Worm Breeder's Gazette 11(1): 20

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.

YAC Rescue of par-2!

D. Levitan, C. Mello and D. Stinchcomb

We have been trying to rescue par-2 mutants using available cloned 
DNA for some time.  The single cosmid which was identified by 
hybridization to flanking sequences of a Tc1 insertion in par-2 failed 
to complement par-2(it5ts).  In addition, an overlapping phage failed 
to rescue the mutant.  We subsequently learned both the cosmid and 
phage had small deletions in the region thought to contain par-2.  The 
only other cloned DNA from this region was the ~80 kb YAC, Y11F11.
We inserted the dominant rol-6 gene into Y11F11, by recombination in 
yeast, in order to have a marker which would identify successful 
injections.  We transformed Y11F11 with a Rol-6 LYS-2 URA-3 vector and 
selected for colonies which could grow on lys- media.  Hybridization 
analysis revealed the vector had integrated into the YAC.  We enriched 
for this YAC by running gently lysed spheroplasts over two sucrose 
gradients and analyzed fractions by hybridization to YAC sequences.  
Appropriate fractions were dialyzed, dried, and injected into N2 and 
par-2(it5ts) animals using the technique of C.  Mello (WBG, this issue)
We obtained one line of transformed rollers out of ~60 injections 
into N2.  This encouraged us that the rol-6 gene would function from 
YAC DNA prepared in this manner.  This line has not been tested for 
the ability to complement par-2.  We then injected par-2(it5ts) 
animals with the same DNA.  Since par-2 is a maternal-effect gene 
whose gene product is required prior to first cleavage (N.  Cheng and 
K.  Kemphues), we placed injected animals at the permissive 
temperature (16 C) to allow the injected DNA time to be expressed.  
When the F1 progeny of the injected animals were at the L3-L4 stage, 
we shifted them to restrictive temperature (25 C), and examined the 
plates for the presence of F2 larva instead of dead eggs.  One out of 
42 injected animals produced F2 progeny at 25 C; some of these F2 
progeny were right rollers.  This transformed line continues to grow 
quite well at restrictive temperature and behaves as if it contains an 
extrachromosomal array--about 20% of the brood produce progeny and the 
remaining 80% are either sterile (a phenotype associated with par-2(
it5ts)) or lay dead eggs.  Non-transformed par-2(it5ts) cannot be 
maintained at 25 C.  We are isolating DNA from these strains in order 
to identify which sequences from this YAC are responsible for the 
rescue.  In addition, we are interested in determining the copy number 
and structure of the transforming DNA.