The maternal
pos-1 mRNA localizes to the posterior half of embryos during the 1st cleavage and localizes to germ lineage in early embryogenesis. To identify the cis-element?for localization, we constructed an in vivo assay system. In this assay system, we first generate transgenic worms, which express fusion mRNAs of tag and
pos-1 sequence, by bombardment. Then, we evaluate distribution of transgene mRNA by using in situ hybridization with anti-tag probe. Firstly, we constructed a vector, which express GFP::
pos-1 3UTR fusion mRNA from
pie-1 promoter. This vector was made from pID 3.01B (a kind gift of G. Seydoux). As expected, transgene mRNA was localized to germlineage cells of late embryo, however, in situ hybridization signals were quite weak, and it was difficult to investigate very early embryos. Thus, secondly, we developed a new vector that has the
pos-1 promoter. Because the expression level of the intrinsic
pos-1 mRNA was high, it was expected that the
pos-1 promoter has strong activity. As expected, in situ hybridization signals of the transgene mRNA were very strong. Thus, we generated transgenic worms that express GFP::
pos-1 3UTR fusion mRNA by using the
pos-1 vector. The signals of the fusion mRNA localized as intrinsic
pos-1 mRNA and localization were clearly observed by the two-cell stage. This result suggests
pos-1 3UTR has major activity for localization. Therefore, we analyzed the
pos-1 3UTR closely. Firstly, we tried in situ hybridization on C. briggsae embyros. Interestingly, C. briggsae
pos-1 mRNA localized as C. elegans
pos-1 mRNA. Thus, secondly, we tested whether C. briggsae
pos-1 3UTR exhibits localization activity in C. elegans. Strikingly, the transgene mRNA localized as the intrinsic
pos-1 mRNA. Though C. briggsae has two orthologs of
pos-1,
Cb-pos-1a and
Cb-pos-1b, the 3UTR of both showed apparently same activity. These results suggest that conserved regions of the 3UTR have the localization activity. Because there were several conserved regions in the 3UTR (J. Konwerski 2005), we further investigated C. elegans
pos-1 3UTR by making deletion constructs. This analysis demonstrated that 122nt stretches (148-269nt of 314nt 3UTR) was necessary and 140nt stretches (1-140nt) was dispensable for localization. The 122nt stretches of 3UTR includes 30nt sequence, which is perfectly conserved among three 3UTR sequence. Therefore, we suppose this is the cis-element for the localization. Finally, we developed a vector, which is optimized for in situ hybridization. This vector contains VENUS coding sequence as tag sequence. And GC content of it is high, 62%. Together with strong activity of
pos-1 promoter, this new tag sequence greatly improved signal intensity and signal to noise ratio of in situ hybridization. Now, we are analyzing 122nt stretches of
pos-1 3UTR by using this improved
pos-1 vector. We will presents the result and discuss about the localizing mechanism at the meeting.