[
Worm Breeder's Gazette,
1994]
Cloning, mammalian expression and knockout of a C elegans serotonin receptor Bjorn Olde, Richard McCombie+, Verena Gobble* and John Flaming* Neurogenetics Section, NINDS, NIH; +Cold Spring Harbor Research Laboratories; *MGH Cancer Center, Dept. of Pediatrics We have cloned and expressed in murine Ltk- cells a C elegans serotonin receptor. The cDNA (SHT-Ce) is 1528 bp long and codes for a peptide of 445 amino acids. The protein displays strong homology to the seven transmembrane family of G- protein coupled receptors especially to the Iymnae, Drosophilo and mammalian 5HT- 1 receptors. The sequence contains the Aspl49, that is part of the DRY-motif conserved in atl aminergic receptors; also conserved is the Ser217 that is fount in att 5HT receptors except for the three Drosophila setotonin receptors. It contains a short carboxy terminal and the presence of a positively enlarged amino acid residue in a conserved position (Arg434) which are structural athnbutes that have been associatod with a negative coupling to adenylate cyclaoe. The sequence of this receptor was compared to all availabte seven transmembrane receptor sequences in the database to determine phytogenetic relationships using a new form of maximum panimony anatysis ( with the help of FranK Kolakowki who has set up a 7-transmembrane receptor database). It was found that this receptor is much closer to the mammatian 5HT-1 receptors than to 5HT-2 or 5HT-5. Within the 5HT-1 cluster it is closest to the Drosophila 5HT-2a and SHT- 2b receptors and to tho Lymnae serotonin receptors. Considering the multiplicity of serotonin receptots in vertebrate as wolt as invertebrate species, it is likely that other serotonin receptor surpes
exi6t in C elegans. When expressod in murine Ltk- celts the 5HT-Ce receptor displays high affinity for the serotonergic radiotigand [125I]-LSD (Kd=0.33nM)) Ergot alkaloids particulary ergoline derivatives (i.e. LSD and tisuride) display high affinity for the receFcor as did the nonspecific sercrconergic antagonist methhthepin. Indolealkylamines which normatty bind to mammalian 5HT-I roceptors with affinitia raoging from 2-400nM all displayed affinities in the micromolecular range from the C elegans receptor. The high affinty for ergot derivatives and the Iow affinity for indoleattcylamines both appear to be common characteristics of invertebrate receptors. Application of serostonin to the colonial cell-line attenuates forskolin stisnulatect cAMP production. The serotonin geno was isotated from a lambda genornic library and sent to the Sanger Centre for fingerprinting where no match with previosly placed DNA was found The gene
i6 6.6 kb in size and is highly interrupted in structure (not intron-less as has been the case with some of the seven transmembrane receptors cloned in other species). The clone did not hybridize to the YAC grid but did weekly hybridize to Yl
l9D3 on the supplementary grit which itself has a somewhat dubious locatiosn left of mgP30 on the left arm of III (around eat- 8). We have screened muator stain MT3126 for tcl insertions in the vicioity csf the receptor by PCR and have identified 4 indepentent lines after sib-selection that require further selection to get down to a single worm. This work was greatly facilitated by the help of Ed Maryon Joel Rothman and Susan Mango. We are currently trying to localize the expression pattern of the receptor using the GFP vectors as a first step towards a screen looking for mutants that effect the placement of this receptor.
[
Worm Breeder's Gazette,
1997]
A number of laboratories have recently described various members of a highly conserved serine/threonine protein kinase family that appears to play a role in chromosome segregation and mitotic spindle dynamics in several different organisms (1,2,3,4). A search of the C. elegans genome database revealed that there are at least two nematode proteins that are highly related to this protein kinase family. We refer to these proteins as AIR-1 and AIR-2 (Aurora/Ipl-1 related). To date, the subcellular location of only one member of this protein kinase family has been reported (3,4). The mammalian protein Iak-1 has been shown to be associated with the centrosomes of the mitotic spindle in tissue culture cells and with meiotic chromosomes in mouse spermatocytes (J. Schumacher, unpublished). By performing immunocytochemistry experiments on fixed C. elegans embryos with antisera raised against specific peptides, we have found that the AIR-1 protein, like its mammalian counterpart, is also found on centrosomes in mitotic cells. The protein is first detectable on centrosomes of the first mitotic division following pronuclear fusion in the one-cell embryo. It is clearly associated with duplicated centrosomes prior to their migration to opposite sides of the nucleus and is found on mitotic centrosomes up to the limits of resolution in two-fold stage embryos. The location of the AIR-2 protein mimics that of Iak-1 in meiotic cells. Like Iak-1 in spermatocytes, AIR-2 is also found on chromosomes undergoing meiotic divisions both in oocytes and spermatocytes. AIR-2 staining is diffuse throughout the cellularized oocytes of the proximal gonad, but becomes localized to the chromosomes in the oocyte that resides next to the spermatheca. The protein persists on these chromosomes throughout meiosis and remains associated with polar body chromatin following these divisions. AIR-2 is also found on meiotic chromosomes during spermatogenesis in C. elegans males. In addition, it is associated with mature sperm present in the spermatheca, but at this stage it doesn!t appear to be localized to the chromatin. Instead, it appears to surround the sperm, suggesting an association with the cellular membrane. In embryos, diffuse AIR-2 staining is found in the cytoplasm, but is also clearly localized to mitotic metaphase chromosomes. The protein may be present on chromosomes at other stages of mitosis, but is difficult to detect on less condensed chromatin. By telophase, AIR-2 is clearly localized to midbody microtubules, and a small dot of staining persists on the cell membrane once cytokinesis is complete. To disrupt the function of each of these proteins during embryogenesis, we injected antisense RNA corresponding to the entire cDNA of each gene into the gonads of C. elegans hermaphrodites. Injection of either RNA resulted in embryonic lethality and the specific loss of each protein as detected by immunocytochemistry. AIR-1 deficient embryos die with greater than 100 cells and are severely aneuploid. Analysis of younger embryos revealed a variety of chromosome segregation defects ranging from the loss of a single chromosome to the missegregation of every chromosome to one daughter cell in a particular division. Some cells also appeared to be severely polyploid and contain multiple centrosomes, suggesting multiple cell cycles that lack an intervening mitotic division. Disruption of AIR-2 resulted in the production of one-cell embryos that contained many nuclei and centrosomes, as well as polar bodies that continue to replicate and divide. One-cell embryos containing anywhere from one to greater than 20 nuclei were found with equally abnormal numbers of centrosomes. This dramatic phenotype again suggests the uncoupling of DNA replication and centrosome duplication from the completion of mitosis. References: 1) Chan, C.S. and Botstein, D. (1993). Isolation and characterization of chromosome-gain and increase-in-ploidy mutants in yeast. Genetics 135, 677-691 2) Glover, D. M., Leibowitz, M. H., McLean, D. A., and Parry, H. (1995). Mutations in aurora prevent centrosome separation leading to the formation of monopolar spindles. Cell 81, 95-105. 3) Gopalan, G., Chan C.S., and Donovan P.J. (1997). A novel mammalian, mitotic spindle-associated kinase is related to yeast and fly chromosome segregation regulators. J Cell Biol 138, 643-656 4) Kimura, M., Kotani, S., Hattori, T., Sumi, N., Yoshioka, T., Todokoro, K., and Okano, Y. Cell cycle-dependent expression and spindle pole localization of a novel human protein kinase, Aik, related to Aurora of Drosophila and yeast Ipl1. J Biol Chem 272, 13766-13771 Research sponsored by the National Cancer Institute, DHHS, under contract with ABL.