Worm Breeder's Gazette 8(1): 49

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.

Attempts at Transformation

D. Eide, J. Kimble, P. Anderson

Using the unc-54 major myosin heavy chain gene as a scorable genetic 
marker, we are trying to develop a system for DNA transformation. We 
have cloned a 14 kb restriction fragment containing a wild-type copy 
of the unc-54 gene into the yeast replicative plasmid YRp7. The unc-54 
restriction fragment is derived from genomic DNA and contains the 
entire coding portion of unc-54 plus approximately 3 kb 'downstream' 
from the mRNA 3'-terminus and 'upstream' from the AUG translation 
initiation codon. The vector, YRp7, is capable of autonomous 
replication in yeast; we hoped that it would behave similiarly in C. 
We micro-injected this plasmid (YRp7(unc-54)) into a 
distal arm of the gonad of unc-54(el301);nuc-1(el392) hermaphrodites. 
el301 is a recessive, temperature-sensitive allele of unc-54; el301 
animals are wild-type when grown at 17 but are paralyzed when grown at 
25  Animals to be injected were grown at 17 and were therefore 
phenotypically wild-type. We successfully injected fourteen 
hermaphrodites; in each case, we verified that the injected gonad arm 
continued to produce zygotes. We grew the Fl progeny at a permissive 
temperature, and shifted the F2 progeny to 25 as eggs. We screened the 
F2 and F3 progeny for phenotypic transformants. No animals were 
isolated that showed a stably-inherited improvement in motility. We 
grew the populations for an additional generationr extracted DNA, and 
analyzed it by Southern blot hybridization.
Most DNA samples contain no detectable traces of the injected 
plasmid. However, DNA prepared from two populations are exceptional: 
each contains sequences homologous to YRp7. We do not fully understand 
the nature of these hybridizing sequences. Rearrangement of the 
injected DNA seems to have occured, since the junction fragments (they 
hybridize both to unc-54 and to YRp7 probes) are of an unexpec size. 
It is unclear whether this rearrangement occured after inject or, 
alternatively, whether a minor component of the injected plasmid DNA 
was preferentially replicated. We shall continue to analyze the nature 
of these 'transformants'. We are also cloning the unc-54 gene into a 
variety of other molecules that we hope will prove to be useful 
transformation vectors for c.