Worm Breeder's Gazette 11(3): 14
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.
The ability to specifically engineer free duplications could have several applications, particularly to mosaic analysis. For analysis of a gene which has already been cloned, the gene could be introduced onto any preexisting Dp containing convenient markers for mosaics. Alternately, a marker construct (such as a -galactosidase fusion) could be put onto a duplication which already contains the gene of interest. Because each duplication contains only a small percentage of the DNA present in the genome, it might be expected that insertion of foreign sequences into Dps would be inefficient and difficult. We wished to investigate the possibility that because of their extrachromosomal nature, free duplications would actually be a preferred target for insertion of foreign DNA. Animals of genotype unc-3 daf-6 lin-15 sup-10(n983); mnDp14 were injected with a mixture of two plasmids: pPD10.46 (an unc-22 antisense plasmid conferring a [dominant] twitching phenotype when present at high copy number, see WBG10#2 p89) and pPD20.97 (a transcriptional fusion between myo-2 [pharyngeal] and -galactosidase). Two sublines which twitch and carry mnDp14 were derived. In both cases the twitching phenotype segregated as a dominant extrachromosomal element unlinked to mnDp14. We selected one line for further study, designated SP1342. As expected, this initial transformant carries both pPD10.46 and pPD20.97 DNA and shows intense staining for - galactosidase in the pharynx. If the extrachromosomal array containing the pPD10.46 construct were to associate with mnDp14 then we would expect to get a strain in which all non-Unc-3 animals were twitchers (i.e., all animals with the composite Dp would twitch). From 270 animals, no example of spontaneous association between the two extrachromosomal elements was found. We then irradiated SP1342 adults with about 3800 rads of gamma radiation [137Cs] and picked 200 animals from the first generation after irradiation. In three of these F1 picks, the twitching phenotype had become linked to mnDp14. In each case the new duplication still carries both lin-15+ and sup- 10+.Chromosomal integration cannot readily be screened in the F1 following irradiation. In a screen of 25 F2 progeny of gamma- irradiated worms, one example of autosomal integration by the transgene array was found. We were surprised at the high frequency of association between the two extrachromosomal elements (Dp and array) following irradiation. Although unproven, it seems likely that the Dp is a preferential target for association with the array. In any case this technique could prove valuable in creating designer duplications.