In total, selleck chem 12 transcriptional fusions with gfp were constructed corresponding to the nine checkpoint genes of interest. For each construct, we generated at least three independent lines that were compared for expres sion pattern consistencies. Due to the mosaicism issues associated with extrachromosomal concatameric arrays, we analyzed at least 30 replicates and recorded GFP expressing cells and tissues that showed expression in at least 50% of the animals at any given developmental stage, as described previously. Our analysis of SAC gene regulatory activities revealed that all of the SAC constructs, except for pczw 1,GFP, confer GFP expression. The 2,101 bp sequence upstream of czw 1 did not drive any detectable GFP expression at any developmental stage in any of the four independent transgenic lines analyzed.
We also exam ined another construct that contained 3 kb upstream sequence of czw 1 and still did not observe any expres sion. Importantly, our analysis of the other eight SAC genes revealed expression that was consistent between the independent lines for every given construct. We have detected GFP at all developmental stages, except for very young embryos, and have identified expressed GFP in all the major tissues, except for germline, likely due to germline silencing of concatameric arrays. Promoters of spindle assembly checkpoint genes drive similar early embryonic expression GFP expression driven by the eight SAC gene upstream regions containing regulatory sequences was commonly observed early in development, well before the comma stage of embryogenesis.
In fact, we were able to detect GFP expression before embryos progressed to gastrulation. Because we observed mosaicism due to mitotic loss of the concatamer arrays, we analyzed many embryos per construct. Our results show that SAC gene promoters drive GFP expression in the major ity of the early embryonic cells. The only construct that did not drive ubiquitous GFP expression in early embryos is the putative promoter of mdf 1, which is in an operon, that extends upstream from the ATG initiator site in the first gene, his 35, of the operon to the adjacent upstream gene. On the other hand, both transcriptional fusions that included an internal mdf 1 promoter revealed the same ubiquitous activities in early embryos. Considering the established role of the mdf 1 checkpoint gene in sur veillance of the metaphase to anaphase transition, as well as the observed antibody localization Carfilzomib in dividing cells in early embryos, we conclude that the mdf 1 containing operon belongs to the hybrid operons class, in which the internal promoter of mdf 1 is neces sary to drive proper expression of this gene in embryo nic cells. The cell cycles of early embryonic cells in C.