Rose AM et al. (1983) Worm Breeder's Gazette "RFD's as probes for cloning."

Beckenbach KA, Rose AM, Mawji NR, Baillie DL, Nelson DW

[Worm Breeder's Gazette, 1983]

We have been screening for restriction fragment differences (RFDs) between the N2 and Bo strains as described by Emmons, et al., 1979. Random N2 fragments with EcoRI and HindIII ends were cloned into pBR322, isolated, and purified. Using these fragments as P nick translated probes, RFDs were detected about one per six tested probes. We are in the process now of accumulating and mapping a set of these probes. It is our hope to establish a reasonably large collection of mapped RFD probes that might be utilized in the cloning of genes with no biochemically identified products. Towards this end we have mapped four such RFDs: One of these pGe s18 has been further mapped to the left of unc-13 ( I). Terry Snutch has screened our Charon 4 library for corresponding lambda clones of this region, which we have restriction mapped and subcloned for chromosome walking. We hope that characterization of the DNA adjacent to pCe s18 will allow us to isolate some of the genes in the unc-15 region described by Rose and Baillie, 1980. Interestingly, probe pCe s18 when hybridized against N2, Bo, and FF shows the N2 banding pattern. We would welcome information on the relationship (if any) between Bo and FF. [See Figure 1]

Johnsen RC et al. (1987) Worm Breeder's Gazette "lin-40 is a hot spot for Tc1 induced mutations."

Baillie DL, Beckenbach KA, Johnsen RC

[Worm Breeder's Gazette, 1987]

A Tc1 mutagenesis screen in the nT1(IV,V) balanced region using mut- 4 RW7037 was performed (McKim et al. C. elegans Abstracts CSH 1987). They isolated 28 lethal mutations, 24 of which mapped to LGV. We substituted the balancer eT1(III,V) for nT1(IV,V). At least two of the 24 lethals on LGV balanced by nT1 were not balanced by eT1, these were not analyzed further. Of the rest: one was too sick to work with; six are still being analyzed; two define new genes (let-448 and let- 449 both of which are on LGV); seven are deficiencies which break in various places, but all of these deficiencies uncover the left most known gene on the chromosome; and six other strains have lethal mutations that fail to complement lin-40. Using the six lin-40 Tc1 mutations we hope to clone the lin-40 gene. To do this we have extracted DNA from the six strains. We then digested the DNA with EcoRI, ran it on a gel, transferred the DNA to a filter and hybridized nick translated Tc1 DNA to it. We noted that three strains show a common band shift and two of these three strains have several shifted bands. This research has been supported by NSERC and MDA (Canada) grants to DLB.

Johnsen RC et al. (1990) Worm Breeder's Gazette "The cloning of lin-"

Johnsen RC, Baillie DL

[Worm Breeder's Gazette, 1990]

From a Tc1 mutagenesis screen in the nT1(IV,V) balanced region using mut-4 RW7037, six lethal mutations that fail to complement lin-40 were recovered (Clark et al. In Press Genome). We have extracted DNA from the six strains containing lin-40 Tc1 mutations. We digested the DNA with EcoRI, ran it on a gel, transfered the DNA to a filter and hybridized nick translated Tc1 DNA to it. Three strains showed a common new Tc1 band. For one allele (s1351) we recovered the new band, isolated DNA flanking the Tc1 sequence and used the flanking sequence to screen T. Snutch's Charon IV library. From the library we isolated a phage (BC#S1001). DNA from BC#S1001 was mapped to LGV(left) on the cosmid map by J. Sulston and A. Coulson (see figure). The location is surprising in that BC#S1001 maps very close to unc-60 molecularly (unc-60 is in F53E2 pers. comm. M. Wakarchuk) but genetically lin-40 is fairly far away (roughly 9 m.u.) to the right of unc-60. We are attempting to rescue lin-40 by microinjection using appropriate cosmids. We are also screening for revertants of some of the Tc1 alleles of lin-40. This research has been supported by an MRC studentship to RCJ and NSERC and MDA (Canada) grants to DLB. [See Figure 1]

La Volpe A et al. (1986) Worm Breeder's Gazette "identification of repetitive DNA families."

Bazzicalupo P, La Volpe A

[Worm Breeder's Gazette, 1986]

224 cosmid clones, representing about one tenth of the Caenorhabditis strain CB1392 H nuc001 (e1392) X ), were screened for the presence of repetitive sequences using as probe total nick-translated genomic DNA. Eighteen clones showed-up as positive in this screening, among these, two were proved to cross- hybridize to the known transposable element Tc1, another represented a ribosomal DNA clone. We have begun to analyze different features of the remaining fifteen clones: none of them shows homology with the MSP gene family and only one, cos repM7, contains a sequence homologous, at low stringency, to the col-1 gene but not in the major repetitive band; none of the clones shows homology to pCe2, pCe5, pCe17 and pCe1006, repetitive DNA clones from S. W. Emmons Laboratory. Two of our clones belong to the same repetitive family we have named B1. The B1 together with other three families, namely C9,F10, and S5 were tested for restriction-sites polymorphisms in genomic DNAs from independently isolated C. elegans wild type strains. They all show polymorphisms for each enzyme used, nevertheless no polymorphisms were detected among laboratory strains derived from N2. They all prove to be abundant in all the strains (more than 50 copies per genome for B1, C9 and the S5, less in the case of F10 that however is of unusual size: more than 20kb). Unlike Tc1, our repetitive families code for abundant transcripts.

Miller DM et al. (1984) Worm Breeder's Gazette "sup-3 and myo-3 are closely linked."

Coulson AR, Sulston JE, Albertson DG, Greenwald IS, Miller DM

[Worm Breeder's Gazette, 1984]

In the last newsletter Donna Albertson (1) reported in situ hybridization data that localized myo-3 to a region of chromosome V encompassing the act 1,2,3 cluster and sup-3. The apparent effect of sup-3 on myo-3 activity (myosin A is elevated)(2) indicated the likelihood that these genes are in close proximity to each other. To test this possibility restriction digests of sup-3 deficiency DNA were probed on Southern blots with myo-3 cosmids. Two restriction fragment differences were detected. DNA was prepared from the semibalanced strain unc-15(e73);sup-3(eDf1)/sma-1(e30) for comparison with N2 DNA. (Homozygous eDf1 segregants die as L1's and e73;e30 homozygotes produce few progeny.)(3) Southern blots were hybridized with nick- translated myo-3 cosmids. The sup-3 DNA exhibits ~15 kb PstI and ~ 8 kb BclI fragments that are not present in N2 or homozygous e30 DNA. Control experiments have verified that the fragments are not partial digestion products nor do they hybridize with cosmid vector sequences. In addition the extra bands are significantly weaker than the wild type bands which is consistent with the approximate half molar amount of sup-3 DNA in this mutant. The precise location of the putative eDf1 deletion has not been determined as yet but it is clear that the myo-3 coding sequence is not altered. These results favor the hypothesis that sup-3 is a cis-acting DNA sequence that modulates the expression of the myo-3 gene.

Albertson DG (1983) Worm Breeder's Gazette "localization of the ribosomal genes in Caenorhabditis elegans by in situ hybridization using biotin-labeled probes."

Albertson DG

[Worm Breeder's Gazette, 1983]

The site of the ribosomal gene cluster on embryonic metaphase chromosomes of Caenorhabditis mapped by in situ hybridization. The clone pCe7, which hybridizes to ribosomal RNA, was nick translated incorporating biotin-labeled UTP. The site of hybridization of this probe DNA was detected by a double-layer fluorescent antibody technique. In the wild type, N2 strain of C. elegans the ribosomal probe hybridized to one end of two embryonic metaphase chromosomes. In order to determine which linkage group carried the ribosomal genes, in situ hybridization was carried out with chromosomes from C. elegans strains carrying cytologically distinct translocation or duplication chromosomes. Linkage groups I, II, IV and X were tested in this way. Since the ribosomal probe was found to hybridize to an internal site, rather than to the end of mnDp10 (X;I) chromosomes, it was concluded that the ribosomal genes are located at the end of the right arm of linkage group I. The free duplication of linkage group I, sDp1 (I;f) also carries the ribosomal genes. The ribosomal genes were not deleted by a deficiency, eDf3 that includes unc54 and maps to the left of unc-54 I. Therefore the ribosomal genes are probably located to the right of unc-54. Chromosomes carrying the lethal mutation, let-209 I (Phil Anderson's balancer chromosome) displayed smaller hybridization signals than wild type, suggesting that these chromosomes carry a partial deficiency of the ribosomal gene cluster. A duplication of the ribosomal genes, eDp20 (I,II) was identified by in situ hybridization. The duplicated ribosomal genes were found to be linked to the right arm of linkage group II.

Prasad SS et al. (1985) Worm Breeder's Gazette "identification and isolation of the coding regions around unc-22."

Baillie DL, Prasad SS

[Worm Breeder's Gazette, 1985]

Our laboratory has been intensively studying the region around unc- 22 gene over the last eight years using conventional genetic analysis. We have obtained cosmids from John Sulston that cover approximately 500 kb. of DNA. These cosmids were identified by means of Tcl RFLD flanking sequences (isolated by Karen Beckenbach) in the region which lies immediately to the left and right of unc-22 gene. We are currently examining the region flanked by dpy-20 on the left and unc-22 on the right. Five overlapping cosmids have been identified which cover this region, (these include D. Moerman's and R. Waterston's unc-22 clones). We are using the method suggested by T. Snutch to identify putative coding regions (Divergent regions between C. elegans and C. briggsae include mostly non-coding DNA sequences whereas the highly conserved regions are likely to code for the proteins essential for nematode growth, development and reproduction). We have restriction mapped and subcloned Hind III and Pst I fragments from these cosmids into pUC19. Nick translated individual fragments have been used to probe Bristol and C. briggsae DNA. This technique helps us to identify the conserved regions or the coding sequences in C. briggsae DNA. We are now in the process of screening B. Meyers's lambda gt10 cDNA library with the probes which show homology to the C. briggsae DNA. We intend to identify and isolate the major coding regions that lie between unc-22 and dpy-20 regions using this approach. The isolation of cDNA clones will be used to study gene expression and regulation during nematode development. The isolated cDNA clones will be sequenced in order to identify the nature of protein coded for by particular gene(s) in this region.

Albertson DG et al. (1977) Worm Breeder's Gazette "Development of the Phasmid Socket"

Anness M, White JG, Sulston JE, Albertson DG

[Worm Breeder's Gazette, 1977]

The phasmid is a sensilla in the tail analogous to the amphids in the head and probably functions as a chemosensory receptor. Like most sensillae it is apparently attached to the hypodermis by a specialized cell, the socket cell. The phasmid socket cell is derived from the T. seam cell after 3 rounds of division that occur in the first larval stage. The sister of the socket cell differentiates into a cell of unknown function which has been nick-named the 'wing' cell. In the hermaphrodite the lineage of the socket cell is T.Paa, whereas the wing is T.PaP. In the male these allocations are reversed. A male and hermaphrodite were fixed and sectioned in L2 lethargus to try to find out the reason for this reversal. It was found that at this stage (i.e. about 5 hours after the socket and wing had been born) both these cells looked like socket cells, one being concentric to the other, both in the hermaphrodite and in the male. Thus it seems that soon after birth these two cells are equivalent and that in the course of development one gets transformed into a wing cell; either may be chosen to do this depending on the sex of the animal. A further animal was fixed about 5 hours after hatching (i.e. before the T. cell had divided) and reconstructed. It was found that in this case the posterior part of the T. seam cell was functioning as a socket cell. The socket and wing cells are ultimately derived from the posterior daughter of the first division of the T. seam cell so that it seems that this differentiated function is propagated through the lineage.

Birchall PS et al. (1993) Worm Breeder's Gazette "FISHing for Complements"

Birchall PS, Albertson DG

[Worm Breeder's Gazette, 1993]

Fluorescence in situ hybridisation (FISH) has been used to detect unc-54 mRNA in post-embryonic stages of N2 . The technique is fast (same-day results) and produces a high resolution signal in about 90% of the sampled nematodes. All washes are done in an eppendorf tube; the worms are spun down, the old wash removed and replaced with 1ml of the next solution. The nematodes are fixed for 6 hours in 3.7% formaldehyde in 0.1M HEPES-KOH (pH 7.5) buffer, permeabilised in 5%- -mercaptoethanol and 50 g/ml Proteinase K and taken into hybridisation buffer (40% formamide, 4xSSC, 0.1M HEPES-KOH (pH 7.0), 80 g/ml sheared salmon sperm DNA, 1.6% SDS and 0.024% SLS). Probes are prepared by one of four methods; PCR, in vitro transcription, nick translation or random priming. In each case, a fluorochrome-conjugated nucleotide is incorporated into the sequence during the polymerase-catalyzed reaction. Hybridization is at 45 C for at least four hours. The nematodes are washed, mounted on a slide, mixed with DABCO (retards fading of the fluorescent signal) and viewed. There is very little background at all (negative controls are hard to find on a slide) and the hybridization signal can be seen with a conventional fluorescence microscope. The nematodes have been simultaneously hybridized with a 1kb ribosomal DNA segment labeled with rhodamine-4-dUTP (Amersham), and with a 1kb unc-54 sequence labeled with fluorescein-12-dUTP (Boehringer Mannheim). In this case, the nucleoli of all the cells appear bright red, the cytoplasm of all the cells appears faintly red and the cytoplasm of the body wall muscle cells appears green, in animals from L1 to adult. These signals are sensitive to RNaseA. An unc-54 -specificsignal is also seen in the vulval and uterine muscles at the L4 /youngadult stage. Embryos are currently being examined. We are extending our investigations to include other genes to gauge the sensitivity and generality of the technique.

Edwards MK (1981) Worm Breeder's Gazette "hybridization of DNA probes to RNA in situ."

Edwards MK

[Worm Breeder's Gazette, 1981]

In situ hybridization should prove useful for analyzing changes in RNA populations during C. elegans development without isolating biochemical quantities of similarly staged embryos or specific tissues. RNA can be detected in various fixed preparations with well- preserved morphology. Embryos, squashed according to the procedure of Gossettand Hecht (1979 C. elegans meeting), maintain their cell boundaries so that the number of cells, and thus the developmental age, is easily determined. Larvae and adults, fixed in ethanol/acetic acid (3:1), frozen in O.C.T. compound from Tissue-Tek, and sectioned at -20 C, retain good tissue morphology. With Nomarski optics, well- preserved muscle, gonad and intestinal tissues can be recognized, and with polarization optics, muscle birefringence and gut granules are visible. Alternatively, adults can be cut open in M9 such that the gonad and intestine extrude as separate tissues from the body of the worm. When squashed in 45% acetic acid, frozen,and then fixed in ethanol/acetic acid, these tissues retain good Nomarski morphology. After hybridization of a radioactive DNA probe to these preparations, followed by autoradiography, grain densities can be determined for particular embryonic cells or adult tissues. I can currently detect abundant RNAs using recombinant DNA probes. In situ hybridizations with cloned nick-translated DNAs containing ribosomal, histone, actin (from Jim Files), and myosin (from John Karn) genes produce strong signals (10 to 200 fold over background after 10- day exposure) under conditions in which non-homologous DNA (Charon 3) gives no signal over background. In addition, three cloned DNAs from our Charon 3 library that plaque-hybridize with C. elegans cDNA also hybridize to RNAs in situ. These three probes show differential hybridization between young embryos (1-50 cells) and old embryos ( comma to hatching) and between two adult tissues (gonad and intestine). Present work involves more precisely characterizing the amount of hybridization as functions of developmental time and tissue specificity for these and other clones.