Worm Breeder's Gazette 10(2): 118

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DNA Sequence Analysis of the unc-5 Gene

Chungyee Leung-Hagesteijn, Brian Stern, Joe Culotti, and Ed Hedgecock

Figure 1

In the last gazette we reported the cloning of a 0.6 KB EcoRI 
fragment from the unc-5 region which appeared to have a Tc1 insertion 
responsible for the unc-5(ev435:Tc1) mutation.  We have identified 
several clones in an EMBL3 library of N2 DNA which hybridize to this 
fragment.  Most clones appear to have part of the 0.6 KB EcoRI 
fragment attached to one of the lambda arms.  Clones NW#11, NW#12 and 
NW#13, however, contain the entire 0.6 KB EcoRI fragment, which 
appears by sequence analysis to be identical to the 0.6 KB EcoRI 
fragment used as probe.  NW#11 and Nw#13 appear identical to each 
other, whereas, clone NW#12 differs from these only slightly.  
Southern blots of EcoRI digested wild type DNA revealed approximately 
23 EcoRI fragments which hybridized to NW#11 DNA.  Polymorphisms in 
several of the unc-5 mutations (ev435, ev440, ev442, h571, rh1036, 
rh1037, and rh1038) isolated in RW7097 were revealed using NW#11 DNA 
to probe Southerns.  Sequence analysis of the 0.6 KB EcoRI genomic 
fragment revealed a relatively GC rich putative open reading frame of 
122 amino acids flanked at 5' and 3' ends by consensus splice acceptor 
and splice donor sites and AT rich DNA.  The Tc1 insertion site of unc-
5(ev435:Tc1) is near the middle of this open reading frame.  We have 
also isolated a 1 KB cDNA by hybridization to the 0.6 KB EcoRI 
fragment.  A portion of the sequence of this cDNA is identical to that 
of the genomic fragment, beginning at the consensus splice acceptor 
site and continuing into the open reading frame.  The hypothetical 
protein fragment of 122 residues encoded by the unc-5 exon comprises a 
tandemly arranged pair of homologous domains each beginning with the 
tryptophan-rich sequence DGGWSXWSXW.  Three similar domains are 
present in human thrombospondin where they are also tandemly arranged (
1).  Thrombospondin (TSP) is a large glycoprotein secreted by blood 
platelets and other cells, which promotes cell aggregation and cell-
substratum adhesion in vitro.  The region of thrombospondin that 
contains these domains is believed to bind to various extracellular 
matrix components including type V collagen, laminin, fibronectin, 
fibrinogen, and plasminogen.  A similar domain occurs twice in 
complement components C8 alpha and C8 beta, once near the amino 
terminus and once at the carboxy terminus.  Only the amino domain 
occurs in complement component C9.  A single related domain occurs 
once in the circumsporozite (CS) proteins of Plasmodium falciparum and 
Plasmodium knowlesi, malarial parasites of humans and Old World 
monkeys (2,3).  It has been suggested that the CS proteins may promote 
parasite adhesion during liver invasion.  The occurrence of this 
common amino acid sequence in apparently unrelated proteins involved 
in cell adhesion suggests that it may fold and function as an 
independent protein domain especially adapted for binding to 
extracellular matrix components.  It may be interesting to learn 
whether each such domain in thrombospondin or the hypothetical unc-5 
protein binds to a distinct ligand.
[See Figure 1]

Figure 1