Worm Breeder's Gazette 8(2): 47
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.
In the last newsletter we reported on a HindIII Tc1 polymorphism, now called stP1, that is 0.5 to 1.0 map unit left of unc-22 on the genetic map. We cloned this polymorphic site into lambda 590 and then subcloned a HindIII/EcoRV adjacent unique DNA segment into pBR322. Using this unique DNA sequence as a probe we found that the stP1 site is empty in the parental Bristol and Bergerac strains, but is occupied by Tc1 in 2 independent unc-22 mutant derivative strains, RW7002 and RW7008 (see last newsletter for strain descriptions). As well, while outcrossing RW7002 to Bristol males to establish the RW7012 strain, a second site, stP2, became occupied by Tc1 and the entire region underwent duplication. The stP2 polymorphism is also present in RW7008 but in this case we do not know the order of occurrence of stP1 and stP2. These observations indicate that Tc1 can transpose in Bergerac or Bergerac/Bristol hybrids. In a third unc-22 mutant strain neither polymorphism is present so we do not think stP1, or stP2, has any causative role in regard to Bergerac spontaneous twitchers. However, we have now identified a third Tc1 polymorphism, called stP3, in 2 independently derived strains which is solely associated with the mutated state of the unc- 22 locus. This Tc1 polymorphism is not genetically separable from these unc-22 mutations even by intragenic mapping. Both mutant strains have Tc1 in the same small (2kb) BglII fragment. We have now examined five intragenic revertants of one strain and all five lack stP3. To investigate the relationship of stP3 to unc-22 we have cloned the stP3 BglII fragment into pBR322 and subcloned a unique 1kb EcoRV fragment from this region. Rehybridization of our Southern's with this unique piece of DNA confirms our earlier result that stP3 is strictly associated with the mutated state of unc-22. Our preliminary conclusion is that this site is part of the unc-22 locus itself. We have used this 1kb fragment to isolate lambda clones from our lambda 1059-N2 library and are using these clones as probes against known deficiencies for the region. A dosage response, or the identification of a fusion fragment due to a deficiency breakpoint would be an independent confirmation that we have molecularly cloned unc-22.A few further comments: Our Bergerac spontaneous twitchers are weak alleles when compared to s32, an unc-22 amber allele. We think there are two possible explanations for this observation, neither of which can be ruled out at the present time. One possibility is that Tc1 may have preference for an intron in unc-22. However, it is also possible that somatic excision of Tc1 may allow enough transcription of unc-22 to allow these mutants to mimic leaky EMS alleles like e105, or s12. The origin of stP1 in RW7002 is clearly by transposition, but its mode of occurrence in RW7008 is unclear. It could be due to transposition, or possibly recombination between the Bristol and RW7002 fourth chromosomes when the RW7008 parental strain was constructed. The 2 stP3 events although independent, are selected transposition events, because they occur simultaneously with the twitching phenotype. The 2 stP2 events, in RW7008,and RW7012, are definitely independent events which have occurred in the same restriction fragment and these events were unselected. This independent recovery of unselected insertion events into the same restriction fragment suggests Tc1 may have strong target preference. Although we have found that germline excision of Tc1 is sensitive to genetic background, i.e. our spontaneous twitchers revert in a Bergerac background, but are stable in a predominantly Bristol background, this is not true for somatic excision of Tc1. The stP1, stP2, and stP3 Tc1 polymorphisms have similar somatic excision frequencies in Bristol and Bergerac. This suggests that excision in these tissue types is controlled differently. Finally, the observation of somatic excision of stP3 led us to examine the muscle cells in our mutants for mosaics. We have used phalloidin-rhodamine staining of F-actin as well as polarized light microscopy to examine cells. To date we have found no evidence for mosaicism but the screen is difficult because of the relatively mild structural disorder of the muscle cells in these animals. Of course, we also do not know whether unc-22 is cell autonomous.