Evidence for a transposon in caenorhabditis elegans

The C. elegans genome contains a 1.7 kb repeated DNA sequence (Tc1) that is present in different numbers in various strains. In strain Bristol and 10 other strains analyzed, there are 20 ± 5 copies of Tc1, and these are located at a nearly constant set of sites in the DNA. In Bergerac, however, there are 200 ± 50 interspersed copies of Tc1 that have arisen by insertion of Tc1 elements into new genomic sites. The interspersed copies of Tc1 have a conserved, nonpermuted structure. The structure of genomic Tc1 elements was analyzed by the cloning of a single Tc1 element from Bergerac and the comparison of its structure with homologous genomic sequences in Bristol and Bergerac. Tc1 elements at three sites analyzed in Bergerac undergo apparently precise excision from their points of insertion at high frequency.     

Analysis of a Mutator Activity Necessary for Germline Transposition and Excision of Tc1 Transposable Elements in Caenorhabditis Elegans

The Tc1 transposable element family of the nematode Caenorhabditis elegans consists primarily of 1.6-kb size elements. This uniformity of size is in contrast to P in Drosophila and Ac/Ds in maize. Germline transposition and excision of Tc1 are detectable in the Bergerac (BO) strain, but not in the commonly used Bristol (N2) strain. A previous study suggested that multiple genetic components are responsible for the germline Tc1 activity of the BO strain. To analyze further this mutator activity, we derived hybrid strains between the BO strain and the N2 strain. One of the hybrid strains exhibits a single locus of mutator activity, designated mut-4, which maps to LGI. Two additional mutators, mut-5 II and mut-6 IV, arose spontaneously in mut-4 harboring strains. This spontaneous appearance of mutator activity at new sites suggests that the mutator itself transposes. The single mutator-harboring strains with low Tc1 copy number generated in this study should be useful in investigations of the molecular basis of mutator activity. As a first step toward this goal, we examined the Tc1 elements in these low copy number strains for elements consistently co-segregating with mutator activity. Three possible candidates were identified: none was larger than 1.6 kb.     

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.     

Structural analysis of Tc1 elements in Caenorhabditis elegans var. Bristol (strain N2)

The transposable element Tc1 in the genome of Caenorhabditis elegans var. Bristol strain N2 is very stable. In order to investigate possible causes of Tc1 immobility in this strain 17 individual isolates have been cloned and characterized with regard to their structure and genomic environment. Ten of 16 elements examined had identical restriction maps, and at least 1 of these (#7) showed a high level of somatic excision. Two of the elements had altered restriction sites, 2 had different internal deletions of about 700 bp, 1 had an 89-bp terminal deletion, and 1 a 54-bp insertion. When DNA sequences flanking the N2 Tc1 elements were used as probes in genomic hybridizations, it was found that most N2 elements are located in regions of repetitive DNA. Furthermore when hybridizations to DNA from N2 and var. Bergerac strain B0 were performed, a major band of the same size was observed in both strains. Two flanking sequences identified strain polymorphic sites hP2(IV) and hP3(IV). In at least one of these cases, a rearranged Tc1 was present in the B0 strain at the same location. The fact that all or most of the Tc1 elements are in the same location in N2 and B0 adds support to the hypothesis that the high copy number B0 strain arose from amplification of Tc1 copies in a N2-like strain. The N2 Tc1 elements are highly conserved; however, intact elements had fewer nucleotide changes than the rearranged elements. These results may indicate that the intact Tc1 elements in N2 are functionally active and subject to selective pressure.     

Spontaneous Unstable UNC-22 IV Mutations in C. ELEGANS Var. Bergerac

This paper describes a mutator system in the nematode Caenorhabditis elegans var. Bergerac for the gene unc-22. Of nine C. elegans and two C. briggsae strains tested only the Bergerac BO strain yielded mutant animals at a high frequency and the unc-22 IV gene is a preferred mutational target. The forward spontaneous mutation frequency at the unc-22 locus in Bergerac BO is about 1 x 10^-4 , and most of these spontaneous unc-22 mutations revert at frequencies between 2 x 10^-3 and 2 x 10 ^-4. Both the forward mutation frequency and the reversion frequency are sensitive to genetic background. Spontaneous unc-22 mutations derived in a Bergerac background and placed in a primarily Bristol background revert at frequencies of <10^-6. When reintroduced into a Bergerac/Bristol hybrid background the mutations once again become unstable.

The mutator activity could not be localized to a discrete site in the Bergerac genome. Nor did mutator activity require the Bergerac unc-22 gene as a target since the Bristol unc-22 homolog placed in a Bergerac background also showed high mutation frequency. Intragenic mapping of two spontaneous unc-22 alleles, st136 and st137, place both mutations in the central region of the known unc-22 map. However, these mutations probably recombine with one another, suggesting that the unstable mutations can occur in more than one site in unc-22. Examination of the phenotypic effect of these mutations on muscle structure indicates that they are less severe in their effect than a known amber allele. We suggest that this mutator system is polygenic and dispersed over the nematode genome and could represent activity of the transposable element Tc1.

     

Isolation and mapping of DNA probes within the linkage group I gene cluster of Caenorhabditis elegans

We have isolated probes for DNA polymorphisms across the linkage group I gene cluster in Caenorhabditis elegans, using Tc1-linkage selection. The probes detect strain polymorphism between the wild-type strains of var. Bristol and var. Bergerac. As a result of mapping the sites hP4, hP5, hP6, hP7, hP9, and sPl, more than 1000 kilobases (kb) of cloned cosmid DNA has been positioned on the genetic map. We found there is more DNA per map unit in the center of the gene cluster than expected on the basis of the genomic average. Furthermore, the amount is not constant across the entire region but reaches a peak in the hP9 unc-13 interval. To find the coding regions, we examined DNA cross-homology between two species, Caenorhabditis elegans and Caenorhabditis briggsae. Approximately one-third of the DNA in the hP5 hP9 interval was examined for coding regions and 21 sequences were identified within 318 kb of DNA.     

Tcl transposition and mutator activity in a Bristol strain of Caenorhabditis elegans

Summary In most strains of Caenorhabditis elegans with a low copy number of Tc1 transposable elements, germline transposition is rare or undetectable. We have observed low-level Tel transposition in the genome of the C. elegans var. Bristol strain KR579 (unc-13[e51]) resulting in an increase in Tc1 copy number and subsequent mutator activity. Examination of genomic blots from KR579 and KR579derived strains revealed that more Tc1-hybridizing bands were present than in other Bristol strains. A novel Tc1-hybridizing fragment was cloned from a KR579-derived strain. Unique sequence DNA flanking the Tc1 element identified a 1.6 kb restriction fragment length difference between the KR579 and N2 strains consistent with a Tc1 insertion at a new genomic site. The site of insertion of this Tel was sequenced and is similar to the published Tel insertion site consensus sequence. Several isolates of KR579 were established and maintained on plates for a period of 3 years in order to determine if Tc1 copy number would continue to increase. In one isolate, KR1787, a further increase in Tc1 copy number was observed. Examination of the KR1787 strain has shown that it also exhibits mutator activity as assayed by the spontaneous mutation frequency at the unc-22 (twitcher) locus. The KR579 strain differs from most low copy number strains in that it exhibits low-level transposition which has developed into mutator activity.     

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.     

Genetic mapping of the 5S rRNA gene cluster of the nematode Caenorhabditis elegans

We have identified a restriction fragment length difference (RFLD) affecting the genomic sequences immediately flanking the 5S rRNA gene cluster in the Bristol and Bergerac strains of the nematode Caenorhabditis elegans. We have used this RFLD as a molecular marker to follow the segregation of the 5S rRNA gene cluster through a series of two- and three-factor interstrain crosses. Our results show that the 5S rRNA gene cluster maps between unc-76 and dpy-21 on the right arm of linkage group V. This genetic localization provides a linkage group V "landmark" with which to localize other cloned sequences by in situ hybridization.     

Mobilization of quiet, endogenous Tc3 transposons of Caenorhabditis elegans by forced expression of Tc3 transposase.

The commonly studied Caenorhabditis elegans strain Bristol N2 contains approximately 15 copies per genome of the transposon Tc3. However, Tc3 is not active in Bristol N2. Tc3 contains one major open reading frame (Tc3A). We have fused this open reading frame to an inducible promoter and expressed it in a transgenic Bristol N2 line. Tc3A expression resulted in frequent excision and transposition of endogenous Tc3 elements. This shows that the Bristol N2 genome contains Tc3 transposons that are cis proficient for transposition, but are immobile because Tc3A is absent. We demonstrate that recombinant Tc3A binds specifically to the terminal nucleotides of the Tc3 inverted repeat, indicating that Tc3A is the Tc3 transposase. Activation of Tc3 transposition in vivo was accompanied by the appearance of extrachromosomal, linear copies of Tc3. These may be intermediates in Tc3 transposition.     

Continuous exchange of sequence information between dispersed Tc1 transposons in the Caenorhabditis elegans genome

In a genome-wide analysis of the active transposons in Caenorhabditis elegans we determined the localization and sequence of all copies of each of the six active transposon families. Most copies of the most active transposons, Tc1 and Tc3, are intact but individually have a unique sequence, because of unique patterns of single-nucleotide polymorphisms. The sequence of each of the 32 Tc1 elements is invariant in the C. elegans strain N2, which has no germline transposition. However, at the same 32 Tc1 loci in strains with germline transposition, Tc1 elements can acquire the sequence of Tc1 elements elsewhere in the N2 genome or a chimeric sequence derived from two dispersed Tc1 elements. We hypothesize that during double-strand-break repair after Tc1 excision, the template for repair can switch from the Tc1 element on the sister chromatid or homologous chromosome to a Tc1 copy elsewhere in the genome. Thus, the population of active transposable elements in C. elegans is highly dynamic because of a continuous exchange of sequence information between individual copies, potentially allowing a higher evolution rate than that found in endogenous genes.     

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.     

Cloning within the unc-43 to unc-31 interval (linkage group IV) of the Caenorhabditis elegans genome using Tc1 linkage selection

The region around the twitcher gene, unc-22, flanked by unc-43 on the left and by unc-31 on the right, has been intensively studied in our laboratory over the period of the last 8 years. In this paper we describe the identification and isolation of probes specific for several restriction fragment length differences (RFLDs) which lie within this region. Many RFLDs in Caenorhabditis elegans are caused by the insertion of a transposable element, Tc1. The method we used involved the isolation of Tc1-containing genomic fragments. These were recovered from a lambda gt 10 library of DNA from a specially constructed genetic strain containing the unc-43 to unc-31 interval from the BO strain and the rest of the genome from N2. Because the BO strain is rich in Tc1 insertion sites and the N2 strain has few, the majority of Tc1-bearing genomic fragments in the constructed strain were derived from the unc-22 region. Of nine such Tc1-bearing genomic fragments isolated, six were found which mapped within the region of interest. The 350 kilobases of genomic sequences isolated as a result of these studies are being used to study the molecular organization of this region. The method described here for Tc1 linkage selection is one that is rapid, general, and may be targeted to any genetically characterized region of the C. elegans genome.     

Tc1 transposase of Caenorhabditis elegans is an endonuclease with a bipartite DNA binding domain.

The Tc1 transposon of Caenorhabditis elegans is a member of the Tc1/mariner family of mobile elements. These elements have inverted terminal repeats that flank a single transposase gene. Here we show that Tc1 transposase, Tc1A, has a bipartite DNA binding domain related to the paired domain of mammalian and Drosophila genes. Both the DNA binding domain of Tc1A and the DNA binding site in the inverted repeat of Tc1 can be divided into two subdomains. Methylation interference studies demonstrate adjacent minor and major groove contacts at the inner part of the binding site by the N-terminal 68 amino acids of the DNA binding domain. In addition, Tc1A amino acids 69-142 are essential for major groove contacts at the outer part of the binding site. Recombinant Tc1A is found to be able to introduce a single strand nick at the 5' end of the transposon in vitro. Furthermore, Tc1A can mediate a phosphoryl transfer reaction. A mutation in a DDE motif abolishes both endonucleolytic and phosphoryl transfer activities, suggesting that Tc1A carries a catalytic core common to retroviral integrases and IS transposases.     

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.     

The Tc5 Family of Transposable Elements in Caenorhabditis Elegans

We have identified Tc5, a new family of transposable genetic elements in the nematode Caenorhabditis elegans. All wild-type varieties of C. elegans that we examined contain 4-6 copies of Tc5 per haploid genome, but we did not observe transposition or excision of Tc5 in these strains. Tc5 is active, however, in the mut-2 mutant strain TR679. Of 60 spontaneous unc-22 mutations isolated from strain TR679, three were caused by insertion of Tc5. All three Tc5-induced mutations are unstable; revertants result from precise or nearly precise excision of Tc5. Individual Tc5 elements are similar to each other in size and structure. The 3.2-kb element is bounded by inverted terminal repeats of nearly 500 bp. Eight of the ten terminal nucleotides of Tc5 are identical to the corresponding nucleotides of Tc4. Further, both elements recognize the same target site for insertion (CTNAG) and both cause duplication of the central TNA trinucleotide upon insertion. Other than these similarities to Tc4, Tc5 is unrelated to the three other transposon families (Tc1, Tc3 and Tc4) that transpose and excise at high frequency in mut-2 mutant strains. Mechanisms are discussed by which four apparently unrelated transposon families are all affected by the same mut-2 mutation.     

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.     

Target sequences for the C. elegans transposable element Tc1.

The target sequences for two independent insertions of the transposable element Tc1 from Caenorhabditis elegans show homology. Because both insertions are at palindromic TA/AT sequences, the exact boundaries of Tc1 cannot be distinguished; Tc1 could be 1610 bp and flanked by a 2-bp duplication of the target site or it could be 1612 bp and without target site duplication. The latter possibility implies a novel manner for insertion of a transposable element.     

High-frequency excision of transposable element Tc 1 in the nematode Caenorhabditis elegans is limited to somatic cells

Tc 1 transposable elements in the nematode Caenorhabditis elegans undergo excision at high frequency. We show here that this excision occurs primarily or entirely in the somatic tissues of the organism. Absence of germ-line excision is demonstrated by showing that Tc 1 elements are genetically stable; elements at particular genomic sites, as well as the overall number of elements in the genome, were stably maintained during a year of continuous, nonselective propagation. Somatic excision is demonstrated by showing that empty Tc 1 sites arise during a single generation of growth of a synchronous population and are not inherited by the next generation. These results suggest that excision of Tc 1 elements is under the control of tissue-specific factors.     

Germline excision of the transposable element Tc1 in C. elegans.

We have examined eight germline revertants generated by the excision of Tc1 from a site within the unc-22 gene of Caenorhabditis elegans. A rich variety of rearrangements accompanied Tc1 excision at this site, including transposon 'footprints', deletions of sequences flanking the insertion site and direct nontandem duplications of flanking DNA. With only modest modification the double-strand gap repair model for transposition, recently proposed by Engles and coworkers (Cell 62: 515-525 1990), can explain even the most complex of these rearrangements. In light of this model rearrangements of the target site accompanying transposition/excision may not be the end result of imprecise excision of the element. Instead, these rearrangements may be the result of imprecise repair of the double-strand gap by the host replication and repair machinery. Sequences surrounding an insertion site influence the fidelity of gap repair by this machinery. This may lead to a number of possible resolutions of a double-strand gap as documented here for a Tc1 site in unc-22.