Rudner A et al. (1993) Worm Breeder's Gazette "A Mutation Which Doubles C. elegans Life Span (Imagine Being 140)"

Tabtiang R, Rudner A, Kenyon CJ

[Worm Breeder's Gazette, 1993]

We have discovered that the strain CB1370 has a mean life span twice that of N2 when raised at 20 . The mean life span of CB1370 is 45 days whereas the mean life span of N2 is 21 days. The increase in life span of this strain is striking. After 31 days, when all the N2 worms have died, 84% of the CB1370 worms are still alive and moving well on their plates. CB1370 contains the mutation daf-2 (e1370).daf-2 (e1370Jis a temperature-sensitive dauer-constitutive mutation. Worms hatched and raised at 25 enter dauer and remain as dauer larvae until shifted to the non-permissive temperature of 20 . Dauer larvae are morphologically different from non-dauer animals and have an increased life span. At 20 CB1370 exhibits increased longevity but not other dauer phenotypes. It eats and produces progeny, and it looks much like a normal non-dauer animal. We have also observed a similar increase in life span when CB1370 worms are hatched at 20 and shifted to 25 as L4 sor early adults. This increase in life span is not as dramatic, however, because a large number of CB1370 worms become bags at 25 . It has generally been assumed the dauer lives longer simply because it is relatively metabolically inert. However, this result suggests that perhaps the longevity of the dauer is not simply due to its being in a state of suspended animation. Instead, there may be a longevity program latent in the non-dauer worm that can, if activated, prolong its life substantially. The longevity program is normally activated along with other aspects of dauer morphogenesis, but the increased lifespan of daf-2 (1370)mutants suggests that it can function independently. Although the mutation in daf-2 is the most likely reason for increased longevity in CB1370 ,there is the possibility that CB1370 contains a second mutation, in an "aging" gene and that daf-2 (e1370)has no longevity phenotype. In order to differentiate between these two possibilities we are currently testing other daf-2 alleles for increased life span. Preliminary findings suggest that daf-2 (sa193),kindly sent to us by Jim Thomas, also lives longer than the wild-type. We conclude that daf-12 wild-type activity is dispensible for the heterochronic expression of the L1 -specificantigen caused by the dauer-constitutive mutations we tested. In contrast, both daf-3 and daf-5 wildtype activity are required for the Srf heterochronic phenotype. Our interpretation of these results is that the daf-c gene products signal to both dauer formation and surface antigen switching, but through downstream steps that eventually split into process-specific branches. Incorporating our results into the pathway hypothesized for dauer formation based on epistasis analysis, one such branch point would be downstream of the daf-c genes (with the exception of daf-2 )and daf-d genes daf-3 and daf-5 .but upstream of daf-12 (see Figure). We would also like to place srf-6 in this model by epistasis analysis. If daf-3 and daf-5 are epistatic to srf-6 for the Srf heterochronic phenotype, it would suggest that the srf-6 product acts upstream of these steps. On the other hand, if srf-6 mutants still express the Srf heterochronic phenotype in the presence of these daf-d mutations, it would suggest that srf-6 acts in a surface antigen-specific way, perhaps downstream of the branchpoint proposed above. We plan to construct the appropriate double mutants to test these hypotheses. To summarize, every daf mutation we have tested so far except for daf-2 and daf-12 mutations also affects the surface antigen switching detected with monoclonal antibody M37 .Because it involves the continued expression of a marker normally expressed on the first larval stage, the Srf heterochronic phenotype is reminiscent of the propensity of mammalian cancer cells to express so-called "carcinoembryonic" or "oncodevelopmental" surface antigens characteristic of embryonic or fetal cells. It remains to be seen whether the effects of the daf genes on surface antigen switching observed in this formal analysis signify a molecular involvement of the gene products in signalling to two distinct processes. We thank Don Riddle and Patrice Albert for encouragement and for gifts of double mutant strains, and Victor Ambros, Gary Ruvkun, and Jim Thomas for discussion.

Winge P et al. (1994) Worm Breeder's Gazette "R-ras 1 and R-ras 2 (TC21) Homologs."

Gobel V, Friend S, Winge P, Fleming JT

[Worm Breeder's Gazette, 1994]

R-ras I and R-ras 2 (TC21) homologs Per Winge*, Vercna Gobel*+, Stephen Friend*, and John Fleming*+. MGH Cancer Center and +DepL of Pediatrics, Boston, MA. Human r-ras 1 and r-ras 2 (TC21) belong to the closer relatives (>50% amino acid identity) of ras in the ras superfamily of GDP/GTP-binding proteins. They are the first members to exhibit transforming potential when mutated at some which render ras oncogenic and make it insensitive to GAP action (Graham & Der, 1994). These recent findings have led to current investigations of their role-in human cancer. Furthermore, r-ras 1 -- by immunoprecipitation and in the yeast-2-hybrid-system -- was shown to interact with bc1-2, the human homolog to ced-9 (Fernandez-Sarabia & Bischoff, 1993) and has thus been implicated as a possible effector of apoptosis. There is evidence that the r-ras proteins participate in some but not all aspects of the ras signal transduction pathway involving upstream tyrosinc kinases and downstream serine/threonine kinases. It has not yet been elucidated in the mammalian system (1) what alternative pathway the r-ras proteins may be utilizing and (2) what functional relevance is represented by the in vitro interaction of r-ras 1 and bc1-2. We are trying to address these questions in C elegans and have cloned the homologs of r-ras I and r-ras 2 using a degeneratc PCR approach. We have screened c-DNA and genomic libraries and obtamed and sequenced full length c-DNA and genomic clones of r-ras 1 and a full length c-DNA clone of r- ras 2. The genomic sequence of r-ras 2 was recently made available by the genome sequencing project. The amino acid comparison shows high homologyrldentity to thc human proteins for r-ras 1 and r-ras 2 (TC21). R-ras 1 was localizcd to chromosome II ncar lin-29, and r-ras 2 maps close to embS on chromosome m. To obtain r-ras germline deletions, we have screened a TCl insertion library which we constructed using the mutator strain MT 3126 (protocols kindly proYided by Jocl Rothman, Susan Mango and Ed Maryon), and have isolated transposon insertions in r-ras 1. We are currently in the proccss of sib sclection to purify the strains. To get some first appreciation of a functional role of r-ras towards apoptosis versus growth stimulating propertics, we have also started to inject a r-ras 1 hcat shock promotor expression construct to generatc strains in which r-ras can be overexpressed Ihis additional approach has been choscn since redundancy may be expected in thc ras related protcin familics and thus thc knockout of one of the proteins may not give clear results. We will screen the overexpressing strains for (1) apoptosis and (2) muv phcnotype. In collaboration with Bob Horvitz's laboratory r-ras GST fusion proteins will be generated to test the in vitro interacion with ccd-9. Finally, we are constructing r-ras 1 and r-ras 2 promotor expression vectors with GFP/betaGAL to define the expression patterns of both genes.

Beitel GJ et al. (1994) Worm Breeder's Gazette "An ACEDB For the Macintosh User Group"

Beitel GJ, Horvitz HR

[Worm Breeder's Gazette, 1994]

An ACEDB for the Macintosh User Group Greg Beitel (BEITEL@MIT.EDU) and H. R. Horvitz, DepL Biology, MIT, Cambndge, MA 02139, USA

Chamberlin HM et al. (1992) Worm Breeder's Gazette "Cell Ablation Experiments and the Characterization of Cell Fate Specification in the / and / Equivalence Groups of the Male B Lineage"

Chamberlin HM, Sternberg PW

[Worm Breeder's Gazette, 1992]

Cell fate determination in the B lineage of the male tail is due, in part, to cell interactions. Two equivalence groups, alpha/ (B.a(l/r)aa) and gamma/delta (B.a(l/r)pp) are evident from the wild-type cell lineage [Sulston & Horvitz, Dev Biol 82:41-55 (1977)]. For instance, in the alpha/ pair, either B.alaa or B.araa (labeled aa in the figure) can adopt a position anterior to the other. The cell that adopts the anterior position will adopt a fate (i.e., undergo an a lineage), and the posterior cell will adopt ,B fate. Thus, although the cells in each pair have equivalent potential, they adopt different fates depending on their relative positions. This abstract summarizes results of cell ablation experiments that begin to characterize cell fate specification in these two equivalence groups. 1) F and U promote anterior (a and y) fates. Ablation of the male specific blast cells F and U in late L1 males results in abnormal a (7/7 animals) and y (5/7) lineages. Specifically, presumptive alpha's often behave like 's, and presumptive gamma's undergo abnormal truncated lineages. lin-3 ,let-23 ,let-60 ,and lin-45 ,genes identified for their role in hermaphrodite vulval induction, also act in these male tail equivalence groups. Mutations that reduce wild-type function of these genes result in the same lineage defects seen in F-U- animals (see WBG 11.2, p. 103). In addition, the F/U signal is redundant. Lineages in F- or U- animals are generally wild-type (F-: 0/4 a abn; 1/4 alpha abn. U-:V4 gamma abn; 0/4 y abn.). 2) Y.p promotes posterior ( and delta) fates. Ablation of Y.p in late L1 males results in abnormal delta (6/7) and occasionally abnormal (2/7) lineages. Specifically, presumptive delta' s undergo an extra round of division. Y.p may play a minor role in providing posterior information for the alpha/ pair. However, it appears to be neither necessary nor sufficient to specify fate (see section 4). 3) lin-12 acts in the gamma/delta equivalence group? The role of lin-12 in the gamma/delta equivalence group shows similarities to its role in the hermaphrodite vulva. lin-12 (0)mutations result in transformation of delta to gamma [Greenwald et al., Cell 34:435-444(1983)]. Although lin-12 (d)mutations result in no gamma/delta phenotype on their own, ablation of F and U in lin-12 (d)animals results in full transformations of gamma to delta (3/3 transformed). Thus, lin-12 specifies delta fate, but the F/U signal can override lin-12 (d)and induce a normal gamma. In contrast, there is currently no evidence that lin-12 plays any direct role in alpha/ cell fate specification. 4) Interactions between other B.a progeny and the alpha/ equivalence group. B.a progeny play a significant role in specification of fate. Ablation of all B.a progeny except B.a(l/r)aa (i.e., B.a(l/r)pp-, B.a(l/r)pa-, and B.a(l/r)apin late L2 )results in both B.alaa and B.araa adopting alpha fate (4/5 both alpha). However, the presence of any left/right pair of B.a progeny is sufficient to correctly specify fate. In other words, wild-type lineages for B.a(l/r)aa were observed in animals with B.a(l/r)p-(3/3 wt), B.a(l/r)pa-and B.a(l/r)ap (3/3 wt), or B.a(l/r)pp-and B.a(l/r)ap- (2/2 wt) ablations, although occasionally division axes were skewed. 5) Interactions between other B.a progeny and the gamma/delta equivalence group. Experiments to test the interactions between other B.a progeny and B.a(l/r)pp are incomplete. Nevertheless, it is evident that the fates of B.a(l/r)pp (or, possibly their progeny), like B.a(l/r)aa, depend on their B.a neighbors. Gamma/delta are wild type in B.a(l/r)a ablated animals (3/3 wt). However, ablation of B.a(l/r)pa and B.a(l/r)aa results in truncated y lineages (4/4 truncated). Perhaps even more curious, ablation of B.a(l/r)ap and B.a(l/r)pa results in three rounds of division with abnormal axes in both presumptive gamma and delta (2/2 abnormal). It is not likely that this is a transformation of delta to gamma fate. One possible explanation is that B.a(l/r)aa (or their progeny) can actively promote cell division in B.a(l/r)pp (or progeny). In unablated animals, this activity is blocked by B.a(l/r)pa and B.a(l/r)ap. Additional experiments may help clarify the roles of B.a neighbors in specification of gamma and delta fate.

Fodor A et al. (1982) Worm Breeder's Gazette "a contribution to the genetic analysis of amanitin-resistant (amaR) mutants."

Sanford TR, Riddle DL, Fodor A

[Worm Breeder's Gazette, 1982]

In the genetic tests performed thus far, all seven mutants behave virtually identically. All are (at least semi-) dominant and closely linked to dpy-13 locus on the linkage group IV. There is no evidence as yet for any maternal effects in the expression of amanitin resistance or sensitivity, Heterozygous (ama(R)/+) males were mated with dpy-8 odites to test X linkage or dominance. The mated hermaphrodites were cloned in microtiter wells in amanitin, The sensitive, self progeny (DpyUnc) did not grow beyond the L1 stage, but ama(R) F1 hermaphrodites with normal movement and body shape developed to maturity along with the DpyUnc males. The results were the same for each of the seven mutants. The production of resistant (ama(R)/+) hermaphrodite progeny shows that ama(R) is dominant. The production of resistant males shows that it is autosomal. Crosses with strains carrying two linked chromosomal markers showed that the ama(R) allele, m118 is not linked to standard reference markers on LG I, II or III and V, but it is closely linked to dpy-13(IV). (The chromosomal markers were the following: dpy-3( DR39)unc-6(e78)(x); 21)unc-3(e151)(x); ) unc-75(e950)(I); )unc-54(e1301)(I); unc-4(II); unc-4(II); daf-2(e1370)unc-32(III); daf-7 I); unc-24(IV); unc-24(IV); unc-22(IV); ; dpy11 unc-51(V).) The preliminary two- factor crosses produced only one dpy-13 ama(R)/dpy-13 + recombinant among 100 homozygous Dumpy segregants. This suggests that ama(m118) locus may be within one map unit of dpy-13. Subsequent crosses revealed that all the seven ama(R) alleles are closely linked to dpy- 13. Three-factor crosses with the m118 in trans to recessive markers, +ama(R)+ unc-22, produced three of the four possible classes of recombinants: Unc-Resistant, tive, and Dpy- Sensitive DpyUnc progeny were invariably Sensitive (40/40 tested). In the total of six recombinants, two were obtained in the interval between dpy-13 and ama(R), and four recombinants were obtained in the interval between ama(R) and unc-22. Obviously, these data are preliminary, however, since all the ama(R) mutants are dominant and all appear to be closely linked to dpy-13(IV) we suspect that they all may be allelic.

Li WQ et al. (1992) Worm Breeder's Gazette "Characterization of the Axonal Guidance and Outgrowth gene Unc-33"

Shaw JE, Li WQ, Herman RK

[Worm Breeder's Gazette, 1992]

Characterization of the axonal guidance and outgrowth gene unc-33 W. Li, R. K. Herman and J. E. Shaw Department of Genetics and Cell biology, University of Minnesota, St Paul, MN 55108

Townsend SR et al. (1994) Worm Breeder's Gazette "C. elegans Molecular Genetics and Long PCR."

Finelli AL, Savage C, Xie T, Padgett RW, Townsend SR

[Worm Breeder's Gazette, 1994]

C. elegans Molecular Genetics and Long PCR Scott R. Townsend, Cathy Savage, Alyce L. Finelli, Ting Xie, and Richard W. Padgett, Waksman Institute and Department of Molecular Biology and Biochemistry, Rutgers University, Piscataway, NJ 08855

Bowerman BA et al. (1994) Worm Breeder's Gazette "The Maternal Gene skn-4 and the Specification of Ventral Blastomere Fates in the Early C. elegans Embryo"

Bowerman BA, Shelton CA, Thorpe CJ, Martin PR

[Worm Breeder's Gazette, 1994]

THE MATERNAL GENE SKN-4 AND THE SPECIFICATION OF VENTRAL BLASTOMERE FATES IN THE EARLY C. ELEGANS EMBRYO Bruce Bowerman, Paula R. Martin, Christopher J. Thorpe, and Christopher A. Shelton. The Institute of Molecular Biology, University of Oregon, Eugene, OR 97403.

Otsuka AJ et al. (1991) Worm Breeder's Gazette "Comparison of the unc-104 Protein 'Motor' Domain with Members of the Kinesin Family of Proteins"

Tang LZ, Whiteaker K, Boontrakulpoontatwee P, Otsuka AJ, Zhang Y

[Worm Breeder's Gazette, 1991]

The unc-104 gene is capable of encoding a kinesin-related protein ( Otsuka et al., Neuron, in press). Comparison of the presumed unc-104 'motor' domain to the ATPase and microtubule-binding region of members of the kinesin family demonstrates the greatest sequence identity in the putative ATP-binding domain [GYN-TIFAYGQTGSGKTYTM-G] and in conserved elements of unknown function, some of which may be involved in microtubule-binding [GIIPR, VK-S(Y/F)-E(I/L)YNE, DLL, R-VA-T--N-- SSRSH-VF-I, SGKL-LVDLAGSE, GAEG-R--E--NINKSL--LG-VI-AL, HIPYR(D/E)SKLT- LLQ-SLGG, N--ET-STL-(F/Y)A-RAK--(R/K)]. In the figure below, residues that are identical in 5 or more sequences are listed below the compilation and asterisks mark residues that are identical in all sequences. [See Figure 1]

Ray C et al. (1995) Worm Breeder's Gazette "Evidence for Proteolytic Processing of a Cuticle Collagen in a Plant-Parasitic Nematode"

Hussey RS, Ray C

[Worm Breeder's Gazette, 1995]

Collagen is the major structural component of nematode cuticles. In Caenorhabditis elegans, the N-terminal regions of cuticle collagen proteins contain the highly conserved motif, R-X-X-R, which has been predicted to contain a potential subtilisin-like proteolytic processing site required for cleavage of the collagen proteins and normal cuticle collagen function (Kramer, 1994, FASEB 8:329-336). Cuticles of adult females of the plant-parasitic nematode Meloidogyne incognita contain a 76 kDa collagen which comprises over 50% of the B- mercaptoethanol-soluble cuticular proteins (Reddigari et al., 1986, J. Nematol. 18:294-302). We determined the N-terminal amino acid sequence of this major collagen and found it to be identical to the predicted amino acid sequence, starting at amino acid number 66, of the M. incognita Lemmi 5 cDNA clone (Van der Eycken et al., 1994, Gene 151:237-242) (Fig.1). A putative subtilisin-like protease recognition site was found immediately upstream of the region of amino acid homology between LEMMI 5 and the N-terminal sequence of the 76 kDa collagen (Fig.1). Our data support previous speculation about the existence of this novel method of collagen maturation and provide further evidence that this mechanism has been conserved during nematode evolution. In addition to protein processing, the expression of the Lemmi 5 gene was transcriptionally regulated: Lemmi 5-specific transcripts were present in adult females but not in eggs or second-stage juveniles. Also, Lemmi 5 analogs were present only in four Meloidogyne species, but not in C. elegans, Heterodera glycines, or tomato. .. PREDICTED LEMMI5 AMINO ACID SEQUENCE .. 1M A T L V V M P Q L Y S Q I N D L N L R V R D G V Q A .. F R V N T D S A W N D L M E L Q V A V T P Q S K P R S * N P F Q S L YR Q K RS L P D Y C I C Q P L E I N79 S L P D Y C I C Q P L E I N ............................................................ CONFIRMED N-TERMINAL AMINO ACID SEQUENCE .............................................................. Figure 1. The partial predicted amino acid sequence (residues 1-79) of the Lemmi 5 collagen gene is shown in roman letters. The putative subtilisin-like protease recognition sequence, R-Q-K-R, is boxed and the first amino acid after the proteolytic cleavage site is indicated by an *. The empirically determined N-terminal sequence of the 76 kDa from adult female M. incognita is italicized.