b274 is the reference allele in WormBase for par-1. PAR-1 is a cell polarity kinase localized to the posterior cortex of the zygote (Guo and Kemphues, 1995). Here we present evidence that b274 is not a null allele.
Two lines of evidence in the literature already suggested that b274 retains some par-1 activity: 1) b274 generates a truncated (86kD) PAR-1 protein (Hurd and Kemphues, 2003) and 2) a par-1-dependent phosphorylation site in MEX-5 is still phosphorylated in b274 (Tenlen et al., 2008).
We have sequenced b274 and have found that it codes for a premature STOP codon at Q814. Consistent with the observed 86kD molecular weight, b274 codes for a truncated protein containing the complete PAR-1 kinase domain but lacking the PAR-1 C-terminus. Using GFP fusions, we have shown that the PAR-1 C-terminus is essential for localization to the posterior cortex of the zygote. GFP:PAR-1 fusions lacking the C-terminus localize throughout the cytoplasm of the zygote.
To determine whether b274 encodes an active but delocalized kinase, we examined MEX-5 mobility. MEX-5 is a cytoplasmic protein that localizes to the anterior of the zygote in a par-1-dependent manner (Schubert et al., 2000). PAR-1 stimulates MEX-5 mobility in the posterior cytoplasm (Tenlen et al., 2008) by direct phosphorylation on two residues (Griffin and Seydoux, unpublished). We found that b274 zygotes show uniformly high MEX-5 mobility. In contrast, par-1(RNAi);par-1(b274) zygotes show uniformly low MEX-5 mobility as do par-1(it51) zygotes (it51 is a missense mutation in the kinase domain- (Guo and Kemphues, 1995). Remarkably, par-1(RNAi), b274 and it51 zygotes all fail to localize MEX-5 and cannot be distinguished by visual inspection of MEX-5 distribution alone.
We conclude that b274 is a not a null allele of par-1. it32 (Q797STOP) and it61 (Q608STOP) also code for premature stops downstream of the kinase domain and may also be non-nulls.
References
Guo S and Kemphues KJ. (1995). par-1, a gene required for establishing polarity in C. elegans embryos, encodes a putative Ser/Thr kinase that is asymmetrically distributed. Cell 81, 611-620.
Hurd DD and Kemphues KJ. (2003). PAR-1 is required for morphogenesis of the Caenorhabditis elegans vulva. Dev Biol 253, 54-65.
Schubert CM, Lin R, de Vries CJ, Plasterk RH, and Priess JR. (2000). MEX-5 and MEX-6 function to establish soma/germline asymmetry in early C. elegans embryos. Mol Cell 5, 671-682.
Tenlen JR, Molk JN, London N, Page BD, and Priess JR. (2008). MEX-5 asymmetry in one-cell C. elegans embryos requires PAR-4- and PAR-1-dependent phosphorylation. Development 135, 3665-3675.
Articles submitted to the Worm Breeder's Gazette should not be cited in bibliographies. Material contained here should be treated as personal communication and cited as such only with the consent of the author.
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