Worm Breeder's Gazette 9(1): 31
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
We used the spontaneous mutant u298 isolated from TR679 to clone at least part of the mec-3 gene by the now-standard 'transposon tagging' method used by Don Moerman to clone unc-22 and Iva Greenwald to clone lin-12. The original mutant was backcrossed to N2 several times, then crossed with unc-24 (0.7 m.u. to the left) and dov-20 (0.3 m.u. to the right). We identified a single Tc1-containing EcoRI fragment which always co-segregated with the mec-3 mutation. From an outcrossed strain, we cloned a Pst-Bam fragment containing the Tc1 insertion into the plasmid pUC8. We also constructed a restriction map of this region of the C. elegans genome. [See Figure 1] A plasmid derivative in which the Tc1 had been excised (pTU1) was used to probe Southern blots of various mutants. All mec-3::Tc1 insertions are at the same site in the same orientation. (As described above, we may only have two independent insertions.) TR679 and a revertant of one of the insertions have the same pattern as N2 for all enzymes tested. The Tc1 elements are identical to the sequenced Tc1 (Rosensweig et al. NAR 11, 4201) in their restriction map as far as we know. (The cloned Tc1 does not have a HindIII site; we don't know about the others.) We can see a faint somatic excision band at the wild-type position in digests of each Tc1 mutant. EMS induced mutations show no change in pattern. We have not yet identified a transcript from this region; nor have John Sulston and Alan Coulson identified a cosmid contig containing our clone (because the cloned DNA is too small). We are doing all the obvious things. Information about the genetic map around mec-3Positions of cosmids. Two cosmids which pick up N2/BO RFLPs were positioned relative to the markers unc-24, mec-3 and dpy-20.[See Figure 2] All 4/4 + mec-3 recombinants derived from eDF18 / + + (BO) heterozygotes had the BO pattern when probed with BO555, so this cosmid maps near or to the left of the right endpoint of eDF18. The CGBP1 cosmid was shown by Don Moerman to contain a hotspot for spontaneous Tc1 insertion. When used as a probe, this cosmid reveals a PstI polymorphism (the polymorphic bands are both >20 kb), which maps as shown, and a cross-hybridizing PstI polymorphic region which maps to the left of unc-24.A third cosmid, BO5I5, sent to us from David Baillie, maps to the right of mec-3. We have not positioned it relative to dpy-20.TR679. When either of the above cosmids is used to examine u298 strains containing this region of chr. IV from TR679, the Bristol pattern is revealed. However, in the course of outcrossing the mec-3::Tc1 insertion described above, it was apparent that there are at least 4 additional Tc1 elements between unc-24 and dpy-20 in this particular TR679 stock. All of these Tc1s are in different sized EcoRI fragments from those in analogously constructed BO/N2 hybrids containing BO DNA around mec-3.