Worm Breeder's Gazette 13(4): 39 (October 1, 1994)

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.

sel-1, a Negative Regulator of lin-12 and glp-1 Activity, Encodes a Novel Extracellular Protein.

Barth Grant, Iva Greenwald

HHMI, Columbia University Dept. of Biochemistry, New York, NY 10032

  The sel-1 gene behaves as a negative regulator of both lin-12 and glp-1 activity
(Sundaram and Greenwald, 1993). loss-of-function mutations in sel-1 can suppress a
wide range of defects associated with partial loss-of-function in lin-12 ,and can
enhance defects associated with lin-12 gain-of-function mutations. Furthermore,
sel-1 mutations suppress the maternal effect lethal phenotype of the glp-1 ( e2142 ).
  In our continuing molecular analysis of sel-1 we have identified the gene as a
member of an operon, determined the complete genomic and cDNA sequence, and
confirmed previous genetic evidence of the null phenotype. As we mentioned in our
last gazette article (WBG 13(1):77), a 10kb HindIII fragment complements a sel-1
mutant for the ability to suppress both lin-12 ( n676 n930 )and glp-1 ( e2142 ).We further
refined the location of the resuing activity to a 7kb HindIII to EcoRV fragment which
we sequenced completely. Combined with cDNA sequence and Northern analysis we
determined that two genes lie very close together in the same orientation within this
fragment, probably forming an operon. Transcripts from the 5' gene are trans-spliced
to SL1 as determined by cDNA sequence and PCR RACE. 3' transcripts are
trans-spliced to one of the rarer SL variants.
  We identified the sel-1 gene as the 5' gene by rescue experiments, Northern
analysis of sel-1 mutant RNAs, and preliminary sequence analysis of sel-1 mutant
alleles. We found that inserting a linker containing stop codons in all frames within
the predicted coding region of the 5' gene abolished rescuing activity, while insertion of
the same linker within the 3' ORF did not. Also we found that deletion of the 3' gene
from the rescuing plasmid still allowed rescue of sel-1 in a lin-12 background
(although not in a glp-1 background - possibly indicating 3' enhancer activity). To
further convince ourselves that sel-1 is the 5' gene, we compared mRNA levels for both
genes in N2 and sel-1 mutant strains by Northern analysis. We found severely
reduced RNA levels from the 5' gene in several sel-1 alleles, while RNA levels from the
3' gene remained unchanged. Finally we have begun sequencing sel-1 mutant alleles
and have identified a predicted W to Stop change in the second exon of sel-1 ( ar167
),which would lead to the production of a severely truncated protein product. In
addition to confirming gene identity, this sequence change supports our genetic
evidence that the sel-1 null phenotype is Sel, i.e. wildtype except for its ability to
influence cell fate decisions in lin-12 or glp-1 mutant backgrounds.
  Analysis of the predicted protein product of the sel-1 gene has provided some
insight into its possible functions. sel-1 is predicted to encode a novel secreted protein
of about 650 amino acids rich in tyrosine and poor in cysteine. In addition to a
predicted N-terminal signal sequence, the extreme C-terminus contains a 16 aa
hydrophobic stretch, in some ways similar to mammalian glycosyl phosphotidylinositol
(GPI) linkage signals. Since we were surprised to find that sel-1 is likely an
extracellular protein, we fused the first 80 aa of sel-1 to 337-galactosidase using two of
Andy Fire's vectors. A fusion containing a sythetic transmembrane domain between
the predicted signal sequence and 337-galactosidase stains intensely, while one without
the transmembrane domain does not. This test supports the functionality of the signal
sequence in vivo, since 337-galactosidase loses activity when secreted, but retains activity
when held just inside the cell by a transmembrane domain (Fire et al., 1990). Because
sel-1 encodes a predicted extracellular protein which behaves genetically as a negative
regulator of lin-12 and glp-1 activity, we believe sel-1 may act on the surface of cells
involved in cell signalling events, interacting directly or indirectly with receptor
molecules or their respective ligands to modulate cell fate decisions.