-
[
International Worm Meeting,
2003]
As part of a consortium of laboratories in Canada and Sweden (see McKay, et al, "Gene expression profiles in cells, tissues and development of C. elegans"), we have developed computational and biological approaches to assist in high throughput analysis of gene expression. Biological source material for SAGE libraries was generated using synchronized populations as well as micro-dissected gut tissue. We are also refining GFP-mediated cell sorting techniques to isolate other cell types from disrupted embryos. SAGE data are generated and tracked in an automated pipeline from sequencing of cloned concatamers through to annotation, quality control and mapping of individual tags. Tags are associated with attributes such as cumulative phred scores, source clone, parent ditag and tag-to-gene mapping data to aid in downstream quality filtering and analysis steps. To facilitate tag-to-gene mapping and comparison of SAGE and Affymetrix GeneChip technologies, a virtual C. elegans transcriptome with both confirmed and estimated untranslated regions (UTRs) for all known and predicted transcripts was constructed using the current gene models and expressed sequence tag (EST) data annotated in wormbase. We are also refining tag-to-gene mapping methods in order to detect previously undocumented transcripts and alternative splice variants. Comparison of the Affymetrix C. elegans chip with the current virtual transcriptome showed that about 97% of the confirmed or predicted transcripts are detected by all or part of at least one probe set. At the time of writing, four developmentally staged SAGE libraries to be used in developmental gene expression profiling have been completed. Preliminary SAGE studies and results of a direct comparison of gene expression levels measured from the same biological source material with SAGE, long-SAGE and Affymetrix GeneChip technologies will be presented. This project is funded by Genome British Columbia and Genome Canada.
-
[
East Coast Worm Meeting,
2000]
Recent reports (see below) showed that high pressure freezing (HPF) followed by freeze substitution is superior to chemical immersion fixation for C. elegans. HPF captures a more "life-like" view of the worm's ultrastructure. We compared HPF and a related technique, rapid freezing onto a metal mirror (MMF). For MMF, live animals on a small piece of filter paper are plunged against a metal mirror in liquid nitrogen. Freezing damage is often a problem, but some animals seem to be well frozen throughout. For HPF, we have tried two methods to concentrate live animals into a small metal planchette (see Lavin and McDonald ref's below). Further processing is the same for both methods. While holding at very low temperatures, the samples are freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. By TEM fast-frozen worms reveal excellent views of membrane events and organelles. For instance, we see active endocytosis events that are not captured by chemical fixation. The microtubule network is better preserved and the basal laminae look strikingly different. Sample images are shown at www.aecom.yu.edu/wormem/new.html. HPF and MMF also hold promise for high resolution immunoEM. By reducing the osmium content and adding a dilute aldehyde fixative to the freeze substitution medium, we can better preserve structure than by our microwave technique (Paupard et al., submitted). We have successfully localized epitopes in thin sections from HPF samples. We are conducting HPF trials with Stan Erlandson and Ya Chen at the U. of Minnesota. MMF equipment is available here at Einstein and elsewhere. HPF machines are available to outside users in Madison, Berkeley, Minneapolis, and Albany. As our skills improve, we will offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/coating.html Martin Muller's website at www.em.biol.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Humana Press) 1999.
-
[
Midwest Worm Meeting,
2000]
Recent reports (see below) showed that high pressure freezing (HPF) followed by freeze substitution is superior to chemical immersion fixation for C. elegans. HPF captures a more "life-like" view of the worm's ultrastructure. We compared HPF and a related technique, rapid freezing onto a metal mirror (MMF). For MMF, live animals on a small piece of filter paper are plunged against a metal mirror in liquid nitrogen. Freezing damage is often a problem, but some animals seem to be well frozen throughout. For HPF, we have tried two methods to concentrate live animals into a small metal planchette (see Lavin and McDonald ref's below). Further processing is the same for both methods. While holding at very low temperatures, the samples are freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. By TEM fast-frozen worms reveal excellent views of membrane events and organelles. For instance, we see active endocytosis events that are not captured by chemical fixation. The microtubule network is better preserved and the basal laminae look strikingly different. Sample images are shown at www.aecom.yu.edu/wormem/new.html. HPF and MMF also hold promise for high resolution immunoEM. By reducing the osmium content and adding a dilute aldehyde fixative to the freeze substitution medium, we can better preserve structure than by our microwave technique (Paupard et al., submitted). We have successfully localized epitopes in thin sections from HPF samples. We are conducting HPF trials with Stan Erlandson and Ya Chen at the U. of Minnesota. MMF equipment is available here at Einstein and elsewhere. HPF machines are available to outside users in Madison, Berkeley, Minneapolis, and Albany. As our skills improve, we will offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/coating.html Martin Muller's website at www.em.biol.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Humana Press) 1999.
-
[
West Coast Worm Meeting,
2000]
Several recent reports (see below) have demonstrated that C. elegans tissues can be very well preserved for electron microscopy by high pressure freezing (HPF) followed by freeze substitution, perhaps substantially better than by standard chemical immersion fixation. HPF shows the potential to capture a more "life-like" view of the worm's ultrastructure. We have been testing both HPF and a related technique, rapid freezing on a metal mirror (MMF) followed by freeze substitution. Both methods obtain similar high quality fixation, although there are some freezing artifacts using the metal mirror device that are eliminated in HPF. For MMF, live animals are concentrated on a small piece of filter paper and plunged against a metal mirror at liquid nitrogen temperature. While freezing damage often occurs about 5-15 microns into the worms, some animals are very well frozen throughout. The frozen samples are held at low temperature and freeze substituted into 1% osmium tetroxide in acetone, then embedded into plastic resin and cured for thin sectioning. For HPF, we have tried two methods to concentrate live animals into small metal planchette, either holding the animals within fine strands of dialysis tubing (C. Lavin, pers. comm.), or mixing them into a slurry of yeast paste to form a space-filling solid support (McDonald, 1999). Examination of fast-frozen specimens by TEM reveals excellent views of membrane events and organelles. For instance, we see many omega figures on coelomocytes which are indicative of active endocytosis, events which are not commonly captured by chemical fixation. Synaptic active zones and vesicles are well preserved, as are their relationships to microtubules. A network of microtubules can also been seen extending to the periphery of hypodermis. Basal laminae look strikingly different, much looser and more mesh-like when compared to chemical fixation. Sample images are shown on our website [www.aecom.yu.edu/wormem/new.html]. These two preparation methods, HPF and MMF, also hold great promise for high resolution immuno-EM. By reducing the osmium content and adding a dilute aldheyde fixation to the freeze substitution medium, we can obtain better resolution than is currently possible by our microwave technique. We have successfully localized epitopes in thin sections from HPF samples. MMF equipment is available here at Einstein campus. We are conducting HPF trials with the help of Stan Erlandson and Ya Chen at the University of Minnesota. As our skills improve, we will be happy to offer such services to the C. elegans community. For further information on HPF, we recommend the following sources: Colleen Lavin's website at www.geology.wisc.edu/~uwmr/caoting.html Martin Muller's website at www.em.bio.ethz.ch/ Kent McDonald, Methods in Molecular Biology, vol 117, pp. 77-97 (Human Press) 1999. In the U.S., there are HPF machines open to the outside users in Madison, Berkeley, Minneapolis and Albany.
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[
International C. elegans Meeting,
2001]
To build upon knowledge gained from the genome of C. elegans , we have begun generating Expressed Sequence Tags (ESTs) from parasitic (and free-living) nematodes. This project will generate >225,000 5' ESTs from 14 species by 2003. Additionally, the Sanger Centre and Edinburgh Univ. will complete 80,000 ESTs from 7 species. Through these combined efforts, we anticipate the identification of >80,000 new nematode genes. At the GSC, approximately 35,000 ESTs have been generated to date including sequences from Ancylostoma caninum, Heterodera glycines, Meloidogyne incognita and javanica, Parastrongyloides trichosuri, Pristionchus pacificus, Strongyloides stercoralis and ratti, Trichinella spiralis, and Zeldia punctata . We will report on our progress in sequence analysis, including the creation of the NemaGene gene index for each species by EST clustering and consensus sequence generation, identification of common and rare transcripts, and identification of genes with orthologues in C. elegans and other nematodes. All sequences are publicly available at www.ncbi.nlm.nih.gov/dbEST. NemaGene sequences and project details are available at WWW.NEMATODE.NET. We would like to thank collaborators who have provided materials and ideas for this project including Prema Arasu, David Bird, Rick Davis, Warwick Grant, John Hawdon, Doug Jasmer, Andrew Kloek, Thomas Nutman, Charlie Opperman, Alan Scott, Ralf Sommer, and Mark Viney. This work is funded by NIH-AI-46593, NSF-0077503, and a Merck / Helen Hay Whitney Foundation fellowship.
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[
International C. elegans Meeting,
2001]
Two web sites have been established to allow easier access to nematode sequences from species other than C. elegans and C. briggsae ; WWW.NEMATODE.NET is maintained by the Genome Sequencing Center (GSC) at Washington University in collaboration with North Carolina State University, and WWW.NEMATODES.ORG is maintained by Mark Blaxter's lab at the University of Edinburgh. Useful features being built for NEMATODE.NET include the following - 1) Searches: All nematode expressed sequence tags (ESTs) generated at the GSC, currently 32,000 from 10 species, and NemaGene clusters built from these ESTs, are available for BLAST and text searching. Searches can be directed by species, library, or nematode clade in a way that is not possible using the NCBI EST database dbEST. 2) FTP: All EST project data can be downloaded for local analysis including FASTA files and sequence trace image files. 3) Trace Viewer: Fluorescent trace representations for each EST can be viewed. Traces can sometimes provide additional sequence information not included in the EST due to quality value cut-offs. 4) Project Updates: Information is available about libraries in construction and sequencing in progress as the project expands toward 235,000 ESTs. 5) Clone Requests: Details on clone availability and ordering procedure are provided. 6) Links: The site includes an up-to-date set of 300 links to information on human, animal, and plant parasitic nematodes. Further plans for NEMATODE.NET include linking of ESTs to their closest C. elegans homologues by DAS third-party curation of Wormbase. This work is funded by NIH-AI-46593, NSF-0077503, and a Merck / Helen Hay Whitney Foundation fellowship.
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[
West Coast Worm Meeting,
1996]
Construction of a chromosome IV cosmid transgenic library of C. elegans strains carrying sequenced cosmids in extrachromosomal arrays has begun as part of our labs' collaboration with the Genome Sequencing Consortium (see abstract by D. Janke et al.). There are 20 chromosome IV cosmids available in transgenic strains at the time of this writing, and we estimate a total of 40-50 will be available to the community by the end of July. In order to enhance the utility of this library, we are also using these strains to localize approximately 50 essential gene mutations, previously identified in the Baillie lab, to cosmids by way of transgenic rescue experiments (for results of similar efforts on chromosomes III and V, see abstracts by Nigel O'Neil et al., Helen Stewart et al., and Brad Barbazuk et al.). Correlation of the genetic and physical maps in this manner will facilitate the selection of candidate cosmids for use in further rescue experiments, and thereby increase the speed with which the connection between genetic mutations and DNA sequence is made. Currently these rescue experiments are focused in the sDf2 region, in particular in the
lin-3 -
let-60 interval, which contains 13 essential gene mutations and 21 overlapping cosmids. The two genes bracketing this interval,
lin-3 and
let-60(ras), were previously placed on the physical map by others. To date, two additional essential genes have been positioned to cosmids on the physical map;
let-70(
ubc2) (in collaboration with Mei Zhen and Peter Candido) and
let-64. It is anticipated that by the time of the meeting several additional mutations will have been positioned to cosmids. This work is being funded by a grant from the Canadian Genome and Associated Technologies Program to Ann Rose and David Baillie.
-
[
International C. elegans Meeting,
2001]
We continue to test alternate methods for preparing worms for transmission electron microscopy. We will describe new protocols, and will demonstrate what makes them better [or different] in comparison to previous methods (Hall, 1995). We still like simple immersion fixation and chopping open the animals by knife blade, and have made minor changes in the starting solutions to get optimum results. For early larval stages, which have never fixed well by immersion, and which are too little to chop open easily, we have adapted a new microwave protocol which gives very good results on intact worms. The resulting fixation looks equivalent to our immersion preparations of adults. Microwave fixation is proving very useful in the analysis of arrested animals from RNAi preparations, and should be excellent for looking at late embryos or dauers. Fast freezing methods offer a quite different approach, and the quality of tissue preservation can be superb. Both metal mirror freezing and high pressure freezing can produce excellent results, and they are achieving wider use over the past few years (Mohler et al., 1998; Rappleye et al., 1999). The inherent contrast after freeze substitution is often much greater, in part because the primary fixation contains only osmium, or a combination of osmium and aldehyde together. These methods allow much more rapid fixation. We can capture more "life-like" views of biological events in action, particularly for events such as vesicle fusions at the plasma membrane. Delicate cytoskeletal elements such as microtubules are also well preserved. We continue to try new combinations of fixatives and solvents to improve the appearance of nerve processes and synapses by fast freezing. Kent McDonald has been very helpful in suggesting improvements to these protocols. Laserhole fixations of embryos are technically rather difficult to accomplish, but can facilitate the passage of fixatives and embedding resins through the eggshell. We are continuing to use the protocol worked out by Carolyn Norris. See our website for details.
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[
International C. elegans Meeting,
1991]
1. UV radiation mutagenesis in C. elegans: Helen Stewart and David Baillie have noted that many UV radiation-induced mutations are chromosomal rearrangements rather than point mutations. We have begun to extend this observation by isolating UV radiation-induced mutations in
fem-3 and
unc-22. As suggested to us by Judith Kimble, and as will be described at the meeting,
fem-3 is particularly well suited for these studies. Mutations have been selected at low and high radiation fluences, as well as in both rad(+) and rad(-) genetic backgrounds. These are currently the subject of both genetic and molecular analyses, with the ultimate hope of elucidating specific sequence contexts which are misrepaired into deficiencies. 2. Cloning a rad gene: We have isolated two allelic mutations in the high-hopper strain TR679 which confer hypersensitivity to methyl methanesulfonate and UV radiation. They map in between
unc-36 and
dpy-17 on linkage group III. Each mutation was crossed 12 times into an N2 background, several of which including forced recombinations in between the mutations and either
unc-36 or
dpy-17. Southern blots were performed, using Tcl as a probe (kindly provided by Phil Anderson) and revealed one novel Tc l- containing fragment in one mutant and three in the other. We are currently cloning these. Since both mutants are hypersensitive only during embryogenesis, we look forward to performing Northern blot analyses in order to determine if this developmental regulation of DNA is exercised at the transcriptional level. 3. The rad mutants are not hypersensitive to 8-methoxypsoralen (8MOP) phototreatment. Extending the observations of Fujita and coworkers (Photochem. Photobiol. 39, 831), survival of three staged populations N2 and four rad mutants was determined after 8-MOP phototreatment. As opposed to their responses after exposure to other DNA-damaging agents, none of the four were hypersensitive. Split-dose experiments indicated that DNA-DNA crosslinks were primarily responsible for lethality. Little if any crosslink repair was observed in N2 using two different assays, explaining why the rad mutants were not hypersensitive to 8-MOP inactivation.
-
[
West Coast Worm Meeting,
1996]
We have isolated 202 new, EMS induced, recessive lethal mutations on the left arm of LGIII. The free duplication sDp3 (III;f) was used to rescue recessive mutations linked to
dpy-17 (recovery rate 1.9%). Of these 202 mutations, 109 have been characterized and analysis of the remaining mutations is in progress. A subset, of 62 recessive lethals, are also marked with
ncl-1 and will facilitate the mosaic analysis of these lethal mutations. We have identified 82 new essential genes
let-700 to
let-790. Of these new essential genes, 13 have multiple alleles, thus we estimated that we have only a 28% saturation of the area for essential genes. As there is about 4.8 Mb of DNA, in the interval between
unc-93 and
dpy-19, we estimate that there will be approximately 293 essential genes, in this region. We intend to generate another set of recessive lethal mutations to facilitate saturation of this area for essential genes. Both gamma rays and ultraviolet light were used to generate a set of overlapping deficiencies. We have shown that duplications can be used to rescue large deletions such as sDf121, sDf125, sDf127 and sDf130, as well as smaller deficiencies. This indicates that there are no "holes" or lethal mutations along the length of sDp3, in the areas uncovered by these large deficiencies. We have shown that duplications, such as sDp3, are valid and useful as balancer systems and have utilized deficiency mapping to resolve the genetic mapping of essential genes. Selected visible mutations have also been complementation tested with our deficiencies. PCR analysis was utilized, with these deficiencies, to correlate the genetic and physical maps and to further define the end points of these deficiencies ( See poster ; Norm Franz et al, this session). As well, work has been progressing on the rescue of essential genes (See poster; Helen Stewart et al, this session), utilizing transgenic strains constructed in this laboratory (see presentation by Diana Janke et al, this session). This work has been funded by a grant from NIH (National Institute of Health), U.S.A. to Ann. Rose; D. Riddle and D.L. Baillie.