Ketting RF et al. (1996) Worm Breeder's Gazette "TARGET SITE SELECTION BY THE Tc1A TRANSPOSASE"

Ketting RF, Plasterk RHA, Fischer SEJ

[Worm Breeder's Gazette, 1996]

One of the key-features of Tc1 transposition is that this transposon always integrates into a TA dinucleotide. Not all TA dinucleotides are equally used, however. Two consensus sequences for integration of Tc1 have been obtained by analysis of respectively 12 and 16 Tc1 insertions (Mori et al. (1988), Proc.Natl.Acad.Sci.USA 85:861-864, Eide and Anderson (1988), Mol.Cell.Biology 8:737-746). These consensus sequences are based on flanking sequences of independent Tc1 alleles of mainly unc-54 and unc-22. Recently a consensus sequence based on 344 endogenous Tc1 insertions was derived (Korswagen et al. (1996), Proc.Natl.Acad.Sci.USA in press). A fourth consensus sequence was derived by van Luenen and Plasterk (van Luenen and Plasterk (1994), Nucl.Acids.Res. 22:262- 269), after analysing 204 de novo Tc1 insertions into the gpa-2 gene. These insertions were detected using PCR after overexpression of the Tc1A transposase. These four consensus sequences are all similar. The consensus sequence derived by Korswagen et al. is depicted in table 1. In vitro studies using gpa-2 as a target gene showed that the distribution of Tc1 elements in this DNA fragment was virtually the same as in vivo; so chromatin structure does not determine the observed preferences. What then determines the hotness of a given TA dinucleotide? To analyse the role of the basepairs flanking the TA dinucleotide, we subcloned a 24 bp fragment, containing the hottest TA dinucleotide of the gpa-2 gene plus two adjacent cold TA sites, into the lacZ polylinker of pUC19. This DNA was then used as target DNA for transposition in vitro. Among 65 independent Tc1 integrations into the lacZ region, 33 were found in the gpa-2 hot-site. The two other TA dinucleotides were never used. When the hot gpa-2 fragment is shortened to 13 bp (with 5 and 6 bp on each site of the TA) again more than 50% of the inserts were found in this gpa-2 hot-site. Although the entire lacZ region counts 27 TA dinucleotides, still 17 out of 32 insertions are found in this particular site. This indicates that whatever makes the site preferred is contained within these 5/6 bp flanks. The simplest mechanistic explanation is that the transposase requires recognition of a TA sequence, and prefers recognition of some additional nucleotides before integration occurs. We are now further shortening and mutating the flanks of the hot TA dinucleotide in order to get a more detailed picture of the target site recognition by the Tc1A protein.

van Luenen HGAM et al. (1993) Worm Breeder's Gazette "Sequence Specificity for Somatic Tc3 Integration"

van Luenen HGAM, Plasterk RHA

[Worm Breeder's Gazette, 1993]

Transposition of the Tc3 transposon has been detected in the high hopper strain TR679 which was initially selected for increased transposition of the related element Tc1 .Both Tc1 and Tc3 insert at a TA dinucleotide. A consensus sequence around this TA dinucleotide has been proposed for Tc1 insertions in the germ line (G A G/T A/G T A C/T G/T T; Mori et al., 1988, Proc. Natl. Acad. Sci. USA 85:86t- 864; Eide and Anderson, 1988, Mol. Cel. Biol. 8:737-746); this consensus sequences is based on the comparison of 16 Tc1 insertions. We have sequenced 96 independent somatic Tc3 insertions to determine the Tc3 integration consensus sequence. This is part of an ongoing study in which we compare Tc1 and Tc3 integration site specificity and in which we investigate the predictive value of the Tc1 integration consensus sequence mentioned above. In the previous WBG (WBG, 1992 12/4:15) we described a transgenic strain in which we could induce the expression of Tc3 transposase in somatic tissues. The transgenic strain was made in a Bristol N2 background in which no Tc3 transposition occurs. After induction, frequent somatic Tc3 transposition events were observed, as witnessed by the appearance of extrachromosomal Tc3 elements and by insertions of Tc3 in a random gene (the gpa-2 gene). Insertions were detected by two rounds of PCR with nested oligo's. To determine the insertion sites, the PCR products were excised from an agarose gel and sequenced according to Craxton (Methods: A companion to methods in enzymology, 1991 3:20-26). The distribution of Tc3 insertions in an 1 kbp region of gpa-2 is depicted in the figure. Each small line on the X axis represents a TA dinucleotide: a potential Tc3 insertion site. The vertical bars represent the number of Tc3 insertions found at a specific TA dinucleotide. [See Figure] From the distribution we conclude that Tc3 insertions are not random. TA dinucleotides which are frequently used for integration are not clustered or regularly spaced. Furthermore, the TA dinucleotide with the most frequent Tc3 insertion (24/96) is flanked by two TA dinucleotides which are used only 1/96 or 0/96 times. This suggests that the local rather than the regional structure of the DNA facilitates insertions. The alignment (according to the 5'-3' orientation of the gpa-2 gene) of all the 96 insertion sites of Tc3 results in the consensus sequence: A(40%)/T(44%) A(60%) A(49%)/C(41%) T A T(79%) T(70%) A(44%)/T(53%). The consensus sequence is not very strict; it does not resemble the Tc1 consensus sequence.

Eide DJ et al. (1985) Worm Breeder's Gazette "Tc1 insertion and excision at unc-54."

Eide DJ, Anderson P

[Worm Breeder's Gazette, 1985]

We have sequenced the insertion sites of nine independent insertions at unc-54. Our purpose is to determine the exact structure of Tc1- induced mutations and to assess the target site specificity of Tc1 transposition. The results of this analysis are summarized below. Each insertion occurs within a TA dinucleotide (underlined). The numbering system is that of Karn et al. (PNAS 80:4253-4257 (1983)). [See Figure 1] All of the insertions are located in exons. The data indicate that Tc1 insertion is highly site-specific. Five of the nine insertions are in the exact same TA dinucleotide. Furthermore, two of the remaining four insertions are located in another identical TA dinucleotide. Combined with the sequences of Rosenzweig et al. for two natural Tc1 insertions in Bergerac (N.A.R. 11:7137-7140), the following consensus sequence emerges: [See Figure 2] It is difficult to judge the significance of parts of this consensus. Only the TA dinucleotide is absolutely conserved. Clearly, the sequences of additional insertion sites are needed, especially those that occur at high frequency. We have determined the sequence of a germ-line, wild-type revertant of unc-54(r323::Tc1). We were surprised to discover that this revertant (TR462) contains a four base pair insertion remaining at the site of excision: This structure (...TATGTA...) is the same as that reported at the worm meeting by K. Ruan and S. Emmons for a somatic excision product of a natural Bergerac Tc1 element. The TG dinucleotide might be derived from the two nucleotides at one end of Tc1 (as indicated by the asterisks). An exon insertion of four base pairs would disrupt the unc-54 reading frame. Therefore, the wild-type phenotype of this revertant is unexpected. One possible explanation is as follows: The target TA dinucleotide in r323 is one base pair removed from the 5' splice site of IVS#3. We suggest that the inserted bases in TR462 generate a new splice site four base pairs 5' of the normal site. In this way, the reading frame is restored. The sequence of the proposed new splice site fits fairly well with the C. elegans splice site consensus sequence compiled by Tom Blumenthal: The suggested new 5' splice is indicated by the vertical line. The wild type splice site is indicated by the apostrophe ('). The similarity of this new site to the consensus may allow sufficient splicing at this location to generate a wild type phenotype. We do not know whether TR462 constitutes a 'typical' excision event. If excisions of this type are the most frequent, mutants having Tc1 inserted within exons may not generally revert at high frequency. It may require 'atypical' excision events for such mutants to visibly revert. We have detected partial revertants for r323::Tc1 (indicating excisions of other types), but their sequences have not been determined. An intriguing feature of this revertant sequence is that the TA target site, coupled with the terminal TG of Tc1 left behind, always yields upon excision four of the highly conserved nucleotides that constitute a donor splice site. Perhaps many Tc1 excisions generate new donor splice sites?

van Luenen HGAM et al. (1993) Worm Breeder's Gazette "Target Choice of the Transposable Elements Tc1 and Tc3"

van Luenen HGAM, Plasterk RHA

[Worm Breeder's Gazette, 1993]

Transposable elements insert at many different positions in the genome, but the integrations are not necessarily randomly distributed over the genome. A detailed analysis of the insertion specificity of a transposon may be revealing with respect to the transposition mechanism of the element, since the integration preference may reflect how the integration complex interacts with the target sequence. We have investigated the target choice of the related transposable elements Tc1 and Tc3 . The exact locations of 204 independent Tc1 insertions and 166 Tc3 insertions were determined. We used a PCR based approach which is sensitive enough to detect transposon insertions represented by a single DNA molecule and which does not involve a phenotypic selection for insertions (and is therefore unbiased for the distribution of insertions). The conditions were chosen such that insertions in an 1 kbp region of the genome were detected and that every insertion represents an independent somatic event. All 204 Tc1 insertions were into the sequence TA. Within the region investigated there are 82 TA's, but not every TA is used as often by Tc1 for integration (some TA's contain many insertions, others contain a few or no insertions; figure 1). The distribution of Tc1 insertions is independent of the orientation of the transposon. This implies that the integration reaction or the target sequence is symmetrical. We favour the first option since the flanking sequences of the TA's at the insertion sites have hardly any discernible symmetry. We considered several explanations for the target site preference. 1) The local DNA structure. If only the local DNA structure makes one TA more attractive that another, we might expect to find a similar distribution for the two related elements Tc1 and Tc3 .Therefore we determined the exact locations of 166 Tc3 insertions. All insertions were into the sequence TA and they are also non-randomly distributed among the TA's in this region. We compared the distribution of Tc1 and Tc3 insertions and found that they are completely different (figure 2). Simple DNA structures (such as bends and kinks) are therefore not very likely determinants in target site selection (unless the two elements recognize them differently) . 2) The donor site. The non-random distribution of insertions could be explained if the original insertion site of the element effects the target choice. To investigate this we compared the distribution of Tc3 insertions to the distribution of a marked Tc3 transposon that jumped from one specific donor site in a transgenic line. The distribution of normal Tc3 insertions is the result of transposition from approximately 15 different donor sites. There is no difference between the two distributions (figure 3) indicating that the donor site has no effect on the target choice. 3) The flanking sequence of the TA dinucleotides. The most likely explanation of local target site preference is the recognition of the sequence flanking the TA dinucleotide by the incoming transposon. We investigated whether we could recognize a pattern of preferred sequences in the close vicinity of the insertion site. No clear consensus sequence was found. These studies focused on somatic transposition. Assuming these studies are also relevant for germ line transposition (and on the basis of germ line insertions studied thus far that seems a reasonable assumption) we do not think it is useful to look for "consensus" Tc1 sites when planning e.g. reverse genetic experiments.

Chamberlin HM et al. (1996) Worm Breeder's Gazette "egl-38 is an essential gene required for normal development of both the hermaphrodite egg-laying system and male rectal epithelium."

Sternberg PW, Thomas JH, Palmer RE, Newman AP, Baillie DL, Chamberlin HM

[Worm Breeder's Gazette, 1996]

Mutations in egl-38 have been recovered in screens based on three different phenotypes: Egl, Mab, and Let. Trent et al. (1983) recovered n578, the first allele of egl-38. n578 hermaphrodites are strongly egg-laying defective, and were originally noted to be defective in vulva morphogenesis. In a screen for mutations that disrupt male tail development, we recovered sy294. sy294 hermaphrodites are Egl, males have severe tail deformities, and both males and hermaphrodites frequently die. sy294 was previously known as lin-50. Finally, in screens for unc-22-linked lethal mutations on LG IV, Denise Clark (1990) identified 24 mutations that were uncovered by mDf7, but complemented sDf2. We found that one of these mutations, s1775, fails to complement both n578 and sy294. We have been investigating the cellular basis for these mutant phenotypes.

Mueller-Immergluck MM et al. (1993) Worm Breeder's Gazette "Combinatorial Activity of lin-39 and mab-5 Controls the Fusion of thePn.p Cells With the Lateral Hypodermis in C. elegans males"

Kenyon CJ, Mueller-Immergluck MM

[Worm Breeder's Gazette, 1993]

In insects and vertebrates, clusters of homeobox genes (HOM genes) specify anteroposterior pattern. Two closely located homeobox genes in C. elegans, lin-39 (B. Wang and C. K, unpubl.; Clark and Horvitz, pers. comm.) and mab-5 ,contain a homeobox most similar to the Antennapedia-class. lin-39 acts in the central (Clark and Horvitz, WBG 11(2), 107 (1990)) and mab-5 in the posterior body region. We found that lin-39 and mab-5 together can specify a cell fate that is different from the fate each gene specifies on its own. Such combinatorial activity in regions of overlap greatly increases the complexity of pattern that can be generated even with a small number of genes involved. In the ventral cord, there are the twelve Pn.p cells P1 .p-P12.p.At the end of L1 ,the descendants of these cells either remain mononucleate or fuse with hyp7 .In hermaphrodites, lin-39 activity defines the "vulval equivalence group" P(3-8).p and allows the production of the vulva (Clark and Horvitz, WBG 11(2),107(1990)). In males, mab-5 defines the "preanal ganglion equivalence group" P(9-11).p and allows the production of the male copulatory apparatus. With the antibody MH27 ,which recognizes a component of septate junctions, we looked which cells fuse in lin-39 and mab-5 single and double mutants. [See Figure] In males, when acting alone (that is, in either mab-5 (-)or lin-39 (-)background) both, lin-39 (+)and mab-5 (+)can prevent cell fusion within their respective domains of function (Fig.1a). In a lin-39 (-)mab-5 (-)double mutant, where both genes are inactive, all the Pn.p cells fuse. In wildtype males, where both genes are active, one might have expected a simple active phenotype, in which all the cells in the combined lin-39 and mab-5 domain [P(3-11).p] would remain unfused. Surprisingly however, the two Pn.p cells within the region of overlap of mab-5 and lin-39 activity, P(7-8).p, adopt the alternative fate, now they fuse. In hermaphrodites, only lin-39 ,but not mab-5 ,influences the fusion decision of the Pn.p cells (Fig.lb). Thus in males, lin-39 and mab-5 together promote a fate (cell fusion), that is different from the fate promoted by either gene acting alone (remain unfused). Fusion also occurs in P1 .pand P2 .p,in which neither lin-39 nor mab-5 appear to function. Our results suggest that, by an unknown mechanism, lin-39 and mab-5 together specify the same fate that is specified if both genes are inactive (cell fusion). We can speculate that the fusion of Pn.p cells to hyp7 probably represents the "hypodermal ground state". The fusion of P(7-8).p in males limits the "preanal ganglion equivalence group" to the unfused P(9-11).p. Apparently, rather than allowing fusion of P(7-8).p by shutting off lin-39 and mab-5 ,evolution selected a combinatorial control mechanism to achieve the same goal.

Vos JC et al. (1995) Worm Breeder's Gazette "Tc1 jumps in vitro"

Vos JC, Plasterk RHA

[Worm Breeder's Gazette, 1995]

Over the last few years, many details about the mechanism of Tc1 and Tc3 transposition have been obtained by studying the process in vivo. Jumping is catalysed by the transposase protein encoded by the element. It generates double strand breaks at the ends of the transposon; this results in a linear excised element with 2 bp staggered 3' overhangs (Cell 79:293-301, 1994). Integration occurs always into a TA dinucleotide. Notably, Tc1 and Tc3 appear to have quite distinctive integration patterns in vivo as shown for a randomly chosen gene area, i.e. a fragment of the gpa-2 gene (NAR 22: 5548-5554, 1994). We have now developed a Tc1 in vitro transposition assay. We make use of a nuclear extract derived from a stable transgenic worm strain with a transposase gene under the control of a heat shock promoter (Genes & Dev. 7 ; 1244-1253, 1993). A donor plasmid with a marked Tc1 element is preincubated with such an extract, and subsequently a target plasmid is added. After the reaction, products were analyzed in both a physical and a genetic assay. In vitro reaction products were directly visualized by Southern blotting and by primer extension. Double strand breaks at one transposon end or at both transposon ends are detectable. The mapping of the ends of the excised element confirms the presence of the 2 bp 3' overhang as previously determined for Tc3 in vivo. Double strand breaks at transposon ends can occur on either a supercoiled or a linear substrate and with substrates containing a single transposon end. Finally, addition of ATP or GTP does not influence the efficiency of Tc1 excision. Experiments to determine the cis-requirements for Tc1 excision in vitro show that only the terminal 26 bp of Tc1 are sufficient. These sequences contain the transposase binding site. Mutation of the transposase binding site abolishes excision. Also, mutation of the terminal 4 base pairs of Tc1, as well as mutation of the flanking TA sequence, which are conserved between Tc1, Tc3 and many other members of the Tc1-family, are detrimental for excision in vitro. In the genetic screen to detect actual integration products, the reaction mix is electroporated into an E. coli strain which allows selection for the Tc1-borne antibiotic resistance and against the donor plasmid. Integration products are obtained which faithfully reflect in vivo transposition events: Tc1 is integrated into a TA dinucleotide, which is duplicated. Among the 80 sequenced independent Tc1 insertions, two exceptions were found where Tc1 had integrated at the sequence TTG or CCT. We included a 1.4 kb gpa-2 sequence in the target plasmid to be able to compare the Tc1 in vitro integration pattern with the one previously determined in vivo. Therefore, 62 independent in vitro integration events in gpa-2 were sequenced. Itappears that hot sites in vivo are hot in vitro and that cold sites in vitro are cold in vivo. Therefore, the integration pattern is not dependent on chromatin structure or the transcriptional status, but rather reflects site-specificity of the transposition complex. The presence of Tc1 related elements in widely divergent phyla has raised the hypothesis that transposition does not require species-specific host factors in addition to the element encoded transposase. Therefore, we expressed transposase in a heterologous system using recombinant baculo virus. Total cell lysates from infected cells were proficient for Tc1 transposition. This shows that no C. elegans specific protein is required besides Tc1 transposase. The next step will be to determine whether purified transposase is sufficient for the whole transposition process or if general host factors are required.

van Luenen HGAM et al. (1993) Worm Breeder's Gazette "Mechanism of Tc Transposition: Starting at the End"

Vos JC, van Luenen HGAM, Plasterk RHA

[Worm Breeder's Gazette, 1993]

The transposable element Tc1 is thought to excises via double strand DNA breaks made at the ends of the inverted repeat (Plasterk (1991) EMB0 J. 7,1919-1925). Excised transposons have been detected as extrachromosomal structures (linear or circular; Rose and Snutch (1984) Nature 311, 485-486; Ruan and Emmons (1984) Proc. Natl. Acad. Sci. USA 81, 4018-4022). Excised, linear elements have also been detected for the related transposon Tc3 (Van Luenen et al. (1993) EMBO J. 12, 2513-2520). We have examined how the Tc1 and Tc3 transposons are excised by determination of the exact double strand cleavage sites. Excision of Tc3 is not very frequent and thus the amount of extrachromosomal Tc3 in "natural" mutator strains is limited. Therefore we have made a stable transgenic line containing the Tc3 transposase under the control of an inducible promoter. Upon induction Tc3 transposase is made and this results in excision and transposition of Tc3 elements (Van Luenen et al. (1993) EMBO J. 12, 2513-2520). The level of excision and thus of the amount of extrachromosomal Tc3 elements is high. The 3' end of the excised Tc3 transposon was determined in two independent ways. A restriction site in the inverted repeat located close to the end of the transposon was used to generate a small fragment containing the end of the transposon. This fragment and a size marker were run on a denaturing polyacrylamide gel. The DNA was Southern blotted and probed with a transposon end specific sequence. The length of the Tc3 fragment was compared to the size marker and from that we conclude that the 3' end of the excised element is the G nucleotide (see figure). Alternatively, the extrachromosomal material was amplified by PCR after 3' tailing with terminal transferase and subsequently the PCR product was sequence. The 5' end of the excised Tc3 element was detected using the same Southern blot approach as used for the 3' end. The results indicated that the excised element ends at the G residue (see figure). In other words: the excised Tc3 element has a 2 nucleotide 3' extension. The same cleavage site is used most likely also for Tc1 excision. This has been observed in an in vitro cleavage reaction using recombinant Tc1 transposase. Cleavage was found directly after the G residue. What are the implications of these results for the mechanism of transposition? Since the sequences of Tc1 and Tc3 were determined it was unclear whether the TA dinucleotides flanking the integrated transposon are part of the transposon or result from target site duplication. Our results show that the TA dinucleotides are the result of target site duplication. The element has to integrate using the 3' end, otherwise the last 2 nucleotides of the transposon will be lost. Integration through a nucleophilic attack of the 3' 0H group is also seen for other transposons. The presence of a 3' OH group at Tc3 ends is indirectly demonstrated by the possibility to add nucleotides to them by terminal transferase tailing. Upon excision the cell is left with a double strand break with a 2 nucleotide 3' extension at both sides. The result of the cellular repair process is seen as a footprint left behind after excision of the element. We have sequenced somatic Tc3 footprints after PCR amplification. The footprints generated by Tc1 and Tc3 are very similar. The footprints are most often 4 basepairs in length (a TA basepair and two other basepair: CA or TG) and we conclude that these result from the 2 nucleotides at the 5' ends of the element which are not co-excised.

Johnsen RC et al. (1990) Worm Breeder's Gazette "The cloning of lin-"

Johnsen RC, Baillie DL

[Worm Breeder's Gazette, 1990]

From a Tc1 mutagenesis screen in the nT1(IV,V) balanced region using mut-4 RW7037, six lethal mutations that fail to complement lin-40 were recovered (Clark et al. In Press Genome). We have extracted DNA from the six strains containing lin-40 Tc1 mutations. We digested the DNA with EcoRI, ran it on a gel, transfered the DNA to a filter and hybridized nick translated Tc1 DNA to it. Three strains showed a common new Tc1 band. For one allele (s1351) we recovered the new band, isolated DNA flanking the Tc1 sequence and used the flanking sequence to screen T. Snutch's Charon IV library. From the library we isolated a phage (BC#S1001). DNA from BC#S1001 was mapped to LGV(left) on the cosmid map by J. Sulston and A. Coulson (see figure). The location is surprising in that BC#S1001 maps very close to unc-60 molecularly (unc-60 is in F53E2 pers. comm. M. Wakarchuk) but genetically lin-40 is fairly far away (roughly 9 m.u.) to the right of unc-60. We are attempting to rescue lin-40 by microinjection using appropriate cosmids. We are also screening for revertants of some of the Tc1 alleles of lin-40. This research has been supported by an MRC studentship to RCJ and NSERC and MDA (Canada) grants to DLB. [See Figure 1]

Jones SJM et al. (1992) Worm Breeder's Gazette "The Characterization of the let-653 Gene in the NematodeCaenorhabditis elegans"

Baillie DL, Jones SJM, Clark DV

[Worm Breeder's Gazette, 1992]

The gene let-653 has been previously mapped and found to lie >0.05 map units from unc-22 and to the right of dpy-20 on linkage group IV (Clark 1992). Two mutant alleles have been isolated, one of which causes developmental arrest at the end of the larval stage L1 .The other allele causes developmental arrest within the larval stage L2 .The developmental arrest occurs just after the appearance of a large vacuole just close to the pharynx. The appearance of the vacuole is consistent with the absence or loss function of the excretory cell or of its pore cell. Although it has been previously thought that the role of this excretory apparatus is one predominantly of osmoregulation, altering the salt concentration of the worm media does not suppress lethality nor the appearance of the vacuole. Germline transformation experiments have demonstrated that the cosmid C29E6 can rescue the lethal phenotype and therefore contains a functional copy of the let-653 gene. The cosmids which overlap C29E6 ,namely B0033 ,C11F2 and C46F3 ,are currently being used in germline transformation experiments; the co-injection of a plasmid bearing the dominant rol-6 mutant allele su1006 acting as positive marker for the injected DNA. This will allow us to define more accurately the position of the let-653 gene.