Worm Breeder's Gazette 10(1): 59

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.

Screening C. elegans Expression Libraries with Nerve Specific Anti-tubulin Antibodies

H. Yasuda and Shahid Siddiqui

C.  elegans, like many other multicellular organisms, has multiple 
tubulin genes.  Linda Gremke has shown that there are at least 4   and 
5 alpha tubulin genes in the nematode, based on homology to the 
chicken tubulin gene probes(1).  Similarly, it has been shown that 
purified tubulin from the nematode can be resolved in to at least 10 
isotypes on isoelectric focusing, including 2 major alpha, 2 major  , 
and about 6 minor alpha and   isotypes(2).  We have screened C.  
elegans cDNA and genomic expression libraries (kindly provided by Bob 
Barstead and Bob Waterston), with a set of anti-tubulin monoclonal 
antibodies, which preferentially stain neurons cytochemically(2).  In 
the present screen 44 clones have been identified, which test positive 
on rescreening.  The insert size of these clones range from 0.6 to 1.7 
kb.  Among these 29 clones are specific to anti-  tubulin, and 14 are 
specific to anti-alpha tubulin antibodies.  Our attention has been 
drawn to the problem of false positives in screening expression 
libraries with antibodies(e.g.  C.  Link and W. Wood, WBG Nov.  1986, 
and Eric Aamodt, personal communication).  These workers have found E. 
coli DNA in the 'positive' clones, as a major artefact.  Affinity 
purification of antibodies with the fusion protein, may not preclude 
the inclusion of false positives (Eric Aamodt, personal communication) 
either.  Therefore, we plan to subclone the inserts in an appropriate 
vector, and determine the nucleotide sequence of the inserts to 
distinguish the true from the false positives.  This is possible for 
the tubulin clones, as the nucleotide sequence of tubulin variants is 
available, including that of C.  elegans   tubulin(1).  We plan to use 
this strategy for other genes for which we have collected 'positives' 
using expression libraries, including N-CAM like antigen, and N-
CADHERIN like antigen of C.  elegans, as these genes have conserved 
sequences, and their nucleotide sequence data are available.