Feinbaum RL et al. (1991) International C. elegans Meeting "NEW ALLELES OF LIN-4."

Ambros VR, Feinbaum RL

[International C. elegans Meeting, 1991]

Based on the phenotype of the single lin-4 allele, (e912), it has been proposed that lin-4 acts at the top of the heterochronic regulatory pathway and controls the activity of lin-14. In order to further understand the role of lin-4 in the temporal control of development we have begun to isolate new alleles of this gene by non- complementation and to identify the null phenotype. Because no new alleles of lin-4 have been identified in numerous general screens for egl or vul animals (where they might have been expected to turn up) and no deficiency of the lin4 locus existed, our first non- complementation screen was aimed at isolating a deficiency of the lin- 4 locus. Wild type N2 males were mutagenized with gamma rays, mated by lin-4(e912)dpy-101mncl; lin-14(nl 79ts) hermaphrodites and the Fl hermaphrodite progeny were screened at 20 C for non-dpy lin-4 like animals. Out of 7,000 Fl hermaphrodites screened, one lin-4 deletion mutant (maDf4) was identified. maDf4 is homozygous inviable and fails to complement lin-4 as well bli-2, clr-l, and unc-85 that map to the left of lin-4. maDf4 does not delete spe-3, a gene that maps only 0.1 mu to the right of lin4. However, Southern blot analysis has shown that maDf4 deletes a 5kb EcoRI fragment (see Lee et al.) that rescues the lin4 mutant phenotype, suggesting that maDf4 does not retain functional lin-4 sequences. Based on analysis using a dissecting microscope, the phenotype of lin4(e912) dpy-l OlmaDf4 animals is indistinguishable from that of lin4(e912)11in4(e912) animals (with the exception of an enhanced sterility of lin4(e912) dpy-lOlmaDf4 animals). These data suggest that the original lin-4(e912) allele is not a gain- of-function allele and indicates that lin4(e912)1null is fertile and viable. Since lin-4(e912)1maDf4 is not significantly different from lin-4(e912)11in-4(e912), lin-4(e912) is probably null or close to null. Encouraged that we knew the expected phenotype of lin4(e912)11in4( null) we have intiated a new series of non-complementation screens to identify EMS induced lin-4 alleles. Out of 8,000 Fl hermaphrodites screened so far we have identified one new lin4 allele, (mal 61). mal 61 looks phenotypically similar to e912 but Southern blot analysis has confirmed that it is in fact a new allele of lin4.

Mains PE et al. (1991) International C. elegans Meeting "MOLECULAR ANALYSIS OF MEI-1, A GENE THAT AFFECTS MEIOSIS AND MITOSIS."

Mains PE, Clark-Maquire SD

[International C. elegans Meeting, 1991]

The one-cell C. elegans embryo must support two distinct patterns of cell division, meiosis and mitosis. These two divisions differ in many respects, but the same cytoplasm must support both, indicating that the activities of the gene products that distinguish meiosis from mitosis are carefully regulated. We have identified maternal-effect mutations in four loci that disrupt either the meiotic or the mitotic divisions. The dominant ts gain-of-function (gf) mutations mei-l (ct46) I and mel-26(ct61) I and recessive loss-of-function (IJ) mutations of zyg-9 II show similar defects in the first mitotic division, and these mutations are strong enhancers of one another's defects. If mutations of mei-l and mei-2 result in recessive meiotic abnormalities but act as dominant suppressors of the mitotic defects of mei-l (ct46gf) and mei-26(ct61gf). We have cloned mei-l, a gene that can be mutated to affect either meiosis or mitosis. This gene maps between two markers on the physical map, lin-10 and lin-28. Fourteen overlapping cosmids spanning the region were each coinjected with the rol-6 plasmid into mei-l (lf)/+ hermaphrodites to establish transgenic lines. Segregants homozygous for mei-l that rolled were then tested for fertility. Two overlapping cosmids, TOlG9 and F54A4 provide mei-l + activity. Injecting F54A4 directly into homozygous mei-l hermaphrodites does not result in even transient rescue. In order to identify restriction enzymes that did not cut within the gene to use for subcloning, one of the cosmids was digested with restriction enzymes, and the digest of each enzyme was injected and tested for rescue. The individual fragments from positive digests were gel-purified and singly injected ( a note of caution: occasional false positives apparently result from a low level crosscontamination present in gel-purified fragments). This analysis was repeated on the rescuing fragment. A 4 kb SnlI/HpaI subfragment containing mei-l has been identihed, but it resulted in a curious partial rescue. From a heterozygous stock, Fl mei-l (IJ) homozygotes that rolled were isolated. These were fertile ( ~ 50% of their progeny hatched) and produced many rolling F2 progeny. However, few if any of the fertilized embryos laid by these F2 rollers hatched. This 'grandchildless' phenotype was common: 25 independent transgenic lines were made, and the homozygous Fl progeny showed good fertility for 19 of them. However, the F2 progeny of all 17 lines that were tested further laid few if any hatching embryos (never enough to maintain a stock). This contrasts with transgenic stocks carrying larger subfragments or the original cosmids, which can be maintained as mei-l homozygotes indefinitely. Apparently, the 4 kb subfragment lacks sequences necessary for complete rescue, but it is hard to explain why it nevertheless rescues well in the Fl.

Speliotes EK et al. (1996) East Coast Worm Meeting "SCREENS TO IDENTIFY NEW GENES INVOLVED IN PROGRAMED CELL DEATH"

Horvitz HR, Speliotes EK

[East Coast Worm Meeting, 1996]

We are conducting two screens to identify new genes involved in programmed cell death. Gain-of-function mutations in ced-9 prevent the programmed cell deaths of cells that would nonnally die, whereas loss- of-function mutations in ced-9 such as nl950 n2161 cause the ectopic programmed deaths of cells that would normally live (Hengartner et al., Nature 356, 494,1992). Homozygous ced-9(nl950 n2161) animals generated by heterozygous mothers survive, but the progeny of these homozygotes die during embryogenesis, suggesting that ced-9 has a maternal effect. Mutations in ced-3 and ced-4 completely rescue this lethality, while mutations in ced-8 delay the onset of this lethality. By screening for supressors of ced-9 loss-of-function lethality in sensitized backgrounds, we hope to isolate not only new mutations in the death- promoting genes ced-3 and ced-4 but also mutations in new genes involved in programmed cell death that were previously missed because their loss interferes only minimally with the ability of a cell to die by programmed cell death. In the first screen we hope to isolate mutations that cannot necessarily rescue the ced-9(nl950 n2161) lethality alone but can rescue this lethality in conjunction with a mutation in ced8. We mutagenized unc- 69(e587) ced-9(nl950 n2161) lqC1;ced-8(nl891) animals and looked at the progeny of 1,425 unc-69(e587 ced-9(nl950n2161); ced-8(nl891) Fl animals for ones that escaped embryonic lethality and were able to propagate as viable strains. We are currently analyzing seven such mutants. In the second screen we hope to isolate mutations in genes that can rescue the lethality caused by weak but not necessarily strong ced-9 loss-of-function mutations. ced-9(nl653)is a weak ced-9 mutation at 20 C (animals are viable and can propagate) and becomes synthetically lethal when combined with an atypical death promoting allele of ced-4, n2273. Although mutations in ced-8 do nest rescue the lethality caused by strong ced-9 loss-of-function mutations, they do rescue the synthetic lethality of ced-4(n2273) ced-9(nl653) double mutants. We mutagenized ced-4(n2273)ced-9(nl653) unc-49 (e382)lqC1 animals and looked at the progeny of 1,200 ced-4(n2273)ced-9(nl653)unc-49(e382) Fl animals for ones that escaped embryonic lethality and were able to propagate as viable strains. We are currently analyzing ten such mutants.

Nirit Assaf-Reizel et al. (2003) International Worm Meeting "How do cells fuse?- new alleles of the fusion gene eff-1"

Nirit Assaf-Reizel, Tamar Gattegno, Benjamin Podbilewicz

[International Worm Meeting, 2003]

Developmental cell fusion is an important process in multicellular organisms. eff-1 is a gene encoding type-1 membrane proteins essential for all epithelial cell fusion events in C. elegans (1). Alternative splicing results in at least four different isoforms of EFF-1. EFF-1A and EFF-1B predicted structures show five putative domains: a signal peptide, a phospholipase A2 site, a fusion peptide, a transmembrane and a cytoplasmic tail, while EFF-1C and EFF-1D are predicted to be secreted proteins containing only the first two domains. Understanding how and where EFF-1 proteins work will help us to better understand the puzzle of the fusion mechanism. Until recently only two eff-1 alleles were known. The more severe allele eff-1(hy21ts) causes total hypodermal fusion arrest and a series of different abnormal phenotypes such as Unc, Dpy, Pvl, Egl and Ste. RNAi experiments and analysis of eff-1(hy21ts)/Df show more severe Eff-1 phenotypes, implying for a correlation between EFF-1 dosage and fusion activity. These experiments also show that hy21ts is not a null allele. To understand the structure and function of eff-1 we looked for new alleles using a non-complementation screen. Young adult ajm-1::gfp males were mutagenized with EMS, mated with eff-1(hy21ts)unc-104(e120)II hermaphrodites and the Fl hermaphrodite progeny were screened at 15 degrees C for non-Unc animals exhibiting Eff-1 phenotypes. Suspected mutants that showed fusion defects and failed to complement eff-1(hy21) were sequenced using eff-1 specific primers. Out of 18,000 Fl hermaphrodites screened, two independent animals were identified as non-complementing eff-1 alleles, both with the same point mutation (hy32 and hy33). The hy32 and hy33 mutation is a C135Y change at the phospholipase A2 site next to the putative aspartic acid active site. This mutation results in Eff-1 phenotypes, more severe than the original hy21ts allele. The hy40 and hy41 alleles of eff-1 were recently isolated in a two-step clonal screen by T. Gattegno. These worms are very Dpy and have a small brood size. hy41 is a change of a conserved G316E. hy40 is a stop codon after aa 224, resulting in a short secreted protein containing the phospholipase A2 site. Another short EFF-1 protein probably exists in the np29 allele, which was recently isolated in a Mos mutagenesis screen by N. Pujol and characterized by J. Novelli (2). We will show a phenotypic comparison between eff-1 alleles as well as the hy21ts/Df strain. We will also show analysis of worms expressing a translational EFF-1A::GFP construct and discuss the implications of these results. 1. Mohler et al, Dev Cell 2002. 2. We thank N. Pujol, J. Novelli and J. Hodgkin for kindly provided np29.

Minniti AN et al. (1991) International C. elegans Meeting "PROGRESS TOWARD CLONING SPE-12."

Ward S, Minniti AN, Varkey JP

[International C. elegans Meeting, 1991]

Mutations in the spe-12 gene cause spermatogenesis to arrest just before activated spermatids form their pseudopod. The spe-12 mutant phenotype is unusual because only hermaphrodites show a sterile phenotype whereas spe-12 mutant males are fertile. Differential spermatid activation observed in vivo is also seen in the presence of various in vitro activators. This suggests that the spe-12 gene product may be involved in initiation of spermiogenesis (Shakes and Ward, Dev .Biol. 134,189-200) . We have isolated five new non- conditional spe-12 alleles in an Fl non-complementation screen. Their phenotypes are similar to each other and to the original allele at the light microscopy level. These results and the phenotypes of some of the mutations over a deficiency suggest that we have identified the null phenotype of spe-12. To further understand the function of the spe-12 gene product we have started cloning the spe-12 gene. The genetic mapping of spe-12 with respect to the previously cloned gene lin-10 enabled us to identify two overlapping cosmids (HHB7 and B0544) that rescued the spe-12 sterile phenotype in germline transformation experiments. We have subcloned restriction fragments from one of the cosmids and used them for transformation rescue. So far, we have located the spe-12 gene to an 18 Kb fragment which rescues the mutation. We are currently narrowing the s p e -12 location and characterizing the mutants in more detail.

Lee JH et al. (1991) International C. elegans Meeting "ROLE OF UNC-101 IN C.ELEGANS VULVAL DEVELOPMENT"

Jongeward GD, Lee JH, Sternberg PW

[International C. elegans Meeting, 1991]

We previously isolated two unc-101 alleles (e.g. syl O8) in a screen for suppressors of vulvaless phenotype of let-23. unc-101 mutations also confer an uncoordinated phenotype as well as subviability, defecation and FITC staining defects. We are interested m the genetics and molecular biology of this locus because of its broad range of phenotypes and lts role in vulval development. We isolated a putative null allele of unc-101. We used trimethyl- psoralen as a mutagen in a screen for mutations that fail to complement unc-l Ol (rh6). One new allele, sy 216, was recovered from 11,000 Fl cross-progeny hermaphrodites. This allele is a homozygous Ll larval lethal. Unlike the visible alleles, the lethality of thls allele cannot be maternally rescued. Animals of the genotype sy2161syl O8 are less viable than animals bearing syl O8 in trans to any visible allele. The sy216 Isyl O8 heterozygote is also a good suppressor of the let-23(syl ) vulval defect. Therefore we consider sy216 a putative null allele of unc-101. We believe that the null phenotype of unc-101 is lethality and suppression of let-23. sy216 does not delete unc-75, ced-l and unc-S9, nor does it overlap eDf3. We are currently trying to clone unc-101 using parallel RFLP mapping with the congenic strain used for cloning ced-l, which was kindly provided by Bob Horvitz's lab. We obtained 37 unc-59 nUnc-75 recombinants from unc-75(e950)ced-1(nlS06) +unc-S9 (e290)1 ++unc-lOl( sylO8) + heterozygotes,and southern hybridization analysis with Tcl as probe is underway. [See Figure]

Perry MD et al. (1991) International C. elegans Meeting "DECIPHERING HER-1'S SEXUAL MESSAGES"

Robertson B, Wood WB, Perry MD, Li WQ, van Auken KM, Trent C, Hageman JM, Ippensen DC

[International C. elegans Meeting, 1991]

The her-l gene, which is required for normal male sex determination, has been cloned using Tcl transposon tagging and shown to have two XO- specific transcripts (1). We are analyzing its functions by determining the structure of the gene and its products. We have sequenced approximately 8 kb of genomic DNA including the her-l gene as well as several cDNAs corresponding to the two transcripts. The first open reading frame in the rare 1.2 kb transcript is predicted to encode a 20 kD polypeptide whose hydrophobic amino terminus could be a secretion signal sequence. The relatively abundant 0.8 kb transcript is predicted to encode the Cterminal (approximately) half of the same polypeptide, with no signal sequence. RNAse protection mapping experiments of the region confirm our earlier evidence that the 1.2 kb and 0.8 kb transcripts include 4 and 2 exons, respectively. DNA- mediated transformation to rescue her-l (lf) mutations has been recalcitrant, and we are now testing smaller and smaller cosmid fragments in hope of removing noxious sequences. To define cisacting sequences in the her-l locus, we have fused different lengths of genomic DNA upstream of her-l to Andy Fire's lacZ constructs and injected them with a rol-6 plasmid. We see poor staining in the resulting Fl male rollers, suggesting that the 2.5 kb of DNA exarnined so far may be insufficient for expression. In an oblique attempt to identify essential elements of the her-l locus, we have used coding region probes in low stringency hybridizations to genomic DNAs and libraries from the male/female species C remanei and WS9-6 and have detected highly conserved fragments. Lack of definitive signals from parallel experiments on the hermaphroditic species C. briggsae suggests that even lower stringencies may be necessary. We are also continuing efforts to obtain anti-her-l antisera using bacterial fusion proteins.

Williams BD et al. (1991) International C. elegans Meeting "essential genes affecting muscle."

Waterston RH, Williams BD

[International C. elegans Meeting, 1991]

Mutations in the myo-3 (Waterston, 1988, EMBOJ 11:3429) and deb-l ( Barstead and Waterston, unpubl.) genes, which encode structural components of body wall muscle, cause a similar lethal phenotype. The embryonic body wall muscle is severely paralyzed, which becomes apparent when the mutant embryos fail start twitching as they reach 1. 5-fold length. 30 minutes later, when the embryos have reached 2-fold length, elongation stops. During subsequent development a cuticle is synthesized, a short but functional pharynx is formed, and the 2-fold length animals hatch and arrest as misshapen Lls. Based on the premise that this 'paralyzed and arrested at two-fold' ( pat) phenotype is caused by a profound failure in body wall muscle cell function, we have performed a genome-wide screen for pat mutants to identify other essential 'muscle affectingU genes. From 3300 Fl clones, 28 recessive lethal pat mutants were recovered by screening first for the elongation defect using the dissecting microscope, and subsequently for the motility defect using time-lapse DIC microscopy. The mutations fall into 13 complementation groups: 2 are deb-l and myo- 3, 3 are genes previously identified by viable alleles that appear to affect muscle (lev-ll, unc-52 and unc-112), and the remaining 8 include several newly identified genes. Immunofluorescence analysis with antibodies to C. elegans muscle components shows that the embryonic muscle cells of the pat mutants have disorganized myofilament lattices. Homozygous lethal unc-52 and pat-3 embryos fail to stain with monoclonal antibodies MH2 and MH25 (Francis and Waterston, 1985, JCB 101:1532 and unpubl.), respectively, consistent with the possibility that these genes code for the corresponding antigens. MH2 recognizes a 200 kD muscle cell antigen associated with the basal lamina, a likely candidate for the unC-52 gene product based on the DNA sequence of this gene (Rogalski and Moerman, 1990, WBG 11[3]:32). The MH25 antigen is associated with the muscle cell membrane and is distributed in a pattern suggesting a role in anchoring elements of the myofilament lattice.

Kim SC et al. (1991) International C. elegans Meeting "lin-24 AND lin-33, GENES THAT CAN MUTATE TO CAUSE DEGENERATION OF Pn. p CELLS"

Kim SC, Horvitz HR

[International C. elegans Meeting, 1991]

Semidominant mutations in the genes lin-24 IV and lin-33 IV can cause degeneration of the Pn.p cells. Since the vulval precursor cells are a subset of the Pn.p cells, these mutations result in a vulvaless ( Vul) phenotype. These degenerations are independent of the functions of the ced-3 and ced4 genes, which are required for programmed cell death. However, these degenerations depend on three of the genes known to be involved in the engulfment of cell corpses: ced-2, ced-S, and ced-10. These results suggest that the changes in the Pn.p cells caused by the semidominant mutations in lin-24 and lin-33 lead to engulfment of the Pn.p cells, which causes their deaths. We have sought the null phenotype of these two genes by mutagenizing mutant hermaphrodites with EMS or X-rays and looking for revertants among the Fl and F2 progeny. We have isolated three intragenic revertants for each gene. We believe that a null mutation in lin-24 results in a wild-type phenotype. We have recently isolated a deficiency, nDf41, that fails to complement markers both left and right of lin-33. Since none of the lin-33 revertants behaves as nDf41 does in trans to lin-33(sd), we do not know the null phenotype of lin- 33. From the revertant screen described above, we isolated a small deletion, n2256, which fails to complement lin-33. We also isolated a duplication, nDpS, which extends from unc44 to unc-26 and fails to complement lin-33 but does complement the lethality of the deletion n2256. Using cosmids from the region of a contig that encompasses lin- 33 as determined by our mapping of N2/N62 RFLPs, we have found a 5kb EcoRI fragment that is missing in the nl O43 n2256; nDpS strain. We are injecting cosmids from this region into a strain of genotype lin- 33(revertant) lin-24(sd). Because a lin-33(revertant) mutation suppresses the lin-24(sd) phenotype, this strain is not Vul. We hoped that by injecting cosmids containing lin-33(+), we would find transformed animals that are Vul. Unfortunately, none of the cosmids injected resulted in Vul transformed animals. Further analysis is under the way.

Tautz TH et al. (1991) International C. elegans Meeting "TOWARD C. ELEGANS INVERSE (CLONE-TO-MUTATION) GENETICS"

Wood WB, Tautz TH

[International C. elegans Meeting, 1991]

Trimethylpsoralen(TMP)-induced unc-22 mutations include a substantial percentage (44%) of <2-kb deletions (see abstract by Yandell et al.). Therefore, this mutagen should be useful in PCRbased screens for deletion mutations in a gene corresponding to a cloned sequence of interest. Such a screen could have advantages over similar screens based on transposon insertion, because TMP mutagenesis is more likely to occur randomly. A deletion screen can be made more sensitive by choosing PCR primers on either side of a unique restriction site and cutting DNA from mutagenized populations with the corresponding enzyme before PCR amplification, so that the majority wild-type sequences present will give no PCR signal. Reconstruction experiments using ctlS6, a well characterized TMP-induced deletion in the S' region of unc-22, indicated that ct156 DNA could be detected by PCR in the presence of <5000-fold excess wild-type DNA A screening protocol was then developed as follows, using unc-22 as the test gene: 1) mutagenize worms with TMP; 2) plate 30-50 Fl worrns per plate onto several hundred plates and grow for one generation; 3) remove half the worms from each plate and combine into pools, placing remaining worms at 10 C; 4) isolate genomic DNA from these pools and cut with the appropriate enzyme; S) amplify by PCR, blot, and look for deletion bands; 6) return to appropriate pool, identify plate containing mutants, and subculture worms for further amplification and probing until a clone of mutant worms is isolated. Reconstruction experiments showed that spiking one plate in a given pool with a single ctlS6 worm consistently allowed detection of the expected deletion band on Southern blots of PCR arnplified DNA. In test screens for new unc-22 mutations with this protocol, two possible deletions in the 5' region were detected, but the mutant animals could not be isolated because of technical problems. We are continuing the unc-22 test screens, as well as preparing to search for mutations in genes corresponding to some of the cloned sequences identified previously as preferentially transcribed in early embryos (I. Schauer and W.B.W. Development 110:1303-1317,1990).