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.     

Population dynamics of miniature inverted-repeat transposable elements (MITEs) in Medicago truncatula

Miniature inverted-repeat transposable elements (MITEs) are small and high copy number transposons, related to and mobilized by some class II autonomous elements. New MITE families can be identified by computer-based mining of sequenced genomes. We describe four MITE families related to MtPH transposons mined de novo in the genome of Medicago truncatula, together with one previously described family MITRAV. Different levels of their intra-family sequence diversity and insertion polymorphism indicate that they were active at different evolutionary periods. MetMIT1 and MITRAV families were uniform in sequence and produced highly polymorphic insertion sites in 26 ecotypes representing a M. truncatula core collection. A subset of insertions was present only in the reference genome of A17 ‘Jemalong’, suggesting that the two families might have been active in the course of domestication. In contrast, all investigated insertions of the MetMIT2 family were fixed, showing that it was not active after M. truncatula speciation. MetMIT1 elements were divided into three clusters, i.e. (I) relatively heterogenous copies fixed in the genome of M. truncatula, (II) uniform but also mostly fixed, and (III) uniform and polymorphic among the investigated accessions. It might reflect the evolutionary history of the MetMIT1 family, showing multiple bursts of activity. A number of MetMIT1 and MITRAV insertions were present within 1 kb upstream or downstream the ORF. A high proportion of insertions proximal to coding regions was unique to A17 ‘Jemalong’.     

The complete chloroplast genome sequence of Taxus chinensis var. mairei (Taxaceae): loss of an inverted repeat region and comparative analysis with related species

Taxus chinensis var. mairei (Taxaceae) is a domestic variety of yew species in local China. This plant is one of the sources for paclitaxel, which is a promising antineoplastic chemotherapy drugs during the last decade. We have sequenced the complete nucleotide sequence of the chloroplast (cp) genome of T. chinensis var. mairei. The T. chinensis var. mairei cp genome is 129,513 bp in length, with 113 single copy genes and two duplicated genes (trnI-CAU, trnQ-UUG). Among the 113 single copy genes, 9 are intron-containing. Compared to other land plant cp genomes, the T. chinensis var. mairei cp genome has lost one of the large inverted repeats (IRs) found in angiosperms, fern, liverwort, and gymnosperm such as Cycas revoluta and Ginkgo biloba L. Compared to related species, the gene order of T. chinensis var. mairei has a large inversion of ~ 110 kb including 91 genes (from rps18 to accD) with gene contents unarranged. Repeat analysis identified 48 direct and 2 inverted repeats 30 bp long or longer with a sequence identity greater than 90%. Repeated short segments were found in genes rps18, rps19 and clpP. Analysis also revealed 22 simple sequence repeat (SSR) loci and almost all are composed of A or T.     

A shotgun approach to discovering and reconstructing consensus retrotransposons ex novo from dense contigs of short sequences derived from Genbank Genome Survey Sequence database records

Retrotransposons constitute the majority of pseudogenic protein coding regions of most eukaryotic genomes. Most genomes carry tens to thousands of retrotransposon copies derived from dozens of distinct families, but most if not all of these copies are non-functional and contain disabling mutations, including large numbers of indels. Until recently, most regions rich in these elements were virtually ignored in all but the most complete genome sequencing projects, and the full extent of their impact on the structure and function of the genomes of higher eukaryotes was under-appreciated. Even when new retrotransposons are encountered and annotated by automated gene finding programs and similarity searches, coding regions are treated as exons and invariably and not surprisingly mistranslated because of numerous frameshift mutations and large indels. Very few functional retrotransposons contain introns, as in silico annotations imply. While many repetitive DNA consensus sequences have been assembled from collections of largely full-length copies using full-length templates, we have shown that repetitive DNA consensus sequence contigs representing long, moderately high copy-number elements can also be generated ex novo in the absence of templates from very short overlapping sequences. We have devised an in silico strategy to recover and reconstruct consensus sequences of elements up to 20,000 bp by building dense contigs of hundreds of overlapping 400 to 900-bp records found in the Genbank Genome Survey Sequence database. The results are hypothetical ancestral sequences that encode elements that appear to be fully functional with intact open reading frames and other conserved features.     

Complete chloroplast genome sequence of Elodea canadensis and comparative analyses with other monocot plastid genomes

Elodea canadensis is an aquatic angiosperm native to North America. It has attracted great attention due to its invasive nature when transported to new areas in its non-native range. We have determined the complete nucleotide sequence of the chloroplast (cp) genome of Elodea. Taxonomically Elodea is a basal monocot, and only few monocot cp genomes representing early lineages of monocots have been sequenced so far. The genome is a circular double-stranded DNA molecule 156,700bp in length, and has a typical structure with large (LSC 86,194bp) and small (SSC 17,810bp) single-copy regions separated by a pair of inverted repeats (IRs 26,348bp each). The Elodea cp genome contains 113 unique genes and 16 duplicated genes in the IR regions. A comparative analysis showed that the gene order and organization of the Elodea cp genome is almost identical to that of Amborella trichopoda, a basal angiosperm. The structure of IRs in Elodea is unique among monocot species with the whole cp genome sequenced. In Elodea and another monocot Lemna minor the borders between IRs and LSC are located upstream of rps19 gene and downstream of trnH-GUG gene, while in most monocots, IR has extended to include both trnH and rps19 genes. A phylogenetic analysis conducted using Bayesian method, based on the DNA sequences of 81 chloroplast genes from 17 monocot taxa provided support for the placement of Elodea together with Lemna as a basal monocot and the next diverging lineage of monocots after Acorales. In comparison with other monocots, the Elodea cp genome has gone through only few rearrangements or gene losses. IR of Elodea has a unique structure among the monocot species studied so far as its structure is similar to that of a basal angiosperm Amborella. This result together with phylogenetic analyses supports the placement of Elodea as a basal monocot to the next diverging lineage of monocots after Acorales. So far, only few cp genomes representing early lineages of monocots have been sequenced and, therefore, this study provides valuable information about the course of evolution in divergence of monocot lineages.     

ArdA proteins from different mobile genetic elements can bind to the EcoKI Type I DNA methyltransferase of E. coli K12

Anti-restriction and anti-modification (anti-RM) is the ability to prevent cleavage by DNA restriction–modification (RM) systems of foreign DNA entering a new bacterial host. The evolutionary consequence of anti-RM is the enhanced dissemination of mobile genetic elements. Homologues of ArdA anti-RM proteins are encoded by genes present in many mobile genetic elements such as conjugative plasmids and transposons within bacterial genomes. The ArdA proteins cause anti-RM by mimicking the DNA structure bound by Type I RM enzymes. We have investigated ArdA proteins from the genomes of Enterococcus faecalis V583, Staphylococcus aureus Mu50 and Bacteroides fragilis NCTC 9343, and compared them to the ArdA protein expressed by the conjugative transposon Tn916. We find that despite having very different structural stability and secondary structure content, they can all bind to the EcoKI methyltransferase, a core component of the EcoKI Type I RM system. This finding indicates that the less structured ArdA proteins become fully folded upon binding. The ability of ArdA from diverse mobile elements to inhibit Type I RM systems from other bacteria suggests that they are an advantage for transfer not only between closely-related bacteria but also between more distantly related bacterial species.     

Identification of multiple binding sites for the THAP domain of the Galileo transposase in the long terminal inverted-repeats

Galileo is a DNA transposon responsible for the generation of several chromosomal inversions in Drosophila. In contrast to other members of the P-element superfamily, it has unusually long terminal inverted-repeats (TIRs) that resemble those of Foldback elements. To investigate the function of the long TIRs we derived consensus and ancestral sequences for the Galileo transposase in three species of Drosophilids. Following gene synthesis, we expressed and purified their constituent THAP domains and tested their binding activity towards the respective Galileo TIRs. DNase I footprinting located the most proximal DNA binding site about 70 bp from the transposon end. Using this sequence we identified further binding sites in the tandem repeats that are found within the long TIRs. This suggests that the synaptic complex between Galileo ends may be a complicated structure containing higher-order multimers of the transposase. We also attempted to reconstitute Galileo transposition in Drosophila embryos but no events were detected. Thus, although the limited numbers of Galileo copies in each genome were sufficient to provide functional consensus sequences for the THAP domains, they do not specify a fully active transposase. Since the THAP recognition sequence is short, and will occur many times in a large genome, it seems likely that the multiple binding sites within the long, internally repetitive, TIRs of Galileo and other Foldback-like elements may provide the transposase with its binding specificity.