Worm Breeder's Gazette 8(1): 50
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.
Using techniques developed by Judith Kimble (1), we have been microinjecting supercoiled plasmid DNA into the gonad distal arm of young adult hermaphrodites. To detect the inheritance of exogenous DNA, progeny of the injected worm are grown for approximately three generations and then harvested. Their DNA is extracted and tested for the presence of the injected sequences by hybridization to the plasmid DNA. To date 11 of 119 injections have shown evidence for the inheritance of foreign DNA. The organization of foreign DNA in worm transformants has been examined by restriction analysis. The exogenous DNA is present as high molecular weight DNA at high copy number (20-100 copies per transformed worm) and arranged predominantly as a head-to-tail tandem array. In one transformant injected with YRp17-Cell0, a plasmid containing sequences with worm homology, the exogenous DNA appears to have integrated into the worm genome by homologous recombination. Two independent transformants containing DNA with no extensive homology to the worm have been tested for the stability of foreign sequences during propagation. The exogenous DNA is lost in these transformants at a fairly high rate. For a majority of subclones of a transformant, only 50% of the progeny in the next generation still contain foreign DNA. By restriction analysis, the foreign DNA in all the subclones of a single transformant appears to be identical high molecular weight concatamers. Thus, the exogenous DNA appears to segregate as a single unit that is not undergoing rearrangements at a high frequency. The high instability of this unit suggests that either the foreign DNA imparts a deleterious phenotype to the worms, or the concatamer is extrachromosomal and is being lost during mitosis or meiosis. We are presently investigating these alternatives. Recently several subclones have shown high stabilities during propagation. We are now trying to determine the cause of this increase in stability. To assess the frequency of homologous recombination of injected plasmid sequences into the worm genome, we have injected both supercoiled and linear plasmids containing portions of the unc-54 gene. Homologous integration of these DNA sequences should result in the production of an unc-54 mutation by disruption of the endogenous gene. We have yet to demonstrate that exogenous DNA can be expressed. Plasmids containing the wild-type unc-54 gene or the amber-suppressing sup-7 allele have been injected to assess complementation of unc-54 mutants or suppression of amber mutants at several loci. In addition, we are constructing hybrid genes that could provide a selection or a visible screen for transformed worms. We have recently injected a - glucuronidase negative worm with a vector carrying the E. coli - glucuronidase gene fused to the col-1 5'- and 3'- sequences. Transformants containing this vector are being tested for - glucuronidase activity. Transformants containing a similar vector carrying the bacterial structural gene for neomycin phosphotransferase are being tested for resistance to the drug G418. Dr. Hans-Borje Jansson, Univ. of Lund, Sweden joined my program in July, 1983. He is supported by a Swedish National Science Foundation Fellowship and a Fulbirght Award. Concurrently Dr. A. Jeyaprakash of India joined us. We are working on trying to characterize surface carbohydrates on C. elegans and attempting to intervene in chemotactic behavior using various experimental manipulations. Bert M. Zuckerman, Professor