Post-Integration Silencing of piggyBac Transposable Elements in Aedes aegypti

The piggyBac transposon, originating in the genome of the Lepidoptera Trichoplusia ni, has a broad host range, making it useful for the development of a number of transposon-based functional genomic technologies including gene vectors, enhancer-, gene- and protein-traps. While capable of being used as a vector for the creation of transgenic insects and insect cell lines, piggyBac has very limited mobility once integrated into the genome of the yellow fever mosquito, Aedes aegypti. A transgenic Aedes aegypti cell line (AagPB8) was created containing three integrated piggyBac elements and the remobilization potential of the elements was tested. The integrated piggyBac elements in AagPB8 were transpositionally silent in the presence of functional transposase, which was shown to be capable of catalyzing the movement of plasmid-borne piggyBac elements in the same cells. The structural integrity of one of the integrated elements along with the quality of element-flanking DNA, which is known to influence transposition rates, were tested in D. melanogaster. The element was found to be structurally intact, capable of transposition and excision in the soma and germ-line of Drosophila melanogaster, and in a DNA sequence context highly conducive to element movement in Drosophila melanogaster. These data show that transpositional silencing of integrated piggyBac elements in the genome of Aedes aegypti appears to be a function of higher scale genome organization or perhaps epigenetic factors, and not due to structural defects or suboptimal integration sites.     

High-Temperature-Induced Transposition of Insertion Elements in Burkholderia multivorans ATCC 17616

An efficient and quantitative method to analyze the transposition of various insertion sequence (IS) elements in Burkholderia multivorans ATCC 17616 was devised. pGEN500, a plasmid carrying a Bacillus subtilis-derived sacB gene, was introduced into ATCC 17616 cells, and 25% of their sucrose-resistant derivatives were found to carry various IS elements on pGEN500. A PCR-based experimental protocol, in which a mixture of several specific primer pairs was used, revealed that pGEN500 captured, in addition to five previously reported IS elements (IS401, IS402, IS406, IS407, and IS408), three novel IS elements, ISBmu1, ISBmu2, and ISBmu3. The global transposition frequency of these IS elements was enhanced more than sevenfold under a high-temperature condition (42°C) but not under oxidative stress or starvation conditions. To our knowledge, this is the first report demonstrating the elevated transposition activities of several IS elements at a high temperature. The efficient experimental protocol developed in this study will be useful in quantitatively and simultaneously investigating various IS elements, as well as in capturing novel functional mobile elements from a wide variety of bacteria.     

Selective Pressures to Maintain Attachment Site Specificity of Integrative and Conjugative Elements

Integrative and conjugative elements (ICEs) are widespread mobile genetic elements that are usually found integrated in bacterial chromosomes. They are important agents of evolution and contribute to the acquisition of new traits, including antibiotic resistances. ICEs can excise from the chromosome and transfer to recipients by conjugation. Many ICEs are site-specific in that they integrate preferentially into a primary attachment site in the bacterial genome. Site-specific ICEs can also integrate into secondary locations, particularly if the primary site is absent. However, little is known about the consequences of integration of ICEs into alternative attachment sites or what drives the apparent maintenance and prevalence of the many ICEs that use a single attachment site. Using ICEBs1, a site-specific ICE from Bacillus subtilis that integrates into a tRNA gene, we found that integration into secondary sites was detrimental to both ICEBs1 and the host cell. Excision of ICEBs1 from secondary sites was impaired either partially or completely, limiting the spread of ICEBs1. Furthermore, induction of ICEBs1 gene expression caused a substantial drop in proliferation and cell viability within three hours. This drop was dependent on rolling circle replication of ICEBs1 that was unable to excise from the chromosome. Together, these detrimental effects provide selective pressure against the survival and dissemination of ICEs that have integrated into alternative sites and may explain the maintenance of site-specific integration for many ICEs.     

The Evolution of Tyrosine-Recombinase Elements in Nematoda

Transposable elements can be categorised into DNA and RNA elements based on their mechanism of transposition. Tyrosine recombinase elements (YREs) are relatively rare and poorly understood, despite sharing characteristics with both DNA and RNA elements. Previously, the Nematoda have been reported to have a substantially different diversity of YREs compared to other animal phyla: the Dirs1-like YRE retrotransposon was encountered in most animal phyla but not in Nematoda, and a unique Pat1-like YRE retrotransposon has only been recorded from Nematoda. We explored the diversity of YREs in Nematoda by sampling broadly across the phylum and including 34 genomes representing the three classes within Nematoda. We developed a method to isolate and classify YREs based on both feature organization and phylogenetic relationships in an open and reproducible workflow. We also ensured that our phylogenetic approach to YRE classification identified truncated and degenerate elements, informatively increasing the number of elements sampled. We identified Dirs1-like elements (thought to be absent from Nematoda) in the nematode classes Enoplia and Dorylaimia indicating that nematode model species do not adequately represent the diversity of transposable elements in the phylum. Nematode Pat1-like elements were found to be a derived form of another Pat1-like element that is present more widely in animals. Several sequence features used widely for the classification of YREs were found to be homoplasious, highlighting the need for a phylogenetically-based classification scheme. Nematode model species do not represent the diversity of transposable elements in the phylum.     

Genome-Wide Distribution of Transposed Dissociation Elements in Maize

The maize (Zea mays) transposable element Dissociation (Ds) was mobilized for large-scale genome mutagenesis and to study its endogenous biology. Starting from a single donor locus on chromosome 10, over 1500 elements were distributed throughout the genome and positioned on the maize physical map. Genetic strategies to enrich for both local and unlinked insertions were used to distribute Ds insertions. Global, regional, and local insertion site trends were examined. We show that Ds transposed to both linked and unlinked sites and displayed a nonuniform distribution on the genetic map around the donor r1-sc:m3 locus. Comparison of Ds and Mutator insertions reveals distinct target preferences, which provide functional complementarity of the two elements for gene tagging in maize. In particular, Ds displays a stronger preference for insertions within exons and introns, whereas Mutator insertions are more enriched in promoters and 5′-untranslated regions. Ds has no strong target site consensus sequence, but we identified properties of the DNA molecule inherent to its local structure that may influence Ds target site selection. We discuss the utility of Ds for forward and reverse genetics in maize and provide evidence that genes within a 2- to 3-centimorgan region flanking Ds insertions will serve as optimal targets for regional mutagenesis.     

Sexual Size Dimorphism in the Color Pattern Elements of Two Mimetic Heliconius Butterflies

Sexual dichromatism and sexual dimorphism of body size are reasonably well studied in butterflies. Sexual size dimorphism of color pattern elements, however, is much less explored. The object of this study is Heliconius, a genus of butterflies well known for the coevolution between mate color preferences and mimicry. Given the sexual role of wing coloration, we investigated the existence of sexual size dimorphism in the wing color elements of a mimetic pair—Heliconius erato phyllis Fabricius and Heliconius besckei Ménétriés—and analyzed the allometric patterns of these traits. Correlation between size of elements in the dorsal and ventral wing surfaces were also estimated. In both species, three out of four elements were larger in males, but the non-dimorphic element was not the same. With regard to the allometric patterns, our most important finding was that smaller males of one species have proportionally larger yellow bars. This is the first study specifically concerning quantitative sexual dimorphism in the coloration of this well-known genus of butterflies and it opens new prospects to investigate sex-related natural selection and sexual selection of color pattern elements.     

Identification of a Class of Chromatin Boundary Elements

Boundary elements are thought to define the ends of functionally independent domains of genetic activity. An assay for boundary activity based on this concept measures the ability to insulate a bracketed, chromosomally integrated reporter gene from position effects. Despite their presumed importance, the few examples identified to date apparently do not share sequence motifs or DNA binding proteins. The Drosophila protein BEAF binds the scs′ boundary element of the 87A7 hsp70 locus and roughly half of polytene chromosome interband loci. To see if these sites represent a class of boundary elements that have BEAF in common, we have isolated and studied several genomic BEAF binding sites as candidate boundary elements (cBEs). BEAF binds with high affinity to clustered, variably arranged CGATA motifs present in these cBEs. No other sequence homologies were found. Two cBEs were tested and found to confer position-independent expression on a mini-white reporter gene in transgenic flies. Furthermore, point mutations in CGATA motifs that eliminate binding by BEAF also eliminate the ability to confer position-independent expression. Taken together, these findings suggest that clustered CGATA motifs are a hallmark of a BEAF-utilizing class of boundary elements found at many loci. This is the first example of a class of boundary elements that share a sequence motif and a binding protein.Chromatin appears to be partitioned into chromosomal domains that are operationally defined by bracketing DNA regions called boundary elements or insulators (10; see reference 34 for a review). Boundary elements are presumably necessary to curtail the potentially promiscuous behavior of enhancers, limiting their action to the domain in which they reside. The biological activity of a boundary element is experimentally measured by either position-independent expression or enhancer-blocking assays. If this view of chromosomal organization is correct, boundary elements play a very important functional role. Yet only a few examples have been identified, and each is so far a unique case, as they do not appear to have notable sequence homologies or to have binding activities in common.The best-characterized boundary elements are the scs and scs′ regions found to bracket the 87A7 hsp70 heat shock puff of Drosophila melanogaster polytene chromosomes (33) and a 340-bp fragment from the gypsy retrotransposon (11). The scs/scs′ and the gypsy-derived elements have a boundary function in both of the assays mentioned above. They confer position-independent expression on a bracketed reporter gene by insulating the transgene from both activating and repressive effects at the site of chromosomal integration, and they block communication between a specific enhancer and promoter when interposed (20, 21, 31). It is important to note that boundary elements do not inactivate promoters or enhancers; they only block communication when interposed (2, 3, 21, 32). For instance, if an enhancer and boundary element are located between two divergently transcribed promoters, the enhancer cannot activate the promoter with the intervening boundary element but can activate the other promoter. Thus, the positional functioning of boundary elements is distinct from the bidirectional repressive effect of silencer elements.The boundary activity of the gypsy-derived element is known to be mediated by the binding of the zinc finger protein su(Hw) to its reiterated binding sites (31). The su(Hw) protein has been studied in some detail, and regions involved in DNA binding, enhancer blocking, and interactions with mod(mdg4) have been identified (8, 13, 22). Interactions between the mod(mdg4) gene product and the su(Hw) protein are necessary for boundary function (9). In addition to loss of enhancer blocking, it has been suggested that some mod(mdg4) mutations lead to an unmasked activity that represses certain promoters (3).To address the boundary activity of scs′ at a biochemical level, we previously characterized two cDNAs encoding the related scs′ boundary element-associated factors BEAF-32A and -32B (14, 38). The BEAF activity in Drosophila nuclear extracts appears to be composed predominantly of trimers of one 32A and two 32B subunits. Interactions between BEAF subunits results in cooperative binding to the three CGATA motifs of the high-affinity binding site in scs′ which, in turn, facilitates binding to the lower-affinity binding site located some 200 bp away (14).Evidence of a role for BEAF in boundary activity derives from an enhancer-blocking assay in Drosophila D1 cells: seven tandem copies of a 48-bp oligonucleotide containing the scs′ high-affinity binding site had enhancer-blocking activity (although less than that obtained by using scs′), while point mutations that eliminated BEAF binding further reduced this activity (38). We immunolocalized BEAF to numerous interbands and puff boundaries on polytene chromosomes, suggesting the existence of a common class of boundary elements in Drosophila and that the band-interband structure of polytene chromosomes could be related to the localization of boundary elements.In this study, we isolated some of these genomic BEAF binding sites and used transgenic flies to demonstrate that the newly isolated sequences tested represent boundary elements. The only homology found between these candidate boundary elements (cBEs) and scs′ are clusters of CGATA motifs. Despite the varied spacing and orientations of the motifs in the different clusters, BEAF interacts with all of the clusters. We also used transgenic flies to directly establish the functional importance of BEAF binding sites by mutagenesis of CGATA motifs. This strongly indicates that the hundreds of BEAF binding sites in the Drosophila genome represent an abundant class of boundary elements, providing the first example of a class of binding elements that share a sequence motif and a binding protein.     

Quantitative Design of Regulatory Elements Based on High-Precision Strength Prediction Using Artificial Neural Network

Accurate and controllable regulatory elements such as promoters and ribosome binding sites (RBSs) are indispensable tools to quantitatively regulate gene expression for rational pathway engineering. Therefore, de novo designing regulatory elements is brought back to the forefront of synthetic biology research. Here we developed a quantitative design method for regulatory elements based on strength prediction using artificial neural network (ANN). One hundred mutated Trc promoter & RBS sequences, which were finely characterized with a strength distribution from 0 to 3.559 (relative to the strength of the original sequence which was defined as 1), were used for model training and test. A precise strength prediction model, NET90_19_576, was finally constructed with high regression correlation coefficients of 0.98 for both model training and test. Sixteen artificial elements were in silico designed using this model. All of them were proved to have good consistency between the measured strength and our desired strength. The functional reliability of the designed elements was validated in two different genetic contexts. The designed parts were successfully utilized to improve the expression of BmK1 peptide toxin and fine-tune deoxy-xylulose phosphate pathway in Escherichia coli. Our results demonstrate that the methodology based on ANN model can de novo and quantitatively design regulatory elements with desired strengths, which are of great importance for synthetic biology applications.     

Defining the Sequence Elements and Candidate Genes for the Coloboma Mutation

The chicken coloboma mutation exhibits features similar to human congenital developmental malformations such as ocular coloboma, cleft-palate, dwarfism, and polydactyly. The coloboma-associated region and encoded genes were investigated using advanced genomic, genetic, and gene expression technologies. Initially, the mutation was linked to a 990 kb region encoding 11 genes; the application of the genetic and genomic tools led to a reduction of the linked region to 176 kb and the elimination of 7 genes. Furthermore, bioinformatics analyses of capture array-next generation sequence data identified genetic elements including SNPs, insertions, deletions, gaps, chromosomal rearrangements, and miRNA binding sites within the introgressed causative region relative to the reference genome sequence. Coloboma-specific variants within exons, UTRs, and splice sites were studied for their contribution to the mutant phenotype. Our compiled results suggest three genes for future studies. The three candidate genes, SLC30A5 (a zinc transporter), CENPH (a centromere protein), and CDK7 (a cyclin-dependent kinase), are differentially expressed (compared to normal embryos) at stages and in tissues affected by the coloboma mutation. Of these genes, two (SLC30A5 and CENPH) are considered high-priority candidate based upon studies in other vertebrate model systems.     

Mobile Antibiotic Resistance Encoding Elements Promote Their Own Diversity

Integrating conjugative elements (ICEs) are a class of bacterial mobile genetic elements that disseminate via conjugation and then integrate into the host cell genome. The SXT/R391 family of ICEs consists of more than 30 different elements that all share the same integration site in the host chromosome but often encode distinct properties. These elements contribute to the spread of antibiotic resistance genes in several gram-negative bacteria including Vibrio cholerae, the agent of cholera. Here, using comparative analyses of the genomes of several SXT/R391 ICEs, we found evidence that the genomes of these elements have been shaped by inter–ICE recombination. We developed a high throughput semi-quantitative method to explore the genetic determinants involved in hybrid ICE formation. Recombinant ICE formation proved to be relatively frequent, and to depend on host (recA) and ICE (s065 and s066) loci, which can independently and potentially cooperatively mediate hybrid ICE formation. s065 and s066, which are found in all SXT/R391 ICEs, are orthologues of the bacteriophage λ Red recombination genes bet and exo, and the s065/s066 recombination system is the first Red-like recombination pathway to be described in a conjugative element. Neither ICE excision nor conjugative transfer proved to be essential for generation of hybrid ICEs. Instead conjugation facilitates the segregation of hybrids and could provide a means to select for functional recombinant ICEs containing novel combinations of genes conferring resistance to antibiotics. Thus, ICEs promote their own diversity and can yield novel mobile elements capable of disseminating new combinations of antibiotic resistance genes.     

Evolutionary Genomics of Transposable Elements in Saccharomyces cerevisiae

Saccharomyces cerevisiae is one of the premier model systems for studying the genomics and evolution of transposable elements. The availability of the S. cerevisiae genome led to unprecedented insights into its five known transposable element families (the LTR retrotransposons Ty1-Ty5) in the years shortly after its completion. However, subsequent advances in bioinformatics tools for analysing transposable elements and the recent availability of genome sequences for multiple strains and species of yeast motivates new investigations into Ty evolution in S. cerevisiae. Here we provide a comprehensive phylogenetic and population genetic analysis of all Ty families in S. cerevisiae based on a systematic re-annotation of Ty elements in the S288c reference genome. We show that previous annotation efforts have underestimated the total copy number of Ty elements for all known families. In addition, we identify a new family of Ty3-like elements related to the S. paradoxus Ty3p which is composed entirely of degenerate solo LTRs. Phylogenetic analyses of LTR sequences identified three families with short-branch, recently active clades nested among long branch, inactive insertions (Ty1, Ty3, Ty4), one family with essentially all recently active elements (Ty2) and two families with only inactive elements (Ty3p and Ty5). Population genomic data from 38 additional strains of S. cerevisiae show that the majority of Ty insertions in the S288c reference genome are fixed in the species, with insertions in active clades being predominantly polymorphic and insertions in inactive clades being predominantly fixed. Finally, we use comparative genomic data to provide evidence that the Ty2 and Ty3p families have arisen in the S. cerevisiae genome by horizontal transfer. Our results demonstrate that the genome of a single individual contains important information about the state of TE population dynamics within a species and suggest that horizontal transfer may play an important role in shaping the genomic diversity of transposable elements in unicellular eukaryotes.     

An Analysis of Male-Recombination Elements in a Natural Population of DROSOPHILA MELANOGASTER in South Texas

Genomes from a group of Drosophila melanogaster collected from a natural population at San Benito, South Texas, in March of 1975 were analyzed for the presence of male-recombination elements. All three autosomes and both sex chromosomes were examined, with emphasis placed on the two major autosomes, the second and third chromosomes. In samples of 16 second and 16 third chromosomes, at least half, but not all, of each were found to carry male-recombination elements. It is suggested, although the data are not conclusive, that some of the fourth, X, and Y chromosomes might also be associated with male-recombination elements.—When a male-recombination element, or elements, was located in the second chromosome, relatively more male recombination was induced in the second than in the third chromosome. This situation was reversed when the element(s) was located in the third chromosome.—Distortion of transmission frequency, one of the characteristics of previously studied second chromosome lines associated with male recombination, was confirmed for these second chromosomes that carried male-recombination elements. Similar, but less pronounced, distortion was observed for the third chromosome lines that carried male-recombination elements.     

Virus-Like Attachment Sites and Plastic CpG Islands: Landmarks of Diversity in Plant Del Retrotransposons

Full-length Del elements from ten angiosperm genomes, 5 monocot and 5 dicot, were retrieved and putative attachment (att) sites were identified. In the 2432 Del elements, two types of U5 att sites and a single conserved type of U3 att site were identified. Retroviral att sites confer specificity to the integration process, different att sites types therefore implies lineage specificity. While some features are common to all Del elements, CpG island patterns within the LTRs were particular to lineage specific clusters. All eudicot copies grouped into one single clade while the monocots harbour a more diverse collection of elements. Furthermore, full-length Del elements and truncated copies were unevenly distributed amongst chromosomes. Elements of Del lineage are organized in plants into three clusters and each cluster is composed of elements with distinct LTR features. Our results suggest that the Del lineage efficiently amplified in the monocots and that one branch is probably a newly emerging sub-lineage. Finally, sequences in all groups are under purifying selection. These results show the LTR region is dynamic and important in the evolution of LTR-retrotransposons, we speculate that it is a trigger for retrotransposon diversification.     

Seasonal Dynamics of Trace Elements in Tidal Salt Marsh Soils as Affected by the Flow-Sediment Regulation Regime

Soil profiles were collected in three salt marshes with different plant species (i.e. Phragmites australis, Tamarix chinensis and Suaeda salsa) in the Yellow River Delta (YRD) of China during three seasons (summer and fall of 2007 and the following spring of 2008) after the flow-sediment regulation regime. Total elemental contents of As, Cd, Cu, Pb and Zn were determined using inductively coupled plasma atomic absorption spectrometry to investigate temporal variations in trace elements in soil profiles of the three salt marshes, assess the enrichment levels and ecological risks of these trace elements in three sampling seasons and identify their influencing factors. Trace elements did not change significantly along soil profiles at each site in each sampling season. The highest value for each sampling site was observed in summer and the lowest one in fall. Soils in both P. australis and S. salsa wetlands tended to have higher trace element levels than those in T. chinensis wetland. Compared to other elements, both Cd and As had higher enrichment factors exceeding moderate enrichment levels. However, the toxic unit (TU) values of these trace elements did not exceed probable effect levels. Correlation analysis showed that these trace elements were closely linked to soil properties such as moisture, sulfur, salinity, soil organic matter, soil texture and pH values. Principal component analysis showed that the sampling season affected by the flow-sediment regulation regime was the dominant factor influencing the distribution patterns of these trace elements in soils, and plant community type was another important factor. The findings of this study could contribute to wetland conservation and management in coastal regions affected by the hydrological engineering.     

Three Groups of Transposable Elements with Contrasting Copy Number Dynamics and Host Responses in the Maize (Zea mays ssp. mays) Genome

Most angiosperm nuclear DNA is repetitive and derived from silenced transposable elements (TEs). TE silencing requires substantial resources from the plant host, including the production of small interfering RNAs (siRNAs). Thus, the interaction between TEs and siRNAs is a critical aspect of both the function and the evolution of plant genomes. Yet the co-evolutionary dynamics between these two entities remain poorly characterized. Here we studied the organization of TEs within the maize (Zea mays ssp mays) genome, documenting that TEs fall within three groups based on the class and copy numbers. These groups included DNA elements, low copy RNA elements and higher copy RNA elements. The three groups varied statistically in characteristics that included length, location, age, siRNA expression and 24∶22 nucleotide (nt) siRNA targeting ratios. In addition, the low copy retroelements encompassed a set of TEs that had previously been shown to decrease expression within a 24 nt siRNA biogenesis mutant (mop1). To investigate the evolutionary dynamics of the three groups, we estimated their abundance in two landraces, one with a genome similar in size to that of the maize reference and the other with a 30% larger genome. For all three accessions, we assessed TE abundance as well as 22 nt and 24 nt siRNA content within leaves. The high copy number retroelements are under targeted similarly by siRNAs among accessions, appear to be born of a rapid bust of activity, and may be currently transpositionally dead or limited. In contrast, the lower copy number group of retrolements are targeted more dynamically and have had a long and ongoing history of transposition in the maize genome.