Worm Breeder's Gazette 9(3): 82

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.

P-Granule Gene Screen

I. Schauer, B. Hall and W.B. Wood

We have used four of S. Strome's anti-P-granule monoclonal 
antibodies, L416, L271, K76, and N123, to screen a modified  gtll 
genomic library from wild-type C.  by R. 
Barstead and R. Waterston. A screen of about 8 genome equivalents 
yielded 37 positive clones that rescreened as positives with one or 
more anti-P-granule antibodies, but were negative with a control IgM. 
Although the antibodies used for the screen had been preadsorbed 
against  gt11-infected E. coli and showed little or no reactivity to E.
coli proteins on 'Western' blots or on immunoblots of negative 
plaques, most of the positive clones were found to contain inserts of 
bacterial DNA. This result suggests that these antibodies react with 
common epitopes that are also found at low abundance in bacteria. The 
result also indicates that the library contains some bacterial DNA in 
addition to nematode DNA. While bacterial DNA has been found in some 
other screens with this library (T. Blumenthal, personal communication)
, it has apparently not been a problem when polyclonal antibodies were 
used (R. Barstead, personal communication). Of 25 positive clones 
tested by Southern blotting, four were found that hybridize to C. 
DNA. Though these could also code for non-P-
granule proteins that share an epitope with a P-granule protein, they 
are being further characterized. Two classes were found. One is 
represented by a single clone, KL3a. It reacts only with the K76 
antibody and hybridizes to a single band on C. 
Southern blots at high stringency. On 
'Northern' blots it hybridizes to a single 2kb mRNA of moderate 
abundance, which appears to be most abundant in adults. Lysogens of 
KL3a make a very unstable fusion protein of about 120 kd (of which 
only 80 kd is  -galactosidase in this modified  gtll vector) that is 
recognized by both K76 and anti- -galactosidase antibody. The insert 
is being subcloned into a better expression vector to allow 
purification of the fusion protein and generation of a polyclonal 
antibody. This antibody will be used initially to determine whether 
the corresponding protein is a P-granule component or simply a cross-
reacting protein. The other class of positive clones containing C. 
epresented by three independent clones, LN4c, 
LN7a, and LN7b, whose inserts cross-hybridize. Proteins produced by 
these clones react with all four antibodies, and their DNA hybridizes 
to about 30 bands on high stringency genomic Southern blots. We have 
not yet identified transcripts for these clones by 'Northern' blot 
analysis. Lysogens of these positive clones, however, produce fusion 
proteins of 120-170 kd, indicating open reading frames of 1-2 kb. One 
of these clones produces a very abundant and stable fusion protein 
which reacts on immunoblots with all four monoclonal antibodies and 
anti- -galactosidase antibody. This fusion protein has been purified 
and used to immunize rabbits. Preliminary results indicate that the 
immune serum contains antibodies against the fusion protein, but fails 
to recognize P-granules. Therefore, the inserts in positive clones of 
this class probably are not derived from genes for P-granule proteins.