Tc8, a Tourist-like transposon in Caenorhabditis elegans.

Members of the Tourist family of miniature inverted-repeat transposable elements (MITEs) are very abundant among a wide variety of plants, are frequently found associated with normal plant genes, and thus are thought to be important players in the organization and evolution of plant genomes. In Arabidopsis, the recent discovery of a Tourist member harboring a putative transposase has shed new light on the mobility and evolution of MITEs. Here, we analyze a family of Tourist transposons endogenous to the genome of the nematode Caenorhabditis elegans (Bristol N2). One member of this large family is 7568 bp in length, harbors an ORF similar to the putative Tourist transposase from Arabidopsis, and is related to the IS5 family of bacterial insertion sequences (IS). Using database searches, we found expressed sequence tags (ESTs) similar to the putative Tourist transposases in plants, insects, and vertebrates. Taken together, our data suggest that Tourist-like and IS5-like transposons form a superfamily of potentially active elements ubiquitous to prokaryotic and eukaryotic genomes.     

Tc7, a Tc1-hitch hiking transposon in Caenorhabditis elegans.

We have found a novel transposon in the genome of Caenorhabditis elegans. Tc7 is a 921 bp element, made up of two 345 bp inverted repeats separated by a unique, internal sequence. Tc7 does not contain an open reading frame. The outer 38 bp of the inverted repeat show 36 matches with the outer 38 bp of Tc1. This region of Tc1 contains the Tc1-transposase binding site. Furthermore, Tc7 is flanked by TA dinucleotides, just like Tc1, which presumably correspond to the target duplication generated upon integration. Since Tc7 does not encode its own transposase but contains the Tc1-transposase binding site at its extremities, we tested the ability of Tc7 to jump upon forced expression of Tc1 transposase in somatic cells. Under these conditions Tc7 jumps at a frequency similar to Tc1. The target site choice of Tc7 is identical to that of Tc1. These data suggest that Tc7 shares with Tc1 all the sequences minimally required to parasitize upon the Tc1 transposition machinery. The genomic distribution of Tc7 shows a striking clustering on the X chromosome where two thirds of the elements (20 out of 33) are located. Related transposons in C. elegans do not show this asymmetric distribution.     

The origin of footprints of the Tc1 transposon of Caenorhabditis elegans.

Mutations caused by the Tc1 transposon in Caenorhabditis elegans can revert by loss of the element. Usually the transposon leaves behind a 'footprint'--a few nucleotides of one or both ends of the transposon. Two possible explanations for the footprints are: (i) imprecise excision or (ii) interrupted repair. Here I report that in a diploid animal having a homozygous Tc1 insertion the reversion frequency is approximately 10(-4), and a Tc1 footprint is found; however when the corresponding sequence on the homologous chromosome is wild-type, the reversion frequency is 100 times higher, and the reverted sequence is precise. Apparently the footprint results from incomplete gene conversion from the homologous chromosome, and not from imprecise excision of Tc1. These results support the following model: Tc1 excision leaves a double-strand DNA break, which can be repaired using the homologous chromosome or sister chromatid as a template. In heterozygotes repair can lead to reversion; in homozygotes Tc1 is copied into the 'empty' site, and only rare interrupted repair leads to reversion, hence the 100-fold lower reversion rate and the footprint.     

Target choice determinants of the Tc1 transposon of Caenorhabditis elegans.

The Tc1 transposon of Caenorhabditis elegans always integrates into the sequence TA, but some TA sites are preferred to others. We investigated a TA target site from the gpa-2 gene of C.elegans that was previously found to be preferred (hot) for Tc1 integration in vivo . This site with its immediate flanks was cloned into a plasmid, and remained hot in vitro , showing that sequences immediately adjacent to the TA dinucleotide determine this target choice. Further deletion mapping and mutagenesis showed that a 4 bp sequence on one side of the TA is sufficient to make a site hot; this sequence nicely fits the previously identified Tc1 consensus sequence for integration. In addition, we found a second type of hot site: this site is only preferred for integration when the target DNA is supercoiled, not when it is relaxed. Excision frequencies were relatively independent of the flanking sequences. The distribution of Tc1 insertions into a plasmid was similar when we used nuclear extracts or purified Tc1 transposase in vitro , showing that the Tc1 transposase is the protein responsible for the target choice.     

Splicing removes the Caenorhabditis elegans transposon Tc1 from most mutant pre-mRNAs.

The transposable element Tc1 is responsible for most spontaneous mutations that occur in many Caenorhabditis elegans strains. We analyzed the abundance and sequence of mRNAs expressed from five different Tc1 insertions within either hlh-1 (a MyoD homolog) or unc-54 (a myosin heavy chain gene). Each of the mutants expresses substantial quantities of mature mRNA in which most or all of Tc1 has been removed by splicing. Such mRNAs contain small insertions of Tc1 sequences and/or deletions of target gene sequences at the resulting spliced junctions. Most of these mutant mRNAs do not contain premature stop codons, and many are translated in frame to produce proteins that are functional in vivo. The number and variety of splice sites used to remove Tc1 from these mutant pre-mRNAs are remarkable. Two-thirds of the Tc1-containing introns removed from hlh-1 and unc-54 lack either the 5'-GU or AG-3' dinucleotides typically found at the termini of eukaryotic introns. We conclude that splicing to remove Tc1 from mutant pre-mRNAs allows many Tc1 insertions to be phenotypically silent. Such mRNA processing may help Tc1 escape negative selection.     

Site-selected insertion of the transposon Tc1 into a Caenorhabditis elegans myosin light chain gene.

We used the polymerase chain reaction to detect insertions of the transposon Tc1 into mlc-2, one of two Caenorhabditis elegans regulatory myosin light chain genes. Our goals were to develop a general method to identify mutations in any sequenced gene and to establish the phenotype of mlc-2 loss-of-function mutants. The sensitivity of the polymerase chain reaction allowed us to identify nematode populations containing rare Tc1 insertions into mcl-2. mlc-2::Tc1 mutants were subsequently isolated from these populations by a sib selection procedure. We isolated three mutants with Tc1 insertions within the mlc-2 third exon and a fourth strain with Tc1 inserted in nearby noncoding DNA. To demonstrate the generality of our procedure, we isolated two additional mutants with Tc1 insertions within hlh-1, the C. elegans MyoD homolog. All of these mutants are essentially wild type when homozygous. Despite the fact that certain of these mutants have Tc1 inserted within exons of the target gene, these mutations may not be true null alleles. All three of the mlc-2 mutants contain mlc-2 mRNA in which all or part of Tc1 is spliced from the pre-mRNA, leaving small in-frame insertions or deletions in the mature message. There is a remarkable plasticity in the sites used to splice Tc1 from these mlc-2 pre-mRNAs; certain splice sites used in the mutants are very different from typical eukaryotic splice sites.     

Insertion and excision of Caenorhabditis elegans transposable element Tc1.

The transposable element Tc1 is responsible for most spontaneous mutations that occur in Caenorhabditis elegans variety Bergerac. We investigated the genetic and molecular properties of Tc1 transposition and excision. We show that Tc1 insertion into the unc-54 myosin heavy-chain gene was strongly site specific. The DNA sequences of independent Tc1 insertion sites were similar to each other, and we present a consensus sequence for Tc1 insertion that describes these similarities. We show that Tc1 excision was usually imprecise. Tc1 excision was imprecise in both germ line and somatic cells. Imprecise excision generated novel unc-54 alleles that had amino acid substitutions, amino acid insertions, and, in certain cases, probably altered mRNA splicing. The DNA sequences remaining after Tc1 somatic excision were the same as those remaining after germ line excision, but the frequency of somatic excision was at least 1,000-fold higher than that of germ line excision. The genetic properties of Tc1 excision, combined with the DNA sequences of the resulting unc-54 alleles, demonstrated that excision was dependent on Tc1 transposition functions in both germ line and somatic cells. Somatic excision was not regulated in the same strain-specific manner as germ-line excision was. In a genetic background where Tc1 transposition and excision in the germ line was not detectable, Tc1 excision in the soma still occurred at high frequency.     

Imprecise excision of the Caenorhabditis elegans transposon Tc1 creates functional 5' splice sites.

Imprecise excision of the Caenorhabditis elegans transposon Tc1 from a specific site of insertion within the unc-54 myosin heavy chain gene generates either wild-type or partial phenotypic revertants. Wild-type revertants and one class of partial revertants contain insertions of four nucleotides in the unc-54 third exon (Tc1 "footprints"). Such revertants express large amounts of functional unc-54 myosin despite having what would appear to be frameshifting insertions in the unc-54 third exon. We demonstrate that these Tc1 footprints act as efficient 5' splice sites for removal of the unc-54 third intron. Splicing of these new 5' splice sites to the normal third intron splice acceptor removes the Tc1 footprint from the mature mRNA and restores the normal translational reading frame. Partial revertant unc-54(r661), which contains a single nucleotide substitution relative to the wild-type gene, is spliced similarly, except that the use of its new 5' splice site creates a frameshift in the mature mRNA rather than removing one. In all of these revertants, two alternative 5' splice sites are available to remove intron 3. We determined the relative efficiency with which each alternative 5' splice site is used by stabilizing frameshifted mRNAs with smg(-) genetic backgrounds. In all cases, the upstream member of the two alternative sites is used preferentially (> 75% utilization). This may reflect an inherent preference of the splicing machinery for the upstream member of two closely spaced 5' splice sites. Creation of new 5' splice sites may be a general characteristic of Tc1 insertion and excision events.     

Somatic excision of transposable element Tc1 from the Bristol genome of Caenorhabditis elegans.

We investigated the ability of the transposable element Tc1 to excise from the genome of the nematode Caenorhabditis elegans var. Bristol N2. Our results show that in the standard lab strain (Bristol), Tc1 excision occurred at a high frequency, comparable to that seen in the closely related Bergerac strain BO. We examined excision in the following way. We used a unique sequence flanking probe (pCeh29) to investigate the excision of Tc1s situated in the same location in both strains. Evidence of high-frequency excision from the genomes of both strains was observed. The Tc1s used in the first approach, although present in the same location in both genomes, were not known to be identical. Thus, a second approach was taken, which involved the genetic manipulation of a BO variant, Tc1(Hin). The ability of this BO Tc1(Hin) to excise was retained after its introduction into the N2 genome. Thus, we conclude that excision of Tc1 from the Bristol genome occurs at a high frequency and is comparable to that of Tc1 excision from the Bergerac genome. We showed that many Tc1 elements in N2 were apparently functionally intact and were capable of somatic excision. Even so, N2 Tc1s were prevented from exhibiting the high level of heritable transposition displayed by BO elements. We suggest that Bristol Tc1 elements have the ability to transpose but that transposition is heavily repressed in the gonadal tissue.     

Computer analyses reveal a hobo-like element in the nematode Caenorhabditis elegans,which presents a conserved transposase domain common with the Tc1-Mariner transposon family

The present report describes the use of computer analyses to reveal a hobo-like element in the genome of Caenorhabditis elegans. This hobo-like sequence is 3039 bp long, contains two inverted terminal repeats of 25–27 bp and probably does not encoded a functional transposase. Sequence comparisons suggest that each transposase of hobo elements probably has a D(D/S)E motif. Thus the transposases of the hAT superfamily of transposons appear to be close to the other transposases and intregrases.     

DNA binding activities of the Caenorhabditis elegans Tc3 transposase.

Tc3 is a member of the Tc1/mariner family of transposable elements. All these elements have terminal inverted repeats, encode related transposases and insert exclusively into TA dinucleotides. We have studied the DNA binding properties of Tc3 transposase and found that an N-terminal domain of 65 amino acids binds specifically to two regions within the 462 bp Tc3 inverted repeat; one region is located at the end of the inverted repeat, the other is located approximately 180 bp from the end. Methylation interference experiments indicate that this N-terminal DNA binding domain of the Tc3 transposase interacts with nucleotides on one face of the DNA helix over adjacent major and minor grooves.     

TcA, the putative transposase of the C. elegans Tc1 transposon, has an N-terminal DNA binding domain.

Tc1 is a transposon present in several copies in the genome of all natural isolates of the nematode C.elegans; it is actively transposing in many strains. In those strains Tc1 insertion is the main cause of spontaneous mutations. The transposon contains one large ORF that we call TcA; we assume that the TcA protein is the transposase of Tc1. We expressed TcA in E.coli, purified the protein and showed that it has a strong affinity for DNA (both single stranded and double stranded). A fusion protein of beta-galactosidase and TcA also exhibits DNA binding; deletion derivatives of this fusion protein were tested for DNA binding. A deletion of 39 amino acids at the N-terminal region of TcA abolishes the DNA binding, whereas a deletion of 108 C-terminal amino acids does not affect DNA binding. This shows that the DNA binding domain of TcA is near the N-terminal region. The DNA binding capacity of TcA supports the assumption that TcA is a transposase of Tc1.     

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.     

Target sequences of Tc1, Tc3 and Tc5 transposons of Caenorhabditis elegans

We report here the consensus target sequence of transposons Tc1, Tc3 and Tc5 of Caenorhabditis elegans. These sequences were obtained by molecular analysis of 1008 random new insertions which have not been exposed to natural selection. This analysis reveals consensus target sites slightly different from those previously reported, and confirms that the mariner elements Tc1 and Tc3 insert in sites which are not preferentially palindromic.     

Transposon Tc1 of the nematode Caenorhabditis elegans jumps in human cells.

The transposon Tc1 of the nematode Caenorhabditis elegans is a member of the widespread family of Tc1/mariner transposons. The distribution pattern of virtually identical transposons among insect species that diverged 200 million years ago suggested horizontal transfer of the elements between species. Thishypothesis gained experimental support when it was shown that Tc1 and later also mariner transposons could be made to jump in vitro , with their transposase as the only protein required. Later it was shown that mariner transposons from one fruit fly species can jump in other fruit fly species and in a protozoan and, recently, that a Tc1-like transposon from the nematode jumps in fish cells and that a fish Tc1-like transposon jumps in human cells. Here we show that the Tc1 element from the nematode jumps in human cells. This provides further support for the horizontal spread hypothesis. Furthermore, it suggests that Tc1 can be used as vehicle for DNA integration in human gene therapy.     

The worm has turned--microbial virulence modeled in Caenorhabditis elegans

The nematode Caenorhabditis elegans is emerging as a facile and economical model host for the study of evolutionarily conserved mechanisms of microbial pathogenesis and innate immunity. A rapidly growing number of human and animal microbial pathogens have been shown to injure and kill nematodes. In many cases, microbial genes known to be important for full virulence in mammalian models have been shown to be similarly required for maximum pathogenicity in nematodes. C. elegans has been used in mutation-based screening systems to identify novel virulence-related microbial genes and immune-related host genes, many of which have been validated in mammalian models of disease. C. elegans-based pathogenesis systems hold the potential to simultaneously explore the molecular genetic determinants of both pathogen virulence and host defense.     

Crystal structure of the specific DNA-binding domain of Tc3 transposase of C.elegans in complex with transposon DNA.

The crystal structure of the complex between the N-terminal DNA-binding domain of Tc3 transposase and an oligomer of transposon DNA has been determined. The specific DNA-binding domain contains three alpha-helices, of which two form a helix-turn-helix (HTH) motif. The recognition of transposon DNA by the transposase is mediated through base-specific contacts and complementarity between protein and sequence-dependent deformations of the DNA. The HTH motif makes four base-specific contacts with the major groove, and the N-terminus makes three base-specific contacts with the minor groove. The DNA oligomer adopts a non-linear B-DNA conformation, made possible by a stretch of seven G:C base pairs at one end and a TATA sequence towards the other end. Extensive contacts (seven salt bridges and 16 hydrogen bonds) of the protein with the DNA backbone allow the protein to probe and recognize the sequence-dependent DNA deformation. The DNA-binding domain forms a dimer in the crystals. Each monomer binds a separate transposon end, implying that the dimer plays a role in synapsis, necessary for the simultaneous cleavage of both transposon termini.     

The structure of the ventral nerve cord of Caenorhabditis elegans.

The nervous system of Caenorhabditis elegans is arranged as a series of fibre bundles which run along internal hypodermal ridges. Most of the sensory integration takes place in a ring of nerve fibres which is wrapped round the pharynx in the head. The body muscles in the head are innervated by motor neurones in this nerve ring while those in the lower part of the body are innervated by a set of motor neurones in a longitudinal fibre bundle which joins the nerve ring, the ventral cord. These motor neurones can be put into five classes on the basis of their morphology and synaptic input. At any one point along the cord only one member from each class has neuromuscular junctions. Members of a given class are arranged in a regular linear sequence in the cord and have non-overlapping fields of motor synaptic activity, the transition between fields of adjacent neurones being sharp and well defined. Members of a given class form gap junctions with neighbouring members of the same class but never to motor neurones of another class. Three of the motor neurone classes receive their synaptic input from a set of interneurones coming from the nerve ring. These interneurones can in turn be grouped into four classes and each of three motor neurone classes receives its synaptic input from a unique combination of interneurone classes. The possible developmental and functional significance of these observations is discussed.