Worm Breeder's Gazette 14(5): 50 (February 1, 1997)
These abstracts should not be cited in bibliographies. Material contained herein should be treated as personal communication and should be cited as such only with the consent of the author.
Section of Genetics and Development, Cornell University, Ithaca New York 14853
We have determined the sequence of a portion of the mRNA encoded by par-4, a gene required for localization of cytoplasmic components in early C. elegans embryos. We found that an allele of par-4, (zu187), isolated in a mutator screen in Jim Priess's lab, carried a novel Tc1-containing band when cut with the enzyme BglII. We isolated a small piece of DNA flanking the insert, but were unable to isolate either a cDNA clone or a additional genomic sequences despite screening over 300,000 plaques from various libraries. Therefore, we used RACE-PCR to amplify 5' sequence of the mRNA and, in total, have obtained about 1200 bases of coding sequence. We used the RACE-PCR product as a probe against other alleles of par-4 and found that five par-4 alleles showed polymorphisms in Southern blots: lw38 (a gamma-induced allele from Jocelyn Shaw) and it120, zu160, zu182 and zu198 (mutator alleles). The putative par-4 DNA also hybridizes to the YAC Y59A8, consistent with the position of par-4 obtained from physical mapping data, and hybridizes to a single 3 kb RNA on a Northern blot. The sequence that we have obtained includes a sequence similar to the C. elegans SL-2 spliced leader followed by an ATG start and open reading frame extending another 870 bases. A BLAST search reveals strong similarity to a C. briggsae cDNA, and both sequences encode a serine-threonine protein kinase. Within the kinase domain, these two proteins share 82% identity. A Xenopus ovary kinase shares 46% amino acid identity with the par-4 sequence and a rat calcium/calmodulin kinase shares 34% amino acid identity. We generated antibodies to 164 N-terminal amino acids upstream of the kinase domain in two rabbits, and found that antibodies from both rabbits recognize an 86 kD protein that is absent in par-4 mutant worms. Indirect immunofluorescence shows that PAR-4, like the other PAR proteins, becomes concentrated at the cortex of cells in early embryos. However, unlike PAR-1, PAR-2 and PAR-3 proteins, PAR-4 is not asymmetric; PAR-4 protein is evenly distributed around the periphery of all cells up to about the 100 cell stage. This protein distribution is not altered by par-1, par-2, par-3, par-5 and par-6 mutations. These observations are consistent with other data suggesting that par-4 may be acting independently of the other par genes.