Somatic Excision of Transposable Element Tcl from the Bristol Genome of Caenorhabditis elegans

Transcription

1 MOLECULAR AND CELLULAR BIOLOGY, May 1986, p Vol. 6, No /86/ $02.00/0 Copyright 1986, American Society for Microbiology Somatic Excision of Transposable Element Tcl from the Bristol Genome of Caenorhabditis elegans L. J. HARRIS AND A. M. ROSE* Department of Medical Genetics, University of British Columbia, Vancouver, British Columbia, Canada V6T I WS Received 23 October 1985/Accepted 18 February 1986 We investigated the ability of the transposable element Tcl to excise from the genome of the nematode Caenorhabditis elegans var. Bristol N2. Our results show that in the standard lab strain (Bristol), Tcl 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 Tcls situated in the same location in both strains. Evidence of high-frequency excision from the genomes of both strains was observed. The Tcls 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, Tcl(Hin). The ability of this BO Tcl(Hin) to excise was retained after its introduction into the N2 genome. Thus, we conclude that excision of Tcl from the Bristol genome occurs at a high frequency and is comparable to that of Tcl excision from the Bergerac genome. We showed that many Tcl elements in N2 were apparently functionally intact and were capable of somatic excision. Even so, N2 Tcls were prevented from exhibiting the high level of heritable transposition displayed by BO elements. We suggest that Bristol Tcl elements have the ability to transpose but that transposition is heavily repressed in the gonadal tissue. The transposable element Tcl exists in the genome of the nematode Caenorhabditis elegans and exhibits a high level of transposition in the Bergerac strain BO (5, 17). This is in contrast to other C. elegans strains, including the common lab strain Bristol (N2). Tcl has been described as a 1,610- base-pair (bp) transposable element with 54-bp perfect inverted repeat termini (8, 13, 22). The behavior of Tcl is very different in the N2 and BO strains. The BO strain has many integrated copies of Tcl in the genome (approximately 300) (8, 13) and contains extrachromosomal copies of Tcl (21, 23). In BO, Tcl transposition occurs at a frequency of 10-4 into the unc-22 locus (17) and at a lower frequency into the unc-54 locus (5). In contrast, N2 has about 30 copies of Tcl (8, 13) and no detectable extrachromosomal forms (21). No N2 Tcls have been demonstrated to possess the ability to transpose into either the unc-54 (4) or unc-22 (<10-7) (17) loci. High-frequency excision of Tcl in the somatic cells of the BO strain has been described previously (7). In this paper, we have studied the ability of Tcl to excise in the N2 strain. We were curious as to whether this trait would also differ between the strains to the great extent that transposition and the presence of closed circular forms do. Two approaches have been taken. To determine whether N2 Tcls retained the ability to excise, Tcls from the N2 strain were cloned. Since some N2 sites are also occupied in BO, it was possible to compare the relative amount of excision of one Tcl in the two strains. Secondly, to test whether excision is a property intrinsic to the element or to the genetic background, the ability of a BO element to excise when transferred to a N2 genome was studied. The latter experiment was performed with the BO element Tcl(Hin) (20). Tcl(Hin) was discovered during the investigation of a restriction fragment length difference (RFLD) which mapped close to dpy-5 on linkage group I (19). This Tcl insertion generated the RFLD spi and has been shown to have a HindIII site not * Corresponding author present in the previously reported Tcl sequence. Tcl(Hin) excises somatically in BO (19), as do other BO Tcls (7). We have studied the excision of Tcl(Hin) from the Bristol genome by genetically crossing this BO Tcl into N2. MATERIALS AND METHODS DNA isolation. C. elegans DNA was prepared by a method modified (19) from that of Emmons et al. (6) and was collected after centrifugation in a cesium chloride gradient in the presence of ethidium bromide (19). Sodium chloride and water-saturated isopropanol were used to remove the ethidium bromide. DNA was precipitated in the presence of 2 volumes of water and 6 volumes of ethanol at -20 C (3). DNA was stored in 10 mm Tris (ph 7.5)-i mm EDTA at 4 C. Plasmid DNA was prepared from transformed Escherichia coli JM83 [ara A(lac-pro) rpsl thi 480d lacz AM15] (16) after chloramphenicol amplification by the modified cleared lysate procedure (11). CsCl density gradients were used to band plasmid DNA. Restriction digests, electrophoresis, and bidirectional transfer. Restriction digests were carried out under conditions recommended by the enzyme suppliers, Bethesda Research Laboratories and Pharmacia, Inc. Restricted DNA was electrophoresed in horizontal agarose gels, and DNA sizes were estimated by comparison with EcoRI-HindIII digests of lambda ci857 Sam7 DNA. DNA fragments were transferred to nitrocellulose by the bidirectional method of Smith and Summers (24). Genomic hybridization and DNA probes. DNA probes were isolated through electrophoresis and electroelution (14) of restricted plasmid fragments. The unique flanking DNA of the N2 Tcl was cut from a Tcl-deleted (described below) plasmid, pceh29, by using EcoRI. The probe for the spi site [BO Tcl(Hin) LGI1 was a 1.6-kilobase (kb) EcoRI-HindIII fragment from pcesl8 (19). a-32p-labeled probe DNA was prepared by nick translation (3). [a-32p]dxtp was purchased from New England Nuclear Corp.

2 VOL. 6, 1986 N2 BO >-.-.4 FIG. 1. Autoradiograph of Tcl excision in N2. EcoRI-digested DNA from N2 (left lane) and BO (right lane) hybridized with the 1.6-kb EcoRI insert from pceh29. This fragment contains unique sequences flanking the N2 Tcl. The parent band is at 3.2 kb, and the somatic excision band is at 1.6 kb. The N2 lane contains more DNA than the BO lane (4 and 3.5 plg, respectively). Size markers from EcoRI-HindIII-digested c1857 Sam7 DNA are shown ( ) at 3.5, 2.0, and 1.6 kb. The band at 2.7 kb is due to low-level puc19 contamination. Tcl SOMATIC EXCISION IN BRISTOL 1783 Nitrocellulose filters were pretreated at the hybridization temperature in 5x SSPE (lx SSPE is 0.18 M NaCl, 0.01 M Na phosphate, and 1 mm EDTA; ph 7.0) and 0.3% sodium dodecyl sulfate. Hybridizations were carried out for at least 20 h at 62 C. Filters were washed extensively at 62 C in 2 x SSPE-0.2% sodium dodecyl sulfate. Library screening. A Charon 4 EcoRI partial digest N2 genomic library, a gift of T. Snutch, was screened with an EcoRV Tcl fragment probe (the inverted terminal repeats of Tcl each contain one EcoRV site 19 bp from the end). Individual phage were isolated and purified by CsCl centrifugation (3). Subeloning. EcoRI restriction fragments from the phage containing Tcl-homologous sequences were subcloned into the vector puc19 (25) and transformed into E. coli JM83. To obtain a unique N2 Tcl flanking sequence probe, the Tclcontaining subclone pceh4 was cleaved with EcoRV and religated, eliminating the central 1,570 bp of Tcl (1) and leaving a 1.6-kb insert of unique flanking sequence. C. elegans strains and culture conditions. C. elegans var. Bristol, strain N2, was obtained from D. Baillie, Simon Fraser University, Burnaby, British Columbia, Canada, and C. elegans var. Bergerac, strain BO, was from D. Hirsh, University of Colorado, Boulder. Some mutant strains used in this study were obtained from S. Brenner, Medical Research Council, Cambridge, England, and the Caenorhabditis Genetics Center, Columbia. Mo. C. elegans was cultured by using nematode growth medium on plates streaked with E. coli OP50 (2). Plates which were used to grow strains for DNA isolation were streaked with an E. coli strain (K-12 thi). RESULTS Excision of an N2 Tcl. To examine the ability of N2 Tcls to excise, we have cloned Tcls from N2. A Charon 4 library of N2 genomic DNA was screened with a BO Tcl probe to obtain N2 Tcl family members. These Tcl-containing phage were digested with EcoRI and subcloned into puc19. Subsequent examination of the subcloned inserts containing Tcl elements showed that most (12 of 15) of these Tcl members were 1.6 kb in length and identical in restriction map to each other and to the published sequence (22). The remaining Tcls (3 of 15) represented variant forms (20) which are currently being characterized (unpublished data). One of the flanking sequences from the plasmid pceh29 hybridized to a single EcoRI fragment of 3.2 kb in both N2 and BO. Figure 1 shows EcoRI-digested genomic DNAs from the BO and N2 strains probed with the pceh29 insert. A minor band can be observed 1.6 kb below the parent band in both N2 and BO and represents excision. Bergerac Tcl excision from the Bristol genome. In the previous experiment, the excision of a Tcl integrated at the same location in the N2 and BO genomes was examined. Although the elements studied were in the same location, there could be structural differences between the N2 and BO elements that would affect their ability to excise. To examine the behavior of the same Tcl in these two strains, the strain KR324 was constructed. Three genetic markers (dpy-5, dpy-14, and unc-13) closely linked to Tcl(Hin) at the spi site were used to recombine the region of BO linkage group I (LGI) that contained Tcl(Hin) into the N2 genome. Two- and three-factor crosses were carried out, followed by extensive backcrossing to N2. The right half of LGI from BO was introduced into the N2 genome (Fig. 2). BO hermaphrodites were mated to dpy- S(e61) dpy-14(el88)1+ + N2 males. The Fl wild-type hermaphrodites were allowed to self-cross, and those demonstrated to be dpy-5 dpy N2-BO hybrids by the progeny they produced also gave the expected Dpy recombinants. Dpy recombinants (dpy-s +Idpy-S dpy-14) were isolated and self-crossed to yield a strain homozygous for the recombinant chromosome. In this way, 11 independent Dpy-5 recombinants were isolated. The portion of LGI that was BO and to the right of unc-13 was eliminated (Fig. 3). One Dpy recombinant was used to establish the strain KR223. KR223 hermaphrodites were crossed to dpy- 14(e188) unc-13(e450)1+ + N2 males. Homozygous Dpy-5 Unc-13 recombinants were generated in a manner similar to that described above for Dpy-5 recombinants. Genomic blots were probed with unique sequence DNA from the spi site to confirm Tcl(Hin) from BO was present. Successive backcrosses to N2 were performed to dilute the BO component of the genome. One Dpy-5 Unc-13 strain containing the spi RFLD was picked and backcrossed to N2 males. After each cross, the hermaphrodites were allowed to self-cross to ensure that they were homozygous. This procedure was repeated seven times to yield the strain KR324. The constructed strain, KR324, contained a chromosome I consisting of BO genome for less than 2 map units (m.u.) between dpy-5 and unc-13 and N2 DNA for the remainder. The remaining chromosomes are predicted to be greater than 99% N2 (0.59) since each backcross of a N2-BO hyrid strain to N2 will reduce the remaining BO component by an estimated 50%. We expected to see one to two BO Tcls

3 1784 HARRIS AND ROSE B A'WWWWv%&vvw \V4Av\ BO BO t3oo Tcls/genome dpy-5 dpy TcIs/genome + N2 + N2 d' MOL. CELL. BIOL B _WVWW BO < dpy-5 dpy-14 + N2 Wildtype: Dpy-5 Dpy44 and Dpy recombinants 3:1 1.5% Dpy-5 recombinants picked FIG. 2. Step 1 in the protocol for crossing the spi site from BO DNA (---) into N2 DNA (.). Due to the tight linkage between dpy-5 and dpy-14 (1.5%), the heterozygous dpy-5 dpy parent is expected to give an overall progeny distribution of three wild types to one Dpy Dpy. remaining in KR324. Genomic hybridizations were carried out to determine whether the BO component had been reduced by the predicted amount. KR324 and N2 EcoRIdigested genomic DNAs were hybridized with a Tcl probe (Fig. 4). Seven additional BO bands were observed. One N2 Tcl was missing in the KR324 DNA. The excision of Tcl(Hin) from the spi site in KR324 was examined and compared with the same event in the BO and N2 strains (Fig. 5). Excision of Tcl(Hin) can be seen in the KR324 strain (Fig. 5, lane 3) at a rate comparable to that in BO (lane 2), although somewhat reduced. C14 -o Z 0. DISCUSSION We have investigated the ability of Tcl to excise from the genome of C. elegans var. Bristol. Previously, Emmons and associates have shown that Tcl elements within the Bergerac strain excise at a high frequency and that this excision occurs predominantly in somatic cells (7). In this Tcl(Hin) -5.3Kb dpy ^mA"VvMvw dpy dpy-14 unc-13 N2 xd + + N2 ^ _4.3Kb -3.4Kb + + dpy-14 unc-13 Dpy Unc-13 recombinants picked and Dpy-5 Unc-13 homozygotes saved dpy-5 + unc-13 dpy-5 + unc-13 FIG. 3. Step 2 in the protocol for crossing the spi site from BO into N2. Symbols: -, N2 DNA;, BO DNA. FIG. 4. Autoradiograph of Tcls in the KR324 strain. EcoRIdigested KR324 (labeled dpy-14 on the figure, as this is the gene the BO sequences are centered around) and N2 DNA hybridized with a 1.6-kb EcoRV Tcl fragment. Tcl bands not common to both strains are marked. Symbols: -*, Tcl band present in KR324 but not N2; +-, Tcl present in N2 but not in KR324.

4 VOL. 6, FIG. 5. Autoradiograph of Tcl(Hin) excision in N2. EcoRIdigested genomic DNA from N2 (lane 1), BO (lane 2), and KR324 (lane 3) probed with the 1.6-kb EcoRI-HindIII fragment from pcesl8 (19). The spi RFLD is shown by the 5.5-kb band in N2 compared with a 7.2-kb band in BO. The minor band in BO and KR324 at 5.5 kb is a result of somatic excision. A 4-p.g sample of DNA was loaded into each lane. paper, we have shown that, in the standard lab strain (Bristol), Tcl excision occurred at a frequency comparable to that in the Bergerac strain. Two approaches were taken. In the first approach, the excision of an element found to be present in the same location in both strains was examined. To do this, individual Tcl elements were cloned from a N2 DNA library. One element (identified by the flanking sequence pceh29) which was found to be present at the same site in N2 and BO enabled us to compare excision of Tcl in the two strains. Our results showed clearly minor bands for both strains in the position expected if they had resulted from Tcl excision (Fig. 1). The Tcls 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, Tcl(Hin), previously described (20). This Tcl which creates the restriction fragment length difference spi was genetically inserted into a N2 LGI by using flanking markers. Tcl(Hin) was also observed to undergo germline excision (unpublished data). The ability of this BO Tcl(Hin) to excise was retained after its introduction into the N2 genome. Some reduction in detectable levels of excision was observed, but this reduction was no more than two- to threefold (Fig. 5). Thus, we conclude that excision of Tcl from the Bristol genome occurred at a high frequency comparable to the frequency of Tcl excision from the Bergerac genome. We assume that this high-frequency excision occurs in the somatic tissue of the Bristol strain, as it does in the Bergerac strain (7). The Bristol genome has 10-fold fewer Tcls than the Bergerac genome, and yet excision was clearly not 10-fold reduced. Thus, somatic excision from the Bristol genome is not dependent on Tcl copy number. Previous experiments by other researchers have shown that Tcl in the Bristol genome is inactive. Liao et al. (13) examined N2 laboratory strains and found no differences in Tcl number and location despite 500 generations of separation. Rose and Snutch (21) were unable to detect in Bristol the extrachromosomal closed circular copies of Tcl that have been detected in BO (21, 23). Eide and Anderson (4) Tcl SOMATIC EXCISION IN BRISTOL 1785 examined 65 spontaneous mutations in N2 at the unc-54 locus, and none were the result of Tcl insertion, in contrast to the 10 of 18 in BO which did result from Tcl insertion. The data presented in this paper may imply that Bristol Tcls are capable of germline excision and transposition. Emmons and Yesner have proposed that excision of Tcl may occur as the first step of the transposition pathway (7). In our laboratory, currently two N2 laboratory strains with altered Tcl patterns have been observed (B. Rattray and J. M. Babity, unpublished data). These results, along with the results described in this paper, encourage us to suggest that Bristol Tcls are capable of somatic and germline tranposition, albeit at a normally low frequency. The frequency of somatic excision from the Bristol genome which we have observed is much higher than the frequency of germline excision from the Bristol strain observed by Moerman and Waterston (17). They have demonstrated that heritable excision from the unc-22 locus is reduced by two or more orders of magnitude in the Bristol genome. For those Tcls which have transposed into the unc-22 locus, the independence of somatic excision with respect to genetic background has been described previously (D. G. Moerman, G. M. Benian, and R. H. Waterston, Proc. Natl. Acad. Sci. USA, in press). In maize (10, 15) and Drosophila melanogaster (9), transposable elements are capable of somatic or germline excision. Recent studies on the Drosophila P element transposase have shown that removal of an intron in the germline is the molecular basis for germline specificity of P element transposition (12). P elements, in constrast to Tcl, excise at high levels in the germline, but not in somatic tissue (9). This raises the question of why high-frequency somatic excision of Tcl from the C. elegans genome occurs. One possibility is that a mechanism exists for the transfer of excised somatic elements to the germline. In the course of our experiments, we have identified six additional BO Tcls within 1.5 m.u. around dpy-14(lgi). If the approximately 300 BO Tcls are assumed to be distributed randomly throughout the 300 m.u. of the C. elegans genome, we would have expected less than two BO Tcl members in the 1.5 m.u. between dpy-5 and unc-13. Our results may imply that there are more than 300 Tcls in BO or that the distribution of Tcls is nonrandom with regard to the genetic map. Baillie and associates isolated six BO Tcls in a 1-m.u. interval to the left of unc-22 compared with one Tcl in the adjacent m.u. to the right (1). The m.u. to the left is gene dense (30 identified genes) in contrast to the m.u. to the right which is gene poor (3 genes) (18). The dpy-14 region is within a gene dense region of LGI (2) and has been predicted on the basis of radiation-induced map expansion to contain more DNA per m.u. than the average for the genome (J. S. Kim, M. S. thesis, University of British Columbia, Vancouver, British Columbia, Canada, 1985). Thus, there are possibly more sites available for Tcl occupation. In conclusion, we have shown that many Tcl elements in N2 are apparently structurally intact (by restriction mapping) as well as functionally intact as assayed by somatic excision. Despite this, N2 Tcls are prevented from exhibiting the high level of heritable transposition displayed by BO elements. We suggest that Bristol Tcl elements have the ability to transpose but are heavily repressed in the gonadal tissue. ACKNOWLEDGMENTS We thank D. L. Baillie for encouragement and discussion, N. Mawji for technical advice, and our reviewers for helpful comments.

5 1786 HARRIS AND ROSE This research was supported by grants to A.M.R. from the Medical Research Council of Canada and the National Cancer Institute of Canada. L.J.H. is supported by a studentship from the Medical Research Council of Canada. Some of the strains used were provided by the Caenorhabditis Genetics Center, which is supported by contract NO1-AG between the National Institutes of Health and the Curators of the University of Missouri. LITERATURE CITED 1. Baillie, D. L., K. A. Beckenbach, and A. M. Rose Cloning within the unc43 to unc-31 interval (linkage group IV) of the Caenorhabditis elegans genome using Tcl linkage selection. Can. J. Genet. Cytol. 27: Brenner, S The genetics of C. elegans. Genetics 77: Davis, R. W., D. Botstein, and J. R. Roth A manual for genetic engineering: advanced bacterial genetics. Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 4. Eide, D., and P. Anderson The gene structures of spontaneous mutations affecting a Caenorhabditis elegans myosin heavy chain gene. Genetics 109: Eide, D., and P. Anderson Transposition of Tcl in the nematode C. elegans. Proc. Natl. Acad. Sci. USA 82: Emmons, S. W., M. R. Klass, and D. Hirsh Analysis of the constancy of DNA sequences during development and evolution of the nematode C. elegans. Proc. Natl. Acad. Sci. USA 76: Emmons, S. W., and L. Yesner High-frequency excision of tranposable element Tcl in the nematode C. elegans is limited to somatic cells. Cell 36: Emmons, S. W., L. Yesner, K. Ruan, and D. Katzenberg Evidence for a transposon in C. elegans. Cell 32: Engels, W. R The P family of transposable elements in Drosophilia. Annu. Rev. Genet. 17: Fincham, J. R. S., and G. R. K. Sastry Controlling elements in maize. Annu. Rev. Genet. 8: Kahn, M., R. Kolter, C. Thomas, D. Figurski, R. Meyer, E. Remaut, and D. R. Helinski Plasmid cloning vehicles derived from plasmids ColEl, F, R6K, and RK2. Methods Enzymol. 68: Laski, F. A., Rio, D. C., and G. M. Rubin Tissue specificity of Drosophila P element transposition is regulated at MOL. CELL. BIOL. the level of mrna splicing. Cell 44: Liao, L. W., B. Rosenzweig, and D. Hirsh Analysis of a transposable element in C. elegans. Proc. Natl. Acad. Sci. USA 80: Maniatis, T., E. F. Fritsch, and J. Sambrook Molecular cloning: a laboratory manual, p Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y. 15. McClintock, B The control of gene action in maize. Brookhaven Symp. Quant. Biol. 18: Messing, J A multipurpose cloning system based on the single-stranded DNA bacteriophage M13. Recombinant DNA technical bulletin, National Institutes of Health publication no , no. 2, p National Institutes of Health, Bethesda, Md. 17. Moerman, D. G., and R. H. Waterston Spontaneous unstable unc-22 IV mutations in C. elegans var. Bergerac. Genetics 108: Rogalski, T. R., and D. L. Baillie Genetic organization of the unc-22 IV region of Caenorhabditis elegans. Mol. Gen. Genet. 201: Rose, A. M., D. L. Baillie, E. P. M. Candido, K. A. Beckenbach, and D. Nelson The linkage mapping of cloned restriction fragment length differences in C. elegans. Mol. Gen. Genet. 188: Rose, A. M., L. J. Harris, N. R. Mawji, and W. J. Morris Tcl(Hin), a form of the transposable element Tcl found in C. elegans. Can. J. Biochem. Cell. Biol. 63: Rose, A. M., and T. P. Snutch Isolation of the closed circular form of the transposable element Tcl in C. elegans. Nature (London) 311: Rosenzweig, B., L. W. Liao, and D. Hirsh Sequence of the C. elegans transposable element Tcl. Nucleic Acids Res. 11: Ruan, K., and S. W. Emmons Extrachromosomal copies of transposon Tcl in the nematode C. elegans. Proc. Natl. Acad. Sci. USA 81: Smith, G. E., and M. D. Summers The bidirectional transfer of DNA and RNA to nitrocellulose or DBM paper. Anal. Biochem. 109: Yanisch-Perron, C., J. Vieira, and J. Messing Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mpl8 and puc19 vectors. Gene 33: