Identification of the Maize Gravitropism Gene lazy plant1 by a Transposon-Tagging Genome Resequencing Strategy

Since their initial discovery, transposons have been widely used as mutagens for forward and reverse genetic screens in a range of organisms. The problems of high copy number and sequence divergence among related transposons have often limited the efficiency at which tagged genes can be identified. A method was developed to identity the locations of Mutator (Mu) transposons in the Zea mays genome using a simple enrichment method combined with genome resequencing to identify transposon junction fragments. The sequencing library was prepared from genomic DNA by digesting with a restriction enzyme that cuts within a perfectly conserved motif of the Mu terminal inverted repeats (TIR). Paired-end reads containing Mu TIR sequences were computationally identified and chromosomal sequences flanking the transposon were mapped to the maize reference genome. This method has been used to identify Mu insertions in a number of alleles and to isolate the previously unidentified lazy plant1 (la1) gene. The la1 gene is required for the negatively gravitropic response of shoots and mutant plants lack the ability to sense gravity. Using bioinformatic and fluorescence microscopy approaches, we show that the la1 gene encodes a cell membrane and nuclear localized protein. Our Mu-Taq method is readily adaptable to identify the genomic locations of any insertion of a known sequence in any organism using any sequencing platform.     

Testing the palindromic target site model for DNA transposon insertion using the Drosophila melanogaster P-element

Understanding the molecular mechanisms that influence transposable element target site preferences is a fundamental challenge in functional and evolutionary genomics. Large-scale transposon insertion projects provide excellent material to study target site preferences in the absence of confounding effects of post-insertion evolutionary change. Growing evidence from a wide variety of prokaryotes and eukaryotes indicates that DNA transposons recognize staggered-cut palindromic target site motifs (TSMs). Here, we use over 10 000 accurately mapped P-element insertions in the Drosophila melanogaster genome to test predictions of the staggered-cut palindromic target site model for DNA transposon insertion. We provide evidence that the P-element targets a 14-bp palindromic motif that can be identified at the primary sequence level, which predicts the local spacing, hotspots and strand orientation of P-element insertions. Intriguingly, we find that the although P-element destroys the complete 14-bp target site upon insertion, the terminal three nucleotides of the P-element inverted repeats complement and restore the original TSM, suggesting a mechanistic link between transposon target sites and their terminal inverted repeats. Finally, we discuss how the staggered-cut palindromic target site model can be used to assess the accuracy of genome mappings for annotated P-element insertions.     

Efficient integration of artificial transposons into plasmid targets in vitro: a useful tool for DNA mapping, sequencing and genetic analysis.

We have developed efficient methods for creating artificial transposons and inserting these transposons into plasmid targets in vitro, primarily for the purpose of DNA mapping and sequencing. A novel plasmid has been engineered to convert virtually any DNA sequence, or combination of sequences, into an artificial transposon; hence, custom transposons containing any desired feature can be easily designed and constructed. Such transposons are then efficiently inserted into plasmid targets, in vitro, using the integrase activity present in yeast Ty1 virus-like particles. A single in vitro integration reaction, which resembles a simple restriction digestion in the complexity of the reaction, gives rise to thousands of recoverable insertion events within DNA target molecules; this frequency approaches one insertion per phosphodiester bond in typical plasmids. Importantly, transposon insertions are recovered from all regions of DNA inserts carried on plasmid targets, indicating that integration is a random or nearly-random process. Because of its versatility, this technology offers a generalized method of generating recombinant DNA molecules of a desired structure. We have adapted this system for DNA sequencing by developing a customized artificial transposon to insert new primer binding sites into internal regions of DNA inserts carried on cloning vectors. Transposon insertions have been generated throughout several different yeast and human DNA inserts carried on plasmids, allowing the efficient recovery of sequence information from these inserts. Our results demonstrate the overall utility of this method for both small and large-scale DNA sequencing, as well as general DNA restructuring, and indicate that it could be adapted for use with a number of additional applications including functional genetic analysis.     

Site-selected insertional mutagenesis of tomato with maizeAc andDs elements

Site-selected insertion (SSI) is a PCR-based technique which uses primers located within the transposon and a target gene for detection of transposon insertions into cloned genes. We screened tomato plants bearing single or multiple copies of maizeAc orDs transposable elements for somatic insertions at one close-range target and two long-range targets. Eight close-rangeDs insertions near the right border of the T-DNA were recovered. Sequence analysis showed a precise junction between the transposon and the target for all insertions. Two insertions in separate plants occurred at the same site, but others appeared dispersed in the region of the right T-DNA border with no target specificity. However, insertions showed a preference for one orientation of the transposon. Use of plants with multipleAc (HiAc) orDs (HiDs) elements allowed detection of somatic insertions at two single-copy genes,PG (polygalacturonase) andDFR (dihydroflavonol 4-reductase). Certain HiDs plants showed much higher rates of insertion intoPG than others. Insertions inPG andDFR were found throughout the gene regions monitored and, with the exception of one insertion inPG, the junctions between transposon and target were exact. SSI analysis of progeny from the HiDs parents revealed that in some cases the tendency to incur high levels of somatic insertions inPG was inherited. Inheritance of this character is an indication that SSI could be used to direct a search for germinalPG insertions in tomato.     

Natural genetic variation caused by transposable elements in humans

Transposons and transposon-like repetitive elements collectively occupy 44% of the human genome sequence. In an effort to measure the levels of genetic variation that are caused by human transposons, we have developed a new method to broadly detect transposon insertion polymorphisms of all kinds in humans. We began by identifying 606,093 insertion and deletion (indel) polymorphisms in the genomes of diverse humans. We then screened these polymorphisms to detect indels that were caused by de novo transposon insertions. Our method was highly efficient and led to the identification of 605 nonredundant transposon insertion polymorphisms in 36 diverse humans. We estimate that this represents 25-35% of approximately 2075 common transposon polymorphisms in human populations. Because we identified all transposon insertion polymorphisms with a single method, we could evaluate the relative levels of variation that were caused by each transposon class. The average human in our study was estimated to harbor 1283 Alu insertion polymorphisms, 180 L1 polymorphisms, 56 SVA polymorphisms, and 17 polymorphisms related to other forms of mobilized DNA. Overall, our study provides significant steps toward (i) measuring the genetic variation that is caused by transposon insertions in humans and (ii) identifying the transposon copies that produce this variation.     

In Vivo Himar1 Transposon Mutagenesis of Burkholderia pseudomallei

Burkholderia psedudomallei is the etiologic agent of melioidosis, and the bacterium is listed as a potential agent of bioterrorism because of its low infectious dose, multiple infectious routes, and intrinsic antibiotic resistance. To further accelerate research with this understudied bacterium, we developed a Himar1-based random mutagenesis system for B. pseudomallei (HimarBP). The transposons contain a Flp recombinase-excisable, approved kanamycin resistance selection marker and an R6K origin of replication for transposon rescue. In vivo mutagenesis of virulent B. pseudomallei strain 1026b was highly efficient, with up to 44% of cells transformed with the delivery plasmid harboring chromosomal HimarBP insertions. Southern analyses revealed single insertions with no evidence of delivery plasmid maintenance. Sequence analysis of rescued HimarBP insertions revealed random insertions on both chromosomes within open reading frames and intergenic regions and that the orientation of insertions was largely unbiased. Auxotrophic mutants were obtained at a frequency of 0.72%, and nutritional supplementation experiments supported the functional assignment of genes within the respective biosynthetic pathways. HimarBP insertions were stable in the absence of selection and could be readily transferred between naturally transformable strains. Experiments with B. thailandensis suggest that the newly developed HimarBP transposons can also be used for random mutagenesis of other Burkholderia spp., especially the closely related species B. mallei. Our results demonstrate that comprehensive transposon libraries of B. pseudomallei can be generated, providing additional tools for the study of the biology, pathogenesis, and antibiotic resistance of this pathogen.     

DNA transposons and the role of recombination in mutation accumulation in Daphnia pulex

Background

We identify DNA transposons from the completed draft genome sequence of Daphnia pulex, a cyclically parthenogenetic, aquatic microcrustacean of the class Branchiopoda. In addition, we experimentally quantify the abundance of six DNA transposon families in mutation-accumulation lines in which sex is either promoted or prohibited in order to better understand the role of recombination in transposon proliferation.

Results

We identified 55 families belonging to 10 of the known superfamilies of DNA transposons in the genome of D. pulex. DNA transposons constitute approximately 0.7% of the genome. We characterized each family and, in many cases, identified elements capable of activity in the genome. Based on assays of six putatively active element families in mutation-accumulation lines, we compared DNA transposon abundance in lines where sex was either promoted or prohibited. We find the major difference in abundance in sexuals relative to asexuals in lab-reared lines is explained by independent assortment of heterozygotes in lineages where sex has occurred.

Conclusions

Our examination of the duality of sex as a mechanism for both the spread and elimination of DNA transposons in the genome reveals that independent assortment of chromosomes leads to significant copy loss in lineages undergoing sex. Although this advantage may offset the so-called 'two fold cost of sex' in the short-term, if insertions become homozygous at specific loci due to recombination, the advantage of sex may be decreased over long time periods. Given these results, we discuss the potential effects of sex on the dynamics of DNA transposons in natural populations of D. pulex.     

<Emphasis Type="Italic">Mutator</Emphasis>-Based Transposon Display: A Genetic Tool for Evolutionary and Crop-Improvement Studies in Maize

Transposable elements account for up to 85% of the maize genome and have significant implications in crop-improvement and evolutionary analyses. The Mutator (Mu) transposon superfamily, a class of DNA transposons, comprises the most complex and active elements in the maize genome, suggesting a special role in plant evolution. Here, we designed a set of Mu-specific primers based on terminal invert repeats and used a transposon display (TD) method for genotyping. We analyzed the distribution pattern of Mu insertions in teosinte (wild relative), sorghum (distant relative), and domesticated maize accessions (dent, sweet, and waxy). The MU-TD analysis suggested the presence of high polymorphic insertions among the species and subspecies, indicating the utility of the method in studying genetic variation and species relationships. Furthermore, we analyzed 80 maize recombinant inbred line populations. Mu-TD generated an average of 60% Mu-anchored polymorphic fragments in which insertions appeared to be segregating in significantly high numbers. The amplification profile was highly reproducible, confirming the utility of Mu elements as a new set of TD markers for developing high-density genetic maps.     

Mos as a tool for genome-wide insertional mutagenesis in Caenorhabditis elegans: results of a pilot study

The sequence of the Caenorhabditis elegans genome contains approximately 19 000 genes. Available mutants currently exist for <20% of these genes. The existence of a Mos-based inducible transposon system in C.elegans could theoretically serve as a tool to saturate the genome with insertions. We report here the results of a pilot study aimed at assaying this strategy. We generated 914 independent random Mos insertions and determined their location by inverse PCR. The distribution of the insertions throughout the genome does not reveal any gross distortion, with the exception of a major hotspot on chromosome I (rDNA locus). Transposons are evenly distributed between the genic and intergenic regions. Within genes, transposons insert preferentially into the introns. We derived the consensus target site for Mos in C.elegans (ATATAT), which is common to Tc1, another mariner element. Finally, we assayed the mutagenic properties of insertions located in exons by comparing the phenotype of homozygous strains to that of known mutations or RNAi of the same gene. This pilot experiment shows that a Mos-based approach is a viable strategy that can contribute to the constitution of genome-wide collections of identified C.elegans mutants.     

Transposon mutagenesis in halophilic eubacteria: conjugal transfer and insertion of transposon Tn5 and Tn1732 in Halomonas elongata

Abstract Molecular genetic studies of halophilic eubacteria have been limited by the lack of a suitable method for mutagenesis. To overcome this, we established a transposon mutagenesis procedure for the ectoine-producing, halophilic bacterium Halomonas elongata . We used suicide plasmids pSUP101 and pSUP102-Gm to introduce the transposons Tn5 and Tn7732 respectively into H. elongata via Escherichia coli SM10 mediated conjugation. Our finding that H. elongata is sensitive to aminoglycoside antibiotics at low salinity enabled us to apply transposons that mediate kanamycin resistance. The insertions of transposon In 1732 occurred at different sites in the chromosome of H. elongata , as proved by Southern hybridization analysis. Phenotypic analysis revealed that different auxotrophic and salt sensitive mutants were generated by mutagenesis with transposon Tn 1732 . To our knowledge this is the first report of a successful application of a transposon for direct generalized mutagenesis in a halophilic eubacterium.     

Amplification dynamics of miniature inverted-repeat transposable elements and their impact on rice trait variability

Transposable elements (TEs) are a rich source of genetic variability. Among TEs, miniature inverted-repeat TEs (MITEs) are of particular interest as they are present in high copy numbers in plant genomes and are closely associated with genes. MITEs are deletion derivatives of class II transposons, and can be mobilized by the transposases encoded by the latter through a typical cut-and-paste mechanism. However, MITEs are typically present at much higher copy numbers than class II transposons. We present here an analysis of 103 109 transposon insertion polymorphisms (TIPs) in 738 Oryza sativa genomes representing the main rice population groups. We show that an important fraction of MITE insertions has been fixed in rice concomitantly with its domestication. However, another fraction of MITE insertions is present at low frequencies. We performed MITE TIP-genome-wide association studies (TIP-GWAS) to study the impact of these elements on agronomically important traits and found that these elements uncover more trait associations than single nucleotide polymorphisms (SNPs) on important phenotypes such as grain width. Finally, using SNP-GWAS and TIP-GWAS we provide evidence of the replicative amplification of MITEs.     

Clonal Expansion Analysis of Transposon Insertions by High-Throughput Sequencing Identifies Candidate Cancer Genes in a PiggyBac Mutagenesis Screen

Somatic transposon mutagenesis in mice is an efficient strategy to investigate the genetic mechanisms of tumorigenesis. The identification of tumor driving transposon insertions traditionally requires the generation of large tumor cohorts to obtain information about common insertion sites. Tumor driving insertions are also characterized by their clonal expansion in tumor tissue, a phenomenon that is facilitated by the slow and evolving transformation process of transposon mutagenesis. We describe here an improved approach for the detection of tumor driving insertions that assesses the clonal expansion of insertions by quantifying the relative proportion of sequence reads obtained in individual tumors. To this end, we have developed a protocol for insertion site sequencing that utilizes acoustic shearing of tumor DNA and Illumina sequencing. We analyzed various solid tumors generated by PiggyBac mutagenesis and for each tumor >10⁶ reads corresponding to >10⁴ insertion sites were obtained. In each tumor, 9 to 25 insertions stood out by their enriched sequence read frequencies when compared to frequencies obtained from tail DNA controls. These enriched insertions are potential clonally expanded tumor driving insertions, and thus identify candidate cancer genes. The candidate cancer genes of our study comprised many established cancer genes, but also novel candidate genes such as Mastermind-like1 (Mamld1) and Diacylglycerolkinase delta (Dgkd). We show that clonal expansion analysis by high-throughput sequencing is a robust approach for the identification of candidate cancer genes in insertional mutagenesis screens on the level of individual tumors.     

Genome‐wide patterns of transposon proliferation in an evolutionary young hybrid fish

Hybridization can induce transposons to jump into new genomic positions, which may result in their accumulation across the genome. Alternatively, transposon copy numbers may increase through nonallelic (ectopic) homologous recombination in highly repetitive regions of the genome. The relative contribution of transposition bursts versus recombination‐based mechanisms to evolutionary processes remains unclear because studies on transposon dynamics in natural systems are rare. We assessed the genomewide distribution of transposon insertions in a young hybrid lineage (“invasive Cottus”, n = 11) and its parental species Cottus rhenanus (n = 17) and Cottus perifretum(n = 9) using a reference genome assembled from long single molecule pacbio reads. An inventory of transposable elements was reconstructed from the same data and annotated. Transposon copy numbers in the hybrid lineage increased in 120 (15.9%) out of 757 transposons studied here. The copy number increased on average by 69% (range: 10%–197%). Given the age of the hybrid lineage, this suggests that they have proliferated within a few hundred generations since admixture began. However, frequency spectra of transposon insertions revealed no increase in novel and rare insertions across assembled parts of the genome. This implies that transposons were added to repetitive regions of the genome that remain difficult to assemble. Future studies will need to evaluate whether recombination‐based mechanisms rather than genomewide transposition may explain the majority of the recent transposon proliferation in the hybrid lineage. Irrespectively of the underlying mechanism, the observed overabundance in repetitive parts of the genome suggests that gene‐rich regions are unlikely to be directly affected.     

Genetic organization of the bacterial conjugative transposon Tn916.

Tn916, which encodes resistance to tetracycline, is a 16.4-kilobase conjugative transposon originally identified on the chromosome of Streptococcus faecalis DS16. The transposon has been cloned in Escherichia coli on plasmid vectors, where it expresses tetracycline resistance; it can be reintroduced into S. faecalis via protoplast transformation. We have used a lambda::Tn5 bacteriophage delivery system to introduce Tn5 into numerous sites within Tn916. The Tn5 insertions had various effects on the behavior of Tn916. Some insertions eliminated conjugative transposition but not intracellular transposition, and others eliminated an excision step believed to be essential for both types of transposition. A few inserts had no effect on transposon behavior. Functions were mapped to specific regions on the transposon.