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.
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.