Gong L et al. (1994) Worm Breeder's Gazette "Probing Touch Cell Biology Using a mec-4/lacZ Fusion Gene"

Gong L, Driscoll MA

[Worm Breeder's Gazette, 1994]

Mec-4 encodes a subunit of an ion channel required for the function of the six touch receptor neurons. Dominant alleles of mec-4 induce degeneration of these neurons. As part of a detailed study of how the MEC-4 (d)protein kills cells we wanted to determine when mec-4 is first expressed in the postembryonic touch receptor neurons AVM and PVM. We have done this by synchronizing worm populations and monitoring expression of an integrated mec-4 /lacZarray. To generate a synchronized population for staining, we picked about 100 gravid worms from plates that contained lots of bacteria (20 C) onto a prewarmed plate. Worms were allowed to lay eggs for one hour and then adults were removed. In theory, all remaining eggs would have been fertilized within an hour of each other. (In practice, a wider window of ages probably occurs as eggs are not all laid at precisely the same moment in development). Larvae were stained for -galactosidase activity at hour intervals. We find that AVM expresses -galactosidase in 50% of animals by 22 hours after the egg is laid (see figure below). PVM expresses -galactosidase in 50% of animals a bit later, by 25 hours. AVM and PVM are generated at about 9 hours after hatching at 20 C, about 19 hours after egg deposition. Thus, mec-4 appears to be expressed very soon after the birth of the touch cell. The fact that we do not see a sharp transition point for the activation of mec-4 expression may reflect the degree of asynchrony in the culture or may result from stochastic activation of mec-4 expression over a three hour period. Interestingly, we have observed that at 27-28 hours the intensity of staining drops dramatically or disappears altogether. According to our calculations, the loss of staining coincides with the time of the L1 -L2molt. We are considering two explanations. First, standard-staining conditions may not be appropriate for molting animals. Alternatively, we wonder if proteins that project into the extracellular space and contact the cuticle are degraded at the time of the molt. The processes of the touch receptor neurons run along the cuticle and are surrounded by an extracellular material called the mantle. MEC-4 is predicted to have an extracellular domain that could be involved in contacting the mantle. Perhaps during molting proteins that associate with the cuticle are temporarily destroyed. Use of mec-4 /lacZto probe the relationship of mec-4 and mec-6 .mec-6 is required for mec-4 (d)-induceddegenerations to occur. We have now looked in detail at staining of mec-4 /1ac-Zin the mec-6 (u450)background. We find that in the wild-type background, mec-4 /lacZstains all six touch receptors intensely. However, in the mec-6 (u450)background, mec-4 /lacZactivity is consistently and significantly lower than in the wild-type background. After one day of incubation with X-gal, staining can barely be observed and after two days of incubation, the touch cells still stain faintly. Lessened staining is clearly not due to a change in the configuration of our array in the mec-6 (u450)strains because when the mec-4 /lac-Zarray is crossed out of the mec-6 (u450)background, intense staining is restored. Our observations indicate that mec-6 mutations may block mec-4 (d)-induceddegeneration by significantly lowering levels of MEC-4 protein. Why this occurs, however, is unclear. Data are consistent with the idea that MEC-6 could be required for high levels of transcription of mec-4 .Data are also consistent with the idea that MEC-6 could be required for insuring the translational stability of MEC-4 .If, for example, MEC-6 was a subunit of the channel complex, its absence could result in turnover of other channel subunits that could not be properly assembled.

Hall DH et al. (1994) Worm Breeder's Gazette "Cytology of Degenerin-Induced Cell Death in the PVM Neuron"

Driscoll MA, Chalfie M, Gu GQ, Hall DH, Gong L

[Worm Breeder's Gazette, 1994]

Cytology of degenerin-induced cell death in the PVM neuron David H. Hall, Guoqiang Gu+, Lei Gong#, Monica Driscoll#, and Martin Chalfie+, * Dept. Neuroscience, Albert Einstein College of Medicine, Bronx, N.Y. 10461 + Dept. Biological Sciences, Columbia University, New York, N.Y. 10027 # Dept. Molecular Biology and Biochemistry, Rutgers University, Piscataway, N.J. 08855

Wadsworth WG et al. (1990) Worm Breeder's Gazette "Searching for Laminin Chain Genes"

Wadsworth WG, Gong L

[Worm Breeder's Gazette, 1990]

Laminin is a large glycoprotein of the extracellular matrix. The comparison of laminins isolated from different species and tissues has demonstrated that a variety of laminin structures may exist and that the expression of different isoforms of laminin may be important for several developmental processes. Laminin is composed of three chains, A, B1, and B2, with possible variants of each chain. In our lab we have been studying structure/function relationships of the B2 chain gene (unc-6). Several other laminin or laminin-related genes from C. elegans have been reported (see Yochem & Greenwald, WBG 11(2) and Rogalski & Moerman, this issue). The sequence of the N-terminal domain (V1) is highly conserved in all three chains. We have been searching for other laminin chain genes by low stringency hybridization using probes derived from this region of the B2 chain. Several genomic clones, distinct from the unc- 6 B2 chain, have been isolated. Currently we are analyzing these clones to determine if any contain new laminin sequences. Using degenerate primers designed for a highly conserved region of domain V1 of the B1 chain, we have amplified and cloned a fragment of a C. elegans B1 chain by PCR. The sequence from this clone is aligned below with other sequenced B1 and B2 chain genes. [See Figure 1]

Kenyon CJ et al. (1994) Worm Breeder's Gazette "A polyray Suppressor"

Kenyon CJ, Maloof JN

[Worm Breeder's Gazette, 1994]

A polyray-l suppressor Julin Maloof and Cynthia Kenyon, Dept of Biochemistry, U.C.S.F, San Francisco, CA 94143 The polyray-l (pry-l) gene is required to keep homeotic selector (HOM-C) genes repressed where their expression has not been initiated (see accompanying abstract and WBG 12(5):39). pry-l mutant worms have numerous homeotic transformations and are very sick, so we were intrigued by a plate of pry-l mutant worms which appeared much healthier than normal. I outcrossed the strain and found F2s with the full pry-l mutant phenotype, suggesting that the "healthy" strain contained both pry-l and an extragenic suppressor. Does the suppressor have a phenotype on its own? Since pry-l causes homeotic transformations due to ectopic HOM-C expression, it seemed possible that the suppressor would cause transformations in the opposite direction, similar to those seen in HOM-C loss of function mutants. Indeed some F2s from the outcross had descendants of the QL neuroblast in the anterior of the worms, a phenotype also caused by loss-of function mutations in the HOM-C gene mab-5. We are calling this newly identified gene spy-l for suppressor of polyray-l. spy-l seems to suppress all of the pry-l mutant-phenotypes: pry-l; spy-l double mutant worms do not have extra rays, are no longer Muv, are not Unc, and have anteriorly placed QR descendants. spy-l could suppress pry-l either by preventing ectopic HOM-C expression, or by preventing ectopically expressed HOM-C genes from having any effect. mab-5-lacZ expression was examined and found to be normal in the p1y-l; spy-l double, so wild-type spy-l is required for the ectopic HOM-C expression seen in pry-l mutant animals. The fact that there is a QL migration defect in worms mutant for spy-l alone suggests that spy-l may normally be required to turn on mab-5 in QL. We compared expression of mab-5-lacZ in wild- type and spy-l mutant worms at 3.5-5 hours and at 8-10 hours after hatching. At 3.5-5 hours 100% (n=20) of the mutant and 95% (n=20) of the wild-type worms had expression in QL. At 8- 10 hours 0% (n=30) of the mutant worms has expression of mab- 5 in the descendants of QL, but 87% (n=30) of the wild-type worms showed expression. It appears that spy-l is not required to initiate mab-5 expression in QL, but that it is required to maintain expression once it is initiated. It is possible that spy-l is required to maintain the ON state of a number of HOM-C genes, in a manner analogous to the brahma and trithorax genes of Drosophila. spy-l is located on X, in a region well covered by cosmids. Hopefully a molecular analysis of spy-l along with the isolation and study of more spy-l alleles will enable us to determine how similar the C. elegans and Drosophila maintenance systems are, and how spy-l and other maintenance genes are used to maintain and specify pattern.

Grant BD et al. (1994) Worm Breeder's Gazette "sel-1, a Negative Regulator of lin-12 and glp-1 Activity, Encodes a Novel Extracellular Protein."

Greenwald IS, Grant BD

[Worm Breeder's Gazette, 1994]

sel-l, a negative regulator of lin-12 and glp-l activity, encodes a novel extracellular protein. Barth Grant and Iva Greenwald HHMI, Columbia University Dept. of Biochemistry, New York, NY 10032

Vladimir V Bakaev et al. (2003) Worm Breeder's Gazette "An attempt to slow aging in C. elegans. 31. No positive effect of glycerol"

Lyudmila M Bakaeva, Vladimir V Bakaev

[Worm Breeder's Gazette, 2003]

The purpose of this study was to investigate the effect of glycerol in water solutions on the nematode life span. In this experiment glycerol was used in following dilutions: 100 g/L, 10 g/L, 1 g/L, 100 mg/L, 10 mg/L, 1 mg/L and 0.1 mg/L. Three adult animals (3-5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and without glycerol) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (without glycerol in medium) every day (one worm in one well) beginning from third day. Then, beginning from 3rd day, these worms were transferred every day in next wells containing medium with glycerol in any concentration. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table.

Yandell MD et al. (1994) Worm Breeder's Gazette "ceg-1 is Very Similar to decapentaplegic."

Wood WB, Yandell MD

[Worm Breeder's Gazette, 1994]

CEG-I IS VERY SIMILAR TO DECAPENTAPLEGIC Mark Yandell and Bill Wood Department of MCD Biology, University of Colorado, Boulder We have cloned a TGF-beta homolog which we have provisionally called ceg-l for C. elegans growth factor-l. ceg-l is a member of the dpp/bmp2-4 subclassl of TGF-beta proteins (see PILEUP diagram) and is very similar to the Drosophila gene decapentaplegic (dpp) and the mouse gene GDF-5, the product of the brachypodism locus, alleles of which cause defects in limb morphologyv.2 We previously reported (WBG 12(5): 82) that ceg-l maps to chromosome V just to the left of the cluster. We have now mapped ceg-l to the genetic map by PCR from single dead embryos homozygous for either the deficiency sDf30 or the deficiency sDf20 and find that it maps to the cluster of lethals near dpy-l l which lie under both deficiencies. Developmental Northern blots show that ceg-l encodes a moderately rare transcript that is present in late embryos and L1 larvae. Sequencing of genomic and cDNA clones reveal that the ceg-l gene has eight introns and encodes a 1.7kb message which is trans-spliced to SL1. We are now preparing to inject heat-shock and LacZ contructs. We are also collaborating with Ronald Plasterk to obtain a Tc1 insertion in ceg-l. Kingsley, D. M., Genes and Development 8: 133-146, 1994. 2 Storm, E. E., Huynh, T. V., Copland, N. G., Jenkins, N. A., Kingsley, D. M., and Lee, S. Nature 368: 639-643. 1994.

Hong K et al. (1992) Worm Breeder's Gazette "A Method for Direct DNA Sequence Analysis of PCR Amplified DNAThat Works Very Well"

Driscoll MA, Hong K

[Worm Breeder's Gazette, 1992]

We have begun DNA sequence analysis of 50 recessive mec-4 mutations. By combining aspects of a few different protocols we have come up with a method for sequencing of PCR products that is reliable and gives high quality data. We hope that this protocol may be of use to others involved in sequencing projects! A. Generation of the template DNA for sequence analysis 1. Amplify genomic DNA in standard PCR reaction. We use 2 g genomic DNA and approximately .5 g each primer (our primers average 21 bp in length) in 100 l reaction volume. 2 Run an agarose gel to assay the concentration of amplified DNA. Ideally, the concentration of DNA should be at least 1 g in 10 l. If the DNA concentration is very low, either ethanol precipitate to concentrate it for the next step or reamplify. Also if large visible amounts of primers remain, it is good to remove them as outlined in Step 4 below. 3 Amplify one strand of DNA. In separate experiments we have used either a new primer internal to the original boundaries of the PCR product or we reused on of the original primers for the single stranded amplification. Both approaches work quite well. Usually we use 10 l of the DNA generated in Step 1 above (About 1 g if possible) and about 200 ng of primer in 100 l volume. 4. Purify the ss amplified DNA away from unincorporated primers by passing over Miniprep Spun Columns form Pharmacia. Alternatively, make homeade columns with Sephacryl S-400. 5. Check DNA concentration on an agarose gel. For certain success, about 2 g of single-stranded DNA is used in each sequencing reaction. If the DNA concentration is too low, DNA can be ethanol precipitated to concentrate the sample or a second single-stranded amplification step is suggested. The amount of DNA template is very important. B. Denaturation of the template and annealing of sequencing primer 1. Add 8 l of template DNA and 1 l of 1 M NaOH to a 1.5 ml microcentrifuge tube; incubate at room temperature for 10 minutes 2. Add 1 l of 1 M HCl to neutralize the solution and then immediately add 2 l (about 200 ng) of sequencing primer (from the complementary strand, of course) and 2 l of sequencing buffer (as in Sequenase kit from US Biochemical). Note: perform this step on each tube individually, ie., if you are doing 10 reactions do not add HCl to each tube and then go back and add primers. Neutralize the first tube and add the appropriate primer and buffer and then start the next tube. This is important. 3. Incubate at 37 C for 15 minutes to allow the primer to anneal to template DNA C. Sequencing reactions Reactions are according to instructions in the USB Sequenase kit with a few changes: Labelling reaction: -14 l annealed DNA (the entire reaction prepared above) -1 l DMSO (added fresh) -1 l DTT. 0.1 M -2 l diluted dITP mix (1:5 dilution) -0.5 l 35S -2 l diluted sequenase* *When diluting Sequenase for reactions the dilution mix is made as follows: enzyme dilution buffer, 6.5 l pyrophosphatase, 0.5 l Sequenase, 1 l as usual, do this just before starting reactions Termination reaction: Although the starting volume is slightly larger than in the USB protocol, still add 4 l of labelled DNA into 2.5 l of each dITP for termination reactions, add 4 l stop solution. Notes: 1. We can read at least 400 bp for each reaction. 2. This procedure works very well for sequencing double stranded plasmid DNA.

Vladimir V Bakaev et al. (2003) Worm Breeder's Gazette "An attempt to slow aging in C. elegans. 32. No positive effect of peroxide"

Vladimir V Bakaev, Lyudmila M Bakaeva

[Worm Breeder's Gazette, 2003]

The purpose of this study was to investigate the effect of hydrogen peroxide in water solutions on the nematode life span. In this experiment hydrogen peroxide was used in following dilutions: 100 mg/L, 10 mg/L, 1 mg/L, 0.1 mg/L, 0.01 mg/L and 0.001 mg/L. Three adult animals (3-5 days old) were kept in microtitre wells containing 0.5 ml of liquid medium (with E. coli and without hydrogen peroxide) during 4 hours, then they were discarded and newborn larvae were transferred in next wells (without hydrogen peroxide in medium) every day (one worm in one well) beginning from third day. Then, beginning from 3rd day, these worms were transferred every day in next wells containing medium with hydrogen peroxide in any concentration. This investigation was carried out in temperature +21C and in the darkness. The obtained results are presented in the following table. Conclusion: If hydrogen peroxide solution was applied to C. elegans, it was not able to increase their mean longevity significantly in comparison with control.

Mains PE (1994) Worm Breeder's Gazette "Interactions Between mei-1 and the unc-116 Kinesin"

Mains PE

[Worm Breeder's Gazette, 1994]

Interactions Between mei-l and the unc-116 Kinesin Paul E. Mains, Dept. of Medical Biochemistry, University of Calgary, Calgary, Alberta, Canada