Worm Breeder's Gazette 14(1): 58 (October 1, 1995)
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.
Dept. of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder CO 80309-0347
In C. elegans, cytoplasmic and nuclear migration may be controlled by separable mechanisms. Mutations in two genes, unc-84 and unc-83, disrupt the migration of the hyp7 and P cell nuclei1,2, indicating an important role for these genes in the nuclear migration process. To molecularly characterize these genes, we have set out to clone both unc-84 and unc-83. We defined a physical region for unc-84 that is duplicated by mnDp25 and mnDp9 but not by mnDp26, mnDp27 and mnDp1 by Southern hybridization. Cosmids in the region were assayed for the ability to rescue unc-84(e1410). Cosmid W01D6 rescued the Unc and Egl phenotypes of this mutant. An 8kb subclone of the cosmid can also rescue this mutant. This subclone hybridizes to a 3.5kb and a 2.5kb transcript present in both early embryonic RNA and L1 RNA, which is consistent with unc-84 function in hyp7 and P cell migration. Using this subclone as a probe, we identified over 30 positive cDNA clones in a screen of approximately 7.5 x 105 plaques of Okkema's early embryonic cDNA library. Five of the cDNA clones have been characterized. Three of the cDNAs are approximately 3.5kb and the other two are approximately 2.5kb, with the difference between the two classes of cDNAs being an additional 1 kb of sequence on the 3' end of the 3.5kb cDNAs. We have determined the intron-exon boundaries using the unpublished genomic sequence in the region. Analysis of the genomic sequence and the Northern data suggest that there is only one complete gene present in the rescuing fragment. The 3.5kb cDNAs contain a long open reading frame that encodes a predicted protein of over 1100 amino acids. This predicted protein has a C-terminal region that is similar to the C-terminal region of the fission yeast protein Sad1. The sad1+ gene is essential and its gene product associates with the spindle pole body throughout mitotic and meiotic cycles3. The predicted UNC-84 and Sad1 proteins also share a putative transmembrane domain. We are collaborating with the Horvitz lab to molecularly analyze the alleles of unc-84 . We are also interested in determining if the two transcripts play a role in the complex complementation observed among unc-84 alleles. We are in the process of cloning the unc-83 gene as well. We originally defined a relatively large physical region with several cosmid gaps for unc-83 but were unable to rescue unc-83(e1408) with cosmids from the region. We have recently defined a much smaller physical region for unc-83. In addition, we have shown that mnDP26 complements unc-83(e1408), suggesting that the mutant can be rescued by arrays that contain the gene. We are currently injecting cosmid and YAC clones in the region. 1Horvitz, H. R. and J. E. Sulston. 1980. Genetics. 96:435-454. 2Fixsen, W. D. 1985. Ph.D. Thesis. MIT. 3Hagan, I. and M. Yanagida. 1995. J. Cell Biol. 129:1033-1047.