Worm Breeder's Gazette 14(2): 62 (February 1, 1996)

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.

Molecular Lesions of lin-3 Mutations

Jing Liu1, Russell Hill2, Paul Sternberg1

1 Division of Biology, 156-29 Caltech Pasadena CA 91125
2 FHCRC, A3-103, 1124 Columbia St., Seattle WA 98104

      lin-3 encodes a member of the epidermal growth factor (EGF) family. 
lin-3 reduction-of-function mutants give rise to a variety of phenotypes
(table), including inviability, sterility, hermaphrodite vulva defect and
male spicule defect.  Some alleles only cause a subset of the above
phenotypes, for example, n378 has a very strong vulva phenotype but is
fertile, n1058  has a weak vulva phenotype but is sterile.  To understand
how it functions in different tissues, we determined the molecular lesions
of nine lin-3 alleles, by performing single-worm PCR and standard
sequencing (figure and table).  
      The results prompt us to make the following conclusions: 1. The EGF
motif is critical to lin-3 function.  s1263 changes one of the conserved
Cys in the EGF motif, resulting in mid-larval lethality.  n1059 is the
null allele and causes L1 arrest.  It has a stop codon in the middle of
the EGF motif.  The null phenotype is either due to a truncated protein, 
or it is the result of drastically reduced amount of protein products.  2. 
sy51 changes a conserved splice donor in the EGF motif, yet its phenotype
is less severe than null.  Therefore changes in the splice donor should
only diminish but not eliminate splicing.  3. We speculate that different
cytoplasmic domains may mediate tissue-specific functions.  The protein
sequence encoded by the last two exons in the cytoplasmic domain is
altered or deleted in n1058, yet n1058 phenotype is sterile but only
partially lethal and Vul, indicating that the last two exons may be more
important to fertility than to viability and vulva dif!
ferentiation.  sy52 and s751 both change the splice acceptor of the first
cytoplasmic exon.  Their mid-larval lethal phenotype may suggest that the
first cytoplasmic exon is essential for viability, if it is not due to a
much lowered protein level.  4. Dosage plays an important role in lin-3
function.  n378 changes the first aa after the signal sequence and might
affect lin-3 localization to the cell membrane.  Its vulva-specific
phenotype suggests that vulva is the tissue most sensitive to the dosage
change.  e1417 may be a promoter mutation.  Its vulva-specific phenotype
can be explained in two ways.  The mutation might moderately decrease the
protein level in all tissues, and the phenotype results from the
requirement for high lin-3 activity in vulva differentiation. 
Alternatively the transcription can be controlled by tissue-specific
elements and e1417 is defective only in the vulva-specific ones.  5. sy53
changes the last nucleotide of an exon in the EGF motif, changing !
Asp to Asn and maybe reducing splicing efficiency.  sy53 causes a lethal
phenotype as severe as n1059 but can complement other alleles for
sterility.  One interesting possibility is that fertility does not require
a perfect EGF motif, but viability does.
      In summary, the functions of lin-3 are regulated in a
tissue-specific manner.  The quantity, the structure of its EGF motif and
cytoplasmic domain all play important roles to ensure its wild type
functions.  Further studies should define the sites of regulation, the
different modes of its signalling in different tissues, and the factors
allele            p  h  e  n  o  t  y  p  e  s              molecular lesions
        lethality   vulva differentiation  sterility     

e1417        +              -                  +            no mutation        
                                                            in coding          

n378         +              -                   +           Glu to Lys         
                                                            at the first       
                                                            aa after the       

n1058       +/-            +/-                  -           disrupts a         
                                                            splice donor       
                                                            in cytoplasmic      

n1059   - (early larval)    n/d                -*           Trp to stop change
                                                            in EGF motif

sy51    - (mid-larval)      n/d                -*           disrupts splice    
                                                            donor in EGF motif

and s751 - (mid-larval)     n/d                 -*          disrupts            
                                                            splice acceptor in
                                                            cytoplasmic region

sy53     - (early larval)   n/d                 +*          disrupts splice    
                                                            donor in EGF motif
                                                            and Asp to Asn in
                                                            EGF motif

s1263    - (mid-larval)     n/d                 n/d         Cys to Tyr in EGF

The s alleles were isolated in D. Baillie's lab, the n alleles were
isolated in R. Horvitz's lab by E. Ferguson, and the sy alleles were
isolated by us.
+: wild-type                        
-: severely affected
*: When the lethality is rescued with transgene         
n/d: not determined