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.
|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 involved. allele p h e n o t y p e s molecular lesions lethality vulva differentiation sterility e1417 + - + no mutation in coding region n378 + - + Glu to Lys at the first aa after the signal sequence n1058 +/- +/- - disrupts a splice donor in cytoplasmic region n1059 - (early larval) n/d -* Trp to stop change in EGF motif sy51 - (mid-larval) n/d -* disrupts splice donor in EGF motif sy52 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 motif 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