Consortium TCeGS (1995) Worm Breeder's Gazette "The C. elegans Genome Sequencing Project: A Progress Report."

Consortium TCeGS

[Worm Breeder's Gazette, 1995]

The C. elegans genome sequencing project: A progress report. The C. elegans Genome Consortium Genome Sequencing Center, Washington University School of Medicine, St. Louis, Missouri, USA and Sanger Centre, Hinxton Hall, Cambridge, UK. We present here another progress report for the C. elegans genome sequencing project. The majority of chromosome I*II is now essentially complete, although some gaps remain where cosmids were not available (Figure 1). While we are rescuing these regions from YACs and lambda clones, we will continue to sequence the cosmid-rich regions of other chromosomes. In the previous issue of the WBG, we reported significant protein similarities (blastx score >100) for several cosmids in the central region of chromosome II. Since then, we have made considerable progress on much of the remainder of this region. Protein similarities for several additional cosmids from chromosome II are presented in Table 1 (all cosmids for which shotgun sequencing has been completed). We also have begun sequencing cosmids near the center of the X chromosome. The St. Louis group started with cosmid C23F12 (near kin-11) and are moving right to left along the physical map. The Sanger Centre group are moving left to right from the same starting point. Similarity data for X chromosome cosmids which have been processed through the initial shotgun phase are also included in Table 1. For each putative genomic locus, the highest blastx score and a brief identifier are listed. Although the cosmids which contain database hits may not be completely sequenced, the Consortium will make preliminary sequence data available to the community with the caveat that it is preliminary and may still contain errors. Finished cosmid sequences are now available by anonymous ftp at: ftp.sanger.ac.uk (directory: pub/C.elegans_sequences). For further information on blastx similarities, please contact LaDeana Hillier (lhillier@watson.wustl.edu) or Steve Jones (sjj@sanger.ac.uk). For information on sequencing plans or estimated completion times, please contact Richard Wilson (rwilson@watson.wustl.edu) or Alan Coulson (alan@sanger.ac.uk). All requests for cosmid clones should be addressed to Alan Coulson. For further information about the C. elegans genome project including our policy statement about sharing both data and sequencing expertise, please contact Richard Wilson.

Thomas JH (1990) Worm Breeder's Gazette "Defecation mutants"

Thomas JH

[Worm Breeder's Gazette, 1990]

Wild-type worms defecate by periodically activating a stereotyped series of muscle contractions. First, the posterior body muscles briefly contract (pBoc) in all four muscle quadrants, squeezing the gut contents anteriorly. When these muscles relax some of the gut contents rush back and collect in a bolus in the preanal region. Just as this relaxation is complete, the anterior body muscles contract ( aBoc) in all four muscle quadrants, driving the pharynx back into the gut and pressurizing the gut lumen. As the aBoc reaches its zenith a set of muscles in the anus contract (Exp) to expel gut contents. This motor program occurs with remarkable regularity in N2, once every 40 to 45 seconds at 23 C in the presence of bacteria. As first found by Steve McIntire for unc-25 and unc-47, most mutants defective in defecation become constipated and are readily recognized using a dissecting microscope. Using a combination of observing existing behavioral mutants and screening directly for new constipated mutants, I have been accumulating mutants defective in defecation. At latest count there are mutations in 23 genes that cause strong defects in some aspect of defecation, including 10 new genes. Viable, fertile mutants for three of the genes appear never to actively defecate, suggesting that the defecation motor program is a dispensable function (instead they appear to rarely release gut contents passively, presumably when gut lumen pressure becomes great enough to force the anus open). The mutants fall, without too much cramming, into five phenotypic classes. Three of the classes have defects specific to one or the other of the three motor steps described above, without affecting the other two. A fourth class has defects in both aBoc and Exp, but not pBoc. The fifth class has defective timing of the cycle period but the motor program, when it occurs, is normal. The most novel mutants are those that affect the cycle period. So far there are three genes in this class, cha-1, dec-1, and unc-75, and each causes the intercycle period to become much longer and more irregular in length than normal. cha-1 encodes choline acetyltransferase, and cha-1 mutants are severely deficient in acetylcholine (ACh). This result implicates ACh, directly or indirectly, in control of the cycle period. Interestingly, unc-75 seems also to be a good candidate for affecting cholinergic function: it's pumping, locomotory, and defecation defects are remarkably similar to those of strong cha-1 alleles. dec-1 mutants are not Unc and pump normally, but have a longer defecation cycle.

Seydoux GC et al. (1997) Worm Breeder's Gazette "Transcriptionally-repressed germline blastomeres lack a specific subpopulation of phosphorylated RNA polymerase II"

Dunn MA, Seydoux GC

[Worm Breeder's Gazette, 1997]

Embryonic germline blastomeres (P1, P2, P3 and P4) fail to express many mRNAs expressed in somatic blastomeres (1). In contrast, rRNAs are expressed in both cell types (2). In principle, this difference could be due to the rapid degradation of newly transcribed mRNAs in germline blastomeres, or to a block in mRNA transcription in the germ lineage. To begin to distinguish between these two possibilities, we have characterized the distribution of phosphorylated isoforms of the large subunit of RNA polymerase II (RNAP II LS) in embryos. For this purpose, we used 2 monoclonal antibodies, H5 and H14, that recognize distinct phosphorylated epitopes on the carboxy terminal domain (CTD) of RNAP II LS (3). Phosphorylation of the CTD has been correlated with transcriptional elongation; indeed, we find that H5 and H14 start to stain somatic nuclei in the 4-cell stage, when these cells are known to begin mRNA transcription. However, no H5 staining, and only weak H14 staining, were detected in germline blastomeres. In contrast, an antibody (4) that recognizes both phosphorylated and unphosphorylated RNAP II showed no difference in staining between somatic and germline blastomeres. These results indicate that germline blastomeres lack the subpopulation of phosphorylated RNAP II recognized by H5, and have reduced levels of the subpopulation of phosphorylated RNAP II recognized by H14. These observations are consistent with a block in RNAP II activity in the early germ lineage. We have previously shown that the germline-specific factor PIE-1 is required to block mRNA production in the embryonic germ lineage (1). To test whether pie-1 is also required for the germline-specific patterns of H5 and H14 staining described here, we stained embryos derived from pie-1(zu154) mothers with H5 and H14. We found that in the absence of pie-1 activity, germ cells stain with H5 and H14 in a pattern identical to that of somatic cells. These results indicate that pie-1 activity is required for the soma-germline differences observed in H5 and H14 staining, and suggest that PIE-1 may function to repress, directly or indirectly, RNAP II activity in the germ lineage. We have also used the H5 and H14 antibodies to stain Drosophila embryos. Remarkably, we find that pole cells (the Drosophila embryonic germ cells) also fail to stain with H5, and stain only weakly with H14. These observations raise the possibility that a block in RNAP II activity may be part of an evolutionarily conserved mechanism that distinguishes germline from soma during early embryogenesis. We thank Miriam Golomb, Steve Warren and Jeff Corden for providing antibodies, and Debbie Andrew for advice on Drosophila handling. 1. Seydoux et al., 1996. Nature 382, 713-716. 2. Seydoux et al., 1996. East Coast Worm Meeting Abstracts, p 31. 3. Bregman et al., 1995. Journal of Cell Biology 129, 287-298; and M. Patturajan and J. Corden, pers. comm. 4. Sanford et al., 1985. Journal of Biological Chemistry 260, 8064-8069

Johnson TE et al. (1990) Worm Breeder's Gazette "Identification of Deficiency Endpoints Surrounding fer-15 and emb-"

Tedesco PM, Johnson TE

[Worm Breeder's Gazette, 1990]

We are in the process of cloning the age-1 gene. The most extreme mutant allele in this locus has an increase in mean life span of 60% and in maximum life span of about 110% at 25 C. In backcrosses to N2, the age-1 locus was tightly linked (<1 map unit) to fer-15 and cosegregated with a four-fold reduction in hermaphrodite fertility ( Brd), as well (Genetics, 118:75, 1988). fer-15 and emb-27 have been mapped to the same interval in the subcluster on chromosome II by deficiency mapping, which we have confirmed. We decided to employ a strategy for cloning age-1 wherein we would identify RFLPs generated by the endpoints of specific deficiency chromosomes and identify fer- 15, d injections. However, recent data suggest that age-1 is separable from fer-15 and nearer to unc-4 (see Hutchinson and Johnson, elsewhere in this Newsletter). Nevertheless, we have decided to finish up our molecular analysis of this region of chromosome II. Three deficiencies have endpoints immediately adjacent to the left of the fer-15, hile eight deficiency endpoints mapped adjacent to the right. We have identified all three endpoints to the right and seven of the eight endpoints to the left. Along the way, we've also identified the other ends of four of these deficiencies and identified a possible deficiency associated with fer- 15(b26) in T13F9. This information is summarized in the figure. We chose cosmids in the Rol-6 contig, which covers most of chromosome II, based on Phil Carter's observation that C26D10 revealed an RFLP in mnDf104 (WBG, 10,2:60) which we confirmed. Since the deficiency chromosomes must be carried in the heterozygous state, we anticipated that an endpoint would be most reliably detected as a new band in a genomic blot. (This is because a band that has a two-fold decrease in intensity is hard to verify without densitometry and likely to be variable for different deficiencies.) Most of these endpoints have been verified as giving new bands with more than one enzyme (to distinguish deletions from single-base changes). In the figure below, the relative position of these endpoints within a cosmid are unordered (we don't yet have a reliable restriction map of the region). We have not yet probed all combinations of deficiencies and restriction enzymes with all overlapping cosmids so we can't localize endpoints to cosmid subregions. As a control for rearrangements, we have been routinely comparing band size of genomic blots with band size of cosmid digests and generally find good correspondence. A few cosmids (e.g F58D3) were difficult to recover intact, often giving identical deletions in different picks. Our current plan is to prove that the fertility deficit is separable from age-1 and to confirm our identification of fer-15 and emb-27 by using T13F9 and adjacent cosmids to rescue these mutants by injection. We hope to get back to cloning of the age-1 gene soon and leave fer-15 and emb-27 to others ( Steve L'Hernault has similar, but not identical data). [See Figure 1]

Stringham-Durovic EG et al. (1992) Worm Breeder's Gazette "Induction of the Heat Shock Response in Individual Cells of C. elegans Using a Laser Microbeam"

Candido EPM, Stringham-Durovic EG

[Worm Breeder's Gazette, 1992]

We have been studying the temporal and spatial expression of the hsp 16 genes of C. elegans by promoter analysis of transgenic animals carrying hsp16 -lacZfusions. Recently, stably integrated strains were derived from previously described strains carrying extrachromosomally inherited hsp 16 lacZ transgenes (Stringham et al., (1992) Molec. Biol. Cell, 3:221-233) by gamma irradiation mutagenesis (Jeff Way, personal communication). When animals carrying the integrated transgene, 48.1 C, are heat shocked and assayed for -galactosidase activity, blue staining is observed in most, if not all, somatic tissues. In contrast, animals which have not been subjected to a heat treatment do not stain at all. Recently, the inducible hsp16 promoters have been employed to achieve tightly regulated expression of various genes in C. elegans including mab-5 (Salser and Kenyon), and mec-3 (Way and colleagues). We have been investigating the feasibility of inducing the heat shock response in individual cells of the worm by directing a laser microbeam to a particular target in an animal carrying an hsp16 -lacZtransgene, 48.1C, and then staining for -galactosidase activity. The intensity of a uv laser beam typically employed for ablation experiments was reduced by placing 14-20 mm of stacked microscope slides in front of the laser to act as a filter. This allows the manipulator to administer continual pulses from the laser to the target over a period of several minutes without killing the cell. Using this technique we have achieved single cell expression of -galactosidase in hypodermal, intestinal, neuronal, pharyngeal and muscle cells. These results demonstrate that a uv laser microbeam normally employed for cell ablation can be adapted to produce a sub-lethal heat shock response in a given cell. The molecular basis for induction by the laser remains to be defined. The common feature of agents or conditions which induce the stress response is thought to be their ability to denature proteins. Laser irradiation might act to damage proteins directly by heating or indirectly by generating free radicals which could result in oxidative damage. Transcription and translation of lacZ appeared to be unhindered by the laser treatment. Thus synthesis of functional proteins encoded by other genes under control of the hsp16 promoter should also be laser inducible in individual cells, offering unique opportunities to the C. elegans investigator who wishes to study cell-cell interactions. To take advantage of the bidirectional nature of the hsp16 promoter, we are currently inserting a polyclonal region into the 5' end of 48.1E1 such that expression of any coding region of interest can be conveniently coordinated with levels of lacZ expression. In this fashion, successful induction of the test transgene in a single cell can be verified by staining for lacZ expression. Alternatively, an integrated hsp16 -lacZtransgene could be crossed into a strain already carrying an hsp16 - fusion of interest. We are currently mapping the sites of array integration in our stable lines to facilitate in this endeavor. We wish to thank Cathy Rankin for use of her laser and Steve Wicks for many helpful discussions. This research was supported by grants from MRC Canada, B.C. Health Research Foundation, and Stressgen Biotechnologies Corp.

Michael Ailion et al. (2001) Worm Breeder's Gazette "All available mutations in flp-1 also delete daf-10 sequences, confounding phenotype interpretation"

Michael Ailion, James H Thomas

[Worm Breeder's Gazette, 2001]

Based on the details explained below, several of the phenotypes previously attributed to flp-1 mutants are more likely to be caused by loss of daf-10 . These include defects in osmotic avoidance (Osm), thermal avoidance (Tav), nose-touch response (Not), wandering behavior, regulation of dauer formation, and possibly regulation of egg-laying behavior in response to food signals. Defects in these behaviors are characteristic of cilium-structure mutants, and could be easily accounted for by loss of daf-10 . In surveying a variety of behavioral mutants for a dauer phenotype at 27deg, we found that both flp-1 deletion mutants, yn2 and yn4 , have a moderate Daf-c phenotype at 27deg and poor dauer recovery. Since many 27deg Daf-c mutants are hypersensitive to dauer pheromone, we tested flp-1(yn2) for response to pheromone at 25deg. Surprisingly, it formed no dauers in response to pheromone. Mutations in the dyf genes similarly lead to a Daf-d phenotype at 25deg and a Daf-c phenotype at 27deg. These mutants have defects in the structure of the ciliated endings of sensory neurons, a defect which can be assayed by the failure of these neurons to fill with fluorescent dyes. Thus, we performed FITC dye-filling assays on flp-1(yn2 ) and flp-1(yn4) and found that both mutants failed to exhibit dye-filling of any of the amphid or phasmid neurons. flp-1 is not expressed in any of the amphid sensory neurons. Thus, we suspected that the flp-1 Dyf phenotype was due to a background mutation present in both flp-1 strains. The two alleles are not independent since they were isolated as deletions from the same parent strain which carries a Tc1 insertion just upstream of flp-1 . We mapped the Dyf phenotype and found that it mapped to the right of unc-5 and was not separable from flp-1 (based on flp-1 movement phenotypes). The only candidate dyf mutant in this region is daf-10 . We found that flp-1(yn2) failed to complement daf-10(e1387) for the Dyf phenotype, indicating that the flp-1 mutant strain also carries a mutation in daf-10 . The flp-1 strains exhibit a more severe dye-filling defect than daf-10(e1387) , indicating that they are stronger alleles. daf-10 is encoded by F23B2.4 (Steve Stone and Jocelyn Shaw, personal communication), the gene immediately upstream of flp-1 . Further inspection of the flp-1 deletions indicates that yn2 and yn4 both delete the daf-10 promoter and from two to four exons of the predicted daf-10 coding sequence. The original Tc1 used to isolate the flp-1 deletions appears to reside in the daf-10 gene, and both flp-1 deletion alleles are likely null for both genes. Although several of the sensory phenotypes of flp-1 mutants can be explained by loss of daf-10 , it is likely that several other phenotypes previously ascribed to flp-1 are bona fide flp-1 phenotypes. Specifically, flp-1 mutants have defects in movement and regulation of the active state of egg-laying that are not easily explained by loss of daf-10 . Furthermore, both of these phenotypes were rescued by flp-1(+) transgenes.

Labouesse M et al. (1990) Worm Breeder's Gazette "Potential zinc finger in lin-26, a gene involved in the control of the asymmetric cell division of the P cells."

Labouesse M, Horvitz HR

[Worm Breeder's Gazette, 1990]

During development, many cells divide asymmetrically to produce daughters that differ in their fates. The mutation lin-26(n156) II disrupts the asymmetry of some cell divisions. This mutation is the only lin-26 allele: it is recessive and is probably not a null allele since it results in lethality in trans to a deficiency (1). lin-26 hermaphrodites are vulvaless. More specifically, the 12 Pn.p cells. which include the six P(3-8).p vulval precursor cells, appear to be transformed to express the fates of their sisters, the 12 Pn.a neuroblasts (2). In wild-type animals, the Pn.a cells undergo up to three rounds of divisions and generate five neural descendants, one of which, the VD neuron, expresses the neurotransmitter GABA. In lin-26 animals, the Pn.a cells are unaffected while the Pn.p cells undergo up to three rounds of divisions and generate descendants with a neural appearance, including extra GABAergic cells (3; Steve McIntire personal communication). These data suggest that lin-26 functions in making the Pn.p cell fate different from the Pn.a cell fate. We have started a molecular analysis of lin-26. The mutation n156 was mapped relative to a set of seven deficiencies spanning part of LGII. The deficiencies mnDf84, nt n156, the deficiencies mnDf97 and mnDf106 partially complement n156, and the deficiencies mnDf88 and mnDf105 do not complement n156 (see genetic map). We physically mapped the left breakpoint of these seven deficiencies using cosmids from the contig spanning most of LGII. The overlapping cosmids C24G10 and C09G7 identify the breakpoint of the deficiencies mnDf97 and mnDf106 (either as polymorphisms or as missing fragments). Using germline transformation, we have been able to rescue n156 by injecting the cosmid C18C9. which overlaps with C24G10 over a 10 Kb region. Subsequent subcloning identified a 9 Kb region sufficient to almost fully rescue n156 (the vulva is slightly protruding, whereas if this 9 Kb fragment is extended on one side the animals are phenotypically wild-type). Northern blot analysis shows that three overlapping RNAs, of 2.2 Kb, 1.5 Kb and 1.45 Kb. are transcribed from the central part of this 9 Kb region. The 1.5 Kb and 1.45 Kb RNAs hybridize to the same set of sub-fragments from the 9 Kb region; most likely they differ by the presence of a small exon or the choice of different 5'/3' ends. We have isolated 14 cDNAs (from 500,000 plaques screened using Stuart Kim's cDNA library): one is a 2.2 Kb cDNA and corresponds to the 2.2 Kb transcript, and one is a 0.75 Kb cDNA and corresponds to the 1.5 Kb/1.45 Kb transcript. The 2.2 Kb cDNA can potentially encode a protein of 473 amino acids. The incomplete 0.75 Kb cDNA contains an open reading frame (ORF) of 131 codons; the 3' untranslated region of the latter cDNA overlaps with the 5' untranslated region and beginning of ORF of the 2.2 Kb cDNA. Database searches (TRANSGEN) indicate that both cDNAs could encode a protein displaying two potential zinc fingers related to, although different from, the Kruppel/mKr2 type of zinc fingers (4; see Figure). We do not yet know which of these three RNAs (if any) are required for lin-26 function. The likely existence of three lin-26 transcripts suggests that alternative splicing and/or alternate use of promoters/terminators plays a role in the expression of lin-26.[See Figures 1 & 2]

Hunter CP et al. (1993) Worm Breeder's Gazette "It's All in the Timing; Initiation and Maintenance of Homeotic Gene Expression"

Kenyon CJ, Hunter CP

[Worm Breeder's Gazette, 1993]

The homeobox gene pal-1 functions in the V6 seam cell to prevent signals from neighboring cells from inhibiting mab-5 function in V6 (Waring and Kenyon 1990;1991). In both mab-5 and pal-1 mutants V6 generates a lineage similar to V1 -V4,producing alae instead of rays. V6 will make rays in pal-1 mutants if seam cells adjacent to V6 are ablated or if mab-5 activity is provided by the mab-5 gain-of-function allele e1751 .Steve Salser (see this WBG) has shown by immunofluorescence antibody staining that, unlike in WT, in pal-1 (e2091 ),mab-5 protein is not expressed in V6 at hatching; a mab-5 -lacZreporter gene is also not expressed in V6 in the embryo. The current model is that signals between seam cells inhibit mab-5 expression in the seam cells and that pal-1 functions in V6 to overcome or bypass this inhibition. We have asked when cell signals, mab-5 ,and pal-1 function. To learn when cell signals function, we ablated the posterior seam cell T, or its seam cell descendants T.a or T.ap, at one hour intervals following hatching in a pal-1 background. V6 descendants produced rays following ablation of T, T.a, and T.ap until about eight hours after hatching. Ablating T.ap after eight hours failed to rescue the pal-1 phenotype. Thus, in a pal-1 mutant, signals from T.ap inhibit mab-5 function in V6 and the effect of these signals becomes "fixed" at about eight hours post hatching. To learn when mab-5 functions in V6 ,we expressed a mab-5 cDNA under heat shock control (Salser and Kenyon 1992). Heat shock-induced mab-5 expression before eight hours post hatching did not significantly rescue pal-1 whereas mab-5 expression after eight hours rescued most animals. That is, mab-5 can only function after eight hours. We know that mab-5 is normally expressed in V6 beginning during embryogenesis (Cowing and Kenyon 1992), and that pal-1 activates mab-5 at this time since a mab-5 -lacZreporter construct is not expressed in V6 in pal-1 (e2091 )embryos. To learn when pal-1 can function, we expressed a pal-1 cDNA under the control of the hsp16 -1heat shock promoter. Heat shocked-induced pal-1 expression anytime during embryogenesis or up to about eight hours after hatching rescued pal-1 mutants. Heat shock treatment after eight hours failed to rescue pal-1 .Since the function of pal-1 is to turn on mab-5 before the effect of the cell signals is fixed, and yet mab-5 functions after that pint, we conclude that the mab-5 gene must be expressed or made expressible before the effect of the cell signals becomes fixed. This observation suggests that homeotic genes not activated early are repressed and raises the possibility that there is a system for maintaining the expression states of homeotic genes in C. elegans, perhaps similar to the polycomb class of genes in Drosophila (see Maloof and Kenyon this WBG). pal-1 may act by either inhibiting the cell signals (by repressing genes required for signal transduction) or by inhibiting their effect (perhaps mediating or preventing a change in the chromatin structure of mab-5 ).To identity other genes involved in this process we have begun isolating extragenic suppressor mutations of pal-1 in a visual screen of F2 clones. In a pilot experiment we isolated seven suppressors from 1500 clones. The three best characterized suppressors cause seam cells anterior to V5 to produce rays and cause anterior expression of a mab-5 -lacZreporter construct.