Gene arrangements and branching orders of gram-positive bacteria

The availability of complete genomic sequence data allows one to develop new methods of reconstructing phylogenetic trees. A simple method of reconstructing branching orders based on gene transposition (or lateral transfer) is presented. It is argued that specific gene arrangements on four different genomes could determine a branching order. A computer search for such gene arrangements was carried out against gene order data of completely sequenced Gram-positive bacteria. Gene arrangements around ribosomal protein S4 gene, murC (UDP-N-acetylmuramate:alanine ligase) gene and dnaE (DNA polymerase III alpha chain) gene each suggest a branching order in which actinobacteria with a high genomic G+C content first branched off from other Gram-positives with a low G+C content and then a split occurred between Mycoplasma species and a group closely related to Bacillus subtilis. A recently sequenced thermophilic bacterium Thermoanaerobacter tengcongensis is suggested to have branched off from the lineage leading to the low G+C Gram-positives prior to the split between the Mycoplasma and Bacillus groups. By contrast to the indel analysis in which a single evolutionary event of insertion or deletion of a signature sequence is assumed, the present method does not necessarily require such a parsimonious assumption of gene transposition.     

Sex chromosome-to-autosome transposition events counter Y-chromosome gene loss in mammals

BackgroundAlthough the mammalian X and Y chromosomes evolved from a single pair of autosomes, they are highly differentiated: the Y chromosome is dramatically smaller than the X and has lost most of its genes. The surviving genes are a specialized set with extraordinary evolutionary longevity. Most mammalian lineages have experienced delayed, or relatively recent, loss of at least one conserved Y-linked gene. An extreme example of this phenomenon is in the Japanese spiny rat, where the Y chromosome has disappeared altogether. In this species, many Y-linked genes were rescued by transposition to new genomic locations, but until our work presented here, this has been considered an isolated case.ResultsWe describe eight cases of genes that have relocated to autosomes in mammalian lineages where the corresponding Y-linked gene has been lost. These gene transpositions originated from either the X or Y chromosomes, and are observed in diverse mammalian lineages: occurring at least once in marsupials, apes, and cattle, and at least twice in rodents and marmoset. For two genes - EIF1AX/Y and RPS4X/Y - transposition to autosomes occurred independently in three distinct lineages.ConclusionsRescue of Y-linked gene loss through transposition to autosomes has previously been reported for a single isolated rodent species. However, our findings indicate that this compensatory mechanism is widespread among mammalian species. Thus, Y-linked gene loss emerges as an additional driver of gene transposition from the sex chromosomes, a phenomenon thought to be driven primarily by meiotic sex chromosome inactivation.

Electronic supplementary material

The online version of this article (doi:10.1186/s13059-015-0667-4) contains supplementary material, which is available to authorized users.     

Physical evidence for a Saccharomyces cerevisiae transposable element which carries the his4C gene.

A Saccharomyces cerevisiae transposable element which carries the his4C structural gene and which is capable of transposition, excision, and mutator activity is described. Physical evidence is presented for transposition of the his4C deoxyribonucleic acid sequences to a new location in the genome and for precise excision of these transposed deoxyribonucleic acid sequences in spontaneous his4C- segregants.     

Generation of rat mutants using a coat color-tagged <Emphasis Type="Italic">Sleeping Beauty</Emphasis> transposon system

A significant barrier to exploiting the full potential of the rat as a biomedical model is the lack of tools to easily modify its germline. Here we show that a tyrosinase-tagged Sleeping Beauty transposon can be used as a simple, efficient method to generate rat mutants in vivo. By making two lines of transgenic rats, one carrying the transposon and another expressing the transposase in germ cells, we are able to obtain bigenic males in which transposition occurs in the germ cells. We show that transposition leads to the appearance of new coat colors in the offspring. Using such bigenic males, we obtained an average of 1.2 transpositions per gamete and identified 19 intragenic integration events among 96 transposition sites that were sequenced. In addition, gene trapping was confirmed and rats with evidence for transposon-induced dominant ocular anomalies were identified. These data suggest that the modified Sleeping Beauty transposon represents a powerful new tool for producing molecularly defined mutagenesis in the rat.     

Gene arrangements characteristic of the phylum Actinobacteria

The availability of genomic sequence data allows new challenges to various biological problems. One of such attempts is the extraction of phylogenetic information from gene order data of genomes. Phylogenetic inferences are most commonly carried out on the basis of 16S rRNA trees, which sometimes produce unresolved or unreliable branching orders. One example for such a low resolution is recognized in the branching pattern among the phylum Actinobacteria. Here, gene arrangements characteristic of the Actinobacteria were identified, based on which Symbiobacterium thermophilum is phylogenetically placed outside that phylum, this being in contrast to 16S rRNA trees and to the current taxonomy in GenBank. Three transposition suggestive arrangements were found which support a notion that Rubrobacter xylanophilus is the earliest diverging species among the completely sequenced Actinobacteria. The gene arrangements identified here serve as a complement to previously reported indels and proteins characteristic of this phylum.     

Excision of Sleeping Beauty transposons: parameters and applications to gene therapy

A major problem in gene therapy is the determination of the rates at which gene transfer has occurred. Our work has focused on applications of the Sleeping Beauty (SB) transposon system as a non-viral vector for gene therapy. Excision of a transposon from a donor molecule and its integration into a cellular chromosome are catalyzed by SB transposase. In this study, we used a plasmid-based excision assay to study the excision step of transposition. We used the excision assay to evaluate the importance of various sequences that border the sites of excision inside and outside the transposon in order to determine the most active sequences for transposition from a donor plasmid. These findings together with our previous results in transposase binding to the terminal repeats suggest that the sequences in the transposon-junction of SB are involved in steps subsequent to DNA binding but before excision, and that they may have a role in transposase-transposon interaction. We found that SB transposons leave characteristically different footprints at excision sites in different cell types, suggesting that alternative repair machineries operate in concert with transposition. Most importantly, we found that the rates of excision correlate with the rates of transposition. We used this finding to assess transposition in livers of mice that were injected with the SB transposon and transposase. The excision assay appears to be a relatively quick and easy method to optimize protocols for delivery of genes in SB transposons to mammalian chromosomes in living animals.     

Characterization of functionally important sites in the bacteriophage Mu transposase protein

The 663 amino acid Mu transposase protein is absolutely required for Mu DNA transposition. Mutant proteins were constructed in vitro in order to locate regions of transposase that may be important for the catalysis of DNA transposition. Deletions in the A gene, which encodes the transposase, yielded two stable mutant proteins that aid in defining the end-specific DNA-binding domain. Linker insertion mutagenesis at eight sites in the Mu A gene generated two proteins, FF6 and FF14 (resulting from two and four amino acid insertions, respectively, at position 408), which were thermolabile for DNA binding in vitro at 43°C. However, transposition activity in vivo was severely reduced for all mutant proteins at 37°C, except those with insertions at positions 328 and 624. In addition, site-specific mutagenesis was performed to alter tyrosine 414, which is situated in a region that displays amino acid homology to the active sites of a number of nicking/closing enzymes. Tyrosine 414 may reside within an important, yet non-essential, site of transposase, as an aspartate-substituted protein had a drastically reduced frequency of transposition, while the remaining mutants yielded reduced, but substantial, frequencies of Mu transposition in vivo.     

Transitory Cis Complementation: A Method for Providing Transposition Functions to Defective Transposons

A genetic complementation system is described in which the complementing components are close together in a single linear DNA fragment; the complementation situation is temporary. This system is useful for providing transposition functions to transposition-defective transposons, since transposition functions act preferentially in cis. The basic procedure involves placing a transposition-defective transposon near the gene(s) for its transposition functions on a single DNA fragment. This fragment is introduced, here by general transduction, into a new host. The transposase acts in cis to permit the defective element to transpose from the introduced fragment into the recipient chromosome. The helper genes do not transpose and are lost by degradation and segregation. The method yields single insertion mutants that lack transposase and are not subject to further transposition or chromosome rearrangement. The general procedure is applicable to other sorts of transposable elements and could be modified for use in other genetic systems.     

Inferring the history of interchromosomal gene transposition in Drosophila using n-dimensional parsimony

Gene transposition puts a new gene copy in a novel genomic environment. Moreover, genes moving between the autosomes and the X chromosome experience change in several evolutionary parameters. Previous studies of gene transposition have not utilized the phylogenetic framework that becomes possible with the availability of whole genomes from multiple species. Here we used parsimonious reconstruction on the genomic distribution of gene families to analyze interchromosomal gene transposition in Drosophila. We identified 782 genes that have moved chromosomes within the phylogeny of 10 Drosophila species, including 87 gene families with multiple independent movements on different branches of the phylogeny. Using this large catalog of transposed genes, we detected accelerated sequence evolution in duplicated genes that transposed when compared to the parental copy at the original locus. We also observed a more refined picture of the biased movement of genes from the X chromosome to the autosomes. The bias of X-to-autosome movement was significantly stronger for RNA-based movements than for DNA-based movements, and among DNA-based movements there was an excess of genes moving onto the X chromosome as well. Genes involved in female-specific functions moved onto the X chromosome while genes with male-specific functions moved off the X. There was a significant overrepresentation of proteins involving chromosomal function among transposed genes, suggesting that genetic conflict between sexes and among chromosomes may be a driving force behind gene transposition in Drosophila.     

A one-time inducible transposon for creating knockout mutants

The maize transposon Ac can move to a new location within the genome to create knockout mutants in transgenic plants. In rice, Ac transposon is very active but sometimes undergoes further transposition and leaves an empty mutated gene. Therefore, we developed a one-time transposon system by locating one end of the transposon in the intron of the Ac transposase gene, which is under the control of the inducible promoter (PR-1a). Treatment with salicylic acid induced transposition of this transposon, COYA, leading to transposase gene breakage in exons. The progeny plants inheriting the transposition events become stable knockout mutants, because no functional transposase could be yielded. The behavior of COYA was analyzed in single-copy transgenic rice plants. We determined the expression of the modified transposase gene and its ability to trigger transposition events in transgenic rice plants. The COYA element thus exhibits potential for development of an inducible transposon system suitable for gene isolation in heterologous plant species. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Alternative conformations of a nucleic acid four-way junction

Sleeping Beauty (SB), a member of the Tc1/mariner superfamily of transposable elements, is the only active DNA-based transposon system of vertebrate origin that is available for experimental manipulation. We have been using the SB element as a research tool to investigate some of the cis and trans-requirements of element mobilization, and mechanisms that regulate transposition in vertebrate species. In contrast to mariner transposons, which are regulated by overexpression inhibition, the frequency of SB transposition was found to be roughly proportional to the amount of transposase present in cells. Unlike Tc1 and mariner elements, SB contains two binding sites within each of its terminal inverted repeats, and we found that the presence of both of these sites is a strict requirement for mobilization. In addition to the size of the transposon itself, the length as well as sequence of the DNA outside the transposon have significant effects on transposition. As a general rule, the closer the transposon ends are, the more efficient transposition is from a donor molecule. We have found that SB can transform a wide range of vertebrate cells from fish to human. However, the efficiency and precision of transposition varied significantly among cell lines, suggesting potential involvement of host factors in SB transposition. A positive-negative selection assay was devised to enrich populations of cells harboring inserted transposons in their chromosomes. Using this assay, of the order of 10,000 independent transposon insertions can be generated in human cells in a single transfection experiment. Sleeping Beauty can be a powerful alternative to other vectors that are currently used for the production of transgenic animals and for human gene therapy.     

Phenotypic effects of short-range and aberrant transposition in Antirrhinum majus

We describe two novel ways in which changes in gene expression in Antirrhinum majus may arise as a consequence of the Tam3 transposition mechanism. One involves excision of Tam3 from the nivea gene promoter and insertion of two new Tam3 copies 3.4 kb and 2.1 kb away, on either side of the excision site. One of the new insertions is in the nivea coding region and completely blocks production of an active gene product. This allele probably arose by a symmetrical double transposition, following chromosome replication. The second case involves a small deletion at one end of Tam3 in the pallida gene, flanked by a sequence typical of a Tam3 excision footprint. This suggests that the end of Tam3 was cleaved at an early step in an attempted transposition and re-ligated back to its original flanking sequence. The alteration restores some expression to the pallida gene, suggesting that the ends of the intact Tam3 element contain components which can actively inhibit gene expression. The implications of these findings for the mechanism of Tam3 transposition and for the effects of Tam3 on host gene expression are discussed.     

DNA gyrase is a host factor required for transposition of Tn5

We show that DNA gyrase is required for transposition of Tn5. Coumermycin, a potent inhibitor of DNA gyrase subunit B, inhibits transposition in a wild-type strain, but has no effect on strains carry ing a coumermycin-resistant allele in gyrB. In addition, strains containing a thermolabile subunit A of gyrase (gyrA43) are defective for transposition at a nonpermissive temperature. The requirement for gyrase is due to a requirement for supercoiled DNA. We showed this by introducing into the gyrA43 strain a deletion of the gene encoding topoisomerase I. The introduction of the second mutation caused an increase in the superhelical density of DNA as well as an increase in the transposition frequency. This also implies that if the DNA is supercoiled there is no further requirement for gyrase. Experiments with coumermycin support this, because the drug does not inhibit transposition if the recipient DNA remains supercoiled. This indicates that if the DNA acting as recipient of the transposon is deficient in supercoils, it will be a poor substrate for transposition. We also describe a system in which a gene on a multicopy plasmid can be efficiently introduced into the Escherichia coli chromosome.     

A mini-Mu transposon-based method for multiple DNA fragment integration into bacterial genomes

A method for construction of bacterial strains with multiple DNA inserted into their chromosomes has been developed based on the mini-Mu transposon and FLP/FRT recombination. Exogenous DNA can be integrated by Mu transposition with an FRT cassette containing selection marker and conditional replicative origin (R6Kγori). Subsequently, with the introduction of a helper plasmid bearing gene of FLP recombinase, drug-resistant selection marker is excised from the chromosome. Cells cured of the helper plasmid can undergo the next cycle of transposition and excision of selection marker. Each cycle can add further foreign gene(s) to the chromosome. As an example, resistance genes of chloramphenicol, tetracycline, and gentamicin were successively integrated into the chromosome of Escherichia coli BW25113 by three cycles of insertion and excision as described above. This method proved to be simple and time-saving, which could be applicable to a variety of microorganisms.     

Rapid generation of gene disruption constructs by in vitro transposition and identification of a Dictyostelium protein kinase that regulates its rate of growth and development

We describe a rapid method for creating Dictyo stelium gene disruption constructs, whereby the target gene is interrupted by a drug resistance cassette using in vitro transposition. A fragment of genomic DNA containing the gene to be disrupted is amplified by PCR, cloned into a plasmid vector using topoisomerase and then employed as the substrate in an in vitro Tn5 transposition reaction. The transposing species is a fragment of DNA containing a Dictyostelium blasticidin S resistance (bs^r) cassette linked to a bacterial tetracycline resistance (tet^r) cassette. After transposition the plasmid DNA is transformed into Escherichia coli and clones in which the bs^r-tet^r cassette is inserted into the Dictyostelium target DNA are identified. To demonstrate its utility we have employed the method to disrupt the gene encoding QkgA, a novel protein kinase identified from the Dictyostelium genome sequencing project. QkgA is structurally homologous to two previously identified Dictyostelium kinases, GbpC and pats1. Like them it contains a leucine-rich repeat domain, a small GTP-binding (ras) domain and a MEKK domain. Disruption of the qkgA gene causes a marked increase in growth rate and, during development, aggregation occurs relatively slowly to form abnormally large multicellular structures.     

Structure-function analysis of the inverted terminal repeats of the sleeping beauty transposon

Translocation of Sleeping Beauty (SB) transposon requires specific binding of SB transposase to inverted terminal repeats (ITRs) of about 230 bp at each end of the transposon, which is followed by a cut-and-paste transfer of the transposon into a target DNA sequence. The ITRs contain two imperfect direct repeats (DRs) of about 32 bp. The outer DRs are at the extreme ends of the transposon whereas the inner DRs are located inside the transposon, 165-166 bp from the outer DRs. Here we investigated the roles of the DR elements in transposition. Although there is a core transposase-binding sequence common to all of the DRs, additional adjacent sequences are required for transposition and these sequences vary in the different DRs. As a result, SB transposase binds less tightly to the outer DRs than to the inner DRs. Two DRs are required in each ITR for transposition but they are not interchangeable for efficient transposition. Each DR appears to have a distinctive role in transposition. The spacing and sequence between the DR elements in an ITR affect transposition rates, suggesting a constrained geometry is involved in the interactions of SB transposase molecules in order to achieve precise mobilization. Transposons are flanked by TA dinucleotide base-pairs that are important for excision; elimination of the TA motif on one side of the transposon significantly reduces transposition while loss of TAs on both flanks of the transposon abolishes transposition. These findings have led to the construction of a more advanced transposon that should be useful in gene transfer and insertional mutagenesis in vertebrates.     

Localization of sequences required in cis for yeast Ty1 element transposition near the long terminal repeats: analysis of mini-Ty1 elements.

In order to identify and characterize sequences within Ty1 elements which are required in cis for transposition, a series of mini-Ty1 plasmids were constructed and tested for transposition. Mini-Ty1s are deletion mutants of the Ty1-H3 element; Ty1 gene products required for transposition are supplied in trans from a helper Ty1 which has intact open reading frames but lacks a 3' long terminal repeat (LTR) and therefore cannot transpose itself. Up to 5 kilobase pairs of internal sequences of the 6-kilobase-pair-long Ty1 element can be deleted without a significant effect on transposition. The smallest mini-Ty1 element capable of transposition contains the 3' LTR and the transcribed portion of the 5' LTR, 285 base pairs (bp) of internal sequence 3' to the 5' LTR, and 23 bp of internal sequence 5' to the 3' LTR. We conclude that Ty1-encoded proteins can act in trans and that cis-acting sequences in Ty1-H3 are all within or near the LTRs. Further deletion of the 285-bp internal sequence adjacent to the 5' LTR significantly reduced transposition frequency, and the mini-Ty1 RNA produced failed to be packaged into the viruslike particles efficiently. Surprisingly, several nonhomologous cellular mRNAs were also associated with viruslike particles.     

Transposition immunity in bacteriophage Mu. The effect of a mutation at the kil gene on the establishment of immunity

Bacteriophage Mu is characterized by a phenomenon similar to the transposition immunity of TnA: the frequency of transposition of Mu or mini-Mu into plasmids containing certain phage sequences is reduced by two orders of magnitude. In order to lend transposition immunity to Mu, the recipient replicon must contain a sequence of phage DNA including a 5.1 kb early region from the c-end of Mu. The product of the kil (or cim) gene takes part in establishing the immunity. The transposition immunity of Mu is connected with the disturbance of cointegrate formation.