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.

The par-4 gene encodes a protein kinase that is cortically distributed in early embryos

Jennifer Watts, Diane Morton, Jennifer Bestman, Su Guo, Lisa Matthews, Ken Kemphues

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
   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.