Worm Breeder's Gazette 10(3): 47

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 daf-1 Gene May Encode a Protein Kinase

Laura Georgi, Miguel Estevez and Don Riddle

Last year [WBG 10(1) 32] we reported Tc1-tagging and cloning daf-1, a 
dauer constitutive gene.  Using the cloned mutant fragment described 
in our previous report, we screened an N2 genomic EMBL4 library 
obtained from the Wood lab and isolated recombinant phage containing 
daf-1 near the center of the clone.  This clone was sent to Cambridge 
and has been assigned to a cosmid contig.  
Fragments subcloned from phage were used to probe northern blots of 
total RNA prepared from an asynchronous population of N2.  A 2.5 kb 
message was identified using a 1 kb BamHI fragment that contains the 
sites of both daf-1-associated transposon insertions.  Flanking 
restriction fragments also hybridized to this message.  Though 
detectable in total RNA, the message was not abundant, being present 
at about a fifth to a quarter the level of the ama-1 message, which 
encodes the large subunit of RNA polymerase II.  
Mutant daf-1(m40) hermaphrodites were injected with an 8 kb fragment 
of N2 genomic DNA subcloned from the EMBL4 phage, and rescued progeny 
were recovered.  Rescued lines continue to segregate mutant progeny, 
so the injected DNA appears to be extrachromosomally inherited.  A 
selected rescued line will be tested for the presence of injected 
Analysis of the genomic sequence has proved inadequate for 
identifying introns in this gene, so a cDNA library (generously 
provided by Bob Barstead) was screened to obtain clones for sequence 
analysis.  This lambda-ZAP library yielded several distinct clones, 
including one that is apparently full length.  Sequencing of these 
clones has thus far defined the 3' end of the message and the limits 
of four introns.  
Examination of the deduced amino acid sequence revealed similarities 
with several protein kinases.  Shared residues included amino acids 
conserved among serine-threonine protein kinases, though the degree of 
similarity was not high enough to assign the daf-1 protein to a 
specific class of these kinases.  Kinase subdomain I (Gly-X-Gly-X-X-
Gly) lies near the 5' end of the 1 kb BamHI fragment mentioned above.  
The two daf-1-associated Tc1 insertions lie between subdomains IV and 
V, and near subdomain IX.  The completed 5' sequence may provide 
additional information on the possible cellular location and function 
of this putative protein kinase.  
We have incomplete sequence data on another interesting open reading 
frame (ORF) less than 2 kb downstream of daf-1.  This ORF appears to 
be part of a collagen like gene; the sequence on hand potentially 
encodes the characteristic collagen triple helical (Gly-X-Y)n.  Since 
daf-1 maps very close to dpy-9, and dpy-13 is a collagen gene, we plan 
to check out the possibility that dpy-9 is in the cloned region by 
performing another mutant rescue experiment.  If anyone has any 
transposon insertion mutations in dpy-9, we might be able to put them 
to good use as well.  
Another closely linked gene is tpa-1, which appears to encode a 
protein kinase [WBG 10(2) 113] that is distinct from the daf-1