Flexibility in MuA transposase family protein structures: functional mapping with scanning mutagenesis and sequence alignment of protein homologues

MuA transposase protein is a member of the retroviral integrase superfamily (RISF). It catalyzes DNA cleavage and joining reactions via an initial assembly and subsequent structural transitions of a protein-DNA complex, known as the Mu transpososome, ultimately attaching transposon DNA to non-specific target DNA. The transpososome functions as a molecular DNA-modifying machine and has been used in a wide variety of molecular biology and genetics/genomics applications. To analyze structure-function relationships in MuA action, a comprehensive pentapeptide insertion mutagenesis was carried out for the protein. A total of 233 unique insertion variants were generated, and their activity was analyzed using a quantitative in vivo DNA transposition assay. The results were then correlated with the known MuA structures, and the data were evaluated with regard to the protein domain function and transpososome development. To complement the analysis with an evolutionary component, a protein sequence alignment was produced for 44 members of MuA family transposases. Altogether, the results pinpointed those regions, in which insertions can be tolerated, and those where insertions are harmful. Most insertions within the subdomains Iγ, IIα, IIβ, and IIIα completely destroyed the transposase function, yet insertions into certain loop/linker regions of these subdomains increased the protein activity. Subdomains Iα and IIIβ were largely insertion-tolerant. The comprehensive structure-function data set will be useful for designing MuA transposase variants with improved properties for biotechnology/genomics applications, and is informative with regard to the function of RISF proteins in general.     

Pentapeptide scanning mutagenesis: encouraging old proteins to execute unusual tricks

Pentapeptide scanning mutagenesis is a facile transposon-based procedure for the random insertion of a variable five amino acid cassette into a target protein. The analysis of a library of proteins harbouring pentapeptide insertions can provide invaluable information on the essential and inessential regions of a target protein, as well as revealing surprising aspects of target protein function and activity.     

In vitro transposition of ISY100, a bacterial insertion sequence belonging to the Tc1/mariner family

The Synechocystis sp. PCC6803 insertion sequence ISY100 (ISTcSa) belongs to the Tc1/mariner/IS630 family of transposable elements. ISY100 transposase was purified and shown to promote transposition in vitro. Transposase binds specifically to ISY100 terminal inverted repeat sequences via an N-terminal DNA-binding domain containing two helix-turn-helix motifs. Transposase is the only protein required for excision and integration of ISY100. Transposase made double-strand breaks on a supercoiled DNA molecule containing a mini-ISY100 transposon, cleaving exactly at the transposon 3' ends and two nucleotides inside the 5' ends. Cleavage of short linear substrates containing a single transposon end was less precise. Transposase also catalysed strand transfer, covalently joining the transposon 3' end to the target DNA. When a donor plasmid carrying a mini-ISY100 was incubated with a target plasmid and transposase, the most common products were insertions of one transposon end into the target DNA, but insertions of both ends at a single target site could be recovered after transformation into Escherichia coli. Insertions were almost exclusively into TA dinucleotides, and the target TA was duplicated on insertion. Our results demonstrate that there are no fundamental differences between the transposition mechanisms of IS630 family elements in bacteria and Tc1/mariner elements in higher eukaryotes.     

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.     

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.     

RISCI - Repeat Induced Sequence Changes Identifier: a comprehensive,comparative genomics-based, <Emphasis Type="Italic">in silico</Emphasis> subtractive hybridization pipeline to identify repeat induced sequence changes in closely related genomes

Background -  

The availability of multiple whole genome sequences has facilitated in silico identification of fixed and polymorphic transposable elements (TE). Whereas polymorphic loci serve as makers for phylogenetic and forensic analysis, fixed species-specific transposon insertions, when compared to orthologous loci in other closely related species, may give insights into their evolutionary significance. Besides, TE insertions are not isolated events and are frequently associated with subtle sequence changes concurrent with insertion or post insertion. These include duplication of target site, 3' and 5' flank transduction, deletion of the target locus, 5' truncation or partial deletion and inversion of the transposon, and post insertion changes like inter or intra element recombination, disruption etc. Although such changes have been studied independently, no automated platform to identify differential transposon insertions and the associated array of sequence changes in genomes of the same or closely related species is available till date. To this end, we have designed RISCI - 'Repeat Induced Sequence Changes Identifier' - a comprehensive, comparative genomics-based, in silico subtractive hybridization pipeline to identify differential transposon insertions and associated sequence changes using specific alignment signatures, which may then be examined for their downstream effects.     

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.     

Genetics in methanogens: transposon insertion mutagenesis of a Methanococcus maripaludis nifH gene.

We designed a transposon insertion mutagenesis system for Methanococcus species and used it to make mutations in and around a nifH gene in Methanococcus maripaludis. The transposon Mudpur was constructed with a gene for puromycin resistance that is expressed and selectable in Methanococcus species. A 15.6-kb nifH region from M. maripaludis cloned in a lambda vector was used as a target for mutagenesis. A series of 19 independent Mudpur insertions spanning the cloned region were produced. Four mutagenized clones in and around nifH were introduced by transformation into M. maripaludis, where each was found to replace wild-type genomic DNA with the corresponding transposon-mutagenized DNA. Wild-type M. maripaludis and a transformant containing a Mudpur insertion upstream of nifH grew on N2 as a nitrogen source. Two transformants with insertions in nifH and one transformant with an insertion downstream of nifH did not grow on N2. The transposon insertion-gene replacement technique should be generally applicable in the methanococci for studying the effects of genetic manipulations in vivo.     

In vitro transposition of Tn552: a tool for DNA sequencing and mutagenesis.

We have explored the potential of the Tn 552 in vitro transposition reaction as a genetic tool. The reaction is simple (requiring a single protein component), robust and efficient, readily producing insertions into several percent of target DNA. Most importantly, Tn 552 insertions in vitro appear to be essentially random. Extensive analyses indicate that the transposon exhibits no significant regional or sequence specificity for target DNA and leaves no discernible 'cold' spots devoid of insertions. The utility of the in vitro reaction for DNA sequencing was demonstrated with a cosmid containing the Mycobacterium smegmatis recBCD gene cluster. The nucleotide sequence of the entire operon was determined using 71 independent Tn 552 insertions, which generated over 13.5 kb of unique sequence and simultaneously provided a comprehensive collection of insertion mutants. The relatively short ends of Tn 552 make construction of novel transposons a simple process and we describe several useful derivatives. The data presented suggest that Tn 552 transposition is a valuable addition to the arsenal of tools available for molecular biology and genomics.     

Probing the alpha-complementing domain of E. coli beta-galactosidase with use of an insertional pentapeptide mutagenesis strategy based on Mu in vitro DNA transposition

Protein structure-function relationships can be studied by using linker insertion mutagenesis, which efficiently identifies essential regions in target proteins. Bacteriophage Mu in vitro DNA transposition was used to generate an extensive library of pentapeptide insertion mutants within the alpha-complementing domain 1 of Escherichia coli beta-galactosidase, yielding mutants at 100% efficiency. Each mutant contained an accurate 15-bp insertion that translated to five additional amino acids within the protein, and the insertions were distributed essentially randomly along the target sequence. Individual mutants (alpha-donors) were analyzed for their ability to restore (by alpha-complementation) beta-galactosidase activity of the M15 deletion mutant (alpha-acceptor), and the data were correlated to the structure of the beta-galactosidase tetramer. Most of the insertions were well tolerated, including many of those disrupting secondary structural elements even within the protein's interior. Nevertheless, certain sites were sensitive to mutations, indicating both known and previously unknown regions of functional importance. Inhibitory insertions within the N-terminus and loop regions most likely influenced protein tetramerization via direct local effects on protein-protein interactions. Within the domain 1 core, the insertions probably caused either lateral shifting of the polypeptide chain toward the protein's exterior or produced more pronounced structural distortions. Six percent of the mutant proteins exhibited temperature sensitivity, in general suggesting the method's usefulness for generation of conditional phenotypes. The method should be applicable to any cloned protein-encoding gene.     

A target specificity switch in IS911 transposition: the role of the OrfA protein

The role played by insertion sequence IS911 proteins, OrfA and OrfAB, in the choice of a target for insertion was studied. IS911 transposition occurs in several steps: synapsis of the two transposon ends (IRR and IRL); formation of a figure-of-eight intermediate where both ends are joined by a single-strand bridge; resolution into a circular form carrying an IRR-IRL junction; and insertion into a DNA target. In vivo, with OrfAB alone, an IS911-based transposon integrated with high probability next to an IS911 end located on the target plasmid. OrfA greatly reduced the proportion of these events. This was confirmed in vitro using a transposon with a preformed IRR-IRL junction to examine the final insertion step. Addition of OrfA resulted in a large increase in insertion frequency and greatly increased the proportion of non-targeted insertions. The intermolecular reaction leading to targeted insertion may resemble the intramolecular reaction involving figure-of-eight molecules, which leads to the formation of circles. OrfA could, therefore, be considered as a molecular switch modulating the site-specific recombination activity of OrfAB and facilitating dispersion of the insertion sequence (IS) to 'non-homologous' target sites.     

Target site choice of the related transposable elements Tc1 and Tc3 of Caenorhabditis elegans.

We have investigated the target choice of the related transposable elements Tc1 and Tc3 of the nematode C. elegans. The exact locations of 204 independent Tc1 insertions and 166 Tc3 insertions in an 1 kbp region of the genome were determined. There was no phenotypic selection for the insertions. All insertions were into the sequence TA. Both elements have a strong preference for certain positions in the 1 kbp region. Hot sites for integration are not clustered or regularly spaced. The orientation of the integrated transposon has no effect on the distribution pattern. We tested several explanations for the target site preference. If simple structural features of the DNA (e.g. bends) would mark hot sites, we would expect the patterns of the two related transposons Tc1 and Tc3 to be similar; however we found them to be completely different. Furthermore we found that the sequence at the donor site has no effect on the choice of the new insertion site, because the insertion pattern of a transposon that jumps from a transgenic donor site is identical to the insertion pattern of transposons jumping from endogenous genomic donor sites. The most likely explanation for the target choice is therefore that the primary sequence of the target site is recognized by the transposase. However, alignment of the Tc1 and Tc3 integration sites does not reveal a strong consensus sequence for either transposon.     

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.     

Efficient transposition of IS911 circles in vitro.

An in vitro system has been developed which supports efficient integration of transposon circles derived from the bacterial insertion sequence IS911. Using relatively pure preparations of IS911-encoded proteins it has been demonstrated that integration into a suitable target required both the transposase, OrfAB, a fusion protein produced by translational frameshifting between two consecutive open reading frames, orfA and orfB, and OrfA, a protein synthesized independently from the upstream orfA. Intermolecular reaction products were identified in which one or both transposon ends were used. The reaction also generated various intramolecular transposition products including adjacent deletions and inversions. The circle junction, composed of abutted left and right IS ends, retained efficient integration activity when carried on a linear donor molecule, demonstrating that supercoiling in the donor molecule is not necessary for the reaction. Both two-ended integration and a lower level of single-ended insertions were observed under these conditions. The frequency of these events depended on the spacing between the transposon ends. Two-ended insertion was most efficient with a natural spacing of 3 bp. These results demonstrate that transposon circles can act as intermediates in IS911 transposition and provide evidence for collaboration between the two major IS911-encoded proteins, OrfA and OrfAB.     

Amplification and detection of transposon insertion flanking sequences using fluorescent muAFLP

The amplification of transposon insertionflanking sequences is the basis of a variety of techniques usedfor the detection and characterization of specific transposon insertion events. We have developed a method for the efficient size determination and quantification of amplified genomic sequences thatflank Mutator (Mu) transposon insertions in maize. Using this detection method, we have been able to optimize Mu insertion site amplification and to assess amplification from increasingly complex templates representing increasing numbers of Mu-active maize plants. This detection method should be applicablefor the characterization of transposon or transgene insertion events in a wide variety of organisms.     

Use of transposon-transposase complexes to create stable insertion mutant strains of Francisella tularensis LVS

Francisella tularensis is a highly virulent zoonotic bacterial pathogen capable of infecting numerous different mammalian species, including humans. Elucidation of the pathogenic mechanisms of F. tularensis has been hampered by a lack of tools to genetically manipulate this organism. Herein we describe the use of transposome complexes to create insertion mutations in the chromosome of the F. tularensis live vaccine strain (LVS). A Tn5-derived transposon encoding kanamycin resistance and lacking a transposase gene was complexed with transposase enzyme and transformed directly into F. tularensis LVS by electroporation. An insertion frequency of 2.6 x 10(-8) +/- 0.87 x 10(-8) per cell was consistently achieved using this method. There are 178 described Tn5 consensus target sites distributed throughout the F. tularensis genome. Twenty-two of 26 transposon insertions analyzed were within known or predicted open reading frames, but none of these insertions was associated with the Tn5 target site. Analysis of the insertions of sequentially passed strains indicated that the transposons were maintained stably at the initial insertion site after more than 270 generations. Therefore, transformation by electroporation of Tn5-based transposon-transposase complexes provided an efficient mechanism for generating random, stable chromosomal insertion mutations in F. tularensis.     

Transposon-induced non-motile mutants of Vibrio cholerae

Non-motile mutants of Vibrio cholerae were isolated after transposon insertion mutagenesis with either Tn5 on a plasmid or Tn10ptac mini-kan in bacteriophage lambda. The physical location and number of transposon insertions was determined. Eighteen Tn5 insertion mutants and 11 Tn10ptac mini-kan insertion mutants had single unique insertion sites. The 18 Tn5 insertions were contained within six different EcoRI fragments and the 11 Tn10ptac mini-kan insertions were contained within eight different fragments of V. cholerae chromosomal DNA. These data suggest that multiple genes are involved in motility. Immunoblot analysis of non-motile mutants with antibody to wild-type flagellar core protein indicated that two of the non-motile mutants made flagellar core protein. Three additional mutants reacted weakly with the antibodies. However, these mutants with immunopositive reactions did not produce any structures which resembled flagella by transmission electron microscopy. In addition, none of the other non-motile mutants produced wild-type flagella. However, five mutants which did not react in the immunoblot produced a structure which resembled a flagellar sheath without the internal flagellar core. In addition to having no filamentous core, the sheaths often extended from the sides of the bacteria, rather than from the poles where the flagellum is normally located. The data suggest that sheath formation is independent of flagellar filament formation, but that proper positioning of the sheath may require the flagellar filament.