide interspersed repeats  (MIRs) are  transposable elements  in the genomes of some organisms and belong to the group of  Short interspersed nuclear elements  (SINEs) .


MIRs are found in all  mammals  (including  marsupials ). [ 1 ]


It is estimated that there are around 368,000 MIRs in the  human genome . [ 2 ]


The MIR  consensus sequence  is 260 basepairs long and has an A/T-rich 3' end. [ 1 ]


Like other  Short interspersed nuclear elements  (SINEs) , MIR elements used the machinery of  LINE elements  for their propagation in the genome, which took place around 130 million years ago. They cannot  retrotranspose  anymore since the loss of activity of the required  reverse transcriptase . [ 3 ]


MIR elements have been first described in human genome 1989-1991  [ 4 ] [ 5 ] [ 6 ]
and were first referred as MB1 family repeats (mirror to sequences of mouse B1 repeat). Then this family repeats were found in other mammalian genomes. [ 7 ]   Then this family was renamed as "Mammalian interspersed repeats" in 1992  [ 8 ]  Later this family was shown to be common for vertebrate genomes. [ 9 ]


Micropia  is the name of a family of LTR  retrotransposons  widespread in the genomes of fruitflies of the genus  Drosophila . [ 1 ] [ 2 ]  Micropia retrotransposons in some species of  Drosophila  express a male germline-specific and meiotic-specific antisense transcript complementary to the reverse transcriptase (RT) and ribonuclease A (RNaseA) genes of the proviral retrotransposon. [ 3 ]  No active transposition of micropia has been registered so far. micropia is likely part of a selfish driver system responsible for the  Drosophila  Y chromosomal lampbrushloop evolution in some species. [ 3 ]


Micropia was discovered after micro-cloning experiments carried out on Y-chromosomal lampbrush loops by Prof. Wolfgang Hennig. Similar loops can be found in lampbrush chromosomes (see  Lampbrush chromosome ) that are characteristic for the female germ cells of most animals, except mammals. The name micropia is an artificial word, i.e. a concoction of " micro cloning" and "co pia -like element".


NM_024554 NM_001258311


PiggyBac Transposable Element Derived 5  is an  enzyme  that in humans is encoded by the  PGBD5   gene . [ 5 ]  PGBD5 is a DNA transposase related to the ancient  PiggyBac transposase  first identified in the  cabbage looper  moth,  Trichoplusia ni. [ 6 ]  The gene is believed to have been domesticated over 500 million years ago in the common ancestor of  cephalochordates  and  vertebrates . [ 7 ]  The putative  catalytic triad  of the protein composed of three  aspartic acid  residues is conserved among PGBD5-like genes through evolution, [ 8 ]  and is distinct from other PiggyBac-like genes. [ 7 ]  PGBD5 has been shown to be able to transpose DNA in a sequence-specific, cut-and-paste fashion. [ 8 ]  PGBD5 has also been proposed to mediate site-specific DNA rearrangements in human tumors. [ 9 ]


Human PGBD5 can mobilize the insect PiggyBac transposons in human cell culture. [ 10 ]


In mature mice brain tissue PGBD5 is found primarily in regions of the  olfactory bulb ,  hippocampus , and  cerebellum . In embryonic mice brain tissue PGBD5 is found not only in the  medial pallium  and  prepontine isthmus , which are embryonic brain areas that give rise to the development of the hippocampus and cerebellum but also in areas in the embryonic brain that give rise to the  hypothalamus  and  medulla . [ 11 ] [ better source needed ]


PGBD5 is expressed in the majority of human pediatric solid tumors. [ 12 ]  It's upregulated in sporadic Creutzfeldt-Jakob disease. [ 13 ]  PGBD5 is associated with frontotemporal dementia, where it gets most expressed in neurons, followed by ogliodendrocytes, mature astrocytes, fetal astrocytes, endothelial cells and then microglia/macrophages. [ 14 ]


The  PiggyBac (PB) transposon system  employs a genetically engineered  transposase  enzyme to insert a gene into a cell's genome. It is built upon the natural PiggyBac (PB)  transposable element  (transposon), enabling the back and forth movement of genes between  chromosomes  and genetic vectors such as  plasmids  through a "cut and paste" mechanism. During transposition, the PB transposase recognizes transposon-specific  inverted terminal repeat sequences  (ITRs) located on both ends of the transposon vector and efficiently moves the contents from the original sites and integrates them into TTAA chromosomal sites. The powerful activity of the PiggyBac transposon system enables genes of interest between the two ITRs in the PB vector to be easily mobilized into target genomes. The TTAA-specific transposon  piggyBac is rapidly becoming a highly useful transposon for  genetic engineering  of a wide variety of species, particularly insects. [ 1 ]  They were discovered in 1989 by Malcolm Fraser at the  University of Notre Dame . [ 2 ] [ 3 ]


The TTAA-specific, short repeat elements are a group of transposons that share similarity of structure and properties of movement. These elements were originally defined in the   Cabbage Looper , [ 4 ]  but appear to be common among other animals as well. They might prove to be useful tools for the  transformation  of insects. The original identification of these unusual TTAA-specific elements came through a somewhat unconventional route relative to most other Class II mobile elements. Spontaneous plaque morphology mutants of  baculoviruses  were observed to arise during propagation of these viruses in the TN-368 cell line. Genetic characterization of these mutations often revealed an associated insertion of host-derived DNAs, some of which appeared to be transposons.


Several different mobile host DNA insertions have been identified within the few-polyhedra (FP) locus of the  baculoviruses  AcMNPV and GmMNPV. The insertions most extensively studied are those now designated as tagalong (formerly TFP3) and piggyBac (formerly IFP2). These insertions exhibit a unique preference for TTAA target sites, whether inserting within the viral FP-locus  or at other regions of the viral genome. Both of these elements are part of a larger family of TTAA-target site specific insertion elements that includes the T. ni derived piggyBac and tagalong elements, the Spodoptera frugiperda derived elements IFP1.6 and 290 bp insertion of Carstens, and the transposon-like insertion within the EcoRI-J,N region of Autographa californica nuclear polyhedrosis virus, whose origin is undefined.


More recently, analysis of sequences obtained from the human genome has revealed what appear to be 100 to 500 copies of a fossil element called LOOPER, which has sequence homology to piggyBac, terminates in 5' CCY....GGG 3', and apparently targets TTAA insertion sites. The LOOPER consensus sequence is on average 77% similar to individual sequences identified in the human genome, indicating it is at least 60 million years old. There are two other TTAA-specific fossil repeat elements, MER75 and MER85 (estimated at 2000 copies per genome) which appear to target TTAA insertion sites and terminate in 5' CCC....GGG 3'. Evidence is accumulating that suggests a superfamily of TTAA-specific mobile elements exists in a diversity of organisms, and that piggyBac-related sequences may be present in a diversity of species. [ 5 ]


The transposon consists of the transposase gene flanked by inverted terminal repeats.


The PB superfamily transposase consists of three domains, a variable N-terminal domain, a catalytic DDE  triad  domain and a C-terminal region with the nuclear localization signal. [ 6 ]


It has apparently been domesticated in a wide range of animals, losing the repeats and thus its mobility. The new functions these copies gain are sometimes significant enough to show signs of positive or purifying selection. In humans, these genes are: [ 7 ]


Hyperactive versions of PiggyBac transposase are suited for genetic engineering purposes. [ 8 ]  A version called mPB was created by optimizing  codon  usage for mammalian (mouse) with a 20x increase in activity, [ 9 ]  and further mutation screening generated hyPB with 10x the activity of mPB. [ 10 ]  PiggyBac system have been successfully employed to express large genetic sequences, such as a doxycicline-inducible CRISPR interference system. [ 11 ]


A novel member of the piggyBac family  hyperactive Mage (MG) transposase (hyMagease) exhibited strong transposability in a variety of mammalian cells and primary T cells and a weaker insertion preference for near genes, transcription start sites, CpG islands, and DNaseI hypersensitive sites in comparison to piggyBac. [ 12 ]


These elements were first identified as insertions in Baculovirus mutants by Dr. Malcolm Fraser, [ 5 ]  professor at the  University of Notre Dame , and were originally named as IFP for Insertions in FP mutants. The name was then changed to TFP for Transposon in FP. Finally the name PiggyBac was adopted to keep the interest of the audience and to bear some resemblance to  Drosophila  gene nomenclature.


Plant transformation vectors  are  plasmids  that have been specifically designed to facilitate the generation of  transgenic plants . The most commonly used plant transformation vectors are  T-DNA binary vectors  and are often replicated in both  E. coli , a common lab  bacterium , and  Agrobacterium tumefaciens , a plant-virulent bacterium used to insert the  recombinant DNA  into plants.


Plant transformation vectors contain three key elements:


A custom DNA plasmid sequence can be created and replicated in various ways, but generally, all methods share the following processes:


Plant transformation using plasmids begins with the propagation of the binary vector in  E. coli.  When the  bacterial culture  reaches the appropriate density, the binary vector is isolated and purified. Then, a foreign gene can be introduced. The engineered binary vector, including the foreign gene, is re-introduced in  E. coli  for amplification.


The engineered binary factor is isolated from  E. coli   and is introduced into  Agrobacteria  containing a modified (relatively small) Ti plasmid. This engineered  Agrobacteria  can be used to infect plant cells. The T-DNA, which contains the foreign gene, becomes integrated into the plant cell genome. In each infected cell, the T-DNA is integrated at a different site in the genome.


The entire plant will regenerate from a single transformed cell, resulting in an organism with the transformed DNA integrated identically across all cells.


A selector gene can be used to distinguish successfully genetically modified cells from unmodified ones. The selector gene is integrated into the plasmid along with the desired target gene, providing the cells with resistance to an  antibiotic , such as  kanamycin ,  ampicillin ,  spectinomycin  or  tetracycline . The desired cells, along with any other organisms growing within the culture, can be treated with an antibiotic, allowing only the modified cells to survive. The antibiotic gene is not usually transferred to the plant cell but instead remains within the bacterial cell.


Plasmids  replicate to produce many plasmid molecules in each host bacterial cell. The number of copies of each plasmid in a bacterial cell is determined by the  replication origin , which is the position within the plasmid molecule where DNA replication is initiated. Most binary vectors have a higher number of plasmid copies when they replicate in  E. coli ;  however, the  plasmid copy-number  is usually lower when the plasmid is resident within  Agrobacterium tumefaciens .
Plasmids can also be replicated using the  polymerase chain reaction  (PCR).


T-DNA contains two types of genes: the  oncogenic genes , encoding for  enzymes  involved in the synthesis of  auxins  and  cytokinins  and responsible for  tumor  formation, and the genes encoding for the synthesis of  opines . These compounds, produced by the condensation between  amino acids  and sugars, are synthesized and excreted by the crown gall cells, and they are consumed by A. tumefaciens as carbon and nitrogen sources.


The genes involved in opine  catabolism , T-DNA transfer from the bacterium to the plant cell and  bacterium-bacterium plasmid conjugative transfer  are located outside the T-DNA. [ 3 ] [ 4 ]  The T-DNA fragment is flanked by 25-bp direct repeats, which act as a cis-element signal for the transfer apparatus. The process of T-DNA transfer is mediated by the cooperative action of  proteins  encoded by genes determined in the Ti plasmid virulence region (vir genes) and in the bacterial chromosome. The Ti plasmid also contains the genes for opine catabolism produced by the crown gall cells and regions for conjugative transfer and for its own integrity and stability. The 30 kb virulence (vir) region is a  regulon  organized in six  operons  essential for the T-DNA transfer (virA, virB, virD, and virG) or for the increasing of transfer efficiency (virC and virE). [ 3 ] [ 4 ] [ 5 ]  Several chromosomal-determined genetic elements have shown their functional role in the attachment of  A. tumefaciens  to the plant cell and bacterial colonization. The loci chvA and chvB are involved in the synthesis and excretion of the b -1,2  glucan , [ 6 ]  the chvE required for the sugar enhancement of vir  genes induction  and  bacterial chemotaxis . [ 7 ] [ 8 ] [ 9 ]  The cell locus is responsible for the synthesis of  cellulose  fibrils. [ 10 ]  The pscA (exoC) locus is involved in the synthesis of both cyclic glucan and acid  succinoglycan . [ 11 ] [ 9 ]  The att locus is involved in the cell  surface proteins . [ 12 ]


A  plasmid  is a small,  extrachromosomal DNA  molecule within a cell that is physically separated from  chromosomal DNA  and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in  bacteria ; however, plasmids are sometimes present in  archaea  and  eukaryotic organisms . [ 1 ] [ page needed ] [ 2 ]  Plasmids often carry useful genes, such as  antibiotic resistance  and  virulence . [ 3 ] [ 4 ] [ 5 ]  While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain additional genes for special circumstances.


Artificial plasmids  are widely used as  vectors  in  molecular cloning , serving to drive the replication of  recombinant DNA  sequences within host organisms. In the laboratory, plasmids may be introduced into a cell via  transformation . Synthetic plasmids are available for procurement over the internet by various vendors using submitted sequences typically designed with software, if a design does not work the vendor may make additional edits from the submission. [ 6 ] [ 7 ] [ 8 ]


Plasmids are considered  replicons , units of DNA capable of replicating autonomously within a suitable host. However, plasmids, like  viruses , are not generally classified as  life . [ 9 ]  Plasmids are transmitted from one bacterium to another (even of another species) mostly through  conjugation . [ 3 ]  This host-to-host transfer of genetic material is one mechanism of  horizontal gene transfer , and plasmids are considered part of the  mobilome . Unlike viruses, which encase their genetic material in a protective protein coat called a  capsid , plasmids are "naked" DNA and do not encode genes necessary to encase the genetic material for transfer to a new host; however, some classes of plasmids encode the  conjugative "sex" pilus  necessary for their own transfer. Plasmids vary in size from 1 to over 400 k bp , [ 10 ]  and the number of identical plasmids in a single  cell  can range from one up to thousands.


The term  plasmid  was coined in 1952 by the American  molecular biologist   Joshua Lederberg  to refer to "any extrachromosomal hereditary determinant." [ 11 ] [ 12 ]  The term's early usage included any bacterial genetic material that exists extrachromosomally for at least part of its replication cycle, but because that description includes bacterial viruses, the notion of plasmid was refined over time to refer to genetic elements that reproduce autonomously. [ 13 ]
Later in 1968, it was decided that the term plasmid should be adopted as the term for extrachromosomal genetic element, [ 14 ]  and to distinguish it from viruses, the definition was narrowed to genetic elements that exist exclusively or predominantly outside of the chromosome, can replicate autonomously, and contribute to transferring mobile elements between unrelated bacteria. [ 3 ] [ 4 ] [ 13 ]


In order for plasmids to replicate independently within a cell, they must possess a stretch of DNA that can act as an  origin of replication .  The self-replicating unit, in this case, the plasmid, is called a  replicon .  A typical bacterial replicon may consist of a number of elements, such as the gene for plasmid-specific replication initiation protein (Rep), repeating units called  iterons ,  DnaA  boxes, and an adjacent AT-rich region. [ 13 ]  Smaller plasmids make use of the host replicative enzymes to make copies of themselves, while larger plasmids may carry genes specific for the replication of those plasmids.  A few types of plasmids can also insert into the host chromosome, and these integrative plasmids are sometimes referred to as  episomes  in  prokaryotes . [ 15 ]


Plasmids almost always carry at least one gene.  Many of the genes carried by a plasmid are beneficial for the host cells, for example: enabling the host cell to survive in an environment that would otherwise be lethal or restrictive for growth.  Some of these genes encode traits for antibiotic resistance or resistance to heavy metal, while others may produce  virulence factors  that enable a bacterium to colonize a host and overcome its defences or have specific metabolic functions that allow the bacterium to utilize a particular nutrient, including the ability to degrade recalcitrant or toxic organic compounds. [ 16 ]  Plasmids can also provide bacteria with the ability to  fix nitrogen . Some plasmids, called  cryptic plasmids , play a crucial role in  horizontal genes transfer , since they carry  antibiotic-resistance  genes. Thus they are important factors in spreading resistance, which can result in antibiotic treatment failures.  [ 17 ]


Naturally occurring plasmids vary greatly in their physical properties.  Their size can range from very small mini-plasmids of less than 1-kilobase pairs (kbp) to very large megaplasmids of several megabase pairs (Mbp).  At the upper end, little differs between a megaplasmid and a  minichromosome .  Plasmids are generally circular, but examples of linear plasmids are also known.  These linear plasmids require specialized mechanisms to replicate their ends. [ 13 ]


Plasmids may be present in an individual cell in varying number, ranging from one to several hundreds. The normal number of copies of plasmid that may be found in a single cell is called the  plasmid copy number , and is determined by how the replication initiation is regulated and the size of the molecule. Larger plasmids tend to have lower copy numbers. [ 15 ]  Low-copy-number plasmids that exist only as one or a few copies in each bacterium are, upon  cell division , in danger of being lost in one of the segregating bacteria. Such single-copy plasmids have systems that attempt to actively distribute a copy to both daughter cells. These systems, which include the  parABS system  and  parMRC system , are often referred to as the  partition system  or partition function of a plasmid. [ 18 ]


Plasmids of  linear  form are unknown among  phytopathogens  with one exception,  Rhodococcus fascians . [ 19 ]


Plasmids may be classified in a number of ways.  Plasmids can be broadly classified into conjugative plasmids and non-conjugative plasmids.  Conjugative plasmids contain a set of  transfer genes  which promote sexual conjugation between different cells. [ 15 ]  In the complex process of  conjugation , plasmids may be transferred from one bacterium to another via  sex pili  encoded by some of the transfer genes (see figure). [ 20 ]  Non-conjugative plasmids are incapable of initiating conjugation, hence they can be transferred only with the assistance of conjugative plasmids.  An intermediate class of plasmids are mobilizable, and carry only a subset of the genes required for transfer. They can parasitize a conjugative plasmid, transferring at high frequency only in its presence. [ 21 ]


Plasmids can also be classified into incompatibility groups.  A microbe can harbour different types of plasmids, but different plasmids can only exist in a single bacterial cell if they are compatible.  If two plasmids are not compatible, one or the other will be rapidly lost from the cell. Different plasmids may therefore be assigned to different incompatibility groups depending on whether they can coexist together.  Incompatible plasmids (belonging to the same incompatibility group) normally share the same replication or partition mechanisms and can thus not be kept together in a single cell. [ 22 ] [ 23 ]


Another way to classify plasmids is by function. There are five main classes:


Plasmids can belong to more than one of these functional groups.


Although most plasmids are double-stranded DNA molecules, some consist of  single-stranded DNA , or predominantly  double-stranded RNA . RNA plasmids are non-infectious extrachromosomal linear RNA replicons, both  encapsidated  and unencapsidated, which have been found in fungi and various plants, from algae to land plants. In many cases, however, it may be difficult or impossible to clearly distinguish RNA plasmids from RNA viruses and other infectious RNAs. [ 30 ]


Chromids are elements that exist at the boundary between a  chromosome  and a plasmid, found in about 10% of bacterial species sequenced by 2009. These elements carry core genes and have  codon usage  similar to the chromosome, yet use a plasmid-type replication mechanism such as the low copy number RepABC. As a result, they have been variously classified as minichromosomes or megaplasmids in the past. [ 31 ]  In  Vibrio , the bacterium synchronizes the replication of the chromosome and chromid by a conserved genome size ratio. [ 32 ]


Artificially constructed plasmids may be used as  vectors  in  genetic engineering .  These plasmids serve as important tools in genetics and biotechnology labs, where they are commonly used to clone and amplify (make many copies of) or  express  particular genes. [ 33 ]  A wide variety of plasmids are commercially available for such uses. The gene to be replicated is normally inserted into a plasmid that typically contains a number of features for their use.  These include a gene that confers resistance to particular antibiotics ( ampicillin  is most frequently used for bacterial strains), an  origin of replication  to allow the bacterial cells to replicate the plasmid DNA, and a suitable site for cloning (referred to as a  multiple cloning site ).


DNA structural instability can be defined as a series of spontaneous events that culminate in an unforeseen rearrangement, loss, or gain of genetic material. Such events are frequently triggered by the transposition of mobile elements or by the presence of unstable elements such as non-canonical (non-B) structures. Accessory regions pertaining to the bacterial backbone may engage in a wide range of structural instability phenomena. Well-known catalysts of  genetic instability  include direct, inverted, and tandem repeats, which are known to be conspicuous in a large number of commercially available cloning and expression vectors. [ 34 ]  Insertion sequences can also severely impact plasmid function and yield, by leading to  deletions  and rearrangements, activation,  down-regulation  or inactivation of neighboring  gene expression . [ 35 ]  Therefore, the reduction or complete elimination of extraneous  noncoding  backbone sequences would pointedly reduce the propensity for such events to take place, and consequently, the overall recombinogenic potential of the plasmid. [ 36 ] [ 37 ]


Plasmids are the most-commonly used bacterial cloning vectors. [ 38 ]  These cloning vectors contain a site that allows DNA fragments to be inserted, for example a  multiple cloning site  or polylinker which has several commonly used  restriction sites  to which DNA fragments may be  ligated . After the gene of interest is inserted, the plasmids are introduced into bacteria by a process called  transformation . These plasmids contain a  selectable marker , usually an antibiotic resistance gene, which confers on the bacteria an ability to survive and proliferate in a selective growth medium containing the particular antibiotics.  The cells after transformation are exposed to the selective media, and only cells containing the plasmid may survive.  In this way, the antibiotics act as a filter to select only the bacteria containing the plasmid DNA.  The vector may also contain other  marker genes  or  reporter genes  to facilitate selection of plasmids with cloned inserts.  Bacteria containing the plasmid can then be grown in large amounts, harvested, and the plasmid of interest may then be isolated using various methods of  plasmid preparation .


A plasmid cloning vector is typically used to clone DNA fragments of up to 15  kbp . [ 39 ]  To clone longer lengths of DNA,  lambda phage  with lysogeny genes deleted,  cosmids ,  bacterial artificial chromosomes , or  yeast artificial chromosomes  are used.


Another major use of plasmids is to make large amounts of proteins. In this case, researchers grow bacteria containing a plasmid harboring the gene of interest. Just as the bacterium produces proteins to confer its antibiotic resistance, it can also be induced to produce large amounts of proteins from the inserted gene. This is a cheap and easy way of mass-producing the protein, for example, utilizing the rapid reproduction of E.coli with a plasmid containing the  insulin  gene leads to a large production of insulin.  [ 40 ] [ 41 ] [ 42 ]


Plasmids may also be used for gene transfer as a potential treatment in  gene therapy  so that it may express the protein that is lacking in the cells.  Some forms of  gene therapy  require the insertion of therapeutic  genes  at pre-selected  chromosomal  target sites within the human  genome . Plasmid vectors are one of many approaches that could be used for this purpose.  Zinc finger nucleases  (ZFNs) offer a way to cause a site-specific  double-strand break  to the DNA genome and cause  homologous recombination . Plasmids encoding ZFN could help deliver a therapeutic gene to a specific site so that  cell damage , cancer-causing mutations, or an  immune response  is avoided. [ 43 ]


Plasmids were historically used to genetically engineer the embryonic stem cells of rats to create rat genetic disease models. The limited efficiency of plasmid-based techniques precluded their use in the creation of more accurate human cell models. However, developments in  adeno-associated virus  recombination techniques, and  zinc finger nucleases , have enabled the creation of a new generation of  isogenic human disease models .


Plasmids assist in transporting  biosynthetic gene clusters  - a set of gene that contain all the necessary enzymes that lead to the production of special metabolites (formally known as  secondary metabolite ). [ 44 ]  A benefit of using plasmids to transfer BGC is demonstrated by using a suitable host that can mass produce specialized metabolites, some of these molecules are able to control microbial population. [ 45 ] [ 46 ]  Plasmids can contain and express several BGCs with a few plasmids known to be exclusive for transferring BGCs. [ 46 ]  BGC's can also be transfers to the host organism's chromosome, utilizing a plasmid vector, which allows for studies in gene knockout experiments. [ 47 ]  By using plasmids for the uptake of BGCs, microorganisms can gain an advantage as production is not limited to antibiotic resistant biosynthesis genes but the production of  toxins /antitoxins. [ 48 ]


The term  episome  was introduced by  François Jacob  and  Élie Wollman  in 1958 to refer to extra-chromosomal genetic material that may replicate autonomously or become integrated into the chromosome. [ 49 ] [ 50 ]  Since the term was introduced, however, its use has changed, as  plasmid  has become the preferred term for autonomously replicating extrachromosomal DNA. At a 1968 symposium in London some participants suggested that the term  episome  be abandoned, although others continued to use the term with a shift in meaning. [ 51 ] [ 52 ]


Today, some authors use  episome  in the context of prokaryotes to refer to a plasmid that is capable of integrating into the chromosome.  The integrative plasmids may be replicated and stably maintained in a cell through multiple generations, but at some stage, they will exist as an independent plasmid molecule. [ 53 ]  In the context of eukaryotes, the term  episome  is used to mean a non-integrated extrachromosomal closed circular DNA molecule that may be replicated in the nucleus. [ 54 ] [ 55 ]  Viruses are the most common examples of this, such as  herpesviruses ,  adenoviruses , and  polyomaviruses , but some are plasmids.  Other examples include aberrant chromosomal fragments, such as  double minute chromosomes , that can arise during artificial gene amplifications or in pathologic processes (e.g., cancer cell transformation).  Episomes in eukaryotes behave similarly to plasmids in prokaryotes in that the DNA is stably maintained and replicated with the host cell.  Cytoplasmic viral episomes (as in  poxvirus  infections) can also occur. Some episomes, such as herpesviruses, replicate in a  rolling circle  mechanism, similar to  bacteriophages  (bacterial phage viruses). Others replicate through a bidirectional replication mechanism ( Theta type  plasmids). In either case, episomes remain physically separate from host cell chromosomes. Several cancer viruses, including  Epstein-Barr virus  and  Kaposi's sarcoma-associated herpesvirus , are maintained as latent, chromosomally distinct episomes in cancer cells, where the viruses express  oncogenes  that promote cancer cell proliferation.  In cancers, these episomes passively replicate together with host chromosomes when the cell divides.  When these viral episomes initiate  lytic replication  to generate multiple virus particles, they generally activate cellular  innate immunity  defense mechanisms that kill the host cell.


Some plasmids or microbial hosts include an  addiction system  or postsegregational killing system (PSK), such as the  hok/sok  (host killing/suppressor of killing) system of plasmid R1 in  Escherichia coli . [ 56 ]  This variant produces both a long-lived  poison  and a short-lived  antidote . Several types of plasmid addiction systems (toxin/ antitoxin, metabolism-based, ORT systems) were described in the  literature [ 57 ]  and used in biotechnical (fermentation) or biomedical (vaccine therapy) applications. Daughter cells that retain a copy of the plasmid survive, while a daughter cell that fails to inherit the plasmid dies or suffers a reduced growth-rate because of the lingering poison from the parent cell. Finally, the overall productivity could be enhanced. [ clarification needed ]


In contrast, plasmids used in biotechnology, such as pUC18, pBR322 and derived vectors, hardly ever contain toxin-antitoxin addiction systems, and therefore need to be kept under antibiotic pressure to avoid plasmid loss.


Yeasts  naturally harbour various plasmids. Notable among them are 2 μm plasmids—small circular plasmids often used for  genetic engineering  of yeast—and linear pGKL plasmids from  Kluyveromyces lactis , that are responsible for  killer phenotypes . [ 58 ]


Other types of plasmids are often related to yeast cloning vectors that include:


The mitochondria of many higher plants contain  self-replicating , extra-chromosomal linear or circular DNA molecules which have been considered to be plasmids. These can range from 0.7 kb to 20 kb in size. The plasmids have been generally classified into two categories- circular and linear. [ 59 ]  Circular plasmids have been isolated and found in many different plants, with those in  Vicia faba  and  Chenopodium album  being the most studied and whose mechanism of replication is known. The circular plasmids can replicate using the θ model of replication (as in  Vicia faba ) and through  rolling circle replication  (as in  C.album ). [ 60 ]  Linear plasmids have been identified in some plant species such as  Beta vulgaris ,  Brassica napus ,  Zea mays , etc. but are rarer than their circular counterparts.


The function and origin of these plasmids remains largely unknown. It has been suggested that the circular plasmids share a common ancestor, some genes in the mitochondrial plasmid have counterparts in the nuclear DNA suggesting inter-compartment exchange. Meanwhile, the linear plasmids share structural similarities such as invertrons with viral DNA and fungal plasmids, like fungal plasmids they also have low GC content, these observations have led to some hypothesizing that these linear plasmids have viral origins, or have ended up in plant mitochondria through  horizontal gene transfer  from pathogenic fungi. [ 59 ] [ 61 ]


Plasmids are often used to purify a specific sequence, since they can easily be purified away from the rest of the genome. For their use as vectors, and for  molecular cloning , plasmids often need to be isolated.


There are several methods to  isolate plasmid DNA  from bacteria, ranging from the plasmid extraction kits ( miniprep to the maxiprep or bulkprep) ,  alkaline lysis , enzymatic lysis, and mechanical lysis  . [ 33 ]  The former can be used to quickly find out whether the plasmid is correct in any of several bacterial clones. The yield is a small amount of impure plasmid DNA, which is sufficient for analysis by  restriction digest  and for some cloning techniques.


In the latter, much larger volumes of bacterial suspension are grown from which a maxi-prep can be performed. In essence, this is a scaled-up miniprep followed by additional purification. This results in relatively large amounts (several hundred micrograms) of very pure plasmid DNA.


Many commercial kits have been created to perform plasmid extraction at various scales, purity, and levels of automation.


Plasmid DNA may appear in one of five conformations, which (for a given size) run at different speeds in a gel during  electrophoresis . The conformations are listed below in order of electrophoretic mobility (speed for a given applied voltage) from slowest to fastest:


The rate of migration for small linear fragments is directly proportional to the voltage applied at low voltages. At higher voltages, larger fragments migrate at continuously increasing yet different rates. Thus, the resolution of a gel decreases with increased voltage.


At a specified, low voltage, the migration rate of small linear DNA fragments is a function of their length.  Large linear fragments (over 20 kb or so) migrate at a certain fixed rate regardless of length. This is because the molecules 'respirate', with the bulk of the molecule following the leading end through the gel matrix.  Restriction digests  are frequently used to analyse purified plasmids. These enzymes specifically break the DNA at certain short sequences. The resulting linear fragments form 'bands' after  gel electrophoresis . It is possible to purify certain fragments by cutting the bands out of the gel and dissolving the gel to release the DNA fragments.


Because of its tight conformation, supercoiled DNA migrates faster through a gel than linear or open-circular DNA.


The use of plasmids as a technique in  molecular biology  is supported by  bioinformatics   software . These programs record the  DNA  sequence of plasmid vectors, help to predict cut sites of  restriction enzymes , and to plan manipulations. Examples of software packages that handle plasmid maps are ApE,  Clone Manager , GeneConstructionKit, Geneious,  Genome Compiler , LabGenius, Lasergene,  MacVector , pDraw32, Serial Cloner,  UGENE , VectorFriends,  Vector NTI , and WebDSV. These pieces of software help conduct entire experiments in silico before doing wet experiments. [ 62 ]


Many plasmids have been created over the years and researchers have given out plasmids to plasmid databases such as the non-profit organisations  Addgene  and  BCCM/GeneCorner . One can find and request plasmids from those databases for research.
Researchers also often upload plasmid sequences to the  NCBI database , from which sequences of specific plasmids can be retrieved.


A  plasmid partition system  is a mechanism that ensures the stable inheritance of  plasmids  during bacterial cell division. Each plasmid has its independent replication system which controls the number of copies of the plasmid in a cell. The higher the copy number, the more likely the two daughter cells will contain the plasmid. Generally, each molecule of plasmid diffuses randomly, so the probability of having a plasmid-less daughter cell is 2 1−N , where N is the number of copies. For instance, if there are 2 copies of a plasmid in a cell, there is  50% chance of having one plasmid-less daughter cell. However, high-copy number plasmids have a cost for the hosting cell. This metabolic burden is lower for low-copy plasmids, but those have a higher probability of plasmid loss after a few generations. To control vertical transmission of plasmids, in addition to controlled-replication systems, bacterial plasmids use different maintenance strategies, such as  multimer resolution systems ,  post-segregational killing systems (addiction modules) , and partition systems. [ 1 ]


Plasmid copies are paired around a  centromere -like site and then separated in the two daughter cells. Partition systems involve three elements, organized in an auto-regulated  operon : [ 2 ]


The centromere-like DNA site is required in  cis  for plasmid stability. It often contains one or more inverted repeats which are recognized by multiple CBPs. This forms a nucleoprotein complex termed the partition complex. This complex recruits the motor protein, which is a nucleotide triphosphatase (NTPase). The NTPase uses energy from NTP binding and hydrolysis to directly or indirectly move and attach plasmids to specific host location (e.g. opposite bacterial cell poles).


The partition systems are divided in four types, based primarily on the type of NTPases: [ 3 ] [ 4 ]


This system is also used by most bacteria for  chromosome segregation . [ 3 ]
Type I partition systems are composed of an ATPase which contains  Walker motifs  and a CBP which is structurally distinct in type Ia and Ib. ATPases and CBP from type Ia are longer than the ones from type Ib, but both CBPs contain an  arginine finger  in their N-terminal part. [ 5 ] [ 1 ] [ 6 ]
ParA proteins from different plasmids and bacterial species show 25 to 30% of sequence identity to the protein ParA of the  plasmid P1 . [ 7 ]
The partition of type I system uses a "diffusion-ratchet" mechanism. This mechanism works as follows: [ 8 ]


There are likely to be differences in the details of type I mechanisms. [ 6 ]


Type 1 partition has been mathematically modelled with variations in the mechanism described above. [ 16 ] [ 17 ] [ 18 ] [ 19 ]


The CBP of this type consists in three domains: [ 6 ]


The CBP of this type, also known as  parG  is composed of: [ 6 ]


For this type, the  parS  site is called  parC .


This system is the best understood of the plasmid partition system. [ 6 ]
It is composed of an actin-like ATPAse, ParM, and a CBP called ParR. The centromere like site,  parC  contains two sets of five 11 base pair direct repeats separated by the  parMR  promoter.
The amino-acid sequence identity can go down to 15% between ParM and other actin-like ATPase. [ 7 ] [ 22 ]


The mechanism of partition involved here is a pushing mechanism: [ 23 ]


The filament of ParM is regulated by the polymerization allowed by the presence the partition complex (ParR- parC ), and by the depolymerization controlled by the ATPase activity of ParM.


The type III partition system is the most recently discovered partition system. It is composed of tubulin-like GTPase termed TubZ, and the CBP is termed TubR.
Amino-acid sequence identity can go down to 21% for TubZ proteins. [ 7 ]


The mechanism is similar to a treadmill mechanism: [ 24 ]


The net result being transport of partition complex to the cell pole.


The partition system of the plasmid R388 has been found within the  stb  operon. This operon is composed of three genes,  stbA ,  stbB  and  stbC . [ 25 ]


The StbA- stbDRs  complex may be used to pair plasmid the host chromosome, using indirectly the bacterial partitioning system.


StbA and StbB have opposite but connected effect related to conjugation.


This system has been proposed to be the type IV partition system. [ 26 ]  It is thought to be a derivative of the type I partition system, given the similar operon organization.
This system represents the first evidence for a mechanistic interplay between plasmid segregation and conjugation processes. [ 26 ]


pSK1 is a plasmid from  Staphylococcus aureus . This plasmid has a partition system determined by a single gene,  par , previously known as  orf245 . This gene does not effect the plasmid copy number nor the grow rate (excluding its implication in a post-segregational killing system). A centromere-like binding sequence is present upstream of the  par  gene, and is composed of seven direct repeats and one inverted repeat.


An environment's  plasmidome  refers to the  plasmids  present in it. [ 1 ]
The term is a portmanteau of the two English words Plasmid and Kingdom.
In biological research, plasmidome may refer to the actual plasmids that were found and isolated from a certain  microorganism  by means of culturing isolated microorganism and investigating the plasmids it possesses or by taking an environmental sample and performing a  metagenomic  survey using  next generation sequencing  methods in order to reveal and characterize plasmid  genomes  that belong to that environment. [ 2 ]


A  prophage  is a  bacteriophage  (often shortened to "phage")  genome  that is integrated into the  circular bacterial chromosome  or exists as an extrachromosomal  plasmid  within the bacterial  cell . [ 1 ]  Integration of prophages into the bacterial host is the characteristic step of the  lysogenic cycle  of  temperate  phages. Prophages remain  latent  in the genome through multiple cell divisions until activation by an external factor, such as UV light, leading to production of new phage particles that will  lyse  the cell and spread. As ubiquitous  mobile genetic elements , prophages play important roles in bacterial genetics and evolution, such as in the acquisition of  virulence  factors.


Upon detection of host cell damage by UV light or certain chemicals, the prophage is excised from the bacterial chromosome in a process called prophage induction. After induction, viral replication begins via the  lytic cycle . In the lytic cycle, the virus commandeers the cell's reproductive machinery. The cell may fill with new viruses until it lyses or bursts, or it may release the new viruses one at a time in an exocytotic process. The period from infection to lysis is termed the latent period. A virus following a lytic cycle is called a  virulent  virus. Prophages are important agents of  horizontal gene transfer , and are considered part of the  mobilome . Genes are transferred via  transduction  as the prophage genome is imperfectly excised from the host chromosome and integrated into a new host (specialized transduction) or as fragments of host DNA are packaged into the phage particles and introduced into a new host (generalized transduction). [ 2 ]  All families of bacterial viruses that have circular (single-stranded or double-stranded) DNA genomes or replicate their genomes through  rolling circle replication  (e.g.,  Caudovirales ) have temperate members. [ 3 ]


During infections by the bacterial pathogen  Clostridioides difficile , spontaneous prophage release from the bacterial  chromosome  is common. [ 4 ]   The presence of  deoxycholic acid  in the intestinal environment can promote induction of  C. difficle   biofilm  formation as well as the induction of prophage release. [ 4 ]


Zygotic induction occurs when a bacterial cell carrying the DNA of a bacterial virus transfers its own DNA along with the viral DNA (prophage) into the new host cell. This has the effect of causing the host cell to break apart. [ 5 ]  The DNA of the bacterial cell is silenced before entry into the cell by a  repressor protein  which is encoded for by the prophage. Upon the transfer of the bacterial cell's DNA into the host cell, the repressor protein is no longer encoded for, and the bacterial cell's original DNA is then turned on in the host cell. This mechanism eventually will lead to the release of the virus as the host cell splits open and the viral DNA is able to spread. [ 5 ]  This new discovery provided key insights into  bacterial conjugation  and contributed to the early repression model of gene regulation, which provided an explanation as to how the  lac operon  and λ  bacteriophage  genes are negatively regulated. [ 6 ]


Bacteriophage λ  is able to undergo a type of  recombinational  repair called prophage reactivation. [ 6 ] [ 7 ]  Prophage reactivation can occur by recombination between a UV-damaged infecting phage λ chromosome and a homologous phage  genome  integrated into the bacterial DNA and existing in a prophage state. Prophage reactivation in the case of phage λ appears to be an accurate recombinational repair process that is mediated by the  recA + and red+ gene products. [ citation needed ]


Lysis of host cells during prophage induction can cause the collapse of a microbial population. [ 8 ] [ 9 ]  On the other hand, induction, transduction and superinfection exclusion mechanisms confer many beneficial functions to the host. Induction of prophages allows hosts to compete in the microbial ecology by infecting and lysing susceptible bacteria. [ 10 ]  Phages also enable the host to pick up and integrate antibiotic resistance genes from nearby cells. [ 9 ] [ 10 ] [ 8 ] [ 11 ]  Additionally, phages can enable the host to acquire virulence and pathogenicity genes. [ 9 ] [ 11 ]  Modulation of biofilm formation is also affected by infection by lysogenic phages. [ 11 ]  Superinfection exclusion, or protection against infection by multiple phages, can be conferred by prophage integration. [ 12 ]  Additionally, phage-mediated recombination mechanisms may remodel the host chromosome and provide new ways for cells to regulate metabolism and gene expression, such as those involved in sporulation and competence. [ 11 ] [ 13 ]


Prophages can tell researchers a lot about the relationship between a bacterium and a host. [ 14 ]  With data from more nonpathogenic bacteria, researchers will be able to gather evidence as to whether or not prophages contribute to the survival value of the host. Prophage genomics has the potential to lead to ecological adaptations of the relationships between bacteria. [ 14 ]  Another important area of interest is the control of prophage gene expression with many of the lysogenic conversion genes ( gene conversion ) being tightly regulated. [ 15 ]  This process is capable of converting non-pathogenic bacteria into pathogenic bacteria that can now produce harmful toxins [ 15 ]  such as in  staph infections . Since the specific mechanisms of prophage are not yet detailed, this research could provide the community with this tool for future research. [ 14 ]


Exotoxins  encoded by prophages cause pathogenic outcomes in  agriculture  and  aquaculture . [ 16 ]


Replicative transposition  is a mechanism of transposition in  molecular biology , proposed by  James A. Shapiro  in 1979, [ 1 ]  in which the  transposable element  is duplicated during the reaction, so that the transposing entity is a copy of the original element. In this mechanism, the donor and receptor DNA sequences form a characteristic intermediate "theta" configuration, sometimes called a "Shapiro intermediate". [ 2 ]  Replicative transposition is characteristic to  retrotransposons  and occurs from time to time in class II transposons. [ 3 ]


Retroposons  are repetitive  DNA  fragments which are inserted into  chromosomes  after they had been  reverse transcribed  from any  RNA  molecule.


In contrast to  retrotransposons , retroposons never encode  reverse transcriptase  (RT) (but see below). Therefore, they are non-autonomous elements with regard to  transposition  activity (as opposed to  transposons ).
Non-long terminal repeat (LTR) retrotransposons such as the human  LINE1  elements are sometimes falsely referred to as retroposons. However, this depends on the author. For example,  Howard Temin  published the following definition: Retroposons encode RT but are devoid of  long terminal repeats  (LTRs), for example  long interspersed elements  (LINEs). Retrotransposons also feature LTRs and  retroviruses , in addition, are packaged as viral particles (virions). Retrosequences are non-autonomous elements devoid of RT. They are retroposed with the aid of the machinery of autonomous elements, such as LINEs; examples are  short interspersed nuclear elements  (SINEs) or mRNA-derived  retro(pseudo)genes . [ 2 ] [ 3 ] [ 4 ]


Retroposition accounts for approximately 10,000 gene-duplication events in the human genome, of which approximately 2-10% are likely to be functional. [ 5 ]  Such genes are called  retrogenes  and represent a certain type of retroposon.


A classical event is the retroposition of a spliced pre-mRNA molecule of the  c-Src gene  into the proviral ancestor of the  Rous sarcoma virus  (RSV). The retroposed c-src pre-mRNA still contained a single  intron  and within RSV is now referred to as  v-Src  gene. [ 6 ]


RetrOryza  is a database of  Long terminal repeat - retrotransposons  for the  rice  genome. [ 1 ]


Retrotransposons  (also called  Class I transposable elements ) are  mobile elements  which move in the host genome by converting their transcribed RNA into DNA through  reverse transcription . [ 1 ]  Thus, they differ from Class II transposable elements, or DNA transposons, in utilizing an RNA intermediate for the transposition and leaving the transposition donor site unchanged. [ 2 ]


Through reverse transcription, retrotransposons amplify themselves quickly to become abundant in  eukaryotic  genomes such as  maize  (49–78%) [ 3 ]  and humans (42%). [ 4 ]  They are only present in eukaryotes but share features with  retroviruses  such as  HIV , for example, discontinuous  reverse transcriptase -mediated extrachromosomal recombination. [ 5 ] [ 6 ]


There are two main types of retrotransposons,  long terminal repeats  (LTRs) and non-long terminal repeats (non-LTRs). Retrotransposons are classified based on sequence and method of transposition. [ 7 ]  Most retrotransposons in the maize genome are LTR, whereas in humans they are mostly non-LTR.


LTR retrotransposons are characterized by their long terminal repeats (LTRs), which are present at both the 5' and 3' ends of their sequences. These LTRs contain the promoters for these transposable elements (TEs), are essential for TE integration, and can vary in length from just over 100 base pairs (bp) to more than 1,000 bp. On average, LTR retrotransposons span several thousand base pairs, with the largest known examples reaching up to 30 kilobases (kb).


LTRs are highly functional sequences, and for that reason LTR and non-LTR retrotransposons differ greatly in their reverse transcription and integration mechanisms. Non-LTR retrotransposons use a  target-primed reverse transcription  (TPRT) process, which requires the RNA of the TE to be brought to the cleavage site of the retrotransposon’s integrase, where it is reverse transcribed. In contrast, LTR retrotransposons undergo reverse transcription in the cytoplasm, utilizing two rounds of template switching, and a formation of a pre-integration complex (PIC) composed of double-stranded DNA and an integrase dimer bound to LTRs. This complex then moves into the nucleus for integration into a new genomic location.


LTR retrotransposons typically encode the proteins  gag  and  pol , which may be combined into a single  open reading frame  (ORF) or separated into distinct ORFs. Similar to retroviruses, the gag protein is essential for capsid assembly and the packaging of the TE's RNA and associated proteins. The pol protein is necessary for reverse transcription and includes these crucial domains: PR (protease), RT (reverse transcriptase), RH ( RNase H ), and INT (integrase). Additionally, some LTR retrotransposons have an ORF for an envelope ( env ) protein that is incorporated into the assembled capsid, facilitating attachment to cellular surfaces.


An endogenous retrovirus is a retrovirus without virus pathogenic effects that has been integrated into the host genome by inserting their inheritable genetic information into cells that can be passed onto the next generation like a retrotransposon. [ 8 ]   Because of this, they share features with retroviruses and retrotransposons. When the retroviral DNA is integrated into the host genome they evolve into endogenous retroviruses that influence eukaryotic genomes. So many endogenous retroviruses have inserted themselves into eukaryotic genomes that they allow insight into biology between viral-host interactions and the role of retrotransposons in evolution and disease.
Many retrotransposons share features with endogenous retroviruses, the property of recognising and fusing with the host genome. However, there is a key difference between retroviruses and retrotransposons, which is indicated by the env gene. Although similar to the gene carrying out the same function in retroviruses, the env gene is used to determine whether the gene is retroviral or retrotransposon. If the gene is retroviral it can evolve from a retrotransposon into a retrovirus. They differ by the order of sequences in pol genes. Env genes are found in LTR retrotransposon types Ty1-copia ( Pseudoviridae ), Ty3-gypsy ( Metaviridae ) and BEL/Pao. [ 9 ] [ 8 ]  They encode glycoproteins on the retrovirus envelope needed for entry into the host cell. Retroviruses can move between cells whereas LTR retrotransposons can only move themselves into the genome of the same cell. [ 10 ]  Many vertebrate genes were formed from retroviruses and LTR retrotransposons. One endogenous retrovirus or LTR retrotransposon has the same function and genomic locations in different species, suggesting their role in evolution. [ 11 ]


Like LTR retrotransposons, non-LTR retrotransposons contain genes for reverse transcriptase, RNA-binding protein,  nuclease,  and sometimes ribonuclease H domain [ 12 ]  but they lack the long terminal repeats. RNA-binding proteins bind the RNA-transposition intermediate and nucleases are enzymes that break phosphodiester bonds between nucleotides in nucleic acids. Instead of LTRs, non-LTR retrotransposons have short repeats that can have an inverted order of bases next to each other aside from  direct repeats  found in LTR retrotransposons that is just one sequence of bases repeating itself.


Although they are retrotransposons, they cannot carry out reverse transcription using an RNA transposition intermediate in the same way as LTR retrotransposons. Those two key components of the retrotransposon are still necessary but the way they are incorporated into the chemical reactions is different. This is because unlike LTR retrotransposons, non-LTR retrotransposons do not contain sequences that bind tRNA.


They mostly fall into two types – LINEs (Long interspersed nuclear elements) and SINEs (Short interspersed nuclear elements). SVA elements are the exception between the two as they share similarities with both LINEs and SINEs, containing Alu elements and different numbers of the same repeat. SVAs are shorter than LINEs but longer than SINEs.


While historically viewed as "junk DNA", research suggests in some cases, both LINEs and SINEs were incorporated into novel genes to form new functions. [ 13 ]


When a LINE is transcribed, the transcript contains an RNA polymerase II promoter that ensures LINEs can be copied into whichever location it inserts itself into.  RNA polymerase II is the enzyme that transcribes genes into mRNA transcripts. The ends of LINE transcripts are rich in multiple adenines, [ 14 ]  the bases that are added at the end of transcription so that LINE transcripts would not be degraded. This transcript is the RNA transposition intermediate.


The RNA transposition intermediate moves from the nucleus into the cytoplasm for translation. This gives the two coding regions of a LINE that in turn binds back to the RNA it is transcribed from. The LINE RNA then moves back into the nucleus to insert into the eukaryotic genome.