Perfect Terminal Inverted Repeats Research Articles

Divergent evolution profiles of DD37D and DD39D families of Tc1/mariner transposons in eukaryotes

DNA transposons play a significant role in shaping the size and structure of eukaryotic genomes. The Tc1/mariner transposons are the most diverse and widely distributed superfamily of DNA transposons and the structure and distribution of several Tc1/mariner families, such as DD35E/TR, DD36E/IC, DD37E/TRT, and DD41D/VS, have been well studied. Nonetheless, a greater understanding of the structure and diversity of Tc1/mariner transposons will provide insight into the evolutionary history of eukaryotic genomes. Here, we conducted further analysis of DD37D/maT and DD39D (named Guest, GT), which were identified by the specific catalytic domains DD37D and DD39D. Most transposons of the maT family have a total length of approximately 1.3 kb and harbor a single open reading frame encoding a ~ 346 amino acid (range 302–398 aa) transposase protein, flanked by short terminal inverted repeats (TIRs) (13–48 base pairs, bp). In contrast, GTs transposons were longer (2.0–5.8 kb), encoded a transposase protein of ~400 aa (range 140–592 aa), and were flanked by short TIRs (19–41 bp). Several conserved motifs, including two helix–turn–helix (HTH) motifs, a GRPR (GRKR) motif, a nuclear localization sequence, and a DDD domain, were also identified in maT and GT transposases. Phylogenetic analyses of the DDD domain showed that the maT and GT families each belong to a monophyletic clade and appear to be closely related to DD41D/VS and DD34D/mariner. In addition, maTs are mainly distributed in invertebrates (144 species), whereas GTs are mainly distributed in land plants through a small number of GTs are present in Chromista and animals. Sequence identity and phylogenetic analysis revealed that horizontal transfer (HT) events of maT and GT might occur between kingdoms and phyla of eukaryotes; however, pairwise distance comparisons between host genes and transposons indicated that HT events involving maTs might be less frequent between invertebrate species and HT events involving GTs may be less frequent between land plant species. Overall, the DD37D/maT and DD39D/GT families display significantly different distribution and tend to be identified in more ancient evolutionary families. The discovery of intact transposases, perfect TIRs, and target site duplications (TSD) of maTs and GTs illustrates that the DD37D/maT and DD39D/GT families may be active. Together, these findings improve our understanding of the diversity of Tc1/mariner transposons and their impact on eukaryotic genome evolution.

Incomer, a DD36E family of Tc1/mariner transposons newly discovered in animals

BackgroundThe Tc1/mariner superfamily might represent the most diverse and widely distributed group of DNA transposons. Several families have been identified; however, exploring the diversity of this superfamily and updating its classification is still ongoing in the life sciences.ResultsHere we identified a new family of Tc1/mariner transposons, named Incomer (IC), which is close to, but distinct from the known family DD34E/Tc1. ICs have a total length of about 1.2 kb, and harbor a single open reading frame encoding a ~ 346 amino acid transposase with a DD36E motif and flanked by short terminal inverted repeats (TIRs) (22–32 base pairs, bp). This family is absent from prokaryotes, and is mainly distributed among vertebrates (141 species of four classes), including Agnatha (one species of jawless fish), Actinopterygii (132 species of ray-finned fish), Amphibia (four species of frogs), and Mammalia (four species of bats), but have a restricted distribution in invertebrates (four species in Insecta and nine in Arachnida). All ICs in bats (Myotis lucifugus, Eptesicus fuscus, Myotis davidii, and Myotis brandtii) are present as truncated copies in these genomes, and most of them are flanked by relatively long TIRs (51–126 bp). High copy numbers of miniature inverted-repeat transposable elements (MITEs) derived from ICs were also identified in bat genomes. Phylogenetic analysis revealed that ICs are more closely related to DD34E/Tc1 than to other families of Tc1/mariner (e.g., DD34D/mariner and DD × D/pogo), and can be classified into four distinct clusters. The host and IC phylogenies and pairwise distance comparisons between RAG1 genes and all consensus sequences of ICs support the idea that multiple episodes of horizontal transfer (HT) of ICs have occurred in vertebrates. In addition, the discovery of intact transposases, perfect TIRs and target site duplications of ICs suggests that this family may still be active in Insecta, Arachnida, frogs, and fish.ConclusionsExploring the diversity of Tc1/mariner transposons and revealing their evolutionary profiles will help provide a better understanding of the evolution of DNA transposons and their impact on genomic evolution. Here, a newly discovered family (DD36E/Incomer) of Tc1/mariner transposons is described in animals. It displays a similar structural organization and close relationship with the known DD34E/Tc1 elements, but has a relatively narrow distribution, indicating that DD36E/IC might have originated from the DD34E/Tc1 family. Our data also support the hypothesis of horizontal transfer of IC in vertebrates, even invading one lineage of mammals (bats). This study expands our understanding of the diversity of Tc1/mariner transposons and updates the classification of this superfamily.

Transposition of the bamboo Mariner-like element Ppmar1 in yeast

The moso bamboo genome contains the two structurally intact and thus potentially functional mariner-like elements Ppmar1 and Ppmar2. Both elements contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase gene. Here we investigated whether Ppmar1 is functional in yeast (Saccharomyces cerevisiae). We have designed a two-component system consisting of a transposase expression cassette and a non-autonomous transposon on two separate plasmids. We demonstrate that the Ppmar1 transposase Pptpase1 catalyses excision of the non-autonomous Ppmar1NA element from the plasmid and reintegration at TA dinucleotide sequences in the yeast chromosomes. In addition, we generated 14 hyperactive Ppmar1 transposase variants by systematic single amino acid substitutions. The most active transposase variant, S171A, induces 10-fold more frequent Ppmar1NA excisions in yeast than the wild type transposase. The Ppmar1 transposon is a promising tool for insertion mutagenesis in moso bamboo and may be used in other plants as an alternative to the established transposon tagging systems.

Two active bamboo mariner-like transposable elements (Ppmar1 and Ppmar2) identified as the transposon-based genetic tools for mutagenesis

Bamboo is one of the most important non-timber forest species in the world, but their molecular breeding lags far behind in contrast to other economic plants. Regarding the difficulties of hybridization and gene modification, the transposon-based insertional mutagenesis might be an alternative, feasible way for molecular breeding of bamboo. A systematic search for potential active transposons identified two full-length mariner-like elements (MLEs) (Ppmar1 and Ppmar2) from moso bamboo in the previous study. Both MLEs contain perfect terminal inverted repeats (TIRs) and a full-length intact transposase. Two transposases contain intact DNA-binding motifs and a DD39D catalytic domain which indicates that Ppmar1 and Ppmar2 are likely active. Here, we deployed a heterologous transposition system of Arabidopsis thaliana to study the transposition activity of Ppmar1 and Ppmar2. The results show that both MLEs could transpose in A. thaliana. Excisions of Ppmar1 and Ppmar2 are usually unperfect as they leave 1–4 bp in excision sites. The reinsertions of both Ppmar1 and Ppmar2 occur at TA dinucleotides and prefer to insert into the TA-rich regions. The insertion sites are dispersed and non-linked. Two active bamboo transposons identified here not only could be applied to construction of the bamboo mutant libraries but also would provide another choice for other plant transposon-based gene tagging.

Ppmar1 and Ppmar2: the first two complete and intact full-length mariner-like elements isolated in Phyllostachys edulis

We report the isolation of two full-length mariner-like elements (MLEs) from a bamboo species (Phyllostachys edulis) by chromosome walking using a modified magnetic enrichment procedure. Ppmar1 is 3472 base pairs (bp) in length with 28-bp perfect inverted terminal repeats (ITRs) and a 1986-bp open reading frame (ORF) encoding transposase, whereas Ppmar2 is 2679 bp in length with the 27-bp perfect ITRs and the 1877-bp ORF encoding transposase. Ppmar1 and Ppmar2 share the typical ITR consensus sequences of plant MLEs (5′-CTC CCT CCR T-3′, where R is A or G) and the typical TA/AT target site duplications. Phylogenetic analysis demonstrated that Ppmar1 belongs to clade A2 and Ppmar2 to clade C of plant MLE subfamilies. The two transposases encoded contain intact DNA-binding motifs and a DD39D catalytic domain and many residues previously shown to be critical for transposase activity are conserved in Ppmar1 and Ppmar2. All the above results imply that both transposons are likely to be naturally active. Cloning of two full-length mariner-like elements would provide a foundation for the investigation of transpositional activity of moso bamboo MLE.

Recent transposition of yabusame, a novel piggyBac-like transposable element in the genome of the silkworm, Bombyx mori

On the W chromosome of the silkworm, Bombyx mori, we found a novel piggyBac-like DNA transposon that potentially encodes an intact transposase (610 amino acid residues), which is flanked by 16-bp perfect inverted terminal repeats and a duplicated TTAA target site. Interestingly, we also identified another intact copy of this transposon on an autosome (chromosome 21), which showed 99.6% identity in the DNA sequence of the transposase (99.3% amino acid identity). These features raised the possibility that this novel piggyBac-like DNA transposon, designated as yabusame, may retain transposition activity. Here we report the identification and characterization of yabusame transposons from the silkworm. We cloned the full length of the yabusame transposon on the W chromosome (yabusame-W) and its autosomal copy (yabusame-1). Southern blot analysis showed that there are interstrain polymorphisms in yabusame elements for their insertion sites and copy number. We also found strong evidence for the recent transposition of yabusame elements in the silkworm genome. Although our in vitro excision assays suggested that the transposition activity of yabusame-1 and yabusame-W has been lost almost entirely, our data will lead to a greater understanding of the characteristics of piggyBac superfamily elements.

Characterization of a new mitochondrial plasmid from Fusarium proliferatum

A 10.3kb linear mitochondrial DNA plasmid designated pFP1 was isolated from Fusarium proliferatum. The DNA sequence of the plasmid consists of 10,336bp with perfect terminal inverted repeats of 400bp. Two major, non-overlapping ORFs were identified on opposite strands, encoding a phage-type RNA polymerase and a family B type DNA polymerase, respectively. One additional minor ORF encoding a putative highly basic protein was also identified. The copy number of pFP1, as determined by RT-PCR, ranged between 1.8 and 3.1 per mtDNA copies depending on the host strain. Real-time PCR analysis of a total of 400 cultures surviving ethidium bromide curing indicated that no plasmid-free strains could be obtained by this treatment. Further single spore selections of the survivors with reduced plasmid content were needed to obtain plasmid-free clones. No phenotypic differences were found between the wild-type strains and their plasmid-free progenies.

The bandit, a New DNA Transposon from a Hookworm—Possible Horizontal Genetic Transfer between Host and Parasite

BackgroundAn enhanced understanding of the hookworm genome and its resident mobile genetic elements should facilitate understanding of the genome evolution, genome organization, possibly host-parasite co-evolution and horizontal gene transfer, and from a practical perspective, development of transposon-based transgenesis for hookworms and other parasitic nematodes.Methodology/Principal FindingsA novel mariner-like element (MLE) was characterized from the genome of the dog hookworm, Ancylostoma caninum, and termed bandit. The consensus sequence of the bandit transposon was 1,285 base pairs (bp) in length. The new transposon was flanked by perfect terminal inverted repeats of 32 nucleotides in length with a common target site duplication TA, and it encoded an open reading frame (ORF) of 342 deduced amino acid residues. Phylogenetic comparisons confirmed that the ORF encoded a mariner-like transposase, which included conserved catalytic domains, and that the bandit transposon belonged to the cecropia subfamily of MLEs. The phylogenetic analysis also indicated that the Hsmar1 transposon from humans was the closest known relative of bandit, and that bandit and Hsmar1 constituted a clade discrete from the Tc1 subfamily of MLEs from the nematode Caenorhabditis elegans. Moreover, homology models based on the crystal structure of Mos1 from Drosophila mauritiana revealed closer identity in active site residues of the catalytic domain including Ser281, Lys289 and Asp293 between bandit and Hsmar1 than between Mos1 and either bandit or Hsmar1. The entire bandit ORF was amplified from genomic DNA and a fragment of the bandit ORF was amplified from RNA, indicating that this transposon is actively transcribed in hookworms.Conclusions/SignificanceA mariner-like transposon termed bandit has colonized the genome of the hookworm A. caninum. Although MLEs exhibit a broad host range, and are identified in other nematodes, the closest phylogenetic relative of bandit is the Hsmar1 element of humans. This surprising finding suggests that bandit was transferred horizontally between hookworm parasites and their mammalian hosts.

Insertion and excision of a transposable element governs the red floral phenotype in commercial petunias

Commercial cultivars of Petunia hybrida with red flowers (red petunias) accumulate cyanidin 3-glucoside as a main floral anthocyanin pigment. The conversion of anthocyanidin 3-glucosides to anthocyanidin 3-rutinosides is catalyzed by a UDP-rhamnose: anthocyanidin 3-glucoside-rhamnosyltransferase (RT). In red petunias, the RT gene is known to be disrupted by the insertion of a transposable element (dTph3). We have cloned the dTph3-like element (called dTph3-C) from a red petunia. Unlike dTph3, dTph3-C harbored a perfect terminal inverted repeat. In addition, an excision product (so-called "footprint") of dTph3/dTph3-C was found in another red petunia. The RT transcripts harboring this footprint could not produce the RT enzyme because a stop codon was created in the footprint sequence. The genotypes of the 42 commercial petunias which exhibit different anthocyanin pigmentation were determined by multiplex PCR. In this technique, the amplified products from normal, dTph3/dTph3-C-inserted, and footprint-retaining RT genes can be separated from one another. Our results indicate that the red-floral phenotype of commercial petunias is governed by insertion and excision events of a transposable element in the coding region of the RT gene.

Characterization of a carbapenemase-producing clinical isolate of Bacteroides fragilis in Scandinavia: Genetic analysis of a unique insertion sequence

In 2003 a Bacteroides fragilis blood culture isolate (K2-28) was recovered from a 61-y-old male with severe general atherosclerosis during treatment with meropenem. K2-28 was shown to possess a functional metallo-beta-lactamase with a reduction in imipenem MIC from 256 to 3 mg/l in the presence of EDTA using the MBL E-test strip. PCR results were for positive for the cfiA gene. Analysis of the cfiA from K2-28 revealed it was 100% identical to previously described cfiA-1 genes. Analysis of the upstream region of cfiA revealed a novel insertion sequence (IS) element, being most similar (94% identity) to IS612 recently described from Japan designating the element within the IS4 family. The element possessed a perfect terminal inverted repeat sequence at the distal ends of the IS element and provided a putative promoter for transcription of the cfiA gene. The distance between the hybrid promoter and the cfiA start codon was 158 base pairs and inserted into a different DNA sequence upstream of cfiA to that previously reported. The -10 promoter region was most similar to that of IS613 (100%) and the -35 promoter region to IS612 (100%), demonstrating the plasticity of these genetic regions.

Molecular characterization ofVulmar1, a completemarinertransposon of sugar beet and diversity ofmariner- andEn/Spm-like sequences in the genusBeta

Transposons of the Tc1-mariner superfamily are widespread in eukaryotic genomes. We have isolated the mariner element Vulmar1 from Beta vulgaris L., which is 3909 bp long and bordered by perfect terminal inverted repeats of 32 bp with homology to terminal inverted repeats of transposons from soybean and rice. According to a characteristic amino acid signature, Vulmar1 can be assigned to the DD39D group of mariner transposons. Vulmar1 is flanked by a 5'-TA-3' target site duplication that is typical for mariner transposons. Southern hybridization revealed that mariner-like copies are highly abundant in Beta species, and sequence analysis of 10 transposase fragments from representative species of the four Beta sections revealed an identity between 34% and 100% after conceptual translation. By fluorescent in situ hybridization, Vulmar1 was detected in distal euchromatin as well as in some intercalary and pericentromeric regions of all B. vulgaris chromosomes. In addition, using PCR, we were able to amplify fragments of the transposase gene of En/Spm-like transposons in the genus Beta. En/Spm-like transposase sequences are highly amplified in four Beta sections and showed a considerable degree of conservation (88.5-100%) at the protein level, while the homology to corresponding regions of En/Spm transposons of other plant species ranges from 49.5% to 62.5%. By fluorescent in situ hybridization, En/Spm-like transposon signals of strong intensity were detected on all chromosomes of B. vulgaris.

Hop, an active Mutator-like element in the genome of the fungus Fusarium oxysporum.

A new type of active DNA transposon has been identified in the genome of Fusarium oxysporum by its transposition into the niaD target gene. Two insertions within the final exon, in opposite orientations at the same nucleotide site, have been characterized. These elements, called Hop, are 3,299 bp long, with perfect terminal inverted repeats (TIRs) of 99 bp. The sequencing of genomic copies reveals a 9-bp target site duplication and no apparent sequence specificity at the insertion sites. The sequencing of a cDNA indicates that Hop does not contain an intron and encodes a putative transposase of 836 amino acids. The structural features (length, TIRs size, and 9-bp duplication), together with the presence of conserved domains in the transposase, strongly suggest that Hop is a Mutator-like element (MULE). Hop is thus the first active member of this family found beyond plants. The high rate of excision observed indicates that Hop is very active and thus represents a promising efficient tagging system for the isolation of fungal genes. The distribution of Hop elements within the Fusarium genus revealed that they are present in different species, suggesting that related elements could be present in other fungal genomes. In fact, Hop-related sequences have been identified in the survey of the entire genome sequence of three other ascomycetes, Magnaporthe grisea, Neurospora crassa, and Aspergillus fumigatus.

Characterization of a unique genomic clone located 5' upstream of the Oshsp16.9B gene on chromosome 1 in rice (Oryza sativa L. cv Tainung No. 67).

Small heat-shock proteins (sHSP) are the most abundant heat stress-induced proteins in plants. In rice, there are at least seven members of class-I sHSP. A 1.6-kb DNA fragment was isolated from the EcoRI-digested rice genomic library probed with the cDNA pTS1 encoding a 16.9-kDa class-I sHSP. This fragment was composed of 365-bp tandem direct repeats (DRs) and 441-bp near perfect long terminal inverted repeats (LTIRs). The DRs contain 123-bp regions with 99% nucleotide identity to the 5' coding region of the Oshsp16.9B gene. Two putative pseudogenes were deduced from the DRs. Using the LTIR as a specific probe, Southern-blotting analysis showed that there was a single copy of this 1.6-kb DNA fragment in the rice genome. By genomic walking, we located this fragment in proximity 5'-upstream of the Oshsp16.9B gene that was mapped on chromosome 1 with other two class-I sHSP genes, Oshsp16.9A and Oshsp16.9C. By comparative analysis of the nucleotide sequences of class-I sHSP genes clustered on chromosome 1 between Tainung No. 67 and Nipponbare cultivars, we confirmed our mapping results of these genes and only the promoter region of Oshsp16.9B was different. However, we found that the expression profile of Oshsp16.9B upon different heat stresses in Nipponbare was not significantly different relative to that in Tainung No. 67.

Characterization of the 450-kb Linear Plasmid in a Polychlorinated Biphenyl Degrader, Rhodococcus sp. Strain RHA1

A strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, has diverse biphenyl/PCB degradative genes and harbors huge linear plasmids, including pRHL1 (1,100 kb), pRHL2 (450 kb), and pRHL3 (330 kb). The diverse degradative genes are distributed mainly on the pRHL1 and pRHL2 plasmids. In this study, the structural and functional characteristics of pRHL2 were determined. We constructed a physical map of pRHL2, and the degradative enzyme genes, including bphB2, etbD2, etbC, bphDEF, bphC2, and bphC4, were localized in three regions. Conjugal transfer of pRHL2 between RHA1 mutant derivatives was observed at a frequency of 7.5 x 10(-5) transconjugant per recipient. These results suggested that the linear plasmid is a possible determinant of propagation of the diverse degradative genes in rhodococci. The termini of pRHL2 were cloned and sequenced. The left and right termini of pRHL2 had 3-bp perfect terminal inverted repeats and were not as similar to each other (64% identity) as the known actinomycete linear replicons are. Southern hybridization analysis with pRHL2 terminal probes suggested that the right terminus of pRHL2 is similar to pRHL1 and pRHL3 termini. Retardation of both terminal fragments in the gel shift assay indicated that each terminus of pRHL2 is linked to a protein. We suggest that pRHL2 has invertron termini, as has been reported previously for Streptomyces linear replicons.

The transposable element IS4712 prevents S-layer gene (sbsA) expression in Bacillus stearothermophilus and also affects the synthesis of altered surface layer proteins.

Cell surface (S)-layer protein synthesis in Bacillus stearothermophilus PV72/p6 is blocked when cells are grown at elevated temperature. From a culture exhibiting the S-layer-negative phenotype, the S-layer deficient mutant T5 (SbsA-) was isolated. Genetic analysis of the S-layer-encoding gene (sbsA) of mutant T5 revealed an insertion element (IS4712) integrated into the upstream regulatory region of the S-layer gene, thereby blocking sbsA transcription. The insertion element consists of 1371 base pairs which are flanked by two perfect inverted terminal repeats. Sequence similarity to other transposases of the IS4 family was detected. DNA-DNA hybridizations demonstrated that multiple homologues of IS4712 were also present within the genomes of several other thermophilic bacillus isolates. Attempts to isolate SbsA+ revertants failed. Instead, cells with altered surface proteins were detected. The synthesis of the altered S-layer proteins was correlated with the presence of IS4712 along with the occurrence of deletions in the sbsA coding region. Furthermore imprecise excision of IS4712 was detected. This work demonstrated that B. stearothermophilus is able to express at least four different S-layer proteins and that blocking of sbsA transcription by the insertion element IS4712 is associated with the expression of altered surface proteins.

Identification and characterisation of IS 1383, a new insertion sequence isolated from Pseudomonas putida strain H

A new insertion sequence (IS 1383) was identified on plasmids from Pseudomonas putida strain H and its nucleotide sequence was determined. IS 1383 contains perfect terminal inverted repeats of 13-bp flanking a 1.4-kb internal sequence. A single significant open reading frame was identified that can encode a 342-amino acid polypeptide which was predicted to be highly basic and to have homology to polypeptides known from several other bacterial insertion sequences. At least six copies of IS 1383 are present on the plasmids pPGH1 and pPGH2, whereas no copy could be detected on the chromosome of P. putida strain H. Target duplications did not flank the inverted repeats of any of the six IS 1383 copies examined. Analysis of the integration sites of IS 1383 revealed hints for a target specificity. Multiple sequence alignments of the transposases, the inverted repeats and the integration sites pointed to the assignment of IS 1383 into a putative new family of insertion sequences defined as the IS 1111 family.

Identification and characterisation of IS1383, a new insertion sequence isolated from Pseudomonas putida strain H.

A new insertion sequence (IS1383) was identified on plasmids from Pseudomonas putida strain H and its nucleotide sequence was determined. IS1383 contains perfect terminal inverted repeats of 13-bp flanking a 1.4-kb internal sequence. A single significant open reading frame was identified that can encode a 342-amino acid polypeptide which was predicted to be highly basic and to have homology to polypeptides known from several other bacterial insertion sequences. At least six copies of IS1383 are present on the plasmids pPGH1 and pPGH2, whereas no copy could be detected on the chromosome of P. putida strain H. Target duplications did not flank the inverted repeats of any of the six IS1383 copies examined. Analysis of the integration sites of IS1383 revealed hints for a target specificity. Multiple sequence alignments of the transposases, the inverted repeats and the integration sites pointed to the assignment of IS1383 into a putative new family of insertion sequences defined as the IS1111 family.

Molecular evolution of an ancient mariner transposon, Hsmarl, in the human genome

A confident consensus sequence for Hsmarl, the first mariner transposon recognized in the human genome, was generated using three genomic and 15 cDNA sequences. It is thought to represent the ancestrally active copy that invaded an early primate genome. The consensus is 1287 base pairs (bp) long, has 30 bp perfect inverted terminal repeats (ITRs), and encodes a 343 amino acid (aa) mariner transposase. Each copy has diverged from the consensus largely independently of the others and mostly neutrally, and most are now defective. They differ from the consensus by an average of 7.8% in DNA sequence and 7.5 indels per kilobase, both of which values indicate that the copies were formed about 50 Myr ago. On average, only 20% of the 73 surmised CpG hypermutable sites in the consensus remain. A remarkable exception to this loss of functionality is revealed by a set of ten cDNA clones derived from a particular genomic copy that has diverged only 2.4% from the consensus, retained 54% of its hypermutable CpG pairs, and which has a full-length transposase open reading frame. The complete sequence of one of these cDNAs (NIB1543) indicates that the transposase gene of this copy may have been conserved because it is spliced to a human cellular gene encoding a SET domain protein. A specific PCR assay was used to reveal the presence of Hsmarl copies in all primates examined representing all major lineages, but not in close relatives of primates. PCR fragments cloned and sequenced from a representative sample of primates confirmed that Hsmarl copies are present in all major lineages, and also revealed another cecropia subfamily mariner in prosimians only, and a third highly divergent mariner present in the greater slow loris Nycticebus coucang. There are about 200 copies of Hsmarl in the human genome, as well as ±2400 copies of a derived 80 bp paired ITR structure and ±4600 copies of solo ITRs. Thus, this transposon had a considerable insertional mutagenic effect on past primate genomes.

Flipper, a mobile Fot1-like transposable element in Botrytis cinerea.

A transposable element, Flipper, was isolated from the phytopathogenic fungus Botrytis cinerea. The element was identified as an insertion sequence within the coding region of the nitrate reductase gene. The Flipper sequence is 1842 bp long with perfect inverted terminal repeats (ITRs) of 48 bp and an open reading frame (ORF) of 533 amino acids, potentially encoding for a transposase; the element is flanked by the dinucleotide TA. The encoded protein is very similar to the putative transposases of three elements from other phytopathogenic fungi, Fot1 from Fusarium oxysporum, and Pot2 and MGR586 from Magnaporthe grisea. The number of Flipper elements in strains of B. cinerea varied from 0 to 20 copies per genome. Analysis of the descendants of one cross showed that the segregation ratio of Flipper elements was 2:2 and that the copies were not linked.

Molecular and cytological analysis of a mariner transposon from Hessian fly.

Degenerate PCR primers for conserved regions of the mariner transposase have been shown to amplify DNA sequences from the Hessian fly (Mayetiola destructor). Using one of these sequences as a hybridization probe, a clone from an M. destructor genomic library in phage lambda was recovered and sequenced. A transposable element, Desmar 1, with perfect inverted terminal repeats and an open reading frame that encodes a mariner class transposase was found. When compared to mariner sequences in the gene database, the transposase proved to be similar to that of the active mariner Mos 1 from the fruit fly (Drosophila mauritiana). In situ hybridization of the transposon DNA sequence to salivary gland polytene chromosomes revealed the general cytological locations of mariner elements. The distribution of sequences with homology to the probe was predominantly, but not exclusively, n paracentromeric regions.