Worm Breeder's Gazette 11(4): 39

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.

col-4 isn't

Yang-Seo Park and Jim Kramer

Of the six blister genes, we have been particularly interested in 
bli-6 IV because Bob Herman has shown that alleles of sqt-1, 
dpy-10 and rol-8 can suppress the dominant 
mutation bli-6(mn4).  Although Dpy is often epistatic to Bli, Bob 
identified non- or very weak Dpy alleles of dpy-2 and dpy-10 that 
suppress mn4.  Since sqt-1, dpy-10 and rol-8 show 
genetic interactions and all except rol-8 have been shown to encode 
collagens (Adam Levy has identified a collagen that is likely to be 
rol-8) there is a good possibility that they may physically interact 
with the bli-6 gene product.
A Bristol/Bergerac polymorphism, identified by hybridizing genomic 
Southern blots with col-2 at low stringency, had been previously 
mapped to the vicinity of bli-6, and was named col-4 (Cox et al.  
Genetics 109:513,1985).  Initial three-factor mapping with unc-5 and 
dpy-20 failed to separate bli-6 and col-4(Berg), so we decided to 
examine col-4 more closely.  Further mapping using unc-44 and unc-24 
showed that col-4 is approx.  0.4 mu to the right of bli-6, but 
concurrent molecular analyses of col-4 produced some unexpected 
The col-4 gene was cloned by identifying a col-2 crosshybridizing 
clone, in a size selected genomic sublibrary, that showed the expected 
Br/Bg polymorphism when probed to genomic Southern blots.  The first 
surprise was that col-4 doesn't encode a collagen.  The first case, 
that we know of, where low stringency collagen cross-hybridization has 
pulled out something other than a collagen.  The col-4 sequence 
predicts a polypeptide consisting primarily of 23 tandem copies of the 
following 15 amino acid 
GAPPSGGPPGPF(D/N)PS     (note: NPS is a potential glycosylation site)

The only variation in the repeats is that about one-half have D and 
one-half have N.  The repeat could have arisen from a collagen triplet 
(Gly-X-Y)s by loss of the second and fifth Gly.  Note also the high 
proline content (40%), also indicative of collagen.  It seemed likely 
that col-4 could encode a novel, collagen-related structural protein.  
High stringency Southern blots showed that the col-4 gene is present 
in a single copy in the Bristol and Bergerac genomes.
The next surprise was that we could not detect any col-4 transcripts.
This result was particularly surprising because the codon usage in 
the repeats (all 6 Pro are CCA, 3/4 Gly are GGA) is that expected for 
coding sequence.  Northern blots and primer extension with several 
fragments flanking the repeats failed to identify transcripts in N2 
RNAs from various stages, including dauer.  RNA from males was not 
examined.  No cDNA clones were identified in Bob Barstead's library.  
The Br/Bg polymorphism is in the region containing the repeats (
Bergerac is approx.  300 bp larger), so we examined Bg RNA but could 
not identify transcripts.  Finally, high-stringency hybridization of 
the repeat region showed that it is not present in the C.  briggsae 
genome.  col-4 has a recognizable 3' structure; 60 amino acids 
following the repeats is a termination codon followed by AT rich 
sequence and even a potential polyadenylation signal.  On the 5' side 
of the repeats, however, it is not obvious where an initiator ATG or 
intron might be.  col-4 might not be expressed at all, or only at very 
low levels, or at some specific time that we did not examine.  
Possibly it is the remains of a once expressed gene.
If col-4 was expressed at some time in the past, as the codon usage 
suggests, what might it have been? One guess is a cuticle surface 
protein, like srf-1 which is not present on the surface of certain 
wild strains of C.  elegans (Politz et al.  Genetics 117 467, 1987).  
Surface protein genes might show a high degree of interstrain 
variability.  Indeed, the col-4 repeat has some similarity to repeats 
in the 22 kD secreted surface protein of Brugia, 5/6 Pro's and 2/4 
Gly's align (Mark Blaxter, personal communication).  We welcome 
comments and suggestions.