The Caenorhabditis elegans Locus lin-15, a Negative Regulator of a Tyrosine Kinase Signaling Pathway, Encodes Two Different Proteins


The Caenorhabditis elegans locus lin-15 negatively regulates an intercellular signaling process that induces formation of the hermaphrodite vulva. The lin-15 locus controls two separate genetic activities. Mutants that lack both activities have multiple, ectopic pseudo-vulvae resulting from the overproduction of vulval cells, whereas mutants defective in only one lin-15 activity appear wild-type. lin-15 acts noncell-autonomously to prevent the activation of a receptor tyrosine kinase/ras signaling pathway. We report here the molecular characterization of the lin-15 locus. The two lin-15 activities are encoded by contiguous genomic regions and by two distinct, non-overlapping transcripts that may be processed from a single mRNA precursor by trans-splicing. Based on the DNA sequence, the 7 1 4 and 1,440-amino acid lin-15 proteins are not similar to each other or to known proteins. lin-15 multivulva mutants, which are defective in both lin-15 activities, contain deletions and insertions that affect the lin-15 genomic region. 
). Three other cells, P3.p, P4.p and P8.p, also have the potential to produce vulval cells. Since all six of these cells are able to express any of three alternative cell lineages (referred to as lo, 2" and 3") and are equivalent in their developmental potential, they are considered to define the vulval equivalence group * ; 

(SULSTON and WHITE 1980; KIMBLE 1981; STERNBERG and HORVITZ 1986). Cells that adopt the 1 O and 2" cell fates generate eight and seven descendants, respectively, that together form the vulva, whereas those that express the 3" fate generate two non-vulval descendants that fuse with the syncytial hypoderm that envelops the animal. 
Cell interactions determine the fates of the cells of the vulval equivalence group (see HORVITZ and STERNBERG (1991) for review). A signal from the gonadal anchor cell induces the nearest Pn.p cells to express vulval cell lineages: P6.p adopts a 1" cell fate, while P5.p and P7.p adopt 2" cell fates (Figure 1B). The more distant cells P3.p, P4.p and P8.p adopt 3" cell fates. The elimination of the anchor cell causes all six cells to express a nonvulval 3" fate, resulting in a vulvaless (Vul) phenotype (KIMBLE 1981) (Figure 1C). Genetic experiments suggest that an inhibitory signal from the syncytial hypoderm prevents the expression of vulval cell fates (HERMAN and HEDGECOCK 1990). The removal of the hypodermal inhibitory signal allows all six cells to express vulval cell fates, resulting in a rnultivulva (Muv) phenotype (Figure 1D). These results suggest that during wild- type development, the anchor cell signal promotes the expression of vulval cell fates by overcoming the hypodermal inhibitory signal. In addition, interactions among the induced Pn.p cells prevent adjacent cells from both expressing a 1" fate (STERNBERG 1988). 
Many mutants with altered vulval cell lineages have been characterized (HORWTZ and SULSTON 1980; FERGUSON and HORVITZ 1985, 1989;BEITEL et al. 1990; I-IAN et al. 1990; KIM and HORVITZ 1990; h o r n and STERNBERG 1991; CLARK et al. 1992; H.m et al. 1993; MILLER et al. 1993). 
Some mutations cause all six cells P3.p-P8.p to express a 3" cell lineage, and no vulva is formed. As this Vu1 phenotype is identical to that of animals lacking the anchor cell, these mutations define genes that could be involved in the signaling process required for vulval induction. Five such Vu1 genes encode proteins similar to those involved in intercellular signaling in other organisms: lin-3 encodes a molecule related to TGFa (HILL and STERNBERG 1992), let-23 encodes a receptor tyrosine kinase ( h o r n et al. 1990), sem-5 encodes an adaptor protein with SH2 and SH3 domains ( C m et al. 1992), let-60 encodes a ras protein (W and STERNBERG 1990) and lin-45 encodes a raf serine/threonine kinase (HAN et al. 1993). Recently, mutations in the mpk-l/sur-l gene, which encodes a mitogen-activated protein ( M A P ) kinase, have been identified as suppressors of activated let-60 ras mutations, suggesting that mpk-l/sur-1 also functions in the signaling pathway required for vulval induction (LACKNER et al. 1994;Wu and HAN 1994). 
Other mutations, including some lin-15 mutations, cause all six cells P3.pP8.p to express 1" and 2" cell lineages, resulting in a multivulva phenotype (FERGUSON et al. 1987). (A) Cell lineages that generate the vulva. Each P3.pP8.p cell expresses one of three lineages, referred to as l", 2" and 3". P6.p normally expresses a 1" cell lineage, which produces eight descendants; P5.p and P7.p normally express a 2" cell lineage, which produces seven descendants; and P3.p, P4.p and P8.p normally express a 3" cell lineage, which produces two descendants. The vulva is formed by the 22 descendants of the 1" and 2" cell lineages, while the 3" cell lineage generates non-vulval hypodermal cells. The lineages are, in part, defined by the axis of the final cell division; L, longitudinal, T, transverse, N, nondividing 
( STERNBERG and H O R ~ 1986). (B) Wild-type. An inductive signal from the gonadal anchor cell causes the nearest Pn.p cells to express a 1" or 2" cell lineage, while the more distant Pn.p cells express a 3" cell lineage. An inhibitory signal from the hypoderm prevents the expression of the 1 and 2" vulval cell lineages and is overridden by the anchor cell inducing signal. Interactions among the induced cells prevent adjacent Pn.p cells from both expressing a 1" cell lineage. (C) Vulvaless. In the absence of the anchor cell signal or of the response to that signal, the hypodermal inhibitory signal prevents all six Pn.p cells of the vulval equivalence group from expressing a 1" or 2" cell fate, so that no vulva is formed. (D) Multivulva. In the absence of the hypodermal inhibitory signal, all six Pn.p cells express a 1 
' or 2" cell lineage, and multiple vulva-like ventral protrusions are formed. Even after the elimination of the anchor cell, multivulva mutants such as fin-15 have a multivulva phenotype. 
The multivulva phenotype of many mutants requires mutations in two genes (HORVITZ and SULSTON 1980; FERGUSON and HORVITZ 1985,1989; J. THOMAS and H. R. 

HORVITZ, unpublished results

). Over 70 such "synthetic Muv" mutations have been identified and divided into two classes, referred to as A and B. Hermaphrodites carrying a class A and a class B mutation have a multivulva phenotype, while hermaphrodites carrying only one mutation or two mutations of the same class have awild-type vulval phenotype. Thus, these class A and class B genes appear to define two functionally redundant genetic pathways both of which must be disrupted to cause the expression of vulval cell fates. 
The first synthetic Muv mutations were discovered when the Muv phenotype of the strain CB1322 was found to require mutations in two unlinked genes, lin-8 11 and lin-9 111 (HORVITZ and SULSTON 1980). Five additional synthetic Muv mutations were fortuitously ob- Of the mutations that caused a synthetic Muv phenotype, six mapped to the same small interval as the gene lin-15, which had been defined previously by single mutations that cause a Muv phenotype (FERGUSON andHORVITZ 1985, 1989). Three of these mutations were in class A, and three were in class B. Complementation tests established that both the class A and class B mutations failed to complement lin-15 multivulva mutations, indicating that lin-15 is a complex locus with three distinct types of alleles: Muv, class A synthetic Muv and class B synthetic Muv. These observations indicate that the lin-15 locus controls two separate activities, one that acts in the class A pathway and one that acts in the class B pathway, and that both of these activities are disrupted in the lin-15 Muv mutants. 
A lin-15 Muv mutant lacking an anchor cell still has a multivulva phenotype, suggesting that its Muv phenotype does not result from the overproduction of the anchor cell signal and that lin-15 functions elsewhere than in the anchor cell to regulate the expression ofvulval cell lineages ( FERGUSON et al. 1987). Genetic mosaic analysis indicates that lin-15 acts noncell-autonomously and likely functions in the syncytial hypoderm that surrounds the Pn.p cells (HERMAN and HEDGECOCK 1990). These observations suggest that lin-15 acts in the hypoderm to inhibit the expression of vulval cell lineages and that the absence of lin-15 function allows the six cells P3.p-P8.p to express vulval cell lineages. Loss-offunction mutations in the receptor tyrosine kinase let-23 gene, the SH3-SH2-SH3 adaptor protein sem-5 gene, let-341, let-60 ras and lin-45 raf suppress the Muv phenotype conferred by lin-15 mutations, indicating that lin-15 acts upstream of these five genes in the genetic pathway for vulval induction (F~RGUSON et aZ. 1987; BEITEL et al. 1990; HAN et al. 1990; CLARK et aZ. 1992, 1993; HAN et aZ. 1993). The action of Zin-15 within the surrounding hypodermis may negatively regulate the let-23 receptor tyrosine kinase signaling pathway. 
We cloned the lin-I5 locus by identifjmg polymorphisms associated with Zin-15 mutations and by rescuing the Zin-15 mutant phenotype by germline transformation. Two adjacent transcripts of 2.3 and 4.6 kb encode the Zin-15 class A and class B genetic activities, respectively. We show that these transcripts are trans-spliced and provide evidence that supports the hypothesis (SPIETH et aZ. 1993) that these transcripts are processed from a single mRNA precursor. The 719-and 1,440amino acid Zin-15 proteins are not highly similar to any known proteins and thus may represent new types of molecules involved in intercellular signaling. 

MATERIALS AND METHODS


Standard methods for culturing and handling C. elegans


Strains:

The following mutations were used in our studies. Linkage group (LG) 11: lin-S(nll1). 
LGIII: lin-9(n112), lin36(n766). 
LGX: lin-I5(el763, n309, ,374, n433, n743, n744, n749, n765, n767, n1139, n1296, n1331, n1344, n2141) nI296, n1331 and n1344 were identifed as spontaneous Muv mutants (KIM and HORVITZ 1990) in the mutator strain TR679 (COLLINS et al. 1987). n1139 was isolated after ?irradiation (FERGUSON and HORVITZ 1985). n2141 (C. BARGMANN, personal communication) as well as the other nine lin-15 mutations (FERGUSON andHORVITZ 1985,1989) were recovered following treatment with ethyl methanesulfonate (EMS). The n433, n 749 and n767 mutations belong to the A class and cause a Muv phenotype in the presence of a class B mutation, such as lin-9(n112) or lin-36(n766). The n374, n743 and n744 mutations belong to the B class and cause a Muv phenotype in association with a class A mutation, such as lin-S(nl11). n 765ts mutants have a temperature-sensitive Muv phenotype because they lack class B function and have a temperaturesensitive defect in class A function. The remaining lin-15 mutants lack both class A and B function and have a Muv phenotype that is independent of other synthetic Muv mutations. 
Analysis of lin-15-associated polymorphisms: Genomic DNA was prepared from Zin-15 mutants raised on 1.5% agarose NGM plates seeded with HBlOl essentially as described (SULSTON and HODGMN 1988). Approximately 5 pg of genomic DNA from each mutant was digested with EcoRI, separated by agarose gel electrophoresis and blotted to nylon filters. The filters were probed with cosmid or plasmid 32P-labeled DNA. All eight lin-I5 multivulva mutants examined and one lin-15 class A mutant, n767, contained EcoRI restriction fragment length polymorphisms. Based on our EcoRI restriction map, we determined the approximate positions and lengths of the deletions and insertions within each fin-15 mutant (see Figure 5). 
Germline transformation and analysis of lin-15genomic region: Germline transformation (MELLO et al. 1991) of lin-8 and lin-15, of lin-9 and lin-15, of lin-36 and lin-15 and of lin-15 Muv mutants was performed by coinjecting test DNA (10-50 pg/ml) and the dominant rol-6(su1008) roller marker (plasmid pRF4 at 80 pg/ml). Transgenic animals typically carry coinjected DNAs as extrachromosomal arrays (MELLO et al. 1991) and are identified by the roller phenotype conferred by pRF4. A rescued F, population was defined as an F, were used (BRENNER 1974). roller population (derived from F, rollers) in which at least 50% of the animals were not multivulva in phenotype. The overlapping cosmids C29B12 and ZC436 each rescued the lin-15(n765) Muv phenotype, suggesting that the lin-15 gene is located in the region common to these cosmids. A number of genomic DNA fragments were subcloned from C29B12 and were similarly tested for the rescue of the Muv phenotype of lin-I5(n765ts), of lin-S(nl1 I); lin-I5(n374) and of lin-9(n112); lin-I5(n433), lin-9(n112); lin-Ij(n749) or lin-36(n766); lin-I5(n767) (see Figure 2B). We determined the DNA sequences of both strands of the 11.7-kb EagI-EcoRI fragment (see Figure 2B) of C29B12 using Sequenase 2.0 (U.S. Biochemical Corp.) or using the AB1 Prism cycle sequencing kit and an AB1 373A DNA Sequencer (Applied Biosystems, Inc.), essentially according to the manufacturers' instructions. Based on the sequences of the two cDNAs (see below), the lin-15 coding regions are completely contained in this genomic DNA fragment. 

Isolation and characterization of lin-15 c D N k

The 10.5-kb BamHI-EcoRI fragment from C29B12 (see Figure 2B) was used to screen approximately 400,000 plaques of a A Z A P cDNA library derived from mixed-stage poly(A)+ RNA (BARSTEAD and WATERSTON 1989). Eleven positive clones were identified; fourwere from the EagI-SalI region, and seven were from the SalI-EcoRI region. We determined the complete DNA sequences of one strand of the longest clone derived from the EagI-SalI region as well as partial sequences of the remaining 10 clones. As none of the cDNAs was full length, we used the method of reverse transcription-polymerase chain reaction (RT-PCR) to clone the 5' regions of the lin-15 transcripts. To amplify the 5' region of the 2.3-kb transcript, we used a primer corresponding to sequences of the lin-15A cDNA (5'CAGAT-TCCATTGACTGGGTAGG) and a primer corresponding to sequences of either the C. elegans trans-spliced leaders SL1 (5'GTITAATTACCCAAGTITGAG) (KRAUSE and HIRSH 1987) or SL2 (5'GGTMTAACCCAGTTACTCAAG) (HUANG and HIRSH 1989). DNA was amplified from 250 ng of mixed-stage RNA using the GeneAmp Thermostable rTth Reverse Transcriptase RNA PCR Kit (Perkin Elmer) and then cloned using the TA Cloning Kit (Invitrogen). We determined the DNA sequences of both strands of two independent clones obtained using the SLl primer and one strand of three independent clones recovered using the SL2 primer. These RT-PCRderived clones contained the trans-spliced leader sequences and lacked putative intron sequences, indicating that they were derived from a processed transcript and not from genomic DNA. The DNA sequence of the 5' region of the 4.6-kb lin-15 B transcript was assembled from the sequences of four overlapping DNA fragments (referred to as B1, B2, B3 and B4) amplified using multiple sets of primers. Fragments B2 and B3 were amplified directly from mixed-stage RNA using RT-PCR, whereas fragments B1 and B4 were generated by a second round of PCR amplification from DNA produced by RT-PCR of RNA. The following primers were used B1, 5'CACACGATGG CITGCAAACG (1st round), 5'CATTGATCGAAGAAGGTGG TCC (2nd round) and 5'GCAGGTTCACCITGGTCTITATGC (both rounds); B2,5'GTGGAATGTCATAGTITGCAACTG and 5'CAAATITAGAAGAATGCAAGm, B3, 5'CATGATGGCTG GCACAACTTGAG and 5 ' A T C T G C G C ; B4, 5'GTlTGTGACGAGACXGITGCT (1st round), 5"- ATITCCGCAGATAMTCGC (2nd round) and SL1 primer (both rounds). We determined the complete DNA sequences of one strand of one independent clone for each fragment as well as partial sequences of additional clones. 

Construction of frameshift mutation clones:

To establish that the lin-15 A and B activities are encoded by the two identified genes, we introduced a frameshift mutation into the predicted coding region of each gene within the SpeI-NruI Subclones were also tested for the rescue of the Muv phenotype of strains defective in either lin-15 A activity [lin-9(n112); lin-l5(n433), lin-9(n112); lin-l5(n749) or lin-36(n766); lin-l5(n767)] or lin-15 B activity [lin-S(nll1); lin-l5(n374)]. Specifically, we used lin-9(n112); lin-l5(n433) for the EagI-Nrul and SnaBI-NruI fragments, lin-9(n112); lin-l5(n749) for the EagI-NheI and SphI-SalI fragments, and Zin-36(n766); lin-15(767) for the SphI-Nrul, SpeI-NruI and SpeI-SalI fragments. The fraction of independently derived transformed F2 populations rescued for the Muv phenotype of lin-lS(n765) animals (A or B Rescue), for lin-15A activity (A Rescue) and for lin-15 B activity (B Rescue) are listed for each clone. +, rescue of lin-15 B activity was observed in 32/33 transformed F, animals, but no stably transformed F, animals were recovered. -, 0/20 transformed F, animals were rescued for lin-15 A activity, and no stably transformed F, animals were obtained. Frameshift mutations (denoted by X ) were introduced in the coding regions of the 2.3-and 4.6kb transcripts present in the SpeI-NruI genomic fragment (see MATERIALS AND METHODS), and these clones were then tested for the rescue of lin-15 A and lin-15 B function. Arrows represent the lengths and directions of transcription of the lin-15A and B transcripts. Abbreviations are as follows: Se ( SpeI), Sp ( SphI) , E (EagI), B ( BamHI) , Sn (SnaBI), S ( SalIj, Nh (NheI) , N (NruI) and R (EcoRI) . 
genomic DNA fragment and tested these altered clones for the rescue of lin-15Aactivity in lin-9(nll2); lin-l5(n433) and B activity in lin-S(nll1); lin-l5(n374) or lin-S(nll1); lin-15(n744) by germline transformation. For the putative lin-15 A coding region, an 8-bp NcoI linker (5'CCCATGGG) was ligated into a MscI site corresponding to nucleotide 757 of the 2.3-kb cDNA (see Figure 4A). This alteration is predicted to produce a truncated lin-15A protein consisting of residues 1-243 of the native protein and an additional 18 amino acids (PWATLEWQLQRKRFVMRG) derived from the linker sequences and the altered reading frame. For the putative lin-15 B coding region, the 8-bp NcoI linker was ligated into a ScaI site corresponding to position 2352 of the 4.6-kb cDNA (see Figure 4B). This alteration is expected to generate a truncated lin-15 B protein containing residues 1-775 of the native protein and an additional 32 amino acids (PWDWPQNYPRRKAHHHQLQLLHKLDPMLPLRN) derived from the linker sequences and the altered reading frame. 
To verify that the failure to rescue the lin-15 gene activity resulted from the frameshift mutation, we restored the reading frame in each modified clone by digesting with NcoI and then filling in the 5' overhang using DNA polymerase I large (Klenow) fragment. This treatment produced a 12-bp insertion (CCCATGCATGGG) at the original site and is predicted to result in the insertion of 4 amino acids (PMHG). The constructs containing the restored reading frames rescued lin-15 gene activity. We confirmed the DNA sequences of the modified region for all four clones. 

RESULTS


Identification of lin-15-specifk polymorphisms:

We cloned the lin-15 gene on the basis of its map position near the sdc-1 gene on the X chromosome (Figure 2A). chromosomes (YACs) (COULSON et al. 1986, 1988; A COUUON and J. SULSTON, personal communication), we obtained cosmids that covered a small region left of the sdc-1 gene (NONET and MEYER 1991). Genomic DNAprepared from four lin-15 Muv mutants (n1139, n1296, nl331, n1344) was probedwith these cosmids to detect possible lin-15-associated DNA rearrangements (see MATERIALS AND METHODS). n l l 3 9 was recovered following mutagenesis by y-irradiation (FERGUSON and HORVITZ 1985), and the other three alleles were isolated as spontaneous mutants in the mutator strain TR679 (KIM and HORVITZ 1990). As y-irradiation often causes chromosomal rearrangements (ROSENBLUTH et al. 1985) and TR679derived mutants often result from transposon insertions (COLLINS et al. 1987), these four mutants seemed likely to have readily detectable lin-15 polymorphisms. All four lin-15 Muv strains were found to contain EcoRI restriction fragment length polymorphisms (RFLPs) in a region covered by two overlapping cosmids, C29B12 and ZK662 (see below). Subsequently, five lin-15 strains ( e l 763, n309, n765, n767, n2141) that were recovered following treatment with EMS were also shown to have EcoRI RFLPs in this region (see below and Figure 5 for description). These results suggest that at least part of the lin-15 gene is located within the region shared by these two cosmids. 
Germline rescue of Zin-15 mutants We tested C29B12,ZK662 and a third cosmid, ZC436, in germline transformation experiments (MELLO et al. 1991) and found that C29B12 and ZC436 each rescued the Muv phenotype of lin-l5(n765ts) animals (Figure 2A). A 14kb genomic fragment ( SphI-NruI) as well as shorter fragments derived from the region common to C29B12 and ZC436 also rescued the Muv phenotype of lin-15(n756ts) mutants (Figure 2B). As the Muv phenotype of lin-15 mutants requires the loss of both A and B activities, the recovery of either A or B activity alone would be sufficient to rescue the Muv phenotype of lin- 15(n765ts) animals or other lin-15 Muv mutants. To assay specifically for lin-15 A function, genomic DNA subclones were tested for the rescue of the Muv phenotype of a double mutant strain containing a class B mutation, lin-9(nll2) or lin-36(n766), and a lin-15 A mutation. As these strains do not lack lin-15 B function, the rescue of the Muv phenotype would indicate that the tested genomic DNA fragment had lin-15 A rescuing activity. We found that the ll-kb EagI-NruI and longer fragments contained lin-15 A activity because they rescued the Muv phenotype of the lin-9(nll2); lin-15(n433), lin-9(n112); lin-l5(n749) or lin-36(n766); lin-l5(n767) double mutant (Figure 2B). No rescued transformants were recovered using a fragment ( SphI-SalI or SpeI-Sun) that lacked the 3.8-kb SalI-NruI region, suggesting that this region is needed for the rescue of lin-15 A function. However, this region alone is not sufficient for lin-15 A rescue, because no rescued transformants were obtained using the 4.9-kb SnaBI-NruI fragment. To assay specifically for the rescue of lin-15 B function, we used a double mutant strain containing a class A mutation, lin-8(nl1 l ) , and a lin-15 B mutation. In contrast to the results for lin-15 A rescue, the EagI-NheI and SphI-SalI fragments rescued the Muv phenotype of the lin-8(nlll); lin-l5(n374) double mutant (Figure 2B). These results indicate that the 7.2-kb region common to these two fragments is sufficient for the rescue of the lin-15 B defect in the lin-8(nlll); lin-15(n374) double mutant and, by inference, in the lin-15(n 765ts) Muv mutant. Together, these data indicate that the 1 l-kb EagI-NruI genomic DNA fragment contains both lin-15A and B gene activities. While the EagI-SalI region is needed for the rescue of both lin-I5 functions, the 3.8-kb SalI-NruI region is essential for only the rescue of lin-I5 A activity. 
Zin-15 encodes two different polypeptides: We determined the DNA sequences of the 11.7-kb EagI-EcoRI genomic fragment and of multiple cDNAs derived from this region (see MATERIALS AND METHODS). As none of the cDNAs isolated from a mixed-stage cDNA library was full length, the DNA sequences of cDNAs obtained by the method of RT-PCRwere also determined (see MATERIALS AND METHODS). From our analysis of the cDNA and genomic DNA sequences, we determined that there are two separate, similarly oriented transcripts that are 2.3 and 4.6 kb in length (Figures 3 and4). cDNAs for the downstream 2.3-kb transcript were generated by RT-PCR using an oligonucleotide primer for either the SLl or SL2 trans-spliced leader (KRAUSE and HIRSH 1987;HUANC and HIRSH 1989). These results show that SL1 and SL2 are used for the processing of the 2.3 kb transcript, but do not indicate the relative abundances of SL1and SL2containing transcripts. cDNAs for the up-processed with SL1 and not with SL2. There are 110 stream 4.6 kb transcript were generated using an oligo-nucleotides between the polyadenylation site of the nucleotide primer for SL1 but could not be recovered 4.6-kb transcript and the splice-leader acceptor site of using a primer for SL2, suggesting that this transcript is the 2.3-kb downstream transcript (Figure 4C). DNA sequences encoding the 4.6-kb transcript are located within the 7.2-kb EagI-SalI fragment sufficient for rescue of lin-15 B function, while DNA sequences encoding the 2.3-kb transcript, except for the first exon and part of the first intron, are present within the 3.8-kb SulI-NmI fragment required only for rescue of lin-15A activity. In addition, the lin-15A mutant n767 contains a 300-bp deletion within the 3-kb EcoRI fragment that contains part of the coding region for the 2.3-kb transcript (see below). These observations sug-gest that the 2.3-kb transcript encodes lin-15 A gene activity and that the 4.6-kb transcript encodes lin-15 B activity. 
T T G T C T C O C A ~~~G A A T G ~C G V S A D E K E E I I A K R K F R M K N V D A M R M S S L A N D R M A F N K K C N T T G A O C T A T O C G A A T G T C M ~A T G T C A A G C T T G G C G A A C O A T C O G A T O A L A M K F V K S A G I G T D A L Q L T ATGCCCTAGCGAT'GAAGTTTGTTAAAAGTGCCGGCATTGGWCGGATGCTCTGCAGCTCA C F Q E L V R H F N P I A A V V V G V K CATGWTCCAGGAGCTCGTCCGOCATTTCAACCCAATTTCAACCCAATTGCCGCAGTTGTTGTTGGTGTCA R E P N S N V Q A E K K T I P X V K T I Q T P T Q S M E S V R L L Q E K K A S A T E E Q S A E S A S I M K H F A N T I P CAACTGAAGAACAATCAGCTGAGTCTGCTTCGATCATGAAGCATTTPGCGAATACAATTC N S T P T Q S V K D V L T A A A S K G Q C A A A C T C R A C A C C A A C G C A A A G C G T G A A G G A ~~~A C G G C ~T G C A A G C ~A C

F K S S A E I F S H F P S E P S P S K P


AATTCARAAGTAGCGCTTCACACTTTCCAAGTGAACCCAGCCCCTCGAAAC


R A T R E G S Q P S D Y T Y C T Y L T P C C C G T G C C A C A C O C G A G ' T G A C T A C A C G T A T T G T A C C T A T C T G A C T C


C I L C E K A L L M R E S I A M T D N E CGTGCATACTCTGeGAGAAOOCPCTTTTTAATGeGAGAAAGCA'RCCAATGACTGATAACG A V K V L M A A V M S G H F R M A T A E A A ~~A A A G T T T I C A T G G C T O A T O O C O O C R O T G A T G T C r C

K A I R H E R L R M C Y D H V D F V Y E M M C D A F E A K T E S E I N E M P P D AAATGATGTGCGACGCCTTTGAAGCTAAAACGGAAAGCGAGARRGCGG R L M R G H D I Y R A L K R V G D L H K G K V T S N T P L Y S F K N S I K S Y Y AAGGGAAGGTTACGTCGAACACTCCGCTTl'ACTCATTCAAGAATTCCATTAAATCTTATT R N H V P R M V N G S L S K P S P K P F ACCGTAACCACGTGCCAAGGATGGTCAAOOATGGTCTCTCAGTAAACCGTCTCCCAAACCGT S E L V A L L Q S V P P S T N L N E L L N H N L S L S D A D K Q E L I Q L I N G TGAATCATAATCTTAGCCTCAGTGATGCTGATAAGCAAOAACTCATTCAACTCATCAATG K D N R F T S R R R K I E D I L D N K F GGAAAGATAATCGTTTCACATCTCGGAGACGGAAAATCGAGGATATTCTXACAACAAGT A A A A A K A Y R D H S E D A P S E P Y TTGCTGCWAGCTGCAUATOCAAAAGCTTATCGAGATCACAGTGAAGAWACCTAGTGAACCGT I P N Q S E M Q N T V E R R K R X L H S ATATTCCCAATCAAAGTGAGAWAGAACACTGTAGAACGCAGAAAGCGAAAGCTTCATT P E Q D D A G S

S S I S W N A K K T X T CGCCTGAACAGGATGATGCTGGATCATCGAGTATCTCATGGAAC-CGAAAA P I D Y V H L A T R V L E G H S I A D E CTCCAATICACTATGTTCACCTAGCAACGCGTGTTTTGGAAGGTCATTCM'NGCCGACG A L L H K S K V S Y A R N A F G E X P S AGGCGCTTTXCACAAATCCAAAGTTTCTTATGCTCGTAATGCATTKGTGAGAAGCCAA S P T P P S

A P L K F C V V N G X X Y L GCTCCCCCACTCCGCCATCGCCACTAAAGTTCTGTGTTGTCAATGGAAAGAAATACT R F E N G T G P P K V V V Q G N V V L R TGCGTTTTGAAAACGGAACEGACCTCCGAAAGTXTAGTTCAAGGGAATGTCGTTCTTC T

N T L K D A L T T A P R A Q N Q P S T G C A C T A A T A C K T T P A A G A C G C A T X A C T A C A O C O C C A C T A


S T D S S S S S E M E G I R Q S F G A P

CGTCCACAGACTCATCAAGCTCATCCGAGATGGAGGGAATACGTCAATCATTTGGGCGC Q K E E E E E E L V P T L L Q N K P T H CTCAAAAGGMGAAGAAGAAGAAGAATTAGTACCTACGCTTCTTC~CAAACCTACCC V E S S S P V E K K P P T K T N V E K P ACGTOULATCTTCGAGCCCCG-CACCAACCAAAACOAACGTAGAGAAAC A V R L G R M L T T A F G S M S Y R T R CAGCGGTCCGTCTAGGAAGAATGTTAACGACTGCATTKGTTCAATGAGTTACCGAACAA K S V E N K T D L L N Q P T S A S P R R GAAAATCGGTAGAGAATAAAACGGATCTCTTAAATCAGCCAACATCTGCCTCACCAAGAC M I K V V R N R N P H L A X Q V A A A P G C A T G A T T A A A G T T G T G A G A A A C C G A A A T C C T C A C C T T G C

S E P K H I P P T H M E K K P E E L L M


CGAGTGAGCCAAAACATATTCCGCCAACGCACATGGARAAGAAGCCGGAAGAGTTGCTCA


D P K P E P I F * TGGATCCAAAGCCTGAOCCAATTTTTTAAACGTTCTTGATAACTTTGATTATTATClTlY A T G T T A C T T G T T P TAGTTCAATTTCATGTTTTTTTTAAACTGTITTTTTCTCATCACAATCGATCTTATAAAT

T C T G G C G T T P T C O T T G T A C G A G C G C A A A A A T G C G A T T T A T C T L A F S I H Y F E D L Y E R K N A I Y L R K L L L T E L D S N S L L T N I R R S CGTCAACAGCTACAGCTTCTCAAATGTCAAGTTTACGAACATTGTGTGTCWCGAAGA V N S Y S F S N V X F T N I V C P N E E GATTTGCAAGCTGGTTGAAGAGAGTGCAGTTGTCAAACGATACAACGTTRXTTCTACAA I C K L V E E S A V V X R Y N V C F Y N T A A T T A T G C A A A C G C T R G A C T T A C A T C AAACCCGOCATTTCAACCCAATCAAT~CGACTTCCAGCAGTAGCAGTGCAA~GCGCAGCAGCAATTCA GCGGAAATTGCTACTTACCG+CTAGACAGCAACAGTCTCCTCACAAACATTCGTCGTTC CTACGTTACCCGTTTTGTGGCCGATPTGATGGATGGAAATTGAAGAGTTTTCCAGTGGGCTGAC Y V T R F V A D L M E I E E F S S G L T ACAA'ITGCGAACATTTGTTCGTTATATGAAACAAAATTCGGATATGTATAGCAAATTTAG Q L R T F V R Y M K Q N S D M Y S K F R ~ ~~~ ~ AAC~AATGCAATTGCAAAAGAATGCGGAACTCGACATTCCGAGTATTGACTCGACATTG GCATTCCACTGCGATTTTCCTCACAAGATGTCTICTTF+ACGACACGTTTACCGAGTT R M Q L Q K N A E

L D I P S I D S G D W H S T A I F L T R C L V W H D T F T E F C G K L D I L H Y I D N E T F N H L I Y

CTGTGGAAAATTAGACATCCTCCACTACATTGACAATGAAACATTCAACCACCTTATATA L Q R L L Q Q C M K H C R

E L S I P N N CCTCCAGCGCCTTCTTCAACAATGCATGAAACATTGTCGAGAGCTCAWATACCGAACAA S I S Q V V P A I M S I R N F I A S N S T T C G A T T T C T C A A G T T G W C A G C C A T C A A T T C G C C T C


M G Y R F Q K R I R D S F T T S F K E I


AATGGGATATCGTTTCCCCGCGATTCGTTTACTACWCATTCAAAGAGAT T S G P S Q D R Y D I A T L L D P R F A TACTTCAGGACCATCACAGGATCOATCGATACGATATTGCTACTCTT'ITGGATCCACGT~

Y R D T V Y T A Q T W R S L E X K V I D CTACAGAGATACGGTTTATACAGCACAAACCTGGAGATCGCTCGAGAWAAAGTCATCGA D F V N S D L Q N D K N F Y Q D I S I L CGACTTTGTAAACTCTGATCTGCAAAATGACAAAAACTTTTATCAAGATATTTCGATCCT N Q E Q R Y D I I X K E F A Y Y R Q T S

GAATCAAGAACAGCGCTATGATATTATTAAAAAGGAGTTIGCTTATTATCGTWCTTC


F V E R P E E N E N S N H W W G M R Q T ATTTGTCGAGCGGCCCGAAGAGAATGAGAATTCGAATCATTGG'TGGGGAATGCGTCAGAC D M E F L A V I A R E Y L A S P A V S I


GGATATGGAATTCTGATTGCTCGTGAATACTTGGCMGTCCTGCAGTTTCTAT


D A G Y Y F G N G G X F Q H I C H T Y S AGACGCTGGGTACTACTMGGGAACGGTGGAAAATTCCAACACATCTGCCACACCTACTC H Q R L E N C L A L A G N Y Q T F R G K TCATCAACGCCTCGAGAACTGCTTGGCACTTGCXGAUCTATCAAACATTTCGTGGAAA G A S V D V I S Q S M I E T L N N T A S AGGAGCATCGGTGGATGTCATTTCTCAATCAATGATTGAGACTTTAAATAACACTGCAAG R L Q K Q V H L G L Y A H G V D N I S S TCGTCTTCAOAAGCAGGTTCACCTTGGTCTTTATGCACACGGAGTTGACAACATCTCTTC D R D V Q S I V G H H Y P P M P T V A N TGATCGAGATGTGCAATCCATTGTCGGCCATCACTACCCTCCGATGCCAACAGTTGCAAA Y D I P H V P K E E E K P P V A N L Q S


CTATGACATTCCACACGTGCCGAAAGAAGAAGAGMGCCTCCAGTGGCTAACCTGCAAAG


T S S P A T S S P T I I R P R A A P P P TACATCTAGCCCAGCGACGTCCTCTCCMCAATCATCC~CCTCGTGCAGCACCTCCACC R T L A Q G R P I P L N G K E L X A V P GAGAACACTGGCTCAAGGAAGACCAATCCCGTTGAAEGGAAAGAACTCAAGGCTGTTCC I R Q I P L Q V R P L P P R P A N V P I


AATCAGGCAGATTCCGCTGCAAGTGAGWCGTTGCCACCGAGACCAGCCAATGTGCCAAT


V P R P T V P Q Q F I K A P A P K P I T TGTGCCAAGGCCAACTGTTCCACAACAGTTTATWGCACCAGCTCCGA&ACCTATCAC


L Q A V V C S I P E K E I K K E T E D V ACTTCAAGCTGTTGTTTOCAGTATTCCAGAAAAGGAAATCAAGAAAGAAACTGAAGACGT A L L E K I K D E P L D E D D F N H P S AGCGCTGCTGGAGAAAATAAAGGATGAACCACTGGATGAAGACGATTTTAATCATCCTTC


GARAATTGTAGTACTGGCCTCAAAACTACCCACGTCGCAAAGCTCATCACCATCAACTGC K I V V L A S K L P T S Q S S S P S T A AACTTCTGCACAAGCTAGATCCCATGTTACCACTGCGCAACTGATCCGATGTGGGCCAAG T S A Q A R S H V T T A Q L I R C G P S T O A l i O G C A C C G T T C C A C A C A T T C A C A C A A C l T T G T G C A G

A A G T ~X W E G T V P Q K I H S H N F V Q K F A Q K Q N F V H K Y A L N S Q D H T G R L N Q AACTGTACCCATGAGAGCTGCGCTGAGGCTTCCAAACAGCGAACAAAAGTCAGGAGCACC T V P M R A A L R L P N S E Q K S G A P TTCTTCGATCAAnGTACAGAGGGATGACTTCmTTGGAACCACTGGACGATW S S I N G K V Q R D D F K L E P L D D F C A A C G G A G A A C C C G A C T A T G A C A A W T A A ' I T G G G G C T C R N G E P D Y D N L I G A Q R L M Y S D N L N D A S A E D A F A R H R V T M E F Q GAAGCGCAGAGCTTGCAACCGTCGCTGTGCAGTATGTGGTCACTTGGAAATCCACGAGCG K R R A C N R R C A V C G H L E I H E R TCTGAAGAATGTGACGATTGAAAATGAAAAGCTGTTGATTATGTTGGGCTGTATTTATCG L K N V T I E N E K L L I M L G C I Y R CGGAGAATTTACTCTAGGACAGGCACAGCTTTTCATGGCGCGTGAGAGCARAACGTACAT G E F T L G Q A Q L F M A R E S K T Y I TTGCCGTCTCCATTTCCTGGAGACCCTCGACGAGATCTACCAGATGCTACGTCTG~TC C R L H F L E T L D E I Y Q M L R
R N N H L R E T P A E L K K L G Q Q Y F TGACTACCGCGAGCCCGAACCTGAGCCAGCCAGAGAGAAACGATGTGGATGAGCAAGAAATCAT D Y R E P E P E P E R N D V D E Q E I I ACCGAAACTCTTTCGCCAGCCACGCAAGCAAGTTCTGGAAGCTGATCAGCATGACGGAAC P K L F R Q P R K Q V L E A D Q H D G T TGTCAAGGTTATAGAGCAGGAAGATTTCAAGCTCCCAACTGTCAAACCGTCTGWTGA V K V I E Q E D F K L P T V K P S E N E GGAATGCGACAATCCGGGCGTTTGCTGCTTCTGTTCGAAACGTGGCGACCGAGGWAAT E C D N P G V C C F C S K R G D R G G M GCTTCGTGTCCCGAGAAGTGAGGAACGTTTGGCTAGATGGGTGGATMGCTTGGACCAGA L R V P R S E E R L A R W V D K L G P E ATTTGAAGCCCGACTTCACACCAATACGGWCCTAATTTGCCGmGCCATTTCCCm F E A R L H T N T E N L I C R S H F P D TGCAGCGTTCAGCAGTCGTGGTAGACTTTTGAAAGGAATGATTCCAGATGCTGCTCCAGA A A F S S R G R L L K G M I P D A A P E AAAAGTTOAGACCACGTATATCATTCAAGGGAACAACTTCCTCWCXAAAGAGCGCAA K V E T T Y I I Q G N N F L K L K E R K GTCTGGAACTGATAAGAATTCAGCCATTGATTTGGCGAATATGTTWCCCCGACGGTGT S G T D K N S A I D L A N M L N P D G V TGAATATACTCAGGAAGAAGAAGAAGAAGAGGAATATGAAGAAATGTCAAGATCCCCAAC E Y T Q E E E E E E E Y E E M S
ACGTGCACTTAAGAAAGGTTACGTACAACTCGAAGACGGAGAAATTGTCGGCGAAGATTG R A L K K G Y V Q L E D G E I V G E D C CGAATACGTGCCTGAAAAAACACCAAGTGGAAGATTGATTCGACAAGCTGTTGCTAGACG E Y V P E K T P S G R L I R Q A V A R R AAGCTTTGCTTTTGCTGATGAGGAAGAGGAGGAGGAAGAGTATGAAGAATCTCCAATTGT S F A F A D E E E E E E E Y E E S P I V K K P K I A G R P V G R P R K D A N K L TMAAAGCCCAAGATTGCTGGAAGGAAGPJCCAGTCGGACGACCGCGCAAAGATGCTAATAAGCT

GCCAACCCCCACACCACCCAGCAACGAATMGTGGAAATATTTTCATCCACCGTTCCTGA TTGTTGTTTTTATATATAATTTTTTCTCCATTGATGTTTTTGATGATTAAGTCATACGCA


TTTAATCATCATTCATCTGAAACCTCACAATGATGAGATGATCTCATCA~TATATTT


P T P T P P S N E ' TAGTTTTACCCTGTTACTTTGATTGCCCTTWTTACCACTTTTATTTTACCTAT"AT AAA T G T A G T T T T A T A T C A G T T T T A T A A A T G T T C A C C G P

To confirm that these two transcripts encode the two lin-15 proteins, we introduced frameshift mutations into the putative lin-15 coding regions within the SpeI-NruI genomic DNA clone and then tested these modified clones for the rescue of lin-15 A and B function by germline transformation (see MATERIALS AND METHODS). We found that a SpeI-NruI genomic clone containing a frameshift mutation within the predicted coding region for the downstream 2.3-kb transcript rescued lin-15 B but not lin-15 A function (Figure 2B). A clone altered to restore the appropriate reading frame rescued lin-15 A function in 6/6 transformed F, populations, indicat- ing that the introduced frameshift mutation within the coding region of the 2.3-kb transcript caused the loss of lin-15 A rescuing activity. By contrast, a SpeI-NruI genomic clone possessing a frameshift mutation within the coding region for the 4.6-kb transcript rescued lin-15 A but not lin-15 B activity. A clone modified to restore the proper reading frame rescued lin-15 B function in 16/16 transformed F, populations, demonstrating that the frameshift mutation within the coding region of the 4.6-kb transcript eliminated the lin-15 B rescuing activity. Together, these results establish that the 2.3-kb transcript encodes lin-15 A activity and that the 4.6-kb transcript encodes lin-15 B activity. 
Based on the DNA sequences, the 2.3kb lin-15A cDNA encodes a 719-amino acid protein and the 4.6-kb lin-15 B cDNA encodes a 1,440-amino acid protein (Figure 4, A and B). The two lin-15 proteins are not obviously similar in sequence to each other, and a search of the Gen-Bank, PIR and SWISS-PROT databases using BLAST (ALTSCHUL and LIPMAN 1990) failed to identify any protein highly similar to either. Thus, the lin-15 proteins define new types of molecules involved in intercellular signaling. 
Zin-I5 Muv mutations are rearrangements: To investigate the molecular basis of the different classes of lin-15 mutations, we analyzed genomic DNA prepared from Zin-15 Muv mutants as well as from lin-15 mutants defective for either class A or class B activity (see MATERIALS AND METHODS). All eight lin-15 Muv mutants analyzed, including four carrying EMS-induced alleles, had chromosomal rearrangements in the lin-15 coding regions: seven mutants ( e l 763, n309, n765ts, n1139, n1296, n1331, n1344, n2141) contained deletions and one, n1296, had a deletion and a 1.6-kb insertion (Figure 5). The lengths of the deletions varied from about 200 bp in the n 765 mutant to at least 15 kb in the e l 763, n309 and n1344 mutants. The e l 763, n309, n1139, n1344 and n2141 Muv mutants had deletions that affected all or most of the genomic region encoding the two lin-15 transcripts. The 4kb deletion present in the nl??l mutant removed part of the lin-15 B coding region and sequences 5' of the lin-15 B gene. Unless a second, undetected mutation exists in the n1??1 strain, this alteration caused the loss of lin-15 A and lin-15 B function. The requirement for the region deleted in the n1??1 strain for the expression of both lin-15 activities is consistent with our germline transformation results, which indicate that this region is essential for the rescue of both lin-15 
A and B function. The n1296 mutant had a 1.6-kb insertion within a region encoding the lin-15 B gene and a 200-bp deletion in the 3-kb EcoRI fragment that contains part of the lin-15 A gene. n1296 was identifed as a spontaneous Muv mutant in the mutator strain TR679 (KIM and HORWTZ 1990). The 1.6-kb insertion might be either a Tcl or a Tc4 transposon, both of which are about 1.6 kb in size (ROSENZWEIG et al. 1983;YUAN et al. 1991). 
The n 765ts mutant has a temperature-sensitive defect in lin-15 A activity and lacks lin-15 B activity, resulting in a temperature-sensitive Muv phenotype (FERGUSON and HORVITZ 1989). The 200-bp deletion in the n 765ts mutant is within the genomic region encoding the lin-15 B transcript and likely disrupts lin-15 B function; it is unclear whether this deletion or another lesion, perhaps a point mutation within the lin-15A gene, confers the temperaturesensitive defect in lin-15 A function. This deletion might lead to a temperaturedependent loss of lin-15 A function if it caused the elimination of the lin-15 B protein and if the lin-15 A protein were unstable at higher temperatures in the absence of lin-15 B protein. If so, the three lin-15class B mutations (n?74, n74?, n744) must not eliminate the lin-15 B protein, as they do not cause a temperaturedependent loss of lin-15 A function. 
In short, the alterations within all lin-15 Muv mutants examined, except possibly n 765ts, affected regions defined by germline rescue experiments to be required for both lin-15 A and B activity. No obvious chromosomal alterations were found in two of three EMS-induced lin-15 class A mutants (n433, n749) or in three E M S induced lin-15 class B mutants (n374, n 74?, n 744) (data not shown). The lin-15 class A mutant n 767 had a 300-bp deletion within the 3-kb EcoRI fragment that encodes part of the lin-15 A gene (Figure 5). DISCUSSION lin-15 is a complex locus that encodes two separate genetic activities involved in the negative regulation of vulval induction ( FERGUSON and HORVITZ 1989). We identified an 11-kb genomic fragment that complemented the loss of both of these activities when maintained as an extrachromosomal array following germline transformation. The analysis of cDNAs and genomic DNA sequences indicated that two distinct, non-overlapping and similarly oriented transcripts of lengths 2.3 and 4.6 kb encode the two lin-15 gene activities. The 4.6-kb transcript encodes lin-15 B function and is processed by the addition of the SL1 trans-spliced leader. The 2.3-kb transcript encodes lin-15 A function, is processed by the addition of the SL1 and SL2 trans-spliced leaders and is located 110 bp downstream of the polyadenylation site of the 4.6-kb transcript. 
The mRNAs of six C. elegans genes are trans-spliced to SL2, and each of these genes is located between 96 and 294 bp downstream of a similarly oriented gene Using the technique of RT-PCR, we recovered cDNAs for the lin-15 A gene using primers for sequences of either SL1 or SL2. These results indicate that the downstream Zin-15 A transcript is processed by trans-splicing using SL1 and SL2, although the relative abundances cannot be estimated from our data. Similar results were reported for transcripts of the downstream gpd-3 gene and were suggested to reflect a variability in the selectivity of the trans-splicing process for polycistronic precursors (SPIETH et al. 1993). The lin-15 A transcript might also be alternatively trans-spliced in this way. On the other hand, the SLl-containing transcripts might originate from another promoter located within the lin-15 B gene and not be derived from a polycistronic precursor. 
The class A and class B synthetic multivulva genes define redundant genetic pathways involved in the negative regulation of vulval induction (FERGUSON and HORVITZ 1989). Although multiple class B genes are located on chromosome 111, the class A and class B genes are not clustered, except for the lin-15 locus. Our analysis provides a molecular basis for understanding the Zin-15 complex locus but fails to explain why the lin-15 A and B genes are clustered and apparently cotranscribed. Perhaps the cotranscription of the lin-15 A and B genes ensures that both genes are expressed within the same cell and at similar levels. 
The Zin-15 null phenotype is Muv: The phenotype caused by lin-15 mutations in trans to a deficiency of the locus suggests that the known lin-15 Muv alleles may not completely eliminate lin-15 gene function (FERGUSON and HORVITZ 1985). Specifically, the phenotype of lin-l5(n765ts)/rnnDf4 animals at 25" is more severe than that of lin-I5(n765ts) homozygotes at 25". lin-15(n 76%) animals raised at 25" have a highly penetrant Muv phenotype similar to that of Zin-I5(n309) and other strong Muv alleles; most n765/mnDf4 animals raised at 25" grow to the size of L3 larvae and are sterile, which is a phenotype only occasionally exhibited by Zin-l5(n765ts) animals grown at 25". At 20", the deficiency does not enhance the lin-15 phenotype. A null mutation in trans to a deficiency uncovering that locus is likely to cause the same phenotype as a homozygous null mutatjon, which suggested that the strong lin-15 alleles may not be null. However, deficiencies often remove large chromosomal regions containing many genes, so haplo-insufficiency could cause strains heterozygous for a particular mutation and a deficiency to have a more severe phenotype than homozygous mutants even if the mutation is null. Our molecular analysis revealed that some Muv mutations delete the complete lin-15 coding region and thus cannot possess any lin-15 function. For example, the e l 763 mutation deletes the entire Zin-15 genomic region, while the n309, n1139, n1344 and n2141 mutations remove most of this region. Thus, e l 763 and most likely the other strong lin-15 mutations are true null alleles. 
Although Preliminary data suggest that let-23 and let-60 are expressed in P3.pP8.p (M. KOGA and Y. OHSHIMA; J. DENT, L. AVERY and M. HAN, personal communications), suggesting that these genes as well as sem-5, let-341 and lin-45 act in these cells to induce the differentiation of vulval cell types. Thus, the action of Zin-15 within The sequences of the 719-and 1,440-amino acid lin-15 proteins do not suggest how lin-15 A and lin-15 B might function. One possibility is that a hypodermal signal involving lin-15 directly inhibits the let-23 receptor tyrosine kinase (Figure 6A). Since neither of the lin-15 proteins contains a hydrophobic signal sequence, this hypothesis would suggest that products of other synthetic Muv genes of both the A and B classes directly mediate these intercellular interactions. The inhibition of let-23 activity could be either directly from the hypoderm or indirectly from within the cells P3.p-P8.p, in which case we expect some synthetic Muv genes to act within these cells. The inhibitory signal from the hypoderm might be similar to the signal involving the Drosophila gene argos (FREEMAN et al. 1992). Mutations in the argos gene cause the overproduction of photoreceptors within the developing fly eye analogously to the proliferation ofvulval cells that occurs in lin-I5 mutants. argos functions non-cell-autonomously, and, based upon its DNA sequence, is predicted to encode a secreted protein. 
Alternatively, the Zin-15 A and B proteins might act in parallel to prevent the hypoderm from expressing a signal that inappropriately causes vulval induction. For example, in the absence of lin-15 function, the hypoderm might release a factor similar to the lin-3 protein, which is related to TGF-a (Figure 6B). 
The lin-15 A and B genes encode unfamiliar molecules involved in intercellular signaling. Receptor ty- rosine kinase/ras signaling pathways highly conserved with the C. elegans vulval induction pathway have been identified in both Drosophila and mammals, suggesting that molecules similar to the lin-15 A and B proteins exist in these organisms. The further study of lin-15 and other synthetic Muv genes may lead to a greater understanding of C. ekgamvulval induction as well as of the regulation of other tyrosine kinase/ras signaling pathways. 

Francisco,

  California 94143. I Current address: Department of Anatomy, University of California, San Genetics 137: 987-997 (August, 1994) 




  FIGURE 1.Vulw.l cell lineages and models for v u l d formation (adapted from SULSTON and HORVITZ 1977; BEITEL et aL 1990). (A) Cell lineages that generate the vulva. Each P3.pP8.p cell 




  FIGURE 2.-Genetic and physical maps of the lin-15 region of the X chromosome. (A) Genetic map with cosmid clones r e p resenting this region shown below. Cosmids were tested for the rescue of the Muv phenotype of the lin-l5(n 765ts) mutant when maintained as extrachromosomal arrays after germline transformation. The fraction of independently derived transformed F, populations rescued for the Muv phenotype of lin-l5(n765) animals is listed for each clone. (B) Restriction map of the central region of C29B12 and structures of genomic subclones. Genomic subclones were tested for rescue of the Muv phenotype of lin-l5(n 765) animals; a subclone containing either lin-15Aor lin-15 B function could rescue the Muvphenotype of lin-l5(n 765).Subclones were also tested for the rescue of the Muv phenotype of strains defective in either lin-15 A activity [lin-9(n112); lin-l5(n433), lin-9(n112); lin-l5(n749) or lin-36(n766); lin-l5(n767)] or lin-15 B activity [lin-S(nll1); lin-l5(n374)]. Specifically, we used lin-9(n112); lin-l5(n433) for the EagI-Nrul and SnaBI-NruI fragments, lin-9(n112); lin-l5(n749) for the EagI-NheI and SphI-SalI fragments, and Zin-36(n766); lin-15(767) for the SphI-Nrul, SpeI-NruI and SpeI-SalI fragments. The fraction of independently derived transformed F2 populations rescued for the Muv phenotype of lin-lS(n765) animals (A or B 




  FIGURE 3.-Zin-15 genomic structure deduced from cDNA and genomic DNAsequences. The protein coding regions are denoted as solid boxes and the 3' untranslated regions are shown as open boxes. SL1 and SL2, the truns-spliced SL1 and SL2 leader sequences, respectively. The downstream Zin-15 A cDNA is also processed with SLl (not shown). ATG and TAA, the predicted start and stop sites of translation, respectively. AAA, poly(A) addition sites. 


FIGURE 4 .

 4 FIGURE 4.-Nucleotide and deduced amino acid sequences (single-letter code) corresponding to lin-15 cDNAs. (A) Nucleotide sequence of a composite lin-I5 A cDNA assembled from sequences of cDNAs obtained by RT-PCR (nucleotides 1-337) and cDNAs isolated from a A cDNA library (nucleotides 338-2,313) (see MATERIALS AND METHODS). The nucleotides derived from the 22nucleotide trans-spliced leader SL2 (HUANG and HIRSH 1989) are underlined. Nucleotides are numbered on the left beginning with the SL2 sequences. Amino acids are numbered on the left in italics beginning with the first predicted methionine. The positions of splice sites as determined by comparison with genomic sequences are marked with vertical lines. The ochre termination codon is denoted as an asterisk. (B) Nucleotide sequence of a composite lin-15 B cDNA assembled from sequences of cDNAs generated by RT-PCR (nucleotides 1-2554) and cDNAs isolated from a A cDNAlibrary (nucleotides 2554-4623) (see Materials and Methods). The nucleotides derived from the 22-nucleotide trans-spliced leader SL1 (KRAUSE and HIRSH 1987) are underlined. The ochre termination codon is denoted as an asterisk. (C) Nucleotide sequence of the lin-15 A and B intragenic region. The sequence shown begins at the polyadenylation site of the .!in-15 B transcript and ends at the splice leader acceptor site of the lin-15 A transcript. 




  Downloaded from https://academic.oup.com/genetics/article/137/4/987/6012608 by Indiana University -Bloomington user on 26 August 2023 




  FIGURE 4.-continued 




  FIGURE 5.-lin-15 Muv mutants contain chromosomal rearrangements that af€ect both the lin-15A and B coding regions. Based on the analysis ofEcoRI restriction fragment length polymorphisms, the approximate position of the deletion and insertion is shown for each Muv mutant and for the lin-15 A mutant n767. For example, the n765ts mutant contains a 200-bp deletion within the 2.1-kb EcoRI fragment; a 200-bp gap is shown within the center of the region, as the precise location within the 2.1-kb fragment was not determined. The genomic region not analyzed is depicted as a dotted line. 


(

  SPIETH et al. 1993). In particular, SPIETH et al. reported the unpublished conclusions of L.HUANG and P. STERNBERG that the lin-15 A gene is located downstream of the lin-15 B gene and that the lin-15 A transcript is trans-spliced to SL2. HUANG et al.(1994) independently cloned and characterized the lin-15 locus and obtained results similar to ours. SPIETH et al. proposed that those gene clusters are transcribed as a single polycistronic mRNA precursor and that the mature transcript for the downstream gene is formed by cleavage at the polyadenylation site and by trans-splicing to SL2.The results of our germline rescue experiments and the site of the deletion in the lin-15 Muv mutant n1??1 are consistent with the cotranscription model proposed by SPIETH et al. for the expression of the two lin-15 transcripts. For example, if the lin-I5 A and lin-15 B genes were transcribed as a single mRNA precursor, the expression of the downstream lin-15A gene would require expression of the upstream lin-15 B gene. We found that rescue of lin-15 A gene activity required the 7-kb region upstream of the lin-15 A coding region that alone rescued only lin-15 B function and contained the lin-15 B gene. A frameshift mutation within the lin-15 B gene eliminated the rescue of only lin-15 B gene function and not the rescue of lin-15 A gene activity, suggesting that Downloaded from https://academic.oup.com/genetics/article/137/4/987/6012608 by Indiana University -Bloomington user on 26 August 2023 lin-15 Encodes Two Proteins 995 the expression of the downstream lin-15 A gene required the transcription but not the translation of the upstream lin-I5 B gene. The deletion present in the Muv mutant n1331 removed part of the Zin-15 B coding region and sequences 5' of the Zin-15 B gene. Unless another lesion exists in the n1331 strain, this deletion confers the loss of both lin-15 A and B activities, indicating that this region is required for the expression of both lin-15 functions. Together, these results support the cotranscription model proposed by SPIETH et aZ. (1993) but do not exclude the possibility that the promoter for the lin-15 A gene is present within the lin-I5 B gene. 




  the class A and class B pathways are redundant for the formation of the vulva, some mutations in the class B pathway affect viability and fertility in the presence of a functional class A pathway, indicating that some genes in the class B pathway may have unique functions outside ofvulval formation (FERGUSON and HORVITZ 1989). In particular, three class B mutations lin-9(nl12), lin-35(n745) and lin-37(n 758) cause a reduction in fertility and body size at 25", and a double mutant carrying lin-9(n112) and Zin-35(n745) is sterile. Bycontrast, the complete elimination of lin-15 A and B activity causes few defects in fertility and body size at 25" and fewer defects at 20". Although lin-15 B activity is required in the class B pathway for vulval formation, Zin-15 B function is largely dispensable in the class B pathway for other processes.Possible roles for the Zin-15A and Zin-15 B proteins:The expression of vulval cell fates depends upon the activation of a tyrosine kinase/ras signaling cascade that during wild-type development is triggered by a signal from the gonadal anchor cell (for review, see HORVITZ and STERNBERG 1991) (Figures1B and 6A). The removal of lin-15 function causes the activation of this signaling pathway even in the absence of the anchor cell inductive signal. A signal involving lin-15 from the surrounding hypoderm might normally prevent the activation of this signaling pathway, since mosaic analysis suggests that lin-15 functions in the hypoderm (HERMAN and HEDGECOCK 1990) (Figures1D and 6A). As loss-offunction mutations in the receptor tyrosine kinase let-23 gene, the SH3-SH2-SH3 adaptor protein sem-5 gene, let-341, let-60 ras and lin-45 raf suppress the Muv phenotype induced by lin-15 mutations, Zin-15 acts upstream of these five genes in the genetic pathway for vulval induction(FERGUSON et al. 1987; BEITEL et aZ. 1990; HAN et al. 1990; CLARK et aZ. 1992 CLARK et aZ. , 1993;; HAN et al. 1993). 




  FIGURE 6.-Two models for the negative regulation of the tyrosine kinase/ras signaling cascade by lin-15. (A) A signal from the hypoderm involving the two lin-15 proteins may inhibit the receptor tyrosine kinase let-23 by direct interactions. The products of other class A and class B synthetic multvulva genes might mediate these intercellular interactions because the lin-15 A and B proteins lack hydrophobic signal sequences. Alternatively, the hypoderm might signal to factors within the Pn.p cells that block the action of let-23. These factors may be encoded by other synthetic multivulva genes. (B) The action of the lin-15 proteins may prevent the hypoderm from producing or releasing molecules related to the lin-3 product that would inappropriately activate the vulval inductive pathway. the hypoderm may negatively regulate the let-23 receptor tyrosine kinase signaling pathway within the cells P3.pP8.p.The sequences of the 719-and 1,440-amino acid lin-15 proteins do not suggest how lin-15 A and lin-15 B might function. One possibility is that a hypodermal signal involving lin-15 directly inhibits the let-23 receptor tyrosine kinase (Figure6A). Since neither of the 




  tained after mutagenesis of a strain that contained an undetected class A mutation (FERGUSON and HORVITZ 1989). Subsequent mutageneses of existing class A and class B single mutants, which are phenotypically wildtype, allowed the systematic isolation of further synthetic Muv mutants (FERGUSON and HORVITZ 1989; J. THOMAS and H. R. HORVITZ, unpublished results). Two genes, lin-8 and lin-38, are defined only by class A mutations, and six genes, including lin-35, lin-36 and lin-37, are defined only by class B mutations (FERGUSON and HORWTZ 1989). 



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Scott G Clark, Xiaowei Lu, H Robert Horvitz, H Wttttaaccca- M L A P A A P A K D V
The Caenorhabditis elegans Locus lin-15, a Negative Regulator of a Tyrosine Kinase Signaling Pathway, Encodes Two Different Proteins