pathway signaling This process gives rise to cells with extra copies of chromosomes, permitting amplification of the genome in specialized cells. In humans, these include hepatocytes, cardiomyocytes and megakaryocytes. In C. elegans, two tissues are polyploid, the hypo dermis and the intestine. Our finding of co expres sion of SAC genes in these tissues may suggest a possible role of these genes in the process of endoredu plication in C. elegans. Furthermore, our findings clearly suggest that SAC genes are differentially regulated at the transcription level at different developmental stages. Conclusion We have examined for the first time in vivo spatiotem poral expression profiles of eight conserved spindle assembly checkpoint genes in C. elegans.
Our compre hensive analysis revealed that all of the SAC gene pro moters displayed common early embryonic activities in the majority, if not all, of the rapidly dividing embryonic cells. Furthermore, we found that all of the SAC gene promoters drive tissue specific postembryonic expres sion. The expression patterns differ between the SAC genes, the majority of the SAC genes co express in hypodermal seam cells and gut cells. These findings sug gest that the SAC components may have distinct roles in postembryonic development which could be different from their role in mitosis. Furthermore, our analysis provides an important starting point for analysis of the checkpoint roles in development of a multicellular eukaryote that may offer explanation for distinct pheno typic consequence upon inactivation of different SAC eration, cell fate determination and cell differentiation in a multicellular organism.
Methods C. elegans strains, alleles and culturing The Bristol strain N2 was used as the standard wild type strain. The following mutant alleles were used in this work, dpy 5, mdf 1, mdf 2, ced 3, unc 26, lin 35, fzr 1 and fzy 1. The wls51 strain JR667 was used to visualize the seam cell nuclei in wild type worms and the mutant backgrounds. The strains were obtained from the Caenorhabditis Genetics Center unless otherwise stated. The following transgenic strains were generated, JNC104, JNC105, JNC106, JNC107, JNC108, JNC109, JNC110, JNC111, JNC112, JNC113, JNC114, JNC115 , JNC116, JNC117. Animals were maintained using standard procedures. Generation of pSAC,GFP transgenic animals The promoter,GFP constructs were generated using the PCR stitching technique.
The PCR experi ments were designed to amplify and fuse 5 sequence immediately upstream of the predicted ATG initiator site for a targeted gene to an adjacent upstream gene. All of the primers were designed semi Brefeldin_A manually with the aid of primer3 and used in standard PCR pro cedures to amplify putative SAC gene promoters from C. elegans N2 single worm lysates. These amplicons were then fused to the PCR products con taining gfp sequence and unc 54 3UTR from pPD95. 75. For fusion PCR reactions we used Phusion high fidelity DNA polymerase.