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.

A Transposon in C. elegans

S. Emmons, L. Yesner, K. Ruan, S. Roberts

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.