The heterochronic gene
lin-42 encodes a protein with high similarity to the PAS domain of the Drosophila circadian rhythm protein PERIOD (dPER). Because the PAS domain of dPER interacts with TIMELESS (dTIM), a second circadian rhythm protein, we investigated the role of TIM-1, a TIMELESS-like protein in C. elegans . Unlike the dtim null mutation, which shows no developmental defects (1), RNAi of
tim-1 results in embryonic lethality. These embryos arrest development as a mass of cells with unevenly-sized nuclei. In early embryos, we observed multinucleate cells by nomarski optics. When stained with DAPI, these embryos showed DNA bridges between forming nuclei, lagging chromosomes, nuclei with abnormal DNA content and fragmented chromosomes. These observations are consistent with a role for TIM-1 in chromosome segregation. Animals that escape the early lethal dsRNA dose show no heterochronic defects and develop into sterile adults. In oocytes of DAPI-stained animals, we observed 11-12 fluorescent "spots", presumably univalents, in contrast to the 6 bivalents observed in wild-type animals. We are using FISH analysis to test whether these spots represent unpaired homologs. Affinity purified antibodies raised against TIM-1 showed nuclear accumulation and enrichment in the region of the kinetochore spindles during metaphase. Preliminary analysis revealed that TIM-1 is abundant in all cells during early embryogenesis and is present in the nuclei of seam cells, the intestine and the developing germ line during larval stages. To identify mutations in
tim-1 , embryonic lethal mutations that mapped near the
tim-1 locus on chromosome III were collected from the CGC. We analyzed candidate alleles by DAPI-staining and nomarski optics, looking for phenotypes similar to the
tim-1 (RNAi) phenotype. We narrowed down the candidates to
csg-5 (
t1545) ( c hromosome s e g regation defective), isolated by Gonczy et al . in a maternal-effect embryonic lethal screen for genes involved in cell division (4). We sequenced
t1545 and found a single base transition, resulting in a Gly924Asp mutation in a region moderately conserved among timeless proteins. A long-range PCR product containing the
tim-1 coding region plus 3.3 kb 5'- and 1.3 kb 3'-flanking sequence fully rescued the embryonic lethality of
t1545. These results indicate that
tim-1 is
csg-5. Recently, the identification of a second Drosophila timeless gene (dTIM2) was reported (2,3). dTIM2 is more closely related to TIM-1 and the single mammalian TIM protein than it is to dTIM. Interestingly, timeless knock-out mice die during early embryogenesis around a period of rapid cell proliferation (3), suggesting that mouse TIM and TIM-1 may play similar roles in the cell cycle and have functions distinct from the functions of the Drosophila clock protein dTIM. 1. Sehgal A, Price JL, Man B, Young MW. Science . (1994) 263 :1603-6. 2. Benna C, Scannapieco P, Piccin A, Sandrelli F, Zordan M, Rosato E, Kyriacou CP, Valle G, Costa R. Curr Biol . (2000) 10 :512-3. 3. Gotter AL, Manganaro T, Weaver DR, Kolakowski LF Jr, Possidente B, Sriram S, MacLaughlin DT, Reppert SM. Nat Neurosci . (2000) 3 :755-6. 4. Gonczy P, Schnabel H, Kaletta T, Amores AD, Hyman T, Schnabel R. J Cell Biol . (1999) 144 :927-46.