[
Worm Breeder's Gazette,
1994]
Expression studies of the putative Caenorhabditis elegans cyclin A and B genes Monique A. Kreutzer, James P. Richards and Karen L. Bennett, University of Missouri-Columbia, Columbia, Missouri 65212. We have cloned putative A, B1 and B2 cyclins from C. elegans. Cyclin B1 and B2 are single copy genes while cyclin A belongs to a multigene family. Cyclin A and B2 each correspond to single transcripts while cyclin B1 recognizes three transcripts. The C. elegans temperature sensitive germline defective mutants
fem-1,
fem-3 gf) and
glp4 were used to analyze cyclin germline expression. When hybridizing C. elegans cyclin A or B2 to the mutant RNAs that produce no oocytes, the cyclin A and B2 messages are significantly reduced. When normal numbers of oocytes are produced, the cyclin A and B2 messages are at wild type levels. Therefore, the cyclin A and B2 transcripts appear to be primarily maternal with some somatic component. Consistent with a somatic component, cyclin A and B2 cDNAs cross-hybridize to Ascans intestinal poly A+ RNA. Hybridization of the cyclin B1 cDNA to these germline defective RNAs suggest that the cyclin B1 transcripts are differentially expressed in the germline. The two larger cyclin B1 transcripts appear to be primarily maternal and the smallest transcript appears to be sperm- specific. The observation that two polyadenylation signals are present in the cyclin B1 3'UTR, as well as results using cyclin B1 probes from specific regions of the B1 3'UTR, suggests that two of the three cyclin B1 transcripts are produced by polyadenylation a~ different sites. There is precedence in mice and flies for producing tissue-specific transcripts by different use of the cyclin B 3'UTRs. It has been shown in mouse that cyclin B1 hybridizes to four differentially expressed transcripts, one of which is testis- specific. Two of these mouse transcripts differ in the choice of polyadenylation sites and therefore in the lengths of their 3'UTRs (Chapman and Wolgemuth (1992) MoL Repro.Dev. 33, 259-269). In Drosophila, the cyclin B cDNA detects two female-specific transcripts that are made by splicing of a region of the cyclin B 3'UTR (Dalby and Glover (1992) Development 115, 989-997). In summary, our initial findings show that the C. elegans cyclin As and Bs are highly expressed in the germline and also suggest that the three cyclin B1 transcripts are differentially expressed in oocytes and sperm.
[
Worm Breeder's Gazette,
1993]
Our goal is to analyze the role that nematode P granules play in germline determination. Whitfield and Glover (Nature, 1989, 338:337-340) have demonstrated in Drosophila that cyclin B mRNA localizes to the germline precursor cells (pole cells) while cyclin A mRNA remains evenly distributed throughout the developing embryo. In order to determine if cyclin B mRNA is similarly localized in nematode embryos. we have cloned Caenorhabditis cyclin genes as reported earlier in the WBG (Richards, et al., January, 1991). To date we have isolated genomic clones and cDNAs for Caenorhabditis cyclin A and B genes. Sequence analysis of the cDNAs has confirmed their identities, with SL1 found on the 5' end of two of the cyclin cDNAs. There appears to be two cyclin B genes and potentially a multigene family of cyclin A's. Multiple cyclin A's are unique to Caenorhabditis in that no other organism has multiple cyclin A's. Further analysis is required in order to determine if all of these genes are functional. Physical mapping localizes one cyclin B to LG IV near
mec-3 and another to LG V near
unc-76 .Two cyclin A's have been physically mapped, one to LG II near
lin-31 and the other to LG III to the right of
lin-12 .Further sequence analysis has revealed some interesting conserved elements in these genes. A conserved proline is present within the potential Caenorhabditis destruction box (required for cyclin protein degradation with each cell cycle). In the 3' untranslated region of the cyclin genes potential adenylation control elements (ACE) are present which have been shown in mice and Xenopus to specify the adenylation state (and thus the translation state) of maternal RNAs in oocytes (Cyclin RNAs have been shown to be abundant maternal messages in clams, Xenopus, and mice.)(Huarte, et al., Cell, 1992, 69:1021-1030). We have also chosen to isolate the cyclin genes from Ascaris, a parasitic nematode whose embryogenic lineages are much like those of Caenorhabditis. We initially chose to use Ascaris for our localization studies by in situ hybridization because it provides the advantage of producing synchronous embryos, permitting easier interpretation of hybridization results. We have used the Caenorhabditis cyclin cDNAs to probe an Ascaris oocyte cDNA library, after first demonstrating cross hybridization to Ascaris genomic DNA, with slightly reduced stringency of 58 and washed to only 0.5XSSPE. We have isolated putative cyclin A and two B cDNAs from Ascaris. Sequence analysis of these cDNAs should provide confirmation. Upon sequence confirmation we plan to use sectioned synchronous Ascaris embryos to demonstrate whether cyclin B RNA localizes to the nematode germline precursor cells while cyclin A RNA remains evenly distributed throughout the embryos. If we detect localization with Ascaris, we intend to repeat the in situ experiments using Caenorhabditis sectioned embryos.
[
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.