Blumenthal TE et al. (1993) Worm Breeder's Gazette "The C. elegans Cleavage and Polyadenylation Signal"

Fields CA, White O, Blumenthal TE

[Worm Breeder's Gazette, 1993]

THE C. ELEGANS CLEAVAGE AND POLYADENYLATION SlGNAL Tom Blumenthal, Department of Biology, Indiana University. Bloomington, IN 47405; Owen White and Chris Fields, Institute For Genomic Research, Gaithersburg, MD, 20878

Ray C et al. (1990) Worm Breeder's Gazette "CORRECTION Isolation of a C. elegans Cysteine Protease Gene"

McKerrow JH, Ray C

[Worm Breeder's Gazette, 1990]

In the previous issue of the WBG (Vol. 11, #3, page 20) we reported the isolation of a cysteine protease clone from a mixed-stage C. elegans cDNA library. This library was originally obtained from Chris Martin and not from Cynthia Kenyon as was reported in the article. Our apologies for any misunderstanding caused by this oversight.

Wallenfang MR et al. (1997) Worm Breeder's Gazette ""IT'S A BOY!" - USING GFP TO SEX EMBRYOS AND YOUNG WORMS"

Wallenfang MR, Seydoux GC, Dunn MA

[Worm Breeder's Gazette, 1997]

Chromosomally-integrated transgenes that express GFP throughout embryogenesis could serve as useful chromosomal markers, enabling worm breeders to genotype embryos, when the use of larval/adult markers is not possible. We have generated such a GFP transgene (pJH1.16) using the pes-10 promoter. Embryonic expression of pJH1.16 is detected as early as the 28-cell stage in all somatic blastomeres; by the 2-fold stage, expression becomes restricted to the gut lineage, and persists there for the remainder of development. We have generated an integrated array containing this transgene using dpy-20 DNA as a selectable marker. By chance, this array integrated on the X chromosome; the resulting strain was outcrossed six times (JH103) and has a mild Dpy phenotype. We realized that this X-linked transgene could be used to determine the chromosomal sex of embryos and young worms before the appearance of any obvious sexual dimorphism. To test this prediction, we mated JH103 males to tra-2(q122) (spermless) hermaphrodites and scored resulting embryos for GFP expression. Green and non-green embryos were transferred to separate plates and allowed to grow to adulthood to score their sex. Since hermaphrodites receive their X chromosomes from both parents, whereas males receive their single X chromosome from their mothers, we expected all green embryos to be hermaphrodites, and all non-green embryos to be males. Results were as follows: Stage at which GFP Green Non-green expression was scored 28-cell to comma 7/7 hermaphrodites 9/9 males comma to 2 fold 3/3 hermaphrodites 3/3 males 3-fold 3/3 hermaphrodites 1/3 males L1 10/10 hermaphrodites 7/7 males TOTAL 23/23 hermaphrodites 20/22 males In summary, 43 out of 45 embryos/L1s were sexed correctly. The 2 embryos which were sexed incorrectly were both 3-fold, a stage at which gut GFP expression is sometimes harder to detect. We conclude that this strain can be used reliably to sex embryos, with the possible exception of 3-fold embryos. The availability of a transgene that expresses reliably throughout embryogenesis should prove to be a useful tool for tracking the segregation of specific chromosomes, and has many conceivable uses besides the specific one described here. Transgene pJH1.16 and strain JH103 are available to anyone interested. (Contact Matt at mwallenf@welchlink.welch.jhu.edu). We are grateful to Andy Fire for creating the GFP used in this study.

Gu GQ et al. (1994) Worm Breeder's Gazette "mec-5 Encodes a Collagen Needed For Touch Sensitivity."

William CM, Chalfie M, Gu GQ

[Worm Breeder's Gazette, 1994]

mec-5 Encodes a Collagen Needed for Touch Sensitivity Guoqiang Gu, Chris William, and Marty Chalfie, Department of Biological Sciences, Columbia University in the City of New York, NY 10027 mec-5 is one of the genes required for mechanosensitivity in C. elegans. Although the touch receptor neurons do not function, they appear morphologically normal in mec-5 mutants. Peanut-lectin binding to the touch cell mantle, however, is absent (M. Chalfie and E. Hedgecock, unpublished data). By transformation rescue, we identified a 6.5 kb BamHI-Kpnl fragment in cosmid E03G2 which completely rescues the mec-5 phenotype. We isolated a 1.2 kb full length cDNA (Northern blots show a 1.2 kb mRNA) from the Chris Martin cDNA library. The cDNA sequence encodes a collagen. The protein (329 aa) has a 20 amino acid putative signal sequence at the N- terminus and 69 Gly-X-Y repeats starting at aa 63. These collagen repeats are followed by 60 additional amino acids. Because collagens are often glycosylated on hydroxyproline residues, the loss of MEC-5 may explain the lack of peanut- lectin binding. To verify that this collagen gene is mec-S, we probed genomic Southern blots with the 6.5 kb fragment and found that the 6.5 kb fragment was deleted in two mec-5 strains, the mut-2 derived strain TU828 and the Y-ray strain TU1031. We are now sequencing other mec-5 mutations and characterizing the expression pattern.

Mounier N (1977) Worm Breeder's Gazette "C elegans gonad staining methods."

Mounier N

[Worm Breeder's Gazette, 1977]

In the meeting, some persons were interested by my stained gonads slides. This technical note describes how I proceed to get them. DISSECTED WORMS Fixative = sublimate acetic : 95 ml sublimate (dissolve 10 g of HgCl2 in 100 ml boiling water-let decant one night) and 5 ml acetic acid. When dessication of dissected worms is completed, deposit a fixative drop on the frottis, then immerse the slide in sublimate acetic for 10 mn. Rinse in water and let one (or more) night in iodine-iodized alcohol (100 ml 70% ethanol, 2g I2, 4g KI). Rinse in a 0.25 % sodium hyposulfite solution (5 mn), then in running water (10 mn). Immerse in HCl for 12 mn in a 56 C water-bath, stop hydrolysis by a room temperature water rinse, immerse in Schiff reagent for 2 hours and rinse in running water (15 mn). Stain finally by a fast passing over in : - 1% light green solution, rinse and dehydrate in 1 x 70, 1 x 95, 2 x 100 alcohols, then 2 x Toluene and mount. - picro indigo-carmine (0.25 g indigo-carmine in 100 ml of picric acid aqueous saturated solution). Rinse directly in 100 ethanol ( twice). I prefer use P.I.C. to light green, because the green staining disappears from cytoplasm in 2 to 3 months ENTIRE WORMS (for sections) Fixative = Zenker fluid - stock solution = HgCl2 : 5 g; K2Cr2O7 = 2. 5 g; Na2SO4 = 1g; distilled water 100 ml - Before use, add 5 ml acetic acid. Background staining = Heidenhain's blue - Dissolve in 100 ml hot

Avery L (1989) Worm Breeder's Gazette "functions of the GREENs: slippery pharynxes and serotonin."

Avery L

[Worm Breeder's Gazette, 1989]

In my initial survey of the pharyngeal neurons, I classified them into three groups: M4, MC, and the 12 remaining anatomical types of pharyngeal neurons, which I call the GREENs. None of the individual GREEN neuron types is necessary for superficially normal pumping, but when all 12 are killed, pumping becomes very abnormal. I have recently made some progress in figuring out what they are doing. During normal pumping, bacteria move posterior in the pharynx when the muscles contract, and are held where they are when the muscles relax again, so that they are efficiently transported towards the intestine. When all the GREENs were killed, the pharynx appeared to become slippery: bacteria slid anterior as well as posterior. When bacteria were abundant, the lumen of a GREEN pharynx became distended with them, and they were ground up and passed to the intestine only slowly. Seymour et al (J Zool 201: 527) showed by microcinematography that in normal pumping the contraction and relaxation of the corpus muscles are not precisely simultaneous along its length: both begin at the front. They proposed this has a function in trapping bacteria in the pharynx. I suspect that in the absence of the GREENs this fine timing goes awry, and as a result bacteria are not held back by the relaxing pharyngeal muscles. The GREEN neuron I understand best at present is M3. When M3 was killed, there was a subtle abnormality in pumping: the contractions lasted longer. Conversely, when all the GREENs except M3 were killed, contractions were very brief. Thus M3 hastens pharyngeal muscle relaxation during a pump, and is opposed by some other(s) of the GREENs. I5 is probably (one of) the GREEN(s) that opposes M3, since pumps became briefer when I5 alone was killed. When both M3 and I5 were killed, pumps were long, just as when M3 alone was killed, suggesting I5 may exert its effects on pump length via M3. In fact, I5 does synapse on M3. There was also a new effect when both M3 and I5 were killed: the isthmus became slippery. The timing of anterior isthmus contraction was abnormal in M3-I5 worms: it contracted just after the corpus contraction, instead of simultaneously with it, and remained contracted longer. M3 is also important for corpus function. In fact, when all the GREENs except M3 were killed, the corpus was not slippery. Other GREENs important for normal corpus function are I2, I6, M1, and possibly I4. M5 may also be important for isthmus function. However, I have not worked out the details for any of these. I have also learned something about GREEN function in a completely different way. I discovered previously that imipramine stimulates pumping in wild-type worms, but not in mutants that have reduced levels of serotonin (WBG 10(2): 39). The only serotonergic neuron in the pharynx is NSM, and the only pharyngeal neurons connected to the extrapharyngeal nervous system are I1 and M1. I therefore killed NSM, I1, and M1, thinking to remove any serotonin from the pharynx, and to sever its communication with extrapharyngeal serotonergic neurons that might be stimulating pumping. Sure enough, imipramine did not stimulate pumping in I1- M1- NSM- worms. I have not yet tested whether it is necessary to kill all three to get the effect.

Ito K et al. (1986) Worm Breeder's Gazette "sperm DNA strands do not co-segregate in the next generation."

Ito K, McGhee JD

[Worm Breeder's Gazette, 1986]

By feeding male worms on bromodeoxyuridine (BrdU) labelled E. coli and using a monoclonal antibody to BrdU, we have been able to identify paternal DNA strands within the next generation of developing embryos. The following is our current protocol, although it is still being improved. A thymidine requiring E. coli strain was grown to saturation in M9 salts supplemented with 0.4 % glucose, 1mM MgSO4, 1.25 g/ml vitamin B1, 9 M BrdU and 1 M thymidine. The culture was concentrated and ampicillin was added so as to prevent contamination of plates with unlabelled bacteria that the worms seem to find tastier. A mixed population of males and hermaphrodites (N2) at dauer larva stage were transferred onto M9-agarose plate with this BrdU-labelled E.coli. After two days at 20 C, male worms were transferred onto a new M9- plate, and were continued being fed on BrdU-bacteria for an additional two days. The incorporation of the label can be seen marching down the gonad over a period of a few days. The labelled males were mated to spermless hermaphrodites, fem-2, (raised at 25 C) on a NGM-plate with non-labelled E. coli (OP50). The hermaphrodites also carried a spontaneous dumpy mutation that turned out to be convenient for separating unlabelled hermaphrodites from any contaminating labelled hermaphrodites inadvertantly brought in with the males. After about five hours of mating, dumpy hermaphrodites were picked up and cross- progeny were obtained by cutting the worms in half followed by gentle hypochlorite treatment (0.4-0.6 % NaOCl in egg-salts, 3 min.). The embryos, from which egg-shells were removed by chitinase-chymotrypsin digestion, were transferred onto a subbed slide and gentle pressure was applied on the cover-slip to spread the cells to avoid cell-cell overlap as well as to permeabilize the embryos. To detect the labelled DNAs, we used monoclonal anti-BrdU antibody conjugated with fluorescein (Becton-Dickinson). Prior to antibody binding, DNA was denatured in 6N HCl for 3 minutes, followed by neutralization in a large excess of 0.4M borate buffer, pH 8.5 for ten minutes. After antibody binding (30 l of a 1:10 dilution per slide), the DNA was stained with DAPI. Distinct green spots appear within the embryos under epifluorescence. Straightforward controls seem to work; green spots are not seen in unlabelled worms; antibody is competed off with BrdU; green spots go away with DNase I. In embryos with total cell number of 100 or more, the number of green spots seems to converge on 10 +/- 2. Our initial conclusions are that: (1) sister chromatid exchange does not appear to occur at high frequency, and (2) sperm DNA strands do not co-segregate, since this would predict only two labelled cells in embryos of all ages. A few preliminary experiments with larval worms suggest that strand segregation could be random, i.e. green spots appeared at different positions in different larvae. We should be able to test far more rigorously whether DNA strand segregation is truly random. Further experiments should be possible by variation on the above technique; for example, following maternal DNA strands, following both paternal and maternal strands in the same embryo, following strand segregation in post-embryonic cell divisions and so on.

Consortium TCeGS (1992) Worm Breeder's Gazette "Update on cDNA Sequencing"

Consortium TCeGS

[Worm Breeder's Gazette, 1992]

This is another interim report on the state of the cDNA sequencing and mapping project that we are carrying out alongside the genomic sequencing effort. We have now sequenced 1222 of the 1743 clones in Chris Martin's library. Of these 27 appear to be duplicates in the strong sense that their sequence looks identical and they come from neighboring micro-titre wells that were most likely cross-contaminated. The following results therefore come from the 1195 left after removing these duplicates. Remember that we are only producing a single gel reading from each clone (5' end of the cDNA insert). Similarities: There are 373 (31%) with BLASTX scores to the public protein databases greater than 100, which is a high enough threshold that these similarities are very likely to be meaningful. A list of these is given on the following pages. [Figures excluded due to low reproducability] Trans-spliced leaders: 166 sequences (14%) had trans-spliced leaders at the 5' end, 149 to Sl1 and 17 to SL2 . Repeat isolations: A number of genes clearly have been cloned more than once. We can unambiguously detect this only when the 5' ends of the inserts overlap, which happens for 334 sequences that fall into 130 overlap groups. This is not out of line with Chris Martin's estimate of repeat frequency. It leaves us with 991 distinct sequences of which 861 are represented only once. However it is likely that there are additional repeats that we don't see this way because we have different parts of the same gene. Hits to previously determined worm sequences: There were exact matches to 13 previously determined C. elegans sequences in the public databases, and to 3 of the 44 predicted genes on the first 4 genomic cosmids that we have sequenced. The latter figure allows us to generate an estimate of (44/3)*991 = 14,500 genes in the worm, but the 95% confidence limits for this are 6,000 to 50,000, so we need more data to make this a useful approach to estimating gene density. Mapping: we are now in the process of mapping all the clones using the YAC "polytene" filters. This data will be in ACEDB and will also be provided if you ask for a clone. All the sequences, similarity and mapping data will be in ACEDB, and any clone is available along with more detailed data from the St. Louis lab.

Chalfie M (1994) Worm Breeder's Gazette "Some Technical Notes on the Use of GFP"

Chalfie M

[Worm Breeder's Gazette, 1994]

The following should be useful to people that are using the Green Fluorescent Protein (GFP) as a marker (see WBG 13 #1 p. 19) I. Vectors A. pGFP10 .1.This is the original GFP plasmid. People using it should not cut out a restriction fragment containing the entire coding sequence. There appears to be something upstream of the coding region that poisons expression. Instead PCR should be used to obtain the sequence. B. Worm vectors. The modified Fire vectors described in the previous Newsletter contribution lack the Kpn I fragment with the SV40 nuclear localization signal. We are in the process of making vectors containing this fragment, but they are not ready yet. II. Photobleachinq A. Effect of Anesthetics. we had originally found that GFP expressed in C. elegans photobleached rapidly when illuminated with light using a 395-440 nm excitation filter (photobleaching with the 450-490 nm excitation filter normally used for FITC was not as rapid). We have now found that the very rapid photobleaching resulted from the use of sodium azide as an anesthetic. The fluorescence is much more long-lived without the azide. Unfortunately, in preliminary experiments phenoxypropanol seemed to eliminate the fluorescence entirely. We have not tried CO(2) as an anesthetic. This fact that azide causes photobleaching may be a useful tool. B. Low Wavelength Excitation. We obtained an interesting result when animals were illuminated with 340-390 nm light (the excitation from the standard DAPI filter set). The green fluorescence rapidly disappeared, but when the animals were subsequently examined with light of the two higher wavelengths (see IIA), the green fluorescence remained. Similarly if the animals were first viewed for a few minutes with light of the higher wavelengths (and showed fluorescing cells) and then examined with the DAPI filter set, no fluorescence was seen. This suggests that there may be two molecular species produced in the animals. Interestingly, the fluorescence at the higher wavelengths seems to be stronger after the 340-390 nm excitation or just a minute or so of excitation at the higher wavelengths. III. Fixation. As other people begin to use GFP, we are learning some interesting things about its properties. One of these was discovered by Shengxian Wang and Tulle Hazelrigg using GFP in Drosophila. They found GFP fluorescence survives formaldehyde fixation (subsequently I learned that there were unpublished results demonstrating that the native protein from jellyfish could also fluoresce after formaldehyde and glutaraldehyde fixation). I have confirmed that formaldehyde fixation does not abolish GFP fluorescence in C. elegans using the uIs4 animals. So fixed as well as living tissues can be examined.

Jin YS et al. (1995) Worm Breeder's Gazette "News on GFP variants"

Horvitz HR, Jin YS

[Worm Breeder's Gazette, 1995]

The use of GFP has enabled the visualization of biological processes in living animals (Chalfie et al., 1994; see reviews in TIG 11 320-329). Recently, several mutations in GFP were found to alter the wavelength spectrum of excitation and emission of the protein and to increase the intensity of fluorescence (Heim et al. 1994; 1995). In particular, Ser65Cys or Ser65Thr, and Ile167Thr mutations resulted in an increased intensity of fluorescence and in red-shifting of the excitation and emission wavelengths. We have made two GFP variants containing mutations at both amino acid 65 and 167: GFP-TT with Ser65Thr and Ile167Thr, and GFP-CT with Ser65Cys and Ile167Thr. These GFP variants are still fluorescent. The intensity of fluorescence of these new GFP variants appeared stronger than that of either the original wild-type GFP or the GFP containing single mutations such as Ser65Cys and Ser65Thr when the expression of each GFP was driven by the promoters of the unc-25 and vab-3 genes. These new GFP variants seemed to have a broader spectrum of excitation and emission than the wild-type GFP and the GFPs with single mutations, since we could detect fluorescence using the rhodoamine filter from these new GFP variants but not from the wild-type or other GFPs when driven by the unc-25 promoter; the maximal intensity was still obtained using the fluorescein filter. These double mutant GFPs also gave stronger signals than the wild-type GFP in premorphogenesis embryos (Andrew Chisholm, personal comunication). We have not performed any biochemical or biophysical analyses of these new GFP proteins. Although GFP-CT and GFP-TT result in similarly strong fluorescence, when fused to the VAMP protein for targeting to synaptic vesicles (Nonet, 1995), GFP-CT produced a much weaker signal than GFP-TT or the wild-type GFP, which suggests that local factors, such as pH, may not be optimal for fluorescence from a Ser65Cys mutant GFP in synaptic vesicles. These GFP variants are available. We would appreciate feedback from anyone who uses these constructs. Chalfie, M., Tu, Y., Euskirchen, G., Ward, W. W. and Prasher, D. C. (1994). Green fluorescent protein as a marker for gene expression. Science 263, 802-805. Heim, R., Cubitt, A. B. and Tsien, R. Y. (1995). Improved green fluorescence. Nature 373, 663-664. Heim, R., Prasher, D. C. and Tsien, R. Y. (1994). Wavelength mutations and posttranslational autoxidation of green fluorescent protein. PNAS 91, 12501-12504. Nonet, M. L. (1995). Visualization of presynaptic terminals using GFP. WBG 13 #5, 40.