Worm Breeder's Gazette 13(2): 87 (February 1, 1994)
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.
Dynein is a high molecular weight, multimeric protein complex involved in microtubule-based intracellular transport. We would like to understand its role in vivo; as a first step in that direction, we have cloned and sequenced genomic DNA containing the dynein heavy chain gene. A 35 kb contig that spans the entire gene, and contains both 5' and 3' flanking sequences has been sequenced on both strands with greater than 6 fold average redundancy. The Unix program Genefinder (L. Hillier and P. Green) was used to find open reading frames and the predicted gene was compared to dynein heavy chain sequences from other organisms in the electronic databases. The region surrounding the hydrolytic ATP binding site is well conserved among all dynein heavy chains, and the carboxy terminal two thirds of all dynein heavy chains contain several regions of strong homology. For the first 1300 (or so) amino acids, the flagellar forms are quite homologous to each other and the cytoplasmic forms are homologous to each other; however, there is very little homology between these two classes of dynein. The functional significance of these differences, if any, remains to be determined.
We are attempting to make cytoplasmic dynein specific antibodies using exon-specific bacterial fusion proteins as antigen. We have produced individual fusions between either exon 1 or exon 4 and the carrier protein, glutathione-S-Transferase (GST) using a derivative of the vector pGEX-1. Both of these exons are in the amino terminal region that is specific to the cytoplasmic forms of dynein heavy chain. While constructing these fusions, we generated a pGEX derivative that might be useful to others. The existing pGEX vectors lacked convenient restriction sites (i.e., sites that were both in frame and did not cut within the desired sequence), so we designed a polylinker (shown below) that contains several sites for rare cutting enzymes and cloned this polylinker into Bam H-I/Eco R-I digested pGEX-1. We designed oligonucleotide pairs that resulted in PCR products with Not-I sites in tails 5' to each exon and Xho-I sites in tails at the 3' ends; these exon specific PCR products were then cloned into our pGEX derivative after Not-I/Xho-I digestion and gel purification. These clones produced fusion proteins which are being used as antigen for polyclonal antibodies.