Otsuka AJ (1990) Worm Breeder's Gazette "ALTERATIONS IN UNC-44 ALLELES"

Otsuka AJ

[Worm Breeder's Gazette, 1990]

Preliminary analysis of six spontaneous unc-44 alleles indicates that alterations occur over a fairly large region in the genomic DNA. Southern analysis of two alleles from E. Hedgecock (rh1013 and rf1042) and four alleles (mn259, mn339, q331, and st200) from R. Herman and other labs (kindly provided by C. Kari, R. Herman, W. Li, and J. Shaw) indicated that restriction fragment differences from wild type are associated with all six alleles (see Franco et al., C. elegans Meeting abstracts, 1989). The construction of preliminary restriction maps of unc-44 cosmid (B0350 and C44A, kindly provided by A. Coulson and J. Sulston) and phage (DD#LRF1 and DD#LRF6) clones allows the approximate positioning of mutant changes. The absolute orientations of the cosmid and phage clones are based on positioning of these clones on the cosmid map. The overlapping of the cosmid and phage clones has not yet been demonstrated. Unfortunately, the cosmid isolate that could quickly clarify the situation (C02C3) deleted during growth. [See Figure 1]

Albertson DG (1990) Worm Breeder's Gazette "The Mutation mn164 is an X;I Translocation"

Albertson DG

[Worm Breeder's Gazette, 1990]

The mutation mn164 recovered after X-ray treatment promotes X chromosomal nondisjunction when homozygous or heterozygous. The mutation was mapped to the left end of the X chromosome and it was proposed that a structural abnormality was affecting segregation of the X chromosome. Equational nondisjunction of the mn164 bearing chromosome, but not its homolog, was observed in heterozygotes (Herman et al. (1982) Genetics 102, 379-400). As most him mutations show reductional nondisjunction, it was of interest to look at the cytogenetics of a him mutation causing equational nondisjunction. The first meiotic division is reductional in C. elegans, and abnormal bivalents have been seen at diakinesis in some of the him mutants that cause reductional nondisjunction. Thus, in mn164, abnormalities were expected at the second meiotic division. However, abnormal chromosome segregation was seen at both meiotic divisions in embryos from mn164/dpy-3 hermaphrodites, and at diakinesis in these hermaphrodites a large trivalent and four normal-sized bivalents were seen. In mn164 homozygotes a univalent was present in addition to the one large and four normal-sized bivalents. The large bivalent is reminiscent of mnT12(IV,X), suggesting that mn164 is a translocation. In situ hybridization of the ribosomal DNA probe (pCe7) to the middle of the large chromosome identified LGI as one member of the translocation. Hybridization of pCe7 and a probe for the right end of the X (mlc-1,2) to embryonic metaphases showed hybridization at 40-50% from one end of the long chromosome (pCe7) and on the end (mlc-1,2). These observations suggested that mn164 is a translocation involving joining of the left end of X to the right end of LGI. Hybridization of pCe7 to the ribosomal DNA appears reduced and therefore the univalent seen in oocytes of mn164 homozygotes, an extra copy of LGI, may be necessary for viability. Since these cytogenetic observations were made on mn164 animals some time after the original description of the mutation, it was possible that the Cambridge stock had changed from the original. Therefore Claire Kari and Bob Herman thawed a stock of mn164, frozen on January 5, 1981 and pseudolinkage between unc-54(I) and dpy-3(X) was confirmed by mating mn164 males with unc-54(I);dpy-3(X) hermaphrodites.

Culotti JG et al. (1991) Worm Breeder's Gazette "Mosaic analysis of unc-5 using an unc-5(+) cosmid array fused to"

Spence AM, Hedgecock EM, Culotti JG

[Worm Breeder's Gazette, 1991]

We have been able to construct a strain suitable for mosaic analysis of unc-5 by fusing the unc-5 cosmid array eEx15/unc-5(+)/ to sDp3/dpy- 1(+) ncl-1(+)/ using the technique described by Herman and Kari ( WBG11#5, p.68). Briefly, we first constructed strain NW705 which carried eEx15 and sDp3 as independently segregating free Dps in a background that was chromosomally mutant for dpy-1 rain segregated wild type, Unc nonDpy, Dpy nonUnc, and Dpy To attempt to fuse eEx15 and sDp3 we treated NW705 adults with 3600 rads of gamma- rays from a [137Co] source. 894 F1 progeny were cloned of which 344 produced broods of greater than 50. Among these we found 9 independently derived strains that behaved genetically as though the cosmid array eEx15 had been fused to sDp3, i.e., they segregated wild type and Dpy tilization progeny, but no Unc nonDpy or Dpy nonUnc progeny. (Some did initially segregate a few Dpy nonUnc progeny, but siblings that only segregated wild types and Dpy Uncs could easily be found, suggesting that extra unfused copies of the cosmid array can hang around). To assess the frequency of mitotic loss of the fused Dp in these 9 strains, we looked for 'gonad mosaic' animals that moved normally, but had 1 or more misreflexed gonad arms (Hedgecock et al., Neuron 4, 61- 85,1990). For 6 strains we failed to see any such mosaic animals and for 2 strains the frequency of such mosaic animals was judged to be too high (approximately 1% or more) to be useful for mosaic analysis. For 1 strain, however, the frequency of mosaic animals was less than 1 in 500, so we chose this strain, NW707 for mosaic studies and named the fused Dp evEx8. This strain has proven to be useful for mosaic analysis of unc-5. By scoring nucleolar size in cells derived from a number of embryonic blast cells, we have found a number of mosaic animals which are all consistent with cell-autonomous action, that is, unc-5 acts in cells that normally exhibit dorsal migrations on the epidermis (motorneurons DA, DB, DD, AS and VD; mesodermal distal tip cells; ectodermal excretory cell). This result is consistent with the hypothesis that the unc-5 protein is an adhesive receptor present on migrating mesodermal cells and neuronal growth cones (CSH Abstracts, 275-276, 1989).

Winter M et al. (1986) Worm Breeder's Gazette "suppressors of bli-6(mn4) IV."

Herman RK, Winter M

[Worm Breeder's Gazette, 1986]

The mutation mn4, which was identified among the progeny of X-ray treated males, confers a blistered phenotype. The mutation is dominant to wild-type with 100% penetrance. The reference allele for bli-6 IV is sc16, which is recessive to wild type. Meredith Kusch has mapped mn4 and another dominant mutation, sc74, close to sc16; and Park and Horvitz (1986, Genetics 113:821) have mapped their EMS-induced dominant mutation n776 very near to sc16. Thus it appears that bli-6 IV is represented by one recessive and three dominant alleles. We have identified 44 independent EMS-induced revertants of mn4. All are extragenic suppressors, and they fall into two general classes: 23 are recessive dumpy mutations and 21 have wild-type morphology. All but one of the dumpy mutations have been assigned to known dpy genes, as follows: 1 allele of dpy-1 III, 3 alleles of dpy-3 X, 4 alleles of dpy-4 IV, 3 alleles of dpy-5 I, 3 alleles of dpy-7 X, 4 alleles of dpy- 8 X, 1 allele of dpy-9 IV, 1 allele of dpy-10 II, and 2 alleles of dpy- 13 IV. Not all dumpy mutations suppress mn4. For example, dpy-6(e14) X, dpy-17(e164) III and dpy-18(e364) III do not suppress mn4 (and no suppressing alleles of these genes have been identified in our reversion screen), but dpy-5(e61) and dpy-1O(e128) do. All but three of the 23 dumpy mutations are recessive suppressors of mn4, i.e., animals of genotype dpy/+; mn4/+ are Bli non-Dpy. In the other three cases, however, dpy/+; mn4/+ animals are non-Bli non-Dpy. The three dominant suppressors are alleles of dpy-4, of these genes, we have at least one recessive suppressor allele; thus in each case the effect is allele specific, not gene specific. An incidental point about another dpy: Claire Kari has shown that the previously unmapped dpy-12(e182) is an allele of dpy-3.Analysis of the wild-type suppressors has not progressed as far, although most have been outcrossed at least twice. Seven of the 21 suppressors are dominant, by the criterion that mn4/+; sup/+ hermaphrodites are non- Bli (males of this genotype are sometimes blistered). Three of the recessive wild-type suppressors appear to be X-linked (mn4/+; sup/O males are non-Bli) and fail to complement each other. We note that Kusch and Edgar (C. elegans Newsletter 9, no. 1. p.51) previously identified a wild-type suppressor of bli-6(sc73) which mapped to LGV and which also suppressed bli-6(sc16).We have put mn4 into a TR679- like background and identified a few spontaneous revertants. One is a dumpy, an allele of dpy-5 I, and we have a spontaneous revertant of it.

Herman RK (1978) Worm Breeder's Gazette "stockkeeping, mapkeeping, and genetic clearing house-keeping."

Herman RK

[Worm Breeder's Gazette, 1978]

With lots of help from the Medical Research Council, especially Marilyn Dew, we now have a core collection of C. elegans E stocks, and we (Claire, Kari & I) hereby offer to supply requested strains. At John Sulston s request, I have also agreed to handle two other functions: maintaining the genetic map and administering the genetic clearing house. At present, we have no special support for this work. As many of you know, Don Murphy at NIA is exploring the possibility of supporting a stock center, which could be here or somewhere else. At any rate, I think it makes sense to have the mapkeeping and clearing house operations affiliated with the stock collection, and on the advice of John Sulston and Bob Horvitz, I have defined what, with your support and help I propose to do, for the time being at least: 1. Stockkeeping: Keep and supply requests for at least one mutant per mapped gene or know where in North America the mutants can be obtained. 2. Mapkeeping: (a) Solicit new data (actual counts, complementation results, etc.) on both old and new genes, and update the official map records as such data become available. (b) Submit new data for publication in the Newsletter. These data would include, besides linkage results, interesting properties of new or old genes. 3. Genetic clearing house-keeping: (a) Register new lab symbols, which, in accordance with Bob Horvitz s nomenclature paper, are one or two letters, used as non-ized upper case prefixes for strain names and ized lower case prefixes for mutation names. (b) Register new gene names and list them in the Newsletter. The laboratory assigned to a gene name is then responsible for issuing gene numbers. I hereby solicit data, comments, suggestions, etc. What follows is a list of E stocks available, some new map data, and a few clearing house assignments. Please note: We have taken a cue from the E. coli stock center and have not attempted to confirm the gentoypes of all stocks. We certainly have checked all the more obvious phenotypes, such as for unc and dpy mutants, and we ve been careful in handling heterozygotes, but we have not confirmed some of the other mutants, such as cat, che and flu. If you request any of the latter class, it will be useful if you inform us, say upon acknowledging receipt of the stocks, how they checked out, and we can record that information. In the list that follows, if no mutation name is given for a homozygous single mutant, it can be derived from the strain name, (i.e. , strain E769 has the following genotype: bli-1(e769)).

Way JC et al. (1991) Worm Breeder's Gazette "Sequences for maintenance of mec-3 expression"

Zhang J, Way JC

[Worm Breeder's Gazette, 1991]

Comparison of the mec-3 DNA sequences from C. elegans and WS9-6 ( another Caenorhabditis species) shows that all the exon sequences are conserved, and in addition, four blocks of nearly identical sequence are found upstream from the start codon (Way, WBG 11, 4). On the assumption that these conserved upstream regions are regulatory sequences, we have used in vitro mutagenesis to investigate their function. Specifically, we have altered various sites in these conserved regions in the mec-3-lacZ fusion plasmid pTU28 (Way and Chalfie, Genes and Dev.3:1823) to see if the conserved sequences are important in determining when and where mec-3 is expressed. Mutations in either Site II or Site III cause a defect in mec-3 maintenance. A disruption of site II causes a severe maintenance defect: the mec-3-lacZ fusion is expressed briefly after the expressing cells are born, then expression is lost for the rest of development. This pattern of expression is the same as when a non- mutant mec-3-lacZ fusion is placed in a strain with a chromosomal mec- 3 mutation. A mutation in site III has a less dramatic effect: expression comes on in all the correct cells, then gradually fades, so that by adulthood, the number of expressing cells is significantly less than for a wild-type mec-3-lacZ fusion. Within the conserved regions are putative binding sites for both mec- 3 and unc-86 (see below and previous newsletters; the mec-3 binding site in Site II is inferred largely from the mutant defect and is only weakly related to the ISL-1 footprint site). Because site II appears to have an unc-86 binding site, we decided to test whether mec-3 expression would depend on unc-86 for maintenance as well as establishment. For ease of strain construction, we first made an integrated derivative of the mec-3-lacZ/unc-22 (antisense) extrachromosomal element uEx4 by gamma-ray treatment (Kari et al. WBG 11, 3, p. 14); this integrated element is termed jeIn2. jeIn2 was placed in a temperature sensitive unc-86 background (n848), so that unc-86 could be inactivated after mec-3 expression had been established. mec-3 does appear to require unc-86 for maintenance. If jeIn2; n848 is shifted to the non-permissive temperature after mec-3-expressing cells are born, they lose expression of mec-3. For example, if this strain is grown at 15 C until the mid-L1 stage, then either stained immediately or shifted to 25 C, the stained L1s express mec-3-lacZ at normal levels. However, if the shifted animals are stained after the L4 stage, expression of mec-3-lacZ is greatly reduced. [See Figure 1]

Hamelin M et al. (1990) Worm Breeder's Gazette "WHAT TURNS ON mec-7? PART II: THE RETURN OF mec-"

Culotti JG, Hamelin M, Way JC

[Worm Breeder's Gazette, 1990]

The mec-7 gene encodes a -tubulin required for the formation of 15- protofilament microtubules (Genes and Dev. 3:870). These large diameter microtubules are found exclusively in the touch neurons (ALML ALMR, AVM, PVM, PLML, and PLMR) (J. Cell Biol.93:15). mec-7 mutants lack the 15-protofilament microtubules and are touch insensitive. We recently reported that a mec-7-lacZ extrachromosomal element, evEx1, is expressed exclusively in the touch neurons, and at a lesser extent, in the FLP neurons (W.B.G.11[3]40). The gene mec-3 encodes a potential transcription factor that is required for the proper differentiation of the touch neurons (including the appearance of 15- protofilament microtubules) (Cell 54:5). Similarly, mec-3 was previously shown to be expressed in the touch neurons and in the FLPs, but also in the PVD neurons (Genes & Dev. 3:1823). An attractive possibility is that mec-3 directly activates mec-7 expression. However, our mec-7-lacZ construct was found to be expressed at almost normal levels in a mec-3 mutant background, using the presumed null allele u298:Tc1. This led us to propose that the mec-3 gene product was not required to trigger mec-7 transcription (W.B.G. 11[3]40). Here we present recent data which suggest that, in fact, mec-3 is the transcriptional activator of mec-7.First, mec-7 may be transcribed in all the cells that were previously shown to express a mec-3-lacZ fusion gene, i.e. the touch neurons as well as the FLPs and the PVDs. We have stably integrated the mec-7-lacZ fusion gene, following the procedure of Kari et al. (W.B.G. 11[3]14). The integrated evEx1 array has been named jeIn1. Using jeIn1-containing N2 animals, we found stronger and more consistent stainings, as compared with evEx1 containing animals -Gal activity was still predominantly confined to the touch neurons, but a significantly higher proportion of larvae stained in the FLPs. PVD neuron staining was also detected in a proportion similar to the FLPs (identities of these cells to be confirmed). mec-7 may therefore be expressed in the exact same subset of neurons as mec-3 (this raises the question as to why 15- protofilament microtubules are not found in the FLPs or the PVDs). Second, and most importantly, the mec-7-lacZ gene expression is greatly reduced in some newly tested mec-3 mutants. We have transferred jeIn1 into the genetic background of 5 more mec-3 alleles ( sent to us by Shohei Mitani). Table 1 shows the results of the X-Gal stainings of these mec-3 transgenics: there is a global decrease, of a similar amplitude in all cells, of mec-7 expression. This decrease is more pronounced in some alleles. This suggests that some alleles of mec-3, in particular u298, are somehow leaky, while others, such as u6 are more severe (but not null?). In the light of these results, the possibility that the mec-3 protein directly activates mec-7 transcription remains a strong one. [See Figure 1]

Schackwitz WS et al. (1992) Worm Breeder's Gazette "Suppressors of daf-11"

Schackwitz WS, Thomas JH

[Worm Breeder's Gazette, 1992]

C. elegans can form an alternative developmental stage called the dauer larva when exposed to high levels of dauer-inducing pheromone. Many genes have been identified that are involved in the formation of a dauer. Mutations in most of these genes are either dauer constitutive (Daf-c) or dauer defective (Daf-d). The genes have been placed in a regulatory pathway based on gene interactions (see Thomas and Birnby, this issue). Although Daf-d mutations in the part of the pathway downstream of daf-7 have been screened for intensively, screens that might identify Daf-d genes just downstream of daf-11 have been very limited. Nearly all such mutations have been discovered fortuitously in other screens. To identify Daf-d genes that lie just downstream of daf-11 ,we screened for EMS-induced suppressors of the Daf-c phenotype of daf-11 (m87).The screen identified 47 independent mutations. Of these, 16 conferred an FITC-filling and osmoticavoidance defect, indicating that they probably affected amphid and phasmid cilium structure. These were sent to C. Kari and R. Herman, who have shown that they include 3 osm-1 mutations, 2 osm-3 mutations, 2 osm-5 mutations, 1 osm-6 mutation, and 1 che-2 mutation. The remaining 31 mutants were normal for either FITC-filling or osmotic-avoidance or both. Tentative double mutants between daf7 (e1372 )and these 31 suppressor mutations were constructed in an attempt to determine where in the pathway the new mutations act. Only one mutation could suppress the Daf-c phenotype of daf-7 ,and it proved to be a new allele of daf-12 .This result suggests that the remaining 30 mutations may affect previously unidentified genes involved in the dauer formation pathway. The remaining 30 suppressor isolates have been tested for response to dauer pheromone. Response was assessed by setting synchronous egg lays (approximately 50-150 eggs) on pheromone-containing plates and M9 -controlplates. The plates were placed at 25 C and scored 55 hours later for the percentage of dauer formation. Nineteen of the mutants were pheromone non-responsive under our assay conditions, one was responsive, and the remaining ten gave inconclusive results. Many of these 30 suppressor mutants have been mapped to a chromosome, and a few have been placed into complementation groups. Sixteen of the mutants have been tentatively mapped to chromosomes using 2-factor crosses. Five appear to map to chromosome I, one to chromosome II, six to chromosome V, and four to the X chromosome. Limited complementation tests have been performed for the mutations that are located on chromosomes I and V. There appear to be at least two complementation groups on chromosome I and at least 3 complementation groups on chromosome V. One of the 30 mutations, sa177 ,is dominant for its suppressor activity. This mutation appears to be closely linked to daf-11 . The suppressor isolate is pheromone responsive, and unlike daf-11 (m87),has a normal response to odorants. Therefore, sa177 appears to be G strong candidate for an intragenic revertant of daf-11 (m87).

Xie GF et al. (1992) Worm Breeder's Gazette "Selection of Mutants Based on Histochemical Staining Patterns."

Hamelin M, Culotti JG, Jones S, Jia Y, Xie GF, Aamodt EJ, Ravi L

[Worm Breeder's Gazette, 1992]

We have used the histochemical staining method described in the accompanying article (G. Xie and E. Aamodt) to screen for mutations that alter the pattern of either carboxylesterase activity in wild-type strain N2 or ,B-galactosidase activity in a strain (NW640) that contains a mec-7 lacZ fusion gene. Wild-type adult C. elegans stain for esterase activity in their gut and in three clusters of cells (neurons?) just behind the nerve ring. We have isolated four mutant strains following EMS mutagenesis of N2 that are also stained for esterase in (as yet unidentified) cells in their heads and in their dorsal and ventral nerve cords (the particular cells stained varies with the mutation). We isolated two other mutant strains that are stained in their pharynx muscle cells. This phenotype is similar to the pharynx expression of the major C. elegans esterase gene (ges-1 )that has had all but 1220 bp of upstream noncoding sequence removed (see Aamodt, Chung and McGhee (1991) Science 252:579) and the esterase staining patterns of wild nematode species (see the accompanying article by G. Xie, L. Ravi and E. Aamodt on esterase expression patterns in wild nematode species). While the ges-1 gene product is the only esterase present in embryos, adults also express several minor esterase species that can be detected on isoelectric focusing gels (McGhee and Cottrell (1986) Mol. Gen. Genet. 202:30). Until we cross these mutations into a ges-1 (0) background, we will not know whether these mutations are affecting the expression of ges-1 or one of the other esterase genes. These mutants have been back-crossed to wild-type once but we do not know yet where they map or whether the mutations are cis or trans to the esterase genes. We have also begun to screen for mutations that alter the expression of a mec-7 lacZ fusion gene constructed by Michel Hamlin and Joe Culotti. This strain has been transformed with plasmid pNW115 which is made with Andy Fire's vector pPD16 .51and contains 852 bp upstream of the mec-7 start codon, 792 bp of mec-7 coding region, the lacZ coding region in frame with mec-7 (the nuclear localization signal was removed), the last 47 bp of the unc-54 coding region and 1276 bp downstream of unc-54 .This construct was transformed into N2 along with Jim Kramer's pRF4 plasmid that contains the dominant mutant rol-6 gene. The extrachromosomal array formed in this way was then linked to chromosome I by the Kari et al. irradiation procedure (WBG 11[3]14) . We are screening EMS mutagenized NW640 for mutations that alter the pattern of expression of the fusion gene. We appear to be isolating mutants at a reasonably high frequency. In the two mutant strains that we have begun to characterize there is greatly increased , -galactosidase expression in the touch neurons, the FLP, PVD and ALN neurons and expression in other nerve cells (as yet unidentified) where the enzyme was not previously detected. We do not know whether the cells that are stained only in the mutants represent new expression in cells where the gene was not previously expressed or simply expression that is now detectable due to the enhanced expression of the gene.