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.
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.