Subterminal Sequences Research Articles

Transposon-mediated telomere destabilization: a driver of genome evolution in the blast fungus.

The fungus Magnaporthe oryzae causes devastating diseases of crops, including rice and wheat, and in various grasses. Strains from ryegrasses have highly unstable chromosome ends that undergo frequent rearrangements, and this has been associated with the presence of retrotransposons (Magnaporthe oryzae Telomeric Retrotransposons—MoTeRs) inserted in the telomeres. The objective of the present study was to determine the mechanisms by which MoTeRs promote telomere instability. Targeted cloning, mapping, and sequencing of parental and novel telomeric restriction fragments (TRFs), along with MinION sequencing of genomic DNA allowed us to document the precise molecular alterations underlying 109 newly-formed TRFs. These included truncations of subterminal rDNA sequences; acquisition of MoTeR insertions by ‘plain’ telomeres; insertion of the MAGGY retrotransposons into MoTeR arrays; MoTeR-independent expansion and contraction of subtelomeric tandem repeats; and a variety of rearrangements initiated through breaks in interstitial telomere tracts that are generated during MoTeR integration. Overall, we estimate that alterations occurred in approximately sixty percent of chromosomes (one in three telomeres) analyzed. Most importantly, we describe an entirely new mechanism by which transposons can promote genomic alterations at exceptionally high frequencies, and in a manner that can promote genome evolution while minimizing collateral damage to overall chromosome architecture and function.

Seamless insert-plasmid assembly at sub-terminal homologous sequences

The engineering of fusion proteins for structural biology and protein nanotechnology often requires seamless DNA assembly with slight variations in the domain boundaries. To improve the molecular biology workflow for such projects, we evaluated the use of sub-terminal homologous sequences (HS) for co-transformation cloning and for T5 exonuclease / Phusion DNA polymerase mediated in vitro assembly. To quantify the effects of different HS-to-ends distances on cloning efficiency, we designed a blue-white-pink screening system that allowed us to easily identify positive clones (blue colonies), negative clones resulting from circular template plasmid (pink colonies) and negative colonies originating from linearized plasmids that have recircularized without an insert (white colonies). Our experiments show that both methods are feasible with HS-to-ends distances up to at least 10 base pairs. Using a combination of co-transformation cloning at sub-terminal HS and nucleotide insertions in non-annealing primer 5′-overhangs, we integrated a fusion protein into the third intracellular loop (ICL) of a G-protein-coupled receptor (GPCR) with nine different linker boundaries, using only a single plasmid linearization reaction. This molecular cloning approach is an invaluable tool for protein engineering, protein nanotechnology and synthetic biology that extends the range of applications of DNA assembly strategies.

Structural Basis of hAT Transposon End Recognition by Hermes, an Octameric DNA Transposase from Musca domestica

Hermes is a member of the hAT transposon superfamily that has active representatives, including McClintock's archetypal Ac mobile genetic element, in many eukaryotic species. The crystal structure of the Hermes transposase-DNA complex reveals that Hermes forms an octameric ring organized as a tetramer of dimers. Although isolated dimers are active in vitro for all the chemical steps of transposition, only octamers are active in vivo. The octamer can provide not only multiple specific DNA-binding domains to recognize repeated subterminal sequences within the transposon ends, which are important for activity, but also multiple nonspecific DNA binding surfaces for target capture. The unusual assembly explains the basis of bipartite DNA recognition at hAT transposon ends, provides a rationale for transposon end asymmetry, and suggests how the avidity provided by multiple sites of interaction could allow a transposase to locate its transposon ends amidst a sea of chromosomal DNA.

DNA binding activities of the Herves transposase from the mosquito Anopheles gambiae

BackgroundDetermining the mechanisms by which transposable elements move within a genome increases our understanding of how they can shape genome evolution. Class 2 transposable elements transpose via a 'cut-and-paste' mechanism mediated by a transposase that binds to sites at or near the ends of the transposon. Herves is a member of the hAT superfamily of class 2 transposons and was isolated from Anopheles gambiae, a medically important mosquito species that is the major vector of malaria in sub-Saharan Africa. Herves is transpositionally active and intact copies of it are found in field populations of A gambiae. In this study we report the binding activities of the Herves transposase to the sequences at the ends of the Herves transposon and compare these to other sequences recognized by hAT transposases isolated from other organisms.ResultsWe identified the specific DNA-binding sites of the Herves transposase. Active Herves transposase was purified using an Escherichia coli expression system and bound in a site-specific manner to the subterminal and terminal sequences of the left and right ends of the element, respectively, and also interacted with the right but not the left terminal inverted repeat. We identified a common subterminal DNA-binding motif (CG/AATTCAT) that is critical and sufficient for Herves transposase binding.ConclusionsThe Herves transposase binds specifically to a short motif located at both ends of the transposon but shows differential binding with respect to the left and right terminal inverted repeats. Despite similarities in the overall structures of hAT transposases, the regions to which they bind in their respective transposons differ in sequence ensuring the specificity of these enzymes to their respective transposon. The asymmetry with which the Herves terminal inverted repeats are bound by the transposase may indicate that these differ in their interactions with the enzyme.

Identification of a high frequency transposon induced by tissue culture, nDaiZ, a member of the hAT family in rice

Recent completion of rice genome sequencing has revealed that more than 40% of its genome consists of repetitive sequences, and most of them are related to inactive transposable elements. In the present study, a transposable element, nDaiZ0, which is induced by tissue culture with high frequency, was identified by sequence analysis of an allelic line of the golden hull and internode 2 ( gh2) mutant, which was integrated into the forth exon of GH2. The 528-bp nDaiZ0 has 14-bp terminal inverted repeats (TIRs), and generates an 8-bp duplication of its target sites (TSD) during its mobilization. nDaiZs are non-autonomous transposons and have no coding capacity. Bioinformatics analysis and southern blot hybridization showed that at least 16 copies of nDaiZ elements exist in the japonica cultivar Nipponbare genome and 11 copies in the indica cultivar 93-11 genome. During tissue culture, only one copy, nDaiZ9, located on chromosome 5 in the genome of Nipponbare can be activated with its transposable frequency reaching 30%. However, nDaiZ9 was not present in the 93-11 genome. The larger elements, DaiZs, were further identified by database searching using nDaiZ0 as a query because they share similar TIRs and subterminal sequences. DaiZ can also generate an 8-bp TSD. DaiZ elements contain a conserved region with a high similarity to the hAT dimerization motif, suggesting that the nDaiZ– DaiZ transposon system probably belongs to the hAT superfamily of class II transposons. Phylogenetic analysis indicated that it is a new type of plant hAT-like transposon. Although nDaiZ is activated by tissue culture, the high transposable frequency indicates that it could become a useful gene tagging system for rice functional genomic studies. In addition, the mechanism of the high transposable ability of nDaiZ9 is discussed.

Human Telomere Structure and Biology

Human telomeric DNA is complex and highly variable. Subterminal sequences are associated with cis-acting determinants of allele-specific (TTAGGG)n tract length regulation and may modulate susceptibility of (TTAGGG)n tracts to rapid deletion events. More extensive subtelomeric DNA tracts are filled with segmental duplications and segments that vary in copy number, leading to highly variable subtelomeric allele structures in the human population. RNA transcripts encoded in telomere regions include multicopy protein-encoding gene families and a variety of noncoding RNAs. One recently described family of (UUAGGG)n-containing subterminal RNAs appears to be critical for telomere integrity; these RNAs associate with telomeric chromatin and are regulated by RNA surveillance factors including human homologs of the yeast Est1p protein. An increasingly detailed and complete picture of telomeric DNA sequence organization and structural variation is essential for understanding and tracking allele-specific subterminal and subtelomeric features critical for human biology.

Sub-terminal Sequences Modulating IS30 Transposition in Vivo and in Vitro

Inverted repeats of insertion sequences (ISs) are indispensable for transposition. We demonstrate that sub-terminal sequences adjacent to the inverted repeats of IS30 are also required for optimal transposition activity. We have developed a cell-free recombination system and showed that the transposase catalyses formation of a figure-of-eight transposition intermediate, where a 2 bp long single strand bridge holds the inverted repeat sequences (IRs) together. This is the first demonstration of the figure-of-eight structure in a non-IS3 family element, suggesting that this mechanism is likely more widely adopted among IS families. We show that the absence of sub-terminal IS30 sequences negatively influences figure-of-eight production both in vivo and in vitro. These regions enhance IR–IR junction formation and IR-targeting events in vivo. Enhancer elements have been identified within 51 bp internal to IRL and 17 bp internal to IRR. In the right end, a decanucleotide, 5′-GAGATAATTG-3′, is responsible for wild-type activity, while in the left end, a complex assembly of repetitive elements is required. Functioning of the 10 bp element in the right end is position-dependent and the repetitive elements in the left end act cooperatively and may influence bendability of the end. In vitro kinetic experiments suggest that the sub-terminal enhancers may, at least partly, be transposase-dependent. Such enhancers may reflect a subtle regulatory mechanism for IS30 transposition.

Unusually short tandem repeats in the chromosome end structure of Rhynchosciara (Diptera: Sciaridae)

The characterisation of chromosome end (terminal and sub-terminal) sequences of Rhynchosciara americana chromosomes was continued with the screening of a plasmid library made of amplified DNA fragments from a microdissected chromosome tip. An insert chosen for analysis hybridised to two chromosome ends and contains two microsatellite arrays in close vicinity to a sequence (named M-47), part of which is significantly similar to minisatellites of Salmonidae that are frequently present in the vicinity of microsatellite arrays. PCR results using a single primer representative of M-47 elements suggest that they are also repetitive in Rhynchosciara genomes. In addition, total single primer PCR products hybridised to the non-telocentric end subset of R. americana chromosomes. Another plasmid microlibrary made of chromosome tips amplified by a single M-47 primer was screened for repeats of Rhynchosciara chromosomes. Selected inserts that hybridised strongly to non-telocentric ends of R. americana and R. hollaenderi have a tandem array of 22 bp repeats (M-22). There is sequence divergence among M-22 repeats but their mean similarity is significantly high in relation to the M-22 consensus sequence derived from the cloned tandem array. M-22 elements lie distal to the 414 bp sub-telomeric satellite array characterised previously as suggested by double labelling for M-22 hybridisation and reverse transcriptase. M-47 elements, formerly identified in Salmonidae, thus contribute to specify unusually short repeats composing the sub-telomeric structure of two Rhynchosciara species.

Tol2: a versatile gene transfer vector in vertebrates

The medaka fish Tol2 element is an autonomous transposon that encodes a fully functional transposase. The transposase protein can catalyze transposition of a transposon construct that has 200 and 150 base pairs of DNA from the left and right ends of the Tol2 sequence, respectively. These sequences contain essential terminal inverted repeats and subterminal sequences. DNA inserts of fairly large sizes (as large as 11 kilobases) can be cloned between these sequences without reducing transpositional activity. The Tol2 transposon system has been shown to be active in all vertebrate cells tested thus far, including zebrafish, Xenopus, chicken, mouse, and human. In this review I describe and discuss how the Tol2 transposon is being applied to transgenic studies in these vertebrates, and possible future applications.

Identification of an active transposon in intact rice plants

A transposable element that is active in intact plants has been identified in rice (Oryza sativa L.). The 607-bp element itself, termed nonautonomous DNA-based active rice transposon (nDart), has no coding capacity. It was found inserted in the gene encoding Mg-protoporphyrin IX methyltransferase in a chlorophyll-deficient albino mutant isolated from backcross progeny derived from a cross between wild-type japonica varieties. The nDart has 19-bp terminal inverted repeats (TIRs) and, when mobilized, generates an 8-bp target-site duplication (TSD). At least 13 nDart elements were identified in the genome sequence of the japonica cultivar Nipponbare. Database searches identified larger elements, termed DNA-based active rice transposon (Dart) that contained one ORF for a protein that contains a region with high similarity to the hAT dimerization motif. Dart shares several features with nDart, including identical TIRs, similar subterminal sequences and the generation of an 8-bp TSD. These shared features indicate that the nonautonomous element nDart is an internal deletion derivative of the autonomous element Dart. We conclude that these active transposon systems belong to the hAT superfamily of class II transposons. Because the transposons are active in intact rice plants, they should be useful tools for tagging genes in studies of functional genomics.

Mx-rMx, a Family of Interacting Transposons in the GrowinghATSuperfamily of Maize

More than half a century after the discovery of transposable elements, the number of genetically defined autonomous elements that have been isolated and characterized molecularly in any one species remains surprisingly small. Because of its rich genetic history, maize (Zea mays) is, by far, the plant with the largest number of such elements. Yet, even in maize, a maximum of only two autonomous elements have been characterized in any transposon superfamily. This article describes the isolation and molecular and genetic characterization of Mx (for mobile element induced by x-rays), a third autonomous member of the hAT transposon superfamily in maize. Mx is 3731 bp long, ends in 13-bp terminal inverted repeats (TIRs), and causes an 8-bp duplication of the target site. Mx and rMx (for responder to Mx), its 571-bp nonautonomous partner, define a classical family of interacting transposable elements. Surprisingly, the TIRs of Mx and rMx are only 73% identical, and the subterminal sequences are even less so, suggesting that Mx and rMx may represent diverging transposable elements still capable of mobilization by the same transposase. Sequences that are closer to the ends of either Mx or rMx are present in the maize genome. Mx is predicted to encode a 674-amino acid protein that is homologous to the Ac transposase. Although Mx and Ac are closely related, they do not interact. Other data suggest that maize may possess at least five families of hAT transposons that do not interact with each other. The possible origin of noninteracting transposon families within the same superfamily is discussed.

Cis- and trans-acting influences on telomeric position effect in Drosophila melanogaster detected with a subterminal transgene.

One model of telomeric position effect (TPE) in Drosophila melanogaster proposes that reporter genes in the vicinity of telomeres are repressed by subterminal telomere-associated sequences (TAS) and that variegation of these genes is the result of competition between the repressive effects of TAS and the stimulating effects of promoters in the terminal HeT-A transposon array. The data presented here support this model, but also suggest that TPE is more complex. Activity of a telomeric white reporter gene increases in response to deletion of some or all of the TAS on the homolog. Only transgenes next to fairly long HeT-A arrays respond to this trans-interaction. HeT-A arrays of 6-18 kb respond by increasing the number of dark spots on the eye, while longer arrays increase the background eye color or increase the number of spots sufficiently to cause them to merge. Thus, expression of a subtelomeric reporter gene is influenced by the telomere structure in cis and trans. We propose that the forces involved in telomere length regulation in Drosophila are the underlying forces that manifest themselves as TPE. In the wild-type telomere TAS may play an important role in controlling telomere elongation by repressing HeT-A promoter activity. Modulation of this repression by the homolog may thus regulate telomere elongation.

Genome-wide analysis of mariner-like transposable elements in rice reveals complex relationships with stowaway miniature inverted repeat transposable elements (MITEs).

Stowaway is a superfamily of miniature inverted repeat transposable elements (MITEs) that is widespread and abundant in plant genomes. Like other MITEs, however, its origin and mode of amplification are poorly understood. Several lines of evidence point to plant mariner-like elements (MLEs) as the autonomous partners of the nonautonomous Stowaway MITEs. To better understand this relationship, we have taken advantage of the nearly complete genome sequences of two rice subspecies to generate the first inventory of virtually all MLEs and Stowaway families coexisting in a single plant species. Thirty-four different MLEs were found to group into three major clades and 25 families. More than 22,000 Stowaway MITEs were identified and classified into 36 families. On the basis of detailed sequence comparisons, MLEs were confirmed to be the best candidate autonomous elements for Stowaway MITEs. Surprisingly, however, sequence similarity between MLE and Stowaway families was restricted to the terminal inverted repeats (TIRs) and, in a few cases, to adjacent subterminal sequences. These data suggest a model whereby most of the Stowaway MITEs in rice were cross-mobilized by MLE transposases encoded by distantly related elements.

P instability factor: an active maize transposon system associated with the amplification of Tourist-like MITEs and a new superfamily of transposases.

Miniature inverted-repeat transposable elements (MITEs) are widespread and abundant in both plant and animal genomes. Despite the discovery and characterization of many MITE families, their origin and transposition mechanism are still poorly understood, largely because MITEs are nonautonomous elements with no coding capacity. The starting point for this study was P instability factor (PIF), an active DNA transposable element family from maize that was first identified following multiple mutagenic insertions into exactly the same site in intron 2 of the maize anthocyanin regulatory gene R. In this study we report the isolation of a maize Tourist-like MITE family called miniature PIF (mPIF) that shares several features with PIF elements, including identical terminal inverted repeats, similar subterminal sequences, and an unusual but striking preference for an extended 9-bp target site. These shared features indicate that mPIF and PIF elements were amplified by the same or a closely related transposase. This transposase was identified through the isolation of several PIF elements and the identification of one element (called PIFa) that cosegregated with PIF activity. PIFa encodes a putative protein with homologs in Arabidopsis, rice, sorghum, nematodes, and a fungus. Our data suggest that PIFa and these PIF-like elements belong to a new eukaryotic DNA transposon superfamily that is distantly related to the bacterial IS5 group and are responsible for the origin and spread of Tourist-like MITEs.

Structure-based mutagenesis of the human immunodeficiency virus type 1 DNA attachment site: effects on integration and cDNA synthesis.

Sequences at the ends of linear retroviral cDNA important for integration define the viral DNA attachment (att) site. Whereas determinants of human immunodeficiency virus type 1 (HIV-1) integrase important for replication in T lymphocytes have been extensively characterized, regions of the att site important for viral spread have not been thoroughly examined. Previous transposon-mediated footprinting of preintegration complexes isolated from infected cells revealed enhanced regions of bacteriophage Mu insertion near the ends of HIV-1 cDNA, in the regions of the att sites. Here, we identified the subterminal cDNA sequences cleaved during in vitro footprinting and used this structure-based information together with results of previous work to construct and characterize 24 att site mutant viruses. We found that although subterminal cDNA sequences contributed to HIV-1 replication, the identities of these bases were not critical for integration. In contrast, the phylogenetically conserved CA dinucleotides located at the ends of HIV-1 contributed significantly to virus replication and integration. Mutants containing one intact CA end displayed delays in peak virus growth compared to the wild type. In contrast, double mutant viruses lacking both CAs were replication defective. The A of the CA appeared to be the most critical determinant of integration, because two different U5 mutant viruses containing the substitution of TG for CA partially reverted by changing the G back to A. We also identified a U5 deletion mutant in which the CA played a crucial role in reverse transcription.

Densely methylated sequences that are preferentially localized at telomere-proximal regions of human chromosomes.

We have constructed a library of densely methylated DNA sequences from human blood DNA by selecting fragments with a high affinity for a methyl-CpG binding domain (MBD) column. PCR analysis of the library confirmed the presence of known densely methylated CpG island sequences. Analysis of random clones, however, showed that the library was dominated by sequences whose G+C content and CpG frequency were intermediate between those of bulk genomic DNA and bona fide CpG islands. When human chromosomes were probed with the library by fluorescent in situ hybridisation (FISH), the predominant sites of labelling were at terminal regions of many chromosomes, approximately corresponding to T-bands. Analysis of the methylation status of random clones indicated that all were heavily methylated at CpGs in blood DNA, but many were under-methylated in sperm DNA. Lack of methylation in germ cells may reduce CpG depletion at some sub-terminal sequences and result in a high density of methyl-CpG when these regions become methylated in somatic cells.

Maize Activator transposase has a bipartite DNA binding domain that recognizes subterminal sequences and the terminal inverted repeats

The mobility of maize transposable element Activator (Ac) is dependent on the 11-bp terminal inverted repeats (IRs) and approximately 250 subterminal nucleotides at each end. These sequences flank the coding region for the transposase (TPase) protein, which is required for the transposition reaction. Here we show that Ac TPase has a bipartite DNA binding domain, and recognizes the IRs and subterminal sequences in the Ac ends. TPase binds cooperatively to repetitive ACG and TCG sequences, of which 25 copies are found in the 5' and 20 copies in the 3' subterminal regions. TPase affinity is highest when these sites are flanked on the 3' side by an additional G residue (A/TCGG), which is found at 75% of binding sites. Moreover, TPase binds specifically to the Ac IRs, albeit with much lower affinity. Two mutations within the IRs that immobilize Ac abolish TPase binding completely. The basic DNA binding domain of TPase is split into two subdomains. Binding to the subterminal motifs is accomplished by the C-terminal subdomain alone, whereas recognition of the IRs requires the N-terminal subdomain in addition. Furthermore, TPase is extremely flexible in DNA binding. Two direct or inverted binding sites are bound equally well, and sites that are five to twelve bases apart are similarly well bound. The consequences of these findings for the Ac transposition reaction are discussed.

Identification of Tnr3, a suppressor-mutator/enhancer-like transposable element from rice.

We isolated members of the retroposon family p-SINE1 in rice and found that one member contained an insertion.Aa 3-bp sequence at the insertion site within p-SINE1 appeared duplicated. The insertion sequence, 1539 bp in length, carried imperfect inverted repeats of about 13 bp at its termini which begin with 5'-CACTA---3'; these repeats are similar to those found in members of the En/Spm transposable element family. These results indicate that the insertion sequence is a transposable element belonging to the En/Spm family and is thus named Tnr3 (transposable element in rice no. 3). In fact, Tnr carried long subterminal regions containing direct and inverted repeats of short DNA sequences of 15 bp, another characteristic of the En/Spm family. The subterminal repeat sequences in Tnr3 are, however, of two kinds, although they share homology with each other. Tnr3 and its relatives were present in multiple copies in rice. considering the length of Tnr3, it cannot represent an autonomous type element, but is a non-autonomous element probably derived by deletion from an autonomous transposon.

DNA organization and polymorphism of a wild-type Drosophila telomere region.

Telomeres at the ends of linear chromosomes of eukaryotes protect the chromosome termini from degradation and fusion. While telomeric replication/elongation mechanisms have been studied extensively, the functions of subterminal sequences are less well understood. In general, subterminal regions can be quite polymorphic, varying in size from organism to organism, and differing among chromosomes within an organism. The subterminal regions of Drosophila melanogaster are not well characterized today, and it is not known which and how many different components they contain. Here we present the molecular characterization of DNA components and their organization in the subterminal region of the left arm of chromosome 2 of the Oregon RC wild-type strain of D. melanogaster, including a minisatellite with a 457bp repeat length. Two distinct polymorphic arrangements at 2L were found and analyzed, supporting the Drosophila telomere elongation model by retrotransposition. The high incidence of terminal chromosome deficiencies occurring in natural Drosophila populations is discussed in view of the telomere structure at 2L.

PCR‐aided genomic sequencing of 5'subterminal sequences of the maize transposable element Activator (Ac) in transgenic tobacco plants

The maize transposable element Activator (Ac) carries subterminal CpG-rich sequences which are essential for the transposition of the element. It has previously been shown that the methylation of certain sequences contained in this region can alter their ability to interact with the Ac-encoded protein. The novel hypothesis that the methylation of subterminal Ac sequences is required for transposition was tested. Approximately 150 bp of the 5' subterminal region of the Ac element was examined for the presence of 5-methylcytosines by the ligation-mediated polymerase chain reaction (LMPCR)-aided genomic sequencing method. The methylation status of 22 and 39 cytosines on either strand of the DNA were analysed in each of five different transgenic tobacco cultures carrying transposable Ac sequences. Ten micrograms of tobacco DNA were used for each base-specific cleavage reaction before amplification by LMPCR. All but one of the cytosines were unmethylated. Only a minor fraction of the Ac molecules was methylated at one cytosine residue. It is concluded that DNA methylation at the tested Ac sequences is not required for the transposability of Ac or Ds elements in tobacco cells.