Worm Breeder's Gazette 11(4): 23
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.
Cytoplasmic dynein is an ATPase involved in intracellular microtubule-based transport. It is a large complex consisting of two copies of an approximately 450 kDa heavy chain and one or more light chains. Immunocytochemical analysis suggests that it is a component of the mitotic spindle. Others have shown that cytoplasmic dynein is involved in fast axonal transport (Schnapp and Reese, PNAS 86:1548- 1522) and intra-cellular vesicle transport (Schroer. et al., CELL 56: 937-946). As the first step in a genetic analysis of cytoplasmic dynein function in C. elegans, we have used both polyclonal antibodies and affinity-purified polyclonal antibodies to select candidate dynein heavy chain (DHC) clones from a lambda gt11 expression vector library ( prepared by Dr. Robert Barstead). One of these clones, 2530B-2, with an insert of approximately 1 kb, may contain sequences derived from the DHC. Fusion protein expressed from 2530B-2 reacts with 2 different anti-dynein polyclonal sera and when used as antigen for affinity purification of antibodies from an anti-dynein antiserum, the eluted antibodies reacted with the DHC. Southern analysis suggests that the 2530B-2 sequence is single copy. We lifted the insert from 2530B-2 using the polymerase chain reaction (PCR), and used the insert as a probe against a YAC polytene filter. This established a physical map position for the sequence on the right end of the unc-73 contig; this sequence is on the left arm of chromosome I, just to the left of the cluster and right of unc-73. We are presently refining this map position relative to deletions in this region, using the PCR assay. With the generous help of Junyi Lei and Guy Benian, who provided us with a Northern blot strip containing high molecular weight RNA, we have been able to determine that our candidate gene is represented by a message of approximately 14 kb; this is large enough to code for a polypeptide of 450 kDa. We are now attempting to clone and sequence the remainder of this candidate DHC gene. We also plan to examine existing mutants mapped to this region for defects in dynein localization and/or accumulation.