Worm Breeder's Gazette 7(2): 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 have isolated and studied the properties of the DNA sequence responsible for the Bristol-Bergerac polymorphisms discovered in our earlier work (Emmons et al., PNAS 76, 1333, 1979). We have found that these polymorphisms arise because the Bergerac genome contains about 200 copies of a 1.7kb transposon inserted at dispersed sites in the DNA, whereas the Bristol genome contains only about 20 copies of the same sequence. We conclude that this sequence is a transposon from the fact that at each site of insertion, in both Bristol and Bergerac, the 1.7kb segments have precisely the same organization. The insertion events have therefore been site-specific with respect to the inserted DNA, but not (highly) site-specific with respect to the target, and it is this property which is characteristic of the transposons that have been studied in other organisms such as bacteria, Drosophila, and yeast. By analogy with the nomenclature adopted in yeast (Ty1), we have named the 1.7kb DNA element Tc1. The arrangement of Tc1 is (probably) different in various wild C. elegans strains we have looked at (see following report). We have further found that Tc1 is not stably inserted, but appears to excise rapidly from three sites of insertion in Bergerac we have studied, lending further support to the conclusion that this is a motile DNA sequence. We isolated Tc1 by screening a Bergerac clone bank in lambda 1059 with a Bristol BamHI fragment that hybridized to polymorphic fragments on a genomic Southern. Similar experiments were carried out in David Hirsh's laboratory using a Dictyosteleum discoideum actin probe ( Newsletter Vol. 7, p.51). So far by several criteria the two inserts isolated by Hirsh's group and by us appear to be identical. The conclusions that Tc1 has a conserved organization and is found dispersed among diverse genomic sequences come from an analysis of Southern hybridizations of genomic DNA using the cloned Tc1 as a probe. When enzymes that do not cut within Tc1 are used a smear of hybridization is seen in Bergerac and a series of bands in Bristol. When enzymes that cut within Tc1 are used, bands due to internal fragments and smears due to the variable external sites are seen, and all the patterns are consistent with the conclusion that every genomic element is 1.7kb in length and has the same internal restriction sites as our cloned copy. By measuring the intensity of hybridization to the internal fragments we concluded that Bergerac has around 200 copies of Tc1 and Bristol around 20. We observe excision of Tc1 when non-Tc1 sequences lying on either side of a site of Tc1 insertion are used as a probe in a genomic Southern. In this case the polymorphic Bristol and Bergerac restriction fragments at the insertion site are seen, the Bergerac fragment being 1.7 kb larger than the Bristol one. In all Bergerac DNA preparations we have examined, we see in addition a small amount of the smaller fragment lacking Tc1. This is true for probes specific for three different Tc1 insertion sites, indicating the excision is likely to be a property of the element and not of the site. To find out how rapidly the excision occurs, we examined separately 8 DNA preparations from grandchildren and great grandchildren of 8 single Bergerac worms. All 8 show the same low level of excision, which we estimate to be about 1%. Excision is therefore very rapid, probably occurring in every worm. To find out whether excision occurs in the germ line, we have been cultivating Bergerac continuously for the past 6 months. DNA from these worms will be examined for complete loss of Tc1 at individual sites. Tc1 is the most highly repeated non-ribosomal sequence in the Bergerac genome and is the only highly repeated sequence present in different amounts in Bristol and Bergerac DNA. We have shown this by hybridizing nick-translated, whole genomic DNA to Bristol and Bergerac clone banks in lambda1059., and comparing the results with those obtained using cloned Tc1 or rDNA as a probe. The only plaques that show a difference with Bristol and Bergerac genomic probes are those that hybridize as well to Tc1. The number of such plaques in the two clone banks is consistent with there being around 200 copies of Tc1 in Bergerac and 20 in Bristol. Finding this transposon has stimulated us to spend a considerable amount of time attempting to demonstrate hybrid dysgenesis in crosses between Bristol and Bergerac. The large number of copies of Tc1 in Bergerac made it seem likely that this strain should be used as the male parent in such crosses. Since Bergerac males were said to be infertile, we first mated Bristol males to Bergerac hermaphrodites. The hybrid sons of this cross were then mated to marked Bristol hermaphrodites (Dpy, E61). Most F1 progeny of this cross are normal. Hermaphrodites give normal broods with the correct proportion of Dpy worms and males are fertile. However, 3 hermaphrodite worms (out of over 100 examined) gave very abnormal broods. They segregated dead embryos in large numbers. Progeny that hatched often died before maturity, or were sterile; 20% were males, and 13% were intersexes of unstable genotype. Hermaphroditic progeny that did mature gave abnormal broods with a similar spectrum of phenotypes. We have not obtained such results with reciprocal crosses, but the numbers of worms involved is too low to draw any conclusions from this. Attempts to increase the frequency of abnormal broods by various means have not been successful. No worms with stable mutant phenotypes were obtained. These results are somewhat encouraging but not very useful so far. We are currently collecting wild strains of C. elegans and will analyze their DNA for Tc1. We are looking for a strain that has no copies at all in the hope that this will be a better maternal parent for a cross with Bergerac.